:i;!':": ■'■'■■ :;; ■.■..•:■■-■ ' '■■■■ -:-;-. -.■■■' ■:-:■-.■■/■■:::. ... .■■■■■'!:.■■■■■.•■.■-'■■.■•■ '•■■■■ - '• :': ■.■■:■'■■"■■■■ ; I ■■'■' WBMm IflRl • ..•■■'■■'■:■•' ■Hi IBi ■M MS ■BR TfiffisaPl llfjBBflSfci mmmm . '; -::.-:;- :- ■•■■ ;-'. ■ - ' ■ : ";!'--::': ■"'■'' .:' ■■'•■ ■ Bra ■■■' THE EUSPORANGIATAE THE COMPARATIVE MORPHOLOGY OF THE OPHIOGLOSSACEAE AND MARATTIACEAE BY DOUGLAS HOUGHTON CAMPBELL PROFESSOR OF BOTANY, LELAND STANFORD JUNIOR UNIVERSITY WASHINGTON, D. C. Published by the Carnegie Institution of Washington 1911 CARNEGIE INSTIT1 ["ION OJ WASHINGTON l'i i.i u \ i ins No. 140 Copies of this Bo»l< wore first issued AOu29 191l PRESS ni ISAAC II BLANCHARD COMPANY NEW YORK PREFACE. The great importance of the eusporangiate ferns as the nearest existing rela- tives of the Paleozoic ancestors of the higher types of flowering plants invests them with an especial interest for the student of plant evolution; and no apology is necessary for presenting at length a summary of our present knowledge of the structure and development of these important plants. For more than twenty years the writer has been much interested in the study of the Eusporangiatae and during this time has published a number of works deal- ing with them. He has had rather unusual opportunities for collecting these forms, and an extensive and representative collection of materials comprising a good many species has been accumulated, so that the time seemed ripe for a comparative study of the group, for the purpose of determining, as far as might be, the relationships existing between the different genera, as well as for throwing some light upon the question of their position in the great series of ferns. The present memoir is an attempt to present the results of these studies, based mainly upon the writer's own materials, but supplemented by a careful study of the work of other investigators who have described the structure and development of the Eusporangiatae. The writer's grateful acknowledgments are due to a number of colleagues who have rendered assistance in various ways. Especially is he indebted to Prof. E. C. Jeffrey, through whose kindness a large number of admirably preserved prothallia and young plants of Botrychium virgmianum were sent the writer, and in addition a number of valuable slides of the same. Without this material the work on Bo- trychium would have been very incomplete. To my colleague in Stanford University, Prof. L. L. Burlingame, thanks are due for valuable assistance in the preparation of the photographic plates, as well as for the use of a number of important slides of Ophioglossum and Helminthostachys. To Prof. J. C. Willis, of the Botanic Gardens at Peradeniya, the writer would express his appreciation of many kindnesses and assistance in collecting during his stay in Ceylon. It was the good fortune of the writer to enjoy the unequaled facilities for collecting material offered by the great gardens at Buitenzorg and Tjibodas in Java, where, through the interest and courtesy of the distinguished director, Professor M. Treub, whose recent death was such an irreparable loss to science, means were afforded for securing the most valuable materials used in the preparation of the present work. Douglas Houghton Campbell. Stanford University, April, 1910. TABLE OF CONTENTS. Preface iij Introduction 3-4 Part I. The Ophioglossales 1. The Gametophyte 6-35 Germination in Ophioglossum 7 The Adult Gametophyte of Ophioglossum ............ 10 The Histology of the Gametophyte 15 The Gametophyte of Botryc hium 16 The Histology of the Gametophyte of Botryckium 18 The Gametophyte of Helminthostachys in The Endophyte 21 The Sexual Organs 22 The Antheridium .............. 22 The Antheridium of Ophioglossum 22 The Antheridium of Botryc hium 24 The Antheridium of Helminthostachys 25 Spermatogenesis 26 The Archegonium 28 The Archegonium of Ophioglossum. . 28 The Archegonium of Botrychium ?o Fertilization ?r Fertilization in Botryckium virgtntanum. .......... 11 Significance of the Endophyte ?2 II. Tin Embryo ... 54-54 The Embryo of Ophioglossum ^4 The Development of the Primary Bud in Ophioglossum moluccanum 40 1 he Embryo of Ophioglossum vulgatum ............ 45 The Anatomy of the Young Sporophyte of Ophioglossum 44 The Embryo of Botrychium 46 The Embryo of Helminthostachys 54 III. Tin- Young Sporophyte 55-84 The "\oung Sporophyte ol Ophioglossum 55 The Young Sporophyte of Botrychium 50 The Young Sporophyte of Helminthostachys " Comparison of the Young Sporophvtes of the ( )phinglossacex Si IV. I in \iui r Sporophyte 85-116 ["he Sporophyte of Ophioglossum 86 The Anatomy of Euophioglossum 89 The Root in Euophioglossum <;,> Anatomy of Ophioderma 94 Anatomy of Cheiroglossa 99 The Sporophyte of Boti ychium 99 The Sporophyte of Helminthostachys 104 The Sporangiophore of the Ophioglossales 108 The Development of the Sporangiophore 1 10 The Development of the Sporangium 112 m CONTENTS Pari II. Tin Mak.vi n \i.i >. I I in Gam i Minn ii "9"I34 I In Prothallium ol Kaulfussta .... m llu Prothallium "i Dana i .... 124 I In Endophytt of the Marattiacea: '-7 I In Sexual Organs ,2° The Antheridium |2° Spermatogenesis ' '9 1'Ir Archegonium '3° Fertilization ' i l 11. lit 1 Embryo Mv'W The Embryo of Marattia '3° I lu I mbryo ol Ingiopteris ' Relationships oi mi Eusforangiatae 20^ CoNCl 1 -l"\ . 21/ Mini iogr vritv 2i<; List of Plati -- i Index 225 THE EUSPORANGIATAE THE COMPARATIVE MORPHOLOGY OF THE OPHIOGLOSSACEAE AND MARATTIACEAE INTRODUCTION. It is pretty well agreed among botanists that most of the seed plants, perhaps all of them, are descendants of some fern-like Paleozoic ancestors. The geological record is remarkably perfect in many respects, and our knowledge of many of these Paleozoic fossils is extraordinarily complete. Among the most important con- tributions made of late years to our knowledge of these Paleozoic fossils is the fact that many of these Paleozoic "ferns" were really seed-bearing plants intermediate in character between the true ferns and the more highly developed flowering plants. At best the fossil record is very incomplete in regard to many extremely impor- tant structural details, and therefore it is especially necessary that these points should be thoroughly studied in such of the existing ferns as, for any reason, seem to be at all closely related to the ancient Paleozoic types. The name Eusporangiatae was proposed by Goebel to include the two very peculiar families of fern-like plants, the Marattiaceae and Ophioglossace.e, which differ in several important respects from the much more numerous and specialized Leptosporangiata?, the predominant ferns of the present day. The Eusporangiatas comprise ioo or more species of widely distributed ferns, of which the Marattiaceae are mainly tropical in their distribution, while the Ophioglossaceae include a good many species of temperate regions as well. The Eusporangiatae are distinguished primarily by the character of the sporangium, which is always much more massive than in the typical ferns, the Eeptosporangiatae. In the latter, the sporangium can almost always be traced back to a single mother cell which usually arises from the surface of the leaf, while in the Eusporangiatae the sporangium is already multi- cellular when it is first recognizable. The Marattiaceae are in general appearance much like the typical ferns, and there is no question of their relationship to the Leptosporangiatae. The resem- blances are less obvious in the case of the Ophioglossaceae, and some students of the ferns have expressed the opinion that the Ophioglossace;e should be separated entirely from the ferns and placed in a distinct class. (See Bower 9.) A careful comparative study, however, of the two families included in the Eusporangiatae shows so many close correspondences in structure, both of the sporophyte and gameto- phyte, that their association together is amply justified. The Marattiaceae are known to be very ancient forms, unmistakable members of this family occurring abundantly in Paleozoic formations. The Ophioglossaceae, on the other hand, are very unsatisfactorily represented in a fossil condition, and for this reason doubt has been thrown on their antiquity, although their structures show strong evidences of an extremely primitive character. Certain of the oldest- known fossil ferns, the Botryopterideae, may possibly prove to be related to the Ophioglossaceae, but the evidence at present is not entirely conclusive. In spite of the unsatisfactory nature of the geological evidence, I am neverthe- less strongly inclined to believe that the Ophioglossace;e, on the whole, represent the most ancient type among the living ferns, this conclusion being based upon a very careful study of the structure of both gametophyte and sporophyte. There are many practical difficulties in tin- way of studying these interesting ferns, and these difficulties undoubtedly account for the comparatively small number of researches that have been made upon their development. A few species, like Ophioglossum vulgatum and several species of Botrychium, are widespread in their 4 INTRODUCTION range and occur in the temperate regions of Europe and America; hut very much thr larger number of these, including all of the Marattiaceae, are tropical or sub- tropical plants and are very seldom seen in cultivation, hence material for their study must be collected in their natural habitat. I he gametophytes, or sexual plants, are seldom encountered unless very special search is made foi them, and they have been but rarely collected; and even where the spores can be made to germinate, the long time necessary for the growth of the gametophyte under artificial conditions, and the difficulties of rearing them to maturity, readily explain the small number of successful attempts to study these plants under artificial conditions. As the result of several visits to the tropics of both the Old and New Worlds, the writer has collected material of all of the eusporangiate genera, except the recently discovered Archangiopteris from southwestern China and Macroglossum from Borneo. It has seemed desirable, with this material as a basis, to make a some- what comprehensive comparative study oi the whole group of the Eusporangiatae. In this study, especial attention will be devoted to the sn ucture and development of the gametophyte and embryo and to the development of the young vegetative organs of the sporophyte. The general anatomy and histology of the mature sporophyte have already been pretty satisfactorily studied and described for most of the genera and a particularly satisfactory account of the development of the sporangium has been given us in the comprehensive memoirs of Professor Bower (Bower 5, 6), and a detailed investigation of these points has hardly seemed necessary, although, so far as possible, the results of the work of previous investigators have been verified. The writer has already published various papers upon the development of both the Marattiaceae and Ophioglossaceae, and these have been freely drawn upon in the preparation of the present monograph; but these earlier studies have been materially extended by further work upon the species previously studied and in- vestigations have been made upon a number of species which hitherto have not been examined at all, or have been studied only very incompletely. This is partic- ularly true of the genus Danaa, which has received comparatively little attention hitherto. An especially fine lot of material of several species of Daniea was col- lected in Jamaica in 1908 and an extended study of the gametophyte and young sporophyte has been made, the complete results of which are now published for the first time. Attention has also been given to the development of the young sporophyte in the other eusporangiates, particularly in the peculiar genus Kaul- fussia, which, like Danaa, has received less attention from the students of these plants than have the genera Marattia and Angioptcris. ( )f late there has been a tendency, especially among students of the fossil ferns, to lay great stress upon the importance of the structure of the vascular skeleton of the ferns in the study of their phylogeny. It is therefore highly important that a careful examination should be made of the origin and development of the vascular system in those forms, i. e., the Eusporangiatae, which, there is good reason to sup- pose, are the nearest relations among living plants of the ancient Paleozoic ferns. The results of the writer's studies on the development of the fibro-vascular bundles of the sporophyte in all of the eusporangiate ferns have led to some rathei unexpected conclusions as to the real nature of the vascular system in these ferns, conclusions decidedly at variance with the views generally accepted at present. These results have seemed sufficiently important to warrant a more extended treat- ment of this subject than was at first contemplated; and this will explain what might otherwise seem to be a disproportionate amount of space devoted to the origin and development of the fibro-vascular system in the young sporophyte. PART I. THE OPHIOGLOSSALES. The Ophioglossales, the "adder-tongue" ferns, include three genera (of which two are practically cosmopolitan) and embrace numerous species, being sometimes further divided into several subgenera; the third genus is a monotypic one, confined to the tropics of the Old World. These three genera are evidently quite closely related among themselves and may without hesitation be referred to a single family, the Ophioglossacea?. The genus Ophioglossum includes several species of the temperate regions of the whole world, and some of the species, like O. vulgatum, are very widespread; the tropical species are much more numerous, but the number of these is very uncertain and much confusion exists as to the limits of certain species. The second genus, Botrychium, belongs mainly to the temperate regions, only a small number of species occurring in the tropics, and these are principally confined to the cooler mountain regions. The third genus, Helminthostachys, with a single described species, H. zeylanica, is a not uncommon fern of the lowland forests of the Indo-Malayan region. The Ophioglossaceae are for the most part quite glabrous plants, of small or moderate size. The smallest species, like Botrychium simplex and some of the smaller species of Ophioglossum (e. g., O. californicum, O. bergianum, and some of the smaller forms of O. molucccuium) may be only 5 or 6 centimeters in height. 1 he largest species of Botrychium and Helminthostachys sometimes attain a height of 50 centimeters or more, with ample, much-dissected leaves, while the long, ribbon- like, pendent leaves of the epiphytic Ophioglossum pendulum, the giant of the family, may reach a length of 1.5 meters. Kxcept for two species of Ophioglossum belonging to the sections Ophioderma and Cheiroglossa, the Ophioglossaceae are terrestrial plants, usually growing in soil abounding in humus, and the gametophyte in all cases is a subterranean structure quite destitute of chlorophyll. The stem is a rhiz.ome, which is short and upright in the terrestrial species of Ophioglossum and in Botrychium, but is dorsiventral in Helminthostachys and the epiphytic species Ophioglossum pendulum. In most of the species of the temperate regions only a single leaf is developed each year, but there are some exceptions to this rule in Ophioglossum, especially in the tropical species, where there is no interruption of the growth. The leaves are usually ample, and may be quite undivided, as in most species of Ophioglossum, or they may be dichotomously divided in 0. palmatum and some forms of 0. pendulum; or they may be much dissected, usually in a ternate fashion, in Botrychium and Helminthostachys. The sporangia are sometimes very large, and are borne upon characteristic spikes, or " sporangiophores," whose morpho- logical nature is a matter of some controversy. The venation of the leaves is reticulate in all species of Ophioglossum, but in the other genera it resembles that of the typical ferns. With the exception of Ophioglossum (Cheiroglossa) palmatum, the sporangio- phore is normally attached to the adaxial side of the leaf, usually near the junction of the lamina and petiole; but sometimes it is inserted much lower down, or it 5 THE OPHIOGLOSSALES may arise close to the base of the petiole and appear to be quite independent of the sterile leaf segment. The sporangia range in number from about half a dozen, in some of the smaller forms of Botrychium simplex and Opluoglossum, to many hundred in such large species as Botrychium virgimanum and Helminthostachys. There is a most interesting series of forms connecting the smaller and simpler types with the large and complicated ones. With the increasing complexity of the sporan- giophores, there is usually a reduction in the size of the sporangia, which, however, become better differentiated than in the simpler types. There is no question about the close relationships existing between the dif- ferent genera of the ( )phioglossacea?, but there is some difference of opinion as to their connection with other Pteridophytes. While they are probably sufficiently distinct to be relegated to a special order, Ophioglossales, their peculiarities hardly warrant separating them entirely from the ferns. As will be seen later, they offer many points of resemblance to the Marattiales, both in regard to the structure ot the adult sporophyte ami that of the gametophyte or sexual plant, and the early phases of embryonic development. These resemblances are too numerous ami too exact to make it at all likely that the two orders are unrelated. I. THE GAMETOPHYTE. The first discovery of the gametophyte of the Ophioglossacea? was made by Hofmeister (Hofmeister 1) who, in 1854, found the gametophyte of Botrychium lunaria; two years later Mettenius (Mettenius 1) described much more fully the prothallium of Ophioglossum pedunculosum, which was cultivated in the botanical garden at Leipzig. No further additions were made to the subject until the writer (Campbell 4) succeeded in obtaining the first germinating stages of 0. pendulum, collected in the Hawaiian Islands, and those of Botrychium virginianum; and the older gametophyte of the latter species was also described. In the year 1898 Jeffrey ( Jeffrey I) published a complete account of the gametophyte and embryo of Bo- trychium virginianum. Later contributions to the subject are those of Lang (Lang 1) on the gametophyte of Ophioglossum pendulum and of Helnuntliostachys; and the papers of Bruchmann (Bruchmann 1, 2) on the prothallium of Botrychium lunaria and Ophioglossum vulgatum. In 1905, Lyon (Lyon 1) published a brief account of the embryo of Botrychium obliquum. In 1906 I collected in Ceylon a few prothallia of a species of Ophioglossum which may have been 0. reticulatum, and shortly afterward, during a stay in Java, a number of prothallia of 0. moluccanum were found, as well as a tew belonging to an undetermined species associated with the latter. During my stay in Java I was fortunate enough to obtain also a large number of prothallia of 0. pendulum. An account of these has already been published (Campbell 8). All species of the Ophioglossaceae that have yet been examined agree in the underground habit and saprophytic nature of the gametophyte, which always has associated with it a peculiar endophytic fungus, or "mycorrhiza," which is un- doubtedly connected with the assimilation of organic food. Mettenius states that chlorophyll may be developed if the prothallium appears above ground, and Bruch- mann found that this was also true in 0. vulgatum; but as a rule the prothallium remains subterranean and quite destitute of chlorophyll. The spores of the Ophioglossacea? are always of the tetrahedral type, and when ripe possess a moderately thick sculptured outer membrane, which is usually colorless or pale yellow, so that the masses of spores are either white or a pale sulphur-yellow tint. The ripe spore as a rule is packed with granular matter, which makes the THE GAMETOPHYTE contents appear opaque and obscures more or less the centrally placed nucleus. A marked exception to the ordinary type of spore was that found in an undetermined species of Ophioglossum collected at Buitenzorg in Java. This was supposed to be 0. moluccanum, with which it was growing, but a comparison with typical specimens of the latter species showed marked differences, the most striking being the spores, which were larger than in the type, had much less dense contents, and were espe- cially notable in that they had regularly two nuclei, a condition unique, so far as I know, among the ferns. (See Campbell 8, fig. 157.) The granular contents of the spores include numerous albuminous granules, together with more or less starch and oil. GERMINATION IN OPHIOGLOSSUM. The first successful attempts to germinate the spores of Ophioglossum were made by me in 1892, when ripe spores of the epiphytic O. pendulum were collected in Hawaii and brought to California. This species was found in Hawaii, growing usually upon the trunks of tree ferns, and the spores were sown upon bits of the bark-like masses of roots, which in the commoner tree ferns of Hawaii (species of Cibotium) cover the trunk with a thick, felted mass. These masses of roots were kept in jars, and the spores were sown upon them, a good many of them germinating in course of time. The germination was very slow, the spores often remaining unchanged for months, and none of these young prothallia developed beyond the stage with three cells. This failure to develop further was undoubtedly due to the fact that they did not become infected with the mycorrhiza. which is essential to the full development of the prothallium. In 1906 ripe spores of the same species were collected in Ceylon and Java. In Ceylon spores were secured at the botanical garden in Peradeniya and in the Barrawa Reserve Forest near Hanwella, where Lang obtained his material. In Java the spores were mostly collected at Tjibodas. In all of these later experiments the spores were sown in humus collected from about the base of the spore-bearing plants. These masses of wet humus were kept in stoppered bottles. As in the earlier experiments, the germination was slow, the first germinating stages being found only after a month or more, and in some cases the spores remained un- changed for a very long period. The bottles containing the spores sown upon the humus were brought back to California, and a recent exami- nation (September 1909) showed a considerable number of apparently normal spores, as well as living three-celled prothallia, presumably the result of comparatively recent germination. Spores sown in Tjibodas on April 18, 1906, were first found germinating at Buitenzorg on May 24, germination at this time being pretty well advanced. On June 3 a number of these had three cells. The germination (fig. 1 ) in all cases corresponded closely with the writer's former observations and closely resembles that which we shall see pres- ently in Ophioglossum moluccanum. In no case could any chlorophyll be detected, A. Germinating spore of Ophioglossum pendulum. X360. B. Optical section of A. C. Three views of a very young gametophyte <>f <>. pendulum, infection b} tnycorrhizal fungus. X360. 8 THE OPHIOGLOSSALES and apparently the prothallium of O. pendulum is strictly saprophytic throughout its existence. The extremely favorable conditions for plant growth at Buitenzorg made this an unusually promising place for studying germination, and very soon after my arrival there a quantity of plants of Ophioglossum moluccanum, or what was sup- posed to be this species, was secured, and the spores were sown. Subsequent study showed that at least three species grew together at Buitenzorg, so that it is not at all certain that all of the germinating spores and the prothallia which were col- lected later really belonged to (). moluccanum. Since in all previous experiments with the Ophioglossaceae germination had been very slow, it was with much surprise that the first lot of spores that were sown, when examined a week later, were found to be germinating very freely and had evidently been growing for some days. New sowings were made, and in some cases the first germination stages were evident within three days from the time the spores were sown. Inasmuch as the spores contain no chlorophyll, this rapid germina- tion is really remarkable. In the most favorable instances the greater part of the spores germinated and many thousand germinating spores were studied. The first sowings were made upon earth taken from where the plants were growing; the earth was placed in small glass dishes and Hooded with water; the spores were then scattered over the surface of the water; some sinking, but the greater part floating on the surface. In later experiments cavities were hollowed out in the earth and these were filled with water, while the rest of the earth was left wet, but not flooded. It was found that the spores germinated more promptly in the water than on the wet earth, and this suggested that probably under natural conditions germination occurs where the spores fall in slight depressions which are filled with water for a time after heavy rains. The prothallia of Hrlminthostachys, to judge from the locality where they were found in the Banawa Forest, occutred only where the forest was subject to inundation, and it may be that immersion in water is necessary for the germination of Ophioglossum moluccanum, or at any rate facilitates germination. The older prothallia of this species which were found in Buitenzorg were growing in low ground between the projecting roots of trees, where water might very well stand for some days in wet weather. The first sign of germination consists in the enlargement of the spore contents, which soon burst the rigid outer membrane along the three lines upon its ventral face, and through the cleft thus formed the spore contents, surrounded by the col- orless inner membrane ("endospore" or "intine") protrude as a blunt papilla (plate I, fig. 2). The first division wall is transverse and is soon followed by a second wall in the upper cell (i. e., the one turned away from the opening). The second wall is at right angles to the first, and the young prothallium now consists of three cells, the basal one, which is partly exposed through the cleft at the upper side of the spore, and the two upper cells, which have more dense contents than the basal one. The basal cell in position corresponds to the first rhizoid in the germi- nating spun of the typical ferns, but it was never found to become extended into a true rhizoid, and no rhizoids were seen in any of the very young prothallia. While Ophioglossum moluccanum and 0. pendulum agree very closely in the early stages of germination, in the latter species no trace of chlorophyll can be detected at any time, but in 0. moluccanum it is not uncommon to find from one to three chloroplasts present. These chloroplasts are very pale in color, but it is certain that a small amount of chlorophyll is present in some cases. While most of the young prothallia ofOphiogioi \um molw < anum do not advance beyond a three-celled stage, a few were found in which there were four cells, but all THE GAMETOPHYTE attempts to cany them beyond this stage failed and after exhausting the spore con- tents they soon died. The small amount of chlorophyll occasionally found in these young prothallia is evidently insufficient for their independent growth, and after a few weeks the granular contents all disappear and the cells soon collapse, showing that the young gametophyte has died from starvation. A number of specimens of the rare Ophioglossum intermedium were collected near Buitenzorg and a few ripe spores were also secured. These spores contain somewhat less dense contents than those of the other species described and have a more delicate epispore. Spores were sown on March 30 in Buitenzorg, and when examined about two weeks later no germinations were found, nor did a second examination about the end of April show any further results. On May 21, however, two three-celled prothallia were found, and subsequently a small number of others, but no later stages were discovered. The young prothallia appeared in every respect like the similar stages in the other species (plate 1, fig. 6). In Ophioglossum pendulum a number of young prothallia were found which had increased very much in size and undergone further division. The first of these were observed on April 3, and had developed from spores sown in Peradeniya on February 9. These young prothallia (fig. 1, c) had from four to six cells. It was found that in each case the mycorrhiza had connected itself with the young prothallium, and evidently had caused a stimulus in its growth. In every case where the young prothallium had more than three cells there was found associated with it the mycorrhiza, which could be easily seen to penetrate into the basal cell. The infection was in all cases due to fragments of mycelium, and in no case to any- thing which could be interpreted as spores. The fungus was apparently growing free in the humus where the spores were sown. This soil, as we shall see, was taken from about the roots of the sporophytes which furnished the spores. The free surface of the basal cell has its wall decidedly thickened, and it was here that the infection took place in all the specimens seen. The branching mycelium of the mycorrhiza was closely applied to the surface of the cell and a haustorium was sent down through the cell wall into the basal cell (fig. 1, m). This haustorium is pointed at first, but after it penetrates into the cell it enlarges and assumes the form of a somewhat thickened worm-shaped body, much thicker than the free mycelium outside. In the cell infected with the fungus, the contents show the peculiar aggre- gated appearance characteristic of the infected cells of the older prothallia. On the 6th of April a specimen with seven cells was found. About a month later, a number of other specimens were observed also, some of these having as many as thirteen cells (plate 1, fig. 9). This was the largest number found in any of the young prothallia. The divisions of these young prothallia are mainly in the lower cells, so that the apex, as in the prothallium of the true ferns, develops mainly from the lower of the two original p roth alii al cells. The basal cell, however, also undergoes divisions, and there is no very marked difference between the. lower and upper ends of the prothallium. At this stage there is a marked resemblance, except for the absence of chlorophyll, to the early germination stages of ' Lycopodium cernuum ( I reub I). The mycorrhiza penetrates the cells adjacent to the one first infected, but leaves the apical region free, and this region probably remains permanently free from the endophyte, as it does in the adult prothallium. The number of young prothallia found was too small to make it possible to determine exactly what may In- considered to be the normal succession of cell divi- sion, and whether at this early stage their is a definite apical cell could not he decided. As will be seen from the figures, there is evidently a good deal of variation in the 10 THE OPHIOGLOSSALES earl\' divisions. In these larger prothallia there is already the beginning of an axial tissue. Whether the cell v ( plate i , fig <;) is to be regarded as an apical cell, it would be hard to say. Owing to my departure from |ava about three months alter the Hrst observa- tions were made, it was impossible to trace the development ol the prothallia further, but this much is certain — without the infection of the fungus, growth will not pro- ceed beyond the three-celled stage, and apparently in 0. pendulum no chlorophyll will develop under any conditions, and the prothallium from its earliest stages must be considered saprophytic in its nutrition. Whether the oval body described as the product of germination is to be considered as a sort of tubercle, such as is found in Lycopodium cernuum, must be decided by further investigations. Lang's descrip- tions and figures of the smallest specimens which he discovered would indicate that this is not the case in 0. pendulum; but the tuberous body usually found at the base of the older prothallia in 0. moluccanutn (and this is true also in 0. vulgaturri) would indicate that in these species it is not impossible that a primary tubercle is first formed and subsequently the fertile branch. THE ADULT GAMETOPHYTE OF OPHIOGLOSSUM. In 1856 Mettenius (Mettenius 1) found the gametophyte of O. pedunculosum Desv., a tropical species, growing spontaneously in the pots where the plants had been cultivated in the botanical garden at Leipzig. He did not succeed, however, in making the spores germinate. These prothallia were slender, subterranean bodies, sometimes branched, sometimes without branches. They ranged in length from 1.5 lines to 2 inches (fig. 3, A, B). There was usually present a basal enlarge- ment or tuber, from which the fertile part of the prothallium extended. The older portions were brownish in color; the growing tips of the branch white. From the surface there grew numerous short brown rhizoids. Archegonia and antheridia grew more or less intermingled and were formed in large numbers. Lxcept for the greater size, these prothallia closely resemble those of O. moluccanum collected by me in Buitenzorg; and as 0. pedunculosum Desv. has been held to be a synonym of 0. moluccanum Schlecht, it is possible that Mettenius' s plants were the same as those found by me growing in Buitenzorg in Java.* The next account of the prothallium of Ophioglossum is that of Lang (Lang 1). He collected in Ceylon specimens of the prothallia of Ophioglossum pendulum. These were found in the Barrawa Reserve Forest, not far from Colombo, and were buried in the humus accumulated between the leaf bases of Polypodtum quercifolium, an epiphytic fern to which O. pendulum is often attached. I visited this same locality in February, 1906, but was unsuccessful in collecting the prothallia, although I obtained numbers of the sporophytes. Some time after, however, when in Java, I found a very large number of prothallia which were growing in much the same way as those collected by Lang, except that in this case the fern to which the Ophio- glossum was attached was the widespread bird's-nest fern, Asplemum nidus. Bruchmann has given a detailed account of the prothallium of the widespread O. vulgatum, which agrees closely in its essential details with O. pedunculosum and 0. moluccanum. Bruchmann's specimens were collected in the Thuringian rorest, in a depression that was subjected at times to overflow, a condition paralleled by the locations where the prothailia of Helminthostachys were collected by Lang and * Cristensen in his recent Index Filicum (loon) regards 0. moluccanum as a synonym of 0. prtlunculowm. Through the kind- ness of K. Goebel, I recently bad an opportunity of examining tome specimens of O. pedunculosum growing in the mil il garden in Munich. These ['Lints were the descendants of the specimens in Leipzig described by Mettenius and certainly wry closely resembled the typical O. moluccanum from Buitenzorg, THE GAMETOPHYTE 11 myself, and also like the locality where most of my specimens of 0. moluccanum were found in Java. This, in connection with my experiments in germinating the spores of 0. moluccanum, makes it not unlikely that actual submersion is a necessary condition for the germination in the terrestrial species of Ophtoglossum. In April 1906, about fifty prothallia of 0. moluccanum were collected by me at Buitenzorg, where, as we have seen, this species is extremely abundant. Only a small number of these were young enough to show the young reproductive organs, and most of them had already developed the young sporophyte. The greater part of these prothallia were found growing together in a slight depression between the projecting roots of a tree. These prothallia were slender bodies, from 5 to 10 millimeters in length, and none of them branched. They showed a more or less conspicuous basal tuber, like that described by Mettenius for 0. pedunculosum, and indeed they very closely resembled his figures of the simpler forms of that species, but are very much smaller. The youngest specimen found (plate 1, fig. 10) consisted of a small, irregular tuberous body of a brownish color, from which grew a short appendage or branch, the tip of which was white. The older ones also showed this basal tuber, but the cylindrical branch was much longer. Owing to their slender form, the prothallia are not always easily distinguishable from roots, and in some cases a microscopic examination is necessary before one can be sure of their real nature. Growing from the surface are scattered short brown hairs like those described by Mettenius for O. pedunculosum. Archegonia and antheridia are formed at an early period, and can be traced to the base of the fertile branch, or in some cases may be found even upon the tuber. In these specimens the reproductive organs seemed to be formed in rather smaller numbers than is the case either in 0. pedunculosum or 0. vulgatum. Among the prothallia was one very much larger than the others which had very large numbers of old archegonia. It is highly probable that this represents a second species, but unfortunately there was no means of determining to which of the two or three forms associated under the name 0. moluccanum it belonged. The cells of the tuber and the lower part of the branch contain the character- istic endophytic fungus, but the greater part of the fertile branch is quite free from this, and the cells appear almost transparent, but they contain numerous small starch granules. The endophyte is much more abundant in the cells of the tuber. The hyph;e, which stain readily with gentian violet, are irregular in outline and branch freely. Very often branches can be seen piercing the walls of adjacent cells. In the living condition the pointed apex of the prothallium is pure white, and even with a hand lens the projection of the antheridia is clearly evident. Mettenius noticed this "varicose" appearance of the smaller prothallia in 0. pedunculosum. A median section of the branch shows that the end is decidedly pointed and has a clearly defined apical cell. Owing to the very small amount of material available, no satisfactory transverse section of the apex could be made, and it can not be told whether the apical cell in transverse section is three-sided, as described by Bruch- mann for 0. vulgatum, or is four-sided, as it is in 0. pendulum. In the smaller speci- mens antheridia were more numerous than archegonia, although several of the latter were present. The antheridia arise, in general, in acropetal succession, but it is not unlikely that secondary ones may be formed also. The archegonia are scattered among the antheridia apparently without any definite order. In some specimens, especially in the larger ones, the old archegonia were found in great numbers, many more than the antheridia. In other specimens a considerable part of the prothallium was quite destitute of either archegonia or antheridia, and in this respect the pro- thallium of O. moluccanum differs from that of 0. pedunculosum or 0. vulgatum. 12 THE OPHIOGLOSSALKS I he gametophyte ol 0. moluccanum shows a greatei or less number of rhizoids both at the base and along the fertile branch (plate 1, fig. 10). In the more slender forms, howevei . these rhizoids are few. They are in some eases two-celled, but more commonly consist of a single elongated cell. It is not unusual to find within this the penetrating filament of the mycorrhiza, as has been described for othei species of Ophioglossum. I he rhizoids are much longei relatively than in 0. pendulum. According to Hruchmann, the rhizoids are quite absent from the prothallia of 0. vulgatum. Mettenius states that in O. pedunculosum the prothallia often appear above the surface of the earth, and they then become somewhat flattened and sometimes divided into several small lobes, and in such cases chlorophyll is developed. Met- tenius, however, does not note any further development of these green lobes. Hruchmann found that chlorophyll might also develop in 0. vulgatum, when the prothallia were exposed to the light, but he did not find any flattening of the apex. Owing to the very small number of growing prothallia found by me, I could not test the power of developing chlorophyll in 0. moluccanum, but the occurrence of chromatophores in the germinating spores makes it highly probable that chlorophyll may be developed in the older prothallia under the stimulus of light. Fig. 2. Gametophytes, fir, and young sporophytes of Ophioglossum moluccanum and allied species, slightly enlarged. C and F from Hakgala, Ceylon; the others from Buitenzorg, Java; k, bud on primary root;/, primary leafj I2, secondary leaf; /, tuberous enlargement at base of gametophyte. The small size of the prothallium in 0. moluccanum and the cessation of growth after the sporophyte is formed indicate that the gametophyte lives only for one season, and this is probably the case also in Helminthostachys. In this respect 0. moluccanum and its allies differ markedly from (). pendulum and 0. vulgatum, where the gametophyte lives foi many years. At Hakgala, in Ceylon, an undetermined species of Ophioglossum of the type of O. retintlatimi is common. After careful search, a few prothallia which closely resembled those of 0. moluccanum were collected (fig. i, (.', /•'). The material was too scanty to make a detailed study possible, but from the external appearance it is likely that the structural details would closely resemble those of 0. moluccanum. I he prothallium of Ophioglossum vulgatum, according to Hruchmann (Hruch- mann I), closely resembles that of 0. moluccanum, but is very much larger, some- times reaching a length of 6 centimeters, and it is not infrequendy branched THE GAMETOPHYTE 13 Fig. 3. A, B. Gametophytes of Ophiogbssum pedunculoium (after Mettenius). C, D. Gametophytes of 0. vulgatum (after Bruchmann). E-G. Gametophytes of 0. pendulum, sp, young sporophyte; r, secondary root. (fig 3, C). There is a more or less conspicuous basal tuber, like that found in 0. moluccanum, and from this extends a branch bearing the reproductive organs; but, unlike the prothal- (ium of 0. moluccanum, ^s there seems to be a complete absence of rhizoids. In general, the form is more irreg- ular than that of 0. moluccanum and more- over it is very long- lived. The branches may become detached and thus form new in- dividuals. From the rate of growth in speci- mens kept under ob- servation for several months, Bruchmann concluded that they might live for twenty years or more. Where the apical growth of a branch is interfered with, there may be a formation of adventitious buds, a phenomenon which is also common in O. pendulum. Ophwglossum (Ophwderma) pendulum, a remarkable epiphytic species wide- spread through the tropics of the Old World and reaching to Hawaii, is the giant of the order, the pendent ribbon-shaped leaves sometimes attaining a length of 1 .5 meters. The sporophyte grows rooted in masses of humus, either upon the rough trunk of a tree fern or palm, or hanging from the mass of humus accumu- lated about the base of certain epiphytic ferns. The bird's-nest fern, Aspic nium nidus, furnishes the favorite substratum for this species in the forest of Tjibodas in Java, where my material was collected. The young prothallium in 0. pendulum is an ovoid body, the somewhat smaller forward portion corresponding to the fertile branch in 0. moluccanum (see Lang I, page 25). The older ones become very much larger than in any other species that has been described, and they branch freely in all directions, except as their growth is controlled by their position. They are always found buried in a mass of humus between the imbricated leaf bases of the fern, and are often much flattened by the pressure of the leaves between which they are confined. Branches extend in all directions, but their growth is to some extent controlled by the inclosing leaf basis and also by the tangled mass of roots of the fern, which grow in all directions through the mass of humus and among which the branches of the prothallium of the Ophio- glossum extend. In one instance several hundred were collected from one large plant of Asplenium nidus. The prothallia closely resemble Lang's figures ami descriptions, but in many cases are very much larger and more extensively branched than any of the specimens collected by him in Ceylon (plate 1, figs. 11 14). I lie larger prothallia may be stellate in form, but they are usually very irregular. I he branches penetrate in all directions between the dense tangle of roots which the Asplenium sends into the humus between its persistent leaf bases, and on pulling these back a mass of fine humus is found, held together by the mat of roots so that it can be removed intact. The prothallia are excessively buttle, and it is practically impossible to remove the larger ones without a loss of some of the numerous branches. 14 THE OPHIOGLOSSALES These break oft at the least touch, and no doubt serve to propagate the gametophyte, which is apparently capable of unlimited growth in this way. It is often impossible to say whether the smaller gametophytes that are found loose in the humus are anything more than branches which have become spontaneously separated from the larger prothallia. The older parts of the gametophyte are dark brown in color, but the tips of the branches are white, as in the other species. The branching, as we have seen, may be very irregular, and old fragments kept moist often send out great numbers of adventitious buds (plate I, tig. n) which apparently in time develop into normal prothallia. A number of the commoner forms are shown in the figures. Plate i, fig. 14, represents the largest one met with. This is by no means complete, as a Fig. 4. A. Longitudinal section of apex of gametophyte of Ophioglossum moluccanum. <5 anthcridia; + archegonia; em, two-celled embryo. B. Apex more highly magnified, showing apical cell, x. C. Transverse section of a branch of the gametophyte of Ophiofdoisum pendulum. D. Apical cell of same. E. F. Short hairs from gametophyte of 0. pendulum; in E may be seen the endophytic fungus filaments. number of branches were unavoidably broken oft" in removing it from among tin tangle of roots in which it was embedded. This specimen measured about 15 millimeters in diameter — more than twice the size of the largest specimen secured by Lang. The surface of the older parts of the p roth allium shows a slightly roughened appearance due to the numerous very short papillate hairs which occur upon it. These are never of the slender form found in O. moluccanum and perhaps are not properly to be considered as rhizoids. Dotted over the surface are pale brown spots, easily seen with the naked eye, and these on examination arc found to be the large, empty antheridia. The branching is sometimes dichotomous, but lateral branches may arise at almost any point, and old fragments ol the prothallia, as already indicated, often develop many adventitious buds, a condition of things which apparently obtains also in the long-lived prothallia of 0. vulgatum. I be- rate of growth of prothallia kept by the writer for more than a year, as well as their THE GAMETOPHYTE 15 position in the humus about the plant, where they are found in the older parts between leaves which must have been dead for many years, indicate that they are very long-lived and, as we have seen, by the ready separation of the branches they are easily propagated. THE HISTOLOGY OF THE GAMETOPHYTE. The younger portions of the growing branches are composed of thin-walled, colorless parenchyma, whose cells have a conspicuous nucleus, and usually numerous small starch granules. In the older portions of the gametophyte the tissue becomes infected with the often-described endophytic fungus. Practically all of the cells of the basal tuber are thus infested, but in the fertile branches the central tissue is usually free from the fungus, and this medulla-like central tissue is surrounded by a more or less definite mantle of cells, in which the endophyte is especially luxuri- ant. The outermost cells are practically free from fungus, although new infections probably may take place there through these outer cells. In O. moluccanutn the endophyte is much less developed than in 0. pendulum, this no doubt being corre- lated with the much briefer duration of the gametophyte in the former species. According to Bruchmann, the endophyte is strongly developed also in O. vulgatum. The endophyte, as in the other forms that have been studied, is quite absent from the apical region of the branches. The limits of the infested zone are not very clearly marked and any cell of the older tissue of the gametophyte may harbor the fungus. Fig. 5. A. Longitudinal section of gametophyte apei of Ofthiogloswm vulpalum. X150. B. Transverse section of gametophyte apei. a, the apical cell. C. Archegonium. X225. D. Two free spermato/oids. X550. (All figures after Bruchmann.) The apical cell in O. pendulum is usually a four-sided pyramid, and not tetra- hedral, as it is in 0. vulgatum. In longitudinal section (fig. 4, /)), the apical cell appears triangular with a fairly regular segmentation, but there is also active division in the adjacent tissue and apparently the segmentation of the apical cell is not ver) rapid. Cross-sections show the apical cell to be approximately four-sided (fig. 16, A), but the sides are not always of equal length and sometimes it is almost triangular in outline; possibly it may be that in some cases, as in 0. vulgatum, it is tetrahedral. There seems to be no absolute rule, however, as to the succession of divisions in the young segments. A more or less definite superficial layer arises from the first periclinal divisions, but anticlinals follow rapidly. K, Till-. Ol'IIHKiLOSSALES THE GAMETOPHYTE OF BOTRYCHI1 M Hofmeister (Hofmeister I), in his studies of the Archegoniates, has included Botrychium lunar i a, whose prothallium he discovered in i S 54. No further contri- butions to om knowledge of the subject are recorded until 1893, when the writer dis- covered at Grosse He, Michigan, a number of old prothallia of />'. uirginianum, with the young sporophytes attached. 