ANNALS OF BOTANY

VOL. XVIII

Ojcforb

PRINTED AT THE CLARENDON PRESS

BY HORACE HART, M.A.

PRINTER TO THE UNIVERSITY

Annals of Botany

ft*

EDITED BY

ISAAC BAYLEY BALFOUR, M.A., M.D., F.R.S.

KING’S BOTANIST IN SCOTLAND, PROFESSOR OF BOTANY IN THE UNIVERSITY AND KEEPER OF THE ROYAL BOTANIC GARDEN, EDINBURGH

D. H. SCOTT, M.A., Ph.D., F.R.S.

HONORARY KEEPER OF THE JODRELL LABORATORY, ROYAL BOTANIC GARDENS, KEW

AND

WILLIAM GILSON FARLOW, M.D.

PROFESSOR OF CRYPTOGAMIC BOTANY IN HARVARD UNIVERSITY, CAMBRIDGE, MASS., U.S.A.

ASSISTED BY OTHER BOTANISTS

%

VOLUME XVIII

With Forty-one Plates and Sixty-one Figures in the Text

Bon&on

HENRY FROWDE, M.A., AMEN CORNER, E.C.

OXFORD: CLARENDON PRESS DEPOSITORY, 116 HIGH STREET

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CONTENTS.

No. LXIX, January, 1904.

PAGE

Lawson, A. A. The Gametophytes, Archegonia, Fertilization, and Embryo of Sequoia

sempervirens. With Plates I-IV . . . . . . . . . 1

Wager, H. The Nucleolus and Nuclear Division in the Root-apex of Phaseolus. With

Plate V 29

Worsdell, W. C. The Structure and Morphology of the e Ovule/ An Historical Sketch.

With twenty-seven Figures in the Text . . . . . . .57

Cavers, F. On the Structure and Biology of Fegatella conica. With Plates VI and VII and

five Figures in the Text 87

Potter, M. C. On the Occurrence of Cellulose in the Xylem of Woody Stems. With

Plate VIII I2i

Williams, J. Lloyd. —Studies in the Dictyotaceae. I. The Cytology of the Tetrasporangium

and the Germinating Tetraspore. With Plates IX and X 141

Benson, Miss M. Telangium Scotti, a new Species of Telangium (Calymmatotheca) showing

Structure. With Plate XI and a Figure in the Text 161

NOTES.

Hem§ley, W. Botting. On the Genus Corynocarpus, Forst. Supplementary Note . . 1 79 Weiss, F. E. The Vascular Supply of Stigmarian Rootlets. With a Figure in the Text . 180 Ewart, A. J. Root-pressure in Trees 181

No. LXX, April, 1904.

Williams, J. Lloyd. Studies in the Dictyotaceae. II. The Cytology of the Gametophyte

Generation. With Plates XII, XIII, and XIV . 183

Bower, F. O. Ophioglossum simplex, Ridley. With Plate XV ...... 205

Parkin, J. The Extra-floral Nectaries of Hevea brasiliensis, Miill.-Arg. (the Para Rubber

Tree), an Example of Bud-Scales serving as Nectaries. With Plate XVI . . .217

Church, A. H. The Principles of Phyllotaxis. With seven Figures in the Text . . . 227

Mottier, D. M. The Development of the Spermatozoid in Chara. With Plate XVII . 245

Weiss, F. E. A Mycorhiza from the Lower Coal-Measures. With Plates XVIII and XIX

and a Figure in the Text ........... 255

Reed, H. S. A Study of the Enzyme-secreting Cells in the Seedlings of Zea Mais and

Phoenix dactylifera. With Plate XX 267

Vines, S. H. The Proteases of Plants 289

NOTES.

Massee, G. On the Origin of Parasitism in Fungi . . . . . . . .319

Salmon, E. S. Cultural Experiments with Biologic Forms of the Erysiphaceae . . . 320

Oliver, F. W., and Scott, D. H. On the Structure of the Palaeozoic Seed Lagenostoma

Lomaxi, with a Statement of the Evidence upon which it is referred to Lyginodendron . 321

VI

Contents .

No. LXXI, July, 1904.

PAGE

Blackman, V. H. On the Fertilization, Alternation of Generations, and general Cytology

of the Uredineae. With Plates XXI-XXIV - 323

Darbishire, O. V. Observations on Mamillaria elongata. With Plates XXV and XXVI . 375

Lawson, A. A. The Gametophytes, Fertilization, and Embryo of Cryptomeria japonica.

