Wtvw ftltlffl\ lijjf fo\t\ Vrt l I'M1 f't t' 11*1 BSKifi* f '»»«{«; ; t it vi. I' ijt{iji|iji : ;■;: n t*rt*i + ' p t • * m Wp J. p. PtII pfearg ^forilf (Eanxlitra Jiiaie Qlolkgs QH58I &5 NORTH CAROLINA STATE UNIVERSITY LIBRARIES S00555440 N Date Due 4e-S — 35 1 - 25 Jail 50' 15Feb'50$ MAR 2 2 , I. Cell in Development and Inheritance, which, though written twenty years ago and with the emphasis primarily on the zoological side, will remain invaluable to all workers for many years to come. The more recent works of Gurwitsch (Morphologie una1 Biologie der Zelle), Heidenhain (Plasma und Zelle), and Buchner (Prakticum der Zellenlehre) are of im- portance especially to the zoologist. The living cell, or protoplast, which represents the organized proto- plasmic unit of structure and function, obviously cannot receive complete description in structural terms. Until a comparatively recent period cytological researches dealt primarily with cell structure, including particulary the conspicuous changes undergone by this structure in connection with the reproduction of the cell (cell-division) and <>! the multicellular organism (maturation and fertilization). A gradual shifting of emphasis has since led to the opening of fruitful fields in other direc- tions, and the important results already achieved have shown wit h increas- ing clearness the need for a closer acquaintance with the physiological aspects of cell activity, not only in metabolism and growth, bu1 also in the reproductive phases of the life cycle. The present work, though dealing mainly with the structural aspects of the subject, may aid indi- rectly in fulfilling the above need by making the prerequisite data ol cell morphology more readily available. Throughout the book, which in many of iis chapters treats chiefly of the plant cell, attention is focussed upon the protoplasi : the fdl wall is given only brief consideration, since it plays :i relatively minor rdle in the processes of particular interest to the cytologisl at the presenl time. vii yjjj PREFACE Because of their fundamental importance in connection with the problems confronting the geneticist, the phenomena of nuclear division, chromo- some reduction, and fertilization arc described with considerable fullness, and their relation to the problems of heredity is taken up in five special chapters. With regard to many of the subjects treated, it has not been found possible to formulate final conclusions, since in many cases nothing more than tentative general statements are warranted by the facts in our possession. In some chapters little more than catalogs of conflicting opinions can be given, but in such a form the state of certain questions is not inaccurately represented. The student entering upon the field of cytology will be impressed by the large number of special points which remain undetermined and general questions which await adequate answers. If he can look upon cytology as a developing science, and if he has reached the stage at which he no longer demands categorical answers to all his questions, this book will be of interest to him as much for the problems it raises as for those it helps to solve. Not the least of its functions is to indicate lines of research along which he can hope to make contributions to the subject. In compiling his materials the author has not hesitated to draw very freely upon the writings of others. In many cases where direct quotation is not made, the language of the originals has been closely followed in order to lessen the liklihood of misrepresentation. His great debt to Professor Wilson's book will be apparent to all those familiar with that admirable work. The majority of the diagrams and a number of the other figures are new. Most of the latter, however, have been redrawn from works cited in the text, not only that the value of the book may be enhanced by the presence of authoritative illustrations, but also that the student may be encouraged to become more familiar with the original papers. The general systematic positions of organisms indicated in the text by their scientific names only may be ascertained by referring to the generic names in the index. The illustrations are largely the work of Miss Mildred Stratton, in whose skill and spirit of cooperation the author has had invaluable assistance. The criticisms of the text kindly given by Professor C. J. Chamberlain of the University of Chicago and Professor R. A. Emerson of Cornell University have been very highly appreciated. Acknowledge- ments are also made to the author's other colleagues for their advice and continued encouragement. Further criticisms looking toward the im- provement of future editions will be welcomed. L. W. S. Ithaca, New York, September 8, 1920. PREFACE ix Note Less than two months after the completion of the texl of tins book the author has received copies of the two now English works on cytology: W. E. Agar's Cytology, With Special Reference to the M< tazoan NucU us and L. Doncaster's An Introduction to the Study of Cytology. Both deal almost exclusively with animal cytology, the firsl being valuable for its account of chromosome behavior in animals, and the second for its discussions of gametogenesis, fertilization, parthenogenesis, and Bex- determination. These works, together with the botanical portions of the present volume, should make an acquaintance with the general field of cytology much more readily attainable. CONTENTS Preface vn CHAPTER I Historical Sketch 1 The discovery of the cell — Preformation and epigenesis Kaily theories 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 Description of the cell — The differentiation of cells — Bibliography 2. CHAPTER III Protoplasm Physical properties — Protoplasm as a colloidal system — Microdissection Chemical nature of protoplasm — Varieties of protoplasm — The plasms membrane — Protoplasmic connections — Vacuoles — Protoplasm aa 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 The centrosome — Occurrence and general characters Individuality Centrosomes in Algae— Fungi — Bryophytes— Conclusion The blepharo- plast— Occurrence — In flagellates— In fchallophytes En byrophytes In pteridophytes — In gymnosperms — In animals— Conclusion Bibliograph CHAPTER VI Plastids and Chondriosomes Plastids — General nature and occurrence— Leucoplasts ( Jhromatophores Starch— The pyrenoid— Elaioplasts and oil bodies The eyespol 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. CHAPTKK VII 1 ■ ; • ; Metaplasm; Polarity Metaplasm— Extruded chromatin- The senescence of the cell Polarity Metabolic gradient Bibliography 7. \i 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 ( )ther Modes of Nuclear Division 202 Cyanophycea? — 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 — Algae — 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. Mendelism and Mutation CONTENTS xm CHAPTER XV Pi Mendelism— A typical case of Mendelian inheritance The cytological basis of Mendelism— Mutation— Mutations accompanied by cl in chromosome number— Bearing on the origin of species and varieties Mutations accompanied by no change in chromosome number < lonclusion. :;.-, ! CHAPTER XVI Sex Experimental evidence for sex-determination — Sex-chromosomes Sex- chromosomes and Mendelism— Experiment :il alteration of the Bex ratio- Metabolic theories of sex — General discussion. CHAPTER XVII Linkage 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 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 XTV-XVIII). Index . . . 427 INTRODUCTION TO CYTOLOGY 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-:^22 B.C.) in his Be Partibus Animalium distinguished the "homogeneous parts'1 and the " heterogeneous parts," the former correspond ing in general to what we classify as tissues (bone, fat, cartilage, flesh, blood, lymph, nerve, membrane, nails, hair, skin, vessels, tendon, etc.), and the lata 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/1 "vein* and "flesh." Aristotle's classification was developed further by Galen (131-201 A.D.) and by his followers. Although we no longer regard tin- 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 workei 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 science, and the compound microscope has since remained an instru- ment of extraordinary value in biological research. 1 >■ * < INTRODUCTION TO CYTOLOGY The Discovery of the Cell.— Cytology may be said to have begun with the discovery of the cell by Roberl Hooke (1635-1703) in 1665. Hooke, who lived in London and lias been described as a man of eccentric appear- ftnce ftnd habits, showed a remarkably varied scientific activity. For a tjIm. ne wafi a professor of geometry, and later became an architect. He rformed many original experiments in mechanics and for a number of - curator of experiments to the Royal Society. His interest in optics led him to examine all sorts of objects with the compound micro- [n charcoal and later in cork and other plant tissues he found small honeycomb-like cavities which he called "cells." He had no dis- ,,,,,. t notion of the cell contents, but spoke of a "nourishing juice," which he interred 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 appear- to us, the Micrographia takes its place as the earliest cytologies! classic. Three ol her names even more prominent in the early history of micro- Bcopy are those of Malpighi, Grew, and Leeuwenhoek. Marcello Mal- pighi i 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 -1 naiome rUmtarum (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 B 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 ( irew (1641-1712) was an English physician and botanist. He began a careful study of plant structure in 1664, and in 1670 read his firsl in i poii ant paper before the Royal Society. Further contributions followed at intervals until 1682, when all of them were published under the till'' Thi 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 1 heory of encasement (emboitement) 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 ISTRODUCTION 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 (1578-1G67; 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- i ion : everything was preformed at the start. The other theory claimed too much postformation: 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 postformation ; it is rather this : How far is post-formation to be explained as the result of pre-formation, 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. ('. 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 problem- 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 Marchantia (1831-1833), 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 gemma?. 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 Halle* (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 3rears later Fontana (1781) saw the rotation of the "sap': in the Characese 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 do name is of greater interest to cytologists than that of Robert Brown (1773 L858). Al- though he is famous chiefly for his great taxonomic monographs and ln- 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 varieyt, 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 organism-. but it remained for Schleiden and especially Schwann to formulate a comprehensive theory embracing the known facts and affording a stall- ing point for further researches. The Cell Theory stated primarily that the body is compos,,! 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 year-. Schleiden was famous not merely because of his own work, but chiefly as the result of the tremendous impetus which he gave to investigation. 8 INTRODUCTION TO CYTOLOGY 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 Wtirzburg and later at Berlin. It was in the latter place that he put forth his statement of the ( 'ell 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, Beitrdge zur Phytogenesis, 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 0 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 "Afifcro- skopische Untersuchungen ilber die Uebereinstimmung in der Struktur und dem Wachslhum der Thieve und Pflanzen." He says: "The elementary parts of all tissues are formed of cells in an analogous, though very di ver- 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.,y And further: "The development of the proposition that there exists one general principle for the formation of all organic productions, and that this principle i- 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 product-, 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 ( 1800-1 87( > . in two works appearing in 1844 on vegetable growing points and tin- growth of internodes, argued for the origin of cells by division. Yon 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'1 (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 Naegli (1807-1891) in 1844 produced an exhaustive treatise on the nucleus, cell-formation, and growth. In algffi and the micro- sporocytes of angiosperms he clearly showed that cells multiply by 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 Niigeli 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 uomnis cell (da e cellula." ( ) pinions 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. Yon 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 embryogeny, 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 lighl 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 hold 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 wore 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. 11. A. dv 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 Kiihne (1864), Cienkowski (1863), and de Bary (1859, L864); and there later followed the well-known structural theories of Klein, Flemming, Altman, and Btitschli. (See Chapter III.) Yon Mohl as early as 1837 held that the plastid is a protoplasmic bod}-. 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 extenl by the organ- ism as a whole (Wager). Such divergence of opinion led to much dis- cussion over the question of organic individuality, which remain- 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 minutiaB, 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 (!. B. Amici (1830) followed the growth of the pollen tube from the pollen main on the Btigma 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 I 1849), and others showed the falsity of this notion, but an acrimonious discussion raged about the subject for a number of years, Schleiden (1842. L844) 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 (178.6) 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 righl 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 Cams (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 0. 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 dioecious 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 aor 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 L858, 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, n. 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.), Butschli (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 (1884-1885) 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 cytotogy, 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., 11th ed. Delage, Y. 1895. La structure du protoplasme et les theories sur l'heredite. 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. Lecons sur la Cellule. Paris. Hertwig, O. 1893. Die Zelle und die Gewebe. Jena. (Engl. Transl. by 11. J. Campbell.) 1900. Die Entwicklung der Biologie 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: 299-319. 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 58: 561-584. 1905. Von Baer and the rise of emb^ology. Ibid. 67: 97-126. 1915. Biology and its Makers. 3d ed. New York. Mark, E. L. 1881. Maturation, fecundation, and segmentation in Limns 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,, 11th ed. Waldeyer, W. 1888. Ueber Karyokinese und ihrc Beziehung zu den Befruchl angs- 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 Generatiohis. Ibid. 1898. Wilson, E. B. 1900. The Cell in Development and Inheritance. 2d ed. Zimmermann, A. 1893-1894. Sammcl-Referate aus dem Gesammtgebiete der Zellenlehre. Beih. Bot. Centr. 3 and 4. (Reviews of early literature). For other reviews of early botanical cell literature Bee Jahresberichl Qber die Fort- schritte der Anatomie und Physiologie, 15-20. 1886-1891'; and Neue Polge, 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 Etiunione degli Bcientiati Ltaliani 2 is INTRODUCTION TO CYTOLOGY tenuta in Padova nel Settembre del 1S42. Padova 1843. See Facchini 1845. See also (liorn. Bot. Ital. Anno 2. ^ubrbach, L. 1874. Organologische Studien. Breslau. von Baku, K. E. L828, 1837. Uber Entwickelungsgeschichte der Thiere. Barry, M. L838-1841. Embryological memoirs in Phil. Trans. Roy. Soc. London 128 -131. Sec also: On the first changes consequent on fecundation in the mam- miferous ovum. Rep. Brit. Assn. Adv. Sci. 1840. de Baby, H. A. 1S02. Uber den Bau und das Wesen der Zelle. Flora 20. 1864. Die Mycetozoen. 2d ed. Leipzig. van Beneden, E. L875. La maturation de l'oeuf, la fecondation et les premieres phases tin developpement embryonnaire des mamiferes d'apres des recherches faites chez le lapin. Bull. Acad. Roy. Belg. 40. 1876. Contribution a l'histoire de la vesicule germinative et du premier noyau embryonnaire. Ibid. 41. 1SN3. Recherches sur la maturation de l'oeuf, la fecondation et la division cellu- laire. Arch, de Biol. 4. Bernhardi, J. J. 1805. Beobachtungen tiber 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 tiber die Erscheinung der Verjiingung 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 Orchidese and Asclepiadeae. 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.) BtiTsCHLi, O. 1875a. Vorl. Mitteilung uber Untersuchungen betreffend die ersten Entwicklungsvorgange im befruchteten Ei von Nematoden und Schnecken. Zeit. Wiss. Zool. 25: 201. 1875/;. Vorl. Mitteilung einiger Resultate von Studien iiber Conjugation der In- f usorien und die Zelltheilung. Zeit. Wiss. Zool. 25 : 426. 1876. Studien iiber die ersten Entwicklungsvorgange der Eizelle, die Zelltheilung, und die Konj ligation der Inf usorien. Abhandl. Senckenb. Naturforsch. Gesell. 10. Camerarius, R. J. 1691. De Sexu Plantarum. 1694. Cares, C. 1824. Von den ausseren Lebensbedingungen der Weiss- und Kalt- blutigen 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 |«) Dutbochet, H. J. 1837. Mcmoires pour servir & I'histoire anatomique el physio- logique des vegetaux et des animaux. Facchini. 1845. Ueber die Amici'sche Ansichl von der Bef ruchtung der Pflanzen ein Beitrag. Flora 28: 193-198. See also 27: 359-360. Flemming, W. 1879a, 1880, 1881. Beitrage zur Kenntniss der Zelle und ihre Lebenserscheinungen. I, II, III. Arch. Mikr. Anat. 16, 19, 20. 1879/>. Ueber das "Verhalten des Kernes bei der ZeUtheilung, usw. Virehow'a Archiv. 77. 1882. Zellsubstanz, Kern und ZeUtheilung. Leipzig. 1887. Neue Beitrage zur Kenntniss der Zelle. Arch. Mikr. Anat. 29. Fol, H. 1873. Die erste Entvvicklung des Geryoniden-Eies. Jen. Zcitsrh. 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. 1S77. Sur le commencement de l'henogenie chez divers animaux. Arch. Sci. Na i et Phys. Geneve 58. 1879. Recherches sur la fecondation et la commencement de l'henogenie. Mem. Soc. Phys. et Nat, Geneve 26: 89-397. Fontana. 1781. Sur la structure primitive du corps animal. In Trait c sur le venin de la vipere. Florence. Gaertner, J. 1788. De fructibus et seminibus plantarum. Gaertner, K. F. 1849. Versuche und Beobachtungen liber die Bastard zeugung. Stuttgart, Gegenbaur, K. 1861. Ueber den Bau und die Entwicklung der Wirbelthiere. Muller'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. b}^ Turner.) Edinburgh. Grew, N. 1682. The Anatomy of Plants. London. Guignard, L. 1899. Sur les antherozoides et la double copulation sexuellC 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. Symbols ad anatomiam villorum intestinalium. 1841. AUgemeine Anatomic Leipzig. Hehtwig, O. 1875. Beitrage zur Kenntniss der Bildung, Befruchtung, und Thril- ling des tierischen Eies, I. Morph. Jahrb. 1. See also 2-4. 1884. Das Problem der Befruchtung und der Isotropic- des Eies, cine Theorie der Vererbung. Jenaische Zeitschr. 18: 276-318. Hofmeister, W. 1849. Die Entstehung des Embryos der Phanerogamen. 1858. Neuere Beobachtungen iiber 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 l'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. Koelreuter, 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. Kuhne, W. 1864. Untersuchungen liber das Protoplasma. Leipzig. La Valette St. George. 1865. Ueber die Genese der Samenkorper. Arch. Mikr. Anat. 1. See also Vols. 2 and 3. van Leeuwenhoek, A. 1673-1723. Brieven. Leiden, Delft. Link, H. F. 1807. Grundlehren der Anatomie und Physiologie der Pflanzen. Malpighi, M. 1675. Anatome Plantarum. Mark, E. L. 18S1. 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 tiber 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 physiologie vegetale. 1808. Exposition et defense de ma theorie de l'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 45. 1837. 1844. Einige Betrachtungen liber 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 Anatomie und Physiologie der vegetabilische Zelle. (Engl. transl. by Henfrey, London, 1852.) Moldenhawer, J. J. P. 1812. Beitrage zur Anatomie 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 l'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. Morphologie der Oedogonieen. Jahrb. Wiss. Bot. 1: 11-81. HISTORICAL SKETCH 21 Remak, R. 1841. Ueber Theilung rother Blutzellen beim Embryo. Med. Ver. Zeit. Muller's Archiv f. Anat. u. Physiol. 1858; 177-188. pi. s 1852. Ueber extracellulare Entstehung thierische Zellen und Qber Vermehrung derselben durch Theilung. Muller's Archiv f. Anat. u. Physiol. 1852; 17 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 dio 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 Phytogenesis. Muller'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. Oberh' 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 einc Zelle zu uenneu hat. Arch. Anat. u. Physiol. 1-27. Schwann, Th. 1839. Mikroskopische Untersuchungen iiber die Uebereinstiinmung in der Struktur und dem Wachsthum der Thiere und Pflanzen. (English transl., Sydenham Soc, 1847). Preliminary statement in Froriep- Nbtizen, 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 l'histoire de la genera t ion des ani- maux et des plantes. Geneva. Sprengel, C. K. 1793. Das neu entdekte Geheimniss der Xatur in Ban und Befruchtung der Blumen. Berlin. Sprengel, K. 1802. Anleitung zur Kenntniss der Gewachs* Straburger, 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, aebsl eiii Anhang uber Befruchtung. Hist. Beitr. 1. Thuret, G. 1854-1855. Recherches sur la fecondation des Fucees, Buivies des observations sur les antheridies des algucs. Ann. Sci. Nat. Bot. [V, 2 and 3. Trevirantjs, L. C. 1806. Vom inwendigen Ban der Gewachse. 1811. Beitrage zur Pflanzenphysiologie. Tulasne, L. R. 1849. Etudes d'embryogenie v6ge"tale. Ann. Sci. Nat. Bot. III. 12: 21-137. pis. 3-7. 22 INTRODUCTION TO CYTOLOGY Unger, F. 1844. Leber das Wachsthum der Internodien, von anatomischer Seite betrachtet. Bot. Zeit. IMeristematische Zellbildung. Ibid. Virchow, II. 1855. Cellular Pathologie. Arch. Path. Anat. Physiol. 8. 1858. Die ( Vllularpathologie, usw. (Transl. by Chance, 1860.) Weismann, A. 1885. Die Kontinuitat des Keimplasmas als Grunglage einer Theorie der Vererbung. Jena. Whitman, C. 0. 1S78. The embryology of Clepsine. Quar. Jour. Micr. Sci. 18: 215-315. pis. 12-15. Wolff, C. F 1759. Theoria Generations. 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 whole-. 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 thai 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 Eooke and the othei 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, straneelv enough, the early worlcers regarded as an 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. J, 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 nOKRTT LIBRARY n PRELIM IS ARY DESCRIPTION OP THE (ELL 25 nucleus, which at least in many respects is the most important of th< organs.1 The cytoplasm, a more or less transparent, viscous, granular Quid, 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 hail of Tradescantia, showing streaming movements in the cytoplasmic strands. K. paren- chyma cell from cortex of Polygonella, showing nucleus, plastids, and scanty cytoplasm. In many cases the cytoplasm forms a system of anastomosing strands thai often show active streaming (Fig. 2, D). Externally the cytoplasm is limited by a layer of different consistency, the ylasma 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 hnin, 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, orkaryo- somes, which represent accumulations of chromatin at certain point- oil the reticulum, and should not be confused with the true nucleoli. 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 Bense. 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 arc usually plastids of one or more kinds in the cytoplasm, the most conspicuous in plant cells being the green chloroplasts. A centrosomt is presenl 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. Ckondriosomes 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 arc 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 alga? 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 DESCRIPTIOX OF THE (ELL 27 of now typos of protoplasmic structure or cellular organization, bul in the development of constantly greater specialization and division of labor between larger and larger groups of colls." ( me 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' !l cells during the course of the ontogeny. In a young vascular plant, for example, definite regions (meristems) consisting of such colls 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 differei it iation, and are separated by no intercellular spaces. They undergo successive divisions very rapidly (hence the use of root tips for the study of mil osifl ; and while some of the products of division become greatly modified in structure in connection with their specialization in function, others retaintheir 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 i , i c i • ,, i. A- • „,m.-f;^,, Osmund, i, Bhowing 1 1 * * - lateral faces, whereas in the root tip, in addition 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 plant - 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 ells (Fig. 2, 28 JSTRODVCTION TO CYTOLOGY E), a number of other types, such as tracheids, vessels, wood fibers, sclerenchyma fibers, and sieve tubes (Eig. 4), all of which are characterized by the peculiar \v;iys 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 fibrillse which have to do with the cell's power of contractility. In certain muscles these fibrillar 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 i tin iiii ..^. .•••' ... • llllllfll • • * • t * » 1 1 • i i ■ < iliHJNMJI ;fiij«'jP'jjj 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 rord, X 75. (After Obersteiner and Hill.) C, nerve cell from the eye. (After L< ithossek.) D. Nerve cell from the earthworm. (After Kowalski.) E, young voluntary musrle cell. F, portion of mature voluntary muscle cell, showing striations. G, Involuntary muscle cell from intestine. (E-G after Piersol.) c Fig. »;. A, connective tissue from the jelly fish, showing branching cells and elastic fibers imbedded in gelatinous matrix. (After Lang.) H. cells of hyaline cartilage imbedded in their secretion. C, blood fell 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 flagellse, 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 I";. 7. — Paramoeeium caudatum. Semidiagrammatic figure showing principal parts. C. V., contractile vacuoles. T, trichocyst. N, n, mega- and micronuclei. P, peri- atomial groove. M, mouth. 0, 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. ( artilage 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 < ELL 3J 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 function- 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 Paramcechmi (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 -tinc- ture for its expression; in fact the most important single step ever taken m 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 sent 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 \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, Ruckert, Waldeyer, Whitman, and Zimniermann. Other works referred to in Chapter II: Hanstein, J. 1880. Das Protoplasma als Trager dcr 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. X. V. 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. Brucke (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. DejBary (1859, 32 PROTOPLASM :;;; 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, Altnian, and Butschli, and known respectively as the " reticular," "fibrillar/1 "granular," and "alveolar' t heories. 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 thai 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, enchyU ma (Hanstein 1880), hyaloplasma (Hanstein), paramitome, and inter-Jltar 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, <>r microsomes. Pfitzner (1883) pointed out that the granules are semi- solid and float in a more fluid ground substance. For Alt man I L886,et< 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 assemblage1 of bioplasts, "like bacteria in a zodglcea," and the bioplasts were believed to arise only by division of others <>t their kind (omne gran alum e granulo!). The alveolar theory, also known as the emulsion, or foam, theory, was elaborated principally by Butschli (1S82, etc.), and is of special interest in view of our present-day notions of protoplasmic structure. According 34 INTRODUCTION TO CYTOLOGY to Butschli protoplasm consists of minute droplets (averaging 1/* 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. Butschli 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 trophoplas?n, 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), Baylies (1915), Hatschek (1916), Bechhold (1919), and Robertson (1920). PROTOPLASM • 35 eye down to single molecules and ions. In the latter case we have a hue 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 rendei visible a body with a diameter less than about 0.20 to 0.25 ju\ 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 Borne 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, but an 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 ISTRODUCTION TO CYTOLOdY 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 1 1 a information 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, dispersing agent. {After Cloives, 1916.) C, surface film of soap or other 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. 1()0M 10fi 600 sq. cm. 10u 109 6,000 sq. cm. 1m 1012 6 sq. m. 100/x/x 1015 60 sq. m. 1(W 1018 600 sq. m. w 1021 6,000 sq. m. The evidence at hand supports the view that protoplasm is essentially a colloidal solution of the emulsion type. It consists of at least three principal phases: a water phase, containing a number of dissolved compo- PROTOPLASM 37 nents; a fat phase, consisting of fats and fat-soluble components; and a complex protein phase. Since the water phase is here the medium of dispersion protoplasm is classed as a "hydrosol' in its ordinary state, or as a "hydrogel" in the set condition. There arc doubtless additional minor phases present, protoplasm being in reality a "complex polyphase colloidal system." The presence of water in protoplasm is a matter of fundamental im- portance. As emphatically stated by Henderson (1913), ". . . the physiologist has found that water is invariably the principal constitu- ent of active living organisms. Water is ingested in greater amounts than all other substances combined, and it is no less the chief excretion. It is the vehicle of the principal foods and excretion products, for most of these are dissolved as they enter or leave the body [across the wall of the intestine and across the epithelia of kidneys, lungs, and sweat glands]. Indeed, as clearer ideas of the physico-chemical organization of proto- plasm have developed it has become evident that the organism itself is essentially an aqueous solution in which are spread out colloidal sub- stances of great complexity [Bechhold 1912]. As a result of these condi- tions there is hardly a physiological process in which water is not of fundamental importance" (pp. 75-77). The amount of water in protoplasm varies greatly under different conditions, but normally it is present in large proportions. It makes up 85 to 95 per cent of the weight of actively streaming protoplasm such is seen in Elodea and Tradescantia, and in actively functioning cells it rarely drops below 70 per cent. In dry spores, however, it may be reduced to 10 or 15 per cent, in which case the protoplasm becomes very viscous. The percentage differs constantly in different parts of the cell; nucleus, cytoplasm, and plastids, though all are composed primarily of protoplasm, contain very different -amounts of water. Since active pro- toplasm is a liquid, the phenomena of surface tension and other propert ies of liquids must enter largely into explanations of its behavior. The colloidal nature of protoplasm is manifested in many of its prop- erties. Its power of adsorption, which lies at the basis of many cell reactions and certain staining processes, is similar to thai of other colloids. Protoplasm, like other colloids, is semi-permeable: a semi-per- meable region is probably present wherever protoplasm comes in contact with other substances, such as water; and the permeability of a vacuolate cell is in general the resultant of the permeabilities of the ectoplast, cytoplasm, and tonoplast. Protoplasm shows most strikingly its colloidal character in t In- changes of physical state which it undergoes as the effects of variations in the external conditions. The alterations due to changes in temperature will serve for illustration. Above a certain temperature the colloid gelatin exists in the sol state it is a hydrosol. If the temperature is sufficiently 38 INTRODUCTION TO CYTOLOGY lowered the gelatin " sets"— it becomes a hydrogel. This setting is a reversible process: if the temperature is again raised the sol state is resumed. On the contrary, egg albumen is a hydrosol at ordinary temperatures and becomes a hydrogel when heated; in this case the change is an actual coagulation and is not reversible. Many of the colloids of the cell are of this non-reversible type. "The evidence that in this colloidal condition the transition from liquid to solid, from sol to gel, tends especially to pass into an indefinite series of gradations gave a basis for the explanation of that mixture of the properties of solids and liquids which has puzzled students of protoplasm" (Harper 1919). Protoplasm is thus easily coagulated, not only by a high temperature, but by a variety of chemical substances. The "fixation'1 of the cell structures by the reagents employed in cytological technique is primarily a coagulation phenomenon, and in the act of coagulation a substance, especially one as complex as protoplasm, undergoes an alteration in physical structure. Although such fixing fluids preserve very well the general structure of the cell, the effects of coagulation should always be borne in mind in interpreting finer details in preparations of fixed cells, and in evaluating the results of those who have made special studies on the ultimate structure of protoplasm. Microdissection.— Much has been added to our knowledge of the physical nature of protoplasm in recent years through microdissection. Certain workers, notably Barber (1911, 1914), Kite (1913), Chambers (1914. 1915, 1917, 1918), and Seifriz (1918, 1920) have developed a technique (fully described by Barber 1914, and Chambers 1918) whereby they have been able to dissect living cells under the high powers of the microscope, thus opening a most promising field for investigation. Kite, working on the cells of several plants and animals, found that protoplasm exists in the form of sols and gels of varying consistency, that of plant cells being as a rule more dilute and less rigid than that of animals. The cytoplasm is commonly somewhat more viscous than is usually thought, having the consistence of a "soft gel," while the nucleus may often be surprisingly firm. (See Chapter IV.) Chambers (1917), who gives a convenient bibliography of the subject, states that in the early germ cells and eggs of certain animals the proto- plasm is in the sol state with a surface layer in the gel state, whereas adult cells are usually gels. He further asserts that the surface gel is readily regenerated after injury, a new gel film being formed over the injured area. As regards the structure of protoplasm, he finds it to consist of a hyaline fluid carrying microsomes and macrosomes, which measure less than 1//. and from 2-4^ in diameter respectively. Upon dis- organization the macrosomes, which are more sensitive to injury than are the microsomes, swell and go into solution, while the hyaline fluid flows out and mixes with water or coagulates, forming a reticular or granular structure. PROTOPLASM 39 Seifriz (1920) has investigated with care the viscosity of the proto- plasms of a number of myxomycetes, algae, pollen tubes, protozoans, and echinoderm eggs. He finds the degree of viscosity to vary widely, from a very watery to a fairly rigid gel condition, not only in the different organs of the cell but also in the protoplast as a whole at different Btag of its development. He warns against accepting viscosity alone as an index of the gel or sol state of the protoplasm, since it is physical structure and not viscosity which determines these states in an emulsion. It is to be hoped that the methods employed by the above investi- gators will be further developed and applied more widely, for through them many misconceptions will undoubtedly be collected. It should be evident from all these considerations thai there is prob- ably no sinlge visible structure characteristic of protoplasm at all times. Any fundamental structure which it may have remains to be discovered in the ultramicroscopic constitution of the colloids and other materials of which it is composed, and in the physical relations which these beat to one another. It should be pointed out, however, that in the idea of protoplasm as a complex colloidal emulsion we have the best hypothesis yet offered as a basis for the interpretation of the behavior of living- substance. Chemical Nature of Protoplasm. — Chemically, as well as physically, protoplasm is exceedingly complex, and the study of its constitution has opened a field of research which is continually broadening. ( )nly a brief summary of some of the more important chemical facts can be presented here; for more detailed accounts special works on the subject must be consulted.1 As already pointed out, the substances of which protoplasm is com- posed are probably not fundamentally different from those found else- where, but show rather a greater complexity and a high degree of organization. Protoplasm is an intricately organized system of water, proteins, enzymes, fatty substances, carbohydrates, salts, and other minor constituents. The often cited analysis by Reinke and Rodewald (1881) of the myxomycete sEthalium septicum (Fuligo) showed the proto- plasm of this form to have the following composition: PER CENT DRY WEIGHT PER CENT DB1 WEIGHT Proteins 40 ( Jholesterin (lipoid I 2.0 Albumins and enzymes 15 Ca salts (except CaCO '» 5 Other N compounds 2 Other salts d 5 Carbohydrates 1- Resins 12 Fats 12 Undetermined . . . . 6 5 The protein matter of protoplasm exists in relative complex forms. "The chief mass of the protein substances of the cells does not consisl <»t' 1 See especially the hooks of Hammareten (1909), Wells (1914), Czapek (1915 . Bayliss (1915). Mathews (1916), Palladia (1918), Robertson L920 , and thereviewby Zaeharias (1909). 40 INTRODUCTION TO CYTOLOGY proteids in the ordinary sense, but consists of more complex phosphorized bodies . . .' (Hammarsten). Such "phosphorized bodies" are the nucleo-proteins, which are "probably the most important constituents of the cell, both in quantity and in relation to cell activity" (Wells). A long series of chemical investigations beginning with the pioneer work of Miescher, Hoppe-Seyler, and Reinke, have shown that these nucleo- proteins are essentially combinations of nucleic acid with proteins, or sometimes with the simpler histones or the still simpler protamines. The nucleus as a rule is free from or very poor in uncombined carbohy- drates, fats, and salts, but is characterized rather by the abundance of a nucleo-protein called nuclein, isolated in 1871 by Miescher, who gave it the formula C29H49N9P3O22. It was shown by Altman (1889) that nuclein, like the other nucleo-proteins, could be split into two substances: nucleic acid and a form of albumin (protein), the two existing in chemical com- bination like an ordinary salt. Nucleic acid from yeast was given the gen- eral formula C10H59N14O22 — 2P205, and that from fish sperm C40H56N11O 2P205. Nucleic acid was further analysed into phosphoric acid and 16 certain bases. The relation of these simple substances to nuclein, and also the relation of nuclein to more complex nucleo-proteins, are shown in the following scheme (mainly from Wells): Higher nucleo- proteins Proteins Nuclein Proteins (albumins, etc.) , Nucleic acid Phosphoric acid Levulinic acid Purin bases Pyrimidins . Pentoses Xanthin Guanin Adenin etc. The nucleo-proteins of the nucleus (chromatin) contain very little of the protein constituent and are thus relatively rich in phosphorus. ( ilaser (1916) accordingly speaks of chromatin as "a conjugated phospho- protein group with a nucleic acid group, the latter group being a complex of phosphoric acid and a nuclein base." Kossel (1889, 1891, 1893) even concluded that in certain instances (during mitosis) chromatin might be simply nucleic acid. In the cytoplasm, on the contrary, the proportion of the protein constituents is relatively high. The cytoplasm probably has no true nuclein, but is rich in nucleo-albumins, albumins, globulins, and pep- tones, which, unlike nuclein, have little or no phosphorus. As a result its reaction is alkaline, in contrast to the acidity of the nucleus. Accord- ing to Hammarsten (1909), "the globulins and albumins are to be con- sidered as nutritive materials for the cell or as destructive products in PROTOPLASM 11 the chemical ( ransformat ion of ) he protoplasm. " ( rranules of " volut in ' formed in the cytoplasm are also looked upon as :i food substance used by the nucleus in the elaboration of chromatin. The fatty components of the cell comprise both ordinary fata and lipoids (fat-like bodies nol decomposed by alkalis); among the Latter lecithin and cholesterin are of great importance, particularly in the cells of animals. The carbohydrates found in protoplasm are chiefly pentoses and hexoses, which are as a rule combined with proteins and with lipoids. Glycogen exists free in the cells of many tissues and serves as a source of heat and energy. The important role played by pentosans in the activity of the plant cell is strongly emphasized by Spoehr (1919) and Macdougal (1920); in fact these authors speak of protoplasm as "an intermeshed pentosan-protein colloid." Inorganic salts are present in considerable variety, as shown by the presence of the following elements in the ash of Fuligo protoplasm: CI, S, P, K, Mg, Na, Ca, Fe. Because of their failure to find any new types of chemical compound- in their analysis of protoplasm Reinke and Rodewald (1881) thought it probable that the peculiarities of protoplasm are due to its structure rather than to its chemical composition. It has since been found, how- ever, that certain of the life processes continue for a time alter the pro- toplasm has been ground up mechanically. Moreover, more refined analytical methods have enabled chemists to isolate from protoplasm certain extremely complex and unstable proteins (the "protoplasmids,J of Etard), which differ greatly in degree of complexity, if not otherwise from proteins encountered elsewhere. Varieties of Protoplasm. — From the foregoing resume it is plain that in protoplasm, because of the many combinations possible among con- stituents present in such great variety, we have a substance which may exist in a vast number of different forms. When it is further recalled that many of the constituents exhibit singly the phenomenon of stereo- isomerism this number is seen to be incalculable. For example, it was shown by Miescher that an albumin molecule with to carbon atoms could have about one billion stereoisomers, and some albumins probably have more than 700 carbon atoms. Albumin, moreover, is only one of many complex substances present in protoplasm. Hence, the state- ment that all living ('('lis are composed of the same substance, proto- plasm, is true only in a general sense. Although they are made up of the same categories of substances existing in the same general type of organization — the hydrocolloidal state -the protoplasms of different organisms vary widely in the relative amounts of these leading con- stituents. For example, the lipoids are much more abundant in the protoplasm of animals than in that of plants, and the carbohydrate- 42 INTRODUCTION TO CYTOLOGY protein ratio also shows notable differences in the two kingdoms. Ana- logous differences also exist between the smaller plant and animal groups, and with these differences in chemical constitution are associated many characteristic diversities in metabolic activity. Thus it is not simply with protoplasm but with protoplasms that the working biologist has to deal. Special emphasis has been placed upon the relation of this great diversity in the constitution of protoplasms to the amazing variety observed among living organisms by Kossel, Reichert, and a number of other writers. As Reichert states, the evidence seems to indicate that "in different organisms corresponding complex organic substances that constitute the supreme structural components of protoplasm and the major synthetic products of protoplasmic activity are not in any case absolutely identical in chemical constitution, and that each substance may exist in countless modifications, each modification being character- istic of the form of protoplasm, the organ, the individual, the sex, the species, and the genus." With regard to the integration of the various protoplasmic constituents, Mathews (1916) says: "Protoplasm, that is the real living protoplast, consists of a gel, or sol, which is composed of the colloids of an unknown nature which include protein, lipin and carbohydrate. Whether these colloidal particles consist of one large colloidal compound in which enzymes, protein, phospholipin and car- bohydrate are united to make a molecule which may be called a biogen [Verworn 1895, 1903], cannot be definitely stated, but it seems probable that something of the sort is the case." The Plasma Membrane. — It was recognized very early that there is at the surface of the protoplast a thin layer of relatively resistant, hya- line protoplasm which Hanstein called ectoplasm, distinguishing it thus from the granular endoplasm within. Pfeffer (1890) employed the cor- responding terms hyaloplasm and polioplasm. The ectoplasmic envelope, which is best seen on "naked" masses of protoplasm, such as amoeba?, myxomycetes, and the zoospores and gametes of algse, has been variously referred to by different writers as the ectoplast, plasma membrane, Haul- schicht, and Plasmahaut.1 The proponents of the reticular and fibrillar theories of the structure of protoplasm looked upon this external layer as a region in which the fibrils are more closely compacted or interwoven, whereas Butschli re- garded its relative firmness as due to a compact radial arrangement of alveolae. Pfeffer (1890) held that such a limiting membrane, which living protoplasm always produces on an exposed surface and which consists mostly or entirely of protein substances, is not itself truly proto- plasmic, whereas the majority of cytologists have thought it to be a 1 A discussion of ectoplasm and endoplasm based upon a large number of ob- servations on Amoeba is given in a new work by Schaeffer (1920). PROTOPLASM 43 special protoplasmic layer: Strasburger, for instance, believed ii to be composed of kinoplasm. The microdissection studies of Kite (1913) and Chambers L917 mentioned above have extended our knowledge of the physical nature of the plasma membrane. Both of these observers describe the ecto- plast of an Amceba as a concentrated gel. Seifriz (1918), as a result of such studies on the Fucus egg and myxomycetes, states that the mem- brane is a definite morphological structure, very elastic and glutinous, and capable of constant repair. He further asserts that membrane form-" ation is a physical process dependent upon the physical state of the protoplasm and not upon that of the medium, and that it does not occur after death. That the formation of such a limiting membrane at the surface of protoplasm is the result of the tendency of colloidal particles to accumu- late on any interface has been pointed out by physical chemists. ( 'it ing, by way of illustration, the film which forms on the surface of cooling milk, Moore (1912) says: "The chief colloid of the milk, on account of its affinities, accumulates on the surface, the accumulation gives increased concentration, the presence of the increased concentration causes the multi-molecules to build together, the larger molecules fall out of solu- tion as particles, and these join to form a close network or film." In a similar manner the unicellular organism or other mass of naked proto- plasm develops its resistant envelope, and the enclosed protoplast of the higher plant its ectoplasmic layer and tonoplast. Permeability. — The physico-chemical nature of the plasma mem- brane has been a subject of much discussion among physiologists. < hi the assumption that the permeability of the cell is a case of solubility in the ectoplasm, E. Overton (1895, 1899, 1900) developed a theory of the constitution of the ectoplast. It was pointed out first, that the ectoplast is not miscible with water; second, that in plant and animal cells the only bodies which are not miscible with water in the ordinary state are fats and oils; third, that the ectoplast is more or less permeable to substances ac- cording as the latter are more or less soluble in fats and oils; and fourth, that any substance insoluble in another substance will not pass through a membrane composed of the latter. It was therefore concluded that the ectoplast is made up of some lipoid compound, such as lecithin, which acts as a semi-permeable membrane. This theory, though very sug- gestive, was effectively opposed by Ruhland I L909, L915) and a number of other investigators, who called attention to many substances which do not behave according to the requirements of the theory stated in so simple a form. A more nearly adequate conception of the constitut ion of the ectoplast has thus been souhgt. Of the more recent theories which have been offered in connection with the problem of permeability the most promising are those which 44 INTRODUCTION TO CYTOLOGY interpret the ectoplasl as an emulsion. According to Czapek (1910, 1911, 1915) the ectoplasl is an emulsion of lipoids, proteins, and other substances, the lipoids forming a suspended phase. " Protoplasm is a colloidal emulsion of lipoids in hydrocolloidal media, the latter containing proteins and mineral salts." Lepeschkin (1910, 1911) advanced the contrary view that the lipoids form the medium of dispersion. In at- tempting to account for changes in permeability Clowes (1916) points out that inversion of phases probably plays an important role, while Spaeth (1916) ascribes changes in permeability to alterations in the degree of dispersion of the colloids, with resulting changes in the vis- cosity of the membrane. A more definitely stated hypothesis of the latter type is that tentatively suggested by Lloyd (1915) and Free (1918). Colloids are known that "have two liquid phases which differ in composi- tion only in the relative proportion of water and of the substance of the colloid" (Free). It is accordingly possible that alterations in permeability may be due to changes in the distribution of water between two such phases present in the plasma membrane. When water passes from the internal (suspended) to the external (continuous) phase, the droplets of the former would become very small; when the move- ment is in the opposite direction they would be- come very large and closely packed. As a result there would be such changes in the physical nature of the membrane as would aid in interpret - „ , ing the behavior of the latter toward substances lie 9. — Amoeba, snow- ing ectoplasm, endo- entering or leaving the cell. It is held that such a plasm and contractile hypothesis accounts more readily for the gradual changes in permeability observed than does the inversion theory of Clowes, according to which the change might be ex- pected to occur suddenly. It is pointed out, however, that both processes are probably involved. Whatever the degree of correspondence between the above inter- pretations and reality may be, it is scarcely open to doubt, especially since the work of Bancroft (1913) and Clowes (1916) on colloids, that in such theories we have our best prospect of reaching an adequate knowl- edge of the plasma membrane,. which, because of its great importance in the life of the cell, is to be regarded as a definite "osmotic organ." Protozoa. — It is in the Protozoa that the ectoplast shows its most elaborate structural differentiations. (See Minchin, 1912, Chapter V.) Here the ectoplast clearly has several functions : protective, motor, excre- tory, and sensory. In most forms other than the Sarcodina there is a resistant envelope of some sort. This may represent (a) the entire ecto- plast modified (the "periplast" of Flagellata); (6) a superficial modified ec t.- PROTOPLASM i:> layer of the ectopias! (the "pellicle" of Infusoria and some Amoebae); (c) a secreted layer ("cell membrane") rather than a modification of the ectoplast. In certain cases definite actively protective organs, the tri- chocysts, are differentiated in the ectoplasm. Among the ectoplasmic structures with a motor function the simpl are the pseudopodia; in the larger ones there is a core of endoplasm Fig. 9), but the more delicate "filose" ones consist entirely of ectoplasm I Fig. 10). The flagellum of Euglena was reported by Butschli to have an elas- v\\ I f Fig. 11. A, flagellum of Euglena, showing endoplasmic core and ectoplasmic sheath. {After Butschli.) B, Trypano- soma tincce, with undulating membrane. {After Min- dopodia composed of ectoplasm, chin.) C, Trypanosoma percoB, showing myonemes. \Jttr {From Minchin, after Schultze.) Minchin.) Z>, flagellum of Euglena, Vfier Dellxnger.) Fig. 10. — Gromia oviformis, showing filose-reticulate pseu- tic endoplasmic core with a contractile ectoplasmic sheath (Fig. 11.1 but the later figure of Dellinger (1909) represents it as composed of tour twisted filaments ending within the animal as a system of branching rootlets (Fig. 11, D). Cilia, which are short and numerous and show rythmic pulsation; cirri, which are formed of tufts of cilia; membranellce, representing fused rows of cilia; and undulating membranes, which are sheet-like extensions of the ectoplasm (Fig. 11. H). arc all essentially ectoplasmic organs. A further motor differentiation is Been in the minute contractile fibrils known as myonemes, which are analogous to a 46 IS 'PRODUCTION TO CYTOLOGY system of muscle fibers (Fig. 11, C). In ciliated forms they run beneath the rows of cilia. Contractile vacuoles, which exercise an excretory function, originate in the ectoplasm, although later they may lie much deeper. A sensory function is performed by the "eyespot," which is sensitive to light, and also by the flagellar and cilia, which are often receptors of tactile stimuli. The eyespot seems in some instances to be plastid-like in character, and will be discussed in Chapter VI. Protoplasmic Connections.- -The fine protoplasmic strands (Plasmo- desmeri) connecting many plant cells through pores in the intervening walls are extensions of the ectoplasm (Fig. 12). Several early workers B C Fig. 12. — Protoplasmic connections in vascular plants. A, B, Pinus pinea: cells of cotyledon. X 375. (After Gardiner and Hill, Phytelephas ("vegetable ivory "): endosperm cells with greatly thickened showing spindle-shaped bundles of connecting strands, m, middle lamella, grammatic. 1901.) C, walls (w), Semidia- suspected the presence of such connections before they were able to see them, and even the coarse strands passing through the sieve plates of sieve tubes, though often observed, were not well known until the time of Hanstein's work in 1864. The finer strands of other plant tissues where described in a large number of researches between 1880 and 1900. Among these may be mentioned those of Wille (1883) and Borzi (1886) on the Cyanophycese; Kohl (1891), Overton (1889), and Meyer (1896) on the Chlorophycese; Hick (1885) on the Fucacese; Hick (1883), Massee (1884), and Rosenvinge (1888) on Floridese; and, on vascular plants, those of Tangl (1879), Russow (1882), Strasburger (1882, 1901), Goros- chankin (1883), Terletzki (1884), Wortman (1887, 1889), Haberlandt (1890), Kienitz-Gerloff (1891), Jonsson (1892), Kuhla (1900), Gardiner (1884, 1897, 1900), Hill (1900, 1901), and Gardiner and Hill (1901). i* With respect to the origin and development of these connecting strands very little is accurately known. Some observers have claimed that the pores through which they pass are present from the time the primary wall is first formed, no wall substance being laid down at these points. Gardiner (1900) believed them to arise directly from the median PROTOPLASM 17 portion of the fibers of the achromal ic figure al I he close of mitosis. 1 1 is observations were made on the endosperm of Lilium and Tamus. < >i hers, on the contrary, have regarded them as secondarily developed si rucl nn Their absence from the walls between CuscvJta and Vixcum and their hosts (Kienitz-Gerloff, Kuhla, Strasburger 1901), and also from many cells which glide over one another during growth, is a fact opposed to the latter interpretation. Although they have been demonstrated in a number of kinds of tissue they probably do not occur so widely as some have supposed; but it may nevertheless be true that in many cases their apparent absence is due to the fact that the special methods often neci sary to their demonstration have not been widely employed. As to their function, it can scarcely be doubted that they may serve to transmit stimuli of one kind or another from cell to cell ( Pfeflfer 1 896 Noteworthy in this connection is their presence in tissues of plant parts known to be particularly responsive to external stimuli, such as the leaves of Mimosa (Gardiner 1884) and Dioncea (Gardiner 1884; Macfarlane 1892), the stamens of Berberis (Gardiner 1884), and the sensitive labellum of the orchid, Masdevallia muscosa (Oliver 1888). Their extensive development in storage tissues, such as the endosperm of seed- (Tangl 1879; Gardiner 1897), would also indicate that they are in part responsible for the readiness with which nutritive materials are translocated insuch specialized tissues. Vacuoles. — Vacuoles in the cytoplasm are more characteristic of plant than of animal cells. They are usually absent in the very young cell, but appear as growth and differentiation progress. In case they are very small and numerous the cytoplasm takes on an alveolar appearance. but more commonly they coalesce to form one lame vacuole which may occupy a volume greater than that of the protoplast itself I Fig. 2 This condition is characteristic of many mature cells of plants, but is comparatively rare in animals. The ordinary vacuole is essentially a droplet of fluid, consisting ol water with differentiation products in solution, surrounded by a delicate limiting membrane. DeVries (1885) developed the theory thai vacuoles are derived from "tonoplasts." The tonoplasts were believed to be small bodies imbedded in the cytoplasm and multiplying by fission. Through the absorption of water they swell and become vacuoles, the vacuole wall thus being made up of the material of the tonoplasl body. We still refer to the vacuole wall as the tonoplast. De Vries looked upon the vacuole as a body with an individuality somewhat similar to thai of a nucleus, since the tonoplast from which it develops was supposed to arise from a preexisting tonoplast by division. The theory wassupported by certain other workers, but it does not enjoy wide acceptance today It has been found by Bensley (1910) and others thai there is in the cytoplasm of certain comparatively young cells a system of fine canals 48 INTRODUCTION TO CYTOLOGY - l48t which later open up to form vacuoles (Fig. 13). The fixing reagents commonly employed in cytological technique destroy these canalicalce; and since Bensley, by using special reagents, demonstrated such canals in the familiar cells of the onion root tip, it is highly probable that they occur very widely. It seems more reasonable oo suppose that the fluid differentiation producos, when they are first forming, gradually come to move along certain paths, forming canals, and later accumulate in the form of vacuoles, than to suppose that the vacuoles originate in such individualized units as the tonoplasts of deVries. Fluids other than water may also occur in the form of vacuoles; oil vacuoles, for example, are not uncommon in oertain cells. If fats, oils, and other products of metabolism take their Fig. 13.