1 he earliest germination stages of this species were also secured. In [895, Prof. E. C. [effrey collected at several points in Canada a large number ot prothallia of this species, and his account ( Jeffrey I ) is much the most satisfactory one which had appeared up to that time. In the spring of 1903 Lyon (Lyon I) discovered prothallia of B. obliquum in Minnesota, and the following year secured a few specimens of B . simplex and B. matricaritefolium, but he has only published a brief note in regard to these species. He states that the reproductive organs 111 B. obliquum "differ essentially from those of B. virginianum," but he does not explain in what these differences consist. Bruchmann, who has added so much to our knowledge of the gametophyte of the European Lycopodiaceae and Ophio- glossaceae, has recently given a very satisfactory account of the prothallium and embryo of B. lunaria, which he found in various parts of German}' and Switzerland. He corrected certain errors made by Hofmeister in his account of the same species. ( Bruchmann 2.) ■ • Fig. b.--llvtr\t liium virginianum. A, B. (»crminating spore (B, optical section). C. Three gametophytes, X3; cm, embryo. D. Section of gametophyte, X 12; the shaded region is that occupied by the endophytc. S anthcridia. E. Apical region of gametophyte, X150. F. Short multicellular hair or paraphysis. Owing to the kindness of Professor [effrey, a large numbei of prothallia and young sporophytes of B. virginianum, together with several slides showing the sexual organs and embryos, were put at my disposal. This has made it possible for me to make a very satisfactory study of the reproductive organs and embryo in this species. The spores of Botrychium, like those of Ophioglossum, are of the tetrahedral cype and arc quite colorless, their contents showing the usual granular appearance, but without any trace of chlorophyll. The early stages of germination are exactly like those of Ophioglossum, the first division wall being transverse, this being then followed by a second wall in the inner cell at right angles to the primary wall (fig. 6, A, B). A few chloroplasts were seen in some of the cells, but this does not seem to be a constant character and perhaps was an abnormality due to the spores having THE GAMETOPHVI I 17 germinated in the light. Bruchmann (Bruchmann 2, page 207) also found the earliest stages in B. lunaria, and these corresponded exactly to those in B. vir- gmianum. The gametophyte of B. virgimanum is a huge, tuber-like body, which grows at a depth of about 10 centimeters below the surface of the earth. 1 he smallest of the specimens found by Jeffrey were 2 millimeters in length by 1.5 millimeters in breadth, exceeding in size the fully developed prothallia of B. lunaria. I he older ones may reach a length of 20 millimeters. I he young prothallia are quite smoothly oval in outline (fig. 6, C), while the older ones are more or less Fir.. 7. A. Young sporophyte of Botrychium virgini- attum attached to gametophyte, f*r. X2. B. A gametophyte with small sporophyte, */>. X4. *P B Fig. S. — Botrychium lunaria (after Bruchmann). A. T\V() NuUIIL; IMNI' 1< ■ ( ' 1 1 \ tc:.. B. An older one, with sporophyte, ., attai X16. C. Surface view of two antheridia, showing opercu- lar cells (shaded). X100. D. A spermatozoid. -:^oo. irregular, and occasionally the anterior end is divided into two equal loins. probably as a result of dichotomy. As in Ophioglossum, the older parts of the gametophyte are brownish in color, while the young apex appears white. In then earlier stages they are covered with numerous rhizoids, which disappear more 01 less completely as the prothallium becomes older. I he forward end is slightly pointed and there is a definite growing point. The antheridia make their appear- ance first, and together with the archegonia are formed only upon the upper surface of the dorsiventral thallus, which is noticeably different in form from the cylindrical radially constructed prothallium of Ophioglossum. 2 18 THE OPHIOGLOSS All S I he antheridia form a more or less evident row, even in the very young prothallium, and later this median row of antheridia is raised upon an elevated ridge, upon whose sides the- archegonia later make their appearance (fig. 6, /)). The gametophyte of B.Iuiuina closely resembles the early stages of B. virgin- ianum. It is very much smaller and rarely exceeds a length of i millimeters, and, like the young stages in B. virgimanum, it is covered with mam long rhizoids (tig. 8, .•/, B.) The smaller prothalha are globular or oval in shape; the larger ones somewhat heart-shaped. I [ofmeister states that the antheridia occur upon the upper surface and the archegonia below; but Bruchmann found that both archegonia and antheridia were confined to the upper surface, as in B. virginianum. As in the latter, also, the dorsal ridge is present, bearing the antheridia upon its crest and the archegonia upon its flanks. I he large prothallia of B. virginianum live for mam years, and Jeffrey even found a plant bearing spores, which was still connected with a prothallium. It is probable that the very much smaller prothallia of B. lunaria have a shorter duration, but in this species also the prothallium persists for some time after the young sporophyte is established. THE HISTOLOGY OF THE GAMETOPHYTE OF BOTRYCHI1 M- In B. virginianum the forward part of the prothallium is made up of colorless tissue, which extends for some distance beyond tin youngest antheridia. The apical meristem in this species (fig. 6, E) lies on the Upper side of the prothallium; in a vertical longitudinal section it shows a group of columnar cells, one of which is Fi<;. 9. A. Transverse section of a gametophyte of G phioglossum pendulum, showing position of endophytic fungus. X20. The shaded region is occupied by the endophyte. B. Endophyte from prothallium of Botrychtum virginianum* C. Sporangium-like enlargements of endophyte. probably the apical cell; but this is not absolutely certain. In B. lunaria (see Bruchmann 2, page 209) the apical meristem occupies the middle region of the anterior surface of the prothallium, and is of very limited extent. As in B. vir- ginianum, its cells appear columnar in form, but neither in longitudinal section nor in a surface view could a single initial cell be recognized. The rhizoids in B. virginianum are from 1 to 4 millimeters in length and may be multicellular, especially those which arise from the crest ami flanks of the pro- thallium, while those which originate from the base are unicellular and longer than these multicellular ones. It is not unlikely that these multicellular structures are rather of the nature of paraphyses than true rhizoids. In B lunaria the rhizoids seem to be, usually at least, unicellular. In both species the rhizoids soon turn brown and their walls become strongly cutinized. Both Jeffrey and Bruchmann THE GAMETOPHYTE 19 observed the penetration of fungus hyphae into the rhizoids, and they believe that the fungus which occurs within the thallus is, mainly at least, due to this method of infection. From a comparison with the early infection of the young prothallium in Ophioglossum, it seems to me more probable that the young prothallium in Botrychium also is infected at a very early period and that the endophyte, once established within its tissues, grows with the development of the gametophyte, the secondary infection through the rhizoids of the older gametophyte being of minor importance. In both species of Botrychium the infected region comprises the greater part of the central tissue, leaving only a comparatively narrow peripheral region free from the endophyte. This uninfected area is thicker upon the upper surface and comprises the whole of the meristematic region, together with the developing sexual organs. As in Ophioglossum, this uninfected tissue contains small starch granules in considerable numbers, but not much else in the way of granular contents. The invasion of the fungus results in the destruction of the starch and the accumulation of large amounts of oil. This oil, according to Jeffrey, is not readily soluble in alcohol, and the cells containing it, both in fresh and stained sections, appear dark- colored. THE GAMETOPHYTE OF HELMINTHOST.H IIYS. The monotypic Hclminthostachys zeylanica is not uncommon throughout the lowlands of the Indo-Malayan region and often occurs in large numbers. The only account yet published of the gametophyte is that of Lang (Lang 1). The prothallia which he describes were collected in part by himself in the Barrawa Reserve Forest in Ceylon in March 1901. Other material studied by Lang was collected at the same place by Mr. Coomara Swamy. I made a visit to the same locality in February 1906, and also found a considerable number of prothallia, but all of these had been fertilized and had attached to them the young sporophyte, so that no young repro- ductive organs were found. The forest where they were collected is subject to inundation from a river which runs through it, and it was in the parts that had been overflowed that the young plants were discovered. This makes it not unlikely that, as in the case of Ophioglossum moluccanum, germination is favored by having the spores immersed in water, and this may be a necessary condition for the first stages of germination. A quantity of ripe spores were collected and various attempts were made to germinate these, but without success, and, as none of the specimens obtained by Lang were very young, the early history of the prothallium still remains to be investigated. As in the other Ophioglossaceae, the prothallia are subterranean, occurring at a depth of from 5 to 6 centimeters in the earth. In form (figs. 10, 11) they are some- what intermediate between Botrychium and Ophioglossum, but are on the whole more like the latter. They are somewhat irregular in outline, with a broad base, recalling the basal tuber of 0. moluccanum, but this tuber is relatively larger and more lobed. From this basal tuberous portion a short upright branch extends, much as in Ophioglossum moluccanum, but it is relatively thicker and shorter. The whole gametophyte is radial in structure, as in Ophioglossum, and thus differs strikingly from the dorsiventral gametophyte of Botrychium. The basal enlarged portion is brown in color and covered with rhizoids, which are mostly absent from the upper, more slender part upon which the reproductive organs are borne. Lang found that there is a tendency to dicecism in the prothallia, some pro- ducing only antheridia (figure 11, A), while in others archegonia predominate, 20 III I OI'IIHK.l.OSS \l 1 S ilthoueh m the latter a few antheridia are almost alw ays developed before tin- arche ii ( >thalhum tin goma appear. In the mah >asal "vegetative portion is relatively small and often is irregularly lobed, these lobes being not ol tin- nature of true branches, but merely the result <>f unequal growth. The female gametophyte (fig. io) lias the basal region relatively larger and is even more irregular in form than the male, while tin- fertile region is shorter and wider. Some i>t the more elongated types of the malt' gametophyte an- quite similar to tlu- simple prothallium ol Ophioglossum moluccanum. As in the other genera, the endophytic fungus is always present and occupies much the same position as in Botrychium. Lang found that the growth is due to the activity of a single apical cell having the form of a four-sided pyramid, much like that found in Ophioglossum pendulum. 1 he lateral segments divide by periclinal walls, and from the superficial cells thus formed originates the layer of tissue from which the sexual organs arise. The inner cells contribute to the axial tissue of the gametophyte. The antheridia in the male plants are evenly distributed about the periphery, so that in cross-section they form a nearly uniform circle. I he axial tissue, especially in the male prothallium, has the cells much elongated, and Lang thinks they are useful in the conduction of plastic material from the basal region of the prothallium to the growing point. I he outer part of the basal region consists of two or three layers ol somewhat flattened cells, which, like the corresponding tissue in Botrychium, are free from the endophyte. Prom some of these superficial cells there are developed unicellular, elongated rhizoids, with markedly cutinized walls. Ibis cutinization extends to the outer walls of the superficial cells, while the inner walls of these cells, as well as those of the inner tissue, show the cellulose reaction. I he formation of the lobes of the basal region are due, not to definite apical growth, but to irregular cell divi- sions in the outer layers, which remain tree from the fungus which occupies the greatei part of the central region of the lobes, as well as the axis of tlu- main shoot of the prothallium. The central tissue is made up of about equal parts of infected and uninfected cells. I In- latter, as in the other cases investigated, contain starch granules which are absent from the cells harboring the fungus. Fungus hyphae were seen in many cases to penetrate the rhizoids, but it is highly probable that in Helminthostachyi also there is a primary infection at an early stage in the germination of the spores. Lang notes that in the older male prothallia the fungus is dead and tin- further growth of the prothallium is dependent upon the amount of reserve food (mainly Fig. io. A. Gametophyte (/>r) of Helminthostachyi zeylanica, with young sporophyte attached; v, primary root. X 4. B. An older sporophyte, with fully-developed set ond leaf. X2. for, rudimentary cotyledon. C. Young second leaf, showing venation. X5. THE GAMETOPHYTE 21 starch) which is left in the cells. It is not clear what role the endophyte plays in the manufacture of the reserve food materials upon which the further growth of the prothallium depends. It is highly probable, however, that some of the necessary organic elements are derived from the destruction of the fungus tissues which serve as food for the further development of the prothallium. While no data are at hand to prove this, the lack of permanent growing tissue in the prothallium and the com- plete destruction of the fungus make it highly probable that the life of the gameto- phyte in Helminthostachys is restricted to a single season, as it is in Ophioglossum moluccanum. Fig. n. A. Male gametophyte of Helminthostachys. X8. C. Surface view of ripe antheridium, showing opercular cells B. Young antheridium. X::;. D. An old archegonium. X about 200. (Figs. A-C, after Lang.) THE ENDOPHYTE. 1 he endophytic fungus which inhabits the gametophyte in all of the Ophio- glossacea? is very much alike in all of the species, differing only slightly in size m different forms (fig. 9). A special study was made of this endophyte in Ophio- glossum pendulum, where it is especially well developed. As we have already seen, the endophyte is absent from the younger parts of the prothallium, but in the older parts it is exceedingly conspicuous. Sometimes fragments of a fungus are found outside the prothallium, growing in the humus, and these are evidently the same forms that infect the very young prothallium when it arises from the germinating spores. These external hyphae in some cases have an occasional septum, and this is also the case in those forms which infect the young prothallium. In all of the hyphae seen within the prothallium, however, these septa seem quite absent. I he infection of the prothallium through the rhizoids has been noted in all the species, but, as we have already stated, it is probable that the endophyte is derived mainly from the first infection of the very young gametophyte. An examination of tin young cells before the entrance of the fungus shows a conspicuous nucleus ami numerous starch grains, which stain very strongly with gentian violet. I he invading mycelium, whether from the outside of the prothallium or from the adjacent cells, penetrates the cell wall and ramifies within the cell, the growth being entirely intracellular. The hyphae are noticeably thicker than those of the external mycorrhiza. The endophyte is perfectly fixed with 1 per cent chromic acid and it stains well with the double stain of gentian violet and safranine. the walls assuming a violet color and the numerous nuclei staining deep led. In the younger lupine, which are of varying sizes, the protoplasm is densely granular, but in the older ones 22 THE OPHIOGLOSSALES the granular appearance disappears to a considerable extent, though the nuclei continue to stain strongly. 1 he fungus is quite variable in form. Sometimes the filaments are nearly straight, running from cell to cell and branching sparingly. Sometimes a cell is completely filled with a dense tangle of hyphae, while in other cases there are sack-like vesicles of very irregular form. Mot infrequently, quite n gular, nearly globular bodies are seen, recalling the obgonia of the Peronosporeae. 1 hese at first contain comparatively few nuclei, scattered through the granular cytoplasm, but in the older ones the nuclei are very numerous and decidedly larger. It looks sometimes as if this were a preparation for the formation of spores, but no certain evidence of the formation of such spores could be seen, although in several instances there was an appearance which might point to this. Structures resembling the "conidia," described by Jeffrey for Botrychium ( fefFrey I, page 12), were seen ami are probably the same thing. As the fungus invades the young cells, irregular, strongly-staining clumps are formed by the aggregation and breaking down of the starch grains. The nucleus of the host cell appears to be but slightly affected by the growth of the fungus, and can usually be found quite unchanged, even in those cells which are almost com- pletely filled by the endophyte. Finally, the thin-walled vesicular growths of the fungus are quite broken down and probably serve to nourish the cells of the gameto- phyte, which is thus parasitic upon the cells of the fungus. The systematic position of the fungus is doubtful. I he varicose, swollen hyphae found at certain stages closely resemble a parasitic fungus, Completoria complens, which is sometimes extremely destructive to green fern prothallia. The oogonium-like organs, often present, suggest Pytluum, a parasitic fungus belonging to the Peronosporeae. Jeffrey ( Jeffrey I, page 13) thinks the endophyte may fairly be regarded as an intermediate form between the two genera, Completona and Pythium, and says thai in this case Completoria should be placed in the Peionospoieie instead of in the Entomophthoreae, where it has been placed by some students of the Fungi. I Ml M \l \l, ORGANS. THE ANTHERIOT1 M The antheridium in all of the Ophioglossaceae is of much the same type and closely resembles that of the Marattiaceae and also Equisetum and Lycopodtum. The mother cell does not project at all above the level of the adjacent tissue, and in all cases the first division separates a superficial or cover cell from an inner one, the latter by further divisions giving rise to a mass of sperm cells which may be very numerous. The cover cell also divides, sometimes only in one plan, sometimes (e. g., in Botrychium) having also periclinal divisions. At maturity the antheridium may project more or less strongly, but this is not always the case. THE ANTHERIDIUM OF OPHIOGLOSSUM The antheridium was first described by Mettenius in Ophioglossum pedunculo- sum, and later Lang described it for 0. pendulum and Bruchmann for 0. vulgatum. The present account is based mainly upon my own studies of O. molui cartum and (). pendulum. 1 he mother cell of the antheridium, which may arise very close to the growing point of the prothallium, lies Hush with the neighboring cells, but later it may become more or less elevated above the surface, forming a prominence which, in slender prothallia, like those of Ophioglossum moluccanum, may give an irregular undulate outline to the branch which bears them (fig. 4, A). In 0. pendulum THE GAMETOPHYTE 23 this projection is less marked, although this may be somewhat decided in the ripe antheridium. The first division in the inner cell is usually vertical, but may be transverse, at least in O. pendulum (fig. 13, A). The second walls intersect the first at right angles, and there are always four nearly equal inner cells resulting (fig. 13, D). The third set of walls is vertical, and the next, in some cases at least., is in the same direction. This is not true, however, of the deeper and narrower type of antheridium. Further divisions occur until the number of spermatocytes is very large. The fully developed mass of spermatocytes is plainly visible to the naked eye, and in O. pendulum may reach a diameter of more than 325 p. 250 cells and upward may be counted in a single section of a large antheridium, and this would mean that there are several thousand in the whole antheridium — perhaps more than in any other Pteridophyte. In O. moluccanum (fig. 12), the number of spermatocytes is much smaller, while in this regard the antheridium of 0. vulgatum appears to be intermediate between that of 0. moluccanum and 0. pendulum. JUG. 12. — O p/no^loaum molurcamtm. A. Ripe antheridium. X180. B, C. Young antheridia. X320. D. Surface view of antheridium, showing opercular cell. The nuclei of the young cells show a conspicuous nucleolus, which becomes less marked in the later stages of the antheridium. According to Mettenius, the outer wall of the antheridium in 0. pedunculosum is composed of two layers of cells; but both Lang and Bruchmann found that the central part of this outer wall of the Fig. 13. — Development of the antheridium in Uphioglossum pendulum. A. Median section of gametophyte, apex, showing apical cell, x, and two young anthcridia, 8 . X 175. B. Transverse section of apex; x, apical cell. C. D. Median sections of young antheridia. E. Median section of a nearly ripe antheridium. F. Surface view of ripe antheridium; 0, opercular cell. 24 THE OPHIOGLOSS \l I - antheridium is but one cell in thickness, and I have verified this for both of the species under consideration. The first wall to be formed in the cover cell is a nearly median one and vertical (fig. 12, />), and this is followed by a second wall which intersects it, as well as one of the lateral walls ol the primary cover cell, so as to include a nearly triangular cell. In this triangular cell there are later formed, as both Bruchmann and Lang showed, a varying number ol segments arranged spirally in the fashion of the segments of a three -si ded apical cell (fig. 13, F). I he same thing occurs in Lycopodium ( Treub I ). Phavc also found a similar condition m the antheridium both of the Marattiaceae and of Equisetum. The last-formed triangular cell is the opercular cell (fig. 1 .;,/-, 0). From the prothallial tissue which adjoins the- sperm cells are cut off flattened cells which surround the sperm cells with a more or less definite layer of "mantle" cells. The limits of the original cover cell are usually plainly visible in both longitudinal and surface sections. THE ANTHERIDIUM or BOTRYCHIUM. The antheridia in Botrychium occur only upon the dorsal surface of the gam- etophyte, which is always monoecious. I he hist antheridia in />'. virginianum (see [effrey I, page M), form a small cluster which is not noticeably raised above the general level of the prothallium, and from this primary cluster extends a single median line of antheridia toward the apex of the gametophvte. Later this median region becomes raised and forms a conspicuous ridge along whose crest the anthe- 1 ul 1:1 are borne. In H. lunaria, according to Bruchmann, the arrangement is much the same. In.. 14. — Development of tin- antheridium in Botrychium virginianum. \ |). Longitudinal sections of young antheridia. ■ ;;o. E II. Transverse sections. 1', O show only the youngs] rlf surface view showing opercular cell,o. I, section of two ripe antheridia. X80. The development of the antheridium is very much like that of Ophioglossum. I In fust division, as in the latter, is a pcriclinal one, separating the primary cover cell from the mother cell of the spermatocytes. I he divisions in the cover cell, however, differ somewhat from those in Ophioglossum, in that periclinal walls THE GAMETOPHYTE 25 are formed in some of these, so that the cover becomes to some extent double. Jeffrey states that the cover is two layers throughout, but Bruchmann found that some of the cells— two and sometimes three — remain undivided and that one of these undivided cells functions as the opercular cell. I have examined this point carefully in Botrychium virginianum and find that there is an opercular cell in this species also. The division walls in the primary cover cell usually intersect at right angles, so that the opercular cell is four-sided, instead of triangular, as in Ophio- glossum (fig. 14, H); but exceptionally it may have the triangular form. In the dehiscence of the antheridium only a single opercular cell is destroyed, not two superimposed cells, as Jeffrey supposed to be the case. The divisions in the central cell are very much the same as in Ophioglossum. Whether the first division is regularly transverse or vertical could not be determined, as no examples of the first division were found. The earliest stages seen after the first separation of the cover cell had four inner cells arranged quadrant-wise, but it was not clear which was the primary wall. The quadrant division is, usually at least, followed by an octant division, and these early divisions may sometimes be recognized for a long while, as there is not much displacement of the cells due to the subsequent divisions. In the later stages the primary divisions can no longer be made out clearly. The number of sperm cells finally formed may be very large — more than a thousand in the largest antheridia- thus nearly or quite equaling the number found in Ophioglossum pendulum, although the sperm cells are very much less in size than in the latter and the antheridium correspondingly smaller. Some- times the antheridium is divided into irregular chambers, apparently due to the persistence of some of the early division walls (fig. 15, B). • •■• Fin. 15. — Botrychium virginianum. A. Longitudinal section of a nearly ripe, hut rather small, antheridium. X about 200. B. Section of a ripe antheridium which has discharged the spermatozoids; some cell-walls have remained, forming irregular chambers, and retaining some of the spermatozoids. C. A surface view of a ripe antheridium, showing square opercular cell. The antheridium of B. lunaria, according to Bruchmann, is smaller than that of B. virginianum, but the size of the spermatocytes and spermatozoids seems to he about the same (fig. 8, D). THE ANTHERIDIUM OF HELMINTHOSTACHYS. The antheridium of H elminthostachys has been studied by Lang, and ir is evident from his account that it resembles that of Botrychium much more than it does that of Ophioglossum. As in the former, the primary cover cell undergoes periclinal divisions as well as anticlinal ones, but there are from two to tour cells which do not divide by periclinal walls, ami one ol these cells Incomes the opercular cell (fig. ii, C). These four undivided cells stain more strongly than the other cells of the cover, but only one of them is broken down ;ir the time of dehiscence. These 26 THE OPHIOGLOSSALES cells, as m the case ol Botrychium lunar i a, are nearly square in outline, most of the walls in tin cover cells being at right angles to each other, as in Botrychium. I he divisions in the central cell are verj similar to those in Ophioglossum and Botrychium, anil the original quadrant divisions are perceptible for some time. 1 he number ol spermatocytes, as in Ophioglossum and Botrychium, may be very large. I'he spermatozoids were not seen by Lang, but from a comparison of the nuclei of the spermatocytes they are apparently of about the same size as those of Botrychium. SPERM VTOGENESIS. Ophioglossum is especially suited to a study of spermatogenesis, owing to the very large size of the spermatozoids. Those of 0. pendulum are probably the largest known among the Pteridophytes. In material fixed with i percent chromic acid, or weak 1* lemming's solution, and stained with gentian violet and aniline safranine, the coloring is beautifully clear and the blepharoplast stains with extra- ordinary sharpness. The spermatozoids of O. pendulum are larger than those ot 0. moluccanum or 0. vulgatum, but the development is very much the same. It the sperm cells are examined previous to the final division to form the spermatocytes (plate i, fig. in), the nucleus will be seen to have a small but distinct nucleolus and a dense reticulum. The whole nucleus stains strongly with safranine. I he cytoplasm is fairly dense, with granules of various kinds in it. In material fixed with !• lemming's solution there are often small black specks, probably fattv bodies, which sometimes interfere somewhat with the study ol the cytoplasm. In well-stained sections the blepharoplasts may be seen as two small, rounded bodies of a \ iolet color ( plate t, fig. 1 6, bt) lying near the nucleus. Several cases of the final nuclear divisions were nut with, but all of these wen- in material fixed with chromic acid, and the blepharoplasts wire not very well differentiated (plate 2, \\^. n). I he nuclear spindle is ver) distinct, and the very numerous chromosomes are so crowded rhat it was quite impossible to determine their number exactly, but it is very large. In the young spermatocytes (plate >, fig. 26) the nucleus shows a more or less con- spicuous reticulum, but the nucleolus has disappeared, as it usually does at this stage in all the forms that have been studied. The further development of the spermatozoid corresponds very closely with that of Equisetum (see Campbell 4 and Belajcff I, 2). One of the blepharoplasts in the primary spermatocyte goes with each daughter cell I the definitive spermatocyte) and can be seen as a distinct rounded body lying near the nucleus (plate 2, fig. 24). In some cases what appeared to be the blepharoplast was King in a depression at the periphery of the nucleus, and looked very much like a nucleolus. Before the nucleus undergoes any marked change, the blepharoplast begins to elongate (plate 2, figs. 26, 27) and assumes the form of a pointed, slender, strongly staining body lying near the nucleus. This body is really somewhat ribbon-shaped, and more pointed at one end than the other, and it is also somewhat curved, even in its earlier stages. A transverse section appeals crescent-shaped. The nucleus now elongates slightly and the reticulate structure becomes very conspicuous (plate 2, fig. 25). There are large, strongly stained granules, which are probably composed of several more or less coherent chromosomes, as the number of these granules is very much less than the number of chromosomes in the nuclear plate of the dividing nucleus. The blepharoplast continues to elongate, and in favorable cases the young cilia can be seen growing from it, but none of my preparations showed the cilia nearly so plainly as Belaieff's figures show7 them in Equisetum and Gymnogramme. There is no doubt, however, that the cilia arise in much the same way as BelajefF describes. The nucleus now becomes indented on one side and assumes a crescent THE GAMETOPHYTE 27 shape, elongating and also becoming more or less flattened. One end becomes narrower and sharply pointed, the other remaining thicker and rounded. The reticulum at this time stains with great intensity and shows a tendency to coalesce, which in the final stage results in an almost homogeneous, deeply-staining mass, composed apparently of the completely fused chromosomes. In successful prepara- tions the nucleus at this stage stains a clear carmine red, in strong contrast to the bright violet of the blepharoplast. With the complete coalescence of the chromo- somes the volume of the nucleus is noticeably decreased (plate I, fig. 19). The blepharoplast forms a spirally coiled narrow band, from which the cilia can be seen to grow, following its curve, but the blepharoplast is not in close contact with the nucleus. The spermatocytes and the nuclei are rather smaller in Ophioglossum moluc- canum than in O. pendulum. In the older stages of the spermatozoid, the nucleus in the former species is decidedly more elongated and more sharply pointed at both ends (plate 1, fig. 18). In this respect it more nearly resembles the spermatozoids of Botrychium and those of the true ferns; while in the larger, comparatively short nucleus, as well as in some other respects, the spermatozoid of O. pendulum is strik- ingly like that of Equisetum. The number of cilia is large, but the exact number could not be determined. Surrounding the spermatozoid and included in its coils is a considerable amount of cytoplasm. I was not successful in obtaining any living spermatozoids, although many attempts were made to do this, but in a number of sections of the opened antheridium some were found in which the spermatozoids had been retained. While these were usually more or less distorted, some were very well fixed ami gave a good idea of the structure of the free spermatozoid (plate 2, fig. 28). The cilia were very much distorted in some cases, but in some the}- were clearly seen. There is one thick posterior coil mainly composed of the nucleus, which is very much largei than that of the sperm cell before it is discharged from the antheridium. The nucleus has the form of a slightly coiled thick band, tapering somewhat at both ends, but more markedly so in front. Beyond this extends a second coil, composed, apparently, mainly of cytoplasm. This second coil extends into a third much smaller one, which seems to be a flattened band along whose upper edge the blepharoplast is closely applied (plate 2, fig. 28). The resemblance of the spermatozoid to that of Equisetum is very strong, but the nucleus is even more shortened than in the latter. The spermatozoid of Ophioglossum pendulum is larger than that of any Pterido- phyte that has yet, been described. The cytoplasmic envelope and vesicle are not very clearly separated and probably are similar to those of the free spermatozoids of 0. vulgatum or of Equisetum. Sometimes this protoplasmic envelope completely surrounds the lower part of the spermatozoid and reminds one somewhat of the peculiar spermatozoids of the Cycads. Owing to their large size, the spermatozoids were often sectioned, and in some of the sections the blepharoplast was free from the body of the spermatozoid and the attachment of the cilia was very easily seen (plate 2, fig. 29, a). Mettenius figures the free spermatozoids of 0. pedunculosum, but his figures are certainly not accurate. Bruchmann, who has figured those of 0. vulgatum (fig. 5, D), shows that they closely resemble the spermatozoids of the true ferns, bur are more massive, and the vesicle which envelops the posterior coils adheres more closely to the spermatozoid than is usual in the ferns. In these respects it resembles the spermatozoid of O. pendulum. Jeffrey examined the development of the spermatozoids in Botrychium vtr- gun/inum, which closely resemble those of Ophioglossum. Owing to the method of 28 THE OPHIOGLOSSALES staining which he employed, the blepharoplast was not clearly evident. I have examined the developing spermatozoids in this species, using the material which Professor Jeffrey was so kind as to send me, hut employing the double stain of safranine and gentian violet, which was used in the study of Ophioglossum. While the differentiation was inferior to that of Ophioglossum, where the material was fixed with Flemming's solution, nevertheless the presence of the blepharoplast was unmistakable. The spermatocytes are only about half as large as those of Ophioglossum pendulum, and the spermatozoids correspondingly smaller; hence less favorable for the study of details. It is evident, however, that the changes in the nucleus and blepharoplast are essentially the same as in Ophioglossum (plate 2, tigs. ^4 to 37). The nucleus, however, becomes much more elongated and the spermatozoids are more slender, in both of which respects it shows a resemblance to the spermatozoids of the typical ferns. The study of the free spermatozoids was confined to those which happened to have become fixed within the open antheridium and within the venter of recently opened archegonia. These were in some cases very satisfactorily fixed and stained, and showed very well indeed the general form of the spermatozoid. The cilia, however, were not as a rule very well fixed, ami while they are evidently numerous their number could not be made out. Bruchmann's figures (fig. 8, /)) of the free spermatozoid of Botrychium lunaria agree closely in form and size with those of B. virginianum. In both species the vesicle attached to the spermatozoid is more distinct than in Ophioglossum, and in this respect also the spermatozoid shows a likeness to the true ferns. THE ARCHEGONH M. The archegonium in the Ophioglossaceae is very much like that of the typical ferns in its general development. The mother cell of tin- archegonium, like that of the antheridium, is first divided bv a pcrielinal wall into an outer cover cell, which later gives rise to the four rows of neck cells, and an inner cell which, as in the typical ferns, divides again by a transverse wall into a lower or basal cell and a central cell, the latter subsequently giving rise to the egg cell and the canal cells. A marked feature 111 the archegonium of the Ophioglossace;e is the inconspicuous character of the ventral canal cell, which very often it is impossible to show, and it is possible that in some cases the ventral canal cell may not be formed at all, although I think this is doubtful. In general the archegonium is much alike in the three genera, but the neck is much shorter in Ophioglossum than in Botrychium or Helmmthostachys. THE ARCHEGONIUM or OPHIOGLOSSUM. My own studies of the development of the archegonium of Ophioglossum were based mainly upon a study of Ophioglossum pendulum. ( )nly a very small number of the young archegonia was secured in 0. moluccanum, and so it was impossible to make a complete study in this species. Lang accurately figures several stages of the archegonium in Ophioglossum pendulum, and Bruchmann has described quite completely its development in 0. vulgatum. Bruchmann tailed to see the two nuclei of the neck canal cell which bang correctly figures tor 0. pendulum. I hese two nuclei are invariably present in both 0. pendulum and 0. moluccanum, ami it is to be expected that they will also be found in 0. vulgatum, as they are constantly present also in Botrychium virginianum and in all of the terns that have been accurately examined. Neither Lang nor Bruchmann saw the ventral canal cell, which is exceedingly difficult to demonstrate. THE GAMETOPHVTE 29 In Of) hi agios sum the young archegonia may be found near the apex of the prothallial branch, but they may also arise at a considerable distance back of it. In general, like the antheridia, they arise in acropetal succession. The mother cell, like that of the antheridium, is sometimes broad, sometimes narrow and deep, and the cover cell is correspondingly shallow or deep. The first division of the inner cell takes place shortly after the cover cell is cut off. The next division is in the cover cell, which first divides by a vertical wall, a second vertical wall being formed almost immediately, intersecting the first and dividing the cover cell into four practically equal cells, arranged quadrant-wise (fig. 16, A). The middle cell next divides by a transverse wall into the primal)' neck canal cell and the central cell (fig. 16, /'"). The canal cell pushes up between the four primary neck cells, which presently divide by nearly horizontal walls, so that there are two tiers of neck cells. One or both of these divide again later, so that each row of neck cells consists of three or four. Rarely there may be five cells in one or more of the rows. Fig. 16. — Development of the archegonium in Ophioglossum pendulum. X180. A. Transverse section of gametophyte apex, showing two young archegonia, 9 > and apical cell, .v. B-G. Successive stages in the development of the archegonium; seen in longitudinal section; n, neck canal cell; b, basal cell. H. Recently fertilized archegonium; sf>, a spermatozoid within the egg nucleus. The neck canal cell is very conspicuous, its base being broad, and the upper part narrower and extending to the uppermost neck cells. The large and conspicu- ous nucleus soon divides into two, but as a rule there is no division wall, although occasionally two distinct neck canal cells may be present. Sometimes both of the nuclei remain in the broad basal part of the cell, and sometimes one is at the base and one nearer the apex. This arrangement seems to depend upon the direction in which the nuclear division takes place. The basal cell divides by a vertical wall at about the same time that the primary canal cell is cut off from the central cell. The basal cell subsequently undergoes further divisions, but its limits are readily distinguishable up to the time that the archegonium is mature (fig. 16, G). In its earlier stages, the archegonium of Ophioglossum pendulum bears a striking resemblance to that of the Maiattiace.e, which the mature archegonium more nearly resembles than it does that of Botrychium. According to Bruchmann's account, 0. vulgatum has a neck somewhat longer, and this is also true of 0. moluc- canum. Even when mature, the neck projects bur little above the surface of the prothallium, although there is some elongation of the cells at the time of dehiscence (fig. 1 6, G). The ventral canal cell is very difficult to demonstrate, and one is sometimes inclined to doubt whether it is formed at all in some cases; neither Lang nor Bruch- 30 THE OIMIIOGLOSSALES mann was able to detect it in 0. pendulum and 0. vulgatum. It is probable that its apparent absence in most of the archegonia is due to the fact that it is formed very late and is extremel) inconspicuous. The same apparent absence of a ventral canal cell in the Cycads and some Conifers lias been shown, on more critical study of the m. m nal, to be due to the small sizeoi the ventral nucleus and to its ven evanescent character. In nearly all of the archegonia examined, just before they opened there was present a vesicular body above the egg, which was probably the ventral canal cell much distended with fluid preparatory to the opening of the archegonium. A small nucleus, or what looked like one, could sometimes he seen, hut it must he said that its nuclear nature was not above suspicion (plate 2, rig. }j). I ust before the archegonium is ready to open, the egg cell, which up to this time is compressed above by the basal wall of the neck canal cell, becomes distended and pushes up the base <>t the canal cell, which thus becomes concave below. It is probably about this time thai the ventral canal cell is cut off. Unfortunately, no cases were found showing mitosis in the central cell, hut then seems no good reason to doubt that a ventral canal cell is, usually at least, cut off. FlG. 17. — Development of the archegonium in Bolrychium virginianum. A-U. Longitudinal sections. X320. E. Ripe archegonium, showing ventral canal cell v. X165. (After Jeffrey.) F. Recently fertilized archegonium, showing spermatozoids within venter. X320. The neck canal cell does not show the complete disorganization which is com- mon, hut retains its form up to the time that the archegonium opens. With the opening of the neck there is some elongation of the outer neck cells, hut there is decidedly less projection above the surface of the prothallium in 0. pendulum than is the case in 0. vulgatum. The nucleus of the egg cell is large, but it does not always stain readily, except the nucleoli, and it may be that the same resistance to stains is the reason win its sister nucleus in the ventral canal cell is so difficult to see. THE ARCHl.taiMI M OF BolKVtllll \I. The archegonia in Botrychium are found usually upon the flanks of the median ridge, upon whose summit are borne the antheridia. In their early stages they closely resemble those of Ophioglossum (tig. 17). As in the latter, there is some variation in the form of the mother cell, which may be quite deep and narrow, or relatively broad and shallow. The subsequent divisions in the central cell and in THE GAMETOPHYTE 31 the cover cell agree very closely with those in Ophioglossum; hut the neck cells are more numerous and at maturity the neck projects much more strongly than is the case in Ophioglossum. There may be as many as seven or eight cells in each row of the neck, which, except that it is quite straight, resembles that of the typical ferns. It is especially like that of Osmunda, in which the neck is also straight. The neck canal cell (fig. 17, D) becomes much elongated, and the nucleus divides, as in Ophioglossum, but in most cases at least there is no division of the canal cell itself. As in Ophioglossum, the ventral canal cell is very inconspicuous, and often impossible to detect. Jeffrey (fig. 17, E) figures a very evident ventral canal cell, but the nucleus is much smaller than that of the egg or neck canal cell. I have also found what seemed to be a ventral canal cell in the archegonium shortly before it opened, but, as in the case of Ophioglossum, this was not absolutely certain. As in the corresponding stage in Ophioglossum, above the egg cell, with its large and conspicuous nucleus, there is a clear space containing a small round body, which showed no evident nucleolus, but otherwise stained very much like the nucleus of the egg, and was probably the nucleus of the ventral canal cell (fig. ij,D,v.) While no certain cases of mitosis of the egg nucleus for the cutting off of the ventral canal cell were encountered, in one case the egg nucleus looked as if it were in the early prophase of division. Bruchmann's figures of the archegonium of Botrychium lunaria closely resemble that of B.virginianum, but he was unable to find a ventral canal cell, nor did he apparently see the division of the nucleus of the neck canal cell. The archegonium of Helminthostachys (fig. 1 1,/)) closely resembles that of Botrychium. FERTILIZATION. In only a few cases were spermatozoids seen within the neck and venter of the open archegonium in Ophioglossum, but twice a spermatozoid was seen within the nucleus of the egg; as there were no other stages obtained, however, the details of nuclear fusion could not be followed (fig. 16, //). The spermatozoid penetrates the nucleus of the egg, where for a time it can be seen distinctly. It is probable that its fusion with the egg nucleus is much like that described by Shaw for Onoclea (Shaw 1, 2). In the mature egg cell the nuclear reticulum is often decidedly con- tracted, but whether this is normal or the result of reagents can not be said. FERTILIZATION IN BOTRYCHIUM VIRGINIANUM. Jeffrey observed a single spermatozoid within the venter of the archegonium and noted that at the time of fertilization the egg developed what he called a "receptive prominence." I have observed the same phenomenon in several cases (plate 2, fig. 46), and in a good many instances have also found one or more sper- matozoids within the venter. In one case, what looked very much like a sperma- tozoid was seen within the egg nucleus itself, and the process of fusion is probably very much the same as that so fully described by Shaw for Onoclea. In the specimen shown in fig. 17, F, one of the spermatozoids looks as if it has partially penetrated the egg, but it is impossible to be certain that it was not simply lying against it. In this case, just above the nulceus of the egg there was a slight break in the granular cytoplasm that looked as if it might be a receptive spot, but the spermatozoid was not entering the egg at this point. In another instance a curious thing was noted, but whether it was normal or not it is impossible to say (plate 2, fig. 46). A spermatozoid had just enn 1. .1 the neck of the open archegonium ami the egg had developed a very conspicuous receptive prominence. The nuclear contents were verj strongly contracted and deeply stained and a portion apparently projected beyond the nuclear membrane .