With Plates XXVII-XXX 417

Gregory, R. P. Spore-Formation in Leptosporangiate Ferns. With Plate XXXI and

a Figure in the Text 445

Massee, G. A Monograph of the genus Inocybe, Karsten. With Plate XXXII . . . 459

Boodle, L. A. On the Occurrence of Secondary Xylem in Psilotum. With Plate XXXIII

and seven Figures in the Text 505

NOTE.

Scott, D. H. On the Occurrence of Sigillariopsis in the Lower Coal-Measures of Britain . 519

No. LXXII, October, 1904.

Engler, A. Plants of the Northern Temperate Zone in their Transition to the High

Mountains of Tropical Africa 523

Trow, A. H. On Fertilization in the Saprolegnieae. With Plates XXXIV-XXXVI . . 541 Lang, W. H. On a Prothallus provisionally referred to Psilotum. With Plate XXXVII . 571 Burns, G. P. Heterophylly in Proserpinaca palustris, L. With Plate XXXVIII . . 579

Ford, Miss S. O. The Anatomy of Psilotum triquetrum. With Plate XXXIX . . *589

Wolfe, J. J. Cytological Studies on Nemalion. With Plates XL and XLI and a Figure

in the Text 607

Ganong, W. F. An undescribed Thermometric Movement of the Branches in Shrubs and

Trees. W7ith six Figures in the Text 631

NOTES.

Wigglesworth, Miss G. The Papillae in the epidermoidal Layer of the Calamitean Root.

With three Figures in the Text 645

Fritsch, F. E. Algological Notes. No. 5 : Some points in the Structure of a young

Oedogonium. With a Figure in the Text 648

Pertz, Miss D. F. M. On the Distribution of Statoliths in Cucurbitaceae .... 653

Hill, T. G. On the Presence of a Parichnos in Recent Plants 654

. *

INDEX.

A. ORIGINAL PAPERS AND NOTES.

Benson, Miss M. Telangium Scotti, a new Species of Telangium (Calymmatotheca) showing

Structure. With Plate XI and a Figure in the Text

Blackman, V. H. On the Fertilization, Alternation of Generations, and general Cytology of

the Uredineae. With Plates XXI-XXIV

Boodle, L. A. On the Occurrence of Secondary Xylem in Psilotum. With Plate XXXIII

and seven Figures in the Text

Bower, F. O. Ophioglossum simplex, Ridley. With Plate XV

Burns, G. P. Heterophylly in Proserpinaca palustris, L. With Plate XXXVIII .

Cavers, F. On the Structure and Biology of Fegatella conica. With Plates VI and VII and

five Figures in the Text *

Church, A. H. The Principles of Phyllotaxis. With seven Figures in the Text . Darbishire, O. V. Observations on Mamillaria elongata. With Plates XXV and XXVI . Engler, A. Plants of the Northern Temperate Zone in their Transition to the High Mountains of Tropical Africa ...........

Ewart, A. J. Root-pressure in Trees

Ford, Miss S. O. The Anatomy of Psilotum triquetrum. With Plate XXXIX .

Fritsch, F. E. Algological Notes. No. 5 : Some points in the Structure of a young Oedogonium. With a Figure in the Text .........

Ganong, W. F. An undescribed Thermometric Movement of the Branches in Shrubs and

Tre6s. With six Figures in the Text .

Gregory, R. P. Spore-Formation in Leptosporangiate Ferns. With Plate XXXI and

a Figure in the Text

Hemsley, W. Botting. On the Genus Corynocarpus, Forst. Supplementary Note

Hill, T. G. On the Presence of a Parichnos in Recent Plants

Lang, W. H. On a Prothallus provisionally referred to Psilotum. With Plate XXXVII Lawson, A. A. The Gametophytes, Archegonia, Fertilization, and Embryo of Sequoia

sempervirens. With Plates I-1V

The Gametophytes, Fertilization, and Embryo of Cryptomeria japonica.

With Plates XXVII-XXX

Massee, G. A Monograph of the genus Inocybe, Karsten. With Plate XXXII .

On the Origin of Parisitism in Fungi . .

Mottier, D. M. The Development of the Spermatozoid in Chara. With Plate XVII Oliver, F. W., and Scott, D. H. On the Structure of the Palaeozoic Seed Lagenostoma Lomaxi, with a Statement of the Evidence upon which it is referred to Lyginodendron . Parkin, J. The Extra-floral Nectaries of Hevea brasiliensis, Miill.-Arg. (the Para Rubber Tree), an Example of Bud-Scales serving as Nectaries. With Plate XVI .

