€ ji CD' 5; eO r- ru AGRICULTURAL AND BIOLOGICAL PUBLICATIONS CHARLES V. PIPER, CONSULTING EDITOR AN INTRODUCTION TO CYTOLOGY °Ms Qraw~3/ill Book (n 7m PUBLISHERS OF BOOKS F O R_/ Coal Age v Electric Railway Journal Electrical World v Engineering News-Record American Machinist v Ingenieria Internacional Engineering 8 Mining Journal ^ Power Chemical & Metallurgical Engineering Electrical Merchandising lllfllllllflllllllllfllllll AN INTRODUCTION TO BY LESTER W. SHARP CORNELL UNIVERSITY "The most important discoveries of the laws, methods and progress of nature have nearly always sprung from the examination of the smallest objects which she contains, and from apparently the most insignificant enquiries." — Lamarck, Philosophic Zuoloyiquc. FIRST EDITION McGRAW-HILL BOOK COMPANY, INC. NEW YORK: 370 SEVENTH AVENUE LONDON: 6 & 8 BOUVERIE ST., E. C. 4 1921 COPYRIGHT, 1921, BY THE MGGRAW-HILL BOOK COMPANY, INC. MAPLE PHE8S TOKK FA . p CD W 00 P° CD CO <0 p p p CD "CD CD COlO H wo 3 I CD O M CD O* CD CD & 3 CD cn CO P- Ol w, H- CD & 0) H' 3 CD ^ O CD CD & B o4 ^3 H- CD 3 CD >-b O ^ CD §, O CD P- O OQ O 3 H- CO 3 M O ^ O r 1 H< O M O "* p 0 *^3 O OD O CD ^^ P C+ f-j'pS <^ O O H" W ^3 ^* * LJD H) n!p 01 O 3 PI , **§? i ., CD **> £ ** o OQ »i ^ P ® M P 25 * P> (OS •STQ P H-1 W P. S5 7Q CD CONTENTS PAGE PREFACE vii CHAPTER I HISTORICAL SKETCH 1 The discovery of the cell — Preformation and epigenesis — Early theories of cell-formation — Early observations on the cell contents — The foundation of the cell theory — Elaboration of the cell theory — The protoplasm doctrine — The new conception of the cell — Fertilization and embryogeny — The be- ginning of the modern period in cytology — Bibliography 1. CHAPTER II PRELIMINARY DESCRIPTION OF THE CELL 23 Description of the cell — The differentiation of cells — Bibliography 2. CHAPTER III PROTOPLASM 32 Physical properties — Protoplasm as a colloidal system — Microdissection — Chemical nature of protoplasm — Varieties of protoplasm — The plasma membrane — Protoplasmic connections — Vacuoles — Protoplasm as the sub- stratum of life— Micromeric theories — Chemical theories — Conclusion — Bibliography 3. CHAPTER IV THE NUCLEUS 59 Occurrence — General characters — Nucleoplasmic ratio — Structure of nucleus — Nuclei of bacteria and other protista — The function of the nucleus — Bibliography 4. CHAPTER V THE CENTROSOME AND THE BLEPHAROPLAST 76 The centrosome — Occurrence and general characters — Individuality — Centrosomes in Algae — Fungi — Bryophytes — Conclusion — The blepharo- plast — Occurrence — In flagellates — In thallophytes— In byrophytes — In pteridophytes — In gymnosperms — In animals — Conclusion — Bibliography 5. CHAPTER VI PLASTIDS AND CHONDRIOSOMES 103 Plastids — General nature and occurrence — Leucoplasts — Chromatophores — Starch — The pyrenoid — Elaioplasts and oil bodies — The eyespot — The individuality of the plastid — Chondriosomes — General nature and occur- rence— Physico-chemical nature — Origin and multiplication — Function — Relation of chondriosomes to plastids — Conclusion — Bibliography 6. CHAPTER VII METAPLASM; POLARITY 133 Metaplasm — Extruded chromatin — The senescence of the cell — Polarity — Metabolic gradient — Bibliography 7. xi xii CONTENTS CHAPTER VIII PAGE SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 143 Somatic mitosis — Preliminary sketch of mitosis — Detailed description of the behavior of the chromosomes in somatic mitosis — Chromomeres — Summary — The individuality of the chromosome — The frequent persistence of visible chromosome limits in the resting reticulum — Prochromosomes — Persistence of parental chromosome groups after fertilization — Size and shape of chromosomes — Chromosome number — Discussion and Conclusions — Bibliography 8. CHAPTER IX THE ACHROMATIC FIGURE, CYTOKINESIS, AND THE CELL WALL 175 The achromatic figure — In higher plants — In animals — Intranuclear figures— Origin of the figure — The mechanism of mitosis — Cytokinesis — In thallophytes — In microsporocytes — In animals — Mechanism of furrowing —The cell wall — The primary wall layer — Secondary and tertiary wall layers — The physical nature of the cell wall — The chemical nature of the cell wall — The walls of spores — Bibliography 9. CHAPTER X OTHER MODES OF NUCLEAR DIVISION 202 Cyanophyceae — Protozoa — Other cases in plants — Amitosis — Amitosis and heredity — Bibliography 10. CHAPTER XI THE REDUCTION OF THE CHROMOSOMES 219 Discovery — The stage in the life cycle at which reduction occurs — The meaning of reduction — Interpretations based on Weismann's theory — Somatic and heterotypic mitoses compared — Modes of chromosome reduc- tion— Scheme A — Scheme B — Comparison of Schemes A and B — Reduction with chromosome tetrads — Numerical reduction without qualitative reduc- tion— Synapsis, or chromosome conjugation — Relationship of the synaptic mates — The stage at which conjugation occurs — The nature of the synaptic union — Chromomeres — Other opinions on the heterotypic prophase — Bibliography 11. CHAPTER XII FERTILIZATION 273 Fertilization in animals — The gametes — The fusion of the gametes — Fertilization in protozoa — The physiology of fertilization — Fertilization in plants — Algse — Fungi — Bryophytes and pteridophytes — Gymnosperms — Angiosperms — Chromosome behavior — Endosperm — Bibliography 12. CHAPTER XIII APOGAMY, APOSPORY, AND PARTHENOGENESIS 311 Apogamy — Apospory — Parthenogenesis in animals — Bibliography 13. CHAPTER XIV THE ROLE OF THE CELL ORGANS IN HEREDITY 323 The law of genetic continuity — The role of the nucleus — The promorphology of the ovum — Plastid inheritance — Aleurone inheritance — General con- clusions. CONTENTS xiii CHAPTER XV PAGE MENDELISM AND MUTATION 336 Mendelism — A typical case of Mendelian inheritance — The cytological basis of Mendelism — Mutation — Mutations accompanied by changes in chromosome number — Bearing on the origin of species and varieties — Mutations accompanied by no change in chromosome number — Conclusion. CHAPTER XVI SEX 354 Experimental evidence for sex-determination — Sex-chromosomes — Sex- chromosomes and Mendelism — Experimental alteration of the sex ratio — Metabolic theories of sex — General discussion. CHAPTER XVII LINKAGE -. . 378 A typical case of linkage — Sex-linkage — Non-disjunction — Linkage groups — The chiasmatype theory — Application of the chiasmatype theory to the problem of linkage — General discussion — Other theories of linkage — Value of chromosome theory of heredity. CHAPTER XVIII WEISMANNISM AND OTHER THEORIES 398 Darwin's hypothesis of pangenesis — DeVries's theory of intracellular pangenesis — Nageli's idioplasm theory — Weismann's theory — Some modern aspects of Weismannism — Non-factorial theories — A chemical theory of heredity — Conclusion — Bibliography 14 (for Chapters XIV-XVIII). INDEX. . 427 CHAPTER I HISTORICAL SKETCH The history of cytology falls naturally into three periods, of which the first begins with the discovery of the cell by Robert Hooke in 1665, the second with the foundation of the Cell Theory by Schleiden and Schwann in 1838-9, and the third with the important researches of Strasburger, Hertwig, Biitschli, and others between 1870 and 1880. In the present sketch attention will be confined almost entirely to the first two periods, the work of the third, or modern, period being dealt with in the other chapters of the book. Prior to the seventeenth century attempts to analyse the structure of organisms were necessarily unsatisfactory. Aristotle (384-322 B.C.) in his De Partibus Animalium distinguished the "homogeneous parts" and the "heterogeneous parts," the former corresponding in general to what we classify as tissues (bone, fat, cartilage, flesh, blood, lymph, nerve, membrane, nails, hair, skin, vessels, tendon, etc.), and the latter being the larger members of the body (head, face, hands, feet, trunk, etc.). Theophrastus, the pupil and successor of Aristotle, taught in his Historia Plantarum that the plant body is composed of "sap," "veins," and "flesh." Aristotle's classification was developed further by Galen (131-201 A.D.) and by his followers. Although we no longer regard the above components as elementary parts, but rather as tissues and organs, the ancients may be pardoned for not carrying the analysis further, for they did not possess the necessary instruments. Something was then known about the refraction of light, but it was not until many centuries later that suitable lenses were available. The first compound microscope was brought out in 1590 by J. and Z. Janssen, spectacle makers of Middle- burg, Holland; and during the first part of the seventeenth century other improved models were designed by other workers. These instru- ments in the hands of men possessed of scientific curiosity soon led to many significant discoveries. A new world was opened to the eye of science, and the compound microscope has since remained an instru- ment of extraordinary value in biological research. 1 2 INTRODUCTION TO CYTOLOGY The Discovery of the Cell. — Cytology may be said to have begun with the discovery of the cell by Robert Hooke (1635-1703) in 1665. Hooke, who lived in London and has been described as a man of eccentric appear- ance and habits, showed a remarkably varied scientific activity. For a time he was a professor of geometry, and later became an architect. He performed many original experiments in mechanics and for a number of years was curator of experiments to the Royal Society. His interest in optics led him to examine all sorts of objects with the compound micro- scope. In charcoal and later in cork and other plant tissues he found small honeycomb-like cavities which he called "cells." He had no dis- tinct notion of the cell contents, but spoke of a "nourishing juice," which he inferred must pass through pores from one cell to another. His many observations were embodied in his Micrographia (1665), a large work illustrated with 83 plates. The chapter containing his re- marks on cells is entitled "Of the schematisme or texture of cork and the cells and pores of some other such frothy bodies." Quaint and crude as it now appears to us, the Micrographia takes its place as the earliest cytological classic. Three other names even more prominent in the early history of micro- scopy are those of Malpighi, Grew, and Leeuwenhoek. Marcello Mal- pighi (1628-1694), an Italian physiologist and professor of medicine at Bologna, Pisa and Messina, is best known for his important pioneer work in anatomy and embryology. Most of'his observations on plants were included in his Anatome Plantarum (1675) and had to do largely with the various kinds of elements making up the body of the vascular plant. Malpighi made a distinct step in advance in studying tissues with the cell as a unit; a clear fore-shadowing of the Cell Theory is seen in his remarks concerning the importance of the "utriculi" in the structure of the body. At Pisa Malpighi was associated with G. A. Borelli, who was one of the first to use the microscope on the tissues of higher animals. Nehemiah Grew (1641-1712) was an English physician and botanist. He began a careful study of plant structure in 1664, and in 1670 read his first important paper before the Royal Society. Further contributions followed at intervals until 1682, when all of them were published under the title The Anatomy of Plants. Like Malpighi, an abstract of whose first work on plants was presented to the Royal Society in 1671, Grew was interested in tissues, and gave particular attention to the combinations of these tissues in different plant organs. He was strongly impressed by the manner in which the cells, which he also called "vesicles" and "bladders," appeared to make up the bulk of certain tissues : " . . . the paren- chyma of the Barque," he said, "is much the same thing, as to its con- formation, which the froth of beer or eggs is, as a fluid, or a piece of fine Manchet, as a fixed body" (p. 64). He further believed the walls of the cells to be composed of numerous extremely fine fibrils : in the vessels HISTORICAL SKETCH 3 or longitudinal elements these fibrils were wound in the form of a close spiral, while the vessels themselves were bound together by a transverse series of interwoven threads. He accordingly compared the structure of the plant with that of a basket, and with "fine bone-lace, when the women are working it upon the cushion" (p. 121). Antony van Leeuwenhoek (1632-1723) of Delft is remembered for his pioneer researches in the field of microscopy. He constructed a number of simple lenses of high power, and with these he was able to see for the first time certain protozoa, bacteria, and other minute forms of life. In the course of his investigations he observed the cells ("globules") in the tissues of higher organisms. His work, in spite of the fact that it was carried on without any definite plan, brought to light a number of important facts, but in general his accomplishments do not bear favorable comparison with those of Grew and Malpighi. Preformation and Epigenesis. — After the death of Leeuwenhoek there ensued a period during which the actual investigation of the cell and the structure of organisms remained practically at a standstill. At that time, however, certain speculations were indulged in which should be recorded here, not because they can be regarded as scientific cytology but because of the influence they exerted upon the formulation of many cytological problems in later years. These speculations resulted in the division of the biologists of the day into two schools, the main question at issue being the manner in which the embryo develops from the egg. The two theories formulated in answer to this question have been Called the Preformation Theory and the Theory of Epigenesis. According to the Preformation Theory, the basis for which was laid in the seventeenth century works of Swammerdam, Malpighi, and Leeuwenhoek, the egg contains a fully formed miniature individual, which simply unfolds and enlarges as development proceeds. Because of this unfolding the theory was also known as the Theory of Evolu- tion, a phrase which has a quite different connotation today. In the eighteenth century the preformation idea was carried to an absurd extreme by Bonnet (1720-1793) and others, who argued that if the egg contains the complete new individual, the latter must in turn contain the eggs and individuals of all future generations successively encased within it, like an infinite series of boxes one within another. This theory of encasement (emboitemenf) was a logical deduction from the since abandoned premise that everything, including organisms for all time, had been formed by one original creation, and that nothing could there- fore be formed anew. The preformationists soon became separated into two groups: the spermists or animalculists, and the ovists. By the former the new individual was supposed to be encased in the sperma- tozoon, and figures were actually published showing a small human figure, or "homunculus," within the sperm head. The ovists, on the contrary, 4 INTRODUCTION TO CYTOLOGY held that the individual is encased in the egg. A bitter strife was carried on over this question by the two groups of preformationists, and various interesting compromises were made. But all extreme forms of preforma- tionism were to disappear in the light of more critical investigations, which went far to support the opposing Theory of Epigenesis. Two of the early champions of the Theory of Epigenesis were William Harvey (1578-1667; Exercitationes de Generatione Animalium, 1651), and Caspar Friedrich Wolff (1733-1794; Theoria Generationis, 1759). As the result of many careful observations on the embryogeny of the chick Wolff was able to show beyond question that development is epigenetic: neither egg nor spermatozoon contains a formed embryo; development consists not in a process of unfolding, but in "the continual formation of new parts previously non-existent as such" (Wilson). Here there was room for the principle of true generation, or "the production of heterogeneity out of homogeneity." The Theoria Genera- tionis is to be regarded as one of the really great contributions to biological science, for the Theory of Epigenesis, to which it furnished substantial support, later became established with modifications as a fundamental principle of embryology, particularly through the work of von Baer in the nineteenth century. In commenting on preformation and epigenesis Whitman (1894) emphasizes the fact that the tendency of modern biology has not been to show the entire falsity of either or both of these views, but to seek out the germs of truth possessed by each, and to relate them to modern biological conceptions. "The two views missed the mark by over-shots in contrary directions," says Whitman. The one theory claimed too much preforma- tion: everything was preformed at the start. The other theory claimed too much postf ormation : everything was formed anew. Our present position, although it excludes both views in their crude original form, involves in a new sense both conceptions. When we say that the egg is organized, possessing an architecture or mechanism in its cytoplasm or nucleus which largely predetermines development, we are making a modernized statement of the preformation idea. When we say that the parts of the individual are in no way delineated in the egg, but are mainly determined by external conditions during the course of development, we are speaking in terms of modern epigenesis. "The question is no longer whether all is preformation or all postf ormation; it is rather this : How far is post-formation to be explained as the result of pre-f ormation, and how far as the result of external influences?^ When it is borne in mind, therefore, that one of the outstanding problems of modern cytology is that of identifying the factors involved in the development of an organ- ized and highly differentiated individual from an organized but relatively undifferentiated egg cell, it is at once evident that our sketch of cyto- logical history would be incomplete without the above reference to the early Theories of Preformation and Epigenesis. HISTORICAL SKETCH 5 Early Theories of Cell-formation. — The researches of Hooke, Malpighi, and Grew in the seventeenth century had shown that "cells," or "globules," are important structural elements in organisms. When attention was again directed to such matters in the eighteenth century there was very soon felt a need for a theory which would account for the origin of cells. We may briefly review some of the suggestions which were offered. One of the earliest theories of cell-formation was that put forward by Wolff in the Theoria Generationis. According to Wolff, every organ is at first a clear, viscous fluid with no definite structural organization. In this fluid cavities (Blaschen; Zellen) arise and become cells, or, by elongation, vessels. These may later be thickened by deposits from the " solidescible " nutritive fluid. The cavities, or cells, are not to be regarded as independent entities; organization is not effected by them, but they are rather the passive results of an organizing force (vis essen- tialis) inherent in the living mass. Three important points in Wolff's theory should be noted because of the relation they bear to subsequent conceptions of the role of cells : the spontaneous origin of the cell, the organization of parts by differentiation in a homogeneous living mass, and the passive role of the cell in this organizing process. This theory was adopted in 1801 by C. F. Mirbel (1776-1854), who further believed that the cells communicate through pores in their walls. K. Sprengel (1766-1833) stated that cells originate in the contents of other cells as granules or vesicles which absorb water and enlarge. Sprengel's observations seem to have been very poorly made, for he evidently mistook starch grains for the "vesicles" which were supposed to grow into new cells. But Sprengel's theory was upheld by L. C. Treviranus (1779-1864) in a work appearing in 1806, and both men fought many years for its support. Kieser (1812) further developed the theory that granules in the latex are "cell germs" which later hatch in the inter- cellular spaces to form new cells. With a much clearer understanding of the nature of the problems involved a number of excellent observations were made by J. J. Bern- hardi in 1805, by H. F. Link and K. A. Rudolphi in 1807, and by J. J. P. Moldenhawer in 1812. It is to be regretted that the deserved attention was not given to their views, for they promised to lead in the right direction. A number of years later Mirbel, in a work on Marc/ianto'a (183 1-1833), distinguished three modes of cell-formation: (1) the formation of cells on the surface of other cells, (2) the formation of cells within older cells, and (3) the formation of cells between older cells. The first mode apparently represented the budding of the germ tube arising from the spore, while the second and third modes were formulated as the result of a misinterpreta- tion of the process of cell-multiplication in growing gemmae. 6 INTRODUCTION TO CYTOLOGY Hugo von Mohl (1805-1872), in spite of his many valuable observa- tions on the growth of algae, in 1835 agreed essentially with Mirbel. He made a step in advance, however, when he described carefully for the first time the division of a cell. We shall see further on that von Mohl's later researches contributed largely to the upbuilding of an adequate theory of the cell. F. J. F. Meyen (1804-1840) held that there are three fundamental forms of elementary organs: cells, spiral tubes, and sap vessels. He noted the wide occurrence of cell-division but did not describe the process in detail. Meyen apparently made the first attempt to distinguish cell- division from the free cell-formation described by previous workers. It has been pointed out by Sachs that if this short step had been clearly taken earlier the peculiar theory of cell-formation later developed by Schleiden would have been impossible. Von Mohl also had made obser- vations ruling out Schleiden's idea, but his excessive caution prevented him from making a decisive statement on the subject. H. J. Dutrochet (1776-1847) in 1837 described the body as being composed of solids and fluids, the former being aggregations of cells of a certain degree of firmness, and the latter, such as blood, being made up of cells freely floating. He believed that although the cell contents may be more or less solid, the highest degree of vitality is compatible only with the liquid condition. He further recognized muscle fibers as elongated cells. To all the above workers the important elementary unit was the "globule." It was customary to refer to this conception as the Globular Theory, in contradistinction to the curious and fanciful Fiber Theory put forth by Haller (1708-1777) many years before (1757), according to which the organism is made up of slender fibers cemented together by "organized concrete." For some the term "globule" stood for the granules seen in the cell contents, whereas for others it meant the cell itself. As observations multiplied and ideas became more definite the Cell Theory of Schleiden and Schwann was more and more distinctly fore- shadowed. Before turning to the Cell Theory, however, we must notice briefly a few observations which had been made on the cell contents. Early Observations on the Cell Contents. — Although the true nature and significance of the contents of cells were not recognized until many years later, a number of early investigators had seen protoplasm and had been impressed by certain of its activities. As early as 1772 Corti, and a few years later Fontana (1781) saw the rotation of the "sap" in the Characeae and other plants. After being long forgotten these facts were rediscovered by L. C. Treviranus (1811) and G. B. Amici (1819), whereupon Horkel, an uncle of Schleiden, called attention to the earlier work of Corti. Protoplasmic circulation of the more complex type was discovered in the stamen hairs of Tradescantia by Robert Brown in 1831, and other workers, especially Meyen, soon added other cases. HISTORICAL SKETCH 7 During the first third of the nineteenth century no name is of greater interest to cytologists than that of Robert Brown (1773-1858). Al- though he is famous chiefly for his great taxonomic monographs and his morphological work, he is known in cytology as the man who is usually given the credit for the discovery of the nucleus, which he announced in 1831. Although it was Brown who was impressed by the probable importance of the nucleus, and who concluded in 1833 that it is a normal cell element, certain other observers, notably Fontana, who described a nucleus in 1781, and Meyen, who saw it in Spirogyra in 1826, should share the honor for its discovery. The phenomenon which has since been known as "Brownian movement" was seen by Brown in 1827. The first period in the development of our subject is seen to have been one in which there was a tendency to indulge in speculation to an extent quite unwarranted by the facts at hand. As we have already pointed out, however, this speculation was of considerable importance to us, in that it had to do with questions which later became central prob- lems of cytology. Carefully made observations were meanwhile in- creasing in number and varie(y$, and the time eventually became ripe for the formulation of a theory which would correlate these data and give a definite trend to cytological investigations. Such a theory was soon forthcoming. The Foundation of the Cell Theory. — The year 1838 marks an epoch in the history of biology. In this and the following year Schleiden and Schwann founded the Cell Theory, which, in view of its enormous in- fluence upon all branches of biological science, may be regarded as second in importance only to the Theory of Evolution. We have seen that cells had been observed by various workers during many years, and had been recognized as being constantly present in the bodies of living organisms, but it remained for Schleiden and especially Schwann to formulate a comprehensive theory embracing the known facts and affording a start- ing point for further researches. The Cell Theory stated primarily that the body is composed entirely of cells and their products, the cell being the unit of structure and function and the primary agent of organization. Subsidiary to this was Schleiden's theory of cell-formation, which should not be confused with the main thesis just stated. Matthias Jakob Schleiden (1804-1881) is one of the most prominent and interesting characters in botanical history. He studied law at Heidelberg, medicine at Gottingen, and botany at Berlin, where he met Schwann and Robert Brown. The association of these men undoubtedly meant much to the future of botany and zoology. Eventually Schleiden became Professor of Botany at Jena, where he remained for 23 years. Schleiden was famous not merely because of his own work, but chiefly as the result of the tremendous impetus which he gave to investigation. He sought to place botany on a scientific footing equal to that of physics and chemistry, and insisted upon accurate observation and developmental studies as the basis of morphology. Sachs says: " Endowed with some- what too great love of combat, and armed with a pen regardless of the wounds it inflicted, ready to strike at any moment, and very prone to exaggeration, Schleiden was just the man needed in the state in which botany then was." Theodor Schwann (1810-1882) was associated as a student with Johannes M tiller, the great physiologist, first at Wiirzburg and later at Berlin. It was in the latter place that he put forth his statement of the Cell Theory. Immediately afterward he went to Louvain, where he was a professor for nine years, and later transferred to Liege. In disposition he contrasted strongly with Schleiden, being described as "gentle and pacific." It is said that Schleiden, while dining with Schwann, discussed with him some of his ideas regarding cells in plants, which he had been studying in his laboratory. Schwann had been making similar observations on animals, and after the meal the two went to Schwann's laboratory, where they came to the conclusion that cells are fundamentally alike in both kingdoms. Schleiden's treatise on the subject, Beitrage zur Phylogenesis, appeared in 1838 and dealt mainly with the origin of cells. Robert Brown had recently discovered the nucleus, and about it Schleiden built up his theory of "free cell-formation," which was essentially as follows: In the general cell contents or mother liquor (" cytoblastema ") there are formed, by a process of condensation, certain small granules (later called "nu- cleoli" by Schwann). Around these many other granules accumulate, thus forming nuclei ("cytoblasts"). Then, "as soon as the cytoblasts have attained their full size, a delicate transparent vesicle appears upon their surface." This vesicle in each case enlarges and forms a new cell, and, since it arise? upon the surface of the cytoblast (nucleus), "the cytoblast can never lie free in the interior of the cell, but is always en- closed [i.e., imbedded] in the cell wall ..." Schleiden thus regarded new cell-formation as endogenous ("cells within cells") rather than the result of cell-division. With respect to the main proposition of the Cell Theory he says in the opening paragraphs: " . . . every plant developed in any higher degree, is an aggregate of fully individualized, independent, separate beings, even the cells themselves. Each cell leads a double life: an independent one, pertaining to its own development alone; and another incidental, in so far as it has become an integral part of a plant. It is, however, easy to perceive that the vital process of the in- dividual cells must form the first, absolutely indispensable fundamental basis, both as regards vegetable physiology and comparative physiology in general; . . ." Schleiden shared the results of his observations, including his errors, HISTORICAL SKETCH 9 with Schwann, who was the one to formulate the Cell Theory in a com- prehensive manner. Schwann announced the theory in concise form in 1838, and in 1839 published a very full account under the title "Mikro- skopische Untersuchungen iiber die Uebereinstimmung in der Struktur und dem Wachsthum der Thiere und Pflanzen." He says: "The elementary parts of all tissues are formed of cells in an analogous, though very diver- sified manner, so that it may be asserted that there is one universal prin- ciple of development for the elementary parts of organisms, however different, and that this principle is the formation of cells." And further: "The development of the proposition that there exists one general principle for the formation of all organic productions, and that this principle is the formation of cells, as well as the conclusions which may be drawn from this proposition, may be comprised under the term Cell Theory . . . " " . . .all organized bodies are composed of essentially similar parts, namely, of cells . . . " Elaboration of the Cell Theory. — The Cell Theory at once became established as one of the main foundation stones of biological research, but it underwent considerable modification as investigations proceeded. The main thesis, that the body is composed of cells and their products, remained, but other ideas associated with this in the minds of Schleiden and Schwann, particularly that concerning free cell-formation, were superseded. Soon after the formulation of the Cell Theory its elabora- tion was begun by Unger, von Mohl, and Nageli, who based their con- clusions on observations of a very high order. Franz Unger (1800-1870), in two works appearing in 1844 on vegetable growing points and the growth of internodes, argued for the origin of cells by division. Von Mohl, in two treatises (1835, 1844), maintained that there are two meth- ods of cell-formation: by division and by the formation of cells within cells. He thought the "primordial utricle" (protoplast) must be ab- sorbed to make way for the two new ones, or, less probably, the old one must divide into two. Like Schleiden, he thought the nucleus must be incorporated in the cell wall, but later (1846) concluded that it lies in the primordial utricle. It was in his paper of 1846 that von Mohl in- troduced the term "protoplasm" in its present sense. Carl von Naj^li (1807-1891) in 1844 produced an exhaustive treatise on the nucleus, cell-formation, and growth. In algae and the micro- sporocytes of angiosperms he clearly showed that cells multiply b}' division, and Schleiden was forced to admit that this might be "a second kind of cell-formation." The continuation of Naegli's researches in 1846 completely overthrew Schleiden's conception of free cell-formation, establishing the significant fact that all vegetative cell-formation is by cell-division. Many similar observations had been made by Unger and von Mohl, but Nageli elaborated a broad theory which took into account all of the data at hand. He distinctly defined cell-division and free 10 INTRODUCTION TO CYTOLOGY cell-formation, and showed that what had been taken for the latter was only a special case of the former. Nageli's conclusions were supported by new evidence furnished by other investigators, who further demon- strated that not only vegetative cells but also those reproductive cells (in thallophytes) which Nageli thought in some cases might be formed freely, originate by a modified process of cell-division. It was now clear that cells arise only from preexisting cells, a conception which had been emphasized by Remak (1841) and which Virchow (1855) expressed in the dictum "omnis cellula e cellula." Opinions concerning the origin of the nucleus and its role in cell- division varied greatly among these workers, reliable observations being as yet insufficient to allow the formulation of any definite conclusion. In 1841 Henle believed with Schleiden and Schwann that the nucleus was formed by the aggregation of "elementary granules," and that it was not constantly present. Goodsir looked upon the nucleus as the reproduc- tive organ of the cell. Von Kolliker in 1845 asserted that nuclear divi- sion precedes the division of the cell, and Remak, as a result of his observations on blood cells in the chick embryo, formulated a definite theory of cell-division (1841, 1858). He believed cell-division to be a "centrifugal" process: the nucleolus, nucleus, cytoplasm, and cell mem- brane were supposed to divide in turn by simple constriction. Just such a process, though evidently very exceptional, has been observed at a more recent date by Conklin (1903). In describing a case of nuclear division Wilhelm Hofmeister (1824-1877) stated that the membrane of the nucleus dissolved, the nuclear material then separating into two masses around which new membranes were formed (1848, 1849). It was generally believed, however, that the origin of nuclei by division was of rare occurrence, and that ordinarily the nucleus dissolved just before cell-division, two new ones forming de novo in the daughter cells. Von Mohl (1851), who in the main agreed with Hofmeister, wrote as follows: "The second mode of origin of a nucleus, by division of a nucleus already existing in the parent-cell, seems to be much rarer than the new produc- tion of them . . . " And again, " . . . it is possible that this process [nuclear division] prevails very widely, since ... we know very little yet respecting the origin of nuclei. Naegli thinks that the process is quite similar to that in cell-division, the membrane of the nucleus form- ing a partition, and the two portions separating in the form of two dis- tinct cells." It was not until many years later, in connection with researches upon fertilization and embrybgeny, that the behavior of the nucleus in cell- division became known in detail, and its probable significance pointed out. In 1879 Eduard Strasburger (1844-1912) announced definitely that nuclei arise only from preexisting nuclei. W. Flemming was led to the same conclusion by his studies on animal cells, and expressed it in the dictum "omnis nucleus e nucleo" (1882). (See footnote, p. 143.) HISTORICAL SKETCH 11 The Protoplasm Doctrine. — The Cell Theory and all of its corollaries were placed in a new light with the development of a more adequate conception of the significance of protoplasm. To its discoverers the cell meant nothing more than the wall surrounding a cavity; they spoke only in the vaguest terms of the "juices" present in cellular structures. The founders of the Cell Theory held a position but little in advance of this; they observed the cell contents but regarded them as of relatively slight importance. Even those who had been impressed by the phe- nomenon of protoplasmic streaming were not aware of the significance of the substance before their eyes. Felix Dujardin (1801-1860) in 1835 described the "sarcode" of the lower animals as a substance having the properties of life. Von Mohl had seen a similar substance in plant cells, and in 1846, as noted above, he called it "Schleim," or "Protoplasma," the latter term having been used shortly before by Purkinje in a somewhat different sense. Nageli and A. Payen (1795-1871) in 1846 recognized the importance of proto- plasm as the vehicle of the vital activity of the cell ; and Alexander Braun (1805-1877) in 1850 pointed out that swarm spores, which are cells, con- sist of naked protoplasm. An important point was reached when Payen (1846) and Ferdinand Cohn (1850) concluded that the "sarcode" of the animal and the "protoplasm" of the plant are essentially similar substances. In the words of Cohn : "The protoplasm of the botanist, and the contractile substance and sarcode of the zoologist, must be, if not identical, yet in a high degree analogous sub- stances. Hence, from this point of view, the difference between animals and plants consists in this; that, in the latter, the contractile substance, as a primordial utricle, is enclosed within an inert cellulose membrane, which permits it only to exhibit an internal motion, expressed by the phenomena of rotation and circula- tion, while, in the former, it is not so enclosed. The protoplasm in the form of the primordial utricle is, as it were, the animal element in the plant, but which is imprisoned, and only becomes free in the animal; or, to strip off the metaphor which obscures simple thought, the energy of organic vitality which is manifested in movement is especially exhibited by a nitrogenous contractile substance, which in plants is limited and fettered by an inert membrane, in animals not so." Protoplasm was now studied more intensively than ever. H. A. de Bary (1831-1888), working on myxomycetes and other plant forms, and Max Schultze (1825-1874), investigating animal cells, demonstrated the correctness of Cohn's view. The work of Schultze was especially important in that it firmly established in 1861 the Protoplasm Doctrine, namely, that the units of organization are masses of protoplasm, and that this substance is essentially similar in all living organisms. The cell, according to Schultze, is "a mass of protoplasm containing a nucleus,, both nucleus and protoplasm arising through the division of the corres- ponding elements of a preexisting cell." The cell wall, upon which the 12 INTRODUCTION TO CYTOLOGY early workers had focussed their attention, turned out to be of secondary importance. The cell was thus seen to be primarily the organized protoplasmic mass, to which Hanstein in 1880 applied the convenient term protoplast. Extensive studies on the physical nature of protoplasm were soon undertaken by Kuhne (1864), Cienkowski (1863), and de Bary (1859, 1864); and there later followed the well-known structural theories of Klein, Flemming, Altman, and Biitschli. (See Chapter III.) Von Mohl as early as 1837 held that the plastid is a protoplasmic body. The classic researches of Nageli (1858, 1863) on plastids and starch grains laid the foundation for our knowledge of these bodies, which was greatly extended in later years by Meyer (1881, 1883, etc.) and Schimper (1880, etc.). (See Chapter VI.) It would be difficult to overestimate the value, both practical and theoretical, of the Protoplasm Doctrine, for its establishment has not only led to knowledge by which the conditions of life have been materially improved, but has also been an important factor in assisting man to a modern, rational outlook on organic nature, in which he has learned to include himself. It is not too much to say that the identification of protoplasm as the material substratum of the life processes was one of the most significant events of the nineteenth century. The doctrine was furnished with a popular expression by Huxley in his well-known essay, The Physical Basis of Life (1868). The New Conception of the Cell. — The conception of the cell had now developed into something quite different from what it had been in the minds of the founders of the Cell Theory. The cell was now recognized as a protoplasmic unit, and the ideas of these men concerning the origin and multiplication of cells had been overthrown. Future researches were to show more clearly the importance of the cell in connection with development and inheritance, and certain limits were to be set to the conception of the cell as a unit of function and organization. To Schlei- den and Schwann the multicellular plant or animal appeared as little more than a cell aggregate, the cells being the primary individualities; the organism was looked upon as something completely dependent upon their varied activities for all its phenomena. "The cause of nutrition and growth," said Schwann, "resides not in the organism as a whole, but in the separate elementary parts — the cells." This elementalistic conception of the organism as an aggregate of independent vital units governing the activities of the whole dominated biology for many years, notwithstanding its severe criticism by Sachs, de Bary, and many other later writers who pointed out that, owing to the high degree of physio- logical differentiation among the various tissues and organs, the cell cannot be regarded merely as an independent unit, but as an integral part of a higher individual organization, and that as such the exercise HISTORICAL SKETCH 13 of its functions must be governed to a considerable extent by the organ- ism as a whole (Wager). Such divergence of opinion led to much dis- cussion over the question of organic individuality, which remains as one of the important problems of modern biology. But in spite of all these changes we should not forget the great service rendered by Schleiden and Schwann in the formulation of the Cell Theory. Huxley (1853) estimated the value of their contribution in the following lines : "Doubtless the truer a theory is — the more appropriate the colligating conception — the better will it serve its mnemonic purpose, but its absolute truth is neither necessary to its usefulness, nor indeed in any way cognizable by the human faculties. Now it appears to us that Schwann and Schleiden have performed precisely this service to the biological sciences. At a time when the researches of innumerable guideless investigators, called into existence by the tempting facilities offered by the improvement of microscopes, threatened to swamp science in minutiae, and to render the noble calling of the physiologist identical with that of the 'putter-up' of preparations, they stepped forward with the cell theory as a colligation of the facts. To the investigator, they afforded a clear basis and a starting point for his inquiries; for the student, they grouped immense masses of details in a clear and perspicuous manner. Let us not be ungrateful for what they brought. If not absolutely true, it was the truest thing that had been done in biology for half a century." Fertilization and Embryogeny. — In Plants. — Although it was known to the ancients that there is in plants something analogous to the sexual reproduction seen in animals, ideas of the organs and processes involved were very vague. Like Grew and others in the seventeenth century, the botanists of antiquity were aware of the fact that the pollen in some way influences the development of the ovary into a fruit with seeds. Definite proof that the stamens are (to speak somewhat loosely) the male organs was furnished in the well-known experiments of R. J. Camerarius (1691). But in spite of the excellent work of J. G. Koelreuter (1761), C. K. Sprengel (1793), and K. F. Gaertner (1849), all of whom proved the correctness of this conclusion, the idea of sexuality in plants was vigor- ously combatted in certain quarters for many years. An important step in advance was made when G. B. Amici (1830) followed the growth of the pollen tube from the pollen grain on the stigma down to the ovule. Schleiden (1837) and Schacht (1850,1858) took up the study and made a curious misinterpretation: they regarded the ovule as merely a place of incubation for the end of the pollen tube, which they supposed to enter the ovule and enlarge to form the embryo directly. The work of Amici (1842), Tulasne (1849), and others showed the falsity of this notion, but an acrimonious discussion raged about the subject for a number of years, Schleiden (1842, 1844) using the most vigorous language in support of his position. After Hofmeister (1849) had fol- 14 INTRODUCTION TO CYTOLOGY lowed the process with his characteristic thoroughness there could remain no doubt concerning the error of Schleiden and Schacht. Hofmeister clearly demonstrated that the embryo arises, as Amici contended, not from the end of the pollen tube, but from an egg contained in the ovule, the egg being stimulated to development by the pollen tube. He was wrong, however, in supposing that the tube did not open, but that a fertilizing substance diffused through its wall. It was in the algae that the union of the sperm cell with the egg cell (the act of fertilization) was first seen in the case of plants. In 1853 Thuret saw spermatozoids attach themselves to the egg of Fucus, and in 1854 he showed that they are necessary to its development. The actual entrance of the spermatozoid into the egg was first observed in 1856 by Nathanael Pringsheim (1824-1894) in (Edogonium. The fusion of the parental nuclei was seen by Strasburger (1877) in Spirogyra, but he thought they thereupon dissolved. This error was corrected shortly afterward by Schmitz (1879), who was thus the first to show clearly that the central feature of the sexual process in plants is the union of two parental nuclei to form the primary nucleus of the new individual. That the same process occurs in fertilization in the higher plants was demonstrated by Strasburger, who in 1884 described the union of the egg nucleus with a nucleus brought in by the pollen tube.. In 1898 and 1899 S. Nawaschin and L. Guignard completed the story by describing the phenomenon of double fertilization, whereby the second male nucleus contributed by the pollen tube unites with the two polar nuclei to form the primary endosperm nucleus. The subsequent work of Strasburger and others on the gymnosperms and angiosperms greatly cleared up the whole matter of fertilization and embryogeny in these plants. This work belongs to the modern period of cytology. In Animals. — It is probable that the spermatozoon was first seen in 1677 by Ludwig Hamm, a pupil of Leeuwenhoek. The credit for the discovery, however, is usually given to Leeuwenhoek, since it was he who brought the matter to the attention of the Royal Society and pursued such studies further. He asserted that the spermatozoa must penetrate into the egg, but it was thought at that time and for many years after- ward that they were parasitic animalcules in the spermatic liquid; hence the name "spermatozoa." Although L. Spallanzani (1786) is usually said to have shown by his filtration experiment that the spermatozoon is the fertilizing element, it is pointed out by Lillie (1916) that Spallanzani did not draw the correct conclusion: he even denied that the spermatozoon is the active element, holding rather that the fertilizing power lies in the spermatic liquid. It was Prevost and Dumas who corrected this mistake and demonstrated the true role of the spermatozoon (1824). The spermatozoon was later shown by Schweigger-Seidel (1865) and La Valette St. George (1865) to HISTORICAL SKETCH 15 be a complete cell with its nucleus and cytoplasm, as von Kolliker had maintained. That Schwann (1839) had been right in considering the egg as a cell was shown by Gegenbaur in 1861. The polar bodies formed at the time the egg matures are said to have been first seen by Carus (1824). Biitschli (1875) showed them to be formed as the result of the division of the egg nucleus, and Giard (1877) and Mark (1881) interpreted them as abortive eggs. The penetration of the spermatozoon into the egg was not actually seen until Newport (1854) observed it in the case of the frog. In 1875 O. Hertwig (b. 1849) announced the important discovery that the two nuclei seen fusing in the fertilized egg are furnished by the egg and the spermatozoon — by the two parents. The role of the nucleus in fertiliza- tion was thus demonstrated in animals only shortly before it was in plants, and it is interesting to note that the first complete description of the union of the germ cells in animals was given by H. Fol in the same year (1879) that Schmitz described clearly the process in plants. It was now evident that fertilization in both kingdoms consists in the union of two cells (gametes), one from each parent (in dioscious forms), and that the central feature of the process is the union of the two gamete nuclei, the new individual therefore deriving half of its nuclear substance from each parent. Although the cleavage of the fertilized animal egg to form the embryo had been seen many years previously, it was first definitely described by Prevost and Dumas in 1824 for the frog. At that time neither the egg nor the products of its division were known to be cells. The true meaning of cleavage was elucidated by M. Barry, who held that the blastomeres are cells and that their division is preceded by the division of their nuclei, and by a number of later writers, including A. von Kolliker, who traced in detail the long series of changes by which the multiplying embryonic cells become differentiated into the various tissues and organs. Embry- ogeny was thus shown to be a process of cell-division and differentiation, the fertilized egg cell initiating a series of divisions giving rise to all the cells of the body, and to the germ cells. The life cycle was now recognized as a cell cycle ; and since the egg is the direct descendant of the egg of the previous generation it became evident, as Virchow pointed out in 1858, that there has been an uninterrupted series of cell-divisions from the beginnings of life on the earth in the remote past down to the organisms in existence today. The statement of this conception is known as the Law of Genetic Continuity. In the words of Locy (1915) : "The conception that there is unbroken continuity of germinal substance between all living organisms, and that the egg and the sperm are endowed with an inherited organization of great complexity, has become the basis for all current theories of heredity and development. So much is involved in this conception that ... it has been designated (Whitman) 'the central fact of modern 16 INTRODUCTION TO CYTOLOGY biology.' The first clear expression of it is found in Virchow's Cellular Pathology, published in 1858. It was not, however, until the period of Balfour, and through the work of Fol, Van Beneden (chromosomes, 1883) Boveri, Hertwig, and others, that the great importance of this conception began to be appreciated, and came to be woven into the fundamental ideas of development.'' The Beginning of the Modern Period in Cytology. — As Wilson (1900, p. 6) points out, the great significance of the many facts brought to light in the early days of cytology lies in the relation which they bear to the Theory of Evolution and to the problems of heredity, though for many years this was only vaguely realized. Darwin, aside from his Hypothesis of Pangenesis, scarcely mentioned the theories of the cell; and not until many years later was the cell investigated with reference to these matters. Researches on the origin of the germ cells, nuclear division, and fertiliza- tion, which brought the Cell Theory and the Theory of Evolution into intimate association, began shortly after 1870 with the works of Schneider (1873), Auerbach (1874), Fol (1875, etc.), Biitschli (1875, etc. ),O.Hertwig (1875, etc.), van Beneden (1875, etc.), Strasburger (1875, etc.), Flemming (1879, etc.), and Boveri (1887, etc.). These men laid the foundations for the work which has followed; and their researches, greatly aided by the development of new refinements in microtechnique, ushered in modern cytology. A powerful stimulus to investigation was given when the zoologists Hertwig, von Kolliker and Weismann, and the botanist Stras- burger, concluded independently and almost simultaneously (1884-J885) that the nucleus is the vehicle of heredity, an idea which Haeckel had put forward as a speculation in 1866. The announcement of this conception led to an even more intensive study of the nucleus and of its role in heredity, a study which is now in progress, and which, more than any other one thing, can be said to characterize the work of our modern period. Bibliography 1 A, Works dealing wholly or in part with the history of cytology, and general works on the cell: AGAR, W. E. 1920. Cytology, with Special Reference to the Metazoan Nucleus. London. BOVERI, TH. 1891. Befruchtung. Ergeb. d. Anat. u. Entw. 1 : 386-485. BUCHNER, P. 1915. Prakticum der Zellenlehre. 1. BURNETT, W. J. 1853. The cell; its physiology, pathology, and philosophy, as deduced from original investigations. Trans. Am. Med. Assn. 6. CHUBB, G. C. 1910. Article on Cytology in Encycl. Brit., llth ed. DELAGE, Y. 1895. La structure du protoplasme et les theories sur I'hereditc. Paris. DONCASTER, L. 1920. An Introduction to the Study of Cytology. London. FLEMMING, W. 1882. Zellsubstanz, Kern und Kerntheilung. Leipzig. 1981-1897. Referate iiber Zelle. Ergeb. d. Anat. u. Entw. 1-7. GURWITSCH, A. 1904. Morphologie und Biologie der Zelle. Jena. HAECKER, V. 1899. Praxis und Theorie der Zellen- und Befruchtungslehre. HEIDENHAIN, M. 1907. Plasma und Zelle. Jena. HISTORICAL SKETCH 17 HENNEGUY, L. F. 1896. Legons sur la Cellule. Paris. HERTWIG, O. 1893. Die Zelle und die Gewebe. Jena. (Engl. Transl. by H. J. Campbell?) 1900. Die Entwicklung der Biologic im 19. Jahrhundert. Jena. HOFMEISTER, W. 1867. Die Lehre von der Pflanzenzelle. HUXLEY, T. H. 1853. The Cell Theory. Brit, and For. Med.-Chir. Rev. 12. Also in "Scientific Memoirs" 1. 1898. JOHNSON, D. S. 1914. The evolution of a botanical problem. The history of the discovery of sex in plants. Science 39 : 2S9-319. KELLOGG, V. L. 1907. Darwinism Today. New York. LILLIE, F. R. 1916. The history of the fertilization problem. Science 43: 39-53. KOERNICKE, M. 1903. Der heutige Stand der pflanzlichen Zellforschung. Ber. deu. Bot. Ges. 21: (66)-(134). LOCY, W. A. 1901. Malpighi, Swammerdam, and Leeuwenhoek. Pop. Sci. Mo. 68 : 561-584. 1905. Von Baer and the rise of embryology. Ibid. 67 : 97-126. 1915. Biology and its Makers. 3d ed. New York. MARK, E. L. 1881. Maturation, fecundation, and segmentation in Limax campes- tris. Bull. Mus. Comp. Zool. Harvard Coll. 6 : 173-625. pis. 5. (Early litera- ture of mitosis and fertilization.) MEVES, FR. 1896, 1898. Referate iiber Zelltheilung. Ergeb. d. Anat. u. Entw. 6, 8. VON MOHL, H. 1851. The Vegetable Cell. (Engl. transl. by Henfrey.) OSBORN, H. F. 1894. From the Greeks to Darwin. RUCKERT, J. 1893. Die Chromatinreduktion bei der Reifung der Sexualzellen. Ergeb. d. Anat. u. Entw. 3: 517-583. VON SACHS, J. 1875. History of Botany. (Engl. transl. 1889.) STRASBURGER, E. 1907. Die Ontogenie der Zelle siet 1875. Prog. Rei Bot. 1. 1910. The minute structure of cells in relation to inheritance. In " Darwin and Modern Science" (Seward, editor). THOMSON, J. A. 1899. The Science of Life. Chapters 9 and 10. TURNER, W. 1890. The cell theory, past and present. Nature 43 : 10-15. TYSON, J. 1878. The Cell Doctrine: its History and Present State. Phila. WAGER, H. 1911. Article on Plants: Cytology, in Encycl. Brit., llth ed. WALDEYER, W. 1888. Ueber Karyokinese und ihre Beziehung zu den Befruchtnngs- vorgangen. Arch. Mikr. Anat. 32 : 1-122. (Engl. transl. in Quar. Jour. Micr. Sci. 30: 159-281. 1889.) (Early cell literature.) WHITMAN, C. O. 1878. The embryology of Clepsine. Quar. Jour. Micr. Sci. 18: 215-315. pis. 12-15. (Early literature of mitosis and fertilization.) 1894. (1) Evolution and Epigenesis. (2) Bonnet's theory of evolution. Woods Hole Biol. Lectures 1894. WHEELER, W. M. 1898. Caspar Friedrich Wolff and the Theoria Generationis. Ibid. 1898. WILSON, E. B. 1900. The Cell in Development and Inheritance. 2d ed. •ZiMMERMANN, A. 1893-1894. Sammel-Referate aus dem Gesammtgebiete der Zellenlehre. Beih. Bot. Centr. 3 and 4. (Reviews of early literature). For other reviews of early botanical cell literature see Jahresbericht uber die Fort- schritte der Anatomic und Physiologic, 15-20. 1886-1892; and Neue Folge, Vols. 1-13. 1892-1907. B. Special works referred to in historical sketch: AMICI, G. B. 1830. Note sur le mode d'action du pollen sur le stigmate. (Extrait d'une lettre de M. Amici a M. Mirbel.) Ann. Sci. Nat. Bot. I. 21: 329-332. Account of Amici's work in Atti della quarta Riunione degli scientiati Italiani 2 18 INTRODUCTION TO CYTOLOGY tenuta in Padova nel Settembre del 1842. Padova 1843. See FACCHINI 1845. See also Giorn. Bot. Ital. Anno 2. AUERBACH, L. 1874. Organologische Studien. Breslau. VON BAER, K. E. 1828, 1837. Uber Entwickelungsgeschichte der Thiere. BARRY, M. 1838-1841. Embryological memoirs in Phil. Trans. Roy. Soc. London 128-131. See also: On the first changes consequent on fecundation in the mam- miferous ovum. Rep. Brit. Assn. Adv. Sci. 1840. DE BARY, H. A. 1862. Uber den Bau und das Wesen der Zelle. Flora 20. 1864. Die Mycetozoen. 2d ed. Leipzig. VAN BENEDEN, E. 1875. La maturation de 1'oeuf, la fecondation et les premieres phases du developpement embryonnaire des mamiferes d'apres des recherches faites chez le lapin. Bull. Acad. Roy. Belg. 40. 1876. Contribution a 1'histoire de la vesicule germinative et du premier noyau embryonnaire. Ibid. 41. 1883. Recherches sur la niaturation de 1'oeuf, la fecondation et la division cellu- laire. Arch, de Biol. 4. BERNHARDI, J. J. 1805. Beobachtungen iiber Pflanzengefasse. BOVERI, TH. 1887a. Ueber die Befruchtung der Eier von Ascaris megalocephala. Sitzungsber. Gesell. Morph. Fhys. Miinchen 3. 18876. Ueber Differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris megalocephala. Anat. Anz. 2 : 688—693. 1887-1890. Zellenstudien 1, II, III. Jenaische Zeitsch. 21-24. BRAUN, ALEX. 1850. Betrachtungen liber die Erscheinung der Verjlingung in der Natur, inbesondere in der Lebens- und Bildungsgeschichte der Pflanze. (English transl., Ray Society 1853.) BROWN, R. 1833. Observations on the organs and mode of fecundation in Orchideae and AsclepiadesD. Trans. Linn. Soc. (Paper read and privately printed in 1831.) Also in: Misc. Bot. Works, Ray Society, 1866. 1866. A brief account of microscopical observations on the particles contained in the pollen of plants; and on the general existence of active molecules in organic and inorganic bodies. Misc. Bot. Works, Ray Society. (Observations made in 1827.) BUTSCHLI, O. 1875a. Vorl. Mitteilung iiber Untersuchungen betreffend die ersten Entwicklungsvorgange im befruchteten Ei von Nematoden und Schnecken. Zeit. Wiss. Zool. 26: 201. 18756. Vorl. Mitteilung einiger Resultate von Studien iiber Conjugation der In- fusorien und die Zelltheilung. Zeit. Wiss. Zool. 25 : 426. 1876. Studien iiber die ersten Entwicklungsvorgange der Eizelle, die Zelltheilung, und die Konjugation der Infusorien. Abhandl. Senckenb. Naturforsch. Gesell. 10. CAMERARIUS, R. J. 1691. De Sexu Plantarum. 1694. CARDS, C. 1824. Von den ausseren Lebensbedingungen der Weiss- und Kalt- bliitigen Thiere, etc. Leipzig. CIENKOWSKI, L. 1863. Zur Entwicklungsgeschichte der Myxomyceten. Jahrb. Wiss. Bot. 3 : 325-337. COHN, F. 1850. On the natural history of Protococcus pluvialis. Nova Acta Acad. Caes. Leop. Carol. Nat. Cur. Bonn 22 : 605-764. (English abst. by Busk, Ray Soc. 1853.) CORTI, B. 1772. Observationi misc. sulla Tremella e sulla circolazione del fluid in una pianta acquaiola. Lucca, 1774. DUJARDIN, F. 1835. Sur les pretendus estomacs des animalcules infusories et sur une substance appelee Sarcode. Ann. Sci. Nat. Zool. II 4 : 364-377. HISTORICAL SKETCH 19 DUTROCHET, H. J. 1837. Memoires pour servir a 1'histoire anatomique et physio- logique des vegetaux et des animaux. FACCHINI. 1845. Ueber die Amici'sche Ansicht von der Befruchtung der Pflanzen, ein Beitrag. Flora 28: 193-198. See also 27: 359-360. FLEMMING, W. 1879o, 1880, 1881. Beitrage zur Kenntniss der Zelle und ihre Lebenserscheinungen. I, II, III. Arch. Mikr. Anat. 16, 19, 20. 1879b. Ueber das Verhalten des Kernes bei der Zelltheilung, usw. Virchow's Archiv. 77. 1882. Zellsubstanz, Kern und Zelltheilung. Leipzig. 1887. Neue Beitrage zur Kenntniss der Zelle. Arch. Mikr. Anat. 29. FOL, H. 1873. Die erste Entwicklung des Geryoniden-Eies. Jen. Zeitsch. 7. 1875. Sur le developpement des Pteropodes. Arch, de Zool. 4. 1876. Sur les phenomenes de la division cellulaire. Comptes Rend. Acad. Sci. Paris 83 : 667-669. 1877. Sur le commencement de I'h6nogenie chez divers animaux. Arch. Sci. Nat. et Phys. Geneve 58. 1879. Recherches sur la fecondation et la commencement de 1'henogenie. Mem. Soc. Phys. et Nat. Geneve 26: 89-397. FONTANA. 1781. Sur la structure primitive du corps animal. In Traite sur le venin de la vipere. Florence. GAERTNEB, J. 1788. De fructibus et seminibus plantarum. GAERTNER, K. F. 1849. Vereuche und Beobachtungen iiber die Bastardzeugung. Stuttgart. GEGENBAUR, K. 1861. Ueber den Bau und die Entwicklung der Wirbelthiere. Mliller's Archiv f. Anat. u. Physiol. p. 451-529. GIARD, A. 1877. Sur la signification morphologique des globules polaires. GOODSIR, J. 1842. On secreting structures. Trans. Roy. Soc. Edinb. 1842. On Peyer's glands. Lond. and Edinb. Mo. Jour. 1845. Anatomical and Pathological Observations. Edinburgh. 1868. Anatomical Memoirs. (Ed. by Turner.) Edinburgh. GREW, N. 1682. The Anatomy of Plants. London. GUIGNARD, L. 1899. Sur les antherozoides et la double copulation sexuelle chez les vegetaux angiospermes. Comptes Rend. Acad. Sci. Paris 128: 864-871. figs. 19. HAECKEL, E. 1866. Generelle Morphologic. Jena. HANSTEIN, J. 1880. Das protoplasma als Trager der pflanzlichen und thierischen Lebensverrichtungen. Heidelberg. HARVEY, WM. 1651. Exercitationes de Generatione Animalium. (English transl., Sydenham Society, 1847.) HENLE, J. 1837. Symbolae ad anatomiam villorum intestinalium. 1841. Allgemeine Anatomic. Leipzig. HERTWIG, O. 1875. Beitrage zur Kenntniss der Bildung, Befruchtung, und Theil- ung des tierischen Eies, I. Morph. Jahrb. 1. See also 2-4. 1884. Das Problem der Befruchtung und der Isotropie des Eies, eine Theorie der Vererbung. Jenaische Zeitschr. 18 : 276-318. HOFMEISTER, W. 1849. Die Entstehung des Embryos der Phanerogamen. 1858. Neuere Beobachtungen liber Embryobildung der Phanerogamen. Jahrb. Wiss. Bot, 1: 82-188. pis. 7-10. HOOKE, R. 1665. Micrographia, or some physiological descriptions of minute bodies made by magnifying glasses. London. HUXLEY, T. H. 1868. The Physical Basis of Life. (Collected Essays.) KIESER. 1812. Memoire sur 1'organisation des plantes. 20 INTRODUCTION TO CYTOLOGY VON KOLLIKER, A. 1845. Die Lehre von der thierischen Zelle. Zeit. Wiss. Bot. 2. 1885. Die Bedeutung der Zellkerne fur die Vorgange der Vererbung. Zeit. Wiss. Zool. 42. KOELRETJTER, J. G. 1761-1766. Vorlaufige Nachricht von einigen Geschlecht der Pflanzen betreffenden Versuchen und Beobachtungen. KOWALEVSKY, A. 1871. Embryologische Studien an Wiirmern und Arthropoden. Mem. Acad. Imp. Sci. de St. Petersburg VII 16 : 13. pi. 4. figs. 24. KtJHNE, W. 1864. Untersuchungen iiber das Protoplasma. Leipzig. LA VALETTE ST. GEORGE. 1865. Ueber die Genese der Samenkorper. Arch. Mikr. Anat. 1. See also Vols. 2 and 3. VAN LEEXJWENHOEK, A. 1673-1723. Brieven. Leiden, Delft. LINK, H. F. 1807. Grundlehren der Anatomic und Physiologic der Pflanzen. MALPIGHI, M. 1675. Anatome Plantarum. MARK, E. L. 1881. Maturation, fecundation, and segmentation of Limax campestris. Bull. Mus. Comp. Zool. Harvard 6: 173-625. pis. 5. MEYEN, F. J. F. 1826. De Primis Vitse Phenomenis in Fluidis. 1830. Lehrbuch der Phytotomie. Berlin. 1837-1839. Neues System der Pflanzenphysiologie. MEYER, A. 1881. Ueber die Struktur der Starkekorner. Bot. Zeit. 39: 841-846, 857-864. pi. 9. 1883a. Ueber Krystalloide der Trophoplasten und liber die Chromoplasten der Angiospermen. Ibid 41 : 489, 505, 525. 18836. Das Chlorophyllkorn. pp. 91. pis. 3. Leipzig. MIRBEL, C. F. 1801. Traite d'anatomie et de physiologic vegetale. 1808. Exposition et defense de ma theorie de 1'organisation vegetale. 1833. Recherches sur la Marchantia. Mem. French Inst. 1835. VON MOHL, H. 1835, 1837. Ueber die Vermehrung der Pflanzenzelle durch Theilung. Dissert. Tubingen 1835. Flora 46. 1837. 1844. Einige Betrachtungen iiber den Bau der vegetabilische Zelle. Bot. Zeit. 2 : 273-277, 289-294, 305-310, 321-326, 337-342. pi. 2. 1845. Vermischte Schriften. 1846. Ueber die Saftbewegung im Inneren der Zellen. Bot. Zeit. 4 : 73-78, 89-94. 1851. Grundziige der Anatomic und Physiologic der vegetabilische Zelle. (Engl. transl. by Henfrey, London, 1852.) MOLDENHAWER, J. J. P. 1812. Bcitrage zur Anatomic der Pflanzen. VON NAGELI, C. 1844, 1846. Zellkerne, Zellbildung, und Zellwachsthum. Zeitschr. Wiss. Bot. 1, 3. (Engl. transl. by Henfrey, Ray Society; London, 1846, 1849.) 1846. On the utricular structures in the contents of cells. Ray Society, 1849. (Engl. Transl. by Henfrey.) 1858. Die Starkekorner. Zurich. NAWASCHIN, S. 1899. Neue Beobachtungen iiber Befruchtung bei Fritillaria und Lilium. Bot. Centr. 77 : 62. (Account in Russian, 1898.) NEWPORT, G. 1851, 1853, 1854. On the impregnation of the ovum in the amphi- bia. Phil. Trans. Roy. Soc. London. PAYEN, A. 1839. Memoire sur I'amidon, etc. Paris. 1846. Memoire sur les developpements des vegetaux, etc. Mem. Acad. Paris 9. PREVOST and DUMAS. 1824. Nouvelle Theorie de la generation. Ann. Sci. Nat. 1 : 1, 167; 2 : 100, 129. PRINGSHEIM, N. 1855. Ueber die Befruchtung und Keimung der Algen und das Wesen des Zeugungsaktes. Monatsber. K. Akad. Wiss. Berlin 1. 1856. Ueber die Befruchtung der Algen. Ibid. 1858. Morphologic der Oedogonieen. Jahrb. Wiss. Bot. 1: 11-81. HISTORICAL SKETCH 21 RKMAK, R. 1841. Ueber Theilung rother Blutzellen beim Embryo. Med. Vcr. Zeit. Miiller's Archiv f. Anat. u. Physiol. 1858; 177-188. pi. 8. 1852. Ueber extracellulare Entstehung thierische Zellen und liber Vermehrung derselben durch Theilung. Muller's Archiv f. Anat. u. Physiol. 1852; 47-57. RUDOLPHI, K. A. 1807. Anatomie der Pflanzen. SCHACHT, H. 1850 Entwicklungsgeschichte des Pflanzenembryon. Amsterdam 1850. In Ann. Sci. Nat. Bot. Ill 15: 80-109. 1851. 1858. Ueber Pflanzenbefruchtung. Jahrb. Wiss. Bot. 1: 193-232. pis. 11-15. SCHIMPER, A. F. W. 1880-1881. Untersuchungen iiber die Entstehung der Starke- korner. Bot. Zeit. 38: 881; 39: 185, 201, 217. pis. 13, 2. 1883. Ueber die Entwicklung der Chlorophyllkorner und Farbkorper. Bot. Zeit. 41: 105, 121, 137, 153. pi. 1. 1885. Untersuchungen iiber die Chlorophyllkorper und die ihnen homologen Gebilde. SCHLEIDEN, M. J. 1837. Einige Blick auf die Entwicklungsgeschichte des vege- tabilische Organismus bei den Phanerogamen. Wiegmann's Archiv. 1: 289. 1838. Beitrage zur Phylogenesis. Miiller's Archiv f. Anat. u. Phys. (English transl., Sydenham Society, 1847.J 1842. Grundziige zur Wissenschaftlichen Botanik. Zweite Aufl. (English transl. by Lankester, 1849.) 1844. Bemerkung zur Bildungsgeschichte des Veg. Embryo. Flora 27: 787-789. SCHNEIDER, A. 1873. Untersuchungen iiber Platelminthen. Jahrb. d. Oberhess. Gesell. Natur-Heilkunde 14. Giessen. SCHMITZ, FR. 1879. Untersuchungen iiber die Zellkerne der Thallophyten. Ver- handl. Naturhist. Ver. Preuss. Rheinl. u. Westf. p. 346. SCHNEIDER, A. 1883. Das Ei und seine Befruchtung. Breslau. SCHULTZE, M. 1861. Uber Muskelkorperchen und das was man eine Zelle zu nennen hat. Arch. Anat. u. Physiol. 1-27. SCHWANN, TH. 1839. Mikroskopische Untersuchungen iiber die Uebereinstimmung in der Struktur und dem Wachsthum der Thiere. und Pflanzen. (English transl., Sydenham Soc., 1847). Preliminary statement in Froriep's Notizen, No. 91: 103, 112. 1838. SCHWEIGGER-SEIDEL, O. 1865. Ueber die Samenkorperchen und ihre Entwicklung. Arch. Mikr. Anat. 1: 309-335. pi. 19. SPALLANZANI, L. 1786. Experiences pour servir a Phistoire de la ge'ne'ration des ani- maux et des plantes. Geneva. SPRENGEL, C. K. 1793. Das neu entdekfce Geheimniss der Natur in Bait und Befruchtung der Blumen. Berlin. SPRENGEL, K. 1802. Anleitung zur Kenntniss der Gewachse. STRABTJRGER, E. 1875. Ueber Zellbildung und Zelltheilung. Jena. 1877. Ueber Befruchtung und Zelltheilung. Jenaische Zeitschr. 11. 1879. Die Angiospermen und die Gymnospermen. Jena. 1884. Neue Untersuchungen iiber die Befruchtungsvorgang bei den Phanero- gamen, als Grundlage fur eine Theorie der Zeugung. Jena. 1888. Ueber Kern- und Zelltheilung im Pflanzenreich, nebst ein Anhang iiber Befruchtung. Hist. Beitr. 1. THURET, G. 1854-1855. Recherches sur la fecondation des Fucees, suivies des observations sur les antheridies des algues. Ann. Sci. Nat. Bot. IV, 2 and 3. TREVIRANUS, L. C. 1806. Vom inwendigen Bau der Gewachse. 1811. Beitrage zur Pflanzenphysiologie. TULASNE, L. R. 1849. Etudes d'embryogenie vegetale. Ann. Sci. Nat. Bot. Ill, 12: 21-137. pis. 3-7. 22 INTRODUCTION TO CYTOLOGY UNGER, F. 1844. Ueber das Wachsthum der Internodien, von anatomischer Seite betrachtet. Bot. Zeit. Meristematische Zellbildung. Ibid. VIRCHOW, R. 1855. Cellular Pathologie. Arch. Path. Anat. Physiol. 8. 1858. Die Cellularpathologie, usw. (Transl. by Chance, 1860.) WEISMANN, A. 1885. Die Kontinuitat des Keimplasmas als Grunglage einer Theorie der Vererbung. Jena. WHITMAN, C. O. 1878. The embryology of Clepsine. Quar. Jour. Micr. Sci. 18: 215-315. pis. 12-15. WOLFF, C. F 1759. Theoria Genera tionis. CHAPTER II PRELIMINARY DESCRIPTION OF THE CELL In a survey of the evolution of biological science it is noticeable that, while diverging lines of inquiry have broadened the field of view, the attention of investigators, speaking generally, has been directed in turn to successively smaller constituent parts of the organism. For many years plants and animals were studied chiefly as wholes. But very early there were made many scattered observations on the various or- gans and tissues composing the body, and from these relatively crude beginnings morphology and histology later arose. Again, when the protoplasmic mass which we know as the cell came to be recognized as the unit of structure and of function, it was evident that the problems it presents should be investigated to a certain extent by themselves, and such investigation is the task of modern cytology. Within the field of cytology itself the focus of attention has gradu- ally shortened. While many workers occupied themselves with a study of the general behavior of the cell nucleus, others devoted their efforts entirely to an investigation of its important constituent elements, the chromosomes. Furthermore, cytologists at present are much interested in knowing whether or not any smaller units, corresponding to the "genes" of the geneticist, can be directly demonstrated, and whether or not the chromatic granules or " chromomeres " are of significance in this respect. In the course of all such studies there are encountered questions which must be referred ultimately to the chemical molecules and atoms and their interactions within the cell, so that biochemistry may in a measure be looked upon as a department of cytology, just as it is to be regarded in other respects as a subdivision of chemistry. The subject of cytology thus occupies an important position in the system of natural sciences. It stands with chemistry and physics on the one hand and the complex phenomena peculiar to living organisms on the other; and the steady mutual approach of the physico-chemical and biological fields is due in large measure to the results of morphological and physiological studies on the cell. For the term cell we are indebted to Robert Hooke and the other microscopists of the seventeenth century, who applied it to the small cavities in the honeycomb-like structure which they discovered in plant tissues. Today the term denotes primarily the protoplasmic "cell contents," which, strangely enough, the early workers regarded as an 23 24 INTRODUCTION TO CYTOLOGY unimportant fluid product. The term protoplast, proposed by Hanstein (1880), is more appropriate and is coming into more general use, but long usage and brevity have probably insured the permanence of the older term. FIG. 1. — Diagram of the cell, showing its principal constituent parts. A, centrosphere, with centrosome and aster. B, nucleolus or plasmosome. C, nuclear membrane. D, nucleus, filled with karyolymph. E, nuclear reticulum, composed of linin and chromatin. F, plastid. G, metaplasmic inclusion. H, chondriosomes. /, vacuole. /, tonoplast or vacuolar membrane. K, cytoplasm. L, ectoplast. Description of the Cell. — The morphology of the cell will here be sketched only in its barest outlines, by way of introduction to the detailed descriptions presented in the subsequent chapters. The two most constant components of the cell (Fig. 1) are the cytoplasm, in which the other cell organs are imbedded, and the PRELIMINARY DESCRIPTION OF THE CELL 25 nucleus, which at least in many respects is the most important of these organs.1 The cytoplasm, a more or less transparent, viscous, granular fluid, may, with its inclusions, occupy the whole volume of the cell. This is generally true of animal cells and the younger cells of plants. If the cell is vacuolate, as is usually the case in the mature plant cell, the cytoplasm may constitute only a thin layer lining the wall, the central vacuole with its cell sap often far exceeding it in volume (Fig. 2, C). FIG. 2. A-C, diagram of a plant cell in three stages of development: the vacuoles increase in volume and the protoplasm becomes limited to the parietal region. D, cell of stamen hair of Tradescantia, showing streaming movements in the cytoplasmic strands. E, paren- chyma cell from cortex of Polygonella, showing nucleus, plastids, and scanty cytoplasm. In many cases the cytoplasm forms a system of anastomosing strands that often show active streaming (Fig. 2, D}. Externally the cytoplasm is limited by a layer of different consistency, the plasma membrane, or ectoplast. Where it comes in contact with the enclosed vacuole it is also limited by a membrane, the vacuole membrane, or tonoplast. The nucleus is bounded by a nuclear membrane and contains an extremely clear fluid, the nuclear sap, or karyolymph. In the karyo- lymph is imbedded the nuclear reticulum, composed usually of linin, an achromatic supporting material, and chromatin, the " nuclear substance par excellence." One or more true nucleoli, or plasmosomes, are commonly present in the nucleus, and may or may not be closely associated with the reticulum. There are often present also chromatin nucleoli, or karyo- somes, which represent accumulations of chromatin at certain points on the reticulum, and should not be confused with the true nucleoli. 1 According to the older usage the extra-nuclear portion of the protoplast was called "protoplasm," which was unfortunate because we now know that the nucleus also is composed of protoplasm, or living substance in its broader sense. It is now the general custom to avoid this ambiguity by employing Strasburger's terms cytoplasm and nucleoplasm (karyoplasm, Flemming). The older usage, however, has not been entirely superseded. 26 INTRODUCTION TO CYTOLOGY There are usually plastids of one or more kinds in the cytoplasm, the most conspicuous in plant cells being the green chloroplasts. A centrosome is present in the majority of animal cells and in those of certain lower plants. It may occupy the center of a visibly differentiated region, the centrosphere or attraction sphere, and at the time of cell- division is the focus of a conspicuous system of radiating astral rays, collectively known as the aster. Chondriosomes have now been demonstrated in the cells of nearly all plant and animal groups. These are minute bodies having the form of granules, rods, or threads, and apparently constitute a group of materials having various functions. Metaplasmic inclusions are accumulations of food materials and differentiation products that are relatively passive. These non-proto- plasmic bodies may exist in the form of droplets or crystals, and those which are not transitory or reserve food materials apparently play a very minor role in the life of the cell. Strictly speaking, the cell wall as at present understood is not a part of the cell proper, or protoplast, but is rather regarded by many as a secretion of the latter. In many cells, particularly those of animals and the motile cells of algae and flagellates, it may be absent. The foregoing is a bare sketch of the general structure of a "typical" cell. It is scarcely necessary to point out that the cell should not be thought of as a static thing with a permanent physical structure: it is rather a dynamic system in a constantly changing state of molecular flux, its constitution at any given moment being dependent upon ante- cedent states and upon environmental conditions. As stated by Moore (1912), "the living cell may be regarded, from the physico-chemical point of view, as a peculiar energy transformer, through which a continu- ally varying flux of energy ceaselessly goes on, and the whole life of the cell is an expression of variations and alterations in rates of flow of energy, and of swings in the balance between various forms of energy." In the words of Harper (1919), the cell is a colloidal system in which the various processes have become progressively localized in certain regions, with the resulting formation of organs, which, with the increasing con- stancy of the processes involved, have come to possess a permanence and individuality of their own. In view of the spatial relationship and definite physiological integration of the various components of the cell, we are to look upon the cell not as a mere mixture of complex substances, but as a definitely organized system. The Differentiation of Cells. — It is a striking fact that in spite of many minor variations the fundamental structure of the cell is essentially similar in nearly all living organisms, and in all the kinds of tissues which go to make up the body of any one of them. As Harper (1919) remarks, "evolution as we know it has not consisted in the production PRELIMINARY DESCRIPTION OF THE CELL 27 of new types of protoplasmic structure or cellular organization, but in the development of constantly greater specialization and division of labor between larger and larger groups of cells." One obvious reason for the fundamental similarity of the cells of widely different tissues is found in the fact that all of them are derived by progressive modification from relatively undifferentiated "embryonic" or "meristematic" cells during the course of the ontogeny. In a young vascular plant, for example, definite regions (meristems) consisting of such cells are present in the root tip and stem tip, and, at a later stage of development in many cases, in the cambium also. As a general rule these meristematic cells are without large vacuoles or other conspicuous products of differentiation, and are separated by no intercellular spaces. They undergo successive divisions very rapidly (hence the use of root tips for the study of mitosis) ; and while some of the products of division become greatly modified in structure in connection with their specialization in function, others retain their embryonic or meristematic character and continue to produce new cells from which new tissues are built up throughout the life of the plant. In the bryophytes and pteridophytes the meristematic activity of the apex (root tip or stem tip, or apical region of thallus) usually centers in a single "apical cell" of definite shape, which cuts off segments (daughter cells) from its various faces with great geometrical regularity. In the Mar- chantiales and Anthoceros the apical cell is cuneate (wedge-shaped) and forms segments from four of its faces; in the anacrogynous Jungermanniales it is sometimes cuneate but more often dolabrate (ax-shaped) and produces segments from its two lateral faces ; and in the acrogynous Jungermanniales and mosses it has the form of a triangular pyramid, cutting off segments from its three lateral faces. This last type is found also in the pteridophytes : in the stem tip it produces segments from its three section of the root tip of lateral faces, whereas in the root tip, in addition Osmunda, showing the triangular pyramidal to these three series of segments, it cuts off from its apical cell, x 144. distally directed face a fourth series, which becomes the tissue of the root cap (Fig. 3). In the higher vascular plants there is no single cell characteristically different from the others of the apical meristematic group. Most of the visible characters which ordinarily serve to distinguish the various kinds of differentiated cells of the vascular plant are found in the cell wall rather than in the protoplast itself; strictly speaking, such characters are histological rather than cytological. Thus we have, besides meristematic and little modified parenchymatous cells (Fig. 2, 28 E), a number of other types, such as tracheids, vessels, wood fibers, sclerenchyma fibers, and sieve tubes (Fig. 4), all of which are characterized by the peculiar ways in which their walls become modified through sec- ondary and tertiary thickenings, and by the form and arrangement as- sumed by the pits. (See p. 191.) The protoplasts may finally disappear completely from wood cells, leaving a tissue or framework composed of lifeless cell walls. FIG. 4. — Differentiated cells from vascular plants. A, wood fiber with thickened wall. B, C, portions of tracheids with spiral and annular thickenings. D, pitted tracheid. E, portion of sieve tube with adjacent companion cells. F, face view of sieve plate shown in section in E. All of this variety of form and structure is conditioned by varied functional activity on the part of different protoplasts : in the process of cell differentiation morphological and physiological changes stand in the closest relationship. All functional differences are accompanied by chemical or physical differences of some sort in the protoplasm, but it is mainly in the non-protoplasmic inclusions and secretions (including the wall) rather than in any conspicuous structural changes in the protoplast itself that cell differentiation is rendered visible in the case of plants. Apart from differences in shape, amount of vacuolar material, accumulated food, and other products of differentiation (see p. 133), protoplasts performing widely different functions may appear much alike. Structural differentiation in connection with division of labor is very striking in animal cells, which are destitute of such walls as plant cells possess. The muscle cell shows many fine longitudinal fibrillae which have to do with the cell's power of contractility. In certain muscles these fibrillse are so segmented that the whole cell, or muscle, fiber, has a transversely striped appearance (Fig. 5, F). The nerve cell (Fig. 5, PRELIMINARY DESCRIPTION OF THE CELL 29 G FIG. 5. — Nerve and muscle cells of animals. A, diagram of a typical neuron: a, axis cylinder process or axon, ending in arborescent system; d, dendrites. (After Obersteiner and Hill.) B, cell from human spinal cord, X 75. (After Obersteiner and Hill.) C, nerve cell from the eye. (After Lenhossek.) D. Nerve cell from the earthworm. (After Kowalski.) E, young voluntary muscle cell. F, portion of mature voluntary muscle cell, showing striations. G, Involuntary muscle cell from intestine. (E-G after Piersol.) I FIG. 6. A, connective tissue from the jelly fish, showing branching cells and elastic fibers imbedded in gelatinous matrix. (After Lang.) B, cells of hyaline cartilage imbedded in their secretion. C, blood cell from chick embryo. 