->_' Mil Ul'HKKJLOSS \1 I S into the receptive prominence. A similar s\ iki psis was noted in several cases. In most instances where free spermatozoids win. present in tin venter of the arche- gonium the egg nucleus presented a curious appearance (plate- i, tigs. 47, 4S). A single large nucleolus was present, but scattered through the nucleus there were sometimes a dozen <»i more of intensely staining rounil bodies, which at hist sight looked like- nucleoli, but on more- careful examination were seen to differ from tin- large- nucleolus in that they appeared less homogeneous and generally showed a central vacuole-like structure. These- granules stain very much like- the- body of the- spermatozoid, and it was thought that possibly they might he- derived from a fragmentation of the bod) of the- spermatozoid that had entered the- nucleus, hut this could not he- satisfactorily demonstrated and the- nature- <>t this phenomenon must remain tor the present uncertain. Fertilization in Melminthostachys has not been observed. SIGNIFICANCE <>l THE ENDOPHYTE. I hat tin presence of the- endophyte is essential to the- existence of the- sapro- phytic gametophyte of tin ( >phioglossaceae is indicated by the- failure- of the germi- nating spores to develop unless they become associated with the fungus. Moreover, the universal occurrence ot a similar endophyte in all humus saprophytes among the seed plants indicates that in all of these chlorophyll-less plants the presence of the fungus is necessary for the existence of the host. Although it has not been directly proved, it is generally assumed that one role of the endophyte is the elaboration of some- of the carbonaceous constituents of the humus. The infrequent communica- tion between the external hyphae and the internal mycelium makes it unlikely that the nutritive products are directly absorbed by the fungus, and it seems much more probable that the rhi/oids of the gametophyte are the direct agents of absorption. How the humus constituents are changed by the action of the fungus so that they are available for the cells of the host is not clear, and it is by no means impossible that some at least of the- necessary carbon may be derived from the- fungus itself, in the digestive process to which it is subjected in the- cells of the host. This seems plausible from the fact that in the green prothallia of certain ferns, where presumably the gametophyte is entirely able to supply its own carbon compounds through photosynthesis, these digestive cells appear to he- wanting; at any rate they were- not observed in a number of forms that I have studied. The experiments of r/ernetz (Charlotte Ternetz I), show that certain fungi, including endophytic mycorrhizae, are- able- to assimilate free nitrogen and confirm the assumption of earlier observers that the fungus is useful to the- host in supplying it with nitrogen compounds; but, while this is probably a very important part of its functions, it seems to me- that if is not perhaps the only one, anel that the carbon also is supplied, directly or indirectly, through the agency of the fungus. In an extended study of the- endophytic mvcorrhi/a of the- saprophytic orchid, Neottia, W. Magnus (W. Magnus I) has shown that two types of mycelium ex- hibited by the endophyte are of very different nature. I he slender, cylindrical hyphae constitute the- active portion of the fungus, which behaves like a parasite toward the- cc-lls which it invades, destroying the starch and probably other constitu- ents of the- cells, but not attacking the- nucleus. The swollen vesicular mycelium, however, is a degenerating structure and is itself destroyed by the cells of the host, which actually digest these fungus mycelia in much the sarin- way that the cells of the leaf of Drosera digest theii prey. Magnus has very graphically shown that the relation of the- two symbionts is mutually antagonistic, each one acting as a parasite on the other; nevertheless the presence of the fungus is essential to the higher CI THE GAMETOPHYTE 33 organism, so long as the latter is destitute of chlorophyll. The explanation of the widespread saprophytism exhibited by so many of the higher plants mav be sought in this attempt to defend themselves against what was probably at first a strictly parasitic organism. Having acquired the power to attack and to feed upon the parasite, the photosynthetic functions were more and more subordinated, until a state of true parasitism (or saprophytism) resulted. The numerous semi-saprophytes like most of the green Kricales and many of the green ( )rchidaceae, are good examples I transition stages, while the characteristic leafless humus saprophytes, such as Monotropa and Corallorhiza, represent the fully developed phase of this peculiar form of symbiosis. We might say that such green prothallia as those of the Marat- tiacea? and Gleichenia, which contain an endophytic fungus, bear somewhat the same relation to the subterranean prothallia of the Ophioglossacea- that the green Ericales do to Monotropa. The occurrence of a similar endophyte has also been noted in a number of liverworts. Cavers (Cavers 1) has studied this association with some care in the common liverwort, Fegatella, as well as in some other Hepaticae. He found in Fegatella that the endophyte is beneficial to the growth of the host, which was more vigorous when the fungus was present. He assumed that this was due to the assist- ance given by the fungus in the assimilation of organic matter from humus or other organic substrata. (See also Humphrey 1). This occurrence of an endophyte in the Hepatica? makes its occurrence in the green prothallia of ferns readily comprehensible. Whether in the latter it is an advantage to the host to have the endophyte present remains to be seen, but it is highly probable that such is the case. Once having acquired the habit of associating itself with the fungus, the gradual evolution of the purely saprophytic subterranean gametophyte of the Ophioglossaceae from green forms similar to those of the Marattiaceae is readily comprehensible. 34 THF. OPHIOGLOSSALES II. THE EMBRYO. The development of the embryo in the- Ophioglossaceae has been more or less completely studied in Ophioglossum pedunculosum, 0. vulgatum, (). moluccanum, (). pendulum, Botrychium virgintanum, B. lunana, and H. obliquum ( Mettenius I, Bruchmann 1 and 2, Lang 1, Campbell 8, [effrey I, Lyon 1). The first division in the young embryo in all of these is usually approximately transverse, although there may be a good deal of variation in this respect. It is probable that in all cases the primary root, the stem apex, and the foot all arise from the epibasal region. The embryo reaches a very large size before the root emerges from the overlying prothallial tissue and all of the organs of the young sporophyte are very late in developing, so that it is not easy to trace their origin back to the early cell divisions in the young embryo. Much the most conspicuous organ of the young sporophyte is the root, which may reach a very large size and an advanced stage of development before any evidence of the other organs is apparent. Indeed, several roots may be developed before the shoot is established. In Ophioglossum vulgatum and Botrychium lunana, according to Bruchmann's account, the young Fig. 18. — 0 pftioglossum moluccanum, \. An old archegonium. X180. B. Two-celled embryo within the archegonium. X180. C. Two sections of an old o. Xi&o. cot, cotyledon; /. h>ot; r,root. sporophyte remains several years under ground before the first green leaf appears above the earth, and it is probable that in Ophioglossum pendulum there is also a long period of underground existence before the first green leaf is developed In Ophioglossum moluccanum, however, and in Botrychium virgintanum, the first leaf developed is a green foliage leaf, which grows rapidly and soon appears above the surface of the ground. "IIIK EMBRYO OK OPHIOGI OSS1 M. The first figures that we have of the embryo of Ophioglossum are those ol Mettenius, but his figures of the embryo of 0. pedunculosum are not at all satis- factory, although he shows correctly sections of the older sporophyte. Lang figures only one embryo, a somewhat advanced one of O. pendulum. Bruchmann figures a two-celled stage and a single more advanced embryo of O. vulgatum, but he describes and figures several stages of the young sporophyte. My own study of Ophioglossum was based mainly upon the development of the embryo in O. pendulum, where a THE EMBRYO 35 fairly complete series of embryos was obtained and the development followed quite satisfactorily. In 0. moluccanum I found two quite young embryos, the youngest consisting of two cells (fig. 1 8, 5), the other much more advanced, with the cotyledon already differentiated and the beginning of the primary root recognizable. In the first embryo the basal wall was nearly transverse to the axis of the archegonium and, to judge from the arrangement of the organs of the older embryo, it looks as if the whole of the hypobasal region formed the foot, the epibasal half giving rise to the cotyledon and root. The older embryo (fig. 18, C) was cut longitudinally in the plane of the cotyledon, which at this time comprises pretty much the whole of the upper part of the embryo, the hypobasal region being occupied mainly by large cells which con- stitute the foot. Unfortunately, in this series the section containing the apex of the leaf was missing, and so it is impossible to say whether at this stage a definite apical cell is present in the cotyledon; but, as in somewhat older stages such a cell seemed to be always present, it is probable that there was an apical cell in the cotyledon of the embryo in question. At the base of the leaf, and almost in the center of the embryo, there was a group of actively growing cells, evidently marking the position of the root apex, which arises deep in the tissue of the embryo, very much as we shall see to be the case in the Marattiaceae; and it seems probable that in O. moluc- canum, as in Danaa, the root grows downward through the foot and in a direction coincident with the axis of the young cotyledon. Fig. 19. — Ophioglossum vulgatum (after Bruchmann). A. Median section of a young embryo. X160. B. An older embryo. X25. C, D. Older embryos, showing beginning of apical bud, b; r, root. XZ5. I he cotyledon grows upward and soon ruptures the overlying prothallial tissue, while the root grows down in the opposite direction and pierces the p roth allium at a point some distarrce below where the leaf emerges. As the root grows downward through the foot the latter becomes unrecognizable, its outer cells remaining, however, as a zone of large cells encircling the equatorial region ot the very much elongated bipolar embryo. The young root very early develops a tetrahedral apical cell, like that of the later roots. I his cell is probably cut out from the central tissue of the embryo, close to the base of the leaf. The cell r in fig. 18, C, to judge from its position and the arrangement of the cells around it, is probably the initial cell of the primary root. The young cotyledon grows rapidly and has fiist a conical form (fig. 23, />'), terminating in a definite apical cell. As it grows ii develops a small oval lamina and a slender petiole, and presently the little green leaf appears above tin surface of the earth. The embryo of 0. vulgatum differs remarkably from th.it of 0. moluccanum in the late development of the leaf. Bruchmann was unable to obtain the youngei stages of the embryo, so that the origin of the different members is still somewhat obscure. In this species it is the root which develops first, and it soon becomes 36 I ll l OPHIOGLOSS \i i i exceedingly conspicuous. Bruchmann considers that the root and fool are both of hypobasal origin, but he bases this on a comparison with the true ferns rather than upon actual study of embryos, as he was unable to obtain embryos sufficiently young to demonstrate this, and all trace of the original divisions disappears before any sign of the stem and leaf is evident. It may be well questioned whether, as in Botrychium and 0. pendulum, the foot dots not take up the whole hypobasal region. It is not impossible that the position of the basal wall may also \ an in 0. vulgatum. From a comparison with the embryo of O. pendulum, I am inclined to assign more of the embryo of the former to the foot region than is done by Bruchmann. In 0. vulgatum there is, finally, a differentiation of the stem apex from the epibasal region, as in Botrychium and the true ferns, while in 0. moluccanum there is no trace of a stem apex in the verj young sporophyte, this developing later as a bud upon the first root. Prom a study of Ophioglossum moluccanum and also of O. pendulum it is evident that the history of the young sporophyte in these species differs strikingly from that of the other Pteridophytes. In both of these species the definitive sporo- phyte always arises secondarily, as a hud upon the root, in the same way that adventitious buds are commonly formed upon the roots of the adult sporophyte. Bruchmann notes in O. vulgatum the very precocious development of the primary Fig. 20. — Ophioglossum pendulum. A. Transverse section of very young embryo. X180. B. Longitudinal section of young embryo. X180. C. Three transverse sections of an older embryo; i-i, basal wall; r, primar D. An older embryo; r, primary root. X180. root and the late appearance of the stem apex and first leaf; but in this species the shoot apex, according to his statement, is derived directly from the epibasal half of the embryo, as it is in most Pteridophytes. In Ophioglossum pendulum, where the development of the embryo seems to offer no check to the further growth of the prothallium, the position of the arche- gonium varies a good deal and it is impossible to tell from a section just what the •position was in the living state, as the branches of the gametophyte extend in all directions and archegonia may be formed at am point upon their surface. To judge from the youngest stages of the embryo that were met with (fig. 20), the basal wall in this species is not necessarily transverse. In both of the rases figured it was oblique, and more nearly longitudinal than transverse. It is likely, however, that it is horizontal, or approximately so. In the four-celled embryo shown in fig. 20, ./, the quadrant walls were at right angles to each other, and th>s was also the ease in the five-celled embryo shown in fig. 20, H. Somewhat older embryos {(■) show that there is a pretty regular octant formation, and Bruchmann starts that this is also the case in 0. vulgatum. While in the typical ferns, and in Botrychium, all of the organs of the young sporophyte can be traced to certain regions of the young embryo, in Ophioglossum THE EMBRYO 37 pendulum only one of the definitive organs, the root, arises in this way, and this becomes differentiated at a very early period. One of the octants next to the arche- gonium at once becomes the apical cell of the young root. This cell is very soon recognizable by its size and shape, and quickly begins its regular segmentation. The primary cap-cell is soon cut off (fig. 20, D), and from now on the young root is very conspicuous. The octant divisions are very clearly marked in this case, and in section 3, which is the uppermost of the three, the large triangular apical cell of the root is very evident. Two types of the embryo were seen in this species. One of these (fig. 21, A) is nearly globular in form; the other (fig. 21, B, C) is elongated. The former looks as if it originated from an embryo in which the basal wall was transverse to the axis of the archegonium; in the other it was probably more or less vertical. It is probable that in the former instance the root initial is one of the epibasal octants, while the whole of the hypobasal portion gives rise to the large foot. In the second type it is difficult to say which half should be considered epibasal and which hypobasal, but, as in the other case, one half may be considered to be root, the other half foot, the Fig. 21. — 0 phio^lossutn pendulum. Older embryos. A. Vertical longitudinal section; /, foot; r, primary mot. B. Horizontal section. X75. C. Older embryo, with single root. D. F.nvbryo with two roots; pr, gametophyte. Shaded regit rcupied by the mycorrhiza. growth of both being nearly in a plane at right angles to that of the archegonium axis, and suggesting the relative positions of cotyledon and foot in the embryo of Opnioglossum moluccanum. In form the first type somewhat recalls the embryo of Botrychium virginianum. The whole lower portion, or hypobasal half, forms the very conspicuous foot, while from the epibasal region flu- primary mot is developing, and already the rudiment of the second root is visible. Whether the latter arises directly from the primary root, or whether it arises independently from flu- second epibasal quadrant, is nor quite certain. 1 he cells of the epibasal region are evidently actively growing, having abundant protoplasm and conspicuous nuclei. The cells of the foot are larger and much more transparent. The second type of embryo (fig. 21, B, (!) resembles very closely the second state of the embryo of Ophioglossum vulgatum, where, as is said by Bruchmann, the embryo is " all root." 38 THE OPHIOGLOSSALES In O. moluccanum the prothallium probably lives for a single season only, and the formation of the sporophyte stops its further development; but in O. pendulum, where embryos are much less frequently found, the large gametophyte continues its growth apparently unchecked by the development of the attached sporophyte, which retains its connection with the gametophyte for a very long time, as in O. vulgatum and Botrychium virgintanum. The embryo reaches a very large size before it breaks through the prothallium. The primary root then emerges as a conical point (fig. 3, /'", sp). The second root remains short for a time. I here seems to be a good deal of difference as to the time of the appearance of the latter. In the globular type of embryo the second root appears very early, and it looks as if it might have been formed quite independently of the primary root. Sometimes, however, the primary root may attain a length of several centimeters, and may even begin to form rootlets before the second root emerges; while in other cases the two roots grow in opposite directions and seem to be of about equal size (fig. 3, £). The leafy shoot in Ophioglossum pendulum does not appear until the root system is well advanced. The primary root, although attaining a length of 10 centimeters or more, in no cases showed any signs of leaf-bearing buds in the specimens that I collected. Rootlets were devel- oped in some cases, and it is not impossible that there may be an extensive development of the root system before the first leafy bud is formed. This is quite in harmony with the large develop- ment of the roots in 0. vulgatum, where Bruch- mann believes that it may be eight or ten years before the first foliage leaf appears above the ground. In 0. vulgatum, however, this leaf arises from the original stem apex derived directly from the embryo, and not from an adventitious bud. Ophioglossum moluccanum and the similar species that grow with it differ very much from Ophioglossum pendulum in the character of the young sporophyte, which, as we have seen, at once develops a green foliage leaf or cotyledon. The sporophyte very closely resembles that of 0. pedun- 1 ulosum described by Mettenius; in fact, the resemblance is so close that it would seem to confirm the close relationship of this species and possibly its identity with some of the forms asso- ciated by Raciborski under the name 0. moluccanum (Raciborski 1). As Mettenius correctly showed in 0. peiunculosum, the first organ to be developed is the cotyledon, which soon pierces the earth and appears as a green foliage leaf. This primary leaf is continued directly into the primary root, but no stem apex is developed, nor is any sheath formed about the leaf base in the young sporophyte, which consists practically of leaf and root alone. The latter often penetrates for some distance into the prothallial tissue before it emerges, so that the central portion of the young sporophyte is surrounded by a sheath formed by the prothallial tissue. A longitudinal section of the sporophyte (fig. 22, A) shows that the tissues of the leaf are continued directly into those of the primary root. A Fit;. 22.— 0 phioglossum moluccanum. \. Median section of young sporophyte before formation of bud; /, cotyledon; r, root. pr, the gametophyte. X15. B. Central region of same, more enlarged; tr, first tracheids. C. An older sporophyte, showing bud, bt de- veloping from primary root. X15. D. E. Lamina of cotyledon, showing venation. X3. THE EMBRYO 39 single axial vascular bundle traverses the whole of the young sporophyte without interruption, and there is no recognizable boundary between the tissues of the leaf base and those of the root. The central region of the embryo is somewhat thickei and its outer cells are enlarged, these outer cells probably belonging to the foot through which the root has penetrated and which at this period can not be clearly recognized. The strictly bipolar character of the young sporophyte and the way in which it perforates the gametophyte resemble most nearly the corresponding stages of Equisetum and the Marattiaceae, and it may be said that Lyon's recent studies on Botrychium obliquum indicate that in this species also there is a similar bipolar arrangement of leaf and root. Mettenius pointed out in O. pedunculosum that the definitive sporophyte arises as an adventitious bud upon the primary root, either close to the leaf base or, more commonly, at some distance from it. This is also the case in O. moluccanum and the other allied species. In one case observed by me two independent sporophytes were found growing from the same prothallium, but this is unusual, and in most of the cases where two sporophytes seem to be present, one of these is really the sec- ondary sporophyte growing from the primary root. Fig. 23. — Ophioglossum moluccanum. A. Young cotyledon, longitudinal section; pr, gametophyte. X80. B. Apex of cotyledon more highly magnified; x, apical cell. C. Young sporophyte in which a rudimentary second leaf (/") is present. X80. D. Cross-section of apex of primary root. X200. While in 0. vulgatum the sporophyte stays under ground for several years, in 0. moluccanum there is every reason to believe that there is only a brief interval between the first formation of the leaf and its appearance above ground. The small size and the character of the gametophyte, as well as the quick germination of the spores and the rapid growth natural to a tropical climate, indicate that the gameto- phyte is an annual and that it dies as soon as the young sporophyte is established. In O. pendulum the primary root as it breaks through the prothallium elongates rapidly, but just how far it grows before the bud is formed upon it could not be determined. These roots are very brittle and easilv broken off, and in no cases were young buds found upon roots which were still connected with the prothallium. The first root sometimes reaches a length of 3 or 4 centimeters before the second root can be seen at all. The growth of the roots in the young sporophyte is in all respects like that occurring in the older ones. There is a large tetrahedral apical cell, the divisions of which are quite regular, and the conspicuous axial vascular cylinder is 40 THE OPHIOGLOSSALES developed at an early period. The stele of the second root joins that of the first where the latter joins the foot (fig. i\, D). 1 he primary root in 0. pendulum is, usually ;it least, diarch. DEVELOPMENT Of THE PRIMARY BUD IN OPHIOGLOSSUM MOLUCCANUM. The several terrestrial species of Ophioglossum growing at Buitenzorg and associated under the name 0. moluccanum differ strikingly from 0. vulgatum in the further history of the young sporophyte as well as in the early development of the first functional leaf. This difference is also found in the young sporophyte of 0. reticulatum ( ?) collected at Hakgala in Ceylon. All of these tropical species agree with 0. pedunculosum, which was correctly descrihed by Mettenius (Mettenius 1), in the origin of the definitive axis which arises as an adventitious hud from the primary root of the young sporophyte. A similar condition of things probably is true also in 0. pendulum, although in the latter no leaf is at first developed from the young sporophyte, which is composed exclusively of one or two roots in addition to the foot, so that the first leaf in O. pendulum is also an adventitious structure. Whether or not the primary leaf in O. pendulum, and probably at the same time the stem apex, arises as a bud from the primary root, or whether (which seems more likelv) it is developed from a secondary root, can not be stated, as no young leafy buds were found in connection with the gametophyte. Fig. 24. — Ophioglossum moluccanum. A. Tangential section of primary root, showing young, endogenous bud, b. X ab mt so. B. C. Two sections of the same bud. B, the first leaf; C, stem apex. X 150. D. Two transverse sections "f .1 ynung bud; 1 passes through stem .i}><-\ U\ 1, through leaf, Mettenius gives no detailed study of the development of the bud upon the root, nor does he state whether he recognized its endogenous origin. The hud a lists much in the same way that a secondary root does, and is not visible upon the out- side of the root until it is far advanced in its development, the young leaf breaking through the overlying tissues in much the same way that a young root emerges. In two instances the rudiment of the young hud could he seen close to the apical meristem of the root, in a position which is exactly the same as that described forthe leaf) buds formed at the apex of the root in 0. vulgatum. More commonly, the hud originated nearer the base of the root, hut it may arise at any point between the hast and the apex. Figure 24 shows sections of the youngest buds that were found. / is .1 tangential section of the primary mot, in which the young hud has formed at a point not very fai hack t mm the root apex, r. The first leaf of the young THE EMBRYO 41 bud and the young stem apex can already be recognized, and these have apparently developed quite independently of each other. The leaf rudiment, B, lies nearer the bundle of the main root and may perhaps have taken its origin from a single endo- dermal cell, but this could not be certainly determined. The young leaf soon forms a slightly projecting conical body, composed of a few cells with conspicuous nuclei, separated from the adjacent root tissue by a small space. At this stage it is not quite clear whether the apical cell has been developed, but later stages show the presence of an unmistakable apical cell of approximately tetrahedral form. Lying nearer the periphery of the root is the young stem apex, composed of a small group of meristematic cells, one of which is unmistakably the initial cell for the stem. This apical cell is truncate below and the base is broader than the outer free face. Above the stem apex can be seen the cavity which separates the outer tissues of the young bud from the cortical tissue of the root. Fig. 24, D, shows two transverse sections of a bud of about the same age as the one just described. No. 1 passes through the stem apex and shows the conspicuous triangular apical cell; No. 2 shows a section of the leaf above the level of the stem apex with the basal tissue of the leaf extending around the stem region. This is the beginning of the hollow stipular sheath which incloses the stem apex of the bud and FlG. 25. — O phio^los^ittn molucctmum. A. Bud about to break through primary root; /, first leaf; K, stem apex, about 80. B. Apex of leaf more highly magnified. C. Transverse section of a young bud passing through base of leaf, /. \ 1 50. D. Section of same passing through stem apex, .v. E. Transverse section of stem apex from an older sporophyte. The apii il cell 1 four-sided. which is so conspicuous a feature in the stem apex of tin- older sporophyte. Probably the large central cell in the section of the leaf shown in No. 2 is the apical cell, but this is not quite certain. A transverse section of a somewhat older bud (fig. 25, (-') shows a narrow cleft at the base of the leaf in front, opening into a cavity within which the stun apex lies. It is impossible to snv exactly how much of this stipular sheath really belongs to the leaf base ami how much is derived directly from the adjacent cortical tissue of the root, since the tissues of the leaf base merge insensibly into the latter. From this time on, each new leaf develops this conical stipular sheath, which incloses the stem apex and the next youngest leaf, and which has so often been described in the older sporophyte. If a median section of the young hud is examined just before the bud breaks through the overlying tissues of the root (fig. 25, A), it can In- clearly seen that the leaf, which now lias the form of an elongated cone, lies above the level of the young stem apex, and by this time the differentiation of the vasculai bundle of the leaf is well advanced. This bundle connects with the vascular bundle of the primary root 42 THE OPHIOGLOSSALES and there is no connection between it and the tissues of the stem apex of the bud. The latter consists of a shallow mass of tissue with the conspicuous apical cell in the center, but below it there is no sign of the development of any procambium. The stun apex lies in a depression formed In- a shallow ridge, which encircles it and forms the beginning <>l the stipular sheath belonging to tin base of the leaf. The exact limits of the basal tissue of the leaf, as we have already Stated, can not be clearly defined. The cavity above tin stem apex is still very evident, but how much of the tissue King above the cavity belongs to the leaf base, and how much to the cortical tissue, is nut clear. 1 he section through the apex of the leaf (fig. 25, H) shows a single large terminal cell which, without doubt, is tin- apical cell. The young bud is now ready to emerge and, very soon after, the rapidly elongat- ing apex of the young leal pushes through the outer root tissue and emerges upon the outside. The stem apex remains buried within the loot tissues and the sheathing base of the leaf. The leaf base is surrounded by a ragged sheath, formed by the ruptured outer tissues of the root. The development of the bud upon the rout in 0. pendulum unfortunately could not be followed. The smallest leaves found were 10 centimeters or more in length, and, although these were probably the primary leaves, they were not recognized as such at the time the plants were collected, and so there was no opportunity of tracing their connection with the original root. The fully developed cotyledon in what may be considered the typical form of 0. moluccanum is more or less lanceolate in outline (fig. 22, D). There is a central vein from which branch secondary veins on either side, connecting with the central vein by anastomosing branches which inclose elongated meshes. In the form with broader leaves, probably another species, the mid-vein is more obscure and tin meshes are broader and more numerous. Three types of the embryo may be recognized in Ophioglossum, represented respectively by 0. moluccanum, 0. vulgatum, and (). pendulum. If, as seems not unlikely, 0. moluccanum is the most primitive of the three, some interesting points arise as to the significance of the peculiarities exhibited by the embryo, which shows only two organs aside from the foot, viz, the cotyledon and primary root, these growing in an almost exactly opposite directum, without any clear line of demarca- tion between them. I have ventured to draw a comparison between the sporophyte of Ophioglossum and that of Anthoceros, assuming that the former has arisen from some bryophytic type not unlike Anthoceros, by the development of a root from the base of the sporogonium and of a special foliar organ from the basal meristem of such a sporogonium. 1 he embryo of (). moluccanum approaches this hypothetical form, as it consists only of leaf and root, and no stem apex is developed from ir, its growth being of limited duration. In this case the definitive sporophyte is a secondary structure developed as a bud upon the primary root. In 0. vulgatum, however, the definitive stem apex, although of very late origin, is apparently a product of the original embryonic tissue, but the first foliage leaf is of much later origin. In 0. pendulum the formation of the leafy sporophyte is also secondary, but neither stem apex nor leaf is developed from the embryo itself. If, as the writer believes, Ophioglossum represents the most primitive type of the fern series, it is quite conceiv able that in (). moluci anum and its allies the embryo represents the condition existing in the ancestral type from which these have sprung. On the supposition that the leafy sporophyte is derived from a large bryophytic sporogonium resembling that ol Anthoceros, there must have been a stage when the sporophyte consisted of two parts only, the upper sporogenous portion, which latei developed into a sporophyll, as represented in Ophioglossum, and the root. Of THE;EMBRYO 43 course, it is quite possible that the peculiar origin of the definitive sporophyte in 0. moluccanum and 0. pedunculosum is secondary, but this is by no means neces- sarily the case. However, it seems highly probable that tbe extraordinary develop- ment of the roots in O. vulgatum and O. pendulum and the protracted subterranean life in these species constitute a secondary phenomenon associated with the pro- nounced saprophytic life of the gametophyte. The apex of the shoot lies in a narrow depression between the base of the leaf and a narrow ridge which extends around it on the side opposite the leaf base. This ridge is the beginning of the conical sheath characteristic of the shoot apex of the older sporophyte. A transverse section of the young apex at about this time shows that the apical cell is triangular in outline. The first root of the young bud does not emerge until the leaf is nearly complete. From this time on the further growth is due to the activity of the stem apex, from which new leaves and roots presumably are developed in the same way as in other species that have been studied. How long it is before fertile leaves are formed was not ascertained, but in the rapidly growing species of a tropical climate it is likely that this takes place before long. The occurrence of very small fertile individuals (fig. 55, B) points to this. THE EMBRYO OK OPHIOGLOSSUM VULGATUM. The embryo of O. vulgatum (Bruchmann I) differs from that of both O. pendulum and O. moluccanum in its development, but in the earlier stages it closely resembles the former species. As in O. pendulum, the root is the first organ to be developed and it reaches a large size before there is any indication of the formation of a stem apex or cotyledon. Bruchmann states that the root apex arises from the hypobasal half of the embryo, but his figure (fig. 19, A) of the young embryo, where the root apex is just visible, closely resembles a corresponding stage in O. pendulum, where the root is certainly of epibasal origin; and, to judge from the elongated form of the two-celled embryo which Bruchmann figures, one is inclined to believe that the basal wall is really at right angles to the long axis of the embryo, and not parallel with it, as he figures, so that the whole of the hypobasal part might be interpreted as the foot, while the root would be of epibasal origin, as it is in O. pendulum. The next stage figured by Bruchmann (fig. 19, B) is very much like the type of 0. pendulum, where only one root was developed at first, and in this case also we are inclined to consider the whole of the enlarged base of the embryo as the foot. Before the cotyledon and stem apex can be recognized the first tracheary tissue of the root has already been developed, and about the same time the apex of the second root can be seen arising near the base of the primary root (fig. 19, C). The cotleydon arises near the base of the root, upon the upper side of the embryo, and forms a small conical protuberance, which grows from a definite apical cell. At the base of the cotyledon, on the side turned away from the root apex, a shallow depression is formed, and in this there may be seen a single large superficial cell, which is apparently the apical cell of the very limited stem apex. Inclosing the cotyledon and the stem apex is an elevated ridge which grows up about them and finally forms a sheath, which rapidly grows so as to include the bud within a cavity completely closed except for a narrow canal which opens outward. While the cotyledon and stem apex are thu.s formed from superficial tissue in 0. vulgatum, their early inclosure in the sheath suggests tliat \\c have t . » do with a condition intermediate between tin completely endogenous shoot apex in 0. moluc- canum and the entirely exogenous shoot in Botrychium. 44 THE OPHIOGLOSSALKS I Ik second root arises quite independently ot the stem apex, being developed below the insertion of the primary mot, and its vascular bundle joins that of the Hist root near its base. A rudimentary vascular bundle is developed within the cotyledon, and also joins the bundle ot the main root near its base, but no bundle at all is developed within the stem. I he cotyledon reaches only a very small size, but the second leaf soon appears nearly opposite the cotyledon. The second leaf also develops at an early period a vascular bundle, which connects with the vascular bundle of the primary root near its point of junction with the second loot, and this exactly resembles the arrangement of the bundles in the bud formed upon the root m 0. moluccanum. It is evident that in the young sporo- phyte of 0. vulgatum, as in 0. moluccanum, the vascular system is made up entirely of the bundles ot the roots and leaves, the stem itself having no proper stele. The second leaf finally emerges and appears above ground as the first sterile green leaf of the young sporophyte. Bruchmann states that the second leaf does not appear above ground until five years after it is first formed, and he believes that the sporophyte, at the time its first leaf appears above ground, is nine to ten years of age. The third leaf may be fertile, but this is not always the case. TIIK ANATOMY OF 1111. YOUNG SPOROPHYTE OF OPHIOGLOSSUM. In Ophioglossiun pendulum the young primary root soon breaks through the prothallium and elongates rapidly, but owing to its brittleness it is easily broken off, and it is impossible to state lure just how far it develops before the bud is formed upon it. The development of the second root varies a good deal. The first loot may reach a length of } or 4 centimeters before the second root can be seen at all. The growth of these earlier roots is in all respects similar to that of the later ones. Th ere is a ■ irge tetrahedral apical cell whose divisions are quite regular, and there is soon visible the axial vascular bundle which extends for some distance into the foot, win-re it ends blindly. 1 he vascular bundle of the second root joins the first at the junction of the latter with the foot (rig. 21, /)). The first trachea ry tissue appears at the point of junction, and is made up of short, somewhat irregular, pointed tra- cheids with reticulate thickenings. From this point the development of the tracheary tissue proceeds toward the apex of the roots. The bundle is diarch, as is plainly seen in cross-sections ( fig. 27. J: ). I he endodermis is very clearly defined and tin characteristic radial markings an- extraordinarily char, especially in sections treated with a double stam of satranme and gentian violet. I he tracheary tissue is also beautifully differen- tiated by this stain. I he bundle is slightly elliptical in form and tin- protoxylem ele- ments appe.11 at the foci of" the elliptical section. I he first appearance of the tracheary tissue is some distance back of the apex and the development proceeds rather slowly. In the oldest part of the roots A. Longitudinal .section of an <>U!er bud of Oftliioglossum moluccanum. X50. j», stem apex; /' first leaf oi bud; '' . h r v t root <>f tin* bud. It. Stem apex ..1 same. X90. THh KMI'.KM) 45 examined the two xylem masses were unequal in size, the largei showing about half a dozen tracheids in cross-section, the smaller two or three. Whether the two protoxylems are ultimately joined hy intermediate tracheal tissue, so as to form a continuous plate, as in the older roots of the adult plant, can not now be stated, but in no cases examined was this true, and it is not unlikely that in the primary root the two xylem masses are permanently separated. The cells of the foot, as usual, are more or less papillate where they arc in contact with the tissue of the gameto- phyte. Ihev early become infected with the endophvte, which probably makes its entrance from the prothallial tissue, and not from the outside. This point, however, is not perfectly clear. The infected area follows the growth of the young root, but leaves the apical tissues free. In O. moluccanum the leaf is the first part of the young sporophyte to develop. In the larger embryos the leaf forms a conical body, merging into a nearly globular basal portion, which is the foot, and within this, probably near the junction of the epibasal and hypobasal halves of the embryo, the apical cell of the root is developed. The leaf now shows a definite apical cell, triangular in section and exhibiting a regular segmentation. The inner cells of the segments form the axial strand of tissue, which is continued through the embryo into the root. The limits of the two Fig. 27. A. Section of petiole of cotyledon of 0. moluccanum, B. Section of young root. C. Vascular bundle of median region of young sporophyte. D. Vascular bundle of primary root. E. Vascular bundle of primary root of 0. pendulum; eti, endodermis. primary organs, the leaf and the root, remain quite indistinguishable. The central tegion, which remains surrounded by the prothallial tissue, is somewhat larger in diameter and the whole of this functions as a foot, although it is composed in part of tissue belonging to the root and the leaf. The young leaf elongates rapidly after it has ruptured the calyptra, and its apex begins to widen out, but still shows a single apical cell. As the upper part widens out there is a division of the original vascular cylinder, and there is developed within the leaf a reticulate system of vascular bundles or veins (fig. 22, D, E). As already indicated, the vascular strand of the young sporophyte is continuous through the cotyledon and root, and sections at different points show essentially the same structure. The petiole of the cotyledon (fig. ij, A), which is traversed by two large lacunae, shows that the axial bundle is decidedly collateral in structure. The xylem consists of a group of about half a dozen tracheids at the inner limit of the bundle, and no endodermis can be recognized. As a section of the bundle is made 46 111 I OPHIOGLOSSALES in the mid-region of the sporoph) te ( fig. i~, C), the only difference noted is a slightly greater development of the xylem. I In- section of the root (fig. 27, D) presents almost exactly the same appearance as that of the leaf. Whether we should call the root bundle "monarch" or "collateral" is merely a question of terms. In the mid- region the endodermis can he clearly seen, and it is then evident that the xvlem is separated from it by a single layer of pericycle cells. Fig. 28. A. Archcgonium of Botrychium virginianum, containing a two-celled embryo. X275. B. Four longitudinal sections of an embryo with 7 cells. X275. THE EMBRYO OF BOTRYCHIUM. The following account of the development of the embryo of Botrychium is based mainly upon material of B. virginianum furnished me through the kindness of Professor Jeffrey. In this species, as in the other Eusporangiatae, there is a marked increase in the size of the fertilized ovum before the first division takes place. At the time of the Fig. 29. -Young embryo ' B ryehium virginianum. Longitudinal sections. X200. b b, basal wall. A. Four-celled embryo. H 1). Oldei stages. hist division the ovum is generally more or less elongated, bur this is not always the case. I his elongation is less marked in B. lunaria (rig. 36), while in B. obliquum the ovum becomes much elongated before the first division occurs (see Bower 9, fig. 266). THE EMBRYO 47 The first division wall, the basal wall, as in the other Eusporangiatae, is, usually at least, transverse to the axis of the archegonium (fig. 28, A). Kach of the cells thus formed is next divided by a vertical wall, so that the embryo is divided into approxi- mately equal quadrants. Jeffrey states that the quadrants are next divided by a third (transverse) wall, so that there is a regular octant formation ( Jeffrey 1, page 16), but adds that this segmentation presents various irregularities and this is confirmed by my own studies of the young em- bryo. In B. lunaria, according to Bruchmann, the octant divisions are very regular. In B. virgimanum the young embryo is often somewhat pointed above, and transverse divisions of such embryos may show that the octant formation is suppressed in the epibasal region, and this tapering upper part of the young embryo suggests a suspensor similar to that de- veloped: in Danaa, but less clearly defined, and probably not determined by the first division of the embryo. Whether the suspensor found by Lyon in B. obliqillim (Lyon 1) Originates in a Fig. 30.— Three longitudinal sections of a young similar fashion remains to be seen. This elonga- re0^ £ ^*£"^f """"" '' ,he tion of the upper part of the embryo, whether it is considered as a suspensor or not, is doubtless, as Jeffrey has suggested, useful in pushing the developing embryo deeper down into the prothallial tissue. For some time, only anticlinal walls are formed, this showing especially plainly in transverse section (fig. 31). Sooner or later periclinal walls are also formed, and it is possible that in some cases the first periclinal wall in one of the large epibasal cells may establish the initial cell of the root, but this is hard to decide. The early stages of the embryo are extremely difficult to embed without shrinkage, and this, together with the difficulty of securing a sufficient number of the right stages and the impossibility of regulating the direction in which they are cut, makes the deter- mination of the origin of the primary organs of the young embryo a peculiarly difficult problem. In., ji. -Four horizontal sections of a young embryo of R. virgitiianum. 1 is next the archegonium; 2 and 3 are nearly median sections. Neither Jeffrey nor Bruchmann was able to recognize a root initial until the embryo had attained a large size, and its origin could not be determined. In the embryo shown in fig. -50, I am inclined to believe that the cell r is really the root initial. Unfortunately, stages between this and the one shown in fig. 32, where the root initial is unmistakable, were wanting, but the position anil state of develop- ment of the root in the latter harmonize with this supposition. My fig. 32 is evi- dently of about the same size as Jeffrey's fig. 46, and probably an examination of other sections of the series from which Jeffrey's figure was drawn would show the root initial, as well as traces of the stem apex and cotyledon. 48 I II I orilMic.l.n.ss VLES Ml ol tin organs cil the young sporophyte arise, .is Jeffrey showed, from the epibasal region, and in tins respect Botrychium virginianum agrees with the Marat- tiaceae and with Ophtoglossum. According to Bruchmann, the embryo in B. lunaria remains quite undifferentiated up to the time it breaks through the calyptra, even the mot apex being unrecognizable at this time. It is therefore impossible to say what nlation the oil;. ins ol the young sporophyte bear to the primary divisions of tin embryo. cot st xc^sr^>. Fig. 32. — Three sections of an older embryo of Botrychium virginianum, cut transverse to long axis of embryo. b b, basal wall; /, f^ot; r, root; rf,stemapex; eo/, cotyledon. 