Pertz, Miss D. F. M. On the Distribution of Statoliths in Cucurbitaceae .... Potter, M. C. On the Occurrence of Cellulose in the Xylem of Woody Stems. With

Plate VIII

Reed, H. S. A Study of the Enzyme- secreting Cells in the Seedlings of Zea Mais and

Phoenix dactylifera. With Plate XX

Salmon, E. S. Cultural Experiments with * Biologic Forms’ of the Erysiphaceae .

Scott, D. H. On the Occurrence of Sigillariopsis in the Lower Coal-Measures of Britain See Oliver, F. W.

Trow, A. H. On Fertilization in the Saprolegnieae. With Plates XXXIV-XXXVI . Vines, S. H. The Proteases of Plants . ..........

Wager, H. The Nucleolus and Nuclear Division in the Root-apex of Phaseolus. With Plate V

PAGE

l6l

323

505

205

579

87

227

375

523

181

589

648

631

445

179

654

57i

4i7

459

319

245

321

217

653

121

267

320

519

54i

289

29

Vlll

Index .

PAGE

Weiss, F. E. A Mycorhiza from the Lower Coal-Measures. With Plates XVIII and XIX

and a Figure in the Text 255

The Vascular Supply of Stigmarian Rootlets. With a Figure in the Text . 180

Wigglesworth, Miss G. The Papillae in the epidermoidal Layer of the Calamitean Root.

With three Figures in the Text 645

Williams, J. Lloyd. Studies in the Dictyotaceae. I. The Cytology of the Tetrasporangium

and the Germinating Tetraspore. With Plates IX and X . . . . .141

Studies in the Dictyotaceae. II. The Cytology of the Gametophyte

Generation. With Plates XII-XIV 183

Wolfe, J. J. Cytological Studies on Nemalion. With Plates XL and XLI and a Figure in

the Text 607

Worsdell, W. C. The Structure and Morphology of the Ovule.’ An Historical Sketch.

With twenty-seven Figures in the Text -57

a. Plates.

I-IV.

V.

VI, VII. VIII. IX, X. XI.

XII-XIV.

XV.

XVI.

XVII. XVIII, XIX.

XX. XXI-XXIV. XXV, XXVI. XXVII-XXX. XXXI. XXXII. XXXIII. XXXIV-XXXVI. XXXVII. XXXVIII. XXXIX. XL, XLI.

B. LIST OF ILLUSTRATIONS.

Sequoia sempervirens (Lawson).

Root-apex of Phaseolus (Wager).

Fegatella (Cavers).

Cellulose in Woody Stems (POTTER). Dictyotaceae (Lloyd Williams).

Telangium Scotti (Benson).

Dictyotaceae (Lloyd Williams). Ophioglossum simplex (Bower).

Hevea brasiliensis (Parkin).

Spermatozoid of Chara (MotTier).

Mycorhiza from Coal-Measures (Weiss). Enzyme-secreting cells (Reed).

Uredineae (Blackman).

Mamillaria (Darbishire).

Cryptomeria japonica (Lawson).

The Reduction-division in Ferns (Gregory). Characters of Hymenium in Inocybe (MASsee). Psilotum (Boodle).

Fertilization in Saprolegnieae (Trow). Prothallus referred to Psilotum (Lang). Proserpinaca palustris (Burns).

Psilotum (Ford).

Nemalion (Wolfe).

1. Figures.

1-27.

28-32.

33*

34*

35-41*

42.

43*

44-50.

5i*

52-57*

58-60.

61.

The Structure and Morphology of the Ovule (Worsdell) 59, 62, 72, 73, 76-78 The Structure and Biology of Fegatella conica (Cavers) 99, 102, 106, 109

Telangium affine (Benson) 164

The Vascular Supply of Stigmarian Rootlets (Weiss) . . . .181

The Principles of Phyllotaxis (Church) . 229, 230, 232-234, 237, 239

A Mycorhiza from the Lower Coal-Measures (Weiss) .... 258

Diagrams of the Reduction-division in Cyclops (Gregory) . . . 454

On the Occurrence of Secondary Xylem in Psilotum (Boodle) . . 509

Cytological Studies in Nemalion (Wolfe) . . . . . .619

Thermometric Movement of Branches in Shrubs and Trees (Ganong)

632, 634, 635, 638, 640

Papillae in the epidermoidal Layer of the Calamitean Root (Wiggles- worth) 645-647

Some points in the Structure of a young Oedogonium (Fritsch) . .651

Vol. XVIII.