30 INTRODUCTION TO CYTOLOGY A-D) typically possesses a single unbranched prolongation (axon) and one or more others (dendrites) which often become very elaborately branched, especially in the ganglion cells of the spinal cord and brain. The cytoplasm of the nerve cell contains fine fibrils, and also granules of chromatic "Nissl substance." Cells specialized in connection with motility. such as spermatozoa (Fig. 103) and the cells of certain epithelial tissues (Fig. 36), show complex structural modifications not only in the flagelke, cilia, and cirri which they bear (p. 45), but also in the other cell organs with which the activities of these motile structures are closely Fia. 7. — ParamcBcium caudatum. Semidiagrammatic figure showing principal parts. C. V., contractile vacuoles. T, trichocyst. N, n, mega- and micronuclei. P, peri- stomial groove. M, mouth. O, oesophagus, with undulating membrane, U. M. F. V., food vacuoles. (After Lang.) ' connected. (See Chapter IV.) Secretory cells are often distinguishable not only by the accumulations of secretion products in their cytoplasm, but also by the peculiar forms assumed by their nuclei (Fig. 17, A, C). The cells of connective tissue (Fig. 6. A) form many long interlacing pro- cesses and lie in a supporting matrix which represents their secretions. Cartilage and bone cells (Fig. 6, B} are likewise imbedded in their secre- tions, which are here produced in relatively enormous amounts and, where present, constitute the main supporting framework of the body. We thus see that the life of the complex multicellular organism is dependent upon the correlated activities of a multitude of cells per- forming many diverse special functions. It is a remarkable fact, that PRELIMINARY DESCRIPTION OF THE CELL 31 all of the functions delegated, as it were, to different cells (contractility, motility, mechanical support, the reception and conduction of stimuli, secretion, and excretion), as well as those general functions common to all cells (nutrition and reproduction by division), may in the protozoa and protophyta be carried on within the limits of a single cell. Such a cell as, for example, the body of a Paramcecium (Fig. 7), exhibits a cor- responding regional differentiation in structure, certain functions being localized in definitely developed organs. Differentiation is therefore something which, fundamentally, does not require multicellular struc- ture for its expression; in fact the most important single step ever taken in differentiation was that which set apart nucleus and cytoplasm, giving the type of organic unit common to all subsequently evolved organisms. It is further evident, however, that the evolution of the higher organisms has unquestionably been very largely conditioned by the multicellular state, and has involved a progressive division of labor in a very real sense. The many functions of a single cell have become distributed among a number of cells in such a way that there has been produced a harmonious whole which is efficient, adaptable, and progressive to a degree not other- wise attainable. Bibliography 2 See Bibliography I A for general works on the cell and for reviews of early cell literature. For the latter see especially the works of Boveri, Flemming, Koernicke, Mark, Meves, Riickert, Waldeyer, Whitman, and Zimmermann. Other works referred to in Chapter II: HANSTEIN, J. 1880. Das Protoplasma als Trager der pflanzlichen und thierischen Lebensverrichtungen. Heidelberg. HARPER, R. A. 1919. The structure of protoplasm. Am. Jour. Bot. 6 : 273-300. MOORE, B. 1912. The Origin and Nature of Life. N. Y. and London. CHAPTER III PROTOPLASM In his famous essay on protoplasm in 1868 Huxley very fittingly referred to it as "the physical basis of life." With a realization of the full significance of this phrase there comes the conviction that protoplasm is the most interesting and important substance to which we can turn our attention, for with it the phenomena of life, in so far as we know them, are invariably associated. In spite of the enormous amount of work which has been done upon protoplasm during many years, our knowledge of it must still be regarded as very superficial and fragmentary. We can scarcely yet say definitely that a given kind of protoplasm is not a single complex chemical com- pound, as is held by one prominent school of biochemists: all ordinary analysis seems to indicate that it represents a somewhat looser combina- tion of substances, many of which are in turn very elaborate in composi- tion; and further that these substances probably differ from those found elsewhere not in any fundamental manner, but only in the degree of their complexity. Proteins, fats, crystalloids, water, and other compounds make up protoplasm, but protoplasm is not a mere mixture of these materials; it is organized — it is a system of complex substances, the activities of which are fully coordinated. Only if we recognize in pro- toplasm an organization can we conceive of it as a physico-chemical substratum for those peculiar orderly activities characterizing living substance, namely, synthetic metabolism, reproduction, irritability, and adaptive response. Physical Properties. — Certain early ideas regarding the physical nature of protoplasm may be briefly reviewed at this point. Protoplasm appeared to its earliest observers merely as a colorless, viscid substance containing minute granules. Two general opinions soon developed : some held that protoplasm consists of but a single fluid, whereas others regarded it as a combination of two fluids. Briicke (1861), who was one of the first to lay emphasis on the fact that pro- toplasm is an organized substance, looked upon the cell body as a con- tractile, semi-solid material through which there streams a fluid carrying granules. Similar to this was the idea of Cienkowski (1863), who be- lieved he saw in the protoplasm of myxomycetes two fluids, one of them hyaline and only semi-fluid (the "ground substance"), and the other a more limpid fluid with granules suspended in it. De Bary (1859, 32 PROTOPLASM 33 1864), on the other hand, regarded protoplasm as a single semi-fluid sub- stance, contractile throughout, but showing many local differences due to varying water content. To this general view the work of Hanstein (1870, 1880, 1882) lent support. Much more prominent have been the structural theories associated with the names of Klein, Flemming, Altman, and Biitschli, and known respectively as the "reticular," "fibrillar," "granular," and "alveolar" theories. The reticular theory, which was formulated by Fromman (1865, 1876, 1884), was developed especially by Klein (1878-9) and supported by van Beneden, Carnoy, Leydig, and others. These workers saw in protoplasm a reticulum or fine network of a rather solid substance (spongioplasm) , which holds a fluid and granules in its meshes. This view was adopted for a time by Strasburger. The fibrillar, or filar, theory, announced by Velten (1873-6) as a result of his observations on Tradescantia and other forms, stated that protoplasm is composed of fine fibrils, which, though often branched, do not form a continuous network. This idea was developed mainly by Flemming (1882), who called the substance of the fibrils mitome and the fluid bathing them paramitome. Some observers asserted that the fibrils are in reality minute canals filled with a liquid, the granules seen by others being merely sections of these canals. An extreme view was that of Schneider (1891), who thought the entire cell might consist of but a single greatly convoluted filament. To the followers of the reticular and fibrillar theories the fluid held between the fibers was known variously as ground substance, enchylema (Hanstein 1880), hyaloplasma (Hanstein), paramitome, and inter-filar substance. The granules were known generally as microsomes (Hanstein). According to the granular theory protoplasm is a compound of in- numerable minute granules which alone form the essential active basis for the phenomena exhibited; the observed fibrillar and alveolar structures are of secondary importance. Martin (1881) held that fibrils and net- works are due entirely to certain arrangements of these granules, or microsomes. Pfitzner (1883) pointed out that the granules are semi- solid and float in a more fluid ground substance. For Altman (1886, etc.), who was the most prominent exponent of the theory, the granules were actual elementary living units, or bioplasts, the liquid containing them being a non-living hyaloplasm. The cell was therefore looked upon not as a unit, but as an assemblage of bioplasts, "like bacteria in a zooglcea," and the bioplasts were believed to arise only by division of others of their kind (omne granulum e granulo!). The alveolar theory, also known as the emulsion, or foam, theory, was elaborated principally by Biitschli (1882, etc.), and is of special interest in view of our present-day notions of protoplasmic structure. According 3 34 INTRODUCTION TO CYTOLOGY to Biitschli protoplasm consists of minute droplets (averaging I/A in diameter) of a liquid "alveolar substance" (enchylema) suspended in another continuous liquid " interalveolar substance." The structure is therefore that of an extremely fine emulsion, and the appearances described by other workers are due to optical effects encountered in examining the minute alveolar structure. Biitschli supported his theory by making artificial emulsions with soaps and oils which showed amoeboid movement and other striking resemblances to living protoplasm. The above four theories have been termed " monomorphic theories," for the reason that each of them stated that protoplasm has a single characteristic physical structure. Strasburger in 1892 and thereafter maintained that the protoplast is regularly composed of two portions; an active fibrillar kinoplasm, concerned primarily with the motor work of the cell, and a less active alveolar trophoplasm, chiefly nutritive in func- tion. It was shown by von Kolliker, Unna (1895), and others, moreover, that one type of structure may be transformed into another. Flemming later adopted the view that no single type characterizes protoplasm, but that the latter may be homogeneous, alveolar, fibrillar, or granular — i.e., it is "polymorphic." Wilson (1899) found that all four states are successively passed through in the echinoderm egg. This observation, which was made upon both living and fixed material, showed in a striking manner the colloidal nature of protoplasm (see below), since it is now known that colloids may assume very diverse structures under the in- fluence of changing environmental conditions. The work of A. Fischer (1899), who treated non-living proteins with cytological fixing reagents and so produced artifacts similar to alveolae, reticula, and granules, should make one cautious in drawing conclusions regarding protoplasmic structure from fixed material. It should be understood that the only trustworthy observations are those which are made at least in part on living material, for it is not difficult to discover all four types of struc- ture in prepared slides: the protoplasm has there been coagulated by fixing reagents, and we know that in the coagulation of such substances as compose protoplasm an entirely new structure may be assumed. Protoplasm as a Colloidal System. — For adequate reasons it is now customary to speak of protoplasm in terms of the physics and chemistry of colloids. Colloids are those glue-like substances which are uncrystal- line, semi-solid, and very slightly or not at all osmotic. They have a high surface tension, coagulate readily, and conduct the electric current very poorly. They are "disperse heterogeneous sytems, i.e., they consist essentially of particles larger than molecules of a substance or substances in a medium of dispersion which may be water or some other fluid" (Child 19151). The particles range in size from those visible to the naked 1 These paragraphs on colloids are based largely upon the convenient summary given by Child (1915, pp. 20 ff.). See also Czapek (19116), Bayliss (1915), Hatschek (1916), Bechhold (1919), and Robertson (1920). PROTOPLASM 35 eye down to single molecules and ions. In the latter case we have a true solution: between colloid and crystalloid the line of demarcation is thus a purely arbitrary one. In many cases the suspended particles are too small to be seen with the ordinary microscope, which will not render visible a body with a diameter less than about 0.20 to 0.25 jw; but with the ultramicroscope, which will reveal particles about one-fortieth of this size, they may be clearly seen. Again, the ultramicroscope is insuffi- cient in the case of certain colloids, in which the presence of suspended particles can still be shown, however, by the Tyndall effect (a milky appearance when a beam of light is passed through them). Most proto- plasmic colloids are of this last type. In a colloidal solution the particles are separate from one another, (sol), whereas in the denser "set" condition (gel) they are more closely aggregated and hence not free to move upon one another. A colloid may be made to pass from the sol to the gel state or vice versa; in some cases this change is reversible, but in others it is not. Colloids are usually classified as suspensoids and emulsoids. Sus- pensoids, in which the particles are solid, are comparatively unstable; are readily precipitated or coagulated by salts; carry a constant electric charge of definite sign; are not viscous; do not show a lower surface tension than that of the medium of dispersion alone; and are mostly only slightly reversible. Emulsoids, in which the suspended particles are fluid, are comparatively stable; are less readily coagulated by salts; are either positively or negatively charged; are usually viscous; have a lower surface tension than the medium of dispersion; form surface mem- branes; and are highly reversible. Most organic colloids are emulsoids, and there can be no doubt that many of the characteristics of living or- ganisms are due to their presence. In an emulsion each physically homogenous constituent is known as a phase. In mayonnaise dressing, to cite a familiar example, there are three phases: a water phase, consisting of water and substances dissolved in it; an oil phase; and a protein phase (egg). These three physically diverse substances are brought into the emulsified state by beating; one of them is the medium of dispersion (external phase) and the others (internal phases) are suspended in it as liquid particles or droplets. In such an emulsion a given phase usually consists of more than one chemi- cal substance: the water phase, for example, is not pure water, butjan aqueous solution of salts and other water-soluble substances. These dif- ferent chemical substances, including the solvent, which make up a single phase, are known as components. It is shown by certain investigators (Bancroft, Clowes) that the drop- lets of a suspended phase in a stable emulsion are bounded by films of different constitution: between the phases of an alkaline water-oil emul- sion, for example, there appear to be delicate films of a soapy nature. 36 INTRODUCTION TO CYTOLOGY These films not only prevent the coalescence of the droplets, but also, through alterations in surface tension, influence the transposition or inversion of phases which occurs under certain conditions, whereby the suspended phase becomes the medium of dispersion and vice versa (Fig. 8). Such inversion probably plays an important role in many cases of transformation of sol into gel and of gel into sol. FIG. 8. — Diagram of a colloidal emulsion, illustrating transformation of emulsion of oil in water to emulsion of water in oil. A, aqueous phase. B, oil or other non-aqueous phase. C, surface film of soap or other dispersing agent. (After Clowes, 1916.) The properties and behavior of colloidal substances in general appear to be due primarily to the enormous extent of the reacting surface be- tween the constituent phases which results from the finely divided state of one or more of them. In the accompanying table is shown the amount of surface which a given mass of matter may expose when subdivided into successively smaller particles. TABLE SHOWING THE INCREASE OF SURFACE WITH THE SUBDIVISION OF 1 c.c. OF MATTER IN THE FORM OF A CUBE (DATA PARTLY FROM HATSCHEK, 1919.) Length of edge of cube Number of cubes Total surface exposed 1 cm. 1 6 sq. cm. 1 mm. 1,000 60 sq. cm. 100M 10" 600 sq. cm. 10u 10" 6,000 sq. cm. IM 1012 6 sq. m. 1(XW 1015 60 sq. m. 1ur 1'interpretation de la fecondation merogonique et sur une theorie nouvelle de la fecondation normale. Ibid. 512-527. 1903. L'heredite et les grandes problemes de la biologic. Paris. VON DERSCHAU, M. 1908. Beitrage zur pflanzlichen mitose: Centern, Blepharo- plasten. Jahrb. Wiss. Bot. 46: 103-118. pi. 6. 1914. Zum Chromatindualismus der Fflanzenzelle. Arch. Zellf. 12: 220-240. pi. 17. DIGBY, L. 1910. The somatic, premeiotic, and meiotic nuclear divisions of Galtonia candicans. Ann. Bot. 24; 727-757. pis. 59-63. DOBELL, C. C. 1909. Chromidia and the binuclearity hypothesis. Quar. Jour. Micr. Sci. 63: 279. See other papers at pp. 183 and 579; also vol. 52. p. 121. 1911. Contributions to the cytology of the bacteria. Ibid. 56: 395-506. pis. 16-19. fig. 1. FISCHER, A. 1894. Untersuchungen iiber Bakterien. Jahrb. Wiss. Bot. 27: 1. 1897. Untersuchungen iiber den Bau der Cyanophyceen und Bakterien. Jena. 1899. Fixierung, Farbung und Bail des Protoplasmas. Jena. 1903. Vorlesungen iiber Bakterien. 2 Aufl. Jena. FLEMMING, W. 1879. Beitrage zur Kenntniss der Zelle und ihre Lebenserschein- ungen. Arch. Mikr. Anat. 16: 302-436. pis. 15-18. GATES, R. R. 1909. The stature and chromosomes of CEnothera gigas, De Vries. Arch. Zellf. 3: 525-552. 72 GERASSIMOW, J. J. 1890. Einige Beuierkungen iiher die Funktion des Zellkerns. Bull. Soc. Sci. Nat. Moscow. 548-554. 1892. Ueber die kernlosen Zellen bei einigen Conjugaten. Ibid. 109-131. 1896. Ueber ein Verfahren kernlose Zellen zu erhalten. Ibid. 477-480. 1899. Ueber die Lage und die Funktion des Zellkerns. Ibid. 220-267. 1901. Ueber den Einfluss des Kerns auf das Wachsthum cler Zellen. Ibid. 185-220. 1904. Ueber die Grosse des Zellkerns. Beih. Bot. Centr. 18: 45-118. pis. 2. GRUBER, A. 1885. Ueber kiinstliche Teilung. bei Infusorien. Biol. Centralbl. 4: 717-722; 6: 137-141. 1886. Beitrage zur Kenntniss der Physiologie und Biologic der Protozoen. Ber. Naturf. Gesell. Freiburg 1. GUILLIERMOND, A. 1907. La cytologie des Bacteries. Bull. Inst. Pasteur 5: 273. 1908. Contribution a 1'etude cytologique des Bacilles endospores. Arch. f. Protistenk. 12 : 9. 1909. Observations sur la cytologie d'un Bacille. Comptes Rendus Soc. Biol. Paris 67 : 102. 1910. A propos de la structure des Bacilles endospores. Arch. f. Protistenk. 19: 6. HABERLANDT, G. 1887. Ueber die Beziehungen zwischen Funktion und Lage des Zellkerns bei den Pflanzen. Jena. 1914. Physiological Plant Anatomy. 4th ed. Transl. by Drummond. HARDY, W. B. 1913. Note on differences in electrical potential in the living cell. Jour. Physiol. 47: 108-111. HEGNER, R. W. 1919. Quantitative relations between chromatin and cytoplasm in the genus Arcella, with their relations to external characters. Proc. Nat. Acad. Sci. 5: 19-22. HEIDENHAIN, M. 1894. Neue Untersuchungen iiber die Centralkorper und ihre Beziehungen zum Kern und Zellprotoplasma. Arch. Mikr. Anat. 43: 423-758. pis. 25-31. HENNEGUY. 1896. Lecons sur la cellule. Paris. HERTWIG, R. 1889. Ueber die Kernkonjugation der Infusorien. Abhandl. Bayer. Akad. Wiss. II 17. 1898. Ueber Kernteilung, Richtungskorperbildung und Befruchtung von Actino- spherium eichornii. Ibid. 19. 1903. Ueber Korrelation von Zell- und Kerngrosse vmd ihre Bedeutung fiir die Differenzierung und die Theilung der Zelle. Biol. Centralbl. 23 : 49-62. 108- 119. 1908. Ueber neue Probleme der Zellenlehre. Arch. Zellf . 1 : 1-32. figs. 9. HUPPE, F. 1886. Die Formen der Bakterien und ihre Beziehungen zu Gattungen und Arten. Wiesbaden. JAHN, E. 1904. Myxomycetenstudien. 3. Kernteilung und Geisselbildung bei den Schwarmern von Stemonitis flaccida Lister. Ber. Deu. Bot. Gesell. 22 : 84-92. pi. 6. 1908. Myxomycetenstudien. 7. Ceratiomyxa Ibid. 26a: 342-352. 1911. Myxomycetenstudien. 8. Der Sexualakt. Ibid. 29: 231-247. pi. 11. JENNINGS, H. S. 1912. Age, death and conjugation in the light of work on lower organisms. Harvey Lectures 1911-1912. Pop. Sci. Mo. 80: 563-577. 1913. The effect of conjugation in Paramcecium. Jour. Exp. Zool. 14: 279-392. figs. 2. KITE, G. L. 1913. See Bibliography 3. KORSCHELT, E. 1896. Ueber die Structur der Kerne in den Spinndriisen der Raupen. Arch. Mikr. Anat. 47 : 500-549. pis. 26-28. THE NUCLEUS 73 KUWAUA, Y. 11)19. Die Chromosomenzahl von Zca Mays L. Jour. Coll. Sci. Tokyo 39: 1-148. pis. 2. LAWSON, A. A. 1903. On the relationship of the nuclear membrane to the proto- plast. Bot. Gaz. 35: 305-319. pi. 15. LILLIE, F. R. 1896. On the smallest parts of the Stentor capable of regeneration. Jour. Morph. 12 : 239-249. LILLIE, R. S. 1902. On the oxidative properties of the cell nucleus. Am. Jour. Physiol. 7:412-421. fig. 1. 1903. On differences in the direction of the electrical connection of certain free cells and nuclei. Ibid. 8 : 273-283. 1913. The formation of indophenol at the nuclear and plasma membranes of frogs' blood corpuscles and its acceleration by induction shocks. Jour. Biol. Chem. 15: 237-248. pi. 1. LOEB, J. 1899. Warum ist die Regeneration kernloser Protoplasmastiicken unmog- lich, usw? Arch. En tw. 8 : 689-693. LOEB, J. and WASTENEYS. 1911. Sind die Oxidationsvorgange die unabhangige Variable in den Lebenserscheinungen ? Biochem. Zeitschr. 36: 345-356. MATHEWS, A. P. 1915. Physiological Chemistry, p. 180. MENCL, E. 1904. Einige Beobachtungen liber die Struktur und Sporenbildung bei symbiotischen Bakterien. Centralbl. Bakt. II 12: 559. 1905. Cytologisches iiber die Bakterien der Prager Wasserleitung. Ibid. 16: 544. 1907a. Eine Bemerkung zur Organisation der Periplaneta-Symbionten. Arch. f. Protistenk. 10: 188. 19076. Nachtrage zu den Strukturverhaltnissen von Bakterium gammari Vejd. Ibid. 8:259. 1909. Die Bakterienkerne und die "cloisons transversales " Guilliermonds. Ibid. 16:62. MIGTJLA, W. 1894. Ueber den Zellinhalt von Bacillus oxalaticus Zopf. Arb Bakt. Inst. Karlsruhe 1. 1897, 1900. System der Bakterien. Jena. 1904. Der Bau der Bakterienzelle. Lafar's Handb. Techn. Mykol. 1 : 48. MINCHIN, E. A. 1912. An Introduction to the Study of the Protozoa. 1916. The evolution of the cell. Am. Nat. 50: 106-118. MINOT, C. S. Senescence and Rejuvenation. Jour. Physiol. 12 : 97-153. 1908. The problem of age, growth, and death. New York. 1913. Moderne Probleme der Biologic. Jena. MONTGOMERY, T. H. 1899. Comparative cytological studies, with special regard to the morphology of the nucleolus. Jour. Morph. 15 : 265-582. (Bibliography of about 450 titles.) NAKAHARA, W. 1917. On the physiology of the nucleoli as seen in the silk-gland of certain insects. Jour. Morph. 29: 55-74. pis. 2. 1918. Some observations on the growing oocytes of the stonefly, Perla immarginata, Say, with special regard to the origin and function of the nucleolar structures. Anat. Rec. 15: 203-216. figs. 9. NAKANISHI, K. 1901. Ueber den Bau der Bakterien. Centr. Bakt. 1 30: 97. OGATA, M. 1883. Die Veranderung der Pancreaszellen bei der secretion. Arch. Anat. Physiol. (Physiol. Abt.) 405-437. pi. 6. OLIVE, E. W. 1907. Cytological Studies on Ceratiomyxa. Trans. Wis. Acad. Sci. 16:754-773. pi. 47. OSTERHOUT, W. J. V. 1917. The role of the nucleus in oxidation. Science 46: 367-369. 74 INTRODUCTION TO CYTOLOGY RKKD, G. B. 1915. The role of oxidases in respiration. Jour. Biol. Chein. 22: 99-111. pi. 1. REED, T. 1914. The nature of the double spirem in Allium cepa. Ann. Bot. 28:271-281. pis. 18, 19. REINKE, FR. 1894. Zellenstudien. I. Arch. Mikr. Anat. 43: 377-422. pis. 22-24. ROSEN, F. 1892. Beitrage zur Kenntniss der Pflanzenzelle. 1. Ueber tinctionelle Unterscheidung verschiedener Kernbestandteile und der Sexualkerne. Cohn's Beitr. z. Biol. d. Pfl. 5 : 443-459 pi. 16. RUZICKA, V. 1908. Sporenbildung und andere biologische Vorgange bei dem Bact. anthracis. Arch. f. Hyg. 64: 219-294. pis. 1-3. 1909. Die Cytologie der Sporenbildenden Bakterien und ihr Verhaltnis zur Chromidienlehre. Centr. Bakt. 11 23 : 289-300. figs. 8. SACHS, J. 1892. Physiologische Notizeri II. Beitrage zur Zellentheorie. Flora 75: 57-67. 1893. Physiologische Notizen VI. Ueber einige Beziehungen der specifischen Grosse der Pflanzen zu ihrer Organisation. Ibid. 77 : 49-81. 1895. Physiologische Notizen IX. Weitere Betrachtungen iiber Energiden und Zellen. Flora (Ergiinzungsband) 81 : 405-434. SCHAUDINN, F. 1902. Beitrage zur Kenntniss der Bakterien und verwandten Organismen. 1. Bacillus Biitschlii, n. sp. Arch. Protistenk. 1 : 306. 1903. II. Bacillus sporonema, n. sp. Ibid. 2:421. SCHULTZE, W. H. 1913. Die Sauerstofforte der Zelle. Verh. Deu. Path. Ges. 16: 161-168. SCHURHOFF, P. N. 1918. Die Beziehungen des Kernkorperchens zu den Chromo- somen und Spindelfiisern. Flora 110: 52-66. figs. 3. SCHWARZ, FR. 1887. Die morphologische und chemische Zusammensetzung des Protoplasmas. Breslau. SPITZER. 1897. Die Bedeutung gewisser Nukleoproteide ftir die oxidative Leistung der Zelle. Arch. f. Ges. Physiol. 67 : 615-656. STRASBURGER, E; 1893. Ueber die Wirkungssphare den Kerne und die Zellgrosse. Histol. Beitr. 5: 97-124. Jena. 1895. Karyokinetische Probleme. Jahrb. Wiss. Bot. 28: 151-204. pis. 2, 3. 1897. Ueber Cytoplasmastrukturen, Kern- und Zelltheilung. Ibid. 30: 375-405. figs. 2. TOWNSEND, C. O. 1897. Der Einfluss des Zellkerns auf die Bildung der Zellhaut. Jahrb. Wiss. Bot. 30: 484-510. pis. 20, 21. VEJDOWSKY, F. 1900. Bemerkungen iiber den Bau und Entwicklung der Bakterien. Centr. Bakt. 11 6 : 577-589. 1 pi. 1904. Ueber den Kern der Bakterien und seine Teilung. Ibid. 11 : 481-496. IpL WHERRY, E. T. 1913. On the metamorphosis of an amoeba, Vahlkampfia sp., into flagellates and vice versa. Science 37 : 494-496. WILSON, E. B. 1900. The Cell in Development and Inheritance. 2d ed. WILSON, E. B. and MATHEWS, A. P. 1895. Maturation, fertilization, and polarity in the echinoderm egg. New light on the "Quadrille of the Centers." Jour. Morph. 10: 319-342. figs. 8. WINKLER, H. 1916. Ueber die experimentelle Erzeugung von Pflanzen mit abwei- chenden Chromosomenzahlen. Zeitschr. f. Bot. 8: 417-531. YATSU, N. 1905. The formation of centrosomes in enucleated egg-fragments. Jour. Exp. Zool. 2: 287-312. figs. 8. ZACHARIAS, E. 1881-1893. See Bibliography 3. THE NUCLEUS 75 ZETTNOW, E. 1891. Uebcr den Bau den Bakterien. Cenlr. Bakt. 10: (590. 1897. Ueber den Bau der Grossen Spirillum. Zeit. Hyg. 24: 72. 1899, 1900. Romanowski's Farbung bei Bakterien. Ibid. 30: 1, and Centr. Bakt. 1 27 : 803. 1908. Ueber Schwellengrebel's Chromatinbander in Spirillum volulans. Centr. Bakt. 146: 193. ZIMMERMANN, A. 1893-1894. Sammel-Referate. 5, 7, 8. Beih. Bot. Centr. 3: 333-342, 401-436; 4: 81-89. (No. 7 reviews 153 papers on nuclei of various plant groups.) CHAPTER V THE CENTROSOME AND THE BLEPHAROPLAST THE CENTROSOME For a full description of the morphology and behavior of the centro- some, based upon the large amount of work done on animal cells prior to 1900, reference should be made to Wilson's book on the cell. In the present account attention will be devoted mainly to centrosome structures in plants. The centrosomes of animal cells will be described only in general terms, their role in cell-division being dealt with in later chapters. Occurrence and General Characters. — The centrosome is an organ which is characteristic chiefly of the cells of animals: in the great majority of these cells it has been found, at least during certain stages. In plants centrosomes are limited to the cells of algae and fungi and the spermato- genous cells of certain bryophytes and pteridophytes. If the blepharo- plast be regarded as a modified centrosome, a question which will be discussed further on, all motile cells (spermatozoids) of bryophytes, pteridophytes, and gymnosperms must be looked upon as possessing centrosomes. During the last decade of the nineteenth century several botanists reported the presence of centrosomes in the cells of a number of angiosperms, but these cases have all failed to stand the test of subsequent more critical research.1 It is scarcely possible to give a description which will apply to all centrosomes, since to any rule there are apparently exceptions. The "typical" centrosome, as seen in animal cells, is a very minute granule, which stains intensely with certain dyes. It is usually situated in the cytoplasm, but in some cases it is found within the nucleus (Fig. 18). It commonly lies in a more or less hyaline mass of material, called the centrosphere (Strasburger 1892), attraction sphere (van Beneden 1883), astrosphere (Fol 1891), or hyaloplasm sphere (Wilson 1901). 2 This centro- sphere may often show two or more concentric zones differing somewhat in structure and appearance (Fig. 60). At certain stages, especially during nuclear division, the centrosome becomes the focus of a system of delicate rays known collectively as the aster (Fol 1877). The aster will 1 For a review of these cases see Koernicke (1903, 1906). 2 There has been much confusion in the application these terms. (See Wilson 1900, p. 324.) 76 THE CENTROSOME AND THE BLEPH AROPLAST 77 receive consideration in the chapter on the achromatic figure.1 Very often there are two centrosomes lying side by side in the centrosphere, I FIG. 18. — Centrosomes in animal cells. A, attraction sphere above nucleus in spermatocyte of Salamandra. (After Rawitz; see also Fig. 59.) B-F, intranuclear centrosome in spermatocyte of Ascaris megalocephala and its behavior during the prophases of mitosis; c, centrosphere; chr, chromosome tetrad. (After Brauer, 1893.) the two having arisen by the division of one, apparently in preparation for the next cell-division (Fig. 19). Von Winiwarter (1912) noticed that in interstitial testicular cells, which may have one, two, or four nuclei, there are re- spectively two, four, and eight rod-like cen- trosomes lying in midst of a granular mass ("idiosome"). In some cells there may be a larger number of smaller "centrioles" rather then one centrosome, and occasionally there are one or more concentric series of granules about the central centrosome. Several such types described by various writers are shown in Wilson's Fig. 152. It is questionable how far these are normal appearances, for Chambers (1917) asserts that several of them may be pro- duced in the animal egg by subjecting the latter to abnormal environmental conditions. Individuality. — The centrosome was dis- covered and described by Flemming (1875) and independently by van Beneden (1876). In 1887 van Beneden and Boveri, as a result of their researches on the thread-worm, Ascaris megalocephala, independently concluded that the centrosome, like the nucleus, is a permanent cell organ maintaining its individuality throughout 1 Because of the relation of the centrosome to the achromatic figure it will be necessary to make constant reference to the latter. Chapter IX should be consulted in this connection. FIG. 19. — Centrosomes in epithelial cells. A, from cornea of mon- key. B, from gastric gland of man. (After Zimmer- mann.) 78 INTRODUCTION TO CYTOLOGY successive cell generations. They observed that, prior to cell-division, the centrosome divides to form two daughter centrosomes, which movo apart to opposite sides of the cell and form the poles between which the mitotic figure is established; and further, that after cell-division is completed the centrosome included in each daughter cell does not disappear, but remains visible in the cytoplasm through the ensuing resting stage. Because of this striking behavior at the time of cell- division (see further p. 177) the centrosome soon came to be known as "the dynamic center of the cell." The above facts seemed to constitute ample ground for the conception of the centrosome as a permanent cell organ, but many obstacles have been found in the way of its acceptance as a theory of universal applica- tion. At certain stages in the history of many animal cells its presence FIG. 20. — Artificial cytasters in the egg of Arbacia. (After M organ, 1899.) cannot be demonstrated, and it is entirely absent from the cells of higher plants. Furthermore, Mead (1898) and Morgan (1896. 1898) found that the formation of centrosomes with asters may be induced in the eggs of certain animals by artifical means (treatment with NaCl and MgCl2 solutions) (Fig. 20), and it has been claimed that centrosomes so formed may function normally in the ensuing division (cleavage) of the egg. Contrary to the opinion of Boveri (1901), Wilson (1901) regarded such "artificial cytasters" as true asters with true centrosomes. Conklin (1912), however, contends that they do not function in mitosis. It is probable that no single conclusion can be drawn concerning this matter which will apply to all cases. There seems to be good evidence for the view that the centrosome in some tissues exists as a permanent cell organ, dividing at each mitosis and remaining visible through the resting stages, THE CENTROSOME AND THE BLEPHAROPLAST 79 at least for a number of cell generations. In other cases it disappears at the close of mitosis, a new one being apparently formed just before the next mitosis. The fact that the formation of centrosomes may be brought about by artificial means suggests that the regular appearance of the centrosome in successive mitoses is closely associated with regularly recurring physiological conditions in the cell; and that its presence in successive cell-divisions does not require an uninterrupted morphological continuity through the intervening stages. Its constant presence in some tissues probably indicates the continuity of some physiological function. Centrosomes in Algae.1 — One of the earliest known centrosomes in plants was that of the diatom Surirella, discovered by Smith (1886-7) and Blitschli, and fully described by Lauterborn (1896) and Karsten (1900). It lies near the nucleus, becomes surrounded by radiations, and divides to form the central spindle of the mitotic figure in a very peculiar manner. FIG. 21. — Centrosomes in algse. A, Stypocaulon. (After Swingle, 1897.) B, Stypocaulon. (After Escoyez, 1909.) C, eentrosphere-like bodies in Polysiphonia. dichotoma. (After Mottier, 1900.)- (After Yamanouchi, 1906.) D, E, Dictyota Centrosomes in the Sphacelariaceae have been described by Humphrey (1894), Swingle (1897), Strasburger (1900), and Escoyez (1909). In the vegetative cells of Sphacelaria, according to Strasburger, the centrosome is situated in a centrosphere at the focus of an aster. Previous to mitosis it divides into two which take up positions at opposite poles of the spindle. In Stypocaulon (Swingle) essentially the same condition exists (Fig. 21). Escoyez later concluded, however, that the asters of Stypocau- lon are formed independently rather than by division, and that the central corpusclse are probably not true centrosomes, but cytoplasmic microsomes. 1 This review of plant centrosomes and also that of the blepharoplast in subsequent pages are based upon similar reviews given by the author in his paper on Spermato- genesis in Equisetum (1912). 80 INTRODUCTION TO CYTOLOGY In the oogonium and segmenting oospore of Fucus Farmer and Williams (1896, 1898) described two centrospheres containing granules and arising independently at opposite sides of the nucleus. Strasburger (1897) reported definite centrosomes with asters all through mitosis. In the sporeling he observed appearances indicating the division of the centrosome, and concluded that the latter represents a permanent cell organ. In a very detailed investigation Yamanouchi (1909) demon- strated in the antheridium and oogonium two very definite centrosomes, which appear independently of each other, become surrounded by con- spicuous asters, and occupy the spindle poles during mitosis (Fig. 61, C). He further showed that when the sperm reaches the egg nucleus a new centrosome appears on the nuclear membrane at the point where the sperm enters. In Dictyota dichotoma Mottier (1898, 1900) states that in the two divisions in the tetrasporocyte, in at least the first three or four cell generations of the sporeling, and in all the vegetative cells of the tetra- sporic plant curved rod-shaped centrosomes with asters occur at the spindle poles, the two having arisen by the division of one during the early phases of mitosis (Fig. 21, D, E). Williams (1904) further reports that the entrance of the sperm causes a centrosome to appear in the egg cytoplasm. Centrosomes in Nemalion were described by Wolfe (1904). In Polysiphonia violacea (Yamanouchi 1906) there are present during the prophases of every mitosis two centrosome-like bodies in the kino- plasm at opposite sides of the nucleus. A little later the small bodies disappear, while the kinoplasm takes the form of two large centrosphere- like structures (Fig. 21, C); during the later stages of mitosis these fade from view. Yamanouchi believes that these structures do not represent permanent cell organs, but are formed de novo at the beginning of each mitosis. Somewhat similar temporary centrospheres, with radiations but no centrosomes, are present in the tetrasporocyte of Corallina (Davis 1898; Yamanouchi). Fungi. — Among the fungi the best known centrosomes are those of the Ascomycetes (Fig. 22). Harper (1895, 1897, 1899, 1905) described granu- lar disc-shaped centrospheres surrounded by asters at the poles of the spindle in the asci of Peziza, Ascobolus, Erysiphe, Lachnea, Phyllactinia, and other genera. He regarded them as permanent organs of the cell. In a recent paper (1919) he speaks of the ascomycete centrosome as a structure differentiated "as a region of connection between nucleus and cytoplasm and for the formation of fibrillar kinoplasm." Harper be- lieved the ascospore walls to be formed by the lateral fusion of the curved astral rays focussing upon the centrosome, a point disputed by Faull (1905) and others. Centrosomes in additional genera were figured by Guilliermond (1904, 1905). InGallactinia succosa (Marie 1905; Guillier- mond 1911) a single centrosome, which arises within the nucleus with a THE CENTROSOME AND THE BLEPHAROPLAST 81 cone of fibrils extending toward the chromatin, divides into two which take up positions opposite each other at the nuclear membrane, at which time asters develop in the cytoplasm. Faull (1905) found centrosomes in Hydnobolites, Neotiella, and Sordaria; in the last named genus they appear to be discoid while the cell is in the resting condition but round and smaller during mitosis. In Humaria rutilans Miss Fraser (1908) observed two centrosomes lying near each other, each at the apex of a cone of fibers and surrounded by a faint aster. These move apart and FIG. 22. — Centrosomes in ascomycetes. A-C, Phyllactinia corylea: division of nucleus in ascus, showing behavior of centro- somes. D, Erisiphe cichoracearum: formation of ascopore wall. (After Harper, 1905.) establish the spindle in the usual manner. Centrosomes are also figured in Ascobolus and Lachnea by Fraser and Brooks (1909); in Otidea and Peziza by Fraser and Welsford (1908); in Microsphcera by Sands (1907); and in Pyronema by Claussen (1912). In the Basidiomycete Boletus (Levine 1913) the centrosomes present during the last mitosis in the basidium attach themselves to the basidium wall, and in close connection with them the daughter nuclei are recon- structed. They mark the points of origin of the sterigmata and eventu- ally pass into the spores. 82 INTRODUCTION TO CYTOLOGY Bryophytes. — The first centrosome known in the liverworts was that of Marchantia described by Schottlander (1893), according to whom the centrosome in the spermatogenous cells divides during the anaphases of mitosis, so that each daughter nucleus is accompanied by two (Fig. 27). In the gametophytic cells certain minute bodies with radiations at the poles of the elongated nucleus and of the spindle were believed by Van Hook (1900) to represent centrosomes. Centrospheres with conspicuous radiations but without true centrosomes were described in the mitoses of the germinating spore of Pellia by Farmer and Reeves (1894), Davis (1901), and Chamberlain (1903). Gregoire and Berghs (1904), however, pointed out that the centrospheres observed by the foregoing writers in Pellia are in reality only appearances due to the intersection of numerous astral rays, and are not distinct bodies. Wftw ^^/tasasy B ""^— . J^' • " FIG. 23. — Centrosomes in Preissia quadrata. A, in fertilized egg just prior to nuclear fusion, B, in cells of young embryo. (After Graham, 1918.) In the cells of Preissia quadrata Miss Graham (1918) has more recently made some observations of much interest. She describes and figures two distinct centrosomes with a few astral rays in the cytoplasm of the fer- tilized egg, at the time when the sexual nuclei are approaching each other and in contact (Fig. 23, A). This, together with Yamanouchi's observa- tion on Fucus and that of Williams on Dictyota, cited above, suggests that in certain plants, as in animals, the formation of centrosomes and asters in the egg cytoplasm is in some way induced by the entrance of the sperm. Similar appearances have been noted by Meyer (1911) in Cor- sinia and by Florin (1918) in Riccardia (Aneura). Centrosomes were also observed by Miss Graham in the four-celled embryo of Preissia (Fig. 23, B), this being one of the only cases in which centrosomes have been seen in non-spermatogenous cells in plants above the algae. Conclusion. — With regard to centrosomes in plants, it may bo con- cluded from the above review that although there is no adequate evidence for their existence in the cells of angiosperms, they are clearly present in many algae, fungi, and probably certain bryophytes, where they perform THE CENTROSOME AND THE BLEPHAROPLAST 83 definite functions in the life of the cell. The question of centrosomes in the spermatogenous cells of bryophytes, pteridophytes, and gymnosperms is dealt with in the following discussion of the blepharoplast. THE BLEPHAROPLAST Occurrence. — The blepharoplast, as indicated by the name given to it by Webber (1897), is the cilia-bearing organ of the cell. Blepharo- plasts of one kind or another are found generally in the motile cells of plants and animals, such as motile unicellular organisms (Flagellata, Ciliata, etc.), swarm spores, spermatozoa, and spermatozoids; and also in cells which, though not freely motile themselves, have motile organs performing other functions, as in the case of ciliated epithelium. In plants blepharoplasts are most conspicuously displayed in the sperma- togenous cells of bryophytes, pteridophytes, and gymnosperms (cycads and Ginkgo), where their striking resemblance to ordinary centrosomes has led to much controversy over their nature. Some cytologists have regarded the blepharoplast as a more or less modified centrosome, where- as others have contended that it is a special kinoplasmic or cytoplasmic organ distinct from the centrosome. In recent years the evidence has tended strongly to support the former view. In the following pages the blepharoplasts of various organisms and the manner in which they function in the development of the motor apparatus will be described in some detail. Attention will be given chiefly to the situations found in plants; the corresponding phenomena in animals will be more briefly considered. Flagellates. — In the flagellates several types of flagellar apparatus are found (see Minchin 1912, pp. 82 ff ., 262-3) : in one series of forms the cell contains a single nucleus and "centriole," the latter functioning both as a centrosome and as a blepharoplast. The centriole may lie either against or within the nucleus, so that the flagellum which grows from it appears to arise directly from the nucleus (Mastigind); in other forms (Mastigella) the centriole is quite independent of the nucleus. In a second series of forms a single nucleus and centrosome are present, and in addition one or more blepharoplasts. * Three conditions have been distinguished here: (a) at the time of cell-division the blepharoplasts and flagella are lost, new blepharoplasts arising from the centrosomes during or after mitosis; (6) the blepharoplasts may persist, dividing to form daughter blepharoplasts from which new flagella arise (Polytomella) ; (c) the centrosome divides to furnish a blepharoplast which subdivides to two: a distal blepharoplast or basal granule of the flagellum, and a proximal blepharoplast or "anchoring granule" at the surface of the nucleus, the two being connected by a delicate strand known as the rhizoplast, rhizonema, or centrodesmose (Peranema trichophorum) . Entz (1918) has recently reinvestigated the structure of Polytoma uvella, first 84 INTRODUCTION TO CYTOLOGY described by Dangeard (1901), and finds the elaborate organization shown in Fig. 24. In a third series of forms two nuclei are present : a principal or trophic nucleus and an accessory or kinetic nucleus. Here there are apparently three conditions : (a) a single centrosome, associated with the kinetonucleus, acts both as a blepharoplast and as a division center; (6) usually both nuclei have centrosomes associated with them, the FIG. 24. FIG. 25. FIG. 24.— Diagram of structure of Polytoma uvella. (After Entz, 1918.) a, end-piece of flagellum. b, uniform portion of flagellum. c, lateronema. d, baso- plast or basal granule, e, contractile vacuole. /, cell envelope, g, eyespot. h, rhizonema. i, karyoplast or anchoring granule, j, centronema. k, nucleolus. I, nuclear membrane. m, starch, n, surface of protoplast. FIG. 25. — Trypanosoma theileri. A, flagellum inserted on basal granule. B, formation of new flagellum from daughter basal granule after division; nucleus dividing. (After Hartmann and Noller, 1918.) one lying near or within the kinetonucleus acting as the blepharoplast; (c) it is possible that in some cases there is a blepharoplast distinct from the centrosomes accompanying the two nuclei. In the trypanosomes (Fig. 25) the recent researches of Kuczynski (1917) and Hartmann and Noller (1918) have shown that the flagellum THE CENTROSOME AND THE BLEPHAROPLAST 85 is inserted on a "basal granule" (centrosome?) very near the "blepharo- plast" (kinetonucleus?). At the time of cell-division the trophic nucleus, blepharoplast, and basal granule all divide, the division of the blepharo- plast showing certain features suggesting mitosis. Although earlier investigators thought the flagellum also split, the above named workers find that the old flagellum remains attached to one of the daughter basal granules while a new flagellum grows out from the other daughter granule. In flagellate organisms, therefore, the centrosome and the blepharo- plast clearly stand in very intimate relationship with one another: in some of the forms they are one and the same organ. Thallophytes. — Among the earliest investigations of the blepharoplast in algae were those of Strasburger (1892, 1900). During the development of the zoospores of (Edogonium, Cladophora, and Vaucheria Strasburger FIG. 26. — Blepharoplasts in Thallophytes. A-D, formation of the cilia-bearing ring in the zoospore of Derbesia. (After Davis, 1908.) E, Stemonitis flaccida: cilia growing from centrosomes during late stage of division in the formation of swarmers. (After Jahn, 1904.) found that the nucleus approaches the plasma membrane, which at that point forms a lens-shaped thickening. From this structure grow out the cilia, and at the base of each a small refractive granule is present. The blepharoplasts of the higher groups were believed by Strasburger to have been derived from such swollen ectoplasmic organs of the algae, and that all of them are morphologically distinct from centrosomes. Dangeard (1898) likewise found a deeply staining granule at the base of the cilia in Chlorogonium. In Hydrodictyon (Timberlake 1902) the cilia are inserted on a small body lying in contact with the plasma membrane and joined with the nucleus by a delicate protoplasmic strand. The possible relationship of this body with the granules seen occupying the spindle poles during the formation of the spore cells was not determined. In the young 86 INTRODUCTION TO CYTOLOGY zoospore cell of Derbesia (Davis 1908) the nucleus migrates toward the plasma membrane, and from it many granules, which are not centrosomes, move out along radiating strands of cytoplasm to the surface of the cell, where by fusion they form a ring-shaped structure from which the cilia develop (Fig. 26, A-D). In the developing spermatozoid of 'Chara (Belajeff 1894; Mottier 1904) the blepharoplast arises as a differentiation of the plasma membrane and bears two cilia. No centrosomes or other granules were seen at the base of the cilia, although Schottlander (1893) had previously reported centrosomes in the cells of the spermatogenous filament. In the zoospore of the fungus Rhodochytrium (Griggs 1904) there is a deeply staining body at the insertion point of the cilia; this is connected by fine cytoplasmic fibers with the nucleus. In the myxomycete Stemo- nitis Jahn (1904) made an observation that is highly suggestive in con- nection with the question of the relationship of the centrosome and the blepharoplast. At the last mitosis in the formation of the swarmers the spindle poles are occupied by centrosomes. and during the anaphases the flagella of the resulting swarmers grow out directly from these cen- trosomes (Fig. 26, E), just as in the spermatocytes of certain insects (p. 95). FIG. 27. — Spermatogenesis in Marchantia. b, blepharoplast; c, centrosome; c. n., " chromatoider Nebenkorper ; " n, nucleus. (After Ikeno, 1903.) Bryophytes. — Among the bryophytes the blepharoplasts of Mar- chantia and Fegatella (Conocephalus) have received much attention. According to Ikeno (1903) a centrosome comes out of the nucleus at each spermatogenous division in Marchantia and divides to form two which separate to opposite sides of the cell, occupy the spindle poles, and disappear at the close of mitosis : it is possible that they are included in the daughter nuclei. After the last (diagonal) division, however, they remain in the cytoplasm as the blepharoplasts, elongating and bearing two cilia (Fig. 27). Another body, the chromatoider Nebenkorper, is THE CENTROSOME AND THE BLEPHABOPLAST 87 also present in the cytoplasm. Similar in most points is the account of Schaffner (1908). Miyake (1905), as the result of his studies on Marchantia, Fegatella, Pellia, Aneura, and Makinoa, believes that such liverwort centrosomes are merely centers of cytoplasmic radiation, and inclines toward the view of Strasburger that the blepharoplast and the centrosome are not homologous structures. Escoyez (1907) finds two ''corpuscles" appearing in contact with the plasma membrane in each cell of the penultimate generation in the antheridia of Marchantia and Fegatella; they occupy the spindle poles and function as blepharoplasts in the spermatids (the cells which transform directly into spermatozoids). Bolleter (1905) believes the cent rosome-1 ike body in Fegatella to arise within the nucleus. In the antheridium of Riccia Lewis (1906) reported centrosome-like bodies in both the early and diagonal divisions. They apparently arise de novo in the cytoplasm prior to each mitosis, showing no continuity through succeeding cell generations except after the last mitosis, when they persist and become the blepharoplasts. ..«r-;:r^:v „ X'.^^v.K - • b FIG. 28. — Spermatogenesis in Blasia. b, blepharoplast; n, nucleus. X 4200. (After Sharp, 1920.) Woodburn (1911, 1913, 1915) has given accounts of spermatogenesis in Porella, Asterella, Marchantia, Fegatella, Blasia, and Mnium. He finds that the blepharoplast is first distinguishable as a special granule in the cytoplasm of the spermatid, and holds that it represents, as Mottier (1904) had formerly suggested, an individualized portion of the kinoplasm arising de novo in certain spermatogenous cells. In a more recent con- tribution (Sharp 1920) it has been shown that in Blasia (Fig. 28) a blepharoplast is present at each spindle pole during all stages of the last spermatogenous mitosis, and that in the spermatid it fragments as it 88 INTRODUCTION TO CYTOLOGY becomes transformed into the cilia-bearing thread after the manner of the blepharoplast of Equisetum, described below. Spermatogenesis in Pellia, Atrichum, and Mnium has been described by M. Wilson (1911). In Mnium and Atrichum the spermatogenous divisions show no centrosomes, whereas in Pellia centrospheres, and probably centrosomes, are present during the later mitoses. The origin of the blepharoplast as here described is very peculiar. In the spermatid of Mnium a number of bodies are said to separate from the nucleolus and pass out into the cytoplasm where they coalesce to form a limosphere, The nucleolus then divides into two masses, both of which pass into the cj'toplasm; one functions as the blepharoplast and the other gives rise to an accessory body. In Atrichum the first body separated from the nucleolus becomes the blepharoplast, a second forms the limosphere, and a third the accessory body. In all three plants the blepharoplast goes to the periphery of the cell and grows out into a thread-like structure along the plasma membrane. The nucleus then moves against this ( hread and the two grow together to form the spirally coiled spermatozoid. Two cilia grow out from the anterior end of the blepharoplast. The most detailed and critical of all researches on the motile cells of bryophytes are those of C. E. Allen (1912, 1917) on Polytrichum (Fig. 29). The first of these papers contains a description of the cyto- logical phenomena accompanying the multiplication of the spermato- genous cells (androgones} up through the last mitosis, which differentiates the spermatids (androcytes) . In the cytoplasm of all the androgones there is a deeply staining kinoplasmic mass; in the early androgones this has the form of a flat plate, while in the later ones it consists of a group of granules (kinetosomes). Prior to each mitosis the plate or group divides to daughter plates or groups which pass to the daugher cells. In the cells of the penultimate generation (androcyte mother-cells) there are no kinetosomes, but instead a spherical "central body" with radia- tions. This body divides into two which move apart and occupy the spindle poles during the last mitosis. Each resulting androcyte therefore has one such body, which functions as the blepharoplast. Allen does not regard the kinetosomes as definite morphological entities, but rather as masses of reserve kinoplasm. The blepharoplast, however, is a definite cell organ, and although Allen inclines toward the view that it is the homologue of a centrosome he regards the question as an open one. Sapehin (1913) looks upon these bodies as plastids. Allen's second paper deals with the transformation of the androcyte (spermatid) into the spermatozoid. The blepharoplast elongates to form a uniform rod and develops two cilia from near its anterior end. The nucleus moves against the middle portion of the blepharoplast and the two elongate together in close union to form the body of the sperma- tozoid, the blepharoplast projecting beyond the anterior end of the THE CENTROSOME AND THE BLEPHAROPLAST 89 nucleus. At about the time the blepharoplast begins to elongate a limosphere appears in the cytoplasm and takes up a position near the anterior end of the blepharoplast. Here it divides, giving rise to a small apical body that remains visible at the end of the blepharoplast until a comparatively late stage. The remaining portion of the limosphere may be seen lying against the nucleus until the maturity of the spermatozoid FIG. 29. — Spermatogenesis in Polytrichum. A-D, androgones, showing behavior of kinoplasmic plates and kinetosomes (fc) during imitoss. E-G, androcyte mother-cells, showing division of central body. H, telophase of last mitosis; each androcyte has a blepharoplast (6). I-K, stages in the transformation of the androcyte into the spermatozoid: a, apical body; /, limosphere; n, nucleus; p, percnosome. L, mature spermatozoid. X 2535. (After Allen, 1912, 1917.) Another body, the percnosome, is also seen in the cytoplasm at certain stages. In the opinion of Allen the limosphere is probably identical with the chromatoider Nebenkorper described by Ikeno in Marchantia, and the percnosome with what M. Wilson (1911) terms the accessory body. The apical body is here described for the first time by Allen. Pteridophytes. — The early papers dealing with the spermatozoid of pteridophytes, such as those of Buchtien (1887), Campbell (1887), Bela- 90 INTRODUCTION TO CYTOLOGY Jeff (1888), Guignard (1889), and Schottlander (1893), give but little information concerning the development of the blepharoplast. Our more definite knowledge of this subject dates from 1897, when Belajeff published three short papers. In the first of these (1897a) it was stated that the fern spermatozoid consists of a thread-shaped nucleus and a plasma band, with a great many cilia growing out from the latter. In the plasma band is enclosed a thin thread which arises by the elongation of a small body seen in the spermatid. In the second paper (18976) the blepharoplast of Equisetum was first described as a crescent-shaped body lying against the nucleus of the spermatid; this body stretches out to form the cilia-bearing thread. The third contribution (1897c) is a short account of the trans- formation of the spermatid into the spermatozoid in Chara, Equisetum, FIG. 30. — Spermatogenesis in Equisetum arvense, showing behavior of blepharoplast (eentrosome) in last spermatogenous mitosis and in transformation of spermatid into sper- matozoid. X 1900. (After Sharp, 1912.) and ferns. In all these forms a small body elongates to form a thread upon which small swellings arise and grow out into cilia. In a comparison with animal spermatogenesis Belajeff here homologized the blepharoplast, the thread to which it elongates, and the cilia of the plant, with the eentrosome, middle piece, and tail (perhaps only the axial filament), respectively, of the animal. In the following year (1898) he figured the details made out in Gymnogramme and Equisetum. In Gymnogramme the two blepharoplasts appear at opposite sides of the nucleus in the spermatid mother-cell, whereas in Equisetum a single blepharoplast is first figured lying close to the nucleus of the spermatid. More recently it has been shown (Sharp 1912) that the blepharoplast of Equisetum (Fig. 30) appears first in the cells of the penultimate generation; there it divides to two which separate and establish between them the achromatic figure after the manner of animal centrosomes. At the close of mitosis the blepharp- 77/7? CENTROSOME AND THE. BLEPHAROPLAST 91 plast in each spermatid fragments into a number of pieces; these later join to form a continuous beaded thread from which the cilia grow out. In Equisetum the elongating nucleus and blepharoplast do not become closely joined, but are held together only by the rather abundant cyto- plasm.. The spermatozoid is multiciliate like that of all other pterido- phytes with the exception of Lycopodium, Phylloglossum, and Selaginella: in these three genera the spermatozoids are biciliate like those of the bryophytes. The most careful work on the blepharoplast of homosporous ferns is that of Yamanouchi (1908) on Nephrodium (Fig. 31). In this form no centrosomes are found. The two blepharoplasts, which arise de novo in the cytoplasm of the sperm- f '"• ... atid mother-cell, take no active part in nuclear division, merely lying near the poles of the spindle. In the spermatid the blepharoplast elongates spirally in close union with the nucleus to form the body of the spermatozoid. In Adiantum and Aspidium Miss R. F. Allen (1911) and Thorn (1899) see the blepharo- plast first in the spermatid. One of the most interesting blepharoplasts is that of Marsilia (Fig. 32), first described by Shaw (1898). According to Shaw a small granule, or "blepharo- plastoid," appears near each daughter nucleus of the mitosis which differentiates the grandmother-cell of the spermatid (the second of the four spermatogenous mitoses). During the next (third) division the blepharoplastoid divides but soon disappears, and a blepharoplast appears near each spindle pole. In the next cell generation (spermatid mother-cell) the blepharoplast divides into two which are situated at the spindle poles during the final mitosis. In the spermatid the blepharoplast gives rise to several granules by a sort of fragmentation; these together form a thread which elongates spirally in close union with the nucleus and bears many cilia. The spermatozoid is of the usual fern type, with several coils and a cytoplasmic vesicle. Shaw saw in the foregoing facts no ground for the homology of the blepharoplast and the centrosome. Belajeff (1899) found that centrosomes occur at the spindle poles during all, excepting possibly the first, of the four divisions which result in the 16 spermatids. He reported that after each mitosis the centrosome divides into two which occupy the spindle poles during the succeeding mitosis, and in the .spermatids perform the usual functions of blepharoplasts. Belajeff regarded this as a strong confirmation of his theory that the blepharoplast and centrosome are homologous organs. FIG. 31 . — Two stages in the sperma- togenesis of Nephro- dium. A, blepharoplasts near poles of spindle in last spermatogen- ous mitosis. B, elon- gation of blepharo- plast near nucleus. Nebenkern at left. (After Yamanouchi, 1908.) 92 INTRODUCTION TO CYTOLOGY The results of Shaw were in the main confirmed by the later work of Sharp (1914), who, however, saw in the achromatic structures accom- panying the blepharoplast striking evidence in favor of BelajefF s view of its homology. In line with this conclusion the suggestion has recently been made (Sharp 1920) that the fragmentation of the blepharoplast in Blasia, Equisetum, Marsilia, and the cycads may be homologized with the normal division exhibited by ordinary centrosomes. FIG. 32. — Spermatogenesis in Marsilia quadrifolia. A, first spermatogenous mitosis; no centrosomes. B, second mitosis, centrosomes present. C, third mitosis; centrosomes present; old centrosome divided and degenerating in cytoplasm. D, penultimate spermatogenous cell; daughter centrosomes separating. E, last spermatogenous mitosis; blepharoplasts (centrosomes) becoming vacuolate. F, frag- mentation of blepharoplast in spermatid. G, transformation of spermatid into spermato- zoid. H, free swimming spermatozoid. X 1400. (After Sharp, 1914.) Gymnosperms. — The first known blepharoplast in plants above the algae was discovered in Ginkgo by Hirase in 1894. He observed two, one on either side of the body cell nucleus, and because of their great simi- larity to certain structures in animal cells he believed them to be attrac- tion spheres. In 1897 Webber observed the same bodies, and noted their cytoplasmic origin. On account of certain differences between these organs and ordinary centrosomes he expressed the opinion that they are not true centrosomes, but distinct organs of spermatogenous cells. The blepharoplast of Ginkgo was later investigated by Fujii (1898, 1899, 1900) and Miyake (1906). THE CENTROSOME AND THE BLEPHAROPLAST 93 In 1897 and 1901 Webber described the blepharoplast of Zamia (Fig. 33). Up to the time of the division of the body cell the two blepharo- plasts, which arise de novo in the cytoplasm, are surrounded by radiations, but they have no part in the formation of the spindle, which is entirely intranuclear. During mitosis they lie opposite the poles, increase greatly in size, become vacuolate, and break up to many granules : these in the spermatid coalesce to form a spirally coiled cilia-bearign band lying just inside the cell membrane. In his full account (1901) Webber gives an extensive discussion of the homology of the blepharoplast. • & ' --*<& tyF*"^**^,'...- FIG. 33. — Spermatogenesis in Zamia. A-E, five stages in the vacuolation and fragmentation of the blepharoplast during the mitosis differentiating the spermatids. F, the two spermatozoids in the end of the pollen tube; prothallial and stalk cells below. Compare Fig. 34. A-D, X 350; E, X 1200. (After Webber, 1901.) In Ikeno's (1898) account of gametogenesis and fertilization in Cycas it was shown that the blepharoplasts appear in the body cell, lie opposite the spindle poles during mitosis, and break up to granules which fuse to form the spiral band in a manner similar to that described by Webber for Zamia. The behavior of the blepharoplast in Microcycas (Caldwell 1907) is essentially the same. Chamberlain (1909) observed in the cytoplasm of the body cell of Dioon (Fig. 34) a number of very minute "black granules" which he was inclined to believe originate within the nucleus. Very soon two undoubted blepharoplasts are present, and are apparently formed by the enlarge- 94 INTRODUCTION TO CYTOLOGY tnent of two of the black granules. Very conspicuous radiations develop about them, and after mitosis they form ribbon-like cilia-bearing bands in the spermatids as in the other cycads. *-;,'•- . .-' ". v^fepSi*stt^ *•-•' •"••'•?••''••••• 'i:''^%^%$$3&& FIG. 34. — Spermatogenesis in Dioon edule. A, "body cell," with black granules in cytoplasm. X 1890. B, two blepharoplasts differentiated. X 1890. C, body cell with two blepharoplasts; prothallial and stalk cells below. X 237. D, fragmentation of blepharoplast in spermatid as spiral band begins to form. X 1890. E, portion of edge of spermatozoid, showing spiral band cut at two points and cilia growing from it. X 945. (After Chamberlain, 1909.) Ikeno in 1898 expressed the opinion that the blepharoplast of Ginkgo and the cycads is a true centrosome, a view shared by Chamberlain (1898) and Guignard (1898). Two additional papers dealing with this subject were published by Ikeno (1904, 1906). In the first of these Ahe made comparisons with analogous phenomena in animals which he believed to THE CENTROSOME AND THE BLEPHAROPLAST 95 sustain the homologies suggested by Belajeff. He pointed out that in Marchantia centrosomes are present in all the spermatogenous divisions, whereas in other liverworts they appear much later, and from this he argued that the bryophytes show various stages in the elimination of the centrosome. He strongly reasserted his belief that blepharoplasts are centrosomes, and spoke of the "transformation of a centrosome into a blepharoplast " in the development of a spermatic! into a spermatozoid. The ectoplasmic blepharoplasts of the algae were also held to be derived from centrosomes. In the second paper he insisted less strongly upon the morphological identity of all blepharoplasts, separating them into three categories : (1) centrosomatic blepharoplasts, including those of the myxo- mycetes, bryophytes, pteridophytes, and gymnosperms; (2) plasmoder- mal blepharoplasts, including those of Cham and some Chlorophyceae; (3) nuclear blepharoplasts, found only in a few flagellates. For a further discussion of this question the student is referred to the present author's papers on Equisetum and Marsilia. The main conclusions reached may be stated in two extracts from the former paper : Although limited to a single mitosis in the antheridium, the blepharoplast [of Equisetum} retains in its activities the most unmistakable evidences of a centrosome nature, and at the same time shows a metamorphosis strikingly like that in the cycads. In thus combining the main characteristics of true centro- somes with the peculiar features of the most advanced blepharoplasts, it reveals in its ntogeny an outline of the phylogeny of the blepharoplast as it is seen developing through bryophytes, pteridophytes, and gymnosperms, from a func- tional centrosome to a highly differentiated cilia-bearing organ with very few centrosome resemblances. The activities of the blepharoplast in Equisetum [Marsilia, and Blasia], taken together with the behavior of recognized true centrosomes in plants and analogous p henomena in animals, are believed to constitute conclusive evidence in favor of the theory that the blepharoplasts of bryophytes, pteridophytes, and gymnosperms are derived ontogenetically or phylogenetically from centrosomes. Animals.— The early researches of Moore (1895), Meves (1897, 1899), Korff (1899), Paulmier (1899), and many other more recent investigators have established the fact that the centrosome (or centrosomes) of the animal spermatid plays an important role in the formation of the motor apparatus of the spermatozoon, the axial filament of the tail growing out directly from it (Fig. 35). Henneguy (1898) even saw flagella attached to the centrosomes of the mitotic figure in the spermatocyte of an insect, an observation which has been often repeated. Wilson (1900, p. 175) concludes that "the facts give the strongest ground for the conclusion that the formation of the spermatozoids agrees in its essential features with that of the spermatozoa . . . " and that the blepharoplast is without doubt to be identified with the centrosome. 96 INTRODUCTION TO CYTOLOGY Although there is comparatively little question that the granule at the base of the flagellum in the flagellates, like the body from which the axial filament of the spermatozoon grows, is of centrosomic nature, the nature of the basal granules in Ciliata and in the ciliated epithelial cells of higher animals is much more difficult to determine. It was held by Henneguy (1897), Lenhossek (1898), Hertwig (1902), and others that these granules, like the basal granules of flagella, are modified centrosomes; whereas certain other investigators (Maier 1903, Studnicka 1899, Schuberg 1905) have found evidence in favor of a contrary interpretation. An extensive FIG. 35. — Spermatogenesis in Helix pomatia, showing growth of flagellum from outer centro- some, and elongation of inner centrosome to form axial filament of middle piece. (After Korff, 1899.) FIG. 36. — Diagram of a ciliated epi- thelial cell. (Constructed from figures of Saguchi, 1917.) discussion of this question is given by Erhard (1911), who concludes that although the basal corpuscles arise from the nucleus in a manner similar to that of the centrosomes of such cells, the evidence is on the whole unfavorable to the theory of Henneguy and Lenhossek. Still more recent are the researches of Saguchi (1917), who describes in great detail the insertion of the cilia in epithelial cells. At the base of each cilium, which itself shows no internal structural differentiation, there is always a basal corpuscle (Fig. 36) . These corpuscles, and hence the cilia, are in parallel rows ; and beneath each row there is a transparent zone in which the rootlets of the cilia are anchored, and through which they pass and become continuous with strands of the cytoplasmic recti- culum. Cilium, corpuscle, rootlet, and cytoplasmic strand form one continuous structure. Saguchi believes that neither the cilium nor the rootlet causes the ciliary movement, but that the kinetic center of this movement is in the basal corpuscles, as Henneguy and Lenhossek thought. Contrary to the opinion of those authors, however, he regards the ciliary THE CENTROSOME AND THE BLEPHAROPLAST 97 apparatus as entirely independent of centrosomes, holding rather that it is produced by the differentiation of chondriosomes, and that the resem- blance of ciliated cells to spermatids, in which centrosomes do produce the motor apparatus, is an accidental one. Conclusion. — In conclusion it may be said that it is highly probable that cilia-bearing structures are not homologous in all plant and animal groups. It is beyond question that in animals the centrosomes of the spermatid produce the motor apparatus of the spermatozoon. That a similar interpretation is to be placed upon the blepharoplasts in the sper- matids (androcytes) of bryophytes, pteridophytes, and gymnosperms appears to be equally well demonstrated. The blepharoplasts of the flagellates are also probably centrosomic in nature, at least in certain cases. In the " plasmodermal blepharoplasts" of motile alga cells we have organs which, in the light of our present knowledge, do not appear to belong to the centrosomic category, but final disposition of them must await further information concerning those algae which possess both cen- trosomes and blepharoplasts. It can scarcely be doubted that the basal corpuscles of ciliated cells represent organs belonging to various categories. It must be left for further research to determine just how far these structures, which are functionally analogous, are homologous with each other and with other cell organs. Bibliography 5 Centrosome and Blepharoplast ALLEN, C. E. 1912. Cell structure, growth and division in the antheridia of Poly- irichum juniperinum Willd. Arch. Zellforsch. 8: 121-188. pis. 6-9. 1917. The spermatogenesis of Polylrichum juniperinum. Ann. Bot. 31: 269-292. pis. 15, 16. ALLEN, R. F. 1911. Studies in spermatogenesis and apogamy in ferns. Trans. Wis. Acad. Sci. 17: 1-56. pis. 1-6. BELAJEFF, W. 1888. Ueber Bau and Entwicklung der Spermatozoiden der Gefass- kryptogamen. Ber. Deu. Bot. Gesell. 7: 122-125. 1894. Ueber Bau und Entwicklung der Spermatozoiden der Pflanzen. Flora 79 : Erganzb.: 1-48. pi. 1. 1897a. Ueber den Nebenkern in spermatogenen Zellen und die Spermatogenese bei den Farnkrautern. Ber. Dea. Bot. Ges. 15 : 337-339. 18976. Ueber die Spermatogenese bei den Schachtelhalmen. Ibid. 15: 339-342. 1897c. Ueber die Aehnlichkeit einiger Erscheinungen in der Spermatogenese bei Thieren und Pflanzen. Ibid. 15 : 342-345. 1898. Ueber die Cilienbildner in spermatogenen Zellen. Ibid. 16: 140-144. pi. 7. 1899. Ueber die Centrosome in den spermatogenen Zellen. Ibid. 17: 199-205. pi. 15. VAN BENEDEN, E. 1876. Recherches sur les Dicyemides, survivants actuelles d'un embranchement des Mesozoaires. Bull. Acad. Roy. Belg. 41: 1160-1205; 42: 35-97. pis. 3. 1883. Recherches sur la maturation de 1'oeuf, la fecondation et la division cellu- laire. Arch. d. Biol. 4. 98 INTRODUCTION TO CYTOLOGY 1887. (van Beneden et Neyt.) Nouvelles recherches sur la fecundation et la division mitosique chez i'Ascaride megalocephale. Bull. Acad. Roy. Belg. Ill 14: 215-295. pi. 1. BOLLETER, E. 1905. Fegatelia (L.> corda. Einc morphologisch-physiologische Monographie. Beih. Bot. Centr. 18: 327-408. pis. 12, 13. BOVERI, TH. 1887a. Ueher die Befruchtung der Eier von Ascaris megalocephala. Sitz-ber. Ges. Morph. Phys. Miinchen 3. 18876. Ueber den Anteil des Spermatozoon an der Teilung des Eies. Ibid. 1888. Zellenstudien. 11. Jenaische Zeitschrift 22: 685-882. pis. 19-23 1901. Zellenstudien. IV. Ueber die Natur der Centrosomen. Jen. Zeitschr. 36 : 1-220. pis. 1-8. BRATJER, A. 1893. Zur Kenntniss der Spermatogenese von Ascaris megalocephala. Arch. Mikr. Anat. 42: 153-212. pis. 11-13. BUCHTIEN, O. 1887. Entwicklungsgeschichte des Prothalliums von Equiselum. Bibliotheca Botanica 8 : 1-49. pis. 1-6. BUTSCHLI, O. 1891. Ueber die sogenannten Centralkorper der Zelle und ihre Bedeutang. Verh. Naturhist.-Med. Ver. Heidelb. 4: 535-538. CALDWELL, O. W. 1907. Microcycas calocoma. Bot. Gaz. 44: 118-141. pis. 10-13. CAMPBELL, D. H. 1887. Zur Entwicklungsgeschichte der Spermatozoiden. Ber. Deu. Bot. Gesell. 5: 120-127. pi. 6. CHAMBERLAIN, C. J. 1898. The homology of the blepharoplast. Bot. Gaz. 26 : 431-435. 1903. Mitosis in Pellia. Decenn. Pub. Univ. Chicago 10: 329-345. pis. 25-27. 1909. Spermatogenesis in Dioon edule. Bot. Gaz. 47: 215-236. pis. 15-18. CHAMBERS, R. 1917. Microdissection studies. II. The cell aster. A reversible gelation phenomenon. Joar. Exp. Zool. 23: 483-504. CLAUSSEN, P. 1912. Zur Entwicklungsgeschichte der Ascomyceten. Zeitschr. Bot. 4: 1-64. pis. 1-6. figs. 13. CONKLIN, E. G. 1912. Experimental studies on nuclear and cell division in the eggs of Crepidula. Jour. Acad. Nat. Sci. Phila. 15: 503-591. pis. 43-49. DANGEARD, P. A. 1896. Memoire sur les Chlamydomonadinees on 1'histoire d'une cellule. Le Botaniste 6: 65-290. figs. 19. 1901. Etude sur la structure de la cellule et ses fonctions. Le Polytoma uvella. Ibid. 8 : 5-58. figs. 4. DAVIS, B. M. 1898. Kerntheilung in der Tetrasporemnutterzelle bei Corallina officinalis L. var. mediterranea. Ber. Deu. Bot. Gesell. 16: 266-272. pis. 16, 17. 1908. Spore formation in Derbesia. Ibid. 22 : 1-20. pis. 1, 2. ENTZ, G. 1918. Ueber die mitotische Teilung von Polytoma uvella. Arch. f. Protistenk. 38: 324-354. pis. 12, 13. figs. 5. ERHARD, H. 1911. Die Henneguy-Lenhosseksche Theorie. Ergeb. Anat. u. Entw. 19: 893-929. (List of 146 papers.) ESCOYEZ, E. 1907. Blepharoplaste et centrosome dans le Marchantia polymorpha. La Cellule 24: 247-256. pi. 1. 1909. Caryocinese, centrosome et kinoplasme dans le Stypocaulon scoparium. La Cellule 25: 181-201. 1 pi. .FARMER, J. B. and REEVES, J. 1894. On the occurrence of centrospheres in Pellia epiphytta, Nees. Ann. Bot. 8: 219-224. pi. 14. FARMER, J. B. and WILLIAMS, J. L. 1896. On fertilization, and the segmentation of the spore in Fucm. Ibid. 10: 479-487. 1898. Contributions to our knowledge of the Fucaceae; their life history and cyt- ology. Phil. Trans. Roy. Soc. London B 190: 623- 645. pis. 19-24. FAULL, J. H. 1905. Development of ascus and spore formation in ascomycetes. Proc. Boston Soc. Nat. Hist. 32: 77-113. pis. 7-11. THE CENTROSOME AND THE BLEPHAROPLAST 9(J FLEMMING, W. 1875. Studien in der Entwicklungsgeschichte der Najaden. Sitz- ber. Akad. Wiss. Wien 71. FLORIN, R. 1918. Das Archegoniurn der Riccardia pinguis (L) B. Gr. Svensk. Bot Tidskr. 12 : 464-470. figs. 4. FOL, H. 1877. Sur le commencement de 1'henogenie chez divers animaux. Arch. Sci. Nat. u. Phys. Geneve 68. 1891. Die " Centrenquadrille," ein neue Episode aus der Befruchtungsgeschichto. Anat. Anz. 6 : 266-274. figs. 10. FRASER, H. C. 1. 1908. Contributions to the cytology of Humaria rutilans. Ann. Bot. 22 : 35-55. pis. 4, 5. FRASER, H. C. I. and WELSFORD, E. J. 1908. Further contributions to the cytology of the ascomycetes. Ibid. 22: 465-477. pis. 26, 27. FRASER, H. C. I. and BROOKS, W. E. ST. J. 190S. Further studies on the cytology of the ascus. Ibid. 23 : 538-549. FUJII, K. 1898. (Has the spermatozoid of Ginkgo a tail or not?) Bot. Mag. Tokyo 12 : 287-290. (Japanese.) 1899. (On the morphology of the spermatozoid of Ginkgo biloba.) Ibid. 13: 260- 266. pi. 7. (Japanese.) 1900. (Account of a sperm with two spiral bands.) Ibid. 14: 16-17. (Japanese.) GRAHAM, M. 1918. Centrosomes in fertilization stages of Preissia quadrata (Scop.) Nees. Ann. Bot. 32 : 415-420. pi. 10. GREGOIRE, V. et BERGHS, J. 1904. La figure achromatique dans le Pellia epiphylld. La Cellule 21 : 193-238. pis. 1, 2. GRIGGS, R. F. 1912. The development and cytology of Rhodochylrium. Bot. Gaz. 63: 127-173. pis. 11-16. GUIGNARD, L. 1889. Developpement et constitution des antherozoides. Rev. G<5n. Bot. 1: 11-27, 63-78, 136-145, 175-194. pis. 2-6. 1898. Centrosomes in Plants. Bot. Gaz. 25: 158-164. GUILLIERMOND, A. 1904. Recherches sur la karyokinese chez les ascomycetes. Rev. Gen. Bot. 16: 129-143. pis. 14, 15. 1905. Remarques sur la karyokine&e des ascomycetes. Ann. Mycol. 3: 344-361. pis. 10-12. 1911. Apercu sur 1'evolution nucleaire des ascomycetes et nouvelles observations sur les mitoses des asques. Rev. Gen. Bofc. 23: 89-121. figs. 8. pis. 4, 5. HARPER, R. A. 1895. Beitrag zur Kenntniss der Kernteilung und Sporenbildung im Ascus. Ber. Deu. Bot. Ges. 13: (67)-(68). pi 27. 1897. Kerntheilung und freie Zellbildung im Ascus. Jahrb. Wiss. Bot. 30 : 249- 284. pis. 11, 12. 1899. Cell division in sporangia and asci. Ann. Bot. 13: 467-525. pis. 24-26. 1905. Sexual reproduction and the organization of the nucleus in certain mildews. Carnegie. Inst. Publ. 37. Washington. 1919. The structure of protoplasm. Am. Jour. Bot. 6: 273-300. HARTMANN, M. und NOLLER, W. 1918. Untersuchungen iiber die Cytologie von Trypanosoma theileri. Arch. Protistenk. 38: 355-374. pis. 14, 15. figs. 6. HENNEGUY, L. F. 1897. Sur les rapports des cils vibratiles avec les centrosomes. Arch. d'Anat Micr. 1 : 481-496. figs. 5. HIRASE, S. 1894. Notes on the attraction spheres in the pollen cells of Ginkgo biloba. Bot. Mag. Tokyo 8 : 359. HUMPHREY, J. E. 1894. Nucleolen und Centrosomen. Ber. Deu. Bot. Ges. 12 : 108-117. pi. 6. HUMPHREY, H. B. 1906. The development of Fossombronia longiseta Austr Ann. Bot. 20: 83-108. pis. 5, 6. figs. 8. 100 INTRODUCTION TO CYTOLOGY IKENO, S. 1898. Untersuchungen iiber die Entwicklung der Geschlechtsorgane und den Vorgang der Befruchtung bei Cycas revoluta. Jahrb. Wiss. Bot. 32 : 557-602. pis. 8-10. 1903. Die Sperm atogenese von Marchantia polymorpha. Beih. Bot. Centralbl. 15:65-88. pi. 3. 1904. Blepharoplasten im Pflanzenreich. Ibid. 24: 211-221. figs. 1-3. 1906. Zur Frage nach der Homologie der Blepharoplasten. Flora 96 : 538-542. JAHN, E. Myxomycetenstudien. 3. Kernteilung und Geisselbildung bei den Schwarmern von Stemonitis flaccida Lister. Ber. Deu. Bot. Gesell. 22 : 84-92. pi. 6 KARSTEN, G. 1900. Die Auxosporenbildung der Gattungen Cocconeis, Surirella, und Cymatopleura. Flora 87 : 253-283. pis. 8-10. KOERNICKE, M. 1903. Der heutige Stand der pflanzlichen Zellforschung. Ber. Deu. Bot. Gesell. 21: (66)-(134). 1906. Zentrosomen bei Angiospermen? Flora 96: 501-522. pi. 5. VON KORFP, K. 1899. Zur Histogenese der Spermien von Helix pomalia. Arch. Mikr. Anat. 54: 291-296. pi. 16. KTJCZYNSKI, M. H. 1917. Ueber die Teilung der Trypanosomenzelle nebst Bemer- kungen zur Organization einiger nahestehender Flagellaten. Arch. f. Protistenk. 38:94-112. pis. 3, 4. LAUTERBORN, R. 1896. Untersuchungen iiber Bau, Kernteilung, und Bewegung der Diatomen. Leipzg. VON LENHOSSEK, M. 1898. Ueber Flimmerzellen. Verh. Anat. Ges. Kiel 12: 106. LEVINE, M. 1913. The cytology of Hymenomycetes, especially the Boleti. Bull. Torr. Bot. Club 40: 137-181. pis. 4-8. LEWIS, C. E. 1906. Embryology and development of Riccia lutescens and Riccia crystailina. Bot. Gaz. 41 : 109-138. pis. 5-9. MAIER, H. N. 1903. Ueber die feineren Bau der Wimperapparate der Infusorien. Arch. f. Protistenk. 2. MAIRE, R. 1905. Recherches cytologiques sur quelques ascomycetes. Ann. Mycol. 3:123-154. pis. 3-5. MEAD, A. D. 1898. The origin and behavior of the centrosomes in the annelid egg. Jour. Morph. 14: 181-218. MEVES, F. 1897. Ueber Struktur und Histogenese der Samenfaden von Salamandra maculosa. Arch. Mikr. Anat. 50: 110-141. pis. 7, 8. 1899. Ueber Struktur und Histogenese der Samenfaden des Meerschweinchens. Ibid. 64: 329-402. pis. 19-21. figs. 16. MEYER, K. 1911. Untersuchungen tiber den Sporophyt der Lebermoose. I. Ent- wicklungsgeschichte des Sporogons der Corsinia marchantioides. Bull. Soc. Imp. Moscou 236-286. MINCHIN, E. A. 1912. An Introduction to the Study of the Protozoa. London. MIYAKE, K. 1905. On the centrosome of Hepaticse. Bot. Mag. Tokyo 19: 98- 101. 1906. The spermatozoid of Ginkgo. Jour. Appl. Micr. and Lab. Methods 5 : 1773-1780. figs. 10. MOORE, J. E. S. 1895. Structural changes in the reproductive cells during spermato- genesis of elasmobranchs. Quar. Jour. Micr. Sci. 38: 275-313. pis. 13-16. MORGAN, T. H. 1896. The production of artificial astrospheres. Arch. Entw. 3: 339-361. pi. 19. 1899. The action of salt solutions on the unfertilized and fertilized eggs of Arbacia and other animals. Ibid. 8: 448-539. pis. 7-10. figs. 21. MOTHER, D. M. 1898. Das Centrosom bei Dictyota. Ber. Deu. Bot. Ges. 16: 123-128. figs. 5. THE CENTROSOME AND THE BLEPHAROPLAST 101 1900. Nuclear and cell division in Dictyota dichotoma. Ann. Bot. 14: 166-192. pi. 11. 1904. The development of the spermatozoid of Chora. Ibid. 18 : 245-254. pi. 17. PAULMIER, F. C. 1899. The spermatogenesis of Anasa Iristis. Jour. Morph. 16 : Suppl. 223-272. pis. 13, 14. RAWITZ, B. 1896. Untersuchungen iiber Zelltheilung. I. Arch. Mikr. Anat. 47: 159-180, pi. 11. SAGUCHI, S. 1917. Studies on ciliated cells. Jour. Morph. 29: 217-279. pis. 1-4. SANDS, M. C. 1907. Nuclear structure and spore formation in Microsphcera. Trans. Wis. Acad. Sci. 16 : 733-752. pi. 46. SAPEHIN, A. A. 1913. Untersuchungen iiber die Individuality der Plastide. Ber. Deu. Bot. Ges. 31 : 14-66. fig. 1. SCHAFFNER, J. H. 1908. The centrosomes of Marchantia potymorpha. Ohio Nat. 9. 363-388. SCHOTTLANDER, P. 1893. Beitragc /ur Kenntniss des Zellkerns und der Sexual- zellen bei Kryptogamen. Cohn's Beitr. Biol. Pflanzen 6 : 267-304. pis. 4, 5. SHARP, L. W. 1912. Spermatogenesis in Equisetum. Bot. Gaz. 64: 89-119. pis. 7,8. 1914. Spermatogenesis in Marsilia. Ibid. 58: 419-431. pis. 33, 34. 1920. Spermatogenesis in Blasia. Ibid. 69: 258-268. pi. 15. SHAW, W. R. 1898. Ueber die Blepharoplasten bei Onoclea und Marsilia. Ber. Dea. Bot. Ges. 16: 177-184. pi. 11. SMITH, H. L. 1886-1887. A contribution to the life history of the Diatomacese. Proc. Am. Soc. Micr. Pts. I and II. STRASBTJRGER, E. 1892. Schwarmsporen, Gameten, pflanzliche Spermatozoiden, und das Wesen der Befruchtung. Hist. Beitr. 4 : 49-158. pi. 3. 1897. Kerntheilung und Befruchtung bei Fucus. Jahrb. Wiss. Bot. 30 : 351-374. pis. 27, 28. 1900. Ueber Reduktionstheilung, Spindelbildung, Centrosomen, und Cilienbildner im Pflanzenreich. Hist. Beitr. 6: 1-224. pis. 1-4. STUDNICKA, F. K. 1899. Ueber Flimmer- und Cuticularzellen mit besonderer Beriicksichtigung der Centrosomenfrage. Sitz-Ber. K. Bohmisch. Ges. Wiss. Math.-Naturwiss. Classe, 36. SWINGLE, W. T. 1897. Zur Kenntniss der Kern- und Zelltheilung bei den Sphacela- riaceen. Jahrb. Wiss. Bot. 30: 296-350. pis. 15, 16. THOM, C. 1899. The process of fertilization in Aspidium and Adiantum. Trans Acad. Sci. St. Louis 9: 285-314. pis. 36-38. TIMBERLAKE, H. G. 1902. Development and structure of the swarm spores of Hydrodictyon. Trans. Wis. Acad. Sci. 13: 486-522. pis. 29, 30. VAN HOOK, J. M. 1900. Notes on the division of the cell and nucleus in liverworts, Bot. Gaz. 30 : 394-399. pis. 2, 3. WEBBER, H. J. 1897a. Peculiar structures occurring in the pollen tube of Zamia. Bot. Gaz. 23: 453-459. pi. 40. 18976. The development of the antherozoid of Zamia. Ibid. 24: 16-22. figs. 5. 1897c. Notes on the fecundation of Zamia and the pollen tube apparatus of Ginkgo. Ibid. 24: 225-235. pi. 10. 1901. Spermatogenesis and fecundation in Zamia. U. S. Dept. Agr. Pit. Ind Bull. 2. pp. 100. pis. 7. WILLIAMS, J. L. 1904. Studies in the Dictyotaceae. II. The cytology of the game- tophyte generation. Ann. Bot. 18: 183-204. pis. 12-14. WILSON, E. B. 1900. The Cell in Development and Inheritance, (p. 175.) 1901. Experimental Studies in Cytology. I. A cytological study of partheno genesis in sea urchin eggs. Arch. Entw. 12: 529-596. pis. 11-17. 102 INTRODUCTION TO CYTOLOGY WILSON, M. 1911. Sperm atogenesis in the Bryophyta. Ann. Bot. 25: 415-457. pis. 37, 38. figs. 3. VON WINIWARTER, H. 1912. Observations cytologiques sur les cellules interstiti- elles du testicule humaine. Anat. Anz. 41 : 309-320. pis. 1-2. WOLFE, J. J. 1904. Cytological studies on Nemalion. Ann. Bot. 18: 607-630. pis. 40, 41. fig. 1. WOODBURN, W. L. 1911. Spermatogenesis in certain Hepaticse. Ann. Bot. 25: 299-313. pi. 25. 1913. Spermatogenesis in Blasia pusilla. Ibid. 27: 93-101. pi. 11. 1915. Spermatogenesis in Mnium affine, var. ciliaris (Grev.), C.M. Ibid. 29: 441-456. pi. 21. YAMANOUCHI, S. 1906. The life history of Polysiphonia violacea. Bot. Gaz. 42 : 401-448. pis. 19-28. 1908. Spermatogenesis, oogenesis, and fertilization in Nephr odium. Ibid. 45 : 145-175. pis. 6-8. 1909. Mitosis in Fucus. Ibid. 47: 173-197. pis. 8-11. ZIMMERMANN, A. 1893-1894. Sammel-Referate. 6. Die Centralkorper und die Kerntheilung. 12. Die Cilien und Pseudocilien. Beih. Bot. Centralbl. 