200. In B. virginianum traits of the quadrant formation are still evident at a com- paratively latr stage, and there seems no reason to doubt the correctness of Jeffrey's conclusion as to the epibasal origin of both the root and the cotyledon. As in Ophtoglossum pendulum and (). vulgatum, the root is especially conspicuous and reaches a large size, while tin stem apex and cotyledon are still inconspicuous. Indeed, in B. lunaria, according to Bruchmann, no trace ol either stem apex or D >i vlcdoti can he made out until the root has broken through the calyptra. At this stage ( fig. 36, C, D) the embi vo of 11. lunaria bears a striking resem- blance to that of Ophto- glossum vulgatum. It was found bv Jef- 1 1 1 \ m B . virginianum that the stem initial was developed before the co- tyledon could be seen, but I have failed to verify this in the specimens I have examined. Fig. ^2 shows three sections of a series taken from an embryo of about the age of [effrey's fig. 4.6. I his embryo wascut transversely to its long axis, and the root apex is thus seen in cross-section. I he root here probably comprises the whole of one of the epibasal quadrants, from the other of which, or from part of it, perhaps a single octant, the stem apex and cotyledon arise in close proximity. It is probable that part of this quadrant goes to form the " suspensor," or that epibasal tissue which is not concerned in the formation of the young organs of the embryo. Fig. .ji, II shows a section passing through the young cotyledon, whose single apical cell is already differentiated. This in section is triangular, and the cell is probably of tetrahedral shape. Fig. }2, C, shows the section passing through the stem apex. It'.;, I '' ium virginianum, cut in the ('Line of the cotyledon. st, stem apex; '.root; /, foot. Xioo. THE EMBRYO 49 Probably the large cell (st) is the apical cell, but this is not absolutely certain. In form and position this cell resembles the apical cell figured by Jeffrey (Jeffrey I, fig. 48) in an older embryo. It may be said that in Jeffrey's figure the beginning of the cotyledon, situated between the stem apex and the root, although not lettered, is evidently present. As the embryo grows the root rapidly increases in length, and together with the foot comprises the greater part of the embryo. The foot is very large, usually nearly hemispherical in form, but not infrequently a good deal elongated (fig. 35), and penetrates deep into the prothallial tissue. I he apical cell of the root is very conspicuous and can be made out without difficulty as a large cell, triangular in form, whether it is viewed in longitudinal or transverse section. Segments are cut off in regular sequence from all of the four faces, and as these are relatively large and contain but little granular contents the apical meristem shows very clearly against the smaller celled and more deeply stained rot Fie. 34. A-C. Three horizontal sections of an old embryo of Botrychium virginianum, X75. D. The stem region. X150. adjacent tissue. In the earlier stages the divisions in the segments cut off from the apical cell do not show absolute uniformity. The first wall in the young segment is probably anticlinal, and seems to be followed by a periclinal wall or walls, cutting off inner cells which contribute to the very large plerome cylinder of the young root. The root cap, which is very massive, is derived in part from segments cut off directly from the apical cell, and in part from cells separated by periclinal walls from the outer part of the lateral segments of the apical cell. The development of the plerome begins very early, and it soon forms a con- spicuous massive strand of procambium cells extending from the root apex to the junction of the root and foot, where it ends abruptly on the lower side, but bends upward on the upper side, and is extended as a simple axial strand of tissue into the cotyledon. The cotyledon, which is first recognizable at an early stage in the development of the embryo, at the stage in question (fig. 35, A) projects slightly as a broad, flat- tened cone, strongly bent away from the root. Fig. 34, A , shows the cotyledon cut 4 50 THE OPHIOGLOSSALES through horizontally. At the slightly projecting apex there is a single apical cell, although it is not always quite certain which is the apical cell and which is its youngest segment. The broad base of the cotyledon is extended laterally, like the stipules of the older leaves, and there is thus inclosed a slightly depressed area, very much as in the embryo of Ophioglossum vulgatum, and within this is situated the stem apex, close to the base of the cotyledon, which bends over it so as to leave only a narrow space above the stem apex. A section across the base of the cotyledon shows a group of small cells indicating the section of the young vascular bundle which lower down (fig. .54, C) joins the young bundle of the primary root. The relative position of the young organs just before the root ruptures the calyptra is best seen in a longitudinal suction. Such a section (fig. 35, A) shows the very large foot, occupying approximately half of the embryo, and deeply embedded in the prothallium. Above this is the root, occupying the major part of the epibasal region of the embryo and varying somewhat in position, perhaps due to the early divisions in the young embryo. It may lie in a plain almost coincident with the cotyledon, or it may make a marked angle with the latter. In the former case the Fig. 15. A. Median section of a young sporophvte of Botr\chium virginianum about toei B. An older stage; I2 , second leaf; r2, second root. X20. X about to. young vasculai cylinder is continued almost straight into the cotyledon; in the latter it binds sharply upward to inter it. The stem apex, which is usually more or less oblique, is small and incon- spicuous, but usually a single initial cell may be made out without difficulty, although sometimes it is not easy to distinguish this from the younger segments which have been cut off from it, as the apical meristem is very shallow, and in longitudinal section has the appearance of a columnar epithelium.' [There seems to be, at least in the earlier stages of development, some variation in the form of the initial cell, which may have a pointed base, or may be truncate below, and while later it usually shows the form of a three-sided pyramid, in these earlier stages it often approximates the truncate form found in Ophioglossum moluccanum. The apical meristem of the stem is very limited in extent and, so far as could be made out, the segmentation at first does not follow any regular scheme. W hile in both root and cotyledon a central strand of procambium is developed at a very early period, no trace of anything which can be interpreted as a "stele' is formed in the young stern. The tissues derived from the further segmentation of the apical meristt 111 remain undifferentiated parenchyma and contribute only to the central pith ot tin hollow woody cylinder which later traverses the axis of the young THE EMBRYO 51 sporophyte. In short, the strand of vascular tissue in the young sporophyte at the time the first root emerges is composed solely of the coalescent strands of the root and leaf exactly as is the case in the young sporophyte ofOphioglossum moluccanum, and there is absolutely no trace of a cauline stele. The foot is composed of large parenchyma cells, which become smaller toward the periphery, where they usually show more or less dense contents and some- times form a quite definite layer of epithelium-like cells, which stain much more deeply than the inner tissue of the foot. This outer layer of cells is presumably active in the absorption of nutriment from the adjacent prothallial tissue. Bruchmann states that previous to the emergence of the root of the young sporophyte of B. lunaria the cells of the embryo are densely packed with granular matter of apparently albuminous nature; but after the emergence of the root starch is developed abun- dantly in the cells of the foot. This appears to be true also of B. virginianum. Compared with B. virginianum, the embryo of B. lunaria (fig. 36) is character- ized by the lesser development of the foot and the later appearance and rudimentary character of the stem apex and the cotyledon. The stem apex in B. lunaria (Bruchmann 2) is first evident as a slight superfi- cial depression near the base of the massive root. A single superficial cell becomes differentiated as the apical cell of the shoot, and a small group of cells is formed before the rudimentary cotyledon develops. Both stem apex and cotyledon remain very inconspicuous. The cotyledon is developed as a scale-like rudiment, which never develops into a foliage leaf. Bruchmann states (Bruchmann 2, page 223) that from seven to nine of these rudimentary leaves are developed before the first green leaf appears above the earth. Hofmeister (Hofmeister I) believed that three of these rudimentary leaves were developed during the first year, and that the second year the first spore-bearing leaf appeared above ground. Bruchmann thinks that this is not the case and that the first spore-bearing leal requires five years for its development, as it does in the older sporophytes, and that only one leaf is formed each year, as in the older plant. The first organ to penetrate the calyptra is, as we have seen, the root, which in B. virginianum is very large and quite overshadows the relatively inconspicuous bud at its base (fig. 7, B). At the time the root first emerges the vascular bundles still have the form of procambium, and it is not until the young root has a length of several millimeters (5 to 20 millimeters, according to Jeffrey) that the Hist tracheary tissue is developed. The first tracheary tissue arises at the base ol tin mot and the development, as usual, proceeds toward the apex. The primary root in Botrychium virginianum is usually diarch, but triarch roots are sometimes found, and [effrey says that the size of the mot has no relation to the number of protoxylems formed. I he first tracheids are short and reticulately marked, as they are in Ophioglossum. The surface of the root is quite destitute of root hairs, as is the case in the adult sporophyte. In 11. lunaria the predominance of the root ovei the shoot is even more marked than in 11. virginianum, for there are several roots, sometimes four to five, developed before the first foliage leaf is formed, and the bud remains extremely small and inconspicuous. A longitudinal section of the cotyledon of B. virginianum, just before it breaks through the overlying calyptra, shows it to hi' strongly curved away from the loot and overhanging the cleft within which lies the stem apex. If the section is exactly a median one, there may be seen near the tip, but lying somewhat tow aid the lower side, a marginal cell of triangular outline, which, from the arrangement of the cells about it, is evidently the apical cell. The cotyledon, instead of being straight as it 52 THE OPHIOGLOSS \l ES is in Ophioglossum and Botrychium lunaria, approaches the- circinate form of that of the Marattiaceae and the typical ferns. The young vascular bundle is now clearly evident, lying somewhat toward the inner side of the leaf, and it can be readily followed downward until it joins the root bundle, with which it is continuous. In B. virginianum, while the cotyledon is still quite small, the second leaf appeals close' to the stem apex and nearly opposite the cotyledon, the sheathing base of which surrounds the stem apex, together with the young second leaf. The growth of the second leaf is also probably from a tetiahcdial apical cell, and this is the case with all of the later leaves. While the second leaf is still very small, there begins the differentiation of the corresponding leaf trace, which joins the bundle of the primary root close to the point at which it gives off another bundle destined for the second root, the apical cell of which is cut out from the tissue near the base of the second leaf, at a point almost directly opposite the cotyledon. Indeed, the second root may be said to bear much the same relation to the second leaf that the primary root dors to the cotyledon (fig. 35, B). Fig. 36. — Botr\chium lunaria (after Bruchmann). A, B. Young embryos. X225. C, D. Older embryos. The cotyledon and stem apex, phioglossaceae, is unfortunately very incompletely known. Lang (Lang I), to whom we owe the only published account of the gametophyte and young sporophyte, was unable to secure any young embryos and only very scanty material of the later stages. Lang says of the youngest forms which he found (Lang I, page 40): "The large hemispherical foot is deeply inserted in the tissue of the prothallium. The upper portion of the embryo had burst through the covering also of the latter; in it can be distinguished the primary root— the first leaf- ami ( covered over by tin- sheath of the first leaf) the depressed apex of the stem. The position of the organs is thus essentially similar to what is found in Botrychium virginianum." Lang's material was collected in the Barrawa Reserve Forest, in Ceylon, and in February iyo6 I visited the same locality and collected a large number of young sporophytes, many of which were still connected with the gametophyte; but none of the gametophytes were young enough to show young embryos. As we have already suggested, it seems very probable that the gametophyte of Helminth- ostachys, like that of Ophioglossum moluccanum, is annual and produces normally a single sporophyte, after which it perishes. The development of the gametophyte is probably dependent upon the annual inundation of the forest, and this perhaps accounts for the fact that all of the young sporophytes were of about the same age. The youngest specimen which I found is shown in fig. 10. The petiole of the leaf was about 1 centimeter in length, and at the tip the minute lamina, strongly bent over like the young leaves of Botrychium virginianum, could be seen, showing the three lobes which characterize the fully expanded leaf. 1 he cotyledon is rudi- mentary in Helminthostachys, the "cotyledon" described by Lang being really the second leaf. At the base, but separated from the root by a short inn-mode, is a swelling which marks the position of the apical bud, inclosed within the hollow sheath at the base of the petiole. The root at this stage is still quite short, the first green leaf seeming to be more precocious than in Botrychium and thus resembling Ophioglossum moluccanum. As the leaf grows the lamina expands and is seen to be ternate in form. Very often the two lateral lobes are of unequal size and show that tin- ternate form is due to an unequal dichotomy, such as is common in many ferns in the early Laves, which are transitional between the dichotomously divided cotyledon and the pinnate leaves of the older sporophyte. The ultimate divisions of the veins, as in Botrych- ium, are dichotomous.* * In a recent note (Lang 2) it is stated that a suvpensor also occuri in Hehninthoilachyt. THE YOUNG SPOROPHYTE 55 III. THE YOUNG SPOROPHYTE. THE YOUNG SPOROPHYTE OF OPHIOGLOSSUM. As we have seen, the single vascular strand in the young sporophyte of Ophio- glossum moluccanum is continuous through the cotyledon and root, and sections at different heights show essentially the same structure throughout. The petiole of the cotyledon (figure 27, A), which is traversed by two conspicuous lacunae, shows that the axial bundle has a perfect collateral structure. The xylem consists of a group of about a half dozen tracheids on the inner side of the bundle, of which the endodermis, however, is not evident. If a section made in the mid-region of the plant is examined, the only difference between its bundle and that within the petiole of the leaf consists in the somewhat greater development of the tracheary tissue. The section of the root bundle also almost exactly resembles that of the leaf. Whether we call the root bundle monarch or collateral is merely a question of terms. r B Fig. 37. A-C. Three sections of a young sporophyte of Oplnovlo;!um moluccanum with the first leaf, I1 , of bud fullv developed; r, primary root of sporophyte; r1, first root of bud; /2, second leaf of bud. D. The stem-apex more highly magnified, showing the third leaf, /3. In the mid-region of the sporophyte the endodermis is well marked, and this is even more the case in the root. In the mid region the xylem is separated from the endo- dermis by a single layer of pericycle cells. About the time that the leaf emerges the first root of the bud begins to develop, and, like the leaf, it seems to arise quite independently of the stem apex. The tetrahedral apical cell is cut out from the cortical tissue of the root some distance below the apex of the young bud, and as soon as the apical cell is established active segmentation begins in the tissue below the bud and a strand of procambium is developed, connecting the young root apex directly with the vascular bundle of the primary root. Thus the vascular bundles of both the first leaf and the first root of the young bud are directly connected with the primary root of the young sporo- phyte and have nothing to do with the tissues in the apex of the stem of the bud, which is still very small. 56 THE OPHIOGLOSSALES The two bundles, belonging respectively to the leaf and the root of the bud, join the bundle of the primary root very near together, and from near this point of junction the development of the tracheary tissue proceeds toward the apices of the young organs. The first tracheids, like those found in the median region of the young sporophyte, are short reticulate ones. The first leaf of the bud grows rapidly and closely resembles the cotyledon in form and size, and at first it often looks as if two young sporophytes of equal size were growing from the prothallium, but a very slight examination shows that the second leaf belongs to the bud upon the primary root. The second leaf of the bud also seems to be formed independently of the stem apex and arises nearly opposite the first one (fig. 37, A). Its development is practically the same as that of the first leaf, and it pushes through the outer tissue of the root at a point removed by an appreciable distance from the base of the first leaf, from which it is separated by a sheath about the base of the latter, formed by the outer root tissues. The vascular bundle in the second leaf is soon developed and can be followed down to the bundle of the first root of the bud, to which it bears somewhat the same relation that the bundle of the first leaf of the bud does to the primary root of the sporophyte. Fig. 37 will show clearly the arrangement of the vascular bundles in a young bud in which the first leaf is full)' developed, while the second and third are pretty well advanced. The latter ( Is ) shows very plainly the conspicuous apical cell and the beginning of the leaf trace. The apical cell of the stem at this time is sufficiently conspicuous, but the amount of tissue surrounding it is quite limited in extent. In fig. 37, D, it is probable that the fourth leaf has begun to develop. Above the apex of the stem the cavity of the leaf sheath can be clearly seen. By this time the first root of the bud has emerged below the first leaf and the tracheary tissue is well developed in its basal region. The second root of the bud originates below the apex of the stem in the vicinity of the base of the second leaf. The vascular strand of the third leaf joins it, and the third leaf seems to have somewhat the same relation to the second root that the second leaf does to the primary root. The studv of the development of the young bud shows that, up to the time of the production of the third leaf, the young organs arise quite independently of each other from the tissue of the primary root. The young sporophyte is, so to speak, made up by the union of several independent members. The third and fourth leaves arise from the stem apex as all of the later ones do, but the first and second leaves and the first root show no recognizable relation to the stem apex. Their vascular bundles are directly connected with the bundle of the primary root of the young sporophyte, and are in no way associated with the tissues which belong to the stem region of the bud. No material was available for the study of the origin of the leafy bud in 0. pendulum, but the first leaf evidently develops very much later than it does in 0. moluccanum. Young leaves 10 centimeters or more in length were found, which, there is some reason to suppose, were the primary leaves of the young plant; but they were not recognized as such at the time they were collected and it is therefore impossible to say whether they really were the first leaves devel- oped. After the study had been made of the young sporophyte in 0. moluccanum, and the secondary origin of the stem apex was made out, it was recognized that these small leaves in 0. pendulum also were probably the primary leaves of the plant, but it was too late then to trace the connection of the roots from which they developed to their connection with the primary root of the young sporophyte. I was unable to determine just how soon the fertile leaves are developed from the young sporophyte in 0. moluccanum. In all of the specimens that were sectioned, THE YOUNG SPOROPHYTE 57 the first three or four leaves were sterile and practically like the cotyledon, and it is not certain which leaf, under ordinary circumstances, first gives rise to the spore- bearing spike. In O. moluccanum and the other tropical species of Ophioglossum growth is continuous, and it is evident that the development of the leaves does not take the long period required in O. vulgatum and other species of temperate regions, where growth is interrupted each year and where only one leaf is developed in the v^SSv Fig. 38. Six of a snirs nl transverse sections of a young sporophyte of Ophioglossum moluccanum, etill attached to primary root, r. Section B passes through the stem apex. X35. season. Mettenius states that O. pedunculosum, which, as we have seen, may per- haps be identical with 0. moluccanum, develops three leaves each season. This was in'^the Botanical Garden at Leipzig, and it is highly probable, under the much more favorable conditions of its native tropical habitat, that this number would be exceeded. There was not time to make investigations in regard to this r point, but it is very certain that several leaves are de- veloped in the course of each year and that the develop- ment of the individual leaf does not require the long pe- riod necessary in the species of cold climates. Figures 38 and 39 show several transverse sections forming a series taken from a young sporophyte with three fully developed leaves and two younger ones. This bud was developed from a small root, but it was not certain that this was the primary root of the embryo. The spirally arranged leaves show the two-fifths diver- gence. The second and third leaves of this bud were successively larger than the first leaf, but the section of the petiole showed a single centrally placed vascular bundle of the same type as that of the primary Fig. 39. A. Section of the sporophyte shown in fig. 38, showing arrangement of leaves; r is the primary root. X about 60. B. Vascular bundle of first leaf, more highly magnified. C. Stem apex, showing youngest leaf trace, /s ; st, apical cell of stem. 58 THE OPHIOGLOSSALES leaf and all of these leaves were sterile, nor could am indication of the develop- ment of a central spike be seen in the development of the fourth leaf. The Hist leaf of the bud, like the cotyledon, showed two large lacun;e in the petiole, but in tin second leaf there was but a single one and this was interrupted at intervals, while the petiole of the third leaf appeared almost solid, there- being only small, irregular, intercellular spaces, such as are always found in any loose parenchyma. All of tin vascular bundles are collateral. In the lower portion of the third leaf eleven tracheids could be- seen in the transverse section of the bundle, but higher up the amount of" tracheary tissue was reduced. Figs. 38, //, and 39, .-/, were cut just above the stem apex and showed clearly the arrangement of the Hist four leaves. The youngest leaf is still entirely surrounded In the conical stipular sheath belonging to the third leaf. Fig. 38,/^, is taken imme- diately above the- stem apex and passes through the very young fifth leaf. This section shows very satisfactorily the arrangement of the first five leaves. Section C lies a short distance- below the stem apex, r is the- root from which the bud has arisen, and the group of tracheids at its junction with the bud marks the point ol union ol the bundles of the first leaf and the first root of the bud. In the center of tin- section may be seen a mass of large-celled parenchyma, the central pith of the stem, which is derived from the- large-celled meristem of the stem apex. 1 he- broken ring of procambium surrounding the pith is composed of obliquely cut traces of the fourth and fifth leaves and the basal tissue of the second root of the bud. The leaf traces from the- second and third leaves are still free in the cortical tissue of the young stem. E and F pass through the base of the bud. The stout bundle of the first root is seen connected with the bundle of the root upon which the bud was developed, and the second leaf trace- (I2) bends in to meet the bundle of the- root but is cut at a level above the point of junction. Close to the second leaf trace is the Iowei part of the fourth leaf trace, which is connected with the trace of the second root of the bud. The steles of both the first and second roots of the bud run horizon- tally across its base before they emerge, and the leaf traces are almost perpendic- ular to these. The second leaf trace at the base of the bud joins the stele of the first root and the short tracheids curve out trom the opposite side of the bundle- of the first root to unite with the base of the third leaf trace, which does not, as might have been expected, show any very obvious relation to the second root. At the point where the first root bends downward to emerge from the overlying cortical tissue the tracheary tissue- of this, with the bases of the leat traces, forms a large- mass of irregular, broad, reticulate tracheids, occupying the center of the base of the young bud. It was probably the presence of this mass 01 tracheary tissue at the base of the- bud which led Rostow/ew to state that the bundle of the- bud is at first solid. It is possible- that the buds formed on the old roots of (). vulgatum may be somewhat different in structure from the- early buds in 0. moluccanum and that there really may In- such a solid stele- at its base. According to Rostowzew (Rostow/ew 1, 2) and Poirault (Poirault 2); the buds upon the older mots of 0. vulgatum usually arise close to the apex, much as they sometimes do in the- formation of the bud upon the primary root of 0. moluccanum. Van Tieghem thought that the apex of the root itself became- changed into the le-af\- shoot, but both Poirault and Rostowzew demonstrated that the young bud originated from a segment of the apical cell of the root anel not from tin apical cell itself; the latter continues its growth and the root thus grows beyond the point of insertion of the young bud. Rostowzew states that an outer cell of the segment divides into two superimposed layers, of which the superior one, or the one nearest the root apex, gives rise to the stem apex of the bud, while from the lower THE YOUNG SPOROPHYTE 59 one, i. e., the one turned toward the base of the root, the first leaf arises. It thus appears that in these adventitious buds in Ophioglossum vulgatum there is much the same arrangement of the first organs of the young bud that we have seen to be the case in O. moluccanum, the leaf and the stem apex being virtually independent organs. From Rostowzew's figures it is evident that the bundle from the first leaf of the bud in O. vulgatum is connected directly with the main root in very much the same way as it is in the primary bud in O. moluccanum; but apparently the first root of the young bud is inserted higher up and does not connect directly with the primary root; this point, however, needs further elucidation. Moreover, according to his statements, there is a good deal of difference shown as to the time of the emer- gence of the leaves and roots, three or four roots sometimes being well developed before the first leaf expands. This reminds one of the behavior of the primary sporophyte, where Bruchmann found that several roots were developed before the first green leaf made its appearance. Fig. 40. — Vertical sections of a young sporophyte of Botrychium virginianum. The sections were cut at right angles to the axis of the root. sh, stipular sheath of cotyledon; E, section of primary root, which in this case was triarch. X20. THE YOUNG SPOROPHYTE OF BOTRYCHIUM. Jeffrey (Jeffrey 1) has described at some length the structure of the young sporophyte of B. virginianum, and Bruchmann (Bruchmann 2) has studied the earlier stages of the sporophyte in B. lunaria. The present account is based mainly upon my own studies of B. virginianum, made from material furnished me through the kindness of Professor Jeffrey. Jeffrey found that from the beginning the con- spicuous stele found in the axis of the young sporophyte was a hollow cylinder, which he assumes to be a really cauline structure, and not made up of leaf traces. The results of my own studies indicate that, as in the case of Ophioglossum, the vascular system of the stem is made up exclusively of confluent leaf traces. Fig. 35, B , shows a longitudinal section of a young sporophyte in which the first two leaves are evident and the continuation into these of the tissues of the vascular bundle of the root is very clear. The large central pith continues up into the stem apex, bur the whole of the fibrovascular tissue is continued into the leaves, there 60 THE OPHIOGLOSSALES being no trace of procambium inside of the leaf traces in the apical region of the stem itself. Fig. 40 shows a sporophyte which was cut at right angles to the primary root, which in this case had a triarch bundle. Near its base the three xylem masses increase in size and form a nearly complete ring of tracheary tissue, which higher up is continued into the leaf traces. The third leaf has already begun to form, but is still very small. Below it, however, can be clearly seen the beginning of its large leaf trace, which can be followed downward to its junction with the second root, which is now just about ready to emerge. The stele of the second root is con- nected with the central cylinder of the axis near its junction with this third leaf trace. The stem apex occupies a very small area, crowded between the base of the second and third leaves. Its deep, narrow, apical cell is not as conspicuous as is often the case. Fig. 41. A-F. Six of a series of horizontal sections of a young sporophyte of Botrychium virginianum. X75. B passes through stem apex, jtf, which is shown more enlarged in G; r2, second root; /2, second leaf. A similar sporophyte is shown in cross-section in tig. 41. This was cut in the plane of the Hist root, which makes a sharp angle with the base ol the cotyledon. The thick stele of the root is much expanded where it joins the leaf traces to form the beginning of the tubular stele of the axis, and at the point of junction there is a large nearly solid mass of short, irregularly disposed, reticulate tracheids. This young sporophvtc was evidently the further development of an embryo of the type shown in fig. 35, A . The trace of the cotyledon makes a right angle with the stele of the root and on the side opposite to its junction with the stele of the root there can be seen the trace belonging to the second leaf, which is already will developed. The apical cell is tetrahedral in Botrychium virginianum and the cells are cut off in regular segments from its three lateral faces. Segmentation is THK YOUNG SPOROI'HYTE 61 evidently slow and there is usually a good deal of difference in the stage of develop- ment in the three segments composing a single cycle. The first division in each of the lateral segments cuts off a small inner cell from a large outer one, and the latter is then divided into two by an anticlinal wall. The divisions were not followed beyond this stage and the limits of the segments comprising the second cycle can not be easily recognized, as by this time the youngest leaf begins to grow and the stem apex is thus crowded into a very small area between the bases of the two youngest leaves. The shallow mass of meristematic tissue comprising the stem apex merges gradually into the large-celled parenchyma which makes up the pith cylinder inside the hollow stele of the axis. Longitudinal sections of a much older sporophyte are shown in fig. 42. Except for the vascular bundles, the whole of the tissue of the young sporophyte is composed of large-celled parenchyma which, especially in the region belonging to the foot, contains large quantities of starch. The form of the young leaves in B. virgtnianum is very different from that in B. lunana. In the former the young leaf is bent forward over the stem apex and the apical cell is at the tip of this bent-over portion. In B. lunana, however, the Fig. 42. Three longitudinal sections of a young sporophyte of Botrychium virginianum, with several leaves. per, absciss layer of periderm; /, /, the youngest leaves; sh, stipular sheath. Xio. bent-over portion becomes the stipular region and the apical cell of the young leaf arises in the convex upper portion, so that the leaf grows straight upward instead of being bent over as it is in B. virginianum. The latter species in this respect resembles the Marattiaceae. The stipules in B. virginianum are lateral structures which extend around the next youngest leaf and the stem apex, but the sheath has a narrow cleft in front, so that we may really speak of two stipules instead of a single stipular sheath (fig. 42, A). In this respect also there is a strong resemblance to the Marattiaceae. Fig. 43 shows a series of transverse sections of a young sporophyte of B. vir- ginianum in which the fourth leaf was just evident. Fig. 43, A and B, are near the base of the fourth leaf and show the arrangement of the first four leaves. The base of the cotyledon (cot) is a good deal flattened and the petiole is seen to be traversed by very conspicuous lacunae, reminding one of those in the cotyledon of Ophioglossum moluccanum. The conspicuous stipules can be seen extending partly around the group of younger leaves, but not completely inclosing them. The two vascular bundles of the petiole are beginning to unite to form the single leaf trace which is found lower down. The xylem in each bundle forms a broad band 62 Mil Ol'HHKILOSSALKS of wood, which is surrounded bv the phloem, although the latter is much less developed upon the inner side than upon tin- outer and the bundles thus approach the collateral condition found in the trace lower down. Small protophloem ele- ments occur in the outer part ot the bundle and are continued part way around toward the lowei side, to the point where the two bundles are beginning to fuse, but here they are no longer visible. The second leaf shows no lacunae and the base of the petiole is more nearly cylindrical than is that of the cotyledon. Like the latter, the petiole contains two vascular bundles which at the level of this section were still quite distinct, and the permanent elements were just beginning to form. The stipules in the second leaf completely inclose the third, but there is a very narrow slit in front where the two stipules meet. The next section figured is taken just above the stem apex and cuts through the youngest I fourth) leaf. The two bundles of the petiole of the third leaf are evidently still quite separate, but closely approximated, and as vet show no per- manent tissue. The stipules of the thud leaf are just becoming evident. At the level of the stem apex the bases of the thud and fourth leaves aie completely united. 0.43. Scriesofti ctiom 0] a j poroph) 1 Botryehium virginianum, showing union of leaf trace: to form vascular cylinder. D and E show rated leaf traces which unite further down; K shows the bundle ol the axis neai 11 tum tion wiili the stele of the primary r-><.r. and tin base "t the second leal is also partially fused with these. I he two bundles from the second leaf are now beginning to unite to foim tin single leaf trace, while in the third leaf the process is complete and a single oval mass of procambium cells marks the position of the single trace of this leaf. On the opposite side of the apical meristem is a similar but less developed mass of procambium, representing the trace from the young fourth leaf. For some distance below the stem apex the two bundles of tin third and fourth leaf respectively are quit< distinct, and ate separated from each other by a mass ol large-celled pith. Lower down the two bundles approach and coalesce, and on the side of tin second leaf trace (which is turned away from the third one) a group of actively dividing cells can be seen which forms the beginning of the fifth leaf trace, although the leaf itself can not be recognized at the summit. These three masses of procambial tissue, viz, the young traces from the third, fourth, and fifth leaves, THE YOUNG SPOROPHYTE 63 make a nearly continuous crescent-shaped mass of procambium. Fig. \\,A,B, shows the section of the central stele; and the space between the horns of the crescent, i. e., the space between the third and fifth leaf traces, constitutes the so-called "foliar gap" which faces the second leaf trace. The central cells of the young stele have already begun to form cambium, which in the older stem constitutes such a char- acteristic feature of the stele. Before any tracheary tissue is formed in the young stele, a few thick-walled cells can be seen in the outer part of the portion of the stele corresponding to the third leaf trace. These are protophloem cells, and similar ones are developed later in the younger parts of the stele. The first tracheary tissue appears in the region of the steel section belonging to the third leaf trace. In the section shown (fig. 44, C) Fig. 44. A, B, C. The young vascular bundles, of sections E, F, G, of the scries shown in fig. 4;- -C150. D. Section of a young sporophytc of Botrychium virginianum, showing junction ol a li al trace with stele of axis. X75. there are three groups of these primary tracheids, separated In a considerable interval and placed at the extreme inner limit of the young stele. In the older portions of the bundle the number of these increases and similar ones make their appearance in the next younger section of the stele, that belonging to the fourth leaf. Last of all, these primary tracheids are formed in the younger region of the stele and new ones arise between these first-formed ones, so that then is developed a nearly complete circle of tracheids marking the inner boundary of the tubular stele. The second leaf trace approaches nearer and nearer the two horns of the crescentic stele section as it is followed downward, and finally joins it, filling up the gap between the regions belonging to the third and fifth leaf traces. Thus, for a 64 THE OPHIOGLOSSALES time the section of the stele appears perfectly circular and continues downward until it reaches the next leaf trace, which marks the point of departure of the trace of the cotyledon. Except for the greater development of the xylem, which is composed ot two to three concentric rows of tracheids, the section of the stele in this region presents much the same appearance that it does in tin- younger pairs nearer the stem apex. The outer part of the stele at this point shows a somewhat broken row ot thick-walled hast fibers within which are large sieve tubes. I he cambium, lying just inside the phloem, is very much less developed in the basal region of the stele than it is higher up and can scarcely he said to he present. The stele in this region ot the stem is quite similar to that of B. lunar i a, figured by Poirault. The endodermis is also much less definite than it is later, and I could not satisfy myself that there was in B. virginianum any trace of the inner endodermis which Poirault states is present in the basal part of the stem in B. lunaria, hut which disappears later. The medullary rays also, which are conspicuous in the stele of the older plant, are very imperfectly developed in this basal legion. In the intermediate region, between the base of the primary root and the trace from the cotyledon, a section shows a thick irregular ring of tracheal)' tissue with no clearly developed medullary rays (fig. 43, L). Indeed, at the lowest part of this region the tracheary tissue forms an almost solid core with only a small amount of parenchyma interspersed, thus forming an inconspicuous and irregular pith. While the endodermis is not clearly delimited, some of the cells in the zone of tissue sur- rounding the stele show the radial thickenings of the walls; but these cells are not all of them in the layer immediately adjoining the stele, being irregularly disposed throughout the three or four layers of cells surrounding the stele. As sections are examined in succession upwards, the pith becomes better defined. Sections taken downward show a ring of tracheary tissue gradually separating into the two or three xylem masses of the primary root. The primary root in the sporophyte which has just been described was diarch. The stele shows a very evident endodermis, within which is the pericycle, for the most part composed of two layers of cells. The xylems are composed of a large group of tracheids, of which the two or three smaller ones next the pericycle repre- sent the protoxylems. Small, deeply staining protophloem elements can be seen toward the outside of the phloem, and within these are the other phloem elements, thick-walled bast fibers, and sieve tubes. The rest of the stele is composed of small, thin-walled parenchyma cells. The outer region of the root is made up of somewhat compressed cells suggesting the periderm cells which are found in parts of the older stem and which are also present in the cortex of the later roots. This outer layer of cells, however, shows no evidence of active division. A cross-section of the petiole of the cotyledon, from the same sporophyte, taken at a point some distance above the stipules, shows within the epidermis about three layers of parenchyma cells with no conspicuous intercellular spaces, but within these are several very large lacuna?, separated by narrow plates of tissue. In the center of the section are seen the two concentric bundles that traverse the petiole. As in the later leaves of this species, these bundles are truly concentric, thus resem- bling those of the Marattiace.e and differing from the collateral bundles of Ophio- glossum and the smaller species of Botrychium. I In phloem is better developed upon the outer side and small protophloem elements can be seen at its outer limit, but these are continued partially around the inner side of the bundle, where the xylem is separated from the outside by about two layers of cells. No definite endodermis can be made out. The two bundles are continued up to the base of the lamina, where one of them passes into each lateral lobe of the ternate leaf. One of THE YOUNG SPOROPHYTE 65 these divides and gives off a branch which passes into the terminal lobe of the leaf (see Jeffrey 1, page 23). In a section of the older stem, about where the fourth leaf trace becomes joined to the fifth and sixth, the section of the stele appears circular. In the portion be- longing to the fifth leaf trace there are present about half a dozen isolated tracheids, arranged in a row, while in the sixth trace only a single small tracheid is developed. The two xylems of the fourth leaf trace are now no longer distinguishable and the wooily part of the trace has the form of an irregular row on the inner side of the trace section, and is continuous with the xylems of the fourth and fifth traces. liven after the fusion of these three traces is practically complete, it is still evident that the ring-shaped section is composed of three parts, the break between the xylems showing the limits of the three component leaf traces. With the increase in the development of the xylem ring the limits of the individual leaf traces are finally completely lost and the section of the stele shows a thick ring of the tracheary tissue interrupted only at intervals by single rows of parenchyma cells in the medullary rays. Outside the ring of tracheids there can be seen a zone of narrow cells arranged in radially disposed rows. This zone constitutes a genuine cambium, precisely similar in appearance to that found in the stems of Conifers and Dicotyledons. This cambium has long been known to occur in the older sporophytes of the larger species of Botrychium, and Jeffrey first noted it in the young sporophyte. The cambium is well developed in the leaf trace for a considerable distance above its junction with the central stele. The primary tracheary tissue is in immediate contact with the pith. Occa- sionally the ring of tracheids is broken by a single parenchyma cell. Outside the tracheary ring the cells are arranged in radial rows and in these the formation of the walls is always periclinal, so that the cambium in these earlier stages presents a very characteristic appearance. The rows of cambium cells continuous with the primary parenchyma cells lying between the tracheids of the primary row do not develop tracheary tissue, but remain parenchymatous and thus give rise to the medullary rays, which later are so conspicuous. The extreme outer portion of the vascular cylinder is composed of a ring of thick-walled cells which are probably bast fibers, but the walls of these do not stain strongly with safranine and are hence much less conspicuous than the tracheids. These may possibly be considered as protophloem elements and evidently corre- spond to the zone of thick-walled cells figured by Poirault for B. lunana (Poirault 2, fig. 1 1). fust outside of this ring of thick-walled cells lies the endodermis, which has not as yet developed the characteristic radial thickenings which aie so easily seen in the older parts of the stele. These thick-walled phloem elements, as is the case with the protoxylems, do not form an unbroken ring, bur are interrupted at the junctions of the confluent leaf traces (fig. 44, C). In the older bundle, a zone of large cells, the young sieve tubes may be recog- nized lying inside the ring of bast fibers. Inside of the zone of sieve tubes lies the cambium, the cells of which gradually pass into the xylem, to which constant but slow additions are made from the cambium; indeed, Jeffrey thinks that we can not speak of primary wood, lie considers that all of the wood owes its origin to the activity of the cambium ring. The radial rows of secondarj wood are inter- rupted at intervals by the medullary rays, ami the resemblance of the stele at this stage to the section of a young coniferous stem is really quite remarkable. I he endodermis has now become exceedingly conspicuous, as the lignificd lateral walls stain very strongly with safranine and stand out strongly in contrast with the cells of the cortex lying outside the endodermis. The inner walls of the endodermal 5 66 THE OPHIOGLOSS \l I S cells also stain more or less strongly with the safranine, indicating that they also are partially lignified. The junction ot the second leaf trace with tin central stele is shown in tig. 43, E, F, G, and similar stages in the fusion of the Hist leaf trace are shown in tig. 43, H, I. The junction ot one ot the early roots, perhaps the third root, with the central stele is shown in hg. 43, /. At the level where the second leaf trace departs from the central stele the xylem ring is composed ot about three rows of cells. In both this second leal trace and the third tin endodermis is very conspicuous upon the outer side of the bundle, but is not developed upon its inner face. \s the trace approaches the central cylinder it can In- seen that the endodermis is interrupted across the leat gap, but as the leat trace approaches, endodermal cells are developed between the endodermis ot the central cylinder and that of the leaf trace. This union takes place first upon one side, the gap being closed sooner on one side than the other, but later a similar process takes place on the other side of the leaf trace and the closing ot the gap is complete, the endodermis now being continued without interruption around the whole ot the circular section. The leaf trace where it joins the central cylinder is exactly like it in size and structure; indeed, it is nothing more than a sector of the hollow cylindrical stele, which is evidently built up exclusively of these fused leat traces. I here is no vestige ot any tissue in the stele which is not referable to the bases of the leaf traces themselves. This becomes very evident when the structure of the leaf trace is examined before it closes the gap where it joins the central stele. An examination of the outer tissues of the older portions of the stem (fig. 42) show that at certain points there is a conspicuous border of periderm (per), forming a layer some four or five cells deep. This border of periderm occupies about a third of the circumference of the section and marks the position of an old leaf base which has fallen away. According to Jeffrey, this periderm is an "abciss" layer and results in the cutting oft of the old leaf bases. He suggests that the layer of cork cells developed from the periderm closes the scar left by the dead leaf and may be efficacious in preventing infection from fungi in the soil. The presence of a periderm in the outer cortex of the ( )phioglossacea? was first pointed out by Russow (Russow 1), and was afterward confirmed by Holle (Holle 1). The periderm in H. virgin- ianum is made up of the usual radially arranged rows of cells (fig. 42, per). All of the tracheary tissue in the young sporophyte is composed of rather short, reticulately marked tracheitis. The secondary tracheids increase in size and the thickened bars on their walls become broader, so that the larger elements have their walls pitted rather than reticulately marked, and these pits, especially in the older plant, are of the bordered type, not very unlike the round bordered pits found in the wood of Conifers. The elongated thick-walled elements in the outer part of the phloem are probably bast fibers. In the older part of the bundle these stain (juite strongly with safranine and show irregular pits upon their walls. The sieve tubes do not show at all clearly in sections mounted in balsam. They are evidently large, but the char- acteristic dotted sieve areas can not be made out and no special study was made of these. Between the large secondary tracheids and the phloem are the narrow, thin- walled cambium cells. These probably contribute only to the wood, and there is no secondary phloem. THK YOUNG SPOROPHYTE 67 THE YOUNG SPOROPHYTE oi HELMINTHOSTACHYS. The only account that has yet been published of the young sporophyte of Helminthostachys is that of Lang (Lang I), who examined at some length the vascular system in the young sporophyte. Lang states that the young sporophyte of Helminthostachys develops its first leaf as a large ternate foliage leaf, and when I first examined the young plants which were collected in Ceylon 1 reached the same conclusion; but on making sections of the young sporophyte it was found that in all of the young plants that were sectioned the supposed cotyledon was really the second leaf, the cotyledon itself being rudimentary and forming an inconspicuous papilla at the base of the second leaf, which was the first to develop a functional lamina. Further examination of a number of other specimens showed that this rudimentary cotyledon was also present and it is probable that the cotyledon in Helminthostachys, as in Botrychium lunaria and Ophioglossum vulgatum, is always a rudimentary organ which never appears above the ground. The youngest sporophytes oi Helminthostachys which were found already had the second leaf well developed and the first root had emerged. The ternate second leaf (fig. 10, C) shows clearly that the ternate form is the result of an unequal dichot- omy, such as is common in the early leaves of many ferns. The second leaf of Helminthostachys closely resembles the cotyledon oi Botrychium virginianum and, like that, usually possesses two vascular bundles extending through the long, slender petiole. At the base of the leaf there is a conspicuous stipular sheath which completely incloses the next younger leaf, as it does in Botrychium lunaria. The two bundles of the petiole contribute to the two lateral lobes of the leaf as they do in the cotyledon of Botrychium virgiiuanum, and one of these, forking again, gives rise to the terminal lobe and smaller lateral lobe. The venation of the second leaf, especially in its younger stages, is almost perfectly dichotomous and is more like the venation in the leaves oi Botrychium lunaria than that of B. virginiaiium. With the enlargement of the leaf segments the venation more nearly approaches the pinnate arrangement found in the later leaves. While the young sporophyte in Helminthostachys in its earliest stages shows a general resemblance to that of Botrychium virginianum, a difference is very soon noted. The young plant, very soon after the formation of the cotyledon, develops a conspicuous internode between the cotyledon and the root, and the stem rapidly elongates as the sporophyte grows, so that it soon shows the characteristic elongated rhizome of the older sporophyte. Unfortunately I was unable to secure any very young sporophytes, my youngest specimens already having the second leaf nearly fully developed. According to Lang, the arrangement of the primary organs of the young sporophyte is the same as in B. virginianum, but I could make no study of the origin of the vascular system in the sporophyte before the first leaf and root had emerged from the prothallium. To judge from longitudinal sections of the youngest plants obtained, the relation of the first leaf and root is the same as in Botrychium virginianum and the base of the young sporophyte closely resembles that of the latter. As in Botrychium, the foot is very large and conspicuous, forming a large, hemispherical body sur- rounded by the prothallial tissue. The large primary root grows out at right angles to the foot and is continued into the axis of the sporophyte, which grows up verti- cally. None of the specimens were young enough to show the relation of the stem apex to the first leaf and root, as in the youngest specimens the axis had already- begun to elongate and an evident internode was formed between the base of the first leaf and the root. 68 III! OPHIOGLOSSALES The longitudinal section of the young sporophyte at tin's time shews that the thick stele of the root bends upward and is continued into the axis, which already is a good deal elongated ami has carried up the young leaves, which an- separated from each other by conspicuous internodes. I In young sporophyte at this staf bud. r, young root. XlOO. C. Stem apex. <2oo. THE YOUNG SPOROPHYTE 71 in the very young leaf, can no longer be recognized and the lamina is divided into two nearly equal lobes, of which one is lateral with reference to the other, but these two lobes probably arise as a result of the dichotomy of the original apex. A group of narrow marginal cells somewhat to one side of the terminal lobe evidently consti- tuted a meristem tissue, and this was preparing for the second dichotomy, by which the ternate form of the young leaf is established. The branching of the lamina at this early period therefore appears to be the result of an unequal dichotomy, as it is in the first foliage leaf. The vascular bundles from each of the two primary leaf seg- ments were just beginning to be recognizable and join into one at the base of the lamina. Surrounding the leaf is the sheath developed from the base of the second leaf. This sheath in the section appears as a closed ring some two or three cells in thickness and looking very much like the sheath of Ophioglossum. The top of the conical sheath is closed and does not show the pore found in Ophioglossum. The sheath becomes thicker lower down, where four rows of cells show in the section. Between the inclosed leaf and the surrounding sheath in the lower portion (fig. 49, B) may be seen the section of flattened scales (sc) which grow from the base of the leaf Fig. 4 V A, B. Two sections of terminal bud of a young sporophyte of Helminthosla C, D. Two similar sections from an older plant, .v, stem apex; sh, petiolar sheath. and from the tissue immediately about the stem apex. At its base the sheath of the third leaf is already completely developed and surrounds the young fourth leaf (/4), which in this section was cut horizontally and shows plainly its large apical cell. The stem apex, which lies directly below the younger leaf, was cut obliquely in this series and did not show the form of the apical cell plainly, but in a young plant of about the same age (fig. 47, C) the apical cell showed that it was regularly trian- gular in form, seen in cross-section. At the level of the stem apex the second leaf shows a single vascular bundle which may be continued undivided upward, or it may divide into two bundles in the petiole. In regard to the structure of the petiole, therefore, Helminthostachys is to some degree intermediate between Botrychium and Ophioglossum. I he young trace of the third leaf can be seen at this level as an oval mass of undifferentiated cam- bium. Immediately below the stem apex the cells are smaller than is the case in the young sporophyte of Botrychium, but one can nor make out the beginning of a central procambium cylinder. The smaller cells in the region immediately underlying the stem apex may be explained by the fact that in these young plants there is no pith developed within the axial stele as there is later on. I he youngest root in this 72 THE OPHIOGLOSSALES specimen was less developed than that in the specimen that we have just described. I his youngest root was cut obliquely, but at its base there could be seen the undif- ferentiated mass of procambium forming its stele, ami the apex showed the tetra- hedral apical cell, presenting much the same hum as the longitudinal section. I he apical portion ot the voting root was somewhat shrunken away from the surrounding tissue, so that there was a very evident space surrounding it. Whethei or nor this was the result of artificial shrinkage was not certain. Below the terminal hud the third leaf trace proceeds downward into the inter- node below, and the tracheary tissue, which is just beginning to develop in the hud legion, increases rapidly in amount and forms a central mass of wood which does In.. 41). — Ten cross-sections from a series of a young sporophyte of Helminthestachys. X25. C. Passes through tin- .stem apex, .v. The round shaded cells are tannin l>. Showi 1I1.