— Cell from root origin in chondriosomes, as some suppose (see tip of Allium cepa, showing Chapter VI), it is not improbable that some- StoftO* UftCr Cham~ thing at least anal°g°us t0 the above mentioned tonoplast behavior may occur in the case of certain substances appearing in the cell. The cell sap and other differentiation products in the cytoplasm will be discussed further in Chapter VII. PROTOPLASM AS THE SUBSTRATUM OF LIFE Since the true significance of protoplasm was first recognized in the middle of the last century many suggestions have been ventured regard- ing the nature of the relation existing between life and its physical basis. A full discussion of this subject obviously cannot be entered upon here, but theories of two types, the micromeric and the chemical, may be cited by way of illustration. Micromeric Theories. — Many years ago there were developed certain speculative " micromeric theories" of the constitution of protoplasm; these became particularly prominent during the latter half of the nine- teenth century. According to these " atomic theories of biology" the principle of life was held to reside in ultimate fundamental particles. The particles were supposed to be for the most part of ultramicroscopic size, capable of independent growth and reproduction, and associated like members of a vast colony in protoplasm. Such vital units were compared by some to chemical molecules, but they were generally regarded as something much more complex. Examples of such units were the " organic molecules" of Buff on, the "microzymes" of Bechamp, the " physiological units" of Spencer, the " plastidules " of Maggi and Haeckel. the "bioplasts" of Altman, the "vital particles" of Wiesner, PROTOPLASM 19 the "gemmules,:i of Darwin, the "biophores" of Weismann, the "pan- gens" of de Vries, and the "ergatules" and "generatules" of Batschek. In a somewhat similar manner a number of the Later investigators occupied with the study of the ultimate structure of protoplasm have often been led to inquire which of the constituents of protoplasm arc the actually living elements. Among those who viewed protoplasm ae reticular structure some held the material of the reticulum to be the true living substance, the liquid ground substance being lifeless, wlierea- others held the reverse to be true. Many of those who saw in protoplasm a granular structure regarded the granules as the ultimate living unit-, and more recently there has even been a tendency on the part of some investigators (Beijerinck, Lepeschkin), who have emphasized the emul- sion nature of protoplasm, to view the droplets of the suspended phase in a similar light. To Butschli the continuous phase was the essential substance. By most modern biologists such attempts to assign the principle of life to any particular constituent unit of protoplasm or of the cell, whether this unit be an observed structural component or a purely imaginary one. are regarded as not in harmony with an adequate modern conception of the term " living." It has been repeatedly emphasized that life should be thought of not as a property of any particular cell constituent, but as an attribute of the cell system as a whole (Wilson 1899); or, as Brooks (1899) put it, not merely as a property but as a relation or adjustment between the properties of the organism and those of its environment. This recalls Herbert Spencer's characterization of life as a "continu- ous adjustment of internal relations to external relations." As Sachs (1892, 1895) and others urged, the various elements in the4 cell should be referred to as active and passive rather than living and lifeless. These elements play various roles in the cell's activity: each contributes to the orderly operation of the whole. When any part fails to function properly, or when the proper adjustment is not maintained, the whole system of correlated reactions, the resultant of which we call life, must become disorganized. As Child (1915) remarks, the theories postulating vital units only transfer the problems of life from the organism to something smaller; the fundamental problem of coordination is no nearer solution than before, and the whole question is placed outside the field of experi- mentation. Harper (1919) also points out that modern cytology DO longer looks upon protoplasm as a substance with a single specific struc- ture, or as one made up of ultimate fundamental units of some kind. but rather as a colloidal system or group of systems of varying structure and composition. "The fundamental organization of living material Is expressed in the structure of the cell." The cell itself, and not some hypothetical corpuscle, is the unit of organic structure. Protoplasm is accordingly not made up of structural units arranged in various ways to i 50 INTRODUCTION TO CYTOLOGY form the cell organs, but is rather a colloidal system in which special processes and functions have become localized and fixed in certain regions; and this in turn has resulted in the evolution of organs possessing more or less permanence. Chemical Theories. — Much more suggestive, if not conclusive, have been certain attempts to place the phenomena of the organism upon a purely chemical basis. With the development of organic chemistry from the time of Wohler's (1828) synthesis of urea onward there has grown up the idea that life processes and chemical reaction not only resemble each other but are actually the same fundamentally. When protoplasm was subjected to chemical anatysis and found to consist chiefly of water and proteins, and when these substances became more intimately known, the task of explaining the activity of protoplasm in terms of the chemis- try of proteins was undertaken. One group of workers developed the hypothesis that peculiarly labile protein molecules are responsible for the organism's reactions, "death,; being primarily a change from the labile to the stable condition on the part of these molecules. Such molecules were called "biogens,; by Verworn (1903). The molecule itself was not thought of as alive, but its constitution was held to be the basis of life, which "results from the chemical transformations which its lability makes possible." Accordingly, "life itself consists in chemical change, not in chemical constitution" (Child 1915). A-dami (1908, 1918) contends that life is thus "the function, or sum of functions, of a special order of molecules." These ultimate molecules of living matter he calls biophores (not to be confused with the biophores of Weismann, which were molecular complexes), and he locates them in the nucleus, the cytoplasm having merely "subvital" functions. They are proteidogenous in nature, i.e., they compose an active substance which takes the form of relatively inert proteins when subjected to chemical analysis. The biophore is conceived by Adami to have the form of a ring or a ring of rings of the benzene type — a ring of amino acid radicles with many unsatisfied affinities or bonds. The biophore grows in a manner analogous to that of the inorganic crystal: ions and radicles from the surrounding medium become attached as side chains to the free bonds of the central ring and take on a grouping similar to that of the latter; in this way the biophoric molecules are multiplied. Since side chains can be detached and new ones of other kinds added, the biophore is changeable and may exist in many different forms. Although the central ring is thought to be relatively stable and fixed, the variety of side chains and their many possible arrangements probably give to each species a distinct kind of biophore. On this hypothesis the molecule of living matter (biophore) is one "of extraordinary complexity, and in a state of constant unsatisfaction, built up by linking on other simple molecules, and as constantly, in the performance of function, giving up PROTOPLASM :,l or discharging into the surrounding medium these and other molecular complexes which it has elaborated" (Adami 1918, pp. 251 2 " Ml vital manifestations are manifestations of chemical change in proteidogen- ous matter, are, in short, the outcome of arrangement of thai matter with the necessary liberation or storing up of energy " | p. 225 }. Accord- ingly, life is "a state of persistent and incomplete recurrent satisfaction and dissatisfaction of . . . certain proteidogenous molecules" flQOS Vol. I, p. r>5). Pictet (1918) also associates the phenomena of life with a Bpecial structure of the organic molecule. Only the arrangement of t he a toms in open chains, he asserts, permits the manifestation of life and its main- tenance; the cyclic structure is that of substances which have losl this faculty; and death results, from the chemical point of view, from a cyclization of the elements of the protoplasm. To the theory that the vital processes are bound up with a special form of protein or protein-like molecule many have objected. For example, Hober (1911) has contended that there are present in the organism only those kinds of proteins which may be formed in the laboratory. He urges that life should not be thought of as a single process, or as dependent upon any particular kind of molecule, but rather that it should be looked upon as the result of many correlated process occurring between many substances under certain conditions. "If we accept this idea," says Child (1915, p. 19), "we must abandon the assumption of a living substance in the sense of a definite chemical compound. Life is a complex of dynamic processes occurring in a certain field or substratum. Protoplasm, instead of being a peculiar living substance with a peculiar complex morphological structure necessary for life, is on the one hand a colloidal product of the chemical reactions, and on the other hand a substratum in which the reactions occur and which influences their course and character both physically and chemically. In short, the organism is a physico-chemical system of a certain kind.'' Harper (1919) is also opposed to theories based upon the conception of protoplasm as a single complex chemical substance, as well as to those which hold protoplasm to be a relatively simple two-phase colloidal system— the alveolar and granular theories, for example. "The crude simplicity and general inadequacy of these . . . concept inn- have done much to bring the whole subject of protoplasmic organization into disrepute. On the other hand the conception of protoplasm as an aggregate of complex compounds, a polyphase colloidal system or system of systems, seems to do much more adequate justice to the observed facts." Conclusion. As stated at the opening of the present chapter it is with protoplasm that the phenomena of life, in so far a- we know them, are invariably associated. The complex behavior of the living organism 52 INTRODUCTION TO CYTOLOGY can receive scientific explanation (i.e., be fitted into an orderly scheme of antecedents and consequents), if at all, only on the basis of the constitution and properties of the materials composing protoplasm; the structural organization of protoplasm; the relation of the reactions and n-sponses of protoplasm in the form of organized units or cells to the environmental conditions; the chain of energy changes occurring in connection with all of the organism's activities; and the correlation of all these conditions and events. It is largely the effort to account for organization and regulatory correlation, and the consequent behavior of the complex organism as a versatile and consistent unit or individual — as something more than a cell aggregate — that has led to certain present day vitalistic theories, as opposed to those which would hold life to be dependent upon "nothing but': the correlated physico-chemical reac- tions and interactions occurring in protoplasm. Whatever our ultimate judgment in this matter shall be — for any decision at present is premature — it is scarcely to be denied that the hypotheses that have thus far been most stimulating to research in biological science and most valuable in analysing the data afforded by this research are those which seek to formulate vital activity in terms of what the physicist for convenience calls matter and energy; and which hold life to be not the manifestation of a super-organic, non-perceptual entity, or even of a distinct perceptual but hypothetical vital energy, but rather the resultant of the many correlated interactions involving only energies of known kinds. The way must not be closed, however, against possible new categories of energy. The description (reduction to order) of our perceptual experience of organic nature, which is the primary task of biological science and which has been scarcely more than begun, must for the present be made as far as possible in terms applicable also to inorganic nature. It is here that achieved results would seem to justify the judicious use of a "mechanistic" working hypothesis, whereby the attempt is made to "describe the changes in organic phenomena by the same conceptual shorthand of notation as suffices to describe inor- ganic phenomena '! (Pearson). To what extent our ultimate biological theory is to show the need of non-mechanical energies or principles will depend very largely upon what this scientific description (orderly formu- lation) turns out to be like as investigation proceeds, and also upon the degree of success with which the physicist will resume the phenomena of inorganic nature in mechanical formulae. Thus, as Professor D'Arcy W. Thompson forcefully says : "While we keep an open mind on this question of vitalism, or while we lean, as so many of us now do, or even cling with a great yearning, to the belief that something other than the physicalforces animates the dust of which we are made, it is rather the business of the philosopher than of the biologist, or of the biologist only when he has served his humble and severe apprenticeship to philosophy, PROTOPLASM 53 to deal with the ultimate problem. It is the plain bounded duty of the bioloig to pursue his course unprejudiced by vitalistic hypotheses, along the road of observation and experiment, according to the accepted discipline of the natural and physical sciences. . . It is an elementary scientific duty, it is a rule thai Kant himself laid down, that we should explain, just as far as we possibly can, all that is capable of such explanation, in the light of the properi tea of matter and of the forms of energy with which we are already acquainted." (Presidential address before the Zoological Sectionof the British Association for the Advance- ment of Science, 1911.) Bibliography 3 Protoplasm Adami, J. G. 1908. Principles of Pathology. 1st ed. 1918. Medical Contributions to the Study of Evolution. London. Altmann, R. 1886. Studien iiber die Zelle. I. Leipzig. 1887. Die Genese der Zellen. Leipzig. 1889. Die Struktur des Zellkerns. Arch. Anat. u. Physiol, p. 109-411. 1890, 1894. Die Elementarorganismen und ihre Beziehung zu den Zellen. Leipzig. 1892. Ueber Kernstruktur und Netzstrukturen. Arch. Anat. u. Physiol. 223-230. 1893. Die Granulartheorie und ihre Kritik. Ibid. p. 55-66. Bancroft, W. D. 1913. The theory of emulsification. Jour. Phys. Chem. 17: 501-520. 1914. The theory of colloid chemistry. Ibid. 18: 549-558. Barber, M. A. 1911a. A technic for the inoculation of bacetria and other sub- stances into living cells. Jour. Infect. Diseases 8: 348-360. 19116. 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(Prelim, note.) 1900. The genesis and development of the wall and connecting threads of the plant cell (Prelim. Comm.) Ibid 66. 186-188. Gardiner, W. and Hill, A. W. 1901. The histology of the cell wall with special reference to the mode of connection of cells. Phil. Trans. Roy. Soc. London B 194: 83-125. pis. 31-35. Gatenby, J. B. 1919. Identification of intra-cellular elements. Jour. Roy. Micr. So,-. London 2: 93-119. figs. 14. 1920. The cytoplasmic inclusions of the germ-cells. VII. The modern technique of cytology. Qiiar. .lour. Micr. Sci. X. S. 64: 267-302. Glaser, O. 1916. The basis of individuality in organisms. Science 44: 219-224. PROTOPLASM •)•) Goroschankin, J. lN. 1903. The rote of diffusion and osmotic pressure in plants. ( ihicago. Lloyd, F. E. 1915. The behavior of protoplasm as a colloidal complex. Yearbook Carnegie Inst. Washington 14: 66-69. Macdougal, D. T. 1920. Hydration and Growth. Carnegie Inst, of Wash., Publ. 297. pp. 176. Macdougal, D. T. and Spcehr, H. A. 192D. The components and colloidal be- havior of plant protoplasm. Proc. Am. Phil. Soc. 59: 150-170. Macfarlaxe, J. M. 1892. Contributions to the history of Dioncea muscipula Ellis. Contrib. Bot. Lab. Univ. Pa. 1: 7-44. pi. 4. Malfatti, H. 1891-1892. Zur Chemie der Zellkerns. Ber. Naturwiss. Ver. zu Innsbruck 20. Manx, G. 1906. Protoplasm, its definition, chemistry, and structure. Oxford. Martin. 1881. Zur Kenntniss der indirekten Kerntheilung. Arch. f. Path. Anat. u. Physiol. 86: 57-67. pi. 4. M assee, G. 1884. On the formation and growth of cells in the genus Polysiphonia. Jour. Roy. Micr. Soc. II 4: 198-200. pi. 6. Mathews, A. P. 1916. Physiological Chemistry. Me yer, A. 1896. Die Plasmaverbindungen unci die Membranen von Volvox globator, usw. Bot. Zeit. 54: 187-215. pi. 8. Miescher, F. 1871. Ueber die chemische Zusammensetzung der Eiterzellen. Med.-Chem. TJnters. Lab. ausgew. Chem. zu Tubingen 4. Berlin. 1874. Die Spermatozoen einiger Wirbelthiere. Verhandl. d. Naturforsch. Gesell. in Basel 6. Mixchin, E. A. 1912. An Introduction to the Study of the Protozoa. London. Moore, B. 1912. The Origin and Nature of Life. New York. Oliver, F. W. 1888. On the sensitive labellum of Masdevallia muscosa Rchb. f. Ann. Bot. 1: 237-253. pi. 12. Overton, E. 1889. Ein Beitrag zur Kenntniss der Gattung Volvox. Bot. Centr. 39: 65, 113, 145, 177,' 209, 241, 273. pis. 1-4. 1895. Ueber die osmotischen Eigenschaften der lebenden Pflanzen- und Thierzelle. Vierteljahrsschr. Nat. Ges. Zurich 40: 159-184. 1899. Ueber die allgemeinen osmotischen Eigenschaften der Zelle, ihre vermuth- lichen Ursachen und ihre Bedeutung fur die Physiologic Ibid. 44: 85-135. 1900. Studien iiber die Aufnahme der Anilinfarben durch die lebende Zelle. Jahrb. Wiss. Bot. 34: 669-701. See Pfliiger's Archiv 92. Palladix, V. I. 1918. Plant Physiology. (Engl, transl., ed. by Livingston.) Pearsox, K. 1892. The Grammar of Science. Pictet, A. 1919. Life and the structure of molecules. (Geneva Arch. Phys. et Xat. Sci.) Engl. Trans, in Sci. American Suppl. 87 : 50-51, 59. Pfeffer, W. 1885. Zur Kenntniss der Kontaktreize. Unters. Bot. Inst. Tubingen 1: 483-535. fig. 1. 1890. Zur Kenntniss der Plasmahaut und der Vacuolen, nebst Bemerkungen .iiber den Aggregatzustand der Protoplasmas und iiber osmotische Vorgange. Abh. Math.-Phys. kl., Sachs. Ges. Wiss. 16. 1896. Ueber den Einfluss des Zellkerns auf die Bildung der Zellhaut. Pfitzxer, W. 1883. Beitrage zur Lehre vom Baue des Zellkerns und seinen Theilungserscheinungen. Arch. Mikr. Anat. 22: 616-688. pi. 25. Prixgsheim, N. 1854. Untersuchungen iiber Bau und Bildung der Pflanzenzelle. Berlin. Reichert, E. T. 1914. The germ plasm as a stereochemic system. Science 40: 649-661. PROTOPLASM 57 Reinke und Rodewald. 1881. Die chemische Zusammensetzung ilea Protoplaan von Mthalium septicum. Unters. Bot. Lab. Gottingen. Reinke, F. 1882. Protoplasma-Probleme. Ibid. 1883. Ein Beitrag zur physiologischen Chemie von Mthalium septicum. Ibid. Robertson, T. B. 1920. Principles of Biochemistry. Philadelphia and New York. Rosenvinge, L. K. 1888. Sur la formation des pores secondairea chez lea Polysi- phonia. Bot. Tidakr. 17: 10. Roskine, G. 1917. La structure des myonemea. Compt. Rend. Soc. Biol. Paria 69: 363-364. Huhland, W. 1909. Beitrage zur Kenntniss der Permeabilita.1 der Plasmahaut. Jahrb. Wiss. Bot, 46: 1-54. figs. 2. 1915. Zur Kritik der Lipoid- und Ultrafiltertheorie der Pla8mahaut, uaw. Biochem. Zeitschr. 54: 59-77. Russow. 1883. Ueber die Perforation der Zellwand und den Zusammenhang der Protoplasmakorper benachbarten Zellen. Sitzber. Naturfor. Gea. Univ. Dorpal 6:5G2. von Sachs, J. 1892. Physiologische Notizen. II. Flora 75: 57-67. 1895. Physiologische Notizen. IX. Weitere Betrachtungen iiber Energiden und Zellen. Ibid. 81: 405-434. (See Wilson 1900, p. 30.) Sch.effer, A. A. 1920. Ameboid Movement. Princeton Univ. Press. Schneider, C. 1891. Untersuchungen iiber die Zelle. Arb. Zool. Inst. Wien 9: 179. Schwarz, F. 1887. Die morphologische und physiologische Zusammensetzung des Protoplasmas. (Rev. in Bot. Zeit. 45 : 576-583.) Seifriz, W. 1918. Observations on the structure of protoplasm by aid of microdis- section. Biol. Bull. 34 : 307-424. figs. 3. (Bibliography.) 1920. Viscosity values of protoplasm as determined by microdissection. Bot. Gaz. 70 : 360-386. Spaeth, R. A. 1916. The vital equilibrium. Science 43: 502-509. Spcehr, H. A. 1919. The carbohydrate economy of cacti. Carnegie [nat. of Wash., Publ. 287. pp. 79. Strasburger, E. 1882. Ueber den Bau und das Wachsthum der Zellhaute. Jena. 1892. Schwarmsporen, Gameten, pflanzliche Spermatozoiden und das We8en der Befruchtung. Histol. Beitr. 4. 1901. Ueber Plasmaverbindungen pflanzlicher Zellen. Jahrb. Wiss. Bot. 36: 493-610. pis. 14, 15. Tangl, E. 1879. Ueber offene Communicationen zwiachen den Zellen des Endo- sperms einiger Samen. Ibid. 12: 170-190. pis. 4-6. Terletzki, P. 1884. Anatomie der Vegetationsorgane von StruthiopU ris gi rmanica und Pteris aquilina. Jahrb. Wiss. Bot, 15: 452-501. pis. 24 26. Unna, P. 1895. Ueber die neueren Protoplaamatheorien, und daa Spongioplasma. Deu. Med. Zeit. 1895. p. 98. Velten, W. 1876. Die phyaikaliache Beachaffenheit dea pflanzlichen Protoplaamas. Sitzber. Akad. Wiss. Wien, Math.-Nat. Kl., 73: 1 131 151. Verworn, M. 1895. Allgemeine Phyaiologie. Jena. 1903. Die Biogenhypotheae. Jena. de Vries, H. 1885. Plaamolytiache Studien iiber die Wand der Vacuolen. Jahrb. Wiss. Bot. 16 : 465-598. Wells, H. G. 1914. Chemical Pathology. 2d edition. Philadelphia. Chapter I. W'ille, N. 1883. Ueber die Zellkerne und die Poren der Wande bei den Phyco- chromaceen. Ber. Deu. Bot. Ge8. 1: 243-246. :»s INTRODUCTION TO CYTOLOGY Wilson, E. B. L899. On protoplasmic structure, in the eggs of echinoderms and some other animals. Jour. Morph. 15: Suppl. 1-23. W ortmann, J. 1887. Zur Kenntniss der Reizbewegungen. Bot Zeit 45-785 801, 817, 833. 1889. Ueber die Beziehungen der Reizbewegungen wachsender Organe zu den normalen AVachsthumerscheinungen. Ibid. 47: 453, 469, 485. Zachamas, E. 1881-1893. Ueber die chemische Beschaffenheit des Zellkerns. Bot. Zeit. 39: 169. Ueber den Zellkern. Ibid. 40: 611. Ueber Eiweiss, Nuclein, imd Flastin. Ibid. 41: 209. Ueber den Nukleolus. Ibid. 43: 257. Beitrage zur Kenntniss des Zellkerns and der Sexualzellen. Ibid. 45 : 281. Ueber Chroma- tophilie. Ber. Deu. Bot. Ges. 11: 188-195. Zimmermann, A. 1893. Sammel-Referate 2, 3. Beih. Bot. Centr 3: 209-217 321-328. CHAPTER EV THE NUCLEUS It is now half a century since the modern period of cytology was ushered in by a series of researches revealing the remarkable behavior of the nucleus during the critical stages of the life cycle Because of the peculiarly intimate relation which this behavior has been shown to have to many outstanding biological problems, including thai of heredity, it is largely in nuclear phenomena that cytological interesl has con- tinued to center throughout the period. The most striking of th< phenomena form the subjects of several subsequent chapters: at this point we shall consider the nucleus only as it appears in the "resting' cell, i.e., in the cell not undergoing division. Occurrence. — The most conspicuous, and in some respects the mosl important of the cell organs is the nucleus. Whether or not we shall Bay that every living cell contains a nucleus will depend upon what we are to include under the term. If the chromatin or chromatin-like substances, no matter whether distributed throughout the cell in the form of granules or aggregated to form a well defined organ, be regarded as constituting a nucleus, then it follows that all plant and animal cells normally have nuclei. If, however, as certain protozoologists prefer, the term nucleus be employed only with reference to a distinctly delimited organ, we must regard those lowly organized cells with scattered chromatic material as devoid of nuclei, although they possess, as all cells apparently do, material which performs at least the nutritive functions of a nucleus. This latter type of organization, which is found in certain members of the Protozoa and Bacteria, and also some Cyanophyceae, will be discussed later on in connection with nuclear structure (p. 66) and cell-division (Chapter X In myxomycetes, where simple and primitive conditions mighl be expected, Jahn (fl)08, 1911) and Olive (1907) have demonstrated the presence of definite nuclei showing mitosis and the phenomenon of chromosome reduction. General Characters. --The vast majority of cells have one nucleus each. A few exceptions may be noted. In tapetal cells, laticiferous ves- sels, the internodal cells of Characeae, and certain other cells there are often several nuclei arising by the division of one. In the Siphone® among green algse (Fig. 14, V) and the Phycomycetes among fungi there arc no cross walls in the filamentous and much branched vegetative body, SO that 59 60 IXTRODUCTION TO CYTOLOGY large numbers of nuclei are associated in one extensive mass of cyto- plasm. Such a body is called a ccenocyte, and the coenocytic condition is found in a number of the lower organisms. In the Uredinese (rusts) the typical life history is made up of two phases, with uninucleate and binucleate cells respectively. In certain Infusoria two kinds of nuclei are regularly present. Thus in Par amcecium caudatum (Fig. 15) there is one small micronucleus which divides by a peculiar form of mitosis, and one large meganucleus (macronucleus) which divides amitotically. In P. aurelia there are two micronuclei and one meganucleus, whereas in Fig. 14. V, portion of body of Vaucheria, showing coenocytic condition; nuclei dark and plastids in outline. C, portion of body of Cladophora, showing semi-coenocytic condition. Fig. 15. — Par amcecium caudatum un- dergoing fission; mega- and micronuclei dividing. {From Minchin, after Biitsehli and Schewiakoff.) Stentor there may be one meganucleus and several micronuclei. In general the meganucleus seems to be a storage organ of the cell; it may disappear and be replaced by a new one. The micronucleus performs the usual nuclear functions. The mammalian red blood corpuscle begins its life as a nucleated cell, but later on the nucleus is lost. The position of the nucleus in the cell is determined largely by physical causes, such as surface tension, the position of the vacuoles, and the relative density of the cytoplasm in different portions of the cell. In a non-vacuolated cell it ordinarily occupies the center of the cytoplasmic mass, whereas in a cell with vacuoles it is imbedded in the cytoplasm even when the latter is reduced to a thin parietal layer; it never lies free in the vacuole. In the Cladophora ceae (Carter 1919) it is regularly imbedded, 77/ A' NUCLEUS 61 Fig. 16. at least partially, in the chloroplast, and this is true even in cells pog i - ing a considerable amount of colorless cytoplasm. lis position is also related to the functions of the cell: in general it lies in the region character- ized by the most active metabolism. For example, in young growing root hairs (Fig. 16, B) and pollen tubes it is commonly found where elongation is taking place, and in thickening epidermal A The thickening of cells (Fig. 16, A) it frequently, though the inner wall of an epi- . ,. .i n dermal cell of Scopolia not always, lies near the wall upon atropoides. H origin of which the thickening material is being root hairs in Pisum sativum. deposited. This relation of position to ' fter a l ' function was emphasized in the works of Haberlandt (1887) and Gerassimow (1890, 1899, 1901). In form the nucleus is typically spherical or ellipsoidal, its shape being determined by a number of physical factors. Under comparatively uniform conditions, as obtain where a small nucleus lies in a relatively large amount of non-vacuolated cytoplasm, a spherical, shape is assumed because of the phenomena of surface tension. Exceptions are often seen in cells with specialized functions. Fig. 17. — Unusual forms of nuclei. A, portion of nucleus from spinning gland of Vanessa urticce, Bhowing irregular form and finely divided state of the chromatin. {After Korschelt, L896.) />. Spirostomum ambiguum, with moniliform nucleus. (After Shin.) C, Nucleus from Balivary gland of Chironomus: the chromatic material exists as a series of discs in a convoluted thread, which ends in two nucleoli. {After Balbiani, 1881.) D, Choenia teres, with chromatic granules scattered throughout the body. (After Oruber, 1884.) /••'. Nucleus from root tip of Afar- silia, showing concentration of chromatic material in the nucleolus. (After Berghs, 1909. In the cells of the spinning glands of Pieris and Vanessa (butterflies the physiological conditions result in the assumption of very irregular forms whereby the nuclear surface is considerably increased (Fig. 17, A). Nuclei seem rather commonly to undergo amoeboid changes in shape; 62 INTRODUCTION TO CYTOLOGY such active movement can be directly observed in the nucleus of the living cycad spermatozoid. In the long, narrow cells of vascular bundles the nuclei, which arc not free to grow in all dimensions, come to be correspondingly elongated. The nucleus may also be passivehT forced into very irregular shapes by the dense accumulation of starch grains and the diminution in the amount of cytoplasm, as in the endosperm cells of maize. Tn Stentor and Spirostomuni the nucleus has the form of a string of beads (Fig. 17, B). In size the nucleus shows a wide variation, ranging in plants from the extremely minute nucleus of Mucor, 1/j, or less in diameter, to the relatively gigantic nucleus of the Dioon egg, with a diameter of 600//,. A similar range is seen in animal nuclei. Although the nuclei of the fungi arc characterized by small size, most of them being less than o/j. in diameter, they may grow to a large size at certain stages. The primary nucleus of Synchytrium, for instance, reaches a diameter of over 60//.. The majority of nuclei, however, fall between 5m and 25ju. In spite of the wide range in the size of nuclei of different organisms, in a given tissue it is comparatively uniform. With respect to the physical nature of the nucleus as a whole, the researches of Kite (1913) and Chambers (1914, 1917) have shown that it ordinarily consists at least in part of a gel of higher viscosity than the cytoplasm, often being so firm that it can easily be handled without injury by means of the dissecting instrument. This obviously would be impossible were the nucleus merely a watery droplet or vesicle in the cyto- plasm. The germinal vesicle (nucleus) of the animal egg Chambers (1917) finds to be a sol droplet with a gel membrane; if it is pinched in two by the dissecting instrument the two halves will reunite if they come in contact. The chemical nature of the nucleus has been dealt with in the preced- ing chapter. With regard to its electrical properties, the nucleus is apparently negative to the cytoplasm. R. S. Lillie (1903) found that free nuclei and the heads of spermatozoa, which are almost entirely nuclear material, pass to the anode in an isotonic cane sugar solution, whereas cells rich in cytoplasm, such as large leucocytes, pass to the cathode. These results have been confirmed by Hardy (1913). Nucleoplasmic Ratio. — Of more importance than the absolute size of the nucleus is the relation of its volume to that of the cytoplasm — the so-called nucleoplasmic or Kernplasma relation. Many years ago it was held by Sachs (1892, 1893, 1895) and by Strasburger (1893) that the size of a meristematic cell in a plant, owing to a supposed limitation of the sphere of influence of the nucleus, maintains a very definite relation to the size of its nucleus. This conception has recently been emphasized anew by Winkler (1916), and parallel views have been expressed by several zoologists (e.g., Hegner on Arcella, 1919). In the case of certain Til E NUCLEUS terminal meristems of plants such a rule may well hold true within limits, but the condition reported by Bailey (1920) in the lateral meristem (cambium) shows clearly Unit it cannot have universal application. The cambial initials may vary enormously in size with no corresponding variation in the size of 1 heir nuclei: two such initials, one of them having many hundreds of times the volume of the other, may possess nuclei of approximately equal size. The nucleoplasmic ratio has figured prominently in discussions of the problem of senescence. R. Hertwig in 1889 advanced the theory that senescence and natural death are associated with an increase in the relative size of the nucleus. He later asserted (1903, 1908) that the nucleo- plasmic relation is self-regulatory within certain limits for each kind of cell, exercising thereby a control over many cell activities, including cell- division. Minot (1891, 1908, 1913), on the contrary, believed that the increase in the relative volume of the cytoplasm, in addition to its differ- entiation, is a fundamental factor in senescence and death. Conklio (1912), as a result of his work on Crepidula, denied the existence of a constant and self-regulatory nucleoplasmic relation, holding rather that changes in this relation are not causes of such cell activities as cell- division, but are results of the metabolic processes by which such cell activities are brought about. Child (1915) points out that in most animal tissues there is an increase in the relative amount of cytoplasm during senescence, whereas in plants, although the cell enlarges through vacuolation, the relative volume of cytoplasm often does not increase. He therefore concludes that the nucleoplasmic relation cannot be regarded as a universal factor in senescence; it is rather an indication of the kind and rate of metabolism. The differentiation of the cytoplasm, apart from its mere change in volume, Child, with many other workers < Minot . Delage, Jennings, etc.), regards as a matter of the greatest importance in senescence. Further discussion of this subject is deferred to ( )hapter VII. Not only has it been held that there is a certain relation between the mass of the nucleus and that of the cytoplasm, whatever the significance of this relation may be, but there also seems to be a size relationship between the nucleus and its contained chromosomes. In L896 Boveri showed that the size of the nuclei in merogonic echinoderm larvse (s p. 325) is dependent upon the number of chromosomes each contain-. In a more extended study (1905) he demonstrated that it is the surface of the nucleus that is proportional to the chromosome number, and also that the size of the cell is proportional to both. ( rates 1 190!)), however, adduced evidence to show that this rule is by no means universal. Structure. — Having reviewed the general features of the nucleus as a whole, we may next give attention to its structure, as -ecu in typical cases. The nucleus is bounded by a distinct nuclear membraru . The nature 64 INTRODUCTION TO CYTOLOGY of this membrane has been a subject of much controversy. Some have regarded it as a precipitation membrane laid down when the newly formed karyolymph comes in contact with the cytoplasm at the time the daughter nuclei are reconstructed during the closing phases of mitosis, while others (Lawson 1903) have interpreted it as merely a denser limit- ing layer of the -cytoplasm. The above cited work of Kite and Chambers, however, leaves no doubt that the membrane is a definite morphological structure belonging to the nucleus: although it is at times very delicate, it remains intact when the nucleus is pushed and pulled about by the dissecting instrument, and is thrown into folds when the karyolymph is withdrawn with a pipette. Within the nuclear membrane is a series of gels of varying consistency. The nuclear sap, or karyolymph, is a highly transparent substance which is generally looked upon as homogeneous, although it has been thought by some workers (Reinke 1894) to be made up of large, pale "cedamatin granules." It may be in the sol or gel state. Imbedded in the karyo- lymph is a network or reticulum, which may be relatively uniform through- out the nucleus or only fragmentary and incomplete. It is usually said to be composed of a gel substance known as achromatin (Flemming 1879) or linin (Schwarz 1887). Supported on the linin reticulum is the chroma- tin (Flemming 1879). This highly stainable substance may exist in the form of small granules or droplets at the nodes of the reticulum, or apparently in many nuclei as a fluid thin enough to distribute itself more or less uniformly throughout the achromatic substance. In the latter case the whole reticulum appears to be composed of a single unevenly stained material, careful examination showing the " chromatic granules'1 and " achromatic support " to be its thicker and finer portions respectively (Fig. 51) (Gregoire and Wyagerts 1903; Gregoire 1906; Sharp 1913, 1920). According to Kite (1913) the granules in the living nucleus con- sist of a very concentrated gel, the supporting reticulum of a somewhat more dilute but not at all fibrous gel, and the karyolymph of a gel which is the most dilute of all. Heidenhain (1894) found imbedded in the colorless linin net two sorts of chromatin in the form of granules: oxychromatin, consisting largely of plastin, poor in phosphorus, and staining with the acid dyes; and basi- chromatin, composed mainly of nuclein, rich in phosphorus, and staining with the basic dyes. These two forms of chromatin apparently may change into each other by the addition or loss of phosphorus. The peri- odic changes in the staining reactions of many nuclei therefore indicate changes in the chemical composition of the chromatin, and these in turn point to the intimate association of the nucleus with the periodic physi- ological processes of the cell. As used by many writers the term oxy- chromatin includes also the linin, so that in much cytological literature linin and oxychromatin are more or less interchangeable terms, while THE NUCLEUS 65 "chromatin" refers to the basichromatin. Oxychromatin appears to be closely similar in composition to the achromatic structures in the cyto- plasm, such as spindle4 fibers and centrosomes. The prominent place occupied by the nucleus in cytology is due in large measure to the con- spicuous behavior of its chromatic substance at the time of cell-divisioo and fertilization, topics which are to receive detailed consideration in subsequent chapters. In many nuclei basichromatin accumulates at certain points in the reticulum, forming karyosomes, also called "net knots' and chromatin nucleoli. These seem to be masses of surplus chromatin elaborated by the nucleus during the resting phase or in some cases chromatin which has flowed to these points from the other parts of the reticulum. During the next mitosis they are distributed with the rest of the chromatin, ks Rosen (1892) long ago showed by his studies of their staining reactions, they differ decidedly in composition from true nucleoli, although they may closely resemble the latter after treatment with certain stains (iron- alum-hsematoxylin) . One or more true nucleoli, or plasmosomes (Ogata 1883), are usually present in the nucleus. A single nucleolus is probably characteristic of most nuclei; there are rarely many, and in some cases there is none. The nucleolus may be in close organic connection with the nuclear reticulum or it may lie entirely apart from it. In composition it consists largely of such oxychromatic substances as plastin and pyrenin, or of nuclei n well saturated with protein (Zacharias). It usually stains with the acid dyes: by a proper selection of stains it may, therefore, be distinguished from the karyosomes, which, being composed of basichromatin, take the basic dyes as a general rule. In structure the nucleolus may appear to be homogeneous throughout, like an oil globule; in other cases it has an outer envelope of different consistency and staining reaction. Very often vacuoles, occasionally containing granules, are presenl in the interior. Crystalloid bodies are also frequently observed in the nucleolus (Digby on Galtonia, 1910; Reed on Allium, 1914; Kuwada on Zca , 1919). In the epithelial cells of the frog intestine Carleton (1920) finds one or more intranucleolar bodies which he calls "nucleolini." These appear to divide and pass to the daughter cells at the time of mitosis, and may possibly initiate the formation of new nucleoli in the daughter nuclei, Montgomery (1899)1 concluded that the nucleolus grows in size by the apposition of smaller particles of nucleolar material on its surface, and by the intussusception of vacuolar substance arising outside the nucleolus. Function of Nucleolus. — Various opinions have been entertained re- garding the function of the nucleolus. By many workers it has been looked upon as chiefly a passive by-produd of no furl her use in the life 1 An exhaustive review of the literature dealing with the nucleolus up to 1890 is given in this paper. 5 66 INTRODUCTION TO CYTOLOGY of the cell (Haecker). Strasburger (1895, 1897), who observed the dis- appearance of the nucleolus at about the time the spindle fibers appear during the prophases of mitosis, concluded that it is a mass of reserve kinoplasm which gives rise indirectly to the achromatic figure. While some have agreed in the main with this conclusion, many have denied the relationship of nucleolus and spindle, contending that the former is rather a reserve constituent for the linin reticulum (Eisen 1900) or the chromatin (Schurhoff 1918). Frequently the bulk of the basichrom- atic material of the nucleus is lodged in the nucleolus at certain stages. In the somatic nuclei of Marsilia (Fig. 17, E), for example, Berghs (1909) shows that it is transferred to the nucleolus during the telophases of mitosis, and returned to the reticulum in the following prophases. This phenomenon, which has an important bearing on the role of the chrom- atin and the individuality of the chromosomes, will be referred to again in Chapter VIII. In many cells, as shown by the work of the zoologists the nucleolus appears to be concerned in the elaboration of secretion and storage prod- utts. In the eggs of certain animals Macallum (1890) showed that the nucleolar material, which appears to differentiate from the chrom- atin, passes into the cytoplasm and there combines with another substance to form the yolk globules. In the cells of the pancreas he further found that material often present in the form of nucleoli func- tions in a similar manner in the production of zymogen. Many other observations of this general nature have been reported. In the silk- gland cells of certain insects it has recently been shown by Nakahara (1917) that some of the nucleoli, which may originally be passive by- products, later pass into the cytoplasm and contribute to the formation of the secretion products. An extreme view of the importance of the nucleolus is that of Derschau (1914), who regards the nucleolus as the real center of the life of the cell. Granules of oxychromatin, he asserts, pass out from the nucleolus through the cytoplasm in the form of chon- driosomes, carrying basichromatin as a building material to the places where it is required. It is highly probable that the nucleolus has various functions in dif- ferent cells, but in general we majr conclude that it is a mass of accumu- lated material which is usually, though not always, utilized in the metabolic processes of the nucleus The Nuclei of Bacteria and Other Protista. — The question of the nucleus in bacteria is one that it appears to be particularly difficult to settle satisfactorily. This is due not only to the minute size of these 'organisms, which makes special methods necessary and observation very difficult, but also to the fact that a variety of conditions seems to be present in the group. That the bacterial cell is devoid of a nucleus has been held by several investigators including Fischer (1894, 1897, 1899, 77/ A' NUCLEI S 67 1903), who looked upon the observed granules as reserve materials rather than nuclear substance. Migula (1894, 1897, L904) regarded the existence of nuclei in bacteria as very doubtful. The majority of workers, on the contrary, have held thai a nucleus or at leasl nuclear material is present in some form. The most striking view is thai which regards the whole cell in some cases as a uaked nucleus (Htippe L886; Zettnow L891, 1897, 1899; Ruzicka 1908, 1909; and, in the case of small bacteria, Biitschli 1890, 1892, 1896, 1902). The evidence advanced in supporl of this hypothesis, however is of very doubtful value. In many bacteria, particularly the larger forms, there is presenl ;i granular substance which has certain characteristics of chromatin, and which in some species exists as a single well defined mass. The "central body" of the sulphur bacterium Btitschh regarded as the homologue of a nucleus, the peripheral portion of the cell being cytoplasm. In a careful study of the entire life cycle of Bacillus Biltschlii Schaudin I 1902) found that, the chromatic material present during most of the cycle as chromidia unites at certain stages to form peculiar spiral figures; in the spores it takes the form of dense masses. Such scattered chromidia and 'spiral filament nuclei" were also observed by Guilliermond (1908, 1909), who has given a review of thesubject (1907). Nakanishi (1901 ), who employed both intra-vitam methods and fixed material, reported the presence of nuclei in the vegetative cells and spores of a number of speci< 3. The nucleus of the large Bacterium gammari was studied by Vejdow- sky (1900), who in 1904 described its division by mitosis. Mend L904, 1905, 1907, 1909) demonstrated by careful methods the nuclei in many species and also reported mitotic division in Bacterium gammari. I )oub1 concerning the systematic position of this form, however, has been raised by some investigators, who think it not improbable that it is a yeast- like fungus rather than a bacterium. Dobell (1908, 1909, 1911), whose review of the subject has been of service in the preparation of this summary, has studied with much care many species of bacteria in their natural culture media. His conclusions are summarized in the following quotation (1911): "All bacteria which have been adequately investigated are like all other Protista — nucleate colls. "The form of the nucleus is variable, not only in different bacteria, but also at different periods in the life cycle of the same species. "The nucleus may be in the form of a discrete system of granules (chromidia); in the form of a filament of various configuration; in the form of one or more relatively large aggregated masses of auclear sub- stance; in the form of a system of irregularly branched or bent short strands, rods, or networks and probably also in the vesicular form char- acteristic of the nuclei of many animals, plants, and protists. "There is no evidence that enucleate bacteria exist." 68 INTRODUCTION TO CYTOLOGY The apparent discrepancy between this view of bacterial organization and that of Minchin, stated below, will be seen to be largely a matter of terminology. It is therefore among the Proitsta that the widest departures from the usual type of nuclear structure are found, certain of them in all prob- ability representing relatively primitive stages in the evolution of the true nucleus. Such an interpretation is evidently to be placed upon the "distributed nuclei1 seen in certain bacteria, protozoans, flagellates, and Cyanophycere (p. 202), which consist of granules of a material akin to chromatin scattered throughout the cell, sometimes with a limiting membrane of some sort but often with none. It is doubtful if granules scattered with no definite limitations throughout the cell, as in Chcenia teres (Fig. 17, D) or Chroococcus turgidus (Fig. 72, A), should be spoken of collectively as a nucleus. As pointed out at the beginning of this chapter, it seems preferable to certain workers to limit the term to those chromatic aggregations which actually have the characters of a definitely localized organ. In discussing the advisability of so restricting the appli- cation of the term Minchin (1912, Chapter VI) points out that "the word ' chromatin' connotes an essentially physiological and biological con- ception . . . of a substance, far from uniform in its chemical nature, which has certain definite relations to the life history and vital activities of the cell. The word 'nucleus,' on the other hand . . . is essentially a morphological conception, as of a body, contained in the cell, which exhibits a structure and organization of a certain complexity, and in which the essential constituents, the chromatin particles, are distributed, lodged, and maintained, in the midst of achromatinic elements which exhibit an organized arranegment, variable in different species, but more or less constant in the corresponding phases of the same species." Ac- cording to this interpretation the term "nucleus" would not be applicable to a mass of granules (chromidia) scattered throughout the cell. Minchin states further that " . . .as soon as a mass or a number of particles of chromatin begin to concentrate and separate themselves from the sur- rounding protoplasm, with formation of distinct nuclear sap and ap- pearance of achromatinic supporting elements, we have the beginning at least of that definite organization and structural complexity which is the criterion of a nucleus as distinguished from a chromidial mass." Those Protista of the lower (bacterial) grade, in which there are only scattered grains of chromatic material, are looked upon as " non-cellular '! in organization by Minchin, who believes that from such a primitive state the "strictly cellular grade of organization has been evolved by concentration of some or all of the chromatin to form a nucleus." In its simplest condition such a nucleus consists of one or more chromatin granules in a sort of vacuole, and is known as a " protokaryon." In other cases the chromatin forms a single large mass at the center of the nucleus 77/ E NUCLEUS 69 ("vesicular nucleus"). Since "the chromatic particles are the only con- stituents of the cell which maintain persistently and uninterruptedly their existence throughout the whole life cycle of living organisms uni- versally," Minchin (1916) believes that the earliest living things, which he calls " Biococci/' were minute particles of a chromat in-like substance. These were the ancestors of the present chromatin grains and find their nearest modern representatives in certain pathogenic Chlamydozoa. Ac- cording to this view the cytoplasm was differentiated later in the evolu- tion of the cell, whereas the more general view probably is that chromatin and cytoplasm were coexistent as two substances in cells from theearliesl known stages (Wilson).1 The Function of the Nucleus. — It may be said without reservation that the nucleus dominates the morphological and physiological changes in the cell. Although the type of organization formed by a nucleus in combination with cytoplasm is required for the carrying on of cell activity, it is nevertheless evident from a huge mass of accumulated observations that in the nucleus is to be found the center of control for both the func- tional activities and for cell reproduction (cell-division). Many years ago Claude Bernard (1878) pointed out that while the cytoplasm is the seat of destructive metabolism, the nucleus is the seat of constructive metabolism, this physiological role offering "the key to its significance as the organ of development, regeneration, and inheritance " (Wilson). The inability of a cell deprived of its nucleus to carry on synthetic metabolism in any complete manner has often been noted, though such a cell may not perish for some time. The mammalian erythrocyte, for example, loses its nucleus at an early stage and may continue to exist in the enucleate state for from 15 to 30 days (Hunter, Quincke). Klebs found that enucleate cells of Spirogyra may continue for some time to form starch. But such cells are apparently unable to divide or to increase their bulk- by the elaboration of new cell substance. Many ordinary act Lvities, such as cell wall formation (Townsend 1897; Gerassimow 1899, 1901), fail to occur. From such observations it is concluded that the nucleus is acc- essary for the synthetic processes associated with growth and reproduc- tion. This conclusion is supported by the facts of regeneration. The role of the nucleus in regeneration was strikingly shown by the well known experiments of Gruber (1885) and F. H. Li Hie (1896) on SU ><- tor. This unicellular organism, which has a nucleus like a string of beads, may be cut into fragments: any fragment containing a portion of the nucleus has the power of regenerating a complete new animal, whereas enucleate fragments, although they may live for a little time, undergo do regeneration and eventually perish. 'For more complete descriptions of the nuclei of Protista the works of Wilson (1900) and Minchin (1912) should be consulted. The behavior <>f such nuclei at the time of coil-division is briefly described in Chapter X of tliis book. 70 INTRODUCTION TO CYTOLOGY A number of biologists (Gruber 1886, Hertwig 1898, Heidenhain 1894, Henneguy 1896, Conklin 1902) concluded that in general the chromo- somes (basichromatm) are concerned chiefly with differentiation and regulation, while the achromatin (oxychromatin) has to do with metabo- lism (Conklin 1917). Metabolism is in reality a great complex of reactions: the reactions are not independent of one another but are closely correlated, and thus constitute an intricately adjusted reaction system. Among these many reactions, according to modern physiology, the most important is oxidation, for the energy utilized by the organism is derived immediately from the union of protoplasm or of its constituent elements with nwgen. Oxidation has been called the "independent variable' (Loeb and Wasteneys 1911) upon which the other reactions largely depend: oxidation is the dominant factor in cell activity, and it is there- fore of the greatest importance to understand as well as possible the rela- tion of the parts of the cell to this process. Following the experiments of Spitzer (1897), who observed thatnucleo- proteins extracted from certain animal tissues have the same oxidizing power as the tissues themselves, it was advocated by Loeb (1899) that the nucleus is the center of oxidation in the cell. Loeb pointed out that this would explain the inability of enucleated cell-fragments to undergo regeneration. This conclusion was supported by R. S. Lillie (1903), who later (1913) showed that rapid oxidation occurs both at the surface of the cell and at the surface of the nucleus, and also by Mathews (1915). Other workers (Wherry 1913, Schultze 1913, Reed 1915) however, have failed to agree. Osterhout (1917), who briefly summarizes the subject, found that "injury produces in the leaf-cells of the Indian Pipe (Monotropa uni flora) a darkening which is due to oxidation. The oxidation is much more rapid in the nucleus than in the cytoplasm and the facts indicate that this is also the case with the oxidation of the uninjured cell." The role of the nucleus in development and inheritance, which has been a subject of so much discussion in recent years, will be dealt with in later special chapters (XIV-XVIII), after the behavior of the nucleus in somatic cell-division, maturation, and fertilization has been described. Bibliography 4 Nucleus Bailey, I. W. 1920c/. The cambium and its derivative tissues. II. Size variations of cambial initials in gymnosperms and angiosperms. Am. Jour. Bot. 7: 355- 367. figs. 3. 1920b. The cambium and its derivative tissues. III. A reconnaissance of cytolog- ical phenomena in the cambium. Ibid. 7: 417-434. pis. 26-29. Balbiani, E. G. 1881. Sur la structure du noyau des cellules salivaire chez Les larves de Chironomus. Zool. Anz. 4: 637-641. figs. 7. Berghs, J. 1909. Les eineses somatiques dans le Mursilia. La Cellule 25: 73-84. 1 pi. THE NUCLEUS 71 Bernard, C 1878. Lecons sur les phenomenes de la vie. Paris. Boveri, Tn. L895. Ueber die Befruchtungs- und Entwicklungsfahigkcil kern lose i Seeigeleier und iiber die Moglichkeh* ihrer Bastardierung. Arch, t. Entw. 2: 35)4-443. pis. 24, 25. 1905. Zellen-Studien. V. Ueber die Abhangigkeii der Kerngrosse und Zellenzahl der Seeigel-Larven von der Chromosomenzahl der Ausgangszellen. Jena. Butschli, O. 1800. Ueber den Ban der Bakterien und verwandter Organismen. Leipzig. 1892. Untcrsuchungen iiber mikroskopische Schaume und das Protoplasma. Leipzig. 1896. Weitere Ausfuhrungen iiber den Bau der Cyanophyceen und Bakterien. Leipzig. 1902. Bemerkungen iiber Cyanophyceen und Bakterien. Arch. f. Protistenk. 1. Carleton, *H. M. 1920. Observations on the intranucleolar body in columnar epithelium cells of the intestine. Quar. Jour. Micr. Sci. 64: 329 344. pi. 17 Carter, X. 1919. The cytology of the Cladophoraceae. Ann. Bot. 33: 167 178 pi. 27. figs. 2. Chambers, R. 1914, 1917. See Bibliography 3. Child, C. M. 1915. Senescence and Rejuvenescence. Chicago. Cox klin, E. G. 1902. Karyokinesis and Cytokinesis in the maturation, fertilization and cleavage of Crepidula and other Gasteropoda. Jour. Acad. Nut. Sci. Phila. 12: 5-121. pis. 1-0. figs. 33. 1912. Cell size and nuclear size. Jour. Exp. Zool. 12 : 1-98. ii^s. 37. 1913. The size of organisms and of their constituent parts in relation to longevity, senescence, and rejuvenescence. Pop. Sci. Mo. 83: 178-198. 1917. Mitosis and Amitosis. Biol. Bull. 33: 39G-436. pis. 10. (Literature. Delage, Y. 1899a. La fecondation merogonique e1 ses resultats. Comptes rendus Acad. Sci. Paris. 18996. Etudes sur la merogonie. Arch. Zool. Exp. 1117: 383-417. 1899c. Sur Interpretation de la fecondation merogonique ei sur une theorie nouvelle de la fecondation normale. Ibid. 512-527. 1903. L'heredite et les grandes problemes de la biologie. Paris. vox Derschau, M. 1908. Beitrage zur pflanzlichen unto-.': Centern, Blepharo- plasten. Jahrb. Wiss. Bot. 46: 103-118. pi. 0. 1914. Zum Chromatindualisnius der Fflanzenzelle. Arch. Zellf. 