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MARINE BIOLOGICAL LABORATORY.
n
Received
Accession No. cpC^.y ^ 2^
Given by //'ax,^L<^<*<^^i'*^^ i>(-<-^^ <^)y^^
Place, ^.X^^ d.J^,
%*No book OP pamphler is to be removed ffom the Uab- opatopy ixiithout the pepmission of the Trustees.
Committee on Publication
Barton W. Evermann Chairman and Editor C. Hart Merriam Bernard R. Green
Frank Baker C. F. Marvin
PROCEEDINGS
OF THE
Washington Academy of Sciences
Vol. VI
1904
WASHINGTON October, 1904-FEBRUARY, 1905
AFFILIATED SOCIETIES.
Anthropological Society of Washington. Biological Society of Washington. Botanical Society of Washington. Chemical Society of Washington. Columbia Historical Society. Entomological Society of Washington. Geological Society of Washington. Medical Society of the District of Columbia. National Geographic Society. Philosophical Society of Washington. Society of American Foresters.
Washington Society of the Arch^ological Institute of America.
PHf 83 OF
The N«w Eh* Printinq Company
LANCASTER, Pa.
CONTENTS.
PAGE
Contributions to the Knowledge of the Life History of Pinus with special reference to Sporogenesis, the Development of the Gametophytes and Fertilization; by Margaret C. Ferguson . i
Studies of Variation in Insects; by Vernon L. Kellogg and Ruby G. Bell ^°3
Hopkins-Stanford Galapagos Expedition.-XVH. Shore Fishes of the Revillagigedo, CHpperton, Cocos and Galapagos Islands; by Robert Evans Snodgrass and Edmund Heller . • -333
Some interesting Beaver Dams in Colorado; by Edward R. Warren ^^^
Organization and Membership of the Washington Academy of Sciences ^^^
Index '^^^
ILLUSTRATIONS
PLATES
FACING PAGE
I-I V. Microsporogenesis 156
V. Development of Pollen-grain 164
VI. Pollination and subsequent Phenomena 166
VII. Growth of the Pollen-tube 168
VIII-XI. Spermatogenesis 170
XII-XIII. Macrosporogenesis 178
XIV. Germination of Macrospore 182
XV. Female Prothallium 1S4
XVI-XVIII. Oogenesis 186
XIX-XXI. Fertilization 192
XXII-XXIII. Development of Proembryo 198
XXIV. Abnormalities 202
XXV. Map of Slate River, Colorado 438
XXVI. Map of Slate River, Colorado 438
XXVII-XXXIV. Thirteen photographs show^ing beaver dams,
houses, etc., in Slate River Valley, Colorado.. 438
TEXT FIGURES
PAGE
I. Fore and hind wings of honey bee (drone) 214
2-5. Fore wing of honeybee (drone) 215
6-7. Hind wing of honey bee (drone) 216
8. Hind wings of honey bee (drone) 217
9. Fore and hind wings of honey bee (worker) 217
10. Fore wings of honey bee (drones) 219
11. Fore wing of honey bee (drone) 222
1 2-20. Frequency polygons of variation in wings of honey
bees 223-330
21. Part of costal margin of hind wing of honey bee
much magnified, to show hooks 231
22-33. Frequency polygons of variation in costal hooks of
wings of honeybees 232-239
34. Fore and hind wings of male black ant 245
35-44. Frequency polygons of variation in costal hooks of
wings of black ants 246-252
45. Wing of ant showing venation 255
46-48. Diagrams showing variation in elytral and pro- thoracic pattern of the convergent lady-bird 258-272
49. Frequency polygon of variation in prothoracic pat- tern in the convergent lady-bird 273
50. Diagram showing variations in elytral pattern of
the flower beetle 274
51-53. Frequency polygons of variation in elytral pattern
of the flower beetle 275-279
54; 55. Diagrams showing variation in the yellow jacket.. 284; 285
56. Leaf hopper, Typhlocyba comes 288
57. Diagram showing variation in pattern of prothorax
of a flower-bug 291
58. Water boatman, Corisa sp 293
59. Parnassian butterfly, Parnassius smintheus 295
60. A\WQ.x\z?ci\ coc\i.xo?Lz\i^ Periplaneta americana 296
61. Red-legged locust, Melanoplus femur-rubruin... 301 62-69. Frequency polygons of variation in the red-legged
locust 302-305
70. Seventeen-year locust. Cicada septendeclm 306
71-78. Frequency polygons of variation in Cicada sep-
tendecitn 307-3 10
79. Antenna of scale insect, Ceroputo yuccce 311
80. Biting bird-louse, Z.z^^Mr?^5 cc/er 314
81. Fredaceous ground-beetle, Pterostichus sp 317
WASHINGTON ACADEMY OF SCIENCES
OFFICERS FOR 1904
President
Charles D. Walcott
Vice-Presiden ts
Fro7ti the Anthropological Society W. H. Holmes
Archceological Society John W. Foster
Biological Society Barton W. E vermann
Botanical Society Frederick V. Coville
Chemical Society C. E. Munroe
Columbia Historical Society W J McGee
Err '^-^olos^ical Society H. G. Dyar
GeoL ciety G. K. Gilbert
Medicu iety W. W. Johnston
National eographic Society A. Graham Bell
Philosophical Society Richard Rathbun
Society of American Foresters Gifford Pinchot
Secretary Treasurer
Frank Baker Bernard R. Green
Managers
Class o/ igoj Class of i gob Class of igoj
L. O. Howard P. W. Clarke Geo. M. Kober
O. H. Tittmann C. W. Hayes Gifford Pinchot
»
Carroll D. Wright G. W. Littlehales F. A. Lucas
PROCEEDINQS
OF THE
WASHINGTON ACADEMY OF SCIENCES
Vol. VI, pp. I-203. Sept. io, 1904.
CONTRIBUTIONS TO THE KNOWLEDGE OF THE
LIFE HISTORY OF PINUS WITH SPECIAL
REFERENCE TO SPOROGENESIS, THE
DEVELOPMENT OF THE GAMETO-
PHYTES AND FERTILIZATION.
By Margaret C. Ferguson, Ph.D., Associate Professor of Botany, Wellesley Cot lege.
Plates I-XXIV. v^
CONTENTS.
Introduction.
Purpose of the study 3
Historical notes 5
Methods.
Collecting 12
Fixing 13
Staining. 15
Chapter I. Microsporogenesis.
The microsporangium.
The wall of the pollen- sac '. 17
The primitive archesporium 18
Tetrad-division.
The definitive archesporium 20
The first nuclear division of the microspore-mother-cell 21
The second mitosis of the mother-cell 30
The problem of reduction 31
Development of the microspore.
The formation of the spore-wall 34
The origin of the air-sacs and liljeration of the microspore .... 36
The growth of the microspore 37
Summary 39
Proc. Wash. Acad. Sci., September, 1904. (i)
2 MARGARET C. FERGUSON
Chapter II. The male gametophyte. The development of the pollen-grain.
The formation of the prothallial cells 41
The mature pollen-grain 44
Pollination.
The ovule at the time of pollination 45
The pollen-chamber 4^
Development of the pollen-tube. The first period of growth.
Germination of the pollen-grain 47
The division of the antheridial cell 4S
The winter condition 50
The second period of growth.
Renewed activities in the macrosporangium 50
Renewed activities in the male gametophyte 51
Division of the generative nucleus 53
Growth of the sperm-nuclei 62
Elongation of the pollen-tube 64
Summary 66
Chapter III. Macrosporogenesis.
The female cone.
The macrosporangium 7°
Formation of the axial row.
The macrospore-mother-cell 7'
The first division of the mother-cell 7-
The second division of the mother-cell 74
Significance of the tetrad-division within the ovule 76
Later history of the axial row.
The fate of the upper cells 79
The growth of the macrospore 80
Summary 81
Chapter IV. The female gametophyte.
Development of the prothallium.
The first period of growth 82
The second period of growth 84
The so-called " spongy tissue."
The first period of growtli 86
The second period of growth 88
The nature and functiftn of this tissue 89
Development of the archegonium.
The early growth of the archegonium 90
The division of the central cell 95
History of the ventral canal-coll 97
Matination of the egg.
The descent and growth of the egg-nucleus 99
Tlie " proteiil vacuoles" 103
Tlie receptive vacuole 109
Suinmarv 110
LIFE HISTORY OF PINUS 3
Chapter V. Fertilization and related piienonicna. Conjugation.
The coming together of the gametoplijtes 113
The union of the sexual nuclei 114
The first division following fecundation.
The prophases of the division 115
Later stages in the mitosis 118
The pro-embryo.
Division of the two segmentation-nuclei 122
The four segmentation-nuclei 124
The development of cell-walls 126
Later mitoses in the formation of the pro-embrvo 127
The fate within the egg of the smaller sperm-nucleus, the stalk- cell, and the tube-nucleus 128
Summary 130
Appendix. Some abnormal conditions.
Supernumerarj' nuclei in the male gametophjte 133
Unusual conditions in the female gametophjte 135
A peculiar method of conjugation 138
Note 139
List of Papers cited 142
Explanation of Plates 154
INTRODUCTION.
There is no chapter in the annals of botanical science more fascinating than that which deals with the history of sexuality in plants. No definite date marks the discovery of the fact that plants, like animals, are male and female ; the idea was rather a growth, as is plainly shown by the writings of Aristotle, Theophrastus, Pliny and others of the early philoso- phers. The fact may, however, be said to have been estab- lished by Camerarius (1694) in his " De sexu Plantarum," but for many years after his time botanists found in this question merely a favorite subject for philosophical speculation. Their ideas remained vague and uncertain, no effort being made to confirm their theories either by observation or experimentation.
It was not until near the middle of the last century that actual investigations were begun along this line. Amici (1830-1846) made certain interesting observations regarding the develop- ment of the pollen-tube and the origin of the embryo in several plants ; but the splendid series of embryological papers pub- lished by Hofmeister (1848-1867) first placed the science upon a sure foundation and marked a new era in the study of sexual
4 MARGARET C. FERGUSON
reproduction in plants. Although the researches of Hofmeis- ter, Strasburger, Warming, Belajeff and others who have con- tributed to our knowledge of this subject, especially during the last decade, have disclosed many facts concerning the structure and development of the pollen-grain, of the ovule and of the embryo, our knowledge of certain phases of spermatogenesis and oogenesis is still ver}'' meager, and not a sufficiently large number of plants have been thoroughly investigated to admit of generalizations. The celebrated discoveries of Hirase, Ikeno and Webber, in 1897, gave a new incentive to this study, par- ticularly in connection with the Gymnosperms, and rendered it highly desirable that fertilization and associated phenomena should be worked out for other members of this group by the more modern methods of investigation.
The present studies were begun in the fall of 1897 with the hope of adding somewhat to our knowledge of this subject. Incidentally, it seemed desirable to determine whether any ves- tiges of the bodies called blepharoplasts by Webber (1897^) still persist in the conifers. As a result of the past embryological studies, a vast number of facts pertaining to the life-history of the gametophytes in the higher plains has accumulated. While many of the conclusions reached are the outcome of serious direct investigations, others are based on the insufficient evi- dence found in a rather superficial study of a large number of plants. What we need to-day is not more facts regarding un- related plants, so much as a careful working out of the details of development in representative genera.
This research is based primarily upon a study of Piiius Strobus, but nearly every observation recorded has been con- firmed for Phius rigida and P. austriaca^ and to a large extent, for P. montana var. uncinata and P. rcsinosa. The descrip- tions given may be understood to refer alike to the five species named above unless otherwise stated in the text. Nearly six hundred paraffin blocks with imbedded material have been made, and more than four thousand slides of serial sections have been stained and studied. Six hundred separate collec- tions of material would seem unnecessarily large if one were studying a plant like Nicotiana in which, according to Guig-
LIFE HISTORY OF PINUS 5
nard (1902), fertilization follows in 2 hours after pollination, but in Phms, where almost 13 months intervene between these two processes, such a number is not excessive. While it is true in cytological studies, as elsewhere, that numbers, or mere mass work, do not signify excellence, it is equally true that the re- sults of investigations based upon a study of a limited amount of material are, at best, unsatisfactory, and, other things being equal, those conclusions will be most valuable which have been formulated after a careful observation of many specimens.'
HISTORICAL NOTES.
In the following brief summary of the literature dealing with the AbicttiiccB, only the more important papers have been noted, and the observations recorded by the various writers have been given without comment.
The tetrad-division in the pollen-mother-cell of Pinns and Abies was studied in 1848 by Hofmeister. He stated that the pollen-mother-cells were already developed in the anthers at the end of November, two special daughter-cells were formed at the close of the first division in the spring, and the four cells resulting from the second division were found to lie either in one plane or at the corners of a tetrad. Three years later (185 1) Hofmeister published the results of his remarkable series of investigations in the higher cryptogams and conifers. He described and figured the pollen-grain in the AbietinecB as con- sisting of a cell-complex, noted the depression in the apex of the nucellus in Piniis at the time of pollination, and the single embryo-sac-mother-cell deep in the interior of the nucellus. It appeared that the pollen-grain rested some weeks upon the nucellus before the pollen-tube was emitted. After the germina- tion of the pollen-grain, the tube grew for several weeks and penetrated nearly to the point of union between integument and nucellus, but it might cease growth before so great a depth was reached.
1 This paper was given especial honorable mention on April 26, 1903, by the Association for Maintaining the American Women's Table at the Zoological Station at Naples and for Promoting Scientific Research by Women. I wish here to express my deep gratitude to Mrs. Ellen H. Richards, Miss Florence Gushing and other members of the above named association through whose generous efforts the publication of this paper in its present form has been made possible.
6 MARGARET C. FERGUSON
He concluded that the embryo-sac remained for a long time as a single cell, its nucleus finally dissolving to be replaced by a number of free nuclei ; in a few days the sac was filled with long cells reaching to the middle ; at the beginning of winter, the walls of this transitory endosperm were greatly thickened ; in the spring, the thickened walls of the endosperm were absorbed and the cells liberated. Each primordial cell thus made free contained, somewhat later, three or four daughter-cells which were, in their turn, liberated by the disso- lution of the mother-wall. Thus the number of cells within the embryo-sac was greatly increased, the embryo-sac itself growing to more than twenty times its previous volume. The cells of the nucellus also multiplied rapidly except in the region previously penetrated by the pollen-tubes. In the middle of May, a layer of cells lined the embryo-sac, cell layers in- creased until they met in the center, then the corpuscula were differentiated. The corpuscula were always separated in the AbietinccB by one or more layers of cells, and the walls enclos- ing the corpuscula were thought to be channelled, thus afford- ing open communication with the surrounding cells. In Pinus from 3 to 5 corpuscula were developed in each ovule, and a corresponding number of funnel-shaped openings occurred in the upper part of the endosperm. When the pollen-tube reached the corpusculum it contained free spherical cells in its lower end. The tube either flattened itself out upon the corpusculum or penetrated a short distance into it. After fertilization the impregnated germinal vesicle increased in size, its nucleus dis- appeared, and soon a large daughter-cell was seen at the base of the corpusculum. By repeated divisions of this cell the pro- embryo was formed.
In 1858 Hofmeister found the usual number of neck-cells in Pinus Strobus to be four, exceptionally three, five, or six, all lying in the same plane. He further demonstrated the vacu- olate character of the contents of the corpusculum during its development. These vacuoles disappeared before impregna- tion, being replaced by free cells — the germinal vesicles, or Keimblaschen. A pit was figured in the apex of the pollen- tube after its entrance into the corpusculum, but it was said that
LIFE HISTORY OF PINUS 7
the tube remained closed until after the formation of the pro- embryo, when it was ruptured by mechanical means. The great abundance of starch in the pollen-tube of the Abictinece was also mentioned at this time. While the " Higher Crypto- gamia" appearing in 1862 was largely a translation of Hof- meister's earlier publications, it likewise presented many new observations. The fact was noted that in Pinus the integument surrounds the nucellus, leaving open above its apex a wide micropylar canal. In all the ConifercB^ after the embryo-sac was entirely filled with cellular tissue, certain cells near the micropylar end ceased dividing but increased markedly in size ; the other cells of the endosperm continued to multiply in num- ber, but remained comparatively small ; thus the corpuscula were differentiated. After the cutting off of the neck-cells in the AbictinecB^ additional cells were developed at the top of the endosperm, giving rise to the depressions referred to in 185 1. Scarcely a day intervened between the approach of the pollen- tube and the formation of a four-celled pro-embryo at the base of the corpusculum, and this occurred contemporaneously in all ovules of all trees growing under similar circumstances.
The works of Strasburger on this subject have been more numerous and complete than those of any other investigator. It is extremely interesting to note how his interpretations have kept pace with the improvements in methods of research. In 1869 he traced the development of the endosperm from the free cells lining the embryo-sac to its maturity, and established the fact that shortly before fertilization the central cell divides to form the canal-cell and the egg-cell. He confirmed Hofmeister's observations regarding the channels in the upper part of the endosperm, and the presence of a closed pit at the apex of the pollen-tube ; but he did not observe the nuclei in the pollen-tube, and remarked that, inasmuch as the sexual organs touch in these plants, spermatozoids would be superfluous and were, in reality, not present. He added, however, that their place was taken by granular protoplasm and starch grains which exercised the same fertilizing effect on the egg as do spermatozoids. After fertilization four nuclei were detected at the base of the corpusculum and a division into a cross took place, these cells
8 MARGARET C. FERGUSON
divided and were separated by cross- walls, the lower four di- vided again making three layers of four cells each, the middle layer then elongated pushing the lowest cells down into the endosperm. In Picea a fourth layer of cells was observed at the base of the central cell.
In 1872 Strasburger stated that the canal-cell loosened itself from the &^^ and hung as a cap just beneath the neck- cells, at the same time the egg-nucleus increased in 'size and moved to the center of the corpusculum. He detected two cells in the pollen-tube of several Gymnosperms, but considered that such cells were extremely rare in the Ad ietmecs, as he had only once found one in this group. The shrunken remains of these cells were seen in the pollen-tube after fertilization. He be- lieved that the pit of the pollen-tube remained closed, and that the exchange-substance was apparently communicated by a vacuole between the apex of the pollen-tube and the egg- nucleus. After fertilization the central nucleus was dissolved, and, in " abnormal " cases, four new nuclei appeared in the central part of the egg, but there was strong evidence that these did not develop into an embryo. Six years later (1878), he observed one or more divisions in the pollen-grain shortly before pollination. The small cells resulting from these divi- sions were interpreted as rudimentary prothallium. Two large primordial cells were demonstrated in the pollen-tube of Pimis and Picca when the tube was just above the archegonium. Ac- cording to Strasburger's interpretation at that time, the nucleus in front was dissolved while the one behind entered the egg and fused with its nucleus. This was a great advance on his previous observations, but he still conceived of the pollen-tube as remaining closed, and fancied that the protoplasmic contents passed through the membrane directly while the starch was dis- solved before its transmission into the ^^^' He was now con- vinced that only a part of the contents of the pollen-tube was taken up by the egg-nucleus, the remaining portion uniting di- rectly with the egg-plasma ; but he was not certain whether the protoplasm active in fertilization came in as a formless mass or in the shape of a nucleus.
Strasburger established the fact, in 1S79, '^^^^'^ ^^ ^^ ^^^ fore-
LIFE HISTORY OF PINUS 9
most of the two sperm-nuclei in the pollen-tube which is instru- mental in effecting fertilization. He reported the presence of an axial row of three cells in Larix^ the lowest cell being the embryo-sac-mother-cell. The generalization was made that the prothallium arises in all the gymnosperms through free cell-division, all the free nuclei dividing at the same time. It was claimed that but a single endosperm was formed in the Abietine(B, that the primary nucleus of the embryo-sac remained undivided during the first year, and that the " transitory endo- sperm " of Hofmeister was in reality the freed cells of the nucel- lus which were destined to be absorbed. It was to these cells that the term spongy tissue was applied. In the following year (1880) Strasburger described and figured the mature archego- nium in Picea and discussed the early stages of endosperm for- mation in Pimis, but he gave little that was new at that time. It was in this same year that Sokolowa (1880) published the results of her researches in the development of the prothallium in the gymnosperms. Cell-walls were laid down between the nuclei imbedded in the peripheral layer of protoplasm, but no cell thus formed was furnished with a wall on its inner free side. These open cells were termed " alveoli." They grew in length until the middle of the embryo-sac was reached, then walls arose at the inner ends and the alveoli were closed ; cell divis- ions followed, and gradually the elongated alveoli gave place to many cells.
Goroschankin (1880 and '83) reported that the protoplasm of the Qgg and of the sheath-cells was in immediate contact through pores in the separating membrane; he saw (1883^) the two sperm-nuclei pass into the egg in Pinus Pumilio^ and believed that both fused with its nucleus ; the great similarity which the spheres in the egg bear to nuclei was commented upon and he questioned the propriety of calling them vacuoles. Stras- burger (1884) confirmed Goroschankin's observations as to the passage of the two sperm-nuclei from the pollen-tube into the egg, but pointed out that only the one in advance fused with the egg-nucleus. As the protoplasmic contents of the central cell increased, the vacuoles decreased, and every transition could be traced between the large vacuoles and the meshes of the proto-
Proc. Wash. Acad. Sci., July, 1904.
lO MARGARET C. FERGUSON
plasm filled with metaplasm. In the pines, a large vacuole often held several smaller ones. The egg-nucleus slowly filled itself with metaplasm during its descent to the center of the cell. Three successive divisions occurred in the large cell of the pollen-grain in Larix, the first two prothallial cells formed were small and soon disorganized, the third one increased greatly in size and divided to form the stalk- and the bod3'-cell.
It was left for Belajeff (1891) to establish the true nature of the cell-complex found in the pollen-grain of the Gymnosperms. He demonstrated the fact that in Taxiis baccata the large nucleus of the pollen-grain is the vegetative or pollen-tube-nucleus, as in the Angiosperms, and that the sperm-nuclei arise b}' the division of one of the smaller cells of the pollen-grain, this smaller cell first dividing to form the stalk- and the generative cell.