- narrow pit, y, between the leal base and the axis. C-J. Show only the vascular bundles. not appeal solid, hut is made up of two groups of tracheids, of which the one nearest the second leaf trace has the larger tracheids. At this stage the bundle somewhat resembles the section of a diarch root. Only a little further down the two xylem masses are joined by the development of tracheids in the middle of the bundle, which thus conns to have a solid xylem con. In the section figured there may be seen a single small isolated tracheid on the side turned toward the second leaf trace, which has now approached so close to the central bundle as to touch it (fig. 50. / |. The central bundle in the internode at this point is beyond question the leaf trace for the third leaf. There is no difficulty in tracing its progress upward to where it enters the leaf and there is no sign of any addition to it from tissue lying outside the original leaf trace. The central stele of the upper part of the second internode is throughout nothing more than the basal portion of the leaf trace from the third leaf. At this point it is concentric in structure. Surrounding the solid xylem core can be seen about three rows of phloem cells, some of the outer ones being small protophloem elements. The large ones lying inside are presumably sieve tubes. \ single layei of rather large pericycle cells separates the phloem from the large endodermal cells, which begin to show very conspicuously lignified radial thickenings, especially on the side of the bundle adjacent to the second leaf trace. 1 he section of the second leaf trace is somewhat extended transversely and the xylem band is composed of about a do/en tracheids. Iii the mid region of the section the xylem is separated from the inner side of the bundle by a single layer of cells, possibly sieve tubes, but at one point the xylem is in contact with the pericycle, which is only imperfect]) developed and wanting at certain points. Upon the dorsal side the phloem may be in direct contact with the endodermis, which is quite wanting at 'bis point upon the ventral suit of the bundle. Higher up, where the trace is quite free, the endo- dermis extends complete!) around it. While the second leal trace, therefore, may THE YOUNG SPOROPHYTE 73 be said to be concentric, it nevertheless approaches closely the collateral type of the other Ophioglossaceae. The fusion of the two leaf traces to form the single axial bundle in the basal part of the internode is very much like that found in the young sporophyte of Bo- trychium (fig. 40, D, F). The endodermis of the two bundles becomes continuous and there may be seen intermediate stages of a single bundle, oval in section, but with two conspicuous xylems, separated by the mass of large, thin-walled tissue which gradually disappears as the bundle is followed downward; and finally, in the lower part of the internode, the section of the stele appears quite circular, the center being occupied by the solid xylem formed by the complete coalescence of the xylems of the fused leaf traces (fig. 49, /). The arrangement of the phloem, pericycle, and endodermis is the same as in the single leaf trace f y^yr'^Vy i '""' in the upper region of the ^VxhSW; internode. The base of the stip- ular sheath can be fol- lowed downward to about the level where the two leaf traces begin to coal- esce. Its anterior free portion, between which and the internode is a narrow slit, finally be- comes quite free and its section is visible as an oblong mass of cells lying quite separate from the section of the internode. Longitudinal sections of a somewhat older sporophyte are shown in fig. 48, A, B; the third leaf is pretty well developed, with the stipular sheath completely overarching the stem apex and the fourth leaf, which already is conspicuous. The third leaf is still completely covered by the large stipular sheath of the second leaf, which apparently forms a closed cavity; but an examination of the adjoining sections showed that the ante- rior margin of the, sheath is quite free in front and that there is a narrow cleft between it and the internode. The real nature of the sheath and its relation to the leaf base is very clearly shown in the median section of the terminal bud from an older plant (fig. 48, C, D), and the resemblance to the terminal bud of Botrychium Inn, nia, except for the dorsiventral arrangement of the parts, is most striking. This is equally marked in the adult sporophyte, as Farmer has already pointed out. The specimen under consideration differed somewhat from the younger one that was described in the relation of the young organs. I he cotyledon was better developed and although the lamina was rudimentary the stipular sheath was large and inclosed the base of the second leaf, while in the youngei specimen discussed no sheath was developed at the base of the cotyledon. I here was a different rela- tion of the roots also, probably associated with the greater development of the cotyledon in the specimen under consideration. The second root, instead of being formed at the base- of the third leaf in the terminal bud, was developed a short dis- tance above the cotyledon, near the base of the second leaf, while in the youngei specimen no root was developed above the primary root until the third leaf had Flo. 5a Two sections of the vascular bundles from the series of Helminlhostachyi shown in fig 40, showing junction of two leaf traces to form central stele, en, endoder- mis. X150. 74 THE OPHIOGLOSSALES begun tn develop; but this second root developed much earlier in its relation to the development of the next leaf than was usually the case. In the present plant the root corresponding to the third leaf, which was the third root in this case, was in a very early stage of development and had not begun to elongate at all. The third leaf in median section was already pretty well advanced and the apical portion was very strongly incurved, as it is in the young leaves of" Botrychium virginianum, which it resembles more than it does that of B. lunaria, and it also suggests the form of the young leaves in the Marattiacc.c. I he basal part of the young leaf, however, is exactly like that of B. lunaria. The stipular sheath forms a thick body, projecting forward and about equal in height to half the total length of the young leaf. It is strongly concave below, and the next leaf is fitted into the cavity (fig. 48, (.'). The forward margin extends downward as a sort of lip which elongates rapidly and keeps pace with the growth of the young leaf inclosed within it, and this is completely concealed until it has reached a large size. The sheath finally forms a long, conical protuberance at the base of the leaf to which it belongs. The cavity below the sheath is extended backward, so that the base of the leaf is hollowed out in front and in section appears much narrower than the part of the petiole above the insertion of the sheath; this brings the stele of the leaf very close to the epidermis at this point. The narrow cleft thus formed between the posterior wall of the stipular cavity and the petiole of the leaf in front is the "canal," which Gwynne-Vaughn described as occurring at the base of the petiole of the older plant and opening above the insertion of the stipule. These canals are easily seen in longi- tudinal sections of the young bud (tig. 48, B) and it is very clear that they are, in truth, nothing but the narrow spaces between the bases of the adjacent leaves. Clwynne- Vaughn's statement that they open above the insertion of the stipules is incorrect unless the stipules of a given leaf are considered to be derived from the sheath sur- rounding the base of the leaf, this sheath properly belonging to the next older leaf. Ibis canal can be seen as a narrow slit in the young leaf, extending below the inser- tion of the next younger one, the young stele of the leaf being separated from the epidermis at this point by only about three rows of cells. I he stele of the young leaf can be traced nearly to its tip, and then continues downward toward the inner side of the leaf into the internode, below where it joins the young stele belonging to the next younger leaf. As in the younger sporophyte, the stem apex is of very limited extent and the apical cell shows the same form. In the section figured (fig 48, C) the youngest leaf is cut almost in a median plane and though externally no differentiation is visible the young stele is already conspicuous and can be followed down without difficulty. Passing on one side- of the stem apex and above the leaf trace, there is no evidence of procambium in the apical region of the stem. In the young sporophyte, therefore, it is perfectly certain that no cauline stele is present. Farmer (Farmer 2) states that in the older sporophyte the stele can be traced above the insertion of the youngest leaves, but we believe a careful study of this point in the older plant would show, as in the younger one, that the stele is developed in the young leaf at an exceedingly earh period and that the stele of the internode is composed entirely of leaf and root traces. The procambium tissue developed on the ventral side of the stele near the apex can be shown, by a study of its transverse sections, to be due entirely to tissue derived from the leaf traces, which are extended downward until they meet on the ventral side of the internode. Near the stem apex are numerous small scales and hairs filling up the space about it and probably associated with the prevention of drying up of the stem THE YOUNG SPOROPHYTE 75 apex, which is doubly protected by these scales and its complete inclosure in the young leaf bases. It is possible that these scales secrete some mucilaginous sub- stance, although there was no evidence of this in the stained sections. In this plant the internodes were quite solid and there was no trace of the large lacunae which were described for the younger plant. Whether the presence of the lacunae is due to any differences in the environment remains to be seen. The speci- mens were all collected under apparently the same conditions, but it is possible that there may have been differences in the amount of water in the soil in which they were growing, and this difference in the amount of water may have something to do with the development of the lacun;e. The root at the base of the third leaf was cut transversely and was in a very early stage of development. The root was still completely buried in the cortex and the stele still imperfectly developed, but showing its connection with the stele of the fourth leaf above the junction of the third leaf trace with the stele of the inter- node. No tracheary tissue was developed and the course of the leaf trace was but little disturbed by the formation of the root. In longitudinal sections taken from a much older sporophyte (fig. 48, C, D) the arrangement of the organs at the apex is seen to be exactly the same as in the younger plants, but of course the parts were all larger. The youngest leaf (/ ' ) still resembles in form and size the corresponding leaf in the younger stem, but the next older leaf is relatively broader than the early leaves of the younger sporophyte, and although the stipular sheath is now well developed the upper portion of the leaf is still quite undifferentiated and the apex is scarcely bent forward at all. The resemblance, therefore, to the young leaf of Botrychium lunaria is even more marked than it is in the younger sporophyte. 1 he apical region of this young leaf is occupied by an epithelium-like layer of columnar cells, and it is doubtful whether any one of these can be certainly denominated the apical cell. Somewhat the same doubt exists also as to the point of a single initial cell in the youngest leaf. In the latter there was some shrinkage in the group of large meristem cells at the apex, which made it still more difficult to decide this point, but a median section showed somewhat indistinctly a single cell, apparently triangular in outline, which from its form and the arrangement of the adjacent cells may very well have been the apical cell. The small group of meristem cells forming the stem apex is crowded in between the base of the youngest leaf and the elongated ridge which surrounds the stem apex upon the ventral side. In consequence of this crowding the outer faces of the apical cell and its youngest segments are very small. The base of the apical cell is more than twice as broad as its free outer face. The ventral face of the apical cell is convex and the young segments cut off from it are strongly curved. The inner or basal wall is oblique, so that the axis of the young cells in the tissue below the apical cell makes an angle with the long axis of the apical cell. Below the apical region may be seen the section of the large central stele of the internode. This is composed, apparently, of two strands of procambium separated by a broad band of pith. As Farmer pointed out, in the older plant there is no question that procambium tissue is developed upon the ventral side of the bundle which extends into the apical region and which is, apparently, not connected with the leaf traces. From this he believes that the stele is really a cauline structure. A study of the cross-sections, however, as will be presently seen, shows that this ventral tissue really does belong to the leaf traces, although it is possible that the basal tissue of the roots may add to it in parr, but I have not been able to satisfy myself that any of this stelar tissue can be properly assigned to the activity of the stem apex. 76 Till- OIMIIOCI.OSS AI.FS As in Botrychium and Ophioglossum, the tissue derived immediately from the activity of the apical meris tern, after the central pith is developed in the stele, contributes only to this central region or pith, which is really not part of the stele proper. < >n the dorsal side of the stele the whole of the procambial strand can easil) be followed into the leaf, and when this is followed downward it is seen to run obliquely through the youngest internode, at the base of which it joins the next leaf. The latter already has its lamina developed and strongly bent over, but is still inclosed in the stipular sheath belonging to the next older leaf. These older leaves have only a single leaf trace, hut it divides into two strands above the level of the stipular sheath. A median section of the thick central stele in Fig. 51. Seven transvei 1 ections from .1 young sporophyte of Helminthostachys. X35. /. petiole "i leaf; t, terminal bud inclosed by stipulai sheath, iA. In G, v is pit between li internode. H, section fa ' af, showing the ternate lamina. the upper region shows conspicuous protoxylem elements made up of much elongated tracheids, whose thickenings are between annular and spiral in form, bur very different from the coarsely reticulate and the conspicuously pitted tracheids of the metaxylem developed in the older parts of the stele. In all cases where truly median sections were seen rlie protoxylem elements occupied the innermost region of the whin and no metaxylem was seen within these, so that the bundle (at Hist, at am rate) is endarch, as in the othei ( >phioglossaceae. If is impossible to see in this sec- tion that metaxylem was developed inside of the protoxylem, as Farmer found to be the case in tin- oldei sporophytes. THE YOUNG SPOROI'HYTE 77 In fig. 48, D, there is shown an insertion of a root upon the stele and its very broadly expanded base with the irregular conjunctive tracheitis. This very broad base of insertion and the obvious continuity of the tracheary tissue of the root with that of the central stele of the stem suggest that the ventral part of the stele might be made up, in part at least, of root traces. Cross-sections of older plants, such as that shown in the series indicated in fig. 51, show essentially the same arrangement of the parts as do the younger ones. At the apex there may be seen in section the three youngest leaves, which we will designate respectively i, 2, and j. No. i being the oldest leaf shown. In this section the oldest leaf shows two vascular bundles in the petiole, and this is true also of the second leaf, which is contained within the stipular sheath of the oldest leaf. The lamina of leaf No. i already shows the characteristic termite form. The young leaves make an angle of about 300 with each other. Section B passes directly through the stem apex and shows the sections of the basal part of the sheath of the two older leaves. The stipular sheath of the youngest leaf partially incloses the stem apex, and surrounding the apical region is a loose mass of cells, derived partly from the tissue immediately around the stem apex and partly from the edges of the sheath of the youngest leaves. These cells are really sections of hairs and scales which perhaps secrete mucilaginous matter, but there was no evidence of this in the sec- tions. Turned toward the dorsal side of the section may be seen the leaf trace from the second leaf and separated from it by a considerable space on the ventral side is a rather vaguely defined mass of young procambium, which marks the trace of the youngest leaf. ( )n the ventral side of the latter is the stem apex itself. Further down, as has already been described for the younger stem, the two leaf traces approach. At the level of the stem apex the trace of the second leaf is clearly defined and nearly oval in outline, but more convex upon the dorsal side. The bundle is inclined toward the side of the stem opposite the insertion of the first leaf. Further down the trace becomes much broader, this being mainly due to the development of the tissue upon one side, which begins to bend downward toward the ventral side of the stem. 1 he trace of the youngest leaf is now somewhat better defined ami appears somewhat kidney-shaped in section, the convexity, like that of the second leaf trace, being turned toward the dorsal side. Ibis trace also begins to show the extension of tissue toward the ventral side of the stem and this is developed on the side opposite to the extension in the next older leaf trace. Ultimately these ventral extensions of the two bundles meet as the two leaf traces come nearer together, and the dorsal ends also finally come into contact, so that the two bundles, the section of each of which is approximately semicircular, form a complete ring inclosing a greater or Kss amount of the ground tissue, w hich thus forms the pith of the hollow stele. 1 he stele resulting from the union of the two leaf traces is not at first perfectly circulai in section, but shows plainly for a long time"that it is composed of two separate bundles (fig. 51, /•'). The first development of permanent tissue in tin older leal trace is evident before it joins the younger one. The first dements are thick-walled cells (which may he called protophloem) in the outer /.one of the phloem, ami a little latei at the inner limit of the bundle a small group of protoxylem elements appears. Very soon after this a similar differentiation takes place in the younger leaf trace, so that when the two leaf traces are completely fused the protoxylems have the appearance of having arisen toward the central part of the bundle, but in reality the component bundles, at least at their earlier stage of development, may be described as endarch and asjiee, therefore, with the bundles of the other Ophioglossaceae. In the leaf traces of the older part of the stem, however, there may generally be found inside 78 1111 OPHIOGLOSSALES the protoxylem .1 few scattered, large tracheary elements, so that the bundle may he s.uil to be mesarch, .is Farmer states is the ease In the older rhizome. During its earliei stages the stele shows no leaf gaps where the leaf traces depart, and the leal gaps are only gradually developed. Soon after the stele has assumed the form of a hollow cylinder the leal gaps are for some time absent, or thev are developed onlv in a \ei\ small degree and close almost immediately upon the departure of the leaf trace. In none of the young plants that I examined could I detect any of the inner endodermis which occurs in the older rhizomes. Farmer, however, states that the inner endodermis is only imperfectly developed and concludes that it is the result of the- invagination of the outer endodermis through the leaf gap; or, to put it in another way, it is the persistence of the inner endodermis of the leaf traces of which the hollow stele is made up. The bundle at this stage most nearly resembles that of Botrychtum lunarta, differing from that of B. virgimanum in the absence of a true Fig. 52. A. Section d( tin ^oung central stele of Hrlminthoitachxs, showing tin' two xylems. X200. IS. Stele from lower part of .in oldei sporophyte, showing junction of a leaf trace with central 6tele. X50. C. Part of central stele, more highly magnified. cambium, the outer wood cells being directly in contact with the inner cells of the phloem. Occasionally, however, there may be seen on the outer edge of the xvlem ring a few imperfectly developed tracheids which probably represent a very rudi- mentary development of secondary wood, but there is no other sign of the definite cambium /one which is so conspicuous in the stele of Botrychtum virgimanum. From this study of the development of the leaf traces, following them from the stem apex downward, it appears that the cylindrical stele in H elmtnthostachyi arises in precisely the same way as that of Botrychtum, viz, by the union of the leaf traces. The appearance of procambium upon the ventral side of the stele, which in longitudinal section appears to he derived directly from the Stem apex, can thus be explained by the ventral extension of the broad leaf traces which meet on the lower side of the stem as well as above, and the cylindrical stele is thus developed. THK YOUNG SPOROPHYTE 79 It is not impossible that the root traces may also contribute to some extent to the development of this ventral portion of the stele. The xylem masses belonging to the two leaf traces remain quite distinct for a long time, the one belonging to the older trace being better developed than that from the younger one (fig. 52, A). The metaxylem consists of scattered tracheids arranged regularly in an arc outside the protoxylem of the older bundle, but there are a few scattered smaller ones lying in the tissue separating the two primary xylems, so that, as we have seen, the bundle may perhaps be described as mesarch, and not endarch as it is in Ophioglossum and Botrychtum. The two xylems of the separate bundles are finally connected by intermediate tracheary tissue, so that a nearly complete ring of wood is formed; but the xylem of the older leaf trace is still clearly recognizable on account of its greater thickness. In the section figured (fig. 52, C) there is no tracheary tissue yet formed inside the protoxylem and there is a well-marked pith occupying the center of the stele. The endodermis is now well developed and inside it are about two rows of pericycle cells. I he protophloem is less conspicuous than in the younger stele and is best developed in the outer portion of the older leaf trace. The rest of the phloem is made up of / Fie. 53. A. Transverse section of apex of first root of Helminthostachys, showing a single tetrahedral apical cell. X200. B. Apex of a later tetrarch root, with apparently no single initial cell. thin-walled cells, the larger of which are presumably young sieve tubes. The section is taken from a much older part of the stem and shows that the bundle has attained practically the same condition as in the adult stem, except for the absence of the inner endodermis. The central pith is surrounded by an unbroken ring of wood, averaging about three cells in width. The outer part of the bundle is essentially the same as in the one that has been described. The canal described by Gwynne-Vaughn for the older sporophyte is plainly evident here and extends backward from the sheath cavity, appearing in cross- section as a narrow cleft between the inner side of the leaf stalk and the internode (fig. 49, D, v). It is evidently not a cavity in the cortex, but merely an imagination of the epidermis, which is continuous with that of the inner surface of the stipular sheath. A peculiar feature of the ground tissue in the young stem and petiole in Helmin- thostachys is the presence of cells containing roundish bodies which stain very strongly with Bismarck brown, and these cells closely resemble the tannin cells of the Marattiaceae, with which they are probably homologous (fig. 49, 6"). Lang figures these cells (Lang I, fig. 65), but makes no mention of them in his text. 80 THE OPHIOGLOSSALES rhe trace foi the second leaf, the Inst functional one, is usually provided with .1 single undivided bundle passing through rlu- petiole, hut sometimes this divides into two, as it does in the cotyledon of Botrychium virginianum. Where a single hunille occurs in the petiole it occupies a nearly nu-dian position and is concentric in structure, although the phloem is somewhat less developed upon the- adaxial side. I he xylem consists of a huge mass oftracheids surrounded by the phloem, which is reduced to about two rows of cells upon the adaxial side. Whethei the largest cells in this portion wen- sieve tubes was not determined. \\ lure two bundles are present they are somewhat smaller and are more nearly circular in outline than the single bundle, which they resemble in structure. In the ground tissue are large inter- cellular spaces which disappear at the hase of the leal, when- the ground tissue in cross-section appears almost solid. 1 his development of lacunas in the first foliage leaf recalls the similar ones in the cotyledons of Botrychium and Ophioglossum. In rhe older part of the rhizome there is a slight development of periderm on the dorsal side, which Farmer found to be the case also in the older rhizome. This periderm is restricted to the dorsal region and is obviousl) associated with the leaf bases, as it is in Botrychium and probably also in Ophioglossum. It presumably I i... 54. Three sections <>f an oldei orophytc <>f Helminthostachys. 8. The stipulai sheath i- ab < i I From tin- third youngest leaf. acts as an absciss layer, such as [effrey demonstrated in Botrychium, and this not only causes tin- separation of tin- old leaves hut perhaps also acts as .1 protective layei to the hat scais. I he absence of periderm from tin ventral side is no doubt connected with the strictly dorsal position of the leaves. I he latei loots, like the first one, grow from a single teiiaheilr.il apical cell, very much like that of Ophioglossum and Botrychium. I he root cap is not ver) prominent and is usuall) somewhat pointed. It apparently owes its origin entireh to the activity of the outei segments of the apical cell. Phe priman segments ol the loot cap undergo periclinal divisions, hut, as in Botrychium, the stratifications of the oldei la\eis is much less marked than it is in the ordinary ferns. Each of the lateral segments of the apical cell divides by a somewhat radially placed anti- clinal, so that a transverse section of tin- three youngest segments shows six cells having a nearly radial arrangement. Periclinal walls maj arise and the inner cells of the segments give use to the central cylinder of rhe root, while from the outei ones is developed rhe cortex. As in tin- othei < >phioglossaceae, no root hairs are formed. THE YOUNG SPOROPHYTE 81 1 he primary root, as Lang pointed out, is usually triarcli, but occasionally diarch roots occur, as they usually do in Botrychium and in Ophioglossum pendu- lum. In this case, however, one of the xylems was rather larger than the other. The later roots are tetrarch and in the older sporophyte, as Farmer showed, the roots generally have six xylem masses, or occasionally seven. This type of root is most like that of the Marattiaceae. The endodermis is pretty well developed, but not so conspicuous as it is in Botrychium. In the cortical region of the first root there is a zone of cells in which occurs an endophytic mycorrhiza, such as is common in the roots of other Ophioglossaceae, and probably the same as the endophyte which is found in the tissue of the prothal- lium. Whether the infection of the primary root is direct from the prothallium or whether there is a new infection from the soil was not determined. Lang states that the mycorrhiza is only developed in the first two or three roots, the fourth root and those formed later not having the endophyte. In these later roots the cortical cells are densely filled with starch. The rhizome continues to grow upright for a good while and it was not deter- mined at just what time it assumes the prostrate position which it has in the adult form. At first there is usually one root formed for each leaf, but in the older plants this regularity is lost, and Farmer states that there may be three or four roots developed for a single leaf. On the other hand, the number of roots may be less than that of the leaves, especially in the younger plants. A curious abnormal form was seen in a young sporophyte, where for some reason several of the earliest leaves had remained in the rudimentary condition of the cotyle- don. Five of these rudimentary leaves could be seen formed in succession. The sheaths were fully developed and there was a long internode between each pair of leaves. Three of these had developed roots, but the others had failed to do so. 1 his rhizome was nearly 3 centimeters in length, but it had hardly increased at all in thickness. A single ternate leaf had expanded at the summit, but whether this was the first functional leaf that the plant had developed could not be determined, as the rhizome was broken off below and there may have been one or more func- tional leaves developed below the first of the rudimentary ones. This repetition of vestigial leaves recalls the condition of things in Ophioglos >///// vulgatum and Botrychium lunarta. For a good while the new leaves are oi the same ternate form as that of the first foliage leaf, but sooner or later, probably depending on the vigor of the plant, the ternate form is gradually replaced by five foliate leaves, the later divisions being the result of an unequal dichotomy of the lateral leaf seg- ments, similar to that by which the second lateral segments of tin- primary leaf are separated from the terminal leaflet. In these five foliate leaves the characteristic "pecopterid" venation of the adult sporophyte is fully attained (fig. 4^, />'). Each lateral vein forks twice, the ultimate veinlets extending to tin margin of the leaflet. A section of the petiole of one of these leaves shows that it contains four vascular bundles, arranged in pairs. The dorsal bundle of each pair is decidedly larger than the ventral one. The base of the petiole is almost perfectly cylindrical, but furthei up it becomes winged, so that a groove appears on its inner face extending for some distance below the junction of the lamina and tin petiole. I here is a \ei\ slight!) developed hypodermal tissue composed of two 01 three layers of cells, the walls of which are colorless and considerably thickened, sunn- of them showing thickened corners like the collenchvma found in the leaves of the Marattiaceae. 82 THE OPHIOGLOSSALES COMPARISON OF THE YOUNG SPOROPHYTES 01 THE OPHIOGLOSSACEffi. If, as we believe, the type of sporophyte found in Ophioglossum moluccanum is reallv primitive, we may assume that the sporophyte at first had a single axial stele, collateral in structure and essentially the same in leaf and root. I his primitive sporophyte had no stem at all, but consisted simply of leaf and root. From the primitive vascular skeleton, composed of a single unbranched strand, we can derive the different types characteristic of the older sporophyte in the three genera. As to the first origin of the stem apex, we can only conjecture; whether it originally arose, as it does in Ophioglossum, as an endogenous structure repeating, as it were, the origin of the primary root, we have no means of knowing, but this seems to be the most probable explanation of the origin of the stem apex in the primitive sporo- phyte from which are descended the different types of the Ophioglossacea?. After the establishment of the stem apex the secondary leaves contributed their quota to the developing skeleton of the sporophyte. In Ophioglossum these leaf traces remain largely free and anastomose only to a limited extent, thus giving rise to the open tubular dictvostele with very large meshes. The structure of the individual strands of the dictyostele is essentially the same as that of the free leaf traces. In Helminthostachys the early leaf traces fuse completely and there is formed a solid stele in the younger internodes with a central xylem core, composed of the united xylems of the two leaf traces. These leaf traces are approximately collateral in structure, although it may be that they have a small amount of phloem upon their inner face. After entering the petiole of the young leaf, however, these assume a distinctly concentric form. As the leaves increase in size then traces become broader and in section appear more or less crescentic, so that when the leaf traces come together there is left between them a certain amount of the ground tissue which after they have united appears as a pith King inside of tin- tubular stele. This pith, however, it must be remembered, is not part of the stele proper, but is merely an included portion of the ground tissue. With tin- complete fusion of these two broad leaf traces the tubular form is established and the wood appears in section as a continuous ring. In Botrychium, especially in the large forms like li. virgin- ianum, the tubular condition which is secondary in Uelminthostachys is established at once; this is probably to be explained by the fact that the vascular bundles of the first leaves are much better developed, there being two strands in the cotyledon and in the second leaf, and the leaf traces belonging to these are correspondingly broad and on fusing include at once a certain amount of tin ground tissue, so that the stele appears tubular from the beginning. Botrychium virgimanum undoubtedly represents the most specialized type of flu * )phioglossaceae, and the development of the cambium with a permanent secondary thickening of the wood is an evidence of a higher degree of specialization in the vascular system than is found in any other living 1'tendophyte. While a very slight indication of this secondary thickening has been found in Ophioglossum, and I have also noted some slight traces of it in Helmintkostai hys, there is never developed in these the genuine cambium ring, such as we find in Botrychium virgimanum. In the development of the spiral protoxylem elements Helminthosta> hyi differs from the other ( )phioglossaceae and suggests the true ferns. In the early development of its vascular system there rare strong sugges- tions of some of the Marattiacex, especially Kaulfussia and Dancea. [Tie develop- ment of concentric bundles in the petiole in Uelminthostachys and Botrychium also suggests the Marartiact ,e. \ssuming that the collateral bundle, which is typical of the stem in all of the Ophioglossace.e and occurs also throughout in Ophioglossum, is primary, the con- THE YOUNG SPOROPHYTE 83 centric bundles as they occur in the petiole of Helminthostachys and Botrychium must be considered as secondary. The monarch root of Ophioglossum moluc- canum and the other members of the section Euophioglossum is to be considered as a relic of the primary condition where the single axial stele, as in the young spo- rophyte of 0. moluccanum, had the single strand of practically uniform structure, extending through the leaf and root. The development of diarch roots, such as those of 0. pendulum and Botrychium, is probably also secondary and perhaps associated with the early development of the second leaf in these forms. The diarch root appears again in the young sporophyte of the Marattiaceae and is permanent in most of the leptosporangiate ferns. The roots of the larger species of Botrychium and especially those of Helminthostachys, with their increased number of xylems, are undoubtedly secondary developments, perhaps associated with the large size of the roots; and we again find this same type developing in the Marattiacea?. In regard to this point, Helminthostachys is the most aberrant of the Ophioglossaceae and approaches nearest to the Marattiaceae. The leaf in the smaller species of Ophioglossum is probably a very primitive structure and the closed stipular sheath — which is not exclusively foliar in origin, but at first owes part of its tissue to that of the cortex of the root, from which the sporophyte arises — is probably an older structure than the strictly foliar, stipular sheaths in Botrychium and Helminthostachys. The simpler types of Botrychium, like B. simplex and B. lunaria, show a transitional condition between the closed sheath of Ophioglossum and the open sheath of B. virginianum, which may really be spoken of as composed of two stipules, in this respect recalling the Marattiaceae. Helminthostachys, in the development of the stipular sheath, agrees exactly with the simpler types of Botrychium. In these forms the sheath is hood-shaped, open- ing by a transverse slit in front and below, and the upper portion of the sheath is broken through when the inclosed leaf emerges, so that the two apparent stipules in Helminthostachys are really secondary, caused by a tearing of this hood-shaped sheath, and are not proper stipules as they are in Botrychium virginianum. The simpler and probably more primitive species of Botrychium, like B. simplex and B. lunaria, are obviously intermediate between Ophioglossum and the larger and more specialized species of Botrychium. This is shown in the form and venation of the leaves, as well as in the character of their tissues. Instead of the pinnate venation found in the leaves of B. virginianum, these more primitive species show no midrib in the leaf, but the veins all radiate from the veins of the leaflet, dividing dichotomously, so that the) are arranged in a fan-like fashion. Were the ends of the veins connected there would result a reticulate venation, exactly like that of Ophioglossum. These leaves, moreover, are fleshy in consistence and have Stomata upon both sides, while in Botrychium virginianum and in Helminthostachys tlie stomata are restricted to the lower surface of the leaf. In all of these particulars Ophioglossum and the simpler species of Botrychium are evidently more primitive than the other genera. In Botrychium virginianum, as we have seen, the cotyledon is extraordinarily developed, more so than that of any other Pteridophyte. The contrast between this highly developed cotyledon and the very rudimentary one in Botrychium lunaria, where the young sporophyte passes several years under ground before the first green leaf emerges, is very striking. It is, however, to be assumed that the rudimentary condition of the leaves in Ophioglossum vulgatum and Botrychium lunaria is a secondary condition, connected with their long life under ground. Helminthostachys, on the whole, approaches more nearly to B. lunaria in its early stages of development than it does to B. virginianum. 1 his is shown in the 84 THE OPHIOGLOSS \l.l S rudimentary condition of the cotyledon and in the venation of the first foliage leaf, which, although it has a tern ate lamina, is quite rudimentary. We have- already referred, however, to the rudimentary leaves <>f B. lunaria, which also sometimes develop a very small ternate lamina. The venation of the first leaf in Helminthos- tachys also approaches the cvclopterid type found in the leaves of B. lunaria. The structure of the stele in tin young stem also, after attaining the tubular form, is more like that ot B. lunaria than like that of B. virginianum. On comparing the early stages of development of the Ophioglossacese and M.i i attiaceae we shall find that they have a good many points in common, which will be discussed more at length aftei we have described the structures of the latter. Ophioglossum, as to the formation of the cotyledon, is very much like Kaulfussia, while Helminthostachyi is more suggestive of Dance a. I he stipular structures of the two families are unquestionably homologous and the entire conical stipular sheath, found in the lower members of the ( Ophioglossaceae, is probably an older type than the free stipules found in Botrychium virginianum and the Marattiaceae. In both the Ophioglossaceae and Marattiaceae the young sporophyte at fiist is made up almost entirely of leaf and root, and the whole vasculai system is composed of the leaf traces with no proper cauhne vascular tissues, so that one might almost say that the stem is made up entirely of the bases of the haves, the dominance of the leaf being the most noteworthy feature in the morphology of these plants. On the whole, probably Helminthostachys most nearly resembles the Marattiaceae. This is true both of the character of the venation of the leaves, the structure of the root bundles, and the development ot tannin cells in the young sporophyte. I hese tannin cells seem to be quite absent from the tissues of both Ophioglossum and Botrychium. THE ADULT SPOROPHYTE 85 IV. THE ADULT SPOROPHYTE. The essential characteristics of the sporophyte are established while it is still quite small and the subsequent differences are mainly an increase in the size of the parts and finally the development of the spore-bearing structures which constitute the peculiar spike or sporangiophore so characteristic of these plants. As the structure of the tissues has been repeatedly studied and described, no attempt will be made here to take up a detailed study of these. The Ophioglossacea? in general, except for the rather elaborate vascular bundles of the stem, especially in Botrychium, are marked by great simplicity in the structure of the tissues. The surface is usually smooth except in the younger plants, where there may be a development of scales and hairs, presumably for the protection of the young tissues of the stem apex. The hard hypodermal tissues and bands of sclerenchyma, so common in the leaves and stems of many ferns and developed to a less degree in the Marattiaceae, are practically entirely absent from the Ophio- glossaceae. Except for the vascular system of the stem the bundles are usually less developed than is common in the more highly differentiated ferns, this being especially the case in Ophioglossum, where the very delicate bundles forming the veins of the leaf run through the spongy green tissue of the leaf without causing any projecting veins at the surface. This condition is true also in the simpler types of Botrychium and in the young leaves of Ht'lminthostachys. The great bulk of the ground tissue in leaf, root, and stem is parenchyma. The development of periderm in the outer tissues of the stem is probably always associated with the leaf bases and serves, as we have seen, both to separate the dead leaf bases and to protect the scar thus left after the leaf has fallen away. The roots are characterized by the complete absence of root hairs. The outer- most layer of cells often has the walls much thickened and they may show the reac- tion of cork. The bulk of the cortex of the root, however, is composed of unmodified parenchyma. The vascular cylinder in the root remains monarch in the section Euophioglossum, but is diarch in the smaller roots of Ophioderma and in the smaller species of Botrychium, and ranges to hexarch in the larger roots of Helminthostachys . The roots, especially in the larger species, are thick and fleshy and as a rule branch sparingly and somewhat irregularly. In the section Euophioglossum no lateral roots are formed and branching is rare. When it does take place it is the result of a true dichotomy of the apex. The root, as in the youngest stages in the plant, grows almost always from a single tetrahedral apical cell, which is much alike in all the genera. The most characteristic feature of the Ophioglossacea? is the peculiar sporangia! spike referred to. There is a certain correlation in the degree of development of this spike and the sterile leaf segment with which it is associated. 