3 : 342-354; 4: 169-171. CHAPTER VI PLASTIDS AND CHONDRIOSOMES PLASTIDS Next to the nucleus, the most conspicuous organ held within the cytoplasm of the plant cell is the plastid. Cytologists have long been aware of the important physiological roles played by plastids of various types in the life of the cell, but it is only recently that an added inter- est has been given these organs by the discovery that certain peculiar characters showing definite modes of inheritance are closely bound up with their behavior. Such problems are complicated by the relation apparently borne by plastids to chondriosomes. In the present chap- ter will be set forth some of the more important facts regarding these two classes of cell elements. General Nature and Occurrence. — Plastids are differentiated portions of the protoplasm, as von Mohl long ago pointed out, ' and represent regions in which certain processes have become localized (Harper 1919). In view of their power of growth and division and their definite relation to certain important physiological functions they are to be regarded as distinct cell organs. Although plastids can be found in the cells of both animals and plants they are chiefly characteristic of the latter, where they are present in one form or another in all groups with the possible exception of bacteria, myxomycetes, and certain fungi. They are abundant only in those plant parts which have to do with specialized physiological functions. Within a single cell there may be regularly but one plastid, as in many algse, Anthoceros, and the meristematic cells of Selaginella (Haberlandt 1888, 1905) ; or two, as in Zygnema; or a /figher number, as in the green tissues/ of most higher plants. They lie imbedded in the cytoplasm and are often closely associated with the nucleus; they are never found normally in the vacuole. The positions which they assume within the cell are fre- quently related in a definite manner to certain external conditions: in the palisade cells of green leaves, for example, the chloroplasts are found near the upper surface if the incident light is weak, whereas they react to strong illumination by taking up less exposed positions along the lateral walls. Plastids may be conveniently classified on the basis of their contained coloring matters. This difference in color, however, is secondary in importance; the fundamental distinction is that based upon the kind of 103 104 INTRODUCTION TO CYTOLOGY physiological work being done, the various pigments being associated in an intimate manner with different reactions occurring within the plastids. Leucoplasts. — Leucoplasts are relatively small and colorless. They are found commonly in the cells of meristematic tissue, and may be retained in some kinds of differentiated cells, such as the glandular hairs of Pelargonium. K lister (1911) states that the leucoplasts of Orchis are very fluid in consistency, undergoing amoeboid changes of shape and multiplying by irregular fission. The smaller leucoplasts appear to represent juvenile stages in the development of plastids of more highly differentiated types, for under certain conditions they develop into the larger and more highly specialized leucoplasts known as amyloplasts, and into the various kinds of chromatophores mentioned below. Chromatophores. — Chromatophores, or chromoplasts, are plastids bear- ing one or more pigments, and having thus a more or less decided color. In green plants the most important of these pigments are chlo- FIG. 37. — Various forms of plastids. A, Draparnaldia. B, Spirogyra. C, Anthoceros. D, chromoplasts of Arisoema. E, cell of Selaginella, showing position assumed by plastid in response to light (direction shown by arrow). A, B, and C show pyrenoids. (E After Haberlandt.) rophyll, carotin, and xanthophyll. Chlorophyll is apparently a combi- nation of two simpler pigments, chlorophyll a and chlorophyll 6. The cells of the Phaeophyceae, Cyanophyceae, and Rhodophyceae are character- ized'respectively by the presence of yellow carotin, blue phycocyanin, and red phycoerythrin, in addition to chlorophyll. The Cyanophyceae exhibit an especially rich variety of pigments, which in many cases do not appear to be held within definite chromatophores.1 Chromatophores are usually spherical, ovoid, or discoid in shape, but many peculiar forms are known, particularly among the green algae. In Ulothrix the chloroplast has the form of a complete or incomplete hollow cylinder; in Draparnaldia (Fig. 37, A), a hollow cylinder with very 1 For the literature pertaining to plant pigments see Palladin 1918. See also Haas and Hill (1913), Willstatter and Stoll (1913), Jorgensen and Stiles (1917), Wheldale (1916), and Beauverie (1919). The distribution of carotin is discussed in an earlier paper by Tammes (1900). PLASTIDS AND CHONDRIOSOMES 105 irregular ends; in dEdogonium, an irregular parietal net; in Spirogyra (Fig. 37, B), a spirally coiled ribbon; and in the desrnids, a series of radiating plates (Carter 1919, 1920). The chromatophore of Antho- ceros (Fig. 37, C) is spindle-shaped, becoming chain-like in the elongated columella cells (Scherrer 1914). The chromatophore of Selaginella may also assume this form (Haberlandt 1888). The chromoplasts of Arisoema (Fig. 37, D) are frequently sharply angular. In the Clado- phoraceae (Carter 1919) the cell is completely lined by a thin chromato- phore which may be entire or fenestrated. In many cells irregular strands pass inward through the cell cavity. Indeed it seems not improbable that in some such cases the plastid may be not at all sharply distinct from the rest of the cytoplasm, the two grading one into the other, and the chlorophyll at certain stages permeating all parts of the cytoplasm. The observations of Timberlake and Harper appear to show that such is the condition in the young cells of Hydrodictyon. Thus the physiological processes show various degrees of localization in the cell, causing manifold degrees of structural transformation and delimitation of the cytoplasmic regions involved (Harper). Of all chromatophores the chloroplasts stand first in importance, for they bear the green pigment, chlorophyll, which, in the presence of light, enables them to combine water from the soil or other surrounding medium with carbon dioxide from the atmosphere to form carbohydrates, the first visible product being starch. The chloroplasts are therefore the world's ultimate food producers. In addition to chlorophyll other pigments, notably xanthophyll, are usually present. Although the body of the chloroplast can be developed in darkness, the chlorophyll will usually not be elaborated unless light is present. Most young seedlings grown in the absence of light show a pale yellowish color, which is due to a substance known as chlorophyllogen, contained in the plastids. When such "etiolated" plants are placed in the light the plastids become green, apparently through an alteration of the chlorophyllogen to chlorophyll ( Monte verde and Lubimenko 1911). Other conditions necessary for the development of chlorophyll are a favorable temperature and the presence of iron, oxygen, and certain carbohydrates. The structure of the chloroplast is an extremely difficult matter to determine, and has been the subject of some controversy. It is generally thought that the body of the plastid is composed of a finely fibrillar meshwork, the stroma, which may be somewhat denser at the periphery, and that the coloring matters are held in the meshes of the stroma in the form of minute droplets. No limiting membrane is definitely known. The included droplets are apparently not composed of the pigments alone : it is probable that they are rather globules of some oily or fatty material containing the pigments in solution. The pigments may easily be dis- solved out with alcohol and other reagents. On the other hand, it has 106 been held by some observers that the stroma is a homogeneous body in which tlie droplets of chlorophyll solution are imbedded, and that the reticular structure so often reported is an artifact due to the reagent employed in removing the chlorophyll. By others the pigment has been thought to form a layer about the plastid. In any case it seems evident that the chlorophyll is not uniformly distributed throughout the stroma. In chromatophores other than chloroplasts the pigments may at times take the form of solid granules or crystals. Starch. — After a period of photosynthetic activity the chloroplast contains starch, the first visible product of that activity, in the form of minute granules. This "assimilation starch" is formed within the body of the chloroplast, as Meyer originally showed (Fig. 38, A, B). It is later transformed through the agency of enzymes into some soluble compound, usually a sugar; in this form it may be carried to growing regions, where, after further changes, it is built into the structure of the plant. Or, it may pass to storage organs where it is transformed into the ordinary "reserve starch," or "storage starch." This de- position of reserve starch is brought about through the agency of amyloplasts, which are leucoplasts capable of changing already elaborated organic materials, such as glucose, into starch (Fig. 38, C). Reserve starch, upon which we depend so largely for food, is a carbohydrate with a composi- tion expressed by the general formula (C6Hi0O5)n, and exists in the form of granules ranging in size approximately from 2//. to 200/j in different plants. Potato-starch grains are usually about 90^ in diameter. The reserve starch grain is formed within the body of the arnyloplast, and is made up of a series of concentric layers successively laid down about a center, or "hilum" (Fig. 39, A).1 In case the grain starts to form near the middle of the amyloplast it may develop sym- metrically, but commonly the developing grain lies near the periphery of the amyloplast, which becomes greatly distended as the grain grows. Material is thus deposited unevenly upon the grain so that the latter becomes very eccentric; in extreme cases the grain ruptures the amylo- plast and remains in contact with it only at one side, where all new material is then deposited. Several grains may start to develop simul- 1 For the structure of the starch grain see the papers of Nageli, Schimper, Meyer, Binz, Dodel, Salter, and Kramer. FIG. 38. — Formation of starch by plastids. A, dividing chloro- plasts of Funaria, with grains of assimilation starch. X 940. (After Strasburger.) B, chloro- plast of Zygnema, with several large starch grains about a central pyrenoid. (After Bour- quin, 1917.) C, leuco- plast (amyloplast) in aerial tuber of Phajus grandifolius with grain of reserve starch. (After Strasburger.) PLASTIDS AND CHONDRIOSOMES 107 taneously in a single amyloplast, later growing together to form :i "compound grain " with more than one hilum. In case the parts making up the compound grain are enveloped in one or more common outer layers the grain is said to be "half-compound." Potato starch is made up of simple, compound, and half-compound grains, whereas in oats and rice all or nearly all of the grains are said to be of the compound type. The successively deposited layers making up the grain differ mainly in water content, the innermost layers being richest and the outermost poorest in water. As a result of this non-uniform composition the grain often splits radially when dried. '•[ FIG. 39. — Reserve starch grains from various plants. A, potato; simple and half-compound grains. B, Colombo starch. C, arrowroot. D, pea. E, maize; intact and partially digested grains. F, rye. G, maize. /, bean. J, rice. K, wheat. (After Tschirch.) H, Euphorbia. As a result of his classic researches Nageli (1858) advanced the theory that the starch grain is made up of ultramicroscopic crystalline particles which he called " micellae/' these being surrounded by water films of varying thickness. It was similarly held by A. Meyer (1883, 1895) that the grain is composed of radially arranged needle-shaped crystals known as "trichites;" these are composed of a- and /3-amylose which turn blue with iodine. In some starch amylodextrin and dextrin are also present, such grains turning red with iodine. Both Nageli and Meyer held the stratification of the grain to be due to the varying numbers of the crystalline units in the successive layers, and Meyer showed that in certain cases it is correlated with the alternation of day and night, and therefore with a periodic activity on the part of the plastid. This con- clusion was confirmed by Salter (1898). The statement made by Schimper (1880) and Meyer (1883, 1895) that starch is always formed by plastids still holds good in its essential feature : so far as is certainly known no primary product of photosynthesis is formed in the cytoplasm apart from plastids, although in some cases, such as the young cells of Hydrodictyon, according to Harper, it is very difficult or even impossible to distinguish the limits of these organs. The 108 INTRODUCTION TO CYTOLOGY granules of paramylum in Euglena and those of "Floridean starch" in the red alga? first appear in the cytoplasm; but, although they arc the first substances which are visible, it is highly probable that they arise through the transformation of a non-visible product (sugar?) of the photosynthetic activity of the plastids, and are not immediately built up from water and carbon dioxide. A similar interpretation may be placed upon corresponding appearances reported in the case of higher plants. Owing to the great difficulty of determining the true cell struc- ture of the Cyanophyceae (see p. 202) it is possible to speak of plastid activity in such forms only with great reserve. If, as Olive (1904) and Gardner (1906) hold, these cells are without plastids, the product of photosynthetic activity, commonly glycogen, must be elaborated in the cytoplasm without their aid. If, on the other hand, the peripheral portion of the protoplast represents a large chromatophore (Fischer 1898), or cytoplasm containing a large number of minute chromatophores (Hegler 1901, Kohl 1903, Wager 1903), the photosynthetic process, although it may result in the production of a different substance, is dependent upon the powers of definite protoplasmic organs much the same as in higher plants. Among bacteria and other low forms in which it seems more certain that plastids and the ordinary pigments are absent, widely different types of metabolism are met with. For further discus- sion of this subject, which lies outside the scope of the present book, more special physiological works should be consulted. The Pyrenoid. — The term pyrenoid was applied by Schmitz (1882) to the refractive kernel-like bodies imbedded in the chromatophores of the algae. Pyrenoids are characteristic of the Chlorophycese especially, being present almost universally in the members of this group. They are known in a few representatives of the IJiodophyceae (Nemalion and the Bangiacese), but apparently do not occur in the cells of the Cyanophyceae, Phaeophyceae, and Characeae. Very rarely they are present in forms above the algae : a conspicuous example is the liverwort Anthoceros. The chromatophore may contain but one pyrenoid, as in Zygnema, or a larger number, as in Spirogyra, Draparnaldia, and many other forms (Fig. 37). As held by de Bary (1858), Schmitz (1884), and Schimper (1885), the pyrenoid appears to be composed of a protein substance with a thick gelatinous consistency. When a single pyrenoid is present in the chroma- tophore it may multiply by fission along with the latter when the cell divides, while in those forms possessing several pyrenoids this multiplica- tion may be much more extensive. Also, as pointed out by Schmitz and Schimper, and more recently by Smith (1914), the pyrenoid may disappear and arise de novo from the cytoplasm or from the plastid protoplasm. With regard to its function, the early workers referred to above ob- PLASTIDS AND CHONDRIOSOMES 109 served that under certain conditions the pyrenoid is closely surrounded by a mass of starch grains, and concluded that it is an organ, or portion of an organ (chromatophore), intimately concerned in the process of starch formation, its action being somewhat similar to that of the amyloplast. The pyrenoid, in fact, has often been likened to a leucoplast imbedded in the chromatophore; Wiesner, for instance, believed the pyrenoid to contain several leucoplast bodies, each of which gave rise to a starch grain. In general, more recent researches have emphasized the close association of the pyrenoid with the starch forming process, although the precise nature of this process remains very much in doubt. Accord- ing to Timberlake (1901) the pyrenoid in Hydrodictyon is differentiated from the cytoplasm and is very active in starch production, segments splitting off from its periphery and forming starch within them. In this way the entire pyrenoid may become a mass of "pyrenoid starch," as distinguished from ordinary, or "stroma starch." McAllister (1913) describes a similar splitting up of the pyrenoid to form several starch grains in Tetraspora. Yamanouchi (1913), however, in his description of a new species of Hydrodictyon, states that some of the chloroplasts give rise to starch while others give rise to pyrenoids, and that the latter have nothing to do with starch formation. A similar diversity of opinion exists with respect to the role of the pyrenoid in Zygnema. Chmielewskij (1896), who looked upon the pyrenoid as a permanent cell organ multiplying only by division, held that starch grains arise wholly from the substance of the pyrenoid, plate-like extensions of the latter being present between and in intimate contact with the developing grains. More recently Miss Bourquin (1917) asserts that the pyrenoid has nothing to do with the appearance of starch, the body of the chromatophore alone being concerned. She observes the starch grains appearing first near the periphery of the chromatophore entirely apart from the pyrenoid, the later formed grains differentiating in positions progressively nearer the pyrenoid (Fig. 38, B). The pyrenoid of Anthoceros (Fig. 37, C) as described by McAllister (1914) is in reality a group of about 25-300 small "pyrenoid bodies" which are probably composed of a protein substance. The outermost bodies become starch, new ones apparently being formed by the fission of those lying on the interior of the group. McAllister states that no pyrenoid is visible in the young sporogenous tissue, starch being formed without its aid. Somewhat later several small bodies appear and aggregate to form the pyrenoid. Cleland (1919) recently reports a close association of the pyrenoid of Nemalion with the formation of Floridean starch. Elaioplasts and Oil Bodies. — In 1888 Wakker discovered in the cells of Vanilla planifolia and V. aromatica certain plastid-like bodies to which he gave the name elaioplasts, since they seemed to be concerned in 110 INTRODUCTION TO CYTOLOGY the elaboration of oil (Fig. 40, A}. They were soon observed in a number of monocotyledons by Zimmermann (1893), Raciborski (1893), and Kiis- ter (1894); and some time later in the flower parts of a dicotyledon, Gaillardia, by Beer (1909). Politis (1914) has found them in monocoty- ledonous plants belonging to 19 different genera, and in five genera of dicotyledons (Malvaceae). There is a considerable lack of agreement in the opinions expressed on the subject of the origin and significance of elaioplasts. Wakker thought it probable that they represent meta- morphosed chloroplasts, which they often closely resemble in structure (Kiister), whereas Raciborski asserted that they arise as small refractive gran- ules in the cytoplasm and multiply by budding. In the zygospores of Sporodinia grandis and Phycomyces nitens Miss Keene (1914, 1919) reports the presence of a number of globular structures with which oil is associated from their earliest stages. These unite to form one or two large reticulate bodies which Miss Keene believes are related to the elaioplasts of higher plants. All of these investigators, with Politis, agree that elaio- plasts are normal cell organs with a special func- tion, namely, the formation of oily substances having a role in nutrition. Beer, on the contrary, states that in Gaillardia they are formed secon- darily by the aggregation of many small degen- erating plastids and their products at one or more points in the cell, all stages of the process being observed. Although the bodies so formed may, if green, produce starch, or, if colorless, an oily yellow pigment, Beer thinks it probable that they have no important special function in the life of the plant. Closely associated with investigations on elaioplasts have been those concerned with the oil bodies found in the cells of many liverworts (Fig. 40, B). These bodies, discovered by Gottsche in 1843, were first carefully described by Pfeffer (1874). Pfeft'er stated that they arise by the fusion of many minute droplets of fatty oil appearing in the cytoplasm of very young cells, and later come to lie in the cell sap; he further believed them to possess a special membrane. Wakker (1888) held them to be analo- gous to leucoplasts and chloroplasts, multiplying by fission at each cell-division, and pointed out that they lie in the cytoplasm rather than in the cell sap. He was inclined to view them as products of elaioplasts, which Ktister (1894) supposed them to resemble in having a spongy stroma containing oil in the form of minute droplets. FIG. 40. A, elaioplast forming oil droplets in epidermal cell of perianth of Poli- anthes tuberosa; nucleus with small plastids at right. (After Politis, 1914.) B, oil bodies in various stages of develop- ment in a cell of Caly- pogeia. (After Gargeanne, 1903.) PL AST IDS AND CHONDRIOSOMES 111 Quite different were the views of Gargeanne (1903). According to him they arise from vacuoles, their limiting membranes thus being the original tonoplasts. While in the juvenile vacuole stage they may multi- ply by division, but when once fully formed they remain unchanged and divide no further. Gargeanne observed small oil droplets moving about freely within the oil body, and hence concluded that the latter has a fluid consistency rather than a spongy stroma as Kiister thought. The most noteworthy recent observations on oil bodies are those of Rivett (1918), who finds them to be very conspicuous in the cells of Alicularia scolaris. Rivett holds that they are in reality only oil vacuoles —that they originate by the coalescence of numerous minute oil droplets secreted by the protoplasm in a manner entirely similar to that in which the ordinary sap vacuole arises (cf. Pfeffer). Although they become very large and project well into the sap vacuole, they continue to be surrounded by a thin film of cytoplasm. The oil body, in the opinion of Rivett, is therefore in no sense a plastid, nor is it formed by any special elaioplast : it is simply an accumulation of ethereal and fatty oils together with some protein substance. The "membrane" observed by Pfeffer is the limiting layer of the surrounding cytoplasm, which may be slightly changed by contact with the oil. Accumulations of oil apparently quite similar to those in liverwort cells have been described in the cells of various angiosperms by a number of writers. To these the term elaiospheres was applied by Lidforss (1893). The published figures of elaioplasts and oil bodies in many cases bear striking resemblance to those of fat- and oil-secreting chondrio- somes (see below), and it is not improbable that the problem of their origin and significance will be brought nearer solution by further studies of the latter class of bodies. The Eyespot. — The so-called eyespot present in the flagellate cell and in the zoospores and gametes of many algae has certain characteristics in common with plastids, and may therefore receive consideration here. This body, which nearly all workers agree is a light-sensitive organ, is an elongated or circular and flattened structure lying in the anterior region of the cell (flagellates) or near its lateral margin, usually in close associa- tion with the chromatophore and the plasma membrane. (Overtoil 1889; Klebs 1883, 1892; Johnson 1893; Strasburger 1900; Wollenweber 1907, 1908). With respect to its mode of origin, it has been variously reported to arise de novo in each newly formed zoospore in several green algae (Overton); to develop from a colorless plastid in the young antheridial cell in the case of the spermatozoid of Fucus (Guignard 1889) ; to arise as a differentiated portion of the plastid in the zoospores and gametes of Zanardinia ( Yamanouchi) ; and finally to multiply by fission at the time of cell-division in flagellates (Klebs 1892). It is generally agreed that the eyespot in many instances consists of 112 INTRODUCTION TO CYTOLOGY a finely reticulate stroma in which an oily red pigment with many of the characteristics of hsematochrom is held in the form of minute droplets or granules (Schilling 1891; Klebs 1883; Franze" 1893; Wager 1900; Wollen- weber 1907, 1908) (Fig. 41, D). As shown by the careful researches of Franze", the stroma may also bear one or more refractive inclusions, which in the Chlamydomonadaceae and Volvocacese consist of starch, and in the Euglenoidese of paramylum (Fig. 41, E). These inclusions were thought by Franze to increase the sensitivity of the eye- spot by concentrating the light at certain points. The eyespot of the zoospore of Cladophora (Strasburger 1900) appears to arise as a swelling of the plasma membrane, and consists of an external pigmented layer beneath which is a lens-shaped mass of hyaline substance (Fig. 41, B}. InGonium and Eudorina (Mast 1916) the lens-shaped portion lies outside with the cup-shaped opaque portion beneath it (Fig. 41, A), an arrangement strongly sug- gesting the primitive eyes of certain higher organisms. In neither portion could any finer structure be detected. Mast has shown that the orientation of the colony is brought about through changes in the intensity of the light As the unoriented swimming colony rotates on its axis, those zooids turning away from the light have the hyaline portion of their eye- -Eyespots of various types. A, zooid of Eudorina; e, eye- spot. (From Mast, After Grave.) B, zoospore of Cladophora, falling upon the light-sensitive substance. (After Strasburger, 1900.) C, anterior end of Euglena viridis, showing eyespot at surface of oesophagus, and in front of it a swelling on one root of the flageiium;face view of eyespot spots shaded by the opaque cup; this sudden reduction in the amount of light energy received brings about an increase in the activity of the flagellae of those zooids, with and crystalloid body. Frame'.) (After at right, showing pigment gran- ules. (After Wager, 1900.) D, eyespot of Euglena velata. (After Franze, 1893.) E, eye- spot of Trachelomonas volvo- dna, with pigment granules the result that the colony as a whole turns more directly toward the source of light. In Euglena viridis the morphological con- nection between the eyespot and the motor apparatus is particularly close. Here Wager (1900) has shown that the eyespot, which is a discoid protoplasmic body containing a layer of large pigment droplets, is situated at the surface bounding the oesophagus in close contact with a swelling on one of the basal branches of the flagellum (Fig. 41, C). In general it may be concluded that the eyespot in some cases bears in its structure, and to a certain extent in its evident function, such a close resemblance to the ordinary plastid that a relationship of some sort PLASTIDS AND CHONDRIOSOMES 113 between the two seems highly probable ; whereas in other cases (Gonium, Cladophora) it appears to represent a differentiation of the ectoplast. It is more than likely that light-sensitive organs have arisen more than once in the evolution of the lower organisms, and that they cannot all be placed in the same category. The Individuality of the Plastid. — It was believed by the early observers, notably Schimper (1883) and Meyer (1883), that plastids never originate de novo but always arise from preexisting plastids by division. Fully differentiated plastids, such as chloroplasts, can readily be seen multiplying in this manner in growing tissues with a frequency sufficient to account for the large number of plastids present in mature plant parts. Since it is known, however, that chloroplasts and other differentiated chromoplasts may arise from leucoplasts through the development of pigments and other characters in the latter, and also that the individual plant arises from sex cells or a spore in which the plastids are usually in a colorless and relatively undifferentiated state, the problem of the individuality of the plastid is mainly one of determining whether these undifferentiated plastids, leucoplasts, or "plastid primor- dia" later developing into chloroplasts and other types are continuous through the critical stages of the life cycle, multiplying only by division, or arise de novo as new differentiations of the cytoplasm. At this point we may review certain cases in which the plastid has been followed through gametogenesis and fertilization. In Zygnema (Kurssanow 1911) each vegetative cell contains one nucleus and two plastids, all of which divide at each vegetative cell- division. In sexual reproduction the entire protoplast, with its nucleus and two plastids, passes through the conjugating tube as a "male" gamete and unites with a similar complete protoplast ("female" gamete) of another filament. The two nuclei fuse, giving the primary nucleus of the new individual (zygospore nucleus), while the two plastids contrib- uted by the "male" gamete degenerate, leaving the two furnished by the "female" gamete as the plastids of the new individual. In Coleochcete (Allen 1905) each vegetative cell and gamete has one nucleus and one plastid: after the sexual union of the gamete nuclei the fertilized egg therefore contains one nucleus and two plastids. These two plastids divide at the first division of the fertilized egg but not at the second, the four resulting cells consequently having one plastid each. In the third cell-division the plastids also divide, so that each cell of the several-celled structure developing from the fertilized egg has its single plastid. Each of the several cells eventually becomes a zoospore which germinates to produce a new Coleochcete body with a single plastid in each cell, the plastid dividing with the nucleus at each cell-division. A somewhat similar regularity in the behavior of the plastid is shown in Anthoceros (Davis 1899; Scherrer 1914). Each gametophytic cell 114 INTRODUCTION TO CYTOLOGY contains a single plastid which divides with the nucleus at each cell- division. The egg likewise contains a plastid, but the spermatozoid has none: the fertilized egg and sporophyte cells which it later forms are therefore characterized, like the cells of the gametophyte, by the presence of one plastid. Although it is difficult to demonstrate the plastid in the young sporogenous cells, every sporocyte shows one very clearly. As shown by Davis (Fig. 42), the sporocyte plastid divides twice during the prophases of the first (heterotypic) division of the sporocyte nucleus, so that each spore of the resulting tetrad receives one. Upon germination the spore produces a gametophyte with one plastid in each cell, and the cycle is complete. FIG. 42. — The behavior of the plastid in the sporocyte of Anthoceroa. A, sporocyte with single nucleus and plastid. B, plastid divided; nucleus in prophase of mitosis. C, plastids divided to four; two nuclei present. D, three of the four spore cells, each of which has a single nucleus and plastid. (After Davis, 1899.) In all of the foregoing examples it is evident that the plastids, as stated by Scherrer for Anthoceros, remain as morphological individuals throughout the whole life cycle, multiplying exclusively by division. A similar claim is made for the plastids of mosses by Sapehin (1915), who has also studied the behavior of the plastids in Selaginella and Isoetes (1911, 1913). In such cases the plastids each possess an individuality com- parable to that of nuclei, from which they differ conspicuously, however, in undergoing no fusion at the time of fertilization. The constancy in number is nevertheless maintained : by the degeneration of the plastids of one gamete in Zygnema; by their failure to divide at one cell-division in Coleochcete; and because of the fact that the male gamete carries no plastid in Anthoceros. It appears to be generally true that while the eggs in all plant groups contain plastids (usually leucoplasts), the latter are present in male gametes in the algae only. Sapehin (1913), however, believes that the blepharoplasts of the higher groups represent plastids. It should be said that only in a comparatively few forms has such a regularity in the behavior of the plastid as that outlined above been demonstrated. A number of investigators, working on a great variety of cells, have been forced to conclude that plastids are either formed de novo as well as by division, or are carried through certain stages of the life PLASTIDS AND CHONDRIOSOMES 115 cycle in some less conspicuous form. If they represent regional trans- formations of the cytoplasm resulting from the localization of certain processes, they might well be expected to differentiate anew as these processes begin in the life of the cell, and to preserve varying degrees of permanence depending upon the processes carried on (Harper). Their individual continuity through certain life cycles would accordingly be interpreted to mean that in such forms there is a persistence of certain types of physiological activity through all stages. In recent years a number of cytologists have described the develop- ment of plastids from minute granular primordia in the cytoplasm, and have attempted to show that these primordia are members of the class of cell inclusions known as chondriosomes. A general theory of the indi- viduality of the plastid must therefore involve the question of the relation of plastids to chondriosomes, and the further question of the origin of the chondriosomes themselves. These matters will be taken up in the fol- lowing pages. CHONDRIOSOMES Notwithstanding the large amount of work which has been done upon chondriosomes during recent years, the condition of opinion as to their origin, behavior, and significance is still so unsettled that little more than a review and partial classification of the more prominent views will here be attempted. Chondriosomes were probably first observed many years ago by Flemming and Altman in the course of their studies on protoplasm. They were first clearly described by La Vallette St. George (1886), who observed them in the male cells of animals and called them "cytomicro- somes." In plants they were first described by Meves (1904) in the tapetal cells of the anthers of Nymphcea (Fig. 43, B). Benda in 1897 and the following years discovered them in cells of many types, notably in the spermatogenous cells of animals, and applied to them the term "mitochondria." It was not until a decade later, through the researches of Meves, Regaud, Faure^Fremiet, Lewitski, Guilliermond, and others that they came into prominence. Since that time they have been very intensively studied by both zoologists and botanists, and a special literature of considerable bulk has developed.1 It now seems evident that the filaments ("fila") of Flemming, the "bioplasts" of Altman, the " plastidules " of Maggi, the " archoplasmic granules" of Boveri, and the "mitochondria" of Benda are all one and the same thing — chondriosomes (Duesberg 1919). General Nature and Occurrence. — Chondriosomes occur in the cyto- plasm of the cell, commonly in the form of minute granules, rods, and 1 Reviews of the subject are given by Duesberg (1911, 1919), Schmidt (1912), Cavers (1914). and Guilliermond (1919). See also Meves (1918). 116 INTRODUCTION TO CYTOLOGY threads, but also in a great variety of irregular shapes (Fig. 43). At present it is customary with the majority of workers to refer to all types as chondrio somes or mitochondria. For those which are definitely rod- and thread-shaped the terms chondriokonts and chondriomites are also used. It is not to be thought that the various forms constitute distinct Masses, for several investigators (N. H. Cowdry; M. and W. Lewis 1915) have observed the chondriosomes undergoing marked changes in shape in living cells, granular ones becoming rod-shaped and filamentous, and vice versa. Schaxel (1911) and Kingery (1917) state, moreover, that in fixed material the shape of the chondriosomes is to a certain extent dependent upon the character of the microtechnical methods employed. FIG. 43. — Chondriosomes in plant and animal cells. A, nerve cell from guinea pig. X 480. (After E. V. Cowdry, 1914.) B, tapetal cell of Nymphaea alba. (After Meves, 1904.) C, living epidermal cell of tulip petal. D, ascus of Pustularia vesiculosa. E, hypha of Rhizopus nigricans. F, portion of embryo sac of Lilium; chondriosomes clustered about nucleus. G, cell of root tip of Allium — (C-F. After Guilliermond, 1918.) Although when first discovered chondriosomes were believed to be rather limited in distribution, they have now been reported in the cells of plants and animals belonging to nearly all of the larger natural groups. It is asserted by N. H. Cowdry (1917) that "in all forms of animals, from amoeba to man, which have been investigated with adequate methods of technique, they occur without exception." They are present, furthermore, in the cells of all tissues. In plants it is probable that they are no less universally present, although it has not yet been possible to demonstrate them with certainty in bacteria, Cyanophycese, and certain Chlorophycese, such as the Conjugate and Confervales (Guilliermond 1915). They are abundant in myxomycetes (N. H. Cowdry 1918), Charales (Mirande 1919), brown and red algae, fungi, and all the higher groups. A critical comparison of the chondriosomes of plants with those of animals has been made by N. H. Cowdry (1917), who concludes, contrary PLASTIDS AND CHONDRIOSOMES 117 to the opinion of Pensa (1914), that there is every reason to regard them as homologous in the two kingdoms. He finds plant and animal chon- driosomes to be practically identical in morphology, reaction to fixatives and dyes, and distribution in resting and dividing cells : any conspicuous differences in arrangement seem to be due to the more pronounced polarity of the animal cell. In both cases they are most abundant in the active stages in the life of the cell. As the cell ages and becomes fully differentiated, i.e., as cytomorphosis proceeds, they diminish in number and may completely disappear. Physico-chemical Nature. — With regard to the chemical and physical nature of chondriosomes, Regaud (1908), Faure-Fremiet (1910), and Lowschin (1913), working respectively on mammals, protozoa, and plants, agree that they are chemically a combination of phospholipin and albu- min. They closely resemble phosphatids, which are combinations of phosphoric and fatty acids, glycerol, and nitrogen bases. Lecithin is such a compound. Since chondriosomes are soluble in alcohol, ether, chloroform, and dilute acetic acid, many of the fixing reagents commonly employed in microtechnique destroy them: this accounts in part for the fact that they were not observed in many familiar tissues until a compara- tively recent date. They are well fixed by neutral formalin, potassium bichromate, osmium tetroxid, and chromium trioxid (chromic acid) ; and these, therefore, are the principal ingredients of the fixing reagents em- ployed in researches upon chondriosomes. Examples of such fluids are those of Altman, Benda, Bensley, Helley, Kopsch, Regaud, and Zenker.1 Besides staining with hsematoxylin and several other dyes commonly employed with fixed material, the chondriosomes show a characteristic affinity for certain intra-vitam stains, such as Janus green B, Janus blue, Janus black I, and diethylsafranin, the reaction with the first of these being especially strong. After certain treatments the chondriosomes may closely resemble the "chromidial substance," or granules of nucleo- protein distributed throughout the cytoplasm in some cells. That the two are not to be confused has been emphasized by Duesberg and by E. V. Cowdry. According to the latter author (1916) chondriosomes are "a concrete class of cell granulations," and may be provisionally defined as "substances which occur in the form of granules, rods and filaments in almost all living cells, which react positively to Janus green and which, by their solubilities and staining reactions, resemble phospholipins and to a lesser extent, albumins." Origin and Multiplication. — The questions of the origin and multipli- cation of chondriosomes are much debated ones. Certain cytologists 1 For convenient summaries of the effects of various reagents upon chondriosomes the student may refer to Kingsbury's (1912) paper on cytoplasmic fixation, E. V. Cowdry's (1914) on vital staining, and N. H. Cowdry's (1917) on plant and animal chondriosomes. 118 INTRODUCTION TO CYTOLOGY believe that they have found good evidence for the view that chondrio- somes may multiply by division, and some (Guilliermond ; Moreau 1914; Terni 1914) have held this to be their sole mode of origin— that they arise only from preexisting chondriosomes and are therefore permanent cell organs. Others are convinced that they may arise de novo in the cyto- plasm, and that the evidence for their division is unsatisfactory (Orman 1913; Lowschin 1913; Scherrer 1914; Miss Beckwith 1914; Chambers 1915; M. and W. Lewis 1915: Twiss 1919; and others). The investiga- tors of the foregoing group, together with Meves (1900), Lewitski (1910), and Forenbacher (1911), hold that the chondriosomes arise from the cyto- plasm, but certain others believe they take their origin from the nucleus. Tischler (1906) and Wassilief (1907), for example, state that they arise from surplus chromatin. Alexieff (1917) thinks that although cyto- FIG. 44. — Examples of regular behavior of chondriosomes in cell-division. A-C, spermatocyte of Gryllotalpa vulgaris, (After Vo'inov, 1916) : A, chondriosomal material in cytoplasm about nucleus; B, heterotypic mitosis, showing chondriosomes (at sides) occupying the spindle with the chromosomes (at center) ; C, stages in the division of a chondriosome. D, Dividing cell of Geotriton fuscus, showing division of individual chondriosomes as cell constricts at equator. (After Terni, 1914.) iplasmic dfferentiation is due to them, they are at least in some cases of nuclear origin; and further that they are not fundamentally different from chromosomes and chromidia, a conclusion contradictory to that of Duesberg and E. V. Cowdry, cited above. Shaffer (1920) believes them to arise as the result of a chemical action of the nucleus upon products of assimilation in the adjacent cytoplasm. Wildman (1913) classifies the cytoplasmic inclusions present throughout spermatogenesis in Ascaris into two main types, both of nuclear origin: "karyochondria," equiva- lent to the mitochondria of other writers, and "plastochondria," which pass into the cytoplasm, form yolk within them, and fuse to form the food supply ("refractive body") of the spermatozoon. That the behavior of the chondriosomes at the time of cell-division is a matter of considerable importance has been generally recognized. In many cases their distribution to the two daughter cells seems to be quite fortuitous, whereas in some tissues more or less definite modes of distribu- tion have been described. According to Faure-Fremict (1910), Terni (1914), Korotneff (1909), and others, the individual chondriosomes divide at the time of mitosis (Fig. 44, D), a conclusion with which many others fail to agree (Orman 1913; Miss Beckwith 1914; etc.). In the cells of PL AST IDS AND CHONDRIOSOMES 1 19 the grasshopper, Dissosteiro, Carolina, Chambers (1915) finds that the chondriosomal material forms a granular net work surrounding the nucleus during the resting stages and the mitotic figure during division. During the later phases of mitosis the strands and granules of this network lengthen into delicate filaments between the two daughter chromosome groups, and finally separate into two granular masses which gradually invest the daughter nuclei. In the mole cricket, Gryllotalpa borealis, the distribution of the chon- driosomes to the daughter cells is accomplished with even greater defin- iteness. According to Payne (1916) they become thread-like and break near the middle, the halves passing to the daughter cells. Voi'nov (1916) states that the "mitochondria" in the spermatocyte of G. vulgaris fuse to form a thread which then segments into a number (70 or more) "chpndrio- somes." These are arranged on the spindle along with the chromo- somes, which they may closely resemble, and divide to form daughter bodies at both maturation divisions, so that they are equally distributed to the four resulting spermatozoa (Fig. 44, A-C}. In certain scorpions also the chondriosomal material is distributed with surprising precision. In a species from Arizona (Wilson 1916) this material in the spermatocyte takes the form of a single ring-shaped body. This ring divides accurately, much like a chromosome, at both maturation divisions, each of the four spermatids, and hence each spermatozoon of the tetrad, receives a quarter of its substance. In a California species (Wilson) there is no ring formed, but instead about 24 hollow spherical bodies. At the two maturation divisions these show no evidence of division, but are passively separated into four approximately equal groups, each spermatid receiving six (occasionally five or seven) . A European species described by Sokolow (1913) agrees essentially with this. Function. — Our knowledge of chondriosomes is yet too incomplete to warrant any categorical statements regarding their functions, but a number of opinions have been expressed, some of them based upon ob- servational evidence and others upon conjecture. Certain of the more prominent opinions may here be reviewed. It was in 1897 that Benda suggested that chondriosomes might be distinct cell organs with a special function. In a series of papers which began to appear ten years later Meves (1907 etc.) put forth and empha- sized the theory that they play an important role in heredity — that they carry the hereditary characters of the cytoplasm. Evidence supporting this view was seen by Meves and Benda in certain experiments of God- lewski which seemed to show that the appearance of certain hereditary characters is dependent upon something present in the cytoplasm rather than in the nucleus. (See Chapter XIV.) This theory has had the support of a number of investigators, among whom are the botanists Cavers (1914) and Mottier (1916). Vomov (1916) also believes that the 120 INTRODUCTION TO CYTOLOGY regular distribution of the chondriosomal substance in Gryllotalpa strongly favors the view that this substance is of some significance in heredity. It is probable, however, that the majority of cytologists regard the evidence brought forward in support of the view as very inadequate. Wildman (1913) points out that the chondriosomes may be largely lost during spermatogenesis, and others have recalled cases in which the nucleus is the only portion of the male gamete which can be seen to enter the egg at fertilization. Meves (1911, 1915) and Benda, on the other hand, show that chondriosomes also enter, at least in the forms studied by them (Fig. 45). In the animal spermatid the chondriosomes appear most commonly to contribute to the formation of the Nebenkern of the spermatozoon (La Vallette St. George 1886; Popoff 1907; Chambers 1915; Shaffer 1920; and others), in some cases later elongating into a sheath around the axial filament of the tail (Shaffer on Cicada). Duesberg (1919) states that although the fate of the chondriosomes of the spermatid varies in different animals, they are nevertheless always present in the sperma- tozoon, and that it has not been clearly shown in any case that they do not enter the egg at fertilization. In many eggs which they do enter, however, they behave with great irregularity during the subsequent cleavage stages (Van der Stricht, etc.). It is not at all improbable that they are in some way concerned in the reactions through which hereditary characters are de- veloped in the individual, but the general opinion is that their apparent variability and indefiniteness in behavior in so many cases are against the view that they take any part in the transmission of factors upon whose presence the development of the characters depends (Gatenby 1918, 1919). The equal distribution of chondriosomes at the time of cell-division is thought to be without any significance in this connection by Harper (1919). It is obvious that much work remains to be done before the possible relation of chondriosomes to heredity and development can be made clear. For the present it is safest to assume, as will be emphasized in later chapters, that hereditary transmission is the function of the nucleus, chiefly if not entirely, since the chromosomes afford a mechanism of precisely the kind required to account for the observed distribution of hereditary characters. Meves (1907a6, 1909) and Duesberg (1909) have also called attention to the close relation of chondriosomes to muscle fibers in the developing FIG. 45. — Fertilization in Filaria papillosa, showing chondriosomes of sperma- tozoon (at top) distributing themselves in the cytoplasm of the egg. (After Meves, 1915.) PLASTIDS AND CHONDRIOSOMES 121 chick embryo. They believe that the chondriosome elongates and directly becomes the young fiber. Gaudissart (1913), on the contrary, shows that the fiber does not arise exclusively from the chondriosome, but that the primary basis is furnished by the plasmatic reticulum with which the chondriosomes cooperate in building up the fiber. Although the chondriosomes thus have a part in the genesis of the muscle fiber, the latter is not a "modified filamentous chondriosome," as Duesberg believed. Hoven (1910a) and Meves have similarly attempted to show that chondriosomes are concerned in the differentiation of neurofibrils and the collagenous fibers of cartilage. Regaud (1911), Guilliermond (1914), FIG. 46. A, formation of fat in cell of rabbit by granular and rod-shaped chondriosomes. (From Guilliermond, after Dubreuil, 1913.) B, formation of needle-shaped crystals of carotin in chromoplasts derived from chondriosomes in epidermal cell of Iris petal. (After Guillier- mond, 1918.) C, chondriosomes and chloroplasts in young cell of Pinus banksiana. X 750. (After Mottier, 1918.) D, transformation of plastid primordia into leucoplasts in root cell of Pisum; some of the leucoplasts contain starch. (After Mottier.) Hoven (19106, 1911), and Lewitski (1914) have thought that the chon- driosomes may in some cases perform a secretory function, and Dubreuil (1913) has associated them with the production of fat (Fig. 46, A). In the oocyte of Cicada Shaffer (1920) finds them transforming into yolk spherules. The activity of bodies called " plastochondria " by Wildman (1913) in the elaboration of the food supply in the spermatozoon of A scam has already been mentioned. Relation of Chondriosomes to Plastids. — One of the most conspicuous views regarding the significance of chondriosomes is that which holds some of them to be the primordia of plastids. After studying the cells of Pisum and Asparagus Lewitski (1910) concluded that the chondriosomes are essential constituents of the cytoplasm, and that they develop into chloroplasts and leucoplasts in the cells of the stem and root respectively. 122 INTRODUCTION TO CYTOLOGY Evidence in support of this conception was contributed by Forenbaelx-r (1911), Pensa (1914), Cavers (1914), and others. Guilliermond (1911- 1920) in particular was led by the results of his extensive researches on the subject to the view that the chondriosomes, arising only from preexist- ing ones by division, persist through the egg and embryonic cells and later become amyloplasts, chloroplasts, and chromoplasts. In this he saw strong evidence for the individuality of the plastid. In 1915 he advanced the opinion that in fungi the chondriosomes function like the amyloplasts of higher plants, forming reserve products as the latter form starch. In this development of chondriosomes into plastids Guilliermond (1913-1915) and Moreau (1914) were able to show that the chondriosomes produce within them certain phenolic compounds which either appear at once as anthocyanin pigments, or as colorless products which may acquire color later through chemical alteration (Fig. 46, J5). Among the most recent researches in this field are those of Mottier (1916, 1918) on the cells of Zea, Pisum, Elodea, Pinus, Adiantum, Antho- ceros, Pallavicinia, Marchantia, and several algae. He finds that leuco- plasts and chloroplasts are derived from small rod-shaped primordia (Fig. 46, C, D) which he regards as permanent cell organs of the same rank as the nucleus. Both primordia and mature chloroplasts multiply by fission. In the cells of Marchantia, Anthoceros, and the seed plants he finds also a second series of bodies, which he calls chondriosomes: these like the plastid primordia, are permanent cell organs multiplying by division, but they do not become chloroplasts or leucoplasts. Further- more, both chondriosomes and primordia are thought by Mottier to be concerned in the transmission of certain hereditary characters. It is also reported by Emberger (1920a6) that in the roots and spor- angia of ferns two kinds of granular elements may be recognized at all times, one of them representing the initial stage of plastid development. Contrary to Mottier's opinion, however, he regards both kinds as true mitochondria. Guilliermond (1920) likewise distinguishes two such types in Iris germanica. P. A. and P. Dangeard (1919, 1920), as a result of their researches on the cells of barley, Selaginella, Larix, Taxus, and Ginkgo, distinguish three classes of cytoplasmic structures differing in .reaction to reagents and in function. In their initial stages all have the granular form. The plastidomes first appear as minute "mitoplasts," which gradually enlarge and develop into plastids. The spheromes are at first recognizable as "microsomes," some of which may be seen to give rise to fat and oil globules while others appear to undergo no change. The vacuomes begin their history as "metachromes;" these elongate and form a peculiar network which later develops into a system of vacuoles. Guilliermond (1920) denies the metachromatic nature of this third class of bodies, and holds them to be quite distinct from mitochondria. PLASTJDS AND CHONDRIOSOMES 123 The existence of such a genetic relationship between chondriosomes and plastids as that described above has been denied by many writers, among whom may be mentioned Lundegardh (1910), Meyer (1911), Rudolph (1912), Lowschin (1913, 1914), Scherrer (1914), Miss Beckwith (1914), Derschau (1914), von Winiwarter (1914), Sa'pehin (1915), Chambers (1915), M. and W. Lewis (1915), and Harper (1919). These workers for the most part hold that chondriosomes are not distinct cell organs at all, but regard them rather as more or less transient visible products of protoplasmic activity. Derschau asserts that they arise neither de novo nor by fission, but that they are merely small masses of plastin and nuclein concerned in nutrition, arising from basichromatin at the surface of the nucleus. Miss Beckwith speaks of them as differ- entiation products of the cytoplasm. Lowschin, who made some ex- periments in the production of artificial chondriosomes, believes them to be due to the emulsified state of the protoplasm and in some instances to the action of fixing agents upon it. To Chambers they appear in living cells not as persistent structures but as temporary physical states of the colloidal substances composing protoplasm. M. and W. Lewis have studied them in tissue cultures and observe that they are continually being formed and used up, and that they show no sharply distinct types. Faure-Fremiet (1910a) distinguishes "mitochondria," which have an individuality of their own and are permanent cell organs, from "lipo- somes," which are temporary accumulations of reserve substance. The almost universal occurrence of chondriosomes in the cells of living organisms, and their frequent alterations in number and appear- ance, suggest a connection with some fundamental process going on almost constantly and common to all living matter. That this process may be oxidation, the chondriosomes being a "structural expression of the reducing substances concerned in cellular respiration" (Kingsbury), has been regarded as highly probable by Kingsbury (1912), Mayer, Rathery, and Schaeffer (1914), N. H. Cowdry (1917, 1918), and others. Evidence favoring this interpretation is seen in the fact that the chondrio- somes occur so widely in the cytoplasm, which acts as a reducing sub- stance; and also in the close similarity between their chemical composition and that of phosphatids, which appear to be capable of auto-oxidation. Conclusion. — From the foregoing review it should be more than plain that the state of our knowledge of chondriosomes is such that almost no definite final statements can be made regarding their origin and function. The evidence at hand apparently indicates that the class of cell inclusions known as chondriosomes comprises a variety of bodies which play differ- ent roles in the life of the cell. It is scarcely open to doubt that some of them are temporary accumulations of substances involved in metabolism, appearing and disappearing in the cell in a manner somewhat analogous to that of starch. The most plausible hypothesis concerning the specific 124 INTRODUCTION TO CYTOLOGY physiological role of such changeable types of chondriosomes is that thoy have to do with the processes of oxidation and reduction — with cellular respiration. It is also becoming increasingly apparent that other chon- driosomes represent the juvenile stages in the development of plastids of various kinds, and that they are in some way concerned in the forma- tion of chlorophyll and other pigments. If this is true they are clearly of the highest importance. Whether or not any of the chondriosomes are to be considered as permanent cell organs is a question to which, in view of the conflicting testimony of competent observers, no final answer can at present be given. To determine whether these minute bodies arise de novo or always multiply by division is a matter of extreme practical difficulty. Until this question is settled it is obviously impossible to come to a deci- sion regarding the individuality of those plastids which appear to take their origin from chondriosomes, or to know what may be the possible relation of chondriosomes to inheritance. With respect to the latter point, the chondriosomes, like all other structures concerned in meta- bolism, may be indirectly associated with the development of hereditary characters, but the view that they transmit or represent differential factors for such characters is as yet unsupported by adequate evidence. From the fact that the chondriosomes may not preserve their indi- viduality at all times, however, it does not follow that they must be denied the rank of cell organs. Their great variability, indifferent behavior at the time of cell-division in so many cases, and their unknown mode of origin are, as Kingsbury (1912) states, against the view that they are cell organs; and it is doubtless true that many chondriosomes should for such reasons be denied such rank. On the other hand, those chondriosomes which seem clearly to perform important and specific functions in the life of the cell should, like centrosomes appearing de novo at each cell-division, be looked upon as cell organs, though not as permanent ones with an uninterrupted continuity. In spite of the fact that the study of chondriosomes has so far raised more problems than it has solved, it has already proved of much value, for it has turned to the cytoplasm some of the attention so long directed almost exclusively to the nucleus, and it appears that many problems of much importance to cytology pertain to the cytoplasm. It has also been of great service in bringing about a closer scrutiny of the effects of fixation and a renewed emphasis upon the importance of the study of living protoplasm. Much has already been learned as the result of this study, but the solution of the principal problems involving chondriosomes must await the results of further research. PL AST IDS AND CHONDRIOSOMES 125 Bibliography 6 Plastids and Chondriosomes ALEXIEFF, A. 1917«. Mitochondries et corps parabasal chez les Flagelles. Comp- tes Rend. Soc. Biol. Paris 69: 358-361. 19176. Mitochondries et role morphogene du noyau. Ibid. 361-363. 1917c. Nature mitochondiale du corps parabasal des Flagelles. Ibid. 499-502. 1917d. Sur les mitochondries a fonction glycoplastique. Ibid. 510-512. ALLEN, C. E. 1905. Die Keimung der Zygoten bei Coleochcete. Ber. Deu. Bot. Ges. 23: 285-292. pi. 13. DE BARY, H. A. 1858. Untersuchungen iiber die Familien der Conjugaten. Leipzig. BEAUVERIE. 1919. The anthocyanin pigments of plants. Sci. Am. Suppl. 87: No. 2244, pp. 2-3, 7. (Transl. from Rev. Gen. Sci. Paris.) BECKWITH, C. J. 1914. The genesis of the plasma structure in the egg of Hydrac- tinia echinata. Jour. Morph. 25: 189-251. pis. 8. BEER, R. 1909. On elaioplasts. Ann. Bot. 23 : 63-72. pi. 4. . BENDA, C. 1897. Neuere Mitteilungen iiber die Histogenese des Saugetiersperma- tozoon. Verh. d. Phys. Gesell. Berlin. 1898. Ueber die Sperm atogenese der Vertebraten und hoherer Evertebraten. 2. Die Histogenese der Spermien. Ibid, 1898. 1902. Die Mitochondria. Ergeb. d. Anat. u. Entw. 12: 743-781. (Review.) BINZ, A. 1892. Beitrage zur Morphologic und Entwicklung der Starkekorner. Flora 76: 34-91. pis. 5-7. BOURQUIN, H. 1917. Starch formation in Zygnema. Bot. Gaz. 64 : 426-434. pi. 27. CARTER, N. 1919a6. Studies on the chloroplasts of desmids. I, II. Ann. Bot. 33 : 215-254, pis. 14-18; 295-304, pis. 12, 20. 1919c. The cytology of the Cladophoracese. Ibid. 33 : 467-478. pi. 27. 1920a. Studies on the chloroplasts of desmids. III. The chloroplasts of Cos- mariuro. Ann. Bot. 34: 265-285. pis. 10-13. 19206. Studies on the chloroplasts of desmids. IV. Ibid. 34: 303-320. pis. 14-16. CAVERS, F. 1914. Chondriosomes (mitochondria) and their significance. New Phytol. 13 : 96-106. (Review of subject.) CHAMBERS, R. 1915. Microdissection studies on the germ cell. Science 41: 290- 293. CHMIELEWSKIJ. 1896. Ueber Bau und Vermehrung der Pyrenoide bei einigen. Algen. (Russian, 10 pp.) See Bot. Centr. 69 : 277-278. 1897. CLELAND, R. E. 1919. The cytology and life history of Nemalion muUifidum Ag. Ann. Bot. 33: 323-352. pis. 22-24. figs. 3. COWDRY, E. V. 1914a. The vital staining of mitochondria with Janus green and diethylsafranin in human blood cells. Internat. Monatsschr. f. Anat. u. Physiol. 31 : 267-286. 19146. The comparative distribution of mitochondria in spinal ganglion cells of vertebrates. Am. Jour. Anat. 17 : 1-29. pis. 3. 1916. The general functional significance of mitochondria. Am. Jour. Anat. 19 : 423-446. (Review of subject.) COWDRY, N. H. 1917. A comparison of mitochondria in plant and animal cells. Biol. Bull. 33 : 196-228. figs. 26. 1918. The cytology of the myxomycetes with special reference to mitochondria. Biol. Bull. 36 : 71-94. 1 pi. 1920. Experimental studies on mitochondria in plant cells. Ibid. 39: 188-206. pis. 3. DANGEARD, P. A. 1919. Sur la distinction du chondriome des auteurs en vacuome, plastidome, et spherome. Compt. Rend. Acad. Sci. Paris 169: 1005-1010. 126 INTRODUCTION TO CYTOUKiY 1920a. Plastidome, vacuome et spherome dans Rrlniiiin-llii Kraussiana. Ibid. 170:301-306. 1 pi. 19206. La structure do la cellule vegetale et son metabolisme. Ibid. 170: TOO- TH. DANGEARD, P. 1920. Sur 1'evolution du systeme vacuolaire chez les gymnospermes. Ibid. 170: 474-477. figs. 8. DAVIS, B. M. 1899. The spore mother cell of Anlhoceros. Bot. Gaz. 28: 89-109. pis. 9, 10. VON DERSCHAU, M. 1914, Zum Chromatindualismus der Pflanzenzelle. Arch. f. Zellf . 12 : 220-240. pi. 77. DODEL, A. 1892. Beitrag zur Morphologic und Entwicklung der Starkekorner von Pellionia Daveauana. Flora 76 : 267-280. pi. 5-6. DUBREUIL, G. 1913. Le chondriome et le dispositif de 1'activite secretaire. Arch. d'Anat. Micr. 16: 53-151. DUESBERG, J. 1907. Der Mitochondrialapparat in den Zellen der Wirbeltiere und Wirbellosen. Arch. Mikr. Anat. 71 : 284-296. pi. 24. 1909. Les chondriosomes des cellules embryonnaires du poulet et leur role dans la genese des myofibrilles, avec quelques observations siir le developpement des fibres musculaires striees. Arch. Zellf. 4: 602-671. pis. 28-30. figs. 10. 1911. Plastosomes, "apparato reticolaro interno," und Chromidialapparat. Ergeb. d. Anat. u. Entw. 20: 567-916. (Extensive review.) 1917. Chondriosomes in the cells of fish embryos. Am. Jour. Anat. 21: 465-493. pis. 1-3. figs. 8. 1918. Chondriosomes in the testicle-cells of Fundulus. Am. Jour. Anat. 23 : 133-154. pis. 2. figs. 21. 1919. On the present status of the chondriosome-problem. Biol. Bull. 36: 71-81. DUESBERG, J. et HOVEN, H. 1910. Observations sur la structure du protoplasme des cellules vegetales. Anat. Anz. B 36 : 96-100. figs. 5. EMBERGER, L. 1920a. Evolution du chondriome chez les cryptogames vasculaires. Compt. Rend. Acad. Sci. Paris 170: 282-284. figs. 5. 19206. Evolution du chondriome dans la formation du sporange chez les fougeres. Ibid. 170 : 469-471. figs. 7. FAURE-FREMIET, M. E. 1910a. Mitochondries et liposomes. Comptes Rend. Soc. Biol. Paris 62 : 537-539. 19106. La continuite des mitochondries a travers des generations cellulaires et le role de ces elements. Anat. Anz. 36: 186-191. figs. 3. FISCHER, A. 1905. Die Zelle der Cyanophyceen. Bot. Zeit. 63: 51-129. pis. 4, 5. FORENBACHER, A. 1911. Die Chondriosomen als Chromatophorenbildner. Ber. Deu. Bot. Ges. 29 : 648-660. pi. 25. FRANZE, R. 1893. Zur Morphologie und Physiologic der Stigmata der Mastigo- phoren. Zeit. Wiss. Zool. 56: 138-164. pi. 8. GARDNER, N. L. 1906. Cytological studies in Cyanophycese. Univ. Calif. Publ. Bot. 2 : 237-296. pis. 21-26. GARGEANNE, A. J. M. 1903. Die Oelkorper der Jungermanniales. Flora 92 : 457-482. figs. 18. GATENBY, J. B. 1918. Cytoplasmic inclusions of the germ cells. III. The sper- matogenesis of some other Pulmonates. Quar. Jour. Micr. Sci. 63 . 197-258. pis. 16-18. figs. 3. 1919. The cytoplasmic inclusions of the germ cells. V. The gametogenesis and early development of Limnaea stagnates (L.) with special reference to the Golgi apparatus and the mitochondria. Quar. Jour. Micr. Sci. 63 : 445-492. pis. 27, 28. figs. 6. (See also Parts I and II in vol. 62, and Part IV in 63.) 1'LASTWS AND CHONDRIOSOMES 127 GAUDISSART, P. 1913. Reseau protoplasmique et chondriosomes daus la genese des myofibrilles. La Cellule 30: 29-43. pis. 1, 2. GOTTSCHE. 1843. Anatomische-physiologische Untersuchangen iiber Haplomilrium Hookeri. Verb. Leop. Carol. Akad. 12 : I, 286. GUIGNARD, L. 1889. .Developpement et constitution des antherozoides. Rev. Gen. Bot. 1: 11; 63, 136, 175. pis. 2-6. GOILLIERMOND, A. 1911. Sur les mitochondries des cellules vegetales. Comptes Rend. Acad. Sci. Paris 153: 199-201. figs. 4. 1912. Recherches cytologiques sur le mode de formation de 1'amidon et sur les plastes vegetaux. Arch. d'Ariat. Micr. 14: 309-428. pis. 13-18. 1913. (a) Sur la signification du chromatophore des algues. Comp. Rend. Soc. Biol. Paris 76 : 85-87. (6) Quelques remarques nouvelles sur la formation des pigments anthocyaniques au sein des mitochondries. Ibid. 478-481. (c) Nouvelles observations sur le choudriome de 1'asque de Pustularia vesiculosa. Ibid. 646-649. (d) Nouvelles remarques sur la signification des plastes de W. Schimper par rapport aux mitochondries actuelles. Ibid. 437-440. 1914a. Etat actuel de la question de 1'evolution et du r61e physiologique des mitochondries. Rev. Gen. Bot. 26: 129-149, 182-210. figs. 16. 19146. Bemerkungen iiber die Mitochondrien der vegetativen Zellen und ihre Verwandlung in Plastiden. Ber. Deu. Bot. Ges. 32: 282-301. figs. 2. 1915a. Nouvelles observations vitales sur le chondriome des cellules epidermiques de la fleur d'Iris germanica. Comp. Rend. Soc. Biol. Paris 67 : 241-249. 19156. Recherches sur le chondriome chez les champignons et les algues. Rev. G£n. Bot. 27: 193, 236, 271, 297, 315. pis. 12. 1917a. Sur la nature et le role des mitochondries des cellules v6g6tales. Comp. Rend. Soc. Biol. Paris 69: 916-924. 19176. Observations vitales sur le chondriome de la fleur de Tulipe. Comp. Rend. Acad. Sci. Paris 164: 407-409. 1917c. Contributions a 1'etude de la fixation da cytoplasme. Ibid. 643-646. 1917rf. Recherches sur 1'origine des chromoplastes et le mode de formation do pigments du groupe des xanthophylles et des carotins. Ibid. 232-234. 1917e. Sur les alterations et les caracteres du chondriome dans les cellules epi- dermique de la fleur de Tulipe. Ibid. 609-612. 1917/. Sur les ph6nomenes cytologiques de la degenerescence de cellules epi- dermiques pendant la fanaison des fleurs. Compt. Rend. Soc. Biol. Paris 69: 726-729. 1918. Sur 1'origine mitochondriale des plastids. Compt. Rend. Acad. Sci. Paris 167 : 430-433. 1919. Observations vitales sur le chondriome des vegetaux et recherches sur 1'origine des chromoplastides et le mode de formation des pigments xantho- phylliens et carotiniens. Rev. Gen. Bot. 31 : 372-413, 446-508, 532-603, 635- 770. pis. 60. figs. 35. 1920a. Sur 1'evolution du chondriome dans la cellule veg4tale. Compt. Rend. Acad. Sci. Paris 170 : 194-197. figs. 4. 19206. Sur les elements figures du cytoplasme. Ibid. 170: 612-615. figs. 5. 1920c. Nouvelles recherches sur 1'appareil vacuolaire dans les ve'getaux. Ibid. 171:1071-1074. figs. 25. HAAS,- P. and HILL, T. G. 1913. An introduction to the chemistry of plant prod- ucts. London. HABERLANDT, G. 1888. Die Chlorophyllkorper dcr Selaginellen. Flora 71: 291-308. pi. 5. 1905. Ueber die Plasmahaut der Chloroplasten in den Assimilationzellen von Selagindla Martensii Spring. Ber. Deu. Bot. Ges. 23 : 441-452. pi. 20. 128 INTRODUCTION TO CYTOLOGY HARPER, R. A. 1919. The structure of protoplasm. Am. Jour. Bot. 6 : 273-300. HEGLER, R. 1901. Untersuchungen tiber die Organization der Phycochromzelle. Jahrb. Wiss. Bot. 36: 229-354. pis. 5, 6. figs. 5. HOVEN, H. 1910a. Sur 1'histogenese du systeme nerveux peripherique et sur le role des chondriosomes dans la neuro fibrillation. Arch, de Biol. 25: 427-494. pis. 15, 16. 19106. Contribution a 1'etude du fonctionnement des cellules glandulaires. Du role du chondriome dans la secretion. Anat. Anz. 37: 343-351. figs. 7. (Note prelim.) 1911. Du role du chondriome dans 1'elaboration des produits de la glande mam- maire. Anat. Anz. 39: 321-326. figs. 4. JANSSENS, F. A., VAN DE PUTTE, E., et HELSMORTEL, J. 1913. Le chondriosome dans les champignons. (Notes prelim.) La Cellule 28: 447-452. pis. 1, 2. JOHNSON, L. M. 1893. Observations on the zoospores of Draparnaldia. Bot. Gaz. 18 : 294-298. pi. 32. JORGENSEN, I. and STILES, W. 1917. Carbon assimilation. New Phytol. Reprint No. 10. London. KEENE, M. L. 1914. Cytological studies of the zygospores of Sporodinia grandis. Ann. Bot. 28:455-470. pis. 35, 36. 1919. Studies of zygospore formation in Phycomyces nitens Kunze. Trans. Wis. Acad. Sci. 19: 1195-1220. pis. 16-18. KINGERY, H. M. 1917. Oogenesis in the white mouse. Jour. Morph. 30: 261-316. pis. 2. KINGSBTJRY, B. F. 1912. Cytoplasmic fixation. Anat. Record 6: 39-52. KLEBS, G. 1883. Ueber die Organization einiger Flagellaten-Gruppen und ihre Beziehungen zu Algen und Infusorien. Unters. Bot. Inst. Tubingen 1 : 233-262. pis. 2, 3. 1892. Flagellaten-Studien. 1,11. Zeit. Wiss. Zool. 65 : 265-445. pis. 13-18. KOHL, F. G. 1903. Ueber die Organization und Physiologic der Cyanophyceenzelle, und die mitotische Teilung ihres Kerns. Jena. KOROTNEFP, A. 1909. Mitochondrien, Chondriomiten, und Faserepithel der , Tricladen. Arch. Mikr. Anat. 74: 1000-1016. pis. 2. KRAMER, H. 1902. The structure of the starch grain. Bot. Gaz. 34: 341-354. pi. 11. KURSSANOW, L. 1911. Ueber Befruchtung, Reifung und Keimung bei Zygnema. Flora 104 : 65-84. pis. 1-4. KUSTER, W. 1894. Die Oelkorper der Lebermoose und ihr Verhaltniss zu Elaio- plasten. Inaug. Dissert., Basel. 1911. Ueber amoboide Formveranderung der Chromatophoren hoher Pflanzen. Ber. Deu. Bot. Ges. 29 : 362-369. figs. 4. LA VALETTE ST. GEORGE, A. 1886. Spermatologische Beitrage. 2. Arch. Mikr. Anat. 27:1-12. pis. 1, 2. LEWIS, M. R. and LEWIS, W. H. 1915. Mitochondria (and other cytoplasmic inclusions) in tissue cultures. Am. Jour. Anat. 17: 339-401. figs. 26. LEWITSKI, G. 1910. Ueber die Chondriosomen in pflanzlichen Zellen. Ber. Deu. Bot. Ges. 28: 538-546. pi. 17. 1911. Die Chloroplastenanlagen in lebenden und fixierten Zellen von Elodea canadensis, Rich. Ibid. 29 : 697-703. pi. 28. 1914. Die Chondriosomen als Secretbildner bei den Pilzen. Ibid. 31: 517-528. Ipl. LIDFORSS, B. 1893. Studien ofver elaiosferer i ortbladens mesophyll och epidermis. Inaug. Dissert., Lund; also in Kgl. Fysiogr. Sallsk. i Lund Handl. 4. PL AST IDS AND CHONDRIOSOMES 129 LOWSCHIN, A. M. 1913. " Myelinformen " und Chondriosomen. Ber. Deu. Bot. Ges. 31: 203-209. 1914. Vergleichende experimental-cytologische Untersuchungen iiber Mito- chondrien in Blattern der hoheren Pflanzen. (Vorl. Mitt.) Ibid. 32: 266-270. pi. 5. LUNDEGARDH, H. 1910. Bin Beitrag zur Kritik zweier Vererbungshypothesen. Jahrb. Wiss. Bot. 48: 285-378. MAST, S. O. 1911. Light and the behavior of organisms, pp. 410. N. Y. 1916. The process of orientation in the colonial organism, Gonium pectorale, and a study of the structure and function of the eyespot. Jour. Exp. Zool. 20, 1-17. figs. 6. MAYER, RATHERY and SCHAEFFER. 1914. Les granulations ou mitochondries de la cellule hepatique. Jour. Physiol. Path. Gen. 16: 607-622. MCALLISTER, F. 1913. Nuclear division in Tetraspora lubrica. Ann. Bot. 27 : 681-696. pi. 56. 1914. The pyrenoid of Anthoceros. Am. Jour. Bot. 1 : 79-95. pi. 8. MEVES, FR. 1904. Ueber das Vorkommen von Mitochondrien bezw. Chondrio- miten in Pflanzenzellen. Ber. Deu. Bot. Ges. 22 : 284-286. pi. 16. 1907a. Ueber Mitochondrien bzw. Chondriokonten in den Zellen junger Embryo- nen. Anat. Anz. 31 : 399-407. 19076. Die Chondriokonten in -ihrem Verhaltnis zur Filarmasse Flemmings. Ibid. 31:561-569. 1908. Die Chondriosomen als Trager erblicher Anlagen. Cytologische Studien am Huhnerembryo. Arch. Mikr. Anat. 72 : 816-867. pis. 39-42. 1909. Ueber Neubildung quergestreifter Muskelfasern nach Beobachtungen am Huhnerembryo. Anat. Anz. 34: 161-165. figs. 3. 1914. Was sind die Plastosomen? Ibid. 86: 279-302. figs. 17. 1915. Ueber Mitwirkung der Plastosomen bei der Befruchtung des Eies von Filaria papillosa. Arch. Mikr. Anat. 87 : II 12-46. pis. 1-4. See also p. 286. 1918. Die Plastosomen theorie der Vererbung. Eine Antwort auf verschiedener Einwande. Ibid. 92 : II, 41-136. figs. 18. (Bibliography.) MEYER, A. 1881. Ueber die Struktur der Starkekorner. Bot. Zeit. 39: 841- 846, 857-864. pi. 9. 1883a. Ueber Krystalloide der Trophoplasten und tiber die Chromoplasten der Angiospermen. Ibid. 41 : 489-498, 505-514. 525-531. 18836. Das Chlorophyllkorn. pp. 91. pis. 3. Leipzig. 1895. Untersuchungen tiber die Starkekorner. Jena. 1911. Bemerkungen zu G. Lewitski: Ueber die Chondriosomen in pflanzlichen Zellen. Ber. Deu. Bot. Ges. 29 : 158-160. MIRANDE, M. 1919. Sur le chondriome, les chloroplastes et les corpuscules nucleo- laires du protoplasme des Chara. Comptes Rend. Acad. Sci. Paris 168 : 283- 286. figs. 7. VON MOHL, H. 1835, 1837. Ueber die Vermehrung der Pflanzenzellen durch Theilung. Dissert. Tubingen 1835. Flora 46, 1837. 1851. Grundziige der Anatomic und Physiologic der vegetabilische Zelle. Engl. Transl. by Henfrey, London 1852. MONTEVERDE, N. A. and LUBIMENKO, V. N. 1911. Recherches sur la formation de la chlorophylle chez les plantes. [Russian.] Bull. Acad. Imp. Sci. St. Peters- bourg VI 6 : 73-100. MOREAU, F. 1914. Le chondriosome et la division des mitochondries chez les Vaucheria. Bull. Soc. Bot. France 61 : 139-142. MOTTIER, D. M. 1918. Chondriosomes and the primordia of chloroplasts and leucoplasts. Ann. Bot. 32: 91-114. pi. 1, 9 130 INTRODUCTION TO CYTOLOGY VON NAGELI, C. 1858. Die Starkekorner. Zurich. OLIVE, E. W. • 1904. Mitotic division of the nuclei of the Cyanophycete. Beih. Bot. Centr. 18: 9-44. pis. 1, 2. ORMAN, E. 1913. Recherches sur les differenciations cytoplasmiques dans les vegetaux. 1. Le sac embryonnaire des Liliacees. La Cellule 28: 365-441. pis. 1-4. OVERTON, E. 1889. Beitrag zur Kenntniss der Gattung Volvox. Bot. Centr. 39: 65-72. 113-118, 145-150, 177-182, 209-214, 241-246, 273-279. pis. 4. PALLADIN, V. I. 1918. Plant Physiology. (Engl. edition by Livingston.) Phila- delphia. PAYNE, F. 1916. A study of the germ cells of Gryliotalpa borealis and Gryliotalpa vulgaris. Jour. Morph. 28: 287-327. pis. 4. figs. 5. PENSA, A. 1914. Ancora a proposito di condriosomi e pigmento antocianico nelle cellule vegetal!. Anat. Anz. 46 : 13-22. PFEFFER, W. 1874. Die Oelkorper der Lebermoose. Flora 57: 2-6, 17-27, 33-43. pl.L POLITIS, I. 1914. Sugli elaioplasti nelle mono- e dicotiledoni. Atti Inst. Bot. Univ. Pavia II 14: 335-361. pis. 13-15. POPOFF, M. 1907. Eibildung bei Paludina vivipara usw. Arch. Mikr. Anat. 70: 43-129. pis. 4-8. fig. 1. RACIBORSKI, M. 1893. Ueber die Entwicklungsgeschichte der Elaioplasten der Liliaceen. Anz. Akad. Wiss. Krakau. p. 259. REGAUD, C. 1908. Sur les mitochondries de l'epithelium seminal. Comptes Rend. Soc. Biol. Paris 65: 660-662. 1910. Etude sur la structure des tubes seminiferes, etc. Arch. d'Anat. Micr. 11 : 291-433. 1911. Les mitochondries, organites du protoplasma consideres comme les agents de la fonction eclectique et pharmacopexique de la cellule. Revue de Medicin. RIVETT, M. F. 1918. The structure of the cytoplasm in the cells of Alicularia scolaris, Cord. Ann. Bot. 32: 207-214. pi. 6. figs. 3. RUDOLPH, K. 1912. Chondriosomen und Chromatophoren. Beitrag zur Kritik der Chondriosomentheorien. Ber. Deu. Bot. Ges. 30 : 605-629. pi. 18. 1 fig. (Review.) SALTER, J. H. 1898. Zur naheren Kenntniss der Starkekorner. Jahrb. Wiss. Bot. 32: 117-166. pis. 1, 2. SAPEHIN, A. A. 1911. Ueber das Verhalten der Plastiden im sporogonen Gewebe. (Vorl. Mitt.). Ber. Deu. Bot. Ges. 29: 491-496. figs. 5. 1913a. Untersuchungen liber die Individualitat der Plastide. [Russian.] pp.133. pis. 17. Odessa. 1913b. Untersuchungen liber die Individualitat der Plastide. (Zweite Vorl. Mitt.) Ber. Deu. Bot. Ges. 31 : 14-16. 1 fig. 1915. Untersuchungen liber die Individualitat der Plastide. Arch. Zellf . 13 : 319-398. pis. 10-26. SCHAXEL, F. 1911. Plasmastructuren, Chondriosomen und Chromidien. Anat. Anz. 39: 337-353. figs. 16. SCHERRER, A. 1914. Untersuchungen liber Bau und Vermehrung der Chromato- phoren und das Vorkommen von Chondriosomen bei Anlhoceros. Flora 107 : 1-56. pis. 1-3. SCHILLING, A. J. 1891. Die Siisswasser-Peridineen. Flora 74: 220-299. pis. 8-10. SCHIMPER, A. F. W. 1880-1881. Untersuchungen liber die Entstehung der Starkekorner. Bot. Zeit. 38: 881-902, pi. 13; 39: 185-194, 201-211, 217-227. pi. 2. PLASTIDS AND CHONDRIOSOMES 131 1883. Ueber die Entwicklung der Chlorophyllkorner und Farbkorper. Bot. Zeit. 41: 105-112, 121-131, 137-146, 153-162. 1 pi. 1885. Untersuchungen iiber die Chlorophyllkorper und die ihnen homologen Gebilde. SCHMIDT, E. W. 1912. Pflanzliche Mitochondrion. Prog. Rei Bot. 4: 163-181. figs. 6. SCHMITZ, FR. 1882. Die Chromatophoren der Algen. Verh. Naturhist. Ver. Preuss. Rheinl. u. Westf. 40. (See also Sitz-Ber. Med. Gesell. f. Nat. u. Heilk. Bonn, 1880.) 1884. Beitrage zur Kenntniss der Chromatophoren. Jahrb. Wiss. Bot. 15: 1-177. pi. 1. SENN, G. 1908. Die Gestalts-und Lageveranderung der Pflanzen-Chromatop- horen. Leipzig. SHAFFER, E. L. 1920. The germ-cells of Cicada (Tibicen) septemdecim (Homoptera). Biol. Bull. 38; 404-474. pis. 9. SMITH, G. M. 1914. The cell structure and colony formation in Scenedesmus. Arch. f. Protistenk. 32: 278-297. pis. 16, 17. SOKOLOW, I. 1913. Untersuchungen iiber die Sperm atogenese bei den Arachniden. I. Ueber die Spermatogenese der Skorpione. Arch. f. Zellf. 9: 399-432. pis. 22, 23. 1 fig. STRASBURGER, E. 1900. Ueber Reduktionstheilung, Spindelbildung, Centrosomen, und Cilienbildner im Pflanzenreich. Hist. Beitr. 6: 1-227. pis. 4. Jena. 1903. Text-Book of Botany (Strasburger, Noll, Schenck, and Schimper). 2d. English edition from 5th German ed. TAMMES, T. 1900. Ueber die Verbreitung des Carotins im Pflanzenreich. Flora 87 : 205-247. pi. 7. TERNI. T. 1914. Condriosomi, idiozoma e formazioni periidiozomiche nella sper- matogenesi degli anfibii. (Ricerche sul Geotriton juscus.) Arch. Zellf. 12 : 1-96. pis. 1-7, TIMBERLAKE, H. G. 1901. Starch formation in Hydrodiclyon utriculatum. Ann. Bot. 15 : 619-635. 1903. The nature and function of the pyrenoid. Science N. S. 17 : 460. TISCHLER, G. 1906. Ueber die Entwicklung des Pollens und der Tapetenzellen bei tfi&es-Hybriden. Jahrb. Wiss. Bot. 42 : 545-578. pi. 15. Twiss, W. C. 1919. A study of plastids and mitochondria in Preissia and corn. Am. Jour. Bot. 6 : 217-234. pis. 23, 24. VOINOV, D. 1916. Sur 1'existence d'une chondriodierese. Comptes Rend. Soc. Biol. Paris 68 : 451-454. figs. 4. WAGER, H. 1900. On the eyespot and flagellum of Euglena viridis. Jour. Linn. Soc. 27: 463-481. pi. 32. 1903. The cell structure of the Cyanophyceae. (Prelim, note.) Proc. Roy. Soc. London 72 : 401-408. figs. 3. WAKKER, J. H. 1888. Studien iiber die Inhaltskorper der Pflanzenzelle. Jahrb. Wiss. Bot. 19: 423-496. pis. 12-15. WASSILIEF, A. 1907. Die Spermatogenese von Blatta germanica. Arch. Mikr. Anat. 70: 1-42. pis. 1-3. 1 fig. WHELDALE, M. 1916. The anthocyanin Pigments of Plants. Cambridge. WIESNER, J. 1892. Die Elementarstructur und das Wachstum der lebenden Sub- stanz. Wien. WILDMAN, E. E. 1913. The spermatogenesis of Ascaris megalocephala with special reference to the two cytoplasmic inclusions, the refractive body and the "mito- chondria:" their origin, nature and role in fertilization. Jour. Morph. 24: 421-457. pis. 3. 132 INTRODUCTION TO CYTOLOGY WILLSTATTER, R. und Stoll, A. 1913. Untersuchungen liber Chlorophyll. Berlin. WILSON, E. B. 1916. The distribution of the chondriosomes to the spermatozoa of scorpions. Science 43 : 539. (Refers also to work of Sokolow.) VON WINIWARTER, H. 1912. Observations cytologiques sur les cellules interstitielles du testicule humain. Anat. Anz. 41 : 309-320. pis. 1, 2. WOLLEN WEBER, W. 1907. Das Stigma von Hcematococcus. Ber. Deu. Bot. Ges. 26:316-320. pi. 11. 1908. Untersuchungen iiber die Algengattung Hcematococcus. Ibid. 26: 238-298. pis. 12-16. figs. 12. YAMANOUCHI, S. 1913. Hydrodictyon Africanum, a new species. Bot. Gaz. 55: 74-79. ZIMMERMANN, A. 1893. Ueber die Elaioplasten. Beitr. z. Morph. u. Physiol. der Zelle 1 : 185-197. 1894. Sammel-Referate. 9. Die Chromatophoren. 10. Die Augenfleck. 11. Elaioplasten, Elaiospharen und verwandte Korper. 15. Die Starkekorner und verwandten Korper. Beih. Bot. Centr. 4: 90-101, 161-165, 165-169, 329-335. CHAPTER VII METAPLASM ; POLARITY In the foregoing chapters we have described successively the various organs of the cell. Our account of the resting cell will now be com- pleted by passing in brief review some of its more conspicuous non- protoplasmic inclusions. We shall also call attention to another characteristic but imperfectly understood attribute of the protoplast, namely, its polarity. Metaplasm. — In addition to their definite cell organs — nucleus, cytoplasm, centrosomes, plastids, and possibly chondriosomes — cells which have undergone any amount of differentiation usually contain a variety of other materials representing products of metabolism. Many of these substances are held in solution in the cell sap, itself a differentia- tion product, while others are present in insoluble form in the cytoplasm, often in special vacuoles. All such non-protoplasmic inclusions, partic- ularly those existing in some visible form, are referred to as metaplasm, a term introduced by Hanstein. Although it has been held by some (Kassowitz 1899) that metaplasm is always inactive and to be sharply set apart from active protoplasm, it is more probable, as Child (1915) contends, that no absolute distinction can be made between the two. Most of the products of differentiation, however, are clearly non-proto- plasmic and relatively inactive. In cells of many types, even in the comparatively undifferentiated cells of the root meristem, there often occur accumulations of chemically complex substances in the form of small globules or irregular masses in the cytoplasm. In many cases these more or less transient bodies, which often stain intensely with the nuclear dyes and are therefore referred to as "chromatic bodies," show reactions indicating a composition closely approaching that of the extra-nuclear granules of nucleo-protein (chro- midia) which R. Hertwig and Goldschmidt interpret as granules of escaped chromatin concerned in cell differentiation. Others resemble the fatty chondriosomes in form and composition. It is therefore a matter of some difficulty to distinguish between these various substances, which, as a matter of fact, probably do not represent sharply distinct classes. The most conspicuous non-protoplasmic inclusions represent food materials in transitory form or in the storage condition; they are conse- quently abundant in cells carrying a supply of reserve foods, such as spores and eggs, and in storage organs, such as many roots and the endo- 133 134 INTRODUCTION TO CYTOLOGY sperm and cotyledons of seeds. In the animal egg the storage material commonly exists in the form of "yolk globules," or "deutoplasm spheres," which consist for the most part of relatively complex protein compounds . Fat or oil globules are usually present with them. In plants the most characteristic storage product is starch, the origin and characters of which were described in Chapter VI along with the plastids by which they are formed. In some organisms, including the fungi, glycogen appears to carry on the function performed by starch and sugar in the higher plants. Fats and oils, usually in the form of droplets but sometimes of soft grains or even crystals (nutmeg), comprise another important class of storage substances: these are especially prevalent in seeds and spores, where light weight is of advantage. In many cases oil may be produced anywhere in the cell, but in certain forms it has been found that special elaioplasts, and FIG. 47. — Crystalline and other inclusions in the cells of various plants. A, cystolith in subepidermal cell of Ficus leaf. B, crystal cells in Arctostaphylos. C, druse in cell of Rheum palmatum. D-K, aleurone grains: D, E, from Myristica; F, from- Datura stramonium; G, from Ricinus communis; H, from Amygdalus communis; I, from Bertholletia excelsa; J, from Faeniculum; K, from Elceis guiniensis. L, raphides in leaf of Agave. M, inulin crystals in preserved cells of artichoke. (B—K after Tschirch.) possibly also chondriosomes, are concerned in this process. The peculiar, oil bodies found in the cells of certain liverworts appear to represent oil vacuoles: these also have been discussed, together with elaioplasts, in Chapter VI. Large masses of intranuclear metaplasm are found charac- teristically in the eggs of gymnosperms. Aleurone grains occur in small vacuoles in the cells of many seeds, particularly in such oily ones as those of Ricinus, Juglans, and Bertholl- etia. In maize and wheat grains they are limited to a single layer of cells, the "aleurone layer." The aleurone grain varies much in. structure and form, several types being described by Pfeffer in 1872. The grain con- sists primarily of an amorphous protein substance, often with an outer, somewhat more opaque shell. Some examples show no greater differen- tiation than this, but many are much more elaborate (Fig. 47, D-K). Those of Ricinus contain within them a single angular crystal of protein (albumen), often referred to as the "crystalloid," and a globule of a double phosphate of calcium and magnesium with certain organic substances METAPLASM; POLARITY 135 called the "globoid" (Fig. 47, (7). The crystalline inclusions sometimes grow to be very large. It has been thought by certain workers that aleurone grains are self-perpetuating bodies with an individuality com- parable to that of nuclei and certain plastids. That this view is correct has been rendered very improbable by the researches of East and Hayes (1911, 1915) and Emerson (1914, 1917) on the inheritance of aleurone characters in maize, and also by the work of Thompson (1912), who suc- ceeded in producing artificial aleurone grains in all essential respects similar to those elaborated by the plant. Crystals occur in great variety in the differentiated cells of plants. They may lie in the cytoplasm, in vacuoles, attached to or imbedded in the cell wall, and even in special cells. They are usually salts of calcium, calcium oxalate being especially prevalent. The bundles of needle- shaped crystals known as "raphides" (Fig. 47, L) found in the leaves of a number of plants are composed of the latter salt, as are also the spherical aggregations called "druses," or " sphserraphides " (Fig. 47, C). The curious clustered " cystoliths " of the Ficus leaf (Fig. 47, A) are made up of cellulose and calcium carbonate. Crystals of silica are very abundant in the thickened walls of wood cells and in many other tissues, such as the outer cells of the Equisetum stem. Crystals of albumen, aside from those found in aleurone grains, are frequently present in the cytoplasm of cells poor in starch, as in the outer portion of the potato tuber. The leucoplast of Phajus often contains a rod-shaped albumen crystal. Protein crystals of various shapes are occasionally observed within the nucleus (Stock 1892; Zimmermann 1893). Cellulose is a common storage material, existing as a rule in the form of laminae deposited upon the original cell wall. As already pointed out, the sap of vacuolated cells may contain a number of differentiation products in solution. The cell sap is usually slightly acid in reaction, owing to the presence or organic acids (malic, formic, acetic, oxalic) and their salts. Inogranic salts are probably always present. Amides, such as glutamin and asparagin, glucosides, sugars, proteins, tannin, and many other substances are of frequent occurrence in the cell sap of various plants. The carbohydrate inulin may be pre- cipitated out of the sap by alcohol: this accounts for the presence of nodules of radiating inulin crystals frequently encountered in preserved material (Fig. 47, M}. Rubber is present in the form of a suspension of minute droplets in the cell sap of Ficus elastica and several other plants. Gutta-percha occurs in a similar state in Isonandra gutta. The cell sap in such cases has a characteristic milky appearance. The cell sap is often colored by red, blue, and yellow anthocyanin pigments (Wheldale 1916; Palladin 1918; Beauverie 1919), some of which change color when the reaction of the sap is altered from acid to basic and vice versa. The striking colors of flowers are due to "(1) the varying color of the sap, (2) 136 INTRODUCTION TO CYTOLOGY the distribution of the cells containing it, and (3) combinations of colored sap with chloro- and chromoplasts." Autumnal coloring is due to the formation of pigments as disorganization products : when cytoplasm and chlorophyll are the main disorganizing substances a yellowish color results, whereas if sugars are present in considerable amounts in the cell sap the brighter pigments are formed. Extruded Chromatin.1 — The actual extrusion of chromatin from the nucleus into the cytoplasm has been reported in a number of instances: in the microsporocytes of various angiosperms by Digby (1909, 1911, 1914), Derschau (1908, 1914), West and Lechmere (1915), and others; in ferns by Farmer and Digby (1910); and in the Ascomycete Helvella crispa by Carruthers (1911). The extruded chromatin commonly takes the form of deeply staining globules or irregular masses in the cytoplasm ; often a clear area suggesting a nuclear vesicle is present about them. In some cases, such as Gallonia candicans (Digby 1909) and Lilium candidum (West and Lechmere 1915), the chromatin may pass through the wall into an adjacent cell, where it forms a rounded mass connected by a chromatic strand with the nucleus from which it originated. The significance of this phenomenon is by no means apparent. It is not at all unlikely that nutritive materials passing from nucleus to cyto- plasm during the normal metabolism of the cell occur at times as visible globules at the nuclear surface. The extrusion of chromatin into neigh- boring cells, on the other hand, in many cases has every appearance of a phenomenon associated with degeneration or some other abnormal physiological condition. West and Lechmere, however, view the process as one which occurs normally at certain stages, and which will probably be found to be more general in plants. Sakamura's (1920) extensive researches on chloralized cells have led him to regard the extrusion of large masses of chromatin as an abnormal phenomenon which occurs as a result of a disturbance of the metabolism of the cell. Its more frequent occurrence in sporocytes than in other cells is attributed to the unusual sensitiveness of the former to disturbing influences. The Senescence of the Cell. — The accumulation of products of metabolism ("differentiation products") has a direct bearing on the problem of protoplasmic senility. As its life progresses the cell gradually "ages," and if nothing occurs to prevent it the process eventually term- inates in death. What shall be taken as an index of the degree of senes- cence has been the subject of much discussion. We have already called attention to the attempts which have been made to correlate senescence with a progressive change in the nucleoplasmic relation, concluding that no constant correlation of the kind has been shown to exist (p. 63). Child (1915) has brought forward much evidence to show that the 1 Extruded chromatin is not metaplasm, but it has been found convenient to treat it at this point along with other inclusions of the cytoplasm. METAPLASM; POLARITY 137 relative rate of metabolism is the main criterion of the cell's physiological age, "young" cells having a high rate and "old" cells a relatively low rate, and a gradual decline in this rate occurring throughout the life of the cell. In embryonic (physiologically young) cells the cytoplasm ap- pears to be comparatively homogeneous and undifferentiated. Older cells, on the contrary, are ordinarily marked by the presence of products of differentiation in the cytoplasm. The true measure of age is there- fore not time, but physiological differentiation. In many cells a rejuvenating process may occur, whereby a high meta- bolic rate is restored and the products of differentiation lost: this is regarded as a "return to the embryonic state" — a real physiological rejuvenescence. "Senescence is primarily a decrease in rate of the dynamic processes conditioned by the accumulation, differentiation, and other associated changes of the material of the colloid substratum. Rejuvenescence is an increase in rate of dynamic processes conditioned by changes in the colloid substratum in reduction and dedifferentiation " (Child, p. 58). Such a rejuvenescence occurs in connection with regeneration, vegetative and other asexual reproduction, and sexual reproduction. In each case the cell which begins the new life cycle — the meristematic regenerating cell, the zoospore, or the zygote — has a high metabolic rate and is comparatively free from the products of differentia- tion. In the lower organisms cell differentiation in this sense is not so great but that almost any cell may retain the power to " dedifferentiate " and begin the development of a new individual vegetatively. In these forms asexual reproduction may occur repeatedly and keep the organism as a whole (in protozoa and protophyta) or the protoplasm of the race (in lower metazoa and metaphyta) physiologically young. Only when the metabolic rate falls very low does sexual reproduction, the most effective of all the rejuvenating agencies, ensue. In the higher plants the retention of the power of dedifferentiation is strikingly shown in the well known cases of Begonia and Bryophyllum, which can regenerate complete new individuals from a few leaf cells. In the higher animals cell differentiation is usually so great that the somatic cells can no longer dedifferentiate and reproduce the organism asexually. Here rejuvenation occurs only after the union of two gametes, which are themselves, unlike the'zoospores of algae, physiologically old. Although local rejuvenescence may occur, as in secretory cells which are "younger" after secretion, and also in wound tissue, the differentiation of the body cells is carried so far that their metabolic rate falls low enough to make a recovery or rejuvenescence no longer possible. Thus it is only the functioning reproductive cells that endure: the ultimate cessation of all life processes in the body cells is the price which is inevitably paid by the complex multicellular organism for the advantages conferred by its high degree of differentiation. 138 INTRODUCTION TO CYTOLOGY Of the highest importance in this connection are the results of at- tempts to maintain the cells and tissues of higher animals in the living condition in artificial culture media outside the body. It has been shown by the remarkable experiments of Carrel, Leo Loeb, Burrows, H. V. Wilson and others that cells may be isolated from any of the highly differentiated essential tissues of the body and kept actively growing and multiplying in vitro for a length of time frequently far exceeding that to which they would have lived in the body. They do not appear to grow old: indeed it is not improbable that in such a constantly favorable environment somatic cells are as "potentially immortal" as the germ cells (see p. 403). In the words of Pearl (1921), "It is the differentiation and specialization of function of the mutually dependent aggregate of cells and tissues which constitutes the metazoan body which brings about death, and not any inherent or inevitable mortal process in the indivi- dual cells themselves." POLARITY Polarity is a feature which is exhibited in some form by the cells of all higher organisms, and in at least many of the simpler ones, as shown by Tobler (1902, 1904) for certain algae; indeed it is probable that it is possessed in some form and degree by ah1 cells. Harper (1919) calls attention to the fact that "in the presence of polarity and the various symmetry relations we have a fundamental distinction between cell organization and that of polyphase colloidal systems as they are com- monly produced in vitro." This polarity has two aspects, the morphological and the physiological. In the first place, the various constituents of the cell may be arranged symmetrically about one or more ideal axes, so that the cell has more or less distinctly differentiate.d anterior and posterior ends. This structural aspect of polarity has been the one chiefly emphasized by certain workers: van Beneden (1883), for instance, looked upon polarity as "a primary morphological attribute of the cell," the axis passing through the nucleus and the centrosome. Later writers, among them Heidenhain (1894, 1895), made this conception of morphological polarity the basis for interpretations of many of the phenomena of cell behavior. (See Wilson 1900, pp. 55-56.) However, as Harper (1919) points out, polarity "is apparently independent of the uni- or multinucleated condi- tion of the cell, which shows that it is in some cases at least a more generalized characteristic of the cell as a whole rather than a mere ex- pression of the space relations of the nucleus and cytoplasm ..." Other investigators (Hatschek 1888; Rabl 1889, 1892) early laid emphasis upon the physiological expression of polarity. The cell shows 'a polar differ- entiation in physiological labor: the processes in one portion of the cell differ from those in another, this difference in the case of tissue cells METAPLASM; POLARITY 139 l>eing due to different environments in the tissue. For these workers this physiological differentiation is the essential element of polarity; any morphological polarity is due secondarily to it. Metabolic Gradient. — The most suggestive physiological conception recently developed in this connection is that of Child (1911-1916). Child has shown in the case of Planaria and other lower animals, as well as in certain algae, that along each of the axes of symmetry there exists a "metabolic gradient," or "axial gradient:" the rate of the physiological processes is highest at one end of the axis and diminishes progressively toward the other end. The anterior end of a planarian, for example, has a higher metabolic rate than the posterior portions. Furthermore, the portions of higher rate dominate and control the development of those portions having a lower rate, with the result that the young indivi- dual soon develops and maintains a definite physiological correlation of anterior and posterior parts. Similarly in individuals with more than one axis of symmetry, there may be a corresponding dorsal-ventral, as well as an axial-marginal, correlation. That polarity is here primarily a physiological matter is indicated by the fact that experimental altera- tions in the metabolic rate in different parts is followed by abnormalities in structural development. As to the means by which the dominance of certain regions over others is exercised, correlating the activities of the various parts of the or- ganism, there are two principal theories in the field. According to one theory chemical substances (hormones) are produced at certain places and transmitted through the body. Although the circulation of such hormones clearly has much to do with correlation in higher complex organisms, Child adduces good evidence in support of the second theory, namely, that the fundamental relations of polarity "depend primarily upon impulses or changes of some sort transmitted from the dominant region, rather than upon the transportation of chemical substances" (p. 224). It cannot at present be said to what extent this conception of polarity is applicable to the single cell. The work of Child shows in a very definite manner the coincidence of the morphological and physiological axes of polarity, which indicates that the two are but different aspects of one and the same polar differentiation. A similar coincidence exists very generally in the case of the single cell. In the cell, as in the organism as a whole, functional and structural differentiation are inseparably connected. In the present state of our knowledge the attempt to determine the real essence of polarity raises questions which cannot yet be answered. Does physiological polarity depend upon a polarized structure which is a fundamental attribute of the cell's ultimate organization? Or does a polarized morphological arrangement follow and depend upon a physio- logical division of labor arising as a difference in intensity or rate in proc- 140 INTRODUCTION TO CYTOLOGY esses originally common to all parts of the cell? If so, to what internal or external factors is the establishment of this difference due in cells having no initial polarity? Analogies with electrical polarity have been resorted to in this connection, concerning which Harper (1919) says: "To pro- vide an adequate basis for understanding the observed facts of polarity, however, it seems to me that the conception of compound aggregate polyphase systems is more suggestive than these attempted analogies . . . In the spatial arrangement and interactions of these systems polar dif- ferences of the most diversified types are bound to arise in the mass as a whole and express themselves in the form and relative rigidity and surface tension of different parts, as well as in the interrelations between the cells of a group in contact." The polarity of the multicellular organism as a whole is closely bound up with the polarities of its constituent cells. Harper has clearly shown (1918) that in Pediastrum the position of the swarm-spores in the colony which they unite to form is directly dependent upon their polarity. This does not mean, however, that the polarity of the multicellular organ- ism is nothing more than the sum of the polarities of its constituent cells, unless we return to Schwann's simple conception of the organism as merely an aggregate of independent cells. (See p. 12.) The higher individuality, the colony, has its own polarity, which may be related to, but is not the same as, that of its individual cells. In the ordinary multi- cellular organism the polarity is an outgrowth of the polarity of the fertilized egg cell rather than of the polarities of the many adult tissue cells. In polarity, then, we encounter another problem which must be brought nearer a solution before we can have any adequate understanding of the relation of the cell to the multicellular organism as a whole, and of the perplexing matter of organic individuality. Bibliography 7 Metaplasm — Senescence — Polarity VAN BENEDEN, E. 1883. Recherches sur la maturation de 1'oeuf, la fecondation et la division cellulaire. Arch, de Biol. 4. BOVERI, TH. 1901a. Ueber die Polaritat des Seeigeleies. Verh. Phys.