Strasburger (1892) showed that Belajeff 's observations on the structure of the pollen-grain and the development of the pollen- tube in Taxtis baccata were, in general, true for the other Gymnosperms. He described the mature pollen-grain in Piims as containing a large tube-cell, a small cell — the third prothallial or antheridial cell — and the remnants of the first two prothallial cells. Pollination was immediately followed by the germination of the pollen-grain, and the nucleus of the large cell wandered at once into the tube. The last formed prothallial cell remained in its place in the pollen-grain until the following spring, when it divided into the stalk- and the body-cell of the antheridium. The division of this cell was not studied, but Strasburger thought it took place at about the same time as the develop- ment of the archegonia. The pollen-grain of Picca was found to correspond exactly with that of Pinus excepting that the an- theridial cell divided while still within the anther. The sperm- cells in Pinus were seen in the apex of the pollen-tube ; the lower cell was the larger ; and each cell was almost entirely filled with its large, coarsely granular nucleus. At the tip of the pollen-tube, the stalk- and the tube-nucleus could no longer be distinguished one from the other. The sperm-nucleus was shown to be smaller than the egg-nucleus, but the two were alike in the amount of active nuclear substance ; and attention
LIFE HISTORY OF PINUS II
was called to the smallness of the first nuclear figure following fecundation in comparison with the size of the conjugating nuclei. The germ-nucleus divided in its original position in the egg, and the two nuclei passed towards the " organic" apex of the archegonium.
Belajeff (1893) worked out the development of the pollen tube in Picea as a type of the AhietinccE. He found that the generative cell divided while still within the pollen-grain and gave rise to two sperm-cells which he figured as of the same size.
Dixon (1894) traced the history of the pollen-grain and the pollen-tube in Piniis sylvcstris from the middle of April to the time of fertilization. He thought that the prothallial cell divided towards the end of April to form a small stalk-cell and a larger body-cell. The body-cell immediately divided into two cells of almost equal size — the male sexual cells. The sperm-cells moved into the pollen-tube followed by the nucleus of the stalk- cell. Pollen-tubes were found to branch freely while in the upper "brown" tissue of the nucellus but only one branch of each tube was continued through the lower part of the nucellus. He noted that the four nuclei, much of the protoplasm, and considerable of the starch of the pollen-tube passed into the oosphere. As a rule, eight chromosomes were found in the nuclei of the female gametophyte.
In giving an account of some work done by his students on the Gymnosperms, Coulter (1897) reported that the work of Dixon " was largely confirmed in the minutest detail " ; and in 1900 he figured the pollen-tube "in pines," when just above the archegonium, showing two sperm-cells of equal size. Atkin_ son (1898) stated that the sperm-mother-cell in Pinus divided into two sperm-cells after having passed into the pollen-tube.
Blackman's excellent treatise on fertilization and related phenomena in Pinus sylvestris was published in 1898. Man y details of development were most carefully worked out, but th e facts recorded are not enumerated here, since they will be duly considered in connection with the observations, as record ed in the body of this paper, that have been made by the writer on other species of pines. Since the appearance of Blackman's monograph, a considerable literature dealing with various stages
12 MARGARET C. FERGUSON
of development in the gametophytes of the Abietinece has been published. The details of these investigations are familiar to all students of the subject. These papers will, therefore, be men- tioned at this point by title only ; they will be referred to again in the discussions which follow. Chamberlain (1899), Oogene- sis in Phiiis Laricio; Wuicizki (1899), Ueber die Befruchtung bei den Coniferen ; Arnoldi (1900), Beitrage zur Morphologic der Gymnospermen, IV; Juel (1900), Beitrage zur Kenntniss der Tetradentheilung ; Murrill (1900), The Development of the Archegonium and Fertilization in the Hemlock Spruce {Tsuga canadensis Carr.); Coulter and Chamberlain (1901), Morphology of the Spermatophytes ; Ishikawa (1901), Reduc- tion Division in Larix ; and the papers published by the writer in 1901.^
METHODS.
Collecting. — On November 15, 1897, and each week there- after until December 25, cones of Pinus Strobtis, P. rigida, P. austriaca^ P. niontana var. tmcinata, and the staminate strobili of P. ausiriaca were collected. Material was brought in occas- sionally during the remainder of the winter. Pistillate cones of the species named, and also of P. resinosa, were collected once each week beginning with April i ; collections were made twice each week throughout the month of May, and three times a week during June. From June 10-30,. a period which was sure to cover fertilization, cones of Pinus Slj'obus were collected every day at about nine o'clock in the morning, and frequently again at four o'clock in the afternoon. Male cones were gath- ered, from those species in which they had appeared, at irregu- lar intervals during the early spring. From the first of May until the time of pollination, which varies by a number of days in the different species, staminate strobili were collected each day. During May and June the young female cones were gathered as well as the more mature ones of the previous year's growth. After July i, the older cones were no longer collected, but the young cones of Pinus Strobus^ P. rigida^ and P. ausiri- aca were collected once each week until November 15. Cones
' See "Note" at close of Appendix.
LIFE HISTORY OF PINUS I3
of Pimis Strobus were again collected regularly, as described above, throughout the spring and early summer of 1899. Collec- tions of the staminate cones of Pinus Strobus and P. rigida were made during May and June 1901, and from May 15 to June 15 of the same year the young pistillate cones oi Pinus ris;ida were gathered daily.
Material was obtained from different trees and different locali- ties. The practice of collecting all one's material from a single tree, as reported by Murrill (1900), Land (1902) and others, does not seem a safe one to follow, for certain peculiarities of develop- ment which are not characteristic of the species may appear in an individual. At the time of each collection, ovules were put up from several cones of each species, these cones being taken not from the tip of one branch but from different branches. The central portion only of the cone was used, the ovules at either extremity being more or less abortive. After collecting, the material was taken at once to the laboratory and preserved. The staminate cones and, in the early stages of development, the pistillate ones were fixed entire or cut into quarters longitu- dinally. Very soon the individual scales of the female cones were removed from the receptacle before fixing, and, when the scales were of sufficient size to admit of such manipu- lation, all superfluous parts were cut away, leaving the two tiny ovules still united by a small portion of the scale. With the renewal of growth in the spring, the ovules were removed from the scales and, as soon as it was feasible, a portion of the integument was cut away* from two or more sides of each ovule, thus bringing the fixing fluid into direct contact with the young gametophyte. For later stages, the endosperm was frequently removed from the integument, but such material did not prove to be as satisfactory as that in which the nucellar cap and a small portion of the coat were left in connection with the prothallium. Throughout the entire mechanical process of preparing material for the fixer, the most extreme care was used, as it was found that a very slight pres- sure was sufficient to cause distortions and thus to render the material worthless for cytological studies.
Fixing. — The methods used in fixing and staining do not
I/j. MARGARET C. FERGUSON
differ materially from those generally employed in cytological work. The fixing fluids tested were chrome-osomo-acetic acid solution, chrome-acetic acid solution, corrosive sublimate in aqueous solution, absolute alcohol, and Carnoy's fluid. The first two were tried with variations in concentration and in length of time. The chrome-osomo-acetic acid solution giving by far the best results, the other fixers were entirely discarded. It was made up according to the following formula :
Chromic acid crystals 1.3 gms.
Osmic acid (in glass bulb) .5 gms.
Glacial acetic acid 83 c.c.
Distilled water 160.0 c.c.
This solution used in one half strength and allowed to act for about 15 hours proved to be most excellent for fixing the pro- thallium at the time when it consists of a wall layer of proto- plasm containing numerous free nuclei. For the development of the pollen-grain and fertilization stages, it was most satisfac- tory when undiluted, and allowed to act for about 24 hours. If the fluid blackened at all, it was poured off after 2 or more hours and fresh added.
After fixing, the material was washed in running water from 2 to 12 hours, but as a rule specimens were not kept in the running water longer than 6 hours. The very convenient piece of apparatus described by Durand ('99) was used for this process. Subsequent to washing, material was dehydrated in 8 grades of alcohol beginning with 15^ and ending with the absolute. It was not allowed to stand in the lower grades for more than 6 hours, and was rarely kept in the absolute alcohol longer than that time ; the latter was changed 3 times, once about every 2 hours, to insure perfect deh3^dration in as short a time as pos- sible. After material had been in 85^ alcohol for 12 hours, it was decolorized in a 35^ solution of hydrogen peroxide, made up in 95^ alcohol, for 24 hours. While material was always bleached in toto, it was frequently found necessary to decolor- ize again on the slide. After dehydration, material was brought gradually, through ascending grades, into pure cedar oil, xylol or chloroform. The best results were obtained with the cedar oil and it was far more commonly used than the others. If it
LIFE HISTORY OF PINUS I5
was desirable to store material for a few days or weeks, pure cedar oil was found to be a much better medium than 75 ^ alcohol, which is commonly used for temporary storing of ma- terial. For the purpose of getting specimens into pure paraffin they were transferred to tiny wire-gauze baskets and carried successively into 25, 50 and 75^ paraffin in cedar oil, and finally into pure paraffin with a melting point of 54°, in which they were at last imbedded. This is a very convenient and eco- nomical method for getting material through the paraffin oven. The grades of cedar oil in paraffin can be kept in the bath a long time and used repeatedly with impunity, and material can be carried in the little baskets from bottle to bottle much more quickly and with less liability to injury than in an}^ other way with which I am familiar. At the time of fixing, a small piece of paper, bearing the number, in pencil, corresponding to the number of the entry in the record book, was placed in each bottle, remained with the material through all the changes which followed, and was finally imbedded in one corner of the paraffin block containing the specimens.
Staining. — A Minot-Zimmermann revolving microtome was used in cutting the material. The sections varied in thickness from 4 to 13.6 microns, but by far the greater number were made 6.3 microns thick. They were fastened to the slide by means of albumen-fixative, and the slides were labelled with glass-ink. In preparing this ink, a paste was made of the best English vermilion in sodium silicate, and sufficient water was added to give the proper consistency for writing. Glass-pen- cils, Higgins' waterproof ink, both with and without collodion, and other methods for marking slides were tried ; but I have never found anything at all comparable, for excellence, with the glass-ink. When properly prepared it is not dissolved dur- ing the process of staining, but can be removed from slides or dishes, when desirable to do so, by heating in a strong solution of potash or in gold dust.
As is usual in cytological studies, considerable experimenta- tion was necessary before satisfactory stains were obtained. Among the stains tested were Rosen's ('92) fuchsin and methy- lene-blue method ; the Ehrlich-Biondi-Heidenhain mixture, as
1 6 MARGARET C. FERGUSON
prepared by Dr. G. Griibler ; Guignard's combination of methyl green, acid fuchsin, and orange G; Flemming's safranin- gentian-violet-orange combination ; and Heidenhain's iron- hasmatoxylin. The last two proved the most satisfactory and were almost exclusively used. The iron-hsematoxylin was fol- lowed by orange G, or, if it was desirable to stain cell-walls, by Bismarck brown. Iron-haematoxylin followed by Flem- ming's triple stain, or by gentian-violet and orange G, brought out the so-called kinoplasmic structures with great definiteness. The best differentiation was obtained with the iron-hgematoxy- lin by allowing the h£ematoxylin to act from 12 to 18 hours, decolorizing in iron-alum, and then washing in running tap- water from 2 to 6 hours. Flemming's triple stain was often used without the safranin with excellent results. Both anilin and aqueous solutions of gentian-violet were used. As a rule, a one-half percent, solution was employed, the slides remain- ing in it from 5 to 20 minutes. The achromatic figures in the divisions of the pollen-mother-cell, especially in Piniis Strobus, were, however, brought out with great difficulty with this stain. The best results were obtained for these stages by allowing the slides to stand from 24 to 48 hours in stender dishes of distilled water to which not more than 10 drops of a one percent, solution of gentian-violet had been added. Pimis sections take the orange with such avidity, that a fraction of a minute was in all cases a sufficiently long time to allow this stain to act. After washing out the superfluous gentian-violet and deh3'drating in absolute alcohol, differentiation was effected by dashing with clove oil. Bergamot oil was used for fixing and clearing, and I have found it expedient to pass the slides from bergamot oil to jars of xylol. They can remain in the xylol for hours, if desirable, without injury, and the xylol is so readily miscible with the balsam that the preparations become clear and more satisfactory for studying in a much shorter time than when car- ried directly to the balsam from the bergamot oil.
LIFE HISTORY OF PINUS 1 7
CHAPTER I.
MiCROSPOROGENESIS. THE MICROSPORANGIUM.
The Wall of the Pollen-sac. — With the exception of Pinus Strobus, the staminate cones, in the pines which I have studied, make their appearance in October or November. I have searched repeatedly in the autumn for the male inflorescences of Pinus Sirobus but have never been able to find them until late April or early May of the following spring. If they are present at all before spring they can be scarcely more than potentially so, for they are not sufficiently devoloped to be detected in the field, nor by careful dissection in the laboratory.
The structure of the microsporangium agrees perfectly with that usually described for the AbietinecB. The wall of the young pollen-sac consists of three or four layers of cells. The cells of the outer layer are nearly isodiametric, while those of the inner layers are smaller and more or less tabular in outline. Just within, and in immediate contact with the archesporium, is the ring of tapetal cells. In the early stages of development the wall-cells are rich in cytoplasm and contain nuclei which are large in proportion to the size of the cells. The microsporangium increases much in size in the spring, and by the time that the microspore-mother-cells are in the prophase of division, considerable change has occurred in the wall-cells of the pollen-sac. The outer layer has lost its nuclei and the cells have become filled with a homogeneously staining resinous sub- stance ; in Pinus Strobus this resinous deposit extends to the second layer of wall-cells as well ; the cells of the inner la3'ers have been considerably flattened out, and their cytoplasmic con- tent has become much reduced. When the pollen-grains are mature, all the wall-cells of the microsporangium, except the outermost layer, have disappeared. They have doubtless been absorbed, their substance contributing to the nutrition of the pollen-grains.
The tapetum cannot be distinguished during the earlier stages of development from the other tissues. It is first clearly differ-
l8 MARGARET C. FERGUSON
entiated in the spring, when the mother-cells are in the early prophase of the heterotypic division. The mitoses leading to development of this layer have not been studied, but there are indications that it is formed from the outer layer of the sporog- enous tissue rather than, as usuall}'" described, from the inner layer of wall-cells. The microsporangium-wall, after the appearance of the tapetum, is composed, as before, of three or four layers of cells ; furthermore, the tapetum is always inti- mately associated with the sporogenous tissue, while it is fre quently found separated from the wall of the pollen-sac, probably as a result of imperfect fixation. The question as to the origin of this tissue in Pimis must, however, await further investiga- tion. During the later stages of division in the pollen-mother- cells, the tapetal cells increase much in size, their cytoplasm becomes very dense and each cell comes to have from one to three nuclei which have been observed in all stages of fusion. Karyokinetic figures have been frequently noted in the tapetal cells indicating that the nuclei of these cells divide mitotically, and the division conforms to the ordinary or typical method of mitosis. When the young microspores become free, these cells have attained to their greatest size, and show a diffuse reaction to stains. From this time they gradually diminish in size and finally disappear altogether. The nutritive function of this tissue is too well understood to require discussion here.
The Primitive Archesporiuni. — With the exception of Pintis Strohus, the primitive archesporium is clearly differentiated in the autumn, but the mother-cells of the microspore do not arise until the latter part of April, and in Piims Strobus not until about three weeks later.
In the younger stages of development, a superficial study shows no sharp demarcation between archesporium and wall, but a careful examination reveals certain differences by which the two can always be distinguished. The cells of the arche- sporium are larger, have larger nuclei, and denser cytoplasm than those of the wall. They are also polyhedral in outline while the wall-cells are somewhat tabular from the first, though not so markedly so as at a later period. During the winter, the nucleus of a primitive archesporal cell contains several nucleo-
I.IFR HISTORY OF PINUS I9
lus-like bodies, of which as many as eleven have been counted in a single section of a nucleus, and a less number than seven is rarely found. The delicate but extensive nuclear reticulum is slightly chromatic and stains scarcely more strongly than the cytoplasm of the cell. Both cytoplasm and nuclear network stain diffusely with gentian-violet during this period of rest
(fig- ^)- . . .
In those species in which the microsporangia make their
appearance in the autumn, the pollen-sacs remain small and the archesporial cells comparatively few in number until the following spring. Hofmeister ('48) found the mother-cells of the pollen-grains in the anthers of Pinus and Abies at the end of November, Belajeff ('94) observed the pollen-mother-cells of Larix in the spireme stage in October, and Coulter and Cham- berlain ('01) have recently figured the ' ' microsporangium of Pinus Laricio in the mother-cell stage in October." The sporogenous tissue, as they have illustrated it, bears a very strong resem- blance to that shown in fig. i of this paper. There is undoubted evidence that these are not pollen-mother-cells in the species of pines which I have studied. In the first place, the number of cells in a single anther in November is far less than the number of microspore-mother-cells which is eventually formed. As the microsporangium enlarges in the spring these cells not only increase in size but multiply in number. During the last of March and first of April karyokinetic figures, representing various stages of division, are seen in all preparations, and in all cases division is proceeding by the typical method character- istic of vegetative or somatic cells. In the latter part of April or first of May (for Pimis Strobus about the middle of May), typical division ceases, and, after a period of growth, the pro- phases characteristic of the heterotypical division are entered upon. The time at which the rest preparatory to the hetero- typic mitosis begins varies by about three weeks in the different species, and by ten or more days in the same species in different seasons. Had Coulter and Chamberlain examined microspo- rangia during the latter part of March they would doubtless have found typic divisions taking place in the archesporial tissue.
20 MARGARET C. FERGUSON
TETRAD-DIVISION.
The Definitive Archesj^orium. — During the period of " rest " preceding the heterot3^pic division, the microspore-mother-cell increases much in size, its nucleus becoming even larger than an entire cell of the primitive archesporium, as is readily seen by comparing figs, i and 2 with figs. 3 and 4. The walls en- closing the spore-mother-cells thicken considerably, and the cytoplasm assumes a fine, almost granular structure which, under high magnification, resolves itself into a delicate, close reticulum. At this stage, only three or four nucleoli are found within the nucleus, but this reduction in number may be only apparent, for the nucleus has enlarged to such an extent that no one section would be liable to contain as many of these structures as would a section of one of the smaller nuclei of the primitive archesporium. No attempt has been made to de- termine the exact number of nucleoli in the nuclei of the arche- sporium at any time in its history, as it is next to impossible to trace accurately the sections in the series of any given cell when each anther contains hundreds of archesporial cells all of which are practically alike in form, structure and staining capacity.
As the nucleus of a pollen-mother-ceil enlarges, its reticu- lum becomes more open, the threads of the net gradually in- crease in thickness, the net-knots or karyosomes become more or less prominent, and numerous smaller granules are distrib- uted irregularly upon the linin. Many cross-threads are with- drawn but no true spireme is formed at this time (fig. 3). The thickening of the threads is more prominent in Pintis Strobiis than in the other species, the net-knots are more conspicuous, and a somewhat imperfect spireme arises, although here, too, many anastomosing threads still persist (fig. 4). A remarkable change has taken place in the attitude of the different elements of the cell towards stains. When the microspore-molher-cells are first formed both cytoplasm and nuclear net stain more or less diffusely with gentian-violet as in the primitive arclie- sporium, but, as growth proceeds, the cytoplasm ceases to react to chromatin dyes and takes the orange G with avidity. The nucleoli are colored far less deeply with the gentian-violet than
LIFE HISTORY OF PINUS 21
formerly, and the nuclear reticulum takes the blue characteris- tic of chromatin. In this condition, the contracted state known as synapsis is entered upon.
The First Nicclear Division of the Mic7'ospore-niother-ceU . — As soon as a microspore-mother-cell has attained full size, cer- tain changes in its nucleus indicate that the prophase of the first division has been initiated. The reticulum gradually draws together, its threads becoming thicker and the meshes smaller (figs. 5 and 6). Contraction continues until the network forms a compact mass at one side of the nucleus. During synapsis the nucleoli may be entirely confined within the contracted sphere or one or more may be partially extruded (fig. 7). Some of the nucleoli still stain deeply with the gentian-violet, but one or more usually take the plasma stain at this time and appear as yellow, porous, or spongy bodies. The same appear- ance has also been obtained with iron-hasmatoxylin followed by orange G.
In Pinus rigida no appearance at all comparable with that known as synapsis is observed until April 21. In material pre- served on this date a few nuclei in all anthers show the begin- nings of contraction as illustrated for P. aiistriaca in fig. 5 and P. Strobus in fig. 6. On April 30 the nucleus of every mother- cell has reached the point of greatest condensation, its contents forming a somewhat spherical, deeply-staining mass at one side of the nuclear cavity — fig. 7 illustrates this stage for P. Strohiis. On May 2 some of the nuclei still retain this structure while others show various stages of recovery. Two days later. May 4, not a vestige of this condition remains, all the nuclei having passed on to more advanced stages in the mitosis. These dates have been given for Pinus rigida, but they would not differ materially in the other species, except that in P. Strobus corresponding phases in this division would occur about 3 weeks later.
Synapsis is not universally recognized as a normal step in the heterotypical division. Guignard ('97), Mottier ('97), Schaffner ('01), and others still look upon it as an artifact caused by im- perfect fixation. On the other hand, Sargant ('97), Wiegand ('99), Duggar('99 and '00), Ernst ('01), Rosenberg ('01) among
2 2 MARGARET C. FERGUSON
botanists, and man}- zoologists consider it a definite characteristic of the early prophase of the heterot3'pic mitosis, several of these investigators having noted it in their material before fixation. I have observed this stage in the fresh material in Pimis, and after carefully studying it in many permanent preparations, I see no reason why this condition, simply because it happens to be one of contraction of the nuclear substance, should be set down as abnormal.
If this appearance were produced artificially why should there be transitional forms both in leading up to and in recovery from it? If it were the result of diffusion currents, as has been suggested, we should expect to find the nuclear substance in all the nuclei of a given anther carried or forced to the same side of the nuclear cavity, but such is not the case. It is doubt- less true, as indicated by Strasburger ('95), that many phenom- ena described as synapsis represent pathological conditions which do not occur under all circumstances, but it seems equally true that this condition of the nuclear substance represents, in some species at least, a characteristic stage in the heterotypic division. Although a contraction comparable with that of synapsis has been reported for somatic cells, I am not aware that anything like so marked an appearance has been described as a usual accompaniment of any but the heterotypical division. The exact significance of this phase is not well understood, but that it is intimately associated with a readjustment of the chromatic and nucleolar substances there can be little doubt.