12: 220 240 pi. 17. Digby, L. 1910. The somatic, premeiotic, and meiotic nuclear divisions of (inil- 16-19. fig. 1. Fischer, A. 1894. CJntersuchungen iiber Bakterien. Jahrb. Wiss. Hot. 27: I. 1S97. Untersuchungen iiber den Bau der Cyanophyceen und Bakterien. Jena. 1S99. Fixierung, Farbung und Ban des Protoplasmas. Jena. 1903. Yorlesungcn liber Bakterien. 2 Anil. Jena. Flemming, \Y. 1879. Beitrage zur Kenntniss der Zelle und ihre Lebenserechein- ungen. Arch. Mik'r. Anat. 16: 302 130. pis. 15 18. Gates, R. R. 19(H). The stature and chromosomes of Oenothera gig as, De Vries. Arch. Zellf. 3: 525 r^J. 72 INTRODUCTION TO CYTOLOGY Gerassimow, J. J. 1890. Einige Bemerkungen iiber die Funktion des Zellkerns. Bull. Soc. Sci. Nat. Moscow. 548-554. 1892. Ueber die kernlosen Zellen l>oi einigen Conjugaten. Ibid. 109-131. 1896. Ueber ein Yerfahren 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 der 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; 5: 137-141. 1886. Beitrage zur Kenntniss der Physiologie und Biologie cler Protozoen. Ber. X a turf. Gesell. Freiburg 1. Guilldbrmond, A. 1907. La cytologic des Bacteries. Bull. Inst. Pasteur 5: 273. 1908. Contribution a l'etude cytologique des Bacilles endospores. Arch. f. Protistenk. 12 : 9. 1909. Observations sur la cytologic 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 Korrelatiori von Zell- und Kerngrosse und ihre Bedeutung fur 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 Stemorritis 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 Spinndrusen der Raupen. Arch. Mikr. Anat. 47 : 500-549. pis. 26-28. THE NUCLEUS 73 Ivuwada, Y. 1919. Die Chromosomenzahl von Zea 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. 189(3. 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 oi 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 Protoplasmastucken unmog- lich, usw? Arch. Entw. 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 iiber die Struktur und Sporenbildung bei symbiotischen Bakterien. Centralbl. Bakt. II 12: 559. 1905. Cytologisches iiber die Bakterien der Prager Wasserleitung. Ibid. 15: 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. Migula, 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: t8. 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 Bilk-gland of certain insects. Jour. Morph. 29: 55-74. pis. 2. 1918. Some observations on the growing oocytes of < he stonefly, P< rla immarginata, Say, with special regard to the origin and function of the nucleolar structun Anat. Rec. 15: 203-216. figs. 9. Nakanishi, K. 1901. Ueber den Bau der Bakterien. Centr. Bakt. I 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. 15: 754-773. pi. 47. Osterhout, W. J. V. 1917. The role of the nucleus in oxidation. Science 46: 367-369. 71 INTRODUCTION TO CYTOLOGY Reed, G. B. 1915. The role of oxidases in respiration. .Jour. Biol. Chem. 22: 99-111. pi. 1. Reed, T. 1914. The nature of the doable spirem in Allium cepa. Ann. Bot. 28: 271-281. pis. L8, 19. Reinke, Fr. 1894. Zellenstudien. I. Arch. Mikr. Anat. 43: 377-422. pis. 22 24. Rosen, F. L892. Beitrage zur Kenntniss der Pflanzenzelle. 1. Ueber tinctionelle Unterscheidung verschiedener Kernbestandteile und der Sexualkerne. Cohn's Beitr. /.. Biol. d. Pfl. 5: 443-459 pi. 16. Ruzicka, V. 1908. Sporenbildung und andere biologische Vorgange bei dem Bad. anthracis. Arch. f. Hyg. 64: 219-294. pis. 1-3. 1909. Die Cytologic der Sporenbildenden Bakterien und ihr Verhaltnis zur ( !hromidienlehre. Centr. Bakt. 11 23: 289-300. figs. 8. Sachs, .1. L892. Physiologische Notizen II. Beitrage zur Zellentheorie. Flora 75 : 57 67. 1893. Physiologische Notizen VI. Ueber einige Beziehungen der specifischen ( trosse der Pflanzen zu ihrer Organisation. Ibid. 77: 49-81. 1895. Physiologische Xotizen IX. Weitere Betrachtungen iiber Energiden und Zellen. Flora (Erganzungsband) 81: 405-434. Schaudinn, F. 1902. Beitrage zur Kenntniss der Bakterien und verwandten Organ ism en. 1. Bacillus Bulschlii, 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 Spindelfasern. Flora 110: 52-66. figs. 3. Schwarz, Fr. 1887. Die morphologische und chemische Zusammensetzung des Protoplasmas. Breslau. Spitzer. 1897. Die Bedeutung gewisser Nukleoproteide fur 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. 1S97. 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. Vej dowsky, F. 1900. Bemerkungen iiber den Bau und Entwicklung der Bakterien. Centr. Bakt. II 6: 577-589. 1 pi. 1904. Ueber den Kern der Bakterien und seine Teilung. Ibid. 11: 481-496. 1 pL 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 7;, Zettnow, E. L891. Ueber den Bau den Bakterien. Centr. Bakt. 10: 690. 1897. Ueber den Bau der Grossen Spirillum. Zeit. Hyg. 24: 72. 1899, 1900. Romanowski's Farbung bei Bakterien. [bid. 30: I. and Centr Bakt. 1 27: 803. 1908. Ueber Schwellengrebel's Chromatinbander in Spirillum minimis. Centr Bakt. 1 46: 193. Zimmermann, A. 1893-1894. Sammel-Referate. 5, 7. 8. Beih. Hot. Centr. 3: 333-342, 401-430; 4: 81-89. (No. 7 reviews 153 papers on nuclei of various plant "roups.) 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 BLEPHAROPLAST i i receive consideration in the chapter on the achromatic figure.1 Very often there arc two centrosomes lying side by side in the centrosphere, Fig. 18. — Centrosomes in animal cells. A, attraction sphere above nucleus in spermatocyte of Salamarulm. (After Rawitz; see also Fig. 59.) B-F, intranuclear centrosome in spermatocyte of Ascaris megaloceptiala 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 (1870). 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 Bhould be consulted in this connection. ■ A Fii in l'.i. ( Jentrosomea epithelial cells. from cornea of mon- key. />'. from gastric gland of man. (After Zimn mm .1 78 INTRODUCTION TO CYTOLOGY successive eell generations. They observed that, prior to cell-division, the centrosome divides to form two daughter centrosomes, which move 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 -'■ ■-■.- ... \ ■•;; — ^'-4|$8 OxO.- Fig. 20. — Artificial cytasters in the egg of Arbacia. (After Morgan, 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 n disappears al 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 thai the regular appearance of the centrosome in successive mitoses is closely associated with regularly recurring physiological conditions in the cell; and thai 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 Butschli, and fully described by Lauferborn (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. • ! tar* t A' B • V1 - i> ■'• • Fig. 21. — Centrosomes in algae. .1. Stypocaulon. (After Swingle, 1897.) H, Stypocaulon. (After Escoyez, 1909 I centrosphere-like bodies in Polysiphonia. (After Yamanouchi, L906.) 1> /•.'. Dictyota dichotoma. (After Mottier, L900.) Centrosomes in the Sphacelariaceae have been described by I [umphrey (1894), Swingle (1897), Strasburger (1900), and Escoyez (1909). In the vegetative cells of Sphacelaria, according to Strasburger, the centrosome is situated in a cent rosphere at t he locus of an aster. Previous t<» mitosis it divides into two which take up positions at opposite poles of tin- spindle. In Stypocaulon (Swingle) essentially the same condition exists (Fig. 21). Escoyez later concluded, however, thai the asters of Stypocau- lon are formed independently rather than by division, and thai the central corpusclse . are probably not true centrosomes, but cytoplasmic microsomes. 1 'This review of plan I cent rosomes and also thai of the blepharoplasl in subsequent pages are based upon similar reviews given by the author in liis paper on Spermato- genesis in Equisetum (1912). 80 INTRODUCTION TO CYTOLOGY In the oogonium and segmenting oospore of Facus 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 Nemation 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). In Gallactinia succosa (Marie 1905; Guillier- mond 1911) a single centrosome, which arises within the nucleus with a THE CENTROSOME AND THE BLEPHAROPLAST M 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 ( 190")) found centrosomes in Hydnobolites, Neoliella, 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 Mi-- 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 If .' t~ ■-■■ ■'. 5**- *•>? fitts&F1** vj', «r&M V , - '■ I ■ % V* I ; / B Wf"> /?< ^y. \ > 1 s ■/•* rt£ n & a*\Aj*f> "A CC" V- to \K (of?; "• g»^^» « W E 2*6 l> Kv*-fV« 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 (Levinc 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. 6 82 ixtkodcctiox 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 colls 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. * 1 ' ' "' : ' - s» 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 be 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. The1 centriole may lie either against or within the nucleus, so that the flagellum which grows from it appears to arise directly from the nucleus (Mastigina) ; in other forms (Mastigella) the centriole is quite independent of the aucleus. 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; (b) the blepharoplasts may persist, dividing to form daughter blepharoplasts from which new flagella arise {PolytomeUa (e) 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' al the surface of the nucleus, the two being connected by a delicate strand known as the rh/zoplast, rhizonema, or centrodesmose {Peranema trichophorum Entz (1918) has recently reinvestigated the structure of Polytoma uvella, firsl 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 AM) THE BLEPHAROPLAST v is inserted on a "basal granule" (centre-some?) 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 earliei 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 aim r '<£> ~ ■■'- <■>- vx -■ , / *WK3 \ HI Fig. 26.— Blepharoplasts in Thallophytes. A-D, formation of the cilia-bearing ring in the zoospore of Derbe&ia. {After Davis, 1908.) E, Stemonitis flaccida: cilia growing from centrosomes during late stage <>f ili\ i-; in the formation of swarmers. (After Jahn, 1904.) isior found that the nucleus approaches the plasma membrane, which at that point forms a lens-shaped thickening. From this structure grow out th< cilia, and at the base of each a small refractive granule is present. Tin blepharoplasts of the higher groups were believed by Strasburger to hav< been derived from such swollen ectoplasmic organs of the alg«, 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 smal] body lying in contact with the plasma membrane and joined with th< 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). W^m '.L-l-^J Fig. 27. — Spermatogenesis in Marchantia. b, blepharoplast; c, centrosome; c.n., " chromatoider Nebenkorper ; " n, nucleus. I 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 77/ A' CENTROSOME AND THE BLEPHAROPLAST also present in the cytoplasm. Similar in mosl points is the account of Schaffner (1908). Miyake (1905), as the resull of his studies on Marchantia, Fegatella, Pellia, Aneura, and Makinoa, believes thai such liverwort centrosomes arc merely centers of cytoplasmic radiation, and inclines toward the view of Strasburger that the blepharoplasl and the centrosome are not homologous structures. Escoyez (1(.)()7) 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 centrosome-like 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 an 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. - 3U !. * '-'••/ ■ i '. xJHf\ • 1 ■y'7^> ': ': ■ • -'•■' \. fete 'v.; ) 9©KJ ■ -• i - Fig. l's. Spermatogenesis in Blasia. b, blepharoplast; rc, nucleus. X 4200. (.!//. percnosome. /,, mature spermatozoid. X 2535. (After Allen, L912, L917 Another body, the percnosome, is also seen in the cytoplasm a1 certain stages. In the opinion of Allen the limosphere is probably identical with the chromatoider Nebenlnrjur described by [keno 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 \\\r developmenl of the blepharoplast. Our more definite knowledge of this subject dates from 1897, when Bela jeff 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 Equisi turn 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 Cham, Equisetum, Fig. 30. — Spermatogenesis in Equisetum arvense, showing behavior of blepharoplast (centrosome) 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 centrosome, 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 blepharo- THE CENTROSOME AM) THE BLEPH VROPL 1ST 91 plasl 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 blepharoplasl do no1 become closely joined, but arc 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 blepharoplasl 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- 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 in Lasii spermatogen- blepharoplast divides into two which are situated at g^ito08fi8'biePw 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 isof the usual fern type, with several coils and a cytoplasmic vesicle. Shaw saw in the foregoing facts no ground for the homology of the blepharoplasl and the centrosome. Belajeff (1899) found thai centrosomes occur al the Bpindle poles during all, excepting possibly the first, of the tour divisions which .result in the 16 spermatids. He reported thai 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 oi blepharoplasts. Belajeff regarded this as a strong confirmation of his theory that the blepharoplast and centrosome are homologous organs. Fig. .'i 1 . T w o stages in the sperma- togenesis of A • /■' ilium. A. blepharoplasts iicai' poles of spindle plasl Dear nucleus. Nebenkern at left. i A/ti r Yamanott 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 Bla.sia, Equisetum, Marsilia, and the cycads may be homologized with the normal division exhibited by ordinary centrosomes. Fig. 32. — Spermatogenesis in Marsilia quadrifolia. A, first spermatogenesis 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 CENTROSOVE AXD THE BLEPHAROPLAST 93 In 1897 and 1901 Webber described the blepharoplasl of Zamia ' 1 ig. 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. Ml w A. - B Wfmi, - c ■ / . i • Fig. 33. — Spermatogenesis in Zamia. A-E, five stages in the vacuolation and fragmentation of the blepharoplasl during the mitosis differentiating the spermatids. F, the two spermatozoida in tht> end of the pollen tube; prothallial and stalk cells below. Compare Fig. 34. A-D, X 350; E, • L200. {After Webber, 1901.) In Ikeno's (1898) account of gametogenesis and fertilization in Cycas it was shown that the blepharoplast s 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 blepharoplasl 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 menl oi two oi 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. rif)r\ ■ ... -\ A B Ctarlei J . Otaml* rUn del. *•-* Fig. 34. — Spermatogenesis in Dioon edule. diff^&fed °ex 18q(»h br°}k fa^!eS £ 7*°^™' X 189°- * two blepharoplasts below V 2qV n f + + y Ce V^ u tW° blePhar«P^sts; prothallial and stalk cells ' X 1890 F T^r ° blepharoplast in spermatid as spiral band begins to m. X 1890. £, portion of edge of spermatozoid, showing spiral band out at two noints and cdia growing from it. X 945. {After Chamberlain. 1909.) P Ikeno in 1898 expressed the opinion that the blepharoplast of Ginkgo a ad 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 firsr of these he made comparisons with analogous phenomena in animals which he believed to THE CENTROSOME AND THE BLEPHAROPLAST 95 sustain the homologies suggested by BelajefT. Be pointed oul thai in Marchantia centrosomes are present in all the spermatogenoua 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 thai blepharoplasts are centrosomes, and spoke of the "transformation <>f ;i centrosome into a blepharoplast" in the development of a spermatid into a spermatozoid. The ectoplasmic blepharoplasts of the algae were also held to be derived from centrosomes. In the second paper he insisted less si rongly upon t he 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 flagellate-. 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 t wo extract s from the former paper: Although limited to a single mitosis in the antheridium, the blepharoplasl fof Equisetum] retains in its activities the most unmistakable evidence- 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 ntogen3r an outline of the phylogeny of the blepharoplast as it i> Been developing through bryophytes, pteridophytes, and gymnosperms, from a func- tional centrosome to a highly differentiated cilia-bearing organ with very lew centrosome resemblances. The activities of the blepharoplast in Equisetum [Marsilia, and Blasia], taken together with the behavior of recognized true centrosomes in plants ami analogous } 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 centre-.. me-. Animals. — The early researches of Moore (1895), Meves I L897, L899 . 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 oul 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 L900, p. 17.'. concludes that "the facts give the strongest ground for the conclusion that the formation of the spermatozoids agrees in its essential featuri with that of the spermatozoa . . . and that the blepharoplasl is without doubt to be identified with the centrosome. 96 1STR0DVCTI0N 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 l- .... /, A \ k\nM\ W* 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. 3(3. — 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 thai it is produced by the differentiation of chondriosonies, and thai 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 gymnosperma 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 musl await further information concerning those alga* which possess both cen- trosomes and blepharoplasts. It can scarcely be doubted that the basal corpuscles of ciliated cells represent organs belonging to various categori It must be left for further research to determine just how far thi structures, which are functionally analogous, are homologous witli each other and with other cell organs. Bibliography 5 C< ntrosome and Blepharoplast Allen, C. E. 1912. Cell structure, growth and division in the antheridia of /'<\w- ber. Akad. Wiss. Wien 71. Florin, R. 1918. Das Archegonium der Riccardia pinguis I. B. < the cytology of Humaria rutilans. Ann. Bot. 22: 35-55. pis. 4, 5. Fraser, H. C. I. and Welsford, E. J. 1908. Further contributions t<> the cytol< of the ascomycetes. Ibid. 22: 465-477. pis. 26, 27. Fraser, H. C. I. and Brooks, W. E. St. J. 1909. Further studies on the cytoli of the ascus. Ibid. 23 : 538-549. Fujii, K. 1898. (Has the spermatozoid of Ginkgo m tail or not?) Bot. M Tokyo 12 : 287-290. (Japanese.) 1899. (On the morphology of the spermatozoid of Ginkgo biloba.) Ibid. 13: '-'tin 266. pi. 7. (Japanese.) 1900. (Account of a sperm with two spiral bands.) Ibid. 14: 16 17. (Japan Graham, M. 1918. Centrosomes in fertilization stages of Preissia tiiiu Scop. Nees. Ann. Bot. 32 : 415-420. pi. 10. Gregoire, V. et Berghs, J. 1904. La figure achromatique dans Le Pellia epiphylla. La Cellule 21 : 193-238. pis. 1, 2. Griggs, R. F. 1912. The development and cytology of Rhodochyirium. Bot . < laz. 53: 127-173. pis. 11-16. Guignard, L. 1889. Developpement et constitution des antherozoides. Rev. Gen. 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 ascomycfct< Rev. Gen. Bot. 16: 129-143. pis. 14, 15. 1905. Rem arques sur la karj'okinese des ascomycetes. Ann. Mycol. 3: 344 361 pis. 10-12. 1911. Apercu sur revolution nuclcaire des ascomycetes et nouvi-llcs observations sur les mitoses des asques. Rev. Gen. Bot. 23: 89-121. figs. 8. pis. I. 5 Harper, R. A. 1895. Beitrag zur Kenntniss der Kernteilung and Sporenbildang 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. Hot. 13: 167 525. pis. _' I 26. 1905. Sexual reproduction and the organization of the uucleus 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 uber die Cytologie von Trypanosoma theileri. Arch. Protistenk. 38: :i.")."» :;7I pis. 11. 15. figs Henneguy, L. F. 1897. Sur les rapports des cils vibratiles avec les centrosomi Arch. d'Anat Micr. 1: 481-496. figs. 5. HiRAsfi, 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. :.. 6. figs 8. 100 INTRODUCTION TO CYTOLOGY Ikeno, S. 1898. Untersuchungen fiber die Entwicklung der Geschlechtsorgane und den Vorgang der Befruchtung bei Cycas revoluta. Jahrb. Wiss. Bot. 32 : 557-602. pis. 8-10. 1903. Die Spermatogenese 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 Korff, K. 1899. Zur Histogenese der Spermien von Helix pomatia. Arch. Mikr. Anat. 54: 291-296. pi. 16. Kuczynski, 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 uber 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 crystaUina. 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. 54: 329-402. pis. 19-21. figs. 16. Meyer, K. 1911. Untersuchungen liber 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. Mottier, 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 Diclyota dichotoma. Ann. Bot. 14; 166 L92 pi. 11. 1904. The development of the spermatozoid of Chora. Ibid. 18:245 _'.">}. pi. 17. Paulmier, F. C. 1899. The spermatogenesis of Anasa trUtis. Jour. Morph. 15: Suppl. 223-272. pis. 13, 11. 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 L'T'.i. pis. 1 1 Sands, M. C. 1907. Nuclear structure and spore format ion in Microsphcera. Trans. Wis. Acad. Sci. 15: 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 polymorpha. Ohio Na1 9. 363-388. Schottlander, P. 1893. Beitrage 7ur Kenntniss des Zellkerns und der Sexual- zellen bei Kryptogamen. Cohn's Beitr. Biol. Pflanzen 6: 267-304. pis. 4, ">. Sharp, L. W. 1912. Spermatogenesis in Equisetum. Bot. Gaz. 54: 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. Deu. Bot. Ges. 16: 177-184. pi. 11. Smith, H. L. 1886-1887. A contribution to the life history of the Diatomaces. Proc. Am. Soc. Micr. Pts. I and II. Strasburger, 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 Cilienbildnei im Pflanzenreich. Hist. Beitr. 6: 1-224. pis. 1-4. Studnicka, F. K. 1899. Ueber Flimmer- und Cuticularzellen init besonderei Beriicksichtigung der Centrosomenfrage. Sitz-Ber. K. Bohmisch. Ges. Wiss. Math.-Naturwiss. Classe, 35. Swingle, W. T. 1897. Zur Kenntniss der Kern- und Zelltheilung bei deD 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 <>t" the swarm sporea of Hijdrodictyon. 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 Zatnia. 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 lube apparatus of Ginkgo. Ibid. 24: 225-235. pi. 10. 1901. Spermatogenesis and fecundation in Zamia. U. S. Dept. Agr. Pit. [nd. 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: L83 204. pis. 12 L4. Wilson, E. B. 1900. The Cell in Development ami 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. Spermatogenesis 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 Hepaticae. Ann. Bot. 25: 299-313. pi. 2:». 1913. Spermatogenesis in Blasia pusilla. Ibid. 27: 93-101. pi. 11. 1915. Spermatogenesis in Mnium affine, var. ciliaris (Grev.), CM. Ibid. 29: 441-456. pi. 21. Yamanouchi, S. 1906. The life history of Polysiphonia violacea. Bot. Gaz. 42: 401-44S. pis. 19-28. 1908. Spermatogenesis, oogenesis, and fertilization in Nephrodiwm. 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 plastic!. Cytologists have long been aware of the important plrysiological 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 thi two classes of cell elements. General Nature and Occurrence.— Plastids are differentiated port ions 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 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 presenl in one form or another in all groups with the possible exception of bacteria. myxomycetes, and certain fungi. They are abundant only in th< se plant parts which have to do with specialized physiological functions. Within a single cell there may be regularly but one plastid, as in many a'gse, Anthoceros, and the meristematic cells of Selaginella (Haberlandl L888, 1905); or two, as in Zygnema; or a ligher 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. 1 he positions which the}- 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. Kuster (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, Drapamaldia. B, Spirogyra. C, Anthoccros. D, chromoplasts of Ariscema. 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 b. The cells of the Phseophycese, Cyanophycese, and Rhodophycese are character- ized respectively by the presence of yellow carotin, blue phycocyanin, and red phycoerythrin, in addition to chlorophyll. The Cyanophycese exhibit an especially rich variety of pigments, which m 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 Drapamaldia (Fig. 37, A), sl hollow cylinder with veiy 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), Wheldalc (1916), and Beauverie (1919). The distribution of carotin is discussed in an earlier paper by Tammes (1900). PLASTIDS AND CH0NDRI0S0M1 105 irregular v\n\s; in Mdogonium, an irregular parietal net; in Spirogyra (Fig. 37, #), a spirally coiled ribbon; and in the desmids, 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 colls (Scherrer 1914). The chromatophore of Selaginella may also assume this form (Haberlandt 1888). The chromoplasts of Ariscema (Fig. 37, D) are frequently sharply angular. In the Clado- phoraceae (Carter 1919) the cell is completely lined by a thin chromato- phore which maybe 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 (Monteverde 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 Btroma 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 INTRODUCTION TO CYTOLOGY been held by some observers that the stroma is a homogeneous body in which the 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 brouj; ht about through the agency of a?nyloplasts, 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 (C6Hio05)n, and exists in the form of granules ranging in size approximately from 2//. to 200//, in different plants. Potato-starch grains are usually about 90/.; in diameter. The reserve starch grain is formed within the body of the amyloplast, 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 a "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 March is mad.' up of simple, compound, and half-compound grains, whereas in ..al- and rice all or nearly all of the grains are said to be of the compound type. The successively deposited Jayers making up the grain differ mainly in water content, the innermost layers being richesl and the outermosl poorest in water. As a result of this non-uniform composition the main often splits radially when dried. -*a4 Fig. 39. — Reserve starch grains from various plant-. A, potato; simple and half-compound grains. B, Colombo starch. D, pea. E, maize; intact and partially digested grains. F. rye. G, maize. /, bean. J, rice. K, wheat. (After Tschirch.) c. arrowroot. H, Euphorbia As a result of his classic researches Nageli (1858) advanced t he 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 L883, L895 that the grain is composed of radially arranged needle-shaped crystals known as "trichites;" these are composed of a- and 0-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 pari of the plastid. This eon- elusion was confirmed by Salter (1898). The statement made by Schimper (1880) and Meyer (1883, L895) that starch is always formed by plasl ids st ill holds good in it - essent ial feature : so far as is certainly known no primary product of photosynthesis is formed in the cytoplasm apart from plastids, although in some cas< 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 are 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 Cyanophyeeae (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 Chlorophyceae especially, being present almost universally in the members of this group. They are known in a few representatives of the IJiodophycese (Nemalion and the Bangiaceae), but apparently do not occur in the cells of the Cyanophyceae, Phseophycese, 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 CHONDRJOSOMES 109 served that under certain conditions the pyrenoid is closely surrounded by amass of starch grains, and concluded thai it is an organ, or portion of an organ (chromatophore), intimately concerned in the process of starch formation, its action being somewhat similar to thai of the amyloplast. The pyrenoid, in fact, has often been likened to a leucoplast imbedded in the chromatophore; Wiesner, for instance, believed the pyrenoid t<> 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 different iated 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 Bonn pun (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 bodies1 which are probably composed of a protein substance. The outermosl bodies become starch, new ones apparently being formed by the fission of those lying on the interior of the group. McAllister states thai 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 18SS Wakker discovered in the cells of Vanilla planifolia and l\ aromatica certain plastid-like bodies to which he gave the name elaioplasts, since they seemed to be concerned in 110 INTRODUCTION TO CYTOLOGY mj^ the elaboration of oil (Fig. 40, A). They were soon observed in a number of monocotyledons by Zimmermann (1893), Raciborski (1893), and Kris- 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 (Klister), 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, Fig. 40. states that in Gaillardia they are formed secon- A, elaiopiast forming darily by the aggregation of many small degen- oil droplets in epidermal 1 x- i i j.i i cell of perianth of Poii- ©rating plastids and their products at one or anthes tuberosa; nucleus more points in the cell, all stages of the process with small plastids at ■, i i \ i j. 1 1 .li i t r 1 right. (After Politis, being observed. Although the bodies so formed 1914.) B, oil bodies in may, if green, produce starch, or, if colorless, an various stages of develop- -i ^ • ^ ,-. . , ., , , , ,, ment in a cell of Caly- 01iv yellow pigment, Beer thinks it probable that pogeia. (After Gar geanne, 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 PfefTer (1874). Pfeffer 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 Kuster (1894) supposed them to resemble in having a spongy stroma containing oil in the form of minute droplets. PLASTIDS AND CHONDRIOSOMES 1 1 1 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 aboul 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 arc 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 (c/. 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 elaioplasl : it is simply an accumulation of ethereal and fatty oils together with souk 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 anriosperms by a number of writers. To these the term elaiospheres was applied by Lidforss f 1X9:-! 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 alga? 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. (Overton 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 Funis \ ( Juignard L889 ; 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 L892 It is generally agreed that the eyespot in many instance- 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 Chlamydomonadacese 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 falling upon the light-sensitive substance. As the unoriented swimming colony rotates on its axis, those zooids turning away from the light have the hyaline portion of their eye- 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 flagellar of those zooids, with 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 -Eyespots of various types. A, zooid of Eudorina; e, eye- spot. (From Mast, After Grave.) B, zoospore of Cladophora, (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 flagellum; face view of eyespot at right, showing pigment gran- ules. (After Wager, 1900.) D, eyespot of Euglena velata. (After Franze, 1893.) E, eye- spot of Trachelomonas volvo- cina, with pigment granules and crystalloid body. (After Franze.) PLASTIDS AND CHONDRIOSOMES 11:; between the two seems highly probable; whereas in oilier cases annum,. 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 thai they cannol 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 plaslids 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 chloropla-i- 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 t he 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 I of another filament. The two nuclei fuse, giving the primary nucleus of the new individual (zygospore nucleus), while the two plastid- 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 1911). Each gametophytic cell 8 114 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 Anthoceros. 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 (usualty 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 L15 cycle in some less conspicuous form. If they represenl regional trans- formations of the cytoplasm resulting from the localization of certain processes, they might well be expected to differentiate anew as thi processes begin in the life of the cell, and to preserve varying degn 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 stage-. 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). Beinhi in L897 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 Bpecial literature of considerable bulk has developed.1 It now seems evident that the filaments ("fila") of Flemming, the "bioplasts" of Ah man, the " plastidules " of Maggi, the "archoplasmic granules" of Boveri, and the "mitochondria" of Benda are all one and the same 1 hing 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, L919), 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 classes, 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. 3 h V Ffc 43. — Chondriosomes in plant and animal cells. A, nerve cell from guinea pig. X 480. (After E. V. Cowdry, 1914.) B, tapetal cell of Nymphoea alba. (After Meres, 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 Confer vales (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 hematoxylin 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 (191-1) on vital staining, and N. H. Cowdry's (1917) on plant and animal chondriosomes. 118 ISTliODUCTION 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;L6wschin 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. AlexiefT (1917) thinks that although cyto- 0 1 6 Fig. 44. — Examples of regular behavior of chondriosomes in cell-division. A-C, spermatocyte of Oryllotalpa 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-Fremiet (1910), Terni (1914), Korotneff (1909), and others, the individual chondriosomes divide at the lime of mitosis (Fig. 44, D), a conclusion with which many others fail to agree (Orman 1913; Miss Beckwith 1914; etc.). In the cells of PLASTIDS AND CH0NDRI0S0M1 119 the grasshopper, Dissosteira Carolina, Chambers (1915) finds thai the chondriosomal material forms a granular network surrounding the nucleus during the resting stages and the mitol ic figure during division. 1 )uring the later phases of mitosis the strands and granules of tins 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. Voinov I 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) "chondrio- 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 191 6) 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 group-, each spermatid receiving six (occasionally five or seven). A European species described by Sokolow (1913) agrees essentially with tin's. 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 mote 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). Voinov (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 Pig. 45. — r ertihzation in . ' r FiiaHa papulosa, showing they are in some way concerned in the reactions chondriosomes of sperma- through which hereditary characters are de- tozoon (at top) distributing , . . themselves in the cytoplasm veloped in the individual, but the general ?oiKhf egg* (After Meves> 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 PLASTIDS AND CHONDRIOSOMES 121 chick embryo. They believe that the chondriosome elongates and directly becomes the young fiber. Gaudissart (1913), on ili«- 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 !':!;< r. 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 . c j&m 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 <>!" carotin in chromoplasts derived from chondriosomes in epidermal cell of Iris petal. {After (mil; mond, 1918.) C, chondriosomes and chloroplasts in young cell of Finns banksiana. X 750. (After Mottier, 1918.) D, transformation of plastid primordia into leucoplasts in i""t cell of Pisum; some of the leucoplasts contain starch. (After M<>tti( r.) 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. li». .1 ). 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 Ascaris has already been mentioned. Relation of Chondriosomes to Plastids.- ( meof the most conspicuous views regarding the significance of chondriosomes is thai 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 Forenbacher (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, B). Among the most recent researches in this field are those of Mottier (1916, 1918) on the cells of Zea, Pisum, Elodea, Pinas, 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 (1920afr) 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 7m 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. PLASTIDS AND CHONDRIOSOMES L23 The existence of such ;i genetic relationship between uhondriosome and plastids :is that described above has been denied by many writers, among whom may be mentioned Lundegardh (1910), Meyer (1911 . Rudolph (1912), Lowschin (1913, 1914), Scherrer ( L91 h. Miss Beckwith (1914), Derschau (1914), von Winiwarter (1914), Sapehin (1915), Chambers (1915), M. and W. Lewis (1915), and Harper L919). These workers for the most part hold thai chondriosomes are qoI distind cell organs at all, but regard them rather as more or less transient visible products of protoplasmic activity. Derschau asserts thai they arise neither de novo nor by fission, but thai they are merely small mass 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 instanci - to the action of fixing agents upon it. To Chamber- they appear in living cells not as persistent structures but as temporary physical states of the colloidal substances composing protoplasm. M. and \Y. Lewi- 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 <»i 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, L918), 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 bet ween their chemical composition and that of phosphatids, which appear to be capable of auto-oxidation. Conclusion. — From the foregoing review ii should be more than plain that the state of our knowledge of chondriosomes is such thai almost do definite final statements can be made regarding their origin and function. The evidence at hand apparently indicates thai 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 thai some of them are temporary accumulations of substances involved in metabolism, appearing and disappearing in the cell in a manner somewhal 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 they 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. PLASTIDS AND CHONDRIOSOMES 125 Bibliography 6 Plastids mitochondria. Biol. Bull. 35 : 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 CYTOLOGY 1920a. Plastidome, vacuome e1 sph6rome dans Selaginella Kraussiana. Ibid. 170: 301-306. 1 pi. 19206. La structure de la cellule vegetale el son metabolisme. Ibid. 170: 70!) 714. Daxgeard, P. 1020. Sur revolution du systeme vacuolaire chez les gymnospermes. Ibid. 170. 474-477. figs. 8. Davis, B. M. 1899. The spore mother cell of Anthoceros. 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 Morphologie und Entwicklung der Starkekorner von Pellionia Daveauana. Flora 75: 267-2S0. pi. 5-6. Dubreuil, G. 1913. Le chondriome et le dispositif de l'activite secretaire. Arch. d'Anat. Micr. 15: 53-151. Duesberg, J. 1907. Der Mitochondrialapparat in den Zellen der Wirbeltierc und Wirbellosen. Arch. Mikr. Anat, 71 : 284-296. pi. 24. 1909. Les chondriosomes des cellules embryonnaires du poulet et leur role dans la gencse des myofibrilles, avec quelques observations sur 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. 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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. Kingsbury, B. F. 1912. Cytoplasmic fixation. Anat. Record 6: 39-52. Klebs, G. 1883. Ueber die Organization einiger Flagellaten-Gruppen and ihre Beziehungen zu Algen und Infusorien. Unters. Bot. Inst. Tubingen 1:233-262. pis. 2, 3. 1892. Flagellaten-Studien. I, II. Zeit. Wiss. Zool. 55: 265-445. pis. 13-18. Kohl, F. G. 1903. Ueber die Organization und Physiologie der Cyanophyceenzelle, und die mitotische Teilung ihres Kerns. Jena. Korotneff, 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. KtisTER, 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. 1 pi. Lidforss, B. 1893. Studien ofver elaiosferer i ortbladens mesophyll och epidermis. Inaug. Dissert., Lund; also in Kgl. Fysiogr. Sallsk. i Lund Handl. 4. PLASTIDS AND CHONDRIOSOMES 129 Lowschin, A. M. 1913. "Myelinformen" und Chondriosomen. Ber. Deu. Bot. Ges. 31: 203-209. 1914. Vergleiehende experimental-cytologiache Unterauchungen Qber Mito- chondrien in Blattern der hoheren Pflanzen. (Vorl. Mitt.) Lbid. 32: 266 270. pi. 5. Lundegardh, H. 1910. Kin Beitrag zur Kritik zweier Vererbungsliypotheseii. Jahrb. Wiss. Bot. 48: 285-378. Mast, S. O. 1911. Light and the behavior of organisms, pp. 410. N. V. 1916. The process of orientation in the colonial organism, G&nium pedoraXe^ 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 mitochondrial de la cellule hepatique. Jour. Physiol. Path. Gen. 16: 007-022. 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 StudieD am Hiihnerembryo. Arch. Mikr. Anat. 72: 816-867. pis. 39-42. 1909. Ueber Neubildung quergestreifter Muskelfasern nach Beobachtungen am Hiihnerembryo. Anat. Anz. 34: 161-165. figs. 3. 1914. Was sind die Plastosomen? Ibid. 85: 279-302. figs. 17. 1915. Ueber Mitwirkung der Plastosomen bei der Befruchtung dea Eiea von Filaria papillosa. Arch. Mikr. Anat. 87: II 12-46. pis. 1-4. See also p. 286. 1918. Die Plastosomentheorie der Vererbung. Eine Antwort auf verschiedener Einwande. Ibid. 92: II, 41-136. figs. 18. (Bibliography.) Meyer, A. 1881. Ueber die Struktur der Starkekorner. Bot. Zcit, 39: 841- 846, 857-864. pi. 9. 1883a. Ueber Krystalloide der Trophoplasten und iiber die Chromoplasteii 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 ct Lea corpuaculea Ducleo- laires du protoplasme des Chara. Comptes Rend. Acad. Sci. Paris 168: 283 286. figs. 7. von Mohl, H. 1835, 1837. Ueber die Vermehrung der Pflanzensellen durch Theilung. Dissert. Tubingen 1835. Flora 45, 1837. 1851. Grundziige der Anatomie und Physiologie der vegetabiliache Zelle. Engl. Transl. by Henfrey, London 1852. Monteverde, N. A. and Lubimenko, V. X. 1911. Recherchea mm- la formation -1«- la chlorophylle chez les plantes. | Russian. 1 Bull. Acad. Imp. Bci. St. Petera- bourg VI 5 : 73-100. Moreau, F. 1914. Le chondriosome et la division dea mitochondriea chei lea Vaucheria. Bull. Soc. l^«»t. France 61: 139-142. Mottier, D. M. 1918. Chondriosofnes and the primordia of chloroplasta and leucoplasts. Ann. Bot, 32 : 91-114. pi. 1. 9 L30 INTRODUCTION TO CYTOLOGY von Nageli, C. 1858. Die Starkekorner. Zurich. Olive, E. W. 1904. Mitotic division of the nuclei of the Cyanophycese. Beih. Bot. Centr. 18: 9-44. pis. 1, 2. Orman, E. 1913. Recherches sur les differenciations cytoplasmiques dans les vegetaux. 1. Le sac embryonnaire desLiliacees. La Cellule 28: 365-441. pis. 1-4. Overton, E. 1889. Beitrag zur Kenntniss der Gattung Volvox. Bot. Centr. 39: 65-72. 113-1 IS, 145-150, 177-182, 209-214, 241-246, 273-279. pis. 4. Palladin, A'. I. 1918. Plant Physiology. (Engl, edition by Livingston.) Phila- delphia. Payne, F. 1916. A study of the germ cells of Gryllotalpa borealis and Gryllotalpa vulgaris. Jour. Morph. 28: 287-327. pis. 4. figs. 5. Pensa, A. 1914. Ancora a proposito di condriosomi e pigniento antocianico nolle; cellule vegetali. Anat. Anz. 46 : 13-22. Pfeffer, W. 1874. Die Oelkorper der Lebermoose. Flora 57: 2-6, 17-27, 33-43. pi. 1. 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 considered comme les agents de la fonction ecleefcique 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 uber die Individualitat der Plastide. [Russian.] pp.133. pis. 17. Odessa. 19136. Untersuchungen liber die Individualitat der Plastide. (Zweite Vorl. Mitt.) Ber. Deu. Bot. Ges. 31: 14-16. 1 fig. 1915. Untersuchungen iiber die Individualitat der Plastide. Arch. Zellf. 13 : 319-398. pis. 10-26. S< haxel, F. 1911. Plasmastructuren, Chondriosomen und Chromidien. Anat. Anz. 39: 337-353. figs. 16. Scherrer, A. 1914. Untersuchungen fiber Bau und Vermehrung der Chromato- phoren und das Vorkommen von Chondriosomen bei Anihoceros. Flora 107 : 1-56. pis. 1-3. Schilling. A. J. 1891. Die Susswasser-Peridineen. Flora 74: 220-299. pis. 8-10. Schimper, A. I'1. \V. L880 L881. Untersuchungen iiber 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 unci Farbkorper. Bot. Zeil 41: 105-112, 121-131, 137-146, 153-162. 1 pi. 1885. Untersuchungen iiber die Chlorophyllkorper und die ihnen homol Gebilde. Schmidt, E. \Y. 1912. Pflanzliche Mitochondrien. Prog. Hci 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. C;«-s«-ll. t. Nat. u. Heilk. Bonn, 1880.) 1884. Beitrage zur Kenntniss der Chroma tophoren. 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 Scenedt Arch. f. Protistenk. 32: 278-297. pis. 16, 17. Sokolow, I. 1913. Untersuchungen liber die Spermatogenese 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 Delia Bper- matogenesi degli anfibii. (Ricerche sul Geotriton juscus.) Arch. Zellf. 12: 1-96. pis. 1-7. Timberlake, H. G. 1901. Starch formation in Hydrodictyon idriculatum. Ann. Bot. 15: 619-635. 1903. The nature and function of the pyrenoid. Science N. S. 17 : M50. Tischler, G. 1906. Ueber die Entwicklung des Pollens und der Tapetenzellen bei ifo&es-Hybridon. 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 l'existence d'une chondriodierese. Comptea Rend. S 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 Cyanophycese. (Prelim, note.) Proc. Roy. 9 London 72 : 401-408. figs. 3. Wakker, J. H. 1888. Studien iiber die Lnhaltskorper 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. Cambridg 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 r61e 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. Wollenweber, W. 1907. Das Stigma von Hcematococcus. Ber. Deu. Bot. Ges. 26: 316-320. pi. 11. 1908. Untersuchungen fiber die Algengattung Hcematococcus. Ibid. 26 : 238-298. pis. 12-16. figs. 12. Yamanottchi, 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 sonic (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 arc therefore referred to as "chromatic bodies," show reactions indicating a composition closely approaching that of the extra-nuclear granules of uucleo-protein (chro- midia) which R. Hertwig and Goldschmidt interprel as granules of escaped chromatin concerned in cell differentiation. Others resemble the fatty chondriosomes in form and composition. It is therefore a mat ter 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- 1 33 134 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 f & 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 Bcrtholletia excelsa; J, from Fceniculum; 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 L35 called the "globoid,JI (Fig. 47, (?)■ The crystalline inclusions sometimes grow to l)c very Large. K has been thoughl by certain workers that aleurone grains arc self-perpetuating bodies with an individuality com- parable lo that of nuclei and certain plastids. That this view is correct has been rendered very improbable by the researches of East and I Lives (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 max 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 plant-. Gutta-percha occurs in a similar state in Tsonandra 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 via 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 dedififerentiation " (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 cycl< — 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 dedifferentiatinn is strikingly shown in the well known cases of Begonia and BryophyUum, which can regenerate complete new individuals from a few leaf cells. In the higher animals cell differentiation is usually so great thai 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. Tim- ii i- only the functioning reproductive ('('lis 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 all 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 differentiated 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 \:\\\ being due to different environments in the tissue. For these work* 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 exist- 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 portion-. 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 t han 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 substances1 (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 t he organism as a whole, functional and structural differential ion are inseparably connected. In the present state of our knowledge the attempt to determine the real essence of polarity raises questions which cannot ye1 be answered. D< 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 l'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. 19016. Die Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus lividus. Zool. Jahrb. (Anat. Abt.) 14: 630-653. pis. 48-50. Carrtjthers, D. 1911. Contributions to the cytology of Helvella crispa Fries. Ann. Bot. 25 : 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 1 11 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 algae. Bot. (laz. 62: 89 111. von Derschau, M. 1908. Beit rage aur 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. DlGBY, L. 1909. Observations on "chromatin bodies" and their relation to the nucleolus in Galtonia candicans Decsne. Ann. Bot. 23: 491-502. pl>. :;.'!. ;i 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: «.)7 1 lo. pis. 8-10. Duesberg, J. 1911. Plastosomen, "Apparato reticolaro interno," und Chromi- dialapparat. Ergelm. Anat. Entw. 20: 567-916. (Review.) East, E. M. and Hayes, H. K. 1911. Inheritance in maize. Oonn. Exp. Sta. Bull. No. 167. 1915. Further experiments in inheritance in maize. Ibid Xo. 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. 57 : 375-439. pis. 2. figs. 35. 19185. 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 fiber 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 Biologic. 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. Hot. 8: 129 574. 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. Experimented Studien iiber die Zell- und Kernteilung mit besonderer Riicksicht auf Form, Grosse und Zahl der Chromosomen. .lour. 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. Hot. Gaz. 54: 336-338. 1 fig. Tobler, F. 1902. Zerfall und Reproduktionsvermogen Thallus einer Rhodo- melacere. Ber. Deu. Bot. Ges. 20: 357 365. pi. Is 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 IAUum candidum, Linn. Ann. Bot. 29: 285-291. pi. 15. Wheldale, M. 1910. 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, Myrosin- und Emulsionkorner. 14. Die Proteinkrystalloide, Rhabdoiden und Stachelkugeln. 15. Die Starkekornrr unci venvandten Korper. Beih. Bot. Centr. 4: 105-109, 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 novo from a mother liquor, or "cytoblastoma," 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 Butschli, 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 the» terms direct and indirect division, which are still used. Still later i ISVJ 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 term- arc 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 'The following additional historical data are of interest. The chroraoson though they appeared in the figures of Schneider L873), were first adequately drawn by Strasburger in 1875. Longitudinal splitting was described by Flemming in 18$ The terms prophase, metaphase, ;my heavy cross pieces repre- senting the portions between the original vacuoles I'm-. .".J and 53, S 154 INTRODUCTION TO CYTOLOGY The materia] constituting the 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 1 ."»."> cated process of mitosis is meaningless unless iln' chromatin is quali- tatively different in the various regions of the nucleus, and thai 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 I L876) and Pfitzner (1881), that the chromatin granules visible in the nuclear rel icu- lum arrange themselves in a series in the chromosome and by their division initiate its splitting, had much to do with the formulal ion of thia hypothesis. That the chromatic granules, or chromomeres (Fol L891), represent the qualities of Roux is a theory which has been widely accepted by cytologists. It was the opinion of Braucr (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. r\A fee? Fig. 54. A, vacuoles in chromosomes at metaphase in Trillium. X 1800. (After Gregoin and Wygaerts, 1903.) B, spiral arrangement of chromatin material within the chromosomes Allium. (After Bonnevie, 1911.) C, D, stages of chromosome splitting in Naja* marina, showing chromomeres. X 2250. (After MiOler, 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 L884, lsss , others have raised serious objections to the theory that they arc significant units or individuals. Gregoire and Wygaerts (1903), .Martins Mano (1904 Gregoire (1906, 1907), Marechal (1907). Bonnevie (1908), Stomps (1910), Lundegardh (1912), Sharp (1913, L920), 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 Muller I L912) the portions of the thread between the chromomeres split first, the division of thechromo- 156 INTRODUCTION TO CYTOLOGY meres then following. It has been pointed out (Sharp 1913) that Muller'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, S). 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 greal i heorel ical 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 ouclear 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 homologies 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 I issue 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 nee sarily the basichromatic fluid held within it, that maintains a structural independence. This view had the support of the earlier observation made by Boveri (1887a, 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 58 INTRODUCTION -TO CYTOLOGY Special emphasis was laid upon this interpretat ion 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 maintairis 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 I.\D/\ TDUALITY I.V.I 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 }<)\^ From this it is evident that the morphological identity <>f the chromo- somes has not been lost between mitoses, although ;i very different type of organization has been assumed. In Carex aquatilis Stout (1912) has found a peculiar condition, line the very small spherical chromosomes, which maintain a serial arranj ment, are visible in the resting state, and can be traced continuously through all stages of the somatic and germ cell divisions witli 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 <»l 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 Cruciferae 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, //), 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 interpret at inn is in all probability a valid one in many of the described cases, but in <>t hers the significance of such chromatic masses is questionable. In Cn , virens de Smet (1914), in harmony with the conclusions of Miss Digby (1914), finds them to be accumulations of material formed during tic resting stages. If such is the case they are to be regarded as karyosom< 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 (Haecker L895; Ruckeri L895 . 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 i- 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 ix) chromosomes of Fundulus and the short (1m) 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, Toxopnenstes, 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 AXD CHROMOSOME INDIVIDUALITY 161 f»f B a 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, beiim drawn pole- wards by the middle, shows both ends, making the number apparently 14. I). K. two successive sections of a heterotypic figure in the mierosporocyte, 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.) B Fig. 56 bis. A, anaphase of somatic mitosis in Crepis virene, showing 2 long, 2 medium Bised, and 2 short chromosomes passing to each pole. (After Rosenberg, L920.) B, the chromosome complement in a somatic cell of Najas major, showing the 7 homologous pairs. {After Tschcrnoyarow, 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 ue-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 1 hree 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 Phrynotettiz (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 Tisehler (1916). For the numbers in animals see Harvey (1916, 1920). SOMATIC MITOSIS AND CHROMOSOME INDIVIDUALITY 163 Bcneden, and Strasburger that the number of chromosomes in any given species is relatively constant. It was largely upon this fad thai the theory of chromosome individuality was originally based: the fad thai the number of chromosomes appearing at every mitosis is almosl 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 thai 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'fi 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, (b) In another experiment the unferl ilized 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 -up- bringing about an irregular distribution of the chromosomes. For ex- ample, a quadripolar spindle was produced, separal ingtheo 1 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 (J91 [) later suggested thai 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 were 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 * I I ii ?♦*••♦♦« B «**4 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 McClunrj, 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 apparenl variations in Dumber by McClung (1917) and Miss Holt (1017) in their researches on multiple chromosomes and chromosome complexes. McClung finds in his analysis of the chromosome groups of the orthopterans Hesperotettix and Mermiria thai temporary associations often occur between various members of a group, wit h t he resull ing forma- tion of "multiple chromosomes" and a consequent decrease in theappar 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 arc 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 <|iin<' 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 th< conditions." Somewhat similar evidence has been brought forward by Hance I 1!»17. 1918a6). Hance finds that the chromosome Dumber in the spermatogo- nia of the pig is regularly 40, wdiereas in the somatic cells it varies from 40 to 57. Similarly in (Enothera scintillans, which ha- 1"> chromosomes in its microsporocytes, there may be from 15 to 21 chromosomes in the somatic cells. Measurements of the members of t he 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 mult iple complexes of ( ' ul< x. 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): 1 . . . 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 the}r 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 latter. The case for discontinuity is strongest in tin- absence of any chromosome order, and becomes progressively weaker with \r< Bananen-Pollens. I. Arch. f. Zellf. 5. 1910. Chromosomenzahl, -Form und -lndividualitat mi Pflanzenreich. Prog. Rei Bot, 5: 164-284. (Review and list of numbers Tschernoyarow, M. 1914 Ueber die Chromosomenzahl in besonders beschaflfene Chromosomen im Zellkerne von Najas major. Ber. Deu. Bot. Ges. 32. ill U6. pi. 10. 174 INTRODUCTION TO CYTOLOGY Waldeyer, W. 1888. Ueber Karyokinese unci ihre Beziehung zu den Befrucht- , ungsvorgangen. Arch. Mikr. Anat. 32: 1-122. figs. 14. (English transl. in Quar. Jour. Micr. Sci. 30: 150-281. 1889.) Wenrich, D. H. 1916. The spermatogenesis of Phrynotettix magnus with special reierence 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 (Stenobothrus) 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. Vlll. 