-Med. Ges. Wiirzburg 34. 19015. Die Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus lividus. Zool. Jahrb. (Anat. Abt.) 14: 630-653. pis. 48-50. CARRUTHERS, D. 1911. Contributions to the cytology of Helvetia crispa Fries. Ann. Bot. 26: 243-252. pis. 18, 19. CHILD, C. M. 1911. Studies on the dynamics of morphogenesis and inheritance in experimental reproduction : I. The axial gradient in Planaria dorotocephala as a limiting factor in regulation. Jour. Exp. Zool. 10: 265-320. figs. 7. 1912. Studies, etc. IV. Certain dynamic factors in the regulatory morphogenesis of Planaria dorotocephala in relation to the axial gradient. Ibid. 13 : 103-152. figs. 46. METAPLASM; POLARITY 141 1913. Studies, etc. VI. The nature of the axial gradients in Planana and their relation to antero-posterior dominance, polarity and symmetry. Arch. Entw. 37: 108-158. figs. 13. 1915. Senescence and Rejuvenescence. Chicago. 1916. Axial susceptibility gradients in alga?. Bot. Gaz. 62: 89-114. VON DERSCHAU, M. 1908. Beitrage zur pflanzlichen mitose: Centern, Blepharo- plasten. Jahrb Wiss. Bot. 46: 103-118. pi. 6. 1914. Zum Chromatindualismus der Pflanzenzelle. Arch. Zellf. 12: 220-240. pi. 17. DIGBY, L. 1909. Observations on "chromatin bodies" and their relation to the nucleolus in Gallonia candicans Decsne. Ann. Bot. 23: 491-502. pis. 33, 34. 1910. The somatic, premeiotic, and meiotic nuclear divisions in Galtonia candicans. Ann. Bot. 24: 727-757. pis. 59-63. 1914. A critical study of the cytology of Crepis virens. Arch. Zellf. 12: 97-146. pis. 8-10. DUESBERG, J. 1911. Plastosomen, "Apparato reticolaro interno," und Chromi- dialapparat. Ergebn. Anat. Entw. 20: 567-916. (Review.) EAST, E. M. and HAYES, H. K. 1911. Inheritance in maize. Conn. Exp. Sta. Bull. No. 167. 1915. Further experiments in inheritance in maize. Ibid No. 188. EMERSON, R. A. 1914. The inheritance of a recurring somatic variation in varie- gated ears of maize. Am. Nat. 48: 87-115. 1917. Genetical analysis of variegated pericarp in maize. Genetics 2. HARPER, R. A. 1918a. Organization, reproduction and inheritance in Pediastrum. Proc. Am. Phil. Soc. 67 : 375-439. pis. 2. figs. 35. 19186. The evolution of cell types and contact and pressure responses in Pedi- astrum. Mem. Torr. Bot. Club 17: 210-240. figs. 27. 1919. The structure of protoplasm. Am. Jour. Bot. 6: 273-300. HATSCHEK, B. 1888. Lehrbuch der Zoologie. HEIDENHAIN, M. 1894. Neue Untersuchungen iiber die Centralkorper und ihre Beziehungen zum Kern und Zellprotoplasma. Arch. Mikr. Anat. 43 : 423-758. pis. 25-31. 1895. Cytomechanische Studien. Arch. Entw. 1 : 473-577. pi. 20. figs. 17. KASSOWITZ, M. 1899. Allgemeine Biologie. Vienna. PEARL, R. 1921. The biology of death. II-The conditions of cellular immortality. Sci. Mo. 12: 321-335; figs. 6. PFEFFER, W. 1872. Untersuchungen liber die Proteinkorner und die Bedeutung des Asparagins beim Keimen der Samen. Jahrb. Wiss. Bot. 8: 429-574. pis. 36-38. RABL, C. 1885. Ueber Zelltheilung. Morph. Jahrb. 10: 214-330. pis. 7-13. figs. 5. 1889. Ueber Zelltheilung. Anat. Anz. 4: 21-30. figs. 2. SAKAMURA, T. 1920. Experimentelle Studien iiber die Zell- und Kernteilung mit besonderer Riicksicht auf Form, Grosse und Zahl der Chromosomen. Jour. Coll. Sci. Imp. Univ. Tokyo 39: pp. 221. pis. 7. STOCK, G. 1892. Ein Beitrag zur Kenntniss der Proteinkrystalle. Cohn's Beitr. Biol. Pflanzen 6: 213-235. pi. 1. THOMPSON, W. P. 1912. Artificial production of aleurone grains. Bot. Gaz. 54:336-338. 1 fig. TOBLER, F. 1902. Zerfall und Reproduktionsvermogen des Thallus einer Rhodo- melacese. Ber. Deu. Bot. Ges. 20 : 357-365. pi. 18. 1904. Ueber Eigenwachstum der Zelle und Pflanzenform. Jahrb. Wiss. Bot. 39 : 527-580. pi. 10. 142 INTRODUCTION TO CYTOLOGY TSCHIRCH, A. 1889. Angewandte Pflanzenanatomie. Wien u. Leipzig. WEST, C. and LECHMERE, A. E. 1915. On chromatin extrusion in pollen mother- cells of Lilium candidum, Linn. Ann. Bot. 29: 285-291. pi. 15. WHELDALE, M. 1916. The anthocyanin pigments of plants. Cambridge. WILSON, E. B. 1900. The Cell in Development and Inheritance. ZIMMERMANN, A. 1893a. Ueber die Proteinkrystalloide. Beitr. z. Morph. u. Physiol. d. Pflanzenzelle 1 : 54-79. pis. 2. 18936. Ueber Proteinkrystalloide. Ibid. 2: 112-158. 1894. Sammel-Referate. 11. Elaioplasten, Elaiospharen und verwandte Korper. 13. Die Aleurone- oder Proteinkorner, My rosin- und Emulsionkorner. 14. Die Proteinkrystalloide, Rhabdoiden und Stachelkugeln. 15. Die Starkekornrr und verwandten Korper. Beih. Bot. Centr. 4: 165-169, 321-335. CHAPTER VIII SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY SOMATIC MITOSIS Since the time when the cell was pointed out as the unit of structure and function it has been recognized that the mode of origin of new cells is a matter of fundamental importance. We have seen in our historical sketch that cells were believed by the founders of the Cell Theory to arise de now from a mother liquor, or "cytoblastema," a misconception removed by later investigations in which it was shown beyond question that cells arise only by the division of preexisting cells. By several early observers the nucleus was seen to have a more or less prominent part in the process, its division preceding that of the cell, but "it was not until 1873 that the way was opened for a better understanding of the matter. In this year the discoveries of Anton Schneider, quickly followed by others in the same direction by Biitschli, Fol, Strasburger, Van Beneden, Flemming, and Hertwig, showed cell-division to be a far more elaborate process than had been supposed, and to involve a complicated trans- formation of the nucleus to which Schleicher (1878) afterward gave the name karyokinesis. It soon appeared, however, that this mode of divi- sion was not of universal occurrence; and that cell-division is of two widely different types, which Van Beneden (1876) distinguished as fragmenta- tion, corresponding nearly to the simple process described by Remak, and division, involving the more complicated process of karyokinesis. Three years later Flemming (1879) proposed to substitute for these terms direct and indirect division, which are still used. Still later (1882) the same author suggested the terms mitosis (indirect or karyokinetic division) and amitosis (direct or akinetic division), which have rapidly made their way into general use, though the earlier terms are often employed. Modern research has demonstrated the fact that amitosis or direct division, regarded by Remak and his followers as of universal occurrence, is in reality a rare and exceptional process;. . . it is certain that in all the higher and in many of the lower forms of life, indirect division or mitosis is the typical mode of cell-division" (Wilson 1900, pp. 64-65). 1 1 The following additional historical data arc of interest. The chromosomes, though they appeared in the figures of Schneider (1873), were first adequately drawn by Strasburger in 1875. Longitudinal splitting was described by Flemming in 1882. The terms prophase, mctaphase, and anaphase were introduced by Strasburger in 143 144 INTRODUCTION TO CYTOLOGY In view of the fact that the phenomena of growth, differentiation, reproduction, and inheritance are now known to be intimately bound up with the process of cell-division, it is obvious that a detailed knowledge of this process is an absolute prerequisite to a solution of many of the problems which confront us. In the present chapter the essential fea- tures of vegetative or somatic nuclear division will be described. After a preliminary sketch of the process of mitosis we shall take up in some detail the behavior of the chromosomes and the question of their individ- uality. In the following chapter attention will be devoted to other features of cell- division: the achromatic figure, the mechanism of mitosis, cytokinesis (the division of the extra-nuclear portion of the cell), and the formation of the cell wall. SOMATIC MITOSIS FIG. 48. — Diagram of a typical case of somatic mitosis in plants. Preliminary Sketch of Mitosis. — The main steps in a typical case of somatic mitosis in plants may be very briefly outlined as follows (Fig. 48): The chromatic material of the "resting" nucleus, as described in Chapter IV, exists in the form of a more or less irregular reticulum. As the process of mitosis begins this reticulum resolves itself into a definite number of slender threads which represent chromosomes. These in w- 1884, and Heidenhain in 1894 first used the term telokinesis (telophase). Lundegardh (19126) added interphase. The chromosome was named by Waldeyer in 1888. Hermann in 1891 distinguished connecting fibers (central spindle) and mantle fibers. That the halves of each split chromosome go to opposite poles was shown by van Beneden for animals and by Heuser for plants in 1884. The achromatic spindle was first figured by Kowalevsky (1871) and Fol (1873), and first carefully described by Butschli (187506). SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 145 many nuclei are distinct from each other from the first, whereas in other cases they may be arranged end-to-end in a more or less continuous thread, or spireme, which later segments transversely into independent chromosomes. The slender threads (chromosomes) now split longitu- dinally throughout their entire length. A progressive shortening and thickening of the split threads ensues, so that the nucleus is eventually seen to have a certain number of chromosomes which have become double through a longitudinal cleavage. While the above changes are occurring, fine fibrils are differentiated in the cytoplasm near the nucleus and become arranged in two opposed groups. The nuclear membrane now disappears and the fibers extend into the nuclear region, where some of them (the "mantle fibers") attach themselves to the double chromosomes, while others (the "con- necting fibers") pass through from one pole to the other. The double chromosomes quickly become arranged in a single plane at the equator of the cell, the fibers meanwhile forming the achromatic figure, or spindle. This stage is known as the metaphase; all the steps leading up to it, be- ginning with the initial changes in the resting reticulum, constitute the prophase. The daughter chromosomes (the halves of the longitudinally split chromosomes) now move apart toward the poles of the achromatic figure, where they soon form two closely packed groups with the central spindle of connecting fibers extending between them. The period during which the daughter chromosomes are thus moving apart is known as the anaphase. The two groups of daughter chromosomes now reorganize the daughter nuclei, in each of which the chromosomes again form a reticulum like that of the original mother nucleus. This reorganization period is called the telophase. During the telophase there is formed upon the connecting fibers (central spindle) a separating wall, which completes the division of the cell. The nucleolus as a rule plays no con- spicuous part in mitosis: it usually disappears during the late stages of the prophase, new nucleoli being formed in the daughter nuclei in the telophase. In rapidly dividing cells the period between two successive mitoses is called the interphase. Mitosis in animals (Fig. 49) is closely similar to that in plants as regards the behavior of the chromosomes. It normally differs in two conspicuous features, namely, the presence of centrosomes and the mode of cytokinesis following the division of the nucleus. During the prophases the centrosome with its aster, if not dlready double, divides. The two daughter centrosomes, each with its own aster, move apart, and a small bundle of fibers extends between them; all these structures together form the amphiaster. The rays on the side toward the nucleus extend into the latter when the membrane dissolves and become attached to the chromosomes, often before the two centro- 10 146 INTRODUCTION TO CYTOLOGY somes have reached polar positions. The centrosomes, surrounded by asters, remain at the poles of the achromatic figure during metaphase, anaphase, and telophase, and after mitosis has been completed they may disappear or remain through the resting stage to function in the next mitosis. (See p. 78.) The division of the cell following nuclear division is commonly brought about in animals by the formation of a cleavage furrow, which grows inward from the periphery as described in the following chapter, rather than by the formation of a wall on the spindle fibers as in plants. FIG. 49. — Diagram of a typical case of somatic mitosis in animals. Although the two above points serve in general to distinguish mitosis in animals from that in plants, the distinction is not a sharp one: cen- trosomes are regularly present in the cells of many lower plants, while cytokinesis by furrowing also occurs in certain cases, as will later be shown. The essential point to be borne in mind is that the significant feature of mitosis — the division of the chromatin and its distribution to the daughter nuclei — is fundamentally the same in both plants and animals. The relative duration of the various phases of mitosis has been studied in a few cases. As an example may be taken the observations of M. and W. Lewis (1917) on the mesenchyme cells of the chick growing in tissue cultures. These investigators summarize the researches of others upon the subject and give the following figures for the chick cells: prophase, 5 to 50 minutes, usually more than 30; metaphase, 1 to 15, usually 2 to 10; anaphase 1 to 5, usually 2 to 3; telophase up to cytokinesis, 2 to 13, usually 3 to 6; telophasic reconstruction of daughter nuclei, 30 to 120; total, 70 to 180 minutes. SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 147 Detailed Description of the Behavior of the Chromosomes in Somatic Mitosis.1 — In the present account we shall depart from the order usually followed in descriptions of mitosis. Instead of commencing with the resting nucleus and tracing the steps leading to the formation of two daughter resting nuclei, we shall begin the description with the fully formed chromosomes as they appear at the metaphase and follow them through anaphase, telophase, resting stage, and prophase to the next metaphase, when they are again clearly seen. This is done in order that the account of the telophasic transformation of the chromosomes to form a resting reticulum, and the prophasic condensation of the latter to form chromosomes, may be given without interruption, which seems advisable in view of the nature of certain questions which are later to be discussed in the light of chromosome behavior. Metaphase (Fig. 50, A}. — As the chromosomes arrange themselves upon the spindle preparatory to their anaphasic separation their double nature is clearly evident. The two halves may lie very close together and in the case of long chromosomes may be somewhat twisted about each other. When they lie a little apart they may often show small connecting strands or anastomoses; in the immediately preceding stages (late prophase) the halves are usually pressed tightly together, so that these anastomoses appear to be due to mutual coherence at certain points when the halves move slightly apart after the disappearance of the nuclear membrane. As the double chromosomes take their places on the spindle, the spindle fibers become attached to them, not to all parts but to a particular portion of each. In the case of long chromosomes the point of attachment is often at about the middle, whereas in shorter ones it is commonly near one end. At their points of attachment to the spindle the double chromosomes all lie with their halves superposed (one half toward each spindle pole) and in a single plane; those portions to which no fibers are attached may extend in various directions with no regular arrangement. Anaphase (Fig. 50, B-D}. — The daughter chromosomes (the halves of the double chromosomes seen at metaphase) now begin to separate, first at the point of insertion, and gradually move away from the equa- torial plane. Owing to the different locations of the points of fiber attach- ment, and also to the fact that the free ends of the chromosomes occupy various positions, the chromosomes, unless they are very short, may now 1 This description is based on the author's accounts of somatic mitosis in Vicia faba (1913) and Tradescanlia virginiana (1920). In these papers, especially in the first, there is presented a more extensive comparison of the results of other investi- gators than can be given here. Comparative studies have shown that in general the present description is widely applicable to mitotic phenomena in plants and ani- mals, although many modifications in detail are known, particularly in forms with small chromosomes. A useful list of works on mitosis in angiosperms is given by Picard (1913). 148 INTRODUCTION TO CYTOLOGY be drawn into a number of peculiar shapes. In the case of long chromo- somes the portions to which the fibers are attached may have reached the poles of the spindle while the other portions are not yet separated at the equatorial plane. As soon as the daughter chromosomes become entirely free from one another they quickly draw apart and contract into two dense masses, which are often actually farther apart than were •0 FIG. 50. — Somatic mitosis in Tradescantia virginiana: metaphase (A), anaphase (B-D), and telophase (E-G). At F are shown cross sections of chromosomes in the stage shown at E. X 1900. (After Sharp, 1920.) the poles of the spindle at metaphase. In these masses the individual chromosomes can be distinguished only with great difficulty or not at all. With this stage, which has been referred to by Gregoire and Wygaerts (1903) as the tassement polaire, the anaphase ends and the telophase begins. Telophase (Fig. 50, E— Fig. 51, 7). — After remaining tightly pressed together for a short time the chromosomes of each daughter group begin to separate, their individual boundaries again becoming visible. As they do so they cohere at various points where their substance becomes drawn out to form anastomoses. It seems clear that the main connec- SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 149 tions between the chromosomes of the reorganizing telophase nucleus are formed in this way, at least in mitoses showing a tassement polaire stage; but it is also probable, as several investigators have pointed out, that other anastomoses may grow out from one chromosome to another in the manner of pseudopodia (Boveri 1904; Gates 1912; Strasburger 1905; Dehorne 1911; Muller 1912; Lundegardh). Reactions taking place between the various chromosomes and espec- ially between them and the cytoplasm now result in the production of the nuclear sap, or karyolymph. Between the outermost chromosomes and the cytoplasm and also within the chromosome group droplets of clear karyolymph appear, and where these come in contact with the cytoplasm a nuclear membrane is formed. As the karyolymph .increases in amount the nucleus enlarges and the chromosomes become more widely separated. The telophasic alveolation of the chromosomes, although it may in exceptional cases begin much earlier, usually commences at about the time the chromosomes first separate from one another in the early reorganization stages of the daughter nucleus. Within each chromosome vacuoles appear, first as obscure though rather sharply delimited circular or elongated areas. They lie not only along the axis but also near and against the periphery. This point is of importance in evaluating the claim advanced by certain investigators (Lundegardh 1910, 1912; Fraser and Snell 1911; Fraser 1914; Digby 1919) that the vacuolation is median and results in a splitting of the chromosomes during the telo- phase, rather than in the prophase. While the vacuoles develop into open spaces through the breaking down of the thin portions bounding them the nucleus increases rapidly in volume, so that each chromosome appears as an irregular net-like band joined to its neighbors by fine anas- tomoses. Careful study of the details of these telophasic changes (see cross sections of chromosomes in Fig. 50, F) shows that the alveolation proceeds with little regularity, and that each chromosome becomes an alveolar and then reticulate body with nothing which can properly be called a longitudinal split. In certain cases these internal changes, which result in the trans- formation of the chromosomes into a reticulum, and which as a rule do not begin until the telophase, may be initiated during the anaphase. In Allium, for instance, Miss Merriman (1904), Lundegardh (1910, 19126), and Nemec (1910) all report that the vacuolation of the chromosomes begins at this time. Even more striking is the case of Trillium (Gre"goire and Wygaerts 1903), in which the unusually large chromosomes may show vacuoles as early as the metaphase (Fig. 54, A~). Internal changes of other types have also been described in anaphase chromosomes, but not with sufficient clearness to warrant their use in general interpre- tations. 150 INTRODUCTION TO CYTOLOGY According to Bonnevie (1908, 1911) the chromosomes of Allium, Ascaris, and Amphiuma each give rise to an endogenous spiral thread during the telophases, this spiral thread persisting through the resting stages until the next prophase, when it again condenses to form a chromo- some (Fig. 54, J5). In his work on Salamandra Dehorne (1911) asserted that each chromosome is represented at telophase by two interlaced spirals arising from an anaphasic split, and further that these double structures are associated in pairs and persist in this condition through the resting stages. These two conceptions have been criticized by Gregoire (1912), Sharp (1913), and de Smet (1914), who have inter- preted such appearances as occasional aspects of the alveolized chromo- somes without the significance attributed to them by Bonnevie and Dehorne. FIG. 51. — Somatic mitosis in Tradescantia virginiana: late telophase (H, I), inter- phase and resting stage (-7, K), and early prophase (L, M). X 1900. (After Sharp, 1920.) In the young telophass nucleus the chromosomes may become ar- ranged in the form of a more or less continuous daughter spireme which is then transformed into the resting reticulum. This spireme stage, however, is not a necessary one; its absence is being reported with sufficient frequency to throw much doubt upon the view that it is a phenomenon of even general occurrence. The nucleolus usually makes its appearance during the early telophase as a small droplet or as several such droplets which may later flow together. It seems to have little direct connection with the chromo- somes, but there can be no doubt that its appearance is closely associated with their physiological activities. As the telophasic changes proceed the chromosomes with their anastomoses gradually form a more and more uniform reticulum, in SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 151 which, however, tho limits of the component chromosomes can !><• distinguished until a very late stage. Inter phase (Fig. 51, «/).— It often happens that in rapidly growing tissue, such as the meristem of the root tip, the mitoses succeed one another so rapidly that the telophasic changes may not proceed far enough to obscure the limits of the chromosomes in the reticulum before the changes of the ensuing prophase begin. In such tissue it is not always possible to tell whether a given nucleus will undergo further telophasic change or will at once enter upon the prophases. Such interphasic nuclei develop nucleoli, but karyosomes (in species which have these bodies) are usually not formed until a more advanced stage. Resting Stage (Fig. 51, J, K). — In slowly growing tissue the successive- mitoses do not follow one another with very great rapidity, and the telophasic changes are carried on until the condition characteristic of the typical resting nucleus is reached: the interphase here becomes the prolonged resting stage. The structure of the resting nucleus has been fully described in Chapter IV. In the reticulum the limits of the con- stituent chromosomes usually become indistinguishable, although it is known that in certain cases such nuclei, if properly sectioned and stained, may reveal heavier and lighter areas in the reticulum which represent respectively the chromosomes and the regions of anastomosis between them. The importance of these facts will be apparent in our treatment of the individuality of the chromosomes. Prophase (Fig. 51, L-Fig. 52). — The first indication that the prophasic changes have begun is seen in the breaking down of the reticulum in certain regions. In the case of nuclei which show heavier and lighter areas in their reticula this breaking down occurs along the light portions. In view of what has been said concerning the origin of the reticulum at telophase it is apparent that the breaking up of the reticulum in the prophase represents in such cases the separation of the constituent chromosomes from each other along the lines of their telophasic union, and it has been inferred that a similar interpretation applies to those nuclei in which the reticulum is perfectly uniform or in which the nuclear material assumes more irregular forms. In this way there are developed from the resting reticulum a number of more or less distinct reticulate units, which, in view of their subsequent behavior, we know to be the chromosomes (Fig. 51, L, M). That these units are essentially the same as those which went to make up the reticulum at the preceding telophase seems highly probable; there can be little doubt on this point when the interphase is short. The material of each reticulate unit (chromosome) now gradually con- denses in a very irregular fashion about its open spaces and cavities. The thinner regions bounding these spaces and cavities become broken down, and the thicker portions remain as a very irregular zigzag thread of 152 INTRODUCTION TO CYTOLOGY uneven thickness, which soon begins to straighten out (Fig. 52, P). At the same time the material composing the thread becomes more evenly arranged throughout its length, so that the chromosome eventually takes the form of a single slender thread. All of these changes — condensa- tion, straightening, and equalization in thickness — may be seen going on simultaneously in different chromosomes of the same nucleus, or even in different portions of a single chromosome. The formation of the slender prophase chromosomes from the retic- ulum in the above manner was first described in detail by Gre"goire and Wygaerts (1903) and Gregoire (1906), and new cases have since been added. The above writers, together with Nemec (1910), Digby (1910), ** .s FIG. 52. — Somatic mitosis in Tradescantia virginiana: prophases. At N are shown cross sections of chromosomes in the stage shown in Fig. 51, M. X 1900. (After Sharp, 1920.) and Miiller (1912), believe that the separated portions of the reticulum may also condense directly into the slender threads without passing through the very irregular zigzag stage above described. It is probable that both methods are followed in different cases, direct condensation possibly being the rule in small nuclei. The view of Bonnevie (1908, 1911) concerning the origin of the zigzag threads has already been mentioned in the paragraphs on the telophase. According to this worker and to certain others (Wilson 1912a6) the chromatic material forms a spiral thread within the chromosome during the telophase, this thread uncoiling and emerging from the chromosome in the following prophase. It is true that the zigzag threads occasionally have a strikingly regular spiral aspect, but in view of the many other aspects observed and the process which is known to give rise to them, it is probable that the SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 153 formation of spirals in the manner described by Bonnevie is at least very exceptional. The true longitudinal splitting of the chromosomes is initiated in the slender threads of the early prophase (Figs. 52 and 53, Q). As soon as a thread becomes sufficiently equalized in diameter small vacuoles appear along its axis and rapidly develop into a more or less continuous split. Not all the threads, nor even all portions of the same thread, undergo the change at the same time. If we consider the whole nucleus at once, the processes of condensation, straightening, equalization, and FIG. 53. — Somatic mitosis in Vicia faba: prophases. Stages P, Q, and S correspond with P, Q, and S of Fig. 52. X 1650. 1913.) (After Sharp, splitting are all going on simultaneously; only in a given small portion of a thread do they follow in definite sequence. Furthermore, as soon as the threads become equalized they at once begin to shorten and thicken, so that when vacuolation and splitting are a little delayed they occur in somewhat heavier threads. The manner in which the vacuoles develop into the complete split should be carefully noted. The vacuoles quickly form openings which extend completely through the chromosome, so that the latter soon takes the form of two parallel strands connected by heavy cross pieces repre- senting the portions between the original vacuoles (Figs. 52 and 53, S). 154 INTRODUCTION TO CYTOLOGY The material constituting tin1 cross pieces gradually moves to the two side strands, the center portion of the cross piece becoming progressively thinner and the material accumulating on the side strands as a pair of chromatic lumps. Although some of the cross pieces may persist until a relatively late stage most of them soon disappear completely, and the material in the two chromatic lumps is gradually distributed more or less evenly along the parallel strands, which represent the daughter chromosomes resulting from the split. The double chromosomes now shorten and thicken, forming the "thick spireme" so conspicuous in prophase nuclei (Fig. 53, T, U). As pointed out in the preliminary sketch of mitosis, the chromosomes in the prophase may form a more or less continuous spireme, but it is becoming increasingly apparent that this is not a universal phenomenon. It is certain that in many cases the chromosomes are separate from the first, and it seems therefore that any association in the form of a continuous spireme is a matter of secondary importance. As the shortening and thickening proceed the split may become obscured by the close association of the halves, but suitable methods reveal its presence. While indications of spindle formation are appearing in the cytoplasm the nucleolus disappears and the nucleus begins to contract, so that the thick double chromosomes become very closely packed together. While the* contraction is at its height the nuclear membrane disappears, after which the chromosomes loosen up as an irregularly arranged group. This contraction stage evidently does not occur in many mitoses: the membrane may disappear while the nucleus has its full size. However, when it does occur it is of very short duration, so that it may take place in more cases than has been supposed. After the disappearance of the nuclear membrane the spindle fibers establish connection with the chro- mosomes, which quickly become arranged with their halves in superposi- tion at the equatorial plane, as described in the paragraph on the metaphase. This brings us to the point with which our description began. It should be added that in many descriptions of mitosis, notably those presented in general text books, the chromosomes are said to split during the metaphase, after they have become arranged upon the spindle. Such a late development of the split may indeed occur in some cases, but it is not improbable that closer examination would often reveal the inception of the process at a much earlier stage. As has been pointed out in the foregoing description, the early formed split frequently be- comes obscured during the later prophases owing to the shortening and thickening of the chromatin threads, and becomes conspicuous again only after the metaphase figure has been established. Chromomeres. — One matter which should receive special attention is that of the chromomeres. It was held by Roux (1883) that the compli- SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 155 rated process of mitosis is meaningless unless the chronudin is quali- tatively different in the various regions of the nucleus, and that the arrangement of the material of the chromosome in the form of a long thread prior to its splitting is a means whereby all these qualities, ar- ranged in a linear series in the thread, are equationally divided and distributed to the daughter nuclei. The theory of Balbiani (1876) and Pfitzner (1881), that the chromatin granules visible in the nuclear reticu- lum arrange themselves in a series in the chromosome and by their division initiate its splitting, had much to do with the formulation of this hypothesis. That the chromatic granules, or chromomeres (Fol 1891), represent the qualities of Roux is a theory which has been widely accepted by cytologists. It was the opinion of Brauer (1893) and many later workers that the granules or chromomeres, rather than the chromosomes themselves, are the significant units in the nucleus, and that their division is an act of reproduction. The division and separation of chromosomes was accordingly regarded as a means of distributing the daughter granules to the daughter cells. That the chromomere is made up of still smaller "chromioles" was held by Eisen (1899, 1900). Strasburger, Allen (1905), and Mottier (1907) also found the chromomere to be composed of smaller chromatic granules. FIG. 54. A, vacuoles in chromosomes at metaphase in Trillium. X 1800. (After Gregoire and Wygaerts, 1903.) B, spiral arrangement of chromatin material within the chromosomes of Allium. (After Bonnevie, 1911.) C, D, stages of chromosome splitting in Najas marina, showing chromomeres. X 2250. (After Midler, 1912.) Although a large number of investigators, particularly those interested in the hereditary role of the chromatin, have placed much confidence in the importance of the chromomeres (Strasburger 1884, 1888), others have raised serious objections to the theory that they are significant units or individuals. Gregoire and Wygaerts (1903), Martins Mano (1904), Gregoire (1906, 1907), Marechal (1907), Bonnevie (1908), Stomps (1910), Lundegardh (1912), Sharp (1913, 1920), and others have found no such definite behavior on the part of the chromatin granules in the dividing chromosomes studied by them, and have suggested other ex- planations for the appearances observed. According to a modification of the chromomere theory adopted by Miiller (1912) the portions of the thread between the chromomeres split first, the division of the chromo- 156 INTRODUCTION TO CYTOLOGY meres then following. It has been pointed out (Sharp 1913) that Miiller's figures (Fig. 54, C, D), which are very similar to the later ones of Stras- burger (1907), may be interpreted as steps in the division of a homogene- ous chromatic thread by the formation of vacuoles, and that the chromomeres in this case are merely the cross pieces between the halves of the incompletely split chromosome, as described in the foregoing account of the prophase (Fig. 53, $). It is becoming increasingly apparent that the distinction between chromatin granules and supporting thread is not so sharp as has been supposed, since the chromatic substance is often very fluid -in consist- ency; and many have felt that the granules when present are far too inconstant in number and behavior to serve as the ultimate units which students of heredity hope to find. On the other hand, it should be said that the constancy in size and position of the chromomeres described by Wenrich (1916) for the grasshopper, Phrynotettix (Fig. 155), argues strongly for the hereditary significance of these bodies, some of which can be seen to retain their identity through the resting stages. But whatever their importance may be, the arrangement of the chromatic material in the form of a long slender thread and its accurate splitting into exactly similar halves are very suggestive in connection with the theory of Roux that many qualities are arranged in a row and all divided at the time of nuclear and cell division. This subject will receive further attention in the chapters dealing with heredity. Summary. — The chromosomes, after having arrived at the poles of the achromatic figure, become irregularly alveolized during the telophase and form ragged net-like structures. These are joined to each other by fine anastomoses and so make up the continuous reticulum of the resting stage. In the next prophase this reticulum breaks up into separate small nets or alveolar units, each of which represents a chromosome. The units condense in a peculiar manner and become long slender threads. These threads undergo a longitudinal splitting. The double threads so formed shorten and thicken, and become the double chromosomes which are arranged on the spindle at metaphase. The two halves (daughter chromosomes) making up each double chromosome separate and pass to opposite poles during the anaphase. The outstanding and significant feature of somatic mitosis is this: each chromosome is accurately divided into two exactly equal longitudinal halves which are distributed to the two daughter nuclei. The two daughter cells thus receive exactly similar halves of the chromatin of the mother cell. Furthermore, as will be shown below, there is good evidence for the view that the chromosomes maintain an individuality of some sort, so that, since all the nuclei of the body arise by the repeated equational division of a single nucleus, all the somatic (body) cells are qualitatively similar in chromatin content: they contain representatives or descendants of each and SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 157 every chromosome present in the first cell of the series. The great theoretical importance of these facts will be apparent when we take up the subject of chromosome reduction, and the application of cytological phenomena to the problems of heredity. THE INDIVIDUALITY OF THE CHROMOSOME In later chapters the question of the significance of the nuclear struc- tures in heredity is to be considered. In connection with this question it is of the highest importance to determine whether or not the chromo- somes to which the reticulum gives rise in the prophase are in any real sense the same as those which went to make up the reticulum at the pre- ceding telophase. That they do preserve their identity as individuals through the resting stage, arise only by division, and maintain therefore a genetic continuity throughout the life cycle, was held by van Beneden (1883), Rabl (1885) and Boveri (1887, 1888, 1891) many years ago, and since that time the idea has received the support of a large number of investigators. We shall now briefly review some of the evidences which have led the majority of cytologists to the view that the chromosomes, "if . . . not actually persistent individuals, as Rabl and Boveri have maintained, . . . must at least be regarded as genetic homologues that are connected by some definite bond of individual continuity from gen- eration to generation of cells" (Wilson 1909). The Frequent Persistence of Visible Chromosome Limits in the Resting Reticulum. — In the foregoing description of the behavior of the chromosomes in mitosis it was pointed out that in rapidly dividing tissue the telophasic alveolation of the chromosomes and their anastomosis to form the reticulum often do not proceed far enough during the interphase to obliterate the boundaries between the chromosomes, which separate again in the ensuing prophase without having lost their visible identity. In such nuclei there can be little doubt that the autonomy of the chromo- some is preserved. In other cases, however, the telophasic transforma- tion of the chromosomes is more complete and the resulting reticulum reacts very weakly to the stains, so that the limits of the constituent chromosomes disappear from view completely. Many workers have therefore objected to the statement that here also the chromosomes are present as individuals, although invisible. Haecker (1902) and Boveri (1904) pointed out that this objection may be met by assuming that it is the achromatic framework of the alveolized chromosome, and not neces- sarily the basichromatic fluid held within it, that maintains a structural independence. This view had the support of the earlier observation made by Boveri (1887o, 1888a, 1891; also 1909) and confirmed by Herla (1893), that the chromosomes in the segmenting egg of Ascaris have a certain arrangement when they build up the nuclear reticulum in the telophase and reappear from the reticulum in the same position at the next prophase. 1 .I INTRODUCTION TO CYTOLOGY Special emphasis was laid upon this interpretation by Marechal (1904, 1907) as a result of his studies on the growth stage of animal oocytes. At this period in the development of the ovum the chromosomes assume a finely branched form (Fig. 86, C, D) and their ordinary staining capacity is lost completely. Although the chromatic fluid may flow from the reticulum to. the nucleolus and vice versa, and may periodically undergo chemical changes which radically alter its staining reactions, the achro- matic chromosomal substratum nevertheless maintains an uninterrupted structural continuity. Such a transfer of the basichromatic material from the persistent reticulum to the nucleolus during the telophase, and to the reticulum again during the succeeding prophase, has also been FIG. 55. — Some evidences for chromosome individuality. A, chromosomal vesicles in Brachystola magna; x-chromosome in vesicle at right. (After Sutton, 1902.) B, chromosomal vesicles in Fundulus embryo. X c. 1800. (After Richards, 1917.) C, chromomere vesicle (c) on chromosome of Chorthippus. X 1500. (After Wenrich, 1917.) D, prochromosomes in Pinguicula. X 4200. observed by Strasburger (1907) and Berghs (1909) in the somatic nuclei of Marsilia (Fig. 17, E}. The chromosome, as Marechal urges, is not simply a mass of chromatin, but rather "a structure periodically chro- matic;" hence the disappearance of stainable substance does not signify the loss of structural continuity on the part of the chromosome. The "chromosomal vesicles" (Fig. 55, A, B) observed by certain investigators constitute valuable evidence in this connection. In the spermatogonia of the grasshopper, Phrynotettix, for example, Wenrich 1916) has shown that each of the alveolizing chromosomes forms its own vesicle about it at telophase, the several vesicles joining to form a common nucleus. In some cases the boundaries between the vesicles do not entirely disappear during the resting stages, and at the next prophase SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 159 the chromatic material of each vesicle organizes in the form of a chromo- some. The same condition is found in the nuclei of Fundulus (Richards 1917), Crepidula (Conklin 1902), and certain fish hybrids (Pinney 1918). From this it is evident that the morphological identity of the chromo- somes has not been lost between mitoses, although a very different type of organization has been assumed. In Carex aquatilis Stout (1912) has found a peculiar condition. Here the very small spherical chromosomes, which maintain a serial arrange- ment, are visible in the resting state, and can be traced continuously through all stages of the somatic and germ cell divisions with the excep- tion of synizesis. The interpretations of Bonnevie (1908, 1911) and Dehorne (1911), according to whom the chromosomes persist through the resting stage as spirals or double spirals, have been mentioned in the description of mitosis. Prochromosomes. — Bodies known as prochromosomes have been described in the nuclei of a number of plants : in Thalictrum, Calycanthus, Campanula, Helleborus, Podophyllum, and Richardia by Overton (1905, 1909) ; in the Cruciferse by Laibach (1907) ; in Drosera and other forms by Rosenberg (1909); in Acer platanoides by Darling (1914); in Musa by Tischler (1910) ; and in a number of other forms. These prochromosomes appear as small chromatic masses in the reticulum (Fig. 55, D), and correspond approximately in number to the chromosomes of the species. They are generally looked upon as portions of chromosomes which have not undergone complete alveolation, and as centers about which the chromosomes again condense at the next prophase. This interpretation is in all probability a valid one in many of the described cases, but in others the significance of such chromatic masses is questionable. In Crepis virens de Smet (1914), in harmony with the conclusions of Miss Digby (1914), finds them to be accumulations of material formed during the resting stages. If such is the case they are to be regarded as karyosomes. Persistence of Parental Chromosome Groups After Fertilization. — In Chapter XII it will be shown that at fertilization there are brought together two sets of chromosomes, one set from each parent; and that in every nucleus of the resulting individual the chromosomes furnished by the two parents are present together, all of them dividing at every mitosis. When the chromosomes of the male parent are similar to those of the female parent it is usually impossible to distinguish them in the nuclei of the offspring. In a number of cases, however, such as Crepidula (Conklin 1897, 1901), Cyclops (Hsecker 1895; Ruckert 1895), and Crypto- branchus (Smith 1919) (Fig. 109), the two parental groups are distinguish- able on the mitotic spindle, and often at other stages, through several embryonal cell generations. It is in hybrids that this phenomenon is shown most strikingly. In hybrid fishes obtained by crossing Fundulus 160 INTRODUCTION TO CYTOLOGY with Menidia Moenkhaus (1904) was able to distinguish easily between the long (2.18 /*) chromosomes of Fundulus and the short (1 ju) ones of Menidia. Here, as in Crepidula and Cyclops, the paternal and maternal chromosomes form separate groups in the mitotic figure. A similar condition was seen by Tennent (1912) in hybrid echinoderms obtained by crossing in various ways Moira, Toxopneustes, and Arbacia. In the later cell-divisions the parental chromosomes mingle more or less, but are nevertheless distinguishable. In Fundulus X Ctenolabrus hybrids (Morris 1914; Richards 1916), as well as in the normally fertilized Cryptobranchus (Smith 1919), the chromatin contributions of the two parents are dis- tinguishable even in the resting nuclei. Size and Shape of Chromosomes.- — One of the most striking evidences favoring the theory of individuality has been found in those plants and animals which show constant differences in size and shape among the various members of each parental chromosome group, so that particular chromosomes are recognizable in the group appearing at each mitosis. Since each parent furnishes a set of chromosomes to the new individual, each kind of chromosome is present in duplicate in the nuclei of this indi- vidual: it is therefore customary to speak of them as being present in pairs, although at most stages of the life history there is ordinarily no actual spatial pairing. Since the description of the chromosomes of Brachystola by Sutton in 1902 (Fig. 101) the reported cases in which the different pairs of the chromosome complement possess different characteristic sizes and shapes have become increasingly numerous. This is notably true of insect cytology, as is evident in a review of the extensive researches of McClung (1905, 1914, 1917), Robertson (1916), Harman (1915), Carothers (1917), and many others. In the sea urchin, Echinus, Baltzer (1909) found that the 36 chromosomes have constant differences in length and shape, some being hooked and some horseshoe-shaped. In the flatworm, Gyrodactylus, (Gille 1914) there are six pairs, all different in length. In Ambystoma tigrinum Parmenter (1919) finds 14 pairs of graded sizes. In plants may be cited the cases of Crepis virens (Rosenberg 1909; de Smet 1914; M. Nawaschin 1915) (Fig. 56 bis, A), which has three pairs of different size; Vicia faba (Sharp 1914; Sakamura 1915), with five short pairs and one long pair (Fig. 56); and Najas (Tschernoyarow 1914), in which there are seven distinguishable pairs (Fig. 56 bis, B) . In Najas the smallest pair is attached to one of the larger pairs: Sakamura (1920) thinks that these together are really a single pair with pronounced constrictions. Not only may certain chromosomes be distinguished on the basis of comparative length, but in some cases there may be other characteristics which serve as marks of identification. In the chromosomes of many plants and animals there are pronounced constrictions in some of the SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 161 D FIG. 56. — The chromosome complement of Vicia faba. A, B, two successive sections of a mitotic figure in the root tip, showing together the 12 split chromosomes, 2 of them about twice as long as the other 10. C, cross section of the group of chromosomes at anaphase: each of the long chromosomes, being drawn pole- wards by the middle, shows both ends, making the number apparently 14. D, E, two successive sections of a heterotypic figure in the microsporocyte, showing the 6 bivalents; the large one is at the left. F, polar view of heterotypic mitosis at metaphase, showing the 6 bivalents. X 1400. (Original.) FIG. 56 bis. A, anaphase of somatic mitosis in Crepis virens, showing 2 long, 2 medium sized, and 2 short chromosomes passing to each pole. (After Rosenberg, 1920.) B, the chromosome complement in a somatic cell of Najas major, showing the 7 homologous pairs. (After Tschernoyarow, 1914.) 11 162 INTRODUCTION TO CYTOLOGY members of the group. It has been shown in certain instances that these constrictions have constant positions in the chromosome. A careful study of this phenomenon has been made by Sakamura (1915, 1920). In Viciafaba, for example; he finds that each of the two long chromosomes ("M-chromosomes") of the somatic group has two constant constrictions, one at the middle and one near the end ("m-constriction" and "e-con- striction") (Fig. 56, A). The m-constriction marks the point of attach- ment of the spindle fibers. There are also end-constrictions in 8 of the 10 short chromosomes. On the basis of the widespread occurrence of con- strictions in the chromosomes of both plants and animals Sakamura has interpreted a number of puzzling phenomena, such as the apparent vari- ation in chromosome number within the species (see below) and certain features of the reduction process (Chapter XI) Such regularly situated constrictions have also been demonstrated in Fritillaria tenella by S. Nawaschin (1914). Here they are present at the middle of the largest chromosomes, nearer one end in the medium-sized chromosomes, and close to the end of the smallest ones. In Crepis virens (M. Nawaschin 1915) there are constrictions near one end in two of the three chromosomes of the haploid group in the pollen grain, in four of the six chromosomes of the diploid group in the somatic cells, and in six of the nine chromosomes of the triploid group in the endosperm cells. Such a definiteness in the location of constrictions was also seen earlier by Agar (1912) in the chromosomes of the fish, Lepidosiren. Somewhat similar evidence has been brought forward by Wenrich (1916), who finds that the chromatic lumps, or chromomeres, have a striking constancy in position as well as in size in the chromosomes of Phrynotettix (Fig. 155). Wenrich (1917) also reports that the small "chromomere vesicles" attached to the chromosomes of- certain orthop- terans always appear at definite points along the chromosome (Fig. 55, C). It therefore appears that the chromosomes of a given group or comple- ment not only maintain a genetic continuity from cell to cell, but are also in some way qualitatively different from one another. They are conse- quently said to have a specificity as well as an individuality, or continuity. The relatively constant positions of the constrictions, chromatic lumps, and chromomere vesicles afford further visible evidences that the chrom- osome may possess some kind of lengthwise differentiation, a fact which, if clearly demonstrated, would be of the highest importance in connection with current views of the role of the chromosomes in heredity. (See Chapter XVII.) The significance of chromosome constrictions in this respect has been emphasized by Janssens (1909), S. Nawaschin (1915), and Sakamura (1920). Chromosome Number.1 — It was long ago noticed by Boveri, van 1 For lists of chromosome numbers in plants see Ishikawa (1916) and Tischler (1916). For the numbers in animals see Harvey (1916, 1920). SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 163 Beneden, and Strasburger that the number of chromosomes in any given species is relatively constant. It was largely upon this fact that the theory of chromosome individuality was originally based: the fact that the number of chromosomes appearing at every mitosis is almost invari- ably the same was taken to mean that the structural identity of the chromosomes is never lost. Certain observers (Fick 1905, 1909) have held that the apparent constancy in number is not due to a structural continuity or individuality of any sort, but rather to the fact that the successive nuclei have a relatively uniform amount of nuclear material, the chromosomes "crystallizing out" of this material in each prophase and going into solution at the close of mitosis. This idea was especially developed by Delia Valle (1909, 1912a&), who described the formation of chromosomes by the aggregation of fluid crystals during the prophase. These chromosomes he held to be in no sense morphologically continuous individuals, but only temporary chromatic accumulations which are in- constant in number and lose their identity in the telophase. Delia Valle's interpretation of chromosome formation has been criticized by a number of writers and his position shown to be untenable by Montgomery (1910), McClung (1917), and Parmenter (1919). Some of the experiments on echinoderm eggs with which Boveri (1895, 1902, 1903, 19046, 1905, 1907) and others supported the theory of chromo- some individuality may be briefly reviewed. Boveri found that if the number of chromosomes is increased or de- creased by artificial means the altered number appears at every mitosis thereafter, (a) An enucleate egg fragment may be entered by a sperma- tozoon, and may then develop into a larva with half the normal number of chromosomes in every cell. (&) In another experiment the unfertilized egg of a sea urchin was caused to undergo division by artificial means, after which a spermatozoon was allowed to enter one of the blastomeres (daughter cells) . A larva resulted in which one-half of the cells had regu- larly 18 chromosomes (half the normal number) while the other half had the normal 36. (c) Two spermatozoa occasionally fertilized one egg: the cells of the resulting larvae had 54 chromosomes, the triploid number. Abnormal mitotic figures were often formed in such dispermic eggs, bringing about an irregular distribution of the chromosomes. For ex- ample, a quadripolar spindle was produced, separating the 54 split chromo- somes (108 daughter chromosomes) into four groups, with 18, 22, 32, and 36 chromosomes respectively (Fig. 127 bis). The resulting abnormal larva ("pluteus") showed these four chromosome numbers in the cells of four different regions of its body. Boveri (1914) later suggested that malignant tumors might be due to such abnormal chromosome distri- bution, (d) The number of chromosomes was doubled by shaking the eggs while the chromosomes were split during the early stages of cell- division. In this manner larvae wore produced with 72 chromosomes, the 164 INTRODUCTION TO CYTOLOGY tetraploid number, in all of their cells, (e) In the threadworm, Ascaris megalocephala, fertilization of an egg of the variety bivalens (two chromo- somes) by a spermatozoon of the variety univalens (one chromosome) resulted in a larva with three chromosomes in all its cells, the chromosome contributed by the male parent being distinguishable from the other two (Boveri 1888a; Herla 1893; Zoja 1895). Results such as the above led Boveri to the conclusion that the number of chromosomes arising from the reticulum in prophase is directly and exclusively dependent upon the number that went to make it up in the preceding telophase. If a nucleus is reconstructed in the telophase by an abnormal number of chromosomes as the result of a disturbance of the 10 9 FIG. 57.— The chromosome complement of Hesperotettix viridis. A, the 12 bivalent chromosomes of the spermatocyte, including the accessory chromo- some (No. 4.) B, complement from another individual, showing two "multiple chromo- somes." Nos. 11 and 12 have united temporarily, as have also Nos. 4 and 9. X 1800. (After McClung, 1917.) mitotic process, the altered number invariably appears in the succeeding prophase: if extra chromosomes are present they are not eliminated in any way during the resting stages, and if chromosomes have been lost during abnormal mitosis they are not replaced. These conclusions have been strikingly confirmed by Sakamura's (1920) work on cells subjected to the influence of chloral hydrate and other agencies causing aberrant chromosome behavior. Variations in Number. — Although the number of chromosomes in a given species is on the whole remarkably constant, departures from nor- mal numbers are occasionally observed. Strasburger (1905) believed that the number, though determined by heredity, is not so rigidly fixed that all variation in the vegetative cells is excluded ; only in the reproduc- tive cells did he hold constancy in number to be necessary. Much light SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 165 has been recently thrown upon such apparent variations in number by McClung (1917) and Miss Holt (1917) in their researches on multiple chromosomes and chromosome complexes. McClung finds in his analysis of the chromosome groups of the orthopterans Hesperotettix and Mermiria that temporary associations often occur between various members of a group, with the resulting forma- tion of "multiple chromosomes" and a consequent decrease in the appar- ent number. In Hesperotettix, for instance, the cells normally have 12 pairs of chromosomes, but because of the formation of such multiple chromosomes individuals with apparently 11, 10, or 9 pairs are frequently found (Fig. 57). For a given individual the number so formed is exactly constant, since the members of a multiple remain together in all the cells of the body; but for the species it is variable within certain limits, owing to the varying numbers of chromosomes which may become involved in such multiple combinations. In all cases the full number of chromosome pairs is present, but some of them are so combined that there is an appar- ent, though not actual, variation in the number. A similar condition is found in other forms by Robertson (1916). In Culex there are three pairs of chromosomes in the somatic cells. During a certain stage in the insect's metamorphosis it has been shown by Miss Holt (1917) that the chromosomes may split repeatedly, giving cells with much larger numbers — up to 72 in some cases. These larger numbers, however, are nearly always multiples of three, indicating that the subdivision of the chromosomes is an orderly process. The daughter chromosomes, moreover, that are formed by the subdivision of each of the original six, remain more or less closely associated as a "multiple complex," which behaves as a single individual in mitosis. It therefore appears that the three pairs of chromosomes "are made up of quite distinct individuals differing from each other to such a degree that chromatin split from one cannot associate itself with that from another pair. . . . Chromosome individuality, alone, can account for these conditions." Somewhat similar evidence has been brought forward by Hance (1917, 1918a6). Hance finds that the chromosome number in the spermatogo- nia of the pig is regularly 40, whereas in the somatic cells it varies from 40 to 57. Similarly in (Enothera scintillans, which has 15 chromosomes in its microsporocytes, there may be from 15 to 21 chromosomes in the somatic cells. Measurements of the members of the various chromosome groups show that the larger numbers are due to a fragmentation, prob- ably of the larger chromosomes, in the somatic cells. Such fragments divide normally, and it appears probable that the fragments of a single original chromosome are held together by colorless portions and behave as a unit, much as do the multiple complexes of Culex. Sakamura (1920) believes that the chief reason for frequently reported 166 INTRODUCTION TO CYTOLOGY inconstancies in chromosome number is to be found in the chromosome constrictions, which under certain conditions become especially pro- nounced and temporarily divide one or more of the chromosomes of the group into loosely connected smaller parts. This suggestion, which Sakamura supports with much direct evidence, is probably one of the most fruitful which has been made in this connection. The theory of chromosome individuality is believed by McClung and Hance to be strengthened, rather than weakened, by such instances of numerical variation as those described above. McClung emphasizes the point that the composition of a given chromosome can be fully under- stood only if something is known of its genetic history, for what appears as a chromosome may often be either an aggregation of two or three chromosomes, or, on the other hand, only a portion of the true chromo- some individual. How widely this interpretation may be applicable to other reported cases of numerical variation and to chromosome structure in general cannot at present be stated, but it promises to lead to signifi- cant results. Discussion and Conclusions. — The author's views on the subject of the individuality of the chromosomes can be most effectively stated in the words of McClung (1917): " . . . the practical matter before us is to decide whether the metaphase chromo- somes of two cells are individually identical organic members of a series because they were produced by the observed reproduction of a similar series of the parent cell, or whether the resemblance is independent of this genetic relation and due to chance association of indifferent materials, or to a reconstituting action of the cell as a whole." "If it were possible for chromosomes to reproduce themselves and still pre- serve their physical configuration unchanged, there would probably be little question of their continuity and individuality — the demonstration would be self- evident. But it happens that the necessities of the case require that each newly produced chromosome should take part in the formation of a new nucleus, through whose activities the cell as a whole and each chromosome, individually, is enabled to restore the volume diminished by the act of division. During this process the outlines of the chromosomes become materially changed and in their extreme diffusion can no longer be traced in many cases. Because of our limitations in observational power they appear to be lost as separate individuals and we are thus deprived of the simple test of observed continuity. Later, in the same cell, there reappears a series of chromosomes severally like those which seemed to disappear during the period of metabolic activity. We confront two alternative explanations for this reintegration of the chromosomes; either they actually persist as discrete units of extremely variable form, or they are entirely lost as individual entities and are reconstituted by some extrinsic agency. There is no other possible explanation and we must weigh the facts for one or the other of the alternatives. All the facts which indicate order and system in chromosome features speak for the former, those which demonstrate variability and indefiniteness, for the SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 167 hitter. The case for discontinuity is strongest in the absence of any chromosome order, and becomes progressively weaker with the establishment of definiteness and precision in form and behavior." "So far as I can see there is no half way ground between the assumption that the chromosomes are definite, self-perpetuating organic structures and the other which presents them as mere incidental products of cellular action. According to one view individual chromosomes are descendents of like elements and possess certain qualities and behavior because of their material descent, the visible mechanism for which is the process of mitosis : according to the other any similari- ties that may exist in the complexes are the result of chance aggregations of non- specific materials. It is a choice between organization and non-organization in the last analysis, at least in terms of cellular structures. To attempt the sub- stitution of a conception of molecular organization, which is beyond the ex- perience of the biologist and which exceeds the present powers of the chemist to analyse, is to cast aside all hope of solving the problem of cellular action, because it is necessary to understand, not only the physical and chemical phenomena involved, but also their different forms in the various parts of the cell." "That the chromosomes do not maintain a compact and easily recognizable form in the interval between mitoses is accepted by many . . . biologists as proof that they no longer exist as entities. All the other manifold indications of char- acter and continuity do not weigh against this apparent loss of identity. Doubt- less it would be more satisfying if we could at all times perceive the chromosomes in unchanging form in all stages of cellular activity, but why we should demand this condition as a test of individuality in the chromosomes when we unhesitat- ingly admit the unity of the organism in all the varied changes of its develop- ment from a single cell, through such complexities of change and metamorphosis as to give rise to doubts of even the phyletic position of some stages, it is difficult to see. Being organic, the chromosomes must change their form, they must suffer division of their substance and they are obliged to restore this loss through meta- bolic changes. Since these changes of substance take place at surface contacts there is an obvious advantage in increased superficies and, in common with other, larger structural elements, the chromosomes become extended and their sub- stances are diffused. In this state their boundaries may not be well defined and this circumstance has been seized upon as a disproof of their continuity." "Since it is not possible to observe directly the action of the chromosome we are obliged to make use of indirect evidence, seeking parallels between elements of structure and action in the chromosomes, and the mass effect of cellular action as exhibited in the so-called body characters. Such a method is justified by all other experience in tracing relations between structure and function in organisms, and while it apparently resolves the organism into parts of greater or less in- dependence, has given us our best conceptions of it as a whole." "What is postulated ... is that the chromosomes are self-perpetuating entities with individual peculiarities of form and function to identify them. Characteristics of form and behavior we see; certain very definite parallels be- tween these and the manifestations of somatic characters exist beyond question ; provision for the perpetuation of the organic unity of the individual chromosomes is found in the process of mitosis; the actual direct result of its operation appears in the uniform conditions of the complex in the individual animal; the extension 168 INTRODUCTION TO CYTOLOGY of this beyond the organism to the group and the means for it in the phenomena of maturation and fertilization are easily established by observation; the age old existence of all these circumstances is revealed by the near approach to uniformity in the chromosome complex of the multitude of species of unnumbered individuals constituting a family. And yet, in the face of this overwhelming mass of evidence indicative of order, system and specific chromosome organiza- tion, some conceive only the action of ordinary chemical forces, or the chance association of indifferent substances, while others, over impressed with the thought of a general coordinating force in the organism, deny significance to the orderly play of its cellular parts." "It is my belief that the observed act of reproduction, by which the organiza- tion of the chromosomes is materially transmitted in each mitosis, together with all facts indicating extensive distribution of given conditions, definiteness of organization, uniformity of behavior and consistence of deviation from the normal, are so many clear indications of the individual character of the chromo- somes. Transmutation of form, even to an extreme degree, can not be held as a valid argument against a persistent individuality. A consideration of the criteria applied to larger organic aggregates well supports this view. Such objects are said to possess individuality when they exhibit a more or less definite unity which is persistent and characterized by peculiarities of form and function. Most clearly defined is this individuality when it may be perpetuated through some form of reproduction to find expression in new units of similar character. The term does not connote unchangeability, and there may be fusions with more or less loss of physical delimitations, followed by separation, even after exchange of substances. The test of individuality is material continuity, but it does not necessarily involve complete or entirely persistent contiguity.1 An organism may bud off new individuals similar to itself, the substance of its body differs from time to time, movements of parts take place, fragmentation occurs, extreme attenuation or extension of substance is found, even separation and recombina- tion of parts may happen and yet the individual maintains itself. What it may have been in the past, what its possibilities of future development are, what potentialities of multiplied individuality it suppresses do not affect the reality of its individuality. It is, as Huxley says, 'a single thing of a given kind.' If one such thing divides into two, there are two individuals; if two unite into one indistinguishably there is a single individual; if a fusion of two things occurs in part, without loss of physical configuration, there are still two individuals in existence. Only when the substance of one thing disappears or becomes in- corporated integrally into the organization of another does its individuality depart. If all these variations of physical state may occur in the history of an organism without sacrifice of individuality, there can be no reason for urging them against a conception of the individuality of the self-perpetuating chromosomes." Bibliography 8 Somatic Mitosis. Individuality of Chromosomes AGAR, W. E. 1912. Transverse segmentation and internal differentiation of chromosomes. Quar. Jour. Micr. Sci. 58: 285-298. pis. 12, 13. 1 Italics ours. SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 169 BALBIANI, E. G. 1876. Sur les phenomenes de la division du noyau cellulaire. Compt. Rend. Acad. Sci. Paris 83 : 831-834. BALTZER, F. 1909. Die Chromosomen von Strongylocentrotus lividus und Echinus microtuberculatus. Arch. Zellf. 2: 549-632. pis. 37, 38. fig. 25. VAN BENEDEN, E. 1876. Recherches sur les Dicyemides, survivante actuelles d'un embranchement Mesozoaires. Bull. Acad. Roy. Belg. 41: 1160-1205; 42: 35-97. pis. 3. 1883. Recherches sur la maturation de 1'oeuf, la fecondation et la division cellu- laire. Arch, de Biol. 4. 1884. (van Beneden et Julin). La spermatogenese chez I'Ascaride megalocephale. Bull. Acad. Roy. Belg. Ill 7: 312-342. 1887. (van Beneden et Neyt). Nouvelles recherches sur la fecondation et la division mitosique chez I'Ascaride megalocephale. Ibid. Ill 14: 215-295. pi. 1. BERGHS, J. 1909. Les cineses somatiques dans le Marsilia. La Cellule 25: 73-84. Ipl. BONNEVIE, K. 1908. Chromosomenstudien. I. Arch. Zellf. 1: 450-514. pis. 11-15. 1911. Chromosomenstudien. II. Chromatinreifung in Allium cepa. Ibid. 6: 190-253. pis. 10-13 BOVERI, T. 1887a. Ueber die Befruchtung der Eier von Ascaris megaiocephala. Sitzber. Gesell. Morph. Phys. Miinchen 3. 18876. Ueber den Anteil des Spermatozoon an der Teilung des Eies. Ibid. 1887c. Ueber Differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris megaiocephala. Anat. Anz. 2:688-693. 1887d. Zellen-Studien. I. Die Bildung der Richtungskorper bei Ascaris megaio- cephala und .Ascaris tumbricoides. Jen. Zeitschr. 21: 423-515. pis. 25-28. 1888a. Zellen-Studien. II. Ibid. 22: 685-882. pis. 19-23. 18886. Ueber partielle Befruchtung. Sitzber. Ges. Morph. Phys. Miinchen 4. 1890. Zellen-Studien. III. Ueber das Verhalten der chromatischen Kernsubstanz bei der Bildung der Richtungskorper und bei der Befruchtung. Jen. Zeitschr. 24: 314-401. pis. 11-13. 1891. Befruchtung. Ergebn. d. Anat. u. Entw. 1 : 386-485. figs. 15. 1902. Ueber mehrpolige Mitosen als Mittel zur Analyse des Zellkerns. Verhandl. Phys.-Med. Ges. Wiirzburg 36. 1904a. Ergebnisse iiber die Konstitution der chromatischen Kernsubstanz. Jena. 19046. (Boveri und Stevens.) Ueber die Entwicklung dispermer Ascariseier. Zool. Anz. 27 : 406-417. 1907. Zellen-Studien. VI. Die Entwicklung dispermer Seeigel-Eier. Jena. 1909. Die Blastomerenkerne von Ascaris megaiocephala und die Theorie der Chromosomenindividualitat. Arch. Zellf. 3: 181-268. pis. 7-11. 1914. Zur Frage der Entstehung maligner Tumoren. BRAUER, A. 1893. Zur Kenntniss der Spermatogenese von Ascaris megaiocephala. Arch. Mikr. Anat. 42: 153-212. pis. 11-13. BUTSCHLI, O. 1875a. Vorl. Mitteilung iiber Untersuchungen betreffend die ersten Entwicklungsvorgange im befruchteten Ei von Nematoden und Schnecken. Zeit. Wiss. Zool. 26: 201. 18756. Vorl. Mitteilung einiger Resultate von Studien iiber Conjugation der Inf usorien und die Zellteilung. Ibid. 25 : 426. CAROTHERS, E. E. 1917. The segregation and recombination of homologous chromosomes as found in two genera of Acrididse (Orthoptera). Jour. Morph. 28: 445-521. pis. 14. figs. 5. CONKLIN, E. G. 1897. The embryology of Crepidulu. Jour. Morph. 13: 1-226. pis. 9. 1919-1920. The mechanism of evolution in the light of heredity and development. Sci. Mo. 9: 481-505; 10: 52-62, 170-182, 269-291. 388-404, 498-515. 170 INTRODUCTION TO CYTOLOGY DARLING, C. A. 1914. Prochromosomes in synapsis. Science 41 : 182-183. DEHORNE, A. 1911. Recherches sur la division de la cellule. 1. La duplicisnir constant du chromosome somatique chez Salamandra maculoaa Lour, et chez Attium cepa L. Arch. Zellf. 6 : 613-639. pis. 35, 36. figs. 2. DELLA VALLE, P. 1909. L'organizzazione della cromatina studiata mediante il numero dei cromosomi. Archivio Zool. Ital. 4: 1-177. 1 pi. 1912a. La morphologia della cromatina dal punto di vista fisico. Ibid. 6 : 37-321. pis. 4, 5. figs. 75. diagrams 9. 19126. Die morphologie des Zellkerns und die Physik der Kolloide. Zeitschr. f. Chemie u. Indus, d. Kolloide 12 : 12-16. DIGBY, L. 1910. The somatic, premeiotic, and meiotic nuclear divisions of Gallonia candicans. Ann. Bot. 24: 727-757. pis. 59-63. 1914. A critical study of the cytology of Crepis virens. Arch. Zellf. 12 : 97-146. pis. 8-10. 1919. On the archesporial and meiotic mitoses of Osmunda. Ann. Bot. 33: 135-172. pis. 8-12. 1 fig. EISEN, G. 1899. .The chromoplasts and the chromioles. Bot. Cent. 19: 130-136. figs. 5. 1900. The spermatogenesis of Batrachoseps, polymorphous cells. Jour. Morph. 17: 1-117. pis. 1-14. FARMER, J. B. and SHOVE, D. 1905. On the structure and development of the somatic and heterotype chromosomes of Tradescantia virginica. Quar. Jour. Micr. Sci. 48 : 559-569. pis. 42, 43. FICK, R. 1905. Betrachtungen tiber die Chromosomen, ihre Individualitat, Reduk- tion und Vererbung. Arch. Anat. u. Phys. Suppl. 179-228. 1909. Bemerkungen zu Boveris Aufsatz liber die Blastomerenkerne von Ascaris und die Theorie der Chromosomen. Arch. Zellf. 3: 521-524. FLEMMING, W. 1880. Beitrage zur Kenntniss der Zelle und ihre Lebenserschein- ungen. II. Arch. Mikr. Anat. 19. 1882. Zellsubstanz, Kern und Kernteilung. Leipzig. 1891-1897. Zelle. Ergeb. d. Anat. u. Entw. 1-7. (Review.) FOL, H. 1873. Die erste Entwicklung des Geryoniden-Eies. Jen. Zeitschr. 7. 1891. Die "Centrenquadrille," ein neue Episode aus der Befruchtungsgeschichte. Anat. Anz. 6: 266-274. figs. 10. FRASER, H. C. I. and SNELL, J. 1911. The vegetative divisions in Vicia faba. Ann. Bot. 26 : 845-855. pis. 62, 63. FRASER, H. C. I. 1914. The behavior of the chromatin in the meiotic divisions of Vicia faba. Ann. Bot. 28 : 633-642. pis. 43, 44. GATES, R. R. 1912. Somatic mitoses in (Enothera. Ann. Bot. : 993-1010. pi. 86. GILLE. K. 1914. Untersuchungen iiber die Eireifung, Befruchtung und Zellteilung von Gyrodaclylus elegans var. Nordmann. Arch. Zellf. 12: 415-456. pis. 32-34. GREGOIRE, V. 1906. La structure de Tenement chromosomique au repos et en division dans les cellules ve'getales. La Cellule 23: 311-353. pis. 1, 2. 1912. Les phenomenes de la me'taphase et de 1'anaphase dans la caryocinese somatique. A propos d'une interpretation nouvelle. Ann. Soc. Sci. Bruxelles 34. GREGOIRE, V. et WYGAERTS, A. 1903. La reconstitution du noyau et la formation des chromosomes dans les cineses somatiques. La Cellule 21 : 7-67. pis. 2. 1907. La formation des gemini h£terotypiques dans les vege"taux. Ibid. 24: 369-420. pis. 2. HAECKER, V. 1896. Ueber die Selbstandigkeit der vaterlichen und mutterlichen Kernbestandteile wahrend der Embryonalentwicklung von Cyclops. Arch. Mikr. Anat. 46: 579-617. pis. 28-30. SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 171 l'.K)2. Ueber das Schicksal dor elterlichen und grosselterlichen Kernanteile. Jcti. Zeitschr. 37: 299-400. pis. 17-20. figs. 16. HANCE, It. T. 1917. The diploid chromosome complexes of the pig (»S'«.« scrofa) and their variation. Jour. Morph. 30. 1918a. Variations in the number of somatic chromosomes of (Enothera scinlillans de Vries. Genetics 3 : 225-275. pis. 7. 19186. Variations in somatic chromosomes. Biol. Bull. 35. HARMAN, M. T. 1915. Spermatogenesis in Paratettix. Science 41 : 440. HARPER, R. A. 1919. The structure of protoplasm. Am. Jour. Bot. 6: 273-300. HARVEY, E. B. 1916, 1920. A review of the chromosome numbers in the Metazoa. Jour. Morph. 28: 1-63; 34: 1-68. HEIDENHAIN, M. 1894. Neue Untersuchungen iiber die Centralkorper und ihre Beziehungen zum Kern und Zellprotoplasma. Arch. Mikr. Anat. 43: 423-758. pis. 25-31. HERLA, V. 1893. fitude des variations de la mitose chez Y Ascaride megalocephale. Arch, de Biol. 13 : 423-520. pis. 15-19. HERMANN, J. 1891. Beitrage zur Lehre von der Entstehung der karyokinetischen Spindel. Arch. Mikr. Anat. 37: 569-586. pi. 31. 2 figs. HERTWIG, O. 1875. Beitrage zur Kenntniss der Bildung, Befruchtung, und Thei- lung des tierischen Eies. I. Morph. Jahrb. 1; see also 2-4. HEUSER, E. 1884. Beobachtungen iiber Zellkernteilung. Bot. Centr. 17: 27-32, 57-59, 85-95, 117-128, 154-157. pis. 2. HOLT, C. M. 1917. Multiple complexes in the alimentary tract of Culex pipiens. Jour. Morph. 29: 607-627. pis. 4. ISHIKAWA, M. 1916. A list of the number of chromosomes. Bot. Mag. Tokyo 30:404-448. (Plants.) JANSSENS, F. A. et WILLEMS, J. 1909. Spermatogenese dans les Batrachiens. IV. La Cellule 26: 151-178. pis. 2. KOWALEVSKY, A. 1871. Embryologischen Studien an Wurmern und Arthropoden. Mem. Acad. Imp. Sci. de St. Petersburg VII 16 : 13. LAIBACH, F. 1907. Zur Frage nach der Individualitat der Chromosomen in Pflan- zenreich. Beih. Bot. Centralbl. 22 : 191-210. pi. 8. LEWIS, W. H. and LEWIS, M. R. 1917. The duration of the various phases of mitosis in the mesenchyme cells of tissue cultures. Anat. Record 13 : 359-368. LUNDEGARDH, H. 1910. Ueber Kernteilung in den Wurzelspitzen von Allium cepa und Vicia faba. Svensk. Bot. Tidskr. 4: 174-196. figs. 11. 1912a. Die Kernteilung bei hoheren Organismen nach Untersuchungen an lebenden Material. Jahrb. Wiss. Bot. 51: 236-282. pi. 2. figs. 8. 19126. Das Karyotin im Ruhekern und sein Verhalten bei der Bildung und Auflo- sung der Chromosomen. Arch. Zellf . 9 : 205-330. pis. 17-19. figs. 9. 1912c. Chromosomen, Nukleolen, und die Veranderung im Protoplasma bei der Karyokinese. Beitr. Biol. Pflanzen 11: 373-542. pis. 11-14. MARECHAL, J. 1904. Ueber die morphologische Entwicklung der Chromosomen im Keimblaschen des Selachiereies. Anat. Anz. 25 : 383-398. figs. 25. 1907. Sur 1'ovogenese des Selachiens. La Cellule 24 : 1-239. pis. 10. MARTINS MANO, TH. 1904. Nucleole et Chromosomes. La Cellule 22: 57-76. pis. 1-4. McCLUNG, C. E. 1905. The chromosome complex of orthopteran sperm atocytes. Biol. Bull. 9 : 304-340. figs. 21. 1914. A comparative study of the chromosomes in orthopteran Spermatogenesis. Jour. Morph. 25: 651-749. pis. 1-10. 1917. The multiple chromosomes of Hesperotettix and Mermiria (Orthoptera). Ibid. 29 : 519-604. pis. 8. 172 INTRODUCTION TO CYTOLOGY MERKIMAN, M. L. 1904. Vegetative cell division in Allium. Bot. Gaz. 37: 178- 207. pis. 11-13. MEVES, FR. 1896, 1898. Zelltheihing. Ergeb. Anat. Entw. 6: 285-390; 8: 430- 542. (Review.) 1911. Chromosomenlangen bei Salamandra, nebst Bemerkungen zur Individual- itatstheorie der Chromosoraen. Arch. Mikr. Anat. 77: 273-300. pis. 11, 12. MOENKHAUS, W. J. 1904. The development of the hybrids between Fundulus heteroditus and Menidia notatus, with especial reference to the behavior of maternal and paternal chromosomes. Jour. Anat. 3: 29-65. MONTGOMERY, T. H. 1910. On the dimegalous sperm and chromosomal variation in Euschistus, with reference to chromosomal continuity. Arch. Zellf. 5: 121- 145. pis. 9, 10. MORRIS, M. 1914. The behavior of the chromatin in hybrids between Fundidus and CtenoLabrus. Jour. Exp. Zool. 16: 501-522. pis. 5. MULLER, H. A. CL. 1911. Kernstudien an Pflanzen I u. II. Arch. Zellf. 8: 1-51. pis. 1, 2. NAWASCHIN, M. 1915. " Haploide, diploide, und triploide Kerne von Crepis virens, Vill." (Russian; cited by Sakamura, 1915, 1920.) NAWASCHIN, S. 1914. "Sur quelques indices de 1'organisation du chromosome." (Russian.) NEMEC, B. 1910. Das Problem der Befruchtungsvorgange und andere Zytologische Fragen. pp. 532. pis. 5. Berlin. OVERTON, J. B. 1905. Ueber Reduktionsteilung in den Pollenmutterzellen einiger Dikotylen. Jahrb. Wiss. Bot. 42 : 121-153. pis. 6, 7. 1909. On the organization of the nuclei in the pollen mother cells of certain plants, with especial reference to the permanence of the chromosomes. Ann. Bot. 23:19-61. pis. 3. PICARD, M. 1913. A bibliography of works on meiosis and somatic mitosis in the angiosperms. Bull. Torr. Bot. Club 40 : 575-590. PINNEY, E. 1918. A study of the relation of the behavior of the chromatin to heredity and development in teleost hybrids. Jour. Morph. 31: 225-292. figs. 88. PFITZNER, W. 1881. Ueber den feineren Bau der bei der Zellteilung auftretenden fadenformigen Differenziemng des Zellkerns. Morph. Jahrb. 7: 289-311. figs. 2. RABL, C. 1885. Ueber Zelltheilung. Ibid. 10: 214-330. pis. 7-13. figs. 5. REMAK, R. 1841. Ueber Theilung rother Blutzellen beim Embryo. Arch. Anat. Physiol. 1858. 1852. Ueber extrazellulare Entstehung thierischer Zellen und iiber Vermehrung derselben durch Theilung. Ibid. RICHARDS, A. 1916. Chromosome individuality in fish eggs. Science 43 : 178. 1917. The history of the chromosomal vesicles in Fundulus and the theory of genetic continuity of chromosomes." Biol. Bull. 32: 249-290. pis. 4. ROBERTSON, W. R. B. 1916. Chromosome studies. I. Jour. Morph. 27: 179- 332. pis. 1-26. ROSENBERG, O. 1909a. Zur Kenntniss von den Tetradenteilung der Compositen. Svensk. Bot. Tidskr. 3: 64-77. pi. 1. 19096. Ueber den Bau des Ruhekerns. Ibid. 3: 163-173. pi. 5. fig. 1. 1918. Chromosomenzahlen und Chromosomendimensionen in der Gattung Crepis. Ark. f. Bot. 15: 1-16. figs. 6. 1920. Weitere Untersuchungen iiber die Chromosomenverhaltnisse in Crepis. Svensk Bot. Tidskr. 14 : 320-326. figs. 5. SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 173 Roux, W. 1883. Ueber die Bedeutung der Kernteilungsfiguren. Leipzig. RticKERT, J. 1895. Ueber das Selbstandigbleiben der vaterlichen und miitterlichen Kernsubstanz wahrend der ersten Entwicklung des befruchteten Cyciops-Eies. Arch. Mikr. Anat. 46 : 339-369. pis. 21, 22. SAKAMURA, T. 1914. Ueber die Kernteilung bei Vicia cracca L. Bot. Mag. Tokyo 28: 131-147. pi. 2. 1915. Ueber die Einschniirung der Chromosomen bei Vicia faba L. (Vorl. Mitt.). Ibid 29 : 287-300. pi. 13. figs. 12. 1920. Experimentelle Studien iiber die Zell- und Kernteilung mit besonderer Riicksicht auf Form, Grosse, und Zahl der Chromosomen. Jour. Coll. Sci. Imp. Univ. Tokyo 39: pp. 221. pis. 7. SCHLEICHER, W. 1878. Die Knorpelzelltheilung. Arch. Mikr. Anat. 16: 248- 300. pis. 12-14. 1879. (Centr. Med. Wiss., Berlin, 1878.) SCHNEIDER, A. 1873. Untersuchungen iiber Platelminthen. Jahrb. Oberhess. Gesell. Natur-Heilk. 14. Giessen. 1883. Das Ei und seine Befruchtung. Breslau. SHARP, L. W. 1913. Somatic chromosomes in Vicia. La Cellule 29: 297-331. pis. 2. 1914. Maturation in Vicia. (Prelim, note.) Bot. Gaz. 57: 531. 1920. Somatic chromosomes in Tradescantia. Am. Jour. Bot. 7: 341-354. pis. 22, 23. DE SMET, E 1914. Chromosomes, prochromosomes, et nucleole dans quelques Dicotylees. La Cellule 29: 335-377. pis. 3. SMITH, B. G. 1919. The individuality of the germ nuclei during the cleavage of the egg of Cryptobranchus alleghanwnsis. Biol. Bull. 37 : 246-287. pis. 9. STOMPS, T. J. 1910. Kerndeeling en synapsis bij Spinacea oleracea. (German transl. in Biol. Centralbl. 31 : 257-320. pis. 3.) STOUT, A. B. 1912. The individuality of the chromosomes and their serial arrange- ment in Carex aquatilis. Arch. Zellf. 9: 114-140. pis. 11, 12. STRASBURGER, E. 1875. Ueber Zellbildung und Zelltheilung. Jena. 1884. Die Controversen der indirekten Kernteilung. Arch. Mikr. Anat. 23 : 246-304. pis. 13, 14. 1888. Ueber Kern- und Zellteilung im Pflanzenreich, nebst einem Anhang tiber Befruchtung. Hist. Beitr. 1. pp. 258. pis. 3. 1900. Ueber Reduktionsteilung, Spindelbildung, Centrosomen und Cilienbildner im Pflanzenreich. Hist. Beitr. 6. pp. 124. pis. 4. 1905. Typische und allotypische Kernteilung. Jahrb. Wiss. Bot. 42: 1-71. pi. 1. 1907o. Apogamie bei Marsilia. Flora 97: 123-191. pis. 3-8. 19076. Ueber die Individualitat der Chromosomen und die Fropfhybriden- Frage. Jahrb. wiss. Bot. 44: 482-555. pis. 5-7. SUTTON, W. S. 1902. On the morphology of the chromosome group in Brachystola magna. Biol. Bull. 4: 24-39. figs. 11. TENNENT, D. H. 1912. Studies in cytology. 1, 11. Jour. Exp. Zool. 12: 391-411. •figs. 21. TISCHLER, G. 1910. Untersuchungen iiber die Entwicklung des Bananen-Pollens. I. Arch. f. Zellf. 6. 1916. Chromosomenzahl, -Form und -Individualitat im Pflanzenreich. Prog. Rei Bot. 6 : 164-284. (Review and list of numbers.) TSCHERNOYAROW, M. 1914. Ueber die Chromosomenzahl in besonders beschaffene Chromosomen im Zellkerne von Najas major. Ber. Deu. Bot. Ges. 32: 411-416. pi. 10. 174 INTRODUCTION TO CYTOLOGY WALDEYER, W. 1888. Ueber Karyokinese und ihre Beziehung zu den Befrucht- ungsvorgangen. Arch. Mikr. Anat. 32: 1-122. figs. 14. (English transl. in Quar. Jour. Micr. Sci. 30: 159-281. 1889.) WENBICH, D. H. 1916. The spermatogenesis of Phrynotellix magnus with special reterence to synapsis and the individuality of the chromosomes. Bull. Mus. Comp. Zool. Harvard Coll. 60 : 55-136. 10 pis. 1917. Synapsis and chromosome organization in Chorthippus (Stendbothrus) curtipennis and Trimerotropis suffusa (Orthoptera). Jour. Morph. 29: 471-516. pis. 1-3. WILSON, E. B. 1900. The Cell in Development and Inheritance. 2d ed. 1909. The cell in relation to heredity and evolution. In "Fifty Years of Darwin- ism." Holt & Co. 1912a. Some aspects of cytology in relation to the study of genetics. Am. Nat 46:57-67. 