No. LXIX.

January, 1904. Price 14s

Annals of Botany

EDITED BY

ISAAC BAYLEY BALFOUR, M.A., M.D, F.R.S.

KING’S BOTANIST IN SCOTLAND, PROFESSOR OF BOTANY IN THE UNIVERSITY AND KEEPER OF THE ROYAL BOTANIC GARDEN, EDINBURGH

D. H. SCOTT, M.A., Ph.D., F.R.S.

HONORARY KEEPER OF THE JODRELL LABORATORY, ROYAL BOTANIC GARDENS, KEW

AND

WILLIAM GILSON FARLOW, M.D.

PROFESSOR OK CRYPTOGAMIC BOTANY IN HARVARD UNIVERSITY, CAMBRIDGE, MASS., U.S.A.

1

ASSISTED BY OTHER BOTANISTS

s ' V ' * ■■ v r II r.friT* . t'V* > .. A , :

ty&j x I /f ■/' .. , - 1\

£ ondon

HENRY FROWDE, AMEN CORNER, E.C.

©jcfo**

CLARENDON PRESS DEPOSITORY, 116 HIGH STREET

1904

Printed, by Horace Hart, at the* Clarendon Press, Oxford.

PAGE

CONTENTS.

Lawson, A. A.— The Gametophytes, Archegonia, Fertilization, and

Embryo of- Sequoia sempervivens. With Plates I-IV . . i

Wager, H. The Nucleolus and Nuclear Division in the Root-apex

of Phaseolus. With Plate V 29

Worsdell, W. C.— The Structure and Morphology of the ‘Ovule.’

An Historical 'Sketch. With twenty-seven Figures in the Text 57 Cavers, F. On the Structure and Biology of Fegatella conica.

With Plates VI and VII and five Figures in the Text . . 87

Potter, M. C. On the Occurrence of Cellulose in the Xylem of

Woody Stems. With Plate VIII ...... 121

Williams, J. Lloyd. Studies in the Dictyotaceae. I. The Cytology of the Tetrasporangium and the Germinating Tetraspore. With

Plates IX and X 1 4 1

Benson, Miss M. Telangium Scotti, a new Species of Telangium (Calymmatotheca) showing Structure. With Plate XI and a Figure in the Text . . . 161

NOTES.

Hemsley, W. Botting.— -On the Genus Corynocarpus, Forst.

Supplementary Note . . . 179

WEISS, F. E. The Vascular Supply of Stigmarian Rootlets. With

a Figure in the Text . .180

Ewart, A. J.— Root-pressure in Trees 181

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The Gametophytes, Archegonia, Fertilization, and Embryo of Sequoia sempervirens.

BY

ANSTRUTHER A. LAWSON, Ph.D.

Instructor in Botany, Stanford University , California, US. A.

With Plates I-IV.

Introduction.

..THOUGH investigations among the Gymnosperms, especially among

the Cycadales and Ginkgoales, have, in the last few years resulted in most important and startling discoveries, this field of research has not received the attention it deserves. The literature on the Coniferales is gradually accumulating and revealing much that is of interest and of importance. These contributions are, however, too fragmentary to deserve the appreciation which they might otherwise receive.

Representatives of every family of the Coniferales have been investi- gated, especially in regard to the gametophyte generation. Of the numerous types selected by the various investigators many phases in the life-history have been revealed which are of great morphological interest and importance. In spite of the large number of forms that have been worked upon, however, Pinus is the only Conifer in which a connected account of the important events completing the life-cycle has been compiled. The works on the other Conifers are nevertheless of great importance, and although they are at present but disconnected chapters in the life-history, their true value will no doubt be fully appreciated as soon as the missing chapters have been written.

The interesting genus Sequoia is represented by two living species, Sequoia gig ante a and Sequoia sempervirens. The former species is confined to very narrow limits in California, while the latter extends along the coast ranges of middle and northern California and for about twelve miles into the State of Oregon. Of the latter species there are at present over one hundred trees growing on the campus of Stanford University. As the majority of these trees are healthy and vigorous, and although young, produce cones every year, and especially as many of them grow in the immediate vicinity of the Botanical Laboratory, excellent opportunity for the daily collections of material was afforded. Taking advantage of these exceptional facilities, I have thought it worth while to work out the morphology of the gametophytes, with the hope of filling in the gaps left

[Annals of Botany, Vol. XVIII. No. LXIX. January, 1904.]