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. Zur Strassen, O. L. 1898. Ueber Riesenbildung bei Ascam-Eieren. Arch. Entw. 7: 642-676. pis. 16, 17. figs. 9. CHAPTKR 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 arc 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 hitter 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 otic pole to the other without being so attached: these 17.-. 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. V A /{■':::Li^7l^''-' Fig. 58. A, spindle beginning to differentiate in kinoplasmic caps at poles of nucleus in Nephro- 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 al the periphery after the fibers have completely disappeared al 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 somewhal differenl from thai 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 thai 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 Ins 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 cent ral spindle port ion < connect ing fibei - 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. Iside 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 Fig. 59. — Mitosis in the spermatocyte of Salamandra. I, prophase, centrosomes in astrosphere substance; latter spread out on nucleus. 77, prophase; bivalent chromosomes formed; centrosomes beginning to diverge; central spin- dle and asters developed. Ill, 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.) )()(). 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 ■ I ■ ■ I ' ■ mix ' ' ■ c Fig. 61. A, B, anaphase and telophase of mitosis in ascus of Laboulbenia chcetophuni. X 1350. (After Faull, 1912.) (See also Fig. 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; 01 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, docs 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 Bee page -00. 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 centre-some. Butschli (1876), who looked upon protoplasm as alveolar in nature, held thai the rays are nol really fibers, but only the lamellae between radially elongated alveolae aboul the cen- trosome. It was the opinion of Wilson (1899) on the other hand, thai 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 fibrillae. Van Beneden (1883) supposed these fibrillae to be derived partly from the intranuclear reticulum, and Rabl I L889) pointed out that they are continuous with the unaltered cytoplasmic mesh work 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 conies from the cyto- plasm, being added to the growing fiber at its end. This was the view of Druner (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 consl it utes t heectoplasl . cent ro- 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 arc simply dragged apart by the contraction of two opposed groups of Bpindle fibers. This theory and its modifications are fully reviewed by Wilson: i1 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), Druner (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 dei 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; th< 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, 1 899 . 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 ( Jhambers 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 L85 the electromagnetic field, he concluded thai 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 pol being of like sign also. The movement of the chromosomes to the pol< 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 r61e 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 thai 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 01 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 Butschli 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 naifH 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 --' ff figi Fig. 63. Only the central part of the cell Fig. 62. Fig. 62. — Cytokinesis by furrowing in Closterium. 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 wali develops uniformly across the THE ACHROMATIC FIGURE, CYTOKINESIS, AM) (ELL WALL 1ST whole equatorial plane at the same time, and not as i 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 Phycomyces, Davis (1903) on Saprolegnia, Kvtz (1907 on Synchytrium, and Harper (1899, 1900, 1914) on Synch \yt num. Pilobol 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 eentrifugally 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) andgTOW inward until they meet at the center, dividing the protoplast simultaneously into four Fn.. 64. t \ tokinesis by furrowing in the micro- sporoc} t<- of Vicol ■ l 100. {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 Butschli (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 FIClTRE, CYTOKINESIS, AND CELL WALL ISO On the contrary, Robertson (1911, 1913) and others all riliute 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 Biitschli's interpretation has been afforded by the studies of Spek (1918). Spck imitated furrowing and division with oil and mercury droplets in water, and showed thai 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 onix 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 Chamb (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 A- 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 long 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 thai the middle lamella itself may increase in thickness by the addition of new material before tin 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 leg 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 thai it- position in the center of p « Fig. r>6. — Longitudinal and transverse Bections of a gymnosperm tra- cheid; />. primary wall or middle lamella; 8, secondary layer; /. 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 (ELL WALL 193 that the wall is made up of ultra microscopic crystalline micella 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 theoldones, a process termed intussusception. Contrasted with this was Si rasburger'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 micell®, 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 t he 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 striatums which both he and von Mohl observed in the wall substance were regarded by Strasburger as due to the linear arrangement of thi 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 solve,]. 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 Dumber 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 Askena (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 difficull problem must await the results of further inquiries by both the cytologisl and the biochemist. The Chemical Nature of the Cell Wall.- -Through the researches of Payen (1842), Fremy (1859), Kabsch (1863), Wiesner I 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 heartwoods 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, THE ACHROMATIC FIGURE, CYTOKINESIS, A ND CELL WALL 1 95 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 t w<> 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 mere; in thickness while they have no immediate contact with the protopls The protoplast now expands, after which a third coat, tin- 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. > m ym (1° • e x. s.rrii 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." 1 ;•■ /,■■>>, L905. Miss Lyon found that the spore coats (S. rupestris) begin to differ- entiate in the midst of the "spore membrane" ("special wall:* Pittii rather than at its inner surface as Fitting thought. The exospore first appears as a double zone, the outer pari of which become- the per- inium (perispore: Fitting) (Fig. 69). The small protoplasi gradually expands and pushes back the undifferentiated inner portion of the Bpore membrane; and while it does so a second coal 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 Mirabilis 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 al«> aid in Bolving 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. Hot. Oaz. 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 fiur. 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 Onagrac>;i\ Beih. Bot. Centr. 19: I 286-313. pis. 3-5. 1909. The development of the spores of Equisetum. New Phytol. 8: 261 '-'06. 1911. Studies in spore development. Ann. Bot. 26: 199-214. pi. 13. van Beneden, E. 1883. Resherches sur la maturation de l'oeuf, la fecondati<»n el la division cellulaire. Arch, de Biol. 4. 1887. (van Beneden & Neyt). Nouvelles recherches sur la fecondati<»n et la division mitosique chez VAscaride megalocephale. Bull. Acad. Roy. Belg. III. 14:215-295. pi. 1. Boveri, Th. 1887. Ueber die Befruchtung der Eier von Ascaris megaloct phala. Sitz.-Ber. Ges. Morph. Phys. Munchen 3. 1888. Zellenstudien. II. Jen. Zeitschr. 22 : 685-882. pis. 19-23. 1895. Ueber das Verhalten der Centrosomen bei der Befruchtung dea Seeigeleies, nebst allgemeinen Bemerkungen liber Centrosomen und Verwandtes. Verh. Phys.-Med. Ges. Wtirzburg, N. F. 29. Butschli, O. 1876. Studien iiber die ersten Entwicklungsvorgange der Eiselle, die Zelltheilung, und die Kunjugation der Infusorien. Senckenb. Naturforsch. Ges. 10. Calkins, G. N. 1898. Mitosis in Noctiluca miliaris and its bearing on the nuclear relations of the Protozoa and Metazoa. Jour. Morph. 15 : 711-770. pis. W 12. Campbell, D. H. 1902. Studies in the gametophyte of Selaginetta. Ann. Hot. 16: 419-428. pi. 19. Chambers, R. 1917. Microdissection Studies. II. The Cell aster. A reversible gelation phenomenon. Jour. Exp. Zool. 23: 183 504. 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, fertilisation 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 Morphologie der Centralspindel. Jen. Zeitschr. 28: 469- 474. 1895. Studien liber 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. Gard. 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 vegetales. 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 karyokinetiques. Ann. Mus. Nac. d. Buenos Aires. 1901. Les croisments des radiations polaires et l'interpretation dynamique des figures de karyokinese. Soc. de Biol. 53. 1906. L'interpretation 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. II. 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. UntersuchungeD iiber die chemische Beschaffenheil der Pflan- zengewebejmitBezugaufdieneuesten Arbeiten Fremya liber diesen GezenafcinH Jahrb. Wiss. Bot. 3 : 357-399. Klebs, G. 1886. Ueberdie Organization der Gallerte bei einigen Algen und FTagel- laten. Unters. Bot. Inst. Tubingen 2 : 333-418. pis. 3, l 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 ZeUteilun* I irch f. Entw. 46: 259-327. pis. 11, 12. figs. 21. Lawson, A. A. 1898. Some observations on the developmeni of the karyokinetic spindle in the pollen mother cell of Cobcea scandens. Proc ( lalif lead Sci I', t 111 1: 169-184. pis. 33-36. 1900. Origin of the cones of the multipolar spindle in Gladiolus Bol Gaz -JO • 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. Rov S„- Fdinb 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. Lutman, B. F. 1911. Cell and nuclear division in Closterium Bot Gaz 51- 401-430. pis. 22, 23. Lyon, 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 vegeHaux. ("our Rend. Acad. Sci. Paris. 107 : 144. 1889. Sur la presence des composees pectiques dans les vegetaux. Ibid. 109 : 579 1890a. Sur la substance intercellulaire. Ibid. 110 : 295. 18906. Sur les reactifs colorants des substances fondamentalea de la membrane Ibid. Ill: 120. 1891. Observations sur la membrane cellulosiq ue. Ibid. 113; lDii'.i. 1893. Sur l'emploi du rouge de ruthenium en anatomie vegetale. Ibid. 116 . 653. McClendon, J. F. 1910a. On the dynamics of cell-division. I. The electric chai on colloids in living cells in the root tips of plants. Arch. Entw Bl: 80-90 nl figs. 2. 19106. On the dynamics of cell-division. II. Changes in perm < -ability 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. An.it. u. Entw. 6: 285 390' 8~- 430-542. (Review.) von Mohl, H. 1853. Ueber die Zusammensetzung der Zellmembran aue I Ssern Bot. Zeit. 11 : 753-762, 769-775. 1858. Die Untersuchungen des Pnanzengewebes mil Bulfe des polaiisierten Lichtes. Ibid. 16: 373-375. 1 pi. Moltsch, H. 1888. Zur Kenntniss der Thyllen, nebsl Beobachtungen QberWund- heilung in der Pflanzen. Sitzber. k. Akad. Wiss. Wien, Math.-Naturw CI I 97 : 264-299. pis. 1, 2. Moreau, F. 1913. Les Karyegamies multiples de la zygospore '!<■ Rhizopua ttitjri- cans. Bull. Soc. Bot. France 60: 121 123. Morgan, T. H. 1899. The action of sail solutions on the unfertilized and fertilized eggs of Arbacia, and of other animals. Arch. Entw. 8: Ms 539. p]< - m 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 liber 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. Memoires sur les developpements des vegetaux. Paris. Pfeffer, W. 1892. Studien der Energetik. Prenant, A. 1910. Theories et interpretations physiques de la mitose. Jour, de l'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 on the chemical mechanics of cell-division. Ibid. 32 : 308- 313. 1913. Further explanatory remarks concerning the chemical mechanics of cell division. Ibid. 35: 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. Experimented 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. 62 : 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 ACHROMATIC FIGURE, CYTOKINESIS, AM) CELL WALL 201 1892. Schwarmsporen, Gameten, pflanzliche Spermatozoiden iind das Weeeo der Befruchtung. Ibid. 4: 49-158. pi. 3. 1897. Ueber Cytoplasmastrukturen, Kern- and Zellteilung. Jahrb. Wise. Bot. 30 : 375-405. figs. 2. 1898. Die pfianzlichen Zellhautc. Ibid. 31 : 534-598. pis. 15, Hi. 1907. Apogamie bei Marsilia. Flora 97: 123-191. pis. 3-8. Swingle, D. B. 1903. Formation of the spores in the sporangia of Rhizop pi- cans and of Phycomyces niiens. 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, 1G. Taylor, W. R. 1920. A morphological and cytological study of reproduction in the genus Acer. Contrib. Bot. Lab. U. of Pa. 5: 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 d cellules vegetales. Amsterdam. Watase, S. 1894. Origin of the centrosome. Biol. Lectures, Woods Hole. Wiesner, J. 1864. Untersuchungen liber das Auftreten von Pectinkorper in dem Geweben der Runkelriibe. Sitzber. k. Akad. Wiss. Wien, Math.-Xaturw. (1. 1150: 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. Xaturwiss. zu Wtirzburg. Williams, C. L. 1899. The origin of the karyokinetic spindle in Passiflora car ah n. Proc. Calif. Acad. Sci. Ill Bot. 1: 189-206. pis. 33-40. Wtilson, E. B. 1899. On protoplasmic structure in the eggs of echinoderms and some other animals. Jour. Morph. 15: 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: 166 191. 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. Butschli (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 those 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 Hoglcr (1901) the nucleus contains granules of chromatin but no nucleolus or nuclear membrane, division occurring by a simple form of mitosis. The coloring mailer exists in the form of mini, granules or cycmoplasts. Two other kinds of bodies are also present: albuminous slime globules and albuminous crystals [cyanophycin granul representing reserve food. KohFs (1904) description of the cell of Tolypothrix I Fig. 70 Is one of the most detailed which has been given in this group of research Kohl shows that the nucleus of this form has extensions reaching outward a \V. *'.'■' ■ ' ' — -* ft \m\ 0©? mill OOC Fig. 70. — Structure and division of the cell of Tolypothrix lanata. A, cell in the vegetative state: c, cytoplasm; n, nucleus;/, fat droplets; />. phycocyanin and chlorophyll granules; s, slime globules; g, granules of cyanophycin. />'. foul cell-division in Tolypothrix, showing transverse division of chromosomi diagram showing 6 stages of cell-division. (After Kohl, 1903.) toward the cell wall, and that they are withdrawn at the time of Qiiclear 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 Fig. 71. — Nuclear division in Oscillatoria Froelichia. 1, 2, 3, 4, four successive stages. {After Olive, 1904.) j Olive (1904) x 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- vides by simple constriction. The vegetative 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 from the nucleus of the higher plants, although it is relatively primitive in several features. In the cytoplasm he finds both cyanophycin 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 Cyanophycese up to 1904 is given by Olive. OTHER MODES OF NUCLEAR DIVISION 2<>:> network with chromatin granules at its nodes, and constricts simultai ously into a large number of daughter nuclei which pass fco the conidia. In Synochocystis aquatilis occurs the "primitive mitosis fl 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 Fig. 72. — The nuclei of various members of the Chroococcacese. A, cell of Chroococcus turgidus with scattered metachromatin granules (to) and plasma- tic microsomes (p) ; division beginning. X 2500. B, cell of Gloeocapsa with chromatic granules. C, M erismopedia elegans, showing two stages of nuclear division. X 1500. D. Chroococcus macrococcus: n, nucleus; m, metachromatin; v, vacuole. X 2-"><>i>. /.. 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 Chlorophyceae and the higher plants." In a more recent investigation of the Chroococcaceae Miss Act on (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 Gloeocapsa 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 Cyanophycese, 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 algaB, 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 stag we have an illustration of "the conception of cell structure which im- * " V B a f> '*%%&' m ,Vff; .■wflftrMp Tit} P*.t>< VSjil ■■m. Nuclear division in Coccidium schubergi. X 2250. {From Minchin, a v haudii C, mitotic division of micronucleus of Paramecium (horizontal figure on Bmaller 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 L919). 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 tropho chromatin 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 XCCLEAK DIVISION 209 The two kinds of chromatin now separate, the "trophochromatiE 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 splitsvinto daughter plates which apparently move to the poles and cooperateVith 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 al the close of the ana- phase the spindle connecting the two groups constricts and complel 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 larf nucleolus, the nuclear reticulum being very delicate and almost invisible in many preparations. According to Berghs (1906), Karstm Minis and Trondle (1912) all the chromosomes which appear in the propha 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, Marechal, 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 Char a (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 L920.) 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 revei 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.1 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 1)V the results of a number of investigations. For instance. Pfeffer (J899 and Nathansohn (1900) found that if Spirogyra filaments are placed in a J£ 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 cytologiste observed nuclear divisions that seemed intermediate in character hit ween mitosis and amitosis (Dixon in the endosperm of Fritillaria, L895; Sargant in the embryo sac of Lilium, 1896; R. Hertwig in Actinosphaerium, L898; Buscalioni in the endosperm of Corydnlis, 1898; and \\ asielewski in tin- roots of Vicia faba, 1902, 1903). Hertwig accordingly concluded that mitosis and amitosis are separated by no sharp boundary line, hut are connected by an unbroken series of transition stages. 1 In a second paper (1918) Nakahara gives a convenienl review of tin' literature of the subject. Fig. 7.">. Amitosis in internodaJ cell of Cham. 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. D F 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 hie 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 colls 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 role 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 mitotieally. Sakamura (1920) is of the same opinion. In a careful study of maturation and cleavage in Crcpidula pla> Conklin (1917) finds that the nuclei divide only by mitosi-. 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 chromosome and their failure to unite into a single nucleus; mitosis without cytokinesis, Lri\ ing cells with two or more nuclei; the failure of certain daughter chromosomi 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 <. inclusive 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 chloral ized 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 t ln- 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 thai descendants of these amitotically dividing nuclei become the nuclei of normally functioning gametes. To gain conclusive evidence for Buch an occurrence it would be necessary to trace the descendants of t he amitol ic- 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 stag 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 Chroococcaceae. Ann. Bot. 28: 433-454. pis. 23, 24. Arber, A. 1914. On root development in Stratiotes aloides L., with special reference to the occurrence of amitosis in an embrj^onic tissue. Froc. Caml>. Phil. Sue. 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. Buscalioni, L. 1898. Osservazioni richerche sulla cellula vegetale. Ann. [net. Bot. Roma 7. Butschli, O. 1890. Ueber den Bau der Bakterien und verwandter Organismen. Leipzig. 1896. Weitere Ausflihrungen iiber den Bau der Cyanophyceen und Bakterien. Leipzig. 1902. Bemerkungen iiber Cyanophyceen und Bakterien. Arch. Protiat. 1. Calkins, G. N. 1901. The Protozoa. New York. Carter, N. 1919. The cytology of the Cladophoracese. Ann. B<>t.33: 167 178. pi. 27. figs. 2. Chambers, R. 1917. Microdissection studies. I. Th- visible structure of the cell protoplasm and death changes. Am. Jour. Physiol. 43: 1 12. tii;-. 2. Child, C. M. 1907a. Amitosis as a factor in normal and regulatory growth. Anat. Anz. 30: 271-297. figs. 12. 19076. Studies on the relation between amitosis and mitosis. Ill VI. Biol. Hull 13: 138-160, 165-184. pis. 2 in. 1911. The method of cell division in Monezia. Ibid. 21: I'm) 296. figs. 16. Chodat, R. 1894. Contenue eellulaire des Cyanophyc€es. Arch. Sri. Phys. Math Geneve 11132: 637-641. Chun, C. 1890. Ueber die Bedeutung der direkten Zelltheilung. Sit/.!- 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 Botaniste 3 : 28-31. pi. 2. Des Cilleuls, J. 1914. Recherches sur la signification physiologique de l'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 Cyanophyceae. 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. Ball. 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 Synchytrimn. Bot. Gaz. 47 : 127-138. pis. 3, 4. Guilliermond, A. 1906. Contribution a l'etude cytologique des Cyanophyc6es. Rev. Gen. Bot. 18 : 392-408, 447-465. 1920. The Yeasts. (Engl, transl. by F. W. Turner.) N. Y. Gurwitsch, A. 1904. Morphologie und Biologie 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 teniceformis. 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 uber 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. Kemteilung und Geisselbildung bei den Schwann ern von Stemonitia flacdda Lister. Her. 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 ZcUkernc von Charafwlida. Bot. Zeit. 39 : 729-743, 745-7* 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 l><-i NephilU, 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 Ehrenbcrg. Zeit. Wise. Zool. 60: 215-235. pi. 11. von Knoche, E. 1910. Experimentelle und andere Studien am Lnsektenovarium. 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 Physiologie 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: 44."> 166. 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. 56: 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: 132- 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 cella of ina and its probable significance. Jour. Morph. 30 : 183-526. pis. 6. figfl 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. L57. Nowikoff, M. 1910. Zur Frage nach der Bedeutung der Amitose. Arch. Zellf. 5 : 365-374. figs. 2. Olive, E. W. 1904. Mitotic division of the nuclei of the Cyanophyce®. Beih. Bot. Centr. 18: 9-44. pis. 1, 2. 1907. Cytological studies on Ceratiomyxa. Trans. Wis. Acad. Bei. 15: ,.".1 T?:^. pi. 47. Patterson, J. T. 190S. Amitosis in the pigeon's egg. Anat. An/. 32: 117 125. figs. 24. Pfeffer, W. 1899. Bericht iiber amitotische Kerntheilung. Ber. d. Math. -PL; 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 Taenia. 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. Saguchi, S. 1917. Studies on ciliated cells. Jour. Morph. 29: 217-279. pis. 1- 4. Sakamura, T. 1920. Experimen telle Studien iiber die Zell- und Kernteilung mit besonderer Rucksicht 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 OsciUatoria and Tolypotkrix. 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 Cyanophyceae. (Prelim, note.) Proc Roy. Soc. London 72: 401-408. figs. 3. von Wasielewski, W., 1902, 1903. Theoretische u. Experimen telle 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 Plasmodiophoracese. 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. \ oung, R. T. 1913. The histogenesis of the reproductive organs of Taenia 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. 2]/ 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. CHAPTEB XI THE REDUCTION OF THE CHROMOSOMES The subject of chromosome reduction is one of the mosl 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 reductioD 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 fad 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 chromosom 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 ;md spermatozoon of A scaris each contain one-half the number <>!' 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 w effecU ens 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 homceotypic mitoses of other organisms, and that the third division is purely vegetative or equational in character. As instances may be cited the work of l'aull I 1905, 1912 1 For a review of sexual reproduction and alternation of generations in tin- algffi see Bonnet (1914). Davis (1916) gives a convenienl summary of tin- life histories of the red algie. Dodge (1914) summarizes and compares the life histories of red alga? and asconivccU's. See Atkinson I I'M.", for a complete review <>t' researches on ascomycetes. For the cytology of the yeasts ruilliermond 1920. Fio. 78. .1, prophase <>f heterotypic division in the tetrasporocyts of /' o I ys i }> h i) ri ia . (After Yamanouchi, 1906.) Ji, proph of heterotypic mit in < oogonium of I (After Yatnai L909.) 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- Fig. 79. — Sexual fusion and maturation divisions in the basidium of Nidularia piriformis . a, two sexual nuclei about to unite, b, 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 Polytrickum 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). A list of numbers of nuclei in the cells of basidiomycetes is given by Levine (1913). THE REDTCTIOX OF THE CHROMOSOMES 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-colls"), 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 arc therefore the primary spermatocyte and the primary oocyte. In most green algffl the gonotokont is the zygote; in the red alga' it is usually the tetrasporo- cyte; in the ascomycetes it is the ascus; in the basidiomyeetes il Is the basidium; and in the bryophytes and vascular plants i1 is the Bporocyfc the microsporocytc 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 mam points of a theory first suggested by Roux I L883) 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 In,. 8 0 — M e k' :i M> >> r «' tetrads in Angiosperms. .1 , tetrad of walled cells in Phy.soslegia viTffiniana', formation of two upper ones just being completed. X 462. {After sharp, 1911.) B, tetrad of megaspore nuclei in Lilium canad* 15 226 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 b}r 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 ^his 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.'1 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 w7as thrown on the problem when Henking (1891), Rtickert (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 pair- oi chromosomes, or hivalent chromosomes, each arising b}r an end-to-end conjugation (synapsis) of two somatic chromosomes. The two parts oi 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 thai 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 thai 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 tin- 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 <>t bivalent pairs, but that both maturation divisions are apparently longi- tudinal (Flemming, Brauer 1S93, Moore L896, Meves L896, Gregoire, etc.). Howt, 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. SOMATIC MITOSIS — EQWATIONAI-- <:-— J> HETEROTYPIC MIT0SI3 - REDUCTIONAU - 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 supposi d to diffi r 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 nucL ust 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 •- EQUATlONAL - 9W 1. Explanation in text. Scheme A. — The first of the two main interpretations of reduction came into prominence in 1900 and shortly after, when von Winiwarl (1900), Gregoire (1904, 1907, 1909), A. and K. E. Schreiner (1904 L908 . and Berghs (1904, 1905) applied it to the phenomena observed by them in several animals and plants. It- essential points are a- follows Figs. 83-88) : At the beginning of the heterotypic prophase the nuclear reticulum, without breaking down into such distinct elementary aets or alveolar units as are seen in the somatic prophase, take- the form of long slender threads (leptothic or leptonema stage).1 During the very early prophi 1 The terms Uptotene, synaptene, pachytene, and there is some enlargement of the cell and nucleus during the stages just describee I, but the chromosomes pass directly from the strepsinema stage to diakinesis. During the relatively enormous growth of the oocyte the chromosomes, which have usually reached the strepsinema 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, is 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 Phrynntf ttix ma . .1. Leptonema; x, chromosome. B, conjuga- tion of " chromosome l . Portions of other uncon- jugated threads ami one Hi her bivalent also pn i n Beef ion. X 21 After Went cA, 1911 on the other hand, 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). i Fig. 86. A, parasynapsis in Allium fistulosum. B, parasynapsis in Osmunda regalis. 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 J^P Fig. 87. — Nuclei from microsporocytes of Viria faba, showing parasynapsis. Synigesis beginning in No. 6. X 1900. Fig. 88. — Heterotypic prophases In spermatocyte of Tomopteris A, pairing of leptotene threads beginning. />'. pairing complete in some threads and only beginning in others. C, conjugation complete; pachynema stage. D, resplitting of pachytene threads (separation of conjugated chromosomes l l. mul K 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 homceotypic 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. Gregoire 1904, '07 Berghs 1904, '05 Rosenberg 1905, '07, Allen 19056c J. B. Overton 1905, '09 Strasburger 1905, '07, Miyake 1905 Tischler 1906 Cardiff 1906 Lagerberg 1906, '09 Yamanouchi 1906, '08, Martins Mano 1909 Lundegardh 1909, '14 Frisendahl 1912 McAllister 1913 Schneider 1913, '14 Weinzieher 1914 Sakamura 1914 de Litardiere 1917 Plants Lilium, Allium, Osmunda Allium, Drosera, Helleborus, etc. '08, '09 Drosera, Compositse Lilium, Coleochcete Thalictrum, Calycanthus, Richardia '08, '09 Lilium, Galtonia, etc. Lilium, Funkia, Iris, Allium, Trades- cantia, Galtonia Ribes Acer, Salomonia, Botrychium, Ginkgo Adoxa '10 Polysiphonia. Nephrodium, Osmunda Funkia Trollius Myricaria Smilacina Thelygonium Xyris Vicia Polypodium THE REDUCTION OF THE CHROMOSOMES 23' 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 M alone 1918 Pratt and Long 1917 Robertson 1916 Rabbit, Man Tunicates, Selachians, Teleosts, Am pin- ox us Tomopieris, OphryotrochOf Zodgonui, Enteroxeno8t Myxine, Salamandra^ Spinax ( 'yclojM Thy8anozoon ZoogoniiA Batracoseps Alytes Planaria Dytiscus Euschislus Hersilia, Enchenopa Phrynotettix, Chorthipptu Cambarus, Cancer Canis Mus Insects RUT HlTCROTIfll niTOSIJ if-UKI) CONTRACTION Honorific riiioiti ;u» sriun ».»«l«tJli Fig. 89. — The method of chromosome reduction according B. Explanation in text. Scheme B. — The second of the two conspicuous interpretations was advanced by Fanner and Moore (1903, L905), and i ntially as follows (Figs. 89-92): In the early heterotypic prophase the reticulum becomes more thready in structure and contracts into a tight knol (synizesis). When this knot loosens up tin' 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 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. ■ 2) m , ■ - ' -s, 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 homceotypic 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 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 telosynapticaUy {metasyndetic- ally) either while the spireme was being formed or when the daughl 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 bivalenl chromosomes, with their split obscured, become much shortened and thickened (diakinesis) and take up their positions on the firsl maturation spindle. In case the original split, instead of being wholly obscured, in- 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 homceotypic 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. Farmer and Moore Farmer and Digby Farmer and Shove Mottier Gregory Lewis Schaffner Digby Fraser Lawson McAvoy Beer Woolery Nothnagel Farmer and Moore Montgomery Moore and Embleton Griggs Zweiger H. S. Davis Nakahara Plants 1903, '05 Lilium, Osmumla, PxUotum, Aneura 1910 Galtonia 1905 Tradescantia 1907, '09, '14 Lilium, Acer, Allium, Podophyllum, Tradescantia, Staphylea 1904 Ferns 1908 Pinus, Thuja 1906, '09 Agave 1910, '12, '14, '19 Galtonia, Primula^ Crepis Osmunda 1914 Vicia 1912 Smilacina 1912 Fuchsia 1912, '13 Equisetum, CrepiSy Tragopogon 1915 Smilacina 1916 Allium Animals 1905 Periplanela, Elasmobrancha 1903, '04, '05, '06, Hemiptera, Amphibia '10 1906 Amphibia 1906 [scoria 1907 Forficula 1908 Insects 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 Os?nunda (see Fig. 92), that the lateral pairing of thin threads in the LAST PRCMtlOTIC MITOSIS HETEROTYPIC MITOSIS : A ANAPHASE SYHIZE.SIS "HOLLOW SPiTtEM" COIOUC'ATION SE.COM3 CONTRACTION 0 V IlMillS.'- ANftTHftStS TELOPHASE («ew sflit) LATE TELOTHAS HonoTVPic niTosis (70 ©. 0 ^> < > d) $r£ MtT«THAS£ ftWflPHflSE TtLOTKflSE 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. 6, 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 24] telophase, and after being temporarily obscured function- in the post- homceotypic division.1 A variation of Scheme B has been observed in (Enothera I rates L908, 1909, 1911; Geerts 1908; B. M. Davis 1909, 1910, L911); 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, an. I Fig. 93. — Diagram showing distinction between Schemes A and H. See text. divide longitudinally (equationally) at the second mitosis, so thai the final result is essentially the same: two of the resulting four nuclei differ qualitatively from the other two in their chromatin content (Fig. '.' 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 Bimple 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 § review <>f Miaa Digl paper on Osmunda by the present author (1920(/ 16 242 INTRODUCTION TO CYTOLOGY case of chromosome tetrads) the split that is to function in the homceotypic mitosis makes its appearance. The doubleness 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, bu1 also thai 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 docs the split develop directly from the early reticulate condition, or thai the telophasic alveolation, though irregular, may not later become so equalized as to constitute the firsl stages of the split; but it does follow thai it is quite unsafe to use the principle of telophasic splitting as a premise from which to draw tin- 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 th< Fig. 94. — Chromosome pair "B" in Phrynotcttix magnus, showing condensation of bivalent pair during the heterotypic prophases to form the compact chromosomes appearing on the spindle at metaphase. >! 1734. (After Wenrich, 1!»16.) 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 firsl 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 L916) as the result of his detailed analysis of the chromosome groups in other Tettigidffi and Acrididae, in which the homologous members can be followed with much certainty because of their frequenl 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 stag* The four chromatids composing the tetrad represent two conjugate. 1 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) for 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. D E >J> S" 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 minis, 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 Till-: CHROMOSOMES 245 mitosis and separate into two haploid groups at the second. To this simple form of reduction Goldschmidl applied the term " Primsertypus." Gregoire (1909a), on the contrary, found parasynapsis and the usual mode of reduction in Zodgonus. The interpretation at one time given by Ruckeri (1893, 1894 Haecker (1895), and vom Rath (1895) for certain copepoda is shown in Fig. 95, E. The continuous spireme splits throughoul 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 oul 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 ai the second mitosis, which is therefore reductions!. In Dicroccelium Gold- schmidt (1908) reported that such tetrads divide reductionally at the first mitosis. Lerat (1905), moreover, has found that in Cyclops strenu 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 I 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 1 1902 Sutton (1902, 1905), Robertson (1908), and others, such tetrads separate reductionally at the second mitosis (postreduction) rather than af 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 iJ, Ci), two chromo- somes conjugate parasynaptically while in the form of slender threads. Instead of remaining unsplit as in most plants, each member then -pin- longitudinally in a plane at right angles to the conjugation plane, thus giving a tetrad composed of four parallel strands (chromatids l> . According to Scheme B (A2-C2), the two chromosomes are at firsl ar- ranged telosynaptically in the spireme and t he lat ter splits t hroughoul it- 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 strand- D The tetrad, by whichever met hod it has arisen, may now undergo a variety of alterations, some of which are shown at /•.' and /'. Tin- chroma- tids may simply shorten and t hicken, t he tel rad at diakinesis maintaining the form of parallel rods (Ei,Fi). They may open out along the plane of 246. INTRODUCTION TO CYTOLOGY conjugation (E2) and take the form of rod tetrads (F2) like those described by Ruckert 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 (Ai-Ci) and Scheme B (A2-C2), and the further transformation of this tetrad into tetrads of five types (Fi-Fb) . 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 CHR0M0S0M1 247 If the conjugated chromosomes remain in contacl al both ends /. : a complete ring results (F4). In certain orthopterans the four chromatids open out along the conjugation plane in some regions and along the plain' of splitting in other regions; this results in the curious compound rii (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 (#5), the tetrad then assuming 1 1n- form of a ring composed of four parts (F-t). 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. STKtPil«tM/« ©Q Fig. 97. — Reduction with chromosome tetrads in Fasdola h, patica, according to Schellenberg (1911). Explanation in text. The four chromatids composing the completed tetrad are in most <• exactly similar in appearance, so that it is a matter of much difficulty to determine along which plane they arc separated at the firsl maturation mitosis. According to the two theories of tetrad origin illustrated in the foregoing diagram, however, the chromatids are supposed in aim* all cases to separate along the plane of conjugation at tin- 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 Sch<>]l(M)l)er- I L91 1 1 for the parasitic flatwopm, I ola 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 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: Buch "tetrads" are seen not only in the oocytes and spermatocytes bul 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 # i at * A Fig. 98. — Chromosome tetrads. A, five stages in the development of the tetrad in the spermatocyte of X 3000. From a preparation by Dr. H. E. Stork. B, tetrads in sporocyte of Chii Enlarged; X 2800. (After Florin, 1918.) C, tetrads in Richardia afrieana. Overton, 1909.) D, false tetrads in somatic cells of Pisum due to action ol chloral hydi 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 Baecker in the copepods constricted chromosomes. The constrictions, according to this writer, represent points of temporary union between non-homologoua element From these considerations it is evident thai 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 Kick | L907, 250 INTRODUCTION TO CYTOLOGY 1908), Meves (1907, 1908, 1911), Giglio-Tos (1908), and Granata (1910), reject the theor}- 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. .i fin (0) (? B C kA © 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 Bonnevie; 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 CHR0M0S0M1 25] An interpretation put forward by Bonnevie (1906, L908) is shown In Fig. 99, C. Here the chromosomes conjugate parasynaptically and come into very intimate union: although fchey appear to undergo a real fusion their identity is maintained for a time. Owing to the fact thai these bivalent chromosomes are inserted upon the Bpindle 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 tin 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 !><• directed to three points: the relationship of the conjugating memfo (the "synaptic mates"), the stage at which the synaptic union taki - place, and the exact nature of this union. Relationship of the Synaptic Mates.— We may first inquire into tin 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 wit h any other chromosome, or does the pairing take place according to more restricting nil It was suggested by Henking (1891) that the two synaptic mat.- 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 fust 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 L909 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 Lamar xkiana (seven chromosomes in gamete) with CE. 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 poionicum (14 chromosomes in gamete) with T. spelta (21 in gamete) there are present in the heterotypic prophase 14 bivalents (poionicum 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 coworkers 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 4^ Fig. 100. — Heterotypic mitosis in Triticum polonicum X T. *j»lta. 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 B magna. {After Sutton, L902. In Najas marina and Najas major (Muller l'.tll; 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 SPORES - / THOBuCt r MMETOFttlTtS An£ f t*ntT£i with 3Anl CHhonoiOMt SET £0DT CLLL SPOROCYTC VICIA FABA SIMILAR PROCESS IN P CREPIS VIRLNS SIMILAR PROCESS I Fig. 102. — Chromosome cycles in Vicia faba and Crepis virens, showing homologous pairing. and pass to the daughter nuclei, bringing about reduction. At the close of the homceotypic mitosis the microspore, and hence the male gamete to which it later gives rise, receives a simplex group of six chromosomes: one long and five short. Since the somatic cells contain each of these in duplicate it is evident that a similar set is contributed by the female gamete. Summing up, we may draw from the above facts certain very impor- tant conclusions: (1) Each parent furnishes the offspring with a set of chromosomes, the members of the two sets being intermingled in all the nuclei of the new individual. This point will receive further attention in the following chapter on fertilization. (2) The two members of each bivalent chromosome formed at synapsis are derived one from each parental set. (3) Each chromosome of the paternal set conjugates with a particular chromosome of the maternal set: the two are in some sense homologous. THE REDUCTION OF THE CHROMOSOMES It should be pointed out thai cytologists and genet Icisl - have generally assumed that each synaptic pair is independent of all the others as regards the manner in which it is oriented on the heterotypic spindle. In some pairs the paternal members are directed toward one pole and in other pairs toward the other pole. It is conceivable that in Bome all the paternal members might go to one pole and all the maternal members to the other. Direct evidence that the assortment of the various chromosome pairs is in this respeel a random one :i- originally assumed has been furnished by Miss Carothers (1913, L917 . In the grasshopper, Trimerotropis, she finds that the components of some of the bivalents are visibly different in size, in the mode of attachment to the spindle fibers, and in the presence of constrictions; and that these differ- ences make it possible to show beyond question that the several pairs an- entirely independent of one another as regards their orientation on the spindle and their consequent distribution to the daughter cells. From the precise manner in which the distribution of chromosomes at the time of reduction and at other stages of the life cycle parallels the distribution of the hereditary characters it is inferred that Buch hom- ologous chromosome pairs represent the material basis for the allelo- morphic pairs of Mendelian characters exhibited by the organism. This subject is to be taken up in Chapter XV. The Stage at Which Conjugation Occurs. — In the great majority of observed cases chromosome conjugation occurs during the prophi of the first maturation division. Since the chromatin threads at some time during these prophases usually take the form of a tightly contracted knot out of which they emerge in an obviously double condition, it was suggested (Moore 1896) that the contraction is an important factor in bringing about the conjugation, and the contraction itself came t<» be called "synapsis." But an examination of the various modes of reduction shows that the conjugation may begin very early, before the contraction (Fig. 83) or, on the other hand, not until the spindle is established (Fig'. 95, D). The conjugation of the chromosomes is therefor.- to be distinguished from the contraction. It has now become customary to refer to the former, at whatever stage it occurs, as synapsis, and to the latter as synizesis. In an increasing number of reported cases the paired a — .nation apparently begins even before the heterotypic prophase. The dim mo- somes have been observed in several instances to undergo pairing during the anaphase and telophase of the hist premeiotic division. Such is the condition in certain Hemiptera (Montgomery L900, 1901 . Onis* (Nichols 1902), Brachystola (Sutton L902), Scolopendra (Blackman 1903, 1905), Pedicellina (Dublin 1905), and a number of more recent cases. Furthermore, the pairing has been stated to begin in the sperm- atogonia several cell generations before maturation in certain Hemiptera 256 INTRODUCTION TO CYTOLOGY and Ascaris (Montgomery 1904, 1905, 1908, 1910), Alytes (Janssens and Willems 1909), Helix and Sagitta (Stevens 1903; Ancel 1903), certain Diptera (Stevens 1908, 1911), and Pediculus (Doncaster 1920). More recently it has been shown that the homologous chromosomes may begin to show a paired arrangement even earlier in the cycle, in some cases directly after the parental groups are brought together at fertilization. In the Diptera, for example, Metz (1916a) has shown that the association, which at certain stages is so close as to constitute a synapsis, begins before the cleavage of the fertilized egg, and that the paired condition is maintained in all cells, somatic and germinal, through- out the life cycle. Metz examined 80 species and in all of them found such a somatic pairing. In Culex (Stevens 1910, 1911; Taylor 1914, 1917), which has six chromosomes, the association can be seen in the nuclei of the segmenting egg, and in the early larval stages there follows an actual parasynaptic fusion, so that the somatic cells thereafter show three bivalent chromosomes rather than six univalents. In the matura- tion divisions the members of each pair separate, the gametes receiving three chromosomes each, just as they would had conjugation begun in the heterotypic prophase as usual. A loosely paired arrangement of the chromosomes in the somatic cells of plants has been reported by Strasburger (1905, 1907, 1910) for Galtonia candicans, Funkia Sieboldiana, Pisum sativum, Melandrium, Mercurialis, and Cannabis; by Sykes (1908) for Hydrocharis, Lychnis, Begonia, Funkia, and Pisum; by Overton (1909) for Calycanthus; by Muller (1909, 1911) for Yucca and other forms; by Stomps (1910, 1911) for Spinacia; by Kuwada (1910) for Oryza; by Tahara (1910) for Morus; and by Ishikawa (1911) for Dahlia. This is another matter that will be considered further in Chapter XII. The Nature of the Synaptic Union. — Because of the manner in which chromosome behavior is at present being applied to the solution of the problems of inheritance, no question concerning chromosome conjugation is more important than that of the exact nature of the synaptic union. In reviewing some of the opinions of this subject it will be convenient to list separately the views of the telosynaptists and the parasynaptists. In such cases as those described by Henking and by Goldschmidt Fig. 95, D) there is only a momentary end-to-end association of the fully formed chromosomes on the spindle of the heterotypic mitosis, there being no real fusion and almost no opportunity for an " interchange of influences." In the other tetrad chromosomes formed by telosynapsis (Fig. 95, E and F; Fig. 90) there is only slightly greater opportunity for such interchange. According to Scheme B (Fig. 89) the synaptic mates are at first arranged end-to-end, and only later, when partially condensed, do they take up a. side-by-side position, allowing a more intimate and extensive union for a short time. THE REDUCTION OF Till-: CHROMOSOMES 257 Generally speaking, the parasynaptists have given more attention to the details of the synaptic union than have the telosynaptisl Al- though cases are on record in which there is only a momentary para- synaptic association of fully formed chromosomes (von Vosa l'.»l 1 the association usually extends over considerable time. Most para- synaptists hold that the conjugation begins with the association of the leptotene threads before or during synizesis, continues through the remainder of the prophase, and ends with the anaphasic separation (Scheme A). The association of the synaptic mates is thus long and intimate. Concerning the closeness of the union, however, opinions differ widely. A few investigators (Vejdowsky 1907; Bonnevie 1906, 1908, 1911; Winiwarter and Sainmont 1909; Schneider 1914) have thoughi that tin- conjugating members fuse completely and lose their individual identity. the "mixochromosome'' so formed then undergoing two true longitudinal splits along new planes at the twTo maturation divisions. In some <•. (Bonnevie; see p. 251) the fusion may not be fully consummated until during the post-meiotic divisions. Others believe the split for the heterotypic mitosis to be along the plane of conjugation (Cardiff 1906, Fasten 1914, and others). Probably the most widely advocated view is that there is no actual fusion of the synaptic threads, the latter main- taining their identity completely. Although their association may at times be so intimate that they seem to constitute a single thick thread, the doubleness, if thus lost to view, reappears during later stages I terghs 1904, 1905; A. and K. E. Schreiner 1905, 1906; Marechal 1907; Overton 1905, 1909; Robertson 1915, 1916) (Fig. 88). Several careful observers have reported that the doubleness can be seen at all stages (Gregoire 1907, 1910; Schleip 1906, 1907; Montgomery 1911; Kornhauser 191 1. 1915; Wenrich 1915, 1917). Gregoire, who has argued strongly for this interpretation, has emphasized the ease with which the closely appressed threads may be mistaken for a single thick structure. One of the most important suggestions which has been made concern- ing chromosome conjugation is embodied in the lt Chiasmatype Hypo- thesis" of Janssens (1909). According to Janssens, the pairing threads, though remaining separate throughout the greater part of their length, fuse at one or more points as they twist about each other. When they again separate a break occurs at each of these fusion point-, but along a new plane, so that each of the two resulting chromosomes is composed of portions of both conjugating members (Fig. 1 19 This interpreta- tion, which has been admitted as possible by several of the investigate named in the preceding paragraph, is significant in that it shows how an orderly evolution of chromosomes with new constitutions may'occur, a point of great importance in connection with current conceptions of tic 17 258 INTRODUCTION TO CYTOLOGY physical basis of heredity. Special attention will be devoted to this question in Chapter XVII. Chromomeres.— An important role has been attributed to the chro- momeres by many students of synapsis. Allen (1905), for example, maintained that the fusion of the leptotene threads in Lilium involves a fusion of their chromomeres, the subsequent division of the fused chro- momeres initiating the resplitting of the pachytene thread. Allen found the chromomeres to be composed of still smaller chromatic elements, and offered various suggestions concerning the manner in which the re- splitting of the pachytene thread might be supposed to effect a redistri- bution of the "idioplasms." That chromosome conjugation is primarily a conjugation of small chromatic elements within the chromosome was held by Strasburger (1904, 1905) and the Schreiners (1906). The visible chromatin granules, or "pangenosomes," were conceived by Strasburger to represent complexes of "pangens" such as were postulated by de Vries, conjugation involving an interchange of these latter units. The chromomere ^ interpretation has been adversely criticised by Gregoire (1907, 1910) on the basis of further evidence obtained from a study of the chromatic structures themselves. This author points out several serious objections to the view that the chromomeres are auton- omous bodies, and concludes that they are rather to be regarded simply as swellings or thicker portions of the chromatin thread, such thick por- tions remaining as the thread undergoes a stretching which is not uni- formly resisted at all points. Their frequently striking correspondence or paired arrangement in the synaptic threads is explained as the result of the response of the two closely associated threads to the same stretch- ing force. This interpretation is also shown to account for the variability in the dimensions of the chromomeres, their tapering form, the often reported absence of correspondence between the chromomeres of the two threads, and various other aspects. Wenrich (1916, 1917), on the other hand, has found that in Phrynotettix (Fig. 155) the chromomeres show a remarkable individual constancy in size and position in a given member of the chromosome complement, not only in the various cells of a given individual, but also in those of different individuals. These facts strongly suggest an autonomy of the bodies in question. Because of their great theoretical importance (see Chapter XVII) it is to be regretted that after such a large amount of research so many points regarding the process of synapsis should remain in such an un- settled state. It is hoped that further refinements in microtechnique may remove some of the obscurity which at present surrounds them. OTHER OPINIONS ON THE HETEROTYPIC PROPHASE Although the phenomena of the heterotypic prophase, particularly synizesis and synapsis, are generally looked upon as normal occurrences THE REDUCTION OF THE CHROMOSOMES of considerable significance, not all investigators concur in this opinion. That synizesis is an artifact due t<» Faulty fixation is an interpretation which, though it may be justified for certain cases in which the contrac- tion may be very slight or absent, is doI of general application. Fixa- tion often serves to accentuate the appearance of contraction, bul the characteristic synizesis figure has been observed widely enough in faith- fully preserved, and even in living material to make it evident that we are dealing here with a normal feature of the heterotypic prophat It may not, however, be of universal occurrence. R. Hertwig (1908) came to the conclusion, as a deduct ion from his theory of the nucleoplasmic relation, that the phenomena of the hetei typic prophase represent an abortive mitosis: the disturbed nucleoplasmic balance is restored to the normal by a multiplication of chromat in without an actual mitosis, the process taking the form of the changes peculiar to the heterotypic prophase. This view of Hertwig, which was denied by Gregoire (19096), is supported by Kingsbury and Hirsch (1912), who state: "According to this view, on the one hand, synizesis represents 'an attempt on the part of the spermatogonia to divide again — which fail-: while on the other hand, the reputed conjugation of chromosomes occurring at about this time i- but the imperfect fission and subsequent fusion of daughter chromosomes of Buch abortive division." The above quoted authors regard synizesis and synapsis as indicat i' of the onset of degeneration. In this conclusion they are supported by Kingery (1917), who, in his investigation of the white mouse, finds synizesis in the primitive germ cells which degenerate, but not in the definitive germ cells. Observations of a similar nature were made by Wodsedalek (1916) in the mule. If, as Kingery (1917) and Popoff (1908) point out, the "heterotypic'1 changes are due to degeneration, they should be found in abnormal somatic cells. Marcus 1907 . in fact, had observed a contraction similar to that of synizesis preceding degeneration in the cells of the thymus gland. Nemec * r.»o:>i and Kemp (1910) also found that in the cells of roots treated with chloral hydrate the nuclei come to have an abnormally high number of chromoson ("syndiploid nuclei"), this number, according to Nemec, being gradually restored to the normal during tin1 subsequent mite--, which -hew phenomena of a heterotypic nature. Strasburger 1 191 1 I, while agreeing with Nemec that the syndiploid condition gradually disappears, denied that any truly heterotypic phenomena are concerned. The 'hetei typic" changes observe* 1 by Xemee he held to be only peculiar vegetative mitoses with a superficial resemblance to genuine reduction divisioi Nemec's conclusion regarding a reduction in chloralized vegetative cells is also contradicted by Sakamura (1920), who has made a particu- larly exhaustive study of these phenomena. Sakamura finds that a 260 INTRODUCTION TO CYTOLOGY variety of agencies/including chloral hydrate, benzene vapor, ether, chlo- roform, and the gall-producing secretions of Heterodera, may be employed to bring about aberrations of the mitotic process. After the chromosomes are divided and partially distributed they may be reorganized in a single :'didiploid': nucleus. In other cases the chromosomes may reorganize as two or more nuclei with various chromosome numbers, and these may often fuse to form "syndiploid" nuclei. To all the kinds of stimuli applied the chromosomes react by becoming shorter and thicker, and thus appear like heterotypic chromosomes. Furthermore, latent or obscure constric- tions are rendered more conspicuous, so that some of the split chromo- somes appear like chromosome tetrads. Sakamura shows that these false tetrads do not represent heterotypic phenomena in any true sense: they are merely the result of the response of split and constricted chromosomes to the abnormal conditions induced in the cell, and have nothing to do with any reducing process. No such autoregulative reduction occurs in these didiploid cells, their gradual decrease in relative number being due to their lowered capacity for, and rate of, division. Child (1915) emphasizes the physiological significance of maturation, and shows that the heterotypic phenomena are associated with a low metabolic rate in the cells, that they may occur occasionally in other cells having a low rate, and that they can be induced artificially with narcotics as Nemec stated. All of these observations are interesting in that they indicate the nature of some of the physiological changes occurring at the time of matur- ation. The description of the heterotypic phenomena upon which will be based our ultimate interpretation of its significance, will not be com- plete until the physiological as well as the morphological changes have been exhaustively examined and correlated. But because it has been found that the onset of the meiotic process is associated with a lowering of the rate of metabolism which, if continued, may result in degeneration; or because appearances similar to those of the heterotypic prophase may occur in other cells with disturbed metabolism; it does not at all follow that the heterotypic phenomena are at bottom phases of a degeneration process, or that they have no other significance in the normal life cycle. These phenomena occur almost universally throughout the whole world of living organisms at a very critical stage in the life cycle and lead to significant results with a high degree of regularity. The lowered rate of metabolism accompanying them offers in the vast majority of cases no check to the normal functioning of the products of the maturation di- visions. It therefore seems more reasonable to regard the observed degeneration as a secondary effect that may occasionally set in during the normal heterotypic prophases because the metabolic rate is already at a relatively low level at that time, than to look upon the heterotypic changes as a part of a degeneration process which is only exceptionally THE REDUCTION OF THE CHROMOSOMES 261 completed — unless, indeed, all changes in the organism which are accom- panied by a fall in the metabolic rate be regarded as degenerative in character. Bibliography 11 Chroviosaim lt', iliicti,, a Allen, C. E. 1904. Chromosome reduction in LUium canadense. Bot. Gaz. 37: 464-469. 