19126. Studies on chromosomes. VIII. Jour. Exp. Zool. 13 : 345-449. pis. 9. ZIMMERMANN, A. 1893. Sammel-Referate 6, 7. Beih. Bot. Cent. 3: 342-354, 401 -436. ZOJA, R. 1895. Sulla indipendenza della cromatina paterna e materna nel nucleo delle cellule embrionali. Anat. Anz. 11: 289-293. figs. 3. ZUE STRASSEN, O. L. 1898. Ueber Riesenbildung bei Ascam-Eieren. Arch. Entw. 7: 642-676. pis. 16, 17. figs. 9. CHAPTER IX THE ACHROMATIC FIGURE, CYTOKINESIS, AND THE CELL WALL THE ACHROMATIC FIGURE The spindle fibers and asters about the centrosomes (when these are present) are collectively termed the achromatic figure, in contradistinction to the chromatic figure, or chromosomes. Compared with the chromo- somes the achromatic figure is relatively little understood, which makes it a very unsatisfactory subject for discussion. We shall first describe the achromatic figure in its more common forms, and after mentioning certain theories which have been propounded to explain its origin and nature we shall briefly review a few of the suggestions which have been made on the subject of the mechanism of mitosis. In Higher Plants. — In somatic mitosis in higher plants the achromatic figure is devoid of centrosomes and asters. Ordinarily it arises and be- haves as follows: While the prophasic changes are taking place within the nucleus the first indications of spindle formation appear in the cyto- plasm in the immediate vicinity of the nucleus. At the two sides of the latter, in the general position of the future spindle poles, there are de- veloped two masses of more or less hyaline material, usually called "kino- plasmic caps." In these two polar caps delicate fibrils soon appear, as if by a process of condensation (Fig. 58, A, B). The nucleus commonly shrinks at this time, while the fibrous areas increase in size and together form a more definitely spindle-shaped figure. After the nuclear mem- brane has shrunken more closely about the chromosomes it goes into solution and the ingrowing fibers attach themselves to the longitudinally split chromosomes. In many cases the membrane disappears without shrinking, the fibers growing considerably in length to reach the chromo- somes. The latter quickly become regularly arranged in the equatorial plane preparatory to their separation (Chapter VIII). The mitotic figure is now established (Fig. 48). The many fibers composing the spindle may focus at a single sharp point at each pole, or they may end indefinitely without converging to a point, forming in the latter case a broad-poled figure which in extreme cases may be as wide at the poles as at the equator (Fig. 74, D). Some of the fibers extend from the poles to the chromosomes, to which they are attached, while others pass through from one pole to the other without being so attached: these 175 176 INTRODUCTION TO CYTOLOGY two sets of fibers are known respectively as mantle fibers and connecting fibers. The latter are also collectively termed the central spindle. It is during the anaphases and telophases that the connecting fibers become most evident; in mitotic figures with many chromosomes it may be impossible to see them at metaphase. At the beginning of the telo- phase they may form a bundle no greater in diameter than the daughter chromosome groups, but as the daughter nuclei reorganize the fibers commonly bend outward at the middle, forming a barrel-shaped phrag- moplast (Fig. 58, C) which in plants usually continues to widen by the addition of new fibers until it comes in contact with the lateral walls of the cell. FIG. 58. A, spindle beginning to differentiate in kinoplasmic caps at poles of nucleus in Ncphro- dium. (After Yamanouchi, 1908.) B, same in Marsilia. (After Berghs, 1909.) C, D, the origin of the cell wall in Pinus: C, connecting fibers between daughter nuclei at telo- phase ; D, thickenings appearing on fibers. E, the continued extension of the cell wall after the completion of mitosis in the endosperm of Physostegia virginiana. X 215. (After Sharp, 1911.) F, multipolar stage of spindle development in microsporocyte of Acer Negundo. X 1125. (After Taylor, 1920.) While the above changes are occurring the new cell wall which is to be formed between the daughter nuclei begins to differentiate. As the central spindle widens the fibers become fainter near the nuclei and more prominent at the equatorial region: this appearance seems to be due to the flow of the material composing the fibers toward the latter region. On the thickened fibers there now appear small swellings (Fig. 58, D) which increase in size until they fuse to form a continuous plate across the equator of the mother cell, thus dividing the latter into two daughter cells. As this cell plate undergoes further changes (see p. 190) the fibers disappear completely, first near the two nuclei and ultimately at the equatorial region near the new wall. If the cell undergoing division is very broad it often happens that wall formation begins near the center of the phragmoplast while the latter is still extending laterally. In THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 177 extreme cases wall formation may still be seen in progress at the periphery after the fibers have completely disappeared at the central region (Fig. 58, E}. Such is notably the case in the tangential divisions of elongated cambium cells (Bailey 1919, 1920). The spindle in many cases has an origin somewhat -different from that described above. The first indication of its differentiation is here the appearance of a weft of fine fibrils in the cytoplasm all around the nucleus (Fig. 84, E}. As these fibrils increase in size and number they may form several distinct groups extending in various directions, thus giving a multipolar spindle (Fig. 58, F). Some of the groups then gradually disappear, while others alter their positions and coalesce, so that a bipolar spindle eventually results. This, in general, is the manner in which the spindle arises in the microsporocytes of angiosperms. For example, Lawson (1898, 1900, 1903) finds that in Cobcea, Gladiolus, and Iris a zone of granular " perikaryoplasm " collects about the nucleus during the prophases of mitosis. When the nuclear membrane dissolves, this substance together with the linin of the nucleus forms a fibrous network which grows out into several cones of fibers, and these later become arranged in two opposed groups. In Animals. — In the majority of animal cells, and in certain cells of lower plants also, the achromatic figure is a much more elaborate struc- ture than that of the higher plants described above. This is due to the presence of centrosomes, which with their asters are very conspicuous at the time of mitosis. Commonly the aster is not present during the resting stages of the cell, but cases are known in which both centrosome and aster are visible, forming with other materials an "attraction sphere" in the cytoplasm. As the process of mitosis begins (Fig. 59), an aster, if not already present, develops about the centrosome. The centrosome divides, and as the daughter centrosomes move apart each is seen to be surrounded by its own aster, and a small group of fibers ("central spin- dle") extends between them. The achromatic figure, made up of the asters and the spindle connecting them, is known both at this stage and later as the amphiaster. As the daughter centrosomes continue to sepa- rate the astral rays increase in prominence. Some of the rays grow into the nucleus when its membrane disappears and become attached as mantle fibers to the chromosomes, while the lengthening central spindle between the asters becomes the central spindle portion (connecting fibers) of the completed mitotic figure (Fig. 49). All the fibers focus upon the centrosomes. During the anaphase the asters remain very conspicuous, but as the telophases progress they gradually fade from view, except in those forms which have a more or less permanent attraction sphere. Aside from the presence of centrosomes and asters the achromatic figure in animal cells differs most conspicuously from that of higher plant cells in its behavior 12 178 INTRODUCTION TO CYTOLOGY II III IV FIG. 59. — Mitosis in the spermatocyte of Salamandra. I, prophase, centrosomes in astrosphere substance; latter spread out on nucleus. //, prophase; bivalent chromosomes 'formed; centrosomes beginning to diverge; central spin- dle and asters developed. ///, late prophase: nuclear membrane dissolved; spindle fibers attaching to chromosomes, centrosomes moving apart. IV, anaphase: connecting fibers prominent. V, telophase: constriction of cell nearly complete; mid-body forming on central spindle or interzonal fibers. (After Meves, 1907.) FIG. 60. — Elaborate achromatic figure in oocyte of Piscinln. X 1000. (After Jnrgcnscn, 1913.) THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 179 during the telophases. Instead of forming thickenings which become an equatorial cell plate, the connecting fibers play relatively little part in cytokinesis. One or more granules may be differentiated on the fibers at the equatorial region, forming the so-called "mid-body," but the actual division of the cell is brought about by the development of a cleavage furrow, as will be described in the section on cytokinesis. Intranuclear Figures. — In the above described cases of mitosis in plants and animals the achromatic figure is derived mainly from the cyto- plasmic region of the cell, the nuclear materials playing a relatively minor part. In a number of forms, both among animals and plants (fungi, for example), the spindle arises entirely in the nuclear region, forming an intranuclear figure which may be completely established before the FIG. 61. A, B, anaphase and telophase of mitosis in ascus of Laboulbenia chcetophora. X 1350. (After Faull, 1912.) (See also Figt 22.) C, intranuclear mitotic figure in oogonium of Fucus. (After Yamanouchi, 1909.) nuclear membrane disappears. Cases are known in which the centro- somes themselves are also intranuclear, but usually these bodies lie in the cytoplasm against the nuclear membrane, so that although the spindle portion of the figure is within the nucleus the asters lie in the cytoplasm. In the division of the nucleus in the ascus of an ascomycete,1 to take a single example, the process is as follows (Figs. 22; 61 A, B): The centrosome, which in ascomycetes is often discoid in shape, lies against the nuclear membrane. As mitosis begins an aster develops in the cyto- plasm about the centrosome, and the latter divides to form two daughter centrosomes. The central spindle, if formed at all, does not persist. From each of the daughter centrosomes, which begin to move apart along the nuclear membrane, a group of fibers extends into the nucleus where the chromosomes are being formed from the reticulum. The centrosomes finally reach opposite sides of the nucleus, and their two 1 For references to the literature of mitosis in ascomycetes see page 290. 180 INTRODUCTION TO CYTOLOGY groups of fibers become arranged in the form of a sharp poled spindle extending through the nucleus with the chromosomes at the equator. The nuclear membrane commonly remains intact until the chromosomes approach the poles at anaphase; it then disappears, allowing the nucleolus, which has remained unchanged, to escape into the cytoplasm nearby. Between the two densely packed daughter chromosome groups there extends a long strand of chromatic material: this soon disappears and the two daughter chromosome groups reorganize two daughter nuclei not separated by a wall. In those cases in which the division of the fungus nucleus is followed by the development of a separating wall the latter is formed by a cleavage furrow independently of the achromatic figure. Origin of the Figure. — Having before us the above examples of the achromatic figure, we may now refer very briefly to some of the ideas which have been advanced regarding the details of its origin in the cell.1 Early observers looked upon the whole mitotic figure — chromosomes, spindle, and all — as a transformed nucleus, all the structures being formed from the nuclear material at each mitosis. Strasburger, who first held this view, later (1888), with Hermann (1891), believed the spindle to arise wholly from the cytoplasm, whereas 0. Hertwig pointed out cases in which the astral rays arise from the cytoplasm and the spindle from the linin reticulum of the nucleus. Flemming (1891) derived the fibers from the linin and the nuclear membrane. It soon became -evident that the spindle, although in some cases arising entirely within the nucleus or wholly from the cytoplasm, is commonly made up of materials derived from both regions, as is evident from the examples described in the fore- going paragraphs. When van Beneden and Boveri announced their view that the centro- some is a permanent cell organ, transmitted by division to daughter cells and directly concerned in the formation of the asters, the theory was adopted that the figure arises from the cytoplasm as a result of the influence of the centrosome. The centrosome therefore came to be known as "the dynamic center of the cell." Although this organ does play a conspicuous role when present, its importance in connection with the achromatic figure was somewhat diminished when it became evi- dent that many centrosomes do not persist from one cell generation to the next, and that such bodies are entirely absent from the cells of higher plants. Rearrangement Theories. — Many attempts have been made to account for the formation of the achromatic fibrils in the cytoplasm. According to some the fibers and astral rays arise as the result of a morphological rearrangement of the preexistent protoplasmic structure, chiefly under 1 Extensive reviews of the early theories are given by Wilson (1900, pp. 72-86 and 316-329) THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 181 the influence of the centrosome. Biitschli (1876), who looked upon protoplasm as alveolar in nature, held that the rays are not really fibers, but only the lamellae between radially elongated alveolae about the cen- trosome. It was the opinion of Wilson (1899) on the other hand, that the rays are actual fibers, though their material is derived from the alveolar walls. Klein (1878) and others who believed protoplasm to be ultimately fibrillar or reticular in structure, regarded the rays as radially arranged fibrillse. Van Beneden (1883) supposed these fibrillae to be derived partly from the intranuclear reticulum, and Rabl (1889) pointed out that they are continuous with the unaltered cytoplasmic meshwork and arise by a direct transformation of the latter. In Passi- flora Williams (1899) found that the nuclear membrane forms a meshwork connecting the linin reticulum with the cytoplasmic reticulum, all three together organizing the spindle. Special Substance Theories. — According to another group of theories the spindle and asters are not formed merely by the rearrangement of a structure already present, but arise from a special substance in the cell. This substance was held by some to be a constantly present constituent of the cell, forming the achromatic figure at the time of mitosis and re- maining in reserve through the resting stages. Boveri's archoplasm hypothesis in its earlier form (1888) was a prominent development of this idea. According to this hypothesis the attraction sphere is composed of a distinct substance called archoplasm, which consists in turn of fine granules or microsomes aggregated about the centrosome as a result of the centrosome's attractive force. The entire achromatic figure was held to arise from this mass of archoplasm, the fibers and astral rays growing out from it like roots, to be withdrawn again into the daughter masses of archoplasm at the two poles during the closing phases of mitosis. In this way each daughter cell was thought to receive half of the archo- plasm. Although other workers (Watase 1894) also held that the fibers are outgrowths of the centrosome or centrosphere substance, it was made evident later that the material composing the fiber comes from the cyto- plasm, being added to the growing fiber at its end. This was the view of Driiner (1894, 1895). Boveri later (1895) modified his archoplasm hypo- thesis, adopting the view that the fiber is formed from the substance of the cytoplasm and not necessarily from a constantly present archoplasm. Another theory based on the idea of a special substance in the cell was that of Strasburger (1892, 1897, 1898). Strasburger held that the cell has two kinds of protoplasm: an active fibrillar kinoplasm and a less active alveolar trophoplasm. The former constitutes the ectoplast, centro- somes, the mitotic fibers, and the contractile substance of cilia and allied structures. The kinoplasm is thus concerned with the motor work of the cell, whereas the trophoplasm has to do chiefly with nutrition. 182 INTRODUCTION TO CYTOLOGY The nucleolus has been thought by some observers to furnish material for the formation of the spindle, because of the fact that it very commonly disappears from view at about the time the spindle begins to differentiate. It is possible that in some cases there may be a connection of this sort between nucleolus and spindle, but it is clear that this cannot serve as a general interpretation of spindle origin. That the achromatic figure may arise from a special substance not constantly present in the cell, but formed anew at each mitosis, is a theory which several workers have advanced. The researches of Devise (1914) and Miss Nothnagel (1916) may be cited for illustration. Devise, as the result of a careful study of the development of the spindle in the microsporocytes of Larix, concluded that the spindle is not formed by the rearrangement of any preexistent nuclear or cytoplasmic structures, but arises from a substance which develops in the nuclear region during the late prophases (after diakinesis). He was not able to decide whether this substance is of purely nuclear origin or is formed when the karyolymph comes in contact with the cytoplasm. The interaction of karyolymph and cytoplasm is emphasized by Miss Nothnagel in her work on Allium. She points out that the contact of newly formed karyolymph with the cytoplasm at telophase brings about the precipitation of the nuclear membrane, and that in an analogous manner an exosmosis of karyo- lymph through the nuclear membrane into the cytoplasm during prophase causes the precipitation of fine fibrils around the nucleus, these fibrils then developing into the spindle. The achromatic figure therefore arises from a special substance, but this substance, as in the case of Larix, is newly formed at each mitosis. Conclusion. — In general it may be said that although the spindle fibers and the motor and contractile elements of the cell appear to have a substantial relationship with one another, the substance common to them is probably "not to be regarded as being necessarily a permanent constituent of the cell, but only as a phase, more or less persistent, in the general metabolic transformation of the cell substance" (Wilson). Indeed the conspicuous tendency on the part of cytologists at present is to regard the achromatic figure neither as a mere rearrangement of a structure previously present, nor as a form assumed by a special spindle substance, but rather as the result of streaming, gelation, and other temporary alterations in the colloidal substratum. This interpretation is strongly supported by the microdissection studies to be cited in a subsequent paragraph. The Mechanism of Mitosis. — Since the phenomenon of mitosis was first described there have been put forward a number of theories to ac- count for the operation of the achromatic structures in bringing about the separation of the daughter chromosomes and for the division of the cell. Many of the suggestions undoubtedly contain elements of truth, but it THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 183 must be admitted that there is no immediate prospect of a satisfactory solution of these problems. Contractility. — One of the simplest and most widely accepted theories was that of fibrillar contractility suggested by Klein (1878) and van Beneden (1883, 1887), according to which the chromosomes are simply dragged apart by the contraction of two opposed groups of spindle fibers. This theory and its modifications are fully reviewed by Wilson: it will be sufficient here to point out that, whereas many facts were cited in its favor, and elastic models made which simulated the supposed contraction and its results (Heidenhain), the further evidence brought forward by Hermann (1891), Driiner (1894, 1895), Calkins (1898), and others led to the general restriction of the role of contractility, until it became appa- rent that this factor, although it may contribute to the general result, must be of minor importance. The contractility factor appeared again in the more elaborate theory proposed by Rhumbler, which may be briefly stated as follows: The centrosome arises as a local solidification of the walls of the alveolae; the denser constituents of the protoplasm collect at this point and form an attraction sphere, driving the less dense constituents to the other parts of the cell where the pressure is lower; this migration of fluid affects particularly those strands of the protoplasmic reticulum which radiate more directly from the centrosomes; these strands or rays, in giving up their fluid, shorten, and thus exert a trac- tive force which draws the daughter chromosomes apart. In this theory, therefore, the main factors are streaming and contractility. Streaming. — The phenomena of streaming and surface tension have been prominent factors in several attempts to explain both karyokinesis and cytokinesis. The role of streaming in karyokinesis has been held to be especially important since Butschli, Hertwig, and Fol showed many years ago that currents exist in the protoplasm. Rhumbler (1896, 1899), Morgan (1899), Wilson (1901), and Conklin (1902) all held that the astral rays are due at least in part to centripetal currents. This inter- pretation has recently been confirmed by Chambers (1917) in his micro- dissection studies on the living cell. With regard to the aster Chambers says: "The formation of the aster consists in the gelation of the hyalo- plasm which comes under the influence of the astral center. A hyaline liquid separates out during the gelation and flows in innumerable centri- petal paths toward the center where it accumulates to form a sphere. This centripetal flow brings about an arrangement of the gelled hyalo- plasm containing the cell-granules into radial strands separated by the hyaline-liquid paths. This produces the astral figure. The strands of gelatinized cytoplasm merge peripherally into the surrounding liquid cytoplasm or reach and anchor themselves in the substance of the gelled surface when the aster is fully formed. The liquid rays merge centrally into the substance of the sphere, the liquid of the rays and of the sphere being thus identical." 184 INTRODUCTION TO CYTOLOGY Sakamura (1920), although holding the fibers to be important agents in the normal separation of the daughter chromosomes, observes that in abnormal nuclear divisions where no fibers are present the chromosomes still show movements which are probably due to streaming of the cyto- plasm and to surface tension phenomena. The relation of streaming and surface tension to cytokinesis will be discussed in the section dealing more particularly with cytokinesis. Osmosis. — In a theory of the mechanics of karyokinesis proposed by Lawson (1911) the principal factor involved is osmosis. Lawson's ex- planation is essentially as follows. During the late prophase karyolymph passes outward through the nuclear membrane by osmosis, this loss of fluid resulting in a contraction of the nucleus. Owing to the fact that the cytoplasmic reticulum is continuous with the nuclear membrane this contraction sets up radial lines of tension in this reticulum on all sides of the nucleus. As the process continues these lines or "fibers" gradually become arranged in two opposed groups, while the nuclear membrane to which they are attached continues to contract until it actually enwraps each double chromosome. To each double chromosome there are thus attached fibers which represent stretched and distorted regions of the cytoplasmic reticulum extending to the two sides of the cell. When the chromosomes become properly arranged at the equatorial plane the fibers, which are under considerable tension, are able to pull the daughter chromosomes apart and draw them to the poles. As the fibers relax they resume their true reticular state. Although the chromosomes are thus drawn apart by the shortening of "fibers" attached to them, Lawson points out that this is not to be regarded as a case of true active contrac- tility, but only as a release of tension set up in the passive but elastic cytoplasmic reticulum as the result of the exosmosis of karyolymph from the nucleus. This theory has been severely criticized by a number of writers, chiefly on the grounds that such an enwrapping of the chromo- somes by the nuclear membrane as Lawson describes cannot be demon- strated in many objects subsequently examined, and that the membrane frequently goes into solution when both it and the growing fibers are still some distance from the chromosomes. Electrical Theories. — The striking resemblance between the achromatic figure and the lines of force in an electromagnetic field early led to at- tempts to account for mitosis on the basis of electrical principles. Several investigators, working with various chemical substances, succeeded in modelling fields of force that illustrated graphically the changes supposed to take place in the dividing cell. In later years the electromagnetic interpretation was again brought into prominence by Gallardo, Hartog, and Prenant. At first Gallardo (1896) believed the two spindle poles to be of unlike sign, but later (1906), as the result of the researches of Lillie (1903) (see p. 62) on the behavior of nucleus and cytoplasm in THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 185 the electromagnetic field, he concluded that the chromosomes and the cytoplasm carry charges of unlike sign: the daughter centrosomes repel each other and move apart because of their like sign, the spindle poles being of like sign also. The movement of the chromosomes to the poles he held to be due to the combined action of two forces: the mutual repul- sion of the similarly charged daughter chromosomes, and the attraction between the oppositely charged centrosomes and chromosomes. The fact that the two centrosomes and hence the two spindle poles are electrically homopolar (Lillie) and alike osmotically at once makes it apparent that the mitotic figure does not represent an ordinary electro- magnetic field, for in the latter the poles are of unlike sign — the field is heteropolar. It has consequently been suggested by Prenant (1910) and Hartog (1905, 1914) that the mitotic figure is the seat of a special force, analogous to electrostatic force but not identical with it, which is peculiar to living organisms. This new force they call "mitokinetism." A large amount of discussion has centered about the possible role of electrical forces in mitosis, and many kinds of normal and abnormal mitotic phenomena have been cited as evidence for various views. So far as conclusive statements are concerned, there is disappointingly little of a definite nature that can be said. Meek (1913) asserts that the only generalization which is at present possible is the negative one that "the mitotic spindle is not a figure formed entirely by the action of forces at its poles." Conclusion. — In conclusion we may emphasize the fact that the achromatic figure depends for its operation upon a variety of interacting factors. Certain investigators have doubtless done good service in em- phasizing the importance of one or another of these factors — streaming, surface tension, contractility, gelation, electrical phenomena, and the like — but it has become increasingly evident that in no one of them alone is the key to the problem of mitosis to be found. In spite of the confi- dence that some progress has been made, at least in the elucidation of certain phenomena which must have a part in any ultimate explanation, it is nevertheless true that the statements made twenty years ago by Wilson (1900, p. Ill) may be taken as an essentially accurate expression of the condition of the subject: "When all is said, we must admit that the mechanism of mitosis in every phase still awaits adequate physio- logical analysis. The suggestive experiments of Biitschli and Heidenhain lead us to hope that a partial solution of the problem may be reached along the lines of physical and chemical experiment. At present we can only admit that none of the conclusions thus far reached, whether by observation or by experiment, are more than the first naive attempts to analyse a group of most complex phenomena of which we have little real understanding." 186 INTRODUCTION TO CYTOLOGY CYTOKINESIS In the foregoing pages discussion has been limited largely to karyo- kinesis. In the present section attention will be directed to cytokinesis, or the division of the extra-nuclear portion of the cell. In plants the wall separating the two daughter cells is formed by two general methods: cell plate formation and furrowing. The first and more common of these methods, by which a wall is formed in close association with the spindle fibers at the close of mitosis, has been briefly described in the foregoing section on the achromatic figure (p. 176) and will be taken up in greater detail in the following section on the cell wall (p. 190). At this point we shall therefore describe the second method, that of furrowing, which in plants is seen most conspicuously in the thallophytes and in the microsporocytes of the higher plants. The review of the subject given by Farr (1916) will be followed. Thallophytes. — In Spirogyra Strasburger (1875) showed that the wall between the two daughter cells appears as a "girdle" or ring-like ingrowth from the side wall of the parent cell. This wall continues to ,\ I*! /•"* — S1' gill FIG. 62. FIG. 63. FIG. 62. — Cytokinesis by furrowing in Closterium. Only the central part of the cell is shown. X 700. (After Lutman, 1911.) FIG. 63. A, Cleavage furrows beginning to form at periphery of sporangium of Rhizopus nigricans. X 1500. B, Cleavage in the sporangium of Phycomyces nitens: intersporal substance in the angular furrows. X 500. (Both after D. -B. Swingle, 1903.) grow centripetally by the addition of new material at its inner edge while the protoplast develops a deep cleavage furrow, the process continuing until the separating wall is completed at the center of the cell. A somewhat similar process occurs in Closterium (Lutman 1911) (Fig. 62). In the brown algae Sphacelaria (Strasburger 1892; W. T. Swingle 1897) and Dictyota (Mottier 1900) the wall develops uniformly across the THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 187 whole equatorial plane at the same time, and not as a progressive in- growth from the periphery. In the fungi Harper and others showed that the two daughter cells are separated by the development of a cleavage furrow in which the new wall is laid down. In large multinucleate masses that become broken up into spores this progressive cleavage is a very complicated process. The manner in which the furrows develop is shown in the studies of Timberlake (1902) on Hydrodictyon, D. B. Swingle (1903) and Moreau (1913) on Rhizopus and Phy corny ces, Davis (1903) on Saprolegnia, Rytz (1907) on Synchytrium, and Harper (1899, 1900, 1914) on Synchytrium, Pilobolus, Sporodinia, Fuligo, and Didymium. In Rhizopus (Fig. 63, A) the cleavage furrows begin to form both at the peripheral membrane of the sporangium and at the columella and work gradually into the multi- nucleate protoplasm, eventually cutting out multinucleate blocks which become the spores. In Phycomyces (Fig. 63, B) small vacuoles appear in the midst of the multinucleate protoplasm, enlarge and become stellate, and cut out spore masses with from 1 to 12 nuclei each. In the myxomycete, Fuligo, the cleavage is from the surface inward, and the multinucleate blocks are subdivided by further furrowing into uninucleate spores. In Didymium the spores are delimited in a similar way by furrows which begin to form along the young capillitium fila- ments in the interior of the multinucleate mass as well as at its periphery. Microsporocytes. — In the microsporocytes of the higher plants it has been shown with great clearness by Farr (1916, 1918) that the quadri- partition to form spore tetrads of the tetrahedral type is brought about by furrowing, previous ac- counts having generally stated that the walls are formed by the cell plate method. Farr finds that after the four microspore nuclei are formed they all become connected by a series of six spindles, or sets of connecting fibers. The two spindles of the second maturation mitosis may persist, four new ones being added, or the two may disappear, six new ones being developed. Although some sporadic thickenings may ap- pear on these fibers they have nothing to do with the formation of the separating walls, there being no centrifugally growing cell plates such as are seen in cells dividing by the cell plate method. Constriction fur- rows appear at the periphery of the cell (Fig. 64) and grow inward until they meet at the center, dividing the protoplast simultaneously into four FIG. 64. — Cytokinesis by furrowing in the micro- sporocyte of Nicotiana. X 1400. (After Farr, 1916.) 188 INTRODUCTION TO CYTOLOGY spores. Any fibers which these furrows encounter as they grow inward are probably incorporated in the new wall, but they play no prominent part in wall formation: the development of the furrows appears to be entirely independent of the fibers present. • In his first paper (1916) Farr states that the microspore tetrads of the bilateral type are usually formed by the cell plate method, a wall being formed across the diameter of the microsporocyte on the connecting fibers after the first maturation mitosis, and the two daughter cells being divided in a similar way after the second mitosis. In his second contribution (1918) he shows that in Magnolia such tetrads also are formed by furrowing. After the first mitosis a cleavage furrow starts to form, but its development is arrested until after the second mitosis, when it resumes its growth toward the center and forms a wall across the diameter of the spherical protoplast. At the same time other new furrows subdivide each hemisphere, so that four uninucleate microspores result. Farr states that no case of bipartition by furrowing is known in the higher plants; bipartition begins in Magnolia, but the furrow ceases to grow until other furrows are formed after the second mitosis, the eventual division occurring by quadripartition. In the lower plants, however, bilateral tetrads may be formed by the cell plate method. It is the opinion of Farr that furrowing in microsporocytes is due to conditions similar to those which bring it about in animal eggs (see below), since both float freely in a liquid. Animals. — In animals there is found nothing corresponding to the formation of a cell plate on the spindle fibers and its development into a thick wall such as is seen in plants. As noted in the section on the achromatic figure, there is often a slight differentiation at this region (the "mid-body"), but it has nothing to do with cytokinesis, which is brought about by simple constriction or furrowing. This process is most easily followed in the segmenting egg. In small eggs, such as those of worms, the daughter cells (blastomeres) round up and become more or less spherical, whereas in larger eggs, such as that of the frog, a cleavage furrow appears at one pole and develops through the egg without altering the shape of the latter, so that the first two blastomeres have the form of hemispheres. It is with animal eggs that most of the researches on the mechanism of cytokinesis by furrowing have been carried out. Mechanism of Furrowing. — Attempts to explain furrowing and the separation of the daughter cells on physico-chemical grounds have been rather numerous. Many years ago Biitschli (1876) advanced the view that as a result of a specific activity on the part of the centrosomes cyto- plasmic currents are set up which flow toward the centrosomes and produce a higher surface tension at the equator of the cell, this in turn bringing about furrowing and cell-division. McClendon (1910, 1913) also reported an increase in surface tension at the region of furrowing. THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 189 On the contrary, Robertson (1911, 1913) and others attribute furrowing rather to a decrease in the equatorial surface tension, this decrease being due to a diffusion of materials toward that region from the daughter nuclei. Evidence favoring Butschli's interpretation has been afforded by the studies of Spek (1918). Spek imitated furrowing and division with oil and mercury droplets in water, and showed that by lowering the surface tension at two poles of the droplet the relatively higher surface tension at the equatorial region could be made to bring about the con- striction and fission of the droplet. In both droplet and dividing egg he found streamings such as Erlangen (1897) had described in the nema- tode egg: an axial movement polewards to the regions of low surface tension and a superficial streaming toward the equatorial region of higher surface tension, the streams turning inward at the furrow (Fig. 65). FIG. 65. — Diagram showing streaming and furrowing in the egg of Rhabditis (A) and an oil droplet (B). (After Spek, 1918.) Although the causes of the initial changes in surface tension in the case of the cell are relatively obscure, these experiments of Spek show beyond question that alteration in surface tension and streaming are very im- portant factors in cell-division of this type. The relation of periodic changes in the viscosity of the egg substance to cytokinesis by furrowing has recently been discussed by Chambers (1919). Immediately after the entrance of the spermatozoon into the echinoderm egg the sperm aster begins to differentiate as a semi-solid region near the sperm head. (See p. 279.) When the aster is most fully developed the egg has its maximum viscosity (Heilbrunn 1915). As the aster disappears the egg again becomes more fluid. Then a second solidification begins at two centers forming the amphiaster, or bipolar figure. The growth of these two semi-solid masses results in the elonga- tion of the egg, and eventually in the development of a cleavage furrow in the more fluid portion of the egg substance separating them. After cleavage is complete the semi-solid masses (asters) revert to a more fluid state. The formation of the cleavage furrow, moreover, may be pre- vented by mechanical means. At the second mitosis in eggs so treated (binucleate eggs) there are four centers of semi-solidification rather than two, and the egg cleaves simultaneously into four blastomeres. An egg cut into two pieces during the amphiaster stage will, provided it does not 190 INTRODUCTION TO CYTOLOGY return to the fluid state, continue to cleave along the normal plane through the equator of the cell as if nothing unusual had happened. All of these observations indicate a close dependence of cytokinesis upon the temporary differentiation of semi-solid masses in the egg cytoplasm, and throw much light upon the question of the true nature of the achromatic figure. THE CELL WALL Probably the most striking difference which meets the eye in a com- parison of animal and plant tissues lies in the relative degree of dis- tinctness with which the limits of the individual cells may be made out. Animal cells as a rule are separated only by very thin limiting membranes which in many tissues are so delicate as to be scarcely discernible, whereas the cells of plants usually possess conspicuous firm walls, which in the case of woody plants become greatly thickened and afford mechanical support to large bodies. The Primary Wall Layer. — Since the time when mitotic cell-division was first carefully studied with the aid of modern methods it has been known that in the cell wall of plants the primary layer, or middle lamella (the "intercellular substance" and "cement" of early writers), is formed in most cases in close connection with the spindle fibers at the close of mitosis.1 The exact manner of its origin, however, has proved to be a very difficult point to determine, and has formed the subject of a Idng continued controversy. (See papers of Timberlake and Allen, 1900 and 1901.) During the telophases of mitosis the spindle fibers con- necting the two daughter nuclei develop thickenings (Fig. 58, D), enlarge until they come in contact with one another and fuse to form a cell plate, or partition, between the daughter cells. For some time it was thought (Strasburger 1875, 1882, 1884) that the cell plate so formed became at once the middle lamella, upon which secondary and frequently tertiary layers were subsequently deposited by the protoplasts on either side. Strasburger here found support for his theory that the cell wall is essen- tially a transformed layer of the protoplast, in opposition to Nageli and von Mohl, who regarded it as primarily a secretion product. As a result of further researches, however, he later (1898) abandoned this view and adopted an interpretation that had been suggested by Treub (1878), namely, that the cell plate formed by the consolidation of the swellings ("microsomes") on the spindle fibers very soon splits to form the plasma membranes of the two daughter cells, and that there is then secreted between these membranes by the protoplasts a substance which becomes the primary layer, or middle lamella. The correctness of this view was confirmed by the careful researches of Timberlake (1900) and Allen (1901). Timberlake pointed out that in the micro- 1 Discussion is here limited to the walls of higher plant tissues. The ectoplast of naked cells has been dealt with in Chapter III. THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 191 sporocytes of Larix and the root cells of Allium the connecting fibers first thicken near the nuclei, then become uniform throughout their length, and finally become swollen at the equatorial region, indicating a transfer toward that region of the material that is to compose the cell plate. Allen was able to show not only that the middle lamella itself may increase in thickness by the addition of new material before the secondary layers begin to be laid down, but also that it consists in reality of two layers representing the secretions contributed by the two daughter protoplasts. Where these two masses of secreted material meet there is developed a median plane of weakness which is ordinarily invisible but along which the lamella invariably splits when inter- cellular spaces are developed by the rounding up of the cells. By the use of proper staining methods it has been found possible to differentiate this " primary cleavage plane." The continuity of the middle lamella is interrupted, if at all, only by the fine pores through which pass the protoplasmic strands connecting adjacent cells. (See p. 46.) Secondary and Tertiary Wall Layers (Fig. 66).— It is probable that the deposition of the secondary layer begins after the cell has reached nearly or quite [its full size, though to this there are ap- parently certain exceptions. The secondary layer, which seems to be formed with considerable rapidity, differs from the primary layer not only chemically (see below) but also in structure, being interrupted by circular or elongated areas in which no secondary substance is deposited, so that the cells at these places are separated only by the delicate primary membrane. Such a wall is said to be "pitted," the primary lamella extending across the pit being termed the closing membrane. The central portion of this membrane sometimes (vascular cells of gymnosperms chiefly) has a more or less conspicuous thickening known as the torus. The portion of the membrane around the torus is pierced by fine pores: in some cases these may become so large and numerous that the torus appears to be suspended on a meshwork (Fig. 67), while extreme cases are known in which it is held in place only by a few strands. In bordered pits (Fig. 68) the second- ary wall overarches the margins of the closing membrane. In this type of pit, characteristic chiefly of water-conducting cells of the gymnosperms, the closing membrane is of such a nature that its position in the center of j r FIG. 66. — Longitudinal and transverse sections of a gymnosperm tra- cheid; p, primary wall or middle lamella; s, secondary layer; t, spiral tertiary thickening. 192 INTRODUCTION TO CYTOLOGY the pit is readily altered. Probably because of changes in pressure it swings to the side of the pit; the torus then lies against the pit opening, or "mouth," and the pit is blocked except for slow diffusion through the rather thick torus. The latter may even be forced tightly into the pit mouth. The secondary wall layer may be even more limited in extent, only a small portion of the primary wall being covered. Such is the case in protoxylem cells, in which the secondary layer is deposited in the form of rings and spirals (Fig. 4). This form of thickening, together with the FIG. 67. FIG. 68. FIG. 67. — Pits in the wood of Larix, showing perforations in pit membrane. X 800. (After Bailey.) FIG. 68. — Diagram of bordered pit of coniferous wood. A, section of pit showing closing membrane supporting the torus, and secondary layers on each side of middle lamella. B, face view of same. C, section- showing torus forced against mouth of pit. (After Bailey.) peculiarly extensible character of their primary walls, allows for the great increase in length of these cells necessitated by the continued growth of the young organs in which they chiefly function. In some cells, notably the tracheids of certain gymnosperms and the vessels of many angio- sperms, a tertiary layer is deposited upon the secondary wall. This ter- tiary layer takes the form of slender spirals, rings, and other figures resembling the secondary thickenings of protoxylem cells. The Physical Nature of the Cell Wall.— Hugo von Mohl (1853, 1858) first expressed the idea that the cell wall grows by apposition, i.e., by.the deposition of material in successive laminae. Although certain other workers (Wigand 1856) supported this view, it became over-shadowed for a time by the theory of Nageli. This investigator, as a result of his classic researches on the wall and on starch grains (1858, 1862, 1863), concluded THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 193 that the wall is made up of ultramicroscopic crystalline micellae, sur- rounded by water films. Growth of the wall in thickness and in area he believed to be due to the intercalation of new micellae between the old ones, a process termed intussusception. Contrasted with this was Strasburger's development of the apposition theory (1882, 1889). Although Stras- burger agreed that the wall had both solid and liquid constituents, he held that the latter were not complex micellae, but only molecules linked together in the form of a reticular framework by their chemical affinities. Growth in area he thought was merely a matter of stretching without the intercalation of additional particles, while increase in thickness was supposed to be accomplished by apposition, or the deposition of layers of new material in the form of small particles, or microsomes. The striations which both he and von Mohl observed in the wall substance were regarded by Strasburger as due to the linear arrangement of these microsomes. That the cell wall is not merely a lifeless secretion of the protoplast, but contains protoplasm in some form, is a view which has often been upheld, and involves problems which are still far from being solved. Prominence was given to the view by Wiesner (1886), who looked upon the growing cell membrane as a living part of the cell. Following Stras- burger's early view, he held the primary layer- to be wholly protoplasmic, and supposed the growing wall to be made up of regularly arranged particles, which he called dermatosomes, connected by fine fibrils of protoplasm. Growth was accomplished by the intussusception of new dermatosomes. Evidence in support of Wiesner's interpretation was brought forward by Molisch (1888), who showed that when tyloses come into contact pits are formed exactly opposite each other in the two abutting walls, a phenomenon which it would be difficult to explain were the walls without living substance. The new intussusception theory of Wiesner was accepted by a number of workers including Haberlandt and Zacharias (1891). The apposition, or lamination, theory of Strasburger also had many supporters, among them being Noll (1887), Klebs (1886), Zimmermann (1887), and Askenasy (1890). According to Pfeffer (1892) both processes, the intussusception of new particles or molecules and the apposition of new material in layers, are concerned in the development of the wall. This view was later adopted by Strasburger (1898), and has received general acceptance. But much work must be done before any final conclusion can be drawn re- garding many points. Especially obscure is the exact relationship of the protoplasm and the wall. The solution of this difficult problem must await the results of further inquiries by both the cytologist and the biochemist. The Chemical Nature of the Cell Wall. — Through the researches of Payen (1842), Fre"my (1859), Kabsch (1863), Wiesner (1864, 1878), and 13 194 INTRODUCTION TO CYTOLOGY particularly Mangin (1888-1893) it has been found that the chief constitu- ents of the newly formed cell walls of plants are pectose and cellulose — that the primary wall or middle lamella consists of pectose, the secondary layer of pectose and cellulose, and the tertiary layer of cellulose. These substances however, rarely exist in the wall in pure and unmodified form. The pectose of the primary layer changes later to insoluble pectates, especially the pectate of calcium, while the secondary and tertiary layers very soon become greatly changed in composition, not alone through the addition of a variety of new substances, but also through an actual trans- formation which in some cases appears to be complete. For example, the secondary and tertiary layers of xylem cells, although at first containing much cellulose, may later become so completely transformed into or re- placed by lignin that they show no reaction whatever to cellulose stains. In some cases the primary wall may undergo a certain amount of lignifi- cation also. The walls of many cells become heavily impregnated with cutin or suberin, the latter substance being responsible for the peculiar character of corky tissues. Infiltration by cutin, or "cutinization," is to be distinguished from "cuticularization," by which is meant the secre- tion of a layer of cutin (cuticle) on the outside of the cell. A variety of mineral substances, such as silica, calcium oxalate, and calcium carbonate, as well as more complex organic compounds, such as tannin, oils, and resins, are often deposited in the walls of old cells. The heart woods of trees owe their qualities largely to the presence of these additional materials. In spite of these modifications, however, it is still true that Cellulose is the substance chiefly characteristic of plant cell walls in general. Al- though cellulose has been identified in certain animals, the membranes of practically all animal cells are composed of other substances, such as keratin, elastin, gelatin, and chitin. In the fungi also the role of cellulose appears to be played in part by chitin. The Walls of Spores. — Special attention has been given to the develop- ment of the elaborate walls, or coats, of the spores of various plants in a number of investigations. Strasburger (1882, 1889, 1898, 1907) con- cluded that such coats arise by two general methods: (1) by the growth in thickness (by apposition) of the original wall of the spore cell through the activity of the protoplast, as in the pollen grains of Malva and other angio- sperms, and (2) by a deposition of material upon the original wall by the tapetal fluid in which the young spores lie, as in the case of the megaspore of Marsilia. The highly specialized coats of the megaspore of Selaginella have been most intensively studied, particularly by Fitting (1900, 1906) and Miss Lyon (1905), whose accounts disagree in several points. At the close of the tetrad division there is formed about each young spore a thick gela- tinous "special wall," at the inner surface of which, according to Fitting, 195 the spore coats begin to differentiate. The exospore first appears, and just outside of it the rough perispore soon begins to develop. Then a second layer, the mesospore, is formed within the exospore. Between the protoplast, which is at this time very small, and the mesospore, and between the exospore and the mesospore, there are developed two cavities filled with a sporangial fluid which furnishes material to the growing coats. Emphasis is placed on the fact that the coats are able to increase in thickness while they have no immediate contact with the protoplast. The protoplast now expands, after which a third coat, the endospore, is formed at its surface. The mature spore thus has three coats according to Fitting's interpretation, which Denke (1902) and Campbell (1902) confirmed. e x. s.m, e n. FIG. 69. — The developing megaspore coat of Selaginella rupestris: p, protoplast with nucleus; en, endospore; s.m., undifferentiated portion of "spore membrane;" ex, exospore: the outer denser portion is the "perinium." (After Lyon, 1905.) Miss Lyon found that the spore coats (S. rupestris) begin to differ- entiate in the midst of the "spore membrane" ("special wall:" Fitting), rather than at its inner surface as Fitting thought. The exospore first appears as a double zone, the outer part of which becomes the per- inium (perispore: Fitting) (Fig. 69). The small protoplast gradually expands and pushes back the undifferentiated inner portion of the spore membrane; and while it does so a second coat is formed at its surface and becomes the endospore (mesospore: Fitting) which increases in thick- ness by lamination. In another species (S. emiliana) the exospore and endospore form simultaneously. Miss Lyon thus finds two coats rather 196 INTRODUCTION TO CYTOLOGY than three, but points out that a portion of the spore membrane which may remain in an undifferentiated condition until a late stage may easily be mistaken for a third coat. The two "spaces" in the immature spore wall she holds to be undifferentiated regions in the spore membrane, and not cavities filled with a foreign fluid; and further urges that the proto- plast is at all times in contact with the gelatinous spore membrane in which the coats are differentiating, opposing the view that the latter have the power of independent growth in thickness. Evidence favoring the view that the spore coats can grow while not in contact with the protoplast has been brought forward by Beer (1905, 1911) and Tischler (1908). Beer asserts that although both the primary wall and the secondary thickening layer of the pollen grain (in certain members of the Onagraceae) originate in intimate connection with the plasma membrane, most of their subsequent growth occurs by intussus- ception while they are completely separated from the protoplast, which secretes the material used. The development of the pollen wall in Ipomoea purpurea has been described in great detail by Beer. Around each young spore immediately after its formation there appears a tem- porary gelatinous "special wall," upon the inner surface of which the protoplast deposits the exine, or outer spore coat. This is at first homo- geneous, but soon differentiates into a thin outer lamella and an inner zone made up of a network of thickenings with the rudiments of spines at its nodes. Both the spines and the small rodlets, which develop in a clear space appearing between the outer lamella and the network of thickenings (mesospore) , undergo most of their development after they are separated from the protoplast. Tischler (1908) reports that the exine of the pollen of sterile Mirdbilis hybrids may continue to increase in thickness after the protoplast begins to degenerate. As an example of the formation of spore coats through the activity of a tapetal plasmodium may be taken the case of Equisetum, described by Beer (1909) and Hannig (1911). The spores of this form have three coats: an endospore, an exospore, and a perispore consisting of several layers including the one which splits to form the "elaters." The young spore cell at first has a simple membrane, the rudiment of the exospore. The walls of the tapetal cells dissolve, allowing the cell contents to flow freely among the spores as a tapetal plasmodium. Upon the spore membrane the plasmodium deposits successively (1) an inner gelatinous layer, (2) the "middle coat," (3) an outer gelatinous layer, and (4) the elater layer. The exospore develops from the original membrane after the middle coat is formed, and the endospore, or innermost coat, is developed last of all. From this brief review, to which other examples might be added, it is evident that spore coats may develop in a variety of ways, but too little is known to warrant any statement as to which method may be the most THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 197 general one. Although cytological interest centers chiefly in other problems, further studies on spore coats would not only contribute to our understanding of cell wall formation, but would also aid in solving the problem of the possible existence of protoplasm in the wall. Bibliography 9 Achromatic Figure. Cytokinesis. Cell Wall. ALLEN, C. E. 1901. On the origin and nature of the middle lamella. Bot. Gaz. 32 : 1-34. ASKENASY, E. 1890. Ueber einige Beziehungen zwischen Wachsthum und Temper- atur. Ber. Deu. Bot. Ges. 8 : 61-94. BAILEY, I. W. 1915. The effect of the structure of wood upon its permeability. 1. Am. Railway Eng. Assn. Bull. 174. 1919. Phenomena of cell division in the cambium of arborescent gymnosperms and their cytological significance. Proc. Nat. Acad. Sci. 5: 283-285. 1 fig. 1920. The cambium and its derivative tissues. 111. A reconnaissance of cytolog- ical phenomena in the cambium. Am. Jour. Bot. 7: 417-434. pis. 26-29. BEER, R. 1904. The present position of cell-wall research. New Phytol. 3: 159-164. 1905. On the development of the pollen grain and anther of some Onagracese. Beih. Bot. Centr. 19: I 286-313. pis. 3-5. 1909. The development of the spores of Equisetum. New Phytol. 8 : 261-266. 1911. Studies in spore development. Ann. Bot. 26: 199-214. pi. 13. VAN BENEDEN, E. 1883. Resherches sur la maturation de 1'oeuf, la fecondation et la division cellulaire. Arch, de Biol. 4. 1887. (van Beneden & Neyt). Nouvelles recherches sur la fecondation et la division mitosique chez I'Ascaride megalocephale. Bull. Acad. Roy. Belg. III. 14:215-295. pi. 1. BOVERI, TH. 1887. Ueber die Befruchtung der Eier von Ascaris megalocephala. Sitz.-Ber. Ges. Morph. Phys. Miinchen 3. 1888. Zellenstudien. II. Jen. Zeitschr. 22: 685-882. pis. 19-23. 1895. Ueber das Verhalten der Centrosomen bei der Befruchtung des Seeigeleies, nebst allgemeinen Bemerkungen liber Centrosomen und Verwandtes. Verh. Phys.-Med. Ges. Wurzburg, N. F. 29. BUTSCHLI, O. 1876. Studien iiber die ersten Entwicklungsvorgange der Eizelle, die Zelltheilung, und die Kunjugation der Infusorien. Senckenb. Naturforsch. Ges. 10. CALKINS, G. N. 1898. Mitosis in Nocliluca miliaris and its bearing on the nuclear relations of the Protozoa and Metazoa. Jour. Morph. 15 : 711-770. pis. 40-42. CAMPBELL, D. H. 1902. Studies in the gametophyte of Selaginella. Ann. Bot. 16: 419-428. pi. 19. CHAMBERS, R. 1917. Microdissection Studies. II. The Cell aster. A reversible gelation phenomenon. Jour. Exp. Zool. 23: 483-504. 1919. Changes of protoplasmic permeability and their relation to cell division. Jour. Gen. Physiol. 2 : 49-68. figs. 14. CONKLIN, E. G. 1902. Karyokinesis and cytokinesis in the maturation, fertilization and cleavage of Crepidula and other Gasteropoda. Jour. Acad. Nat. .Sci. Phila. 12:5-121. pis. 1-6. figs. 33. DAVIS, B. M. Oogenesis in Saprolegnia. Bot. Gaz. 35 : 233-249, 320-349. pis. 9, 10. DENKE, P. 1902. Sporenentwicklung bei Selaginella. Beih. Bot. Centr. 12: 182- 199. pi. 5. 198 INTRODUCTION TO CYTOLOGY DEVISE, R. 1914. Le fuseau dans les microsporocytes du Larix. (Note prelim.) Comptes. Rend. Acad. Sci. Paris 158 : 1028-1030. DRUNER, L. 1894. Zur Morphologic der Centralspindel. Jen. Zeitschr. 28 : 469- 474. 1895. Studien iiber den Mechanismus der Zelltheilung. Ibid. 29: 271-344. pis. 4-8. VON ERLANGEN, R. 1897. Beobachtungen iiber die Befruchtung und ersten Teil- ungen an den lebenden Eiern kleiner Nematoden. Biol. Centr. 17 : 152-160, 339-346. figs. 25. FARR, C. H. 1916. Cytokinesis of the pollen-mother-cells of certain Dicotyledons Mem. N. Y. Bot. Card. 6: 253-317. pis. 27-29. 1918. Cell-division by furrowing in Magnolia. Am. Jour. Bot. 5: 379-395. pis. 30-32. FAULL, J. H. 1912. The cytology of Laboulbenia chcetophora and L. Gyrinidarum. Ann. Bot. 26: 325-355. pis. 37-40. FITTING, H. 1900. Bau und Entwicklungsgeschichte der Makrosporen von Isoetes und Selaginella und ihre Bedeutung fur die Kenntniss des Wachsthums pflanz- lichen Zellenmembranen. Bot. Zeit. 581:107-164. pis. 5, 6. 1906. (Review of paper by Lyon, 1905). Ibid. 64: 42-43. FLEMMING, W. 1891. Neue Beitrage zur Kenntniss der Zelle. II. Arch. Mikr. Anat. 37 : 685-751. pis. 38-40. FREMY, E. 1859a. Recherches chimiques sur la composition des cellules ve"getales. Comptes Rend. Acad. Sci. Paris 48: 202-212. 18596. Recherches chimiqies sur la cuticle. Ibid. 667-673. 1859c. Recherches sur la composition chimique du bois. Ibid. 862-868. GALLARDO, A. 1896a. La carioquinesis. Ann. Soc. Cientif. Argentina 42. 18966. Essai d 'interpretation des figures karyokine'tiques. Ann. Mus. Nac. d. Buenos Aires. 1901. Les croisments des radiations polaires et 1'interpre'tation dynamique des figures de karyokinese. Soc. de Biol. 63. 1906. L'interpre"tation bipolaire de la division karyokinetique. Ann. Mus. Nac. d. Buenos Aires 13 : 259. 1909. La division de la cellule phenomene bipolaire de caractere electro-colloidal. Arch. Entw. 28: 125-154. Figs. 9. HANNIG, E. 1911. Ueber die Bedeutung der Periplasmodien. I. Die Bildung des Perispors bei Equisetum. II. Die Bildung der Massulse von Azolla. Flora 102 : 209-278, pis. 13, 14. figs. 17. HARPER, R. A. 1899. Cell division in sporangia and asci. Ann. Bot. 13 : 467-525. pis. 24-26. 1900. Cell and nuclear division in Fuligo varians. Bot. Gaz. 30 : 217-251. pi. 14. 1914. Cleavage in Didymium melanospermum (Pers.) Macbr. Am. Jour. Bot. 1: 127-144. pis. 11, 12. HARTOG, M. 1905. The dual force of the dividing cell. I. The achromatic spindle figure illustrated by magnetic chains of force. Proc. Roy. Soc. London 76 B: 548-567. pis. 9-11. 1914. The true mechanism of mitosis. Arch. Entw. 40: 33-64. figs. 16. HEILBRUNN, L. V. 1915. Studies in artificial parthenogenesis. 11. Physical changes in the egg of Arbacia. Biol. Bull. 29: 149-203. HERMANN, J. 1891. Beitrage zur Lehre von der Entstehung der karyokinetischen Spindel. Arch. Mikr. Anat. 37: 569-586. pi. 31. JORGENSEN, M. 1913. Zellenstudien. II. Die Ei- und Nahrzellen von Piscicola. Arch. Zellf. 10: 125-160. pis. 13-18. THE ACHROMATIC FIGURE, CYTOKINESIS, AND CELL WALL 199 KABSCH, W. 1863. Untersuchungen uber die chemische Beschaffenheit der Pflan- zengewebe; mit Bezug auf die neuesten Arbeiten Fremys uber diesen Gegenstand. Jahrb. Wiss. Bot. 3: 357-399. KLEBS, G. 1886. Ueber die Organization der Gallerte bei einigen Algen und Flagel- laten. Unters. Bot. Inst. Tubingen 2 : 333-418. pis. 3, 4. KLEIN, E. 1878, 1879. Observations on the structure of cells and nuclei. Quar. Jour. Micr. Sci. 18: 315-339, pi. 16; 19: 125-175. pi. 7. KUHN, A. 1920. Untersuchungen zur kausalen Analyse der Zellteilung. I. Arch. f. Entw. 46: 259-327. pis. 11, 12. figs. 21. LAWSON, A. A. 1898. Some observations on the development of the karyokinetic spindle in the pollen mother cell of Cobcea scandens. Proc. Calif. Acad. Sci. Bot. Ill 1: 169-184. pis. 33-36. 1900. Origin of the cones of the multipolar spindle in Gladiolus. Bot. Gaz. 30: 145-153. pi. 12. 1903. Studies in spindle formation. Ibid. 36: 81-100. pis. 15, 16. 1911. Nuclear osmosis as a factor in mitosis. Trans. Roy. Soc. Edinb. 48: 1. 137-161. pis. 1-4. LILLIE, R. S. 1903. On differences in the direction of the electrical connection of certain free cells and nuclei. Am. Jour. Physiol. 8: 273-283. LTJTMAN, B. F. 1911. Cell and nuclear division in Closterium. Bot. Gaz. 61: 401-430. pis. 22, 23. LTON, F. 1905. The spore coats of Seiaginella. Bot. Gaz. 40 : 285-295. pis. 10, 11. MANGIN, L. 1888. Sur la constitution de la membrane des v^getaux. Comptes Rend. Acad. Sci. Paris. 107 : 144. 1889. Sur la presence des compose'es pectiques dans les ve'ge'taux. Ibid. 109 : 579. 1890o. Sur la substance intercellulaire. Ibid. 110 : 295. 18906. Sur les r^actifs colorants des substances fondamentales de la membrane. Ibid. Ill : 120. 1891. Observations sur la membrane cellulosique. Ibid. 113 : 1069. 1893. Sur 1'emploi du rouge de ruthenium en anatomic vege'tale. Ibid. 116. 653. McCLENDON, J. F. 1910a. On the dynamics of cell-division. I. The electric charge on colloids in living cells in the root tips of plants. Arch. Entw. 31 : 80-90. pi. 3. figs. 2. 19106. On the dynamics of cell-division. II. Changes in permeability in develop- ing eggs to electrolytes. Am. Jour. Physiol. 27: 240-275. 1913. The laws of surface tension and their applicability to living cells and cell division. Arch. Entw. 37 : 233-247. figs. 10. MEEK, C. F. U. 1913. The problem of mitosis. Quar. Jour. Micr. Sci. 68 : 567-592. MEVES, FR. 1896, 1898. Zelltheilung. Ergeb. d. Anat. u. Entw. 6: 285-390; 8: 430-542. (Review.) VON MOHL, H. 1853. Ueber die Zusammensetzung der Zellmembran aus Faserii. Bot. Zeit. 11 : 753-762, 769-775. 1858. Die Untersuchungen des Pflanzengewebes mit Hiilfe des polarisierten Lichtes. Ibid. 16: 373-375. 1 pi. MOLISCH, H. 1888. Zur Kenntniss der Thyllen, nebst Beobachtungen liber Wund- heilung in der Pflanzen. Sitzber. k. Akad. Wiss. Wien, Math.-Naturw. Cl. I 97 : 264-299. pis. 1, 2. MOREAU, F. 1913. Les Karyogamies multiples de la zygospore de Rhizopus nigri- cans. Bull. Soc. Bot. France 60: 121-123. MORGAN, T. H. 1899. The action of salt solutions on the unfertilized and fertilized eggs of Arbacia, and of other animals. Arch. Entw. 8: 448-539. pis. 7-10. figs. 21. 200 INTRODUCTION TO CYTOLOGY MOTTIER, D. M. 1900. Nuclear and cell-division in Dictyota dichotoma. Ann. Bot. 14: 166-192. pi. 11. VON NAGELI, C. 1858. Die Starkekorner. Zurich. 1863a. Ueber die chemische Zusammensetzung der Starkekorner und Zellmem- bran. Ibid 1863. (See also Nageli's Bot. Mitt. 1, 2.) 18636. Die Anwendung der Polarisationsmikroscops auf die Untersuchungen der organischen Elementartheile. Beitr. z. Wiss. Bot. 3: 1-126. pis. 1-7. 1864. Ueber den innern Bau der vegetabilischen Zellmembran. Sitzber. k. b. Akad. 1864. NOLL, F. 1887. Experimentaluntersuchungen uber d. Wachsthum d. Zellmembran. NOTHNAGEL, M. 1916. Reduction divisions in the pollen mother-cells of Allium tricoccum. Bot. Gaz. 61: 453-476. pis. 28-30. fig. 1. PAYEN, A. 1842. M6moires sur les d6veloppements des v6getaux. Paris. PFEFFER, W. 1892. Studien der Energetik. PRENANT, A. 1910. Theories et interpretations physiques de la mitose. Jour, de 1'Anat. et Phys. 46. RABL, C. 1889. Ueber Zellteilung. Anat. Anz. 4: 21-30. figs. 2. RHUMBLER, L. 1896. Versuch einer mechanischen Erklarung der indirekten Zell- und Kerntheilung. I. Cytokinese. Arch. Entw. 3 : 527-623. pi. 26. figs. 39. 1897. Stemmen die Strahlen der Astrosphare oder ziehen sie? Ibid. 4: 659-730. pi. 28. figs. 27. 1898. Die Mechanik der Zelldurchschnurung nach Meves' und nach meiner Auffassung. Ibid. 7 : 535-554. pi. 12. figs. 5. 1899. Furchung des Ctenophoreneies nach Ziegler und deren Mechanik: usw. Ibid. 8 : 187-238. figs. 28. 1903. Mechanische Erklarung der Aehlichkeit zwischen magnetischen Kraftlinien- system und Zellteilungsfiguren. Ibid. 16: 476-535. figs. 36. ROBERTSON, T. B. 1909. Note on the chemical mechanics of cell-division. Arch. Entw. 27:29-34. 1911. Further remarks onjthe chemical mechanics of cell-division. Ibid. 32 : 308- 313. 1913. Further explanatory remarks concerning the chemical mechanics of cell division. Ibid. 36 : 692-707. figs. 3. RYTZ, W. 1907. Beitrage zur Kenntniss der Gattung Synchytrium. Centr. f . Bakt. 11 18: 635-655, 799-825. 1 pi. figs. 10. SAKAMURA, T. 1920. Experimentelle Studien liber die Zell- und Kernteilung mit besonderer Riicksicht auf Form, Grosse und Zahl der Chromosomen. Jour. Coll. Sci. Imp. Tokyo 39: pp. 221. pis. 7. SHARP, L. W. 1911. The embryo sac of Physostegia. Bot. Gaz. 52 : 218-225. pis. 6,7. SPEK, J. 1918a. Oberflaschenspannungsdifferenzen als eine Ursache der Zellteilung. Arch. Entw. 44: 1-113. figs. 25. 19186. Die amoboiden Bewegungen und Stromungen in den Eizellen einiger Nematoden wahrend der Vereinigung der Vorkerne. Arch. Entw. 44: 217-255. figs. 15. STRASBURGER, E. 1875. Ueber Zellbildung und Zelltheilung. Jena. 1882. Ueber den Bau und das Wachstum der Zellhaute. Jena. 1884. Die Controversen der indirekten Kernteilung. Arch. Mikr. Anat. 23 : 246- 304. pis. 13, 14. 1888. Ueber Kern- und Zellteilung im Pflanzenreich, nebst einem Anhang iiber Befruchtung. Hist. Beitr. 1. pp. 258. pis. 3. 1889. Ueber das Wachstum vegetabilischer Zellhaute. Ibid. 2. THE A CHROMA TIC FIG URE, C YT 0 KINESIS, A ND CELL WALL 201 1892. Schwarmsporen, Gameten, pflanzliche Spermatozoiden und das Wesen der Befruchtung. Ibid. 4: 49-158. pi. 3. 1897. Ueber Cytoplasmastrukturen, Kern- und Zellteilung. Jahrb. Wiss. Bot. 30 : 375-405. figs. 2. 1898. Die pflanzlichen Zellhaute. Ibid. 31 : 534-598. pis. 15, 16. 1907. Apogamie bei Marsilia. Flora 97: 123-191. pis. 3-8. SWINGLE, D. B. 1903. Formation of the spores in the sporangia of Rhizopus nigri- cans and of Phycomyces nitens. U. S. Dept. Agric. Bur. Pit. Indus. Bull 37. SWINGLE, W. T. 1897. Zur Kenntniss der Kern- und Zelltheilung bei den Sphace- lariaceen. Jahrb. Wiss. Bot. 30 : 296-350. pis. 15, 16. TAYLOR, W. R. 1920. A morphological and cytological study of reproduction in the genus Acer. Contrib. Bot. Lab. U. of Pa. 6: pp. 30. pis. 6-11. TIMBERLAKE, H. G. 1900. The development and function of the cell plate in higher plants. Bot. Gaz. 30 : 73-99. 154-170. 1902. Development and structure of the swarm spores of Hydrodictyon. Trans. Wis. Acad. Sci. 13 : 486-522. pis. 29, 30. TISCHLER, G. Zellstudien an sterilen Bastardpflanzen. Arch. Zellf. 1: 33-151. TREUB, M. 1878. Quelques recherches sur le role du noyau dans la division des cellules vegetales. Amsterdam. WATASE, S. 1894. Origin of the centrosome. Biol. Lectures, Woods Hole. WIESNER, J. 1864. Untersuchungen iiber das Auftreten von Pectinkorper in dem Geweben der Runkelriibe. Sitzber. k. Akad. Wiss. Wien, Math.-Naturw. Cl. 11 50: 442-453. 1886. Untersuchungen iiber die Organization der vegetabilischen Zellhaut. Ibid. I 93 : 17-80. figs. 5. WIGAND, A. 1856. Ueber die feinste Struktur der Zellenmembran. Schriften d. Ges. z. Beford. d. ges. Naturwiss. zu Wiirzburg. WILLIAMS, C. L. 1899. The origin of the karyokinetic spindle in Passiflora ccerulea. Proc. Calif. Acad. Sci. Ill Bot. 1 : 189-206. pis. 33-40. WILSON, E. B. 1899. On protoplasmic structure in the eggs of echinoderms and some other animals. Jour. Morph. 16 : Suppl. 1-23. 1900. The Cell in Development and Inheritance. 1901. Experimental studies in cytology. I. A cytological study of parthenogene- sis in sea urchin eggs. Arch. Entw. 12: 529-596. pis. 11-17. figs. 12. ZACHARIAS, E. 1888. Ueber Kern und Zellteilung. Bot. Zeit. 46: 33-40, 51-62. pi. 2. 1891. Ueber das Wachstum der Zellhaut bei Wurzelhaaren. Flora 74: 466-491. pis. 16, 17. ZIMMERMANN, A. 1887. Die Pflanzenzelle. 1893. Sammel-Referate. 6. Beih. Bot. Centr. 3: 342-354 CHAPTER X OTHER MODES OF NUCLEAR DIVISION In accordance with the well established principle which states that only through the simpler organisms can an adequate understanding of those higher in the scale of complexity be approached, search has been made for primitive modes of nuclear division with the hope that light may thereby be thrown upon the origin and significance of the elaborate karyokinetic process which is so universally found in the cells of higher animals and plants. It is to be acknowledged that such a phylogenetic explanation of mitosis is very far from being reached, but many of the observations recorded are nevertheless of a very suggestive nature. To botanists the most interesting of these have been made upon the Cyano- phyceae, which have long been a subject of controversy in this connection. Cyanophyceae. — For many years the nature of the "central body" of the cells of such blue-green algae as Oscillatoria remained very obscure. Biitschli (1890), Dangeard (1892), Scott (1888), and others believed it to be a nucleus of a somewhat primitive type, whereas other investigators, among them Zacharias (1892) and Chodat (1894), denied its nuclear nature. Zukal (1892) held that the peripheral portion of the cell repre- sents a chromatophore, the central body consisting of cytoplasm with a number of minute nuclei imbedded in it. One of the first critical accounts based partly on the study of sections was that of Fischer in 1897. Fischer concluded that the central body, in which he found no chromatin, is the main portion of the cytoplasm, and not to be regarded as the forerunner of the nucleus or indeed as an independent organ at all. He also investigated the nature of the periph- eral portion of the protoplast. By treating the plants with 10 per cent hydrofluoric acid he dissolved away the other parts of the cell, leav- ing this portion intact; and as the result of comparative studies on other plants he concluded, in harmony with Zukal, that it is a single large chromatophore. Since Fischer's work the most important contributions are tKose of Hegler, Kohl, Olive, Phillips, Gardner, and Miss Acton. Contrary to the view of Fischer, all of these cytologists interpret the central body as a nucleus, and the first three regard its division as essentially mitotic. The opinions of these workers with respect to the organization of the cell of the Cyanophyceae and the behavior of its nucleus are summarized below. 202 OTHER MODES OF NUCLEAR DIVISION 203 According to Hegler (1901) the nucleus contains granules of chromatin but no nucleolus or nuclear membrane, division occurring by a simple form of mitosis. The coloring matter exists in the form of minute granules or cyanoplasts. Two other kinds of bodies are also present: albuminous slime globules and albuminous crystals (cyanophycin granules) representing reserve food. Kohl's (1904) description of the cell of Tolypothrix (Fig. 70) is one of the most detailed which has been given in this group of researches. Kohl shows that the nucleus of this form has extensions reaching outward FIG. 70. — Structure and division of the cell of Tolypothrix lanata. A, cell in the vegetative state: c, cytoplasm; n, nucleus; /, fat droplets; p, phycocyanin and chlorophyll granules; s, slime globules; g, granules of cyanophycin. B, four stages of cell-division in Tolypothrix, showing transverse division of chromosomes. C, diagram showing 6 stages of cell-division. (After Kohl, 1903.) toward the cell wall, and that they are withdrawn at the time of nuclear division. The nucleus, which contains chromatin, also includes a num- ber of large Zentralkorner, or slime globules, while in the cytoplasm are fat droplets, cyanophycin granules of reserve albumen, and granules of chlorophyll and phycocyanin. The nucleus, which is very rarely in the resting state, divides as follows : the chromatic material forms a spireme which segments into a definite number of chromosomes; these lie in the direction of the long axis of the cell and break transversely as the separat- ing wall grows inward from the periphery. Their halves are thus in- cluded in the two daughter cells, where they form daughter nuclei without membranes. 204 INTRODUCTION TO CYTOLOGY Olive (1904) l finds that the nucleus of Oscillatoria (Fig. 71) consists of a fibrous achromatic framework with a number of very small chroma- tin granules, and is nearly always in some stage of division. A spireme is formed carrying 16 chromatin granules (8 in Glceocapsa and 32 in one species of Oscillatoria), each representing a chromosome. The spireme and its chromatin granules are split longitudinally, and the daughter spiremes with the daughter granules separate, a distinct central spindle extending between them. The dividing wall is formed as a centripetally growing partition. In Glceocapsa the cell di- F- ';A,Li * , U*4l-:-;^ vides by simple constriction. The vegetative )'•:'•.'• f\n^^^- — 4^4 nuclei of Oscillatoria very rarely approach the resting condition, but in spores and heterocysts they soon pass into this state, a nuclear membrane and vacuole being de- veloped. In the heterocyst the protoplast disorganizes. Olive regards the central body of the Cyanophycese as not essentially different FIG. 71.— Nuclear division from the nucleus of the higher plants, although in Oscillatoria Froeiichia. 1,2, ft [s relatively primitive in several features. 3, 4, four successive stages. . (After Olive, 1904.)] -In the cytoplasm he rinds both cyanophycm granules and slime globules, but no cyano- plasts, the coloring matters being diffused in the peripheral portion of the protoplast. Fischer (1905), in reply to the claims of Kohl and Olive, reasserted his view that the central body is not a nucleus, but rather an accumulation of carbohydrate materials. The glycogen formed as a result of assimila- tory activity gathers in the central body where it is transformed into another carbohydrate, anabcenin, which assumes the form of sausage- shaped structures. At the time of cell-division these masses of reserve material are distributed by a process of " pseudomitosis " to the daughter cells. Fischer therefore regards the mitotic figures observed by others as significant in connection with nutrition rather than with the functions usually attributed to nuclei. Gardner (1906), investigating a number of species, found nuclei of three kinds, which he called the diffuse type, the net karyosome type, and the primitive mitosis type respectively. The "diffuse type" of nucleus, which has no very definite delimitation from the peripheral portion of the protoplast, contains an indefinite number of chromatin masses. As the cell divides this central aggregation of chromatic material divides into approximately equal portions. In the "net karyosome" type, found in Dermocarpa, the distinction between the nucleus and the surrounding cytoplasm is much clearer. The nucleus has an achromatic 1 A very convenient tabulation of the results of researches on cell structure in the Cyanophyceae up to 1904 is given by Olive. OTHER MODES OF NUCLEAR DIVISION 205 network with chromatin granules at its nodes, and constricts simultane- ously into a large number of daughter nuclei which pass to the conidia. In Synochocystis aquatilis occurs the "primitive mitosis" type: here Gardner found the only case of anything approaching mitotic behavior. A spireme develops and segments into three pieces which arrange them- selves parallel to the long axis of the cell and divide transversely; the daughter pieces then separate and a centripetally growing cell wall completes the division of the cell. Gardner thus finds in the Cyano- phyceae "a series of nuclear structures, beginning with a very simple m P 7) FIG. 72. — The nuclei of various members of the Chroococcacese. A, cell of Chroococcus turgidus with scattered metachromatin granules (m) and plasma- tic microsomes (p) ; division beginning. X 2500. B, cell of Gloeocapso. with chromatic granules. C, Merismopedia elegans, showing two stages of nuclear division. X 1500. D. Chroococcus macrococcus: n, nucleus; m, metachromatin; v, vacuole. X 2500. E, dividing nucleus of Chroococcus macrococcus. X 2500. (After Acton. 1914.) form of nucleus scarcely differentiated from the surrounding cytoplasm and dividing by simple direct division" and passing "by very gradual steps to a highly differentiated form of nucleus which in dividing shows a primitive type of mitosis, and in structure approximates the nucleus of the Chlorophycese and the higher plants." In a more recent investigation of the Chroococcaceae Miss Acton (1914) finds that the nucleus is in general much simpler than that of the higher plants. Like Gardner, however, she points out a series beginning with a form in which definite organization is almost entirely lacking and ending with one in which the structure of the higher plant nucleus is closely approached (Fig. 72). In Chroococcus turgidus the protoplast is 206 INTRODUCTION TO CYTOLOGY made up of a ground substance with a reticulum bearing bodies of two sorts: granules of metachromatin closely similar to chromatin in reaction, and cyanophycin granules, or plasmatic microsomes. Although there is no definitely delimited central region in the cell the metachromatin is found mostly at the center and the cyanophycin mostly nearer the pe- riphery. When the metachromatin granules become numerous division sets in, a centripetally growing wall cleaving the protoplast into two daughter cells. In Glceocapsa the central region is somewhat more definite and may often show a spireme-like appearance such as Olive describes; but this may possibly be an artifact. In Merismopedia elegans there is a definitely delimited nucleus, not like that of the higher plants but merely an accumulation of chromatin or chromatin-like material which divides just before the cell constricts into two portions. In Chroococcus macro- coccus, finally, the nucleus and cytoplasm are sharply distinct, the former having a reticulum with chromatin granules at its nodes and dividing by a sort of constriction at the time of cell-division. As a result of these observations Miss Acton advances a theory of the evolution of nucleus and cytoplasm, which is briefly as follows. The excess food elaborated by the protoplast with its pigments was first stored as plasmatic microsomes composed of a carbohydrate, cyanophy- cin. As the reserve material became more complex in nature the nucleo- protein metachromatin was elaborated; this became aggregated at the center of the cell, insuring its equal distribution in cell-division, as in Merismopedia. There thus arose in the cell a physiological and mor- phological differentiation, the nucleo-protein with its portion of the supporting reticulum becoming a stable nucleus, as in Chroococcus macrococcus, and the ground substance remaining as the cytoplasm. Summary. — In the Cyanophyceae, therefore, although these forms in all probability had nothing directly to do with the evolution of the higher plants, we see a series of stages such as may well have occurred in the evolution of the nucleus and its complicated mitotic division. In the simplest forms the material concerned with those cell activities which in higher organisms are associated with the nucleus, is scattered through- out the cell without the morphological distinctness characteristic of an organ in the strict sense. It is passively distributed to the daughter cells when the cleavage wall is formed at the time of cell-division. In other cases this material reacts more strongly like true chromatin and may form a more or less definite aggregation separating into two masses as the cell divides. This metachromatin, which is a nucleic acid com- pound, has also been observed in other algse, in Protozoa, and in fungi, including the yeasts. It appears to represent a reserve material, though it may also have other functions. Finally, definite and well organized nuclei are present in certain of the forms described in the foregoing pages, and although these nuclei may lack some of the features exhibited OTHER MODES OF NUCLEAR DIVISION 207 by the nuclei of higher organisms, they show in the division and distribu- tion of their chromatic elements many of the characteristics of true mitosis. In the evolution of the nucleus through such a series of stages we have an illustration of "the conception of cell structure which im- FIG. 73. — Nuclear division in Protozoa. A, one form of mitosis in Amoeba diplomilotica. (From Minchin, after Aragao.) B, Nuclear division in Coccidium schubergi. X 2250. (From Minchin, after Schaudinn.) C, mitotic division of micronucleus of Paramcecium (horizontal figure on smaller scale than others.) (From Minchin, after Hertwig.) plies differentiated regions of a colloidal system in which special processes have become localized and tend to remain fixed" (Harper 1919). Protozoa. — Such an apparent derivation of mitosis from a simpler, more indefinite division of a less sharply delimited mass of special 208 INTRODUCTION TO CYTOLOGY chromatic substance is seen also in the protozoa. Here the chromatic material in a number of species is held together in loose granular aggrega- tions, and the achromatic figure is seen in curious, relatively simple manifestations. In many cases, however, even among the Rhizopoda, there occurs a very advanced type of mitosis, with spindle, centrosomes, asters, and a definite number of chromosomes. One of the simplest types of nuclear division found among the pro- tozoa is known as "chromidial fragmentation." Here the nucleus is resolved into a large number of chromatin granules, or chromidia, which reassemble in two or more groups and form new nuclei. In nuclei of the "vesicular type," found commonly among the Microsporidia, the chroma- tin is concentrated in a single large body, or karyosome, which becomes dumbbell-shaped and divides, the rest of the nucleus then dividing also. In other forms, such as Coccidium (Fig. 73, B], the nucleus contains in addition a second kind of chromatin which is approximately halved in nuclear division. It is but a short step from such non-mitotic division as this to the simplest types of mitosis ("promitosis") seen in many protozoa. Within the group are found all gradations in complexity from such primitive modes of division up to the advanced types showing a complete achromatic figure with chromosomes which are regular in form, number, arrangement, and division, just as in the higher animals.1 Both Metcalf and Kofoid (1915) have emphasized the fundamental similarity of protozoan and metazoan nuclei. The process of mitosis has the same succession of phases in the two cases, though many minor variations occur. In some representatives of all the main groups of protozoa are found elongated chromosomes which Metcalf regards as linear aggregates of chromatin granules, and which split longitudinally, giving exact equivalence to the daughter-nuclei. Although the cell mechanism of Mendelian inheritance is thus held to be present in members of each great protozoan group and to operate as in the metazoa at the sexual stages, Metcalf believes this mechanism is not kept intact through the vegetative phases as it is in the higher groups. Other Cases in Plants. — With regard to the myxomycetes, the researches of Strasburger (1884), Harper (1900), Jahn (1904, 1911), Olive (1907), and Winge (1912) have shown that nuclear division is essentially mitotic, and that in some cases the chromosomes are not only definite in number but undergo a reduction prior to spore formation. As an example of an exceptional condition may be taken Sorodiscus (Fig. 74, A), in which Winge describes two sorts of chromatin: vegetative trophochromatin and generative idiochromatin, the two forming a single mass at the center of the nucleus. As nuclear division begins this mass takes the form of three or four bodies very similar to chromosomes. 