B

2 Lawson . The Gametophytes, Archegonia, Fertilization ,

by Arnoldi, Shaw, and Strasburger, the only writers who have contributed to our knowledge of the gametophytes of this interesting Conifer.

It is therefore the object of the present work to give a connected account of the events leading to the development of the gametophytes, sexual organs, fertilization, and embryo, thus completing as far as possible the life-history of Sequoia sempervirens.

While the observations recorded by Shaw and Arnoldi are of great interest, they are by no means complete, and as we shall see later, some are even inaccurate.

Shaw (1896) has given a description of how the male and female flowers develop in Sequoia sempervirens. When very young he finds that the macrosporangium is about as long as it is broad. The integument at this time consists of an epidermis and two layers of hypodermal cells. The integument develops rapidly and soon comes to be about twice as long as the nucellus. Soon after pollination the upper and inner layers of epidermal cells enlarge and by their elongation finally close the micropyle. By this means the pollen-grains are completely enclosed in a subconical cavity at the apex of the nucellus. Within the nucellus several sporogenous cells now make their appearance. Shaw reports that these cells divide twice, each one giving rise to four macrospores. Upon germination the macrospores develop a number of female prothallia. As the embryo-sacs increase in size they contain several nuclei. The further development of the prothallia was not observed. The archegonia were found to be very numerous and distributed over the upper portion of the prothallium and each one has but a single neck-cell. At the time of pollination each microspore consists of two cells, a large central cell with a large nucleus and a much smaller cell. As the pollen-tube develops the nucleus of the larger cell moves forward and enters the tube. The tube extends down between the nucellus and the integument and as often as not it branches. The further course of the tubes was not followed. Later a number of long suspensors bearing the young embryos on their tips were formed in the endosperm.

Arnoldi (1900) has given a description of the manner of endosperm- formation in Sequoia sempervirens. According to this description the form of the embryo-sac may vary considerably. When young it consists of a very large central vacuole surrounded by a parietal layer of cytoplasm in which numerous nuclei are found. As the sac develops the parietal layer increases and the protoplasm accumulates in great abundance in the lower end and at the same time the free nuclei divide repeatedly. In the central region a portion of the vacuole remains, and here the cellular endosperm is formed by means of Alveolen 5 as Sokolowa describes for other Conifers, while the remaining endosperm is formed by ordinary free cell-formation. The development of the archegonia is confined to the tissue derived from the ‘Alveolen/ so that the prothallium has a distinct generative region.

Embryo of Sequoia sempervirens. 3

In the development of the endosperm in Sequoia , Arnoldi sees a striking similarity to that which occurs in Gnetuni.

In the same year (1900) Arnoldi published some observations on the archegonia and pollen-tubes in Sequoia sempervirens . He finds that the archegonia arise from peripheral endosperm-cells and are present in large numbers and may appear singly or in groups. Each archegonium has two neck-cells but none contain a ventral canal cell. The position taken by the pollen-tubes is between the nucellus and the endosperm and they eventually lie opposite the archegonia.

In his more recent work, Arnoldi (1901) touches upon fertilization and the development of the embryo. In addition to Sequoia this short paper also discusses these phases in the life-history of Taxodium , Cryptomeria , Cunninghamiay Glyptostrobus, and Sciadopitys . In Sequoia sempervirens , which concerns us more particularly, he finds that the pollen-tube eventually contains two male cells and two free nuclei, of which one is the tube nucleus. At the time of fertilization the male cell becomes elongated or even spirally twisted. The male and female nuclei fuse in the middle of the egg and then move to the base of the archegonium, where the first segmentation-spindle is developed. Following this division two cells are organized, one behind the other. The lower of these divides again so that the embryo now consists of a row of three cells. The lower cell of the first division functions no further and soon becomes disorganized.

On the sporophyte of Sequoia , Peirce (1901) has contributed some interesting and important observations on fasciation, albinism and vegeta- tive reproduction.

Methods.

There are few groups of plants that offer more difficulties in the way of cytological research than the Coniferales. The structures that are of greatest cytological interest are usually buried deep in the other tissues, thus requiring very careful dissection before being placed in the killing fluids. Then, again, if resin is present, as is usually the case, a rapid penetration of the fluid is impossible.