1905a. Nuclear division in the pollen mother cells of LUium canaden e. Ann. I 19: 189-258. pis. 6-9. 19056. Das Verhalten der Kernsubstanzen der Synapsis in den Pollenmutter- zellen von LUium canadense. Jahrb. Wiss. Bot. 42: 72-82. pi 2 1905c. Die Keimung der Zygoten bei Coleochcnte. Ber. Den. Bot. Gee 23: 285 292. pi. 13. Ancel, P. 1903. Histogenese et structure de la glande hermaphrodite . 1904. Ergebnisse iiber die Konstitution der chromatischen Kernsubstani. Jena. 262 INTRODUCTION TO CYTOLOGY Brauer, A. 1893. Zur Kenntniss der Spermatogenese von Ascaris megalocephala. Arch. Mikr. Anat. 42: 153-212. pis. 11-13. Calkins, G. N. 1897. Chromatin-reduction and tetrad-formation in Pteridoptrytes. Bull. Torr. Bot. Club 24: 101-115. pis. 295, 296. Carothers, E. E. 1917. The segregation and recombination of homologous chromosomes as found in two genera of Acrididae (Orthoptera). Jour. Morph. 28: 445-521. pis. 14. figs. 5. Carruthers, D. 1911. Contributions to the cytology of Helvetia crispa Fries. Ann. Bot. 25: 243-252. pis. 18, 19. Child, C. M. 1915. Senescence and Rejuvenescence. Chicago. Claussen, P. 1912. Zur Entwicklungsgeschichte der Ascomyceten. Pyronema confiuens Zeitschr. f. Botanik 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. Dahlgren, K. V. O. 1915. Der Embryosack von Piumbagella, ein neuer Typus. Ark. f. Botanik 14: 1-10. figs. 5. Davis, B. M. 1909. Pollen development of Oenothera grandiflora. Ann. Bot. 23:551-571. pis. 41, 42. 1910. Thereduction division of (Enothera biennis. Ibid. 24: 631-651. pis. 52, 53 1911. Cytological studies on (Enothera. III. A comparison of the reduction divisions of (Enothera Lamarckiana and. (E Gigas. Ibid. 25 : 941-974. pis. 71-73. 1916. Life histories in the red algse. Am. Nat. 50: 502-512. Davis, H. S. 1908. Spermatogenesis in Acrididse and Locustidse. Bull. Mus. Comp. Zool. Harvard Coll. 53: 159-212. pis. 9. Debaisieux, P. 1909. Les debuts de l'ovogenese dans le Dytiscus marginalis. La Cellule 25 : 207-236. pis. 2. ,Deton, W. 1908. L' "etape synaptique" dans le Thysanozoon Brochii. La Cellule 25:133-146. 1 pi. Dietel, P. 1911. Versuche liber die Keimungsbedingungen der Teleutosporen einiger Uredineen. Centr. Bakt. II 31: 95-106. Digby, L. 1910. The somatic, premeiotic, and meiotic nuclear divisions of Galtonia candicans. Ann. Bot. 24: 727-757. pis. 59-63. 1912. The cytology of Primula Kewensis and of other related Primula hybrids. Ibid. 26 : 357-388. pis. 41-44. figs. 2. 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. Dodge, B. O. 1914. The morphological relationships of the Florideae and the ascomycetes. Bull. Torr. Bot. Club 41: 157-202. figs. 13. Doncaster, L. 1920. On the spermatogenesis of the louse (Pediculus corporis and P. capitis), with some observations on the maturation of the egg. Quar. Jour. Micr. Sci. 64: 303-328. pi. 16. 1 fig. Dublin, L. I. 1905. The history of the germ cells in Pedicellina americana (Leidy). Ann. N. Y. Acad. Sci. 16: 1-64. pis. 1-3. Duesberg, J. 1908. La spermatogenese chez le rat (Mus decumanus Pall., varietc albinos). Arch. f. Zellf. 1: 399-449. pi. 10. 1 fig. 1909. Note complementaire sur la spermatogenese du rat. Ibid. 3: 553-562. Farmer, J. B. 1894. Studies in the Hepaticse: On Pallavicinia decipiens. Ann. Bot. 9: 35-52. pis. 6, 7. 1895. On spore formation and nuclear division in the Hepatica?. Ibid. 9: 469-523. pis. 16-18. THE REDUCTION OF THE CHROMOSOMES 263 Farmer, J. B. and Williams, J. L. 1898. Contributiona to our knowledge of the Fucaceae; their life history and cytology. Phil. Tran R - London R 190: G23-645. pis. L9-24. Farmer, J. B. and Moore, J. E. S. L903. New investigations ,,, the reduction phenomena of animals and plants. Proc. Roy. Soc. London 72: mi pis 6 figs. 1905. On the meiotic phase (reduction divisions) in animals and plai Quar Jour. Micr. Sci. 48: IV 489-557. pis. 34 1 1 . Farmer, J. B. and Shove, D. 1905. On the structure and development of the somatic and heterotype chromosomes of Tradeacantia virginica Quar Four Micr. Sci. 48: 559-569. pis. 42, 43. Farmer, J. B. and Digby, L. 1910. On the somatic and heterotype mitoses in Gallonia candicans. Rep. Brit. Assn. Sheffield, p. 77s Farmer, J. B. 1912. Telosynapsis and parasynapsis. Ann. Bot. 26: 623 824 Fasten, N. 1914. Spermatogenesis in the American crayfish, Cambarua ririlit and Cambarus immunis (?) with special reference to synapsis and the chromatoid bodies. Jour. Morph. 25: 587-649. pis. 1-10. 1 fig. 1918. Spermatogenesis of the Pacific coast edible crab, Cancer magister Dana Biol. Bull. 34: 277-306. pis. 1-4. Faull, J. H. 1905. Development of ascus and spore formation in ascomyoel Proc. Boston Soc. Nat. Hist. 32. 77-113. pis. 7-11. 1912. The cytology of Laboulbenia chcetophora and /.. Gyrinidarum \nn Bol 26 : 325-355. pis. 37-40. Federley, H. 1913. Das Verhalten der Chromosomen bei der Spermatogenese der Schmetterlinge Pygaera anachoreta, curtula und pigra sowie einiger Hirer Bastarde Zeitschr. Ind. Abst. Vererb. 9: 1-110. pis. 1-4. figs. 5. Fick, R. 1905. Betrachtungen iiber die Chromosomen, ihre [ndividualitat Reduc- tion und Vererbung. Arch. Anat. u. Physiol.. Anat. Abt., Suppl. 179 228 1907. Vererbungsfragen, Reduktions- und Chromosomenhypothesen, Bastard- regeln. Ergeb. Anat. u. Entw. 16: 1-140. (Review.) 1908. Zur Konjugation der Chromosomen. Arch. Zellf. 1: 604 611. Fitzpatrick, H. M. 1918. The cytology of Eucronartium muscicola \m Jour Bot. 5: 397-419. pis. 30-32. Flemming, W. 1887. Neue Beitrage zur Kenntniss der Zelle. Arch. Mikr Inal 29 : 389-463. pis. 23-26. Florin, R. 1918. Cytologische Bryophytenstudien. I. CJeber Sporenbildung bei Chiloscyphus polyanthus (L) Corda. Ark. f. Bot. 15: 1 II). 1 pi. Foot, K. and Strobell, E. C. 1905. Prophases and metaphase of the firs! matura- tion spindle of Allolobophora jcetida. Am. Jour. Anal. 4: 199 243, pi- i 1909. The nucleoli in the spermatocytes and germinal vesicles of E uc) ' . .■ larius. Biol. Bull. 16: 215-238. 1 pi. 1910. Pseudo-reduction in the oogenesis of AUqlobqphora foetida. Arch. Zellf 5- 149-165. pis. 11, 12. 1 fig. 1912. A study of chromosomes and chromatin nucleoli in Etuchistv Ibid. 9: 47-62. pis. 2-4. Fraser, H. C. I. 1907. On the sexuality and development of tin- ascocarp Lachnea stercorea Pers. Ann. Bot. 21: Ml'.) 360. pis. 29, 1908. Contributions to the cytology of Humaria rutilans. Ibid. 22. j,U 4,5. 1914. The behavior of the chromatin in the meiotic divisions of l Fdba. [bid. 28 : 633-642. pis. 43, 44. Fraser, H. C. I. and Welspobd, K. .J. 1908. Further contributions t<> the cytoli of the ascomycetes. Ann. Hot. 22: lii.". 177. pis. 26, 27, 264 INTRODUCTION TO CYTOLOGY Fraser, H. C. I. and Brooks, W. E. 1909. Further studies in the cytology of the ascus. Ann. Bot. 23 : 538-549. Fraser, H. C. 1. and Snell, J. 1911. The vegetative divisions in Vicia faba. Ann. Bot. 25 : 845-855. pis. 62, 63. Fries, R. E. 1911. Zur Kenntniss der Cytologic von Hygrophorus conicus. Svensk. Bot. Tids. 5: 241-251. pi. 1. Frisendahl, A. 1912. Cytologische und entwicklungsgeschichtliche Studien an Myricaria germanica Dear. Kgl. Svensk. Vet. Handl. 48. Gates, R. R. 1908. A study of reduction in (Enothera rubrinervis. Bot. Gaz. 48 : 1-34. pis. 1-3. 1909. The behavior of chromosomes in (Enothera lata X 0. gigas. Ibid. 48 . 179-199. pis. 12-14. 1911. The mode of chromosome reduction. Ibid. 51: 321-344. Giglio-tos, E. e Granata, L. 1908. I mitocondrii nelle cellule seminali di Pam- phagus marmoratus, Burm. Biologica 2 : No. 4. Goldschmidt, R. 1905. Eireifung, Befruchtung und Embryonalentwicklung des Zoogonus mirus Lss. Zool. Jahrb. 21: 607-654. pis. 36-38. 1908c. Die Chromatinreifung der Geschlechtszellen des Zoogonus mirus Lss. und die Primartypus der Reduktion. Arch. Zellf . 2 : 348-370. pis. 24, 25. figs. 6. 1908a. Ueber das Verhalten des Chromatins bei der Eireifung und Befruchtung des Dicrocailium lanceolatum Stil. et Has. (Distomum lanceolatum.) Ibid. 1: 232-244. pi. 7. 19086. 1st eine parallele Chromosomenkonjugation bewiesen? Ibid. 1: 620-622. Goldsmith, W. M. 1919. A comparative study of the chromosomes of tiger beetles (Cicindelidie). Jour. Morph. 32: 437-487. pis. 1-10. Granata, L. 1910. Le cinesi spermatogenetische di Pamphagus marmoratus, Burm. Arch. Zellf. 5. Gregoire, V. 1899. Les cineses polliniques chez les Liliacees. La Cellule 16: 235-297. pis. 2. 1904. La reduction numerique des chromosomes et les cineses de maturation. Ibid. 21: 297-314. 1905. Les resultats acquis sur les cineses de maturation dans les deux regnes. Ibid. 22: 221-376. (Review.) 1907. La formation des gemini heterotypiques dans les vegetaux. Ibid. 24: 369- 420. pis. 2. 1909a. La reduction dans le Zoogonus mirus Lss. et le "Primartypus." Ibid. 25: 245-285. pis. 2. 1909b. Les phenomenes de l'etape synaptique represent-ils une caryocinese avortee? Ibid. 25 : 87-99. 1910. Les cineses de maturation dans les deux regnes. L'unite essentielle du pro- cessus meiotique. Ibid. 26: 223-422. figs. 145. 1912. La verite du schema heterohomeotypique. Compt. Rend. Acad. Sci. Paris 155: 1098-1100. Gregory, R. P. 1904. Spore formation in leptosporangiate ferns. Ann. Bot. 18: 445-458. pi. 31. 1 fig. Griggs, R. F. 1906. A reduction division in Ascaris. Ohio Nat. 6: 519-528. pi. 33. Guignard, L. 1891. Nouvelles etudes sur la fecondation. Ann. Sci. Nat. Bot. VII 14: 163-296. pis. 9-18. 1899. Le developpement du pollen et la reduction dans le Naias minor. Arch. d'Anat. Micr. 2 : 455-509. pis. 19, 20. Guilliermond, A. 1910. La sexualite chez les champignons. Bull. Sci. France et Belg. 44: 109-196. THE REDUCTION OF THE CHROMOSOMES 265 Haecker, V. 1S90. Ceber die Reifungsvorgange bei Cyclops. Zoo] \nz 18:551- 558. 1 fig. 1892. Die Eibildung bei Cyclops und CarUhocamptus. Zool. Jahrb 5- 211-f,l^ pi. 19. 1895a. The reduction of the chromosomes in the sexual cells. Ann Bot 9' 95-102. 18956. Die Vorstadien der Eireifung. Arch. Mikr. Anat. 45: 200 272 pis 14-17. 1897. Weitere Uebereinstimmungen zwischcn den Fortpflanzungsvorgange del Thiere und Pflanzen. Biol. Centralbl. 17: G89-705, 721-745. Sgs. 36. 1899. Die Reifungserscheinungen. Ergebn. Anat. u. Entu. 8 : sir <.»_>_>. f,- _>_\ Harman, M. T. 1920. Chromosome studies in Tettigidae. II. BioL Hull. 38: 213-230. Harper, R. A. 1900. Sexual reproduction in Pyronema confluens and the morphol- ogy 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. Wash. Publ. 37. von Henking, H. 1891. Untersuchungen liber der ersten Entwicklungsvorg&nge in den Eiern der lnsekten. Zeit. Wiss. Zool. 51: 685-736. pis. :;.") :;7. 1892. Same title. 111. Ibid. 54: 1-274. pis. 1-12. figs. 12. Hertwig, R. 1908. Ueber neue Probleme der Zellenlehrc. Arch. Zellf. 1: 1-32. figs. 9. He user, F. 1884. Beobachtungen uber Zellkerntheilung. Bot. Centralbl. 17: 27, 57, 85, 117, 154. pis. 1, 2. Ikeda, T. 1902. Studies on the physiological functions of antipodals and related phenomena of fertilization in Liliacese. 1. Tricyrtis hiria. Bull. ( Soil. Sci. Agr. Tokyo 5. Ishikawa, M. 1911. Cytologische Studien uber Dahlien. Bot. Mag. Tokyo 25: 1-8. 1 pi. Jahn, E. 1908. Myxomycetenstudien. 7. CeraUomyxa. Ber. Deu. Bot. ( jSes. 26a : 342-352. Janssens, F. A. 1905. Spermatogenese dans les batrachiens. III. Evolution dee auxocytes males du Batracoseps attenuatus. La Cellule 22: 379-42"). pis. 7 1909. La theorie de la chiasmatypie. Ibid. 25: 389-411. pis. 2. Janssens, F. A. et Willems, J. 1909. Spermatogenese dans les batraciens. IV. Ibid. 25: 151-178. pis. 2. Juel, H. O. 1898. Die Kerntheilungen in den Basidien und die Phylogenie del Basidiomyceten. Jahrb. Wiss. Bot, 32: 361-388. pi. 1. Karsten, G. 1908. Die Entwicklung der Zygoten von Spirogyra jugalis, Kt/g Flora 99: 1-11. pi. 1. Kemp, H. P. 1910. On the question of the occurrence of "heterotypical reduction" in somatic cells. Ann. Bot. 24: 775-803. pis. 66, 67. Kihara, H. 1919. Ueber cytologische Studien l»ei einigen Getreidearten. Bot. Mag. Tokyo 32: 17-38. figs. 21. King, H. D. 1907. The spermatogenesis of Bufo lentiginostu. Ain. Jour. An.it. 7: 345-388. pis. 3. 1908. The oogenesis of Bufo tenUginosus. Jour. Nforph. 19: 369 138. pis. I. Kingery, H. M. 1917. Oogenesis in the white mouse. Ibid. 30: 261 316. 5 pis. Kingsbury, B. F. and IIirsch, P. E. 1912. The degeneration of th< mdary spermatogonia of Desmognathus fusca. Jour. Morph. 23: 231 253. pis Kniep, H. 1911. Ueber . 7. 1910. On the dimegalous sperm and chromosomal variation in EuschUtu^ with reference to chromosomal continuity, [bid. 5: 1-1 145. pis. 9, 10. 1 fig. 1911. The spermatogenesis of an Hemipteran, Euschistus. .lour. Morph. 22: 731-798. pis. 1-5. 268 INTRODUCTION TO CYTOLOGY Moore, A. C. 1905. Sporogenesis in Pallavicinia. Bot. Gaz. 40: 81-96. pis. 3,4. Moore, J. E. S. 1896. On the structural changes in the reproductive cells during the spermatogenesis of the Elasmobranchs. Quar. Jour. Micr. Sci. 38 : 275-314. pis. 13-16. Moore, J. E. S. and Embleton, A. L. 1906. On the synapsis in Amphibia. Proc. Roy. Soc. London 77 : 555-562. pis. 20-23. Mottier, D. M. 1897. Beitrage zur Kenntniss der Kerntheilung in den Pollen- mutterzellen einiger Dikotylen und Monokotylen. Jahrb. Wiss. Bot. 30: 169-204. pis. 3-5. 1903. The behavior of the chromosomes in the spore mother-cells of higher plants and the homology of the pollen and embryo-sac mother-cells. Bot. Gaz. 35: 250-292. pis. 11-14. 1905. The development of the heterotypic chromosomes in pollen mother-cells. Bot. Gaz. 40: 171-177. 1907. The development of the heterotypic chromosomes in pollen mother-cells. Ann! Bot. 21 : 309-347. pis. 27, 28. 1909. Prophases of heterotypic mitosis in the embryo-sac mother-cells of Lllium. Ibid. 23: 343-352. pi. 23. 1914. Mitosis in the pollen mother-cells of Acer negundo, L., and Staphylea trifolia, L. Ibid. 28: 115-133. pis. 9, 10. Mottier, D. M. and Nothnagel, M. 1913. The development and behavior of the chromosomes in the first or heterotypic mitosis of the pollen mother-cells of Allium cernuum Roth. Bull. Torr. Bot. Club 40: 555-565. pis. 23, 24. Muller, H. A. Cl. 1909. Ueber karyokinetische Bilder in den Wurzelspitzen von Yucca. Jahrb. Wiss. Bot. 47: 99-117. pis. 1-3. 1911. Kernstudien an Pflanzen. I u. II. Arch. Zellf. 8: 1-51. pis. 1, 2. Nakahara, W. 1919. A study of the chromosomes in the spermatogenesis of the stonefly, Perla immarginata Say, with special reference to the question of synapsis. Jour. Morph. 32 : 509-530. pis. 3. 1920. Side-to-side versus end-to-end conjugation of chromosomes in relation to crossing over. Science 52 : 82-84. Nemec, B. 1903. Ueber die Einwirkung des Chloralhydrats auf die Kern- und Zell-teilung. Jahrb. Wiss. Bot. 39: 645-730. figs. 157. Nichols, G. E. 1908. The development of the pollen of Sarracenia. Bot. Gaz. 45:31-37. pi. 5. Nothnagel, M. 1916. Reduction divisions in the pollen mother-cells of Allium Iricoccum. Bot. Gaz. 61: 453-476. pis. 28-30. fig. 1. Oehlkers, F. 1916. Beitrag zur Kenntniss der Kernteilung bei den Characeen. Ber. Deu. Bot. Ges. 34 : 223-227. Olive, E. W. 1907. Cytological studies on Ceratiomyxa. Trans. Wis. Acad. Sci. 15 : 754-773. pi. 47. Osterhout, W J. V. 1897. Ueber Entstehung der karyokinetischen Spindel bei Equisetum. Jahrb. Wiss. Bot. 30: 159-168. pis. 1, 2. Overton, E. 1893. On the reduction of the chromosomes in the nuclei of plants. Ann. Bot. 7 : 139-143. Overton, J. B. 1905. Ueber Reduktionsteihmg in den Pollenmutterzellen einiger Dikotylen. Jahrb. Wiss. Bot. 42: 121-153. pis. 6, 7. 1906. The morphology of the ascocarp and spore formation in the many-spored asci of Theocatheus pelletieri. Bot. Gaz. 42: 450-492. pis. 29, 30. 1909. On the organization of the nuclei in the pollen mother cells of certain plants, with special reference to the permanence of the chromosomes. Ann. Bot. 23: 19-61. pis. 1-3. THE REDUCTION OF THE CHROMOSOMES 269 Paulmier, F. C. 1898. Chromosome reduction in the Hemipters. Anat. Vnz 14: 514-520. figs. 19. 1899. The spermatogenesis of Anasa triads. Jour. Morph. 16: SuppL 223 272. pis. 13, 14. Picard, M. 1913. A bibliography of works on meiosis and somatic mitosis in the angiosperms. Bull. Torr. Bot. Club 40: 575-590. Popoff, M. 1908. Experimentelle Zellstudien. Arch. Zellf. 1 : 246-380. figs. 18. Pratt, B. H. and Long, J. A. 1917. The period of synapsis in the egg of the white rat, Mus norvegicus albinus. Jour. Morph. 29: 441-458. pi. 1. figs. 2. vom Rath, O. 1892. Zur Kenntniss der Sperm atogenese von Gryllotalpa vulgarii Latr. Arch. Mikr. Anat. 40: 102-132. pi. 5. 1893. Beitrage zur Kenntniss der Spermatogenese von Salamandra maculosa. Zeitschr. Wiss. Zool. 67: 97-185. pis. 7-9. 1895. Neue Beitrage zur Frage der Chromatinreduktion in der Samen- und Eireife. Arch. Mikr. Anat. 46 : 168-238. pis. 6-8. Robertson, W. R. B. 1916. Chromosome studies. 1. Jour. Morph. 27: 179-M pis. 1-26. Rosenberg, O. 1907. Zur Kenntniss der praesynaptischen Entwicklungsphasen der Reduktionsteilung. Svensk. Bot. Tidskr. 1. 1909a. Zur Kenntniss von den Tetradenteilungen der Compositen. Ibid. 3: 64-77. pi. 1. 19096. Cytologische und morphologische Studien an Drosera longifolia X rotundir folia. Kgl. Svensk. Vet. Handl. 43 : 3-64. pis. 4. figs. 33. 1918. Chromosomenzahlen und Chromosomendimensionen in der Gattung Crepis. Ark. f. Bot. 15: 1-16. figs. 6. Roux, W. 1883. Ueber die Bedeutung der Kerntheilungsfiguren. Leipzig. Ruckert, J. 1892. Zur Entwicklungsgeschichte des Ovarialeies bei Selachiern. Anat. Anz. 7: 107-158. figs. 6. 1893. Die Chromatinreduktion bei der Reifung der Sexualzellen. Ergeb. d. Anat. u. Entw. 3: 517-583. (Review.) 1894. Zur Eireifung der Copepoden. Ibid., Anat. Hefte. 4: 261-352. pis. 21 25 Sabaschnikoff, M. 1897. Beitrage zur Kenntnis der Chromatinreduktion in der Ovogenesis von Ascaris. Bull. Soc. Nat. Moscow 1. Sakamura, T. 1914. Ueber die Kernteilung bei Vicia cracca. Bot. Mag. Tokyo 28 : 131-147. pi. 2. 1915. Ueber die Einschnurung der Chromosomen bei Vicia Faba L. [bid. 29: 287 300. pi. 13. figs. 12. 1920. Experimentelle Studien liber die Zell- und Kernteilung mil besonderei Riicksicht auf Form, Grosse und Zahl der Chromosomen. Jour. Coll. Bei. Imp. Univ. Tokyo 39: pp. 221. pis. 7. Schaffner, J. H. 1909. The reduction division in the microsporocytes of Agon virginica. Bot. Gaz. 47: 198-214. pis. 12-14. Schleip, W. 1906. Die Entwicklung der Chromosomen im Ei von Planaria ij<>no- cephata Dug. Zool. Jahrb. (Anat. Abt.) 23: 367 380. pis 23, 24. 1907. Die Samenreifung bei Planarien. Ibid. 21: 129 174. pis. 11. IV Schellenberg, A. 1911. Ovogenese, Eireifung und Befruchtung von Fatciola hepatica, L. Arch. Zellf. 6: 443-484. pis. 24 26. Schneider, H. 1914. Ueber die Prophasen der ersten Reifeteilung in PoUenmutb zellen, inbesondere bei Thelygonium CynocrambeL, Arch. Zellf. 12: ri. pi. 28. Schreiner, A. u. K. E. 1904. Di«' Eleifungsteilungen bei den Wirbeltieren. An.it Anz. 24: 561-578. figs. 24. 270 INTRODUCTION TO CYTOLOGY 1905. Ueber die Entwicklung der mannlichen Geschlechtszellen von Myxine glu- tinosa (L.). Arch. d. Biol. 21: 183-355. pis. 5-14. 1906. Neue Studien uber die Chromatinreifimg der Geschlechtszellen. Arch. d. Biol. 22: 1-70, pis. 1-3; 419-492, pis. 23-26; Anat. Anz. 29: 465-479. figs. 17. 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. 1920a. Mitosis in Osmunda. (Review). Ibid. 69: 88-91. 19206. Somatic chromosomes in Tradescaniia. Am. Jour. Bot. 7: 341-354. pis. 22, 23. Stevens, N. M. 1903. On the ovogenesis and spermatogenesis of Sagitta bipunctata. Zool. Jahrb. 18: 227-240. pis. 20, 21. 1908. A study of the germ cells of certain Diptera, with reference to the hetero- chromosomes and the phenomena of synapsis. Jour. Exp. Zool. 5: 359-374. pis. 4. 1911. Further studies on the heterochromosomes of the mosquitoes. Biol. Bull. 20:109-120. figs. 38. Stomps, T. J. 1910. Kerndeeling en synapsis bij Spinacia oleracea. pp. 162. pis. 2. See Biol. Centr. 31 : 257-320. pis. 1-3. 1911. Strasburger, E. 1888. Ueber Kern- und Zellteilung im Pflanzenreich, nebst einem Anhang uber Befruchtung. Hist. Beitr. 1. pp. 258. pis. 3. 1894. The periodic reduction of chromosomes in living organisms. Ann. Bot. 8: 281-316. 1897. Kerntheilung und Befruchtung bei Fucus. Jahrb. Wiss. Bot. 30: 351-374. pis. 27, 28. 1900. Ueber Reduktionsteilung, Spindelbildung, Centrosomen und Cilienbildner im Pflanzenreich. Histol. Beitr. 6. pp. 224. pis. 4. 1904a. Anlage des Embryosacks und Prothalliumbildung bei der Eibe nebst anschliessenden Erorterungen. Festschr. f. Hseckel. Jena. 19046. Ueber Reduktionsteilung. 8itzber. Berlin. Acad. Wiss., phvs.-math.Kl. 18 : 587-614. figs. 9. 1905. Typische und allotypische Kernteilung. Jahrb. Wiss. Bot. 42: 1-71. pi. 1. 1907. Ueber die Individuality der Chromosomen und die Propfhybriden-Frage. Ibid. 44 : 472-555. pis. 5-7. 1908. Chromosomenzahlen, Plasmastrukturen, Vererbungstrager und Reduktions- teilung. Ibid. 45 : 479-568. pis. 1-3. 1910. Ueber geschlechtsbestimmende Ursachen. Ibid. 47: 427-520. pis. 9, 10. 1911. Kernteilungsbilder bei der Erbse. Flora 102: 1-23. pi. 1. Sutton, W. S. 1902. On the morphology of the chromosome group in Brachystola magna. Biol. Bull. 4: 24-39. figs. 11. Svedelius, N. 1914a. Ueber die Tetradenteilung in den vielkernigen Tetraspor- angiumanlagen bei Nitophyllum punctatum. Ber. Deu. Bot. Ges. 32 : 48-57. pi. 1. 1 fig. 19146. Ueber Sporen an Geschlechtspflanzen von Nitophyllum punctatum, usw. Ibid. 32: 106-116. pi. 2. 1 fig. Sykes, M. G. 1908. Nuclear division in Funkia. Arch. 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Chromosomen im Zellkerne von Najas major. Her. Deu. Hut. < ,■ 32: 411-416. pi. 10. Vandendries, R. 1913. Le nombre des Chromosomes dans la BpermatogeDJ Polytrichum. La Cellule 28: 257-261. figs. 11. van Leeuwen-Reijnvaan, D. 1907. Ueber eine zweifache Reduktion bei einigen P oly trichum- Ar ten. Rec. Trav. Bot. Xeerl. 4. 1908. Ueber die Spermatogenese der Moose. Ber. Deu. Hot. ('n>>. 26a: 301 308. pi. 5. Vejdowsky F. 1907. Neue Untersuchungen liber 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. Intracellular 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 ranis Werner. Jour. Morph. 30: 527-604. pis. 9. 1 fig. Weinzieher, S. 1914. Beitrage zur Entwicklungsgeschichtr vi>is suffusa (Orthoptera). .lour. Morph. 29: 171 516 pis. 1-3. Williams, J. L. 1904. Studies in the Dictyotaceae. 11. The cytology of the game- tophyte generation. Ann. Bot, 18: 183 204. pis. L2 11. Wilson, E. B. 1900. The Cell in Development and Inheritance. 2d ed. von Winiwarter, H. 1900. Recherches but I'ovogenese e! I'organogenese de l'ovaire des mammiferes (Lapin et Homme). Arch. d. Biol. 17: 33 199 pis. 3-8. von Winiwarter, H. et Sainmont. 1909. Nouvelles recherchea Bur I'ovof I'organogenese de I'ovaire des Mammiferes (('hat'. Chapter IV. [bid. 24: 1-142, 165-276, 373-432, 627 652. pis. 11. Wodsedalek, J. E. 1916. Causes of sterility in the mule. Biol. Bull. 80: 1 Wolfe, J. J. 1904. Cytological studies on Nemalion. Ann. Hot. 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. 45 : 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 doI dis- covered until 1883, some of the grosser features of fertilization had been made out many years previously (Chapter I). Bu1 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'e parallel discoveries in plants following in 1877 (Spirogyra) and L884 (angiosperms). As the finer details of fertilization and the significance of its results become better understood, the aptness of Huxley's 18^8 often quoted simile, in which he compares the organism to "a web <»f which the warp is derived from the female and the woof from the mail 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 t he 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. KM , The /<<• upon Professor F. R. Lillie's admirable and concise presentation of tin- subject, Problems of Fertilization (1919). 18 -'73 274 IN TRODVC TION TO C Y TOLOG 1 ' 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. G 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 do part in the formation of the chromosomes when division ensui 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. L06 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 tip nucleus ("female pronucleus"), rendering the egg ready for the sexual fusion. Fig. 104. Fig. 105. Fig. 104. — Diagram of typical flagellate spermafc P, perferatorium; A, acrosomc; A", nucleus; M, middle piece; /. ixial fUamci cytoplasmic sheath; E, end piece. (After Wilson.) Fig. 105. — The differentiation of tin- oocyte in Hyd A, very young oocyte lying betweeri ectodermal cells al right. B .\th period, with yolk globules, X 500. {After Downing, 1909. The time relation of the mat mat ion of i hi and t he en1 ranee of t he spermatozoon varies considerably in differenl animals. In echinoderms and some other forms maturation is complete. 1 before the spermato penetrates. In some other animals it proceeds as far as the metaphase of the heterotypic mitosis (Chcetopterus, Cerebratulw of the propha the homceotypic mitosis (many vertebrates), bul does do! go further unless penetration occurs. In the marine annelid. A i . finally, tl 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 fiagellum 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 277 and tail being left on the egg Burface. The process in \, , as de- scribed by LiUie (1912, 1919) is as foUows. The ofth rmha 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 alveola* of the cortical layer. This layer dow takes the form of a zone traversed by radial protoplasmic plates representing the walls of the alveolae, A transparent "fertilization cone" extends from the inner part of the egg across this zone and touches the membrane a1 the poinl where the spermatozoon is beginning to penetrate. The perforatorium 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 of penetration lies with the egg rather than with the spermatozo Since only the head enters the egg in Nereis it seems clear thai 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 __ 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 /< rtilization n 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. Thes 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 f>v 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 thi 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 sijnkaryon, 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. mmm ?mm C M^ r^-^tiM^^^S 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 organize- when the nuclear membranes dissoh 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 ferl ilizai ion, in bol h animals and plant each parent furnishes the offspring with a haploid sit of chromosome the two intermingled sets constituting the diploid set of th( m w dual. Since every chromosome divides equatio natty at every subsequent somatic mitosis, every cell of the body receives half of its chromosome complement from each parent. The cardinal importance of this fad 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 (Ruckert 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 ! thai 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 fT.) 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 appea 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 lias been that the newly appearing centrosome is in reality that of the spermatid. \\ 'hat ever its origin, it soon divides to form the two which fund ion in I he firsi cleavi 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 Bpermatoaoon has a centrosome but not sufficient cytoplasm in which toact. 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 nun's. Another early view of the origin of the cleavage centrosomes was that of van Beneden (1887) and Wheeler (1895, L897 , 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 sug d 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 (Ascaris) 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- FERTILIZATinx 281 acters. Several observations, however, fail <<> harmonize with this view. In some echinoderms the middle piece, which contains the chondriosomal material, is not distributed to the daughter cells during the cleavaj divisions, but remains in one of them. It is unlikely thai 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 a& yet no adequate reason to regard them as important in the transmission of the factors of inheritance. Fig. 110. — Ohromidiogamy in Arcella. (From Minchin, after Swarasewaky.) Somewhat modified. Fertilization in Protozoa. — Among the protozoa there arc ton ml several modes of sexual union very different from that described above for the higher animals. Four illustrative cases may be cited Bee Minchin 1912). In Arcella, a rhizopod with a hemispherical shell, the protoplast 1 two "primary nuclei" and many scattered chromidia. Two individuals come together with their shell openings opposite one another I Fig. 1 1' 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 granul< 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 organised uninucleate amcebulse which escape from the shell and give rise to Dew 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. sh:C\ Fig. 111. — Copulation in Actinophrys sol. X 850. (From Minchin, after Schaudinn.) In Actinophrys sol (Schaudinn 1896) (Fig. Ill) two individuals, each with 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 Nagler.) 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 FERTIUZATIOX 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 Mvrgan, 19] other. It then completes its division to form the two gamete nuclei, or pronuclei, while the four reduction nuclei degenerate. Ifter lying apart for some time the two pronuclei approach each other and fuse; sexual union thus takes place between Bister nuclei. The complicated nuclear behavior occurring at t he t ime of conjugal ion m. Paramcecium caudatum (Fig. 113) may best be described in the words 284 INTRODUCTION TO CYTOLOGY of Wilson (1900, pp. 224-226)0 In "Paramecium caudatum, 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 laj of the egg. Although in some forms (starfish) this one treatmenl is sufficient to bring about successful development, in mosl sea urchin) the eggs become sickly and die. Loeb found thai 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 th< 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 Buch parthenogenetic development has two phases: the inducement <>! 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 thai it carries two substances: first, a lysin which brings about membrane format ion 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 eg,L>- 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 wTould 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 irate of oxida- tion, etc.) of the egg. Following Warburg (1914) Loeb suggests thai the cytolysis releases the catalyzer by breaking down the cortical emulsion; this results in an increase in the rate of oxidation and other react ions, 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, L917), McClendoD I 1912), Loeb and Bancroft (1913), and particularly Bataillon ( L910), 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 Btarfish eggs may be made to develop parthenogenetically by exposing them to high temperatures for definite periods. (See V. R. Lillie. I'M!*. I Ihapter 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 FERTILIZA TION 287 of those reactions within the egg system upon which development depends." As a result of his direct analysis of the gametes during 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-fertilizinr." As a rule it is only during the period ;it 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 1 he egg. That the protection is thus a physiological rather than a mechanical one is indicated by the fact that membraneless egg fragment- 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 t he 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 (Loch 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 substance-. 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, B), 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 Conjugatae) 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 Polysiphoiiia a non-motile male gamete FERTILIZAWtX 289 (spermatium) comes in contact with a prolongation (trichogyne) of the female sex organ (carpogonium). Solution of the intervening walla allows the nucleus of the spermatium to pass into the 1 richogyne and down to I lie 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 i his : .'■<■ - • .. . ■ i :■■'".•'■■ "-'■' *. V 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 " ccenocentrum " (larger dark body. I A ntheridial 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 I wo princi- pal forms, which serve to divide the group into two main divisions: Oomycetes and Zygomycetes. In the Oomycetes the cytologica! phenomens 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 Writ 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 Pythium (Trow 1901 ; Miyake 1901) a single uninucleate egg is produced, the fertilization process closely resembling that in Albugo Candida. In the Zygomycetes, represented chiefly bytheMucoracese, 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 Ascobolus furfuraceus 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 Helvetia crispa 1911; Blackman and Welsford on Poly stigma rvbrum L912 , P>.\ the above investigators this early fusion was regarded as a sexual one, thai in the ascus being vegetative in nature; and some described a "double reduc- tion" in the ascus to compensate for the two nuclear fusions. 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 thai occurring in the ascus: with Dangeard they saw in this union the sexual act. Fur- thermore, no "double reduction'' was found in the ascm. Among the researches supporting this view, which now appeals to be the more probable, may be cited the following: Claussen on Pyroncma confliu Fig. 116. A, nuclear fusion in the ascus of Peziza vesiculosa. fusion in aeciospore sorus of Phragmidium speciosinn. - B {After Dangeard, 1894 AfU r Christman, L90S B. cell 1907, 1912; Schikorra on Monascus 1909; \V. H. Brown on Pyronema confluens 1909, Lachnea scutellata 1911, and Leotia 1910; Faull on Lab- oulbenia 1911, 1912; Blackman on ColUma pulposum 1913; Nienburg on Polystigma rubrum 1914; Ramlow on Ascophanus cdrneus and Ascobolus immersus 1914; Brooks on Gnomon in erythrostoma 1910; McCubbin <>n Helvetia elastica 1910; H. B. Brown on Xylaria terUaculaia L913; and Fitz- patrick on Rhizina undulata 1918'/. As the two nuclei fuse in the young ascus Harper I L905 observed in the case of Phyllactinia corylea that not only the chromatin Bystems 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 aucleus,' undergo* - 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 hyphse arise as the result of cell fusions ("plasmogamy;" "pseudogamy") between uninucleate hyphse 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 Riccia, Meyer (1911) on Corsinia, Graham (1918) on Pn and Woodburn (1920) on Reboulia. It appears thai in bryophytea the body of the biciliate spermatozoid, which consists mainly of nuclear material, undergoes in the egg cytoplasm a 1 ransformal ion into ,-i pel iculate nucleus before fusing with the egg nucleus (Fig. 117). The fat-' of the non- nuclear structures (cytoplasm, blepharoplast, and filial i< not known with certainty, but it is probable that they arc absorbed in Up 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 •£■•■ . ; '-ft* £ 'J'-f-i ■v. ■ ? %> -,'•-• *^' <^w !r Fig. 117. Fio. 118. Fig. 117. — Fertilization in Anthoceros. Male and female pronuclei about c> fuse in lower part of egg in venter of archegonium; elongated plastid above them. Gametopl cells show one nucleus and one plastic! each. X 10.50. Fig. 118. — Fertilization in Nephrod&utn. A, spermatozoid entering egg nucleus. B, spermatosoid becoming retioulate in n of female reticulum. {After Yamanouchi, 1908.) Nephr odium (Fig. 118). In Nephr odium the multiciliate Bpermatosoid enters bodily into the egg nucleus with do 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 Bingle fine-meshed network. Gymnosperms. — Among living gymnosperms the Cycadales and Ginkgoales are characterized by the possession of motile spermatoaoids. 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 Zamia floridana by 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.) FERTlLIZMIdS 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), Torrcya californica (Robertson 1901). To,,-, yQ taxifolia (Coulter and Land 1905), Cephalotaxus Fortunei (Coker L907 . Ephedra (Berridge and Sanday 1907; Land 1907), PhyUocladus (Kildahl 1908 Juniperus (Nichols 1910), Agathis (Eamea L913), and Taxus (Dupler 1917). Chromosome Behavior. — The behavior of the chromosomes duri 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 Fig. 120 — Fertilization in I' inns. A, male nucleus pressing into female nucleus. X 140. B, first embryonal mitt showing separate paternal and maternal chromosome groups. X 472. {A/U /'■ 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 (Blackmari L898; ( 'hani- berlain 1899; Ferguson 1909, 1904) (Fig. 120), Larix (Woyciki L899 Tsuga candensis (Murrill 1900), Juniperus communis (Nore*n L907 . Cunninghamia (Miyake 1910), and Abies (Hutchinson L915 , In Sequoia, on the other hand, Lawson I 1904) reports thai the two nuclei form a common reticulum in which the male and female constituents cannot be distinguished. With regard to the firsl 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. Tin- type of behavior was described for the chromosomes of Pinus by M Ferguson (1904) and at once came to be regarded as general tor .(.in- fers, as it had been for other organisms. A new interpretation differing in certain fundamental point.- from the above has been more recently suggested by Hutchinson 1 1915 . as B n suit 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. ABk £5 I* • •«•) FUSION NVCklUt TWISTING I OOPinC. SttMfcltTflTION HX StontNTS fIRST TWO CnSBTO NUCLII (i* • it.) If. TO torn TOLl jTIG< 12i. — 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 29' mitosis, a reduction, qualitative as well as Dumerical, in all respe< 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 pail 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 jusl which ones pass to each pole. If both halves of one transversely divided chromosome p to one pole (see Fig. 122), that daughter nucleus only, and not the oth< will receive the kind of chromatin carried by thai chromosome, so that CLf.AVA&£ MITOSIS LQUATlONAL A- 0 J& CD & FUSION NUCltUi V St&fJLNTATlON V W CLCAVA&C. MlTOJli DlffCRCNTlAl Fig. 122. — Diagram showing the behavior of the chromosomes in fertilisation and tin- first embryonal mitosis as usually interpreted (upper part) and according to Hut. Inn- interpretation (lower part). the two nuclei will be qualitatively different. A qualitative reduction will have occurred, but without a change in the Dumber of chromoson* since each old chromosome has become two new oni It. on the other hand, the two halves of the transversely segmented chromosome regularly pass to opposite poles, each daughter oucleua will receive a hah' of each and every parental chromosome: thus if there are just as many kinds of chromatin as there are chromosomes, these nuclei will I..- qualitatively alike, just as they would be had the division hern 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 Dot 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 philade.1- 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, B). 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 in one male nucleus than in the other, bo thai the two ma} appear quite unlike during the later stages. Miss Welsford also in the male cytoplasm certain granules which she thinks may represent the vestig of blepharoplasts. I . 1895. Second memoire sur la reproduction sexuelie dee Ascomycetes. Il.id. 6: 245-284. figs 17. Davis, B. M. 1896. The fertilization of Batrackospermum. Ann. Bot. 10: 19 76 pis. 6, 7. 1900. The fertilization of AUmgo Candida. Bot. Gaz. 29: 297-311. pi 22 1903. Oogenesis in Saprolegnia. Ibid. 85: 233-249. 320-349. plfl. 9, 10. 1905. Fertilization in the Saprolegniales. Ibid. 39: t',1 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 gamctophytes of Taxus canadi < Marsh. 1 /,. 64: 115-136. pis. 11-14. Eames, A. J. 1913. The morphology of Agathis australis. Ann Bot. 27: 1 pis. 1-4. 92 figs. Ernst, A. 1902. Chromosomenreduktion, Entwicklung des Embryosackes und Befruchtung bei Paris qvadrifolia 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 Fucaceae; their life history and cytology. Phil. Trans. Roy. Soc. London B 190: 623-645. pis. 19-24. Fatjll, J. H. 1911. The cytology of the Laboulbeniales. Ann. Bot. 25: 649 664. 1912. The cytology of Labovlbenia choetophora and L. Gyrinidarutn. 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 - 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. 66: 601 502. Fitzpatrick, H. M. 1918a. Sexuality in Rhizina undutala Fries. Bot. Gas. 65: 201-226 pis. 3 4. 19186. The cytology of Eucronariium muscicola. Am. Jour. Bot 5: 397 119. pis. 30-32. Focke, W. O. 1881. Die Pflanzen-Mischlingc. Berlin. Fol, H. 1891. Die "Centrenquadrille," ein neue Episode aus der Befruchtun| geschichte. Anat. Anz. 6: 266-274. figs. 10. Fraser, H. C. I. 1907. On the sexuality and development of the Lachnea stercorea Pers. Ann. Bot. 21: 349-360. pis. 29, 30. 1908. Contributions to the cytology of Humaria rutilant. Hud. 22. pis. 4, 5. Fries,R.E. 1911. ZurKenntniss der Cytologic von Hygrophoru* em Svensk. Bot. Tids. 5: 241-251. pi. 1. Garber, J. F. 1904. The life history of Ricciocarpua nutans. Bot. G 37. 101 177. pis. 9, 10. Graham, M. 1918. Centrosomes in fertilization stagesof P Nees. Ann. Bot. 32:415-420. pi. 10. Guignard, L. 1899. Sur les antherozoids el la double copulation sexuelie ch vegetaux angiospernies. Comp. Rend. Acad. Set Paris 128 : 864 871 figfl 1900. L'appareil sexuel et la double fecondation dans lee Tulipes. Ann. 8 Nat. Bot. Vlll 11: 365 387. pis. 9 11. 1901. La double fecondation chez I- - Renonculaceee. Jour. Botanique 15: 394-408. figs. 16. 1902. La double fecondation chez les Sola nr. <. Ibid. 16 : 146 167. BgS. 46, 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 mutterlichen 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 Ascus. Ber. Deu. Bot. Ges. 13: (67)- (78;. pi. 27. 1896. Ueber das Verhalten der Kerne bei den Fruchten. 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 mechanisme de la parthenogenese 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 l'embryogenie du Ginkgo biloba. Jour. Imp. Coll. Sci. Tokyo 8: 307-322. pis. 31, 32. 1898. Etudes sur la fecondation et l'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 liber die Entwicklung der Geschlechtsorgane und der Vorgang der Befruchtung bei Cycas revoluta. Jahrb. Wiss. Bot. 32 : 557-602. pis. 8-10. 1901. Contributions a l'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 Phijllocladus alpina. Bot. Gaz. 46: 339-348. pis. 20-22. Koltzoff, N. K. 1906. Studien uber die Gestalt der Zelle: I, Untersuchungen iiber die Spermien der Decapoden. Arch. Mikr. Anat. 67: 364-572. pis. 25-29. figs. 37. FERTILIZATION Land, W. J. G. 1900. Double fertilization in Composite. Bol Gai 30- j 260. pis. 15, 16. 1902. A morphological study of Thuja. Ibid. 34: 249 259. pis. 6 v 1907. Fertilization and embryogeny of Ephedra trifurca. [bid. 44: \ pis. 20-22. L.AWSON, A. A. 1904*/. The gainetopliN tc. mclirgonia, fertilisation and cinl»: of Sequoia sempervirc us. Ann. Bot. 18: 1 28. pis. I L 19046. The gametophyte, fertilization and embryo of Crypi Ibid. 18: 417-444. pis. 27-30. 1907. The gametophytcs, fertilization and embryo of Cephaiotaxv Ibid. 21. 1-23. pis. 1-4. Levine, M. 1913. The cytology of Hymenomycetes, especially the Boletl Bull Torr. Bot. Club 40: 137-181. pis. 4-8. Lillie, F. R. 1901. The organization of the egg in Unto based on a study ol maturation, fertilization and cleavage. Jour. Morph. 17: 227 292. pis. 24 27. 1912. Studies of fertilization in Nereis. 111. The morphology of normal fertilisa- tion in Nereis. IV. The fertilizing power of portions of the spermatoa Jour. Exp. Zool. 12- 413-476. pis. 1-11. 1914. Studies of fertilization. VI. The mechanism of fertilization in Arba 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 ita action. Jour. Exp. Zool. 5 : 375-428. 1915. On the conditions of activation of unfertilized starfish eggs under tin- influence of high temperatures and fatty acid solution. Biol. Hull. 28. 26 Loeb, J. 1910. Die Hemmung verschiedener Giftwirkungen auf das befruohb Seeigelei durch Hemmung der Oxidationen indemselben. Biochem. Zeitschr 29. 1911. Auf welche Weise rettet die Befruchtung das Leben dee 1 ii< - ' Arch. 31: 658-668. 1912. The Mechanistic Conception of Life. ( hicago. 1913. Artificial Parthenogenesis and Fertilization. Chicago. Loeb; J. and Bancroft, F. W. 1913. The sex of a parthenogenetic tadpole and fi Jour. Exp. Zool. 14: 275-277. Loeb, J. and Wasteneys, H. 1910. Warum hemmi Natrium cyanide die Gift- wirkung einer Chlornatriumlosung fur das Seeigelei? Biochem. Zeitsch. 2. 1911. Sind die Oxidationsvorgange die unabh&ngige Variable In den I^-l scheinungen? Ibid. 36: 345-356. 1912. Die Oxidationsvorgange im befruchteten und unbrfrurhtrten Srrstrrnri. Arch. Entw. 35 : 555-557. Luther, A. 1904. Die Eumesostominen. Zeit. Wise. Zool. 77: 1 273. p figs. 16. MacCurdy, II. M. 1919. Division, nuclear organisation and conjugation in Arcella vulgaris. Mich. Acad. Sci. Rep. 21: 111 113 Macormick, F. a. 1912. Development of zygospore in Hkiao B Gaz. 63: 67-68. McClendon, J. J. 1912. Dynamics of cell division. Artificial parthenog«iuM^ in Vertebrates. Am. Jour. Physiol. 29: 21 8-301. McCubbin, W. A. 1910. Development of the Helvellineae Bol Qai 49: ID 206. pis. 11 16 Meves, Fr. 1899. Ueber Struktur und Elistogenese der Samenfaden dec Meersch- weinchens. Arch. MikT. Anat. 64: 329-402. pis. lfr-21. figs. H 308 INTRODUCTION TO CYTOLOGY 1908. Die Chondriosomen als Trager erblicher Anlagen. Cytologische Studien am Huhnerembryo. Ibid 72 : 816-867. pis. 39-42. 1915. Ueber Mitwirkung der Plastosomen bei cler Befruchtung des Eies von Filaria papiUosa. Ibid. 87 : 11 12-46. pis. 1-4. 1918. Die Plastosomentheorie der Vererbung. Ibid. 92: II 41-136. figs. 18. (Bibliography). Meyer, K. 1911. Untersuchungen liber die Sporophyt der Lebermoose. 1. Entwickkingsgeschichte 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 Pythvum deBaryanum. Ann. Bot. 15: 653-667. pi. 36. 1910. The development of the gametophytes and embryogeny in Cunningham ia 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 Entwickkmg 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. (Esterreich. Bot. Zeitschr. 59: 457. 1910. Naheres tiber 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 Entwickkingsgeschichte von Polystigma 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. Vet.-Akad. 3. pp. 11. 1907. Zur Entwickkingsgeschichte 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 ZelU-heilung. Anat. Anz. 4: 21-30. figs. 2. FERTILIZATION Ramlow, G. I'M I. Beitrage zur Entwicklungsgeschichte der Ascobota Mycol. Ccntralbl. 6: 177 198. pis. 1, 2. figs. 20. Robertson, A. L904. Studies in the morphology of Torreya cali II. The sexual organs and fertilization. New Phytol. 8:205 216. pi- 7 Rom i kit, M. p.m. i,;, spermiogenese chez VA carta mfyalocfrphala. Arch Zellf 6: 254-325. pis. 1 l 17. Ruckkrt, J. ls<.,."). Ueber die Selbst&ndigbleiben der vaterlichen mid mQtterlichen Kernsubstanz wahrend der ersten Entwicklung des befruchteten l l Arch. Mikr. Anat. 45: :>;;!> 369. pis. 21, 22. Sargant, E. 1896. The formation of the sexual nuclei in Lilium Ma lagoi I Oogenesis. Ann. Hot. 10: I 15 177. pi-. 22, 23 1897. Same title. II. Spermatogenesis. Ibid. 11: 187 224. pi-. 12, 13 Sawyer, M. L. 1917. Pollen tube and spermatogenesis in Iris. Bot. Gaz. 64: 159-164. figs. 18. Sax, K. 1916. Fertilization in FHHUaria 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 Actinopkrys sol. Sitzbei kcad. Wiss. Berlin. Scheben, L. 1905. Beitrage zur Kenntniss des Spermatozoons von Ascai I >- cephala. Zeit. Wiss. Zool. 79: 396 431. pis. 20, 21. :: figs. Schikorra, W. 1910. Ueber die Entwicklungsgescnichte von U Zeitschr. f. Bot. 1: -T79-410. Sharp, L. W. 1912. Spermatogenesis in Equisetwn. Bot. Gai. 54: 89 l pi pis. 7, 8. 1914. Spermatogenesis in Marsilia. Ibid. 58: U9 131. |>1-. :;:;. :;i 1920. Spermatogenesis in Blasia. Ibid. 69: 258-268. pi. 15. Smith, B. G. 1919. The individuality of the germ-nuclei during the clca^ . f the egg of Cryptobranchus aUeghaniensis. Biol. Bull. 37. 246 287. Steil, W. X. 1918. Method for Btaining antherozoid of fern. Bot. Gaz. 65: 562-563. 1 fitf. Steykns, F. L. 1S99. The compound oosphere of AH"/,/,, blili. Bot. Gaz. 28: 149-176. pis. 11-15. 1901. Gametogenesis and fertilization in .\U>u{i<>. Ibid. 32: 77 98 pis l I Strasburger, E. 1S77. Ueber Befruchtung und Zelltheilung. Jen. Zeitachr. 11. 1884. Neue Intersuchungen liber die Befruchtungsvorgang bei den Phan< gamen, als Grundlage ftir eine Theorie der Zeugung. Jena. 1892. Schwarmsporen, Game ten, pnanzlichen Spermatozoiden, und da* \Vo«en Befruchtung. Histol. Beitr. 4: 19 158. pi. 3 1897. Kerntheilung und Befruchtung bei Fucus. Jahrb. Wiss. Bol 30: .;.". i pis. 27. 28. P.ioo. Binige Bemerkungen zur Frage nach der "doppelten Befruchtung' den Angiospermen. Bot. Zeit. 58: 293 316 1901. Ueber Befruchtung. Ibid. 59: II. Trow, A. II. 1895. The karyolog] "i Saprol inn Bol 9: • i 24, 25. is'.M). Observations on the biology and cytology of anew variety of Aehl cana. Ibid. 13. 131 179. pis 8 10. 1901. Biology and cytologj of Pythium iiUimum, n. -p Inn. Bol 15 312. pis. 15, 16. 1904. On fertilization in the Saprolegniacea?. Ibid. 18: .Ml 310 INTRODUCTION TO CYTOLOGY Wagek, 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 Befrueht- 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 iiber die Oxidationsprozesse im Seeigelei. Zeitschr. Physiol. Chem. 57. 1910. Ueber die Oxidationen im lebenden Zellen nach Versuchen am Seeigelei. Ibid. 66. 1911. Untersuchungen iiber die Oxidationsprozesse im Zellen. Miinchener Med. Wochenschr. 57. 1914. Beitrage zur Physiologie der Zelle, inbesondere iiber 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. Welsford, 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 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. 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.) Yamanouchi, S. 1906. The life history of Polysiphonia violacea. Bot. 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 gametophytej which arises from the spore and produces gametes; and the sporophyte, which arises from the fusion produel of two gametes and produces spores. In such a normal life cycle the Dumber of chromosomes in the nuclei is doubled at the union of the gametes and reduced to the original number at sporogenesis ; t he gametophyte is I herefore t he 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 algae and fungi also, so that the general conception of alternation of generations has been extended to these lower group-. 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 Dumber is broken, arc now known. the conspicuous examples being found among the fern- and certain angios perms. The very convenient classification of Buch abnormalities drawn up by Vines (1911) is given as the basis for the present portion of the chapter. All dates and t he matter included within square brack* have been added by the present author. "In the first place, the sporophyte may be developed either after an abnormal sexual art, or without any preceding sexual act a; all, a con- dition known as apogamy. In the second, the gametophyte may be developed otherwise than from ;i po>t -meiot ie Bpore, :i condition known as apospory.1 1 [Apogamy in ferns was discovered by Farlo* in 1874. Vpospor} was discovered in mosses by Pringsheim in L876 and in ferns by Druer} in 1884 I ons of these phenomena are given bj Winkler 1908 and Strasburgei I •' >.] i 1 312 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 Uredineae (Caoma 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. poly dactyl a. X 500. {After Fanner and Digby, 1907.) B, section through gametophyte, showing young sporophytic tissue (s) "engrafted" into surrounding gametophytic tissue (g). (After Farmer and Digby.) C, sporophyte arising apogamously from gametophyte in Pier is cretica: b1, 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 Pow, Puccinia Poarum) : male cells (spermatia) present but function- less. APOGAMY, APOSPORY, AND PARTHENOGENESIS 313 id) There is no female organ : fusion takes place bet ween i wo 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, &, and c: observed I Farmer [and Digby] L907) in the prothallium of certain ferns I Lastrcea p«< udo- mas, var. polydactyla) [Fig. L24, A]: male organs (and sometimes female) present l)in functionless. Another such case is thai of Humaria rutilans (ascomycete), in which nuclear fusion m^AJ'n J * 1950. I B. ^-40Z/™ 1911 ) B, incomplete nuclear division in sporangium ol Nephrodium htrtijx ■ \-> ■ (After Steil 1919.) C, apogamy and sporophytic budding in the embryo sac o I Ua pastorates: egg developing apogamously below; cell of aucellus forming an embryo above; two polar nuclei and one synergid nucleus al center. [After Murbeck, IWJ has been observed (Fraser L908) in hyphse of the hypothecium: the asci are developed from these hypha, and in them meiosis takes place; there are no sexual organs. [A similar condition has been reported in Helvetia crisp*, (Carruthers L911) 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 asms, 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 elata. Part henogeneti 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).] (j8) 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 (b) The gametophyte is diploid (sec under Apospory): (a) The sporophyte is developed from the diploid oosphere: observed in some Pteridophyta, viz. certain ferns I Farmer 1907), Athyrium Filix-fcemina, var. clarissima, Scolopend- Hum vulgare, var. crispum-Drummond(Bf and Marsilia (Strasburger 1907); also in some Phanerogams, viz., Compositas (Taraxacum, Murbeck 1904 : .1 nti nnaria alpina, Juel 1898, 1900; sp. of Hieracium, Rosenberg L906): Rosacese (Eu-Alchemilla sp., Murbeck L901, 19043 Stras- burger 1905 [Fig. 125, C]): RanunculaceaB {Thalictrum purpurascens, Overton 1902). [Also in the lily, Atamosco (Pace 1913), and Burmannia (Ernst 1909). Besides tin- 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 eases the chromosome number in apogamous species is about twice as large as that of nearly related forms reproducing sexually (Rosenberg 1909).] (/?) The sporophyte is developed vegetatively from the gameto- phyte: observed (Farmer [and Digby] 1907) in the tern 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 (2a0) 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, 80 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 Composite {Taraxacum and AnU nnaria alpina, Juel 1898, 1900, 1904), some Rosacea (Eu-AlckemiUa, 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 Hieradum it has been found (Rosenberg 1900) 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 angularc var. pulcherrimuvi; sp, sporangia. X 70. (After Bower.) B, gametophyte with archegonia arising from tip of pinnule in Pohjstichu X 10. {After Bower.) in . 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 Fucaceae 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) Withoul meiosis: the gametophyte is developed upon the sporo- phyte by budding; thai 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 El. and fan. Marchal (1909. 1912) were able to product^ in Mnium, Bryum, Phascunij 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-fcemina, Scolopendrium vulgare, Lastrcea pseudo-mas, Polystichum angulare, and in the species Pteris aquilina and Asplenium dimorphum), the gametophyte (prothallium) 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 eases 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 alter 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 \\ quite unfitted for independeni repro- duction, rather than from any inherent difference hot ween 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 Ascaris, 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) t wo 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, AFOSPORY, AND PARTHENOGEh ESIS 3 1 9 develop into workers or into queens with the diploid number (32) of chromosomes; those not fertilized develop into drones with flic 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 lenticular is, 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 zygogenetie 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 zygogenetie 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 tlii- respect it resembles the less highly specialized cells of other tissue- 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 purposeonly 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 Uredineae. 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. 1. 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 liber 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. 11. 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-fcemina). 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 coelistris 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: 101-199. pis. 16-20. GOLDSCHMIDT, R. 1920. Arch. Zellf. 15 : 283. Hertwk;, P. 1920. Haploidc und diploide Parthenogenese. Biol. Zentralbl. 40: 145-174. Juel, H. 0. 1898. Parthenogenesis bei Antmnaria alpina. (L.) R. Br. Bot. Centr. 74: 309-372. 1900. Vergleichende Untersuchungen iiber typische und parthenogenetische Fort- pflanzung bei dor Gattung A>tl< mtaria. Ilandl. Svensk. Vet. Akad. 33: pp. .V.t. 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 fiber die Entwicklungsgeschichte der Phaeophyceen. 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 sexualitc chcz les mousses. II. Bull. Acad. Roy. Belg. 1249-1288. 