1 For a description of mitotic phenomena in protozoa see Minchin (1912, Chapter VII). OTHER MODES OF NUCLEAR DIVISION 209 The two kinds of chromatin now separate, the "trophochromatin placing itself in the center and the generative or idiochromatin lying like a thin equatorial plate around it." As the nucleus elongates the tropho- chromatin body becomes dumbbell-shaped and breaks into two, while the idiochromatin plate splits into daughter plates which apparently move to the poles and cooperate with the trophochromatin in the forma- tion of the daughter nuclei. FIG. 74. A, two stages of mitosis in Sorodiscus. (After Winge, 1912.) B, anaphase of nuclear division in Euglena. Chromosomes grouped about dividing "nucleolo-centrosome." (After Keuten, 1895.) C, chromosomes developing from nucleolus in Spirogyra. X 1335. (After Berghs, 1906.) D, Mitosis in Spirogyra crassa. (After Merriman, 1913.) A process with much the same appearance at certain stages is seen in the flagellate, Euglena (Keuten 1895) (Fig. 74, B). Here the chromo- somes group themselves about the large nucleolus which soon takes the form of a dumbbell-shaped "central spindle" or "centrodesmose." The nucleolus completes its division, the chromosomes meanwhile separating into two groups which pass to the poles and reorganize the daughter nuclei. In certain other flagellates Kofoid (1915) reports a split spireme and a definite number of chromosomes which differ markedly in size and shape. In Cladophora (Carter 1919) nearly all the chromatin is contained in one or more large chromatin nucleoli, or karyosomes. After the numer- ous chromosomes have arrived at the two poles at the close of the ana- phase the spindle connecting the two groups constricts and completes the division of the nucleus. Another unusual condition is found in Spirogyra (Fig. 74, C, D). In this form nearly all of the chromatic material is lodged in the large nucleolus, the nuclear reticulum being very delicate and almost invisible in many preparations. According to Berghs (1906), Karsten (1908), and Trondle (1912) all the chromosomes which appear in the prophase and split as usual are derived from this nucleolus, most of its material 14 210 INTRODUCTION TO CYTOLOGY being used in their formation. In the opinion of Miss Merriman(1913) the chromatic bodies observed by the above workers are not true chromo- somes, but are rather more indefinite chromatic aggregations which are variable in number and appearance, and which are irregularly pulled apart as mitosis proceeds. She finds here "no evidence throughout the karyokinesis of an equational division of autonomous bodies." In Zygnema both Escoyez (1907) and van Wisselingh (1914) find that the reticulum, and not the nucleolus, gives rise to all the chromosomes. Although the nucleolus furnishes no morphological element, chromatic material may flow from it to the chromosomes as they develop from the reticulum. Much the same condition is found in Marsilia (Strasburger 1907; Berghs 1909). Strasburger points out that in the somatic nuclei (in the cells of the root and the young prothallium) most of the chromatic substance is held in the nucleolus during the resting stages (Fig. 17, E), and that the material of the reticular framework, which is very delicate, is to be regarded as the substance of importance in heredity. Berghs shows that the nucleolus consists of an achromatic substratum which appears independently of the reticulum in the telophase and soon becomes impregnated with chromatic material transferred to it from the chromo- somes. In the next prophase the chromatic material flows back to the delicate reticulum, from which the chromosomes gradually develop. As the chromosomes increase in distinctness the nucleolus becomes paler, and when the nuclear membrane breaks down the nucleolus dissolves in the protoplasmic liquid. It is therefore clear that in Marsilia the nu- cleolus is not a mere aggregation of the chromosomes of the telophase, as might at first be supposed. The chromosomes arise from the reticulum as usual, and not from the nucleolus as reported for Spirogyra. In these observations we have additional evidence favoring the view of Haecker, Boveri, Mare'chal, and others (see Chapter VIII) that it is the achromatic substratum of the chromosome, and not the chromatic substance which it carries, that should be regarded as. the persistent structural unity representing the basis of inheritance. Amitosis. — In amitotic or direct nuclear division the nucleus simply constricts and separates into two portions while in the "resting" condi- tion, no condensed chromosomes, centrosomes, spindle, or asters being formed. As a general rule such a division of the nucleus is not followed by a division of the cell; cells with two or more nuclei therefore commonly result. As examples may be cited the tapetal cells in the anthers of angiosperms, the internodal cells of Chara (Fig. 75) (Johow 1881), and certain glandular cells of animals. The presence of more than one nucleus cannot by itself be regarded as evidence that amitosis has occurred, however. Amitosis appears to be of rather frequent occurrence among the lower organisms, some of which show other methods of divi- sion also. For example, amitosis occurs regularly in budding yeasts, OTHER MODES OF NUCLEAR DIVISION 211 though the divisions giving rise to the ascospore nuclei have been shown to be mitotic in certain cases. (See Guilliermond 1920.) Amitosis was once believed to be the normal mode of nuclear division, mitosis being looked upon as very exceptional. The true condition, so far as higher organisms are concerned, has turned out to be quite the reverse: it is evident that amitosis occurs frequently in certain kinds of cells, but the mitotic method of division has been found to be almost universal. What the physiological significance of amitosis may be is not well known. It was once suggested (Chun 1890) that it aids the processes of metabolism by increasing the nuclear surface in the cell, since it is of such frequent occurrence in cells with a dis- tinctively nutritive function. This view has recently been restated by Nakahara (1917) as a result of his work on the larva of Pieris. l The most generally held opinion regarding amitosis in the higher organisms was for many years that expressed by Flemming (1891), namely, that it represents a degeneration phenomenon or aberration of some kind, which would explain why it is so often found in degenerating and pathological tissues. In the words of vom Rath (1891), "when once a cell has undergone amitotic division it has received its death-warrant; it may indeed continue to divide for a time by amitosis, but inevitably perishes in the end." That the view of vom Rath must be modified has been indicated by the results of a number of investigations. For instance, Pfeffer (1899) and Nathansohn (1900) found that if Spirogyra filaments are placed in a ^ to 1 per cent solution of ether the nuclei divide by amitosis only, and that when the filaments are returned to pure water the mitotic method of division is resumed, with no evidence of degeneration. Haecker, how- ever, working on the eggs of Cyclops, came to view such artificially in- duced behavior not as true amitosis but rather as a much modified mitotic division, which he termed "pseudoamitosis." Other cytolpgists observed nuclear divisions that seemed intermediate in character between mitosis and amitosis (Dixon in the endosperm of Fritillaria, 1895; Sargant in the embryo sac of Lilium, 1896; R. Hertwig in Actinosphcerium, 1898; Buscalioni in the endosperm of Corydalis, 1898; and Wasielewski in the roots of Vicia faba, 1902, 1903). Hertwig accordingly concluded that mitosis and amitosis are separated by no sharp boundary line, but are connected by an unbroken series of transition stages. 1 In a second paper (1918) Nakahara gives a convenient review of the literature of the subject. FIG. 75. — Amitosis in internodal cell of Chara. X 413. 212 INTRODUCTION TO CYTOLOGY As a result of his recent researches on chloralized cells (Fig. 76) Sakamura (1920) interprets all such unusual types of nuclear division as those described by Hertwig and Wasielewski as the effect of disturbed mitotic division, but denies the claim of those authors that such types of division represent actual transition stages between amitosis and mitosis. True amitosis he regards as a fundamentally different process, and as essentially a degeneration phenomenon. FIG. 76. — Abnormal mitosis in chloralized root cells of Vicia. A, chromosomes distributed irregularly in cell. B, scattered chromosomes beginning to assume nuclear form. C, nucleus reconstructed by scattered chromosomes. D, scattered chromosomes reconstructing 3 separate nuclei. E, chromosomes reconstructing 2 nuclei connected by bridge. F, amitosis-like appearance resulting from condition shown in E. (After Sakamura, 1920.) On the contrary, Des Cilleuls (1914) reports that in the rabbit periods of amitosis and mitosis succeed each other regularly in the same cell lineage without affecting the vitality of the cells. In his opinion, therefore, amitosis does not necessarily place the stigma of senescence upon the cell. A similar conclusion is reached by Arber (1914), who finds amitosis supplementing mitosis in the early growth stages of the leaves and adventitious roots of Stratiotes aloides; and by McLean (1914), who asserts that it is the sole method of nuclear division in the cortical parenchyma of several aquatic angiosperms. Saguchi (1917) likewise states that the nuclei in the ciliated cells of vertebrates divide by amitosis only. Amitosis and Heredity. — One of the most important theoretical ques- tions raised by the phenomenon of amitosis is that of the effect which the process may have upon the hereditary mechanism of the cell. Ac- cording to the chromosome theory of heredity and development in its usual form it has been thought that, although amitosis may occur in connection with an altered metabolism in cells not to undergo further differentiation, mitosis must occur exclusively in the germ cell lineage, in order that the chromosomes and the hereditary elements they con- OTHER MODES OF NUCLEAR DIVISION 213 tain shall be properly distributed to the reproductive cells; and also in developing tissues and organs, so that differentiation may proceed nor- mally. On the other hand, several workers (Meves; Flemming in his later papers) admit that amitosis may not affect any hereditary powers which the nuclei concerned may possess. Child (1907, 1911), who re- ports amitosis in both the somatic and germ cells of certain animals, where it appears to play an important role in the developmental cycle, strongly urges that such facts render the hypothesis of chromosome in- dividuality highly improbable, and that our conceptions of the r61e of the cell organs in heredity must be greatly altered. The hopelessly unsettled state of opinion on this question may be illustrated by the list of authors and their views cited by Conklin (1917). That amitosis frequently occurs in the process of normal cell differ- entiation, and therefore constitutes evidence against the chromosome theory, has been held by Nathansohn (1900), Wasielewski (1902, 1903), Gurwitsch (1905), Hargitt (1904, 1911), Child (1907, 1911), Patterson (1908), Glaser (1908), Jordan (1908), Jorgensen (1908), Maximow (1908), Moroff (1909), Knoche (1910), Nowikoff (1910), and Foot and Strobell (1911). Several of these investigators, together with R. Hertwig (1898), Lang (1901), Calkins (1901), Herbst (1909), Godlewski (1909), and Konopacki (1911), see no principal distinction between amitosis and mitosis, believing that both may occur without interfering with normal differentiation. Haecker (1900), Nemec (1903), and Schiller (1909) dissented from the above view, which was also strongly contested by Boveri (1907) and Strasburger (1908). Richards (1909, 1911) and Harman (1913) failed to confirm the results of Child on amitosis in cestodes, but Child (1911) reasserted his view, which was supported by Young (1913). Schurhoff (1919), working on Podocarpus, emphatically states that a nucleus which has once undergone true amitosis is incapable of dividing mitotically. Sakamura (1920) is of the same opinion. In a careful study of maturation and cleavage in Crepidula plana Conklin (1917) finds that the nuclei divide only by mitosis. There are many apparent cases of amitosis, but upon careful examination they all prove to be only various modifications of the regular mitotic process. Such modifications are these : the scattering of the chromosomes and their failure to unite into a single nucleus; mitosis without cytokinesis, giving cells with two or more nuclei; the failure of certain daughter chromosomes to pull apart, leaving a chromatic bridge between the daughter nuclei; the persistence of the nuclear membrane, with a division of the chromo- somes by mitosis and of the nuclear vesicle by constriction. Conklin concludes as a result of his many observations and an examination of the evidence offered by others, that there is not known a single conclusive case of true amitosis in a normally differentiating cell, and that all attacks 214 INTRODUCTION TO CYTOLOGY upon the chromosome theory on the ground of amitosis have signally failed. The results obtained by Sakamura (1920) in his study of modi- fied mitosis in chloralized plant cells are strikingly similar to those of Conklin, and his conclusions regarding the chromosome theory are es- sentially the same. From the foregoing it is evident that the problem of the effect of amitosis upon the differentiation of the tissues in which it occurs and upon the hereditary powers of the nucleus is by no means easy of solution, and that much care must be used in interpreting supposed amitotic phe- nomena in fixed preparations. The work of Conklin and Sakamura has shown clearly that many of the phenomena reported as amitosis are in reality aberrations of the mitotic process, and that the opinions of many writers are undoubtedly due to a failure to recognize this fact. Should it be proved, however, that true amitosis may occur in the lineage of normally functioning germ cells a serious obstacle would be placed in the way of the chromosome theory of inheritance in its current form, for this theory requires that, no matter what happens in cells not in the direct line of the germ cells, nuclear division in this line must be exclusively mitotic in order that the hereditary mechanism in the nucleus shall be preserved. This mechanism, as we shall see in later chapters, is supposed to be of such a nature that amitosis would seriously derange its organization., In each daughter nucleus of an amitotic division some of the elements necessary for normal functional activity would presumably be lacking, owing to the simple mass division of the chromatin. With reference to this point it has been contended by Child that the nucleus is a dynamic system capable of regenerating its lost parts and "producing a whole" after amitosis. But it is a well established fact that when chromosomes are lost in abnormal mitotic division they are not regenerated by the daughter nuclei (non-disjunction; Chapter XVII). In this connection an experiment performed by Chambers (1917) is of interest. This investigator succeeded in pinching the nucleus of an animal egg into two pieces. The two "amitotic" nuclei so produced reunited upon touching, after which the egg was fertilized and passed through the early cleavage stages in the normal manner. It is known that the character of these early stages is largely independent of the nuclei present, being the outgrowth of an organization already present in the egg cytoplasm. (See Chapter XIV.) The later stages, in which the effects of the hereditary constitution of the nucleus appear, were not reached in the present experiment. Moreover, the entire chromatin outfit was present in the reunited nucleus, which is not supposed to be true of a daughter nucleus of an amitotic division. From this experiment, therefore, it can only be concluded that whatever disturbance of the spatial arrangement of the nuclear elements . may have been caused by the temporary separation of the nucleus into two parts, it had no serious OTHER MODES OF NUCLEAR DIVISION 215 effect on the nutritive functions performed by the nucleus during the early cleavage stages. Development did not proceed far enough to warrant any conclusion regarding the effect upon the role of the nucleus in differentiation and inheritance. Although what probably represents amitosis has been observed in young germ cells, it has not been shown with certainty in any case that descendants of these amitotically dividing nuclei become the nuclei of normally functioning gametes. To gain conclusive evidence for such an occurrence it would be necessary to trace the descendants of the amitotic- ally dividing nuclei through to particular gametes or spores and then to note the effect upon the individuals produced by them. This would be a matter of extreme experimental difficulty, and not at all possible in most organisms. If it were successfully accomplished and the individuals were found to be normal in every respect, not only in the cleavage stages but throughout development, the revision of the chromosome theory which various workers have advised would at once become necessary. Bibliography 10 Other modes of nuclear division ACTON, E. 1914. Observations on the cytology of the Chroococcacese. Ann. Bot. 28: 433-454. pis. 23, 24. ARBER, A. 1914. On root development in Straliotes aioides L., with special reference to the occurrence of amitosis in an embryonic tissue. Froc. Camb. Phil. Soc. 17 : 369-379. pis. 2. BERGHS, J. 1906. Le noyau et la cinese chez le Spirogyra. La Cellule 23: 53-86. pis. 3. 1909. Les cineses somatiques dans le Marsilia. Ibid. 25: 73-84. 1 pi. BOVERI, TH. 1907. Zellen-Studien VI. Die Entwicklung dispermer Seeigeleier. Jena. BTJSCALIONI, L. 1898. Osservazioni richerche sulla cellula vegetale. Ann. Inst. Bot. Roma 7. BUTSCHLI, O. 1890. Ueber den Bau der Bakterien und verwandter Organismen. Leipzig. 1896. Weitere Ausftihrungen liber den Bau der Cyanophyceen und Bakterien. Leipzig. 1902. Bemerkungen liber Cyanophyceen und Bakterien. Arch. Protist. 1. CALKINS, G. N. 1901. .The Protozoa. New York. CARTER, N. 1919. The cytology of the Cladophoracese. Ann. Bot. 33 : 467-478. pi. 27. figs. 2. CHAMBERS, R. 1917. Microdissection studies. I. The visible structure of the cell protoplasm and death changes. Am. Jour. Physiol. 43: 1-12. figs. 2. CHILD, C. M. 1907a. Amitosis as a factor in normal and regulatory growth. Anat. Anz. 30: 271-297. figs. 12. 1907&. Studies on the relation between amitosis and mitosis. Ill- VI. Biol. Bull 13: 138-160, 165-184. pis. 2-10. 1911. The method of cell division in Monezia. Ibid. 21: 280-296. figs. 16. CHODAT, R. 1894. Contenue cellulaire des Cyanophyce*es. Arch. Sci. Phys. Math Geneve 11132: 637-641. t CHUN, C. 1890. Ueber die Bedeutung der direkten Zelltheilung. Sitzber. Schr. Phys.-Oekon. Ges. Konigsberg. 216 INTRODUCTION TO CYTOLOGY CONKLIN, E. G. 1903. Amitosis in the egg follicle cells of the cricket. Am. Nat. 37: 667-675. figs. 8. 1912. Experimental studies on nuclear and cell division in the eggs of Crepidvla. Jour. Acad. Nat. Sci. Phila. 15 : 503-591. pis. 43-49. 1917. Mitosis and amitosis. Biol. Bull. 33 : 396-436. pis. 10. DANGEARD, P. 1892. Le noyau d'une Cyanophycee. Le BotanisteS: 28-31. pi. 2. DBS CILLETJLS, J. 1914. Recherches sur la signification physiologique de 1'amitose. Arch. d'Anat. Micr. 16 : 132-148. pis. 7, 8. DEXON, H. H. 1895. Note on the nuclei of the endosperm of Fritillaria imperialis. Proc. Roy. Irish Acad. Ill 3: 721-726. pi. 24. ESCOYEZ, E. 1907. Le noyau et la caryocinese chez le Zygnema. La Cellule 24: 355-366. 1 pi. FISCHER, A. 1897. Untersuchungen liber den Bau der Cyanophyceen und Bakterien. Jena. 1905. Die Zelle der Cyanophyceen. Bot. Zeit. 63 : 51-129. pis. 4, 5. FLEMMING, W. 1890. Amitotische Kerntheilung im Blasenepithel des Salamanders. Arch. Mikr. Anat. 34: 437-451. pi. 27. 1891. Neue Beitrage zur Kenntniss der Zelle. II. Arch. Mikr. Anat. 37: 685- 751. pis. 38-40. FOOT, K. and STROBELL, E. C. 1911. Amitosis in the ovary of Protenor belfragi and a study of the chromatin nucleolus. Arch. Zellf. 7 : 190-230. pis. 12-20. GARDNER, N. L. 1906. Cytological studies in Cyanophycese. Univ. Calif. Publ. Bot. 2 : 237-296. pis. 21-26. GLASER, O. 1908. A statistical study of mitosis and amitosis in the entoderm of Fasciolaria tulipa var. distans. Biol. Bull. 14: 219-248. GODLEWSKI, E. 1909. Das Vererbungsproblem, usw. Vortrage u. Aufsatze Entw. Mech. 9. GRIFFITHS, B. M. 1915. On Glaucocystis Nostochinearum, Itz. Ann. Bot. 29: 423-432. pi. 19. GRIGGS, R. F. 1909. Some aspects of amitosis in Synchytrium. Bot. Gaz. 47: 127-138. pis. 3, 4. GUILLIERMOND, A. 1906. Contribution a l'6tude cytologique des Cyanophyce'es. Rev. Ge"n. Bot. 18: 392-408, 447-^165. 1920. The Yeasts. (Engl. transl. by F. W. Turner.) N. Y. GURWITSCH, A. 1904. Morphologic und Biologic der Zelle. Jena. HAECKER, V. 1900. Mitosen im Gefolge amitosenahnlicher Vorgange. Anat. Anz. 17:9-20. figs. 16. HARGITT, C. W. 1904. The early development of Eudendrium. Zool. Jahrb. 20: 257-276. pis. 14-16. 1911. Some problems of Coelenterate ontogeny. Jour. Morph. 22: 493-550. pis. 3. HARMAN, M. T. 1913. Method of cell division in the sex cells of Tcenia tenieeformis. Jour. Morph. 24: 205-244. pis. 8. HARPER, R. A. 1900. Cell and nuclear division in Fuligo varians. Bot. Gaz. 30: 217-251. pi. 14. 1914. Cleavage in Didymium melanosporum (Pers.) Macbr. Am. Jour. Bot. 1: 127-144. pis. 11, 12. 1919. The structure of protoplasm. Am. Jour. Bot. 6: 273-300. HEGLER, R. 1901. Untersuchungen tiber die Organization der Phycochromzelle. Jahrb. Wiss. Bot. 36: 229-354. pis. 5, 6. figs. 5. HERBST, C. 1909. Vererbungsstudien VI. Arch. Entw. 27: 266-308. pis. 7-10. HERTWIG, R. 1898. Ueber Kerntheilung, Richtungskorperbildung und Befruchtung von Actinosphcerium eichornii. Abh. Bayer. Akad. Wiss. 19. 1908. Ueber neue Probleme der Zellenlehre. Arch. Zellf. 1 : 1-32. figs. 9. OTHER MODES OF NUCLEAR DIVISION 217 JAHN, E. 1904. Myxomycetenstudien. 3. Kernteilung und Geisselbildung bei den Schwarmern von Stemonitis flaccida Lister. Ber. Deu. Bot. Ges. 22: 84-92. pi. 6. 1908. Myxomycetenstudien. 7. Ceratiomyxa. Ibid. 26a: 342-352. 1911. Myxomycetenstudien. 8. Der Sexualakt. Ibid. 29: 231-247. pi. 11. JOHOW, F. 1881. Die Zellkerne von Charafcetida. Bot. Zeit. 39 : 729-743, 745-753. pi. 7. JORDAN, H. E. 1908. The accessory chromosome in Aplopus mayeri. Anat. Anz. 32 : 284-295. figs. 48. JORGENSEN, M. 1908. Untersuchungen iiber die Eibildung bei Nephilis, usw. Arch. Zellf . 2 : 279-347. pis. 20-23. figs. 4. KARSTEN, G. 1908. Die Entwicklung der Zygoten von Spirogyra jugalis Ktzg. Flora 99: 1-11. pi. 1. KEUTEN, J. 1895. Die Kerntheilung von Euglena viridis Ehrenberg. Zeit. Wiss. Zool. 60: 215-235. pi. 11. VON KNOCHE, E. 1910. Experimentelle und andere Studien am Insektenovarium. Zool. Anz. 35: 261-265. figs. 3. KOFOID, C. A. 1915. The evolution of the protozoan nucleus and its extranuclear connections. Science 42 : 658. KOHL, F. G. 1903. Ueber die Organization und Physiologic der Cyanophyceenzelle, und die mitotische Teilung ihres Kerns. Jena. KONOPACKI, M. 1911. Ueber den Einfluss hypotonischen Losungen auf befruchtete Echinideneier. Arch. Zellf. 7: 139-184. pis. 9-11. LANG, A. 1901. Lehrbuch der vergleichenden Anatomic. II Aufl. Jena. MACKLIN, C. C. 1916. Amitosis in cells growing in vitro. Biol. Bull. 30 : 445-466. MAXIMOW, A. 1908. Ueber Amitose in den embryonalen Geweben bei Saugtieren. Anat. Anz. 33: 89-98. figs. 11. MCLEAN, R. C. 1914. Amitosis in parenchyma of water plants. Proc. Camb. Phil. Soc. 17 : 380-382. 1 fig. MERRIMAN, M. L. 1913. Nuclear division in Spirogyra crassa. Bot. Gaz. 66: 319-330. pis. 11, 12. METCALF, M. M. 1915. Chromosomes in protozoa. Science 42: 658. MINCHIN, E. A. 1912. An Introduction to the Study of the Protozoa. London. MOROFF, TH. 1909. Oogenetische Studien. I. Copepoden. Arch. Zellf. 2 : 432- 494. pis. 34-36. NAKAHARA, W. 1917. Preliminary note on the nuclear division in adipose cells of insects. Anat. Record 13: 81-86. figs. 11. 1918. Studies in amitosis: Its physiological relations in the adipose cells of insects, and its probable significance. Jour. Morph. 30: 483-526. pis. 6. figs. 36. NATHANSOHN, A. 1900. Physiologische Untersuchungen iiber amitotische Kernteil- ung. Jahrb. Wiss. Bot. 35: 48-78. pis. 2, 3. NEMEC, B. 1903. Ueber die Einwirkung des Chloralhydrats auf die Kern und Zellteilung. Jahrb. Wiss. Bot. 39: 645-730. figs. 157. NOWIKOFF, M. 1910. Zur Frage nach der Bedeutung der Amitose. Arch. Zellf. 6 : 365-374. figs. 2. OLIVE, E. W. 1904. Mitotic division of the nuclei of the Cyanophyceae. Beih. Bot. Centr. 18: 9-44. pis. 1, 2. 1907. Cytological studies on Ceratiomyxa. Trans. Wis. Acad. Sci. 15: 754-773. pi. 47. PATTERSON, J. T. 1908. Amitosis in the pigeon's egg. Anat. Anz. 32: 117-125. figs. 24. PFEFFER, W. 1899. Bericht iiber amitotische Kerntheilung. Ber. d. Math.-Phys. Kl. d. Kgl. Sachs. Ges. Wiss. 218 INTRODUCTION TO CYTOLOGY PHILLIPS, O. P. 1904. A comparative study of the cytology and movements of the Cyanophyceae. Contrib. Bot. Lab. Univ. Pa. 2 : No. 3. VOM RATH, O. 1891. Ueber die Bedeutung der amitotischen Kerntheilung im Hoden. Zool. Anz. 14: 331, 342, 355. figs. 3. RICHARDS, A. 1909. On the method of cell division in Tcenia. Biol. Bull. 17: 309-326. 1911. The method of cell division in the development of the female sex organs of Monezia. Ibid. 20: 123-178. 8pls. SAGTJCHI, S. 1917. Studies on ciliated cells. Jour. Morph. 29: 217-279. pis. 1-4. SAKAMURA, T. 1920. Experimentelle Studien iiber die Zell- und Kernteilung mit besonderer Riicksicht auf Form, Grosse und Zahl der Chromosomen. Jour. Coll. Sci. Imp. Univ. Tokyo 39: pp. 221. pis. 7. SARGANT, E. 1896. Direct nuclear division in the embryosac of Lilium martagon. Ann. Bot. 10: 107-108. SCHILLER, I. 1909. Ueber kunstliche Erzeugung "primitiver" Kernteilungsformen bei Cyclops. Arch. Entw. 27 : 560-609. figs. 62. SCHURHOFF, P. N. 1915. Amitosen von Riesenkernen im Endosperm von Ranun- culus acer. Jahrb. Wiss. Bot. 55: 499-519. pis. 3, 4. 1919. Das Verhalten des Kerns in den Knollchenzellen von Podocarpus. Ber. Deu. Bot. Ges. 37 : 373-379. SCOTT, D. H. 1888. On nuclei in Oscillatoria and Totypothrix. Jour. Linn. Soc. Bot. 24: 188-192. pi. 5. figs. 1-4. STRASBURGER, E. 1884. Zur Entwicklungsgeschichte der Sporangien von Trichia fallax. Bot. Zeit. 42: 305-316, 321-326. pi. 3. 1907. Apogamie bei Marsilia. Flora 97: 123-191. pis. 3-8. 1908. Chromosomenzahlen, Plasmastrukturen, Vererbungstrager und Reduktion- steilung. Jahrb. Wiss. Bot. 45 ; 479-568. pis. 1-3. TISCHLER, G. 1900. Verh. Naturhist. Med. Ver. Heidelberg 6. TRONDLE, A. 1912. Der Nucleolus von Spirogyra und die Chromosomen hoherer Pflanzen. Zeit. f. Bot. 4: 721-747. pi. 9. WAGER, H. 1903. The cell structure of the Cyanophycese. (Prelim, note.) Proc Roy. Soc. London 72: 401^408. figs. 3. VON WASIELEWSKI, W., 1902, 1903. Theoretische u. Experimentelle Beitrage zur Kenntniss der Amitose, I, II. Jahrb. W. Bot. 38: 377-420, pi. 7; 39: 581, 606. figs. 10. WINGE, O. 1912. Cytological studies in the Plasmodiopho races;. Arkiv. for Botanik 12 : 1-39. pis. 3. VAN WISSELINGH, C., 1914. On the nucleolus and karyokinesis in Zygnema. Rec. Trav. Bot. Neer. 11 : 1-13. YOUNG, R. T. 1913. The histogenesis of the reproductive organs of Tcenia pisifor- mis. Zool. Jahrb. 35: 355-418. pis. 18-21. ZACHARIAS, E. 1887. Beitrage zur Kenntniss des Zellkerns und der Sexualzellen. Bot. Zeit. 45 : 297-304. pi. 4. 1890. Ueber die Zellen der Cyanophyceen. Ibid. 48: 1-. pi. 1. 1892. Ueber die Zellen der Cyanophyceen. Ibid. 50: 617-624. 1903. Jahrb. Hamb. Wiss. Anst. 21. 1907. Ueber die neuere Cyanophyceen-Literatur. Bot. Zeit. 65: 264-287. ZUKAL, H. 1892. Ueber den Zellinhalt der Schizophyten. (Vorl. Mitt.) Ber. Deu. Bot. Ges. 10: 51-55. CHAPTER XI THE REDUCTION OF THE CHROMOSOMES The subject of chromosome reduction is one of the most important to be met with in the study of cytology. Many of the problems, both theo- retical and practical, upon which biological investigators are expending their most intense efforts seem to be bound up directly or indirectly with the reduction of the chromosomes. The essential feature of reduction is relatively simple in nature, and must be thoroughly grasped in order that the discussions in the following chapters may be intelligible. The entire process by which reduction is accomplished, on the other hand, is very complicated and extremely difficult to observe and interpret with any degree of confidence. In spite of the enormous amount of work already done there still exists much difference of opinion regarding some of the significant steps in the series of changes undergone by the nuclear material. In the present chapter a number of these opinions will be reviewed, but our main purpose will be to make clear the fundamental feature of chro- mosome reduction. We have seen that all the cells of the body in a given species are char- acterized by the presence of a certain number of chromosomes in their nuclei, and that this number is held constant throughout development by an equational division of every chromosome at every somatic mitosis. When we speak of "reduction" we ordinarily refer to the fact that at a certain stage in the life history of the organism the number of chromosomes is reduced one-half. This mere change in the number of chromosomes, though very important, is not in itself the essential feature of the reducing process, as will be seen further on. The whole number is restored at the time of fertilization, when two nuclei, each with the reduced number, unite. In all organisms reproducing sexually reduction and fertilization thus represent the two most critical stages in the life cycle so far as the chromosomes are concerned ; hence the exhaustive researches on these two processes. Discovery. — The discovery of reduction was made by van Beneden, who in 1883 announced that the nuclei of the egg and spermatozoon of Ascaris each contain one-half the number of chromosomes found in the body cells. Although van Beneden and other early workers believed that the change in number was brought about by the simple casting out of half the chromosomes during the growth of the germ cells, it was soon shown that this view was incorrect, and that "reduction is effected by a rearrange- 219 220 INTRODUCTION TO CYTOLOGY ment and redistribution of the nuclear substance without loss of any of its essential constituents" (Wilson 1900, p. 233). In plants the discovery of reduction came somewhat later. Stras- burger in 1888 showed that in angiosperms the number of chromosomes in the egg and male nuclei is fixed by a reduction occurring in the mother- cells of the embryo sac and pollen respectively. This was at once con- firmed by Guignard (1889, 1891). E. Overton (1893) found that the female gametophyte cells in the cycad, Ceratozamia, have half the number of chromosomes found in the cells of the sporophyte. He further sug- gested that reduction probably occurs in the sporocytes in mosses and ferns. In the liverwort, Pallavicinia, Farmer (1894) found the gametophyte cells to have four chromosomes and the sporophyte cells eight. That Overton's theory of a reduction in the sporocytes of bryophytes and pteridophytes was correct was demonstrated by Strasburger (1894), who postulated the occurrence of a periodic reduction of the chromosomes in all organ- isms reproducing sexually. The Stage in the Life Cycle at which Reduction Occurs. — The reduc- tion of the chromosomes is accomplished during the course of two nuclear divisions which, since in animals they have to do with the maturing of the gametes, early came to be known as the maturation divisions. Because of its peculiar character the first of these divisions was termed the hetero- typic by Flemming (1887), while the second, which is essentially like a somatic division, was called the homceotypic (sometimes written homo- typic) . Although the essential act of reduction usually occurs at the first division, the entire process, to which the name meiosis has been applied, is of such a nature that the second division is normally necessary for its completion. As a result of the two divisions the "reduced " nuclei or cells are formed in groups of four, or tetrads, though all members of a tetrad may not function. The point in the life cycle at which these divisions take place in various organisms will now be noted. In animals, almost without exception, reduction occurs at gameto- genesis (Fig. 77). In the male those cells (spermatogonia) in the testes whose ultimate descendants are to become spermatozoa multiply by divisions of the ordinary equational type until a certain number are produced. These cells, now called primary spermatocytes, enlarge a little and quickly undergo two successive divisions: the first division in each is heterotypic and results in two cells called secondary spermatocytes; the second is homceotypic and divides the two secondary spermatocytes into four spermatids, each of which becomes transformed into a spermatozoon. The four spermatozoa are therefore the immediate result of the two maturation divisions. In the female the situation is somewhat different: here nearly all of the differentiation of the gamete is accomplished before the nuclear divisions bringing about reduction actually occur. The primary oocytes (ovocytes) are the descendants of a number of generations THE REDUCTION OF THE CHROMOSOMES 221 of oogonia (ovogonia). The oocyte, usually while its nucleus is in the prophases of the first maturation division, enlarges greatly ("growth period"), becomes filled with stored food, and develops the general fea- tures characterizing the egg. The oocyte is now called the "ovarian egg," and it actually is an egg in all respects save one of much importance : its nucleus still has the full number of chromosomes. At a comparatively late stage, in many cases even after the spermatozoon has entered the egg at fertilization, the oocyte nucleus (germinal vesicle), having passed through some of the prophasic changes characteristic of the heterotypic ANIMAL FIG. 77. — Diagram showing the history of the chromosomes in the ordinary life cycles of animals and plants. mitosis before and during the growth period, gives rise to a mitotic figure which is often surprisingly small for the volume of the nucleus. The spindle takes up a position perpendicular to the surface of the cell, and at telophase the chromosomes passing to the outer pole are included in the first polar body, a small cell budded off at this point. (See Fig. 106.) A second spindle is rapidly formed about the chromosomes remaining in the egg (called at this stage the secondary oocyte) and the second matura- tion mitosis occurs, one daughter nucleus being included in the second polar body. In the course of these two divisions chromosome reduction is accomplished. The first polar body may divide to form two, thus completing the tetrad of cells corresponding to the tetrad of spermatozoa in the male. Although the polar bodies are normally functionless they are 222 INTRODUCTION TO CYTOLOGY generally looked upon as eggs historically: the maturation divisions probably resulted formerly in a tetrad of eggs, whereas now only one relatively large and highly differentiated egg is produced at the expense of the other three cells, which remain small and functionless. Among the protozoa (see Minchin 1912) it has been found that in those forms which appear to have their chromatin aggregated into no definite number of chromosomes, there often occur two successive nuclear divisions suggestive in certain respects of maturation divisions, a part of the products then degenerating. Some have regarded this as a "casting out of effete vegetative chromatin," an interpretation which was at one time placed upon the maturation process generally. In many cases this "reduction" of the chromatin occurs immediately prior to syngamy (sexual union),1 and so agrees with reduction in higher forms in taking place at gametogenesis ; but in other cases it immediately follows syngamy, as in certain alga3 mentioned below. Other protozoa have been shown to have a definite chromosome number which is regularly reduced in a man- ner essentially comparable to that in the metazoa. In plants it is among the members of the lower groups (thallophytes) that a striking diversity is shown in the stage of the life cycle at which reduction takes place : in the groups above the thallophytes it is regularly accomplished at sporogenesis. In the myxomycete, Ceratiomyxa, it has been shown by Olive (1907) and Jahn (1908) that spore formation is accompanied by a chromosome reduction. In the green alga3 it is in the first two divisions of the zygote (either a zygospore or a fertilized egg) that reduction occurs: this has been definitely established in Spirogyra (Karsten 1908; Trondle 1911), Zygnema (Kurssanow 1911), Coleochcete (Allen 1905c), and Chara (Oelkers 1916). In a number of other forms, such as Uloihrix, (Edogonium, Sphceroplea, and Closterium, in which the chromosomes are not well known, it is probable that the same condition holds, since the zygote upon germination gives rise with considerable regularity to four cells; in some cases ((Edogonium) these four cells are zoospores. In the BROWN ALG.E Cutleria (Yamanouchi 1912), Zanardinia (Yama- nouchi), and Ectocarpus (Kylin 1918a) reduction occurs in connection with zoospore formation. In Fucus, however, an exceptional condition is found: here reduction takes place in the antheridium and oogonium initials, in the first two divisions following the one delimiting the stalk cell (Fig. 78, B). Since there are only three divisions in the oogonium, which thus produces eight eggs, the eggs are but one division removed from the four products of the maturation mitoses, a condition closely approaching that in animals. That reduction in Fucus is associated with gametogenesis was inferred by Strasburger (1897) and Farmer and Williams (1898) and demonstrated by Yamanouchi (1909). 1 See the cases of Actinophrys sot and Amoeba albida, Chapter XII. THE REDUCTION OF THE CHROMOSOMES 223 In the RED ALG^E reduction occurs in the two divisions differentiating the nuclei of the tetraspores when the latter are present in the life history. Such is the case in Polysiphonia (Yamanouchi 1906) (Fig. 78, A), Gri- ffithsia (Lewis 1909), and Corallina (Yamanouchi). The brown algae Dictyota (Williams 1904) and Padina (Wolfe 1918) also conform to this scheme. In Nemalion, which has no tetraspores, it was long supposed (Wolfe 1904) that reduction occurs in connection with carpospore forma- tion, .but Cleland (1919) has recently shown that it takes place at the time the zygote germinates, as in so many green algse.1 In the ASCOMYCETES reduction occurs in the course of the first two of the three mitoses initiated by the primary ascus nucleus (Figs. 22, 61) and re- sulting in the eight ascospore nuclei. It was for a long time generally thought that there were two nuclear fusions in the life history — one in the archicarp and one in the ascus (see p. 290), and the three di- visions in the ascus were accordingly regarded as a process whose function was to reduce the "quadri- valent" chromosomes to the univalent condition (Harper 1905; Overton 1906). Such a double reduc- tion was described by Miss Fraser (1907, 1908) for Humaria rutilans: the first mitosis she found to be heterotypic, the second homceotypic, and the third "brachymeiotic," the last bringing about a further reduction by the separation of the chromosomes into two smaller groups. This was also reported for Otidea aurantia and Peziza vesiculosa (Fraser and Welsford 1908), Lachnea stercorea, Ascobolus furfura- ceus, and Humaria granulata (Fraser and Brooks 1909), and Helvella crispa (Carruthers 1911). Harper (1900, 1905), although he thought two fusions occurred, found no double reduction, holding rather that the fusion of the two ascus nuclei and their chromsomes is so complete as to render the quadrivalent character of the latter entirely invisible. Other investigators also find no double reduction in the ascus. They show rather that the first two mitoses correspond to the heterotypic and homosotypic mitoses of other organisms, and that the third division is purely vegetative or equational in character. As instances may be cited the work of Faull (1905, 1912) 1 For a review of sexual reproduction and alternation of generations in the algae see Bonnet (1914). Davis (1916) gives a convenient summary of the life histories of the red algse. Dodge (1914) summarizes and compares the life histories of red algae and ascomycetes. See Atkinson (1915) for a complete review of researches on ascomycetes. For the cytology of the yeasts see Guilliermond 1920. B FIG. 78. A, prophase of heterotypic division in the tetrasporocyte of Polysiphonia. (After Yamanouchi, 1906.) B in oogonium of Fucus. (After Yamanouchi, 1909.) 224 INTRODUCTION TO CYTOLOGY on Hydnobolites, Neotiella, and Laboulbenia, and that of Claussen (1912) on Pyronema. Furthermore, it is becoming increasingly apparent (see p. 291) that there is but one fusion in the life cycle — that in the ascus, so that the necessity for a second reduction is removed. In the BASIDIOMYCETES it has been shown by the researches of Juel (1898), Maire (1905), Guilliermond (1910), Kniep (1911, 1913), Levine (1913), and others on the hymenomycetes, and by those of V. H. Black- man (1904), Dietel (1911), Fitzpatrick (1918), and others on the rusts,1 that reduction occurs in the two mitoses giving rise to the four basidio- 6 I? FIG. 79. — Sexual fusion and maturation divisions in the basidium of Nidularia pisiformis. a, two sexual nuclei about to unite. 6, prophase of heterotypic division in fusion nucleus, c, heterotypic mitosis, d, homceotypic mitosis, e, the four basidiospore nuclei. X 1800. (After Fries, 1911.) spore nuclei (Fig. 79). As in the ascomycetes, it thus follows immediately upon the nuclear fusion: in the basidium in hymenomycetes and in the teleutospore in rusts. An exception is reported in the case of Hygro- phorus conicus, in which Fries (1911) finds in the basidium neither a nuclear fusion nor a reduction. In the BRYOPHYTES reduction, so far as known, is universally brought about by the two mitoses which differentiate the four nuclei of each spore tetrad. It was at one time reported (van Leeuwen-Reijnvaan 1907) that in Polytrichum there is a second reduction at spermatogenesis and oogenesis: the sporophyte was said to have 12 chromosomes, the spore and gametophyte six, and the gametes three. This double reduction was thought to be compensated for by the fusion of the ventral canal cell with the egg, raising the number in the latter to six, in combination with the entrance of two sperms into the egg at fertilization, making the sporophytic number 12. This interpretation has been shown to be false by both Vandendries (1913) and Walker (1913), who find the life cycle normal in every respect: a reduction from 12 to 6 occurs at sporo- genesis but no second reduction follows at gametogenesis. 1 A summary of researches on rusts is given by Maire (1911). nuclei in the cells of basidiomycetes is given by Levine (1913). A list of numbers of THE REDUCTION OF THE CHROMOSOMES 225 In VASCULAR PLANTS reduction in all normal life cycles in both homo- sporous and heterosporous forms occurs uniformly in the divisions differ- entiating the spore tetrads (Fig. 77). The sporocytes, particularly the microsporocytes ("pollen mother-cells"), of the higher plants have long been favorite objects for the study of reduction. Since the gametophyte generation in the higher plants is so abbreviated, reduction closely pre- cedes fertilization in these forms. In the ordinary angiosperm embryo sac in which the eight nuclei are derived from a single megaspore of the tetrad, the egg nucleus is removed from the product of reduction (mega- spore nucleus) by only three mitoses. In some cases, of which Lilium is the best known example, walls fail to form be- tween the four megaspore nuclei (Fig. 80, B], leaving them in a common cavity (embryo sac) where they undergo but one further division to produce the eight nuclei of the female gameto- phyte. The egg here is consequently removed from the product of reduction by a single mitosis. In one known case, Plumbagella (Dahlgren 1915), the four reduced nuclei, formed as in Lilium, divide no further, one of them functioning directly as the egg nucleus. Here, therefore, the condition characteristic of animals has been reached : the gamete nucleus is itself the direct product of reduction, and the haploid generation usually produced by the spore is eliminated. The male gametophyte also has undergone much abbreviation in higher plants, but the male nucleus is still removed from the reduction product (microspore nucleus) by two mitoses. In no known case does the micro- spore nucleus function directly as a gamete nucleus. The term gonotokont was introduced by Lotsy (1904) to designate any cell, whatever its origin or position in the life cycle, in which the reduction process is initiated. In animals the gonotokonts are therefore the primary spermatocyte and the primary oocyte. In most green algs& the gonotokont is the zygote; in the red algae it is usually the tetrasporo- cyte; in the ascomycetes it is the ascus; in the basidiomycetes it is the basidium ; and in the bryophytes and vascular plants it is the sporocyte — the microsporocyte and megasporocyte in the case of heterosporous forms. The Meaning of Reduction. — In order that the true meaning of reduc- tion may be appreciated it will be necessary to indicate the main points of a theory first suggested by Roux (1883) and later developed particu- larly by Weismann (1887, 1891, 1892). It had been believed by the earlier workers that reduction was merely a process whose function was "to prevent a summation through fertilization of the nuclear mass and of 15 A FIG. 80. — Megaspore tetrads in Angiosperms. A, tetrad of walled cells in Physostegia virginiana; formation of two upper ones just being completed. X462. (After Sharp, 1911.) B, tetrad of megaspore nuclei in Lilium canadense. 226 INTRODUCTION TO CYTOLOGY the chromatic elements" (Hertwig 1890). But the chromatic mass is actually quartered at reduction, whereas the number of chromosomes is halved. Moreover, great changes in nuclear volume occur with no change in the number of chromosomes. This careful guarding, so to speak, of the chromosome number was siezed upon as a most significant fact by Roux, who "argued that the facts of mitosis are only explicable under the assumption that the chromatin is not a homogeneous substance, but differs qualitatively in different regions of the nucleus; that the collection of the chromatin into a thread and its accurate division into two halves is meaningless unless the chromatin in different regions of the thread represents different qualities which are to be divided and dis- tributed to the daughter cells according to some definite law. He urged that if the chromatin were qualitatively the same throughout the nucleus, direct division would be as efficacious as indirect, and the complicated apparatus of mitosis would be superfluous."1 Upon this conception Weismann based his remarkable theory, the starting point of which was "the hypothesis of De Vries that the chromatin is a congeries or colony of invisible self-propagating vital units or biophores, somewhat like Darwin's 'gemmules,' each of which has the power of determining the development of a particular quality. Weismann conceives these units as aggregated to form units of a higher order known as 'determinants,' which in turn are grouped to form 'ids,' each of which ... is assumed to possess the complete architecture of the germ-plasm characteristic of the species. The 'ids' finally, which are identified with the visible chromatin-granules, are arranged in linear series to form 'idants' or chromosomes. It is assumed further that the 'ids' differ slightly in a manner corresponding with the individual variations of the species, each chromosome therefore being a particular group of slightly different germ-plasms and differing qualitatively from all the others. "We come now to the essence of Weismann's interpretation. The end of fertilization is to produce new combinations of variations by the mixture of different ids. Since, however, their number, like that of the chromosomes which they form, is doubled by the union of two germ- nuclei, an infinite complexity of the chromatin would soon arise did not a periodic reduction occur. Assuming, then, that the 'ancestral germ- plasms' (ids) are arranged in a linear series in the spireme thread or the chromosomes derived from it, Weismann ventured the prediction (1887) that two kinds of mitosis would be found to occur. The first of these is characterized by a longitudinal splitting of the thread, as in ordinary cell-division, 'by means of which all the ancestral germ-plasms are equally distributed in each of the daughter-nuclei after having been divided into halves.' This form of division, which he called equal division (Aequationstheilung), was then a known fact. The second 'This and the following quotations are from Wilson (1900, pp. 245-246). THE REDUCTION OF THE CHROMOSOMES 227 form, at that time a purely theoretical postulate, he assumed to be of such a character that each daughter-nucleus should receive only half the number of ancestral germ-plasms possessed by the mother-nucleus. This he termed a reducing division (Reduktionstheilung), and suggested that this might be effected either by a transverse division of the chromo- somes, or by the elimination of entire chromosomes without division. By either method the number of 'ids' would be reduced; and Weismann argued that such reducing divisions must be involved in the formation of the polar bodies, and in the parallel phenomena of spermatogenesis." Reduction in Weismann's sense, then, is a reduction of the number of kinds of germ-plasm or ancestral hereditary qualities present, this reduction being brought about by means of a redistribution, half of the qualities to one daughter nucleus and the remainder to the other daughter nucleus. The change in the number of chromosomes is a consequence of the manner in which this redistribution is accomplished, as we shall see. Interpretations Based on Weismann's Theory. — As would be ex- pected, there were announced certain interpretations of chromosome be- havior based on Weismann's idea. Several cytologists thought that they found the chromsomes actually dividing transversely at one or the other of the two maturation mitoses. This interpretation, however, proved to be incorrect. Much light was thrown on the problem when Henking (1891), Ruckert (1891, etc.), Haecker (1890-9), vom Rath (1892-3), and others showed that the double chromosomes appearing in the reduced number on the spindle at the first maturation mitosis are not split chromosomes like those seen in somatic divisions, but are pairs of chromosomes, or bivalent chromosomes, each arising by an end-to-end conjugation (synapsis) of two somatic chromosomes. The two parts of each bivalent then separate at the first or second maturation division, the entire chromosomes thus being segregated into two groups, each with the reduced number. Thus it appeared unnecessary that a single chromosome, representing a linear series of different qualities, should be transversely divided in order for Weismannian reduction to occur: it was only necessary to assume that the whole chromosomes differ qualita- tively from one another, so that when the two members of a bivalent pair separate there would be a segregation of different qualities. It is in the light of this bivalent chromosome conception that we are to interpret the many early reports of a transverse division of the chromosome during maturation. What was called a transverse division was merely the separation of two entire chromsomes placed end-to-end. A number of workers soon found that in many cases there is nothing even simulating a transverse division, either of single chromosomes or of bivalent pairs, but that both maturation divisions are apparently longi- tudinal (Flemming, Brauer 1893, Moore 1896, Meves 1896, Gregoire, etc.). How, then, is there any Weismannian reduction if there is neither 228 INTRODUCTION TO CYTOLOGY a transverse division of the chromosome nor a transverse separation of bivalents? Montgomery (1901), von Winiwarter (1900), Sutton (1902), Boveri (1904), and a number of others showed that here the chromo- somes conjugate side-by-side rather than end-to-end. Thus when they separate there is an appearance of a longitudinal division, but reduction is nevertheless accomplished, since entire somatic chromosomes suppos- edly qualitatively different, and not the longitudinal halves of split chromosomes, are separating. The appearance of a longitudinal division may also be present after an end-to-end conjugation, for the two members may bend around to a side-by-side position before finally separating. FIG. 81. — Diagram showing essential difference between somatic and heterotypic mitoses. As a matter of fact, the maturation divisions in nearly all cases, especially those studied by botanists, are both longitudinal in appearance. End- to-end conjugation later came to be called telosynapsis or metasyndese, and side-by-side conjugation parasynapsis or parasyndese. Somatic and Heterotypic Mitoses Compared. — Before taking up a more detailed account of the process of reduction as it has been described by various investigators it is of the utmost importance to fix clearly in mind the essential difference between somatic and heterotypic mitosis in order to realize what constitutes the cardinal feature of reduction, and thereby to detect the significant points of the various theories. This essential difference is illustrated in Figs. 81 and 82. In a somatic or vegetative mitosis every chromosome is split into two exactly similar longi- tudinal halves which are distributed to the two daughter nuclei. The daughter nuclei are therefore like each other and like the mother nucleus in the quality THE REDUCTION OF THE CHROMOSOMES 229 of their substance. In the heterotypic mitosis, the first of the two matura- tion mitoses, the chromosomes conjugate two by two during the prophase to form the reduced number of bivalent chromosomes, which take their place on the spindle. The members of each pair, which are supposed to differ qualitatively from each other, separate and pass to the two daughter nuclei. These nuclei are therefore qualitatively unlike each other, having different members of the full chromosome group; and also unlike the mother nucleus, since each of them has only half as many chromosomes as the latter. The second maturation mitosis (not shown in the diagrams) is essentially a vegetative mitosis in most cases: each chromosome splits longitudinally SOMATIC MITOSIS FIG. 82. — Diagram showing essential difference between somatic and hetero- typic mitoses. and the halves are distributed to the daughter nuclei. The four nuclei, and consequently the four cells, resulting from the two maturation divi- sions are therefore of two kinds : two of them have half of the chromosomes of the original nucleus and the other two have the remaining ones. Assuming, then, that the chromatin of the nucleus represents the principal physical basis of inheritance (see Chapter XIV), reduction is essentially this: a reduction in the number of kinds of hereditary units by the separation and distribution of qualitatively different masses of chromatin to different cells and eventually into different hereditary lines, rather than an equational division and distribution of all the qualities as in somatic mitosis. As has already been stated, the change in the number of chromosomes ("numerical reduction") is a consequence of the method by which this qualitative reduction is brought about, this method being the distribution of entire chromosomes, each representing one or more particular qualities, to different cells. 230 INTRODUCTION TO CYTOLOGY Another important feature of the reduction process should be noted before proceeding further. In many cases, chiefly among animals, the chromosomes appearing on the spindle of the first maturation mitosis are not merely double, but quadruple. This is due to the fact that each of the conjugating chromosomes is already longitudinally split, giving the bivalent chromosome the form of a chromosome tetrad (not to be confused with tetrads of cells or nuclei) . The four constituents of the chromosome tetrad are known as chromatids, and are distributed by the two matura- tion mitoses to the four resulting cells. It is thus seen that in the case of chromosome tetrads the lines of separation for both maturation mitoses are marked out in the prophase of the first. It should be borne in mind that one of these lines represents a plane of chromosome conjugation, and the other a plane of true longitudinal splitting. When the chroma- tids separate along the conjugation plane reduction occurs, whether this be at the first or second mitosis, and when separation along the plane of splitting occurs the mitosis is equational, as in somatic division. . MODES OF CHROMOSOME REDUCTION In all cytology there is scarcely a subject upon which there has been entertained so great a variety of opinion as upon the question of the exact behavior of the chromosomes during the meiotic phases. Entirely aside from the theoretical interpretations placed upon the process of maturation, cytologists have yet failed to arrive at any universally accepted conclusion regarding all the structural changes which occur. This diversity of opinion is due in part to the complexity of the process and the difficulty of interpreting its various stages, some of which fail to stand out clearly in preparations made by our available methods. On the other hand, a great variety of organisms have been studied, and these undoubtedly differ considerably in the details of the reduction process, so that agreement in all particulars is not to be expected. The attempt has too often been made to apply universally an interpretation founded upon a study of one or two organisms. Certain essential fea- tures of meiosis may be expected to show close agreement in all organisms reproducing sexually, as Strasburger pointed out, but it is evident that there is no full correspondence as regards the exact manner in which the essential changes are accomplished. In the following pages are given brief descriptions of a few representative interpretations advanced by various cytologists.1 The two interpretations of reduction which have been most conspicu- ous in the literature of recent years are diagrammed in Figs. 83 and 89. 1 No attempt can be made in a work of this scope to give a complete summary and classification of all the interpretations that have been put upon the maturation phenomena. Only enough will be presented to afford a starting point for a study of this complex subject. For a review and criticism of all views expressed up to 1910 see Gr6goire's two invaluable works (1905, 1910). A useful list of works on somatic and heterotypic mitosis in angiosperms is given by Picard (1913). THE REDUCTION OF THE CHROMOSOMES 231 Nearly all of the accounts of reduction now appearing, especially those given by botanists, conform in general to one or the other of these two schemes, though they vary greatly in detail. Both theories have been upheld by competent observers, and it may be possible that both modes of reduction actually occur; but the same objects have been so differently described by the two opposing schools that it seems very probable that interpretation is chiefly responsible for the persistent diversity of opinion. For convenience the two theories will be referred to as Scheme A and Scheme B. UL7TONEMA STfltPSINLMA FIG. 83. — The method of chromosome reduction according to Scheme A. Explanation in text. Scheme A. — The first of the two main interpretations of reduction came into prominence in 1900 and shortly after, when von Winiwarter (1900), Gregoire (1904, 1907, 1909), A. and K. E. Schreiner (1904-1908), and Berghs (1904, 1905) applied it to the phenomena observed by them in several animals and plants. Its essential points are as follows (Figs. 83-88) : At the beginning of the heterotypic prophase the nuclear reticulum, without breaking down into such distinct elementary nets or alveolar units as are seen in the somatic prophase, takes the form of long slender threads (leptotene or leptonema stage).1 During the very early prophase 1 The terms leplotene, synaptene, pachylene, and diplotene were proposed by von Winiwarter (1900); 'leptonema, zygotene, pachynema, and strepsinema by Gre'goire (1907); amphitene by Janssens (1905); strepsitene by Dixon (1900); diakinesis by Haecker (1897); synapsis by Moore (1896); synizesis by McClung (1905); and meiosis by Farmer and Moore (1905). The terms ending in -tene are ordinarily used as adjectives. 232 INTRODUCTION TO CYTOLOGY these threads conjugate in pairs side-by-side (parasynaptically ; para- syndetically) . The association does not take place at all regions of the threads at once: it begins at one or two points, commonly at one end, FIG. 84. — Reduction in sporocyte of Nephrodium. A, leptonema. B, recovery from synizesis; parallel conjugation consummated during synizesis. C, pachynema. D, second contraction of bivalent spireme. E, diakinesis. F, anaphase of heterotypic mitosis. G, interkinesis. H, homoaotypic mitosis. /, two of the four spore cells. (After Yamanouchi, 1908.) and gradually involves all portions, so that at stages when the process is yet incomplete the two threads may be closely paired at some points and widely divergent at others, giving an appearance very unlike that of the halves of a longitudinally split chromosome in a somatic cell. This is THE REDUCTION OF THE CHROMOSOMES 233 known as the zygotene, zygonema, synaptene, or amphitene stage. Usually before the union is complete the nucleus enlarges somewhat and the threads contract, forming a tight knot at one side of the nucleus. This stage, formerly called synapsis (see p. 255), is now more properly known as synizesis. The pairing threads come into very close association during synizesis, which, though variable in time, usually ensues at about this stage. When the closely paired threads recover from the synizesis con- traction they extend more uniformly throughout the nucleus ("open spireme"), and are now seen to be much thicker t(pachytene; pachynema). In many cases they may again contract into a loose knot with loops extending from it ("second contraction"). As they continue to decrease in length and increase in diameter the members of each pair twist more or less tightly about each other for a short time (strepsitene; strepsinema; diplotene). Eventually they become very short and thick, and the various pairs (gemini; bivalent chromosomes), present in the haploid or reduced number, lie scattered throughout the nucleus (diakinesis) . The two components of each geminus may now separate slightly at one or both ends or at the middle, which gives them the form of Ys, Vs, Xs, and Os. The bivalent chromosomes are now fully formed and ready to take their places on the spindle, which soon forms. In the case of the animal egg the "growth period" introduces a complication. In the sporocytes of plants and the sperm atocytes of animals there is some enlargement of the cell and nucleus during the stages just described, but the chromosomes pass directly from the strepsinema stage to diakinesis. During the relatively enormous growth of the oocyte on the other hand, the chromosomes, which have usually reached the strepsinema stage when the enlargement begins, become greatly modified in form. Their achromatic framework takes the form of fine threads extending out in all directions, giving the chromosome an irregular brush- like form (Fig. 86, C, D), while the chromatic substance either may flow into the nucleolus, leaving the chromosome framework uncolored and very difficult to observe, or by loss of its staining capacity through chem- ical change it may disappear from view completely. As the growth period comes to an end, however, the original staining capacity returns and the chromosomes again assume the compact form and pass into the diakinesis stage. FIG. 85. — Parasynapsis in Phrynotettix magnus. A, leptonema; x, sex- chromosome. B, conjuga- tion of "chromosome A." Portions of other uncon- jugated threads and one other bivalent also present in section. X 2000. (After Wenrich, 1916.) 234 INTRODUCTION TO CYTOLOGY In the case of most animals, and apparently in certain plants also, the split which is to function in the homceotypic mitosis may develop dur- ing diakinesis or even much earlier, the result being the formation of chromosome tetrads. This introduces another element of complication which will be touched upon later (p. 243). B FIG. 86. A, parasynapsis in Allium fistula sum. B,pa,rsisynapsismOsmundaregalis. C, nucleus of oocyte of Scyllium canicula (Selachian) in "growth stage." D, single chromosome in growth stage, showing the fine subdivision of its substance. (A and B after Gregoire, 1907, C and D after Marechal 1907.) The diakinesis stage is terminated by the dissolution of the nuclear membrane and the formation of the spindle, upon which the bivalent chromosomes, whether secondarily split or not, now become arranged. Because of the peculiar form and consistency of the heterotype chromo- somes the mitotic figure presents a striking contrast in appearance to the ordinary figure of somatic cells. This is especially true as the chromo- somes are drawn into various curious shapes as their anaphasic separation begins. The two univalent components of each bivalent chromosome eventually become free from each other and pass to the two daughter nuclei, bringing about reduction. During the anaphase the separating THE REDUCTION OF THE CHROMOSOMES 235 FIG. 87. — Nuclei from microsporocytes of Vicia faba, showing parasynapsis. Synigesis beginning in No. 6. X 1900. Fia. 88. — Heterotypic prophases in spermatocyte of Tomopteris onisciformis. A, pairing of leptotene threads beginning. B, pairing complete in some threads and only beginning in others. C, conjugation complete; pachynema stage. D, resplittirig of pachytene threads (separation of conjugated chromosomes.) (After A. and K. E. Schreiner, 1905.) 236 INTRODUCTION TO CYTOLOGY univalents, if not already double, rapidly develop a longitudinal split, in some cases even before they are entirely free from each other. The resulting halves tend to open out along this split; chromosomes being drawn endwise to the poles thus take the form of simple Vs, while those to which the fibers are attached at the middle appear as double Vs. After reaching the poles the split chromosomes begin the reconstruction of the daughter nuclei. As a rule this does not proceed very far, since the horn ceoty pic mitosis follows very quickly upon the heterotypic. Well organized daughter nuclei are often formed, whereas in the animal egg there may be no reconstruction whatever, the daughter chromosomes of the first mitosis at once taking their places on a newly formed spindle for the second mitosis. In the homceotypic mitosis the chromosomes, if there has been an inter- vening interkinesis of any length, usually appear much longer and thin- ner than in the heterotypic mitosis, and separate along the longitudinal line of fission seen in the preceding anaphase. The homceotypic mitosis is therefore equational in character, and differs from an ordinary somatic mitosis only in the number of its chromosomes and the precocity of their splitting. In each of the four nuclei resulting from the two maturation mitoses there is now the haploid number of univalent chromosomes, and meiosis is complete. The foregoing interpretation of reduction has been widely accepted from the first by both botanists and zoologists. The following is a partial list of works in which it has been described. PLANTS Gr6goire 1904, '07 Lilium, Allium, Osmunda Berghs 1904, '05 Allium, Drosera, Hetteborus, etc. Rosenberg 1905, '07, '08, '09 Drosera, Composite Allen 19056c Lilium, Coleochcete J. B. Overton 1905, '09 Thalictrum, Calycanthus, Richardia Strasburger 1905, '07, '08, '09 Lilium, Galtonia, etc. Miyake 1905 Lilium, Funkia, Iris, Allium, Trades- cantia, Galtonia Tischler 1906 Ribes Cardiff 1906 Acer, Salomonia, Botrychium, Ginkgo Lagerberg 1906, '09 Adoxa Yamanouchi 1906, '08, '10 Polysiphonia, Nephrodium, Osmunda Martins Mano 1909 Funkia Lundegardh 1909, '14 Trollius Frisendahl 1912 Myricaria McAllister 1913 Smilacina Schneider 1913, '14 Thelygonium Weinzieher 1914 Xyris Sakamura 1914 Vida de Litardiere 1917 Poly-podium 237 ANIMALS von Winiwarter Marechal 1900 1904, '05, '07 A. and K. E. Schreiner 1906, '07, '08 Lerat 1905 Deton 1908 Gregoire 1909 Janssens 1905, '09 Janssens et Willems 1909 Schleip 1906, '07 Debaisieux 1909 Montgomery 1911 Kornhauser 1914, '15 Wenrich 1916, '17 Fasten 1914, '18 Malone 1918 Pratt and Long 1917 Robertson 1916 Rabbit, Man Tunicates, Selachians, Teleosts, Amphi- oxus Tomopleris, Ophryotrocha, Zoogonus, Enteroxenos, Myxine, Salamandra, Spinax Cyclops Thysanozoon Zoogontis Batracoseps Alytes Planaria Dytiscus Hersilia, Enchenopa Phrynotettix, Chorthippus Cambarus, Cancer Cants Mus Insects "^ FIG. 89. — The method of chromosome reduction according to Scheme B. Explanation in text. Scheme B. — The second of the two conspicuous interpretations was advanced by Farmer and Moore (1903, 1905), and is essentially as follows (Figs. 89-92) : In the early heterotypic prophase the reticulum becomes more thready in structure and contracts into a tight knot (synizesis). When this knot loosens up the chromatic material has 238 INTRODUCTION TO CYTOLOGY assumed the form of a continuous spireme which is double. This double- ness is believed to represent a true longitudinal split, and although it usually disappears from view during the later prophases it is thought to A B c FIG. 90. — The heterotypic prophases in Lilium, according to Mottier (1907.) A, synizesis knot loosening up; threads splitting; note chromomeres. B, hollow spireme. C, second contraction. D, diakinesis. X 900. FIG. 91.— Maturation mitoses in microsporocyte of Vicia faba. A, anaphase of heterotypic mitosis; split for second mitosis evident in separating daughter chromosomes. B, one daughter nucleus in early telophase of heterotypic mitosis. C, later telophase. D, metaphase of homoeotypic mitosis. E, anaphase of same, showing portions of both spindles. F, three of the four microspore nuclei. X 1335. (After Fraser, 1914.) persist and reappear at a much later stage. After extending loosely throughout the nucleus ("open spireme"), the double spireme, now con- siderably thickened and twisted (strepsinema) , contracts again and is THE REDUCTION OF THE CHROMOSOMES 239 thrown into loops (" second contraction"). These loops then break apart from one another through a segmentation of the spireme; each of them is composed of two split chromosomes arranged end-to-end. Chro- mosome conjugation has thus occurred telosynaptically (metasyndetic- ally] either while the spireme was being formed or when the daughter spiremes were formed in the preceding telophase. The two members of each pair are brought around to a side-by-side position by the looping at the second contraction, usually but not always remaining closely connected at the original point of conjugation. The resulting bivalent chromosomes, with their split obscured, become much shortened and thickened (diakinesis) and take up their positions on the first maturation spindle. In case the original split, instead of being wholly obscured, is visible at this time or earlier, chromosome tetrads are evident. In the heterotypic anaphase the bivalents are separated into their component univalents, bringing about reduction. During the anaphase the uni- valents often widen out along the line of fission which had been tempo- rarily obscured, giving them the form of simple or double Vs as described for Scheme A. They remain through interkinesis in the double condi- tion, and in the homoeotypic mitosis separate along this line of fission. The following is a list of the principal works in which this theory of eduction has been advocated. PLANTS Farmer and Moore 1903, '05 Lilium, Osmunda, Psilotum, Aneura Farmer and Digby 1910 Gallonia Farmer and Shove 1905 Tradescantia Mottier 1907, '09, '14 Lilium, Acer, Allium, Podophyllum, Tradescantia, Staphylea Gregory 1904 Ferns Lewis 1908 Finns, Thuja Schaffner 1906, '09 Agave Digby 1910, '12, '14, '19 Galtonia, Primula, Crepis, Osmunda Fraser 1914 Vicia Lawson 1912 Smilacina McAvoy 1912 Fuchsia Beer 1912, '13 Equisetum, Crepis, Tragopogoh Woolery 1915 Smilacina Nothnagel 1916 Allium ANIMALS Farmer and Moore 1905 Periplanela, Elasmobranchs Montgomery 1903, '04, '05, '06, Hemiptera, Amphibia '10 Moore and Embleton 1906 Amphibia Griggs 1906 A scans Zweiger 1907 Forficula H. S. Davis 1908 Insects Nakahara 1920 Perla 240 INTRODUCTION TO CYTOLOGY Some of the above named investigators, notably Miss Digby (1910, 1912, 1914, 1919), Miss Fraser (1914), and Miss Nothnagel (1916), have laid emphasis upon the view that the split seen in the early hetero- typic prophase has its origin in the telophase of the last premeiotic divi- sion, each chromosome persisting through the intervening resting stage in the double condition. It is consequently held, as fully stated by Miss Digby (1919) in her account of the archesporial and meiotic phases of Osmunda (see Fig. 92), that the lateral pairing of thin threads in the LAST PRtHtlOTlC IC MITOSIS FIG. 92. — Diagram showing behavior of chromosomes in premeiotic and meiotic phases in Osmunda, according to Digby (1919). a, split which originates in telophase of premeiotic mitosis, persists (though obscured at times) through heterotypic prophases, reappears in heterotypic anaphase, and becomes effective in homceotypic mitosis, b, split which originates in heterotypic telophase, persists obscured through homceotypic prophases, reappears in homceotypic anaphase, and becomes effective in post-homceotypic division, x, plane of conjugation. heterotypic prophase which the advocates of Scheme A have regarded as a conjugation of entire chromosomes is in reality only the reassocia- tion of the two halves of one chromosome which had been split in the preceding telophase. Such a reassociation is thought to occur in every prophase, somatic and heterotypic, since these workers regard chromo- some splitting as regularly a thlophasic phenomenon. The split which forms in the last premeiotic telophase functions in the homceotypic mitosis : the homceotypic division is therefore looked upon as the continua- tion of the premeiotic division, the heterotypic mitosis being an inter- polated process bringing about numerical reduction. Not only does this premeiotic split reappear in the anaphase of the heterotypic mitosis to function in the homceotypic, but a new split develops in the heterotypic THE REDUCTION OF THE CHROMOSOMES 241 telophase, and after being temporarily obscured functions in the post- homceotypic division.1 A variation of Scheme B has been observed in (Enothera (Gates 1908, 1909, 1911; Geerts 1908; B. M. Davis 1909, 1910, 1911); in Fucus and Cutleria (Yamanouchi 1909, 1912) ; in Bufo (King 1907) ; and in a few other forms. Here the spireme in the heterotypic prophase does not become double, the split for the second division appearing first in the heterotypic anaphase. Comparison of Schemes A and B. — According to both of the fore- going prominent theories of reduction the conjugated chromosomes separate at the first maturation mitosis, thus causing reduction, and v@o v A (!S V (il*. FIG. 93. — -Diagram showing distinction between Schemes A and B. See text. divide longitudinally (equationally) at the second mitosis, so that the final result is essentially the same: two of the resulting four nuclei differ qualitatively from the other two in their chromatin content (Fig. 93). The distinction between the two interpretations is nevertheless an im- portant one, and may be emphasized in the following summary. According to Scheme A the double character of the chromatin spiremes of the early heterotypic prophase is due to a lateral pairing of simple threads each representing an entire somatic chromosome, the second con- traction not being significant as regards pairing. The bivalent chromo- somes so formed, after much shortening and thickening, are separated in the heterotypic mitosis, during the anaphase of which (or earlier in the 1 A more detailed summary of this view may be found in a review of Miss Digby's paper on Osmunda by the present author (1920a). 16 242 INTRODUCTION TO CYTOLOGY case of chromosome tetrads) the split that is to function in the homoeotypic mitosis makes its appearance. The double ness in the heterotypic pro- phase is therefore not homologous with that in the somatic prophase: in the former it is due to a conjugation and in the latter to a split. According to Scheme B the doubleness of the heterotypic prophase is due to a true splitting as in the case of somatic division. In both cases, moreover, the split may have its origin in the preceding telophase. The bivalent chromosome is formed by the association in pairs (often at first end-to-end in the spireme but later side-by-side) of segments of this split spireme at the time of the second contraction. The two split univalents composing the bivalent are separated in the heterotypic mitosis, while in the homceotypic mitosis the separation is along the line of the split originating in the last premeiotic telophase and seen in the spireme of the early heterotypic prophase. The doubleness of the early heterotypic prophase is therefore regarded as homologous with that of the somatic prophase: in both cases it represents a true split. It cannot yet be said what the outcome of this controversy is to be. The advocates of Scheme A believe that those of Scheme B have mis- interpreted the changes occurring in the early heterotypic prophase and in all telophases, while the latter charge the former with a neglect of the second contraction stage. Scheme B as fully elaborated by Miss Digby has certain advantages: it allows one interpretation to be placed upon the double spireme in both somatic and heterotypic prophases, irrespective of the exact time at which the split originates, and it also helps to explain the sudden appearance of the split for the second maturation mitosis in the anaphase of the first. Scheme A, on the other hand, is preferred by geneticists because of the earlier and much longer continued association of the conjugating chromosomes, which allows a greater opportunity for "crossing-over" to occur. The significance of this point will be brought out in Chapter XVII. This question, however, must be settled primarily by direct evidence. It is obvious that its solution depends upon the exact manner in which the telophasic transformation of the chromosomes and the derivation of the latter from the reticulum in the prophase are accomplished. It is granted by both schools that the alveolar or reticulate condition in which the chromosomes are found in late telophase is continuous with the similar condition seen in the succeeding prophase. If, then, it is true (1) that the telophasic transformation (alveolation) represents a true splitting, and (2) that the early prophasic reticulate condition passes directly into the double spireme, it follows that this doubleness in every prophase is due to the split originating in the preceding telophase. But workers on mitosis are not at all agreed that the evolution of the chromosomes is that stated in (1) and (2). It has been shown in Viciafaba (Sharp 1913), Tradescantia (Sharp 19206), and a number of other instances (see Chapter THE REDUCTION OF THE CHROMOSOMES 243 VIII) not only that the telophasic alveolation is too irregular to be regarded as a splitting, but also that the reticulate condition of the pro- phase, instead of developing directly into the definitive split, gives rise to simple thin threads in which a new split is developed. From this it cannot be concluded that in no form does the split develop directly from the early reticulate condition, or that the telophasic alveolation, though irregular, may not later become so equalized as to constitute the first stages of the split; but it does follow that it is quite unsafe to use the principle of telophasic splitting as a premise from which to draw the conclusion that the approximation of thin threads in the early heterotypic prophase represents the reassociation of the halves of a single split chromosome. It is well to emphasize the possible importance of the premeiotic telophase, but any ultimate solution of this perplexing prob- lem must be reached mainly through a more refined analysis of those FIG. 94. — Chromosome pair "B" in Phrynotettix magnus, showing condensation of bivalent pair during the heterotypic prophases to form the compact chromosomes appearing on the spindle at metaphase. X 1734. (After Wenrich, 1916.) prophasic changes which have led a long list of investigators to the con- clusion that the early heterotypic association of slender threads represents a conjugation of entire chromosomes which separate in the first matura- tion mitosis. One of the most convincing pieces of direct evidence favoring Scheme A is found in Wenrich's recent work on Phrynotettix (1916). Wenrich is able to trace a single pair of chromosomes, distinguishable by their peculiar form and the arrangement of their chromatic accumulations or chromomeres, through every stage from the spermatogonia to the sperma- tids. During the heterotypic prophase the two members of the pair conjugate parasynaptically while in the form of slender filaments. Simi- larly strong arguments are advanced by Robertson (1916) as the result of his detailed analysis of the chromosome groups in other Tettigidse and Acrididse, in which the homologous members can be followed with much certainty because of their frequent inequality in size. Reduction With Chromosome Tetrads. — As already pointed out, the marking out of the lines of separation for both maturation divisions during the heterotypic prophase, with the resulting formation of chromo- some tetrads, increases in no inconsiderable manner the difficulty of interpreting the essential changes at these stages. The four chromatids composing the tetrad represent two conjugated chromosomes each of which is longitudinally split. Because of the variety of ways in which 244 INTRODUCTION TO CYTOLOGY these may arrange themselves with reference to one another — in the form of simple or compound rods, crosses, and rings — their distribution to the daughter nuclei, as well as the manner of their origin, is very difficult to follow with certainty. The accompanying diagrams will serve to illus- trate the more common modes of behavior described for chromosome tetrads, which are found chiefly in the cells of animals. Figure 95, D represents an exceptional method of tetrad formation described by Henking (1891) f or Pyrrochoris and by Korschelt (1895) for Ophryotrocha. The continuous spireme segments to form the diploid number of chromosomes,1 which then split longitudinally and shorten. FIG. 95. — Reduction with chromosome tetrads. D, in Pyrrochoris (Henking) and Ophryotrocha (Korschelt.) E, in certain copepods (Riickert, Haecker, and vom Rath.) F, in Anasa and Allolobophora (Paulmier; Foot and Strobell). No conjugation occurs until the metaphase, when the split chromosomes come together end-to-end, forming tetrads. They at once separate in the anaphase, bringing about reduction. In the second mitosis they divide along the original split, so that each of the four resulting nuclei receives the haploid number of chromosomes, two of the nuclei thus differing from the other two as the result of the separation of entire (though secondarily split) chromosomes at the first mitosis. According to Goldschmidt (1905), the chromosomes of Zoogonus mirus, after thus undergoing 'no prophasic conjugation, divide longitudinally at the first 1 For the sake of uniformity and clearness the diploid number is represented as 6 in all of these diagrams. THE REDUCTION OF THE CHROMOSOMES 245 mitosis and separate into two haploid groups at the second. To this simple form of reduction Goldschmidt applied the term "Primsertypus." Gregoire (1909a), on the contrary, found parasynapsis and the usual mode of reduction in Zoogonus. The interpretation at one time given by Riickert (1893, 1894), Haecker (1895), and vom Rath (1895) for certain copepods is shown* in Fig. 95, E. The continuous spireme splits throughout its length and then breaks into the haploid number of segments. These again break trans- versely, forming chromosome tetrads, each composed of two split chromo- somes arranged end-to-end. In some species the chromatids open out to form four-parted rings, whereas in others they maintain the rod form. A separation occurs along the line of the original split at the first mitosis, which is therefore equational, and along the plane of conjugation at the second mitosis, which is therefore reductional. In Dicrocodium Gold- schmidt (1908) reported that such tetrads divide reductionally at the first mitosis. Lerat (1905), moreover, has found that in Cyclops strenuus, one of the forms used by the earlier workers, the tetrads arise by a parallel conjugation of thin threads which later split. A third mode of tetrad behavior is that reported by Paulmier (1899) for Anasa tristis and by Foot and Strobell (1905, 1907) for Anasa and Allolobophora foetida (Fig. 95, F). Here the chromosomes conjugate end-to-end, the bivalents so formed then splitting longitudinally, giving tetrads which take on a cross or ring form. At the first mitosis the sepa- ration is along the plane of conjugation, effecting reduction, and at the second it is along the plane of splitting. According to McClung (1902), Button (1902, 1905), Robertson (1908), and others, such tetrads separate reductionally at the second mitosis (postreduction) rather than at the first (prereduction) in certain orthopterans studied by them. Figure 96 illustrates the origin of chromosome tetrads of five charac- teristic types by the two prominent modes of reduction described in detail in foregoing pages. According to Scheme A (Ai-Ci), two chromo- somes conjugate parasynaptically while in the form of slender threads. Instead of remaining unsplit as in most plants, each member then splits longitudinally in a plane at right angles to the conjugation plane, thus giving a tetrad composed of four parallel strands (chromatids) (D). According to Scheme B (Az-Cz), the two chromosomes are at first ar- ranged telosynaptically in the spireme and the latter splits throughout its length. The two conjugating members then take up a side-by-side position, and their split, instead of becoming obscured as usually occurs in plants, remains open, giving the tetrad of parallel strands (D). The tetrad, by whichever method it has arisen, may now undergo a variety of alterations, some of which are shown at E and F. The chroma- tids may simply shorten and thicken, the tetrad at diakinesis maintaining the form of parallel rods (Ei, FI). They may open out alongjhe plane of INTRODUCTION TO CYTOLOGY conjugation (E£) and take the form of rod tetrads (F2) like those described by Riickert and Haecker. While opening out in this manner the longi- FIG. 96. — Diagram showing the origin of the tetrad of chromatids (D) according to Scheme A (A\-C\) and Scheme B (Az-C*), and the further transformation of this tetrad into tetrads of five types (Fi-F$). tudinal halves of each chromosome may diverge where the two chromo- somes remain in contact (£"3), the tetrad eventually taking the form of a cross (F3) as in the cases described by Paulmier and by Foot and StrobelL THE REDUCTION OF THE CHROMOSOMES 247 If the conjugated chromosomes remain in contact at both ends (#4) a complete ring results (F4). In certain orthopterans the four chromatids open out along the conjugation plane in some regions and along the plane of splitting in other regions; this results in the curious compound rings (Fig. 156) found in the cells of these insects. Finally, the chromatids may open out from one end along the conjugation plane and from the other end along the splitting plane (EB), the tetrad then assuming the form of a ring composed of four parts (F5). In all cases the tetrads usu- ally condense into compact quadruple bodies by the time they take their places on the spindle of the heterotypic mitosis. FIG. 97. — Reduction with chromosome tetrads in Fasciola hepatica, according to Schellenberg (1911). Explanation in text. The four chromatids composing the completed tetrad are in most cases exactly similar in appearance, so that it is a matter of much difficulty to determine along which plane they are separated at the first maturation mitosis. According to the two theories of tetrad origin illustrated in the foregoing diagram, however, the chromatids are supposed in almost all cases to separate along the plane of conjugation at the first mitosis, and this conclusion is supported by the behavior of those bivalent chromo- somes which are not divided into tetrads of chromatids. A further interpretation of reduction involving chromosome tetrads has been given by Schellenberg (1911) for the parasitic flatworm, Fasciola hepatica (Fig. 97) . The chromatin in the heterotypic prophase takes the form of a long slender filament which splits longitudinally soon after synizesis. This double thread then segments into the haploid number of 248 INTRODUCTION TO CYTOLOGY pieces, each representing two chromosomes end-to-end; these have the form of loops with a definite orientation ("first boquet stage"). Each segments again, giving the diploid number of split chromosomes, which again assume the form of oriented loops ("second boquet stage"). The halves twist tightly about each other, shorten to form the double bodies seen at diakinesis in the diploid rather than the haploid number, and then conjugate to form the haploid number of chromosome tetrads. The conjugating members (each split) separate at the first mitosis, bringing about reduction; at the second mitosis the separation is along the line of the original split. According to this interpretation, therefore, the double- ness of the early heterotypic prophase is due to a split, as in Scheme B, but the chromosomes arranged end-to-end in the spireme soon become separated and do not conjugate again until diakinesis. For a number of years it was thought (Carnoy 1886; Boveri 1887; Hertwig 1890; Brauer 1893) that the chromosome tetrad in Ascaris megalocephala was exceptional in being formed by two longitudinal fis- sions of a primary chromatin rod, there being as a consequence no quali- tative reduction in the two maturation divisions unless the organization of the chromatin were different from that of other organisms. But it has since been shown that they arise as in other organisms by the conjugation of two split chromosomes (Sabaschnikoff 1897; Tretjakoff 1904; Griggs 1906). In the oogenesis Griggs reports telosynapsis with prereduction, whereas in the spermatogenesis Tretjakoff describes parasynapsis followed by postreduction. In Ascaris canis (Marcus 1908; Walton 1918) the four chromatids each show a transverse constriction, the chromosomes on the first maturation spindle having the form of octads. Although the formation of well differentiated chromosome tetrads occurs very commonly in animals, it appears to be very rare in plants. Farmer (1895) described tetrads in Fossombronia, and they have since been reported in at least three other bryophytes: Pallavicinia (Moore 1905), Sphagnum (Melin 1915), and Chiloscyphus (Florin 1918). They have also been described in a few vascular plants : Equisetum (Osterhout 1897), Pteris (Calkins 1897), Ariscema (Atkinson 1899), Tricyrtis (Ikeda 1902), Thalictrum, Calycanthus, and Richardia (Overton 1909) (Fig. 98), Spinacia (Stomps 1911), Primula (Digby 1912), and Lopezia (Tackholm 1914). According to Gregoire (1905) such structures in plants are not true tetrads, but resemble them because the chromosomes are often bent and have their material accumulated largely at their ends. Sakamura (1920) interprets them as conjugated constricted chromosomes, and denies that the quadripartite condition has anything to do with reduction in such cases. He likewise accounts for the metasynaptic rod tetrads (Fig. 95, E) described by several investigators of maturation in animals, holding that they represent two constricted chromosomes conjugated THE REDUCTION OF THE CHROMOSOMES 249 parasynaptically rather than two split ones placed end-to-end. In support of this contention he cites the following observations: such "tetrads" are seen not only in the oocytes and spermatocytes but also in oogonia, spermatogonia, and somatic cells; the supposed telosynaptically conjugated members are often very unequal in size; such tetrads are sometimes divided in the transverse plane at neither maturation mitosis ; not only tetrads, but also octads and hexads are often observed, even in FIG. 98. — Chromosome tetrads. A, five stages in the development of the tetrad in the spermatocyte of Anasa tristis. X 3000. From a preparation by Dr. H. E. Stork. B, tetrads in sporocyte of Chiloscyphus. Enlarged; X 2800. (After Florin, 1918.) C, tetrads in Richardia africana. (After Overton, 1909.) D, false tetrads in somatic cells of Pisum due to action of chloral hydrate on constricted chromosomes. (After Sakamura, 1920.) the same cell, and these are plainly due to the presence of additional accentuated constrictions. Robertson (1916) also interprets such telo- synaptic rod tetrads as those observed by Haecker in the copepods as constricted chromosomes. The constrictions, according to this writer, represent points of temporary union between non-homologous elements. From these considerations it is evident that constrictions have much to do with the appearances assumed by chromosomes, and that they should be taken into account in interpreting the chromosome tetrad. Numerical Reduction Without Qualitative Reduction. — Figure 99 illustrates the behavior of the chromosomes in maturation according to three not widely accepted views. A few workers, including Fick (1907, 250 INTRODUCTION TO CYTOLOGY 1908), Meves (1907, 1908, 1911), Giglio-Tos (1908), and Granata (1910), reject the theory of chromosome individuality and specificity, and therefore do not regard the chromosomes which are distributed to the four cells at maturation as at all identical with those of the divisions im- mediately preceding, except in so far as they are composed of the same nuclear material. Accordingly they, recognize no qualitative reduction, but only a numerical one. This reduction in number results from the fact that the spireme formed in the heterotypic prophase (Fig. 99, A) segments into the haploid number of pieces instead of the diploid number, these pieces being simply divided longitudinally at both maturation divisions, and the four resulting nuclei being qualitatively similar. FIG. 99. — Diagram showing three reported modes of numerical reduction with- out qualitative reduction. A, according to Fick et al. B, according to Vejdowsky; complete fusion of conjugating members. C, according to Bonne vie; bivalents arranged on spindles in juxtaposition; fusion of conjugating members eventually becomes complete. According to Vejdowsky (1907) (Fig. 99, B} the chromosomes appear in diploid number in the heterotypic prophase and conjugate parasynapti- cally. The members of the pair fuse completely and lose their individual identity, so that the chromosomes appearing on the first maturation spindle in haploid number are new entities, and not merely temporary pairs of somatic chromosome individuals. At both divisions these bodies split longitudinally, giving equivalence to the four resulting nuclei. Here, as in the foregoing example, there is no definite qualitative reduc- tion in Weismann's sense, though a numerical reduction is brought about by means of a complete fusion at the time of chromosome conjugation. THE REDUCTION OF THE CHROMOSOMES 251 An interpretation put forward by Bonnevie (1906, 1908) is shown in Fig. 99, C. Here the chromosomes conjugate parasynaptically and come into very intimate union: although they appear to undergo a real fusion their identity is maintained for a time. Owing to the fact that these bivalent chromosomes are inserted upon the spindle with their halves in juxtaposition (side-by-side with respect to the poles) rather than in superposition (one toward each pole), the members of a conjugated pair separate neither at the first division nor at the second. As a result each of the four cells receives the haploid number of chromosomes, all of which are bivalent, and no qualitative reduction occurs. Bonnevie believes that the conjugating members of each pair finally fuse completely in the subsequent stages. In this case, therefore, as in the preceding one, numerical reduction is supposed to result from a complete fusion of the chromosomes in pairs. Whether any confidence is to be placed in such interpretations or not — and according to most cytologists none should be — they at least serve to show how it is possible that numerical reduction may occur without effecting any qualitative reduction, and that the essential feature of the reduction of the chromosomes is something other than the mere change in their number, as pointed out at the beginning of the chapter. SYNAPSIS, OR CHROMOSOME CONJUGATION The phenomenon of chromosome conjugation, or synapsis, which we have seen above is such an important feature of the reduction process, must now be somewhat more closely examined. Attention will be directed to three points: the relationship of the conjugating members (the "synaptic mates"); the stage at which the synaptic union takes place, and the exact nature of this union. Relationship of the Synaptic Mates. — We may first inquire into the relationship which may exist between the two chromosomes pairing to form a given bivalent chromosome: is any chromosome of the duplex group (the two intermingled parental chromosome sets in the individual's nuclei) present in the gonotokont free to pair with any other chromosome, or does the pairing take place according to more restricting rules? It was suggested by Henking (1891) that the two synaptic mates are ultimately derived from the two parents at the previous fertilization, one from the father and the other from the mother: the chromosomes of one parental set pair with those of the other parental set to form the haploid number of bivalent chromosomes appearing on the first matura- tion spindle. This idea was later emphasized and developed by Mont- gomery (1900-4), Sutton (1902), Boveri (1901), and others, who found for it much supporting evidence in organisms with chromosomes differ- ing in size and shape. An observation made by Rosenberg (1909) on Drosera hybrids is significant in this connection. When Drosera rotundi- 252 INTRODUCTION TO CYTOLOGY folia (20 chromosomes) ' is crossed with D. longifolia (40 chromosomes) there results a hybrid with 30 chromosomes, of which 10 are contributed by rotundifolia and 20 by longifolia. When synapsis occurs preparatory to reduction in this hybrid only 10 bivalents are formed, 10 chromosomes remaining unpaired. This was taken by Rosenberg to mean that the 10 rotundifolia chromosomes pair with 10 of the longifolia ones, leaving the other 10 of longifolia without synaptic mates. Had any chromosome of the duplex group of 30 been free to pair with any other, 15 bivalents would have been produced. Other instances of this phenomenon may be mentioned. By crossing (Enothera Lamarckiana (seven chromosomes in gamete) with (E. gigas (14 in gamete) individuals with 21 chromosomes are obtained. Geerts (1911) found that, preparatory to reduction, the seven Lamarckiana chro- mosomes pair with seven of the gigas chromosomes, leaving the other seven of gigas unpaired. On the contrary, however, Gates (1909) found that the 21 chromosomes in a lata-gigas hybrid simply separate into two approximately equal groups, usually of 10 and 11 chromosomes re- spectively. Kihara (1919) reports that in some 35-chromosome wheat hybrids formed by crossing Triticum polonicum (14 chromosomes in gamete) with T. spelta (21 in gamete) there are present in the heterotypic prophase 14 bivalents (polonicum conjugated with spelta) and seven univalents (spelta). The 14 bivalents are arranged on the spindle and separate as usual, whereas the seven unpaired spelta chromosomes split longitudinally at the first mitosis and distribute themselves irregularly at the second (Fig. 100). An analogous condition is found in Pigcera hybrids by Federley (1913). A very significant additional suggestion with respect to synapsis was made by McClung (1900) and Sutton (1902): not only are the two chromo- somes which conjugate derived from the two parents, but they are hom- ologous— each chromosome of one parental set pairs with a particular chromosome of the other parental set, the two members of the resulting bivalent being presumably of corresponding hereditary value, as will be shown in Chapter XV. The evidence for this important hypothesis was found chiefly in Brachystola (Fig. 101) and a number of other insects having chromosome complements made up of members with constant characteristic differences in size and shape. Many such cases have been subsequently discovered, especially by McClung and his eoworkers in their extensive researches on insect spermatocytes. As examples among plants may be cited Crepis virens, Najas major, N. marina, and Vicia faba. Crepis virens (Rosenberg 1909) (Fig. 102) has six chromosomes: two long, two medium sized, and two short. When synapsis occurs the like chromosomes pair, forming bivalents of three sizes. The members of each pair separate and pass to the daughter cells at the first maturation THE REDUCTION OF THE CHROMOSOMES 253 mitosis, each microspore (after the second mitosis) having as a result three chromosomes: one long, one medium sized, and one short. Since the gamete receives such a simplex group of three chromosomes, and the Fio. 100. — Heterotypic mitosis in Triticum potonicum X T. spelta. A, the 21 chromosomes (polar view). B, 14 bivalents separated into component univalents; 7 unpaired spelta chromosomes have split and are about to be distributed. (After Kihara, 1919.) somatic cells of the new individual show six (two of each length), it is evident that the other gamete furnishes a similar simplex group of three. FIG. 101. — The chromosome complement in the spermatocyte of Brachystola magna. (After Sutton, 1902.) In Najas marina and Najas major (Miiller 1911; Tschernoyarow 1914) the duplex group of 14 chromosomes is made up of seven visibly different pairs (Fig. 56 bis). In the heterotypic prophase these conjugate selectively to form seven bivalents, the reduced nuclei therefore receiving 254 INTRODUCTION TO CYTOLOGY a set of seven visibly different chromosomes. Sakamura (1920) holds the number here to be six rather than seven. (See p. 160.) In Vicia faba (Sharp 1914; Sakamura 1915, 1920) there are in the somatic cells 12 chromosomes, two of them being about twice as long as the other 10 (Figs. 56 and 102). At synapsis in the microsporocyte there are formed six bivalents, one of them having about twice the length of the other five. Hence it is clear that the two long chromosomes pair with each other. In the heterotypic mitosis the synaptic mates separate SIMILAR PROCESS IN . Ueber Sporen an Geschlechtspflanzen von NitophyUum punctatum, usw. Ibid. 32: 106-116. pi. 2. 