These and many other difficulties probably account for the frag- mentary nature of the work that has been done. A brief statement of the methods adopted in the following work on Sequoia may be useful to others working in this field. The fixing fluids experimented with were as follows :

1. Flemming’s weak solution

25 c.c. of 1 °/o chromic acid 10 c.c. of 1 °/o acetic acid 10 c.c. of 1 °/o osmic acid 55 c.c. distilled water.

4 Lawson . The Gametophytes , Archegonia , Fertilization , <2/^

Flemming’s strong solution.

3. Chrom-Acetic mixture.

4. Chromic Acid 1 °/o sol.

5. Alcohol Acetic.

Of these Flemming’s weak solution probably gave the best results although equally satisfactory fixation was generally obtained by the Chrom-Acetic and one per cent Chromic. The Alcohol Acetic solution proved to be a failure.

The fixing fluids were always taken into the field and the material deposited in them immediately. In the very early stages of the ovules and also of the pollen no dissection was necessary. On account of the air present in them these structures had a tendency to float. This diffi- culty was, however, overcome by sinking the material in the fluid by means of cotton plugs.

In the very early stages the entire ovules were removed and im- mediately killed without further dissection, but in all the later stages it was found necessary to remove the integument. This, however, was not resorted to until after the pollen-tube had penetrated the nucellus. To insure rapid fixation most of the dissections were made with the material immersed in the fluid. The ovules were removed one by one, placed in a watch-glass containing the fixing reagent and while in the fluid the in- tegument was immediately removed by means of a sharply pointed scalpel and forceps. With a little experience this may be accomplished very rapidly.

The material was allowed to remain in the fixing fluid from ten to twenty-four hours and then washed in running water from four to six hours. Care was now taken in transferring the material to alcohol. For this purpose Schleicher and Schull’s diffusion shells were used. The shells were cut to the height of small beakers and the material placed in the bottom, and 95% alcohol placed in the beakers. Water was now poured in the shell in sufficient quantity to make the combined solutions about 7o°/o alcohol. By placing the shell, containing the material and water, in the beaker containing the 95 °/o alcohol, a gradual diffusion took place which was not sufficiently rapid to cause shrinkage. In two or three hours the material was transferred directly to 95 °/o alcohol. I found the shells much more convenient than ordinary parchment paper.

In preparation for imbedding the material was thoroughly dehydrated in absolute alcohol. Bergamot oil was used to precede the infiltration of paraffin. After dehydration the material was placed in a mixture of 1 part absolute alcohol and 1 part bergamot oil ; then into pure bergamot oil ; then into a mixture of 1 part Bergamot oil and 1 part melted paraffin ; and finally into pure paraffin.

Minot’s wheel microtome was employed for cutting and the sections varied from 2 //, to 8 /x in thickness according to the detail desired.

5

Embryo of Seqttoia sempervirens.

It was found very desirable to use albumen instead of alcohol as a fixative. When the staining was not satisfactory, the sections fixed on the slide with albumen were bleached and restained without trouble. With the alcohol method, however, restaining was impossible, as the sections were invariably washed off the slide. By restaining, many valuable demonstrations were restored.

The triple stain safranin, gentian, and orange G., was found to be the most satisfactory in differentiating the various cell-structures.

The Male Gametophyte.

The reduction-division of the microspore mother-cell leading to the formation of the tetrads takes place during the first week in December. The first division is rapidly followed by the second, and within a week or ten days after the first division the tetrads have separated and the pollen-grains formed.

The microspores remain within the sporangium at least three weeks before pollination takes place. During this period they become larger, spherical in form, and surround themselves with a hard thick wall. The cytoplasm is very granular and contains a small amount of starch. The nucleus is comparatively small and is always centrally situated (PL I, Fig. i). While yet in the sporangium and about a week before pollination the nucleus of the microspore enlarges and divides ; so that at the time the pollen is shed there are two nuclei in each grain. Sections made before and after pollination showed a considerable difference in the size of the nuclei, the one being about twice the size of the other. The larger one was centrally situated, while the smaller one was invariably found near the spore wall. The smaller nucleus was surrounded by a sharply differen- tiated zone of very granular cytoplasm, which suggested the presence of a membrane between the two nuclei as shown in Fig. 2. The chromatin of the smaller nucleus was in the form of small granules closely packed together ; it consequently stained more deeply than the larger nucleus, where the meshes of the chromatin network appeared to be much more loosely arranged. A study of the further history of these nuclei has convinced me that the larger nucleus is the so-called tube-nucleus and the smaller one represents the generative cell.