As the nucleus slowly recovers from synapsis, it soon becomes apparent that the reticular structure has been replaced by a broad, closely coiled band which stains more deeply than did the net- work prior to the contracted stage. The coils of the thread gradually open out until the nuclear cavity is filled with a spireme, which consists of a broad linin band, so irregularly studded with chromatin-granules that it has a much roughened, almost minutely echinulate, appearance. These granules soon collect into indefinitely outlined masses which remain connected by clear, faintly staining portions of the linin thread. The chro- matin-groups never assume the definite disk-like form figured by Mottier ('97) for Lilitun and IlcUcborus, and by Duggar (00)
LIFE HISTORY OF PINUS 23
for Symflo car-pus^ but they remain always irregular and jagged in outline (figs. 8 and 9). Whether there is one continuous thread or more than one could not be determined with certainty, as the coil is at first very densely massed, and free ends might be obscured. When the loose skein fills the nuclear cavity more than one spireme can usually be detected, but the indica- tions are that this effect has been produced by the microtome knife. At certain places the coils of the spireme run together and appear to be more or less anastomosed. Such a point of contact alwa3's indicates the position of a nucleolus which has become almost obscured by the massing of the thread about it, figs. 9, 13 and 15. Not all the nucleoli are found thus associ- ated with the skein, but in those cases in which they are free from the coils of the nuclear thread their capacity for staining has generally been greatly reduced (figs. 9, 11 and 15).
As soon as the chromatin-band has become loosely wound about the entire nuclear cavity, longitudinal splitting occurs, and the segmentation of the spireme becomes apparent (fig. 10), but transverse fission is not completed until the longitudinal division has taken place (fig. 11). The segments are long, coiled, and present various appearances. Whether they correspond in number to the number of chromosomes eventu- ally formed, I could not ascertain with any degree of certainty, since they are so long and closely intermingled in the nucleus (fig. 11). Most of those shown in figs. 12 and 12, «, were taken from sections through the edge of nuclei, and, while they rep- resent the looped and twisted condition of the chromatic seg- ments at this time, they have in many instances been cut during sectioning so that only a portion of most of the segments appears. From a study of many nuclei containing chromatic threads similar to these, it is evident that the looped figure has not been formed by the bending on itself of one of the longi- tudinal halves of a segment. There are no indications that the sister-halves of any portion of the nuclear band ever become entirely disassociated. They may separate widely at one or both extremities, but at some point along the thread, an inti- mate relation is permanently maintained. The loop arises, therefore, by the complete fusion of the sister-threads at one of
24 MARGARET C. FERGUSON
their free ends (fig. 12, «, r, J, e). Even in such a late stage of fission as that represented in fig. 13 the sister threads can ahnost invariably be traced, but not always, as some are out of focus and others are doubtless in another section.
The stages immediately following longitudinal splitting and segmentation of the nuclear spireme are somewhat different from any that I have seen described by other writers. So puzzling were they to me when the study of microsporogenesis was first undertaken in 1899 that a paper, partially prepared at that time, was laid aside until a larger experience with cell structures could be brought to bear upon this, which is to me at once one of the most intricate and interesting problems con- nected with the activities of the cell. As stated in the intro- duction, new material was collected in 1901 and fixed with great care. Many slides were subsequently prepared, and the phases in the tetrad-division were found to accord perfectly with those observed during the first period of study. The interpretation of the phenomena noted is, however, much more satisfactory now than formerly, although there is still much that is obscure. Sporogenesis has not been studied in Pimis montana var. un- ct7iata, but there is complete accord, except in such details as have already been mentioned, in the other four species.
Longitudinal division is scarcely more than completed when the double skein begins to contract, the two halves of each seg- ment twisting upon each other to a greater or less degree and gradually fusing. As the segments contract the sister-halves may frequently become more or less twisted upon each other ; they may appear as parallel threads ; the half segments may separate at both ends, remaining united at the middle only ; or, having fused at both extremities, they may open out, forming rings (figs. 12 and 12, a). Fusion invariabl}^ begins first about those nucleoli which have still retained, although in a less degree than prior to S3'napsis, the power to react to chromatin-stains (fig. 13). Contraction and fusion continue until a coarse, more or less anastomosing structure is formed in which only traces of the earlier longitudinal division re- main evident (fig. 14, plate II), and a little later all signs of fission, both longitudinal and transverse, disappear (fig. 15).
LIFE HISTORY OF PINUS
As the thread thickens and broadens it becomes irregular in outline, the irregularities increase, those from neighboring por- tions of the threads meeting and fusing. Soon afterwards a transverse division again becomes apparent (tig. i6). The segments continuing to shorten and thicken gradually draw away from one another, finally remaining united only by delicate threads ; the connecting fibers are at last severed and the chromosomes lie free in the nuclear cavity. The usual number of segments formed is twelve, although thirteen, four- teen, and, in rare instances, as many as sixteen have been counted (figs. i6, 17, 18, a-c, and 20).
The chromosomes thus arise from an incompletely reticu- lated structure rather than directly from the spireme. While this suggests the condition in magnolia w^here, as recently described by Andrews ('01), the chromosomes arise directly from the resting reticulum without the intervention of a spireme, it is, in matter of fact, very different. We have here not a nuclear reticulum in the ordinary acceptation of that term, but a somewhat reticulated structure formed by the anastomosing with each other, at certain points of contact, of adjacent por- tions of a previously longitudinally split spireme. As the chromosomes separate out almost every conceivable form may be found, not only the X's, Y's and V's of Belajeff, but rings, parallel rods, eights open and closed, L's, U's and irregular- shaped bodies (fig. 19, a-l).
In my earlier study of this phenomenon, I supposed the chromosomes to be the equivalents of the long, coiled segments first formed, and with such an hypothesis the whole series of events following longitudinal fission was inexplicable. But after again considering not only such stages as those represented in figs. 10-17, but every transitional form connecting them, I am convinced that this assumption was incorrect and that each seg- ment consists, rather, of two distinct chromosomes standing side by side, each half of the double chromosome represent- ing two sister-segments which were formed by the earlier longi- tudinal fission but have now fused. If such be the origin of these chromosomes, and I no longer have any hesitancy in affirming that they have thus arisen, the phases following the
Proc. Wash. Acad. Sci., July, 1904.
26 MARGARET C. FERGUSON
longitudinal and transverse divisions of the skein are no longer unintelligible. The sister-threads formed by the longitudinal splitting not only unite again, but adjacent portions of the double threads draw together and become more or less fused, giving rise when transverse fission again becomes apparent to the one half number of chromosomes. The forms of the resultant chromosomes are exactly what would be expected from such an origin. In fig. i8, h^ for instance, adjacent portions of double segments have fused at the ends, trans- verse division has followed, and three chromosomes — parallel rods, a U, and a Y, are seen in the act of separation. When the component chromosomes have fused at both ends only, the ring, or, if a twist follows, the closed eight results ; if fusion has occurred at but one extremity the V, U, or open eight is formed ; if the segments remain attached at the middle point the X occurs ; when the constituents of the double chromosomes have united end to end and the bend has not taken place at the point of their union the L results and so on. The structure or composition of the X, Y and V forms of chromosomes as found in plants have been explained in much the same way as the above by Belajeff ('97 and '98), but he did not trace their development from the closed spireme and considered these three forms as the typical or characteristic ones whereas, in Pinus, the other forms named have been quite as frequently observed. When the chromosomes first become apparent, irregular fragments of the chromatic substance are frequently left at various points (fig. 17), but these are ultimately absorbed, doubtless being appro- priated by the growing chromosomes (fig. 20). ^
At the time when the chromosomes are being differentiated, they often appear as if pulling away from the nucleoli, and may be seen still connected with them b}-- delicate threads (figs. 18, a and c). The nucleoli now have a spongy or porous appearance and fail almost absolutely to take either nucleolar or chromatic stains. With the final separation of the chromosomes they dis- appear altogether. The history of these nucleoli from the primitive archesporium up to the time of their dissolution leads irresistably to the conclusion tiiat here, at least, there is a very
' See " Note " at close of Appendix.
LIFE HISTORY OF PINUS 2*J
intimate relation between nucleolar and chromatic substances. Whether the nucleoli are actual reservoirs of chromatin which is given out passively to the chromatic thread, or whether they are actively engaged in furnishing nourishment to the chromatic substance, I have not been able to determine, but, from certain observations to be described in a later chapter, I am inclined to consider them more than passive elements of the cell.
Coordinate with the formation of the chromosomes the nuclear membrane resolves itself into a weft of threads which crowd into the nuclear cavity, together with delicate granular fibers from the cytoplasm. The latter are evidently formed by a re- arrangement of the granules of the cytoplasmic reticulum. Up to this time the cytoplasm has remained close meshed in the region of the nucleus but has become less dense at the periphery of the cell. As the nuclear membrane disappears, coarser reticulations arise in the cytoplasm and extend towards the nucleus, doubtless contributing to the forming spindle. When the achromatic figure is fully developed, the cytoplasm again becomes uniform in structure throughout the cell, but there seems to have been an actual loss in granular substance, the meshes of the network being much larger now than formerly (figs. 20 and 21). A few delicate fibers may be seen in the cytoplasm just before the dissolution of the nuclear membrane, but, although I have searched repeatedly for cytoplasmic phe- nomena such as that described by Mottier ('97 and '98), Duggar ('00), Juel ('00) and others, I have never been able to detect anything at all comparable with the structures figured by these authors. If they are present in Pinus, I have not been able to differentiate them with any of the stains used.
The spindle is almost invariably tripolar in origin, but it may arise as a multipolar diarch. In either case, its ultimate form is that of a sharply pointed bipolar spindle (Figs. 21-24). Belajeff ('94) describes this spindle as many poled in origin in Larix, and Mottier ('97) makes the same statement for Pinus; but in the many thousands of karyokinetic figures observed for this division, I have never found one that showed more than three poles. A few scattering fibers have occasionally been seen to pass from all sides towards the nucleus but achromatic threads have not been found to converge at more than three points.
26
MARGARET C. FERGUSON
longitudinal and transverse divisions of the skein are no longer unintelligible. The sister-threads formed by the longitudinal splitting not only unite again, but adjacent portions of the double threads draw together and become more or less fused, giving rise when transverse fission again becomes apparent to the one half number of chromosomes. The forms of the resultant chromosomes are exactly what would be expected from such an origin. In fig. i8, h, for instance, adjacent portions of double segments have fused at the ends, trans- verse division has followed, and three chromosomes — parallel rods, a U, and a Y, are seen in the act of separation. When the component chromosomes have fused at both ends only, the ring, or, if a twist follows, the closed eight results ; if fusion has occurred at but one extremity the V, U, or open eight is formed ; if the segments remain attached at the middle point the X occurs ; when the constituents of the double chromosomes have united end to end and the bend has not taken place at the point of their union the L results and so on. The structure or composition of the X, Y and V forms of chromosomes as found in plants have been explained in much the same way as the above by Belajeff ('97 and '98), but he did not trace their development from the closed spireme and considered these three forms as the typical or characteristic ones whereas, in Pmus, the other forms named have been quite as frequently observed. When the chromosomes first become apparent, irregular fragments of the chromatic substance are frequently left at various points (fig. 17), but these are ultimately absorbed, doubtless being appro- priated by the growing chromosomes (fig. 20).^
At the time when the chromosomes are being differentiated, they often appear as if pulling away from the nucleoli, and may be seen still connected with them b}*" delicate threads (figs. 18, a and c). The nucleoli now have a spongy or porous appearance and fail almost absolutely to take either nucleolar or chromatic stains. With the final separation of the chromosomes they dis- appear altogether. The history of these nucleoli from the primitive archesporium up to the time of their dissolution leads irresistably to the conclusion that here, at least, there is a very
' See " Note " at close of Appendix.
^^tik.
a. €t''4v t^ i"» r i"'i''a*"A
LIFE HISTORY OF PINUS
27
intimate relation between nucleolar and chromatic substances. Whether the nucleoli are actual reservoirs of chromatin which is given out passively to the chromatic thread, or whether they are actively engaged in furnishing nourishment to the chromatic substance, I have not been able to determine, but, from certain observations to be described in a later chapter, I am inclined to consider them more than passive elements of the cell.
Coordinate with the formation of the chromosomes the nuclear membrane resolves itself into a weft of threads which crowd into the nuclear cavity, together with delicate granular fibers from the cytoplasm. The latter are evidently formed by a re- arrangement of the granules of the cytoplasmic reticulum. Up to this time the cytoplasm has remained close meshed in the region of the nucleus but has become less dense at the periphery of the cell. As the nuclear membrane disappears, coarser reticulations arise in the cytoplasm and extend towards the nucleus, doubtless contributing to the forming spindle. When the achromatic figure is fully developed, the cytoplasm again becomes uniform in structure throughout the cell, but there seems to have been an actual loss in granular substance, the meshes of the network being much larger now than formerly (figs. 20 and 21). A few delicate fibers may be seen in the cytoplasm just before the dissolution of the nuclear membrane, but, although I have searched repeatedly for cytoplasmic phe- nomena such as that described by Mottier ('97 and '98), Duggar ('00), Juel ('00) and others, I have never been able to detect anything at all comparable with the structures figured by these authors. If they are present in Piniis^ I have not been able to differentiate them with any of the stains used.
The spindle is almost invariably tripolar in origin, but it may arise as a multipolar diarch. In either case, its ultimate form is that of a sharply pointed bipolar spindle (Figs. 21-24). Belajeff ('94) describes this spindle as many poled in origin in Larixy and Mottier ('97) makes the same statement for Pinus ; but in the many thousands of karyokinetic figures observed for this division, I have never found one that showed more than three poles. A few scattering fibers have occasionally been seen to pass from all sides towards the nucleus but achromatic threads have not been found to converge at more than three points.
28 MARGARET C. FERGUSON
As the spindle-tibers press into the nuclear cavity, the chro- mosomes take up their position at the equatorial plate. They are now verv regular in outline, apparently homogeneous, and the X. Y, V, O, etc., forms can still be clearly distinguished (hg. 24, plate III). Each segment is oriented with its longer axis perpendicular to the axis of the spindle, the free limbs ex- tending outward. The spindle-fibers are attached at one ex- tremity of the parallel rods, and ordinarily at or near the point of union of the constituents of the dual chromosomes. In the Y-shaped chromosomes the achromatic threads may become at- tached at the point where the two limbs become free or at the free end of the fused chromosomes, but, whatever the shape of a segment, the spindle-fibers are never attached at the extremi- ties of its free limbs.
The line of cleavage at the equatorial plate is not such as to separate the two chromosomes but is rather such as to effect a longitudinal splitting, the two half chromosomes of each pair passing together to opposite poles. During metakinesis the daughter-chromosomes become very irregular in outline and in- crease much in size, the half chromosomes apparently exceeding in volume the undivided ones (figs. 25-2S). This augmentation of the segments maybe due to actual addition of new substance, but from the fact that in the telophase they are unquestionably smaller than in the late prophase, it is probable that this is merely an amplification without actual or permanent growth. The parts of the spireme separated during the longitudinal fis- sion following synapsis have so completely fused again that they are now disunited with difficulty. The appearance of the dividing chromosomes indicates that they are being subjected to great strain. Under this tension they are flattened out and rendered irregular in outline ; the irregularities result from the unequal stretching of the chromatic substance at different points, just as a poor rubber band when greatly extended be- comes more or less moniliform. The complete separation of the half chromosomes may sometimes be greatly delayed, when the stretched segments extend nearly the entire length of the spindle, the achromatic figure being almost obscured, in some instances, by the chromosomes (figs. 25, 26, 2S and 29). That
LIFE HISTORY OF PINUS 29
these segments are actually flattened out is further shown by the fact that the arms which remain united and elongated stain much less deeply than do those which, having become free, have contracted to nearly their former length. This would seem to indicate that the chromatic spireme is a plastic or viscid body. Lloyd ('02) describes a similar action, though much less marked, in Crticianella. While the position of the retreat- ing half chromosomes is such as to give ordinarily the appear- ance of V's or U's, other figures occur with sufficient frequency to establish the reality of their persistence after the close of the metaphase of the division. This point will be considered more fully later.
The achromatic figure increases but little in length as the chromosomes pass to the poles so that the movement here must be due in large measure to a pull exerted by the contracting fibers and not to any great extent to a push brought about by the growth of the central spindle. If the force which seems necessary to effect the separation of the half chromosomes is furnished by the achromatic fibers, we should expect to find the poles of the spindle firmly buttressed as described by Stras- burger ('00) for Larix ; but no strengthening fibers are devel- oped, and, although the apices of the spindle are usually inserted in the ectoplasm, they not infrequently end blindly in the cytoplasm. It is possible that the force exercised by the growing fibers of the central spindle just equalizes the counter force exerted by the mantle fibers in drawing the chromosomes to the poles, the equilibrium thus established giving rigidity and rendering a support for the poles unnecessary. By the time the pairs of daughter-chromosomes have reached the poles they have become much reduced in size and regular in contour (figs. 27 and 30).
After the chromosomes reach the point where the daughter- nuclei are to arise, they do not at once fuse end to end to form a continuous spireme, but as the chromosomes lie side by side they lose their clear outline and gradually assume a diffuse reaction to stains. In this condition the halves of the longi- tudinally split pairs of chromosomes are doubtless fused, after which fusion the adjacent segments unite by their ends to
30 MARGARET C. FERGUSON
form a coiled, somewhat moniliform thread (figs. 30-32). Immediately upon the formation of the skein a delicate nuclear membrane appears, the coils loosen somewhat and branch freely thus giving rise to a reticulum. Extensive growth follows and a large " resting" nucleus is formed (figs. 33 and 34). The nuclear net consists at first of delicate achro- matic linin threads bearing scattered chromatin-granules and uniting large irregularly branched chromatic portions. Distri- bution of the chromatin continues until there is a delicate linin reticulum with chromatin granules of varying sizes imbedded in it (figs. 33-35). These nuclei have the form of a plano- convex lens the flat side of each nucleus being perpendicular to the axis of the spindle and facing the other daughter-nucleus. It is obvious from the foregoing that a definite resting nucleus is formed in Pinus at the close of the heterotypic division. This accords with the recent observations on the formation of the microspore by Duggar ('99) in Bignonia, Strasburger ('01) and Gager ('02) in Asclc^ias and Andrews ('01) in Magnolia. A true nucleolus has not been observed in the daughter-nuclei. Contrary to the observations of Hofmeister ('51), no cell-wall is laid down and in only a very few instances has a slight thickening of the spindle fibers in the region of the cell-plate been observed.
The Second Mitosis of the Mothei'-cell. — The resting daugh- ter-nuclei are scarcely more than established before the initial steps of the second division are instituted, as evidenced in the readjustment of the nuclear reticulum. The more delicate threads of the net are withdrawn, the nuclear membrane fades out, the chromatin loses its granular aspect and becomes evenly distributed upon the linin, and there issues forth a heavy, homo- geneous, deeply-staining band which is more or less coiled and branched (fig. 36). The chromatin-thread, which now lies free in the cytoplasm of the mother-cell, continues to thicken, the branches or cross fibers disappear, and in an almost incredi- bly short time, the delicate nuclear net has given place to a broad, somewhat spirally coiled skein (fig. 37).
Achromatic threads arise in the cytoplasm forming a multi- polar diarch spindle. The fibers are not abundant and always
LIFE HISTORY OF PINUS 3 1
arise in a plane perpendicular to the axis of the primary spindle. Harper (bo) makes the statement that in Larix^ where no cell- wall follows the first division of the pollen-mother-nucleus, the spindle-fibers of the primary mitosis are utilized in the formation of the spindle for the second division, lam unable to trace any such connection in the pollen-mother-cells of Pinus^ all traces of the first karyokinetic figure having been lost to view before the inception of the spindle for the second division.
As the kinoplasmic fibers appear the chromatin-band forms a double row of loops extending across the spindle-threads in the plane of the equatorial plate. The longitudinal splitting is now clearly apparent. The loops continue to shorten, and in this position transverse fission occurs, segmentation almost always taking place at the outer free ends of the loops (figs. 38 and 39, plate IV). The sister-halves of each V- or U-shaped chromo- some entirely separate, undergo readjustment, and finally come to stand in a double row with their free ends in the line of the nu- clear plate and their angles towards their respective poles (figs. 38-41). The spindle-fibers become attached to the chromosomes at their point of bending, and the half chromosomes pass to the poles (figs. 42-43). The dissociation of the sister-halves of each segment is so complete before the beginning of the separation at the equatorial plate that the figure during metakinesis is such as to give the impression of whole chromosomes passing to the poles, but a study of the prophases of the division shows clearly that each represents the half of a double chromosome. In the telophase of the division the chromosomes unite end to end to form a spireme (fig. 44). The nuclear membrane appears, and the chromatic band branches, giving rise to the reticulum of the resting nucleus (figs. 44 and 45).
The Probleju 0/ Reduction. — Here as in all studies of spore- formation at the present time the question of reduction demands consideration. As already indicated, the reduction in the num- ber of chromosomes takes place, as is the rule, during the so- called resting stage of the spore-mother-cell, the one half num- ber of chromosomes appearing in the prophase of the hetero- typical division. But the inquiry concerning the presence or absence of a qualitative reduction is not so easily answered.
32 MARGARET C. FERGUSON
With few exceptions, botanists of to-day follow the present lead of Strasburger and accept the view of a double longitudinal splitting of the chromosomes in the first division of the spore- mother-cell. According to this interpretation, reduction, in the sense in which Weismann uses the term, does not occur in plants. Among the exponents of a qualitative or true reduction in plants, Atkinson ('99), Belajeff ('97, '98), Calkins ('97), Ishikawa ('97, '01), and Schaffner ('97, '01) are almost alone to-day in not having retracted their earlier published conclusions regarding this subject.