1912. Recherches cvtologiques sur le genre " Amblystegium.1' Ibid. 51: 189-203. lpl. 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 mil bezug auf eingebettete Antheridien und Apogamie. Jahrb. Wiss. Bot. 56: 0.~t 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, 1901. pp. 285-290. 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). Areh. Zellf. 11: 169-241. pis. 7-10. Nathansohn, A. 1900. Ueber Parthenogenesis hci Marsilia und ihre Abhangigkeit von der Temper;. tur. Her. Deu. Hot. Ges. 18: 99 1<)<». figs. 2. Osawa, J. 1913. Studies <»n the cytology of some species of Taraxacum. Arch. f. Zellf. 10: 450-4G9. pis. 37, 38. -1 322 INTRODUCTION TO CYTOLOGY Overton, 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. 56: 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 und seinen Anschluss an den Generationswechsel der Moose. (Vorl. Mitt.) Monat- schr. Akad. Wiss. Berlin, 1876. p. 869. 1878. Ueber Sprossung der Moosfruchte 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. 1919a. 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. Stokey, 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., 11th Ed. Weismann, A. 1886. Richtungskorper bei parthenogenetischen Eiern. Zool. Anz. 9 : 570-573. 1887. Ueber die Zahl der Richtungskorper und liber 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, Helene 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. Y \manouchi, S. 1908. Apogamy in Nephrodium. Bot. Gaz. 45: 289-318. pis. «). 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 ontogenetic 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 then1 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). 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 (ELL ORGANS IN HEREDITY 325 plants, Guignard, Nawaschin (1910), and Welsford (19] \ pointing out that in Lilium only the male nucleus enters the egg, its accompanying cytoplasm being rubbed off and left behind. (Sec p. 299.) Certain ingenious experiments of Boveri (1889, L895j also L909 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 granulans undergoing artificially induced cleavage 1 1 1 i t « .- i ~ ; spermatozoon of Strongylocentrotus lividus lias entered and taken the form of a chromosome group. B, cytokinesis beginning; one blastomere will have a purely maternal nucleus, ami the other ;i hybrid nucleus. (Diagrammed after Herbst, !!><)<».) 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 thai it is the sperm nucleus alone, and not the egg cytoplasm, that transmits the hereditary characters from one generation to the next in tin's case. Other experiments of a similar nature, however, tinned oul differently. as will presently he 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 larva? 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 Parcel) i mis (sea urchin) were fertilized by sperm of THE ROLE OF THE ('ELL ORGANS IX HEREDITY 327 Antcdon (crinoid) the Larvae so produced, contrary to BoverPs 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 larvae. 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 that 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 aform that easily escapes observation, but which can exert its usual influence on development. In accordance with his earlier observations, enucleate egg fragments of Sphcerechinus 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 larva1 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 larva1 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 larva? 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 Ascaris (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, thai 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 !»<• 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. CTENOPHORE. TUKBELLATIIAN ECHINODERn A5CIDIAN Fig. 128. — Eggs of various animals, showing the patterns assumed by the materials 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.) Substances 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 arc 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 191"), p. lis . If such eggs arc 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 (ELL ORGANS IN HEREDITY 331 general type of development and the sperm and egg nuclei supply only the details" (p. L76). Plastid Inheritance. Certain cases of 'plastid inheritance'1 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 thai tin- 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 alga? in which a swarm spore germinate- to produce ;i multicellular individual {Ulothrix etc.), or associates with others of it- kind to form a colony {Hydrodictyon, Pediastrum; Harper 1908, I918a6), the color of the successive colonies or multicellular individuals is a charac- ter that is transmitted directly by the repeated division of chloroplast-. 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 tin-! organs. Abnormalities in chlorophyll coloring are accordingly held to be due to an abnormal condition or behavior of the chloroplast s. 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 mat (anal. 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 tin4 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 bothjparents. 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 finding 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 progressal Cornell University on variegated races of maize points in this direction. One of the most interesting problems in cytology and genetics a1 presentis 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 o\ granular primordia, which multiply by fission and develop into aleurone bodies of various tvpes and colors. But since aleurone and other endo- sperm characters are inherited in Mendelian fashion, as shown by Eas1 and Hayes (1911, 1915), Collins (1911), and Emerson (1918), and since there has been adduced in support of the supposed sort ing out of primor- dia no evidence approaching in cogency that upon which t he chromosome theory has been built up, geneticists generally are of the opinion thai the chromosomes with their well known mechanism of segregation oiler 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, rhose characters which are inherited according to Mendelian rules, to anticipate a conclusion based on evidence to be presented in the nexl chapter, mall probability owe their repeated appearance in successive generations to "factors" of some sort which are transmitted by tin- chromosomes oi the nucleus. This applies also to those characters which, while Men- delian in distribution, depend lor their expression upon the presence of other cell organs (plastids) which may have an individuality oi the,,- 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 (ELL 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 response 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 eit her 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 XVII 1. CHAPTER XV 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 \y2 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 invisible 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 (F2) 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 at. 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 1919a; Thomson 191.3; East and Jones 1919. 336 MENDELISM AND Ml TATlo.X 337 by T) which makes the resulting planl tall. The germ cells of the dwarf plant carry something (/) causing the dwarf condition. In the firsl PARENTS r, L Fig. 12<). — 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 the3:l ratio of tall plants to dwarfs in the second hybrid generation (/•' ■■>. \t the right is shown the corresponding distribution of tin- Mendelian factors for tallness (T) and dwarfness generation not only three genotype- 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 77. 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 AA Bbcc is homozygous for the characters represented by .1.1 and re, and heterozygous for those represented by Bh. 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 Menclelian 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 ABCD 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 FERTILIZATION Union of simplex groups CLEAVAGE Duplex group i ABCD abed SOMATIC DIVISIONS Duplex groups Aa Bb Cc Dd SYNAPSI S GERM CELLS Simplex groups Fig. 131. — Diagram showing tho 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. MENDELISM AND MUTATION 341 It will be recognized at once thai the above is precisely the sort of distribution shown by i he characters in Mendel'* s experiments: two groups PARENTS / «k KQ,^l- Fig. 132.— Mendelian inheritance in black and albino gui guinea pigs. Fig. 133.— Chromosome history in the cross represented in Fig. L32 showing th» Parallehsm , twl,M1 t distribution of a single homologous pair of chr ome° M of a single allelomorphic pair of Mendelian characters. of (factors for) characters are brought together al fertilization and -ire 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 F2 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 Fx 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 (F^) 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 MEXDEIJSM AND MUTATION 343 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 it 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 arc 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 emphasized by Boveri and by Sutton, which has led geneticists generally to the view that the characters arc actually Fig. 1.34. — Diagram showing the 16 genotypic constitutions which may be present in the gametes of an organism with only 4 pairs of factors. (After Wilson, L913 represented in the chromosomes by material factors, or genes, which in some unknown manner control the development of the characters in the body. The earlier view that each character is thus represented by a single material unit or determiner has now given way to the more fully devel- oped Factorial Hypothesis, according to which, on the one hand, a character may be due to the cooperative action of two or more factors ("duplicate" or " cumulative " factors); and, on the other hand, a single factor may have "manifold effects," influencing the development of several characters. The factors, or genes, are thought by some to constitute a complex reaction system, interactions between genes having a marked effect upon their activity in producing characters.1 "The factorial 1 A simple and brief explanation of the effects of cumulative factors is given by Coulter and Coulter (1918). 344 INTRODUCTION TO CYTOLOGY hypothesis does not assume that any one factor produces a particular character directly and by itself, but only that a character in one organism may differ from a character in another because the sets of factors in the two organisms have one difference." "It can not . . . be too strongly insisted upon that the real unit in heredity is the factor, while the charac- ter is the product of a number of genetic factors and of environmental conditions" (Morgan et at, 1915, pp. 210, 212). The abundant opportunity for the formation of new factor combina- tions should be noted in this connection. An organism with four pairs of chromosomes in its body cells, and only one pair of factors in each chromo- some pair, could form, as the result of the independent distribution of the four pairs of chromosomes, gametes with as many as 16 different geno- typic constitutions (Fig. 134). Such a diversity being present in the gametes of both sexes, this means that more than 200 different com- binations are possible at fertilization. The 12 pairs of chromosomes in man may in the same way form several million such combinations. Since there is good reason to believe that each chromosome carries more than one factor the number of variations actually produced by these means is almost incalculable. This subject will be pursued further in the chapter on Linkage (Chapter XVII), where the evidence for the presence of many factors in a single chromosome will be presented and the consequences of this condition pointed out. MUTATION Although opinion is divided over the question of the real nature of the phenomenon of mutation, particularly in (Enothera Lamarckiana, one school (deVries et at.) holding that it represents the actual origin of new forms, and another (Bateson, Davis, Lotsy) regarding it as the result of segregation in an organism of hybrid constitution, the observed facts in either case are nevertheless very significant with respect to the chromo- some theory of heredity. The mutations observed to arise from (Enothera Lamarckiana fall into two general classes: first, those accompanied by alterations of the normal chromosome number (seven pairs), and second, those in which the number undergoes no change.1 Mutations Accompanied by Changes in Chromosome Number. — It is to be noted first of all that the mutants belonging to this class do not behave in a typically Mendelian fashion when bred to other forms, and that this is correlated with the serious disturbance of the chromosome mechanism. (Enothera mutants with many abnormal chromosome num- bers have been observed; Gates, for example, found them with 15, 20, 21, 22, 23, 27, 28, 29, and 30 chromosomes. 1 Our knowledge of the cytology of the Oenotheras is due mainly to the researches of Gates, Davis, Stomps, and Miss Lutz. MENDEL/SU AM) MUTATION 345 The 2$-chromosome Mutants (gigas group). In the mutanl tonus of tin's group, of which (Enothera gigas is :i member, the somatic number of chromosomes is 28 ra1 her 1 lian I 1 ; t he plants are te1 raploid I Fig. 135, B). How this condition arises is not certainly known. Stomps (1912 believed it to he the result of 1 he union of 1 wo unreduced gametes, whereas Gates (19096) suggested thai 'the doubling in the chromosome number had probably occurred as the result of a suspended mitosis in i he feii ilized egg or in an early division of the young embryo." Strasburger (19106) also adopted the latter view. *»y e Fk;. L35. — Chromosomes in (Enothera mutants. A, interkinesis in (E. Lamarck i ana; 7 split chromosomes. />', same in (E. gigas; 11 split chromosomes. C, somatic cell of CE. semilata; 1") chromosomes. I), tnetaphase of homceotypic mitosis in CE. biennis lata, showing 8 chromosomes on one spindle and 7 on the other. Spores and gametes with these numbers will result. E, the 21 chromosomes in a mutant from (E. Lamarckiana. (A and Ii after Davis, 1911; C and I) after Gates ami Thomas, 1914; E after Lute, 1912.) The mutants of the gigas group are characterized chiefly by an unusu- ally large size, not only of the plant as a whole but also of its anatomical eoustit uents. In t he tet raploid mutant (Enothera stenona res, tot instance, Tupper and Bartletl (1916) found that the change from the diploid to the tetraploid condition is concomitant with a 50 per cent increase in the length of the vessel, a 150 per cent increase in t he area of its cross section, a 50 per cent increase in the length and diameter of the tracheids, an increase in t he t hree dimensions of t he medullary ray cells, and a break- ing up of the tall multiple ray into a number of thin simple cays. 346 INTRODUCTION TO CYTOLOGY' A further significanl observation on mutants of this type was thai of Gregory (1914) on a tetraploid Primula. He showed by breeding experi- ments thai twice the normal number of Mendelian factors are present: thus when the chromosome number is tetraploid the number of factors is also tetraploid; each allelomorphic pair is represented twice. It should be stated that not all cases of gigantism are accompanied in this manner by an increase in the chromosome number. In Phragmites communis, for example, Tischler (1918) finds abnormally large size asso- ciated with an increase in the size of the chromosomes, but not in their number. Stomps (1919) points out that among gigas mutants of (Eno- thera, Narcissus, and Primula there are diploid as well as tetraploid individuals, which must mean that the altered chromosome number is not the sole cause of such mutation but is rather one of the characters of the mutant. The lb-chromosome Mutants {lata group). — The presence of an extra chromosome in the cells of (Enothera lata and other members of this group is due to the fact that the members of one pair of chromosomes in (Eno- thera Lamarckiana fail to separate at the reduction division, both of them going to one daughter cell. This phenomenon is known as non-disjunc- tion. As a consequence there are gametes with eight and six chromosomes rather than the normal seven; and a union of an 8-chromosome gamete with a normal 7-chromosome gamete results in an individual with 15 chromosomes instead of the normal 14 (Fig. 135, C). In her study of 15-ehromosome mutants Miss Lutz (1917) found 11 or 12 types belonging to this group; only two of them were of the usual lata type. This condition may be accounted for on the hypothesis that it is sometimes one pair of chromosomes and sometimes another which fails to separate at the time of reduction, so that the extra chromosome is not in all cases the corresponding one of the complement. If the various chromosomes of the complement differ in hereditary value, as there is much reason to believe, it is evident that this would allow for a great variety of mutants with the same aberrant chromosome number. In (Enothera scintillans Hance (1918) has shown by careful measurements that the extra chromosome can be distinguished from the regular 14. Two classes of gametes are formed, some with seven chromosomes and some with eight (Fig. 135, D) The union of two 7-chromosome gametes gives (Enothera Lamarckiana, the form from which (E. scintillans sprang as a mutant; whereas a union of a 7-chromosome gamete with an 8- chromosome gamete gives (E. scintillans. Hance therefore points out that the scintillans characters are plainly associated with the extra chromosome. . one from each gamete, and this individual develops into a female. In Ancyracanthus (Mulsow 1912) (Fig. L38) the male has a single heterochromosome which, since it has do homologue with winch to pair, passes to half the sperms, while in the female there arc two such elements, every egg receiving one. The two types of union result in individuals of the two sexes, as in Ascaris. In Ancyracanthus Mulsow stale- thai the five and six chromosomes can actually be counted in the living spermatozoa. ASCARIS ANCYRACANTHUS LYGAEOS PRlONIDUS Fig. 138. — The behavior of the sex-chromosomes in Ascaria (Boveri), AncyraeatUhua (Mulsow, 1912), Lygaws (Wilson, 1905), and Prionidua (Payne, 1909). In Lygcens (Wilson 1905) (Figs. 138; 139, .4) there are in the male two heterochromosomes, one small and one large (an UXY ' pair); in the female there are two large ones ("XX"). Half the sperms receive the X and half the F, and every egg has an X. Fertilization by an X sperm results in a female (XX), and by a Y sperm in a male (A')'). In Prionidus (Payne 19091) (Figs. L38; L39, B) the male has three small heterochromosomes and also a much Larger one. At the time of reduction the three small ones behave as a unit and pair with the large one: half of the sperms therefore carry the former and half the latter. In the female there are six small heterochromosomes, and the eggs are all alike in having three each. Fertilization now results in females with six small elements and males with three small and one large. 1 In this paper Payne gives diagrams of several other types of heterochromosomes. 360 INTRODUCTION TO CYTOLOGY In the fruit fly, Drosophila melanogaster (Stevens 1907; Morgan 1911; Metz 1914) (Fig. 140), there are four pairs of chromosomes, including in the male an 17 pair and in the female an XX pair. Reduction in #* B f\\ \ D ' . '•/ M h, E K •♦.♦ k F : W Fig. 139. — Sex-chromosomes in various insects. A, spermatocyte of Lygceus, showing the X-chromosome at left and y-chromosome above, both split. X 2250. (After Wilson.) B, prophase in spermatocyte of Prionidus, showing sex-chromosomes enclosed in plasmosome. X 2294. (After Payne.) C, pro- phase in spermatocyte of Protenor. X 2250. (After Wilson.) D, metaphase of hetero- typic mitosis in spermatocyte of Protenor. X 2250. (After Wilson.) E, anaphase of heterotypic mitosis in spermatocyte of Musca domestical h, heterochromosomes. X 1500. (After Stevens.) F, the two daughter chromosome groups in the anaphase of the hetero- typic mitosis in the oocyte of Phragniatobia fuliginosa, showing 28-29 distribution. X 4080. (After Seiler.) spermatogenesis gives sperms of two sorts : all contain four chromosomes, but in half of them one of the four is the X, and in the other half it is the Y. Since every egg contains an X, two kinds of union are possible at fertilization: an X with a Y, giving a male fly, and an X with an X, SEX 361 giving a female. In this case, a typical example of the A')' form of sex inheritance, extensive researches have shown thai the F-chromosome carries no factors for sex; the presence of one ^-chromosome is associated with maleness and t hat of t wo A'-chroinosonies with femaleness. Morgan (1914, 19196) and Morgan and Bridges ( 1919) find that gynandromorph- ism frequently appears in Drosophila females as the resull of the elimination of one sex-chromosome in abnormal mitosis. DBOSOPHILA / \ / \ MAN PhAA&MATOBIA Igpr Tig. 140. — The behavior of the sex-chromosomes in Drosoph ila (Stevens, Morgan, Metz), Man (Wieman), Phragmatobia (Seiler), ami the fowl (Guyer). Malone (1918) reports that there are present in the spermatocyte of the dog 10 pairs of autosomes and one large unpaired X-chromosome. The X passes undivided to one pole in the first mitosis ami divides in the second, so that half of the spermatids, and hence spermatozoa, receive an X while half do not. When measurements of these spermatozoa are plotted a bimodal curve results, showing that the chromosome difference is correlated with a size dimorphism. The same condition, except for the number of autosome pairs, is reported for the spermatozoa of horses, pigs, and cattle (Wodsedalek 1913, 1914, 1920). In the case of man also the evidence at hand indicates a digametic condition on the part of the male, hut certain striking discrepancies in the findings of various investigators have afforded a puzzle which up to the present time has not been satisfactorily solved. Plemming (1898) counted 24 chromosomes in the cells of the cornea, and Duesberg ( L906 found 12 in the spermatocytes. The same numbers were found by Montgomery (1912). In 1910 Guyer reported that the spermatogonia of the negro contain 22 chromosomes; these in the spermatocyte form 10 bivalents and two distinguishable accessories. At the tirst maturation mitosis both of the latter go to one pole and at the second miiosi< both divide, so that half of the sperms have 10 chromosomes and half have L2; the two accessories in the latter case are visible as " ehromal in nucleoli ' in the resting stage. This difference in the gametes Guyer regarded as probably associated with sex-determination. Gutherz (1912) failed to 302 IXTRODFCTIOX TO CYTOLOGY confirm ( hiyer's report of a dimorphism among the sperms, though he also observed the accessories. Guyer in 1914 reasserted his conclusions of 1912, but added thai he was finding a much larger chromosome number in the cells of the white man. Yon Winiwarter (1912), also working upon the white man, found in the spermatogonia and spermatocytes 47 chromosomes, including one accessory. Since this accessory passes undivided to one pole in the first mitosis and divides in the second, half of the sperms receive 23 and half 24 chromosomes. In the cells of the female there are 48. From these data von Winiwarter logically concluded that the egg has 24 chromosomes, and that it develops into a male when fertilized by a sperm with 23 chromosomes, and into a female when fertilized by one with 24. Such a large number is found in the white man by Evans1 also, but he finds 48 chromosomes rather than 47 in the spermatogonia, indicating the presence of an XY pair as in Drosophila. M- Fig. 141. — Sex-chromosomes in man. A, primary spermatocyte, negro. B, same, white. C, metaphase of heterotypic mitosis, negro. D, interkinesis, white. XY, the sex-chromosomes; P, plasmosome. (After Wieman, 1917.) Wieman (1917), using both negro and white material, finds 24 chromo- somes in the spermatogonia, two of them being distinguishable as an unequal XY pair which remains condensed while the autosomes form the reticulum (Figs. 140, 141). In the spermatocyte 12 pairs are evident, including the XY pair. At the first maturation mitosis the 11 autosome pairs separate into univalents as usual, but the X and Y divide longitud- inally; thus each daughter cell (secondary spermatocyte) receives 11 auto- somes and an XY pair. At the second mitosis the 11 autosomes divide longitudinally in the normal fashion and the X and Y separate. As a result all of the sperms receive 12 chromosomes : in half of them one of the 12 is the X and in the other half it is the Y. Although for a time it ap- peared that the white man had twice as many chromosomes as the negro, a difference ordinarily great enough to mark them as distinct species, Wieman shows clearly that in his material the two have the same num- ber, and is inclined to regard von Winiwarter's material as in some way abnormal. Sex inheritance in man is evidently of the XY type, as Wieman's researches and genetic data indicate with considerable clearness ; but why some material should plainly show twice as many chromosomes 1 Unpublished work cited by Babcock and Clausen, p. 538. SEX 363 as other materia] is a question which only future investigation can answer. It is not improbable, however, thai a segmentation of the chromosomes at points of constriction may be mainly responsible for this condition. In all of the cases reviewed above the male produce- two sorts of spermatozoa differing visibly in chromatin content: in the language of Mendelism, the male is "heterozygous for sex." The female produces but one kind of egg; she is "homozygous for sex." The sex of the off- spring is clearly dependent on the kind of spermatozoon which timet ion-, and is therefore definitely correlated with the chromosome mechanism. Female Heterogametic.- -Thwc are also on record a number of cases chiefly among moths and birds, in which the female produces two kinds of eggs differing in chromosome content, while the male produces but one kind of spermatozoon: the female is heterozygous for sex, and therefore heterogametic, while the male is homozygous. Certain cases of this type will now be reviewed. In the moth, Phragmatobia, Seiler (1913) has described the following condition. In the male the somatic number of chromosomes is 56, including 54 autosomes and two Z-chromosomes1 (Figs. 139 F\ 110). Eaeh sperm receives 28 (27 + Z). In the female the somatic number is likewise 56, but includes a ZW pair instead of the ZZ pair of the male. Half the eggs receive 27 + Z and the other half 27 + Ww (the ^-chromosome breaks temporarily into two parts during maturation). An egg with 28 (27 + Z) chromosomes fertilized by a sperm with 28 (27 + Z) develops into a male moth with 56 (54 + ZZ). An egg with 29 (27 + Ww) chromosomes fertilized by a sperm with 28 (27 + Z) develops into a female moth with 57 (54 + ZWw). The W and w subsequently reunite to form a single W, both sexes then having the same number, 56. Since some embryos show more than the normal number of chromosomes Seiler thinks it probable that the /-chromo- some is compound and may under certain conditions subdivide into smaller elements. The same investigator has recently (1919) reported a digametic condition in the female in two other moths. Taloeporia tubulosa and Fumea casta. In the former the eggs have 29 and 30 chromosomes, and in the latter 30 and 31. In the moth, Abraxas grossulariata (Doncaster 1914), sex inheritance is apparently of the WZ type, though there is often an aberrant behavior on the part of the chromosomes which has not been entirely explained. In the common fowls Guyer (1909, 1916) has made observations which he interprets as follows (Fig. 140.). In the male there are IS chromo- somes: 16 autosomes and two accessories. Both of the latter go to one pole in the first maturation mitosis, and in the second mitosis they sepa- 1 It is customary to refer to the Bex-chromosomes in species with sexually hetero- zygous females as W and /. instead of )' and A' as in (lie more common sexually heterozygous males. 364 INTRODUCTION TO CYTOLOGY rate. Half the spermatids, and therefore sperms, receive nine chromo- somes (8 + 1 accessory), while the other spermatids failing to receive an accessory apparently degenerate. In the female there are 17 chromo- somes: 16 autosomes and one accessory. Since the accessory passes undivided to one pole at the first maturation mitosis and divides at the second, half of the eggs receive nine chromosomes (8 + 1 accessory) and half receive eight. An egg with nine fertilized by a sperm with nine develops into a male with 18 (16 + 2 accessories). An egg with eight fertilized by a sperm with 9 develops into a female with 17 (16 + 1 accessory). Cases of Parthenogenesis. — The cytological phenomena in those animals reproducing in part by parthenogenesis (see p. 357) are of much interest in this connection. In the honey bee the male, which develops from an unfertilized egg, has the haploid number of chromosomes in his cells, whereas the female, arising from a fertilized egg, is diploid. Similar in some respects is the case of the gall-fly (Neuroterus), in which eggs that have undergone reduction develop into haploid males, while other eggs are formed without reduction and develop into diploid females. In the male-producing eggs of Phylloxera there are two sex-chromosomes, two others being lost in the polar body; in the female-producing egg all four are present. Two kinds of sperms are produced, half of them with a sex-chromosome and half of them without it. The latter kind degen- erate, leaving only the former functional. All eggs, if fertilized, develop into females. In Hydatina senta those eggs producing one polar body and developing into females are diploid, whereas those giving off two polar bodies and developing into males are haploid. In all of these cases maleness accompanies the haploid, and femaleness the diploid condition1. Plants. — Up to the present time a visible chromosome difference between the two sexes in plants has been established only in Sphcero- carpos, the genus of dioecious liverworts in which Douin (1909) and Stras- burger (1909) found two of the spores of a single tetrad to be male and the other two female. In Sphcerocarpos Donnellii (Allen 1917, 1919) (Figs. 142, 143) there are in the cells of the female gametophyte seven autosomes which differ somewhat in length, and one very large X-chromosome. In the cells of the male gametophyte there are seven autosomes and a very small F-chromosome. The sporophyte therefore has eight pairs: seven autosome pairs and the XY pair. Although all the stages in the divisions at sporogenesis have not been seen, the evidence is sufficient to show that the X and Y separate in the heterotypic mitosis and divide longitudinally in the homceotypic. Two spores of a tetrad therefore receive an X- chromosome in addition to the seven autosomes; these spores develop into female gametophytes. The other two spores of the tetrad receive 1 Compare the case of the frogs developing by artificial parthenogenesis, page 319. si-:.\ 365 the Y in place of i !,<• X and develop into male gametophytes. The same condition has been reported for Sphoerocarpos tezanus (Miss Schacke 1919). Although the situation in Sphoerocarpos suggests the XY type of sex inheritance in Drosophila and other forms, it differs in several respects, iiiiiii Male gametophyte IIIIIII' Female gametophyte Fertilized egg Sporophyte Sporocyte Maturation divisions SPHAE.K0CARPU5 IIIIIII Male gametophyte I I Hill' Female gametophyte 6 &> Fig. 142. — The history of the chromosomes in the life cycle of Sphoerocarpos. I data of C. K. Allen, 1917, 1919.) as Allen (1919) points out. In Sphoerocarpos the separation of the XY pair results in the production of two kinds of spores which develop directly into haploid organisms (gametophytes) of two sexes, whereas in Droso- phila the corresponding separation results in two sorts of male gametes which determine the sexes of the diploid organisms developing from the eggs they fertilize. Furthermore, in animals wit h sex-chromosomes some forms show the presence of XX to be correlated with femaleness and X or XFwith maleness (male heterozygous for sex), while in other forms XX ™** :l"d [TnL^m^An^r E •'» _ Sphavrocarpos Doumlhi. ifter < . /. is correlated with maleness and X AUen, 1919.) or XY with femaleness (female heterozygous for sex1)- There is evidence to show thai the K-chromo- some carries no sex-factors in these cases,2 though its absence may result in sterility (Bridges). (See Chapter XVII.) In Sphoerocarpos, on the 1 Sex-chromosomes referred to in this case as Z and W rather than X and Y, 2 £oe in this connection Castle 1921. Fig. l 13. ( Jhromosome groups from 366 INTRODUCTION TO CYTOLOGY oilier hand, 1 lie* presence of X is correlated with femaleness, Y with maleness, and XY with the non-sexual condition of the sporophyte. Here the Y is apparently as important as the X in the transmission of sex-factors. Allen suggests that secondary sexual differences of the gametophytes, such as size, may be connected with the relative amounts of chromatin in the nuclei: the female gametophyte, having the large X-ehromosome and therefore a distinctly greater mass of chromatin, develops more rapidly and becomes much larger than the male gameto- phyte with its small F-chromosome. The primary sexual differences he regards as due to other factors. Although Sphoerocarpos affords the only known example of hetero- chromosomes in plants it is not improbable that other cases will be discovered. Conclusion. — In all of the organisms included in the foregoing review the individuals of the two sexes differ visibly in their chromosome com- plements. Moreover, in most of them the sexual differences are definitely correlated with special distinguishable chromosomes, which are accord- ingly known as sex-chromosomes. The distribution of these bodies at the time of reduction results in the production of two kinds of male gametes or two kinds of female gametes, and in at least one case two kinds of spores. In all of these the chromosome differentiation in the cells correponds to the sexual differentiation of the organisms into which they develop. The conclusion appears unavoidable that the differentia- tion of the sexes is here determined by a cell mechanism, and that the hetero- chromosomes have a definite causal relationship with sex. How close this relationship may be, and to what degree it is a fixed one, are as yet by no means clear, but it is beyond question that the heterochromosomes are not the sole determining cause of sex, as some workers have hastily concluded. To this question we shall subsequently return. Sex -chromosomes and Mendelism. — The heterocbromosome phe- nomena are intimately bound up with the whole matter of Mendelian inheritance. According to the Mendelian interpretation the sexes are due, like other heritable characters, to factors carried by the chromo- somes— by the heterochromosomes where these are present. The ap- proximate 1 : 1 ratio of the sexes in most organisms is accounted for in the following manner. Referring to our typical Mendelian pair of charac- ters in the pea, tall and dwarf, it is found that when a plant heterozygous for tallness (Tt) is crossed with a pure recessive (tt) the resulting off- spring are half tall (Tt) like one parent and half dwarf (tt) like the other, a 1 : 1 ratio. If it is assumed in a similar manner that there is a pair of factors for sex, one sex (the male, Correns; the female, Bateson) being- heterozygous and the other a homozygous recessive, a 1 : 1 ratio of the sexes results. Largely because of the observed behavior of sex-chromosomes the SEX 36' Mendelian interpretatioD has been restated as follows. There is :i single factor for sex. In some organisms the presence of two of these factor- is correlated with femaleness and one with maleness (Fig. 111. A I: the male, having only one sex-factor (S), is heterozygous and produces gametes of two kinds, with and without the fact or; the female, having two sex-factors, is homozygous and produces eggs of one kind, with one sex- factor each. Two types of union are here possible, giving males and females with one and two sex-factors respectively. This interpretation is directly applicable to those cases in which the male has one sex-chromo- some and the female two (Ancyracanthus, Ascaris), and also to those having an XY pair in the male and an XX pair in the female (Droso- phila, Lygceus). Each sex-factor is thus thought to be located in an A- O0O0 \ X / OQ / \ / \ OQD0 Fig. 144. — Mendelian interpretations of sex-inheritance. Explanation in text. chromosome. In other organisms (moths and birds) these conditions are reversed (Fig. 144, C), the presence of two sex-factors being correlated with maleness and one with femaleness. The female is thus heterozygous and produces eggs of two kinds, with and without the factor. In the next generation the male receives two sex-factors (in the Z-chromosomes of the egg and sperm) and the female one (in the Z-chromosome of the sperm; the W-chromosome of the egg carries no sex-factors). In view of these two contrasted conditions as regards the quantitative relationship between factors and sex, it is probable that the sex-factors carried by the X-chromosomes are in some manner different from those in the /-chromo- somes. It has been suggested that in some cases ( Fig. 144, B) the male may have no sex-factors at all, the heterozygous female thus having one more sex-factor than the male, as in the homozygous females of Fig. 144, A. Experimental Alteration of the Sex Ratio. Although most organisms approximate closely the I : 1 sex ratio called for on the basis of the 368 INTRODUCTION TO CYTOLOGY Mendelian theory of sex, constant deviations from this ratio are fre- quently found. Still more significant is the fact that in many cases the ratio can be markedly altered by changing the environmental condi- tions. Thus R. Hertwig (1906, 1912) and Kuschakewitsch (1910) found that if the eggs of the frog are allowed to become over-ripe before fertilization, in which case they take up an abnormal amount of water, the resulting individuals show an unusually high percentage (even 100 per cent) of males. Conversely, Miss King (1907-1912) lowered the water content of toad eggs, and with a mortality of only a little over 6 per cent obtained 80 per cent females.- In Dinophilus, as already noted, there are two kinds of eggs laid: large ones developing into females and small ones developing into males. Malsen (1906) fouDcl that by altering the temperature the relative pro- portion of the two sexes could be changed, but this effect was brought about through an influence on the laying of the eggs: both kinds were produced as usual, but the laying of one kind was hindered. The rotifer, Hydatina senta (Whitney 1914, 1916), if scantily fed on Polytoma, continues to produce generations of parthenogenetic females, but when copiously fed on Euglena females appear which lay male-pro- ducing eggs, and sexual reproduction then occurs. According to Shull and LadofT (1916) the percentage of males is here correlated with the supply of oxygen which counteracts certain agencies (accumulated substances in the water) tending to decrease male production. Whitney (1919), however, contends that oxygen is not a factor affecting sex in Hydatina. In interpreting such results as these considerable care should be exercised in distinguishing an actual determination of the sex of an individual from a number of other phenomena which, though they may appear like sex-determination, are not to be regarded as such in the strict sense. In many cases in which changed environmental factors have been shown to have an influence on the sex ratio it is clear that the results are not due to an actual reversal or determination of the sex in any individual, but rather to the fact that the new conditions imposed have caused a greater mortality among the eggs or embryos of one sex, so that those of the other sex preponderate. Although the ratio of the sexes may here be subject to an experimental control, the sex of no given individual is actually determined or altered. Indirect control of another type is seen in organisms whose sex is dependent upon the form of reproduction (zygogenetic or partheno- genetic). Environmental conditions may in such cases influence the form of reproduction resorted to, and therefore the sex of the animals resulting; but the sex of no individual, once started, is altered. Morgan points out that the change of diet in Hydatina, instead of altering sex directly, induces the formation of a new type of female which may either function SEX 369 sexually or produce eggs which develop parthenogenetically into males. Another case in point is that of Dinophilus, in which abnormal temper- ture conditions prevenl the laying and development of eggs from which the individuals of one sex normally arise. As Thomson ( L913, p. 502 remarks, ". . .if nutritive and other environmental influences .in- operative, it is, in the main, by affecting the production and survival of sexually-predestined germ cells." A clear case of sex-determination in the strict sense would be one in which a given parthenogenetic egg, fertilized egg, spore, or young individ- ual, in a unisexual organism, could be made to develop at will into either sex;or in which such an organism, already showing the essenl ial characters of one. sex, could be made to develop into the other sex (sex-reversal). Evidence that in certain cases such a determination or reversal can actually be accomplished has had much to do with the development of recent metabolic theories of sex. Metabolic Theories of Sex — Animals. — According to the metabolic theories of sex, the difference between the two sexes is primarily one oi metabolism, the difference being not necessarily in the kind of metabolic processes, as Geddes and Thomson thought, but more probably in their rate or level. One of the most prominent exponents of this type of theory is Riddle (1912, etc.), who has continued the important researches on pigeons begun by Whitman many years ago. At each laying these birds produce a pair, or clutch, of eggs. Under normal condition- the first egg laid develops into a male and the second into a female. By a careful analysis of a large number of eggs Riddle has been able to show that the yolk of the first egg is the smaller, and has the smaller amount of storage material (fats and phosphorus-bearing compounds), the higher water content, and the higher oxidizing capacity, or higher metabolism. The second or female-producing egg therefore differs in having a Larger yolk, more storage material, less water, and a lower metabolism. The chromosomes of the pigeon are not accurately known, but it is probable, as indicated by the behavior of the sex-linked factors (see next chapter), that the WZ type of sex-chromosome differentiation found in other bird- is present here. Whatever their cytological differences may be. Ih< two sexes are characterized in the egg stage by different levels of metabolism^ tin male having the higher level and thefemaU the lower; and this difference has been shown (as indicated by the metabolism of the blood) to persist into the adult stage. The seasonal change in the amount of storage, and consequently in the level of metabolism, is definitely con-elated with the percentage of a given sex and the degree of it> manifestation. As the season advances both eggs of the clutch -tore more energy-cunt aining materials, and the birds developing from the second (female-producing) egg, while often 24 370 INTRODUCTION TO CYTOLOGY quite "masculine,: in their secondary sex characters early in the season, become increasingly " feminine " toward the end of the season. Moreover, the total percentage of females increases. By influencing storage, water content, and the general metabolic condition of these eggs Riddle has been able to induce experimentally an actual reversal of their natural sex tendencies. Hence he concludes that sex is a quantitative, modifiable, and "fluid" character; the two sexes do not represent two qualitatively distinct, mutually exclusive properties, but are rather two conditions of one general property — two levels in a continuous series of metabolic states passing gradually one into another. Accordingly, if the two levels normally maintained can be sufficiently altered a series of intergrades between the two sexes and an alteration of the sex itself should be possible, and this Riddle has apparently accomplished. On the basis of this metabolic theory of sex Riddle interprets the results obtained by Miss King with toad eggs, by Hertwig with those of t he frog, by Whitney and Shull with rotifers, and by other investigators to be mentioned below. The correlation of high and low water content with maleness and femaleness respectively in toads and frogs, and that of change of food and increased oxygen supply with maleness in rotifers, are held to be in harmony with similar correlations which have been shown to exist in pigeons. The theory that sex is a quantitative, reversible state closely asso- ciated with metabolic conditions is strongly supported by the researches of Goldschmidt (1916a6, 1917), Banta (1916), and Lillie (1917). In the gypsy moth, Lymantria dispar, which is cytologically heterogametic, Goldschmidt has been able to induce experimentally a large series of "sex intergrades " between the male and female conditions. It appears that an individual of either sex, after beginning its development, may be made to develop the characters of the other sex partially or completely, the degree of alteration depending upon the time at which the change sets in. Gold- schmidt concludes that normal individuals of both sexes must have both sex capabilities, the sex manifested depending upon the relative strength with which they are caused to act by certain conditions. He thinks it prob- able that the hereditary characters have their material basis in enzymes or substances of a similar nature, those associated with femaleness and maleness being termed " gynase " and " andrase " respectively. Although for a time he interpreted the behavior of the sexes in Lymantria on the basis of Mendelian factors, he is now inclined to view this form of explanation as inadequate. The chromosome behavior in these moths is not well known, but breeding experiments with an intersexual female functioning as a male show the results which would be expected on the assumption thai she had the PTZ-chromosome constitution. This would mean that the moth in question had had its sex reversed without any visible change in its chromosome1 complement, but this intrepretation awaits cytological confirmation. SEX ;;;, In the phyllopod crustacean, Simocepkalus vetulus, Banta 1 1916) finds that under certain experimental conditions". . . the same individual even the same sex gland, may develop eggs and sperms at 1 lie same time or sperms at one time and eggs at another time." Banta looks upon as not a fixed or definite state but rather "a purely relative thing" dependent upon the general physiological state of the organism which in turn is under the influence of environmental factors. Be also reports sex intergrades in Daphnia longi spina (1918). Lillie (1917abc), as the result of a study of twins in cattle, concludes that "sex-determination in mammals is zygotic, but it does not imply an irreversible tendency to the indicated sex-differentiation. Intensification of the male factors of the female zygote from the time of onsel of sexual differentiation by action of sex hormones may bring about very exten- sive reversal of the indicated sex-differentiation" (1917c). Intersexes have recently been described in Drosophila simulant by Sturtevant (1920). All the intersexual flies are modified females and show the same grade of intersexuality. Sturtevant believes t he condit ion to be due to the action of a mutant gene. If this interpretation is applied generally to the phenomenon of intersexuality it is evidenl that certain genes at least are modifiable by environmental conditions, for the reason that such conditions so often evoke the intersexual state. A very striking instance of the control exercised by environmental factors over the sex of a given individual is found in Bonellia (Baltzer 1914). In this gephyrean worm the male is very small and degenerate, and lives parasitically upon the long proboscis and in the nephridium of the much larger female. If young individuals are kept in an aquarium by themselves they develop into females, but if they are placed with mature females they settle upon the probosces of the latter and develop into males. By allowing them to remain on the probosces for varying lengths of time Baltzer was able to obtain many intergrades between the typical male and female conditions. It is plain that the proboscis exercises an influence over the sex of the young animal, but whether this is through a secretion or some other agency is not known. A somewhat similar case is that of Crepidula plana, described by Gould (1917). This mollusk is hermaphroditic but completely protan- dric, i.e., the male and female phases are entirely separate in time, the former developing first. The development of the male phase is dependent upon the presence of a larger individual (not necessarily a female) of the same species. If a male is removed from the neighborhood of a Larger individual the male organs degenerate, and after a period of sexual inactivity the animal becomes a female. The nature of the stimulus exerted by the larger individual has not been ascertained. Plants- Experiments of a corresponding nature on sex modification in plants are on the whole less conclusive than those on animals, for the 372 INTRODUCTION TO CYTOLOGY reason that many of them have been carried out with angiosperms, in which hermaphroditism is so common and which are regarded by some botanists as all potentially bisexual. As a case illustrating the latter point may be cited the hemp plant, Cannabis sativa. This species is normally unisexual, but if the flowers are removed it will produce flowers of the other sex (Prit chard 1916). Intersexes of many grades between the normal plants in dioecious angiosperms are frequently found, as in Myrica Gale (Davey and Gibson 1917), Cannabis sativa, Salix amygdaloides, and Morus alba (Schaffner 1919ab), whereas the relative proportions of maleness and femaleness in hermaphroditic forms are apparently very easily influenced by the environment. In Plantago lanceolata (Bartlett 1911; Correns 1908; Stout 1919) the stamens and pollen are developed in various degrees in different flowers, or even in the same flower; in some cases they are only rudimentary, leaving the flowers functionally female. Stout inclines to the view that dicecism results from the suppression of femaleness or maleness in organisms originally monoecious, and points out that the many " degrees of maleness" in Plantago fill the gap completely in this case. He accordingly concludes, in agreement with Riddle, Banta, and Goldschmidt, that sex is a labile, reversible character; that maleness and femaleness, both present in the somatic cells of all sporophytic individuals, are relative and not absolute conditions; and that " sex- determination, at least in hermaphrodites, is fundamentally a phe- nomenon of somatic differentiation that is ultimately associated with processes of growth, development, and interaction of tissues, and subject to modification or even complete determination by them." The sex- chromosome theory is held to be inadequate in the case of hermaphro- dites: sex is an "epigenetic," not a " preformed" character. Yampolsky (1919, 1920) thinks it probable that male and female gametes with graded potencies are produced in Mercurialis annua, and that the sexes cannot be due to a segregation of sex-factors at reduction. The sex of the haploid phase (gametophyte) of the plant life cycle, when this phase is unisexual (dioecious), behaves as a much more nearly irreversible character, as shown by the experiments on mosses, Marchantia, and Phycomyces cited early in the present chapter. How widely this holds true in thallophytes and bryophytes is not known. Allen regards the quantitative theory of sex as quite inapplicable to the case of Sphcerocarpos. In the homosporous pteridophytes the gametophytes are commonly monoecious, and the fact that here, as in many liverworts, the male organs appear early and the female organs later suggests a physiological basis of sex-determination. Some fern gametophytes, on the contrary, are dioecious under all ordinary conditions. In Onoclea (Wuist 1913), such a normally dioecious form, the female gametophytes under certain experimental conditions produced antheridia in addition SEX 373 to their archegonia, but the male gametophytes could n« >i be made to develop archegonia. In Osmunda regalis japonica and Asplenium nidus which are monoecious, Nagai (1915) found thai the concentratioa of the Knop's nutrient solution used has a controlling influence over the kind of sex organ appearing, the number of antheridia in general decreasing with the concentration. These results, taken together with the fact that many gametophyto especially those of homosporous pteridophytes. are monoecious, -how thai the capabilities of both sexes can be present in the haploid nucleus as Strasburger thought, although in many forms the visibly developed sexes may be automatically determined by some cell mechanism. General Discussion, — We have now reviewed some of the evidenc which have led to two general theories of sex and its determination. ( me theory represents an attempt to account for the phenomena in question on the basis of a morphological cell mechanism whereby Mendelian or other factors are distributed in a definite and fixed manner, whereas on the other theory it is held that they are the results of a physiological differentiation manifesting itself chiefly in alterable levels of metabolism. Although these two conceptions may appear to be mutually exclusive if expressed in too uncompromising a form, both must contain elements of truth. It is beyond question that the two manifestations of sexual differentiation, the physiological and the morphological, are both of importance and cannot be ultimately irreconcilable: our task i- to determine their relative significance and to discover the nature and degree of their mutual interdependence. It seems clear that the digam- etic condition when present in dioecious forms does regulate the ratio of the sexes: under all ordinary circumstances the sex of the individual is here dependent upon the kind of gamete which gives rise to it (or the kind of spore in the case of certain gametophytes). Hut it i> to be emphasized that the dimorphism shown by such gametes or spores i- not entirely a morphological one, or even mainly so: in many cases no morpho- logical difference can be detected although the two are clearly different in physiological behavior, as shown by the spores of Phycomyces and certain bryophytes. It is generally inferred that here a structural difference, although invisible, is nevertheless present. In this connection it may be recalled that the differences between the male and female gametes, irrespective of any different ial ion which may !"