A very careful search was made with the hope of finding a vestige of the vegetative tissue of the gametophyte. One or more vegetative cells have been reported for the Cycads Ginkgo and Pinus> but a most searching examination failed to reveal even a vestige of such a cell or nucleus in Sequoia. I am strongly inclined to believe that the develop- ment of these evanescent structures has been entirely suppressed.

Observations of two years indicated that pollination takes place during the first week in January, just about the time the female flowers

6 Lawson . The Gametophytes , Archegonia , Fertilization ,

make their appearance. During this time the trees are constantly en- veloped in a cloud of pollen, so that it would be almost impossible for any of the exposed ovules to escape the reception of at least a few of the grains. At this time the integument of the ovule is about on a level or a little above the apex of the nucellus, and from four to six pollen-grains are here deposited. The grains remain in this position for three or four weeks without further germination, when the integument grows over them and closes the micropyle in the manner described by Shaw (1896).

The first indication of the further germination of the pollen-grains was the splitting off of the hard thick wall. (If some ripe grains are examined in water under the microscope, it will be seen that the casting off of the outer wall takes place suddenly and with considerable force, leaving a thin delicate membrane underneath.) The pollen-tubes now push out over the tops of the nucellus, and one or two of them may grow down between the nucellus and the integument, as shown in Fig. 3. Material collected during the first week in March frequently showed the pollen-tubes extending more than halfway down the side of the nucellus. In such cases the tube-nucleus was invariably near the tip of the tube, with the generative nucleus considerably in the rear. Both nuclei were found in the central axis of the tube, suspended in a broad strand of cytoplasm which contained an abundance of starch grains.

While one or two of the tubes may follow the course between the nucellus and the integument, others may penetrate the nucellus immediately at the top, as shown in Fig. 4. The penetration of the tube is accompanied by a breaking down and a probable absorption of the cells of the nucellar tissue through which the tube forces its way. From a study of longi- tudinal and cross-sections in series it became quite evident that the course taken by the pollen-tubes may vary considerably. At a later stage cross- sections of the endosperm showed the tubes in various positions. Usually three or four tubes develop and become functional. In the majority of cases they are found situated between the female prothallium and the remaining tissue of the nucellus. Many were found partially surrounded by endosperm, while others were completely surrounded. In no case was I able to find any evidence of branching of the tube, although Shaw (1896) reports that 4 quite as often as not it branches.’

Just about the time the tube penetrates the wall of the nucellus, the generative nucleus, having increased to quite the size of the tube-nucleus, divides. As shown in Fig. 4, there are now three nuclei in the tube, one large one and the two smaller ones. The largest of these, situated nearer the tip of the tube, is no doubt the tube-nucleus, while the other two are the stalk- and body-nuclei. Of these latter two there is a slight difference in size. As the larger one appears to be preparing for further activity, I regard it as the body-nucleus and the smaller one as the stalk-

7

Embryo of Sequoia sempervirens.

nucleus. During the further development of the pollen-tube, the three nuclei remain in close proximity to each other. During these changes the size of the tube-nucleus remained the same, while that of the stalk-nucleus increased slightly. The body-nucleus, however, increased to at least three or four times the size of the stalk-nucleus, as shown in Fig. 5. During its development the body-nucleus surrounds itself with a dense zone of granular cytoplasm. This zone increases until it is about half the diameter of the nucleus in thickness, when it becomes shut off from the rest of the cytoplasm in the tube by a distinct membrane. The tube now contains one large cell and two free nuclei (Fig. 6).

Previous to the formation of the body-cell, the free nuclei lie close together suspended in the same strand of cytoplasm, which contains an abundance of starch. The most of the starch was present in the vicinity of the body-nucleus, and it later becomes confined within the cytoplasm of the body-cell. The stalk-nucleus remains close to the body-cell (Fig. 5), even up to the time the male cells are formed.

The changes resulting in the organization of the body-cell showed considerable variation as to the time of their occurrence. In some cases the mature body-cell was found in material collected early in May, while others were found as late as the middle of June. This irregularity as to the time of the changes was also noticeable in all later changes in the development of the male prothallium, even in the matter of fertilization.