1 fig. SYKES, M. G. 1908. Nuclear division in Funkia. Arch. Zellf. 1: 381-398. pis. 8, 9. 1 fig. TACKHOLM, G. 1914. Zar Kenntniss der Embryosackentwicklung von Lopezia coronala Andr. Svensk. Bot. Tidskr. 8. TAHARA, M. 1910. Ueber die Kernteilung bei Morus. Bot. Mag. Tokyo 24: 281-289. pi. 9. TAYLOR, M. 1914, 1917. The chromosome complex of Culex pipiens. Quar. Jour. Micr. Sci. 60: 377-398. pis. 27, 28; 62: 287-302. pi. 20. THE REDUCTION OF THE CHROMOSOMES 271 TISCHLBR, G. 1906. Ueber die Entwicklung des Pollens und der Tapetenzellen bei flibes-Hybriden. Jahrb. Wiss. Bot. 42 : 545-578. pi. 15. 1916. Chromosomenzahl, -Form und -Individualitat in Pflanzenreich. Prog. Rei. Bot. 6: 164-284. (Bibliography). TRETJAKOFF, D. 1904. Die Spermatogenese bei Ascaris megalocephala. Arch. Mikr. Anat. 65; 383-438. pis. 22-24. 1 fig. TRONDLE, A. 1911. Ueber die Reduktionsteilung in den Zygoten von Spirogyra und iiber die Bedeutung der Synapsis. Zeitschr. f. Bot. 3 : 593-619. ' 1 pi. 20 figs. 4 TSCHERNOYAROW, M. 1914. Ueber die Chromosomenzahl und besonders beschaf- fene. Chromosomen im Zellkerne von Najas major. Ber. Deu. Bot. Ges. 32 : 411-416. pi. 10. VANDENDRIES, R. 1913. Le nombre des Chromosomes dans la spermatoge'nese des Polytrichum. La Cellule 28 : 257-261. figs. 11. VAN LEEUWEN-REIJNVAAN, D. 1907. Ueber eine zweifache Reduktion bei einigen Polytrichum-Arten.. Rec. Trav. Bot. Neerl. 4. 1908. Ueber die Spermatogenese der Moose. Ber. Deu. Bot. Ges. 26a: 301-308. pi. 5. VEJDOWSKY F. 1907. Neue Untersuchungen iiber Reifung und Befruchtung. Kgl. Bohm. Ges. Wiss. Prag. 1912. Zum Problem der Vererbungstrager. pp. 184. pis. 12. Prag. 1911-2. VON Voss, H. 1914. Cytologische Studien an Mesostoma Ehrenbergi. Arch. Zellf. 12:159-194. pis. 12-14. figs. 5. DEVRIES, H. 1889. Intracellulare Pangenesis. Jena. WALKER, N. 1913. On abnormal cell-fusion in the archegonium; and on spermato- genesis in Polytrichum. Ann. Bot. 27: 115-132. pis. 13, 14. WALTON, A. C. 1918. The oogenesis and early embryology of Ascaris cams Werner. Jour. Morph. 30: 527-604. pis. 9. 1 fig. WEINZIEHER, S. 1914. Beitrage zur Entwicklungsgeschichte von Xyris indica L. Flora 106 : 393-432. pis. 6, 7. figs. 10. WEISMANN, A. 1887-1902. Ueber die Zahl der Richtungskorper und liber ihre Bedeutung fur die Vererbung. Jena 1887. Essays upon Heredity. Oxford 1891-2. Das Keimplasma. 1892. (Engl. 1893.) The Evolution Theory. 1902. WENRICH, D. H. 1915. Synapsis and the individuality of the chromosomes. Science 41 : 440. 1916. The spermatogenesis of Phrynotetlix magnus, with special reference to syn- apsis and the individuality of the chromosomes. Bull. Mus. Comp. Zool. Harvard Coll. 60: 55-136. 10 pis. 1917. Synapsis and chromosome organization in Chorthippus (Stenobothrus) curtipennis and Trimerotropis suffusa (Orthoptera). Jour. Morph. 29: 471-516. pis. 1-3. WILLIAMS, J. L. 1904. Studies in the Die tyotacese. II. The cytology of the game- tophyte generation. Ann. Bot. 18 : 183-204. pis. 12-14. WILSON, E. B. 1900. The Cell in Development and Inheritance. 2d ed. VON WINIWARTER, H. 1900. Recherches sur I'ovog6nese et I'organoge'nese de 1'ovaire des mammiferes (Lapin et Homme). Arch. d. Biol. 17: 33-199. pis. 3G — O. VON WINIWARTER, H. et SAINMONT. 1909. Nouvelles recherches sur I'ovogSnese et 1'organogenese de 1'ovaire des Mammiferes (Chat). Chapter IV. Ibid. 24: 1-142, 165-276, 373-432, 627-652. pis. 11. WODSEDALEK, J. E. 1916. Causes of sterility in the mule. Biol. Bull. 30: 1-56. WOLFE, J. J. 1904. Cytological studies on Nemalion. Ann. Bot. 18: 607-630. pis. 40, 41. 1 fig. 272 INTRODUCTION TO CYTOLOGY 1918. Alternation rnd parthenogenesis in Padina. Jour. Elisha Mitchell Sci. Soc. 34:78-109. WOOLERY, R. 1915. Meiotic divisions in the microspore mother-cells of Smilacina racemosa (L) Desf. Ann. Bot. 29 : 471-482. pi. 22. 1 fig. YAMANOUCHI S. 1906. The life history of Polysiphonia violacea. Bot. Gaz. 42: 401-449. pis. 19-28. 1908. Sporogenesis in Nephrodium. Ibid. 46 : 1-30. pis. 1-4. 1909. Mitosis in Fucus. Ibid. 47: 173-197. pis. 8-11. 1910. Chromosomes in Osmunda. Ibid. 49 : 1-12. pi. 1. 1912. The life history of Cutleria. Ibid. 54 : 441-502. pis. 26-35. figs. 15. ZWEIGER. 1907. Die Sperm atogenese von Forficula auricularia. Jen Zeitschr. 42 : 143-172. pis. 11-14. CHAPTER XII FERTILIZATION We have already pointed out that reduction and fertilization con- stitute the two principal cytological crises in the life cycles of all organisms reproducing sexually. Although the first of these processes was not dis- covered until 1883, some of the grosser features of fertilization had been made out many years previously (Chapter I). But the central feature of this process — the union of the two parental nuclei — was not known until 1875, when O. Hertwig discovered it in animals, Strasburger's parallel discoveries in plants following in 1877 (Spirogyra) and 1884 (angiosperms). As the finer details of fertilization and the significance of its results become better understood, the aptness of Huxley's (1878) often quoted simile, in which he compares the organism to "a web of which the warp is derived from the female and the woof from the male," becomes increasingly striking. We shall first describe the morphology of the fertilization process as it is typically shown in many animals, after which attention will be given to some of its physiological aspects. The second half of the chapter will be devoted to a review of fertilization in the various groups of the plant kingdom. FERTILIZATION IN ANIMALS1 The Gametes. — The spermatozoa of different animals exhibit a surprising variety of form and structure (Fig. 103). What may be referred to as the "typical" spermatozoon consists of three fairly distinct parts: head, middle piece, and tail or flagellum (Fig. 104). The head represents the nuclear portion of the sperm cell : it consists almost wholly of an extremely compact mass of chromatin. It has an envelope of cytoplasm which in few forms is very conspicuous and in many cases is scarcely distinguishable. Anterior to the nucleus there may be an acrosome, and the end of the head often has the form of a sharp point, the perferatorium. Posterior to the head is the middle piece; this is made up of cytoplasm in which are located the centrosomal structures, together with chondriosomes and other inclusions, such as the "Golgi bodies." The flagellum, or tail, consists of a slender axial filament, 1 In the preparation of this portion of the chapter the author has drawn very freely upon Professor F. R. Lillie's admirable and concise presentation of the subject, Problems of Fertilization (1919). 18 273 274 INTRODUCTION TO CYTOLOGY which grows out from the centrosome in the middle piece or in some cases apparently from the base of the nucleus, and a cytoplasmic sheath which usually extends not quite to its end. The sheath sometimes has the form of an undulating membrane. The spermatozoa of crustaceans and nematodes are non-flagellate, and in other groups various departures from the "typical" form and structure are known. A few of the many known types are shown in Fig, 103. FIG. 103. — Various types of spermatozoa. A, Triton (salamander). (After Ballowitz.) B, Nereis (annelid). (After Lillie, 1912.) C, guinea pig. (After Meves.) D, Phyllopneuste (bird). (After Ballowitz.) E, sturgeon (After Ballowitz.) F, Vesperugo (bat). (After Ballowitz.) G, Castrada hofmanni (turbel- larian). (After Luther.) H, Pinnotheres veterum (crustacean) . (After Koltzoff.) I,Homa- rus (lobster). (After Herrick). J, Ascaris (nematode) ; a, apical body; n, nucleus; r, "re- fractive body." (After Scheben.) The ovum undergoes nearly or quite all of its elaborate differentiation before the maturation divisions occur. Certain cells in the ovary gradu- ally become greatly enlarged (Fig. 105), and during this "growth period" the cytoplasm may not only differentiate into visibly distinct regions but may also become stored with energy-containing materials ("food"), which in the case of some animals, such as birds, is present in relatively enormous amounts. The "ovarian egg" or primary oocyte, as the egg cell is called before the maturation mitoses take place, may have a definite limiting membrane at its surface, but in many forms this cannot be demonstrated. The nucleus of the primary oocyte is known as the FERTILIZATION 275 germinal vesicle: it is very large and contains in addition to its chromatin a considerable amount of material which appears to take no part in the formation of the chromosomes when division ensues. After the cyto- plasmic differentiation is complete and the oocyte has reached its full size — even after the spermatozoon has entered in many cases — the oocyte nucleus undergoes two divisions in rapid succession at the periphery of the egg, which at this point buds off two small nucleated cells, the polar bodies (Fig. 106). The first polar body may or may not divide again. The details of chromosome behavior in these two mitoses have been described in the preceding chapter. The reduced or haploid number of chromosomes left in the egg organize the egg nucleus ("female pronucleus"), rendering the egg ready for the sexual fusion. FIG. 104. FIG. 105. FIG. 104. — ^Diagram of typical flagellate spermatozoon. P, perferatorium; A, acrosome; N, nucleus; M, middle piece; F, axial filament; S, cytoplasmic sheath; E, end piece. (After Wilson.) FIG. 105. — The differentiation of the oocyte in Hydra. A, very young oocyte lying between ectodermal cells at right. B, oocyte after growth period, with yolk globules, X 500. (After Downing, 1909.) The time relation of the maturation of the egg and the entrance of the spermatozoon varies considerably in different animals. In echinoderms and some other forms maturation is completed before the spermatozoon penetrates. In some other animals it proceeds as far as the metaphase of the heterotypic mitosis (Chcetopterus, Cerebratulus) or the prophase of the homceotypic mitosis (many vertebrates), but does not go further unless penetration occurs. In the marine annelid, Nereis, finally, the 276 INTRODUCTION TO CYTOLOGY germinal vesicle undergoes no change unless the spermatozoon has entered the egg. FIG. 106. — Maturation and fertilization in Ascaris. A, spermatozoon about to enter egg. B, spermatozoon inside; first maturation mitosis in progress. C, first maturation mitosis completed; first polar body budded off. D, second maturation mitosis, forming second polar body; sperm nucleus below. E, male and female pronuclei, each with 2 chromosomes, meeting. F, first cleavage mitosis, showing 2 paternal and 2 maternal chromosomes. (After Hertwig.) FIG. 107. — Fertilization in Physa (snail.) Sperm head and amphiaster at right, with long flagellum extending toward left. Second maturation mitosis in progress. (After Kostanecki and Wierzyski, 1896.) The Fusion of the Gametes. — In most cases the whole spermatozoon enters the egg (Fig. 107). In some sea urchins only the head and middle piece enter, while in Nereis the head alone passes in, the middle piece FERTILIZATION 211 and tail being left on the egg surface. The process in Nereis as de- scribed by Lillie (1912, 1919) is as follows. The egg of this worm has a tough vitelline membrane, an alveolar cortical layer, many yolk and oil droplets, and a large central germinal vesicle (nucleus). If many spermatozoa are present in the vicinity a large number attach themselves to the egg, but usually all but one are carried away by an outflow of jelly from the alveolae of the cortical layer. This layer now takes the form of a zone traversed by radial protoplasmic plates representing the walls of the alveola?. A transparent "fertilization cone" extends from the inner part of the egg across this zone and touches the membrane at the point where the spermatozoon is beginning to penetrate. The perferatorium pierces the egg membrane and becomes attached to the transparent cone. The latter is now withdrawn, carrying the head of the spermatozoon into the egg with it. Thus it appears that the initiative for the final act of penetration lies with the egg rather than with the spermatozoon. Since only the head enters the egg in Nereis it seems clear that the only necessary portion of the spermatozoon in the actual union is the nucleus : the middle piece and tail are accessory and function only as locomotor organs. The immediate visible effects of the entrance of the sperm are seen chiefly in changes in the appearance of the cortical region of the egg. If a vitelline membrane is present, as in vertebrates, a " perivitelline space" usually appears between the membrane and the egg; and this space may in some cases (frog) be great enough to permit the rotation of the egg within the membrane. In the sea urchin a fertilization mem- brane is formed as the result of fertilization : it first appears at the point where the spermatozoon is attached and spreads over the egg with great rapidity. It seems probable that a delicate membrane already present is raised and thus made more conspicuous. In Ascaris, which is parasitic in the intestine of the horse, this membrane becomes very thick and later acts as a protection against the digestive juices of the host. These cortical changes do not depend upon the actual entrance of the sperma- tozoon into the egg: in Nereis they occur before the slow penetration can be completed, or even if the spermatozoon is shaken loose shortly after penetration has begun. In describing the remarkable transformation undergone by the spermatozoon within the egg the behavior of its different organs will for the sake of clearness be considered separately. The Nucleus. — Immediately after gaining entrance to the egg (Fig. 108) the sperm head begins to enlarge and assumes the usual form and structure of a nucleus. Meanwhile it advances toward the egg nucleus. As Lillie points out, both male and female pronuclei pass toward a posi- tion of equilibrium in a cell preparing to divide and consequently meet : the assumption of an attractive force between them is unnecessary. By 278 INTRODUCTION TO CYTOLOGY the time they meet the male pronucleus has usually, but not always, become equal in size and appearance to the female pronucleus. The union of the two pronuclei to form a fusion nucleus, or synkaryon, usually FIG. 108. — Diagram of fertilization and cleavage in an animal. It is assumed that in this case the egg has undergone maturation before the penetration of the spermatozoon. FIG. 109. — -Independence of parental chromatin contributions in the cleavage of -the egg of Cryptobranchus. A, first cleavage mitosis. B, C, prophase and metaphase of fourth cleavage mitosis (After Smith, 1919.) occurs at once after they meet. In a great many cases there may be no actual fusion of the pronuclei at all: as they come close to one another each passes through the prophase stages and gives rise independently to FERTILIZATION 279 its group of chromosomes, the two groups arranging themselves on a common spindle which organizes when the nuclear membranes dissolve. The first cleavage mitosis (first embryonal division) then ensues, and the two daughter nuclei receive longitudinal halves of each and every chromosome. Thus in the act of fertilization, in both animals and plants, each parent furnishes the offspring with a haploid set of chromosomes, the two intermingled sets constituting the diploid set of the new individual. Since every chromosome divides equationally at every subsequent somatic mitosis, every cell of the body receives half of its chromosome complement from each parent. The cardinal importance of this fact in connection with current theories of heredity will be apparent in subsequent chapters. The two groups of chromoosmes, paternal and maternal, can often be distinguished not only on the spindle of the first cleavage division, but in several divisions thereafter. As examples may be cited Cyclops (Riickert 1895; Hsecker 1895), Crepidula (Conklin 1901), and Crypto- branchus (Smith 1919) (Fig. 109). This phenomenon is especially evident in hybrids (p. 160). There is much reason to believe that the chro- matins of the two parents, although intermingled in the nuclei of the offspring, never actually fuse, unless it is at the time of synapsis in the next maturation; and it has already been pointed out (Chapter XI) that they may not fuse even then. This fact also has an important bearing on the chromosome theory of heredity. The Centrosome.— (See Wilson 1900, pp. 208 ff.) Shortly after the entrance of the spermatozoon into the egg (Figs. 106-108) an aster devel- ops at the base of the sperm head, and in the aster a centrosome appears. Since the centrosome thus arises in the position of the middle piece, and since the centrosome of the spermatid is included in the middle piece dur- ing spermatogenesis, a widely accepted theory has been that the newly appearing centrosome is in reality that of the spermatid. Whatever its origin, it soon divides to form the two which function in the first cleavage mitosis. These facts had much to do with the formulation of a theory of fertilization set forth by Boveri (1887, 1891), who was much impressed by the conspicuous part played by the centrosomes in cell-division. Accord- ing to Boveri's theory the egg is not able to undergo division because of the lack of any centrosome to initiate the process, while the spermatozoon has a centrosome but not sufficient cytoplasm in which to act. Through the union of the gametes all the organs necessary for division are brought together and cleavage proceeds. This theory has recently been recalled by Walton (1918) in his work on Ascaris canis. Another early view of the origin of the cleavage centrosomes was that of van Beneden (1887) and Wheeler (1895, 1897), who believed them to be the centrosomes of the egg cell. The theory that the cleavage centrosomes arise from both egg and spermatozoon is of some historic interest. It was suggested by Rabl 280 INTRODUCTION TO CYTOLOGY (1889) that if the centrosome is a permanent cell organ the conjugation of the gametes must involve not only a union of nuclei but also a union of centrosomes (Wilson, p. 210). Fol (1891), in his work on echinoderm eggs, thought that he observed just such a process, which he termed "The Quadrille of the Centers." The egg centrosome and the centrosome brought in by the spermatozoon were both supposed to divide, the prod- ucts then fusing in pairs to form the two cleavage centrosomes. A simi- lar thing was reported by certain other investigators, but none of the cases stood the test of later work. Another theory now abandoned was that advanced by Carnoy and Lebrun (1897), who also attempted to derive one centrosome from each gamete. The cleavage centrosomes were thought to arise de novo and separately, one inside each pronucleus, to migrate thence into the cytoplasm. Much less confidence is now placed in the persistence of the spermatid or egg centrosomes through the fertilization stages. Since the middle piece, which is thought to contain the centrosome, does not enter the egg at all in Nereis, it seems probable that the male nucleus in some way induces the formation of asters and centrosomes by the egg cytoplasm. Lillie found that even a portion of the sperm head will bring about this effect. In Unio (Lillie 1897, 1898) and Crepidula (Conklin 1897) it seems not unlikely that each pronucleus causes the formation of one cleavage centrosome. In the sea urchin Wilson (1901) concluded that the cleav- age centrosomes in all probability arise by the division of one which orig- inates de novo at the nuclear membrane. In almost every case there are gaps in the known history of the centrosome in fertilization, and it seems very doubtful whether the cleavage centrosomes are continuous with those of either gamete. This conclusion is supported by the fact that the formation of asters with centrosomes in the egg cytoplasm can be arti- ficially induced by treating the eggs with certain chemicals, such as weak MgCl2. It is possible that the spermatozoon carries a substance which brings about centrosome formation in a similar way. However this may be, the importance of the centrosome undoubtedly lies in its relation to cleavage rather than to fertilization. Cytoplasm and Chondriosomes. — In some cases (Nereis} no cytoplasm can be shown to enter the egg with the spermatozoon, whereas in others (A scam) a relatively large amount is brought in. Its great indefiniteness in behavior makes it seem probable that it has no special significance in the fertilization process. The importance of the chondriosomes in fertilization has been empha- sized by Meves (1911, 1915), who finds that many of these bodies are present in the large cytoplasmic mass accompanying the sperm nucleus in Ascaris, and that they mingle with the chondriosomes of the egg. Meves (1908, 1915, 1918), together with other writers, accordingly thinks that they are concerned in the transmission of certain hereditary char- FERTILIZATION 281 acters. Several observations, however, fail to harmonize with this view. In some echinoderms the middle piece, which contains the chondriosomal material, is not distributed to the daughter cells during the cleavage divisions, but remains in one of them. It is unlikely that hereditary material would behave in this manner. Furthermore, in the worm, Nereis, the middle piece does not even enter the egg. In Peripatus (Montgomery 1912) the chondriosomal mass is thrown out of the spermatid. Wildman (1913) points out that in Ascaris also the chon- driosomes may be largely lost during spermatogenesis. Although their almost constant presence in the spermatozoon indicates that they have a special physiological role comparable to that in other cells, there is as yet no adequate reason to regard them as important in the transmission of the factors of inheritance. FIG. 110. — Chromidiogamy in Arcella. (From Minchin, after Swarczewsky.) Somewhat modified. Fertilization in Protozoa. — Among the protozoa there are found several modes of sexual union very different from that described above for the higher animals. Four illustrative cases may be cited (see Minchin 1912). In Arcella, a rhizopod with a hemispherical shell, the protoplast has two "primary nuclei" and many scattered chromidia. Two individuals come together with their shell openings opposite one another (Fig. 110), and the protoplasm of one flows almost entirely into the other shell, where it mingles with that of the other individual. The primary nuclei degenerate, while the chromidia become divided up into fine granules. The protoplasm with the fine chromidia now becomes evenly distributed in the two shells, after which the latter separate. In each individual the chromidia now form "secondary nuclei;" around these are organized uninucleate amcebulse which escape from the shell and give rise to new Arcella individuals. This process, in which the chromatin chiefly con- 282 INTRODUCTION TO CYTOLOGY cerned is that of the chromidia and not that of the larger nuclei, is quite rare and is known as chromidiogamy. Arcella also has the other form of sexual union, karyogamy. jk-Uf'i V\ ,A~.y str4 /4/v^. 'tB^^^i V ^ /" t \ 4i>55fcJ^t \ TJ x _/ i r/ i \ jf FIG. 111. — Copulation \n-Actinophrys sol. X 850. (From Minchin, after Schaudinn.) In Actinophrys sol (Schaudinn 1896) (Fig. Ill) two individuals, each -vith a single nucleus, approach each other and become enclosed in a common cyst. In each of them the nucleus now undergoes two pre- liminary mitotic divisions, at each of which a small "reduction nucleus" is expelled from the body in a manner very similar to the expulsion of chromatin into the polar bodies of higher animals. The two individuals, FIG. 112. — Autogamy in Amoeba albida. (From Minchin, after Ndgler.) or gametes, as they may now be called, fuse completely, their nuclei uniting to form a synkaryon. Soon the synkaryon divides mitotically, this being followed by the division of the cell to form two individuals which escape from the cyst and resume the vegetative state. In Amoeba albida (Nagler 1909) (Fig. 112) a peculiar process known as autogamy occurs while the organism is in the encysted state. The single FERTILIZATION 283 nucleus divides, forming a large vegetative nucleus and a smaller gener- ative nucleus. The former moves to the surface of the cell and degener- ates, while the latter gives rise to the gamete nuclei in the following manner. After becoming elongated and incompletely divided it buds off four "reduction nuclei" — two from one end and then two from the FIG. 113. Conjugation in Paramcecium. (After Morgan, 1913.) other. It then completes its division to form the two gamete nuclei, or pronuclei, while the four reduction nuclei degenerate. After lying apart for some time the two pronuclei approach each other and fuse ; sexual union thus takes place between sister nuclei. The complicated nuclear behavior occurring at the time of conjugation in Paramcecium caudatum (Fig. 113) may best be described in the words 284 INTRODUCTION TO CYTOLOGY of Wilson (1900, pp. 224-226), In "Paramcecium co.udatum, which possesses a single macronucleus and micronucleus, . . . conjugation is temporary and fertilization mutual. The two animals become united by their ventral sides and the macronucleus of each begins to degenerate, while the micronucleus divides twice to form four spindle-shaped bodies. Three of these degenerate, forming the 'corpuscles de rebut,' which play no further part. The fourth divides into two, one of which, the 'female pronucleus,' remains in the body, while the other, or 'male pronucleus,' passes into the other animal and fuses with the female pronucleus. Each animal now contains a cleavage-nucleus equally derived from both the conjugating animals, and the latter soon separate. The cleavage-nucleus in each divides three times successively, and of the eight resulting bodies four become macronuclei and four micronuclei. By two succeeding fissions the four macronuclei are then distributed, one to each of the four resulting individuals. In some other species the micronuclei are equally distributed in like manner, but in P. caudatum the process is more complicated, since three of them degenerate, and the fourth divides twice to produce four new micronuclei. In either case at the close of the process each of the conjugating individuals has given rise to four descendants, each containing a macronucleus and a micronucleus derived from the cleavage-nucleus. From this time forward fission follows fission in the usual manner, both nuclei dividing at each fission, until, after many generations, conjugation recurs." The Physiology of Fertilization. — The principal results of fertiliza- tion are two: the activation of the egg, and, in dioecious organisms, biparental heredity. Both of these have their physiological as well as their morphological aspects, and in the present section the first of them will be considered with special reference to its physiology. What is the nature of the physiological reactions through which the development of the egg is initiated? In the terms used by Child (1915), how does fertilization bring about the rejuvenation of the egg, which is a physio- logically old cell? The attack upon this problem has been carried out along two main lines: by a study of artificial parthenogenesis and by a direct analysis of the chemical constitution of the gametes at these stages. Artificial Parthenogenesis. — This line of attack has been followed particularly by Loeb, who has found a number of methods by which the parthenogenetic development of unfertilized eggs may be artificially induced.1 As stated in the foregoing pages, the first externally visible effect of fertilization is in many animals the formation of a fertilization membrane. The formation of this membrane, which seems to be a condition necessary to the future development of the egg, Loeb was able to induce in the California sea urchin by placing the eggs for 2 minutes in a solution made up of 50 c.c. of sea water and 3 c.c. of a tenth-normal 1 For a convenient summary of such methods see Harvey (1910). FERTILIZATION 285 fatty acid (butyric, propionic, or valerianic), and then back into pure sea water: the membrane then forms by a cytolysis of the cortical layer of the egg. Although in some forms (starfish) this one treatment is sufficient to bring about successful development, in most cases (sea urchin) the eggs become sickly and die. Loeb found that this sickli- ness may be prevented, allowing normal development, by either of two second treatments. If, after membrane formation, the eggs are placed for 20 minutes in hypertonic sea water or other solution with an osmotic pressure 50 per cent above that of ordinary sea water, they will develop normally when returned to pure sea water. The same effect may be brought about, though not always so successfully, by placing the eggs for 3 hours in sea water free from oxygen, or into sea water with a trace of KCN. It is therefore concluded by Loeb that the stimulus to such parthenogenetic development has two phases: the inducement of mem- brane formation by cytolysis, and the subsequent effect of the hyper- tonic solution. In rare cases the first treatment alone is sufficient for normal development, but in all cases it at least starts the egg into activity. As a result of these experiments Loeb has interpreted the action of the spermatozoon in normal fertilization on the assumption that it carries two substances: first, a lysin which brings about membrane formation by cytolysing the cortical layer of the egg, and which can act even if the spermatozoon does not enter the egg; and second, a substance which produces an effect similar to that of the hypertonic sea water employed in the experiments. The quite different explanation offered by Lillie will be mentioned further on. How it is that cytolysis of the cortical layer of the egg brings about activation Loeb attempts to explain in the following manner. A calcium lipoid compound forms a continuous layer just beneath the surface of the egg, and the solution of this layer would probably result in the destruc- tion of the cortical emulsion. It is assumed that in this cortical region there is a catalytic agent which increases the metabolism (rate of oxida- tion, etc.) of the egg. Following Warburg (1914) Loeb suggests that the cytolysis releases the catalyzer by breaking down the cortical emulsion; this results in an increase in the rate of oxidation and other reactions, and development proceeds. That the process of activation is bound up primarily with reactions occurring in the cortical region of the egg is shown further by the experi- ments of Guyer (1907), Herlant (1913, 1917), McClendon (1912), Loeb and Bancroft (1913), and particularly Bataillon (1910), who have shown that the egg of the frog may be made to develop by pricking it with a needle, especially if some blood enters the egg with it; and also by the researches of R. S. Lillie (1908, 1915), who finds that starfish eggs may be made to develop parthenogenetically by exposing them to high temperatures for definite periods. (See F. R. Lillie, 1919, Chapter VII.) 286 INTRODUCTION TO CYTOLOGY Heilbrunn (1920) shows that the egg of Cumingia can be induced to undergo maturation by agencies which release the fluid cytoplasm from the restraint of the tough vitelline membrane. If the membrane be swollen, elevated above the egg surface, ruptured, or removed the maturation changes begin at once. The sickliness and death of those eggs given only the first treatment Loeb thought to be due to the continued action of the cytolytic agent. Against this conception it is urged by F. R. Lillie that since any activated egg not developing normally cytolyzes sooner or later from internal causes, it is more probable that the sickliness and death are due to some internal cause resulting from activation, and points out that such a conclusion is supported by the cytological phenomena in eggs activated by Loeb's method. To these phenomena we may turn for a moment. Eggs which have been given the first treatment alone do not begin to disorganize for many (12 to 24) hours. During this period Herlant (1917) has observed the following events. After the formation of the mem- brane and a hyaline zone, alterations cease, and the nucleus becomes the seat of a series of conspicuous changes. The nuclear membrane dissolves, and around the chromosomes there is formed a monaster (one-poled group of achromatic fibers), but no amphiaster develops. The chromo- somes divide but do not separate, and although the cytoplasm becomes active no cytokinesis ensues. The chromosomes then return to the resting condition. This process is repeated several times, the nucleus increasing in bulk each time, but it soon becomes very irregular and the egg ultimately breaks down by general cytolysis. The second treatment (Loeb's method) in some way gives the egg the capacity to divide regu- larly. Morgan (1899) and Wilson had long before shown that such treatment with hypertonic sea water causes aster formation in the unfertilized sea urchin egg. Herlant shows that one of these asters and a second aster formed in connection with the egg nucleus together form an amphiaster, normal division then ensuing. In the light of these facts it seems evident that the death of the egg after the first treatment alone is not due to the continued action of the cytolytic agent employed, but rather to irregularities in the activation processes aroused by the cortical changes in the absence of a proper coordination of nuclear and cell division. The second treatment pro- duces a regulatory effect, partly through aster formation, resulting in normal development. This recalls Boveri's morphological theory of normal fertilization. Direct Analysis of the Fertilization Process. — In contrast to the theory that the spermatozoon contributes organs (Boveri) or substances (Loeb) necessary for the activation, Lillie (1919, Chapter VII) regards the egg itself as an "independently activable system." "The egg possesses all substances needed for activation; the spermatozoon is an inciting cause FERTILIZATION 287 of those reactions within the egg system upon which development depends;" As a result of his direct analysis of the gametes durirg the fertilization period Lillie has identified a substance in the egg which he calls fertilizin. This substance is present in the egg for a short time only; its formation usually begins at about the time the germinal vesicle begins to break down, and immediately after fertilization its production ceases, possibly through the neutralizing action of a second substance, called "anti-fertilizin." As a rule it is only during the period at which fertilizin is present that spermatozoa will enter the egg; the egg re- mains fertilizable for but a short time. Hence it seems clear that it is not the fertilization membrane that prevents the entrance of other spermatozoa, as Fol thought, but rather the physiological state of the egg. That the protection is thus a physiological rather than a mechanical one is indicated by the fact that membraneless egg fragments without fertilizin are not entered by spermatozoa. Fertilizin has two effects: it first acts by causing an agglutination of the spermatozoa at the surface of the egg, and later causes the activa- tion of the egg. It may thus be said to stand between the spermatozoon and the activation reactions in the egg. Being present in the egg secre- tion at a certain period it binds the spermatozoon to the surface of the egg, and the spermatozoon, without necessarily penetrating the egg at all, by means of a substance which it bears releases the activity of the fertilizin within the egg, which results in development. In brief, the activating substance is already present in the egg and is not brought to it by the spermatozoon. It may be incited to activity by the sperm- atozoon, but by other agencies as well. In concluding this sketch of the physiological features of fertiliza- tion we may state briefly the immediate physiological consequences of the process as summarized by Lillie (1919, Chapter V). The rate of oxida- tion increases in most cases in which it has been investigated. In the sea urchin egg (Warburg 1908-1914) this rate increases as much as six- or seven-fold ; in Strongylocentrotus, four- or five-fold (Loeb and Wasteneys, 1912, 1913); in the starfish, apparently not at all. The egg membrane becomes more permeable to oxygen, CO2, pigment, water, alkalis, intra- vitam stains, and a number of other substances. The protoplasm be- comes less fluid after fertilization (Heilbrunn 1915). This gelation effect Chambers (1917) believes to center upon the sperm aster. The volume of the egg decreases and its electrical conductivity rises. The most conspicuous chemical change is seen in the loss of the fertilizin, and with it the loss of capacity for further fertilization reaction. FERTILIZATION IN PLANTS Although the central act of the process of fertilization is regularly the union of two sexually differentiated nuclei, the morphological 288 INTRODUCTION TO CYTOLOGY features associated with this fusion are more varied in plants than in animals. This is especially true of the algae and fungi. Algae. — In Ulothrix fertilization consists in the complete union of two morphologically similar, motile biciliate gametes (Fig. 114, A). In Fucus the two gametes are very dissimilar: the male (spermatozoid) is small, laterally biciliate, and actively motile (Fig. 114, 5), while the female (egg), though discharged from the oogonium, is large and passive, as in all higher plants and animals. In (Edogonium (Fig. 114, D, E) the FIG. 114.- — Spermatozoids of plants. A, Ulothrix: , 1, gamete; 2, gametes fusing (isogamy); 3, zygospore. B, Fucus. (After Guignard.) C, Zamia. (After Webber.) D, bit of filament of (Edogonium; spermatozoids escaping from antheridial cells below; spermatozoid about to enter egg above. (After Coulter.) E, spermatozoid of (Edogonium. F, Chara. (After Belajeff.) G, Onoclea. (After Steil.) For figures of spermatozoids of Blasia, Polytrichum, Equisetum, and Marsilia, see Figs. 28, 29, 30, and 32. egg is not shed from the cell which produces it, but is fertilized in situ, a condition which is retained in all the higher plant groups. The sperm- atozoid in this genus has a crown-like ring of cilia. In Spirogyra (and other Conjugates) certain vegetative cells, without further morphological differentiation, function as gametes. The entire contents of such a cell pass through a conjugation tube to a similar cell in an adjacent filament, where the two unite to form the zygospore. The two nuclei fuse, but the chloroplasts furnished by the contributing ("male") gamete may event- ually degenerate (Zygnema). In Polysiphonia a non-motile male gamete FERTILIZATION 289 (spermatium) comes in contact with a prolongation (trichogyne) of the female sex organ (carpogonium). Solution of the intervening walls allows the nucleus of the spermatium to pass into the trichogyne and down to the female nucleus in the base of the carpogonium. In Polysiphonia we have one of the few cases among lower plants in which the fusion of the sexual nuclei has been minutely described. According to Yamanouchi (1906) the male. nucleus, by the time it has reached the female nucleus, has resolved itself into a group of 20 chromosomes (Fig. 115, A). In this FIG. 115. A, fertilization in Polysiphonia. Group of male chromosomes about to enter female nucleus. (After Yamanouchi, 1906.) B, fertilization in Albugo Candida. Female nucleus lying in center of ooplasm near the "coenocentrum" (larger dark body.) Antheridial tube about to discharge a male nucleus; another male nucleus in neck of tube. Additional nuclei in periplasm surrounding the ooplasm. (After Davis, 1900.) condition it enters the female nucleus while the latter is yet in the reticu- late state. Soon the female reticulum becomes transformed into 20 chromosomes, which arrange themselves with the 20 paternal chromo- somes upon the spindle as the fusion nucleus divides. Fungi. — In the PHYCOMYCETES sexual reproduction occurs in two princi- pal forms, which serve to divide the group into two main divisions: Oomycetes and Zygomycetes. In the Oomycetes the cytological phenomena are best known in the Peronosporales and Saprolegniales. In the former there is differentiated in the oogonium a single large egg into which the contents of an antheri- dium are discharged through a penetrating tube. In Albugo bliti and A. portulaccce (Stevens 1899, 1901) the egg has a large number of nuclei, 19 290 INTRODUCTION TO CYTOLOGY and after the entrance of the antheridial nuclei about 100 sexual fusions occur. In Albugo Candida (Cystopus candidus) (Wager 1896; Davis 1900), Peronospora parasitica (Wager 1900), Albugo tragopogonis and A. ipomceae (Stevens 1901) the mature egg has but one nucleus, which fuses with a single male nucleus discharged into the egg by an antheridium (Fig. 115, .B). In all cases an oospore results. In the Saprolegniales, as shown by the researches of Davis (1903, 1905), Miyake (1901), Trow (1895-1905), and Claussen (1908), there are two general conditions. In Saprolegnia (Trow, Davis, Claussen) from 10 to 15 uninucleate eggs are formed within an oogonium. One or more antheridia send in conjugating tubes and deliver a male nucleus to each egg, in which a single sexual fusion then occurs. In Pyihium (Trow 1901 ; Miyake 1901) a single uninucleate egg is produced, the fertilization process closely resembling that in Albugo Candida. In the Zygomycetes, represented chiefly by the Mucoraceae, the sexual process consists in the union of the contents of two similar (except oc casionally in size) multinucleate gametangia, the result of the fusion being a zygospore. As shown by Blakeslee (1904) these two gametangia are borne on the same mycelium in some species ("homothallic" species), whereas in other species ("heterothallic" species) they are regularly borne on different mycelia, no zygospores being formed in the latter spe- cies on a mycelium arising from a single spore. Owing to the extremely minute size of the nuclei their behavior at these stages is not well known. By some investigators (Macormick on Rhizopus nigricans, 1912) it is held that only one fusion occurs, the remaining nuclei degenerating. Others (Keene on Sporodinia grandis, 1914) think it probable that al- though some degeneration occurs, the nuclei nevertheless fuse in pairs in considerable numbers. Until further researches have been carried out very little of a definite nature can be said concerning the nuclear history of the Zygomycetes. In the ASCOMYCETES (see Atkinson 1915) the fusion of two nuclei in the ascus was first described for several species by Dangeard (1894) (Fig. 116, A), who regarded it as a sexual fusion and the ascus as an oogo- nium. The matter soon became complicated when a number of cytolo- gists, beginning with Harper (1895 etc.), found what they believed to be a nuclear fusion at an earlier stage in the life cycle. This fusion was described as occurring (a) in the archicarp when fertilized by the contents of an antheridium (Harper on Sphcerotheca castagnei, 1895, 1896, Erisiphe 1896, Pyronema confluens 1900, and Phyllactinia 1905; Blackman and Fra- ser on Sphcerotheca 1905; Claussen on Boudiera 1905) ; (6) in the archicarp when the antheridium is functionless or absent (Blackman and Fraser on Humaria granulata 1906; Fraser on Lachnea stercorea 1907; Welsford on A scobolus furfur aceus 1907; Dale on Aspergillus repens 1909); or (c) in the vegetative cells when the archicarp is functionless or absent (Fraser FERTILIZATION 291 on Humaria rutilans 1907, 1908; Carruthers on Helvella crispa 1911; Blackman and Welsford on Polystigma rubrum 1912). By the above investigators this early fusion was regarded as a sexual one, that in the ascus being vegetative in nature; and some described a "double reduc- tion" in the ascus to compensate for the two nuclear fusions. (See p. 223.) In a series of somewhat later researches another group of observers found the evidence for an early fusion to be very unsatisfactory, and' concluded that the only nuclear union in the life cycle is that occurring in the ascus: with Dangeard they saw in this union the sexual act. Fur- thermore, no "double reduction" was found in the ascus. Among the researches supporting this view, which now appears to be the more probable, may be cited the following: Claussen on Pyronema confluens FIG. 116. A, nuclear fusion in the ascus of Peziza vesiculosa. (After Dangeard, 1894.) B, cell fusion in seciospore sorus of Phragmidium speciosum. After Christman, 1905.) 1907, 1912; Schikorra on Monascus 1909; W. H. Brown on Pyronema confluens 1909, Lachnea scutellata 1911, and Leotia 1910; Faull on Lab- oulbenia 1911, 1912; Blackman on Collema pulposum 1913; Nienburg on Polystigma rubrum 1914; Ramlow on Ascophanus carneus and Ascobolus immersus 1914; Brooks on Gnomonia erythrostoma 1910; McCubbin on Helvella elastica 1910; H. B. Brown on Xylaria tentaculata 1913; and Fitz- patrick on Rhizina undulata 1918a. As the two nuclei fuse in the young ascus Harper (1905) observed in the case of Phyllactinia ccfrylea that not only the chromatin systems but also the nucleoli and "central bodies" (centrosomes), upon which the chromatin strands converge, unite. In the Ascomycetes generally the fusion nucleus, or "primary ascus nucleus," undergoes three successive mitoses to form the eight ascospore nuclei, the spore walls in each case being formed in association with the curving astral rays which focus upon the centrosome. (See p. 80.) 292 INTRODUCTION TO CYTOLOGY In certain yeasts it has been shown (see Guilliermond 1920) that the production of ascospores is preceded by a copulation of two cells with a fusion of their nuclei, the fusion nucleus dividing to form the spore nuclei. A somewhat similar copulation of the ascospores themselves has also been observed in a few cases. Among the BASIDIOMYCETES the nuclear phenomena are best known in the case of the rusts, owing to the researches of Blackman (1904), Christman (1905), and a number of later writers. In the typical rust life cycle there is a fusion of uninucleate cells at the base of the aecial sorus (Fig. 116, B}. The binucleate cells thus arising produce the binu- cleate aeciospores; and these upon germination form a mycelium with binucleate cells, the two nuclei dividing in unison ("conjugately") at each cell-division. After producing a series of crops of binucleate uredospores this mycelium eventually bears teliospores which may con- sist of one or more cells. In each cell of the teliospore the two nuclei delivered to it as the result of the conjugate divisions throughout the binucleate mycelium finally unite, initiating the uninucleate phase of the life cycle. Here the fusion of sexual cells and the fusion of their nnclei — two events which in most organisms occur very near each other in time — are widely separated in the cycle. The two nuclei dividing conjugately constitute together a synkaryon in many respects equivaleng to a diploid nucleus. Since there is as yet no evidence to show in what degree the two effects of fertilization (the stimulus to development and the mixing of hereditary lines) are brought about in the rusts by the fusion of the sexual cells on the one hand and by the final union of their nuclei on the other, it seems best to regard the two fusions as two phases of the fertilization process in spite of their wide separation in the life history. In the Hymenomycetes it has been known for some time that a fusion of two nuclei occurs in the basidium, itself the terminal cell of a binucleate hypha, prior to the formation of the four basidiospore nuclei (Fig. 79). The origin of the binucleate condition in the mycelium which has ap- parently arisen from a uninucleate spore has long been an obscure point. It has recently been shown by Miss Bensaude (1918) in the case of Coprinus fimetarius that the binucleate hyphae arise as the result of cell fusions ("plasmogamy;" "pseudogamy") between uninucleate hyphsB arising from different spores, and that no carpophores are produced upon a uninucleate mycelium arising from a single spore. Thus it appears that in at least some hymenomycetes the sexual process is initiated by a fusion of two cells of different strains ("plus" and "minus"), as in the heterothallic molds. Bryophytes and Pteridophytes. — In bryophytes and pteridophytes the details of the union of the motile spermatozoid with the egg in the archegonium have been described in very few cases. In the former group may be cited the works of Garber (1904) and Black (1913) on FERTILIZATION 293 Riccia, Meyer (1911) on Corsinia, Graham (1918) on Preissia, and Woodburn (1920) on Reboulia. It appears that in bryophytes the body of the biciliate spermatozoid, which consists mainly of nuclear material, undergoes in the egg cytoplasm a transformation into a reticulate nucleus before fusing with the egg nucleus (Fig. 117). The fate of the non- nuclear structures (cytoplasm, blepharoplast, and cilia) is not known with certainty, but it is probable that they are absorbed in the egg cyto- plasm. In the liverwort, Preissia quadrata, Miss Graham has found two centrosomes with weakly de- veloped asters in the cytoplasm of the egg at the time the two pronuclei are about to fuse (Fig. 23, A). It is not known what relation their ap- pearance may have to the entrance of the spermatozoid. The most detailed account of fer- tilization in a pteridophyte is that given by Yamanouchi (1908) for FIG. 117. FIG. 118. Fia. 117. — Fertilization in Anthoceros. Male and female pronuclei about to fuse in lower part of egg in venter of archegonium; elongated plastid above them. Gametophyte cells show one nucleus and one plastid each. X 1050. FIG. 118. — Fertilization in Nephrodium. A, spermatozoid entering egg nucleus. B, spermatozoid becoming reticulate in midst of female reticulum. (After Yamanouchi, 1908.) Nephrodium (Fig. 118). In Nephrodium the multiciliate spermatozoid enters bodily into the egg nucleus with no previous alteration into the reticulate state. Here it gradually becomes reticulate and irregular in shape, until finally its limits are indistinguishable, the chromatic material contributed by the two gametes apparently forming a single fine-meshed network. Gymnosperms. — Among living gymnosperms the Cycadales and Ginkgoales are characterized by the possession of motile spermatozoids. 294 INTRODUCTION TO CYTOLOGY These spermatozoids are very much alike in structure and behavior in the two groups, and are unusually large, being easily visible to the naked eye. The body is made up of a large nucleus surrounded by a thin cytoplasmic layer in which is imbedded a long, spirally coiled blepharo- plast bearing many cilia (Fig. 114, C). The behavior of the spermatozoid in fertilization has been studied in Ginkgo by Hirase (1895, 1918) and Ikeno (1901); in Cycas revoluta by Ikeno (1898); in Zamiafloridanaby Webber (1901); and in Dioon edule, Ceratozamia mexicana, and Stangeria paradoxa by Chamberlain (1910, 1912, 1916). In all cases the entire spermatozoid penetrates into the egg cytoplasm, where the nucleus frees itself from the cytoplasmic sheath with its blepharoplast and cilia and advances alone to the egg nucleus, with which it fuses (Fig. 119). The behavior of the chromatin during the fusion is not well known in either Ginkgo or the cycads. In the Coniferales and Gnetales the male cells have no motile apparatus. Each consists of a nucleus surrounded by a more or less sharply delimited mass of cytoplasm. In most cases this cytoplasm remains intact until after the male cell has entered the egg, but in other forms, such as Pinus, it mingles with the cyto- plasm of the pollen tube, so that only male nuclei, rather than completely organized male cells, are delivered to the egg. All the nuclei present in the pollen tube — stalk nucleus, tube nucleus, the two male nuclei, and in certain species free prothallial nuclei — may be dis- charged into the egg. All but the functioning male nucleus usually degenerate at once, but in some cases they have been observed to undergo division. When a complete male cell enters the egg the cytoplasm of the former shows two general modes of behavior. In some species it may be left behind in the peripheral region of the egg as the male nucleus frees itself and advances alone to the female nucleus. This type of behavior has been reported in Pinus (Ferguson 1901, 1904), Thuja (Land 1902), Juniperus (Noren 1904), Cryptomeria (Lawson 1904), and Libocedrus (Lawson 1907). In Sequoia (Lawson 1904) the male nuclei escape from their cytoplasm before their discharge from the pollen tube, and enter the egg alone. In a second group of species the male cytoplasm remains intact and invests the fusing sexual nuclei, being clearly distinguishable from the cytoplasm of the egg. The pollen tube cytoplasm often plays FIG. 119. — Fertilization in Zamia. Male nucleus uniting with egg nucleus at center; cytoplasmic sheath with spiral blepharoplast above. Another sperm outside egg. X 25. (After Webber, 1901.) FERTILIZATION 295 a conspicuous part in the formation of this "mantle." This phenomenon, the significance of which can only be conjectured, is found in Taxodium (Coker 1903), Torreya calif ornica (Robertson 1904), Torreya taxifolia (Coulter and Land 1905), Cephalotaxus Fortunei (Coker 1907), Ephedra (Berridge and Sanday 1907; Land 1907), Phyllocladus (Kildahl 1908), Juniperus (Nichols 1910), Agathis (Eames 1913), and Taxus (Dupler 1917). Chromosome Behavior. — The behavior of the chromosomes during the fusion of the sexual nuclei and the first embryonal division has been described in a number of conifers. As a general rule, to judge from the data at hand, the chromatin contributions of the two pronuclei do not become intimately associated in the fusion nucleus, but remain distinguishable until the first embryonal mitosis occurs. Each of the pronuclei then gives rise to its complement of chromosomes which B f FIG. 120 — Fertilization in Pinus. A, male nucleus pressing into female nucleus. X 140. B, first embryonal mitosis, showing separate paternal and maternal chromosome groups. X 472. (After Ferguson, 1904.) become arranged, often as two separate groups, upon a common spindle. Such an independent formation of the male and female chromosome groups has been observed in Pinus (Blackman 1898 ; Cham- berlain 1899; Ferguson 1909, 1904) (Fig. 120), Larix (Woyciki 1899), Tsuga candensis (Murrill 1900), Juniperus communis (Noren 1907), Cunninghamia (Miyake 1910), and Abies (Hutchinson 1915). In Sequoia, on the other hand, Lawson (1904) reports that the two nuclei form a common reticulum in which the male and female constituents cannot be distinguished. With regard to the first embryonal mitosis the general opinion has been that all the chromosomes, paternal and maternal, split longitudinally, the daughter chromosomes being distri- buted to the daughter nuclei as in any other somatic mitosis. This type of behavior was described for the chromosomes of Pinus by Miss Ferguson (1904) and at once came to be regarded as general for coni- fers, as it had been for other organisms. A new interpretation differing in certain fundamental points from the above has been more recently suggested by Hutchinson (1915), as a result 296 INTRODUCTION TO CYTOLOGY of his work on Abies balsamea. According to Hutchinson (Fig. 121) there appear in the fusion nucleus two groups of chromosomes, each containing the haploid number (16). A spindle is differentiated about each group; and the two spindles soon unite to form one, thus bringing the two chromosome groups, representing the two parental contributions, into closer association. The chromosomes now approximate two by two to form 16 pairs. The members of each pair twist about each other and become looped; each of them becomes transversely segmented at the apex of the loop, forming 32 (2x) pairs of segments; these pairs separate to form 64 (4x) chromosomes; a new spindle is formed and 32 (2x) chromosomes pass to each pole. TWUTINC LMNN IMMMMTMD tnnto KUCUI (»*•») FIG. 121. — The behavior of the chromosomes in fertilization and the first embryonal mitosis in Abies, according to Hutchinson. (1915.) This interpretation of chromosome behavior at fertilization is remark- able not only because it indicates features resembling those of the hetero- typic prophase, but chiefly because it actually calls for a qualitative reduction of the chromatin at the first embryonal mitosis if the chroma- tin is not qualitatively the same throughout the nucleus. This impli- cation has not been discussed by the advocates of the new theory. The chromosomes pair and twist about one another in a way that parallels closely their behavior during the prophase of a reduction division. That the doubleness seen is due to a pairing and not to a splitting as has heretofore been held is supported by the assertion that the pairs are present in the haploid number, rather than in the diploid number as would be the case were a splitting of all the chromosomes occurring. If the two members of each pair were to separate at the first embryonal FERTILIZATION 297 mitosis, a reduction, qualitative as well as numerical, in all respects similar to that accomplished in the regular heterotypic mitosis, would be brought about if the pairing members are qualitatively different. But instead of such a separation, each member of each pair segments transversely, giving 4x segments which are equally distributed to the two daughter nuclei, each of the latter receiving the diploid number. Since the 4x segments become more or less intermingled before their distri- bution it is probably impossible to determine just which ones pass to each pole. If both halves of one transversely divided chromosome pass to one pole (see Fig. 122), that daughter nucleus only, and not the other, will receive the kind of chromatin carried by that chromosome, so that CLUWAfrE MITOSIS EQUftTIONAL FUSION NUCLEUS V SEGMENTATION FIG. 122. — Diagram showing the behavior of the chromosomes in fertilization and the first embryonal mitosis as usually interpreted (upper part) and according to Hutchinson's interpretation (lower part). the two nuclei will be qualitatively different. A qualitative reduction will have occurred, but without a change in the number of chromosomes, since each old chromosome has become two new ones. If, on the other hand, the two halves of the transversely segmented chromosome regularly pass to opposite poles, each daughter nucleus will receive a half of each and every parental chromosome: thus if there are just as many kinds of chromatin as there are chromosomes, these nuclei will be qualitatively alike, just as they would be had the division been longitudinal instead of transverse. But, as has been stated in the chapter on reduction and will be developed at greater length in Chapter XVII, there is a considerable body of evidence which indicates that each chromosome is not only qualitatively different from its fellows, but possesses a linear differen- 298 INTRODUCTION TO CYTOLOGY tiation of some sort; so that the separation of the two halves of a trans- versely divided chromosme would constitute a qualitative reduction. If such actually is the condition of the chromatin, and if the chromosomes do behave as Hutchinson supposes, a qualitative reduction must immedi- ately follow each fertilization, and half of the resulting body cells must have a constitution differing from that of the other half. Since there are known no chromosome fusions in which a restoration in the number of qualities is known to occur, the number of these qualities in a single chromosome would in a few generations be reduced to one: in view of the large number of past generations this must have already occurred. This new interpretation of chromosome behavior at fertilization and the ensuing mitosis is thus seen to offer a direct challenge to those theories of heredity that are based upon the idea of chromosomes carry- ing linear series of differentiated units. It has now been put forward by Hutchinson (1915) for Abies balsamea, by Chamberlain (1916) for Stangeria paradoxa, and by Miss Weniger (1918) for Lilium philadel- phicum and L. longiflorum. Consequently several investigators have renewed the study of fertilization, and evidence contradictory to the new theory has been found by Miss Nothnagel (1918) and Sax (1918), whose researches are summarized in the following section on the angiosperms. Angiosperms. — The angiosperms are characterized by the occurrence of "double fertilization," a phenomenon discovered independently by Nawaschin (1898) and Guignard (1899). One .of the two male nuclei formed by the male gametophyte and brought into the embryo sac by the pollen tube, enters the egg and fuses with its nucleus, thus forming the primary nucleus of the embryo, while the other male nucleus fuses with the two polar nuclei to form the primary endosperm nucleus (Fig. 123, 5). As the male nuclei pass down the pollen tube they are usually unaccompanied by any specially differentiated .cytoplasm: the male gametes are naked nuclei and not complete cells. In some cases, how- ever, male cells have been reported (Fig. 123, A). When they are liberated in the embryo sac by the rupture of the end of the pollen tube any such cytoplasm is indistinguishable from that of the sac and that discharged from the pollen tube. The male nuclei may appear in all respects similar to other nuclei, or they may be distinctly vermiform, as was observed by Mottier (1898) and later by many other workers (Fig. 123, E). That such vermiform nuclei have the power of inde- pendent movement has been held by Nawaschin (1899, 1900, 1909, 1910) for Lilium and Fritillaria, by Guignard (1900) for Tulipa, and by Blackman and Welsford (1913) and Miss Welsford (1914) for Lilium Martagon and L. auratum. The vermiform condition may persist until the time of fusion, but in other cases, such as Fritillaria (Sax 1916), it gives way to the ordinary shape. This change may occur more rapidly FERTILIZATION 299 in one male nucleus than in the other, so that the two may appear quite unlike during the later stages. Miss Welsford also sees in the male cytoplasm certain granules which she thinks may represent the vestiges of blepharoplasts. B FIG. 123. — Fertilization in angiosperms. A, end of pollen tube from basal portion of style of Lilium auratum, showing two male cells and tube nucleus. X 250. (After Welsford, 1914.) B, double fertilization in Lilium canadense: male and female nuclei about to fuse in egg; second male and two polar nuclei fusing at center of embryo sac; s, synergids, one degenerated; a, antipodals X 250. C, fusion of sexual nuclei in egg of Lilium philadelphicum. X 1000. (After Weniger, 1918.) D, the second male and two polar nuclei in Lilium Martagon. X 750. (After Nothnagel, 1918.) E, vermiform male nucleus in contact with egg nucleus in Triticum durum. X 600. (After Sax, 1918.) F, spireme stage of triple fusion nucleus in Triticum durum, showing distinctness of three chromatin contributions. X 750. (After Sax, 1918.) G, inclusion of cytoplasm in fusing sexual nuclei of Peperomia sintenesii. (After Brown, 1910.) Fusion in Egg. — As already stated, one male nucleus passes into the egg and fuses with the egg nucleus. So far as observations enable one to say, only the male nucleus, and no cytoplasm, enters the egg, a point of much importance in connection with the transmission of heredi- tary characters from the male parent. It would be a matter of extreme difficulty, however, to demonstrate conclusively that in passing through the egg membrane the male nucleus is absolutely freed of all adhering cytoplasm or chondriosomes; and it must be admitted that such a demonstration has not yet been given in any case. The fusion of the two sexual nuclei probably occurs in most cases very soon after they come in contact, though in certain forms the actual fusion is known to be 300 INTRODUCTION TO CYTOLOGY considerably delayed. The chromatin of the two nuclei at the time these unite may be in the reticulate (resting) condition, the male and female chromatins being indistinguishable in the fusion nucleus. This situation was described by most of the earlier workers, including Stras- burger (1900, 1901), Mottier (1904), Nawaschin (1898, 1899), and Ernst (1902). It has also been reported by Sax (1916) in his recent work on Fritillaria. In other cases, as early reported by Guignard (1891), the chromatin has already reached the spireme stage characteristic of the prophase, the male and female elements being distinguishable on the spindle in the ensuing division of the fertilized egg. Such is the condi- tion, for instance, in Calopogon (Pace 1909), Trillium (Nothnagel 1918), and Lilium (Weniger 1918). That the same species may show con- siderable variation in this respect is indicated by the situation in Fritillaria, in which Sax (1916, 1918) finds that fusion, though it usually occurs in the resting stage, sometimes takes place after the spiremes have been developed. Miss Weniger (1918) reports that in Lilium philadelphicum and L. longiflorum the egg nucleus is in the resting con- dition and the male nucleus in the spireme stage at the time of union. Chromosome Behavior. — With regard to the behavior of the two par- ental groups of chromosomes, it has been generally held that, whatever their condition at the time of the nuclear union, all of them, both pater- nal and maternal, split longitudinally at the first division of the fer- tilized egg, the daughter chromosomes so formed being distributed to the two resulting nuclei, just as in all the subsequent somatic divisions. Recently, however, Miss Weniger (1918) has reported a condition in Lilium similar to that described by Hutchinson (1915) for Abies: the maternal and paternal chromosomes form pairs and divide transversely into daughter segments which pass to the poles. (See p. 296.) With this conclusion other recent investigations of fertilization in angiosperms are not in agreement. Miss Nothnagel (1918) finds in Trillium no such pairing and cross segmentation as Hutchinson and Miss Weniger de- scribe, and states that each chromosome splits longitudinally as held by Miss Ferguson for Pinus and by cytologists in general. Sax (1918) also shows that each paternal and maternal chromosome in Fritillaria divides longitudinally, the diploid number (24) passing to each pole. In Trillium he reports an essentially similar state of affairs, the diploid number here being about 28. He therefore holds that the first mitosis in the fertilized egg is like any other somatic mitosis, and that no mech- anism for the segregation of factors of inheritance, such as occurs at reduction, exists here. The outcome of this controversy is awaited with much interest because of its great theoretical importance. Endosperm Fusion. — The fusion of the second male nucleus with the two polar nuclei of the embryo sac to form the primary endosperm nucleus may be carried out in a variety of ways. The most commonly FERTILIZATION 301 reported method is that by which the two polars fuse to form an "em- bryo sac nucleus" before the entrance of the pollen tube, the male nucleus later being added. Ernst (1902), for example, found this to be the method in Pan's quadrifolia. Less frequently the male nucleus meets and fuses with the polar nucleus of the micropylar end of the sac, the other polar then fusing with the product. This is the method de- scribed by Nawaschin (1898, 1899) in his account of the discovery of double fertilization in Lilium Martagon and Fritillaria tenella. The simultaneous fusion of all three nuclei appears to be a common occurrence; it has recently been described in some detail by Miss Noth- nagel (1918) for Trillium and Lilium. Just as in the case of the union of the first male nucleus with the egg nucleus, the chromatin of the second male and two polar nuclei may be either in the reticulate or the spireme condition as they come together. In Lilium Martagon (Noth- nagel 1918) (Fig. 123, Z>) it is in the form of fine strands, intermediate between the resting and spireme stages. Although the three nuclei often appear exactly alike, it is frequently possible to distinguish the male from the polars, not only by its shape and smaller size, but by the condition of its chromatin: in Lilium longiflorum (Weniger 1918), for example, the male nucleus is in the spireme stage while the polar nuclei are still in the resting condition. The membranes of the three nuclei may persist for some time after they come into intimate contact, and even after they have dissolved the chromatic elements of the three con- stituent nuclei may in many cases be distinguished if the section has been made in a favorable plane. When fusion occurs in the resting stage this is not so apparent, but when it occurs in the spireme stage the three chromatic groups are made out with little difficulty. Endosperm. — As the division of the endosperm nucleus approaches the spiremes of its three constituent nuclei become increasingly dis- tinct, even' if one or more of the nuclei have fused in the resting stage. Nothnagel (1918), Weniger (1918), and Sax (1918) in their recent studies all report this condition (Fig. 123, F). As the spiremes are being developed into completed chromosomes all of them (3x in number) split longitudinally, no observer reporting such a pairing as some have thought to occur between the chromosomes of the egg and first male nuclei. Miss Nothnagel describes the formation of a tripolar spindle about the chromosomes; the bipolar condition soon develops from this. How frequently this may occur is not known. Eventually in any case the mitosis proceeds along the usual lines and the two daughter nuclei receive 3x chromosomes each. This number is characteristic of all the cells of the endosperm formed by the repeated division of these nuclei. An exceptional condition has been noted by Sax (1918) in Fritillaria. Here the lower polar nucleus, because of an irregularity in the mitosis giving rise to it, has 24 (2x) chromosomes instead of the normal 12 302 INTRODUCTION TO CYTOLOGY (x). Consequently the female parent contributes 3C chromosomes (24 in one polar nucleus and 12 in the other) to the endosperm, while the male parent contributes only 12; thus the endosperm has 48 (4x) chromo- somes instead of the normal 36. Although in the great majority of known examples endosperm is formed by the repeated division of a triple fusion nucleus, cases are known in which it is produced by the polar fusion nucleus (embryo sac nucleus) alone without the male, or by the fusion product of the male and one polar, or by one polar alone. Combinations of these three methods may be found in the same embryo sac. The development of the endosperm may be initiated by the formation of a number of free nuclei which are parietally placed and in later mitoses become separated by walls, or by the formation of walled cells from the start. (See Coulter and Cham- berlain, 1903.) The term xenia was applied by Focke (1881) to the effect of foreign pollen on the endosperm of the resulting seed in angiosperms. Thus if maize of a certain strain which produces seeds with white endosperm when self-pollinated, is pollinated with pollen from a plant whose seeds have red endosperm, the endosperm of the resulting hybrid seeds is red like that of the pollen parent. No satisfactory explanation of this phenom- enon was at hand until the discovery of double fertilization by Nawas- chin and Guignard in 1898-9. It then became clear that the endosperm, which was formerly supposed to contain only maternal nuclear material, may show endosperm characters of the parent furnishing the pollen for the reason that the latter contributes a nucleus to the primary endosperm nucleus, so that every endosperm cell contains some nuclear material from the pollen parent. Normally the endosperm cells are all alike in containing two chromo- some sets from the female parent and one set from the male, and the nor- mal inheritance of endosperm characters as well as all ordinary cases of xenia can be understood on this basis. Mottled or mosaic effects in the endosperm of maize hybrids were attributed by Webber (1900) to such abnormal modes of endosperm origin as were referred to in a foregoing paragraph: some of the cells may have been formed by polar nuclei of purely maternal constitution while other cells were the result of the independent division of the second male nucleus. Although this explana- tion may fit some cases, it is becoming apparent from the work of Emerson and others that most of them can be better accounted for on the basis of aberrant chromosome behavior, such behavior having been observed in certain other organisms. Additional evidence is found in all these phenomena for the theory that the nuclear substance in some way represents the physical basis of inheritance. The second male nucleus not only is concerned in the initiation of the development of the endosperm, but xenia shows that it FERTILIZATION 303 also transmits parental characters. Here, therefore, as in the sexual fusion in the egg, the two principal effects of fertilization may be recognized. Bibliography 12 Fertilization ALLEN, C. E. 1917. The spermatogenesis of Polytrichum juniper inum. Ann. Bot. 31 : 269-292. pis. 15, 16. ATKINSON, G. F. 1915. Phylogeny and relationships in the ascomycetes. Ann. Mo. Bot. Garden 2: 315-376. figs. 10. BALLOWITZ, K. (Many papers on spermatozoa.) See Anat. Anz.'l, 1886; 27, 1905; 28, 1906; Arch. Mikr. Anat. 32, 1888 (birds); 36, 1890 (fishes, amphibia); 65, 1904; 70, 1907; Zeitschr. Wiss. Zool. 50, 1890 (insects); 52, 1891 (mammals); 91, 1908; Arch. f. Ges. Physiol. 46, 1890. BATAILLON, E. 1910. L'embryogenese complete provoquee chez les amphibiens par piqure de 1'oeuf vierge, larves parthenogenetiques de Ranafusca. Compt. Rend. Acad. Sci. Paris 150 : 996-998. VAN BENEDEN, E. 1883. Recherches sur la maturation de 1'oeuf, la feconda- tion et la division cellulaire. Arch. de. Biol. 4. 1887. (van Beneden et Neyt) Nouvelles recherches sur la fecondation et la divi- sion mitosique chez 1'Ascaride m£galocephale. Bull. Acad. Roy. Belg. Ill 14: 215-295. 1 pi. BENSATJDE, M. 1918. Recherches sur la cycle evolutif et la sexualit6 chez les Basisiomycetes. Diss. Nemours, pp. 156. pis. 13. (Review in Bot. Gaz. 68:67-68. 1919. Also in Bot. Absts. 3 : 49. 1920.) BERRIDGE, E. M. and SANDAY, F. 1907. Oogenesis and embryogeny in Ephedra distachya. New Phytol. 6: 128-134, 167-174. pis. 3, 4. BLACK, C. A. 1913. The morphology of Riccia Frostii, Aust. Ann. Bot. 27: 511- 532 pis. 37, 38. BLACKMAN, V. H. 1898. On the cytological features of fertilization and related features in Firms sylvestris L. Phil. Trans. Roy. Soc. London B 190: 395-426. pis 12-14. 1904. On the fertilization, alternation of generations, and general cytology of the Uredinese. Ann. Bot. 18: 323-369. pis. 21-24. BLACKMAN, V. H. and FRASER, H. C. 1. 1906. Further studies on the sexuality of the Uredinese. Ibid. 20: 35-48. pis. 3, 4. BLACKMAN, V. H. and WELSFORD, E. J. 1912. The development of the perithecium of Polystigma rubrum. Ibid. 26: 761-767. 2 pis. 1913. Fertilization in Lilium. Ibid. 27: 111-114. pi. 12. BLAKESLEE, A. F. 1904. Sexual reproduction in the Mucorinea?. Proc. Am. Acad. Arts and Sci. 40: 205-319. 1920. Sexuality in the Mucors. Science 51 : 375-382, 403-409. figs. 4. BOVERI, T. 1887a. Ueber die Befruchtung der Eier von Ascaris megaiocephcda. Sitzber. Gesell. Morph. Physiol. Munchen 3. 18876. Ueber die Anteil des Spermatozoon an der Teilung des Eies. Ibid. 1888. Zellenstudien. II. Jenaische Zeitschr. 22 : 685-882. pis. 19-23. 1890. Zellenstudien. III. Ibid. 24: 314-401. pis. 11-13. 1891. Befruchtung. (Review.) Ergebn. Anat. Entw. 1 : 386-485. 1902. Das Problem der Befruchtung. pp. 48. figs. 19. Jena. BROOKS, F. T. 1910. The development of Gnomonia erythrostomum Pers. Ann. Bot. 24 : 585-605. pis. 48, 49. 304 INTRODUCTION TO CYTOLOGY BROWN, H. B. 1913. Studies in the development of Xylaria. Ann. Mycol. 11: 1-13. pis. 1, 2. BROWN, W. H. 1909. Nuclear phenomena in Pyronema confluens. J. H. U. Circ. 6:42-45. 1910a. The development of the ascocarp of Leotia. Bot. Gaz. 50: 443-459. figs. 47. 19106. The exchange of material between nucleus and cytoplasm in Peperomia Sintenesii. Ibid. 49 : 189-194. pi. 13. 1911. The development of the ascocarp of Lachnea scutellata. Ibid. 62: 275-305. pi. 9. figs. 51. CARNOY, J. B. et LEBRUN, H. 1897. La fecondation chez I'Ascaris megalocephala. La Cellule 13 : 63-195. pis. 2. CARRUTHERS, D. 1911. Contributions to the cytology of HelveUa crispa Fries. Ann. Bot. 25 : 243-252. pis. 18, 19. CHAMBERLAIN, C. J. 1899. Oogenesis in Pinus Laricio. Bot. Gaz. 27: 268-280. pis. 4-6. 1910. Fertilization and embryogeny in Dioon edule. Ibid. 50: 415-429. pis. 14-17. 1912. Morphology of Ceralozamia. Ibid. 63: 1-19. pi. 1. figs. 7. 1916. Stangeria paradoxa. Ibid. 61 : 353-372. pis. 24-26. 1 fig. CHAMBERS, R. 1917. Microdissection studies. II. Jour. Exp. Zool. 23 : 483-504. CHILD, C. M. 1915. Senescence and Rejuvenescence. Chicago. CHRISTMAN, A. H. 1905. Sexual reproduction in the rusts. Bot. Gaz. 39: 267- 274. pi. 8. 1907. The alternation of generations and the morphology of the spore forms in the rusts. Ibid. 44: 81-101. pi. 7. CLAUSSEN, P. 1907. Zur Kenntniss der Kernverhaltnisse von Pyronema confluens. Ber. Deu. Bot. Ges. 25: 586-590. 1908. Ueber Entwicklung und Befruchtung bei Saprolegnia monoica. Ibid. 26 : 144-161. 1912. Zur Entwicklungsgeschichte der Ascomyceten. Pyronema confluens. Zeit. f . Bot. 4 : 1-64. pis. 1-6. figs. 13. CLELAND, R. E. 1919. The cytology and life history of Nemalion multifidum, Ag. Ann. Bot. 33 : 323-352. pis. 22-24. figs. 3. COKER, W. C. 1903. On the gametophytes and embryo of Taxodium. Bot. Gaz. 36: 1-27, 114-140. pis. 1-11. 1907. Fertilization and embryogeny in Cephalotaxus Fortunei. Ibid. 43 : 1-10. pi. 1. figs. 5. CONKLIN, E. G. 1897. The embryology of Crepidula. Jour. Morph. 13: 1-226. pis. 1-9. 1901. The individuality of the germ-nuclei during the cleavage of Crepidula. Biol. Bull. 2 : 257-265. COULTER, J. M. and CHAMBERLAIN, C. J. 1903. Morphology of Angiosperms. 1910. Morphology of Gymnosperms. Chicago. COULTER, J. M. and COULTER, M. C. 1918. Plant Genetics. Chicago. COULTER, J. M. and LAND, W. J. G. 1905. Gametophytes and embryo of Torreya taxifolia. Bot. Gaz. 39 : 161-178. pis. A, 1-3. CUTTING, S. M. 1909. On the sexuality and development of the ascocarp in Ascophanus carneus Pers. Ann. Bot. 23 : 399-417. pi. 28. DALE, E. 1909. On the morphology and cytology of Aspergillus repens. Ann. Mycol. 7: 215-225. pis. 2, 3. DANGEARD, P. A. 1894a. M6moire sur la reproduction sexuelle des Basidio- mycdtes. Le Botaniste 4: 119-181. figs. 24. FERTILIZATION 305 18946. La reproduction sexuelle des Ascomycetes. Ibid. 4: 21-58. figs. 10. 1895. Second memoire sur la reproduction sexuelle des Ascomycetes. Ibid. 5 : 245-284. figs 17. DAVIS, B. M. 1896. The fertilization of Batrachospermum. Ann. Bot. 10: 49-76. pis. 6, 7. 1900. The fertilization of Albugo Candida. Bot. Gaz. 29: 297-311. pi. 22. 1903. Oogenesis in Saprolegnia. Ibid. 35: 233-249, 320-349. pis. 9, 10. 1905. Fertilization in the Saprolegniales. Ibid. 39: 61-64. DOWNING, E. R. 1909. The ovogenesis of Hydra. Zool. Jahrb. (Anat. Abt.) 28: 295-324. pis. 11, 12. 2 figs. DUPLER, A. W. 1917. The gametophytes of Taxus canadensis Marsh. Bot. Gaz. 64: 115-136. pis. 11-14. EAMES, A. J. 1913. The morphology of Agathis australis. Ann. Bot. 27: 1-38. pis. 1-4. 92 figs. ERNST, A. 1902. Chromosomenreduktion, Entwicklung des Embryosackes und Befruchtung bei Paris quadrifolia L. und Trillium grandiflorum Salisb. Flora 91.1-46. pis. 1-6. FARMER, J. B. and WILLIAMS, J. L. 1898. Contributions to our knowledge of the Fucacese; their life history and cytology. Phil. Trans. Roy. Soc. London B 190: 623-645. pis. 19-24. FAULL, J. H. 1911. The cytology of the Laboulbeniales. Ann. Bot. 25: 649-654. 1912. The cytology of Laboulbenia chcetophora and L. Gyrinidarum. Ibid. 26: 325-355. pis. 37-40. FERGUSON, M. C. 1901. The development of the pollen tube and the division of the generative nucleus in certain species of Pinus. Ann. Bot. 15: 193-223. pis. 12-14. 1904. Contributions to the knowledge of the life history of Pinus. Proc. Wash. Acad. Sci. 6 : 1-202. pis. 1-24. 1913. Included cytoplasm in fertilization. Bot. Gaz. 56 : 501-502. FITZPATRICK, H. M. 1918o. Sexuality in Rhizina undutata Fries. Bot. Gaz. 65: 201-226 pis. 3 4. 19186. The cytology of Eucronarlium muscicola. Am. Jour. Bot. 6: 397-419. pis. 30-32. FOCKE, W. O. 1881. Die Pflanzen-Mischlinge. Berlin. FOL, H. 1891. Die "Centrenquadrille," ein neue Episode aus der Befruchtungs- geschichte. Anat. Anz. 6 : 266-274. figs. 10. FRASER, H. C. I. 1907. On the sexuality and development of the ascocarp of Lachnea stercorea Pers. Ann. Bot. 21 : 349-360. pis. 29, 30. 1908. Contributions to the cytology of Humaria rutilans. Ibid. 22: 35-55. pis. 4, 5. FRIES, R. E. 1911. Zur Kenntniss der Cytologie von Hygrophorus conicus. Svensk. Bot. Tids. 5: 241-251. pi. 1. GARBER, J. F. 1904. The life history of Ricciocarpus natans. Bot. Gaz. 37: 161- 177. pis. 9, 10. GRAHAM, M. 1918. Centrosomes in fertilization stages of Preissia quadrata (Scop.) Nees. Ann. Bot. 32 : 415-420. pi. 10. GUIGNARD, L. 1899., Sur les antherozoids et la double copulation sexuelle chez les ve'ge'taux angiospermes. Comp. Rend. Acad. Sci. Paris 128: 864-871. figs. 19. 1900. L'appareil sexuel et la double fe"condation dans les Tulipes. Ann. Sci. Nat. Bot. V1I1 11: 365-387. pis. 9-11. 1901. La double f4condation chez les Renonculace'es. Jour. Botanique 16: 394-408. figs. 16. 1902. La double f^condation chez les Solanees. Ibid. 16 : 145-167. figs. 45. 20 306 INTRODUCTION TO CYTOLOGY GUILLIERMOND, A. 1910. La sexualite chez les champignons. Bull. Sci. France et Belg. 44: 109-196. 1920. The Yeasts. (Engl. transl. by F. W. Turner.) N. Y. GUYER, M. F. 1907. The development of unfertilized frogs' eggs injected with blood. Science N. S. 25: 910-911. HAECKER, V. 1895. Ueber die Selbstandigkeit der vaterlichen und miitterlichen Kernbestandtheile wahrend der Embryonalentwicklung von Cyclops. Arch. Mikr. Anat. 46 : 579-617. pis. 28-30. 1899. Praxis und Theorie der Zellen- und Befruchtungslehre. HARPER, R. A. 1895. Beitrag zur Kenntniss der Kerntheilung und Sporenbildung im ASCU&. Ber. Deu. Bot. Ges. 13: (67)- (78). pi. 27. 1896. Ueber das Verhalten der Kerne bei den FruchteR. ntwicklung einiger Asco- myceten. Jahrb. Wiss. Bot. 29: 655-685. pis. 11, 12. 1900. Sexual reproduction in Pyronema confluens and the morphology of the ascocarp. Ann. Bot. 14: 321-396. pis. 19-21. 1905. Sexual reproduction and the organization of the nucleus in certain mildews. Carnegie Inst. Publ. 37. HARVEY, E. N. 1910. Methods of artificial parthenogenesis. Biol. Bull. 18: 269-180. HEILBRUNN, L. V. 1915. Studies in artificial parthenogenesis. 11. Physical changes in the egg of Arbacia. Biol. Bull. 29: 149-203. 1920. Studies in artificial parthenogenesis. 