It has seemed best to record the details of the observations made in studying the tetrad-division in Piiiiis, before entering upon any discussion of the significance of the phenomena noted, but in so doing some reiteration is inevitable.
Strasburger's statement that certain forms of chromosomes occurring in the anaphase of the heterotypic division are inex- plicable on any other assumption than that of a double longi- tudinal splitting is, doubtless, correct when those forms have been derived from V-shaped chromosomes. But, while it may be true that such figures are due to a double longitudinal fission when derived from other than V-shaped chromosomes, it is like- wise true that, in such cases, the phenomena are capable of rational explanation on other grounds. The V with the three arms, for instance, may result from the attachment of the spindle fibers at the middle point of a Y, the stem of the Y bending down as it moves to the poles (fig. 30, a, plate HI), and a double V might be derived in the same way from an X-shaped chromo- some (fig. 30, c). In fig. 26 the second chromosome on the left represents a Y opening out from its lower extremity, and the next chromosome shows parallel rods just separating. Occasionally an X or Y figure becomes apparent in the late anaphase of this division (figs. 28, 29). Such appearances are doubtless to be attributed to an early straightening out of the segments. If the constituents of the double chromosomes are disunited in this mitosis, then such chromosomes as those illustrated in figs. 28, d^ and 30, a, c^ and «?, might result from the more or less com- plete longitudinal fission of the sister-segments. Should this prove to be the case, and if my interpretation of the origin of
LIFE HISTORY OF PINUS 33
these chromosomes is correct, then both a quantitative and a qualitative reduction of the chromosomes would occur in the first or heterotypic division, and whole chromosomes, each representing the half of a dual chromosome, would pass to opposite poles. I am aware that such a phenomenon has been described by Atkinson and a few others, but after long and care- ful study there does not seem to me the least doubt, that, in the case of the pines investigated, a longitudinal fission, and not a transverse one, occurs in this first mitosis ; and X-, Y-, and ring-shaped segments, as well as V's, pass to the poles, although, as Belajeff has pointed out, they usually, because of their posi- tion, have the form of V's in the anaphase of this divison.
Most writers on sporogenesis, and especially those who are advocates of the true reduction, have not found a resting nucleus intervening between the heterotypic and the homotypic divisions. As already stated a resting nucleus is clearly demonstrated at this point in Pinus. The spireme formed from this nucleus shows signs of longitudinal division before segmentation, and, while lying at the equatorial plate, the two halves of each seg- ment separate entirely, in most instances at least, before their final orientation on the spindle. Now the question arises as to whether or no this homotypic division effects a qualitative reduc- tion. If the theory of the so-called " individuality of the chromo- somes " is without foundation then it certainly does not ; but, if the possibility of the complete rehabilitation of the chromosomes be accepted, a qualitative reduction very probably does occur. For under such conditions, the skein preceding the homotypic division would consist of the daughter-chromosomes, formed as a result of the heterotypic mitosis, fused end to end. These daughter-chromosomes, it will be remembered, arose by the longitudinal fission of a double chromosome and each, therefore, consists of a pair of half chromosomes. Thus the second, apparently longitudinal, splitting would effect the separation of the half chromosomes of each pair, rather than the longitudinal fission of a single chromosome. Reduction would thus take place in the true or Weismann's sense. Because of certain phenomena to be described in connection with the development of the pro-embryo, I am inclined to believe that the chromo-
34 MARGARET C. FERGUSON
somes retain their individuality through succeeding cell-genera- tions. I am, therefore, disposed to regard the tetrad-division in Pinus as a true reducing division ; in this way only does the complicated process just described find satisfactory explanation. No positive statement can, however, be made either way, in connection with this division in Pinus, until we are in posses- sion of greater knowledge than at present of the origin and ulti- mate destiny of chromosomes.
Guignard ('97) expresses the opinion that the regularity of the chromosomes in certain forms has been overestimated. Be that as it may, I am conscious that there is recorded in this paper a greater variation in the forms of the chromosomes than has been described in a single genus by other writers. It has been my purpose to note not only that which is in accordance, or at variance, with the observations of other investigators, but to give as faithful a record as possible of the conditions found in the preparations studied. And may we not yet find that here, in the divisions preceding spore-formation in plants, as in many other instances, there is greater variation in matters of detail than was formerly supposed to be the case?
DEVELOPMENT OF THE MICROSPORE.
The For7nat{on of the Sforc-zvall. — Hofmeister ('51) de- scribed four " special " cells, each with its own wall, within the pollen-mother-cell in the AhietinccB, and Juranyi ('72 and '82) devoted particular attention to the formation of the wall of the microspores in many Gymnosperms and Angiosperms. He described the development of a wall separating the two nuclei after the first division. This wall was soon absorbed and during the second division the entire cell was filled with connecting fibers stretching between the four nuclei. Delicate walls were then laid down between the nuclei giving rise to the four microspores. These dividing walls thickened and united with the inner wall of the spore-mother-cell : thus a portion of each spore-wall was formed from the inner mother-wall. After a period of rest the outer mother-wall was burst and the " pollen- cells " became free. If there is any recent literature of value on this subject, I have failed to find references to it.
LIFE HISTORY OF PINUS 35
As already indicated, no wall separating the daughter-nuclei is formed at the close of the heterotypical division in Piniis. During the late telophase of the second mitosis in the microspore mother-cell, a readjustment of the spindle-fibers occurs giving rise to the complex figure that has been described as character- istic of spore-formation in many plants. The development of the archoplasmic structures connecting the nuclei of the tetrad is much less marked than in PodofhylliLm (Mottier '97) and in many other phanerogams (fig. 44). By the time the nuclei have reached the resting stage, a division has occurred in the cytoplasm giving rise to four cells which are surrounded by delicate clear walls. A prominent thickening of the wall of the spore-mother-cell takes place, and at the same time a thick wall, continuous with the inner portion of the mother-wall, appears between the daughter-cells.
This wall frequently attains remarkable thickness. Whether it constitutes an inner wall, or is merely a thickening of the primary wall by the deposition of new material on its inner sur- face, I am unable to say. The outer, primary wall stains more deeply and is frequently seen separated from the inner broad portion (figs. 44-47). This inner wall, which is continuous with the broad walls separating the young microspores, stains deep yellow with orange G, if the orange is allowed to act from one to two minutes ; it appears a pale rose when treated with safranin, but fails altogether to stain with iron-haematoxylin. In a few instances, slight evidences of stratification have been observed, but ordinarily the wall appears perfectly homogene- ous, giving the impression of a liquid or viscid substance in which the spores are imbedded ; but the fact that it is often separated from the outer wall by a clear space, and also that it is left behind as a definitely outlined wall after the escape of the spores militates against the probability of its fluid nature. After the spores have grown for a certain period the mother- wall is ruptured and the spores are liberated. At this time the empty mother-cell with its four chambers is often met with (figs. 48, 49).
In so far as I am aware, this permanent division of the mother-cell into four compartments by thick cellulose walls has
36 MARGARET C. FERGUSON
not been previously described. A broad open space, repeatedly figured between the daughter-spores and the mother-wall, has been invariably attributed to shrinkage ; but it is probable that, in some cases at least, it represents this thickened wall which has failed to be differentiated with the stains used. Wiegand ('99) says that the spores of Potamogeton are as if imbedded in a ground mass of some viscid substance, but he does not figure it and makes no statement regarding the development of cell-walls between the microspores.
Origin of the Air-sacs. — As soon as the young microspores have become enclosed, each within its own special chamber of the mother-cell, it is evident that a special wall has been de- veloped about each spore. This is doubtless secreted by its own cytoplasm and is not, as Juranyi thought, derived from the inner wall of the microspore-m other-cell. The spore-wall while still very delicate becomes differentiated into an inner and an outer layer corresponding to the intine and extine of the pollen- grain. The young microspores are characterized by the rela- tively large size of their nuclei, the nucleus filling almost the entire cell just prior to the discharge of the spores. The cyto- plasm which fills the remainder of the cell is in the form of a loose reticulum (figs. 46, 47).
As time goes on the outer wall of the microspore expands at two points on opposite sides of the spore. A resistance is met with in the thick wall of the spore-mother-cell and the plastic inner wall of the microspore responding to this new pressure becomes indented along the surfaces corresponding to the ex- tended portions of the outer spore-wall. Thus a clear open space having in section the form of a biconvex lens is formed between the extine and the intine on either side of the microspore. These are the beginnings of the wings or air-sacs that are so conspicuous in the mature pollen-grain of the AbietinccB. Finally the pressure becomes so great that the mother-wall is ruptured and the spores are liberated (figs. 47, 48). Coulter and Chamberlain ('01) noted the fact that the wings make their appearance in Pinus Laricio while the microspores are still within the mother-cell, but they recorded no observations regard- ing the origin and development of these sacs. Strasburger and
LIFE HISTORY OF PINUS 37
Hillhouse ('oo) consider that these bladder-like appendages con- sist of the outer part only of the extine, the extine having under- gone cleavage at these two points. In studying the develop- ment of these organs from their earliest beginnings, it appears to me that the line of cleavage lies rather between the two coats of the young spore. If it is not, then at the time that the micro- spore leaves the parent-cell, the intine has not been developed, or, if present, is so delicate that I have not been able to detect it (fig. 48).
Growth of the Microspore. — After its escape from the mother-cell the microspore undergoes rapid growth, and the outer surface of the spore becomes beautifully marked by the formation of delicate, irregular ridges over the entire inner sur- face of the extine, except along that portion which connects the two wings on the concave or ventral side of the pollen-grain. It is at this point that the pollen-tube later makes its exit, and there is here no appreciable thickening of the spore- wall. These ridges continue to grow and extend inward forming a very pretty reticulated structure which is most distinctly apparent on the walls of the wings ; along the convex or dorsal side of the pollen-grain the reticulations are closer and the extine forms a broad, deeply staining layer (figs. 50-54, plate V). This irregular thickening of the extine is an admirable adaptation for securing strength with slight increase in weight.
When the young microspore attains to its mature-size, a par- tial wall, extending along the back and for a longer or shorter distance down the sides of the spore, becomes apparent within the intine (fig. 54). It consists of a broad, homogeneous- appearing band which gives precisely the same staining reac- tions as the thick wall developed within the spore-mother-cell after the formation of the young microspores. These immature pollen-grains, after treatment with Flemming's triple combination or with the gentian-violet and orange G alone, afford the most brilliant effect that I have observed with these stains. The extine presents a very intense, clear blue, the inner homogeneous wall an equally vivid yellow, while the protoplasmic elements take the colors characteristic for these dyes. The fact that this third partial wall fails entirely to respond to some stains doubtless
38 MARGARET C. FERGUSON
accounts for its haviug been overlooked by previous writers. It is not shown at all in the series of figures, recently published by Coulter and Chamberlain ('01), illustrating the development of the pollen-grain in Pinus Laricio.
The various tests commonly used in determining the nature of the cell-wall have been applied to the young pollen-grains as well as to the special spore-mother-walls. These tests show that the outer wall of the pollen-grain is clearly of the nature of cutin, as has been demonstrated by Strasburger. Both the innermost wall of the microspore, and of the pollen-grain, as also the wall of the special spore-mother-cells, respond to the reaction for cellulose, but not in a very marked manner. If they are of the nature of cellulose there would seem to be an admixture of some other substance, but I have not succeeded in obtaining entirely satisfactory results regarding the nature of these inner, prominent walls. Tests thus far have been made with "fixed" material only; further experimentation along this line will be made when fresh material is at hand.
During the season of growth, the nucleus of the microspore always remains close against the convex or dorsal side of the spore, occupying a central position along this wall. As is usual in cell-development, the microspore-cell attains full size before any mitoses occur within it, and there is never any fur- ther increase in the size of this cell after the inception of the first division. The fully developed microspore is, therefore, the exact counterpart, so far as size is concerned, of the mature pollen-grain. Compare fig. 54, plate V, with fig. 65, plate VI. During the development of the microspore, the cytoplasm which at first was uniformly distributed in a rather loose net work, becomes more closely reticulated and at the same time less abundant in proportion to the size of the cell. At the maturity of the spore the cytoplasm is largely distributed about the nucleus from which strands extend outward in a radial man- ner and end in the ectoplasm. In 1898 the microspores of Pinus Strobns were ready to leave the mother-cells on May 30, they had attained full size on June 7, and on June 10 the pollen- grains were fully mature.
LIFE HISTORY OF PINUS 39
SUMMARY.
In Finns rigida, P. austriaca and P. resinosa the primitive archesporium is well developed before the approach of winter, but the microspore-mother-cells do not arise until the end of the following April. The male inflorescence does not appear in Pinns Strobus, until the end of the April preceding pollination, and the definitive archesporium is differentiated in this species about the middle of May. The nuclei of the primitive arche- sporium are characterized by several deeply staining nucleoli and a fine, close-meshed reticulum which responds but slightly to chromatic dyes.
The wall of the pollen-sac consists in all cases of from three to four layers of cells. The tapetum is not clearly distinguished until spring and there are indications that it may be derived from the outer layer of sporogenous tissue. The nuclei of this tissue multiply mitotically and the cells reach their maxi- mum size about the time when the microspores become free. At this period each cell has from one to three nuclei which pre- sent all stages of fusion. When the pollen-grains are mature the tapetum has entirely disappeared and the wall of the micro- sporangium consists of a single layer of cells, or at most of not more than two.
Synapsis is recognized as a normal stage in the prophase of the heterotypical division in the pollen-mother-cell of Piniis. It is not preceded by a definite spireme, but a broad skein con- taining irregular masses of chromatin separated by clear portions of the linin thread issues from the contracted nuclear mass.
The chromatic spireme splits longitudinally and breaks up by transverse fission into several segments. The loosely coiled, delicate threads resulting from the longitudinal division soon draw together and fuse, double threads also come into contact at various points and fuse more or less perfectly. These threads always anastomose most freely in the region of the nucleoli, some of which still stain deeply while others stain but faintly after synapsis.
Fission occurs at various points in the now irregularly con- tracted and anastomosed threads, and the separate chromosomes,
40 MARGARET C. FERGUSON
in the reduced number, become apparent. These segments are at first irregular and jagged in outHne showing distinctly the points at which each has separated from neighboring segments, but they gradually diminish in size and become more regular in contour. The chromosomes thus formed are in the form of X's, Y's, V's, U's, L's, parallel rods, rings, and indefinitely-shaped bodies. Each segment consists of two chromosomes fused side by side.
The spindle-fibers arise both from the nuclear membrane and from the cyto-reticulum. The achromatic figure ma}'- originate as a multipolar polyarch of three poles or as a broad multipolar diarch spindle. At the close of the prophase of the heterotypic division the spindle has become sharply bi-polar and its extremities may be imbedded in the ectoplasm or they may end blindly in the cytoplasm.
The chromosomes are separated at the equatorial plate with difficulty giving the appearance of a plastic substance under tension. Their separation may be so delayed that the daughter- chromosomes stretch from pole to pole. They ordinarily have the form of V's or U's during the anaphase of the mitosis, but other forms are not infrequent. The first division effects a longitudinal splitting of the chromosomes into daughter-seg- ments of the same form as the parents.
A resting nucleus is established at the close of the first mitosis but the daughter-nuclei are not separated by a cell-wall. The daughter-reticulum soon gives rise to a more or less spirally coiled chromatic band which loops itself at the equatorial pl^te and splits longitudinally before segmentation.
The chromosomes have the form of U's and are oriented at the equatorial plate in two rows with their free ends touching and the bent portion of each segment directed towards the poles, the complete fission of the segments having been completed before their migration to the poles begins. The writer inclines to the view that these are the half chromosomes of the daughter- pairs which were separated in the first division. If this hy- pothesis be correct, the homotypic mitosis in Piiius effects a true or qualitative reduction of the chromosomes.
The wall of the microspore-mother-cell increases markedly in thickness and its protoplasmic contents is separated into four
LIFE HISTORY OF PINUS 4I
parts by prominent cross walls which are continuous with the inner portion of the mother-wall. The microspores are then developed each in its own particular chamber of the mother- cell.
A double wall is quickly developed about each spore and the air-sacs become apparent while the spores are still within the mother-wall. They arise by the separation of the extine from the intine at two definite points on opposite sides of the spore. By the growth of the spore, and more especially by the expan- sion of the air-sacs, the spore-mother-wall is ruptured and the spores set free.
Growth ensues, the extine becomes irregularly thickened on its inner surface except at the concave side of the spore, and a broad partial wall is laid down just within the intine and along the back and sides of the microspore. During the growth of this cell its nucleus maintains a position at the central point of its dorsal side. Before the germination of the microspore it attains to the full size of the mature pollen-grain.
CHAPTER II. The Male Gametophyte.
the development of the pollen-grain.
Formation of the ProthaUial Cells. — So much confusion has arisen in the application of terms used to designate the various cells of the male gametophyte in Gymnosperms that it is desirable, if not almost necessary, that one should define at the outset the nomenclature adopted. Throughout this paper, the first two cells cut off from the larger cell are known respectively as the first and second prothallial cells, and the third small cell formed represents the antheridial or third prothallial cell. The large cell, so long as it continues to divide, is designated as the apical cell, but after division ceases in this cell it is referred to as the tube-cell and its nucleus constitutes the tube-nucleus. The antheridial cell divides to form the stalk-cell and the gen- erative cell, the latter giving rise to the binucleated sperm-cell.
Proc. Wash. Acad. Sci., July, 1904.
42 MARGARET C. FERGUSON
As soon as the microspore has reached maturity, there arises within its nucleus one of the most beautiful, homogeneous, loosely-looped and coiled spireme-bands that I have ever seen in any dividing nucleus (fig. 54)« The material studied showed every stage in the first division, and all succeeding mitoses which occur within the microspore, but they offer nothing especially instructive from a cytological point of view, since they conform to the typic method of division. I shall, there- fore, describe and figure only such phases as are of interest in tracing the development of the pollen-grain. It is interesting to note that in the late prophase of all the mitoses which occur in the development of the male gametophyte the achromatic figure presents a very characteristic appearance, being sharply monopolar at its outer or lower extremity and broadly multi- polar at the opposite end. It thus forms a fan-shaped body rather than one resembling a spindle. During the telophase it usually becomes bluntly bipolar, though the upper pole often remains to the last somewhat broader than the lower pole (figs. 55, 56 and 60, and plate V. A similar method of karyokinesis has been noted by Wiegand ('99) in the development of the pollen-grain in Potamogeton^ by Duggar ('00) in Symploca7'pus^ and by Coker ('02) in Podocarpus. This mode of division will be referred to again in connection with certain phases in the development of the female gametophyte.
In all the divisions which occur within the wall of the micro- spore the nuclear substance is divided equally, the cytoplasm unequally. The nucleus of the first prothallial cell, however, never equals in size that of the apical cell and always stains more or less diffusely, thus showing signs of disintegration from the time of its organization (fig. 57). Fig. 58 shows one of the very largest and most nearly normal of all the prothallial cells observed. The nucleus of the apical cell enters the complete resting stage, instituting a definite network within the meshes of which one or more faintly staining nucleoli become apparent, but this reticulum at once resolves itself into a homogeneous, spireme exactly similar to the one first formed. When the nucleus of the apical cell has reached the spireme-stage of the second division, the first prothallial cell is invariably found
LIFE HISTORY OF PINUS 43
pushed against the dorsal side of the spore-wall, not a vestige of its cytoplasm is left, and the nucleus has become greatly flattened, although there is still a faint suggestion of its former reticular character (fig. 59). When the telophase of the divi- sion is reached this nucleus has lost all traces of its former structure and persists only as a deeply staining, linear body lying against the spore-wall (fig. 60). During the following division it becomes scarcely more than a line so that it is fre- quently detected with difficulty. Coulter and Chamberlain ('01) figure this cell in Pinus Laricio as still projecting into the cytoplasm of the apical cell when that cell is in the telophase of the second division, but I have never found it in such a state of preservation at so late a date. The second prothallial cell is invariably smaller than the first, and during the third mitosis of the apical cell, which follows immediately the formation of the second prothallial cell, it exactly repeats the history of the first cell (figs. 61-63).
The partial, broad, innermost wall, described in connection with the development of the microspore, persists throughout the entire history of the pollen-grain, and a comparatively broad wall, continuous with it and having exactly the same staining capacity, invests both the first and second prothallial cells as shown in figs. 57-63. The presence of the remnants of the prothallial cells imbedded apparently in the inner wall of the mature pollen-grain (fig. 63) was very perplexing before the histor}'^ of these cells was studied. But in tracing their develop- ment it is clearly demonstrated that the remnant of each cell is pushed back against the wall of the spore and remains perma- nently covered on its outer side by its own wall. That the remains of these cells come to lie nearer the intine than when first formed would again suggest the somewhat plastic nature of the partial or incomplete membrane against which the pro- thallial cells are pressed (figs. 57-64). These observations con- firm the statement of Strasburger, Noll, Schenck and Schimper ('97) that the two prothallial cells formed in the pollen-grain of the Gymnosperms are invested with cellulose-walls. Coulter and Chamberlain ('01) make no mention of the formation of walls in connection with the development of these cells in Pinus
44
MARGARET C. FERGUSON
Lartcio, and Coker ('02) says that in Podocarjbus " as in other cases " no cellulose-wall is formed. The small cell cut off by the third and last division of the apical cell persists as a perma- nent feature of the mature pollen-grain. Its cytoplasm is dis- tinctly differentiated from that of the tube-cell, but no cellulose- wall has been observed in connection with this cell, its boundary being marked by scarcely more than a condensation of its periph- eral cytoplasm.
The Mature Pollen-grain. — During the development of the male gametophyte the cytoplasm of the large cell gradually increases in amount, the vacuoles becoming smaller from the region of the nucleus outward, and finally disappearing alto- gether. The pollen-grain has the same size, form, and, so far as the wall is concerned, the same structure as the microspore just prior to its germination. The thick, innermost, partial wall described in connection with the microspore still persists as a very prominent characteristic of the mature pollen-grain. With the expansion of the wings, certain protoplasmic portions of the microspore-cell are left with no support except the delicate endo- spore ; it therefore seems probable that this broad, incomplete wall extending along the back and down the sides of the pollen- grain has been developed for the purpose of strengthening these weakened points in the spore-wall, and as an additional support to the dorsal side of the pollen-grain.