• present among those of either kind, are both physiological and morpho- logical. The primary characters <>t" sex are those possessed by the gametes themselves, and the principal distinction between the male and female gametes seems to be a physiological one which is manifested in their mutual attraction and fusion. Any visible morphological differ- entiations that they may possess an1 to be regarded as secondary adapta- tions to unlike functions, because of the fact thai in many of the lower 374 INTRODUCTION TO CYTOLOGY organisms there is no discernible structural difference between the male and female gametes, and further because structural differences may be annulled in certain instances (some gregarines). Any material differences present in visibly similar gametes are more probably chemical in nature, the structural difference being of a molecular order. Indeed, there is probably no physiological difference without a structural differ- ence of this sort. If the term structure be extended to include molecular constitution the discussion over the relative priority of structural and physiological differentiation becomes futile, for at this level the two are aspects of one and the same change. It is only when we restrict the term structure to the grosser, visible features that we can speak of physiological differentiation as preceding alteration in structure. Ultimately structural and functional changes are indistinguishable. Just as in the gametes of the two sexes, so also in the unisexual individuals which the gametes produce there may be striking differences of both morphological and physiological natures; but if we use the term morphological only with reference to visible features the primary distinction between the sexes in organisms of all grades is apparently one of physiological state, this distinction and its result (sexual reproduction) being of the greatest biological importance. Taking into consideration all organisms, low and high, it seems probable that any dimorphism among the gametes of one sex or the other has in some way been developed in connection with the maintenance of the above mentioned difference in physiological state in organisms of a certain level of advancement. Different organisms show all degrees in the differentiation among the gametes of one sex: some are marked by an absence of any visible difference either in the gametes or in the her- maphroditic individuals produced; in others the gametes (of one sex) are visibly similar but result in male and female individuals in regular ratio; and finally there are those in which the gametes are of two kinds both physiologically and morphologically, the two kinds controlling the pro- duction of individuals of the two sexes. It is in organisms of the last type especially that the question of sex-determination finds the adherents of the chromosome-Mendelian theory and those of the metabolic theory in disagreement. Is the sex of the individual inevitably dependent upon the type of gamete functioning (usually the kind of sperm fer- tilizing the egg), or is it possible to overcome the effect which the chromosome mechanism may have by influencing sufficiently the metabolism of the organism? If the sex of an individual is so changed, does the chromosome mechanism undergo a corresponding alteration? Those who have developed the chromosome-Mendelian theory have perhaps too often held that the two sexual states, maleness and female- ness, are in their ultimate analysis mutually exclusive — that they are two fundamental and qualitatively different alternative characters depending SEX 375 unalterably upon unit factors. The hereditary factors m genes are usually regarded by geneticists as unmodifiable excepl by sudden muta- tions. Failure to distinguish between (he modification of genes and ill- modification of the interaction of genes during ontogenesis has led many to the view that the sex of the individual must be rigidly fixed al fertiliza- tion in digametic and dioecious forms, or at reduction in the case of dioe- cious gametophytes like those of Sphcerocar pos. It is very difficult to reconcile any inflexible theory of this nature with the great diversity of situations known without resorting to hypotheses of somatic segregation of factors, alterations in dominance, and other assumptions not well supported by observational evidence. Such difficulties are encountered in the common hermaphroditic condition of gametophytes, which are haploid; in the possibility of causing the development of the second sex in gametophytes normally unisexual (Onoclea); and especially in the numerous cases of sex intergrades, in which it is possible not only to con- trol the relative amounts of maleness and femaleness in hermaphroditic forms, but also to produce all intermediate grades between male and female individuals, and furthermore to reverse the sex in unisexual forms, even in certain species with sex-chromosomes (moths and probably pigeons). If, in accordance with the ideas of many biologists, sex is held to be a quantitative, " fluid" character associated with a continuous series of physiological states which may pass into one another, the way i- open for the explanation on a common basis of all cases of hermaphrodit ism in both haploid and diploid individuals, of sex intergrades, and of the experi- mental modification of sex. At the same time the influence of the sex- chromosomes or even of smaller factors within them may be allowed. As Riddle (1917) states, organisms have had the problem of producing germs of two metabolic levels, and in some cases this has led to the establishment in the two sexes of two amounts of chromat in or even of t \\ <> different chromosome complements. The sex-chromosomes, or units contained within them, act with others in the maintenance of two diverse levels of metabolism in the gametes and in the offspring, and with these levels are correlated the two conditions which we distinguish as male and female. Even if the sexes in such cases do not differ in the quality of their chromatin, they at least differ quantitatively in this respect, in agreement with the theory that sex isaquantitat ive character. The chromosome difference being only one factor in .1 complex system producing the two sexual states, and no single element in tin- System being the sole determiner of sex, it is not impossible that the r\)\'r\ of this one factor should be annulled by sufficiently altering the other factors and thus modifying the action of the factorial system as a whole. Tin- same is to be said of other characters also. What an organism inherits is not simply this or that character or sex, but rather a tendency to 376 INTRODUCTION TO CYTOLOGY develop a definite group of characters, including- a particular sex, under a given set of environmental conditions. In those organisms possessing heterochromosomes the sex of the individual under all ordinary circumstances is dependent upon the kind of sperm (or, in some cases, the kind of egg) functioning at fertilization, and does not change thereafter. Furthermore, it apparently cannot be changed by many methods commonly supposed to be efficacious in this respect. So far the chromosome theory is valid; but it does not follow from this that the sex which is characterized by a certain physiological state and is correlated with a certain type of chromosome complement and a variety of secondary sexual characters, is so firmly fixed that it cannot be altered by any extraordinary means. The metabolic state, even though its regulation may be accomplished in part through a visible mechanism, is the resultant of a complex series of reactions which may be interfered with at many points. In some cases this metabolic state has been artificially altered to a degree sufficient to bring about an actual reversal of the sex. It is admitted that other heritable characters de- finitely associated with constant genes are greatly modified in the manner and degree of their expression by environmental influences, and the evidence now at hand indicates that no exception to this rule can be made in the case of sex. In criticizing the results and interpretations of Riddle, Morgan (1919a) points out that the behavior of a certain sex-linked character worked out by Strong (1912) indicates that the females which were " changed into males" have the male chromosome complement, and that sex is as much a matter of chromosomes here as elsewhere. He declares further that there is as yet no known case in which the sex determined by a chromo- some mechanism has been changed by other agencies in spite of the chromosome arrangement. The evidence here points to the conclusion that when an alteration of the sex is induced, this does not occur with- out a corresponding alteration in the chromosome mechanism. In Droso- phila, however, Sturtevant (1920) finds that the intersexes observed by him are modified females with the usual two X-chromosomes. Here, therefore, certain male characters at least are present in an organism with the female chromosome complement. The number of instances of change of sex in forms normally controlled by a chromosome mechanism will probably increase as the nature of sexual differentiation becomes more fully known and as experimental technique improves; but it is also probable that in animals the sex of which we are most desirous of controlling, practical difficulties may pre- vent the attainment of satisfactory results. Slight differences in the responses of the two kinds of male gametes (or female gametes) might conceivably make possible a control over the kind functioning, but it seems more probable that the sex of the individual will be found to be sex 377 more easily influenced, it" al all, after fertilization. Bui ii is quite un- known whether or not an individual which had undergone a reversal of sex would be as successful biologically as a normal one. Unusual fea- tures may be expected in a sexually fund ional organism wit h I be chromo- some complement normally accompanying the other sex. it such an organism should be found; but such interesting questions can only be answered by the facts yielded by further research. All questions of sex control are secondary to the main problem of the ultimate nature of sex, a problem which reveals itself with increasing clearness as primarily a physiological one. The question of the origin and significance of sex is one which lies outside the scope of this work. Bibliography at end of Chapter XVIII. CHAPTER XVII LINKAGE In Chapter XV attention was directed to the remarkable parallelism which exists between the distribution of the Mendelian characters and that of the chromosomes. A vast number of breeding experiments with both plants and animals have shown that new combinations of characters are formed at the time of fertilization, when two parental sets of chromo- somes are brought together, and that a segregation of characters occurs at the time of reduction, when the chromosomes are sorted out into two groups. Moreover, the distribution of a single allelomorphic pair of Mendelian characters parallels precisely that of a single homologous pair of chromosomes. These facts indicate clearly that chromosomes and characters are in some manner causally related. This conclusion is strongly supported by the cytological aspects of sex inheritance, maleness and femaleness in a large number of reported cases being definitely corre- lated with the activity of certain distinguishable chromosomes. It has also been pointed out that the hypothesis upon which these phenomena are generally interpreted is that the characters are repre- sented in the chromosomes by material factors, or genes, which in some way control the development of characters in the individual. Since, now, an organism usually has many more Mendelian character pairs than it has chromosome pairs, one pair of chromosomes must as a rule carry genes for more than one pair of characters. Furthermore, the different pairs of chromosomes are entirely independent of one another in distribution. It would therefore follow that if two allelomorphic character pairs have their genes located in different chromosome pairs, they will be quite independent of each other in their inheritance through a series of genera- tions; whereas, if their genes are located in the same pair of chromo- somes, they will be inherited together. The latter condition — the persistent association of characters belonging to different allelomorphic pairs through a series of generations — actually exists and is known as linkage. The phenomenon of linkage was discovered in 1906 by Bateson and Punnett in the sweet pea. They found flower color to be linked with the shape of the pollen grain: purple flowers nearly always had long grains, while red flowers had round grains. The possible relationship between linkage and the chromosome hypothesis was pointed out by Lock in the same year. Linkage relations have since been worked out in a consider- 378 LINKAGE 379 able number of plants and animals, and are especially well known in the case of the fruit, fly, Drosophila melanogaster, owing to the exhaustive researches of Morgan and his coworkers. A Typical Case of Linkage. — As a typical example may be taken tin- following case of linkage in Drosophila (Fig. 1 15). A male fly with white eyes and yellow body is mated to a female with red eyes and gray body. linkage: in drosophila ^0 Fig. 146. — Sex-linkage in DrosophUa. Three successive generations al left; red • shown in black. The history of the sex-chromosomes through these generations sho* D a1 right; X- chromosome of original male shown in black. (Adapted from Morgo In such cases as the above it is evident that characters other than sex may be referred to certain chromosomes of the complement: if is possible not only to tell which chromosomes have to do with sex, but also to identify the ones concerned in the production of red and white eye colors. A large number of such sex-linked characters haw been identified in DrosophUa, and several have been found in other animals. Human colorblindness is a character which is inherited in a manner analogous to that of sex-linked characters in Drosophila, and its me- chanism is apparently the same. The presence of this detect more commonly in men than in women, and its appearance in so few individ- uals in affected lines, are due to the tact that it is both a recessive and a i Sex-linked characters are not to be confused with Bex-limited characters. The latter are those found exclusively in one sex, and are now referred to as secondary .sexual characters. 382 INTRODUCTION TO CYTOLOGY sex-linked character, precisely like white eyes in Drosophila. It occurs in a woman only if both of her X-chromosomes bear factors for it, which means that such a factor must have been received from each parent ; whereas one such factor is sufficient to produce colorblindness in a man, because his F-chromosome carries no factors which might domi- nate it. Furthermore, since the X-chromosome of the male is always derived from the mother, a man can inherit colorblindness from his mother but not from his father. From these facts it follows that a colorblind woman transmits the defect to all of her sons and to half of her grandsons and granddaughters; whereas a colorblind man transmits the defect to none of his children and only to one-half of his grandsons.1 The first sex-linked character known in plants was that of narrow rosette leaves in the red campion, Lychnis dioica (Shull 1910, 1911), a plant which appears to have the XY type of sex inheritance, but in which no distinguishable sex-chromosomes have been identified. Non-disjunction. — The chromosome interpretation of sex and of sex-linkage has received an interesting confirmation in a phenomenon discovered by Bridges (1913). In a certain strain of Drosophila the white-eyed females were observed to give rise to a certain proportion of " unexpected " forms. Most of their offspring (92 per cent) were red-eyed females and white-eyed males, as expected in such an experiment, but some of them (8 per cent) were white-eyed females and red-eyed males. A long series of crosses showed that these results could be accounted for if it were assumed that in the original white-eyed female both of the X- chromosomes passed together to one pole in the reduction division instead of separating as they should. This was termed non-disjunction (Fig. 147). As a result the eggs, instead of having the normal single X-chromosome, would have either two or none, and the distribution of the sexes and the sex-linked characters would be altered in the manner observed. In a cytological examination of the flies in which these abnormal phenomena appear Bridges showed that this non-disjunction of the X-chromosomes does occur occasionally in the female (Fig. 148). The chromosome theory thus received confirmation. "An abnormal distribution of the sex-chromosomes goes hand in hand with an abnormal distribution of all sex-linked factors" (Morgan). Additional genetic and cytological data have since been furnished by Bridges (1916) and Safir (1920). Linkage Groups. — An extensive series of studies on linkage relations in various plants and animals has brought out the fact that the Mendel ian characters of a given organism fall into a certain number of "linkage groups," the members of each group being linked to one another in various degrees but showing no linkage with the members of other groups. It appears further, when the relationships of enough characters have been worked out, that the number of linkage groups is the same as that 1 This case is fully explained by Babcock and Clausen (1918, p. 197). LI \ KAGE of the chromosome pairs. Drosophila melanogaster, in which Linkage relations have been most fully analysed, has lour pairs of chromoson (Fig. 148): two large "euchromosome" pairs, one pairofsex-chromosom< DROSOPHILA MOM Pi ' UM( ; X KIN J J Of 'X' f HINDS toos /* 0"~" ^ T ) ( *tY )( ,Xt j f tt j Fig. 147. — Non-disjunction and its results in Drosophila. The two large circles in first row represent male and female flies producing sperms and eggs respectively. Non- disjunction in the female gives 2 kinds of eggs, with XX and with no Bex-chromoson instead of the normal single kind with one X. At fertilization there are possible 1 combi- nations rather than 2, as shown in the large circles of second row. I ►wing to th<' wveral ways in which her 3 sex-chromosomes may be distributed at maturation, tin female repre- sented by the third circle produces 4 kinds of eggs. When mated to a normal male (below horizontal line) with his 2 kinds of sperms, 8 combinations are possible (las! row). Nos 1, 4, and 5 are normal flies and give the usual types of progeny. Nos. 2, 6, and 7. owing to the presence of 3 sex-chromosomes, give exceptional results when bred. Types N and No. 8 do not appear in the cultures, probably because they die very early. 1 la- original male has red eyes and the original female white eyes. Red eyes "•'! by dots) appear in every fly bearing the X-chromosome of the original male as in 1 Compare Morgan 1919a' Figs. 93 and 94. (Diagram based on n< lanogasU r :i- they appear during mitosis in a female, a male, and a non-disjunctional female. I I Morgan.) and one pair of very small " m-chromosomes." The Mendelian charac- ters in Drosophila fall into four linkage groups, and it is noteworthy that one of these groups contains only two known characters, Each 384 INTRODUCTION TO CYTOLOGY chromosome pair therefore seems to be responsible for a certain group of characters. It has been shown above that one of these groups, the sex- and sex-linked characters, can be definitely assigned to the pair of sex-chromosomes; and Morgan further believes that the factors for the two characters of the small linkage group are located in the m-chromosomes. The two remaining linkage groups, which contain many characters each, are assigned to the large euchromosome pairs. Each chromosome is accordingly regarded as a body containing the fac- tors or genes for a considerable number of characters; and on the basis of the evidence to be presented below it is concluded that these genes, differing thus in their hereditary potencies, are arranged in the chromo- some in a linear series as suggested by Roux many years ago. In plants the best known cases of linkage are in Zea Mays, in which Emerson and his students at Cornell have identified six linkage groups, and Pisum, which has so far shown four linkage groups (White 1917). Since maize has 10 pairs of chromosomes, four more groups may be expected, while in Pisum, which has seven pairs, three more groups will probably be established; in fact seven independently inherited characters are known. It is an interesting fact that Mendel, in his famous researches on Pisum, happened to select for study seven pairs of characters belonging to the seven different groups, and so did not detect the phenomenon of linkage. From the foregoing considerations there arises an interesting and very important question. If two homologous chromosomes, each carry- ing factors for a certain group of characters (those of one group allelo- morphic to those of the other group), separate into different gametes (or spores) at the time of reduction, how does it happen that occasionally there appears an individual with some of the characters of each group? And if a single chromosome carries a series of factors for a certain group of characters, how shall we account for the occasional individual with some of these characters but not the rest? To state the problem in the terms of linkage, if each group of linked characters is represented by a series of genes in a given chromosome, how is the linkage broken in a certain percentage of cases, with the resulting formation of new link- age groups, as shown by the exceptional red-yellow and white-gray flies in the experiment described at page 379? A solution to this problem has been offered in the Chiasmatype Theory. The Chiasmatype Theory. — In our discussion of chromosome con- jugation it. was pointed out (p. 257) that various opinions have been entertained regarding the nature of the association between the members of the synaptic pair. Some workers have held that the chromosomes fuse completely and lose their identity, and that the two chromosomes appearing on the first maturation spindle are not to be looked upon as identical with those which entered into conjugation. On the contrary, LINKAGE 385 there are those who deny any fusion a1 all between the members of the pair, holding rather that their identity is do1 impaired in any way during Fig. 149. — The behavior of the conjugating homologous chromosomes according t-> the Chiasmatype Theory of Janssens. Single crossing over at left; double crossing over at right. (Adapted from Babcock and Clausen.) CHIA3I1ATYPC • JAN33EM5 I90». 0 v> 0 00 Fig. 150. — Crossing over between 2 of the 4 chromatids of the chromosome tetrad, giving 2 crossover and 2 non-crossover gametes. (Adapted from Janssens, 1909 Fig. 151. — Chromosomes of Balracose.ps attenuatns, Bhowing chiasmas. (After Janssens.) their intimate association: the two chromosomes appearing on the first maturation spindle are exactly the same as those which conjugated. 386 INTRODUCTION TO CYTOLOGY Between these two extremes lie other views, the most suggestive of them being that proposed by Janssens (1909).1 The theory of Janssens in its simplest possible form may be stated as follows. The members of the conjugating pair twist about each other and come into very intimate association at certain points. When they again separate a break occurs at the point or points of closest contact, but along a new plane, so that each of the two separating chromosomes is made up of portions of both conjugating members (Fig. 149). This process is known as chiasmatypy or crossing over. Such a behavior might occur at various stages in the heterotypic prophase: most probably it takes place at an early stage, when the conjugating chromosomes are in the form of simple thin threads (Fig. 83). In other cases it may take place at a later stage, when, in the case of animals, each of the chromo- somes has split preparatory to the second mitosis, forming a tetrad of chromatids. Here the crossing over may occur between only two of the four chromatids (Fig. 150, Janssens's typical case; see also Fig. 151), or between all four. If only two of the four chromatids are concerned, only two of the resulting gametes (or spores) will be " crossover gametes" (or spores), as in Fig. 150; whereas, if the crossing over takes place between all four of the chromatids, or between the two yet unsplit threads in the earlier prophase, all four of the gametes (or spores) will be " crossover gametes" (or spores). Application of the Chiasmatype Theory to the Problems of Linkage. — It is the above interpretation of the nature of chromosome conjugation that lies at the basis of the work of Morgan and his students on Drosophila. As already pointed out, these workers have found good evidence for the conclusion that each chromosome is responsible for a certain group of characters, the members of the group showing a strong tendency to remain associated because their genes are borne by a common carrier. They further believe that the evolution of new character groupings has been brought about not only through the crossing of different hereditary strains, but also through the evolution of chromosomes with new con- stitutions by the process of crossing over. On the basis of the frequencies in which the new types of grouping occur the relative positions (loci) of the genes for the different characters have been plotted in the chromosomes. The above points are illustrated in Fig. 152, which summarizes what is supposed to have occurred in a certain series of crosses between flies with yellow body, white eyes, and miniature wings, and flies with gray body, white eyes, and long wings. In the oells of the hybrid there is 1 Janssens has recently (1919«l>) published an outline of his views of the maturation phenomena in Orthoptera in which he again makes use of the chiasmatype interpreta- tion. His results are discussed in some detail from the cytological and genetic points of view by,, Wilson and Morgan (1920), LINKAGE 387 a chromosome from one parenl bearing the genes for the three linked characters, Y, W, and M, and a chromosome from the other parenl with the genes for the three linked characters, G} /.'. and L. The first three characters are allelomorphic to the last three respectively, and the two chromosomes are homologous.1 [JpoD breeding from the females of these hybrids it is found that in the majority of cases (firsl column) the F2 flies show the same genetic consitution as do the grandparents with respect to these particular characters). This is taken to mean that the two homologous chromosomes forming the synaptic pair and separating at reduction have maintained their substance intact- -there has been DROSOPHILA YELLOW »0»Y WHITt CVCt MINIATum WIN&S &WAY »0»Y KID VtU LON» WINfrj ^ o ^ V K? V 1361 20S9. ^ ^ ^ 817 887 23 17 O Fig. 152. — Diagram illustrating the evolution of new linkage groups through crossing over. Explanation in text. (Adapted from Morgan.) no crossing over. In a certain number of cases (second column) Dew groupings of the characters in question are observed in the Pi flies: some have Y, W, and L, while others show G, R, and M. This is interpreted on the assumption that a break has occurred along a new plane (dotted line) at a point of contact, so that at reduction some of the garnet and hence the F2 flies to which they give rise, receive a chromosome with Y, W, and L, while others receive G} /?, and M . In a smaller Dumber of cases (third column) the new combination- YRL and QWM arc formed in a similar manner, the break and reunion occurring ;ti another point. In a very small number of cases (fourth column) the combinations YRM and GWL appear, which can be explained on 1 he basis of a double 1 The hybrids have gray bodies, red eyes, and long win^s, because a, B, and L are dominant over )', W, and M. (Ordinarily t he genes have other designations.) CHROMOSOME I 0.0 \ «4 /yellow, scute 0.0 + A lethal 7 //, broad l/A prune 1.5 — — white, etc. 3.0-—: - — notch, facet abnormal 5.5 echinus 7.3 ; 7.5-" bifid " - ruby 13.7 \6.7 20.0 21.0 27.5 33.0 36.0- 36\1- 37.5 38.0 43.0 44.4 53.5 5^.5 56 57, 58, 59 65.0 croesTeinlese club cut singed tan Term! lion •tiny-bristles -miniature dusky furrowed sable garnet small-wing rudimentary forked bar small-eye fused cleft CHROMOSOME IT 2.0 telegraph 0.0 1.0 ^.0 expanded 9.0 11.0 13.0 14.0 85.0 88.0 100.0 101.0 103.0 104.5 105.0 105.5 22.3 28.0 29.0 33.0 35.5 44.0 46.5 46.7 48.5 52.5 58.0 61.0 65.0 66.5 67.0 70.0 71.0 73.5 75.0 77.0 95.0 97.5 98.5 star aristaless truncate gull pink-wing streak cream-b flipper dachs ski squat minute-6< black jaunty apterous purple eafranin trefoil Testigial telescope dash lobe minute-5II curved dacheoug roof tninute-2 humpty purpleoid aro plexu6 lethal-IIa brown blistered morula speck Mkloon CHROMOSOME III 0.0 roughoid CHftOMOSOVE IV 25.3 25.8 32.0 I- 33.5 34.0 35.0 38.3 38.5 40.5 41.5 43.0 !*4.0 •+5.0 45.5 53.8 54.0 55.0 56.0 59.0 60.0 60.5 61.0 63.5 65.5 67.5 68.0 70.0 72.0 86.5 89.0 95.7 0.0 bent eyeless 0.0 + sepia hairy divergent cream-III dwarfoid scarlet tilt dichaete ascute deformed maroon curled dwarf pink tv/o-bristle spineless bi thorax tithoraxoid glass kidney giant spread delta hairless ebony band CIII white-ocelli rough beaded tlaret raim»t* hio. lo3. — The chromosome map of Drosophila melanogaster, showing the loci of the genes as determined by Morgan and his associates. Correcte'd to November. 1920. Figure kindly furnished by Professor Morgan. LINKAGE crossing over us shown in the diagram. Mosi of the Ft flies show the same combinations as their grandparents, which indicates thai do crossing over has occurred in the majority of the gamete-forming cells. Since there are many more F2 individuals with YWL or GRM second column than with YRL or GWM (third column), ii is believed thai the distance between the genes W and M (and between Rand /.) in the original chromo- some must be greater than that between Fand W (and between G and R so that there is more chance for crossing over to occur in the lower pan than in the upper. Thus if two linked characters arc often separated (i.e., have their linkage broken) their genes are thought to lie relatively far apart in the chromosome, whereas linked characters separated only rarely are supposed to have their genes located very near each other. Recombinations involving a double or even more complex crossing over (fourth column) would be expected very rarely. In this way the gen for the various characters have been assigned to their loci in the chromo- somes on the basis of the frequencies in which the various new combi- nations in F2 appear. In Fig. 153 is shown the "map™ of the chromosomes of Drosophila as determined by Morgan and his associates, each factor being placed a certain number of units of distance from the end. Many other known genes are not shown in the diagram. As the unit of distance is la ken that space separating two factors whose linkage is broken (i.e., between which crossing over occurs) in 1 per cent of the cases. Thus crossing over occurs between "yellow" and "bifid" in 7.3 per cent of the observed cases; the factors are therefore placed 7.3 units apart (first chromosome Furthermore, if some linkage relations are known, it is possible to calcu- late certain other linkages in advance. For example, if it were known that crossing over occurred between "sepia" and "pink*' (third chromo- some) in 20.2 per cent of the flies, and also that "sepia" and "kidney' showed a 34.7 per cent crossing over, the prediction that "pink' and "kidney' would show a 14.5 per cent crossing over (not including the modifying effects of double crossing over, should this occur) would In- borne out by experimental results. Such an agreement of the results of new crosses with predictions made on the basis of known linkages has occurred over and over again in the experiments of Morgan and his students. The chiasmatype hypothesis as thus elaborated obviously fits the observed facts remarkably well. Interference. — Another piece of evidence broughl forward in suppoii of the hypothesis that the factors have a linear arrangemenl within the chromosome is the phenomenon of interference, which has been elucidated bySturtevant (1913), Weinstein (1918), and particularly by Muller. It the factors or genes are arranged in a series as supposed on the above hypothesis, it would be expected t hat when crossing over occurs at a given point in a pair of chromosomes, the regions immediately on either side of 390 INTRODUCTION TO CYTOLOGY d f if this point would be prevented from crossing over at the same time, for the reason that the twisting of the chromosomes about each other is not close enough to allow two crossovers so near each other. Thus in Fig. 154, if crossing over occurred between the two factor pairs Dd and Ee, breaking the linkages between D and E and between d and e, there would be at the same time no such break between Cc and Dd or between Ee and Ff, since for mechanical reasons a second crossing over could not take place at either of these points simultaneously with that between Dd and Ee. Crossing over at one point would thus interfere with crossing over which might otherwise occur at nearby points. The amount of this interference would progressively decrease at points farther and farther from the first crossover point, until at a certain distance (measured by the length of the loops usually formed by the twisting chromosomes), as a,tLM, it would vanish entirely; here crossing over would occur with its normal frequency irrespective of any crossover at DE. * Muller has found that the characters behave accord- H ing to these expectations. If two characters have their linkage broken in a certain percentage of cases, this . percentage is noticeably lowered if breaks in linkage occur between two other characters having normally a fairly close linkage with the first two. In other words, one linkage break interferes with other linkage breaks within the same linkage group ; and the degree of this interference varies from a high value in the case of a closely linked series of characters to zero in the case of characters very loosely linked. This, it is pointed out, P is just what should occur among characters represented by a linear series of genes in chromosomes which un- dergo crossing over, but which cannot twist about one another with more than a certain degree of closeness. The phenomenon of interference thus indicates another point in which the chiasmatype hypothesis as developed by Morgan fits the experimental facts. A further point is of interest in this connection. In Drosophila it is only in the females that crossing over takes place; it is in the eggs, and not in the spermatozoa, that new factor combinations appear as the result of this process. The absence of crossing over in the male may be asso- ciated with the fact that the F-chromosome carries no known factors;1 the male is heterozygous for sex. In the fowl, in which the female rather than the male is heterozygous for sex, it has been shown that crossing over occurs in the male but not in the female. Crossing over for ^ee, however, Castle (1921). V M K >*\ H Fig. 154.— Dia- gram illustrating interference. Ex- planation in text. LINKAGE 393 some reason is limited in these cases and some others to the Bex which is heterozygous Tor the sex-factors. < >n the contrary, in the grasshopper, Apotettix, Nabours (MM!)) has shown thai some crossing over occurs in both sexes, and the same appears to be 1 rue in Primula (( Iregorj ; AJten- burg 1916), the rat (Castle and Wright 1916), and Zea (Emerson . In Paratettix, in which no crossing over lias been demonstrated, Miss Harman (1920) reports that the homologous chromosomes do do1 conjugate until the end of the prophase, and suggests thai their indepen- dence during the early stages may account for the absence of crossing over. General Discussion. — In the foregoing pages a brief account has been given of the main points in the theory developed by those who have mad. the most thoroughgoing attempt to relate the phenomena of heredity bo a visible cell mechanism. To follow out the details of its applied ion does not lie within the scope of this chapter: it is here intended only to furnish a starting point for cytological studies in this field by indicating the common ground upon which cytology and genetics meet. It is import- ant, however, to differentiate between evidence which is genetical and that which is cytological in nature; and further to remind ourselves to what extent observed fact and hypothesis respectively have been woven into the theory. Caution is particularly necessary in this latter regard. since the general nature of many of our ideas of inheritance is traceable in part to the speculative theories of Weismann. Weismann's theories of heredity and development, which are summarized in the next chapter. were primarily "corpuscular" or "particulate" theories: the phenomena of heredity and development were referred to distinct material units which in some way were able to bring about the development of I In- heritable characters in the individual and their transmission from one generation to the next. Bearing in mind the phenomena of inheritance reviewed in the preceding chapters, especially the behavior of the Men- delian characters, it is difficult to escape the conclusion that differential factors of some sort, which in an unknown manner initiate the series of reactions resulting in the several characters, are carried in the nucleus. To determine the nature of these factors and to discover the real relation existing between them and the developed characters are among our greatest problems. That the factors or genes are discrete units is a hypothesis which is not only plausible, but has also proved itself to be most useful. If such factors exist, the chromosomes afford a means of precisely the kind required to account for the observed distribution of characters throughout a series of generations. Hence from Roux and Weismann onward the factors have been lodged in the chromosomes. But it is when these factors are directly sought with the aid of the microscope that disappointment is met. The frequently observed granules or chromomeres in the chromatin thread or chromosome are 392 INTRODUCTION TO CYTOLOGY accepted by some geneticists as the desired material genes; but, as pointed out in the chapters dealing with somatic mitosis and reduction, many cytologists are very uncertain as to the morphological status and signifi- cance of these bodies, which seem to them to be far too inconstant in number and behavior to represent the units in question. Although it is tempting to look upon the chromatic granules as the units which current theories of heredity seem to require, it must be admitted that the observational evidence is insufficient to warrant the categorical state- ments frequently made to the effect that the chromosome is composed of a definite number of more elementary visible chromatic units, which have definite space relations and are the significant units in the cellular mechanism of heredity. On the other hand, the careful observations of Wenrieh (1916) have shown that in the grasshopper, Phrynotettix (Fig. 155), the chromatic granules are relatively constant in size and position c * e f g h i & -^ fc Fig. 155. — Chromosome pair "B" in conjugation from the spermatocytes of 13 differ- ent individuals of Phrynotettix magnus, showing constancy in size and arrangement of the principal chromomeres. The same constancy is shown in the different cells of a single individual. X 1500. {After Wenrieh, 1916.) in a given member of the chromosome complement, even in different individuals; and they furthermore show a close correspondence in the two homologous chromosomes as they pair at synapsis. This is one of the most striking pieces of direct cytological evidence yet brought forward in support of the theory that the chromosome is a "chain of factorial beads '; (Harper), and heightens the probability that the postulated units of inheritance will turn out to be more than purely conceptual ones. Whatever may be the value of the chromatic granules, one can hardly fail to recognize the highly suggestive nature of the arrangement of the chromatin in a thin thread, its frequently beaded appearance, and its accurate longitudinal fission into two equal parts at the time of cell- division. In the absence of direct and convincing cytological evidence for the presence of various "qualities'3 arranged in a series along the thread, we may still look hopefully for the support which it would seem that the theory of Roux must sooner or later have. It must be admitted that at present the evidence for the existence of genes is in the main genetical rather than cytological. LINKAGE Similarly unsatisfactory is the cytological evidence for the breaking and reunion of the chromal in 1 hreads required by 1 !)<■ crossing over hypo- thesis. Since the hypothesis was pu1 forward byJanssens L909) adequate and convincing descriptions of this process have been singularly wantii A B KJ A1 I I I «• ft v B e C t I B1 r\ I I I B I Fig. 156. — Diagrams illustrating various possibilities concerning tin- compound ring tetrads in Orthopteran spermatocytes, following the outlines <>f Janssens'a figures, bu1 showing also the relations of the chromatids. At the left in each of the upper figures i- the longitudinal tetrad-rod from which the riim-series arises, showing results of assumed early cross-overs in Bl and C1. A, the compound ring as conceived by Mc< Hung. Robertson, etc., with the four resulting chromatids at .1' (no cross-overs) . H, a compound ring, Buch as might follow a two-strand cross-over at each code, giving the results shown in IV. I compound ring giving the results shown in Janssens'a diagrams, resulting from a two- strand cross-over between two pairs of threads, in regular alternation at successive nodes. The result (i'x) is four classes of chromatids, as shown in Cl. (Ft(.>, which have been copied from Conklin (1919-1920). 408 INTRODUCTION TO CYTOLOGY Notwithstanding the fact that many changes have been made in its details, Weismann's theory of heredity proved to be of much greater value than his theory of development, Morgan (Morgan et al. 1915, pp. 223- 227) points out that Weismann made three contributions to the study of genetics, which may be stated in three propositions: (1) The germ-plasm GERtt CE.LL V SOMATIC CELLS Crthn CELL SOMATIC CELLS Fig. 159. — The behavior of the hereditary units in ontogenesis according to Weismann (A) and the current interpretation (B). In A the determinants in the nucleus (-1, 2, 3, 4) are supposed to be distributed differentially to the various somatic cells. In B the genes (1, 2, 3, 4) are distributed equally to every cell, but the cytoplasm is distributed differen- tially. The same genes working upon different cytoplasms produce different results in various somatic cells. {Diagrams and legend from Conklin, 1919-1920.) contains independent elements which may be substituted one for another without undergoing change; (2) a segregation of maternal and paternal factors, pair by pair, occurs at one period in the history of the germ cells; (3) the behavior of the chromosomes is specifically applicable to the problems of heredity. In these principles are found "the basis of our WEISMANNISM AND OTHER THEORIES 409 present attempt to explain heredity in terms of the cell," for upon them is founded the Factorial Hypothesis, now supported by a large mass ol experimental evidence. In our conception of the uature of the heredity units or factors we have departed widely from Weismann. For him each of the ids arranged in a series in the chromosome represented the sum of the characters of a complete organism; the smaller pails were represented by the smaller units (determinants) composing the id, and these units in turn were made up of biophores, which were ultimate and independent living particles. According to our modern hypothesis each of the Berially arranged factors or genes exerts an influence on the development of one or more characters, but does not stand for a complete organism as did the id, or for a pari of it as did the determinant. Moreover, it is generally regarded as a mass of some complex chemical substance whose activities are due to its defi- nite though imperfectly known physico-chemical properties, rather than to forces exerted by hypothetical vital units. In justice to Weismann it should be pointed out that the frequently made criticism that his theory was a vitalistic one is warranted only to a limited extent. Although his ultimate hereditary units, the biophoi were regarded as actually living particles, Weismann stated that "they are not composed in their turn of living particles, but only of molecules, whose chemical constitution, combination, and arrangement are such as to give rise to the phenomena of life." He was careful to point out that in spite of the fact that it cannot be proved that no peculiar vitalistic principle exists, we should hold fast to a purely physico-chemical basis of life "until it is shown that it is not sufficient to explain the tact-, thus following the fundamental rule that natural science must not assui in- unknown forces until the known ones are proved insufficient ... We can quite well believe that an organic substance of exactly proportioned composition exists, in which the fundamental phenomena of all life combustion with simultaneous renewal — must take place under certain conditions by virtue of its composition" (1902, lecture :>(>). The manner in which hereditary factors are segregated at gameto- genesis has been found to be different from t hat conjeet ured by Weismann. As indicated in the chapter on reduei ion, he supposed it to occur t hrough a transverse division of the chromosome, whereas it is now known thai it is accomplished by the disjunction of pairs of entire chromosomes, the separating members of each pair being qualitatively different. The "reduction" predicted by Weismann was found to occur, but not in the manner he supposed. As shown above, his idea of a further qualitative segregation of units of a lower order in the somatic divisions has Q01 been substantiated. Notwithstanding the abandonment of his theory of development and the changes made in his theory of heredity, Weis- mann's influence on both cytology and genetics was enormous, largely 410 INTRODUCTION TO CYTOLOGY because of his emphasis upon the need for careful studies of the cell mechanism at the critical stages of the life history, and upon the idea that this mechanism is in some way bound up with the phenomena of heredity. "It has been Weismann's great service to place the keystone between the work of the evolutionists and that of the cytologists, and thus bring the cell-theory and the evolution-theory into organic con- nection" (Wilson 1900, p. 13). We may further point out, with Morgan (1915), that the factorial hypothesis assumes only three things about the factors: they are constant, they are usually in duplicate in each body cell and immature germ cell, and they usually segregate in the maturing germ cells. The hypothesis, and the Mendelian theory in general, therefore have to do only with heredity : they do not attempt to explain the causes of development. They seek rather to account for the initial resemblances or differences in here- ditary potentiality which are observed to exist between the germ cells from which successive generations arise. Between the materials com- posing the initial factors and the fully expressed characters of the or- ganism "lies the whole world of embryonic development," to which the application of the theories under consideration has not yet been extended in any systematic or satisfactory manner. Nevertheless many investi- gators, though realizing the failure of Weismann's attempt to explain development in terms of representative particles, are strongly inclined to the view that since the Mendelian characters appearing toward ma- turity behave as though associated with discrete units in the germ, the course of ontogenetic development in its earlier stages must also be due in large part to the activity of factors carried by the nucleus. Development is thus held to be predetermined or controlled by an internal mechanism : external agencies act only by affecting the operation of this mechanism. The factors control the character and behavior of the cells, and upon these in turn the organism, which is a cell aggregate, is alone dependent for its characters and activities. In place of the early hypothesis on which it was supposed that the development of characters is controlled by the migration of determiners or pangens from the nucleus into the cytoplasm at precisely the right times and places, we now have the theory that the factors in the nucleus probably produce their effects by initiating series of chemical reactions which involve all parts of the cell. As Mor- gan (1920) states, " Granting that differences may exist in the nuclei of different species, different end products are expected. The evidence that such differences may be related to specific substances in the nucleus is no longer a speculation but rests on the analytical evidence from Men- delian heredity. In what way and at what times the nuclear materials take part in the determination of characters we do not know. The essential point is that we are in no way committed to any interpretation. Stated negatively we might add that there is nothing known at present to preclude the possibility that the influence is a purely chemical process." 1 1 ' E ISM A X Y TSM AND OTH E R Til Et > R I ES 1 1 1 Non-factorial Theories. -The above theory <>! the dependence <»! the course of development upon the operation of an internal factorial mech- anism is essentially an "elementalistic" conception: the attempl ie made to explain the organism in terms of its constituenl parts, namely, the cells and smaller elements contained by them. A> noted in our historical sketch, a number of botanists and zoologists many yeara ago called attention to the fact that limits must I"- Bel 1<> the conception «»t' the cell as the unit of struct ure and function; and they have been followed by a school, made up largely of experimental embryologists, which holds that organization is not the result of cell formation, bu1 rather precedes and regulates the latter. From this "organismar standpoint the or- ganism as a whole, and not one or another of its elementary parts, is regarded as the primary individual. This individual is something more than the cell aggregate pictured by Schleiden and Schwann: it dominates the activity of its constituent members from the beginning of the life cycle onward, and behaves as a unit irrespective of the manner and degree of its subdivision into special centers of action, the cells. The condition present in ccenocytic plants is especially noteworthy in this connection, as are also those cases among animals in which a derangement of the early embryonic cells does not prevent the eventual attainment of the normal form. As urged with much force by Ritter (1919), " the organism in its totality is as essential to an explanation of its elements as its elements are to an explanation of the organism." •The factorial theory may also be said to represent preformationism in a very modern form. "We are sailing nearer the preformation coast than at any time since the modern study of development began under von Baer" (Conklin 1913). Directly opposed to corpuscular and fac- torial theories of development are those which seek to explain the course of ontogenesis not by an internal mechanism but rather as the result ot the influence of external agencies and the physiological responses Bhown by protoplasm in the form of cells to such influence, development is held to be truly epigenetic. The control exercised by environmental factors during the organism's early developmental stages, and the effects of various tropisms and tactisms between the component cells upon the type of organization resulting, have been especially emphasized bj Hertwig, Hartog, Roux, Herbst, Driesch, and others. The most sug- gestive recent work of this nature in plants is thai of Harper (1908, 1918o6) on colonial alga*. In Hydrodictyon and Pediastrum a number of free-swimming cells come together and build up colonies of very definite forms, and a series of experiments has shown that the position in the colony of any given cell is in no way predetermined. A> already pointed out in Chapter XIV, Harper contends i hat the type of multicellular organiza- tion thus built up in successive life cycles is to be explained as the result of physico-chemical interactions between independent cells organized as 412 INTRODUCTION TO CYTOLOGY swarm sixties, and nol as the product of the activity of a system of spatially arranged factors in a special germ-plasm. In this connection the name of Driesch (1907-8, 1914) has become particularly prominent, not only because of his great experimental ingen- uity, but also because of his decision that the facts of ontogenetic devel- opment cannot be accounted for on the basis of any mechanical theory, either now or in the future. As a result he takes the unscientific step of assuming the existence of a non-mechanical, non-spatial, non-psychic, non-energetic "entelechy," which presides over and controls develop- ment. Such non-experiential agencies, manufactured for the purpose of solving difficult problems, lead to experimental indeterminism and tend only to obscure the points at issue : they may furnish convenient names for great gaps in our knowledge, but they never give more than pseudo- explanations. Nevertheless, in spite of his tendencies to mysticism, as Harper (1919) remarks, Driesch has shown the impossibility of an exact parallelism in spatial configuration between the germ-plasm and the multicellular organism as a whole: there can be no strict preformation in development. On the other hand, the work of the Mendelians shows clearly that development cannot be completely epigenetic: nothing seems clearer than that development is at least in part dependent upon the orderly operation of an internal organization or mechanism. Wilson (1909, pp. 106 ff.), in discussing the relation of the chromatin to heredity and development, writes as follows: 'But do we really need to employ the pangen symbolism in the consideration of this question? It seems a sufficient basis for our present attack on the problem to assume that the control of the cell-activities is at bottom a chemical one and is effected by soluble substances that may pass from nucleus to protoplasm and from protoplasm to nucleus. Certainly it is to such a view that very many of the chemical and physiological studies in this field are now unmistakably pointing. The opinion is gaining ground that the control of development is fundamentally analogous, perhaps closely similar, to the control of specific forms of physiological action by soluble ferments or enzymes . . . We are thus led to something more than a suspicion that the factors of determination, and therefore of heredity, are at bottom of chemical nature . . . The conclusion thus becomes highly probable that the characteristic differences of metabolism between different species, including those involved in development, are traceable to initial chemical differences in the germ cells. In so far as the chromatin theory expresses the truth, the primary basis of these differences may be sought in the nuclear substance." A Chemical Theory of Heredity. — Among the theories based on the conception of the idioplasm as a substance with a special chemical consti- tution, rather than as a system of determinants, may be mentioned that of Adami (1908, 1918). As indicated in Chapter III, Adami attributes the phenomena of life to the activities of a protein-like "biophoric mole- WEISMANNISM AND OTHER THE0RI1 s II cule," which is made up of a chain or ring of amino-acid radicles to which side-chains of various kinds may become attached, \\nli regard to individual development it is supposed thai 'in the ovum there is one common idioplasm of simple type, to which, when distributed in the various cells derived from that ovum, different side-chains become attached, according to the relationships assumed by those cells, so thai the cells of different orders arc controlled and formed around proto- plasmic or idioplasmic molecules composed of those central rings plus varying series of side-chains" (p. 14.")). With Driesch ii is held thai 't lu- st ructure of the cells in a multicellular organism is a function of their position," since ''the position of the cell determines the modification under- gone by its idioplasm." Furthermore, " the greater t lie change impressed upon the idioplasm of these cells, and the longer that idioplasm is sub- jected to the conditions inducing this change, the more permanently will the daughter cells exhibit the peculiar alteration in the idioplasm, wit h con- sequent modified structure wherever they find themselves in the economy. We have, in short, to recognize that two orders of forces determine the structure of every cell in the body: (1) the previous influences act ingupon its idioplasm and causing it to be of a particular chemical constitution; and (2) the position in which the cell finds itself, and the forces acting momentarily and immediately upon its idioplasm. Or, briefly, these two series of forces are inheritance and environment, and inheritance and environment determine the constitution of the idioplasm and the struc- ture of the cells" (p. 151). "In terms of this theory, therefore, inheritance essentially d< pends upon the chemical constitution of the idioplasm or the life-bearing or biophoric protoplasm of the germ cells, not upon the number of the sepa- rate ids or biophores or ancestral plasms or pangens contained in the idio- plasm; and variation, whether slight and individual, or extensive and leading to the production of new species, is ultimately the expression of modification in the constitution of that idioplasm brought about by envi- ronment. Whereas Weismann's theory lays stress upon relative fixity in the constitution of the idioplasm, this theory admits lively the capacity for change in structure of the same. So long as I he surrounding condi- tions are unaltered the idioplasm is unchanged; alter these conditions and the idioplasm is liable to variation in constitution' (pp. 152 3 Adami cites certain calculations of the probable size of inorganic and organic molecules to show that the existence of a system of determinants or other representative particles of the Weismannian type is a physicial impossibility. He also points out thai since the idioplasm musl increase enormously in bulk by the addition of new material and become repeat- edly subdivided as ceils and individuals multiply, there can be no actual continuity of the germ-plasm through countless generations: what is eternal is rather a potential continuity of molecular arrangement and 414 INTRODUCTION TO CYTOLOGY constitution, i.e., the physical and chemical properties of the germ-plasm rather than the substance itself. Conclusion. — In the foregoing pages we have touched upon some of the most important biological problems toward the solution of which cytology must make her further contributions. With regard to individual development it must be determined on the one hand to what extent the course of ontogenesis is dependent upon the operation of an internal cell mechanism and how this mechanism brings about its results, and on the other hand how far it is controlled by external environmental agencies: a way must be found between the "Scylla of preformation and the Charybdis of epigenesis'; (Conklin 1913). Furthermore, the manner and the causes of the progressive modification of the hereditary mechan- ism must be better known in order that evolutionary advance may be accounted for. With respect to both development and heredity the roles of the two individualities, the cell and the organism as a whole, must be more fully ascertained and correlated. It is obvious that no adequate solution of any of these problems can be reached until the physico-chemical constitution of protoplasm, especially that of the idioplasm or inheritance material, is more clearly disclosed. Only further research can show whether we shall continue to regard the idioplasm or chromatin as a heterogeneous system of discrete molecules or molecular complexes (factors or genes) with a definite spatial arrangement, as is supposed on our current Mendelian theories, or shall come to look upon it as a single enormously complex chemical substance in which varying side-chains or other portions of the molecule are responsible for the variety of results observed. It is at any rate a striking fact that ain the Mendelian phenomenon we see a synthesis, splitting apart, and recombination of determinative factors that is singularly like that of chemical elements or radicles" (Wilson 1909, p. 108); and nothing appears more clearly evident than the truth of Wilson's assertion that " . . .in the union of cytology and biochemistry lies our greatest hope of future advance." Bibliography 14 Heredity; Sex Adami, J. G. 1918. Medical Contributions to the Study of Evolution. London. Allen, C. E. 1917. A chromosome difference correlated with sex differences in Sphcerocarpos. Science 46: 466-467. 1919. The basis of sex inheritance in Sphcerocarpos. Proc. Am. Phil. Soc. 58: 289-316. figs. 28. Altenburg, E. 1916. Linkage in Primula sinensis. Genetics 1: 354-366. Amma, K. 1911. Ueber Differenzierung der Keimbahnzellen bei den Kopepoden. A rcli. Zellf. 6: 497-576. figs. 25. pis. 27-30. Babcock, E. B. and Clausen, R. E. 1918. Genetics in Relation to Agriculture. 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A biological and cytological study of sex determination in phylloxerans and aphids. Jour. Exp. Zool. 7: 239-352. 1 pi. 23 figs. 1909c. The effects produced by centrifuging eggs before and during development. Anat. Rec. 3: 157-161. 9 figs. 1910a. The chromosomes in the parthenogenetic and sexual eggs of phylloxerans and aphids. Proc. Soc. Exp. Biol, and Med. 7. 19106. The method of inheritance of two sex-limited characters in the same animal. Ibid. 8. 1910c. Sex-limited inheritance in Drosophila. Science 32 : 120 122. 1910^. Chromosomes and heredity. Am. Nat. 44: 449-496. 1910*?. Cytological studies on centrifuged eggs. Jour. Exp. Zool. 9: .">'.•.: 655. pis. 8. 1911. An attempt to analyse the constitution of the chromosomes on the basif sex-limited inheritance in Drosophila. Ibid. 11: 365-413. pi. 1. 1912. The elimination of the sex-chromosomes from the male] producing e| of pylloxerans. Ibid. 12 : 479-498. figs. 29. 1913. Heredity and Sex. New York. (Bibliography. 1914. Sex-limited and sex-linked inheritance. Am. Nat. 48: 577 58 1919a. The Physical Basis of Heredity. Philadelphia. (Bibliography.) 19196. Several ways in which gynandromorphism in insects may arise, Anat. Record 15: 357. 1920. Whitman's work on the evolution of the group of pigeons. Science 51: 73-80. Morgan, T. H. and Bridges, C. B. l(.»lti. Sex-linked inheritance in Drosophila. Carnegie Inst.' Wash. Publ. '237. 1919. Contributions to t he genetics of Drosophila melanogeater, I. The origin of gynandromorphs. Carnegie lust. Wash, Publ, 278 422 INTRODUCTION TO CYTOLOGY Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C. B. 1915. The Mechanism of Mendelian Heredity. 1st ed. New York. Muller, H. J. 1916. The mechanism of crossing-over. Am. Nat. 50: 193-221, 284-305, 350-366, 421-434. figs. 13. Muller, H. J. and Altenburg, E. 1919. The rate of change of hereditary factors in Drosophila. Proc. Soc. Exp. Biol, and Med. 17: 10-14. Mulsow K. 1912. Der Chromosomenzyklus bei Ancyracanthus cystidicola Rud. Arch. Zellf . 9 : 63-72. pis. 5, 6. figs. 5. Nabours R. K. 1919. Parthenogenesis and crossing over in the grouse locust Apotettix. Am. Nat. 53: 131-142. Xachtsheim, H. 1912. Parthenogenese, Eireifung und Geschlechtsbestimmung bei der Honigbiene. Sitzber. Ges. Morph. Physiol., Miinchen. 1913. Cytologische Studien iiber die Geschlechtsbestimmung bei der Honigbiene (Apis mellifica), Arch. Zellf. 11: 169-241. pis. 7-10. 1919. Zytologische und experimentelle Untersuchungen iiber die Geschlechts- bestimmung bei Dinophilus apatris Korch. Arch. Mikr. Anat. 93: II, 17-140. pis. 2-5. figs. 5. Nagai, I. 1915. On the influence of nutrition upon the development of sexual organs in the fern prothallia. Jour. Coll. Agr. Univ. Tokyo 6: 121-164. pi. 10. figs. 7. von Nageli, C. 1884. A Mechanico-physiological Theory of Organic Evolution. (Engl, transl.) (See Wilson 1900, p. 401.) Nawaschin, S. 1910. Naheres liber die Bildung der Spermakerne bei Lilium Mar- tagon, Ann. Jard. Bot. Buit. 2: Suppl. 