1 he fertile leaves may arise very early in the history of the sporophyte. Bruchmann states that the first leaf to appear above ground in Botrychium lunaria is already a fertile one and in Ophioglossum vulgatum the second green leaf to be developed usually bears spores. This early development of the fertile leaf is probably an indication of the primitive nature of these plants, as we must assume that the ancestral form must have at once developed a sporangia! structure on the Hist leaf, or what corresponded to that in the embryo. In Botrychium lunaria Bruchmann figures young spore-bearing plants which are still connected with the piothallium, and Jeffrey states that in B.virgini- ,11111111 he once found a fruiting plant with which the piothallium was still connected. 86 THE OPHIOGLOSSM I S THE SPOROPHYTE 01 OPHIOGLOSSUM . ( )f the three existing genera of the Ophioglossaceae, the genus Ophioglossum is much the largest and most widespread. The number of species, however, is very imperfectly understood, as there is great confusion in the nomenclature, owing to the inadequate study of the tropical species, of which the number is undoubtedlv much greater than has generally been supposed. The smaller terrestrial species look very much alike and it is evident that collectors have often failed to discriminate among them. My attention was especially called to this while I was collecting ma- terial of Ophioglossum in Java, where there is evidently a considerable number of species. Raciborski (Raci- borski 1), who has described the Pteridophytes of this region, mentions only a single species as occurring in the neighborhood of Buitenzorg, where most of my collect- ing was done. But in that immediate neighborhood 1 collected at least three very distinct species, and two were collected at Tjibodas, some 4,000 feet above Buit- enzorg. Raciborski, however, mentions only a single species as occurring at Tjibodas. There is no doubt that a critical study of these plants from other regions where they abound will greatly increase the number of species to be recorded. The great majority of the species of Ophioglossum belong to the subgenus Euophioglossum, which should probably be separated completely from the very differ- ent forms which comprise the other subgenera. The species of Euophioglossum are all small or moderate- sized plants with undivided leaves which are generally lanceolate or broadly oval in outline and have the sporangial spike borne on a very long stalk attached to the sterile part of the leaf near the base of the lamina. These plants are always terrestrial, the leaves growing from an upright rhizome, which is sometimes a good deal enlarged, sometimes more slender in form. Prom the rhizome there extend numerous roots which are sometimes developed one for each leaf, but this is by no means always the case. The sporophyte of Euophio- glossum seldom exceeds a height of 30 to 40 centime- ters and some of them are verv much smaller (fig. 55, also plate 3). Except for their larger size the later leaves do not differ essentially from the primary leaf of the young sporophyte. I In venation is always reticulate, usually without any definite midrib, although sometimes there is a central vein which is slightly stronger than the others. In many species, e. g., 0. moluccanum, 0. vulga- tum, small branches ending blindly are found within the large meshes. In other species, e. g., O. lusitanicum, there are no free veins within the areoles. In many of the species but one leaf is formed each year, but in others, especially main' of the tropical species, there may be as many as four or five formed in a 1 . .. 55. Two specimens of Ophioglossum moluc- canum (Schlrcht), slightly reduced. The larger one is the typical 0. moluccanum ; the smaller one is probably a second speci-' THE ADULT SPOROPHYTE 87 single season. In O. vulgatum the development of the leaf is very slow, the leaf remaining three years inclosed in the bud and emerging in the fourth season. So far as I know, no study has been made of the development of the leaves in those species of the temperate climates where more than one leaf is developed in the season, and it remains to be seen whether the leaves which unfold in the same season are of the same age or not. In the rapidly growing tropical species it is exceedingly unlikely that more than a few months are necessary for the complete development of the leaf, which unfolds as soon as it is mature, but no data are available on this subject. While the spike in Euophioglossum is usually inserted at the base of the sterile lamina it may be attached much further down. This is especially noticeable in the small species O. bergianum, from South America (see Bower 9, page 435). In 0. bergianum the leaves do not show a clear separation into lamina and petiole and the spike is inserted close to the base of the linear leaf, so that it appears to be an en- tirely distinct structure. In nearly all of the species of Euophioglossum the later roots, like the primary ones, are monarch, but in 0. bergianum they are diarch. In most cases at least, as Holle (Holle 1) showed, only one root is formed for each leaf. This seems to be the case in O. moluccanum, which was investigated with some care. Ophioglossurn pendulum (plate 4, B) is the best-known representative of Ophio- derma, the second section of the genus. This differs a good deal in its general char- acters from the small terrestrial species of Euophioglossum. It is not uncommon throughout the eastern tropics, where it grows as an epiphyte upon the trunks and branches of various trees. The rough stems of certain tree ferns and some palm- like species of Phoenix form a favorite habitat for this fern in certain regions and, as we have pointed out in the study of the gametophyte, it is frequently found rooting in the masses of humus between the old leaf bases of some epiphytic ferns like Asplcnium nidus. The stem, instead of being upright, is markedly dorsiventral, but is short, with the leaves crowded together near the end and all growing upon the dorsal side of the rhizome, as they do in Helminthostachys (fig. 63). The leaves are very much elongated and the ribbon-like lamina merges very gradually into the extremely stout petiole. These leaves in large specimens may reach a length of 1.5 meters, or even more, and the long, strap-shaped leaves hanging down from the boughs of the trees present a very characteristic appearance. In larger specimens it is not uncommon to find the lamina dividing dichotomously (see plate 4, B) and it is said that sometimes this dichotomy may extend to the petiole, in which case each segment of the leaf bears a separate spike. The venation of the leaf is reticulate, the veins inclosing long, narrow meshes with no free veins, and the venation thus resembles the type represented in Euophio- glossum by Ophioglossum lusitanicum. The larger plants always have several leaves which are expanded at the same time and growth goes on uninterruptedly, The leaves are crowded together and there is no evident internode between them, but it is clearly seen that they are arranged in two rows on the flanks of the short rhizome (fig. 63, A). The remains of the stipular sheaths surround the bases of the leaves and these sometimes present the appearance of two small stipule-like organs. As in Euophioglossum, adventitious buds are frequently formed upon the roots. As a rule they are not terminal, but lateral structures and probably do not differ essentially in their development from those oi 0. moluccanum. The rhizome is more or less buried in the humus and the roots ramify through this. Even in the very young plant the roots are branched and this branching becomes very -marked in the larger sporophyte. There seems to be some difference 88 THE OPHIOGLOSSAL1 S in the number oi routs, hut in must cases two were found connected with each leaf, instead of one as I stated in my earlier description of the plant (Campbell 4). The roots are very stout, sometimes attaining a diameter oi over 3 millimeters. The smaller routs are diarch, but in the large roots there may be three, tour, or even five xylems. Ophioglossum pendulum is much the largest member of the Ophioglossacea and the sporangial spike as well as the individual sporangia far exceeds in size those of any other species (plate 4, B, 3-6). The spike is attached by a short peduncle and hangs down from the pendent lamina. In large specimens the- spike may reach a leiis'th of 20 centimeters 01 more with a breadth of about a centimeter. Above the insertion of the spike the leaf is thin, but the slender peduncle is ion tinned downward into a thick, flattened midrib which merges gradually into the petiole, so that the spike has very much the appearance of being a terminal structure with the sterile lamina adherent to it. The young leaves in (). pendulum emerge while they are still very young and the sporangial spike is in an extremely rudimentary condition I he general devel- opment of the leaf is therefore verj <. .is\- to follow in this species, as these young leaves are entirely free. The second species of the section Ophioderma is the rare (). intermedium (fig. 69; plate 4, A). 1 his for a long time was known only from one locality in Bunco, but has lately been collected at other points in the Malayan region. The specimens figured were collected by the writer near Buitenzorg in |ava. In the account of the Ophioglossacea; given in Engler and 1'rantl's "Naturliche Pflanzen- familien" (Bitter 2) it is suggested that (). intermedium is only a terrestrial form of 0. pendulum; but there is no doubt at all that it is a very distinct species. The plants described here were growing in masses of humus at the base of old clumps of bamboo. Ilu stem is usually very short, forming a small tuberous body, from which, in most cases, only a single leaf was growing, although two were sometimes found. A careful examination of this short rhizome showed that, like Ophioglossum pendu- lum, it is dorsiventral. The roots were short and in all cases observed were without branches. In one of the specimens a bud very similar indeed to the corresponding buds in 0. pendulum was found growing from one of the roots. Ophioglossum intermedium differs from 0. pendulum in being rigidly upright. I he peduncle is longer and the lamina of the leaf much smaller and more clearly differentiated from the petiole. As in 0. pendulum, however, the petiole is prolonged into the peduncle of the spike with the same midrib-like thickening, caused by the coherence of the basal part of the peduncle with the lamina. This plant is exceedingly variable. In the larger specimens, except for the much shorter lamina, the plant a good deal resembles small specimens of (). pendulum. In others, however, the lamina is almost completely suppressed and this condition closely approaches the third member of this section, Ophioglossum simplex (fig. 71 ). This latter species is at present known only from one locality in Sumatra, and is dis- tinguished from all the other species of Ophioglossum by the practically complete suppression of the sterile portion of the leaf (see BowerS). The form of the rhizome and the habit of" the plant in 0. simplex most nearly resemble 0. intermedium, to which it is probably not very distantly related. Whether or not we regard the absence of the lamina of the leaf as a case of reduction, the fertile leaf in 0. simplex certainly very closely resembles what one would assume to have been the primitive condition of the leaf in the ancestors of the Ophioglossacea'. I he third section, Cheiroglossa, contains a single very peculiar species, 0. palmatum I plate 5). This occurs throughout tropical America, but does not seem t.. be .1 common plant. It has been reported from Florida, where, however, it is THE ADULT SPOROPHYTE 89 very rare. It has been collected in various parts of South America and in the West Indies, and apparently the same species is known also from the Island of Bourbon and from the Seychelles in the Indian Ocean. The specimens from which the figures and descriptions given here were made were collected in the summer of 1908 in the Blue Mountains of Jamaica, and were found in one place only, growing upon a rotten tree stump. The rhizome in Ophioglossum palm at um is an almost globular tuber, which is radially constructed and in this respect resembles that of Euophioglossum more than that of Ophioderma. The leaf bases are covered with long brown hairs, which give a characteristic appearance to the rhizome. The roots are numerous and more slender than those of Ophioglossum pendulum. They are sparingly branched and the branching may have the appearance of a true dichotomy, but the material available did not allow ot a critical investigation of this point. However, since in Euophioglossum the branching of the roots is actually dichotomous, it is not impossible that the same may be true in Cheiroglossa. The smaller roots, as in 0. pendulum, are diarch and, as in all of the other forms, a niycorrhiza is developed in the cortical region. In sections of one of the largest roots available the bundle was triarch. This root was rather young and the tracheary tissue was only partially lignified. The mycorrhi/.a was also less evident than in the smaller roots taken from a younger plant. Two young plants were found which probably had arisen from buds upon the roots of the older sporophyte. One of these is shown in figure 70, A . It had developed a single nearly lanceolate leaf very much like the early leaves of O. pendulum. A single root had developed near the base of the leaf and this was joined to the small globular rhizome. A second root could be seen above this, but whether this second root belonged to the leaf or was part of the root upon which the bud had arisen could not be determined. The older leaves may reach a length of 60 centimeters or more (plate 5). They are more or less deeply palmately cleft into narrow segments, which give the leaf a curious superficial resemblance to certain kelps, such as Laminarta digitata. 1 he base of the fan-shaped lamina is abruptly narrowed into a nearly cylindrical petiole, about equal in length to the lamina itself. The venation is rather of the type of O. vulgatum, having numbers of free veins in addition to the elongated closed meshes. In some of the smaller leaves (plate 5, 3) there is present a single small median spike, which, like that of 0. pendulum, has a short peduncle, but it is inserted below the lamina, in this respect again being more like Euophioglossum. In the larger leaves there are several sporangial spikes arranged in a series on either side of the petiole, usually below the insertion of the lamina, but sometimes some of them are inserted above the junction of the lamina and the petiole. Bower (Bower 9, page 436) has figured a series of specimens of this species showing the great variation in the number and arrangement of the spikes. He emphasizes the fact that although these spikes are apparently marginal, in reality they always arise from the adaxial side of the leaf. He shows that in this species also there may be a branching of the individual spikes similar to that which occurs in Ophioglossum pendulum and less frequently in certain species of Euophioglossum. THE ANATOMY OF EUOPHIOGLOSSUM The stem apex of the adult plant has been repeatedly studied in the widespread Ophioglossum vulgatum, but there is little information in regard to this point in other species. From a somewhat careful examination of 0. moluccanum it is evident that this species agrees closely with 0. vulgatum, and a study by one of my students, 90 THK OI'IUOCLOSSALKS Mr. C. S. Morris, <>t this species and also ot a broad-leaved form from Ceylon, probably O. reticulatum, showed that the same characters prevail in the species he examined as in O. vulgatum. It therefore seems likely that all species of Euophio- glossum agree in the main in the structure of the stem apex and the development of their tissues. We have seen that in the young sporophyte the stem apex is completely inclosed in a small cavity formed by the hood-like stipular sheath of the youngest leaf. This condition, once established, is permanent (fig. 56) and there is no essential difference in the appearance of the stem apex between a young plant in which two or three Fie. 56. Longitudinal sections of an older sporophyte of Ophioglossum moluccanum, showing arrangement of vascular bundles; .v, stem apex; ip, young sporophvlls. D shows apical region more enlarged. leaves have been developed and the full-grown sporophyte. The stem apex is of limited extent, crowded in between the bases of the young leaves, and in O. moluc- 1 ,niiim the single apical cell is of the same form as that which was found in the early condition of the young bud; i. e., in longitudinal section it is four-sided, with a broad base and a narrower outer face. According to Holle (Holle I), the apical cell in O. vulgatum is pointed below; but Rostowzew ( Rostowzew 2) in his figures shows the same form as the apical cell in O. moluccanum. It is not at all impossible, however, that both forms may occur, as in cross-sections the apical cell in O. mol- uccanum may be either three-sided or four-sided (figs. 39 and 56, /)). The apical THE ADULT SPOROPHYTE 91 cell in O. moluccanum may therefore be described as a three-sided or four-sided prism or a truncate pyramid. As in the young plant, the stipular sheath of each new leaf is formed mainly from the basal tissue of the leaf itself, but includes also tissue from the margin of the stem apex. The sheath is open above by a narrow pore through which it communicates with the space between it and the next stipular sheath. In O. moluccanum, as in O. vulgatum, three leaves of different ages can generally be seen inclosed in the bud and sometimes the first indication of the fourth young leaf can B Fig. 57. Six of a series of cross-sections from a sporophvte of Ophioglossum molurxanum. sp, young sporophyll; x, stem apex. also be distinguished (fig. 56, C,D). A cross-section of the youngest sheath shows the pore as a minute opening, separated from the stem apex below by a very narrow space. A careful study of the older stem gives no reason for assuming that the tissues of the very open reticulum forming the fibrovascular system of the rhizome is in any part due to additions from the apical tissue of the stem. Whether longitudinal or transverse sections are examined it is perfectly clear that the strands of this reticulum are made up entirely of leaf traces which can be readily followed into the young leaves or the tissue immediately below. The immediate apical region of the stem, as in the younger sporophyte, is unmodified parenchyma which adds only to the large central pith, if pith this can be called, as the separate bundles of which the vascular reticulum is composed run free for long distances through the ground tissue 92 THE OPHIOCLOSSA1.IS of the stem, which is identical in appearance in the cortical and the central regions. I'he bundle from each young leaf can be traced to a junction with a root stele and from this point of junction it extends through the ground tissue of the stem, running almost horizontally until it joins the trace from the next older leaf. In this way is built up the open, large-meshed vascular cylinder. So far as could he determined in 0. moluccanum only one root was formed tor each leaf. The tissues of the root base are continued upward to connect with the young leaf and downward to join the stele from an older one. No endoderrnis can be detected in the bundles of the stem in 0. vulgatum and the same is true tor (). moluccanum, hut in 0. bergianum, 0. capense, and 0. ellip- tic am, according to Poirault (Poirault 2), both an inner and outer endoderrnis occur in the older part ot tin rhizome; these however, disappear in the younger region higher up. A transverse section ot the mature rhi- zome (fig. ^7) in Ophioglossum moluccanum shows the widely separated sections of the strands of the vascular cylinder as a circle of small collateral bundles without any endoderrnis, the mass of wood being in immediate contact with the thin-walled parenchyma of the ground tissue or sepa- rated from it at most by a single row of pericycle cells. In some of the smaller species the leaf traces are relatively broader and there is an approximation to the ring- shaped section presented by the cylindrical stele of Botrychium or Helminthostachys, and sometimes the same appearance may be approached in sections ot the older rhizome which happen to pass through a region where there are numerous anastomoses of the bundles foi mini; the reticulum (tig. 57, E). I xcept for the vascular bundles, the tissue of the stem is made up almost ex- clusively of simple parenchyma. The de- velopment <>f periderm, which takes place to a limited extent in the outer region, is doubtless associated with the old leaf bases, as it is in Botrychium. I he leat structure ot knof'luoglossum is exceedingly simple. The mesophyll is made up of thin-walled green cells, practically uni- form throughout; and through this spongy mass of mesophyll the delicate veins pass without forming any projections at the surface. Both sides of the leaf are provided with a simple epidermis, stomata being developed on both sides of the leaf. In those species in which the leaf lies mote or less horizontally, as it does, for example, m 0. reticulatumt the stomata are less numerous upon the upper surface. The arrangement of the bundles in the petiole has been already studied in the commoner European species, 0. vulgatum and 0. lusitanuuin. In all ot the species belonging to the section Euophioglossum there is given off from the vascular system ot the rhizome a single leaf trace, which divides at the base of the leaf into two Fig. 58. Longitudinal section of a sporophyte of Ophioglossum moluccanum, older than the one shown in tig. 56. X20. THE ADULT SPOROPHYTE 93 strands. Each of these strands may divide, or only one of them (fig. 60). In some of the smaller species there are only three bundles in the petiole, and in these forms there are no anastomoses of the bundles above the base. In the larger species these branches divide further and the number of bundles in the section is greater. Thus, each of the two ventral bundles may divide again in the upper region of the petiole so that a cross-section at this point would show five bundles, a large dorsal one and two pairs of smaller ventral ones. A similar condition of tilings is found in the Fig. 59. A. Vascular bundle from an adult rhizome of Ophiogloisum moluccanum. X 1 50. B. Section of an adult root showing monarch vascular cylinder. X}C. The shaded zone is occupied by the mvcorrhiza. small species, O. californicum. As sections are examined below tin1 junction of the lamina and the petiole, it can be seen that further forking of the bundles has taken place preliminary to their entering the lamina itself. Some of the species have the tissues of the petiole quite compact, others show a greater or less development of lacunae or air-spaces in the petiole. THE ROOT IN EUOPHIOGLOSSUM. The early roots in 0. moluccanum grow from a large tetrahedral apical cell showing a fairly regular segmentation, much like that of the typical ferns; but the root cap is not so well developed nor does it show as definite a stratification. In the c Fig. 60. Nine of a series of sections of a young sporophvll of 0 phioglo 1 moluccanum. sp, the sporangiophorc. early roots the whole of the root cap seems to be derived from the outer segments of the apical cell. In the later and larger roots there seems to be somewhat less regularity in the segmentation and no sections were found which showed the apical 94 THK OPHIOGLOSSALES cell and its segmentation as clearly as in the younger roots; however, only a small number of these later roots were sectioned and it may be that they were not all quite normal. A section of an adult root is shown in fig. 5c;, 11. The cortical region is made up of simple parenchyma and the mycorrhizal zone is very conspicuous. 1 he endo- dermis is not very clearly defined, less so than in the earlier roots, and its limits are in.. 61. Five sections of a full-growD sporophyl] of 0 phioglouum woluccanutt K, m-iNhum! pr,luiuic of spike; /. lacun.i' not easily determined. The cortical cells inside the mycorrhizal zone an- densely filled with starch. More than half of the section of the bundle is occupied by the huge solid mass of wood. This is separated from the endodermis by a single layer of pericyele cells. The single mass of phloem King next the wood is not much more than half as large. \\ \K)\1V OF OPHIODERMA. Owing to the dorsiventral position of the rhizome and to the more rapid de- velopment of the leaves, the section of the stem apex in Ophioglossum pendulum differs a good deal from a similar section of Euo phi 0 gloss um (fig. 64). The base of' the leaf is bent sharply upward, this being the case also in the very young leaf which is inclosed within the bud. The leaves break throujdi the inclosing sheath in a vcrv rudimentary condition, although of such size that a section through the base of the youngest visible leaf shows usually but a single younger leaf inside the sheath, instead of the three 01 four that are seen in a similar section of tin bud in Euophioglossum. Within the sheath at the base of the youngest visible leaf there is found a cavity in which is the growing point of the stem and the youngest leaf. The apex of the stem forms a strongly inclined, nearly plane surface and, as in Euophioglossum, the growth is from a single apical cell which, so far as could be deter- mined, has the form of a three-sided pyramid, whos< lateral faces are more or less strongly convex and whose apex may be truncate. From the few cases observed it is not possible to say definitely whether basal seg- ments are regularly cut off as well as the lateral ones. The segments cur oft from the lateral faces are large and the divisions irregular. Owing to the large size of the sporophyte there are practical difficulties con- nected with the study of the vascular system of the rhizome from a series of micro- tome sections. The numerous bundles in the leaf base do not unite into a single lie. 62. A|'c\ i>f root of Ophioefossum moiuc- (attum, showing dichotomy. i THE ADULT SPOROPHYTE 95 leaf trace, but pass downward through the cortex separately, and even if it were possible to make a satisfactory series of the sections of the large rhizome the task of following the course of the very numerous individual bundles would be a difficult one. From free-hand sections of the large base of the leaf, however, one can see without difficulty the general plan of the vascular skeleton. The stout petiole is slightly contracted at its base, but a section presents very much the same appear- ance as one made higher up. In specimens that have been preserved in alcohol so as to become decolored, the tissues of the thick leaf base are sufficiently transparent to show quite clearly the course of the bundles even without sectioning, by simply slitting the petiole through the middle. The bundles are seen to anastomose freely at the extreme base of the leaf, very much as they do in the lamina. If such a section is examined where the leaf base joins the rhizome (fig. 63, D) the circle of bundles can be seen to become Fig. 63. — Ophioglcssum pendulum L. A. Stem showing dorsiventral form and branched roots. /, bases of leaves. B. Buds attached to a root fragment. The older bud, 61, has developed a young sporophyll, ., and two secondary roots. C. Base of a large leaf, showing arrangement of vascular bundles. D. Section of same. smaller as the bundles enter the cortex, but the bundles remain lor the most part quite separate, although there may be an occasional anastomosing of these, as occurs in the petiole itself. This group of bundles composing the leaf trace — if such it can be called — forms a circle about the opening in the vascular cylinder of the stem where it joins the latter. A section of the vascular cylinder within the rhizome itself presents the appearance of a nearly complete ring, which is probably formed by fus- ing of the bundles derived from tbe leaf bases. The much greater development of woody tissue in the stem of O. pendulum as compared with that of Euophtoglossum is doubtless due to the very much larger size of the leaves and the correspondingly greater number of vascular bundles contributed by them to the woody cylinder of the rhizome. The very young leaf, such as is shown in fig. 63, B, has a thick, fleshy leaf base, terminating in a very small and pointed lamina strongly bent over as it is, e. g., in the young leaf of Botrychium virginianum. This differs from the species of Euophioglossum, in which the leaf is straight in the bud. Under the arching hood formed by this folded-over lamina is the young spike, almost equal in length at this time to the lamina itself. A somewhat older stage is shown in fig. 82, B. The leaf has now become somewhat flattened, but as yet there is no sharp distinction between the lamina and the petiole. In this early stage of development the inter- pretation of the spike as a terminal structure seems very plausible. An interesting 96 THE OI'HIOGLOSSALKS case is shown in rig. 82, E, where the lamina is almost entirely suppressed and the terminal character of the spike is very evident. As the leaf develops, there is a very great increase in size in the lamina, which, as we have seen, may reach a great length. The anatomical structure of the leaf Fig. oa. — Ophioglossum pendulum* A. Section of stem apex, X3. 1). The stem apex, more enlarged. B. Young sporophvll. sp, sporangiophore. X35. K. Very young sporophyll. sp, sporangiophore. 9 C. Stem apex, .v, arul youngest leaf, /. F. Cross-section of young sporangiophore. closely resembles that of Euophioglossum, hut the stomata are much largci and around the stoma is a concentrically arranged series of cells, indicating that probably the formation of the mother cell of the stoma is preceded by a series of preliminary I 1 ' -Vasculai bundle from petiole ol '' sum pendulum* I ,...66. \. Section ol !• nole of a sporophyll of 0. pendulum. It. Section ncai ba 1 ol lamina. Sybase of spike. C-E. Section) ol peduncle of spike. C ami 1) are from the same leaf; K from a laigcr one . divisions in the epidermal cell. The tissues in the petiole are very like those in the lamina of the leaf except that they are somewhat more compact. The walls of the ground tissue in this region are very deeply pitted. The structure of the vascular bundle of the petiole (rig 65), is much like that in Euophioglossum, except that there is a rather greater development of tissue upon the THK ADULT SI'OROPHYTE 97 inner side of the wood, so that there is a slight suggestion, at least, of a concentric structure of the bundle, such as is found in the petioles of Botrychium. The xylem is composed entirely of tracheids with reticulate markings. The rest n[ the bundle is made up of sieve tubes, mingled irregularly with smaller, thin-walled elements. It could not be certainly deter- mined whether any sieve tubes were developed on the inner side of the wood. The number of bundles in the petiole, especially in large specimens, is much greater than in any of the species of Euophioglossum. In a spe- cimen of medium size (fig. 66) eighteen bundles could be seen, of which probably seven or eight are destined to sup- ply the spike. In the fertile leaves these form a complete circle, but in sterile leaves there are no bundles on the adaxial side of the section. In the free portion of the peduncle, which is relatively slender, the number of bundles is much less than in the broad basal portion. In the smaller specimen figured there were but three bundles in the middle part of the peduncle. The complete absence of the adaxial strands in the petiole of the sterile leaf, even at its base, is a strong confirmation of the view suggested by both the older leaf and the younger stages, that the peduncle is really an independent structure whose bundles are joined directly to the rhizome. a B Fig. 67. A, longitudinal; B, transverse section of the root apex of 0 phioglossum pendulu -v ■-J-.-;.-'--.... -■■■■■:'■ :' . Fig. 68. A. Diarcli root-bundle of Ophioglossum pendulum. B. Tetrarch root of the same. Xzo. Dotted area is mvcorrhizal zone. 0. intermedium (fig. 69) closely resembles 0. pendulum in its anatomy, like the latter developing stomata upon both sides of the leaf, and these stomata are very large and show accessory cells similar to those found about the stomata of 0. pendulum. Except for the difference in size, the arrangement of the vascular bundles in the peduncle and their structure is closely similar to that in 0. pendulum. 98 THE OPHIOGLOSSALES The anatomy of 0. simplex, so far as it has been investigated (see Bower 8) corresponds closely with that of the other species of the section OphioAerma. The roots of 0. pendulum also show the tetrahedral apical cell (tig. 67), but the divisions arc much less regular than in 0. moluccanum and probably the other species ot tin- kuopluoglossum. 1 he segments divide slowly and increase a good ileal in size before tin- first divi- sions take place, so that the youngest segments may equal the apical cell in size, and there is thus a certain resem- blance to the apical meristem in the root of the Marattiaceae. It is prob- able that, as in the Marattiaceae, the lateral segments of the apical cell also contribute to the root cap, which is rather better developed than it is in 0. moluccanum. There is a good deal of difference in the structure of the root bundle (fig. 68). Ibis is diarch in the primary root, as we have seen, and also in the smaller ones of older sporophytcs; but in the larger roots, which may reach a diameter of over 3 millimeters, it is 1 ■'" ■• '")■ tiiarch; tetrarch bundles are also corn- Five specimens of o. intermedium Hk., slightly reduced. mon. and in one very large root which C and D have a very much reduced lamina. , ■ i i i ' 1 1 1 1 examined the bundle was pentarch, but one of the xvlem rays was shorter than the others, the xylem forming a solid central mass having the form of an irregular five-pointed star. Fig. 70. \. Small plant of Ophioglossum palmalum L., growing as a bud upon a root fragment B. Hairs from base of leaf. X35. C. Section of petiole of a large leaf. X7.5. THE ADULT SPOROPHYTE 99 ANATOMY OF CHEIROGLOSSA. Material was lacking for a study of the anatomy of the rhizome in 0. palmatum (tig. 70), but the structure of the petiole is very much like that of O. pendulum and probably the same relation of the leaf bundles to the bundles of the rhizome obtain as in O. pendulum. The difference in the venation has already been pointed out and the leaves differ also from those of O. pendulum in the fact that stomata are developed only upon the lower side. A feature of this species is the presence at the base of the leaves of very conspicuous multicellular branching hairs, quite different from those found in any other species of Ophioglossum (fig. 70, C). I find that the cells are much more elongated in my specimens than those figured in the account of the Ophioglossaceae given in the Naturliche Pflanzenfamilien.* THE SPOROPHYTE OF BOTRYCHIUM. There is much the same uncertainty as to the number of species of Botrychium that there is in Ophioglossum. Some of the species are exceedingly variable and their limits are difficult to determine. Bitter, in his account of the Ophioglossaceae in I'.ngler and Prantl's "Naturliche Pflanzenfamilien," states that there are sixteen species. Underwood (Underwood 1) says "about thirty," of which fifteen occur in the United States. Christensen in his Index Filicum recognizes thirty-four species. Many of the species are very widespread, being most abundant in the North Temperate Zone. According to Christ (Christ I ), B. lunaria is not only widespread throughout the colder parts of the Northern Hemisphere, but occurs again in the corresponding regions of the Southern Hemisphere, having been reported from Pat- agonia, southern Australia, Tasmania, and New Zealand. A few species occur in the mountain regions of the tropics; thus B. htnuginosum is not uncommon in the highlands of Ceylon, where I collected it at an altitude of about 7,000 feet. The simplest member of the genus is Botrychium simplex, a very variable species occurring at various places in North America, northern Asia, and Europe. Its smaller forms closely resemble a small Ophioglossum (fig. 72, A, B), except that the sporangia are more distinct. From this primitive type, obviously not very remote from Ophioglossum, there is an interesting series of forms leading up to the large species like B. virginianum and B. silaifolium. This latter species, which grows in the mountains of western North America, is perhaps the largest of the genus (plate 7). The plant is sometimes 1 feet high, the leaf nearly a foot in width, and the very large panicle of sporangia 6 inches or more in length. In the evolution of the leaf in Botrychium, the sporangiophore shows a devel- opment parallel with that of the sterile leaf. In the forms of li. simplex, having an entirely undivided sterile lamina, the sporangiophore has the- form of a simple spike with a single row of large sessile sporangia on either side ( fig. ~ji, //) and vei \ closely resembles a small Ophioglossum. As the sterile leaf segment becomes more and more dissected, there is a corresponding branching of the sporangiophore which, * Professor Bower, at the List meeting of the British Association lot the Advancement of Science, read .t paper on tin Vnatom) of Ophioglossum palmatum. This has nol ye\ been published, Inn Professor Bower has kindly furnished me with an abstract ol his paper from which the following extract is quote,!. "It was thought probable that (>. palmatum (the only species of section Cheiroglossa) would share with the species named (i.e., the species of Ophiaderma) the character of a divided (leaf) trace, and material collected in Jamaica has shown that it does. The axis is much distended by parcnchvni.it , sporangiophore. C. Apex of sporangiophore. X1Z5. I). Transverse section 01 a verv young sporophyll, showing dichotomy ol apex. ■ IJJ, sporophyll, and the two branches (viz, the sterile segment and the sporangiophore) are structures of equal rank. It would thus appear that the old view put forward by Mettenius of two equal branches of the fertile leaf is probably the correct one. It is probable that younger stages than those figured by Bower for 0. vulgatum and 0. reticulatum would show the same condition of affairs that we pointed out in THE ADULT SPOROPHYTE 111 0. moluccanum and 0. pendulum, and the conclusion that the young sporangiophore is an outgrowth of the sterile part of the leaf is the result of the subsequent rapid growth of the upper sterile portion of the leaf, which thus carries its apex far beyond the apex of the young spike. The young sporophyll in (). pendulum (fig. 64, E) resembles that of 0. moluc- canum, except that all the parts are much larger. The apex of the young sporangio- phore appears upon the adaxial side of the very broadly conical young sporophyll. Bruchmann's studies on Botrychium lunaria indicate that in this species the separation of the fertile and sterile segments of the sporophyll is the result of a dichotomy of the apex o{ the sporophyll at a very early stage in its development. Fig. Si. A. Young sporophyll of Ophioglossum pendulum, sft, the sporangiophore. X2, B. An older sporophyll. Xi. C. A still oliler stage, showing venation oi lamina. I). Base of a full-grown sporangiophore. E. Sporophyll with rudimentary lamina, /. I he sporophyll, however, is larger at this time than is the case in Ophioglossum moluccanum (Bruchmann 2, fig. 57). The earliest stages in the development of the sporangiophore have nut been studied in Helmtnthostachys. Bower says (Bower i), page 4^5): "The origin of it is similiar (i. e., to Ophioglossum) and its early stages nut unlike. It appears at first as an outgrowth on tin- adaxial side of tin- sterile frond, and it is curved over while young so that the actual apex is pointed downward." In the latter respect it might verv wt being merely an adaxial appendage <>t the sporophyll. rhese changes in tin- relative importance of the points can be very well shown by a study of the very large sporophylls of O. pendulum. In this species the young leaf emerges from the sheath while tt is still in a very earlv stain ol development. These young leaves (fig. Si) have .1 very thick, Heshy leaf base, ending in the small pointed lamina, bent over and almost hiding the young sporangiophore which is attached to its inner surface. I his curved-over form of the young sporophyll differs from that of Euophioglossum} where the young leaf is straight, and in this respect then' is a suggestion of Botrychium or of the true ferns. The young spike at this stage is almost equal in length to the sterile lamina. If the very young sporophyll of Ophioglossum moluccanum is examined, it will be seen that the vascular bundle leaving it passes downward and joins a young root trace. Very soon after the bundle enters the young leaf it divides into two branches, one of which passes into the base of the spike and the other and larger oneinto the sterile section of the sporophyll. This latter bundle now divides again into two, one of which passes to the dorsal side of the sterile segment while the other is turned toward the ventral side of the leaf, which probably represents tissue belonging to the sporangiophore. In the cross-sections of older leaves the petiole, which includes the completely fused base or peduncle of the sporangiophore, shows three distinct bundles, a large abaxial one and two smaller adaxial bundles. I he former remains undivided for some distance above the base of the insertion of the peduncle of the sporangiophore. The two adaxial bundles divide into several branches, this begin- ning just below the point where the lamina of the sterile leaf becomes differentiated from the petiole. The lamina has its edges infolded so that it has a conical form, open in front and inclosing the young spike ( rigs. 56, 57). 1 he successive branching of the two adaxial bundles follows in quick succession, and the outer bundles con- tribute to the veins of the lamina; the inner ones pass upward into the peduncle of the spike. In a previous paper (Campbell 7) the conclusion was reached from the study of a series of sections at different heights, taken from the full-grown sporophyll, that the two adaxial bundles belonged exclusively to the spike. A study of the devel- opment of the young sporophyll shows that this statement is not entirely correct and that a considerable part of the vascular system of the sterile lamina owes its origin also to branches from the two adaxial bundles before the latter enter the peduncle of the sporangiophore. Of the two adaxial bundles, one represents the original bundle of the sporangiophore derived from the primary forking of the leaf trace; the other is derived secondarily from a subsequent division of the second branch of the leaf trace, this also giving rise to the large abaxial bundle of the petiole. THE DEVELOPMENT ()!•' THE St'ORANGIl M . Much the most complete account of the development of the sporangium of the ( >phioglossaceae is that of" Bower ( Bower 5), who has made a very exhaustive exam- ination of the development of the sporangium in all three genera. My own studies have been for the most part confined to Ophioglossum pendulum, 0. moluccanum, and Botrychium virginianum. Burlingame (Burlingame 1) has more recently examined the development of the sporangium in a species of Ophioglossum from the Philippines, which was assumed to In- 0. reticulatum. Beers ( Beers 1) has also published recently a somewhat incomplete account of the sporangium in Hel- THE ADULT Sl'UKOI'H\ I 1 113 mmthostachys. Th rough the kindness of Prof. L. I.. Burlingame I have also had an opportunity to examine some of his preparations of Ophioglossum reticulatum and Helminthostach ys. An examination of both longitudinal and cross sections of the young spike in (). moluccanum (and this is true also ot 0. pendulum) shows that they agree entirely with Bower's account of the development in the species studied by him. There Fig. 83. A. 'transverse section of a very young sporangiophore of Ophioglossum moluccanum. X150. B. Section of an older sporangiophore. X75. The shaded areas mark the " sporangiogenic " bands. C. Part of B, more highly magnified. The nucleated cells mark the sporangial region. seems to be no question that, as Bower first pointed out, there is formed a continuous band of tissue on each side of the sporangiophore, this band being the so-called "sporangiogenic" band, from which the individual sporangia are differentiated later. The sporangia arise from this sporangiogenic band at more or less definite intervals, these fertile areas being separated by bands of sterile tissue. In the sporangial areas periclinal walls are formed by which an inner mass of tissue, the Development of the sporangium in Ophioglossum pendulum. The darkly shaded nuclei in A mark tin- archesporium; 1. tapetuni; sp, sporogenous tissue. A is a longitudinal section; B, a cross-section; C, part ol B, more highly magnified; l>. mature sporangia. X8. archesporium, is separated from the outer cells which are to form the wall of the future sporangium. The inner cells constituting the archesporium later give rise to the masses of spores. Between the young sporangia there lie partitions or septa formed from the intervening sterile cells of the sporangiogenic band. I he nil groups which form the archespcria and the sterile septa are derived from sister cells 114 ormoci ,oss \i i s of the original sporangiogenic bands. Bowei thinks that .ill of the sporogenous tissue can not be traced back to a single primary archesporial cell, and that spore- bearing tissue is formed secondarily In periclinal divisions in the cells outside of the original archesporium. In some of the very young sporangiophores of 0. pendulum there can sometimes be seen a single large epidermal cell which may possibly be the mother cell of a young sporangium, bur it is ven difficult to determine this point satisfactorily. After the archesporium is differentiated there is a rapid division in its cells and there is thus formed a very large mass of cells, whose limits, however, are not always very clearly marked. Later on the contents of these cells become denser and are more easily distinguished from the surrounding sterile tissue. The cells King outside the archesporium divide rapidly both by longitudinal and transverse walls anil give rise to the thick outer wall of the sporangium. In lon- gitudinal sections through the sporangium two rows of cells may he seen extending from the mass of archesporial cells to the outside of the sporangium. These two rows of cells mark the point where the transverse cleft arises by which the spo- rangium opens at maturity. The outercells derived from the archesporial complex Fig. 85. A, B. Young sporangia; C, an older sporangium of Bolr\chium virgtnianum. In C, the sporogenou tissue is shaded; the nucleated cells adjoining form the tapelum. do not develop into spores, but constitute the tapetum, which later becomes dis- organized and forms a sort of plasmodium extending among the growing spore mother cells, and is, no doubt, of great importance in the further development of these. Bower thought that some of the inner cells of the archesporial tissue contrib- uted to this plasmodium, but a further study has led him to the conclusion that this is not the case and that all of the inner cells of the archesporium develop into spores. This view is confirmed by the recent paper of Burlingame ( Burlingame I ), who investigated the development of the spores in 0- reticulatum. At maturity the sporangium opens by a transverse cleft whose position is already evident in the younger stages of the sporangium. As the cells shrink with the drying of the ripe sporangium the spores are crowded out through this cleft, hut there is no special mechanism like the annulus found in the sporangia of the higher ferns which facilitate the dispersal of the spores. Our knowledge of the development of tin sporangium of Botrychium has been based largely upon the study of B. lunaria. I have investigated with some care the development in B. virginianum, which differs mainly from B. lunaria in the smallei i hi \Di i.i spoRopm 1 1 115 size of the sporangium and the fact that it has a well-marked though short pedicel. In the simplest forms of Botrychium, such as H. simplex, the sporangia are much larger and are sessile. In B. virginianum (fig. 85) the development of the individual sporangia begins just about a year previous to their ripening, and if the plants are taken up about the time the spores are shed the earliest stages of the sporangia will be found in the leaf which is to expand the following season. At this time the sporangiophores in the larger specimens are thrice pinnate, and the youngest recognizable sporangia are borne at the tips of the branches. These young sporangia form slight elevations which become smaller as they approach the tip of the segments, and if an exact median section is made of one of tbese young sporangia it will be seen to have at its apex a large pyramidal cell with a truncate base. Holtzman (Holtzman 1), states that the whole sporangium may be traced back to a single cell and that the divisions at first are like those of a three-sided apical cell. I have not been able to satisfy myself as to the accuracy of this statement, but the youngest stages which I have been able to find would not forbid such an interpretation, although there seems no question that the basal part of the sporangium is derived, in part at least, from the surrounding tissue. Fig. 86. A. Sporangiophores of Helminihosiachys. The two lower figures seen Irom above. /, sterile appendages. X3. B. Section of a young sporangium, showing sporogenous tissue, sp; tapetum, t; and wall, ir. (From preparation made by Prof. I,. L. Burlingame.) i he terminal cell of the sporangium is divided by a periclinal wall into a single inner cell which forms the archesporium, and an outer one which contributes to the wall of the sporangium. The outer cell rapidly divides and similar divisions occur in the archesporium. By active growth in the basal part of the sporangium it pro- jects more and more until it assumes the form of a projecting globular body with a short stalk, thus differing strikingly from the deeply sunken sporangia of Ophio- glossum. It is impossible to detect any definite succession of divisions in the arche- sporium, which ultimately becomes changed into a large globular mass of cells with the usual dense contents, separated from the outside of the sporangium by half a do/en or more layers of sterile cells. The cells immediately adjoining the sporo- genous tissue constitute the tapetum. Bower states that the tapetum is exclusivelj derived from this outer sterile tissue, but it must be said that, in Botrychium vir- ginianum at least, the limits between the sporogenous tissue and the tapetum are by no means easy to detect. The wall of the ripe sporangium has from four to six layers of cells and the dehiscence is by a vertical cleft, whose position may sometimes 1 Id TH1 OPHIOGLOSSAL1 S be recognized in the young sporangium by cells arranged very much as they are in the sporangium of Ophioglossum. 