But, while the wall of the mature pollen-grain is identical with that of the microspore, the essential or protoplasmic part of the spore has undergone marked changes, as we have already seen. One or two deeply staining lines, more often one than two in the mature pollen-grain, lie on the dorsal side of the pollen-grain apparently imbedded in its innermost wall. Extending from this wall at its middle point is a strongly convex cell, the antheridial cell, with delicately reticulated cytoplasm and a comparatively large nucleus. Just below and always in contact with this cell is the nucleus of the tube-cell. The cyto- plasm of the tube-cell is closely reticulated and slightly more dense than that of the antheridial cell. Imbedded in its cyto- plasm are numerous starch-grains. In this condition the pol- len-grain of Finns awaits pollination (tigs. 64, 65, plate VI).
LIFE HISTORY OF PINUS 45
Starch-grains have been found in the large cell from an early date in the development of the pollen-grain, but they are more abundant after maturity is reached than at any previous time. According to Coker ('02) the pollen-grains of Podocarfiis con- tain large starch-grains from the beginning of the first division. With such variations in details as have been noted above, this description of the development of the pollen-grain in Pinus agrees with that given by Strasburger in 1892 and Coulter and Chamberlain in 1901.
' POLLINATION.
The Ovule at the Time of Pollination. — In the vicinity of Cornell University, 42^° north latitude, the pollen-grains of Pinus Strolms are ready for dispersion late in May or early in June, but in the other species studied pollination takes place during the latter part of May. At this time the axis of the female cone elongates, thus separating the ovuliferous scales which now make an angle of about thirty-five degrees with the rachis. After pollination the fruit scales draw together and, according to Strasburger and Hillhouse ('00), their edges are consolidated by the ingrowth of papillae. The presence of two ovules at the base of each scale, each ovule with its apex extend- ing downwards, that is towards the base of the scale, and out- wards, is too familiar a fact to need more than a passing men- tion here.
As pointed out by Hofmeister ('62) the integument is con- tinued above the nucellus into two long arms which curve out- ward before pollination and lead below to a wide mycropylar canal. The degree of development which the ovule has obtained at the time when the pollen-grains reach the nucellus is shown in fig. 66. Deep within the central portion of the ovule, at its chalazal end, a single cell is distinguished from the others by its greater size and larger nucleus, this is the macrospore ^ of Hof- meister ('51). The so-called "spongy "tissue of Strasburger is already well differentiated when pollination takes place (figs. 66^ plate VI, and 124, plate XII). Somewhat later the integu-
1 In 1901, I stated that, at the time of pollination, there was in the nucellus an axial row of cells. I know, now, that this condition has rarely been reached at so early a date, and should be noted as very exceptional rather than as normal.
46 MARGARET C. FERGUSON
ment has closed over the pollen-grains and the macrospore mother-cell has divided giving rise to an axial row of cells the lowest of which becomes the functional macrospore (fig. 69, plate VI).
The Pollen- cha7iib er . — The pollen-grains fall upon a scale and slip down to its base where they come into contact with the extended arms of the ovule. These prolongations of the integu- ment now straighten and partially draw together thus bringing the pollen-grains down into the wide micropylar canal (fig. 123, plate XII, and fig. 66, plate VI). The free limb of the integument is seen in section to consist, at this time, of three layers of cells. As soon as the pollen-grains have found their way into the lower portion of the micropylar canal and some, at least, have come into contact with the tip of the nucellus, the cells constituting the middle layer of the arms, at a point slightly above the apex of the nucellus, elongate rapidly. The bulge or protuberance thus formed extends inwards from all sides and meets, closing the opening above the pollen-grains (figs. 66 and 67). As soon as the opening has been closed and the pollen-grains secured, these elongated cells give rise by division to many smaller ones (fig. 68). By the rapid elongation of these cells the safety of the pollen-grains is as- sured in a very short time, and then cell multiplication follows leisurely. This very pretty mechanism by which the final clos- ing of the micropyle is effected has not been previously described for any Gymnosperm, unless it be noted in Shaw's ('96) state- ment, unaccompanied by figures, that the micropyle in Sequoia is closed by the radial elongation of the cells about it.
The depression in the apex of the nucellus in the Abielinece at the time of pollination, described by Hofmeister in 185 1, and since noted by man}?^ writers, has, it seems to me, been greatly exaggerated so far as Pinus is concerned. The expression " cup-like depression " is not infrequent in literature, but, in so far as my observations go, saucer-like is as strong a term as one is justified in using (figs. 66, 67 and 69, plate VI, and 75, plate VII). At the time of pollination the upper concave portion of the nucellus terminates in a row of more or less elongated cells, which are not closely united at their free extremities, but
LIFE HISTORY OF PINUS 47
stand up, as it were, like so many fingers to catch the pollen- grains ; they also serve to facilitate the entrance of the pollen- tubes into the tissue of the nucellus (fig. 75, plate VII). A little later this depression may become more prominent, both by the slight disintegration of some of the superficial cells of the nu- cellus, due to the action of the pollen-tubes, and by the incon- siderable growth, after pollination, of the peripheral layer of cells of the nucellar tip. The deep cup-like depression some- times observed is invariably the result of abnormal disintegration. The pollen-chamber in Pinus, then, consists of a space bounded on the bottom by the more or less concave upper surface of the nucellar tip, and arched above by the ingrowth of the free por- tion of the integument. Later a resinous substance is secreted which securely seals the opening by which the pollen-grains entered.
DEVELOPMENT OF THE POLLEN-TUBE. THE FIRST PERIOD OF GROWTH.
Germination of the Pollen-grain. — Germination of the pollen- grain follows immediately after pollination. Ovules of Pimis Strobus that were fixed on June 6, 1898, had not been pollinated, but on June 13 pollination had occurred and the pollen-tubes had been emitted ; similar evidence could be given for the other species studied, but exact data on this point are at hand ior Pinus rigida only. Dispersion of the pollen occurred in this species in the vicinity of Wellesley College in 1902 on May 27, and in material fixed two days later. May 29, the first stages of germi- nation are clearly evident. It is probable that the time is not longer in the other species. This confirms Strasburger's ('92) statement that germination takes place in Pinus at once after pollination. Hofmeister ('5 1) was doubtless unable to detect the early stages in the germination and hence was led to the con- clusion that pollination and germination were separated by several weeks in the AbiciinccB.
The pollen-grain increases slightly in size, the ventral or concave portfon of the wall becomes convex, then bulges out, the exospore is ruptured, and the endospore is gradually pro- longed into a tube. Immediately upon the formation of the
48 MARGARET C. FERGUSON
pollen-tube the tube-nucleus, as shown by Strasburger ('92) moves away from the antheridial cell and into the pollen-tube (figs. 75, 76, plate VII). According to Coulter and Chamber- lain ('01, page 92), the tube-nucleus does not enter the tube until the following April. That the tube-nucleus should at once loose its association with the antheridial cell and accompany the growing point of the pollen-tube is exactly what we should expect from what we know, through the investigations of Haberlandt ('87) and others, regarding the relation of the nucleus to growth ; and, also, judging from the standpoint of analogy, from the remarkable migrations of the tube-nucleus in order to be near the growing point of the pollen-tube in Cycas (Ikeno '98) and in Zamia (Webber '01).
Division of the Antheridial Cell. — Strasburger ('92) described the antheridial cell in Pinus sylvestris as remaining unchanged until the archegonia are formed in the following spring. Dixon states that it divides about a month before fertilization, but from a careful reading of the text one is given the impression that this was an inference on his part rather than a demonstrated fact, as he did not study material that was preserved earlier than April 24 and did not find the karyokinetic figure for this division. And, in so far as I am aware, this mitosis has not been observed in Pinus. Strasburger describes and figures it in Picea while the pollen-grain is still within the anther.^
I have found great variation in the time at which the anther- idial cell divides, not only in different species but in the same species. It is rather interesting that Pinus Strohus, which invariably lags somewhat behind the other species in all other developmental phases studied, is remarkably precocious as regards this step. Figs. 78, 80, and 81 were all taken from material of Pinus Strohus which was collected and pre- served on August 4, 1898, barel}' two months after pollina- tion. In the same material, other pollen-grains were observed in which the division of the antheridial cell had not yet taken place ; but in material fixed somewhat later it was rarely found undivided. The division of this cell has not been observed in Pinus austriaca, but two cells have been found in the pollen- grain in the middle of November and in February, and in such
' See note at close of appendix.
LIFE HISTORY OF PINUS 49
instances the tube-nucleus can invariably be detected in the pollen-tube. As pollen-grains containing but one cell were also observed in this species on these dates, it might be suggested that in the case of two cells the second prothal- lial cell had persisted. The two cells, however, are exactly similar to the stalk and the generative cell in their young con- dition, and I see no reason for considering that they are not these cells. On and after March 8 the antheridial cell of P. austriaca is almost never found undivided. This date is given for 1899; it would probably fluctuate in different years. Fig. 79 shows the prophase of this division in Finns rigida. Mi- totic figures for this species have been found from April 21 to May 13 of the same season. The division of the antheridial cell in Pinus resinosa has been observed but once, this division occurring on April 11. All that can be said at present regard- ing this mitosis in Pinus montatia var. uncinata is that the gener- ative cell and the stalk-cell are found as early as April 9. When they are formed has not been determined.
In one preparation of Pinus Strobus two of the three pollen- tubes which have almost reached the prothallium are furnished with sperm- and stalk-cells, while in the third only the tube- nucleus is found. On the apex of the nucellus there is a pollen-grain which at this late date contains one cell, the antheridial cell, still undivided (fig. 73). The nucleus of this pollen-grain (fig. 74) is large, plump, and to all appearances perfectly normal, and it is possible, though scarcely probable, that it might still have divided. That one cannot trace a defi- nite connection between the pollen-tube containing only the tube-nucleus and this pollen-grain signifies little, for those who have studied the pollen-tube of Pinus know that it is the excep- tion rather than the rule when a given pollen-tube can be traced through the lacerated dead tissue of the upper portion of the nucellus to the pollen-grain from which it proceeded. Such a condition as that described is rarely met with at so late a date ; but occasionally during the summer and fall pollen-grains of Pinus Strobus are found in which no cell-division has taken place since pollination, although in the great majority of cases
50 MARGARET C. FERGUSON
the stalk- and the generative cell have been formed before the middle of August.
These observations indicate that, while the division of the antheridial cell takes place comparatively soon after the pollen- grain has germinated in Pimis Strohus^ and in some instances, at least, before the winter's rest in P. austriaca, it is deferred until the following spring in Pinus rigida and P. I'esinosa. Furthermore, the time during which this cell may divide in a given species may extend over several weeks, and in some cases the division may never take place at all.
The Winter Condition. — A vertical section of an ovule of Pinus Strobtis collected on January 4 is represented in fig. 70, plate VI. The spongy tissue surrounds a cavity crossed by irregular strands of cytoplasm in which the free nuclei of the prothallium are imbedded. In this instance the prothallium has doubtless been displaced during fixation as it consists, normally, at this stage, of a uniform layer of cytoplasm surrounding the gametophytic vacuole and containing several nuclei. The stalk- and the generative cell are enclosed within the pollen- grain, and the tube-nucleus is near the apex of the irregularly branched pollen-tube. This pollen-tube is shown more highly magnified in fig. 83, plate VIII. At this time the pollen-tubes have penetrated the nucellus almost to the point at which it joins the free limb of the integument. The greatest depth to which the tubes may have grown is not indicated in the illustration, but this section was figured because it shows more clearly than any other section in the series the cells of the pollen-grain and the tube-nucleus. Other sections of the same ovule would have shown pollen-tubes which had pierced to a greater depth into the nucellus. The conditions of development as figured for Janu- ary coincide perfectly with those which exist during the latter part of October.
THE SECOND PERIOD OF GROWTH.
Renewed Activities in the Macrosporangitim. — Growth is very slow during the first period of development following pol- lination, but with the renewed activities of spring the ovule increases rapidly in size ; the central cavity of the nucellus
LIFE HISTORY OF PINUS 5^
becomes greatly enlarged and is lined with the growing endo- sperm. The cells of the nucellar cap which are penetrated by the pollen-tubes during the previous season do not again become active, but remain as deeply staining, thick-walled, dead cells. The cells just beneath them, however, multiply rapidly, and become literally packed with large starch-grains. A few of the cells from this portion of the nucellar cap represented in fig. 73, plate VII, are shown more highly magnified in fig. 89, plate VIII. By the growth and increase of these cells, the dead top of the nucellus with its pollen-tubes is lifted far above the developing endosperm, so that the pollen-tubes, once so near their goal, are now removed from it by a considerable distance (figs. 70-72, plate VI).
Renewed Activities in the Male Gametofhyte. — During the rapid development of the ovule in the spring, the pollen-tube increases little, if at all, in length, renewed activities in the male gametophyte being first indicated by a further development of the cells within the pollen-grain.
The stalk-cell increases in size and its cytoplasm assumes a vacuolate character. The growth of the generative cell is still more marked, and its cytoplasm on the contrary becomes dense and deeply staining. (Compare fig. 83, January 4, with fig. 84, May 3, plate VIII.) In Pinus syhestris, as studied by Dixon ('94) and confirmed by Coulter ('97) in Pimis Laricio, the gen- erative cell divides while it is within the pollen-grain. In the species of pines which I have investigated, this division does not occur until the generative and the stalk-cell have entered the pollen-tube and the stalk-cell has passed below the gen- erative cell. As the generative cell increases in size it stretches out towards and into the neck of the pollen-tube, drawing after it the stalk-cell, or possibly being forced out by that cell, the two passing into the tube together.
Dixon states that only the naked nucleus of the stalk-cell enters the polfen-tube, and in so far as I am aware, no writer has described the entrance of the entire stalk-cell into the pollen- tube in Pinus. The material which I have studied shows con- clusively that the nucleus does not '* slip out " of its cytoplasm (figs. 83-86). The entire cell can be identified in the tube and
52 MARGARET C. FERGUSON
later in the egg- During the time that this cell is moving over the generative cell its C3^toplasm cannot always be differentiated from that of the latter ; but when once the stalk-cell has passed the generative cell, its nucleus surrounded by a sphere of very vacuolate cytoplasm, scarcely more than a peripheral la3'er, is again distinctly demonstrated (figs. 90 and 91). After pass- ing the generative nucleus, the stalk-cell ordinarily takes up a position between the generative cell and the tube-nucleus (fig. 92), but occasionally it may pass the tube-nucleus (fig. 93). This phenomenon is always accompanied by a great in- crease in the starch content of the pollen-tube, the tube being in some instances almost filled with starch in the region of the generative cell (fig. 91).
When the generative cell leaves the pollen-grain, its nucleus is situated near the top of the cell, but the nucleus of this cell evidently moves faster than its cytoplasm, and at the time when the stalk-cell is passing over the generative nucleus this nucleus has come to lie at or below the center of its cell (fig. 84, 90 and 91). Shortly after this the generative nucleus is again observed at the uppermost part of its cytoplasm.
During its passage into the tube, the generative cell increases much in size ; it has no definite cell-wall, and its cytoplasm forms a large, irregular tongue about the nucleus. This cytoplasm in no way suggests the alveolar structure of Butschli ('94) but is distinctly reticular, differing in appearance from the nuclear net only by its greater delicacy. This is shown more clearly at a somewhat later stage.
The tube- and generative nuclei are now very similar in structure, though each is sufficiently characteristic to be readily recognized by one who is familiar with them. The tube-nucleus has one large, usually homogeneously staining nucleolus, rarely one or more smaller nucleoli, and it is furnished with a rather scanty, delicate reticulum which is apparently poor in chromatin. Either it is in a state of partial collapse, or, what is more prob- able, it is very hard to fix at this period in its history, for its outline is, as a rule, quite irregular at this time. The genera- tive nucleus has one large, hollow or vacuolate nucleolus, and commonly two smaller ones; its reticulum, though more abun-
LIFE HISTORY OF PINUS 53
dant than that of the tube-nucleus, is still delicate and often shows a weak reaction to nuclear stains. The stalk-nucleus has a very decided individuality which it maintains throughout its entire history. It bears a strong resemblance from the first to the nuclei of the nucellar tissue ; rarely, if ever, contains a true nucleolus ; and its close-meshed reticulum is conspicuous for its comparatively large net-knots or karyosomes.
Division of the Generative Nucleus. — Comparatively few students have occupied themselves with the growth of the pol- len-tube in the Abietincce, and no one, in so far as I have been able to determine, has described the cytological features attend- ing the formation of the sperm-nuclei in this group.
Dixon ('94) describes this division in Pinus sylvestris as tak- ing place about a month before fertilization^ while the genera- tive cell is still ivithin the pollen-grain ; and Coulter ('97) states, as already mentioned, that in his study of Pinus Laricio he has been able to confirm Dixon's observations in the minutest detail. At this time, as pointed out by Dixon, the nuclear and cytological phenomena are very greatly obscured by the pres- ence in the pollen-tube of large quantities of starch (fig. 91). The starch, which resists the microtome knife and is therefore easily displaced by it, not infrequently falls out and carries away with it the free cells of the pollen-tube. The dead, deeply staining tissue of the nucellus, representing that portion of the nucellar cap which was penetrated by the pollen-tubes during the previous season, and in which the generative nucleus divides (fig. 72, plate VI) is also very troublesome. Furthermore the dense cytoplasm of the generative cell has a great affinity for stains, so that when the archegonia and other portions of the ovule are well stained, this cell often appears merely as a deeply stained mass showing no differentiation of parts. Considering the fact that I was led not only to expect this division to take place within the pollen-grain but to search for it some weeks earlier than it actually occurs in the species of pines studied, together with the difficulties of staining, it is not surprising that seven hundred slides of serial sections were made, which means that more than two thousand pollen-tubes were studied, before any definite clue was obtained as to the
54 MARGARET C. FERGUSON
true sequence of events in the development of the pollen-tube. When once the mitotic figure was observed in the ^ollen-ttibcy scarcely more than a week before fertilization, and the fact noted that special staining was necessary in order to study this mitosis satisfactorily, further research was prosecuted with comparative ease. I find no authority in Dixon's paper for the statement recently made by Coulter and Chamberlain ('oi) which reads as follows: "The liberation and descent of the body cell into the tube," etc., " has recentl}?^ been described in detail by Dixon." What Dixon ('94) does affirm is this : " Verj' shortly after this it is found that the body-cell has broken free from the stalk-cell and has divided into two cells, which are almost equal in size. These cells are the male sexual cells. During this process the wall of the stalk-cell is ruptured and its nucleus follows the two cells resulting from the division of the body-cell which move into the pollen-tube." And throughout Dixon's paper there is no sentence that could be interpreted as implying that the body-cell ever passes into the pollen-tube before dividing to form the male sexual cells.
After the generative cell has passed into the pollen-tube but while it is still in the upper dead portion of the nucellus, it gives rise to the sperm-nuclei by a division which presents some new and interesting features, although it resembles to a greater or less degree certain mitoses described by various cytologists ^ during the past few years.
When the generative nucleus has again come to lie in the extreme upper portion of its cell, certain changes in the cyto- plasm indicate that division is being initiated. At some little distance below the nucleus the cytoplasm shows a finely granu- lar structure which is not at this stage dense nor deeply stain- ing. From this region irregular granular threads arise which extend outward tow^ards the periphery of the cell, those extend-
' Of the long list that might be mentioned I have noted only the following : Rosen ('95) in the root-tip of hyacinth; Ostcrhout ('97) in Equisetnm ; Swingle ('97) in Sp/iacelariacece ; Schaffner ('98) in root-tip of Allium Ccpa ; Mottier ('98) in the embryo-sac of Lilititn ; Fulmer ('98) in pine seedlings: Ilof ('98) in Ephedra and other plants; Nawaschin ('99') in Plasmodiophora ; Nemec ('98 and '99) in various plants; Strasburger ('00) in Vicia Faba ; Mottier ('00) in Dictyota; and Murrill ('00) in Tsuga. Of animal cytologists I mention but one, Hertwig, R. ('98) in Actinospkccrium.
LIFE HISTORY OF PINUS 55
ing in the direction of the nucleus forming a hollow cone over its lower portion (fig. 94, plate VIII). Gradually the granular area increases in density and in staining capacity, at the same time drawing nearer to the nucleus which is separated from it by a hyaline court. Into this court delicate granular threads pass (fig. 95, plate IX). When these threads reach the nuclear mem- brane, the nucleus is forced so closely against the peripheral layer of cytoplasm that its wall is frequently indented on the upper side, while the condensation from which the so-called kinoplasmic threads arise withdraws, or is forced by the growth of the threads, further from the nucleus. A great number of delicate anastomosing threads now extend, in the form of a solid cone, from a point within the granular condensation up towards and against the nucleus. The outer threads of the cone pass over the lower portion of the nucleus and appear in sections of the cell as closely packed against either side of the nucleus. At the same time the entire cytoplasmic reticulum has assumed a more or less radial arrangement about the condensed area in which the spindle-fibers arose and from which some of the more delicate threads extend into the surrounding cytoplasm (fig. 96).
Coordinately with these changes in the cytoplasm, the chro- matin of the nuclear net collects in spherical or irregular masses on the reticulum, and sooner or later gives rise to a broad spi- reme, along which the chromatic disks are distributed at regu- lar intervals (figs. 94-98). After the segregation of the chro- matin, there remains a delicate achromatic reticulum distributed throughout the nucleus. This reticulum is also granular like the chromatic network, but whether or not these granules rep- resent the oxychromatin-granules of Heidenhain ('93 and '94) I am unable to say. Webber ('01) has recently described and figured a similar achromatic network in the generative cell in Zamia. Whether the formation of the spireme precedes or fol- lows the penetration into the nuclear cavity of the achromatic threads seems to depend upon the length to which these threads attain. They may become very long when their entrance into the nucleus is delayed ; but more frequently a portion of the nuclear membrane gives way, and some of the achromatic
56 MARGARET C. FERGUSON
fibers pass into the nuclear cavity before the spireme is estab- lished (fig. 100). Rarely, the nuclear membrane appears pushed in irregularly along its entire lower margin, as indicated in figs. 96 and 98 ; as a rule, however, there seems to be one deep, sharp indentation along one side of which the nuclear wall first gives way (figs. 99 and 100). With the initial steps in the disappearance of the nuclear membrane the nucleolus is either not apparent or, if still demonstrable, it stains but feebly. When the membrane disappears along the entire lower portion of the nucleus, the kinoplasmic threads press so closely against it that it can not be definitely demonstrated whether it passes into the cytoplasmic and the nuclear reticulum or becomes fib- rous and contributes to the formation of the achromatic threads (figs. loi and 102). The threads which have been packed so closely against the wall of the nucleus now press into the nuclear cavity and mingle with those which have entered from below. And the dense, granular, cytoplasmic area from which the threads diverge is gradually dissipated (fig. 103).