Soon after the body-cell has been fully organized, its nucleus enlarges and prepares for division. By extreme good fortune the spindle of this division was found. As this is the division which results in the formation of the two male cells, it demands a careful examination. It was during the division of the body-cells in Cycas , Zamia , and Ginkgo (Hirase, 1898 ; Ikeno, 1896-8; Webber, 1897-1901) that the centrosome-like bodies known as blepharoplasts were discovered. It was thought probable that a vestige of such an organ might be found in Sequoia, , but an examination of the cytoplasm surrounding the spindle failed to reveal a trace of any body that might be interpreted as a blepharoplast. It must be remembered, however, that the blepharoplasts are only concerned with the development of the cilia, and as these latter structures have never been found in con- nexion with the male cells in Conifers, it is not surprising that the organs responsible for their formation should also be missing. Fig. 7 shows the spindle dividing the body-nucleus with the daughter-nuclei at the poles. The formation of the cell-plate that separates the nuclei and divides the body-cell into two was not actually observed. But an examination of Fig. 7 where the connective fibrils curve out laterally, and of Fig. 8 where the two daughter-cells are lying side by side, makes it obvious that the cell-plate is developed in the usual way. After the wall separating the male cells has been formed, the latter remain close together for some

8 Lawson . The Gametophytes , Archegonia , Fertilization ,

time, and a section of the two together has the outline of an ellipse that has been cut in half. They are rounded on one side and flat on the other. They are of equal size, and, as we shall see later, are both functional. Just before the male cells separate from each other, the nucleus in each has increased to about twice its original size. The chromatin is in the form of a network which contains a large irregularly shaped nucleolus.

At the time of fertilization the male cells become almost spherical, and are perfectly similar in regard to their size and structure. Arnoldi (1901) has reported that the male cells may become elongated, and he figures one that has a spirally twisted form. I was unable to find such conditions, and feel confident that they are abnormal or due to shrinkage by poor fixation. In all the cases I have examined the male cell was spherical, and, as we shall see later, its spherical form may persist for some time after its nucleus has been injected into the egg.

As we shall point out later, under the head of fertilization, only the nucleus of the male cell enters the archegonium, the rest of the male cell remaining outside in the pollen-tube. The nucleus is first liberated and, with but a small film of protoplasm surrounding it, passes between the neck-cells of the archegonium and immediately fuses with the egg-nucleus. In all other Gymnosperms in which observations have been recorded, at least one male cell enters the archegonium, so that in this respect the spermatogenesis of Sequoia is unique. According to the following diagram, Coulter and Chamberlain have compared the spermatogenesis of the Cycads with that of Isoetes.

Isoetes. Cycads . Sequoia sempervirens.

0 Generative cell @ Generative cell . . a

It will be observed that the male gametophyte is complete with the organization of the male cells, and this is true for all other gymnosperms where spermatogenesis has been worked out. If, however, we construct a similar diagram for Sequoia , the additional step of the discharge of the male cell-nucleus suggests more strongly the spermatogenesis of the Pteridophytes than even that of the Cycads, although the male cells of the later are ciliated.

Embryo of Sequoia semper virens.

9

The Female Gametophyte.

Shaw (1896) has given an accurate account of the development of the macrosporangium and the integument, but has, however, given a very meagre description of the sporogenous cells and the macrospores. There may be as many as five or six macrospore mother-cells organized from the hypodermal cells of the sporangium. Many preparations were made of this stage in the development of the sporangium, and a special study was made of the mother-cells as soon as they became differentiated from the surrounding sterile cells. They first become recognized as mother-cells by their large deeply staining nuclei. In the beginning they are not much larger than the other hypodermal cells, but they very soon enlarge, and their cytoplasm becomes very dense and granular and stains very readily with orange G. They are further characterized by the absence of large vacuoles. They are situated just about the centre of the sporangium ; about five or six layers of cells lying between the uppermost of them and the epidermis at the apex. A careful study was made of the number of mother-cells formed, and there seems to be a slight variation in this respect. Six was the largest number found. In all the sporangia studied five or six was the prevailing number, but in no case were fewer than four found.

Shaw (1895) has reported that these sporogenous cells divide twice, each cell giving rise to four spores. As there are nearly always five or six of these sporogenous cells developed, and if each one gave rise to four spores, this would result in the formation of twenty or twenty-four macrospores. We shall show later that no such large number of macrospores were formed, but that, on the contrary, ten or twelve were the prevailing numbers