111. Cortical changes and the inia- tion of maturation in the egg of Cumingia. Biol. Bull. 38: 317-339. HERLANT, M. 1913. Le me'chanisme de la parthenoge"nese esperimentelle. Bull. Sci. France et Belg. VII 50 : 381-404. 1917. Etude sur les bases cytologiques du mecanisme de la parthenogenese experi- mentelle chez les amphibiens. Arch. d. Biol. 28: 505-608. HERTWIG, O. 1875. Beitrage zur Kenntniss der Bildung, Befruchtung, und Thei- lung des tierischen Eies, I. Morph. Jahrb. 1. HIRASE, S. 1895. Etudes sur la fecondation et 1'embryogenie du Ginkgo biloba. Jour. Imp. Coll. Sci. Tokyo 8: 307-322. pis. 31, 32. 1898. Etudes sur la fecondation et 1'embryogenie du Ginkgo biloba. (Second memoire.) Ibid. 12: 103-149. pis. 7-9. 1918. Further studies on the fertilization and embryogeny in Ginkgo biloba. Bot. Mag. Tokyo 32: No. 378. HOYT, W. D. 1910. Physiological aspects of fertilization and hybridization in ferns. Bot. Gaz. 49: 340-370. figs. 12. HUTCHINSON, A. H. 1915. Fertilization in Abies balsamea. Bot. Gaz. 60: 457- 472. pis. 16-20. fig. 1. HUXLEY, T. H. 1878. Evolution in Biology. IKENO, S. 1898. Untersuchungen ilber die Entwicklung der Geschlechtsorgane und der Vorgang der Befruchtung bei Cycas revoluta. Jahrb. Wiss. Bot. 32 : 557-602. pis. 8-10. 1901. Contributions a 1'etude de la fecondation chez le Ginkgo biloba. Ann. Sci. Nat. Bot. VIII 13: 305-318. pis. 2, 3. JONES, W. N. 1918. On the nature of fertilization and sex. New Phytol. 17 : 167-188. KEENE, M. L. 1914. Cytological studies of the zygospores of Sporodinia grandis. Ann. Bot. 28: 455-470. pis. 25, 26. KILDAHL, N. J. 1908. The morphology of Phyllocladiis alpina. Bot. Gaz. 46: 339-348. pis. 20-22. KOLTZOFF, N. K. 1906. Studien liber die Gestalt der Zelle: I, Untersuchungen iiber die Spermien der Decapoden. Arch. Mikr. Anat. 67: 364-572. pis. 25-29. figs. 37. FERTILIZATION 307 LAND, W. J. G. 1900. Double fertilization in Corapositse. Bot. Gaz. 30: 252- 260. pis. 15, 16. 1902. A morphological study of Thuja. Ibid. 34: 249-259. pis. 6-8. 1907. Fertilization and embryogeny of Ephedra trifurca. Ibid. 44: 273-292. pis. 20-22. LAWSON, A. A. 1904a. The gametophyte, archegonia, fertilization and embryo of Sequoia sempervirens. Ann. Bot. 18: 1-28. pis. 1-4. 19046. The gametophyte, fertilization and embryo of Cryplomeria japonica. Ibid. 18: 417-444. pis. 27-30. 1907. The gametophytes, fertilization and embryo of Cephalotaxus drupacea. Ibid. 21 ; 1-23. pis. 1-4. LEVINE, M. 1913. The cytology of Hymenomycetes, especially fche Boleti. Bull. Torr. Bot. Club 40: 137-181. pis. 4-8. LILLIE, F. R. 1901. The organization of the egg in Unio based on a study of its maturation, fertilization and cleavage. Jour. Morph. 17:' 227-292. pis. 24-27. 1912. Studies of fertilization in Nereis. III. The morphology of normal fertiliza- tion in Nereis. IV. The fertilizing power of portions of the spermatozoon. Jour. Exp. Zool. 12 r 413-476. pis. 1-11. 1914. Studies of fertilization. VI. The mechanism of fertilization in Arbacia. Ibid. 16: 523-590. 1919. Problems of Fertilization. Chicago. LILLIE. R. S. 1908. Momentary elevation of temperature as a means of producing artificial parthenogenesis in starfish eggs and the conditions of its- action. Jour. Exp. Zool. 6 : 375-428. 1915. On the conditions of activation of unfertilized starfish eggs under the influence of high temperatures and fatty acid solution. Biol. Bull. 28. 260-303. LOEB, J. 1910. Die Hemmung verschiedener Giftwirkungen auf das befruchtete Seeigelei durch Hemmung der Oxidationen in demselben. Biochem. Zeitschr. 29. 1911. Auf welche Weise rettet die Befruchtung das Leben des Eies? Arch. Entw. 31:658-668. 1912. The Mechanistic Conception of Life. Chicago. 1913. Artificial Parthenogenesis and Fertilization. Chicago. LOEB, J. and BANCROFT, F. W. 1913. The sex of a parthenogenetic tadpole and frog. Jour. Exp. Zool. 14: 275-277. LOEB, J. and WASTENEYS, H. 1910. Warum hemmt Natrium cyanide die Gift- wirkung einer Chlornatriumlosung fur das Seeigelei? Biochem. Zeitsch. 2. 1911. Sind die Oxidationsvorgange die unabhangige Variable in den Lebenser- scheinungen? Ibid. 36: 345-356. 1912. Die Oxidationsvorgange im befruchteten und unbefruchteten Seesternei. Arch. Entw. 36 : 555-557. LUTHER, A. 1904. Die Eumesostominen. Zeit. Wiss. Zool. 77: 1-273. pis. 9: figs. 16. MAcCtTRDY, H. M. 1919. Division, nuclear organization and conjugation in Arcella vulgaris. Mich. Acad. Sci. Rep. 21: 111-113. MACORMICK, F. A. 1912. Development of zygospore in Rhizopvs nigricans. Bot. Gaz. 63: 67-68. McCLENDON, J. J. 1912. Dynamics of cell division. Artificial parthenogenesis in Vertebrates. Am. Jour. Physiol. 29: 228-301. McCuBBiN, W. A. 1910. Development of the Helvellineae. Bot. Gaz. 49: 195- 206. pis. 14- 16 MEVES, FR. 1899. Ueber Struktur und Histogenese der Samenfaden des Meersch- weinchens. Arch. Mikr. Anat. 64: 329-402. pis. 19-21. figs. 16. 308 INTRODUCTION TO CYTOLOGY 1908. Die Chondriosomen als Trager erblicher Anlagcn. Cytologische Studien am Hiihnerembryo. Ibid 72: 816-867. pis. 39-42. 1915. Ueber Mitwirkung der Plastosomen bei der Befruchtung des Eies von Filaria papillosa. Ibid. 37 : 11 12-46. pis. 1-4. 1918. Die Plastosomentheorie der Vererbung. Ibid. 92: II 41-136. figs. 18. (Bibliography). MEYER, K. 1911. Untersuchungen iiber die Sporophyt der Lebermoose. 1. Entwicklungsgeschichte des Sporogons der Corsinia marchantioides. Bull. Soc. Imp. Moscow 236-286. MINCHIN, E. A. 1912. An Introduction to the Study of the Protozoa. London. MIYAKE, K. 1901. The fertilization of Pylhium deBaryanum. Ann. Bot. 15: 653-667. pi. 36. 1910. The development of the gametophytes and embryogeny in Cunninghamia sinensis. Beih. Bot. Centr. 27: 1-25. pis. 5. MORGAN, T. H. 1899. The action of salt solutions on the unfertilized and fertilized eggs of Arbacia, and of other animals. Arch. Entw. 8: 448-539. pis. 7-10. figs. 21. 1913. Heredity and Sex. New York. MOTTIER, D. M. 1898. Ueber das Verhalten der Kerne bei der Entwicklung des Embryosacks und die Vorgange bei der Befruchtung. Jahrb. Wiss. Bot. 31 : 125-158 pis. 2, 3. 1904. Fecundation in Plants. Carnegie Inst. Publ. 15. MURRILL, W. A. 1900. The development of the archegonium and fertilization in the hemlock spruce (Tsuga canadensis, Carr.) Ann. Bot. 14: 583-607. pis. 31, 32. NAGLER, K. 1909. Entwicklungsgeschichtliche Studien iiber Amoben. Arch. f. Protist. 15 : 1- NAWASCHIN, S. 1899. Neue Beobachtungen iiber Befruchtung bei Fritillaria und Lilium. Bot. Centr. 77: 62. (Russian account in 1898.) 1909. Ueber das selbstandige Bewegungsvermogen der Spermakerne bei einigen Angiospermen. CEsterreich. Bot. Zeitschr. 59: 457. 1910. Naheres iiber die Bildung der Spermakerne bei Lilium Martagon. Ann. Jard. Bot. Buit. 12 : Suppl. Ill, 871-904. pis. 33, 34. NEMEC, B. 1910. Das Problem der Befruchtungsvorgange. Jena. 1912. Ueber die Befruchtung bei Gagea. Bull. Internat. Acad. Sci. Boheme, 1-17. figs. 19. NICHOLS, G. E. 1910. A morphological study of Juniperus communis var. depressa. Beih. Bot. Centr. 25: 201-241. pis. 8-17. figs. 4. NIENBURG, W. 1914. Zur Entwicklungsgeschichte von Polystigrna rubrum DC. Zeit. f. Bot. 6 : 369-400. figs. 17. NOTHNAGEL, M. 1918. Fecundation and formation of the primary endosperm nucleus in certain Liliacese. Bot. Gaz. 66: 143-161. pis. 3-5. NOREN, C. O. 1904. Ueber Befruchtung bei Juniperus communis. (Vorlauf. Mitt.) Arkiv. Bot. Svensk. \et.-Akad. 3: pp. 11. 1907. Zur Entwicklungsgeschichte des Juniperus communis. Uppsala Univ. Arsskrift, pp. 64. pis. 4. OSTERHOUT, W. J. V. 1900. Befruchtung bei Batrachospermum. Flora 87: 109- 115. pi. 5. OVERTON, J. B. 1913. Artificial parthenogenesis in Fucus. Science 37: 841. 844. PACE, L. 1909. The gametophytes of Calopogon. Bot. Gaz. 48: 126-137. pis. 7-9. RABL, C. 1889. Ueber ZeUtheibuig. Anat. Anz. 4: 21-30. figs. 2. FERTILIZATION 309 RAMLOW, G. 1914. Beitrage zur Entwicklungsgeschichte der Ascoboleen. Mycol. Centralbl. 5: 177-198. pis. 1, 2. figs. 20. ROBERTSON, A. 1904. Studies in the morphology of Torreya californica. 11. The sexual organs and fertilization. New Phytol. 3: 205-216. pis. 7-9. ROMIEU, M. 1911. La spermiogenese chez I'Ascaris megalocephala. Arch. Zellf. 6: 254-325. pis. 14-17. RUCKERT, J. 1895. Ueber die Selbstandigbleiben der vaterlichen und miitterlichen Kernsubstanz wahrend der ersten Entwicklung des befruchteten Cyclops-FAes. Arch. Mikr. Anat. 45: 339-369. pis. 21, 22. SARGANT, E. 1896. The formation of the sexual naclei in Lilium Marlagon. I. Oogenesis. Ann. Bot. 10 : 445-477. pis. 22, 23. 1897. Same title. II. Spermatogenesis. Ibid. 11: 187-224. pis. 12, 13. SAWYER, M. L. 1917. Pollen tube and spermatogenesis in Iris. Bot. Gaz. 64: 159-164. figs. 18. SAX, K. 1916. Fertilization in Frilillaria pudica. Bull. Torr. Bot. Club 43: 505-522. pis. 27-29. 1918. The behavior of the chromosomes in fertilization. Genetics 3. 309-327. pis. 1, 2. SCHAUDINN, F. 1896. Ueber die Copulation von Actinophrys sol. Sitzber. Acad. Wiss. Berlin. SCHEBEN, L. 1905. Beitrage zur Kenntniss des Spermatozoons von Ascaris megalo- cephala. Zeit. Wiss. Zool. 79: 396-431. pis. 20, 21. 3 figs. SCHIKORRA, W. 1910. Ueber "die Entwicklungsgescnichte von Monascus. Zeitschr. f . Bot. 1 : 379-410. SHARP, L. W. 1912. Spermatogenesis in Eguisetum. Bot. Gaz. 64: 89-119. pis. 7, 8. 1914. Spermatogenesis in Marsilia. Ibid. 68: 419-431. pis. 33, 34. 1920. Spermatogenesis in Blasia. Ibid. 69 : 258-268. pi. 15. SMITH, B. G. 1919. The individuality of the germ-nuclei during the cleavage of the egg of Cryptobranchus alleghaniensis. Biol. Bull. 37 . 246-287. STEIL, W. N. 1918. Method for staining antherozoid of fern. Bot. Gaz. 66 : 562-563. 1 fig. STEVENS, F. L. 1899. The compound oosphere of Albugo blili. Bot. Gaz. 28: 149-176. pis. 11-15. 1901. Gametogenesis and fertilization in Albugo. Ibid. 32: 77-98. pis. 1-4. STRASBURGER, E. 1877. Ueber Befruchtung und Zelltheilung. Jen. Zeitschr. 11. 1884. Neue Untersuchungen tiber die Befruchtungsvorgang bei den Phanero- gamen, als Grundlage fur eine Theorie der Zeugung. Jena. 1892. Schwarmsporen, Gameten, pflanzlichen Spermatozoiden, und das Wesen der Befruchtung. Histol. Beitr. 4: 49-158. pi. 3. 1897. Kerntheilung und Befruchtung bei Fucus. Jahrb. Wiss. Bot. 30 : 351-374. pis. 27, 28. 1900. Einige Bemerkungen zur Frage nach der "doppelten Befruchtung" bei den Angiospermen. Bot. Zeit. 68: 293-316. 1901. Ueber Befruchtung. Ibid. 59: 11, 353-368. TROW, A. H. 1895. The karyology of Saprolegnia. Ann. Bot. 9: 609-652. pis. 24, 25. 1899. Observations on the biology and cytology of a new variety of Achlya ameri- cana. Ibid. 13: 131-179. pis. 8-10. 1901. Biology and cytology of Pylhium ultimum, n. sp. Ann. Bot. 15: 269- 312. pis. 15, 16. 1904. On fertilization in the Saprolegniacese. Ibid. 18: 541-569. pis. 34-36. 310 INTRODUCTION TO CYTOLOGY WAGER, H. 1896. On the structure and reproduction of Cystopus candidus Lev. Ann. Bot. 10: 295-342. pis. 15,16. See also pp. 89-91. 1899. The sexuality of fungi. Ibid. 13: 575-597. WALDEYER, W. 1888. Ueber Karyokinese und ihre Beziehung zu den Befrucht- ungsvorgangen. Arch. Mikr. Anat. 32: 1-122. figs. 14. (Engl. transl. in Quar. Jour. Micr. Sci. 30: 159-281. pi. 14. 1889.) WALTON, A. C. 1918. The oogenesis and early embryology of Ascaris canis Werner Jour. Morph. 30: 527-604. pis. 9. fig. 1. WARBURG, O. 1908. Beobachtungen tiber die Oxidationsprozesse im Seeigelei. Zeitschr. Physiol. Chem. 57. . 1910. Ueber die Oxidationen im lebenden Zellen nach Versuchen am Seeigelei. Ibid. 66. 1911. Untersuchungen tiber die Oxidationsprozesse im Zellen. Mtinchener Med. Wochenschr. 57. 1914. Beitrage zur Physiologie der Zelle, inbesondere tiber die Oxidationsgesch- windigkeit in Zellen. Ergeb. d. Physiol. 14. WEBBER, H. J. 1900. Xenia, or the immediate effect of pollen in maize. U. S. Dept. Agr., Div. Veg. Path, and Physiol., Bull. 22: pis. 4. 1901. Spermatogenesis and fecundation in Zamia. U. S. Dept. Agr. Bur. Pit. Ind. Bull. 2. pp. 100. pis. 7. \VELSFORD, E. J. 1907. Fertilization in Ascobolus furfuraceus. New Phytol. 6: 156. 1914. The genesis of the male nuclei in Lilium. Ann. Bot. 28: 265-270. pis. 16, 17. WENIGER, W. 1918. Fertilization in Lilium. Bot. Gaz. 66: 259-268. pis. 11-13. WHEELER, W. M. 1895. The behavior of the centrosomes in the fertilized egg of Myzostoma glabrum Leukart. Jour. Morph. 10: 305-311. figs. 10. 1897. The maturation, fecundation and early cleavage of Myzostoma glabrum Leukart. Arch. d. Biol. 15: 1-77. pis. 1-3. WILDMAN, E. E. 1913. The spermatogenesis of Ascaris megalocephaia with special reference to the two cytoplasmic inclusions, the refractive body and the "mito- chondria": their origin, nature and role in fertilization. Jour. Morph. 24: 421-157. pis. 3. WILSON, E. B. 1900. The Cell in Development and Inheritance. 2d ed. 1901. Experimental studies in cytology. I. A cytological study of artificial parthenogenesis in sea urchin eggs. Arch. Entw. 12: 529-596. pis. 11-17. figs. 12. WINGE, O. 1914. The pollination and fertilization process in Humulus lupulus L. and H. Japonicus. Comp. Rend. Trav. Lab. Carlsberg 11. WOODBURN, W. L. 1920. Preliminary notes on the embryology of Reboulia hemis- phcerica. Bull. Torr. Bot. Club. 46: 461-464. pi. 19. WOYCICKI, Z. 1899. (On fertilization in Coniferse.) pp. 57. pis. 2. (Russian.) YAMANOTTCHI, S. 1906. The life history of Polysiphonia violacea. Bofc. Gaz. 42: 401-449. pis. 19-28. 1908. Spermatogenesis, oogenesis, and fertilization in Nephrodium. Ibid. 45: 145-175. pis. 6-8. CHAPTER XIII APOGAMY, APOSPORY, AND PARTHENOGENESIS APOGAMY AND APOSPORY The life cycle in all bryophytes and vascular plants is characterized by a regular alternation of two well marked phases or generations: the gametophyte, which arises from the spore and produces gametes; and the sporophyte, which arises from the fusion product of two gametes and produces spores. In such a normal life cycle the number of chromosomes in the nuclei is doubled at the union of the gametes and reduced to the original number at sporogenesis ; the gametophyte is therefore the haploid generation and the sporophyte the diploid generation, their limits being marked by the two cytological crises, fertilization and reduction. Such an alternation of haploid and diploid phases has been discovered in the life cycles of many alga? and fungi also, so that the general conception of alternation of generations has been extended to these lower groups. This, however, is not the place for a discussion of the homologies implied. It should be added that gametophyte and sporophyte may arise not only from each other, but either generation may also multiply by vegetative means. Many instances in which the above typical life cycle is departed from, and in which the correlation between the alternation of two generations and periodic changes in chromosome number is broken, are now known, the conspicuous examples being found among the ferns and certain angiosperms. The very convenient classification of such abnormalities drawn up by Vines (1911) .is given as the basis for the present portion of the chapter. All dates and the matter included within square brackets have been added by the present author. "In the first place, the sporophyte may be developed either after an abnormal sexual act, or without any preceding sexual act at all, a con- dition known as apogamy. In the second, the gametophyte may be developed otherwise than from a post-meiotic spore, a condition known as apospory.1 1 [Apogamy in ferns was discovered by Farlow in 1874. Apospory was discovered in mosses by Pringsheim in 1876 and in ferns by Druery in 1884. General discussions of these phenomena are given by Winkler (1908) and Strasburger (19096).] 311 312 INTRODUCTION TO CYTOLOGY Apogamy. — The cases to be considered under this head may be arranged in two groups: 1. Pseudapogamy: sexual act abnormal. — The following abnormalities have been observed: (a) Fusion of two female organs: observed (Christman 1905) in certain Uredinese (Cceoma nitens, Phragmidium speciosum, Uromyces Caladii) where adjacent archicarps fuse: male cells (spermatia) are present but functionless. (6) Fusion between nuclei of the same female organ: observed in the ascogonium of certain ascomycetes, Humaria granulata (Black- FIG. 124.- — Apogamy in ferns. A, nuclear migration in gametophyte cells of Lastrcea pseudo-mas var. polydactyla. X 500. (After Farmer and Digby, 1907.) B, section through gametophyte, showing young sporophytic tissue (s) "engrafted" into surrounding gametophy tic tissue (g). (After Farmer and Digby.) C, sporophyte arising apogamously from gametophyte in Pteris cretica: bl, first leaf; v, stem apex; w, root. (After de Bary.) man 1906), where there is no male organ; Lachnea stercorea (Fraser 1907), where the male organ (pollinodium) is present but apparently functionless. [A similar condition has been reported in Ascobolus furfuraceus (Welsford 1907), Aspergillus repens (Dale 1909), and Ascophanus carneus (Cutting 1909).] (c) Fusion of a female organ with an adjacent tissue-cell: observed (Blackman 19046) [Blackman and Fraser 1906] in the archicarp of some Uredineae (Phragmidium violaceum, Uromyces Poce, Puccinia Poarum) : male cells (spermatia) present but function- less. APOGAMY, APOSPORY, AND PARTHENOGENESIS 313 (d) There is no female organ: fusion takes place between two adjacent tissue-cells of the gametophyte ; the sporophyte is developed from diploid cells [''grafted tissue"] thus produced, but there is no proper zygote as there is in a, b, and c: observed (Farmer [and Digby] 1907) in the prothallium of certain ferns (Lastrcea pseudo- mas, var. polydactyla} [Fig. 124, A] : male organs (and sometimes female) present but functionless. Another such case is that of Humaria rutilans (ascomycete), in which nuclear fusion ••• v--«*v*x /wi. & * • •" '• i >Mm** Jf- ••• FIG. 125. A, cell fusion in the sporangium of Aspidium falcatum. X 1950. (After R. F.Allen 1911.) B, incomplete nuclear division in sporangium of Nephrodium hirtipes. X 1250. (After Steil, 1919.) C, apogamy and sporophytic budding in the embryo sac of Alchemilla pastoralis: egg developing apogamously below; cell of nucellus forming an embryo above; two polar nuclei and one synergid nucleus at center. (After Murbeck, 1902.) has been observed (Fraser 1908) in hyphse of the hypothecium : the asci are developed from these hyphae, and in them meiosis takes place; there are no sexual organs. [A similar condition has been reported in Helvella crispa (Carruthers 1911) and Polystigma rubrum (Blackman and Welsford 1912). It has already been pointed out (p. 291) that many students of the ascomycetes deny the existence of a nuclear fusion in the archicarp or vegetative cells, holding rather that the only 314 INTRODUCTION TO CYTOLOGY fusion in the life cycle is that observed in the ascus, and that this fusion is the real sexual act.] [(e) Fusion of two haploid sporocytes: In Aspidium falcatum (R. F. Allen 1911) a haploid sporophyte arises by vegetative apogamy from a haploid gametophyte. In the sporangium the 16 haploid sporocytes fuse in pairs, producing eight diploid cells (Fig. 125, A}. In these cells reduction occurs, 32 haploid spores resulting.] 2. Eu-apogamy: no kind of sexual act. (a)" The gametophyte is haploid: (a) The sporophyte is developed from the unfertilized haploid oosphere : no such case of true parthenogenesis has yet been observed. [Kusano (1915) has observed the division of the haploid nucleus of an unfertilized egg in a few excep- tional cases in the orchid, Gastrodia data. Parthenogeneti c development proceeds no further. The unfertilized egg of Fucus has been made to begin development by artificial means (Overton 1913), but the cytological facts are not known here. Motile gametes of certain other algae have been observed to develop without conjugation, as in Ectocarpus tomentosus (Kylin 1918).] (/3) The sporophyte is developed vegetatively from the gameto- phyte and is haploid : observed in the prothallia of certain ferns, Lastrcea pseudo-mas, var. cristata-apospora (Farmer and Digby 1907), and Nephrodium molle (Yamanouchi 1908). [In the gametophytes of Nephrodium molle, which has antheridia but no functional archegonia, Yamanouchi found no nuclear migrations such as Farmer described in Lastrcea (see Id} ; but there was haploid grafted tissue, from which a haploid sporophyte developed. In Nephro- dium hirtipes (Steil 1919) a haploid sporophyte arises by vegetative apogamy from a haploid gametophyte. When there are eight sporogenous cells in the sporangium there is an incomplete nuclear and cell division (Fig. 125, B), each nucleus coming to have the diploid number of chromo- somes. These eight diploid cells function as sporocytes and produce 32 haploid spores. Steil at first (1915) adopted Allen's interpretation (le) for his material, but later decided that the phenomenon observed was one of in- complete division, and not one of fusion. In this case, as in Aspidium falcatum, apogamy is offset not by apospory but by an abnormal course of events in the sporangium. In Aspidium falcatum the sporophyte arises as in the examples mentioned in this paragraph, but because of APOGAMY, APOSPORY, AND PARTHENOGENESIS 315 the presence of a cell and nuclear fusion it is classified under le.] (6) The gametophyte is diploid (see under Apospory): (a) The sporophyte is developed from the diploid oosphere: observed in some Pteridophyta, viz. certain ferns (Farmer 1907), Athyrium Filix-fcemina, var. clarissima, Scolopend- rium vulgare, var. crispum-Drummondce, and Marsilia (Strasburger 1907); also in some Phanerogams, viz., Composite (Taraxacum, Murbeck 1904; Antennaria alpina, Juel 1898, 1900; sp. of Hieracium, Rosenberg 1906): Rosacese (Eu-Alchemilla sp., Murbeck 1901, 1904, Stras- burger 1905 [Fig. 125, C]): Ranunculacese (Thalictrum purpurascens, Overton 1902). [Also in the lily, Atamosco (Pace 1913), and Burmannia (Ernst 1909). Besides this form of apogamy ("ooapogamy" or "generative apo- gamy") Antennaria may also develop embryos from diploid synergids ("vegetative apogamy") and from cells of the nucellus ("sporophytic buddirg"). A similar variety of embryo origins is found in certain other angio- sperms. In many cases the chromosome number in apogamous species is about twice as large as that of nearly related forms reproducing sexually (Rosenberg 1909).] (/3) The sporophyte is developed vegetatively from the gameto- phyte: observed (Farmer [and Digby] 1907) in the fern Athyrium Filix-foemina, var. clarissima. In all cases enumerated under Eu-apogamy, apogamy is associated with some form of apospory except Nephrodium molle, full details of which have not yet been published. [It is possible that a behavior like that in Aspidium falcatum (le) or in Nephrodium hirtipes (2a/3) may occur in Nephrodium molle.] Many other ferns are known to be apogamous, but they are not included here because the details of their nuclear structure have not been investigated. Apospory. — The known modes of apospory may be arranged as follows : 1. Pseudapospory: a spore is formed but without meiosis, so that it is diploid — observed only in heterosporous plants, viz. certain species of Marsilia (e.g. Marsilia Drummondii) where the megaspore has a diploid nucleus (32 chromosomes) and the resulting prothallium and female organs are also diploid (Strasburger 1907); and in various Phanerogams, some Compositse (Taraxacum and Antennaria alpina, Juel 1898, 1900, 1904), some Rosacese (Eu-Alchemilla, Strasburger 1905), and occasionally in Thalictrum purpurascens (Overton 1902), where the megaspore ([and] embryo-sac) is diploid; in some species 316 INTRODUCTION TO CYTOLOGY of Hieracium it has been found (Rosenberg 1906) that adventitious diploid embryo-sacs are developed in the nucellus: these plants are also apogamous. [In Marsilia Drummondii, which Shaw (1897) and Nathansohn (1909) had shown to be apogamous, Stras- burger (1907) found that, although normal reduction occurs in some of the megasporocytes, giving spores with 16 chromosomes, other megasporocytes undergo two divisions neither of which is reductional: the first division is homceotypic in character and the second is an additional vegetative mitosis without a homologue FIG. 126. A, gametophyte with antheridium (anth.) and rhizoids (r) arising aposporously from tissue of sorus in Polystichum angulare var. pulcherrimum; sp, sporangia. X 70. (After Bower.) B, gametophyte with archegonia arising from tip of pinnule in Polystichum. X 10. (After Bower.) in the normal cases. The resulting spores are therefore diploid, and ooapogamy follows.] 2 Eu-apospory: no spore is formed — of this there are two varieties: (a) With meiosis: this occurs in some Thallophyta which form no spores; the sporophyte of the Fucacese bears no spores, con- sequently meiosis takes place in the developing sexual organs. The Conjugate Green Algae also have no spores, meiosis taking place in the germinating zygospore which develops directly into the sexual plant. APOGAMY, APOSPORY, AND PARTHENOGENESIS 317 (b) Without meiosis: the gametophyte is developed upon the sporo- phyte by budding; that is, spore-reproduction is replaced by a vegetative process: for instance, in mosses it has been found possible to induce the development of protonema, the first stage of the gametophyte, from tissue cells of the sporogonium: [In this way fil. and £m. Marchal (1909, 1912) were able to produce in Mnium, Bryum, Phascum, and Amblystegium diploid gametophytes; these in turn produced tetraploid sporophytes which bore diploid spores. In one case (Ambly- stegium) a tetraploid gametophyte was regenerated from cells of the tetraploid sporophyte.] Similarly, in certain ferns (varieties of Athyrium Filix-foemina, Scolopendrium vulgare, Lastrcea pseudo-mas, Polystichum angulare, and in the species Pteris aquilina and Asplenium dimorphum), the gametophyte (prpthallium) is developed by budding of the leaf of the sporo- phyte [commonly from the margin of the leaf or from the tissue of the sorus (Fig. 126)], and in some of these cases it has been ascertained that the gametophyte so developed has the same number (2x) of chromosomes in its nuclei as the sporophyte that bears it — that is, it is diploid. Apospory has been found to be associated frequently with apogamy [in the life cycle]; in fact, in the absence of meiosis, this association would appear to be inevitable." PARTHENOGENESIS IN ANIMALS1 The natural development of an egg without having been fertilized by a male gamete is a phenomenon which is apparently of much more frequent occurrence in animals than in plants. The best known examples are found among the rotifers, crustaceans, and insects, parthenogenesis being the regular mode of reproduction in some species. Other modes also usually occur in such organisms under certain conditions or after a certain number of generations. Parthenogenesis is reported in some protozoa (Plasmodium vivax, Schaudinn 1902), where the macrogamete, after certain nuclear changes, continues the life cycle without fusing with a microgamete. Moreover, as has already been described in the preceding chapter, parthenogenesis may be artificially induced in the eggs of other animal groups, notably echinoderms, mollusks, and amphi- bians, and around this fact centers much of the significant work of modern experimental biology. In commenting upon parthenogenetic develop- ment Minchin (1912, p. 137) points out that "... the gamete which has this power is always the female ; but this limitation receives an explanation from the extreme reduction of the body of the male gamete and its 1 The cytological results of researches on maturation and development in cases of parthenogenesis have recently been summarized by Paula Hertwig (1920). 318 INTRODUCTION TO CYTOLOGY feeble trophic powers, rendering it quite unfitted for independent repro- duction, rather than from any inherent difference between the two sexes in relation to reproductive activity." Many normally parthenogenetic animal eggs are known to have the diploid chromosome number as the result of a failure of reduction, a condition paralleling that known as ooapogamy in plants. On the contrary, there are some which, unlike any known vascular plant, are haploid, reduction having taken place in the normal fashion. Partheno- genesis is often associated with certain irregularities in the behavior of the polar bodies, as will be noted in the following descriptions of some well known examples. In the majority of recorded cases the partheno- genetic egg produces but one polar body; in some, however, two are formed as in all zygogenetic eggs (those developing after having been fertilized). It was long ago noticed by Blochmann (1888; see Wilson 1900, pp. 281-4) that in Aphis both zygogenetic and parthenogenetic eggs are produced; the former produce the usual two polar bodies while the latter have but one. It was also seen that the polar bodies are not budded off as separate cells, but remain within the membrane of the egg. Weismann (1886, 1887), working on rotifers, concluded that the second polar body has something to do with parthenogenetic develop- ment; and Boveri (1887d, 1890), who had seen the chromosomes of the second polar body transform themselves into a nucleus in the egg of A scam, made the suggestion that this second polar body might unite with the egg nucleus and so initiate development. Brauer (1894) an- nounced that this is precisely what occurs in Artemia, a phyllopod crustacean. In this organism two types of parthenogenesis are found. In some cases the nucleus of the second polar body, with 84 chromosomes, actually does unite with the egg nucleus, likewise with 84, causing "fertilization" and the resulting development of an individual with the diploid number (168) of chromosomes. In other cases only one polar body is produced, but reduction is accomplished in the division forming it, and the resulting haploid egg develops parthenogenetically into an individual with only 84 chromosomes. In Phylloxera carycecaulis (Morgan 1906, 1908, 1909, 1910, 1915) only one polar body appears, but here no reduction occurs: the diploid egg develops parthenogenetically. In Nematus lacteus (Doncaster 1906) two polar bodies are produced, but reduction fails and the diploid egg proceeds to develop as in Phylloxera. It has long been known that the eggs of the honey bee, Apis mellifica, will develop either zygogenetically into females or parthenogenetically into males. It has been shown in both cases that there are two polar bodies (Blochmann) and that a normal reduction in the number of chromosomes occurs (Nachtsheim 1912, 1913). The fertilized eggs APOGAMY, APOSPORY, AND PARTHENOGENESIS 319 develop into workers or into queens with the diploid number (32) of chromosomes; those not fertilized develop into drones with the haploid number (16). (At the time of spermatogenesis in the drone no further reduction in chromosome number occurs: the spermatozoa retain the number present in the body cells (16).) In the gall-fly, Neuroterus lenticularis, Doncaster (1910-1911) has shown that there are two classes of parthenogenetic females. The egg of the first class gives off no polar bodies, retains the diploid number (20) of chromosomes, and develops parthenogenetically into a sexual female. The egg of the second class gives off two polar bodies, retains the reduced number (10) of chromosomes, and develops parthenogenetically into a male. (The offspring of the sexual females and males constitute the next generation of parthenogenetic females.) There are thus several organisms in which both zygogenetic and parthenogenetic eggs are produced. In some of them, such as the bee, in which the same egg can develop in either way, the two classes of eggs show no morphological differences. In other forms, such as a species of Melanoxanthus (a plant louse) and Sida crystallina (crustacean), they may differ considerably. The parthenogenetic egg, for example, may contain much less yolk than the zygogenetic one : it is less highly differen- tiated, and "still retains the capacity to initiate dedifferentiation and reconstitution independently of union with a male gamete. In this respect it resembles the less highly specialized cells of other tissues rather than the gametes" (Child 1915, p. 408). It has recently been shown that frogs which have been induced to develop parthenogenetically from punctured eggs (Bataillon's method) are of both sexes (Loeb 1921). The chromosome number in the females has not been determined, but both Parmenter (1920) and Goldschmidt (1920) report the diploid number in males so derived. The origin of this diploid condition has not been satisfactorily explained. Parmenter suggests that it may be due to the retention of one polar body, or to a premature division of the chromosomes without cytokinesis just before the first cleavage. This promises to be an interesting case in connection with the mechanism of sex-determination. Conclusion. — To review the various theories which have been advanced to account for the origin of parthenogenesis, its relation to other forms of reproduction, and its significance in the life history, is a task which lies be- yond the scope of the present work: it has been our purpose only to indicate some of the outstanding cytological facts in certain conspicuous instances of the phenomenon. The cytological features have been accurately ascertained in only a very few cases, and these show little agreement. Furthermore, it is in artificially induced rather than in natural partheno- genesis that the physiological conditions are best known. In view of these facts it appears more than probable that many more cytological 320 INTRODUCTION TO CYTOLOGY and physico-chemical data must be secured before any theory ad- equately harmonizing all the observed phenomena of parthenogenesis can be formulated. Bibliography 13 Apogamy; Apospory; Parthenogenesis (For papers of Bataillon, Harvey, Herlant, McClendon, F. R. Lillie, R. S. Lillie, Loeb, and others on artificial parthenogenesis see Bibl. 12; also for papers of Black- man, Carruthers, Cutting, Dale, Fraser, and Welsford on ascomycetes.) ALLEN, R. F. 1911. Studies in spermatogenesis and apogamy in ferns. Trans. Wis. Acad. Sci. 17 : 1-56. pis. 1-6. BLACKMAN, V. H. 1904a. On the relation of fertilization to "apogamy" and "parthenogenesis." New Phytol. 3: 149-158. 19046. On the fertilization, alternation of generations, and general cytology of the Uredinese. Ann. Bot. 18: 323-369. pis. 21-24. 1906. On the sexuality and development of the ascocarp of Humaria granulata Quel. Proc. Roy. Soc. London Bot. 77: 354-368. pis. 13-15. BLACKMAN, V. H. and FRASER, H. C. I. 1906. Further studies on the sexuality of the Uredineae. Ann. Bot. 20 : 35-48. pis. 3, 4. BLOCHMANN, F. 1884. Ueber eine Metamorphose der Kerne in den Ovarialeiren und iiber den Beginn der Blastodermbildung bei den Ameisen. Verh. Nat.-Med. Verein Heidelberg. 3 : 243-246. 1886. Ueber die Eireifung bei Insekten. Biol. Cent. 6: 554-559. 1887. Ueber die Richtungskorper bei Insekteiren. Morph. Jahrb. 12 : 544-574. pis. 26, 27. See also Biol. Cent. 7: 108-111. BOVERI, T. 1887. Zellen-Studien. I. Die Bildung der Richtungskorper bei Ascaris megalocephala und Ascaris lumbricoides. Jen. Zeitsch. 21: 423-515. pis. 25-28. 1890. Zellen-Studien. II. Ueber das Verhalten der chromatischen Kernsub- stanz bei der Bildung der Richtungskorper und bei der Befruchtung. Ibid. 24 : 314-401. pis. 11-13. BRAUER, A. 1894. Zur Kenntniss der Reifung des parthenogenetisch sich en- twickelenden Eies von Artemia salina. Arch. Mikr. Anat. 43 : 162-222. pis. 8-11. BROWN, E. D. W. 1919. Apogamy in Camptosorus rhizophyllus. Bull. Torr. Bot. Club 46: 27-30. pi. 2. CHILD, C. M. 1915. Senescence and Rejuvenescence. Chicago. CHRISTMAN, A. H. 1905. Sexual reproduction in the rusts. Bot. Gaz. 39: 267-274. pi. 8. DONCASTER, L. 1907. Gametogenesis and fertilization in Nematus ribesii. Quar. Jour. Micr. Sci. 51: 101-114. pi. 8. 1908. Artificial Parthenogenesis. Sci. Progress.' 1910-1911. Gametogenesis of the gall fly, Neuroterus lenticularis (Spathegaster bac- carum). Proc. Roy. Soc. London B 82: 88-113. pis. 1-3; 83: 476-489. pi. 17. DRUERY, C. T. 1884. Observations on a singular mode of development in the lady- fern (Athyrium Filix-faemina) . Jour. , Linn. Soc. Bot. 21: 354-357. Further studies on a singular mode of reproduction in Athyrium Filix-fcemina. Ibid. 358-360. 2 figs. ERNST, A. 1909. Apogamie bei Burmannia ccelistris Don. Ber. Deu. Bot. Ges. 27 : 157-168. pi. 7. FARLOW, W. G. 1874. An asexual growth from the prothallium of Pteris cretica. Quar. Jour. Micr. Sci. 14 : 266-272. pis. 10, 11. See also Bot. Zeit. 32 : 180-183. FARMER, J. B., MOORE, J. E. B., and DIGBY, L. 1903. On the cytology of apogamy and apospory. I. Proc. Roy. Soc. London 71 : 453-457. figs. 4. APOGAMY, APOSPORY, AND PARTHENOGENESIS 321 FARMER, J. B. and DIGBY, L. 1907. Studies in apospory and apogamy in ferns. Ann. Bot. 21: 161-199. pis. 16-20. GOLDSCHMIDT, R. 1920. Arch. Zellf. 15: 283. HERTWIG, P. 1920. Haploide und diploide Parthenogenese. Biol. Zentralbl. 40: 14.5-174. JUEL, H. O. 1898. Parthenogenesis bei Antennaria alpina. (L.) R. Br. Bot. Centr. 74: 369-372. 1900. Vergleichende Untersuchungen iiber typische und parthenogenetische Fort- pflanzung bei der Gattung Antennaria. Handl. Svensk. Vet. Akad. 33: pp. 59. pis. 6. • figs. 5. 1904. Die Tetradenteilung in der Samenanlage von Taraxacum. Ark. f. Bot. 2: 1-9. 1905. Die Tetradenteilung bei Taraxacum und anderen Cichoraceen. Kgl. Svensk. Vet. Akad. 39: 1-20. pis. 1-3. KUSANO, S. 1915. Experimental studies in the embryonal development in an angiosperm. Jour. Coll. Agr. Tokyo 6: 7-120. pis. 5-9. figs. 28. KYLIN, H. 1918. Studien iiber die Entwicklungsgeschichte der Phseophyceen. Svensk. Bot. Tids. 12: 1-64. LOEB, J. 1921, Further observations on the production of parthenogenetic frogs. Jour. Gen. Physiol. 3: 539-545. figs. 3. MARCHAL, EL. and EM. 1909. Aposporie et sexualite chez les mousses. II. Bull. Acad. Roy. Belg. 1249-1288. 1912. Recherches cytologiques sur le genre " Amblystegium." Ibid. 51: 189-203. Ipl. MINCHIN, E. A. . 1912. An Introduction to the Study of the Protozoa. London. MORGAN, T. H. 1906. The male and female eggs of phylloxerans of the hickories. Biol. Bull. 10: 201-206. figs. 4. 1908. The production of two kinds of spermatozoa in phylloxerans. Proc. Soc. Exp. Biol. and Med. 5. 1909a. Sex determination and parthenogenesis in phylloxerans and aphids. Science 29. 19096. A biological and cytological study of sex-determination in phylloxerans and aphids. Jour. Exp. Zool. 7 : 239-352. 1 pi. figs. 23. 1910. The chromosomes in the parthenogenetic and sexual eggs of phylloxerans and aphids. Proc. Soc. Exp. Biol. & Med. 7. 1913. Heredity and Sex. New York. MORGAN, T. H., STURTEVANT, A. H., MULLER, H. J., and BRIDGES, C. B. 1915. The Mechanism of Mendelian Heredity. New York. MOTTIER, D. M. 1915. Beobachtungen iiber einige Farnprothallien mit bezug auf eingebettete Antheridien und Apogamie. Jahrb. Wiss. Bot. 66: 65-84. MURBECK, S. 1901. Parthenogenetische Embryobildung in der Gattung Alche- milla. Lunds Arsskr. 36: pp. 41, pis. 6. 1904. Parthenogenesis bei den Gattungen Taraxacum und Hieracium. Bot. Not., Lund, 1904. pp. 285-296. NACHTSHEIM, H. 1912. Parthenogenese, Eireifung und Geschlechtsbestimmung bei der Honigbiene. Sitzber. Ges. Morph. u. Phys., Miinchen. 1913. Cytologische Studien iiber die Geschlechtsbestimmung bei der Honigbiene (Apis mellifica). Arch. Zellf. 11: 169-241. pis. 7-10. NATHANSOHN, A. 1900. Ueber Parthenogenesis bei Marsilia und ihre Abhangigkeit von der Temperatur. Ber. Deu. Bot. Ges. 18: 99-109. figs. 2. OSAWA, J. 1913. Studies on the cytology of some species of Taraxacum. Arch. f. Zellf. 10: 450-469. pis. 37, 38. 21 322 INTRODUCTION TO CYTOLOGY OVEBTON, J. B. 1902. Parthenogenesis in Thalictrum purpurascens. Bot. Gaz. 33 : 363-375. pis. 12, 13. 1913. Artificial parthenogenesis in Fucus. Science 37: 841, 844. PACE, L. 1913. Apogamy in Atamosco. Bot. Gaz. 66: 376-394. pis. 13, 14. PARMENTER, C. L. 1920. The chromosomes of parthenogenetic frogs. Jour. Gen. Physiol. 2: 205-206. PRINGSHEIM, N. 1876. Ueber den Generationswechsel der Thallophyten tind seinen Anschluss an den Generationswechsel der Moose. (Vorl. Mitt.) Monat- schr. Akad. Wiss. Berlin, 1876. p. 869. 1878. Ueber Sprossung der Moosfriichte und den Generationswechsel der Thallo- phyten. Jahrb. Wiss. Bot. 11 : 1-46. ' pis. 1, 2. ROSENBERG, O. 1906. Ueber die Embryobildung in der Gattung Hieracium. Ber. Deu. Bot. Ges. 24: 157-161. pi. 11. 1909. Ueber die Chromosomenzahlen bei Taraxacum und Rosa. Svensk Bot. Tidskr. 3: 150-162. figs. 7. SCHAUDINN, F. 1902. Krankheitserregende Protozoen. II. Plasmodium vivax. Arb. d. k. Gesundheitsamte, Berlin, 19 : 169. SHAW, W. R. 1897. Parthenogenesis in Marsilia. Bot. Gaz. 24: 114-117. STEIL, W. N. 1915. Some new cases of apogamy in ferns. (Prelim. Note.) Science 41 : 293-294. 1918. Studies of some new cases of apogamy in ferns. Bull. Torr. Bot. Club 45 : 93-108. pis. 4, 5. 1919o. Apogamy in Nephrodium hirtipes Hk. Ann. Bot. 33: 109-132. pis. 5-7. 19196. Apospory in Pteris sulcata, L. Bot. Gaz. 67: 469-482. pis. 16, 17. figs. 4. STOREY, A. G. 1918. Apogamy in the Cyatheacese. Ibid. 65: 97-102. figs. 10. STORK, H. E. Studies in the genus Taraxacum. Bull. Torr. Bot. Club 47: 199-210. STRASBURGER, E. 1905. Die Apogamie der Eualchemillen und allgemeine Ge- sichtspunkte, die aus sich ergeben. Jahrb. Wiss. Bot. 41 : 88-164. pis. 1-4. 1907. Apogamie bei Marsilia. Flora 97: 123-191. pis. 3-8. 1909a. Die Chromosomenzahlen der Wikstroemia indica (L.) C. A. Mey. Ann. Jard. Bot. Buit. II 3: suppl. 13-18. figs. 3. 19096. Zeitpunkt der Bestimmung des Geschlechts, Apogamie, Parthenogenesis, und Reduktionsteilung. Hist. Beitr. 7. VINES, S. H. 1911. Article on Reproduction in Plants. Encyl. Brit., llth Ed. WEISMANN, A. 1886. Richtungskorper bei parthenogenetischen Eiern. Zool. Anz. 9: 570-573. 1887. Ueber die Zahl der Richtungskorper und iiber ihre Bedeutung fur die Verer- bung. Jena. Also in Essays upon Heredity, 1889. WINKLER, H. 1904. Ueber Parthenogenesis bei Wikstroemia indica (L.) C. A. 'Mey. (Vorl. Mitt.) Ber. Deu. Bot. Ges. 22 : 573-580. 1906. Ueber Parthenogenesis bei Wikstroemia indica. Ann. Jard. Bot. Buit. 5 : 208-276. pis. 20-23. 1908. Ueber Parthenogenesis und Apogamie im Pflanzenreiche. Prog. Rei Bot. 2 : 293-454. WORONIN, HELENS W. 1907. Apogamie und Aposporie bei einigen Farnen. (Vorl. Mitt.) Ber. Deu. Bot. Ges. 25 : 85-86. 1908. Same title. Flora 98: 101-162. figs. 72. YAMANOUCHI, S. 1908. Apogamy in Nephrodium, Bot. Gaz. 45 : 289-318. pis, 9, 10. (Bibliography.) CHAPTER XIV THE ROLE OF THE CELL ORGANS IN HEREDITY The chief interest of cytology at the present time probably lies in the relation which it bears to the subject of heredity. From the time when the problems of cell research first began to take definite shape, especially since a connection between the activities of the cell and the phenomena of inheritance was suggested, the efforts of most cytologists have con- tributed directly or indirectly to the solution of two great and closely interrelated problems of biology: the problem of ontogerietic develop- ment and the problem of heredity. The aid which cytology has afforded in these respects has been invaluable. Not only has it been able to discover a large number of the significant facts of individual development, or ontogeny, but it has also thrown a flood of light upon many obscure matters in the field of heredity, and has so come to be an important factor in the study of phylogeny and evolution. The Law of Genetic Continuity. — "The most fundamental contribu- tion of cell-research to the theory of heredity," says Wilson (1909), "is the law of genetic continuity by cell-division. Cells arise only by the division of preexisting cells ... In each generation the germinal stuff runs through the same series of transformations; hence that reappearance of the same traits in successive generations that we call heredity." It is by the light of the above law that we are enabled to see some- thing of the nature of the material continuity which exists between suc- cessive stages of the ontogenetic development, and also between success- ive generations. It is to be remembered, in the first place, that all the cells of the adult multicellular organism are derived by repeated division from the single cell (ordinarily a zygote or a spore) with which develop- ment starts, so that the causes of events occurring at any particular stage are to be sought largely in the reactions of cells at earlier stages; and, in the second place, that the material link connecting two successive gen- erations is a single organized cell, usually a gamete or a spore, which means that the heritage of a long ancestry is in some way represented in this single cell and its capabilities. "The conception that there is an unbroken continuity of germinal substance between all living organisms, and that the egg and the sperm are endowed with an inherited organiza- tion of great complexity, has become the basis for all current theories of heredity and development" (Locy, 1915, p. 224). 323 324 INTRODUCTION TO CYTOLOGY Cytological studies have therefore centered mainly about the general organization of the egg (chiefly that of animals) as related to the character of the organism arising from it (the problem of development), and about the roles played by the various cell organs of the gamete in the transmis- sion of heritable characteristics from one generation to the next (the problem of heredity). The character of the principal modern theory of heredity to which these studies have led is due in no small measure to the influence of a number of earlier hypotheses, such as those of Darwin and deVries, and especially that of Weismann. These hypotheses will be reviewed in Chapter XVIII, where their relation to the modern cytolog- ical interpretation of heredity, set forth in this and the following three chapters, will be discussed. It is obvious that an account of the physical basis of heredity would require for completeness not only a description of the structural changes by which visible materials are transmitted and distributed during game- togenesis, fertilization, and development; but also a review of many phy- siological processes which accompany these changes, and through which many characters are brought to expression. In these chapters attention will be limited largely to the structural aspects of the problem. Among the physiological changes those occurring at the time of fertiliza- tion are best known, and have already been discussed in Chapter XII. The R6le of the Nucleus.— It was Ernst Haeckel (1866) who first advanced the hypothesis that "the nucleus of the cell is the principal organ of inheritance." Cytological evidence in support of this view, announced by Haeckel as a speculation, was brought forward by O. Hertwig (1875 etc.), Strasburger (1878, 1884), van Beneden (1883 etc.), and a number of other investigators, who described the behavior of the nucleus in the various stages of the life cycle, particularly in somatic cell-division, maturation, and fertilization. Two of these workers, O. Hertwig and Strasburger, who had discovered the fusion of the gamete nuclei at the time of fertilization in animals and plants respectively, definitely announced the theory, now supported by a considerable body of observational and experimental evidence, that the nucleus is the "vehicle of heredity." They held that hereditary transmission is through the nuclei of the gametes, and that the chromatin is the special inheritance material, or "idioplasm," about which there had been so much speculation. This view was at once widely adopted by biologists. The efforts' of many cytologists were now directed toward the further elucidation and verification of this nuclear hypothesis of heredity, and many observations and experiments apparently demonstrated its essen- tial correctness. It was noted that, so far as could be discerned, the spermatozoon in many cases brings nothing but nuclear material into the egg, so that hereditary transmission from the male parent must be through the nucleus alone. A similar condition was later reported in THE ROLE OF THE CELL ORGANS IN HEREDITY 325 plants, Guignard, Nawaschin (1910), and Welsford (1914) pointing out that in Lilium only the male nucleus enters the egg, its accompanying cytoplasm being rubbed off and left behind. (See p. 299.) Certain ingenious experiments of Boveri (1889, 1895; also 1909 and 1918) led to the same conclusion regarding the nucleus. Boveri induced the fer- tilization of enucleated fragments of Sphcerechinus eggs (a phenomenon FIG. 127. A, egg of Sphcerechinus granularis undergoing artificially induced cleavage mitosis; spermatozoon of Strongylocentrotus lividus has entered and taken the form of a chromosome group. B, cytokinesis beginning; one blastomere will have a purely maternal nucleus, and the other a hybrid nucleus. (Diagrammed after Hcrbst, 1909.) FIG. 127 bis. — Diagram showing the irregular distribution of the chromosomes by a quadripolar mitotic figure. (After Boveri.) known as merogony) by spermatozoa of Echinus, and obtained larvae which were purely paternal in character. From this it was argued that it is the sperm nucleus alone, and not the egg cytoplasm, that transmits the hereditary characters from one generation to the next in this case. Other experiments of a similar nature, however, turned out differently, as will presently be noted. Certain echinoderm hybrids, furthermore, show paternal larval characters even when the egg nucleus has not been removed. 326 INTRODUCTION TO CYTOLOGY A strong piece of evidence supporting Boveri's conclusion was fur- nished by Herbst (1909). By treating eggs of Sphcerechinus with valeri- anic acid Herbst caused them to undergo cleavage artificially. While the cleavage mitosis was in progress a spermatozoon of Strongylocentrotus was allowed to enter the egg, where it at once gave rise to its group of chromosomes (Fig. 127). These, however, arriving too late to join regularly in the mitosis, were incorporated in neither of the daughter nuclei of the first cleavage : they resumed the form of a nucleus, and this was included in one of the blastomeres. This blastomere therefore contained two nuclei, one maternal and one paternal, which combined during subsequent stages, whereas the other blastomere had a maternal nucleus only. Herbst regarded such nuclear behavior as responsible for the frequently found larvae which are hybrid in character on one side and purely maternal on the other. This experiment has been held to show not only that it is the nucleus of the spermatozoon which brings in the paternal characters, but also that it is the chromosomes alone that are responsible. It is assumed, though perhaps without sufficient evidence, that the other nuclear materials (karyolymph etc.) which may be present, as well as any cytoplasmic elements, have opportunity to mix generally with the egg cytoplasm, since the membrane of the male nucleus breaks down and leaves the chromosomes lying free before the egg divides into the two blastomeres. The paternal characters, however, appear only where the chromosomes come to be located — that is, in the cells compos- ing one-half of the organism. In his work on multipolar mitoses in di- spermic eggs (Fig. 127 bis; see also p. 163) Boveri (1902, 1907) was able to show further that abnormal chromosome distribution is associated with abnormalities in development in a very definite way; and that if isolated blastomeres resulting from such abnormal divisions be made to develop 'independently, completely normal larvae result only where there is statistical reason to believe that a full complement of the qualitatively different chromosomes is present. The nuclear theory had its opponents from the beginning. Verworn, Waldeyer, Rauber, and other early investigators held that the cytoplasm as well as the nucleus must be concerned in the hereditary process, since the spermatozoon in many cases does bring cytoplasm into the egg, and also because neither nucleus nor cytoplasm can function independently of the other. This view received support in certain experiments which seemed to discount the power of the nucleus in controlling heredity. Loeb (1903) found that when a sea urchin egg was fertilized by a starfish sperm the resulting larva possessed purely maternal characters, the sperm nucleus exerting no visible hereditary effect. The same thing was noted by Godlewski (1906) in crosses between sea urchins and crinoids. Godlewski made the further significant observation that when enucleated egg fragments of Parechinus (sea urchin) were fertilized by sperm of THE ROLE OF THE CELL ORGANS IN HEREDITY 327 Antedon (crinoid) the larvae so produced, contrary to Boveri's results, were maternal in character: they were like the mother, which had pre- sumably contributed cytoplasm only, and not like the father, which had furnished the nucleus. Fertilization by a spermatozoon had here pro- duced a developmental stimulus but no amphimixis (the combining of hereditary lines), so far as could be judged from the appearance of the larva?. Even if the male cytoplasm were admitted to have no heredi- tary role, it nevertheless seemed that the cytoplasm of the egg was clearly so concerned. In his work on sea urchin hybrids Baltzer (1910) was able to show why it is tha.t some such larvae are maternal in character while others have the characters of both parents. When an egg of Strongylocentrotus fertilized by a spermatozoon of Sphcerechinus undergoes its first cleavage division the paternal chromosomes behave irregularly ; they fail to become incorporated in the daughter nuclei and are lost. Those individuals which develop far enough show maternal skeletal characters. In the reciprocal cross, on the contrary, all of the chromosomes behave normally and the resulting larvae are truly hybrid in character. Thus in the first cross, in which the paternal chromosomes are lost, the spermatozoon furnishes only a developmental stimulus and has no appreciable effect on the character of the new individual; whereas in the second cross, in which the paternal chromosomes are included in the blastomere nuclei, the spermatozoon not only furnishes the developmental stimulus but also contributes paternal characters to the new individual. This is particularly convincing evidence in favor of the view that the chromo- somes are in some way responsible for the development of parental characters in the offspring. In a posthumous paper Boveri (1918) reported an additional observa- tion which, he believed, goes far toward explaining the conflicting results of different investigators. He found that egg fragments, and even whole eggs, may often have chromatin in a form that easily escapes observation, but which can exert its usual influence on development. In accordance with his earlier observations, enucleate egg fragments of Sphoerechinus fertilized by spermatozoa of Strongylocentrotus may develop into purely paternal larvae. Most of them, however, are intermediate in charac- ter, resembling the maternal parent also in certain features. Having previously (1895, 1905) demonstrated that the size of the nuclei in merogonic larvae is proportional to the number of chromosomes they contain (see Chapter IV), Boveri was able to show that the nuclei of the intermediate larvae are diploid rather than haploid, so that it is clear that the supposedly enucleate fragments in such cases must have contained chromosomes. It is probable, Boveri believed, that the maternal larvae obtained by Godlewski may be accounted for in a similar fashion. It was pointed out by Strasburger (1908), who had come to believe in 328 INTRODUCTION TO CYTOLOGY the complete monopoly of the nucleus in the transmission of hereditary characteristics, that the maternal character of Godlewski's larvae could be explained on the assumption that the early developmental stages do not require the expression of the hereditary capabilities of the nucleus, but are dependent more directly upon mechanical causes. Boveri (1903, 1914), as a result of his hybridization experiments, strongly emphasized the view that the spermatozoon has an influence upon all of the larval char- acters; but he pointed out that the larval stages by themselves are not sufficient grounds upon which to establish complete conclusions regarding the respective roles of nucleus and cytoplasm, since the general course of the early developmental stages in such organisms is immediately dependent to a very large extent upon the general organization of the egg. This brings us to a brief consideration of the "promorphology" of the highly organized animal egg, and of the relation which exists between this organization and the character of the organism developing from it. Of the large amount of work done in this field only a hint can be given here. The Promorphology of the Ovum. — There arose very early two views regarding the organization of the egg which recall the older theories of preformation and epigenesis (Chapter I). According to one, most fully expressed in W. His's Theory of Germinal Localization (1874; see Wilson 1900, p. 397), the embryo is prelocalized in the general cytoplasm of the egg — not preformed in the old sense of Bonnet, but having its various parts represented by substances with definite relative positions. This view found support in those cases in which a single isolated blastomere of the two-celled stage develops into a half-larva instead of a complete smaller larva (Roux on the frog, 1888; Crampton on the marine gastropod, Ilyanassa, 1896) ; and especially in Beroe, a ctenophore, which produces an incomplete larva even if a portion of the unsegmented egg be removed (Driesch and Morgan, 1895). Opposed to the above view was that which held the egg to be isotropic and without any predetermination of embryonic parts. Certain well known experiments appeared to bear out this conclusion. It was found in the frog (Pfluger 1884; Roux 1885), the sea urchin (Driesch 1892), an annelid (Wilson 1892), and A scarfs (Boveri 1910) that very abnormal types of cleavage can be artificially induced, but that normal larvae nevertheless result. A number of cases were also described in which complete embryos arose from single isolated blastomeres of the two-celled stage (Fundulus, Morgan 1895; and other forms), or even from those of the sixteen-celled stage (Clytia, Zoja 1895). Had there been any prelocalization of parts in the egg it is difficult to see how normal or complete embryos could have arisen in such abnormal ways as these. It appears that the eggs of different animal species vary greatly in the degree and fixity of their internal differentiation. In some cases THE ROLE OF THE CELL ORGANS IN HEREDITY 329 the egg is virtually isotropic, and through several succeeding cell gene- rations the blastomeres are equipotential, that is, equally capable of developing into any part of the body or even into the whole of it. Thus the embryonic parts, and hence many of the individual's characters, are not definitely marked out until a comparatively late stage. On the contrary, there are forms in which the axis of polarity and certain funda- mental embryonic parts are roughly delimited in the egg cytoplasm in such a way that an alteration in the relative positions of the egg materials brings about a corresponding alteration in the character of the resulting individual. As an illustration of such internal differentiation may be taken the case of Styela, an ascidian, described by Conklin (1915). In the egg of Styela there are four or five distinct kinds of plasma arranged in a definite order and distributed in a regular manner as cleavage pro- ceeds, each kind eventually giving rise to a certain portion of the embryo. CTENOFHOflE TURBELLAKIAN ECHINOBERM A5CIBIAM FIG. 128. — Eggs of various animals, showing the patterns assumed by the material s which give rise to the various body regions. In the first three the egg has undergone division, and the plasmas becoming ectoderm, mesoderm, and endoderm are represented in clear white, cross-hatching, and parallel ruling respectively. In the fourth egg two divisions have occurred, and several definitely arranged substances are distinguishable (After Conklin, 1915.) Substancss which are yellow, gray, slate-blue, and colorless give rise respectively to muscle and mesoderm, nervous system and notocord, endoderm, and ectoderm. " Thus within a few minutes after the fertiliza- rion of the egg, and before or immediately after the first cleavage, the anterior and posterior, dorsal and ventral, right and left poles are clearly distinguishable, and the substances which will give rise to ectoderm, endoderm, mesoderm, muscles, notocord and nervous system are plainly visible in their characteristic positions" (Conklin 1915, p. 118). If such eggs are placed in a centrifuge the various substances may be made to assume an entirely abnormal stratified arrangement, which in turn "may lead to a marked dislocation of organs; the animal may be turned inside out, having the endoderm on the outside and its skin and ectoderm on the inside, etc." (p. 321). Such a behavior emphasizes the determinative character of the cytoplasmic pattern clearly present in many eggs. It has further been noted that the eggs of various animal phyla are characterized by distinct patterns in the arrangmeent of their visibly different materials (Fig. 128). "The polarity, symmetry and 330 INTRODUCTION TO CYTOLOGY pattern of a jellyfish, starfish, worm, mollusk, insect or vertebrate are foreshadowed by the characteristic polarity, symmetry and pattern of the cytoplasm of the egg either before or immediately after fertilization" (Conklin, p. 172-5). That the arrangement of the embryonic parts is not solely dependent upon such visible egg substances is shown by the observations of Morgan (1909c, 1910e) and Boveri (19106) on centrifuged eggs of Arbacia, Ascaris, the frog, and other forms. Here it is found that the displacement of the various substances does not necessarily cause a dislocation of the body parts of the embryo: hence the setting apart of the embryonic regions must be dependent upon a polarity in the egg which at least in many cases is not disturbed by the experimental alteration in position of the visible egg substances. But in either case differentiation appears to be related to a cytoplasmic organization. From this it would appear that the characters which such an organism inherits from the preceding generation do not belong to one category and are not transmitted in the same way. There are first those general characteristics of organization which are the direct outgrowth of a corre- sponding organization in the egg cytoplasm. Secondly, there are the Mendelian characters which appear later in the ontogeny and which there is every reason to believe are represented in some way in the chromosomes of the gamete nuclei (Chapter XV). Boveri thus distinguished two periods after fertilization : an early one in which the course of develop- ment is dependent on the organization of the egg cytoplasm, only general metabolic functions of the chromosomes being active; and a later one in which the specific hereditary powers of the chromosomes are brought to expression, the right chromosomal combination then proving to be necessary for normal development. The question naturally arises as to how much the cytoplasmic organ- ization may be due in turn to the activity of the nucleus during the differentiation of the egg — as to whether the general characters which are the direct outgrowth of this organization may or may not be ultimately dependent, as are the clearly Mendelian characters, on nuclear factors. Conklin (1915) comments upon this point as follows: " In this differentia- tion and localization of the egg cytoplasm it is probable that certain influences have come from the nucleus of the egg, and perhaps from the egg chromosomes. There is no doubt that most of the differentiations of the egg cytoplasm have arisen during the ovarian history of the egg, and as a result of the interaction of nucleus and cytoplasm; but the fact remains that at the time of fertilization the hereditary potencies of the two germ cells are not equal, all the early stages of development, includ- ing the polarity, symmetry, type of cleavage, and the pattern, or relative positions and proportions of future organs, being foreshadowed in the cytoplasm of the egg cell, while only the differentiations of later develop- ment are influenced by the sperm. In short the egg cytoplasm fixes the THE ROLE OF THE CELL ORGANS IN HEREDITY 331 general type of development and the sperm and egg nuclei supply only the details" (p. 176). Plastid Inheritance. — Certain cases of "plastid inheritance" have been brought forward to show that the character of an organism may not be entirely due to factors delivered to it by the gamete or spore nuclei. It has been pointed out that two successive generations of cells repro- ducing by division resemble each other for the obvious reason that the organs of any given cell may actually become the corresponding organs of the daughter cells. Thus in the case of a unicellular green alga the daughter individuals are like the mother individual in being green because the chloroplast of the mother cell is divided and passed on directly to them. In those algae in which a swarm spore germinates to produce a multicellular individual (Ulothrix etc.), or associates with others of its kind to form a colony (Hydrodidyon, Pediastrum; Harper 1908, 1918a6), the color of the successive colonies or multicellular individuals is a charac- ter that i's transmitted directly by the repeated division of chloroplasts. Thus, as Harper urges, the nucleus is not required here to account for the resemblance between successive generations of cells or individuals, so far as this character is concerned. A similar interpretation has been placed by some geneticists upon the inheritance of "chlorophyll characters" in the higher plants, the supposi- tion being that plastids, multiplying only by division, are responsible for the distribution, in the individual plant and through successive generations, of those characters which manifest themselves in these organs. Abnormalities in chlorophyll coloring are accordingly held to be due to an abnormal condition or behavior of the chloroplasts. Such a case is that of Mirabilis jalapa albomaculata, described by Correns (1909). In plants of this race there are some branches with normal green leaves, some with white leaves, and some with "checkered" (green and white) leaves. Flowers are borne on branches of all three types. In all cases crosses between unlikes result in seedlings with the color of the maternal parent: inheritance is strictly maternal. For instance, if a flower on a green branch is pollinated with pollen from a flower on a white branch the offspring are all green. In the reciprocal cross the offspring are all white, and soon die because of the lack of chlorophyll. In neither case does the pollen affect the color of the resulting individual. The explanation offered by Correns for the color- less condition is that it is due to a cytoplasmic disease which destroys the chloroplasts. It is therefore delivered directly to the next generation in the egg cytoplasm, and is not transmitted by the male parent because no male cytoplasm is brought into the egg at fertilization. If it had been due to nuclear factors it would have been transmitted by both parents, since the nuclear contributions of the two are equal. This condition is analogous to that occasionally found in animals, in which bacteria may 332 INTRODUCTION TO CYTOLOGY be carried from one generation to the next in the egg cytoplasm, causing a direct inheritance of the disease. But such pathological cases are not to be confused with, or thought to contradict, normal Mendelian heredity, which, as will be seen in the following chapter, is closely bound up with nuclear phenomena. They are rather to be regarded as examples of repeated reinfection. Results differing from those of Correns were obtained by Baur (1909) in his researches on Pelargonium zonale albomarginata. This form, which is characterized by white-margined leaves, often has pure green and pure white branches, as in Mirabilis. Crosses either way between flowers on these two kinds of branches result in every case in mosaic (green and white) offspring: inheritance is here not purely maternal as in Mirabilis. Although Baur admits for this case the possibility of a Mendelian inter- pretation if a segregation of factors for greenness and whiteness in the somatic cells be allowed, he thinks it more probable that inheritance in this instance is not a matter of chromosomes and Mendelism at all, but is rather due to a sorting out of green and colorless plastids, themselves permanent cell organs, in the somatic cells. In order to account for inheritance through both the male and the female, Baur assumes that primordia of plastids are brought in through the male cytoplasm as well as the egg cytoplasm, a conclusion directly contradictory to that of Correns. Ikeno (1917), working on variegated races of Capsicum annum. obtained results similar to those of Baur on Pelargonium, and concluded that transmission of variegation is not through the nucleus, but through plastids contributed by both parents. Although the results and interpretations of Correns and Baur are at present irreconcilable except on the basis of assumptions not warranted by known facts, they agree in the conclusion that plastid inheritance is not Mendelian, but is due rather to extra-nuclear factors. Baur reports corroborative evidence in Antirrhinum (1918). Opposed to this con- clusion is that of Lindstrom (1918), who has clearly shown in the case of certain variegated races of maize that the inheritance of characters due to unusual plastid behavior is strictly Mendelian. This means that the distribution or degree of prominence of the plastids, although these may be organs with their own individuality, depends upon the activity of Mendelian factors in the chromosomes, which represent the only known cell mechanism in which there is at present any hope of rinding an expla- nation for the distribution of Mendelian characters (Chapter XV). In Lindstrom's plants plastid inheritance appears to be as much a nuclear matter as the inheritance of any other character manifested in the extra- nuclear portion of the cell. On the basis of the data at hand the tentative conclusion seems fully justified that all cases of chlorophyll inheritance do not belong to one category. Some of them are clearly to be accounted for on the same basis THE ROLE OF THE CELL ORGANS IN HEREDITY 333 with other Mendelian characters, whereas others appear to require an explanation of another kind. The results of work in progress at Cornell University on variegated races of maize points in this direction. One . of the most interesting problems in cytology and genetics at present is that concerning the manner in which extra-nuclear bodies, such as plas- tids and their primordia, may account for certain types of inheritance, and the extent to which their behavior may be influenced by the nucleus. Aleurone Inheritance. — We may here refer to the attempt which has been made to explain the inheritance of aleurone color in maize endosperm on the basis of a somatic segregation of special cell organs in the form of granular primordia, which multiply by fission and develop into aleurone bodies of various types and colors. But since aleurone and other endo- sperm characters are inherited in Mendelian fashion, as shown by East and Hayes (1911, 1915), Collins (1911), and Emerson (1918), and since there has been adduced in support of the supposed sorting out of primor- dia no evidence approaching in cogency that upon which the chromosome theory has been built up, geneticists generally are of the opinion that the chromosomes with their well known mechanism of segregation offer the best promise of an explanation of the inheritance of aleurone characters, though all admit that other organs may play a part in bringing these characters to expression. Furthermore, the case for the self-perpetuity of the aleurone grain is much weakened by the fact of their artificial pro- duction by Thompson (1912). The theory that chondriosomes are concerned in heredity has been discussed in Chapters VI and XII. General Conclusions. — In conclusion the statement may again be made that as genetical researches multiply it becomes increasingly clear that the characters in which an individual resembles that from which it- sprang are not in every case transmitted to it in the same manner. Those characters which are inherited according to Mendelian rules, to anticipate a conclusion based on evidence to be presented in the next chapter, in all probability owe their repeated appearance in successive generations to "factors" of some sort which are transmitted by the chromosomes of the nucleus. This applies also to those characters which, while Men- delian in distribution, depend for their expression upon the presence of other cell organs (plastids) which may have an individuality of their own. All or nearly all of the hereditary contribution made by the male parent must in most organisms be in the above form, since the male gamete consists almost exclusively of nuclear material. The female gamete, or egg, in addition to the clearly Mendelian characters repre- sented by factors in its nucleus, may at least in the case of many animals 334 INTRODUCTION TO CYTOLOGY contribute certain general characters, such as polarity, symmetry, and general type of early development, which are the direct outgrowth of an elaborate organization present in the egg cytoplasm. It is true that this organization is the result of processes in which the nucleus cooperates during the differentiation of the egg, and those who hold to the universal applicability of the Mendelian interpretation would assume that the type of organization must depend upon Mendelian factors carried in the nucleus. However this may be, the fact remains that the two gametes at the time of fertilization are not equal in hereditary potency, as Conklin states. So far as the clearly Mendelian characters are con- cerned, however, all evidence goes to show that they are precisely equal. The direct inheritance of metidentical characters, such as the above mentioned green plastid color in Pediastrum, and the indirect inheritance of colony characters in the same form, afford other examples of hereditary transmission otherwise than through the nucleus. With respect to colony characters, Harper has shown in a striking manner, both in Hydro- dictyon and Pediastrum, that the characteristic form and type of organiza- tion assumed by the colony are the results of interactions between the form, polarities, adhesiveness, surface tension, etc. of the free-swimming swarm spores which aggregate to build it up. The swarm spore has an individual organization of a particular type, but its capabilities show it to be devoid of any arrangement of its protoplasmic parts corresponding either to its future position in the colony or to the arrangement of the cells in the colony as a whole. The character of the colony thus depends upon the interactions of its component units and is in no way represented in any one of them. Consequently it is held by Harper that no system of spatially arranged factors in a special germ plasm is required to account for the regular reappearance of such cell and colony characters in these organisms, and that such facts must be reck- oned with in attempting to explain heredity and development in terms of the cell. By whatever means they are transmitted, it is evident that most characters must be brought to expression through the activity of the cell system as a whole, the process involving a long series of reactions in which all or nearly all of the cell constituents play their parts. At the present time little or nothing is known of the real nature of the " factor" or of the manner in which it may influence the development of a character. In general, then, we may say that the heritage bequeathed by an indi- vidual to its offspring is in most organisms transmitted mainly through the nucleus, since it is very largely upon this organ that the development or non-development of particular characters in the organism depends; but also that the development of the characters in the offspring, however these THE ROLE OF THE CELL ORGANS IN HEREDITY 335 may be transmitted, involves all of the cell organs as well as a complicated and orderly series of intercellular reactions and responses. These two phases, the transmission of a heritage of factors and the develop- ment of the organism's characters as the result of their influence, must both be very much more fully known before either can be adequately understood. To some of the more cogent evidence upon which these general con- clusions are based we shall now turn. Bibliography at end of Chapter XVIII. MENDELISM AND MUTATION MENDELISM The classic researches carried out by Mendel a half-century ago on the hybridization of garden peas are now so well known that a detailed description of them would be superfluous here. Moreover, since the main principles of Mendelism are illustrated in the results of the simplest of Mendel's experiments, a review of one or two of the latter will for our purposes- be sufficient.1 A Typical Case of Mendelian Inheritance. — Mendel crossed plants of a pure bred race of tall peas (6 to 7 feet in height) with plants of a pure bred dwarf race (^ to 1% feet in height) (Fig. 129). All the plants of the first hybrid generation (Fi) were tall like one of their parents. When these tall hybrids were self-fertilized or bred to one another, it was found that the second hybrid generation (Fz) comprised individuals of the two grandparental types, tall and dwarf, in the relative numerical proportion of 3:1. It was further found that the tall individuals of this generation, though alike in visible characters, were unlike in genetic con- stitution: one-third of them, if bred for another generation, produced nothing but tall offspring, showing that they were "pure" for the character of tallness; whereas the other two-thirds, if similarly bred, produced again in the next generation both tall and dwarf plants in the proportion of 3 : 1, showing that they were hybrids with respect to tallness and dwarfness. The dwarf plants of the second hybrid generation (Fa) produced nothing but dwarfs when interbred; they were "pure" for dwarfness. From these facts it was evident that the plants of the F2 generation, although they formed only two visibly distinct classes, were in reality of three kinds: pure tall individuals, tall hybrids, and pure dwarfs, in the relative numerical proportions of 1 :2 :1. The explanation offered by Mendel for these phenomena may be briefly stated as follows (Fig. 129). The germ cells produced by the pure tall plant carry something (now termed a factor, represented here 1 Detailed accounts of the many facts of Mendelism may be found in more special works on the subject. See Morgan et al. 1915, Chapters 1 and 2; Bateson 1913; Castle, Coulter et al. 1912; Castle 1916; Coulter and Coulter 1918; Babcock and Clausen 1918, Chapter 5; Punnet 1919; Darbishire 1911; Morgan l&19a; Thomson 1913; East and Jones 1919. 336 MEN DELI SM AND MUTATION 337 by T) which makes the resulting plant tall. The germ cells of the dwarf plant carry something (t) causirg the dwarf condition. In the first PMENT5 T T 00 -© ©00 # m FZ FIG. 129. — A typical Mendelian cross between tall and dwarf peas, showing dominance of the tall over the dwarf condition in the first hybrid generation (Fi), and the 3: 1 ratio of tall plants to dwarfs in the second hybrid generation (Ft). At the right is shown the corresponding distribution of the Mendelian factors for tallness (T) and dwarf ness (t). hybrid generation (F\) both factors are present, T coming from one parent and t from the other, but T "dominates" and prevents the expression of 22 338 INTRODUCTION TO CYTOLOGY the "recessive" t, so that the plants of this generation are all tall. When the hybrid (Fi) produces germ cells the two factors for tallness and dwarfness separate, half of the germ cells receiving T and the other half receiving t. Each gamete therefore carries either one or the other of the two factors in question, but never both: a given gamete is "pure" either for T or for t. This segregation in the germ cells of factors pre- viously associated in the individual without their having been altered by this association is the central feature of the entire series of Mendelian phenomena, and is often referred to as Mendel's first law. Since, now, the gametes, both male and female, produced by the hybrid plants of the Fi generation are of two kinds (half of them bearing T and half bearing ft MIRABIUS JALAPA FIG. 130. — Blending inheritance ("incomplete dominance") in Mirabilis jalapa, showing 1:2:1 ratio of three genotypes in Ft. (Adapted from Correns.) four combinations are possible : a T sperm with a T egg, a T sperm with a t egg, a t sperm with a T egg, and a t sperm with a t egg. These four combinations result respectively in a tall plant (pure dominant, TT), two tall hybrids (Tt and tT), and a dwarf plant (pure recessive, tt). It is obvious that in the long run these three types will occur in the ratio of 1 :2:1. Mendel's researches on peas included also a study of six other pairs of heritable characters (now known as allelomorphic pairs), the two members of each pair behaving toward each other in a manner similar to that described above for tallness and dwarfness. He further observed that the seven pairs are entirely independent of each other in inheritance (Mendel 's second law; now modified; see p. 384). All these phenomena he interpreted on the basis of the hypothesis that each character is in some way represented by a factor in the cells, new combinations of factors MEN DELI SM AND MUTATION 339 being formed at fertilization and the members of each allelomorphic pair of factors separating when the germ cells are formed. The Mendelian proportion of pure forms and hybrids is more easily followed in cases of "incomplete dominance," the pure dominants here being visibly distinguishable from the hybrids. Such a case is that of Mirabilis jalapa, the four-o'clock (Fig. 130). If plants bearing pure red flowers (var. rosea) are crossed with those bearing pure white flowers (var. alba) the result is an F} generation of intermediate pink-flowered plants. When these pink hybrids are bred among themselves the result- ing FZ generation comprises plants of three visibly different types: pure dominants with red flowers, hybrids with pink flowers, and pure recessives with white flowers, in the numerical ratio of 1:2:1. Terminology. — We may here introduce certain terms prominent in the literature of genetics. The genotype is the entire assemblage of factors which an organism actually possesses in its constitution, irrespective of how many of these may be expressed in externally visible characters. The phenotype is the aggregate of externally visible characters, irrespective of any other factors, unexpressed in characters, which may be present in the organism. For illustra- tion: in the case of the tall and dwarf peas there are in the second hybrid genera- tion (F2) three genotypes (with respect now only to the single character pair discussed): TT, Tt, and tt; represented respectively by pure tall plants, tall hybrids, and dwarfs; but there are only two- phenotypes : tall and dwarf, because of the fact that the complete dominance of tallness over dwarfness renders the hybrids externally indistinguishable from the pure tall individuals. Thus one phenotype (tall plants) here includes individuals with two genotypic constitu- tions, and the two can be distinguished only by a study of their progeny. In Mirabilis, however, there are in the F% generation not only three genotypes represented, but also three phenotypes, since the incomplete dominance renders the hybrids externally unlike either of the pure forms. An individual is said to be homozygous for a given allelomorphic character pair if it has received the same factor from the two parents — a pea, for example, with the constitution TT or tt. If it has both members of the pair, such as Tt, it is said to be heterozygous. It may be homozygous for some allelomorphic pairs and heterozygous for others, or it may conceivably be either homozygous or heterozygous for all of its characters. Thus an organism with the genotypic constitution AABbcc is homozygous for the characters represented by A A and cc, and heterozygous for those represented by Bb. It is thus a pure dominant with respect to A and a, a pure recessive with respect to C and c, and a hybrid with respect to B and b. The phenotypic appearance of the organism would be determined by the dominant factors A and B and by the recessive c; a given dominant factor dominates only its recessive allelomorph, and not the recessive factors belonging to other pairs. It is a common practice to represent dominant factors or characters by capital letters and their respective recessive allelomorphs by the corresponding small letters.; The Cytological Basis of Mendelism. — Having before us some of the principal facts of Mendelism and Mendel's interpretation of them, we 340 INTRODUCTION TO CYTOLOGY may now turn to the cytological basis of the Mendelian phenomena, and inquire what visible mechanism there is in the cell which will in any way help us toward an understanding of the striking behavior of the Mendelian characters. The behavior of the chromosomes at the critical stages of the life cycle as described in the chapters on reduction and fertilization must first be recalled. (See Fig. 131.) It has been shown that their history is as follows. Each parent furnishes the offspring with a set of chromo- somes, the two sets (represented in the diagram by A BCD and abed) being associated in all the cells of the offspring. When gametes (or spores followed later by gametes in the case of higher plants) are to be FEHTILIZATIOH Union of simplex groups CLEAVAGE Duplex groups ABCD abed SOMATIC DIVISIOMS Duplex groups REDUCTION DIVISION GERM CELLS Simplex groups FIG. 131. — Diagram showing the history of the chromosomes in the typical life cycle of animals. (After Wilson, 1913.) See also Fig. 77. formed by the new individual the chromosomes pair two by two (synap- sis), the two homologous members of each pair coming from the two parental sets. In the first maturation division (usually) the two members of each pair separate and enter different daughter cells: this is reduction, or the separation of entire chromosomes, presumably qualitatively differ- ent, instead of qualitatively similar halves of chromosomes as in somatic division. In the second maturation division all the chromosomes split longitudinally (equationally), so that as the result of the two divisions there are four gametes (or spores), two of them differing from the other two in chromatin content. The somatic chromosomes are therefore segregated into two unlike groups : each gamete (or spore) has a single set of chromosomes, the set being composed of one member of each of the pairs formed at synapsis. This set represents the contribution made to the following generation. MEN DELI SM AND MUTATION 341 It will be recognized at once that the above is precisely the sort of distribution shown by the characters in Mendel's experiments: two groups PARLNT5 FIG. 132. — Mendelian inheritance in black and albino guinea pigs. FIG. 133. — Chromosome history in the cross represented in Fig. 132, showing the parallelism between the distribution of a single homologous pair of chromosomes and that of a single allelomorphic pair of Mendelian characters. of (factors for) characters are brought together at fertilization and are associated in the body of the offspring. When the germ cells are formed 342 INTRODUCTION TO CYTOLOGY the (factors for the) two characters forming each allelomorphic pair separate and pass to different gametes (or spores). Thus the chromo- somes and the characters alike form a duplex group in the body cells and a simplex group in the gametes (or spores) : the chromosomes, like the characters, form new combinations at fertilization and are segregated when the gametes (or spores) are formed. In the diagram the letters ABCDabcd stand equally well either for chromosomes or for characters. In view of these facts it appears extremely probable that chromosomes and Mendelian characters have a definite causal relationship of some kind: it is scarcely conceivable that the exact and striking parallelism that they show can be without significance. The precise nature of this correspondence between chromosome be- havior and character distribution can be even more clearly shown by a consideration of the history of a single homologous pair of chromosomes in a typical Mendelian cross. If a pure white (albino) guinea pig be mated to an individual of a pure black strain the offspring are all black ; black is completely dominant over white (Fig. 132). If these black hybrids are bred among themselves they produce in the Fz generation three black animals to one white, or, more precisely, one pure black to two black hybrids to one pure white. Let us now follow a single pair of chromo- somes of each of the original animals through these two generations. At the left in Fig. 133 are represented the two animals, pure black and pure white, their chromosomes being drawn in solid black and outline respectively. In the black animal the two chromosomes pair at synapsis and separate to the two daughter cells at the first maturation mitosis, and split longitudinally at the second, so that each of the gametes re- ceives a single chromosome representing a longitudinal half of one of the original pair. A similar process occurs in the white individual. Unions between the gametes of the two animals now result in the F\ hybrids, each of which has one chromosome from its black parent and one from its white parent (not counting the chromosomes of other pairs) . When these hybrids form gametes, as is seen at once in the diagram, the pa- ternal and maternal members of the chromosome pair separate, with the result that half the gametes receive one of them and half the other. There are thus two kinds of spermatozoa and two kinds of eggs, one kind carrying the paternal chromosome and the other carrying the maternal one. Chance combinations now result in a generation (F2) of animals, one-quarter of which have derived both chromosomes of the pair in question from the black grandparent, one-half of which have derived one chromosome of the pair from each grandparent, and one-quarter of which have derived them both from the white grandparent. Moreover, these animals are respectively pure black, hybrid black, and pure white, in the proportion of 1:2:1. Thus it is seen that there is a direct paral- lelism, not only between chromosome sets and character groups, but also MEN DELI SM AND MUTATION between the distribution of a given homologous pair of chromosomes and thai of a single allelomorphic pair of Mendelian characters. This is exactly the condition which would result if two material units, each representing one of the characters of an allelomorphic pair, were located in two homologous chromosomes that pair and separate at reduction. The chromosomes afford precisely the type of mechanism required to account for the distribution of characters if the latter are associated with a definite material basis. It is this parallelism between the behavior of the chromosomes in reduction and that of Mendelian factors in segregation, first emp