With the disappearance of the wall along the lower part of the nucleus, the achromatic nuclear network seems to undergo a partial rearrangement. A portion of it is resolved into granu- lar threads of more or less regularity which, in general, assume a position parallel to the threads entering the nuclear cavity ; some of them become attached directly to the ends of these fibers, lose their granular appearance and doubtless contribute to the growth of the elongating spindle-threads.
As the spindle-fibers proceed in their development across the nucleus the chromatic spireme collects in the region of the future equatorial plate, and becomes more or less massed together. At the same time it assumes an homogeneous aspect and gives rise by segmentation to the chromosomes (figs. 101-104). Some of the ingrowing spindle-threads may extend across the nucleus to the nuclear membrane, which is still present on the upper side of the nucleus, but by far the greater number unite some distance below this membrane to form several poles, thus giving rise to a diarch spindle which, like the karyokinetic figures occurring during the development of the pollen-grain is multi- polar at its upper extremity and unipolar, or nearly so, at its
LIFE HISTORY OF PINUS 57
lower end. Gradually the poles of the upper portion draw together, while the spindle is somewhat shortened Dy the lower extremity of the threads being again resolved into granules. Finally a true bipolar diarch spindle is formed with the V-shaped chromosomes oriented at the equatorial plate. Each pole termi- nates in a slight granular condensation. The upper pole has never been observed to reach the nuclear membrane, but fre- quently coarse granular threads extend from the pole to the membrane of the nucleus, and apparently act as supports for the upper pole (fig. 105, plate X). These are evidently formed by a rearrangement of the linin reticulum. The nuclear mem- brane persists along the upper side of the nucleus until the late telophase of the division (figs. 101-103, plate IX, and 104-107, plate X).
As the chromosomes pass to the poles the central spindle elongates, so that the daughter-nuclei are separated, as a rule, by a greater distance than the length of the original spindle. While this is characteristic of cell-division in general, it is occa- sionally much exaggerated here, the daughter-nuclei being apparently forced apart with considerable energy. The nucleus which occupies the position nearest to the micropylar end of the ovule often shows a deep indentation along its upper surface as if a resistance had been met with in the peripheral layer of cytoplasm (figs. 11 1, plate X, and 113, plate XI). Not infre- quently the upper nucleus is found almost entirely separated from the cytoplasm (fig. 112). This, however, maybe due to mechanical rupture during sectioning and staining. No cell-wall is ever formed, and in only one instance was a condensation of the spindle-threads in the region of the cell-plate observed (fig. no). The spindle may contract at or near its center during its dissolution, thus presenting the appearance of an hour-glass, or it may give rise to such a condition as that shown in fig. 113. These appearances, with various modifications, are not uncommon in this mitosis in Pinus. Hertwig ('98) describes and figures a very similar lengthening of the spindle-fibers in ActinosfhcBrium. He also finds that the elongating spindle finally bends along its median line so that the daughter-nuclei come to lie near together in very much the same way as that
Proc. Wash. Acad. Sci., July, 1904.
58 MARGARET C. FERGUSON
shown in fig. 113. I am unable to trace definitely the origin of this figure, but it is not improbable that it is caused by a con- traction of the cytoplasm resulting from the cessation of the force which effected the separation of the daughter-nuclei ; or it may be produced by the resistance which the peripheral layer of cytoplasm, along the outer surface of the upper nucleus, offers to the growing fibers, thereby forcing them back upon themselves as shown in the figure. When all traces of the spindle have disappeared, the two sperm-nuclei are surrounded by a common mass of cytoplasm, and there is never throughout the later history of this cell the least suggestion of a dividing wall.
The mitosis just described seems to be unique as regards the origin and development of the achromatic spindle. Hertwig's ('98) fig. 3, plate V, illustrating an early stage in the division to form the first polar body in AclinosphcBriti7Ji, bears a striking resemblance to the prophase of this mitosis as illustrated in fig. 95, plate IX, of this paper ; but the origin of the figure shown by Hertwig, and the later history of the division are very dissimilar to that of the karyokinesis under consideration. The most exaggerated instances of asymmetry in spindle-formation which I have found recorded as occuring in plants is that described and figured by Nemec ('99^) in Solamim tuberosum, and more recently by Murrill ('00) in the division of the central cell in Tsuga. In both these instances the nucleus lies at one side of the cell, and the spindle-fibers are very much more prominent on the free side of the nucleus than on the side adjacent to the cell-wall. In another paper Nemec ('99^) shows by experimenta- tion that the form of the figure which gives rise to the extra- nuclear spindle depends upon external forces or conditions. In obedience to the law established by Haberlandt ('87) we should expect to find the generative nucleus in that part of its cell which is nearest the growing point of the pollen-tube, rather than at the end more remote from it, and it may be that its passage from the lower to the upper side of the cell is due to the fact that the forces, instrumental in effecting the division, first become active at a point below the nucleus, and exert a repelling action on it. But I have at present no adequate explanation or theory to offer
LIFE HISTORY OF PINUS 59
regarding the position of this nucleus at the time of its division. Whether it is due to the origin of the karyokinetic figure, or whether the unusual method of division is attributable to the very eccentric position of the nucleus, I have not been able to determine. It is evident, hov^ever, that the position of the cfenerative nucleus at the time of its division is such that the spindle if extranuclear in origin must of necessity be unipolar, since there is no cytoplasm, or almost none, above the nucleus from which fibers could arise.
The blending of the linin reticulum with the cytoplasmic network after the disappearance of the lower portion of the nuclear membrane, and the relation of certain portions of the achromatic nuclear reticulum to the ingrowing fibers are such as to suggest an intimate relation between these structures. That the spindle-fibers which originate in the cytoplasm and apparently grow by a differentiation of its network are later fed by the linin of the achromatic nuclear reticulum, there seems little room for doubt. In fact, all the phenomena connected with this division indicate that we are dealing, not with per- sistent cell-constituents, but with different manifestations of one and the same thing. In a word, we find no evidence here of the presence in the cell of a definite kinoplasmic substance. I am aware that these observations are directly opposed to the views of the students of the Bonn laboratory, and many others of the highest authority ; but the relations of nucleus, spindle, and cytoplasm, not only in this division but in those to be described in connection with fertilization, are such, it seems to me, as to render no other conclusion in the case of these divis- ions in Piniis possible. In 1895 Farmer arrived at a similar decision regarding the origin of the spindle in spore-formation in the Uepaiicc^, and Farmer and Williams ('98) in a study of Fitctis " do not regard the kinoplasm as a persistent proto- plasmic structure, but as forming the visible expression of a certain phase of protoplasmic activity." Hertwig ('98) expresses himself as opposed to the view of a special spindle-forming sub- stance in the protoplasm, while Wilson ('99 and '00) states that the astral rays " grow by a progressive differentiation out of the general cytoplasmic meshwork," and he finds in the echino-
6o MARGARET C. FERGUSON
derm's egg " no ground for a specific kinoplasm." The term, however, is a convenient one and may be employed consistently, as suggested by JNIottier ('oo), by those who do not find in kino- plasm a morphological constituent of the cell, as descriptive of that portion or manifestation of the protoplasm which is active in spindle-formation.
Nothing has been said regarding the nature of the granular, cytoplasmic condensation from which the achromatic spindle takes its origin. It never has a definite boundary, though it is often very clearly differentiated by its dense granular appear- ance and its strong affinity for stains ; but at certain stages in the division it may be inconspicuous or fail entirely of demonstration. Such a vast amount of literature has accumulated during the past decade regarding the nature and existence of the centrosome and the centrosphere that one feels inclnied to avoid the subject alto- gether. Yet the question may very properly be asked : Is this condensation which forms the center of a system of radiating fibers a centrosphere? It certainly is as clearly an attraction- sphere as some bodies which have been described as such ; but if we accept Wilson's ('oo) definition of the centrosphere, the body under consideration cannot be so denominated, as no cen- trosome has been observed at its center. More deeply staining granules may sometimes be present within the condensation, but these are not considered of any special significance as such granules may be found anvwhere in the cytoplasm.
Karsten ('93) describes the nucleoli in Psilotum as passing out of the nucleus and assuming the role of centrosomes, and Strasburger ('00) considers that the nucleoli not only contribute material for the formation of kinoplasmic threads, but that they also make active the spindle-forming substance in the cytoplasm — in other words, they act as the kinetic centers of the cell. There seems to be no evidence that such is the case here, for the nucleoli, after the condensation has arisen and the spindle- threads have attained considerable length, are morphologically the same as they were before the inception of the spindle. Neniec ('99') remarks that in the higher plants, where the cen- trosome is not demonstrablv present, the entire nucleus may exercise llie function of the centrosome. The idea of a diffused
LIFE HISTORY OF PINUS 6l
centrosome in the cells of the higher plants was suggested by Guignard in 1897 and was again hinted at by Le Dantec in 1899. If we may accept Guignard's suggestion, then the kinetic center of the cell in the higher plants is no longer indicated by the presence of a definite organ, the centrosome, but the power of this organ has become dissipated throughout the entire cell. When that phase of cell-activity which has to do with spindle- formation comes into play, the points at which it is centered would naturally be indicated by a greater accumulation of the microsomes, and thus an aster of more or less definiteness would be formed, as when the individualized centrosome is present. In the division of the generative nucleus in Pmits, the position of the nucleus is such that the energy active in spindle-forma- tion must perforce, if external to the nucleus, be centered at some point below it. Such a centering of the activity would naturally result in an attraction-sphere of unusual prominence ; and there would be no occasion for its division since there is not sufficient space above the nucleus for the organization of kino- plasmic threads.
When these studies were undertaken, it was thought that it would be interesting to determine whether any suggestions or remnants of a cilia-forming body (called blepharoplast by Webber in Zamid) still persist in the Conifers. Somewhat later, after the present research was begun, MacMillan ('98) pointed out the desirability of such a study both in Conifer<^ and Gnetales. I have seen no indication of a structure which might be regarded as a reduced blepharoplast, or as suggestive of a cilia-forming body of any sort in connection with the formation of the sperm- nuclei in Pinus. Inasmuch as spermatozoids do not exist here, such an organ, if present, must be functionless. But the cyto- plasmic radiations which accompany the division of the genera- tive nucleus in its early stages seem to differ in degree only from those found by Webber ('97) in the generative cell of Zamia. If we compare figs. 3 and 5 of Webber's paper with figs. 96 and 97, plate IX, of this paper, the question may be raised whether in this cytoplasmic figure we may not have still persisting in the cell the last vestiges of such an organ as that described by Webber.
62 MARGARET C. FERGUSON
The endosperm has become a solid mass of tissue at the time when the generative nucleus divides. The archegonia are still comparatively small and quite vacuolate and the central cell has not yet divided (fig. 72, plate VI).
Grozvth of the Sperm-nuclei. — After the mitotic figure has entirely disappeared, the sperm-nuclei are separated by a con- siderable distance. The form assumed by the cytoplasm sur- rounding them seems to vary with the shape of the pollen-tube. Gradually the two nuclei approach each other until they come to lie in the extreme uppermost part of their cytoplasm (figs. 112, plate X, 117, 118, plate XI). There is now considerable differ- ence in their size. This inequality in size could be detected as far back as the formation of the daughter-nuclei (figs. 109, no, plate X). Belajeff ('91) was the first to figure and describe bi- nucleated sperm-cells in the Gymnosperms. Coulter and Cham- berlain ('01), page 94, cite Belajeff as having observed an unequal division of the generative cell in Taxtis^ the larger male cell func- tioning, the smaller one remaining in the tube. But if I translate the German correctly, what Belajeff says is that the nucleus of the generative cell divides forming two nuclei which are about one-half as large as the nucleus from which they were derived ; one nucleus becomes larger and occupies a central position in the plasma, the other nucleus is flattened and remains at the periphery of the cell on its upper side ; the flattened nucleus was never found surrounded by its own plasma, but in the same plasma with the spherical nucleus. This is exactly the condi- tion shown in Belajeff's figures, one of which is reproduced by Coulter and Chamberlain. Jager ('99), however, has shown two dissimilar sperm-cells in Taxus, the larger one in advance, but he finds that occasionally the nucleus of the smaller cell may exceed in volume that of the larger one. Jaccard ('94) found two sperm-nuclei of the same size in Ephedra both sur- rounded by the same mass of cytoplasm, and Coker ('02) has re- cently described the sperm-cell in Podocarpus as binucleated, the smaller nucleus being above the larger and " thrust almost out of the cell." No one, I believe, except the writer (1901''""'^), has recorded the presence of a single binucleated sperm- cell in the AbietinecB. In his earlier studies of the Gymno-
LIFE HISTORY OF PINUS 63
sperms, Strasburger ('6g-^2) was unable to demonstrate, satis- factorily to himself, the character of the cells found in the pollen-tube in Pinus, and he has not recently investigated the male gametophyte in the Abietinece. Coulter ('97) described two sperm-cells which were of the same size until within the arche- gonium. Blackman ('98) stated that each sperm-nucleus was clearly seen in the pollen-tube surrounded by its own cytoplasm, but he did not figure them.^ Chamberlain ('99) figured the sperm-nuclei, in Pinus Lartcto, of equal size in the pollen-tube, and showed them lying together in the cytoplasm of the tube. Not having seen these cells within the archegonium before the conjugation of the sexual nuclei, he accepted Coulter's state- ment for the growth of one of them after their entrance into the egg. According to Coulter ('00) the " male cells in pines " are alike in size. The same figures are reproduced by Coulter and Chamberlain ('01).
As stated by the writer in 1901, two sperm-cells have not been observed in any of the pines which I have studied ; but the sperm -nuclei, which are of unequal size from a very early date, remain, while in the pollen-tube, surrounded by a common cyto- plasmic body (figs. 109-112, plate X; 113-118, plate XI, and 119-120, plate XII). As Strasburger ('92) observed, the larger nucleus is always ahead, that is, on the side nearest the apex of the pollen-tube. The smaller nucleus remains close against the upper boundary of the cytoplasm, and suggests the condition in Cycas (Ikeno '98) and Ginkgo (Hirase '98), where the stalk- nucleus is forced entirely out of the cytoplasm surrounding the generative nucleus. In the case of the smaller sperm-nucleus in Pimis, the action is not carried to so great an extent. Webber ('01) has recently shown that such an interpretation as that re- corded above for Cycas and Ginkgo is not true as regards the stalk-nucleus in Zamia. One very interesting preparation which I have obtained shows the smaller sperm-nucleus in advance of the larger (fig. 114). Here it will be seen that the entire order of arrangement has been changed, the stalk-cell and the tube- nucleus being above the sperm-cell. But this abnormal arrange- ment is onl}'- apparent, for it was found that the ^^^ which had
* See note at close of Appendix.
64 MARGARET C. FERGUSON
been approached by this pollen-tube had already been fertilized, and the pollen-tube had turned aside and was passing up over the top of the endosperm, as if seeking for another egg. The position of the various elements of the pollen-tube is therefore normal, the larger sperm-nucleus being in reality in advance of the smaller. This suggests that, w^hen a pollen-tube has con- jugated with the egg, a substance may be secreted which repels other pullen-tubes, as has been described in case of spermato- zoids in the Bryophytes and Pteridophytes.
The formation of the sperm-nuclei shows most beautifully the manner of the development of the nuclear reticulum. The chromosomes unite end to end, giving rise to a homogeneous, coiled band, before the nuclear membrane is formed. When the nuclear-wall has been differentiated, the coil expands about the periphery of the nucleus, while the band broadens, at the same time becoming irregularly jagged along its margins. These irregularities increase in length until finally those from adjacent threads meet and fuse, thus giving rise to the reticulum (figs. 107-110, plate X). When the sperm-nuclei have nearly or quite come into contact they have as a rule reached their ma- ture size. More than a year has now elapsed since pollination.
Elongation of the Pollen-tube. — Up to this time the pollen- tube has elongated very slowly, having penetrated as yet little, if any, beyond the nucellar tissue of the previous year's growth. In this upper portion of the nucellar cap the tube may become very broad, or it may branch freely (figs. 71, 72, plate VI, and 83, 87, plate VIII). When the sperm-nuclei have attained their full size, the downward growth of the tube is exceedingly rapid, travelling in from eight to ten days more than twice the distance traversed during the entire preceding year. The path pursued during this rapid growth is comparatively straight and the tube is unbranched (fig. 73, plate VII). In Pinus Strobus^ P. rigida and P. mistriaca about ten days intervene between the division of the generative nucleus and fertilization ; in Pinus ino7itana unci^iata, the two processes are separated by an even shorter space of time.
The sperm-nuclei which at first present a very beautiful, rather delicate reticulum (figs. 112, plate X, 117, plate XI), become
LIFE HISTORY OF PINUS 65
more dense as the pollen-tube advances through the nucellus. Strasburger ('92) describes them as coarsely granular ; but, with a high power, the presence of a reticulum which is sometimes coarse and interrupted can invariably be made out in well pre- pared material. By the time that these nuclei have reached in their downward course the central portion of the nucellar cap they have usually become very dense in structure (figs. 115 and 116), and frequently stain intensely, though they may show at this time a weak reaction to dyes. The reticula of the two nuclei may present the same appearance, or they may differ as in the figures referred to above. The nucleolus, if it be present at this time, is usually obscured by the dense network. Arnold! ('00) described the sperm-nuclei in Cephaloiaxus as being grad- ually filled with metaplasm. I find no evidence of such a proc- ess in the development of these nuclei in Pinus.
Archoplasmic areas similar to those figured by Chamberlain ('99) have been observed in connection with the sperm-nuclei, but as such granular accumulations may occur at any point in the cytoplasm of the sperm-cells no importance is attached to them.
When the pollen-tube reaches the egg-, its apex is abundantly supplied with cytoplasm, in the upper part of which the tube- nucleus lies. The sperm-cell is just above with the stalk-cell still in contact with the lower portion of its cytoplasm (fig. 120, plate XII). Still higher up the tube may contain many starch- grains. There is never any doubt at this time as to the identity of the stalk-cell and the tube-nucleus in the material which I have studied. Yet Dixon ('94) states that they cannot be distin- guished, and Coulter ('97) describes them as having lost their original outline.
As many as six pollen-tubes have been found making their way through the same nucellus, but, as a rule, not more than three pollen-tubes renew their growth during the second season, and frequently only two penetrate to the endosperm. The effect of the pollen-tubes upon the upper part of the nucellar tissue is very marked. The cells in the immediate vicinity of the branched pollen-tubes early lose their protoplasmic contents and their walls become crushed and broken. Those cells more
66 MARGARET C. FERGUSON
remote from the tubes do not suffer so severely, and retain their protoplasm for a much longer time. Finally all the cells representing the first year's growth of the nucellar tip loose their content to a greater or less degree, and their cell-walls become thickened and dead. During the rapid growth of the pollen-tubes through that portion of the nucellar cap which develops the second season, the effect of the tubes on the sur- rounding tissue is less marked, though here, too, the cells with which they come into contact are crushed and destroyed (fig. 73, plate VII). I have made no physiological investigations regarding the action of these tubes on the tissue of the nucellus, but, judging from the disappearance of the starch in the cells just in advance of the tubes and the gradual disintegration of those cells, it seems very probable that the destruction of tissue is not due to mechanical reasons alone, but to the action of some ferment or digestive substance as well. Various views have been expressed concerning the action of the pollen-tube and the directive agent in its growth by Molisch ('93), Miyoshi ('94), Lidforss ('99) and others, but we are still far from a clear under- standing as to the controlling factor in the movement. The pollen-tube cannot be guided to the egg in Pimis by any peculiar attraction existing between the sexual cells, for it grows with normal rapidity when no sperm-cells are formed, and also when the archegonia are in a state of disintegration.
SUMMARY.
Upon the germination of the microspore, three divisions fol- low in rapid succession giving rise to the pollen-grain. At the close of the prophase of each division the karyokinetic figure is pointed at its lower extremity and very broad at the extremity in contact with the dorsal side of the young pollen-grain. The inner, incomplete, thick wall formed in the development of the microspore persists as a part of the mature pollen-grain. It probably serves as a strengthening layer, particularly at those points at which the wall has been weakened b}"^ the expansion of the exospore. When the telophase of the second division is reached the first prothallial cell has become flattened against the convex side of the spore-wall, its cytoplasm has been withdrawn,
LIFE HISTORY OF PINUS 6'J
andthe nucleus has lost all signs of its former structure remain- ing as a much flattened, deeply staining mass. At the close of the third division, the second prothallial cell has suffered a simi- lar fate. Both prothallial cells are furnished with cellulose-walls.
In the mature pollen-grain the prothallial cells are usually represented by two broken, dark lines along the dorsal side of the pollen-grain, but all vestiges of the first cell may have dis- appeared. The antheridial cell projects from the convex side of the spore at its middle point, and the tube-nucleus is always directly below but in contact with the antheridial cell. Starch is found in the pollen-grain at maturity and during its develop- ment.
Pollination takes place between 42° and 43° north latitude during the latter part of May or the first ten days in June. At this time the macrospore-mother-cell is distinctly visible in the center of the ovule, but slightly nearer its basal end.
In the young ovule the free portion of the integument, above the tip of the nucellus, consists in cross-section of three layers of cells. After pollination the arms of the integument become erect, thus bringing the pollen-grains into the wide micropylar canal. Then the inner layer of cells just above the pollen- grains elongates rapidly, extending inwards and meeting at the center. The pollen-grains having thus been made secure, the elongated cells become divided into many small cells. It is felt that the pit in the apex of the ovule in Piniis has been ex- aggerated. There is rarely more than a slight concavity before pollination. Through the action of the pollen-tubes it may be somewhat deepened, but in normal conditions it does not become " cup-like."