3, 871-904. pis. 33, 34. Newman, H. H. and Patterson, J. T. 1909. A case of normal identical quadruplets in the nine-banded armadillo, and its bearing on the problems of identical twins and sex determination. Biol. Bull. 17: 181-187. figs. 3. 1910. The development of the nine-banded armadillo from the primitive streak stage to birth; with special reference to the question of specific poly embryony. Jour. Morph. 21: 359-424. pis. 9. figs. 15. Osborn, H. F. 1917. The Origin and Evolution of Life. N. Y. (Chapter V.) Parmenter, C. L. 1920. The chromosomes of parthenogenetic frogs. Jour. Gen. Physiol. 2 : 205-206. Paulmier, F. C. 1898. Chromatin reduction in the Hemiptera. Anat. Anz. 14: 514-520. 19 figs. 1899. The spermatogenesis of Anasa tristis. Jour. Morph. 15: Suppl. 223-272. pis. 13, 14. Payne, F. 1909. Some new types of chromosome distribution and their relation to sex. Biol. 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Eine Monographic auf wissenschaftlicher und praktischer Grundlage. pp. 355. figs. 32. Jena. Zoja, R. 1895. Sullo sviluppo dei blastomeri isolati uova di alcune meduse (e di altri organismi). Arch. Entw. 1 : 578-595. pis. 21-23; 2 : 1-37, pis. 1-4. INDKX Bold-face numbers indicate pages bearing illustrations A Abies (fir), 295 Abraxas (moth), 363 Accessory body, 88, 89 chromosome, 358 ff. Acer (maple), 159, 176, 236, •_):'>,.>. 356 Achromatic figure, 66, 145 in animals, 111 ff. in higher plants, 17") ff. operation of, 182 ff. origin of, 180 Achromatin, 64 Acids, organic, 135 Acquired characters, inheritance of, 399, 403 Acrididse, 243 Acrosome, 273, 275 Actinophrys (protozoan), 282 Actinosphcerium (protozoan), 211 Activation of egg, 284 ff. Acton, 202, 205 Adami, 50, 412 ff. Adiantum (fern), 91, 122 Adoxa (angiosperm), 236 Adsorption, 37 JFAhalium (slime mold), 39 Agar, ix, 162, 403 Agathis (conifer), 295 Agave (century plant), 134, 239 Age, 136, 137, 138 Akinetic division, 143 Albugo (fungus), 289, 290 Albumen crystals, 135 Albumin, 40, 41, 117 Alchemilla (angiosperm), 313, 315 Aleurone, 134, 135, 333 AlexiefT, 118 Alicularia (liverwort), 111 Allelomorphic pairs, 338 ff., 378 ff. Allen, C. E., cell plate, 190 chromomeres, 155, 39 1 plastids, 113 reduction, 222, 236 sex-determination, 357, 364 365 spermatogenesis, 88, 89 Allen, R. I ■'., 91, 313, 31 l Mhum (onion achromal i<- figure, l s- canalicul®, 48 cell wall, L91 chondriosomes, 116 nucleolus, 65 reduction, 234, 23d, 2 somal i<- chromosomes, I 19, l"-11 AUolobophora (annelid worm . 244, Altenburg, 391 Alternation of general i Amici, 6, 13 Amides, 135 Amitosis, 210 if. and heredity, 212 215 criticism of evidence, 213 215 Amma, 405 Amoeba (protozoal, 42, 43, 44, 207, 222 Amphiaster, 1 15, 177. 178, 286 Amphibia, 239 Amphimixis, 403 .1 mphtoxus (lancelel \, 237 Amphitene, 231, 2 .1 mphiuma (amphibian I, 150 Amygdalus (angiosperm I, 134 Amylodextrin, 107 Amyloplast, 104, L06, 122 Amylose, 1<>7 \ii:il>a'iiili. 20 1 Analysis of gametes, 2 Anaphase heterotypic, 234, 236, 238, 239, 242 somatic, 144, 1 r>. 1 17 Anasa (bug), 244, 245, 249 Anastomoses, I 17. 148, 1 19, 150 Ancel, 256 AncyracanthiM (nematode worm), 367 127 428 INDEX Anderson, 395 Androcyte, 88, 89 Androgone, 88, 89 Aneura (liverwort), 82, 87, 239 Angiosperms fertilization, 298, 299 sporogenesis, 225 Antedon (echinoderm), 327 Antheridium, 87, 222, 289, 290 Anthoceros (liverwort) apical cell, 27 chondriosomes, 122 fertilization, 293 plastid, 103, 104, 105, 113, 114 pyrenoid, 104, 108, 109 Anthoryanin pigments, 122, 135 Anti-fertilizin, 287 Antirrhinum (snap dragon), 332 Aphis (aphid), 318 Apical body, 89 Apical cell, 27 Apical growth, 27 . Apis (bee), 318 Apogamy, 311 ff.} 312 v Apospory, 311, 315 ff., 316 Apotettix (grasshopper), 391 Apposition, 192 Arbacia (sea urchin), 78, 160, 330 Arber, 212 Arcella (protozoan), 62, 281 Archicarp, 223, 290, 312, 313 Archoplasm, 115, 181 Arctostaphylos (angiosperm), 134 Arisanna (angiosperm), 104, 105, 248 Aristotle, 1 Armadillo, 357 Arnold, 239 Artemia (crustacean), 318 Artichoke, 134 Artificial cytasters, 78, 280 Artificial parthenogenesis, 284 ff. Ascaris (nematode worm) centrifuged egg, 330 centrosome, 77 chondriosomes, 118 chromatin diminution, 404, 405 chromosomes (somatic), 150 cleavage, 404, 405 fertilization, 276, 277, 280, 281 hybrids, 164 individuality of chromosome, 157, 164 polar body, 318 Ascaris (nematode worm) reduction, 219, 239, 248, 256, 276 sex-chromosomes, 358, 359, 367 spermatozoon, 274 Ascidian egg, 329 Ascobolus (fungus), 80, 81, 223, 290, 291 Ascogonium, 312 Ascomycetes, centrosomes, 80 fertilization, 290 ff. mitosis, 179 reduction, 223 Ascophanus (fungus), 291, 312 Ascospore wall, 80, 291 Asexual reproduction, 137 Askenasy, 193 Asparagus, 121 Aspergillus (fungus), 290, 312 Aspidium (fern), 91, 313, 314, 373 Assimilation, 106 Aster, 26, 76, 177, 178, 183, 189, 279, 280, 286 Asterella (liverwort), 87 Astrosphere, 76, 177, 178 Atamosco (lily), 315 Athyrium (fern), 315, 317 Atkinson, 223, 248, 290 Atoms and factors, 353 Atrichum (moss), 88 Attraction sphere, 26, 76, 177 Auerbach, 16 Autogamy, 282 Autosome, 358 Autumnal colors, 136 Avena (oats), 348 Axial filament, 273, 275 gradient. 139 Axon, 29, 30 B Bacillus Butschlii, 67 Bacteria, 3, 66, 108 Bacterium gammari, 67 Bailey, 63, 177, 192 Balbiani, 61, 155 Ballowitz, 274 Baltzer, 160, 327, 371 Bancroft, 35, 44 Banta, 370, 371 Barber, 38 Barley, 122 Barry, 15 Bartlett, 372 I Shi. \ 129 de Bary, 11, 12, 32, L08, 312 Basal granules or corpuscles 96, 97 Basichromatin, 64, 66, 70, 158 Basidiomycetes, centrosomes, SI fertilization, 292 reduction, 22 I Basidiospores, 22 t Basidium, 224 Bataillon, 285, 319 Bateson, 344, 360, 395 Bateson and Punnett, 378 Butracoseps (salamander), 237, 385 Baur, 332 Beauverie, 135 Bechamp, 48 Bechhold, 37 Beckwith, 118, 123 Bee, 318, 319, 357 Beer, 110, 196, 239 Begonia (angiosperm), 137, 256 Beijerinck, 49 Belajeff, 86, 90, 95, 288 Benda, 115, 119 van Beneden, achromatic figure, 181, 183 attraction sphere, 76 centrosome, 77, 180 early work on mitosis, 143, 144 fertilization, 279 individuality of chromosome, 157 polarity, 138 reduction, 219 Bensaude, 292 Bensley, 47 Berberis (barberry), 47 Berghs, nucleus and mitosis, 61, 66, 158, 176, 209 reduction, 231, 236, 257 Bernard, 69 Bernhardi, 5 ' Beroe (ctenophore), 328 Berridge and Sanday, 295 BerthoUetia (Brazil nut), 134 Biochemistry, 23, 414 Biococci, 69 Biogen, 42, 50 Biophore (Adami), 50, 412$'. Biophore (Weisinann), 49, 226, 400 ff. Bioplasts, 33, 48 Birds, sex-determination, 363, 367, 369 Bivalent chromosomes, 161, 227, 233, 239 Black, 292 Blackman, M. W., 255 Blackman, \ II 224, 291, 29 312 Blackman and Fi er, 290, 312 Blackman and \\ elaford, 291 l.; Blakeslee, 290, 35 Bla "/ liverwort . 87, Q Blastomere, 188, 325 Blending inheritance, 338 Blepharoplast, 83 ff. relation to centrosome, '»l Blochmann, 3 1 s Blood cells, 29, tin Boletus I mushroom . 81 Bullet, r. s7 Bone, 30 Bonellia (gephyrean worm . 37 1 Bonnet . ( '.. 3 Bonnet. J., 223 Bonnevie somatic chromosomes, 150 L52, 155, 159 reduction, 250, 251, 257 Boquet stage, 247, 248 Bordered pit, 191, 192 Borelli, 2 Borzi, 46 Botrychium (fern . 236 Boudiera (fungus), '-".»<> Bourquin, lot'.. 109 Boveri, amitosis, 213 archoplasm, 115, l si cent rosome, 77. 78, ls<> chromosome number, 163, 16 l experiments on echinoderms etc., Hi:;. 164, 325 330 fertilization, 279, 286 germ cells of Ascaris, mi. i<>:. hereidtary substance, 210 individuality of chromosome, 157 nuclear size, 63 polar body in Ascarti, 31 s reduction, 228, 248, 251 somatic chromosomes, l 19 Bower, 316 Brachymeiosis, 223 Brachystola (grasshoppei . 158, 160, 252, 253, 255 Brauer, 77. L55, 227, 248, 318 Braun, 1 1 Bridges, 382 Brooks, !■'. 'I'.. 291 Brooks. \\ . K . 10 Brown, H. B.. ^j\ Brown R., ti. 7 Brown W. II.. 291, 29 430 INDEX Brownian movement, 7 Bryonia (angiosperm), 356 BryophyUum (angiosperm), 137 Bryophytes, blepharoplast, 86 centrosome, 82 fertilization, 292, 293 reduction, 224 sex-determination, 3.55, 356, 364 Br yum (moss), 317 Buchtien, 89 Bud sport, 351 Buffon, 48, 398 Bufo (toad), 241 Burrnannia (angiosperm), 315 Buscalioni, 211 Biitschli, achromatic figure, 181 bacteria, 66 blue-green algae, 202 centrosome, 79 ectoplasm, 42 furrowing, 188 polar bodies, 15 protoplasm, 33, 49, 183 C Cceoma (rust fungus), 312 Calcium salts, 135, 194 Caldwell, 93 Calkins, 183, 213, 248 Calopogon (orchid), 300 Calycanthus (angiosperm), 159, 236, 248, 256 Calypogeia (liverwort), 110 Cambarus (crayfish), 237 Cambium, 27, 63, 177 Camerarius, 13 Campanula (angiosperm), 159 Campbell, 89, 195 Canaliculse, 48 Cancer (crab), 237 Canis (dog), 237, 361 Cannabis (hemp), 256, 372 Capsicum (angiosperm), 332 Carbohydrates, 41 Cardiff, 236, 257 Car ex (sedge), 159 Carloton, 65 Carnoy, 33, 248 and Lebrun, 280 Carothers, 160, 255 Carotin, 104, 121 Carpogonium, 289 Carrel, 138 Carruthers, 136, 223, 313 Carter, 60, 105, 209 Cartilage, 29, 30 Carus, 15 Castle, 390 and Wright, 391 Castrada (flatworm), 274 Catalyzer in egg, 285 Cattle, sex-determination, 361, 371 Cavers, 119, 122 Cell, description, 24 discovery, 2 evolution of, 69 a system, 26 Cell-division, Chaps. VIII-X Cell-formation, early views, 5 Cell plate, 176, 190 sap, 135 Theory, 7, 9, 12 wall, 26, 28, 190 ff. Cellulose, 135, 194 Central spindle, 177, 178 Centrifuged eggs, 329, 330 Centriole, 77, 83 Centrodesmose, 83, 209 Centronema, 84 Centrosome, 26, 76 ff. individuality, 77 in fertilization, 279 in mitosis, 145, 146, 177, 178, 180 in plants, 79 ff. Centrosphere, 26, 76 Cephaloiaxus (conifer), 295 Ceratiomyxa (slime mold), 222 Ceratozamia (cycad), 220, 294 Cerebratulus (nemertean worm), 275 Chaznia (protozoan), 61, 68 Chatopterus (annelid worm), 275 Chamberlain, 48, 82, 93, 94, 294, 295, 298 Chambers, observations on living cell, 38, 43, 62, 77, 118, 119, 120, 123, 183, 189, 214, 287 Chara (stonewort) and other Charales, 86, 90, 95, 108, 116, 210, 211, 222, 288 Chemical theory of development, 412 of heredity, 412 theories of life, 50 Chiasmatype hypothesis, 257, 384 ff. Chick cells in tissue cultures, 146 Child, amitosis, 213 I \ DE\ Child, colloids, 3 I germ-plasm, 1<>7 germ-track. J()."> metabolic gradient, 139 metabolism in maturation, 260 metaplasm, 133 nucleoplasmic ratio, 63 parthenogenesis, 319 physiology of fertilization, 284 polarity, 139 senescence, 136, 137 vital activity, 49, 50, 51 Chitoscyphua (liverworl I, 248, 249 Chimera, 351 Chironomus (midge), 61, 405 Chitin, 194 Chlamydomonadaceac, 112 Chloralized cells, 136, 104, 248, 249, 259, 260 Chlorogonium (green alga), 85 Chlorophyll, 104, 105, 124 inheritance, 331 Chlorophyllogen, 105 Chloroplast, 104, 105, 113 in heredity, 331-334 Chmielewskij, 109 Chodat, 202 Cholesterin, 41 Chondriokont, 116 Chondriomite, 116 Chondriosomc, 26, 48, 97, 111, 115 jf., 134 division, 118 in fertilization, 120, 280 microtechnical methods, 117 other functions, 121 and oxidation, 123 relation to plastid, 121 jf., 124 rank as cell organ, 124 role in inheritance, 119, 122, 121, 279 Chorthippus (grasshopper), 158, 237 Christman, 291, 292, 312 Chromatid, 230, 246, 247 Chromatin, 25, 40, 64 contributions of parents, 279, 340 diminution, 404, 405 extrusion, 136 qualitatively varied, 226 Weismann's conception of, 400 Chromatoider Nebenkorper, 86, 89 Chromatophore, 104, 136, 202 Chromidia, 67, 68, 117, 118, L33, 208 < liKMiinli.il fragmentation, Jus ( Shromidiogamy, 281, 282 < Ihromioles, 155 ( Ihromomere \ esicle, 158, 162 Chromomeres, 154 jf.. 165, 238, 243, 258, 391, 392 ( Shromosomal i esicle, 158 Chromosomes, aberrranl behavior, 302 alveolation, 148, I 19 m artificial parthenogei bivalent, 227. 233 in cleavage division, 278, 2 conjugation, 227. 233, 251 jf. in ( lyanophycese, 203, Jul early work on. 1 1.; in endosperm, 301 equational division, 156, 226 in fertilization, 277 279, 295 fj . 340 in heredity, 326, 330, 396 individuality, 1">7 His in life cycle, 221, 340 linear organization, 162, --*> in man, 361, 362 map. 388, 389 in mitosis, 1 1 \ jf. multiple chromosomes, 164, 165 and mutation, 3 1 1 jf. number, 162 .//".. :; 17 jf. pairing before maturation, 255 pairs. 160, 161, 227, 251 in parthenogenesis, '■'< I 8, 319, 36 1 qualitative division. 227 reduction of, 219 261 sex, 358 jf. size and shape, loll. J.YJ jj. and species. ."> 17 if. specificity of, 162, J-">0 splitting of. | I:,. 152, 153, ! tetrads, 230, 233, 243 Jf. transverse division, 227 Chroococcua (blue-green alga 68 205, 206 Chrysanthemum (angiosperm . -Is Chun. 21 1 Cicada (inseel , 120, 121 ( 'ienkow Bki, 12, 32 Cilia. 30. i:». 96 Ciliated cells, 96, 212 Cirri, 30, 15 Cladophora (green alga . 60, 85, 105, 112, 209 432 INDEX Claussen, 81, 224, 290, 291 Cleavage centrosomes in, 279, 280 chromosomes in, 278, 295 ff. division, 278, 329 furrowing, 146, 186 ff. Cleland, 109, 223 Closing membrane, 191, 192 Closterium (desmid), 186, 222 Clowes, 35, 36, 44 Clytia (coelenterate), 328 Coagulation, 38 Cobcea (angiosperm), 177 Coccidium (protozoan), 207, 20S Ccenocentrum, 289 Coenocytes, 60, 411 Cohn, 11 Coker, 295 Coleochoste (green alga), 113, 222, 236 Collema (lichen), 291 Collins, E. J., 357 Collins, G. N., 333, 349 Colloids, 34 Colonial alga?, 140, 331, 334, 411 Colorblindness, 381 Components (of colloids), 35 Compositae, 236, 315 Coniferales, fertilization, 294 f. Conjugate, 288, 316 Conjugate division of nuclei, 292 Conjugation in Paramoecium, 283, 284 Conklin, achromatic figure, 183 amitosis, 213-214 centrosomes, 78 fertilization, 279, 280 individuality of chromosome, 159 nucleoplasmic ratio, 63 performationism, 411, 414 promorphology of ovum, 329, 330 role of chromatin, 70 Weismannism, 408 Connecting fibers, 145, 176 Connective tissue, 29, 30 Conocephalus (liverwort), 86 Constrictions in chromosomes, 160, 161, 162, 166, 248, 249, 255, 260 Continuity of chromosomes: see chromo- somes, individuality of germ-plasm, 402, 403, 413 Contractile vacuole, 30, 44, 46, 84 Contractility in achromatic figure, 183 Copepods, 245, 249, 405 Coprinus (mushroom), 292 Corallina (red alga), 80, 223 Cork, 2, 194 Corpuscles de rebut, 283, 284 Corpuscular theories, 391, 398 jf. Correlation, 139 Correns, 331, 332, 338, 356, 366, 372 Corsinia (liverwort), 82, 293 Corti, 6 Corydalis (angiosperm), 211 Coulter. 288 and Land, 295 Cowdry, E. V. 116-118 Cowdry, N. H., 116, 117, 123 Crampton, 328 Crepidula (mollusk), amitotic appear- ances, 213 fertilization, 279, 280 individuality of chromosome, 159 nuclear size, 63 sex-determination, 371 Crepis (angiosperm), 159-162 reduction, 239, 252, 254 species and chromosomes, 349 Crinoid, 326, 327 Criss-cross inheritance, 380 Crossing over, 385, 386 f. Cruciferee, 159 Cryptobranchus (amphibian), 159, 160, 278, 279 Cryptomeria (conifer), 294 Crystalloid, 35, 65 Crystals, 134, 135 Ctenolabrus (fish), 160 Ctenophore, 328, 329 Cuenot, 355 Culex (mosquito), 165, 256 Cumingia (mollusk), 286 Cumulative factors, 343 Cunninghamia (conifer), 295 Cuscuta (angiosperm), 47 Cuticle, 194 Cuticularization, 194 Cutin, 194 Cutinization, 194 Cutleria (brown alga), 222, 241 Cutting, 312 Cyanophycese, cell-division, 202 ff. nucleus, 68 plastids, 108 Cyanophycin granules, 203, 206 Cyanoplasts, 203 Cycas (cycad), 93, 294 Cyclops (copepod), 159, 160, 237, 245, 279 INDEX I.;.; Cystolith, 134, 135 Cytoblast, 8 Cytoblastema, 8, 1 i:; Cytokinesis, 186 ff, by cell plate, 176, 190 by furrowing, L86 Cytolysis, 285, 286 Cytomorphosis, 1 17 < Jytoplasm, 25, 40 in fertilization, 280, 294, 299 in heredity, 326, 328, 330 pattern in ovum, 329 reducing action, 123 Czapek, 44 D Dahlgren, 225 Dahlia (angiosperm,), 2.56 Dale, 290, 312 Dangeard, 84, 85, 86, 122, 202, 290, Daphnia (copepod), 371 Darling, 159, 356 Darwin, 16, 49, 398, 402 Datura (angiosperm), 134 Davey and Gibson, 372 Davis, B. M., blepharoplast, 85 eentrospheres, 80, 82 cytokinesis, 187 fertilization, 289, 290 (Enothera mutants, 344, 345 plastid, 113, 114 reduction, 241 Davis, H. S., 239 Death, 136, 138 Debaisieux, 237 Dedifferentiation, 137 Degeneration, 2\\ ff., 259 Dehorne, 150, 159 Delage, 63 and Goldsmith, 400 Delia Valle, 103 Dellinger, 45 Dendrite, 30 Denke, 195 Derbesia (green alga), 85, 86 Dermatosome, 193 Dermocarpa (blue-green alga), 201 Derschau, 66, 123, 136 Des Cilleuls, 212 Desmid, 105, 186, 222 Determinant. 226, 400 ff. Deton, 237 28 I teutoplasm, L3 l Development (Ontogen. 15, I in chemical <-<»nt rol of, 1 1 j non-factorial 1 1 1 1 1 R i ismann'fl I heory of, mi . 107 Developmental mechanics, 1 ! 1 Devi-.'. 182 Dextrin, 107 Diakinesis, 231, 232, 2 Dicroccdium (flatworm . 245 Dictyota (brown alga . cell wall, l centrosome, 79, so. 82 reduction, 223 Didiploid nuclei, 260 Didymium (slime mold . 1^7 Dietel, 22 I Differentiation of cells, 26, 137 products. 133 Jf. Digametic condition. :;.">s /f. 291 Digby, extruded chromatin, L36 nucleolus, 65 prochromosomes, L59 reduction, 239, 240, 248 somatic chromosomes, 1 19, 152 DinophUtts (annelid worm), :;.".7. 368 Dionoea (angiosperm . 17 Dioon (cycad), 02. '.»:;. 94, 294 Diplotene, 231, 233 Diptera, 256 Dispermy, 163, 326 LH8808teira (grasshopper), 119 Dixon, 211 Dobell, 67 Dodge, 22:; Dog, 361 I >ominance, .;.'i7. 339 Doncaster, i\. 319, 363 Double fertilization. 14, 298, 299, 302 Double heterotypic spireme, 241 243 Double reduction, 223, 224, 291 Douin, •">•">•">, 36 l Downing, 275 Draparnaldia (green alga . 104, 108 Driesch, 328, U2 and Morgan. 328 Drosera (angiosperm 159, 236, 251, 252 Drosophila (fruit fly), chromosome com- plement, 350, 383 map, 388, 389 crossing over. 3^7 jf. intersexes, 37 1 . 376 linkage, "'7'.i 434 INDEX Drosophila (fruit fly), non-disjunction, 382, 383 sex-chromosomes, 360 ff. sex-linkage, 380 Drosophilidse, 349, 350 Druery, 311 Druner, 181, 183 Druse, 134, 135 Dublin, 255 Dubreuil, 121 Duesberg, 115-120, 361 Dujardin, 11 Dupler, 295 Duplex chromosome group, 340 Duplicate factors, 343 Dytiscus (beetle), 237 E Eames, 295 and Hayes, 135, 333 East and Jones, 352 Echinoderm hybrids, 160, 163, 325-327 Echinus (sea urchin), 160, 325 Ectocarpus (brown alga), 222, 314 Ectoderm, 329 Ectoplasm, 42, 44, 45 Ectoplast, 25, 43, 44, 85 Egg, segmentation in animal, 188, 189 fragments, 287 Eisen, 66, 155 Elasis (palm), 134 Elaioplast, 109, 110 Elasmobranchs, 239 Elastin, 194 Electrical charge of nucleus and cytoplasm, 62, 184 phenomena in egg, 287 theories of mitosis, 184 Elementalism, 12, 411 Elodea (angiosperm), 37, 122, 356 Emberger, 122 Embryo sac, 299 Embryogeny (see Development) historical, 13, 15 Embryonal mitosis, 278, 295 ff. Embryonic cells, 137 Emerson, 135, 302, 333, 384, 391 Emulsion, 34-36, 44 Emulsoid, 35 Enchenopa (bug), 237 Enchylema, 33, 34 End piece, 275 Endoderm, 329 Endoplasm, 42, 44 Endosperm, development of, 302 mosaic effects, 302 nuclear behavior in, 300-301 Endospore, 195, 196 Energy change in cell, 26, 52, 70 Entelechy, 412 Enteroxenos (mollusk), 237 Entz, 83, 84 Enucleated cells, 69, 70 Environment and development, 411, 413 Enzyme, 39 Ephedra (gymnosperm), 295 Epigamic sex-determination, 357 Epigenesis theory, 4 Epithelial cells, 30, 83, 96 Equational division of chromatin, 156, 226, 228, 401 Equisetum (pteridophyte), chromosome tetrad, 248 dicecism, 356 spermatogenesis, 90-92, 95 spore coat, 196 stem, 135 Ergatule, 49 Erhard, 96 von Erlangen, 189 Ernst, 300, 301, 315 Erysiphe (fungus), 80, 81, 290 Escoyez, 79, 87, 210 Etiolation, 105 Euchlcena (angiosperm), 349 Eudorina (green alga), 112 Euglena (flagellate, eyespot, 112 flagellum, 45 mitosis, 209 paramylum, 108 Euschistus (bug), 237 Exine, 196 Exospore, 195, 196 Extruded chromatin, 136 Eye color in Drosophila, 380 Eyespot, 46, 84, 111 #., 112 Factor (see Gene), 333, 334, 336, 353, 409, 410 Factorial hypothesis, 343, 410 Farlow, 311 Farmer, 220, 248 and Digby, 136, 239, 312-315 and Moore, 237, 239 I.\ DEX Farmer and Reeves, 82 and Shove, 239 and Willi:. ins, so, 222 Fair, 186-188 Fasciola (flatwonn), 'J 17 Fasten, 237, 257 Fats, 41, 134 and chondriosomes, 121, 122 I a i ill, 80, 81, 179, 223, 291 Faurc-Fremiet, 115, 117, lis Fedorley, 252 Fegatella (liverwort), 86, 87 Ferguson, 295 Fertilization, animals, 273 jj. centrosome in, 279 cone, 277 historical, 13 membrane, 277, 284 parental chromosome groups in, 159, 254, 276, 278, 279, 295, 300, 340 physiology of, 284 ff. plants, 287 ff. relation to maturation of ovum, 275 Fertilzin, 287 Fiber theory, 6 Fibrillar theory of protoplasm, 33 Fick, 163, 249, 250 Ficus (rubber plant), 134, 135 Filaria (roundworm), 120 Fischer, 34, 66, 108, 202, 204 Fish hybrids, 159, 160 Fitting, 194, 195 Fitzpatrick, 224, 291 Fixation, 38, 117 Flagellates, blepharoplast, 83, 111 Flagellum, 45, 84, 96, 112 of spermatozoon, 273-275 Flemming, amitosis, 211, 213 cell-division, 143 centrosome, 77 chondriosomes, 115 chromatin, 64 chromosomes in man, 361 nuclei by division only, 10 protoplasm, 33, 34 reduction, 220, 227 spindle, ISO Florideae, 108, 116, 223 Floridean starch, 108, 109 Florin, 82, 248, 249 Flower colors, 135 Focke, 302 /■ / niculum (angiosperm . 134 Fol, aster and asl roephen . 76 Cell-division, 1 |.; cent rosomes in fertilizatioi chromomeres, 155 fertilization, l"> -i reaming of protoplasm, 1 Fontana, 6 I 'ood materials, l Foot and Strobell, 213, 244, 245 Forenbacher, lis, 122 Forficula ^earwig . -'•!'.» Fo88ombronia (liverworl , 248 Fowl, 361, 363 Krauze, 112 Fraser, brachymeiosis, 223 centrosome, si fusion in ascomycetes, 290, 312, '>\ '- reduction. 238, 239, 240 somatic chromosomes, l 19 Fraser and Brooks, si, 223 and Snell, 149 and Welsford, 81, 223 Free, 44 Fremy, 193 Fries, 224 Friesendahl, 236 FrUiUaria (angiosperm . amitosis, 211 constricted chromosomes, 162 fertilization. 298, 300, '.<>1 Frog, artificial parthenogenesis, 319 blastomeres, 328 centrifuged eggs, :;:;,) cleavage, 188 organization of egg, 330 sex-determination, 368, 370 Fromman, 33 Fuchsia (angiosperm l, 21 Funis, centrosome, s". B2 rv.-pot, 111 fertilization, l 1 gametes, 288 parthenogenesis, 31 1 plasma membrane, 43 reduction, 222. 223, 24] spindle, 179 Fujii, 92 Fuligo (slims mold . 39, 1 1 . 1 s~ Fuim a | moth Fund rin mOSfl . 106, ■'>■'<> Function and si ructure, 37 I Fumlulus (fish . 158, 159, 160, 328 Funkia (angiosperm . 236, 266 436 INDEX Furrowing (cytokinesis), 186 jf. Fusion, centrosomes, 280/291 chromosomes, 257, 394 gametes, 276 ff. nuclei, 15, 219, 211 ff., 299, 301 sporocytes, 313, 314 three nuclei in endosperm, 299, 301 G Gaertner, 13 Gaillardia (angiosperm), 110 Gall-fly, 319, 364 Gallactinia (fungus), 80 Gallardo, 184 Galton, 399, 402, 404 Galtonia (angiosperm), extrusion of chromatin, 136 nucleolus, 65 reduction, 236, 239 somatic pairing, 256 Gametes, 137, 273 ff., 288, 293 ff., 333 ff. Gametophytes, sex-determination in, 355, 356, 364, 372 Garber, 292 Gardiner, 46, 47 and Hill, 46 Gardner, 108, 202, 204 Gargeanne, 110, 111 Gastrodia (orchid), 314 Gatenby, 120 Gates, 63, 149, 241, 252, 344, 345 and Thomas, 345 Gaudissart, 121 Geddes and Thomson, 355, 369 Geerts, 241, 252 Gegenbaur, 15 Gel, 35 Gelatin, 37, 194 Gelation, 38 Gemini, 233 Gemmule, 49, 226, 398 Generative apogamy, 315 Generatule, 49 Genes, 343 cytological evidence for, 391 ff. in development, 410 linked, 380, 386 modification of, 351-353 nature of, 352, 391, 409 Genetic continuity, 15, 323 Genotype, 339 Geotriton (salamander), 118 Gerassimow, 61, 69 Germ cells and soma cells, 402 ff. Germ-plasm, 226, 334 and soma-plasm, 407 Weismann's conception of, 400 ff. Germ-track, 403 ff. Germinal localization theory, 328 selection, 404 vesicle, 62, 221, 275 Giard, 15 Gigantism, 346 Giglio-Tos, 250 Gille, 160 Ginkgo (gymnosperm), 92, 122, 236, 294 Gladiolus (angiosperm), 177 Glaser, 40, 213 Glceocapsa (blue-green alga), 204, 205, 206 Globular theory, 6 Globulin, 40 Glucose, 106 Glucoside, 135 Glycogen, 108 Gnetales, 294 Gnomonia (fungus), 291 Godlewski, 119, 213 echinoderm hybrids, 326-328 Goldschmidt, chromidia, 133 parthenogenesis, 319 reduction, 244, 245, 256 sex-determination, 370 Gonium (green alga), 112 Gonotokont, 225 Goodsir, 10 Goroschankin, 46 Gottsche, 110 Gould, 371 Grafted tissue in apogamy, 312-314 Graham, 82, 293 Granata, 250 Granular theory of protoplasm, 33, 49 Grasshopper, 156, 158, 162, 233, 237, 243, 258, 351, 391, 392 Gregoire, chromomeres, 258 nuclear reticulum, 64, 150, 152, 155 reduction, 227, 231, 234, 236, 245, 248, 257, 259 Gregoire and Berghs, 82 and Wygaerts, 64, 149, 152, 155 Gregory, 239, 346, 391 Grew, 2 Griffithsia (red alga), 223 Griggs, 239, 248 / \ DEX 137 Gromia (protozoan), 45 (1 round substance, ll'A Growth period in oocyte. 158, 221, 233, 234, 274, 275 Gruber, 61, 69, To Gryllotalpa (mole cricket), 118, 119 Guignard, blepharoplast, '.*<), 94 eyespot, 111 fertilization, 14, 298, 300, 325 reduction. 220 Guilliermond, bacteria, 67 eentrosome, NO chondriosomes, 115 122 reduction, 224 yeasts, 211, 292 Guinea pigs, 274, 341, 342 Gurwitsch, 213 Gutherz, 361 Gutta percha, 135 Guyer, 285, 361, 363 Gyrrmogramme (fern), 90 Gyrodactylus (flat worm), 160 II Baberlandt, 46, 61, 103, 104, 105, 193 Haeckel, 16, 48, 324, 402 Haecker, fertilization, 279 hereditary substance, 210 individuality of chromosome, lf>7. 159 Keimbahn-plasma, 405 nucleolus, 66 pseudoa mitosis, 211, 213 reduction, 227, 244, 245 sex-determination, 357 Hamiatochrom, 112 Haller, 6 Hamm, 14 Hammarsten, 40 Hance, 165, 346 Hannig, 196 Hanstein, 12, 24, 33, 46, 133 Hardy, 62 Bargitt, 213 Barman, 160, 213, 391 Harper, centrosomes in a8CU8, NO. 81 chondriosomes, 123 cleavage furrow, 187 development in colonial algse, 334, 411 evolution of cell structure. 207 fertilization in ascomycetes, 290 Harper, inheritance in colonial alga?, • 334 mitosis in myxon 208 plastids, 103, 105, L07, lb". polarity, 138, l 10 protoplasm, 38, 19, ">i reduction in ascomyceti Hart niann and Noller, s \ Bartog, KM. 185 Harvey. E. E., 162 Barvey, E, V. 284 Harvey, Win.. 1 Hasper, 105 Batschek, 36, 19, 138 Bautschicht, 12 Heart wood, 194 Hegler, 108, 202, 203 Begner, 62, 104, 105 Beidenhain, 64, 70, 138, III. 18 Beilbrunn, 189, 286, 287 Helix (snail . 96, 256 HeUeboru8 (angiosperm . 159 Helvetia (fungus . L36, 223, 291, 313 Bemiptera, 239, 255 Hemp, 372 Benking, 227. 244, 251, 256, 358 Henle. 10 Benneguy, 7o. 95, 96 Berbst, 213, 325, 326 Heredity, cell organs in, 323 ff. chemical theory of, 112 in colonial algS, 331 . 33 1 early theories of, ">-,s Jf. Mendelian. 336 ff. non-factorial theories of, 1 1 1 Weisniann's theory of, 101 Berla, 157. 164 Berlant, 285, 286 Hermann, 1 1 1. 180, 183 Berrick, 27 l //. rsiliti (copepod . 237 I bit win, < )-. basal granul< cell-division, 1 13 fertilisation, 15, 273, 276 nucleus in heredity, l'». 32 I spindle, ls<> Bertwig, R., amitosia, 211, 213 chromidia, 133 division in protOSOa, 207 heterotypic prophase an abort mitosis, 259 Ducleoplasmk rat i<>. 63, 70 Bex-determination, 368, 37( I 438 INDEX Hesperotettix (grasshopper), 164, 165 Heterochromosomes, 358 ff. Heterodera (fungus), 260 Heterogametic females, 363 males, 358 ff. Heterotypic mitosis, 220, 231 ff. compared with somatic mitosis, 228 and degeneration, 259-260 Heterozygous state, 339 Heuser, 144 Hexads, 249 Hick, 46 Hieracium (angiosperm), 315, 316 Hill, 46 Hilum, 106 Hirase, 92, 294 His, 328 Hober, 51 Hofmeister, 10, 13, 14 Holt, 165 Homarus (lobster), 274 Homoeotypic mitosis, 220, 231 ff. Homologous chromosome pairing, 161, 252-254, 340, 342 Homozygous state, 339 Homunculus, 3 Hooke, 2, 23 Hoppe-Seyler, 40 Hormones, 139 Horse, 361 Hoven, 121 Humaria (fungus), 81, 223, 290, 291, 312, 313 Hi'ippe, 67 Hutchinson, 295 ff. Huxley, 12, 13, 168, 273 Hyaloplasm, 33, 42, 183 sphere, 76 Hybrid echinoderms, 160, 163 fishes, 159, 160 Hydatina (rotifer), 357, 364, 368 Hydnobolites (fungus), 81, 224 Hydra (coelenterate), 275 Hydrocharis (angiosperm), 256 Hydrodiclyon (green alga), 85, 105, 107, 109, 187 development of, 334, 411 inheritance in, 331, 334 Hydrogel, 37 Hydrophilus (beetle), 239 Hydrosol, 37 Hygrophorus (fungus), 224 Hymenomycetes, 224, 292 Hypertonic solutions, 285 Hypothesis, role of, 396-397 Id, 226, 400 ff. Idant, 226, 401 Idiochro matin, 208, 209 Idiochromosome, 358 Idioplasm, 258, 324, 400 chemical conception of, 412 theory of Nageli, 399 Idiosome, 77 Ikeda, 248 Ikeno, 86, 89, 93, 94, 294, 332 llyanassa (mollusk), 328 Immortality of certain cells, 138, 403 Independence of chromosome pairs, 255 Individuality, aleurone grain, 135 alga colony, 140 centrosome, 77 chromosome, 157 ff., 168 organism, 168 plastid, 113 Infusoria, 60 Inheritance of acquired somatic changes, 399, 403 colony characters, 331, 334 Mendelian characters, 336 ff. plastid characters, 331 ff. sex, 354 ff. Insects, 160, 239, 359 ff. Inter-alveolar substance, 34 Interference, 389, 390 Inter-filar substance, 33 Interkinesis, 232, 236 Interphase, 145, 150, 151, 236 Intersexes, 370 ff. Intracellular pangenesis, 399 Intranuclear spindle, 179 Intra-vitam stains, 117, 287 Intussusception, 193 Inulin, 134, 135 Inversion of phases, 36, 44 Ipomcea (angiosperm), 196 Iris (flag), 121, 122, 177 Ishikawa, 256 Isoetes (pteridophyte), 114 Isonandra (angiosperm), 135 Isotropy of egg, 328 Jaeger, 402 Jahn, 59, 208, 222 INDEX 139 Janssen, 1 Janssens, 237, 257, 385, 386, 393 and Willems, 237, 256 Jennings, »'»:;. 396 Johnson, 1 1 1 Johow, 210 Jonsson, li> Jordan, 213 Jorgensen, 178, 213 Juel, 224, 315 Juglans (walnut, butternut), L34 .1 iini pi rns juniper i. 294, 2,.».") K Kabsch, 193 Kahle, 404 Karsten, 79, 209, 222 Karyochondria, 118 Karyogamy, 282 Karyokinesis, 143 (sec Mitosis) Karyolvmph. 25, 64, 149, 182, 184 Karyoplasm, 25 Karyoplast, 84 Karyosome, 25, 65, 151, 209 Kassowitz, 133 Keene, 110, 290 Keimbahn, 403 -determinants, lo<> -plasma, 406 Kemp, 259 Keratin, 19 1 Kernplasma relation, 62 Keuten, 209 Kienitz-Gerloff, 4<>, 47 Kieser, 5 Kihara, 252, 348 Kildahl, 295 Kinetonucleus, 8 1 Kinetosomes, ss King, 241, 368 Kingery, 116, 259 Kingsbury, 117, L23, 124 ami Hirsch, 259 Kinoplasm, '■> 1. t3, l sl Kinoplasmic caps, 1 75 Kite, 38, 13, 62, 64 Kiel,., in. L12, L93 Klein. :;:;. 181, 183 Kniep, 22 1 Knoche, 213 Koelreuter, 13 Kofoid, 208, 209 Kohl, Mi. His. 202, 203 von Kolliker, lo, l.",. 16, :;i Koltzoff, 274 Konopacki, 213 Korff, it."). 96 Kornhauser, 237, 257 Korotneff, 1 In Korechelt, 61, 24 1 Kossel, in. 12 Kostanecki and Wierzyski, 276 Kowalevsky, I 1 1 Kuczynski, 8 1 Kuhla, 1»>, 17 Knline, 12 Kiirss.-inou , 1 ).;. 222 Kuschakewitsch, 368 Kusano, 314 Kuster, 101, 110 Kuwada, <»">, 256, ■'> Is. '■'< 19 Kylin, 222. ill 1 Laboulbenia (fungus), 179, 22 1. 291 Lachnea (fungus), 80, 81, 223, 290, 291, 312 Lagerburg, 236 Laibach, 159 Land. 294 Lang, 213 Larix (larch . 122. L82, L91, 192, 2 Lastrcea (fern), 312, 313, 314, 317 Laticiferous vessel. 59 Lauterborn, 7'.' La Valette St. George, 1 1. 1 15, IS Lawson, fertilization. 294, 295 nuclear membrane 6 1 reduction, 239 spindle. 177. 1M Lecithin, 11 van Leeuwenhoek, 3 win Leeuwen-Reijnvaan, 221 Leguminose, •'> Is Lenhossdk, 96 Leolia ^fungus), 291 Lepeschkin, 1 1 19 Li pidoi ■ li-li . 162 Leptonema, 231 233 Leptotene, 23 1 Lerat, 237, 245 Leucoplast, 104, L06, 109, 113, 121, L22 Levine, 81, 22 1 440 INDEX Lewis, C. E., 87 Lewis, I. F., 223 Lewis, I. M., 239 Lewis, M. and W., 116, 118, 123, 146 Lewitski, 115, 118, 121 Leydig, 33 Libocedrus (conifer), 294 Lignin, 194 LiUum (lily), amitosis, 211 chondriosomes, 116 extruded chromatin, 136 fertilization, 298-301, 325 megaspore nuclei, 225 protoplasmic connections, 47 reduction, 236, 238, 239 Lillie, F. R., fertilization, 273 ff., 286 regeneration, 69 sex-determination, 370, 371 Lillie, R. S., 62, 70, 184, 185, 285 Limosphere, 88, 89 Lindstrom, 332 Linin, 25, 64, 180, 181 Link, 5 Linkage, 378 ff. calculation of, 389 groups, 382 ff. Lipoid, 41, 43, 44 Liposomes, 123 de Litardiere, 236 Liverworts, blepharoplast, 86 centrosome, 82 fertilization, 293 sex-chromosomes, 364, 365, 372 Lloyd, 44 Lock, 378 Locy, 323 Loeb, J., 284, 319, 326 Loeb, L., 138 Loeb and Bancroft, 285 and Wasteneys, 70, 287 Lopezia (angiosperm), 248 Lotsy, 225, 344 Lowschin, 117, 118 Lundegardh, 123, 144, 149, 155, 236 Luther, 274 Lutman, 186 Lutz, 345, 346 Lychnis (angiosperm), 256, 382 Lycopodium (pteridophyte), 91 Lygoeus ^bug), 359, 360, 367 Lymantria ^moth), 370 Lyon, 194, 195 Lysin, 285 M Macallum, 66 Macdougal, 41 Macfarlane, 47 Macormick, 290 Macronucleus, 30, 60 Macrosomes, 38 Maggi, 48, 115 Magnolia (angiosperm), 188 Maier, 96 Maire, 80, 224 Maize, chlorophyll inheritance, 332 crossing over, 391 hybrid nature? 349 linkage groups, 384 Makinoa (liverwort), 87 Male cells and nuclei, 294, 298, 299 cytoplasm in fertilization, 294 Malone, 237, 361 Malpighi, 2 Malsen, 357, 368 Malva (angiosperm), 194 Man, centrosome, 77 colorblindness, 381 reduction, 237 sex-chromosomes, 361, 362 Mangin, 194 Manifold effects of factor, 343 Mantle fibers, 145, 176, 177 Marchal, 317, 355 Marchantia (liverwort), blepharoplast, centrosome, and spermatogene- sis, 82, 86 ff. cell-formation, 5 chondriosomes, 122 sex-determination, 355 Marcus, 248, 259 Marechal, 155, 158, 210, 234, 237, 257 Mark, 15 Marsilia (water fern), apogamy, 315 316 blepharoplast and centrosome, 91, 92, 95 nucleus, 61, 66, 210 spindle, 176 spore coat, 194 Martin, 33 Martins Mano, 155, 236 Masdevallia (Orchid), 47 Massee, 46 Mast, 112 Mastigella (flagellate), 83 INDEX 141 Mastigina (flagellate . s;i Mathews, 42, 70 Maturation divisions, 220 ff. Maxiiimw , 213 Mayer, Rathery, and Schaeffer, 123 McAllister, 109, 236 McAvoy, 239 McClendon, iss, 285 McClung, chromosomes in insects, 351 chromosome individuality, L60, 163 -168 reduction, 245, 252, 393 sex-chromosome, 358 McCubbin, 291 McLean, 212 Mead, 78 Mechanism of cytokinesis, L88 of karyokinesis, 182 Mechanistic hypothesis, 52 Meek, 185 Meganucleus, 30, 60 Megaspore, Lilium, 225 Marsilia, 194 Physostegia, 225 Selaginella, 195 Meiosis, 220 Melandrium (angiosperm), 256 Melanoxanthus (plant louse), 319 Melin, 248 Membranellse, 45 Mencl, 67 Mendelism, 336-344 and sex, 366 Mi nidia (fish), 160 Mercurialis (angiosperm), 250, 356, 372 Meri.smopcflia (green alga), 205, 206 Meristem, 27, 63, 137 M< nutria (grasshopper), 165 Merogony, 63, 325 Merriman, 149, 209, 210 Mesoderm, 329 Mesospore, L95 Metabolic gradient , 139 rate or level, 137, L39, 260 theories of sex, 369 ff. Metabolism, 69, 70. 260 Metachromatic 205, 206 Metachrome, 122 Metaphase, 144, 145, 1 17, 1 18 Metaplasm, 26, 133 jf. .-Hid Benescence, 136 Metasyndese, 228 Metcalf. 208 Met/. 256, 349, l. 395 Meves, amitosis, 213 cent rosome in Bperm, chondriosomes, 1 15 1 2 1 in fertilization, 120, 280 reduction, 227. 250 spermatozodn, 274 spindle, 178 Meyen, 6 Meyer, A., if.. 106, L07, L13, u I Meyer, K . 82, 2 Miastor [fly . MM 106 Micella*, L07, L93, 399 Microcycas (cycad . 93 Microdissection, 38, 62 Micromeric theories of protoplasm, iN Micronucleus, 30, 60 Microscope, 1 Microsome. 33, 122, 190 Microsphoera [fungus . M Microsporocvte, cytokinesis, 187 reduction. 225. 235, 238 Microzyme, 18 Mid-body, 178, 179, 188 Middle lamella. 190, 191, 192, 194 piece, 273, 275, 281 Miescher, 40, 41 Migula, 67 Mimosa [angiosperm I, 17 Minchin, 14, 45. 60, 68, 83, 207, 208, 222, 281, 282, 317 Minot, 63 MirdbMs (angiosperm), plastid inheri- tance, 331 Mendelian behavior, 338, 339 spore coat, 196 Mirande, 116 Mirbel, 5 Mitochondria, 115 ff. Mitokinetism, ls5 Mitome and paramitome, 33 Mitoplast, 122 Mitosis, duration of phases, l M'> heterotypic. 220, 231 ff. mechanism of, L82 jf. somatic, 1 h"> jf. compared with heterotypic, 228 summarised, 156 in ( Jyanophyces, 202 ff. in other thallophytes, 1 79 m protozoa, 207 ff. Mixochromosome, 257 Miyake, 87, 92, 236, 290 295 442 INDEX Mnium (moss), 87, 88, 317 Moenkhaus, 160 von Mohl, cell-formation, 6. 9, 10 cell wall, 190, 192 plastid, 12, 103 Moira (echinoderm), 160 Moldenhavver, 5 Molisch, 193 Monascus (fungus), 291 Monaster, 286 Monkey, eentrosomes, 77 Monotropa (angiosperm), 70 Monteverde and Lubimenko, 105 Montgomery, 65, 163, 228, 237, 239, 251, 256, 257, 281, 358, 361 Moore, A. C, 248 Moore, B., 43 Moore, J. E. S., 95, 227, 255 Moore and Embleton, 239 Moreau, 118, 122, 187 Morgan, achromatic figure, 183 blastomeres, 328 centrifuged eggs, 330 conjugation in Paramoecium, 283 crossing over, 379 ff. cytasters, 78, 286 factorial hypothesis, 344, 410 linkage, 379 ff. sex-determination, 357, 360, 361, 368, 376 on Weismann, 408 Moroff, 213 Morphoplasm, 400 Morris, 160 Morus (mulberry), 256, 372 Mosaic endosperm, 302 leaves, 331 ff. Mosses, regeneration, 317 sex-determination, 355 ff. spermatogenesis, 88 ff. Moths, sex-determination, 363, 367 Motile cells, 83 Mottier, blepharoplast, 86, 87 cell plate, 186 eentrosomes, 79, 80 chondriosomes, 119 jf. chromomeres, 155, 238 fertilization, 298, 300 reduction, 238, 239 Mucor (mold fungus), 62 Muller, 389, 390 and Altenburg, 352 Muller, 149, 152, 155, 156, 253, 256 Mulsow, 359 Multiple chromosomes, 164, 165 complex, 165 Multipolar mitosis, 163, 325, 326 stage of mitosis, 176, 177 Murbeck, 313, 315 Murrill, 295 Mus (mouse), 237 Musa (banana), 159 . Musca (fly), 360 Muscle cells, 28, 29, 120-121 Mutation, 344-352 and chromosome number, 344, 351 of gene, 351-353 rate of, 352 vegetative, 351 Myoneme, 45 Myrica (angiosperm), 372 Myricaria (angiosperm), 236 Myristica (angiosperm), 134 My .vine (fish), 237 Myxomycetes, chondriosomes, 116 cytokinesis, 187 mitosis, 208, 209 nucleus, 59 protoplasm, 32 reduction, 222 N Nabours, 391 Nachtsheim, 318, 357 Nagai, 373 von Nageli, cell-formation by division, 9 cell wall, 190, 192 idioplasm theory, 399 plastid and starch, 12 starch grain, 107 Nagler, 282 Arajas (angiosperm), 160, 161, 253 Nakahara, 66, 211, 239 Nakanishi, 67 Narcissus (angiosperm), 346 Narcotics, 259, 260 Nathansohn, 211, 213, 316 Nawaschin, M., 160, 162 Nawaschin, S., 14, 162, 298, 300, 301, 325 Nebenkern, 91, 120 Nemalion (red alga), controsomes, 80 pyrenoid, 108, 109 reduction, 223 Nematus (saw-fly), 318 INDEX 1 1 3 Nernee, 149, 152, 213, 259 NeoHetta (fungus), 81, 224 Nephrodium (fern), apogamy, 313 315 fertilization, 293 reduction, 232, 236 spermatogenesis, 91 spindle, 176 Nereis (annelid worm), 274 277, 280 Nerve cells, 29, 30 Neuroterus (gall-fly), 319, Mill Newman and Patterson, 357 Newport, L5 Nichols, 255 Nichols, G. E., 295 Nicotiana (tobacco), 187 Nidularia (fungus), 224 Nienburg, 291 Nissl substance, 30 Noll, 193, 355 Xon-disjunction, 346, 348, 382, 383 Non-factorial theories of heredity and development, 411 Noren, 294, 295 Nothnagel, achromatic figure, 182 fertilization, 298-301 reduction, 239, 240 Nowikoff, 213 Nuclear membrane, 63, 149, 154 migration, 312 Nucleic acid, 40 Nuclein, 40, 64 Nucleolini, 65 Nucleolo-centrosome, 209 Nucleolus, 25, 65, 150, 154, 181 and chromosomes, 209, 210 Nucleoplasm, 25 Nucleoplasmic ratio, 62 Nucleo-proteins, 40 Nucleus, division, 143^". discovery, 7 distributed nucleus, 59, 68 early views on division, 10, 143 evolution of, 206 in fertilization, 277 functions, 69 in heredity, 16, 324 ff. kinetic nucleus, 84 nuclear membrane, ti.'!. 1 19, 154 in protista, tit; relation to metabolism, *'»'.• sphere of influence, 62 si ructure, 63, 150 trophic nucleus, s l Nucleus^ vesicular, 69 Numerical reduction, 229 2 19 Nussbaum, t02 \ ympkcea water lilj . l 15, 116 t > Octads, 249 (Edamatin, 6 1 (Edogonium (green alga . blepharopla 85 fertilization, 1 1, 288 reduction, 222 (Elkers. 222 GEnothera, cytology of mutants, ill jF. fragmentation of chromosomes, I synapsis in hybrids, 252 Ogata, 65 Oil, 110, 111, 122, 134, I'M body, 110, 134 droplet, division of. 189 vacuole, 48, 111, 134 Olive, 59, 108, 202. 204, 208, 222 Oliver, 17 Oniscus (crustacean), 255 Onoclea (fern), 288, 356, 372, 375 Ontogenesis, 323, 324, 398, L01, L07, 111 ( >6apogamy, 315 Oocytes or ovocytes, 220, 233, 274, 275 Oogenesis, 221, 271. 275 Oogonia or ovogonia, 221 Oogonium (of plants), 179, 222. 223, 288 Oomycetes, 289 Open spireme, 238 Ophryotrochd (annelid worm . 237, 211 Orchis (orchid). 104 Organic acids, 135 Organism as a whole. 12. 52, 1 M), 11 1 Orman, 118 Orthogenic i- . in 1. U0 Orthoptera. 386, 393 Oryza (rice), 256 OsciUatoria (blue-green alga . 202, 204 ( temosis in mitosis, Is I Osmunda i fern . apical cell, 27 reduction, 234, 236, 239, 240 -c\-(lcterininal ion, 373 ( teterhout, 70, 248 Otidea (fungus), Bl, 21 ( >\ -arian egg, 221, 27 1 Overton, E . 13, W, ill. 220 Overton, J. IV. parthenogenesis, 314, 315 prochromosomes, 159 444 INDEX Overton, reduction, 223, 236, 248, 249 somatic pairing, 256, 257 Ovum, 221, 274, 275, 276 activation of, 284 ff. organization of, 328 ff. Oxidation, and chondriosomes, 123, 124 in egg, 285, 287 and nucleus, 70 and sex-determination, 368 Oxychromatin, 64, 66, 70 Pace, 300, 315 Pachynema, 231, 232, 233 Pachytene, 231, 233, 258 Padina (brown alga), 223 Palladin, 135 Pallavicinia (liverwort), 122, 220, 248 Pangenesis hypothesis, 398, 399 Pahgenosomes, 258 Pangens, 258, 399 Parallelism of chromosomes and characters, 342-343 Paramitome, 33 Paramecium, 30, 60, 207, 283 Paramylum, 108, 112 Parasynapsis, 228, 231, 233, 234, 235, 243, 248, 250, 251, 257 Parasyndese, 228 Paratettix (grasshopper), 391 Parechinus (sea urchin), 326 Parental chromosome sets, 254, 279, 340 Paris (angiosperm), 301 Parmenter, 160, 163, 319 Parthenogenesis, 314, 317, 364 artificial, 284 ff. Passifiora (angiosperm), 181 Patterson, 213 Paulmier, 95, 244, 245 Payen, 11, 193 Payne, 119, 359, 360 Pea, Mendelism, 336, 337 linkage, 378, 384 Pearl, 138 Pearson, 52 Pectates, 194 Pectose, 194 Pediastrum (green alga), 140, 331, 334, 411 Pedicellina (flatworm), 255 Pelargonium (angiosperm), 104, 332 Pellia (liverwort), 82, 87, 88 Pellicle, 45 Pensa, 117, 122 Pentose and pentosan, 41 Peperomia (angiosperm), 299 Peptone, 40 Peranema (flagellate), 83 Percnosome, 89 Perforatorium, 273, 275, 277 Perikaryoplasm, 177 Perinium, 195 Peripatus (arthropod), 281 Periplaneta (cockroach), 239 Periplasm, 289 Periplast, 44 Perispore, 195, 196 Perivitelline space, 277 Perla (stone-fly), 239 Permeability, 37, 43, 287 Peronospora (fungus), 290 Peziza (fungus), 80, 81, 223, 291 Pfeffer, aleurone grain, 134 amitosis, 211 cell wall, 193 ectoplast, 42 oil body, 110 protoplasmic connections, 47 Pfitzner, 33, 155 Pfliiger, 328 Phaeophycese, 108, 222 Phajus (orchid), 106 Phascum (moss), 317 Phases in colloids, 35 of mitosis, 145 Phenotype, 339 Phillips, 202 Phosphatid, 117 Phospholipin, 117 Phosphoric acid, 40 Photosynthesis, 105, 107, 108 Phragmatobia (moth), 360, 361, 363 Phragmidium (fungus), 291, 312 Phragmites (angiosperm), 346 Phragmoplast, 176 Phrynotettix (grasshopper), 156, 158, 162, 233, 237, 243, 258, 392 Phycocyanin, 104 Phycoerythrin, 104 Phycomyces (fungus), 110, 186, 187, 355, 373 Phycomycetes, 289 Phyllactinia (fungus), 80, 81, 290, 291 Phyllocladus (conifer), 295 Phylloglossum (pteridophyte), 91 PhyUopneuste (bird), 274 /\ l>i:\ H" Phylloxera (aphid), -'Us. 357, 364 Phy8a (snail), 276 Physiological units, 18, 398 Physiology of fertilization. 284 ff. of sex, 369 #. Physostegia (angiosperm), 176, 225 Phi/teh phus (angiosperm >, 46 Picard, 147. 230 Pictet, .",i Pieris (butterfly), 61, 21 1 Pig, chromosomes, 165, 301 Pigcera (butterfly). ,_>.7_* Pigeons, sex-determination, 369, 376 Pigments. 104. 105, 122. 13."). 136 Pilobolus (fungus), 187 Pinguiada (angiosperm), 158 Pinney, 159 Pinnotheres (crustacean), 274 Pin us (pine), cell plate, 176 rhondriosomes, 121, 122 fertilization, 294, 295 protoplasmic connections, 46 reduction, 239 Pisciola (leech), 178 Pisum (pea), 61, 121, 122, 249, 256, 34* linkage in, 378, 384 Pits. 28, 191, 192 Pin a aria (flat worm), 139, 237 Plantago (angiosperm), 372 Plasma membrane, 25, 42 Plasmahaut. 42 Plasmatic microsomes, 205, 200 Plasmodermal blepharoplast, 95, 97 Plasmodesmen, 46 Plasmodium (protozoan), 317 Plasmogamy, 292 Plasmosome, 25, 65 Plastid, 26, 103 ff. individuality of, 113 inheritance, 331-334 primordia, 115, 122, 124. 332, 333 Plastidome, 122 Plastidule, L8 Plastochondria, 1 18, 121 Plough, 395 Plumbaqella (angiosperm), 225 Podocarpu8 (conifer), 213 Podophyllum (angiosperm ». 1 .">'.», 231 1 Point mutation, 351 Polar bodies, 15, 221, 275, 276 in parthenogenesis, 318, 319, 364 Polar nuclei, 299, 300, 301 Polarity, L38jf. 329, 334 PoHanthet (angiosperm , 110 Polioplasm, 12 Politis, 110 Pollen grain uall. r.ti; tube, 294, 298, 299 Polygon* lla (angiospei m . 25 Polypodium fern . 236 Polysiphonia (red alga . centrosph 79, 80 fertilization. 289 reduction, 223, 236 PolysHchum (fern I, 316, :;i7 Polystigma (fungus), 291 Polytoma (green alga), 83, 84 Polytomella (green alga . Polytrichum (mo- . 88, 89, 221 Popoff, 120, 259 Porella (liverwort), 87 Postreduction, 245, 248 Potato starch. 107 Pratt and Long. 237 Preformation theory. :; Preissia (liverwort), 82, 293 Prenant, 184, 185 Prereduction, 245, 2 1s Prevost and Dumas, 14, 15 Primaniypus, 245 Primula (primrose), 239. 248, 346, 391 Pringsheim, 14, 311 Prionidus (bug), 359, 360 Pritchard, 372 Prochromosomes, 158, 159 Progamic sex-determination. 357 Promitosis, Jus Promorphology of ovum, 328 jf. Pronucleus, 275, 276, 277 Prophase, somatic. 144, 11."). 150 153 heterotypic, 231 ff. Protandry, 371 Protein, 39, 40. .".1. 134 crystals, 135 Protenor (bug . 360 Prothallial auclei, 294 Protokaryon, 68 Protoplasm, chemical uatui r. 50 colloidal nature. .", I ff.t ."» 1 doctrine, 1 I early obeerval ions, 6, 32 ff. physical properties, 32 senescence, 136 structural theori< substratum of life, 32, Is .//". 446 INDEX Protoplasm, a system, 32, 49, 52 varieties of, 41 Protoplasmic connections, 46 Protoplasmic! , 41 Protoplast, 12, 24 Protoxylem, 192 Protozoa, ectoplast, 44 mitosis, 207 ff. nucleus 68 reduction 222 Pseudapogamy, 312 Pseudapospory, 315 Pseudoamitosis, 211 Pseudogamy, 292 Pseudomitosis, 204 Pseudopodium, 45 Psilotum (pteridophyte), 239 Pteridophytes, fertilization, 292, 293 sex-determination, 356 spermatogenesis, 89 ff. Pteris (fern), 248, 312, 317 Puccinia (rust fungus), 312 Punnett, 395 Purkinje, 11 Pustidaria (fungus), 116 Pyrenoid, 104, 108 Pyronema (fungus), 81, 224, 290, 291 Pyrrochoris (bug), 244 Pythium (fungus), 290 Q Quadrille of centers, 280 Quadripolar division, 325 Quadrivalent chromosomes, 223 Qualitative division of chromosome, 227, 229, 297, 401 Quantitative theory of. sex, 370 ff. R Rabbit, 212, 237 Rabl, 157, 181, 279 Raciborski, 110 Ramlow, 291 Random assortment of chromosome pairs, 255 Ranunculacese, 315 Raphides, 134, 135 Rat, 391 vom Rath, 211, 227, 244, 245 Rauber, 326, 402 Rawitz, 77 Reboulia (liverwort), 293 Recessive, 338, 339, 351 Recombination of factors, 343, 344 Reducing action of cytoplasm, 123 Reduction of chromosomes, in animals, 220 ff. with chromosome tetrads, 243 ff. . denned, 229 discovery, 219; 220 modes of, 230 ff. numerical, 229 in plant groups, 222-225 in somatic cells? 259 Weismann's theory of, 226 without qualitative change, 249 Reduplication hypothesis, 395 Reed, 65, 70 Refractive body, 118, 274 Regaud, 115, 117 Regeneration, 69, 137, 406 Regional mutation, 351 Reichert, 42 Reinke, 64 and Rodewald, 39, 41 Rejuvenescence, 137 Remak, 10, 43 Reserve starch, 106, 107 Resin, 194 Respiration, 123, 124 Resting stage of nucleus, 144, 150, 151, 157 Reticular theory of protoplasm, 33, 49 Reticulum of nucleus, 25, 64, 144, 150, 157 Rhabdites (nematode worm), 189 Rheum (angiosperm), 134 Rhizina (fungus), 291 Rhizonema, 83, 84 Rhizoplast, 83 Rhizopus (mold fungus), 116, 186, 187, 290 Rhumbler, 183 Ribes (gooseberry), 236 Riccardia (liverwort), 82, 236 Riccia (liverwort), 87, 293 Richardia (angiosperm), 159, 248, 249 Richards, 158, 159, 213 Ricinus (castor bean), 134 Riddle, 369, 375 Ritter, 411 Rivett, 111 Robertson, A., 295 Robertson, T. B., 189 INDEX h; Robertson, \\ . R. B., 160, L65, 237, 245, 249, 257, 351, 393, 394 Rosa (rose . 3 Is Rosaces, 315, 3 Is Elosen, 65 Rosenberg, L60, 1 « > 1 apogamy, 3 i~>. 316 chromosomes in Crepis, 3 19 reduction, 236, 251, 252 Rosenvinge, 16 Rotifers, 318, 364, 368, 370 Roux, 154, 156, 328, 384, 392 Roux-Weismann theory, 225, 391 Rubber, 135 Ruckert, 159, 227. 244. 245, 27'.' Rudolph, 123 Rudolphi, 5 Ruhland, 43 Kussow, 46 Rusts, 60, 224, 292 Ruzicka, 07 Rytz, 187 S Sabaschnikoff, 2 Js Sachs, 12, 49, 62 Safir, 382 Sagitta, 256 Saguchi, 96, 212 Sakamura, chromosomes and specie-. 348 deranged mitosis, 212, 214, 259 extruded chromatin, 136 false tetrads, 248, 249, 259 reduction, 236, 254 somatic mitosis, 160, 162, 164, L65 Salamandra (salamander), achromatic figure. 178, IM attraction sphere, 77 chromosomes, 150 reduction, 237 Salix iwillow i. 372 Salter, 107 Salts, 135 Sands, 81 Sap in cell, 135 Sapehin, 88, 114. 123 Saprolegnia fungus . 187, 2 Sarcode, 1 1 Sargant, 211 Sax. 298 -300 Schacht, 13 Schacke, 365 Schaeflfer, 12 - banner, 87, 239, 372 Schaudinn, 67, 207, 282, 317 Schaxel, 1 16 Scheben, 27 1 Schellenberg, 2 17 Scherrer, L05, 113, lis. I Schikorra, 291 Schiller. 213 Schilling, 1 12 Schimper, 12. 107, 108, 1 13 Schleicher, 1 13 Schleiden, 7. 8, 13 Schleip, 237, 257 Schmitz, 14, ids Schneider, A., 33, 143 Schneider, II.. 236, 257 Schntt lander. S2, 86, (Ml Schreiner, 231, 235, 237, 257, 258 Schuberg, 96 Schultze, M.. 11 Schultze, W. H., 70 Schulz, 34'.) SchiirhorY, 66, 213 Schwann, 8, 9, 1 5 Schwarz, 6 1 Schweigger-Seidel, 1 I Scolojn ndra (centipede . 255 Scolopemlrium (fern . 315, -!17 Scopolia (angiosperm . 61 Scorpion, 119 Scott, 202 Sea urchin, artiticial parthenogenesis 284, 287 blastOmeres, 328 hybrids, 325 -127 >rr>,u<\ contraction, 232, 233, 238, 2 Secondary thickening of cell wall. 191 Secretions, 28, 30, I Secretory cells, 30 3 gmentation, of chromoson in fertilization, 296 of egg, 188 Of spireme. 1 15, 239 Segregation, of chromosomi Mendelian, 5 108, U Bomal ic, 352, 395 Seifrii . 38, 39, 13 Seiler, 360, 361, 363 Selachians, 237 Selaginella, plastids, 103, 104, 105, 1 1 ; 122 448 INDEX Selaginella, spore coat, 194, 195 Senescence, 63, 136 ff. Sequoia (conifer), 294, 295 Sex, chromosomes, 358^"., 380 ff. determination of, 354 ff., 373 ff. intergrades, 370 ff. linkage, 380 ff. Mendelian interpretation of, 366 practical control of, 376 quantitative theory of, 370 ratio, alteration of, 367 ff. reversal, 370 ff. Sex-limited characters, 381 Sex-linked characters, 381 Sexual reproduction and rejuvenescence, 137 Shaffer, 118, 120, 121 Sharp, 64, 87, 90, 92, 147 ff., 160, 176, 225, 242, 254 Shaw, 91, 316 Shull, A. F., and Ladoff, 368 Shull, G. H., 382 Sida (crustacean), 319 Sieve tube, 28 Silica, 135, 194 Silk gland, 66 Simocephalus (crustacean), 371 Simplex chromosome group, 340 de Sinety, 358 Siphonese, 59 Slime globules, 203 de Smet, 150, 159, 160 Smilacina (angiosperm), 236, 239 Smith, B. G., 159, 160, 278, 279 Smith, G. M., 108 Smith, H. L., 79 Sokolow, 119 Sol, 35 Somatic mitosis, 143 ff. comparison with heterotypic, 228 pairing of chromosomes, 256 segregation, 352, 395 Sordaria (fungus), 81 Sorodiscus (slime mold), 208, 209 Spallanzani, 14 Species, origin of, 347 Specificity of chromosomes, 162, 250 Spek, 189 Spencer, 48, 49, 398 Spermatia, 312 Spermatid, 87, 220 Spermatocyte, 220, 235 Spermatogenesis, in animals, 95, 96, 220 Spermatogenesis, chondriosomes in, 119, 120 in man, 361, 362 in plants, 86 ff. Spermatogonia, 220 Spermatozoids, 86 jf., 288 ff., 293, 294 Spermatozoon, discovery, 14 in fertilization, 277 ff. in heredity, 324, 328 structure, 273, 275 types, 274 Sphacelaria (brown alga), 79, 186 Sphaerechinus (sea urchin), 325-327 Sphcerocarpos (liverwort), 355, 364, 365, 372, 375 Sphceroplea (green alga), 222 Sphcerotheca (fungus), 290 Sphserraphides, 135 Sphagnum (moss), 248 Spherome, 122 Spinacia (angiosperm), 248, 256 Spinax (fish), 237 Spindle, 145, 147, 154, 175 Spiral stage of chromosome, 150, 152, 155 wall thickening, 28, 192 Spireme, 145, 150, 153, 154, 232, 238 in fertilization, 300, 301 in heterotypic prophase, 241-243 Spirogyra (green alga), amitosis, 211 conjugation, 288 cytokinesis, 186 enucleated cells, 69 mitosis, 209 nuclear fusion, 14, 273 plastid, 104 pyrenoid, 104, 108 reduction, 222 Spirostomum (protozoan), 61, 62 Spitzer, 70 Splitting of chromosomes, in first em- bryonal division, 295, 300; in matu- ration, 236, 239, 242, 258; in somatic mitosis, 153, 242, 392 Spoehr, 41 Spore membrane, 195, 196 tetrads, 223-225, 355 walls, 194 Sporocyte, 225, 232 Sporodinia (mold fungus), 110, 187, 290 Sporogenesis, in various plant groups, 222 ff. behavior of plastids in, 114 without reduction, 315, 316 INDEX 149 Sporogenesis, and Bex-determination, :;.").") 356 Sporophytio budding, 312, 315 Sprengel, ( '. K., L3 Sprengel, K., 5 Stains, 117 Stangeria (cycad I, 294, 298 Staphylea (angiosperm), 239 St a nli. 106, 107, 112, 134 Starfish, 286, 287 Steil, 28S, 313, 314 Stein, 61 Stemonitis (slime mold), 85 Stentor (protozoan), 60, 62, 69 Stevens, F. L., 289, 290 Stevens, N. M., 256, 358, 360, 361 Stomps, 155, 248, 256, 345-347 Storage, 66, 106, 133 Stout, 159, 372 Strasburger, amitosis, 213 apogamy and apospory, 311, 315, 316 blepharoplast, 85 cell-division, 143 cell wall, 190, 193 centrosphere, 76, 79, 80 chromomeres, 155, 156, 258 chromosome number, 163, 164 chromosomes (somatic), 149, 158 cytokinesis, 186, 190 ectoplast 43 eyespot, 111, 112 fertilization, 300 mitosis in myxomycetes, 208 nuclear fusion, 14, 273 nuclei by division only, 10 nucleolus, 66 nucleoplasmic ratio, 62 nucleus in heredity, 16, 324, 327 of Marsih'd. 210 protoplasm, 34 protoplasmic connections, 47 reduction, 220. 222. 236 Bex-determination, 355, 356, 364 somatic pairing, 256 spindle, L80, 181 spore wall, 19 1 starch, 106 tct raploidy, 345 Siratiotes (angiosperm), 212 Streaming in cell-division, ls;!. 189 Strepsinema, 231, 233, 238 Strepsitene, 231, 233 29 Stroma, 105 starch, 109 Strong, 376 Strongyloa ntroi ■ i urchin . 287, 327 Structure and function, 37 I Studnicka, 96 Sturgeon, 274 Sturtevanl . 371 . 376, 389 Styela (ascidian . 329 Stypocavlon (brown al ii-.i . 79 Suberin, 194 Sugars, 106, 135. 136 Supernumerary chromosomt >fi jj. Surface tension in cytokinesis, lss Surirella (diatom), 7'.) Suspensoid, 35 Sutton, 158, 160. 228, 245, 251, 252, - 255 Swarczewsky, 2s 1 Swarm spores and colony, 1 lo. 331, 334, 111 Swingle. I). B., L86, 187 Swingle. W. T., 79, 186 Sykes, 256 Symmetry, 139, 329, 334 Synapsis. 227. 233, 251 ffi. and degeneration, 259 nature of union, 256, 384, 394 Synaptene, 231, 233 Synaptic mates, relationship, 251 251 Synchytrium (fungus), 62, ls7 Syndiploid nuclei. 259, 260 Synergids. 299 Syngamic sex-determination, 357 Syngamy, 222 Synizesis, 231, 233, 237, 238, 255 in somat ic cells? 259 Synkaryon, 27s. 282, 292 Synochocystis (blue-green alga . 205 T T&ckholm, 248, 348 Tahara, 256 Tamus (angiosperm I, 17 Tangl, 16, 17 Tannin, P'l Tapetal cells, 59, 210 Plasmodium, 194, 196 Taraxacum (dandelion . 3 15 Tassemenl polaire, 149 Taxodium (conifer . 295 450 INDEX Taxus (conifer), 122, 295 Taylor, M., 256 Taylor, W. R., 176 Teleosts, 160, 237 Teliospore or teleutospore, 292 Telophase, somatic, 144, 145, 148 Telophasic splitting? 149, 240, 242 Telosynapsis, 228, 239, 244, 245, 248 Tennent, 160 Teosinte, 349 Terletzki, 46 Terni, 118 Tertiary thickening of wall, 191 Tetrads, of cells, 220/. of chromosomes, 230, 234, 239, 243 Jf.; in plants, 248, 249 Tetraploidy, 317, 345 Tetraspora (green alga), 109 Tetraspores, 223 Tettigidse, 243 Thalidrum (angiosperm), 159, 236, 248, 315 Thelygonium (angiosperm), 236 Theophrastus. 1 Thorn, 91 Thompson, 52, 333 Thomson, 369, 404 Thuja (conifer, arbor vitse), 239, 294 Thuret, 14 Thysanozoon (flat worm), 237 Timberlake, 85, 105, 109, 187, 190 Tischler, chondriosomes, 118 gigantism, 346 prochromosomes, 159 reduction, 236 spore coat, 196 Tissue cultures, 138, 146 Toad eggs, 368, 370 Tolypothrix (blue-green alga), 203 Tomopteris (annelid worm), 235 Tonoplast, 25, 47, 111 Torreya (conifer), 295 Torus, 191, 192 Townsend, 69 Toxopneustes (sea urchin), 160 Tracheid, 28, 191, 192, 345 Trachelomonas (protozoan), 112 Tradescantia (angiosperm), protoplasm, 6, 25, 33, 37 reduction, 236, 239 somatic mitosis, 147, 148, 150, 152, 242 Tragopogon (angiosperm), 239 Transverse division of chromosome, 227 Tretjakoff, 248 Treub, 190 Treviranus, 5, 6 Trichites, 107 Trichocyst, 30, 45 Trichogyne, 289 Tricyrtis (angiosperm), 248 Trillium (angiosperm), 149, 155, 300, 301 T rimer otropis (grasshopper), 255 Triple fusion in angiosperms, 299, 301 Triploidy, 347 Triticum (wheat), chromosomes and species, 348 fertilization, 299 synapsis in hybrids, 252, 253 Triton (salamander), 274 Trollius (angiosperm), 236 Trondle, 109, 222 Trophochromatin, 208, 209 Trophoplasm, 34, 181, 400 Trow, 290, 395, 396 Trypanosoma (flagellate), 45, 84 Tschernoyarow, 160, 161, 253 Tschirch/ 107, 134 Tsuga (conifer, spruce), 295 Tulasne, 13 Tulipa (angiosperm), 298 Tumors, 163 Tunicata, 237 Tupper and Bartlett, 345 Turbellarian egg, 329 Twins, 357 Twiss, 118 Tyloses, 193 Tyndall effect, 35 U Ulothrix (green alga), 104, 222, 288, 331 Undulating membrane, 45, 274 Unger, 9 Unio (mollusk), 280 Unna, 34 Uredineje, 60, 224, 292 Uromyces (rust fungus), 312 Vacuole, 25, 47, 60, 111, 135 contractile, 30, 44, 46, 84 Vacuome, 122 INDEX 451 Vandendries, 224 Vanessa (butterfly), 61 Van Hook, 82 Vanilla (angiosperm I, !<)*.» Varieties, origin of, 3-17 Vaucheria (green alga), 60, 85 Vegetative apogamy, 314, 315 nnil at ion, 351 Vejdowsky, 67, 250, 257 Velten, 33 Vermiform nuclei, 298, 299 Verworn, 42, 50, 326 Vesper ago (bat), 274 V tela (horse bean), chromosome comple- ment, 160, 161 deranged mitosis, 211, 212 reduction, 235, 236, 238, 254 somatic mitosis, 147, 153, 242 Vines, 311/. Virchow, 10, 15 Viscosity of protoplasm, 39, 62, 189 Viscum (mistletoe), 47 Vital units, 48, 398 ff. Vitalism, 49, 52, 409, 412 Vitelline membrane, 286 Vo'inov, 118, 119 Volutin, 41 Volvocaceae (green algae), 112 von Voss, 257 de Vries, intracellular pangenesis, 399 (Enothera mutants, 344, 347, 349 pangens, 49, 258, 399 tonoplast, 47 W W-chromosome, 363 Wager, 13, 108, 112, 290 Wakker, 109, 110 Waldeyer, 144, 326 Walker, 224 Wall of roll, 26, 28, 190 jf. Walton, 248, 279, 405 Warburg, 285, 287 Wasielewski, 211, 212, 213 Wassilief, 118 Watase, 181 Water in protoplasm, 37 Webber; 83, 92, 93, 288, 294, 302 Weinstein, 389 Weinzieher, 236 Weismann, 16, 49, 225 parthenogenesis, 31 8 Weismann, reduction, 226 theories of heredity ami develop- ment, 391, loo ]T. Wells, H» Welsford, 290, 298, 299, 312, 325 Weniger, 298 :;<)1 Wenrich, 156. 158, 162, 233, 237, 243, J.-.7. lios. 392, West and Lechmere, 136 Wheeler, 279 Wheldale, 135 Wherry, 70 White, 384 Whitman, 369 Whitney, 357, 36s. 370 Wieman, 361, 362 Wiesner, 48, 109, 193 Wildman, lis. 120. 121. 281 Wille, 46 Williams, C. L., 1M Williams, J. L., 80. s2. 223 Wilson, E. B., abnormal eleavage, 328 achromatic figure, 181-1 v"» chemical nature of gene, 112. 114 chondriosomes, 119 chromatin and cytoplasm, 69 chromosomes and heredity, 396 397 conjugation in Paramcecium, 284 crossing over, 393, 394 genetic continuity, 323 hyaloplasm sphere, 76 individuality of chromosome, 157 protoplasm, 34, 49 sex-chromosome-. :;."i^ ff. somatic chromosomes, 152 spermatozoon, 275 Wilson, H. V., 1 38 Wilson, M., 88, 89 Wilson and Morgan, 386, 393 Winge, 208, 209 von Winiwarter, centrosomes, 77 chondriosomes, 123 chromosomes in man. 362 reduction, 228, 231, 237 von Winiwarter and Sainmont, 257 Winkler, 62, Ml I win Wisselingh, 210 Wodsedalek, 361 Wdhler, 50 Wolfe, 80, 223 WolflF, i. 5 Wollenweber, ill. 112 Woodburn, 2 452 INDEX Woolery, 239 Wort man, 56 Woyciki, 295 Wuist, 372 X X-chromosome, 359 jf., 380 ff. Xanthophyll, 104, 105 Xenia, 302 Xylaria (fungus), 291 Xyris (grass), 236 Y-chromosome, 359 ff., 380 #., 390 Y.i manouchi, achromatic figure, 176, 179 apogamy, 314 blepharoplast, 91 centrosomes, 79, 80, 82 eyespot, 111 fertilization, 289, 293 pyrenoid, 109 reduction, 222, 223, 232, 236 Yampolsky, 372 Yeasts, 206, 210, 292 Yolk, 134 Young, 213 Yucca (angiosperm), 256 Z-chromosome, 363 Zacharias, 65, 193, 202, 404 Zade, 349 Zamia (cycad), 93, 288, 294 Zanardinia (brown alga), 111, 222 Zea (maize), 65, 122 aleurone inheritance, 333 chlorophyll inheritance, 332 chromosomes, 349 crossing over, 391 linkage groups, 383 Zentralkorner, 203 Zettnow, 67 Zigzag stage of chromosomes, 151, 152, 153 Zimmermann, 110, 193 Zoja, 164 Zoogonus (worm), 237, 244, 245 Zoospores, 85, 86, 112, 137, 222 Zukal, 202 Zweiger, 239 Zygnema (green alga), fertilization, 113 mitosis, 210 plastid, 103, 106, 108, 109 pyrenoid, 109 reduction, 222 Zygogenetic eggs, 318, 319 Zygomycetes, 290 Zygonema, 231, 233 Zygospore, 113, 222 Zygote, 222, 223 Zygotene, 231, 233 Zymogen, 66 rHOffiMT LIBRARY ft C. State frUe* utfufln !:fei;j;ih;i mm m\ '■'• '.l ,'$W IrinMTI