1 he stalk is traversed by a short vascular bundle which can first be recognized when the sporogenous cells arc- pretty well advanced in their development. The sporangia ol Melminthostachys on the whole more resemble those * > t Botryi Iniirn than those ol Ophioglossum. In the very young sporangiophore there is developed a band of tissue on either side much like that in Ophioglossum (set Bowei 9, page 455)- transverse sections ol the fertile spike show that the spo- rangiophores originate from certain groups ol cells ol this sporangiogenic hand. These young sporangiophores, however, project above the surface and later form crowded projections which may undergo a greater or less degree ol branching, so that the number ol sporangia borne upon a single one of these secondary sporangio- phores may be considerable. 1 hese sporangiophores become irregularly lobed and the final divisions of these develop into the sporangia in a manner which is very similar indeed to that of Botrychium, but the individual sporangia are usually less distinct. Bower's account of the further development may be quoted as follows: "It has already been noted that the position and number of the sporangia which they bear are inconstant. In early stages it is impossible to distinguish the cells which will give rise to the sporangia, but from rather older stages it ap- pears that the sporogenous groups, together with the superficial cells which cover them are referable in origin to the segmentation of a single superficial cell. More- over, the first penchnal division of the cells defines the whole of the sporogenous tissue from the protective wall. As the sporangia grow older they project from the surface of the sporangiophore. The sporogenous mass increases rapidly in bulk, while the cells surrounding the sporogenous mass to the extent of several layers assume the character of a tapetum which gradually becomes disorganized; finally the sporogenous cells separate and divide into tetrads. As the sporangia approach maturity the upper part of the sporangiophore may grow out into an irregular rosette of laciniae of vegetative tissue." A study of the older sporangia of Helminthostachys shows that the tapetum is extraordinarily developed, constituting a very thick layer surrounding the mass of spores (fig. 86). The clusters of fully developed sporangia are often very regular in form (fig. 86, A), and remotely suggest the svnangium of a Marattiaceous fern. Part II. THE MARATTIALES. The members of the second order of the Lusporangiatae — the Marattiales — resemble quite closely the typical Leptosporangiata?. Both in the form and venation of the leaves and in the development of the sporangia upon their lower surface they recall the common ferns. But the structure of the sporangium is very different, and both in its structure and development the sporangium shows certain evident resemblances to that of the Ophioglossaceae. The Marattiaceous type is a very old one, and this fact lends a special interest to these few survivors of the ancient Paleozoic fern flora. Like the Ophioglossaceae, the leaves of the Marattiaceae are thick and fleshy in texture, but usually firmer than is the case in the Ophioglossaceae. Some of the smaller species of Dancra are only 2 or 3 decimeters in height, but the larger species of Marattia and Angiopteris are among the largest of the ferns. The latter, which is common in the eastern tropics and extends to Australasia, is a noble fern whose great caudex, covered with the persistent leaf bases, is nearly as big as a barrel, and the gigantic leaves reach a length of 5 or 6 meters, with stalks almost as thick as a man's arm. In the dense, wet forests of Tjibodas in Java, for example, the development of this fern is espe- cially luxuriant, and groups of them form one of the most striking features of this rich, tropical flora. The Marattiaceae frequent, for the most part, the moist, shady forests of the tropics, where they form a characteristic feature of the vegetation. Along the steep banks of moist, shady ravines or streams they sometimes occur in profusion. The number of living Marattiaceae* is small, some authorities recognizing only about thirty species. There is, however, a good deal of difference of opinion as to the number of species in some of the genera. This is especially true of Angiopteris^ which many authorities consider to have only one single, extremely variable species, while others recognize man}- species. One of the genera, Marattia, is widespread throughout the tropics of both hemispheres, where there are about 25 species. (See Bitter 1, Christensen I.) One of these, M. douglasii, extends to the Hawaiian Islands, where ir is a common and conspicuous fern. Angiopteris is widespread throughout the tropics of the old world and reaches beyond the tropics into Australia and southern fapan. Archangioptens, with a single species, A. henryi, is a recent!}' discovered form occurring in southwestern China. Kaulfussia, also a monotypic species, occurs throughout the Indo-Malayan region and extends as far as the Philippines. The genus Dancea is exclusively neo-tropical and com- prises about 20 to 25 species, most of which occur in the West Indies, northern South America, Central America, and Mexico. A sixth genus, Macroglossum, from Borneo, has recently been described (Copeland 1). Of these, Kaulfussia is the most aberrant, differing markedly from the other genera in the form and venation of the leaves, as well as in the shape and position of the synangia. Kaulfussia and most species of Dan a- a have dorsiventral rhizomes, while the other genera have an upright, radially-constructed caudex. The gametophyte, in all of the investigated species, is a large, dark-green, fleshy thallus, much resembling superficially such a liverwort as Pelha. The re- productive organs are very much like those of Ophioglossum, and chlorophyll is formed in abundance in the gametophyte. The large liverwort-like gametophyte * Christensen (I) recognizes 62 species of Angiopteris, I of Archangiopteris, 26 of Danaea, 1 of Kaulfussia, and 28 of Marattia. 117 1 IN THE MARA I I I \l . I S and the character of the reproductive organs are marked indications of the primitive nature o) these ancient ferns. 1 he embryo also shows many points in common with that of the Ophioglossaceae. As in the latter, the organs of the young sporophyte are all of epibasal origin and the vasculai system in the young sporophyte belongs exclusively to the leal and root, the stem being entirely destitute of a proper stele. in Danaa a short suspensor is usually developed, hut the other genera have as yet shown no examples of this. The sporophyte is very much alike in its early development in all of the Marat- tiacea?. The young plant consists at first mainly of the primary leaf and root, which are traversed by a single axial vascular strand as in Ophioglossum moluccanum, hut a stem apex is developed at an early period, although it remains relatively incon- spicuous. Kaulfussia, Danaa, and Archangwpteris are all ferns of moderate size and comparatively simple structure; but Marrattia, Angiopteris, and Macroglossutn are very large, and the arrangement of the vascular bundles in the stem becomes exceedingly complex, corresponding to the numerous bundles in the enormously- developed leaves. The form of the leaf in the Marattiaceae ranges from a perfectly simple leaf in Dmiiv/i simplicifoha to the gigantic decompound leaves of Angiopteris and Marattia. Most of the species of Danaa (and this is true also of Archangiopterii and Macro- glossum) have simply pinnate leaves, while in Kaulfussia the leaves are palmatelv divided into from three to seven leaflets, the larger leaves looking curiously like a horse-chestnut leaf in outline, whence the specific name. The ternate form, which is often found in Kaulfussia, recurs in the early leaves of the other genera and is sometimes retained in quite large leaves in Marattia (plate 12, B). This recurrence of the ternate leaf form suggests Helminthostachys and the ternate species of Botryi Ilium. The leaves are usually quite smooth, but in the earlier stages then is sometimes a sparing development of bans and scales. The lattei are especially noticeable upon the leaves of the young sporophyte in Dana-a- The leaves are furnished at the base with very conspicuous fleshy stipules which remain adhering to the stem after the leaves fall away, and these leaf bases, with their attached stipules, more or less completely cover the surface of the stem. As the leaves fall away they leave a characteristic scar marked by the remains of the vascular bundles. 1 he leaf base as well as the- stalks of tin leaflets shows a more oi less marked enlargement, recalling rlu- pulvinus which occurs so commonly in the Leguminosa?. It is at this point that the leaf stalk separates, the smaller divisions of the leal often breaking away from the main or secondary rachis, in the same fashion as the main leaf stalk falls. In the large species of Marattia and Angiop- teris this enlarged leaf base with the two thick, fleshy stipules curiously resembles in shape and size the hoof of a horse. The leaves when young are coiled up in the same fashion as those of the typical ferns and their venation in general is decidedly fern-like, the ultimate veins being dichotomously branched and the venation very much like that of Helminthostachys or certain species of Botrychium. In Kaulfussia, however, the venation is reticulate and strikingly like that of the typical Dicotyledons. In its earlier stages, however, there is a marked resemblance to the venation of Ophioglossum. The fleshy leaves do not, as a rule, show a very large development of mechanical tissues; but there is developed, especially in the larger leaves, a thick layer of hypo- dermal tissue, which is usually sclerenchyma, somewhat like that of the typical ferns, but may be thick-angled tissue or collenchyma, such as is common in many flow- ering plants. A marked anatomical feature is the presence of large mucilage ducts. THE GAMETOPHYTE 119 I. THE GAMETOPHYTE. The first account of the germination and development of the gametophyte in the Marattiacea? was published by Luerssen (Luerssen 2), who studied the germination in Marattia ctcutafolia and in Angiopteris. Not long afterward the Dutch botanist, Jonkmann, published an account of the development of the prothal- lium in both Marattia and Angiopteris. His original paper (published in Dutch, but afterward translated into French) gives an extremely satisfactory account of the germination and development of the prothallium and reproductive organs (Jonk- mann 1). Somewhat later he also published a preliminary account of the germina- tion in Kaulfussia, but apparently the work was never published in full (Jonkmann 2). In 1892 Farmer (Farmer I) described the gametophyte and embryo in Angi- opteris evecta and in 1894 the writer (Campbell 3) gave an account of the prothallium and embryo in Marattia douglasii collected in Hawaii. Two years later Brebner (Brebner 2) described the prothallium and embryo in Dancea simplicifolia. In 1908 the writer published an account of the prothallium and young sporophyte of Kaulfussia (Campbell 9) and in 1909 a preliminary note was published in regard to Dancea (Campbell, 10). The following account of the germination is based mainly upon the work of Jonkmann. I have, however, examined the early germination stages in Marattia douglasii for comparison with Jonkmann's account, and, so far as my experiments went, it agrees entirely with the species described by jonkmann. The ripe spores of the Marattiaceae are small and may be either of the bilateral type or tetrahedral (radial). According to Jonkmann, the bilateral spores are much more abundant in Marattia than the radial spores, but in Angiopteris the radial spores predominate. The wall of the spore (see Jonkmann 1, pp. 203, 204) shows a differentiation into an inner membrane orendospore and a middle layer, theexospore, which is often found divided into layers. There is a very thin external coat, the perispore or epispore, which is generally thrown off in the early stages of germination or even before germination begins. All of the membranes except the epispore are colorless, while the latter is a more or less pronounced yellowish brown tint. The surface of the spore is roughened by small papilla' which arise from the exospore. The spores contain no chlorophyll, but there is a considerable amount of oil present, which appears as drops of varying size, and there are also other granular contents — starch and albuminous granules. The nucleus lies in the center of the spore and is connected with the peripheral protoplasm by delicate protoplasmic filaments. Germination begins quite promptly under favorable conditions and within about a week the spores, which hitherto were quite colorless, begin to show a greenish tint, due to the development of chlorophyll. Jonkmann states that the chlorophyll appears first as flocculent masses near the nucleus, but these apparently amorphous masses are really composed of very small, faintly tinted chromatophores, which lie between the large oil drops and rapidly increase in size and depth of color as germination proceeds, their number increasing by the usual division. 1 he chloro- plasts later become very conspicuous and are distributed in the periphery of the now very much enlarged spore, the outer membranes of which are ruptured so as to expose the endospore, containing the nucleus and numerous large and conspicuous chloroplasts. Starch granules are also to be seen in most cases. The cell remains undivided until it has attained a size many times greater than that of the spore. The first division wall, which is formed about a month after the spores are sown, is transverse both in Angiopteris and Marattia, and, like that in the germinating spore of Ophioglossum, divides the primary cell into two nearly 120 l II 1 MAR VTT1 \l.l s equal parts (fig. 87, A). A rhizoid may be an of! either before or after this first transverse \\ ;< 1 1 is developed, but frequently no rhizoids are formed until a much later period, as in Ophioglossum. I hi- primar] rhizoid, when present, is formed much as in the typical ferns, the papilla from which it develops being cut off from the largei cell, and it contains little 01 no chlorophyll. I- ach of the primary prothal- lial cells divides, in typical casts, h\ a longitudinal wall, so that the young gameto- phyte consists of four cells, arising quadrant-wise ( fi<;. XX, A), and closely resembling corresponding stages 111 Ophioglossum, except for the absence of chlorophyll in the latter. Where the young plants are crowded or light is deficient, as for instance when the germination occurs within the sporangium, then- is a tendency to the development of a filament, a phenomenon often met with also in the typical ferns. Usually one of the upper pan of cells in the four-celled stage assumes the role of an apical cell, and for some time, as in the typical ferns, there is growth from a two-sided apical cell (fig. XX, C). As soon as the apical cell is established, it »rows I 1,.. s \. Two germinating spores ol Marattia frax inea Smith. ■ 200. The remains oi the spore meml li. Young gametophyte ol ame 1 -;. (A, B, after Jonkmann). i . Marattia tambucina Blume. X r.c. I) II. M.douglasii Baker. 1)1', . 1.,-; 1; , H, x-,. G,H, show the young sporophytc. I) and E represent the same prothallium aftei an interval of about .1 pear. F 1^ tin- ventral riew of E. k, adventitious bud. very much as it does in such a liverwort as Aneura and produces a thallus of the same form and structure. But as the prothallium grows older a periclinal wall is formed in the apical cell, and in the outermost of the two cells thus produced there is a longitudinal wall dividing it into two equal cells, and from this time on it is impossible to recognize a single apical cell in the prothallium, the apex of which is occupied by a group of apparently similar marginal initial cells. At hist tin- prothallium has a spatulate form, but before the single apical cell is replaced by the group of marginal initials the outer cells of the younger segments grow more rapidly than the inner ones, so that they project beyond the apical cell, which thus comes to he in a depression between the two lobes, and the familiar heart-shaped form so commonly found in the prothallium of most ferns is established . 1 he marginal initial cells vary in number with the width of the depression in which they In. In a horizontal section they appear oblong in form, but in the vertical sections made they have a semicircular outline (fig. XX, D, E). THE GAMETOPHYTE 121 Basal segments are cut off by a wall extending the whole depth of the prothallium and the segment thus cut off is divided at once by a horizontal wall into a dorsal and ventral cell of nearly equal size (fig. 88, E, d, v). Cell divisions are more active in the ventral segments, more manifestly so at some distance back from the apex. It is due to this more active cell division in the ventral segments that the strongly projecting cushion-like mass of tissue is formed upon the ventral surface of the prothallium, upon which the archegonia later make their appearance. The superficial cells of both surfaces of the prothallium have a thick cuticle which often makes it difficult to embed the p roth alii a without bad shrinkage. From the under side of the prothal- lium numerous rhizoids are developed, which in the case of Marattia and Atigiopteris are unicellular and thin-walled, but in Dancea become divided into several cells. Sometimes there seems to be no definite apical growth in the early stages, but on the other hand Jonkmann states that both of the superior cells may function as apical cells, thus inaugurating the early dichotomy of the young prothallium, and even a third branch may arise from one of the inferior quadrants, which assumes the character of a third apical cell. Among the leptosporangiate ferns, the forms which most nearly resemble the Marattiacea? in the development of the gametophyte are the Osmundaceae, especially Fig. SS. A-C. Threr young gametophytes "t Atigiopteris. !■ I)A\ V.A. In |ulv, 1908, the prothallia of three species oiDanaa were collected in Jamaica. \s the genus Dante a has received comparatively little attention, especially as regards the gametophvte, it seemed very desirable to secure as complete a series of the pro- thallia and young sporophytes as could be done, and to this end the trip was made to the West Indus. The genus Dancea is confined to the American tropics and comprises, according to Christensen, 26 species of extremely characteristic ferns. The type is evidently an old one, as some of the fossil Marattiaceae are closely allied to the living genus Dancea and may possibly be referred to it. The only account of the prothallium hitherto published, so far as I am aware, is the paper of Brebner (Brebner 2) on D. simplicifolia, a species from British Guiana. In 1897 ' collected a small number of specimens of prothallia of Datura, prob- ably D. jenmani, and in the summer of 1908 the same locality was visited, as well as some others in the same district, and material of three species u as secured. These O'vv C-A—^s^ \. Three gatnetophyies of Dan.ea jenmani Underwood. X2. B. Three young gametophytes of D. jamaicensis Underwood. \j. C. Four large gametophytes of D.jamaicensis. C, 1, shows four groups of archegonia, 9 • collections were all made in the vicinity of Cinchona, a mountain station at an eleva- tion o{ about 5,000 feet. As I have found in collecting other Marattiaceae, the most favorable collecting ground for the prothallia is upon moist, clayey banks where the fruiting plants are growing. In the shady crevices in such positions a careful search will usually be rewarded by the discovery of numerous young plants, and with these there are often associated prothallia in various stages of development. Brebner described the prothallia of D. simplicifolia as being nearly circular in outline, but very few of the prothallia of the species collected by nu showed this form; but they were usually decidedly elongated, sometimes very strongly so, and were, as a rule, very much larger than the specimens of D. simplicifolia described by Brebner. I he three species collected by me were D. jamaicensis I nderwood, D. jenmani Underwood, and D. ellipttca Smith. In all three species the larger prothallia are usually decidedly elongated and very irregular in outline, often showing conspicu- ous leaf-like marginal lobes, like those occurring in the prothallia of ()s munda and Gleichenia. THE GAMETOPHYTU 125 The germination of the spores and the early stages are quite unknown in Daticea, but probably resemble those of the other genera which have been studied. To judge from the younger stages collected, there is a good deal of variety of form, as is the case also in the other Marattiacea?. The youngest specimens collected belonged to D. jamaicensis. These were very broad in outline and unsymmetrical, one wing of the prothallium being much better developed than the other (fig. 91, B). Fk.. 01.— Foul gametophytes of D. elliptica Smith. XI. B-D, seen from belov, . Fig. 92 shows some older specimens of D. elliptica which were greatly elongated, the very much attenuated posterior region being quite thin and delicate in texture, with no midrib and the archegonial cushion being confined to a small region just back of the growing point. The marginal region in these young prothallia was composed of a single layer of cells and a considerable portion was made up of but Fig. 93. A. Young gamctophyte, probably developed from an adventitious bud "I Datura jamaicensis. X20. ff antheridia. B. Large gametophyte of D. elliptica, with two sporophytes. Xi. C. Multicellular rhi/.oid. Xioo. D. A cell of the rhizoid, showing nucleus, if. X210. two layers, while the mid region, where the antheridia and later the archegonia are developed, was not more than four to five cells in thickness. In general the prothallia of Daneea a re ]m ore delicate in texture than those of the other Marattiacea? that have been studied. 126 I II I M \K \ II I \l I S A midrib usually begins to form at an early period and in the oldei prothallia may become very conspicuous, sometimes reaching a thickness "I eight to ten cells. The margin, as we have seen, is always more or less irregulai in outline and often develops large leaf-like lobes which are particular!) conspicuous in I), \amaicensh (fig. 91, (-')• but are noticeable also in the othei species. The large prothallia, which are sometimes nearly •; cm. in length, are often branched (fig. 92, /)); the branching Fig. 94 . A, archegonium; B, antheridium of Marattia douglasii. >i, nrt the cell remains intact. I III SEXU \l ORGANS. 1 he w 1 111 uinn \i. I hi development ol the antheridium, except foi the details of spermatogenesis, was correctly described In' Luerssen and fonkmann for Marattia and Angiopteris. The other genera agree closely with these in the essential structure of the antherid- ium. The development of the antheridium in the Marattiaceae agrees very closely indeed with that of Ophioglossum. Fig. 97. — Development of the antheridium in Danaa. A. Section of prothallium, bearing antheridia on both surfaces. X8o. B-H. Longitudinal sections. X300. E-H, D.elliptica; the others, D. jamaicenyh. The mother cell of the antheridium, as in Ophioglossum, divides first by a periclinal wall into an outer cell, the primary cover cell, and an inner one, from which the mass of spermatocytes is developed (figs. 96, 97). The mother cell of the anther- idium shows much the same variation in form as that of Ophioglossum, sometimes being relatively broad and shallow and at other times deeper and narrower (fig. 96, B, C). The first division in the inner cell is usually transverse, but in the broader type of antheridium this first wall may he longitudinal. The primary wall is followed by a second one at right angles to it and the four cells thus formed are again divided so that there result eight nearly equal cells. The first wall occasionally is somewhat oblique, but even in such cases the regular quadrant and octant walls arise at right I III GAMETOPHYT1 l29 angles to the primary division wall. I he next divisions are, usually al least, anti- clinals (Hg. 97, G), but before long the periclinal walls also are developed and sub- sequent divisions do not show any recognizable regularity in their sequence; there seems to be a good deal ot variation in this respect, even in the same species. The number of cells ultimately formed varies a good deal, but the number of spermato- cytes finally developed is probably never so great as that found in some of the ( )phioglossaceae. Kaulfussia, both in the size of the antheridium and thai of the spermatozoids, approaches nearest to Ophioglossum. I he number of spermatozoids may reach several hundred, fifty or more being visible in a single section of a large antheridium, and nearly or quite as many may sometimes be found in Dancea, where, however, the spermatozoids are much smaller than in Kaulfussia. In the cover cell the divisions are all anticlinal and horizontal sections or surface views show that the successive walls are arranged spirally in a way suggesting the segmentation of a three-sided apical cell. The last-formed wall cuts out a small, nearly triangular cell, the opercular cell (fig. 95 D, E, 0). In most cases, at least. Fie. yv. Ripe antheridium of /). jamaicensis. m, mantle nils. A. Transverse section of three jroung antheridia of D.jamaici ■ i 0, opercular cell. X350. B. Surface view of the two youngest ones, a, opercular cell. X380. this opercular cell Js thrown off when the antheridium opens, leaving a small aperture through which the spermatozoids are ejected. Surrounding the mass of spermatocytes is a layerof mantle cells cut off from the adjacent cells of the prothal- lium. These mantle cells, at the time of the opening of the antheridium, become very much distended and project strongly into the cavity of the empty antheridium (fig. 100, /?, m). They no doubt play an important part in the dehiscence ol tin ripe antheridium. SPERM \ loci, \ |s|s. (Plate :, figs ^-44.) Of the Marattiaceas, Kaulfussia is the most satisfactory foi studying the details of spermatogenesis, owing to the much larger size ot the spermatozoid. I he development of the spermatozoids agrees very closely in its details with that of Ophioglossum. If the spermatocyte is examined just before the final division the two blepharoplasts can be seen and the division of the cell into the two spermatocytes proceeds very much as in Ophioglossum. After the final division the nucleus ot the spermatocyte appears coarsely granular ami, as in other cases, no nucleolus can In- seen. In favorable cases the blepharoplast is visible as a round body, stained 9 \M) in i MARATTIALES rather strongly and lying neai the nucleus. The blepharoplast soon takes on the curved form and becomes much extended and the cilia begin to develop from it before the nucleus lias materially changed its shape. The nucleus now becomes slightlj pointed .it one end and begins to stretch out so as to appear somewhat crescent-shaped, very much as in Ophtoglossum and as it does in other ferns that have been described. With this change in the form of the nucleus, the blepharoplast becomes still more elongated and strongly colored and the cilia increase in length. The nucleus of the spermatozoid in Kaulfussia is less elongated than is usual in the ferns, and in this respect, as well as in its larger size, it more nearly resembles Ophtoglossum than it does the other Marattiacea?. I he granular appearance of the nucleus is maintained until the spermatozoid is almost fully developed, when there seems to he a fusion of the chromosomes so that it appears almost homogeneous; this is accompanied by a noticeable diminution in the size of the nucleus. The nucleus occupies only the large posterior coil of the spermatozoid, while the anterior portion, which shows about two coils, is composed of the blepharoplast with probably a certain amount of other cytoplasmic matter. Fig. 100. A. Cross-section of a ripe antheridium of D.jamaicensis. B. Cross-section of an empty antheridium; m, mantle cells. C-D. Surface views, showing opercular cell. All figures X 350. In Marattia and Angiopteris (plate 2, tig. 4+) the nucleus of the spermatocyte becomes much more extended and the whole spermatozoid is more slender than in Kaulfussia. Indeed there is very little difference between the appearance of the spermatozoids of Angiopteris and Marattia and those of the typical leptosporangiate ferns. Some observations were made also upon Datura, in which the spermatozoid is somewhat intermediate in character between that of Angiopteris and kaulfussia (plate 2, figs. 42, 43). In size the spermatozoids are more like those of Angiopteris, but the nucleus is much less elongated and the general form of the spermatozoids is more like that of Kaulfussia. THE VRCHEGONIl M. The archegonium in the Marattiaceae, like the antheridium, very much resembles that of Ophtoglossum, but the neck of the archegonium is even less developed than in the latter. Jonkmann ( Jonkmann I) has given a fairly complete account of the development in Marattia and Angiopteris, and Farmer (Farmer 1) has described and figured the archegonium of the latter genus. Usually, at least, the archegonium is developed only upon the cushion of tissue back of the apex, the young archegonia arising in acropetal succession. Jonkmann THE G \MI inl'in I I suites, however, that In- has found them also upon the upper surface of the prothal- lium, hut none of the specimens I have examined have shown this. The mother eell of the young archegonium is scarcely distinguishable in form from the young antheridium and like it is first divided by a periclinal wall into an outer cover cell and an inner cell, the latter usually, but not always, divided subsequently into a central and a basal cell (figs. 101-103), as in Ophioglossum and in the typical ferns. Sometimes the mother cell of the archegonium is seen in transverse section to have been cut out very much as the axial row of cells arises in the archegonium of the Hepaticae. I here seems no question that the so-called mother cell of the archego- nium in all the ferns is really homologous only with the axial row of cells of the bryophyte archegonium, the four rows of neck cells being a further development of the terminal cap cell of the liverwort archegonium. 1 he inner of the two primary cells, as we have already stated, may have a basal cell cut oft from it before the further divisions arise, by which the egg cell and the canal cells are divided. The neck canal cell is very broad and may become divided into two cells, but usually the division is confined to the nucleus, which probably divides in all cases. The ventral canal cell cut off in the usual fashion from the egg is, with the exception of Dancea, very large and conspicuous, thus differing from Fig. 101. Archegonia of Angiopterii. X275. b, basal cell; o,egg; v, ventral canal cell. Ophioglossum, where the ventral canal cell is so difficult to demonstrate; but in Dancea there is the same imperfect development of the ventral canal cell that is found in Ophioglossum. As the archegonium approaches maturity a layer of mantle cells, much like those which surround the antheridium, is cut oft" from the tissue surrounding the venter of the archegonium. The archegonia of Marattia douglasu are confined to the lower side of the midrib and begin to form at some distance back of the grow- ing point; so far as can be determined, any superficial cell of the apical meristem can develop into an archegonium. The mother cell divides, as we have seen, into three superimposed cells, of which the lowest, b, usually divides later by vertical walls, and forms the base of the archegonium. From the central one, by transverse divisions, are formed the canal cells and egg, and from the uppermost the neck. Compared with the typical ferns, the most striking differences are the short neck and the very broad canal cells. The cover cell undergoes division into four, by two intersecting vertical walls, as in Ophioglossum, and each of these four cells then undergoes division bv nearly horizontal walls, but the cells remain short, so that the neck projects very little and there are only three or four cells in each row; occasionally there may be only two. Jonkmann states that, as a rule, two of the rows of the neck contain three cells and two contain tour, hut that there may occa- sionally be as many as five. The neck canal cell often shows a trace of a division and there may be an actual division wall formed (fig. 101, D), but in Marattia douglasu 132 I II I \1 \K A I I I \l I S this is not ordinarily the case. In one instance in this species I observed a division In a vertical wall, so that two neck canal cells \\cn formed, placed side by side, in away recalling very strongly the division of the neck canal cell described by Jeffrey in certain species of Equisetum ( feffrey 2). The central cell is divided again by a hori- zontal wall into two nearly equal cells, the lower one being the egg, the upper one the ventral canal cell. The mature egg is nearly elliptical in form, the upper third being almost homogeneous and quite colorless, forming the so-called receptive spot. I he nucleus is of moderate size and does not stain very strongly. 1 he archegonium of Angiopteris closely resembles that of Marattia, but is perhaps somewhat narrowei (fig- ioi). Jonkmann figures the archegonium ot M . cicuttefoha, which shows that this species also is, in the relative size of the canal and neck cells, very much like Angi- opteris. Farmer thought that a basal cell was usually, it not always, present, and my own studies tend to confirm this. D' E Archegonia of K auifussia. X210. b, basal cell; o,egg; is ventral canal cell; «.c, neck canal cell. In Kaulfussia (fig. 102) the archegonia form, as a rule, only after the antheridia have ceased to develop. Compared w7ith the other Marattiace;e they are decidedly large, and in this respect Kaulfussia approaches Ophioglossum. Like the mothei cell ot the antheridium, there is a good deal of variation in the width of the young archegonium in Kaulfussia. Some of the narrower types recall the archegonium of Anthoceros and emphasize the resemblances between the archegonium of the Anthocerotacea- and the eusporangiate terns. The neck of the archegonium is very short, each ot the tour original neck cells often dividing only once, so that there may be but two cells to each ot the four rows. More commonly, however, there is a Fig. 10;. Young archegODia of Dan0 The archegonium in Dance a, while resembling that of the other Marattiaceae, in its position and early development shows some marked differences, the signifi- cance of which is not quite clear. The division of the mother cell into the primary neck cell and the central cell follows in the same way as in the other forms, but the inner cell, usually at least, does not have a basal cell separated from it, but develops at once into the egg cell and canal cells. I he absence of the basal cell is by no means unknown, however, among the other Marattiaceae. I he primary neck cell gives rise to the usual four cells, each of which divides into three or four, and exception- ally into five. There is a marked elongation of the inner cell before its separation into the central cell and the neck canal cell (fig. 103, D). Fk.- 104. A, young, B, r. early mature, archegonium of D.jamaicensit. X360. C, I), en ss-section of a young archegonium of same species. D shows the four priman neck cells. Up to this point there is nothing peculiar in the development of the archegonium in Datuea, but while in most of the other Marattiaceae a conspicuous ventral canal cell is formed, its sister cell being the egg, in Daucea the formation of a definite ventral canal cell could not be satisfactorily demonstrated. In a number of cases (fig. 105; plate 2, fig. 4.5) :i small nucleus-like body could be seen in a large, cleai Fn,. 105. Three mature archegonia of Danaa elliptica. X350. A and V> slu.w trace; <■( .1 ventral canal cell; :', in B, a division of the neck canal cell. space just above the somewhat contracted mass of protoplasm, with its large and conspicuous nucleus, which constitutes the egg cell; but this ventral canal nil nucleus, if such it is, is very different in appearance from the large and conspicuous one found in the other Marattiaceae, and in the absence of any division stages its nuclear nature must for the present remain somewhat doubtful. There is somewhat the same uncertainty in regard to the primary division of the neck canal cell. This possesses a large and conspicuous nucleus which in large and apparently mature archegonia was still undivided. In ;i few cases a division of the protoplasm in the neck cells was observed; in other casts, without any such division, 134 THE MARATTIALES a second body of rather indefinite outline and staining much less strongly than the lower nucleus could be made out; but up to the present time I have not been able to satisfy myself that the division o{ the neck canal cell nucleus, which always occurs in the other Marattiacea?, takes place here. The very uncertain nature of the ventral canal cell recalls strongly the condition of affairs in Opfuoglossum, where it is equallv difficult to demonstrate satisfactorily the presence of a true ventral canal cell. FERTILIZATION. It has not been possible to follow in detail the process of fertilization in the Marattiaceae, but several stages were found in preparations of Marattia douglasii. The entrance of the spermatozoid into the archegonium was not seen, but in a number of cases the material had been killed immediately after, and twice spermato- zoids were found which had penetrated into the egg. In these cases the spermato- zoid was quite unchanged in form, but had not yet entered the nucleus itself. One case was observed where there were apparently two nuclei in close contact, but the egg nucleus was much contracted and it is doubtful whether this was really a normal appearance. It is probable that the details of fertilization are quite similar to those observed in other ferns. THE EMBRYO 135 II. THE EMBRYO. The study of" the embryogeny of the Marattiaceae offers many difficulties. Fertilization does not seem to be of common occurrence and a very large number of prothallia must be examined before even a small series of embryos can be secured; moreover, the earlier stages of the embryo are peculiarly liable to shrinkage in preparing them for sectioning and it is exceedingly difficult to secure really satis- factory preparations of these early stages. A marked peculiarity of the young sporophyte, which was first shown by Luers- sen and Jonkmann for Marattia and Angwpteris, is the orientation of the primary organs of the young sporophyte with reference to the archegonium. The primary or basal wall in the embryo is always transverse, as it usually is in Ophioglossutn, instead of being vertical as in most of the typical ferns. The first leaf, instead of being formed from the portion of the embryo nearest the archegonium, as it is in the common ferns, arises from the half of the embryo which is turned away from the archegonium, and grows straight upward, bursting through the prothallium upon its upper surface, instead of appearing upon the lower side of the prothallium and curving upward. The external organs of the young sporophyte are differentiated cot I'x.. 106. Marattia douglasii. A. Two longitudinal sections of a young embryo, b b, basal wall. X200. B. A similar section of an older embryo, cot, cotyledon; r, root initial. C. Three transverse sections of a much older embryo, showing junction of the two first leaf traces. X50. much later than is the cast- in the Leptosporangiates, and the Marattiacea? in this respect closely approach the ( )phioglossacea?. It has been generally assumed that, as in the leptosporangiate ferns, the cotyle- don and stem are of epibasal origin, the root and foot hypobasal. This conclusion was reached by both Jonkmann and Farmer, and my early studies on Marattia douglasii led me to the same conclusion. A further study of this species, however, as well as an examination of the embryos of Angiopteris, Kaitlfiis.ua, and Datura, has shown that this is not the case, but that the whole of the hypobasal region is devoted to the formation of the foot, and the root is developed secondarily from the epibasal region, from which are also derived the stem apex and the cotyledon. While in the typical ferns the young organs of the embryo at a very early stage show a definite apical growth, the apical cells being readily traceable to the primary octants of the embryo, this is by no means so readily shown in the Marattiacea?. In the later stages, such initial cells can be seen in the root and stem at least, but the relation of these initial cells of the root and stem to the early divisions of the embryo is very difficult to determine. In M. douglasii (Campbell 3) I stated that there 136 THE MARATTIAI.ES was probably a single initial in the stem and in the primary root, and Farmer also thought that a single root initial was always present in Angiopteris, but concluded that such ;m initial cell was not present in the stem of the young sporophyte. Breb- in i i Brebner 2) says that in Dancea simplicifolia such a single initial cell seemed to be always present in the stem, and my studies on Angiopteris, Kaulfussia, and Danan indicate that a single initial is developed ;it an early period in the stem apex and persists until the sporophyte has developed several leaves. Before any division occurs in the embryo the fertilized cell increases markedly in size, aftei which there is formed the horizontal basal wall (rigs. 106, 109, l> b). This is probably followed at once by the median wall (except in Datura), so that the embryo at this stage is divided into four approximately equal quadrants (see fonk- mann 3, tig. 9). The genus Danaa, at least this is true for D. jamaicensis and /). elliptica, differs in the early divisions of the embryo from the other Marattiaceae. The egg cell after fertilization elongates in a way which closely resembles that found in Botrychium obliquum ( Lyon I ). I he primary hypobasal cell either divides no further or only once and forms a short suspensor, so that all the organs of the young embrj 0, including the foot, are really ot epibasal origin. As yet no trace of such a suspensor has been found in the other Marattiaces?. THE EMBRYO OF MARATTIA. Luerssen (Luerssen 2) found the one-celled embryo and young plants of Marattia cicutafoha, but was not able to procure the older embryos. The writer (Campbell 3) succeeded in procuring several stages of the embryo in Marattia douglasii, and somewhat later Luerssen described and figured some of the earlier stages in M. fraxinea and M. weinmannttefolia. I hese, so far as I am aware, complete the list of records upon the embryo of Marattia. The fertilized ovum in Marattia douglasii becomes much enlarged before it divides and completely fills the venter of the archegonium. The granular contents of the egg cell become evenly distributed without any apparent increase in quantity as the fertilized ovum grows, so that the one-cell embryo contains comparatively little granular contents, but the nucleus is ver) conspicuous. At the time of tin- first division the young embryo is almost perfectly globular in form. I was unable to find the stages immediately following this, but fonkmann has figured an eight- cell embryo of M. fraxinea (see Jonkmann 3, fig. 9). The basal wall is transverse and this is followed successively by the median and transverse walls, so that the globular embryo is divided into approximately equal octants. From the hypobasal half, which is nearest the archegonium, there is developed the foot alone, while all of the other organs arise from the epibasal portion, which is turned away from the archegonium. The youngest embryo (except tin- one-celled stage) which I found in \/. douglasii, is slmwn in fig. \oh,A. This is about the same stage as the one shown in fonkmann's figs. 11 and 13. Unfortunately this embryo was rather badly shrunken in the process of embedding and the division walls were a good deal distorted, so that it is rather hard to determine exactly their correct relation to each other; but probably tin- wall b b represents the basal wall and tn the median wall. The embryo has now lost its original globular form and become oval, the long axis lying transversely. The secondary divisions in the octants are mostly anticlinals, and the first periclinals have just appeared in a few of the cells. Figure 106, A, shows two consecutive median sections. It will be seen that cell division is more active in the epibasal portion than in the hypobasal region. THE EMBRYO 137 In my original study of Marattia I concluded that the primary divisions resulted in the establishment of the organs of the young sporophyte in a manner similar to that in the Leptosporangiatae, i. e., that the stem and leaf were derived from the epibasal quadrant, the root and foot from the hypobasal ones. Farmer thought that this was true for Angtopteris and Jonkmann assumes that it is the case also in Marattia. A further study of my preparations, however, has led me to believe that the whole of the hypobasal region is devoted to the foot, while the root, together with the leaf and stem apex, are of epibasal origin. A large cell (fig. 106, A) occupies approximately the same position as the stem apex in the older embryo and it is possible that this may be the apical cell of the stem, but in the absence of the inter- mediate stages this can not be positively asserted. For a long time the embryo retains the oval form and there is scarcely any sign of the young organs of the sporophyte which in the leptosporangiate ferns are so early manifest; indeed, Jonkmann states that there is no differentiation at all, but a careful study of the older embryo shows unmistakable indications of the definitive organs. The devel- opment of the tissue in the epibasal region is not uniform, but somewhat to one side of the center (fig. 106, B) there may be seen a group of columnar superficial cells, which mark the position of the future growing point of the stem. Whether one of these superficial cells is the definitive apical cell it is impossible to determine, but from a comparison with the older stages, as well as with corresponding stages of the embryo in the other genera, it seems proba- ble, at least, that this is the case, and perhaps this initial cell may be traced back to the centrally placed cell noted in the younger embryo. A considerable portion of the epibasal tissue is not included in the meri- stematic region, which is derived mainly, at least, from only one of the original epibasal quadrants. The tissue adjacent to this meristematic region is made up of large cells with less dense contents and smaller nuclei and differs in no way from the large cells of the foot, into which this tissue insensibly merges and of which it may be said to constitute a part. In the embryo figured, it was not quite certain whether the root apex had begun to form or not. It seems probable that the cell r is really the apical cell of the young root, but except for its position it was not noticeably different from the cells adjacent; however, as in Angioptnis and Darura there is no question that the root originates in this position, it is probable that this cell is really the initial tor the young root and is cut out from the base of the epibasal tissue near its junction with the foot. This marked endogenous formation of the root is very much like that in the embryo of Ophioglossum moluccanum, but differs entirely from the superficial origin of the apical cell of the root in the embryo of the Leptosporangiates. The stem apex in the older embryo is of very limited extent, consisting of only a few cells, of which one, as we have said, is probably the definite apical cell, although it must be said that this point is very difficult to decide, as all of tin central cells of this apical group look a good deal alike, but a careful study of both transverse and longitudinal sections seems to point to one of these as tin apical cell, which in shape is a good deal like that in the stem apex of Ophioglossum. In longitudinal Fig. 107. Two cross-sections of an old embryo oi Marattia A passes through the stem apex, J/, and base ,>t COtyli don, B passes through root apex, r. X200. 