Two days after pollination, in Pinus rigida, the pollen-tubes have been emitted. In the other species germination has been shown to take place in less than a week after pollination, but more exact data have not been obtained for these species. As soon as the pollen-grain has germinated, the tube-nucleus severs its connection with the antheridial cell and moves into the elon- gating tube.
The division of the antheridial cell takes place in Pinus Strobus during the first week in August. It sometimes divides
68 MARGARET C. FERGUSON
during the summer and fall in P. aush'iaca^ but, as a rule, the division takes place in this species very early in March. This mitosis has been observed in P. resinosa during the second week of April, and in P. rigida from the middle of April to the middle of May. It is evident that this cell does not always divide at a definite and fixed time, but that in a given species the time during which it may divide extends over a considerable period.
During the first season the pollen-tube grows very slowly, and it may be broad and irregular in outline or it may branch freely.
Shortly before fertilization the generative cell, followed by the stalk-cell, moves into the pollen-tube. The stalk-cell soon passes the generative cell and takes up a position near the tube- nucleus. These changes and those immediately following are frequently much obscured by the presence in the pollen-tube of large quantities of starch.
When the macrosporangium enters upon the winter's rest, the pollen-tubes have penetrated nearly to the line at which the in- tegument becomes free from the nucellus and the tube-nucleus maintains its position in the apex of the pollen-tube.
The generative cell is never limited by a well-defined cell- wall, and consists at the time of its division of an irregular pro- toplasmic body in the upper part of which the nucleus lies.
In the division of the generative nucleus the spindle is extra- uuclear arid unipolar in origin, a unique and heretofore unob- served method of division.
The formation of the spindle indicates that the cytoplasmic network and the nuclear reticulum have essentially the same structure, and the spindle-fibers are apparently formed by a transformation of both. The nuclear membrane persists along the upper part of the nucleus until the earl}^ stages in the forma- tion of the daughter-nuclei. This division takes place a little more than a year after pollination and from a week to ten days before fertilization, nearly thirteen months elapsing between pol- lination and fertilization.
Two sperm-cells are never formed, but the sperm-nuclei remain surrounded by a common mass of cytoplasm. An in-
LIFE HISTORY OF PINUS 69
equality in the size of these nuclei is very early apparent, and becomes more pronounced as they reach maturity. The sperm- nuclei soon come to lie together in the upper part of their cyto- plasm and quickly attain their full size, the larger one being invariably in advance. The nuclear reticulum, at first delicate, soon becomes very dense, but there is no evidence of the pres- ence in these nuclei of a special metaplasmic substance.
During the division of the generative nucleus the ovule in- creases much in size, and the nucellar cap becomes several times deeper than during the first season, thus carrying the upper portion of the nucellus with its pollen-tubes far above the endosperm.
At the time when the sperm-nuclei come into contact, or nearly so, the pollen-tube has penetrated little, if at all, beyond the nucellar tissue of the first year's growth. Now, however, it again begins to elongate, and its downward course through the new nucellar tissue is extremely rapid. The destruction of the nucellar tissue through which the pollen-tubes travel, ap- parently results not only from mechanical disturbances, but from the entire dissolution of some of the cells through the action of a ferment.
When just above the egg, the apex of the pollen-tube is filled with cytoplasm. The tube-nucleus lies in the upper part of the cytoplasm, and near it is seen the stalk-cell still in contact with the lower portion of the cytoplasm which surrounds the sperm- nuclei.
The existence of the diffused centrosome is suggested in con- nection with the division of the generative nucleus, and there is a possibility that, in the prominent cytoplasmic figure from which the spindle takes its origin, we may have represented, in its vestigial state, the cilia-forming body found In the lower Gymnosperms.
70 MARGARET C. FERGUSON
CHAPTER III. Macrosporogenesis. the female cone.
The Macrosforangiuni. — During this investigation I have made no attempt to study the early development of the ovule except to note definitely the date of its origin. The pistillate strobili cannot be detected in Pimis Strobiis with the most careful examination until the last of April or the first of May. In the other species studied they are about one and one-half milli- meters long at the middle of March, and it is possible that in these species they were organized in the autumn, but I have not been able to find any evidence that such is the case. I have recently, November 25, 1902, attempted to discover the young cones of Pinus rigida and P. atcstri'aca, but, as formerly, the search was futile. I was led to look again for these strobili in the autumn by the recent statement of Coulter and Chamberlain ('01). On page 79 of their book on the morphology of the Gymnosperms, I find this sentence, based on a study of Pinus Lai'icio : ** In June the archegonia are ready for fertilization, which occurs about the first of July, at least twenty-one months after the first organization of the ovule." This by a very simple mathematical calculation places the " organization of the ovules " on October i.
I have not only been unable to detect the pistillate cones before the approach of winter, but in tlie tin}^ cones of Piniis rigida^ P. aitslriaca and P. montana uncinata, fixed on March 14 there is not the least suggestion of ovules, the entire cone consisting in each case of a broad axis on the margin of which are slight elevations or papillaj — the beginnings of the bracts which subtend the ovuliferous scales (fig. 121, plate XII). The first indications of the ovules are found in these species about the last of April or the first of May. In material of Pinus Strobus fixed on May 31, 1898, the position of the ovule can be detected only by a slight bulge on the inner sur- face of the ovuliferous scale, the integument not 3'et having been differentiated. One week later, June 6, the ovule is
LIFE HISTORY OF PINUS 7 1
found fully organized and nearly ready for the reception of the pollen-grains (ligs. 122, and 123). The evidence is conclusive that the ovules are not organized in the species of pines studied by the writer until about three weeks or less before pollination, and seven months later than in Pimis Laricio as recorded by Coulter and Chamberlain. This is the more surpris- ing when we consider that P. aiistriaca is at least a variety of P, Laricio, and, according to some authorities, it is a synonym for that species.
It is not my purpose to enter into a discussion of the origin and cellular development of the female cone, nor yet of the homologies of its parts. These points have been fully investi- gated by Celakovsky, who has frequently published papers on this subject from 1879 to the present time, and the many theories advanced by different writers regarding these structures have recently been brought together and reviewed by Worsdell ('00).
FORMATION OF THE AXIAL ROW.
The Macrosfore-iuother-cell. — The origin of the sporog- enous tissue from a hypodermal cell or cells was described by Strasburger for several Gymnosperms in 1879, and this idea without further confirmation has come down to the present time. While this may be true for many Gymnosperms, and possibly for Pimis, I find no evidence, direct or indirect, that the macro- spore-mother-cell is derived from a hypodermal cell in the pines investigated. When the mother-cell is sufficiently differentiated to be distinguishable from the other cells of the surrounding tissue, it is found to lie deep within the nucellus ; and there are no rows or axial strands of cells lying above it to suggest its derivation from a hypodermal cell. On May 8, 1902, the ovules of Pinus rigida were sufficiently developed to show clearly the separation into nucellus and integument, and a like condition was found to exist in P. Sirobits on June 6, 1898. In both instances, so far as one is capable of determining, every cell of the nu- cellus is exactly like every other cell (fig. 123), and the same condition obtains in the other species at this time. One week later, as illustrated for Pinus rigida, the macrospore- mother-cell can first be distinguished, and the so-called spongy
72 MARGARET C. FERGUSON
tissue is clearly differentiated about it (fig. 124). The mother- cell in this instance has relatively the same position in the ovule as that shown in fig. 66, plate VI, which was taken from an ovule collected twelve days later. If this cell be the direct de- scendant of a hypodermal cell, it has now become deep-seated by the addition of cells above it ; but there is nothing in the arrangement of the cells of the nucellus either before the appearance of the mother-cell or after it to denote such a course of development.
The mother-cell is first detected by its larger size and b}- its failure to stain as deeply as do the other cells of the nucellus. In the first stages of growth the nucleus almost fills the cell (fig. 125), and its weakened capacity for staining is doubtless due to its rapid growth without a proportional increase in the amount of nuclear substance. The nucleus contains in this young stage a delicate reticulum with a varying number of larger and smaller net-knots, and from two to four small nucleoli, not differing materially, except in size and staining power, from the nuclei of the adjacent tissue. This cell in- creases considerably in size before its division so that it becomes very conspicuous in the nucellus, its reticulum taking the chro- matin-stains with greater avidity than at an earlier period. The season of growth for the macrospore-mother-cell may extend over about three weeks. The early stage shown in figs. 124 and 125 represent its size on May 15, 1902, and the spireme stage illustrated in fig. 126 indicates the condition of this cell on June 5 of the same year.
First Division of the Macrosporc-moiher-ccU. — After the mother-cell has attained its full size, the reticulum of the resting nucleus gradually becomes more open, the chromatic granules become more prominent and there arises a beauti- ful, regularly moniliformed, more or less interrupted skein, but a true spireme is not formed until after synapsis (lig. 126). This somewhat branched thread is very delicate, the chro- matic discs are uniform in size and distributed upon the linin with great regularity. It is probable that these apparently homogeneous discs, which have doubtless been derived from the fusion of the smaller chromatic granules, would, under
LIFE HISTORY OF PINUS 73
greater magnification, be resolved into slightl}' irregular and roughened bodies, as in the prophase of the heterotypical mitosis in the microspore-mother-cells, but with the powers of the micro- scope at my command, I have no evidence that such is the case.
The phenomenon of synapsis is as marked here as in the primary mitosis of the microspore-mother-cell, but the contracted mass is less dense, probably because of the smaller size of the nucleus and the consequent diminution in nuclear substance (fig. 127, plate XIII). With the recovery from synapsis the linin thread is seen to have increased in thickness, and the chromatin- granules are irregularly distributed upon the continuous spireme, which gradually comes to fill the entire nuclear cavity with its open uninterrupted coils (figs. 128 and 129). The chro- matic substance again collects into definite areas of varying dimensions, which are united by clear portions of the linin- band, and the longitudinal splitting now becomes apparent. Condensation and segmentation follow, and the distinct chro- mosomes, in the reduced number, become evident (figs. 130, 132 and 133). The forms of the chromosomes are similar to those already described in connection with the division of the microspore-mother-cell (figs. 132-136). Because of the com- paratively small size of these nuclei, the steps by which the irregularly shaped chromosomes are derived could not be traced with the same degree of confidence as in the microspore-mother- cell ; but the entire phenomenon is such as to indicate very con- clusively that the process is practically the same in both.
The spindle, at first a multipolar diarch, early becomes bi- polar and during metakinesis it is very sharply so. The poles do not reach the walls of the cell, but a few threads sometimes radiate from them and extend to the ectoplasm. There may be a slight granular .condensation in the neighborhood of the poles but it is never prominent and often does not appear at all. The chromatic segments become short and broad at the equa- torial plate, and their separation into daughter-chromosomes presents the figure characteristic of the heterotypic division. Unsplit ends of the chromosomes extend outward in the plane of the equatorial plate, thus giving rise to dark clumps of chro- matic substance along the median line (figs. 134-137). The
Proc. Wash. Acad. Sci., July, 1904.
74 MARGARET C. FERGUSON
passage of the one-half chromosomes to the poles has not been observed. Resting nuclei are formed during the telophase of the mitosis, and a cross wall divides the mother-cell into two compartments (fig. 138).
From the foregoing it is evident that the first division which takes place in the macrospore-mother-cell is heterotypic in nature, and agrees in all essentials with the primary mitosis within the microspore-mother-cell. This is in accordance with the conclusions reached by all other investigators who have recently studied the tetrad divisions occurring within the ovules of various Phanerogams.
Second Division of the Macrospore-juother-cell. — Beginning with the telophase of the first division considerable variation may occur in the subsequent steps in the formation of the axial row. A cell-plate is always formed between the daughter- nuclei though it may remain very delicate, consisting of little more than a condensation of the ectoplasm. The daughter- cells may be very similar in appearance, excepting that the lower one is usually the larger, and in such instances both nuclei enter the resting stage, presenting a clear, definite reticulum (figs. 138, and 141). More often, however, the lower cell is much larger than the upper one and the nucleus of the upper cell does not enter into the complete resting stage, but early shows signs of disintegration. The chromosomes may unite to form a spireme as usual, but development may then cease without the organization of a network, and the diffuse reaction of the nucleus to stains shows that disintegration has begun (figs. 139, 140).
I have but a single preparation showing the second division of the macrospore-mother-cell, and I can therefore offer no con- clusions of any value regarding the nuclear phenomena accom- panying the mitosis. From this figure it appears that the spindle originates as a multipolar diarch as in the first division, and both nuclei in this instance are dividing at the same time. During the initiation of the spindle the chromosomes are short and thick, somewhat irregular in outline, and apparently in the forms of U's, V's and rings. The reduced number of chromo- somes occurs in both of the dividing nuclei (fig. 142).
LIFE HISTORY OF PINUS 75
The state of disintegration referred to above is always con- fined to the upper of the two daughter-cells and never occurs in the lower one, except in those cases in which the whole ovule is undergoing destruction. The lower cell invariably divides again and the basal cell thus formed constitutes, in every instance observed, the functional macrospore. The lack of constancy in the division of the upper cell would naturally give rise to some axial rows of four cells and some of three, and this is exactly what we find (figs. 144, 145, plate XIV). Fig. 143 shows the second division of the lower cell just completed, and it is evi- dent from the structure and appearance of the uppermost nucleus that it would never have divided. In the axial row presented in fig. 144 some time has elapsed since the mitosis was completed, as evidenced by the increase in size of the lowest cell of the row. The upper of the two cells formed as a result of the first mitosis still remains undivided, and, moreover, it would not have divided later, judging both from its appearance and from the fact that the rapid growth of the initial cell of the female gametophyte would soon have been instrumental in effecting its obliteration. Juel ('00) finds that these cells do not divide simultaneously in Larix^ but he does not find the division completed in the lower cell before it begins in the upper one. In the single preparation showing the second division in the macrospore-mother-cell, both nuclei are dividing, and both are in the same stage of the prophase, but this does not necessarily mean that when both cells divide they always do so synchronously. This lack of uniformity in the number of cells in the axial row is not peculiar to Pinus ; it has been observed by many investigators in a large number of plants including both Gymnosperms and Angiosperms.
Coulter and Chamberlain ('01) figure an axial row of four cells in Piniis Laricio, and, as above indicated, such an axial row is frequently met with in the species of pines which I have studied, but it is much more common in Piniis ausiriaca than in the other species (figs. 145, plate XIV, 142, plate XIII, and 261, plate XXIII). There is no doubt whatever, after a study of many preparations showing the axial row, that in the great majority of cases in Pinus Strobus and P. rigida the upper cell remains undivided and that the usual axial row in these species
76 MARGARET C. FERGUSON
consists of three cells. The axial row represented in fig. 144, for instance, is a beautiful object, clearly and definitely differentiated from the surrounding tissue, 3^et there is not the least ground for supposing that the upper cell has ever divided. Such a figure as this represents the characteristic axial row in Pinus Strobiis and P. rtgida, while the axial row of four cells illus- trated in fig. 145 is typical for P. austriaca. This point has not been sufficiently studied in the two other species to admit of generalizations for them. The axial row, then, varies from three to four cells in the same species, but there is a tendency in some species to form three and in others to form four cells.
Significance of the Tetrad DivisionWithin the Ovule. — We have observed that at a certain point in the development of the ovule in Pinus a centrally located cell becomes differentiated from those surrounding it by its greater size and the more vacuolate character of its cytoplasm. This cell after under- going a period of growth and rest gives rise to the reduced number of chromosomes by a peculiar method of division known as the heterotypical division, and this mitosis, as is characteris- tic in spore formation, is quickly followed by a second division, at least in the lower cell. The basal cell resulting from this last division passes through a season of growth extending over several weeks, as we shall shortly see, and finally, by repeated divisions, gives rise to the female gametophyte. The process of division is in all essentials exactly similar to that which takes place within the microspore-mother-cell, and results, as there, in spore-formation. Nuclear phenomena attending the early development of the female gametophyte have not been carefully investigated until comparatively recent times, but wherever studied the conclusion has been unhesitatingly drawn that in the ovule, as within the anther, a true spore-formation takes place.
The essential character of a spore is, manifestly, not that it should have a certain arrangement relative to its sisters within the mother-wall, neither is the presence or absence of a wall of vital importance to its existence unless, indeed, the spore is to be disseminated. Rosenberg ('01) finds the pollen-grains to be filiform in Zostcra and arranged side by side ; Strasburger ('01)
LIFE HISTORY OF PINUS 77
and Gager ('02) show that the descendants of a pollen-mother- cell in Asclcpt'as have a linear arrangement ; while Juel ('00) discovers that in the Cyperaccce three young pollen-grains or microspores abort and the fourth remains permanently within the microspore-mother-wall. Yet from the standpoint of origin alone, no one hesitates to call the young pollen-grains of these plants microspores. Juel ('00) affirms that the heterotypic divi- sion must be the criterion by which we decide whether or no we have a true tetrad-division, and he concludes that in Larix the embr3^o-sac-mother-cell is homologous with a spore or a micro- spore-mother-cell. Schniewind-Thies ('01) reaches the same conclusion for Angiosperms ; and Lloyd ('01) asserts that the division of the embryo-sac-mother-cell in the RubiacecB is a true tetrad-division, and the four resultant cells are spores. Other instances where similar conclusions have been reached might be cited, but the above is sufficient to demonstrate that the most recent studies along this line point conclusively to a normal spore-formation within the ovule, and do not confirm Campbell's ('02) statement that a true tetrad-division is usually absent in the ovule of spermatophytes.
For many years botanists have been involved in a contention regarding the true nature of the embryo-sac in Phanerogams. A paper was published by Atkinson in 1901 reviewing the interpretations made by earlier writers and suggesting as a solution of the difficulty that spores, no longer being necessary in the higher plants, had dropped out of the cycle of develop- ment in these plants. That is, the female gametophyte arises in the higher plants without the intervention of spores. While the results of recent investigations do not serve to strengthen this view, the theory is a most interesting one and the paper has further served an excellent end in stimulating thought and research along this line. Mottier observed one instance in which the first division of the embryo-sac-mother-cell was homo- typic, or, if we use Strasburger's ('00) term adopted through- out this paper, typical, and the number of chromosomes was not reduced. Juel found the same to be true normally in Anten- naria alpina, a species of Antennaria in which the embryo develops parthenogenically. In both instances we have an
78 MARGARET C. FERGUSON
illustration of development within the embryo-sac without the intervention of a spore, but these are apparently isolated and exceptional cases.
The whole difficulty seems to me to lie in the fact that all along we have been endeavoring to make a morphological unit out of that which is primarily a physiological unit, and not necessarily a morphological one, although it may be so. It has been shown conclusively that in Larix and Pinus among the Gymnosperms a true macrospore is formed which germinates within the macrosporangium and gives rise to the female gamet- ophyte — both a morphological and a physiological unit. But as we advance to the Angiosperms there is a shortening of on- togeny in the female gametophyte, the most extreme case being represented by Lilitim. Mottier ('98) demonstrated the fact that the division of the embryo-sac-mother-cell in Lilhim is a true tetrad division and we cannot, therefore, it seems to me, escape the conclusion that the resultant four cells are spores. But once rid ourselves of the idea descended from Hofmeister, that the mother-cell of the embryo-sac is always a macrospore, and the product of its development, therefore, always a single gametophyte, and many difficulties vanish. Lloyd ('02), in his recent discussion of this subject, accepts the heterotypical divi- sion as the criterion for spore formation, and then explains the condition in Lilium, where the first four cells of the embrj'o- sac are spores, by " regarding the gametophyte as an individ- ual by coalescence.'" It appears to me not only more simple but more plausible to consider that we have here four gameto- phytes each reduced to two cells. The embryo-sac is still here as elsewhere (with the exception of parthenogenic plants), a physiological unit whose function is to give rise to a new plant through the sexual process, but it is morphologically a complex made up of several individuals. Whether all eight cells thus formed are considered as potential eggs is immaterial, practi- cally, but one retains the power to respond to the sperm-cell, though the others have been shown to be capable of fertiliza- tion in some instances. Ordinarly, however, they remain ster- ile and have come to have a vegetative or nutritive function only. All work together for one end and in that sense may
LIFE HISTORY OF PINUS 79
make " an individual by coalescence," that is, they are physi- ologically one.
This is not the place to enter into a detailed discussion of the homologies of the embryo-sac, but I believe that the suggestion herein made will form an interesting working basis, and it may bring us nearer to a true conception of these structures than we have yet attained. But whatever our opinion regarding the ele- ments within the embryo-sac, it is clear that we cannot longer use the terms macrospore and embryo-sac interchangeably as so many writers have done. We now know that a tetrad division may occur within the ovule and it has been shown that the embryo-sac may result from the germination of a single macro- spore, that it may be formed directly from the macrospore- mother-cell, or that it may have its origin in one of the daughter- cells formed as the result of the heterotypical division. In any case would it not be far less confusing if we should designate the multicellular bodies, developed within the macrosporangium and the microsporangium of the higher plants, as embryo-sac and pollen-grain, or female and male gametophyte, respectively, and should retain the terms macrospore and microspore for the true spores in their one-celled stage?
LATER HISTORY OF THE AXIAL ROW.
The Fate of the Uffer Cells. — Whether the number of cells in the axial row of Pinus be three or four the female gameto- phyte is always the product of the lowest cell. Very shortly after the second division is completed, the upper cells of the axial row give evidence of disintegration, while the basal cell increases much in size, its nucleus becoming very large. The nuclei of the four spores in Larix are very similar, Juel ('oo), fig. i8, but in Pinus the basal cell is markedly different from the others at a very early date (figs. 144, 145, plate XIV). The upper cells of the axial row gradually disintegrate, and are crowded to one side by the growth of the macrospore, remain- ing for a time as deeply staining, amorphous masses which finally disappear altogether (figs. 69, plate VI and 147, 148, plate XIV). Instances in which one of the upper cells of the axial row in Angiosperms becomes the functional macrospore
8o MARGARET C. FERGUSON
are not rare. Campbell ('oo) has recorded such a condition in the AracccB, Lloyd ('oi) in certain Rubiacece, and Karsten ('02) in the JuglandacecR. But, so far as investigated, the sequence of events following the establishment of the axial row in the AbietinecB results in the obliteration of all but the lowest cell. I have avoided using the term " potential macrospore " in con- nection with the upper cells of the axial row, because the upper of the two cells first formed does not always divide and in such instances it cannot properly be designated as a spore since development ceased before spore formation was completed.
Growth of the Macrospore. — Starch is sometimes found within the cells of the axial row, though never in such abundance as in the cells of the adjacent tissue (fig. 143). It may become very abundant within the macrospore during its period of growth, and is sometimes found pressed so closely against the nucleus as to actually produce indentations in its membrane (fig. 146).
The reticulum of the nucleus of the functional spore is very scanty during its growth period, but later it presents the appear- ance of an ordinary resting nucleus. The cytoplasm, never abundant, forms at an early date a loose, granular network. Later the nucleus is connected with the ectoplasm by delicate strands which are gradually withdrawn into the peripheral cyto- plasm, until there is thus formed in the one-celled stage a definite layer of cytoplasm lining the wall of the macrospore, and inclosing a large central vacuole. The nucleus moves to one side of the cell, usually the upper side, imbeds itself in the cytoplasm and awaits further development (figs. 147, 148).
The organization at so early a period of this definite peripheral layer of cytoplasm has not, I believe, been demonstrated for any of the other Gymnosperms. Finding the cavit}' containing the developing endosperm crossed b}^ irregular strands of cyto- plasm as illustrated in fig. 70, plate VI, I had the impression for a long time after these studies were begun, as stated in an earlier paper (1901''), that such a condition, as that described above for the resting macrospore, did not obtain until the beginning of the second period of growth. This layer of cyto- plasm is very easily displaced by the action of the fixing fluid, but with care it may be obtained in an apparently normal con-
LIFE HISTORY OF PINUS 8 1
dition. I now have an abundance of preparations which show not only that the wall layer is instituted in the one-celled stage, but that it persists as long as free cell-formation continues in the endosperm. The only reference which I find regarding the establishment of the wall-layer of cytoplasm in any of the Gymnosperms is the following statement made by Coulter and Chamberlain ('oi), with reference to Pinus : " Probably when but two or three free nuclei have appeared the nuclei become imbedded in a parietal, cytoplasmic layer."
SUMMARY.
The female cones can be distinguished early in March, excepting in Pinus Strohiis where they do not appear until the very last of April. The ovules cannot be detected until about three weeks before pollination.
There is no evidence that the macrospore-mother-cell arises from a hypodermal cell. When first differentiated it is cen- trally placed nearer the chalazal end of the ovule.
The division of the macrospore-mother-cell is a true tetrad- division and the cell which gives rise to the female gametophyte is a true spore.
Of the two cells formed as a result of the heterotypic division the lower one always divides again, the upper one may. An axial row of three cells seems to be the rule in Pinus Strohus and P. rigida, and one of four cells the rule in P. austriaca, though neither is constant in any of the species. The lowest cell of the axial row always becomes the functional macrospore.
The two or three upper cells of the axial row begin to disin- tegrate very soon after they are formed and are finally absorbed by the enlarging macrospore.
The lower cell passes through a long period of growth during which the cytoplasm is withdrawn from the central portion of the cell and forms a uniform layer lining the wall of the macro- spore. The nucleus moves towards the upper side of the cell and imbeds itself in the peripheral layer of cytoplasm.
The suggestion is made that the embryo-sac may or may not be a morphological unit, but that it is essentially a physiological unit, existing for the purpose of sexual reproduction. Such a
82 MARGARET C. FERGUSON
conception of the embryo-sac seems to the writer to form a more satisfactory basis for a rational explanation of the structure, or composition, and homologies of the embryo-sac than do any of the existing theories regarding the nature of this body.
CHAPTER IV.
The Female Gametophyte. development of the prothallium.
The First Period of Growth. — We are indebted to Hof- meister ('51) for our first definite knowledge regarding the life history of the female gametophyte in the Gymnosperms. It is true some errors in observations were made, but they were inter- mingled with much that has stood the test of the most modern research. In 1879 Strasburger declared the " transitory endo- sperm " described by Hofmeister to be a fallacy, but he himself fell into quite as grave an error, though in the opposite direction, when he stated that the primary nucleus of the embryo-sac remained undivided during the first year, an observation since corrected by himself.
As already stated, the young macrospore immediately organ- izes a peripheral layer of cytoplasm and passes through a period of growth which continues for six weeks or more. The degree of development which has been attained by P. austriaca on June 13, 1898, is shown in figs. 145 and 147 ; the first division of the macrospore-nucleus in this species occurred on July 29 of the same year, as illustrated in figs. 149 and 150. The germinating macrospore had now enlarged to such an extent that it was found necessary to reduce the scale of mag- nification at this point so that a comparison of the figures does not present, visually, the amount of growth which ensues between the organization of the macrospore and its first division. Pinus differs substantially in respect to the very marked growth of the macrospore before the first division of its nucleus from Larix where two nuclei are formed before there is any con- siderable increase in size of this cell (Juel ('00) plate xv, figs.
LIFE HISTORY OF PINUS 83
18-20). The persistence of the potential megaspores in Larix at this time is also in very striking contrast to Pimcs, where the other cells of the axial row have become entirely absorbed before the germination of the macrospore occurs (figs. 147-149).
The third division of the macrospore-mother-cell, or the first division of the macrospore-nucleus, takes place during the very last of July or the first of August in all the species studied, and is of the ordinary or typic method. It differs from the mitoses occurring in the vegetative tissue of the sporophyte only in presenting the one-half number of chromosomes (fig. 150). The daughter-nuclei may remain at one side of the pro- thallial cavity, but more frequently they pass to opposite sides as in the development of the embryo-sac in Angiosperms (fig. 151). The second mitosis follows rather quickly, and is already completed in Pinus Strobus on August 4 (fig. 152). Nuclear divisions follow until several free nuclei have been formed. The observations of Strasburger ('79), and of all later students of the Gymnosperms, upon the simultaneous division of the free nuclei of the endosperm have been confirmed. On October 12, 1898, sixteen nuclei were observed in the cytoplasmic layer, all being in the spireme stage of division. On October 15 of the same year sixteen nuclei, all presenting the equatorial plate- stage of mitosis were found in the cytoplasm of the prothallium, (figs. 153-155). The karyokinetic figure is sharply bipolar, each pole ends in a slight condensation of the cytoplasm, and the chromosomes are clearly of the reduced number.
I find no evidence that any further divisions occur during the first period of growth and it is probable that the thirty-two nuclei which result from the division just described pass into the resting stage and remain inactive during the winter. But I have not examined a sufficiently large number of preparations with this point in mind to affirm that the prothallium of Pinus invariably enters upon its long period of rest in the thirty- two nucleated stage. The number may not be fixed even in the same species, but it is certain that it is never large. The pro- thallium, therefore, at the close of its first season of growth is a spherical body composed of an ectal layer of cytoplasm in which are imbedded, in many instances at least, thirty-two free
84 MARGARET C. FERGUSON
nuclei. This thin cytoplasmic shell encloses a large central vacuole which is reported by Strasburger, Arnoldi and others to be filled with a fluid substance. I have made no observa- tions regarding the cell-sap of this large vacuole and can neither affirm nor den}-^ its presence.
The Second Pei'iod of Growth. — It has been seen that the ovular development in Pinus is very slow during the period imme- diately subsequent to pollination, but with the renewal of growth in the spring development becomes much more rapid. Coor- dinately with the enlargement of the ovule already described, the endosperm cavity increases in size until it occupies almost the entire basal and central portions of the nucellus, presenting in longitudinal section the figure of an ellipse (fig. 71, plate VI). The thin peripheral layer of cytoplasm with its free nuclei persists until the latter part of May, and free nuclear division continues to take place within it until a large number of nuclei are formed. Jager ('99) estimated that there are 256 free nuclei formed in Taxus, and Hirase ('95) made the same observation in Ginkgo. The number is certainly much larger in Pinus. More than 500 free nuclei are present early in May and about 2,000 have been counted in Pinus Strobus at the time when the nnclei are being separated by the development of dividing walls.
The free nuclei are considerably larger in surface view than the nuclei of the nucellar tissue, but in side view they often appear somewhat flattened. They have the structure of typical resting nuclei (figs. 156-159, plate XV). Each contains, almost invariably, two rather large nucleoli surrounded by clear areas. The reticulum is close and studded with irregular granules, but the net-knots are not so prominent as in the nuclei of the nucel- lus. They simulate very closely the nuclei of the sheath-cells at certain stages in the development of the archegonia. The cytoplasm in surface view presents a pseudo-alveolar structure consisting of a coarse, granular reticulum enclosing numerous vacuoles (fig. 156). During the late telophase in the division of the free nuclei of the prothallium the complicated karyokinetic figure characteristic of free nuclear division becomes very con- spicuous, and is evidently formed as a result of the rearrange- ment of the cyto-reticulum (fig. 159).
LIFE HISTORY OF PINUS 85
At some time during the latter part of May in Pinus Strobus and about the middle of the month in the other species free nuclear division ceases and cell-walls are developed between the nuclei. The development of the prothallium from this point on was studied by Sokolowa ('80), and her observations have in general been confirmed by all more recent writers, with the exception of Jager ('99) in Taxus. I find the development of cell-walls in the prothallium of Pinus to agree perfectly in its early stages with that described by Sokolowa. Walls are formed perpendicular to the wall lining the prothallial cavity, thus each nucleus with its proper portion of the cytoplasm is separated from all the other nuclei. No wall is laid down on the inner sides of these cells, so that in radial section the cells appear as uncovered boxes, the opening extending towards the center of the prothallial cavity. In surface view the cells are more or less isodiametric, polygonal in outline and very uniform in size. A layer of densely reticulated cytoplasm surrounds each nucleus, and delicate strands radiate from it to the ectal layer of cytoplasm, thus giving a very different aspect to the cytoplasm than it had prior to the development of cell walls (figs. 160 and 161). Jager described the presence of walls on the inner face of these cells in Taxus when the cells were first organized, but other students have not confirmed his observa- tions.
According to Sokolowa these cells grew inwards forming long open tubes which extended to the center without division, a wall was then formed at the inner end and the cells became divided by cross walls. To these long cells the name alveoli was applied. Only those from the sides extended clear to the center before being closed, those from the extremities becoming more or less wedge-shaped. Jaccard ('94) notes that \x\ Ephedra ^otcvq of the alveoli may divide before reaching the center, but many do not, while Arnold! ('99 and '01) finds that no division occurs in Sequoia until after the alveoli have met at the center and their ends have become closed by walls. The development sub- sequent to the formation of the open cells varies considerably in Pinus from that described by these writers for other Gymno- sperms. No cell has ever been observed to extend from the
86 MARGARET C. FERGUSON
circumference to the center of the prothallial cavity. The cells are long, it is true, the walls delicate and wavy in outline, but a ring of tissue composed of longer or shorter cells is formed rather early in the inward growth of the prothallium. The cells of the innermost row always remain open on their outer free sides, their cytoplasm is more abundant than in the other cells of the prothallium and their nuclei invariably retain a position near the open side of the cells (fig. 162). As observed by Jaccard ('94), and Jager ('99), the nuclei of the prothallium cease to divide synchronously after individual cells have been organ- ized. When the center is reached the cells close and thus, one year after pollination, the endosperm becomes a solid mass of tissue.
The prothallium grows rapidly after it has become a con- tinuous cellular body and in a few days it fills all the central and lower portion of the ovule. Above it is the prominent nucellar cap, while only a few cells of the nucellus remain along the sides separating the gametophyte from the integument (fig. 73, plate VII). Cell-divisions continue to take place, and the cytoplasm becomes more abundant, though the prothallial cells are never richly supplied with cytoplasm. Strasburger ('80), Jager ('99), and several more recent students have noted many nuclei in the endosperm cells. I have not observed multi- nucleated cells in the prothallium of Piniis up to the time when the suspensor has elongated and carried a several celled embr3^o to a considerable depth into the endosperm. Later stages than this have not been studied. There is often an appearance of more than one nucleus in a cell, but careful study never fails to demonstrate a delicate cell-wall between the nuclei. At an early stage in prothallial development the cell-walls are very delicate, scarcely more than condensations of the ectoplasm, so that they might easily be mistaken, in Pinus^ for strands of cytoplasm. Doubtless the cells become plurinucleated during a more advaned stage in embryo formation.
THE SO-CALLED SPONGY TISSUE.
The Fh'st Period of Growth. — When tiie macrospore- mother-cell first becomes apparent it is surrounded by a group
LIFE HISTORY OF PINUS 87
of cells, three to five cells in thickness, which are more or less clearly delimited from the surrounding tissue by their slightly larger nuclei, their somewhat radial arrangement about the macrospore-mother-cell as a center, and, in some instances, by a rather indefinite and broken space which separates this group of centrally lying cells from the adjacent nucellar tissue (fig. 124, plate XII). At the close of the tetrad-division these cells have become much more conspicuous by the increase in the size of their nuclei, the somewhat greater density of their cytoplasm, and by the presence just exterior to them of an interrupted layer of tabular cells which are evidently undergoing disintegration. The disintegrating cells usually appear on one side first then at other points about equally distant from the young gametophyte (figs. 66, 69, plate VI ; 124, plate XII, 148, and plate XIV). It was to this tissue, immediately surrounding the young endo- sperm, together with the disintegrating cells just exterior to it, that Strasburger gave the name " spongy " tissue, and for convenience I shall use this term in speaking of it.
Ovules are frequently found during the summer and fall which, so far as external appearances go, are perfectly normal, but, when prepared for study, reveal the fact that either the macrospore-mother-cell has never divided or the macrospore, if formed, has not developed. Such ovules do not renew their growth in the following spring. In those cases in which the development of the mother-cell or of the young gametophyte is arrested, very characteristic changes occur in the spongy tissue. These cells grow and become rich in cytoplasm even when the mother-cell does not divide, or when the macrospore fails to germinate. But after a time they, too, become inactive, their cytoplasm is gradually lost, their nuclei become dense and deeply staining, and their cell-walls are very greatly thick- ened (fig. 163, plate XV). This state of disintegration may enter in at any time during the first period of growth but it is more common before any divisions have occurred in the macro- spore. When the mother-cell fails to divide, the cells of the spongy tissue may grow until they almost equal it in size before showing signs of breaking down. In such instances they bear a very striking resemblance to the mother-cell, and might easily
88 MARGARET C. FERGUSON
be taken by one not familiar with the history of this tissue for a group of macrospore-mother-cells (fig. i68, plate XVI). In fig. 148, plate XIV, the slightly reduced cytoplasm of the cells of the spongy tissue and the prominence of their cell-walls are sure evidences that pathological conditions have entered in, though all other parts of the ovule are still perfectly normal the process of disintegration having only just begun. Had this ovule been left in connection with the sporophyte for a longer time, the spongy tissue would undoubtedly have assumed later the character shown in fig. 163.
It is this abnormal appearance which I believe led Hofmeister to conclude that there were two prothallia formed in the pines, one for each season of growth. Strasburger thought that Hof- meister mistook the normal spongy tissue for endosperm, and Coulter and Chamberlain have recently expressed the same view. Now the walls of the normal spongy tissue are never thickened but remain even less prominent than those of the nucellus. Hofmeister was surely too accurate a student of cells as cells to have fallen into such an error. It is a well- known fact that many ovules are organized in Pinus that never reach maturity and they are very frequently found in the autumn and late winter in the condition just described ; but with the renewed growth of the healthy ovules in the spring, these fail to develop farther and are soon detected by their smaller size. Shortly afterward they become brown and dead. Having found this thick-walled abnormal condition in the autumn and winter, and in the spring finding within the ovules then developing the large central cavity, it is not surprising that Hofmeister should have concluded that a thick-walled transitory endosperm was formed in the fall.
The Second Period of Growth. — When growth is renewed in the spring the cells of the spongy tissue become organized for the first time into a definite zone from two to three cells thick which forms a hollow prolate spheroid immediately surrounding the endosperm, and limited on its outer surface by a thin stratum of disintegrating nucellar tissue. The cells and their nuclei are not only somewhat larger than those of the nucellus, but their most distinguishing characteristic is to be found in the greater
LIFE HISTORY OF PINUS 89
density of their C3'toplasm^ which is almost identical with that of the prothallium, while the cells of the nucellus are scantily supplied with cytoplasm. These cells divide karyokinetically, and, as they increase in number, they press against the adjacent cells of the nucellus which become flattened against this con- stantly advancing tissue, and are absorbed, only to give place to other cells which meet a similar fate. Sometimes absorption seems to precede the outward march of the spongy tissue, so that this tissue is separated from the normal nucellus by a clear space made up of cells of the nucellus which have lost all their protoplasmic content, but which have not as yet suffered collapse (figs. 157, 158, plate XV). The parietal layer of cytoplasm which constitutes the endosperm remains always in closest con- tact with the inner surface of this tissue (fig. 71, plate VI).
The cells of the spongy tissue are still prominent when the endosperm becomes a solid multicellular body. Soon after- wards, however, they show signs of disintegration, and at the time of fertilization they have, as a rule, entirely disappeared as cells, only the remnants of the cell-walls remaining. The spongy tissue is then represented by a deeply staining fibrous body of no definite structure which persists between the gametophyte and the nucellus (figs. 162, plate XV, and 72, plate VI; 73, plate VII).
The Nature and Function of the Spongy Tissue. — The prominent character of the cells surrounding the prothallium in certain Gymnosperms has been commented upon, in a general way, by all students of the Ahieti^iece ; but, as was noted by the writer in 1900 and 1901 and confirmed by Coker in Taxodium^ 1902, the true nature and function of these cells seem to have escaped entirely the notice of previous writers, as they have in- variably been described as tissue showing evidence of breaking down. After a preliminary note regarding the nature of this tissue was sent to press in 1900, Lang ('00) described a similar layer of cells about the endosperm in Stangeria. He designated them as sporogenous cells and " possibly tapetal in nature."
As recently stated (1903),^ these cells may possibly represent sporogenous tissue, each cell being a potential macrospore-
1 See note at end of Appendix.
Proc. Wash. Acad. Sci., August, 1901.
90 MARGARET C. FERGUSON
mother-cell, but there is no evidence from the standpoint of origin that such is the case in Pinus. They arise directly from a nucel- lus in which a few days before their appearance every cell was apparently like every other cell. This alone is not conclusive, as the functional macrospore-mother-cell has a similar origin, so far as one can see. But, what is more conclusive, the divisions in this tissue are according to the typic method and present the number of chromosomes characteristic of the sporophyte (figs. 164-167). If these cells were once, in some remote ancestor, sporogenous in nature, they have entirely lost their primitive function and have acquired a new and important function in connection with the development of the endosperm. This is not then a layer of disintegrating tissue, as described by all earlier students of the AbietinccB, but rather as alread}'^ noted by the writer (1901^) a definite zone of physiological tissue which is intimately connected with the nutrition of the young gametophyte. It doubtless not only passes on to the endosperm the nutrititive substances derived from the nucellus, but is itself active in the manufacture of food, as numerous starch grains are often found within its cells. It is probable, too, that it performs an important mechanical role in the wa}^ of protection. It not only forms a support for the prothallium in its multinu- cleated state, but gradually receding, it pushes before it, as it were, the tissue of the nucellus thus making room within for the growth of the delicate gametophyte.
Though we now know that this is a far more important tissue than it was formerly thought to be, it does not seem to me wise to apply to it the name tapetum or to suggest a new name by which to designate it. Strasburger's term " spongy" tissue, although given when the nature of this tissue was not understood and being a misnomer so far as its structure and function are con- cerned, has obtained a wide usage in the literature of the Gym- nosperms, and should be retained, just as the term cell is still retained in all biological literature.
DEVELOPMENT OF THE ARCHEGONIUM.
The Ea7'ly Grozvih of the A?'chci>-o)iii(i)i. — The archegonia first become apparent during the latter part of May or the very first
LIFE HISTORY OF PINUS 9I
of June, the time varying somewhat with the species and with the season. The degree of development which the prothalHum has attained when the archegonia-initials make their appear- ance also varies not only in the different species but in the same species. The differentiation of the archegonia may be deferred until the prothallial cells have united to form a continuous tis- sue ; but it quite as frequently happens that, while there still remains a comparatively large, open space at the center of the prothallial cavity, certain cells at the micropylar end of the pro- thallium divide by periclinal walls more rapidly than do the other cells of the endosperm and become comparatively rich in cyto- plasm ; several of the superficial cells in this region do not so divide, but continue to grow, and are distinguished from the adjacent cells by their greater size, larger nuclei and more vacuolate cytoplasm. These are the initial cells of the arche- gonia (fig. 162, plate XV, and 169-171, plate XVI).
In less than a week after an archegonium-rudiment has ap- peared, and while it is still quite inconspicuous, it divides, giving rise to a small upper cell, the mother-cell of the neck, and a large, lower cell which forms the venter of the archegonium (figs. 171, 172, plate XVI). The small cell immediately divides by an anticlinal wall, and the two cells thus formed divide by walls that are perpendicular to the first, the resulting four cells all lying in the same plane. These constitute what may be called the normal neck in Pimis StrohtLS (figs. 173, 177, 180). Con- siderable irregularity in the number and arrangement of the neck-cells has, however, been noted even within the same spe- cies. Frequently two of the four cells divide again, as figured by Strasburger for Piniis Strobiis in 1869, the six cells being arranged in a single layer (figs. 178, 183, plate XVI, and 212, plate XIX). Occasionally all four cells divide by anticlinal walls, the neck then consisting of eight cells, all of which lie in the same plane (figs. 179, plate XVI, and 213, plate XIX). In rare instances the four cells divide by periclinal walls, when the eight cells which compose the neck of the archegonium are dis- posed in two tiers of four cells each (fig. 187, plate XVII). This last represents the structure of the neck in Pinus sylvcstris as figured by Mottier ('92) and Blackman ('98), and