138 I III MAK.VI HALES sections this is somewhat wedge-shaped, the narrow end turned outward and the broad base below. In cross-section this cell is nearly square in shape (rig. 107, A,x) and there may be seen a fairly regular series of four segments cut from the lateral faces. From the broad truncate base of the cell, segments are also cut off which contribute to the inner tissue of the stem.* Much the greater part of the epibasal meristem contributes to the cotyledon, which is soon evident as a broadly conical protuberance, somewhat flattened on the side adjacent to the stem apex and merging gradually on the outer side into the large- celled tissue which adjoins the foot. To judge from the limited number of young embryos which I could examine, it seems that the growth of the cotyledon is not due to the activity of a single apical cell, but this point was somewhat uncertain. By the time that the cotyledon is established, growth has progressed in the young root, which now is seen to have a conspicuous apical cell, which divides rapidly so that the root quickly elongates in a direction opposite to that of the cotyledon (tig. 108). The apical cell of the primary root in M. doiiglam is not triangular in outline, but more or less quadrilateral, whether seen in longitudinal or cross sections (fig. 107, B). In form and segmentation it perhaps more nearly resembles that of Dancea than it does the tetrahedral apical cell which occurs in the young primary root oi'Angiopteris. Active cell divisions take place also in the tissue of the foot, which completely incloses the young root and becomes practically merged with it, so that it is quite impossible to say, at the time the root emerges, just how much of the tissue of its outer portion really belongs to the root itself and how much is derived from the original tissue of the foot. The latter is now no longer recognizable as such, the young sporophyte apparently at this time being composed almost entirely of the cotyledon and the very huge root, with the stem apex, King near their junction. From a comparison with younger stages, however, it is perfectly evident that the I 1 . 10S. V Section of an advanced embryo o\ Xtttratti ■ ■m e "t cot) ledon. If. The cot) ledon, more enlarged. jusi before larged region of the embryo at this time is composed mainlv of tissu belonging to the foot, which is, so to speak, perforated by the root in its downward growth. The development of the vascular bundles at a very early period is first evident in the cotyledon, which almost as soon as it can be recognized at all is seen to have a strand of procambium extending below it. If this procambium strand is traced <1<>\\ nward, it is seen to continue without interruption into the base of the root, exactly as it does in Ophioglossum moluccanum. No trace of a procambial cylinder can be found extending into the stem apex, which gives rise only to the parenchyma of the central pith. This early development of the vascular bundles in the cotyledon and ioor was correcdy observed bv Jonkmann and Farmer, both for Marattia and * In a very recent paper in the Botanical Gazette (Feb. 191 1), Miss Charles states that the apical cell of the stem in Marattia alata is at first triangular in cross-section. In older sporophvtcs the single apical cell was replaced by a group of initial cells. THE EMBRYO 139 Angiopteris, but thev seem to take it for granted that a vascular strand is also developed in the stem; at any rate they make no reference to the absence of such a vascular strand from the stem region. In my earlier study of Marattta, I supposed that a strand was developed which belonged to the stem itself, but a further exam- ination of many plants, after a study of this point in the other genera, has made it pretty clear that this supposed stem bundle really belongs to the second leaf, the rudiment of which appears at a very early period. THE EMBRYO OF ANGIOPTERIS. In Angiopteris the embryo very early becomes more greatly elongated trans- versely than is the case in Marattia, so that in a longitudinal section it appears as a very much depressed oval (fig. 109); the epibasal region is larger than the hypobasal Fig. 109. A. Two sections of a young embryo of Angiopteris. The cells were badly shrunken. B. Diagrams showing arrangement of cells of same embryo. C. An older embryo, b b, basal wall; sr, stem apex; cot, cotyledon; f, foot. and the quadrant divisions are often still evident. Whether octant divisions are formed in all the quadrants could not positively be determined, but Jonkmann states that such is the case. There can usually be found in the young embryo a nearly centrally placed large cell (tig. log, C, si), which probably is the initial cell of the young stem. The position of this cell is not unlike that which occurs in the Fig. 1 10. Two longitudinal sections of an older embryo of Angiopteris. X200. The young root, r, is shown in /?. st, stem apex; cot, cotyledon; /, foot. embryo of Equisetum, and at this stage there is also a certain resemblance to the em- bryo of Botrycht um virginianum. As the embryo grows it tends to assume a more nearly globular form (fig. no). The basal wall can still be imperfectly followed, the hypobasal portion of the embryo being made up of the large cells forming the foot, while above the basal wall the cell divisions are more active and the rudiments of 140 THE MARATTIALES the stem and cotyledon can be recognized, although the embryo still retains its oval outline. As in the embryo of Marattia, growth is most active in the central region of the epibasal part of the embryo and there can generally he recognized a large central cell, which is presumably the single initial of the stem apex, although, as in the case of Marattia, it is not absolutely certain that a single initial cell is always present. Cell division is especially active on one side of the stem apex, and this area marks the position of the young cotyledon. The limits of this growth area are Fig. hi A. Two longitudinal sections of an old embryo of Angioptgrh. X75 B. Stem region of same embryo. X160. C. Root apex. X160. not very sharply defined and it is difficult to say whether it can be traced back to a single quadrant or octant cell, but it is probably not always constant in its position; indeed, the stem looks as if the stem apex and the cotyledon both arose from the same quadrant, the second epibasal quadrant contributing, at least in part, to the foot. As seems to be true in all the Marattiacese, the root makes its appearance at a comparatively late period and it is evident that the root, as in the embryo of Marattia, is a strictly endogenous structure. The first indication of the mot is the development Fi<;. 1 12. A. Longitudinal section of an advanced embryo <>f Angiopttrh, tut in tin- plane <>l tin* lamina of the cotyledon. X50. B. Cotyledon of same embryo, showing dichotomy "t .iy<-\. ■ 220. C. Root apex of same. X2io. of a group of actively dividing cells, almost in the center of the embryo below the stem apex. This meristematic region is probably always of epibasal origin, but it is close to the basal wall and it is possible that sometimes it may arise below it. The apical cell, which in the early stages almost always appears triangular in section, becomes conspicuous and was recognized by Farmer in his study of the embryo of Till IMIiKMi 141 Angiopteris (Farmei I ). I lit root initial now divides by regular segmentation and the root apex pushes rapidly down through the underlying foot and ultimately emerges on the lower side of the prothallium. In the meantime the cotyledon grows actively and there is a rapid elongation of the whole embryo in a vertical direction. As in the case of Marattia, it is difficult to prove that the cotyledon grows from a single initial cell. The cotyledon has very much the form of that in Marattia, growth being more active on the outer side, so that it curves over the stem apex very much as the cotyledon does in the embryo of Botrychium virginianum. About the time that the cotyledon is ready to emerge, the apex becomes flattened out and (sometimes, at any rate) there is a true dichotomy of this apex (see fig. 112, B). In the primary root there seems to be no question of the presence of the single initial cell which, at first at least, has the tetrahedral form, but later on is apt to have the base truncate, although it usually has three series of lateral segments. I have not been able to confirm Farmer's statements that the single apical cell is later replaced by a group of similar initials, as in the later roots of the sporophyte, although it is not at all impossible that such may sometimes be the case. The development of the embryo of the vascular bundles in the young sporophyte is exactly the same as in the corresponding stages of the embryo in Marattia. THE EMBRYO OF KAULFUSSIA. So far as I am aware, no account has been published of the embryo in Kaul- fussia, except one of my own (Campbell 1 1). Only two young embryos were found, so that it was impossible to follow in detail the early history of the young sporophyte. The basal wall, as in the other Marattiaceae, is transverse and, to judge from a comparison of similar stages of the embryo in Marattia and Angiopteris, all of the organs of the young sporophyte in Kaulfussia, except the foot, are also of epibasal origin. Figure 113, A, shows a nearly median longitudinal section of the young embryo. This is very much elongated transversely, and to judge from the position of the cells the basal wall is probably followed by the median walls, forming nearly equal quadrants. The large cell (st) in one of the epibasal quadrants corresponds in position to the similar cell found in the embryos of the other genera and very likely may represent the primary initial cell of the stem. The embryos, however, Fig. 113. — Young embryos of Kaulfussia. X275. V Longitudinal section. R. Three cross-sections of a similar embryo, b b, basal wall; 11, quadrant wall. were too young to make clear the relation of the cotyledon and primary root to the stem. Three nearly transverse sections of an embryo of about the same age are shown in fig. 113, B. To judge from the structure of the older sporophyte at the time when it first emerges from the prothallium, Kaulfussia agrees in the main with Marattia and Angiopteris in the origin of the young organs of the sporophyte. u: I ll l MARATTIALES THE EMBRYO < >F l)\\ I \. Brebnei (Brebnei 2) h;is described the oldei embryo oi Datieea simplicifolta, but did not secure the earlier stages. 1 he youngest specimens he figures closely resemble in form a corresponding stage in the species studied by me, and the slightly pointed basal region suggests the possibility of the presence of a short suspensor in D. simplicifolia like that which I have found in D. jamaicensis and D. elliptica. While my own collection of young embryos is not as complete as might be wished, still enough stages wen secured to show that at least /). jamaicensis and D. elliptica Fig. 114. Danaa jamaieenui A. Archegonium containing .1 one-celled embryo. B. Outline of next section of same embryo. C. Three-celled embryo, showing suspensor, ju.<. D. Outline of next section of tin- same embryo. I.. Three longitudinal sections of a four-celled embryo, F. An older embryo, which was shrunken. O. Diagram of F, showing probable arrangement of cells. differ remarkably from the other Marattiaceae that have been studied in the develop- ment of a short suspensor, thus showing an interesting analogy with the embryo of Botrychium obliquum, described by Lyon (Lyon I). Whether D. jenmani shows the same peculiarity was not determined, on account of the failure to obtain the young embryo of this species; but as in its later development it corresponds very closely to the other species, it is highly probable that a suspensor is developed. Fig. 115 A. A nearly median section of an older embryo of D.jamaicenth. The suspensor does not show in this section. B. Three sections of a young embryo of D. tlliptica. juj, suspensor. Xzoo. Before the first division takes place in the embryo, the fertilized ovum enlarges to several times its original size and becomes decidedly elongated. The first division wall, as in the other Marattiaceae, is transverse, but of the two primary cells thus formed the hypobasal one divides no further, 01 only once, and forms a short sus- pensor which pushes up into the neck of the archegonium whose cells become more or less completely disorganized (fig. 114, A). The next division wall is a nearly median one in the epibasal cell, and this is soon followed by a second wall in each THE EMBRYO 143 of the epibasal cells, so that this portion of the embryo is divided into torn nearly equal quadrants. There is some evidence that these are followed by horizontal octant walls, so that the epibasal region is thus divided into octants in much the same fashion as obtains in the whole embryo in other Marattiaceae and in the lepto- sporangiate ferns. How far these divisions are constant can only be conjectured, owing to the small number of young embryos which were available. The elongated pear-shaped embryo in Dana-a appears very different indeed, in these early stages, from the broadly elliptical and much depressed embryos of corresponding stages in the other Marattiaceae (rig. 1 14, C, D, E). Fig. 116. — D. jamakensis. Three sections of an older embryo. X200. Section B is a nearly median section ; A is the next section in the series; C shows the suspensor, sus. A further study of the embryos shows that all of the lower half of the epibasal region, probably that derived from the four lower octants — i. e., the four octants that were in contact with the suspensor — develops into the foot, while all of the other organs of the embryo (leaf, stem, and root) arise from the four terminal octants. Quite early in the development of the embryo, there appears the same centrally placed large cell which we have observed in the embryos of the other genera and which, as we have seen, probably represents the primary stem initial. Brebner concluded that a single initial was present in the stem of D. simplicifolia and this is true of the three species examined by me. The initial of the stem in Danaa becomes Fig. u-j.—D.elliplica. Three sections of a large embryo. X150. jus, suspensor; 5/, stem apex; rot, cotyledon; r, root initial. very easily seen in the older stages and is perhaps more clearly defined than it is in any other of the Marattiaceae. Whether or not this cell can be traced hack to one of the original terminal octant cells is difficult to say, but it is quite possible. No single initial could be made out for the cotyledon, and it seems quite likely that the position of the cotyledon is not always exactly the same, being determined perhaps by the position of the embryo with reference to the light or to some other factor. The cotyledon, as in Marattia and Angiopteris, first appears as a slight prominence close to the nearly centrally placed stem apex, and there is soon visible 144 I 111 MARATTIALES .1 group acti\ 1 1 \ dividing cells which constitute its growing point, bul no one <>f these can be certainly named as the apical cell (fig, 117, 3). I In- embryo now rapidly increases in breadth until the apex is almost H;ir, except for the slight pro- jection in rlu- center, where are situated the stem apex and the young cotyledon. The whole embryo at this stage may be described as "top-shaped." '1 he stem and cotyledon occupy a comparatively small portion of this broad terminal area and are surrounded by a ring of large absorbent cells, physiologically, at least, belonging to the foot ami merging insensibly into the similar cells which make up the lowei halt of tin embi yo. FiG« 118. — D.jamaicensis. A, B. Two sections of a large embryo. X150. col, cotyledon; si, stem; /.foot. C. Root apex of same embryo. X360. As in the other Marattiaceae, the very young embryo shows no trace at all ol the root and this appears only after the embryo has reached a comparatively large size. The origin of the root is exactly the same as in the other genera and it is a strictly endogenous structure. The single initial cell arises deep down in the tissue of the embryo, usually below the cotyledon and probably from the same octant as that from which the cotyledon is formed, but owing to the displacement of the original division walls this can not be determined positively and it may be that the position of the root is not always exactly the same. The apical cell of the root soon 1 1 -. 119. D.jamaicensis. A. Two sections of an advanced embryo. 100. B. Stem apex of the same embryo. 220. becomes conspicuous, but it does not usually show the triangular form as seen in a longitudinal section, hut appears more nearly square, as in Marattia. 1 his root initial cell is quite variable in form, hut more commonly it appears in longitudinal section with a truncate base (fig. 117, ;;. In transverse section it approaches the triangular form, but is more or less irregular in outline, 4 he lateral segments cut off from the initial cell are large and contribute later to the root cap as well as to the inner root tissues, and it is not impossible (as Farmer believes to be the case also THK EMBRYO 145 in Angiopteris) that sometimes more than one of these terminal cells function as the apical cell. As soon as the root apex is established its growth is very rapid and, as in the other genera, there is a rapid elongation of the whole embryo, whose vertical diameter very soon becomes greater than the transverse diameter, and ultimately the whole embryo becomes very much elongated. In the axial region of the embryo just below the stem apex there is a marked elongation of the central cells which might at first sight be taken to represent the central vascular cylinder, but these elongated cells do not give rise to vascular elements, but remain as elongated parenchyma and belong really to the pith. The part of the foot which lies below the growing root apex acts as a very massive root cap and is pushed down with the growth of the root until the latter emerges from the lower side of the prothalhum. The cotyledon in the meantime grows actively upward and finally penetrates the prothallium, emerging Fig. 120. — D. jamatcrruis. Five of a series < f transverse sections from an advanced embryo. X200. I passes through stem apex; 5 shows apex of root . on the upper side. The growth of the stem is slight and the cotyledon and root form nearly a straight line, as in the other Marattiacea? and in Op/uoglossum, so that the young sporophyte may be described as bipolar. The equatorial region is surrounded by the large absorbent cells in contact with the prothallium and all of these may be said to function as the foot, although it is impossible to say how much of this tissue is derived from the original foot. Til \N\1()MV AND HISTOLOGY OK THE YOUNG SPOROPHYTE. The young sporophyte, at the time the root emerges from the prothallium, is very much alike in all of the genera (figs. 108, ill, I2l). The cotyledon at this stage is a thick, conical protuberance, strongly curved inward over the stem apex, owing to the more rapid growth upon its outer side. Close to its base lies the very limited growing region of the stem, adjacent to which is the rudiment of the second leaf, which at this time projects very slightly above the level of the stem apex. The cells of" the latter, and also those of the young leaves, are evidently actively growing cells with conspicuous nuclei and are quite different in appearance from the large, transparent cells that compose the enlarged mid-region of the young sporo- phyte. This is largely made up of the original tissue of the foot, but this tissue merges insensibly into the basal region of the cotyledon and the upper part of the root, these two organs forming in their growth almost a straight line. \\ hether or not we term this middle region of the young plant the "stem," it must be borne in mind that it does not arise from the activity of the extremely limited meristem, forming the true stem apex. The young sporophyte is traversed by a single conspic- uous vascular bundle which extends through the cotyledon and mot without inter- 10 146 Till MARATTIALKS ruption, and it is quite impossible to say just exactly where the point of junction is. In Angiopteris elongated tannin cells, which stain very strongly, accompany the vascular bundle, both in the rout and cotyledon; bur these are either entirely wanting or bur slightly developed in Marattia and are quite absent from the young sporophyte at this stage in the other genera. In the relation of the primary root and leaf, there- fore, the embryo of the young sporophyte in the Marattiaceae shows a most striking resemblance to the condition found in Ophioglossum moluccanum. 1 he cotyledon, as. in Ophioglossum, is not to be looked upon as an appendage of the stem, but as an organ sui generis. Almost as soon as the second leaf is recognizable, there is evident, connecting it with the primary vascular strand, a short group of procambium cells, and the stem apex is seen to occupy the space between these two leaf traces. No procambium is developed in the stem above the junction of the leaf traces, but the inner cells, derived from the apical meristem, contribute solely to the medullary tissue of the sporophyte (fig. 121, A). Fig. I2i. Dantea jamaicensss. A. Large embryo, cot, cotyledon; /2, second leaf. X 50. B. Median section of cotyledon; h, epidermal scale. X95. C. Second leaf. X150. D. Trichomcs from apical region. The first tracheary tissue arises in the mid-region of the young sporophyte and consists of a group of short reticulate tracheids. From this point the development of the tracheary tissue proceeds upward into the cotyledon and downward into the root. The single strand of tracheary tissue in the cotyledon is seen to be continuous with one of the xylem masses of the diarch root. The second xylem of the root arises somewhat later and is connected with the xylem of the second leaf. This second xylem mass is decidedly smaller than the first one, at least in Danaa, where I have studied this point carefully, and an examination of the other genera points to a similar inequality in the xvlems of these as well, but this may not always be the case. 1 in. COTYLEDON. The cotyledon at a very early period bends strongly over the stem apex, very much as it does in Botrychium virginianum, and very soon afterwards begins to flatten out, so as to indicate the separation of the lamina from the petiole. The flattening of the apex is followed, in many cases at least, by a true dichotomy, which is soon repeated, so that the lamina becomes fan-shaped, with a strictly dichotomous venation (fig. 87, G, H; fig. 127). This shows especially well in Marattia iouglasti. In M. sambuctna (fig. 87, C) the cotyledon is more nearly orbicular than in M. douglasti, but the venation indicates a similar early dichotomy of the apex. In Angiopteris, the form of the cotyledon is extremely variable (fi^. 124). Farmer states that it has a distinct midrib extending to the apex of the cotyledon THE YOUNG Sl'OROl'HYTE 147 and that the secondary veins are pinnately arranged with reference to the midrib. These later veins, however, may show dichotomous branching. I made an examina- tion of a considerable number of young sporophytes collected at the same place where Professor Farmer secured his specimens, but very few of the plants that I collected showed this pinnate venation in the cotyledon, although it is usually conspicuous Young sporophyte oi Marattia douglasii still attached to thr gametophyte, pr; Natural si/r. Fig. 12^. A. Young sporophyte of Kaulfussia attached to gametophyte, pr. X2. B. Cotyledon from another plant. X2. C A later leaf; si, stipule. Xl. in the second leaf. Figure 124, C, shows drawings of a number of the forms from my collection, showing the variation from a strictly pinnate to a perfectly dichoto- mous venation. Many of them show an intermediate venation, but with very few exceptions, even where a midrib was present, this was forked at the apex, indicating F10. 124. A. Young sporophyte of Angiopttris, with three leaves, i, the cotyledon. Xz. B. Young cotvlcdon. X20. C. Three cotyledons, showing the variation in form. X3. that there had been an early dichotomy. This primary dichotomy of the apex was well shown in a section through a very young leaf (fig. 1 12, B) where there was no question that such a dichotomy was taking place. In Dancea there is much the same variation in the form of the cotyledon as in Angiopteris. Brebner's figures of D. simplicifolia show that the cotyledon in this species has a midrib with two lateral veins near the base, but sometimes the midrib forks at the summit, indicating again the early dichotomy of the cotyledon. In one 14N I 111 MAR VITI \I IS ot his figures, where there is no forking ol the mid-vein, the lateral veins seem to follow the margin ot the leaf and rejoin the central vein, thus inclosing two large areoles like those found in the cotyledon of Kaulfussia; no mention of this, however, is made in the text. D. jamaii ensis (fig. 125, A) shows a very similar form and vena- Fig. 12c. A. Four young sporophytes still attached to the gamctoplntc of Danaa jamaicensis. ■ :. B. Young cotyledon of D. ell! plica. X15. C. An older cotyledon of D. clliptica. X3.5. tion, but the cotyledon is larger and there is sometimes a forking of the lateral veins. In this species also there may sometimes be found a fan-shaped lamina with true dichotomous venation like that of Marattia, but as I had little material of this species I can not say how common this form is. It is probable that an examination Fig. ij6. A. Young sporophytc "t 0- jamaicensh, with four leaves, slightly enlarged. 1. the cotyledon. B. Base of the fourth leaf, showing stipules, ft, and peltate scale, jr. X20. of a large series of young plants would show examples of the same type of cotyledon as in the other species. The cotyledon of D. elliptica (tig. 125, B, (.') is of much the same form as that of /). jamaicensis, but the cotyledon is larger as a rule and the venation may be more complex, although conforming to the same general ripe. THE YOUNG SPOROPHYTE 149 The cotyledon in Kaulfussia (fig. 123) is spatulate in form and not unlike that of Daneea in shape and also closely resembles in outline the broader leaf forms of Ophioglossum moluccanum. The resemblance to the latter is much increased by the venation, which is reticulate and extraordinarily like that of the cotyledon of Ophioglossum. As in Ophioglossum, there is usually a mid-vein, but this is not, however, noticeably thicker than the laterals. The latter, instead of extending free to the edge of the leaf, are connected at their distal ends with the mid-vein so as to inclose more or less elongated areoles. The central vein does not extend to the tip but, as in Datura, divides into equal branches which join above so as to inclose a fi Fir.. 127. A. Lamina of young cotyledon of Marattia douglasii. B. Section of lamina of cotvledon. X300. X260. a terminal areole. A careful examination of the secondary veins shows that their divisions are really dichotomous, as in the lateral veins of the other Marattiacea?, and some of the ultimate branches may reach the margin of the leaf, or more rarely they may end freely within the areoles. As the cotyledon develops there is more or less elongation of the petiole, which, however, very seldom exceeds the lamina in length. The petiole is usually more or less deeply channeled upon its inner face. In Marattia douglasii (fig. 133) the base of the cotyledon is much enlarged just above the level of the stem apex, which is Fig. 12S. A. Section of lamina «f the mother cell of the stoma (tig. 12c;, C.). The complete stoma appears, then-ton-, surrounded by a series of somewhat concentrically arranged accessory cells (tig. 1 20, /)). Similar accessory cells, but much less developed, can be recognized in Marattia also. The stomata of Kaulfitssiti have long been known for their gnat size and the fact that they form permanently open pores, a peculiarity aheadv developed in the stomata of the cotyledon. These are very much larger than those of the other genera and can be readily recognized by the naked eye as little dots. I he guard cells become very large (tig. 128) and strongly curved, so that the stoma appears almost circular in outline, with a very large permanently open central pore. I his hum pon is surrounded In concentrically arranged series of cells, suggesting that the mother cell of the stoma is cut off very much as in Daneea, but material was want- mi; tni a thorough investigation of this point. THE YOUNG SPOROPHYTE 153 THE STEM OF THE YOUNG SPOROPHYTE. The stem apex projects very little or not at all, and occupies but a limited area close to the base of the cotyledon. Its outer surface is nearly horizontal, except in Angiopteris, where it is generally strongly inclined (fig. 134). There probably is always a single apical cell. Segments are cut off from the sides, and very often also inner segments, where the base of the apical cell is truncate. 1 he lateral segments also have, cut off from their inner faces, cells which contribute to the growth in length of the stem, which remains, however, very short in the young sporophyte, the leaves being very much crowded together. As we have already stated, no central cylinder properly belonging to the stem can be demonstrated at this time, all of the procambial Fig. 135. — Young sporophyte of Angiopteris. A. Stem apex, showing single apical cell. X150. B. Second leaf, /".of same sporophyte. Fig. 1 }6. A. Nearly median section of a ymmg sporophyte of Kaulfussia. B. Another section of same, showing continuity of bundle: "I pr, game- cotyledon and root. / , second leaf ; m, mucilage ducts; stt stem apex; tophyte. X about 20. C. The stem apex. tissue belonging either to the cotyledon or to the mot. In the older embryo of Mar alt 1 a douglasii the apex of the stem (fig. 139, B) is occupied by a group of rela- tively large cells, which at first sight seem to be pretty much alike; but a careful examination of these makes it probable that one of them may be considered as the real initial cell. This, in cross-section, appears almost square and shows a fairly regular segmentation. While it must be said that the segmentation is not so regular that one can assert without question that all of the meristem is really due to the activity of a single cell, I believe that this is probably the case. My own investigations of the stem apex in Angioptern have led to the conclusion that there is a single initial cell here also in the young sporophyte, although Farmer 154 THE MARATTIALES in his earlier studies of this plant failed to find a single initial cell; however, in a more recent paper (farmer 3) he has decided that a single apical cell is present in the young sporophyte of Angiopteris, and he gives figures of this. In longitudinal section (fig. 134) the apical cell appears oblong, with a markedly truncate base, while in transverse section it is imperfectly triangular in outline. I he apical cell of the stem in Kaulfussia (fig. 136, C) is usually somewhat broader than it is in Angiopteris, and closely resembles that of Ophioglossum moluccanum. It has a broadly truncate base and is somewhat narrowed above. In transverse section it approaches the triangular outline of the apical cell in Ophioglossutiu but the seg- mentation is less regular and in this respect it is not unlike that of Angiopteris. The stem apex was examined in three species of Datura, all of which seemed to agree Fig. 137. — Kautju A, B. Two transverse sections of a young sporophyte, above C. Transverse section of petiole of cotyledon. Xoo. level of stem apex, show'ng closed sheath formed by D. Vascular bundle of cotyledon, more enlarged, base of cotyledon. I1, second leaf. X50. E. Second leaf. closely in the form of the apical cell, which is much more definite in Dancea than it is in any of the other genera. Brebner's account and figure of the apical cell in D. simplicifolia agree closely with my own preparations of D. ellipttca, D. jenmam, and D. jamaicensts. The apical cell (fig. 140, A), seen in longitudinal section, is very deep and, unlike that of the other genera, is not usually, at least, truncate below, but pointed, so that it closely resembles in form that of Botrychium or llelmmthostachys. In transverse section it also usually appears triangular, so that it is tetrahedral like that of Botrychium. While the segmentation is somewhat less regular than in Botrychium, nevertheless the limits of the younger segments can be followed without much difficulty, especially in longitudinal sections, although the exact sequence of the divisions in the segments themselves did not seem to be absolutely regular. In all of the species the tissue derived from the inner cells of the segments of the apical cell, or cut off from the base of the apical cell itself, remains as undif- ferentiated parenchyma and contributes only to the central pith of the stem. Except THE YOUNG SPOROPHYTE 155 that the leaves arise from the segments of the apical cell of the stem, and their tissues are therefore indirectly derived from the stem apex, we may say that the stem apex takes no part in the differentiation of the nbro-vascular system, which in the young sporophyte is composed entirely of the leaf and root traces. THE ROOT. Only a small number of sections of the primary root were obtained in Marattia Jouglasii, and these showed some variation, so that it is not possible to state posi- tively what is the typical form of its apical cell, but there is no question that the primary root grows from a single initial cell, as in the other genera. In longitudinal section it appears oblong, with a broadly truncate base from which segments arise, as well as from the lateral faces. There seem to be regularly four sets of lateral cot Fig. i A. Nearly median section of a young sporophyte of Danga jamaicensis, passing thruugh stem apex. X about 40. B. Another section of same, passing through second leaf, I2 C. Section of another^ similar sporophyte cut at right angles to that shown in A and B, and showing continuity of bundles of cotyledon and primary root, pr, gametophyte; tc, scales. segments, although cross-sections of the apical cell sometimes appear almost trian- gular. The root cap is formed in part from segments derived from the outer face of the apical cell, in part from similar segments cut off from the outer portion of the youngest lateral segments of the apical cell. The central cylinder of the root is formed in part from the basal segments of the apical cell, but the lateral seg- ments also contribute to it (fig. 141, D). In Angiopteris (fig. in, C) the apical cell in the earlier stages of the root may appear triangular in longitudinal section, but in the later stages it is usually more or less truncate. The primary root in Kaulfussia, in the few cases where satisfactory sections were made of it, did not show the tetrahedral apical cell, but this was truncate at the base and in cross-section appeared four-sided (fig. 142). In this respect it seems to approach Marattia. The primary root in all of the Marattiacea- is ordinarily diarch; 156 THE MARATTIALES the statement (Campbell 3, page 14) that in Marattia douglasii tin- primary root is tetrarch I have found to be based upon a mistake, the root which was so de- scribed not being the primary root, but evidently one of the later ones. I have occasionally found very strong plant- lets m Dan, in, where the primal \ root was triarch, as it sometimes is m Botryi hium, and very commonly is m tielminthostachys; but such spe- cimens are not common. The root bundle is bounded by a conspicuous endodermis, whose cells show very plainly the characteristic radially thickened bands (rigs. 14}, en; 144). The endodermis gradually loses its definite appearance at the junction of the root and leaf bundles, and I have not been able to recognize it above the level of the base of the cotyledon, although it is not at all impossible that with proper staining it might be traced further up. THE SECOND LEAF. The second leaf begins to develop while the cotyledon is still small, and by the time the latter ruptures the overlying prothallium tissue the second leaf is clearly evident as a flattened cone, closely resembling the early stage of the cotyledon. In Kaulfussia the second leaf arises quite close to the cotyledon and on the same side of the stem apex, so that the dorsiventral character of the stem is already indi- cated at this early stage of development. In the other genera the second leaf arises nearly opposite tin- cotyledon, bur not I'IG. 39- A. Transverse section of a young sporophyte of Marattia tlouglmi st, stem apex. X150. B. The stem apex. X360. Fig. 140. A. Stem apex of a y< phyte of Danaa jamaicen is, showing single apical cell B. Se< ond leaf from same. C. D. Set lions i>i primary roots, showing apical meristem, 1S0. i» exactly so, and the subsequent leaves form a spiral, and for a rime at hast, except in Kaulfussia, the anatomy of the shoot is radial. Ibis radial arrangement is retained permanently in Marattia and Angiopteris and also in some species of THK VOL' NO SI'OKOPHYI 1 157 Daneea, e.g., /). elliptica; but in I), jenmani and in I), ^amatcensts, as well as in most of the other species, the stem finally becomes dorsiventral, as it is from the firsr in Kaulfussia. The development of the second leaf is marked by an increased activity in the apical meristem, quite close to the apical cell, and it is clear that the leaf arises from the younger segments, but it is impossible to state exactly what the relation of the young leaf is to the younger segments of the apical cell, or to say whether the whole Fig. 141. A-C. Three transverse sectiuns of primary root of Mtirattia douglatii. D Longitudinal section of apex of primary root. of the leaf is the derivative of a single segment. When the young leaf can first be distinguished it is elevated very slightly above the level of the stem apex and its growing point is composed of a group of columnar cells. Sometimes, especially in Datura, there may be recognized near the center of this group of cells a single, somewhat larger one, which may be interpreted as a single apical cell, but this is not always recognizable. Fig. 142. A, longitudinal, M. transverse, sections of apex of primary root of Kaulfussia. X200 C. Apex ot second root. D Transverse section of root apex. Even before there is any noticeable projection ol the leaf rudiment, then is developed beneath it the beginning of the short strand of procambium which is to form its vascular bundle, and this can be followed downward to a point below the stem apex, where it joins the trace from the cotyledon. Above this junction there is no evidence of procambium tissue extending into the stem apex, but the whole tissue lying between the junction of these two primary vascular bundles and the apical meristem of the stem is made up of undifferentiated parenchyma. The outer surface of the stem apex may be nearly horizontal or (especially in Angiopteris) it may be strongly inclined and a single fairly conspicuous initial cell 158 IHI MARATTIALES may usually be recognized without difficulty. I his cell is usually quite deep and in all of the genera except Danaa has a truncate base in longitudinal section. In Dance a, however, the base is usually pointed ami the cell closely resembles the corre- sponding cell in Botrychium, or the typical leptosporangiate ferns. I he growth of the stem is usually rather slow ami the leaves of the young plant are much crowded. In all of the forms examined, the tissue derived from the inner cells of the segments and from the basal segments of the apical cell, when- such segments \\> r< formed, remained as undifferentiated parenchyma and contributed only to the internal pith of the stem. Except that the leaves arise from the segments of the apical cell of the stem and all of their tissues are therefore indirectly derived from it, the stem apex of the young sporophyte takes no part at all in the development of the fibrovaseular system of the stem, which, except for the bundles of the roots, is entirely composed of the leaf traces. The second leaf in Angiopteris usually shows a pretty well-defined but slendei midvein, which may, however, fork at the apex, and this is true also of the third leaf. It is in the third leaf, both in Marattia and Angiopteris, that the characteristic- stipules are first developed. These appear as lateral wing-shaped bodies close to the Fig. 143. \. Section of primary root of Vanxa jamaicensis. X70. B. The vascular bundle. X175. en, endodcrnu C. Triarch bundle from a primary root of D. elliptica. base of the petiole, partially inclosing the next youngest leaf and the stem apex. The third leaf in Angiopteris and several others succeeding it assume a more ami more pronounced lanceolate form with a prominent midrib and pinnately arranged lateral veins which usually fork once. The margin of the leaf is serrate. After several of these lanceolate leaves have been developed, leaves are formed in which the base is provided with auricles, and these gi adually pass into tin next tvpeof leaf, which is ternate, the terminal leaflet being very much larger than the lateral ones (plate 13, figs. 4, 5). These ternate leaves then pass by intermediate stages into the pinnate form of the leaves of the adult plant. In Kaulfussia the second and third leaves closely resemble the primary one, except that they are somewhat larger (fig. \ii,). The second leaf develops well- marked stipules which are found in all of the later leaves. The earlier leaves do not show a definite midrib, though there is usually a delicate midvein at the base of tin lamina, but this vein forks usually about half-way up. In the older leaves (fig. 123, C) there is developed a stout midrib, from which extend distinct pinnatelv arranged lateral veins, much as in Angiopteris, but these lateral veins are connected by a system of anastomosing veins, so that the leaf most strikingly resembles that of a typical Dicotyledon. It is not until a very late period that the leaves in Kaulfussia assume the compound form of the older plant (plate 1 1, fig. 1). THK YOUNG SI'OROPHYTK 159 In Dancra a dozen or more leaves may be formed before there is any branching of the lamina. While the cotyledon may show an approach to the pinnate venation of the older leaves, there is almost always a dichotomy of the apex of the slender midvein, and this is generally true also for a number of the subsequent leaves, although in these later leaves the midrib becomes well marked and the lateral veins are developed apparently monopodially. In D. simplicifolia the leaves remain permanently unbranched, but in all of the other species the later leaves are once pinnate. The intermediate leaves have the ternate form found in Marattia and Angiopteris, and after a few of these, about two or three, have been formed, five foliate leaves appear and the number of leaflets gradually increases, as new leaves are formed, until the full number of leaflets is developed. The early ternate leaves often have the terminal leaflet much larger than the lateral ones, and the leaf Fig. i 1-5. Transverse sections from a series of a young root of Danaa jarnaicrnsis. 2 shows apical cell. 6. Section of bundle fr&m fully developed primary root, en, endodermis. closely resembles the corresponding stages in Angiopteris. The petiole in most species is more or less obviously winged. In Dancea the stipules are (sometimes, at least) not formed until the fourth leaf. In the fourth leaf (fig. 126, B), however, they are well developed, appearing as two conspicuous wing-like organs with senate edges. In all of the genera there is a root formed for each leaf in the earlier stages of the sporophyte, but how late this continues was not determined. In the older sporophyte more than one root may be formed for each leaf. In Kaidfussia the second root is developed earlier than in the other genera, beginning to develop while the second leaf is still very small, and in general the roots in Kaulfussia seem to develop earlier than in the other genera. The second root is especially late in developing in Duntea, the very first rudiment of the root making its appearance between the first and second leaves, after the latter is already quite well advanced. 160 THE MARATT1 VLES III. I III oi.DI R SPOROPHY II I'hf vascular system ol the adult sporophyte in the Marattiaceae is extreme!) complex, especially in the larger species of Mar atti a and Angiopteris. In Datura and Kaulfussia it is simpler, hut in these also the arrangement of the bundles in the adult stem is by no means easy to trace. A thorough study of the condition in the earlv stages of the sporophyte is therefore specially important for understanding the much more complex arrangement of the bundles in the older plant. In view of the conditions that we have found to exist in the ( )phioglossaceae, and particularly the nature of the vascular system in the voting sporophyte of Ophioglossum moluccanum, a careful reinvestigation of the developmental bistorj of the vascular system in the young sporophyte of the Marattiace;e, especially the simpler and presumably more primitive genera Danaa and Kaulfussia, was very much needed. To this end series of sections were made of young sporophytes of both of the latter genera, ranging from individuals in which the second leaf was still undeveloped to those in which a dozen or more leaves had been formed. These series were fairly complete and made it possible to trace quite satisfactorily the evo- lution of the vascular system in the young sporophyte of both Datura and Kaulfussia. Brebner ( Brebner 3) has given an accurate account of the distribution of the bundles in the young sporophyte of Datura simplicifoha, which agrees quite closely with the arrangement of the bundles as I have found them in the species studied by me. Jeffrey ( |effrey 3) has described and figured a few specimens of D. alata ( ?), but his material was too incomplete to make the study at all satisfactory. The available material of Mar atti a was much less complete, although a numbei of stages were secured. Farmer has made an exhaustive study of the vascular system in the young sporophyte of Angiopteris (Farmer 3), and an extended study of An- giopteris did not seem necessary, therefore, except for a study of the early stages to determine the origin of the vascular bundles. All of the writers mentioned con- cerned themselves almost exclusively with the fully developed bundles, beginning their study at the base of the stem and working upward. No attempt was made to follow out the development of the vascular bundles from the meristematic region of the apex and proceeding downward, a method of study which we think would have materially modified the conclusions drawn by these observers in regard to the relation of the different parts of the vascular system. In my studies as to the origin ol tin- vascular system in the young sporophyte of the Marattiaceae the course of the bundles has been carefully followed in series of microtome sections, passing downward from the apical meristem to the base of the sporophyte, where tbe axial primary bundle passes into the primary root. These series of transverse sections were then compared carefully with series of longitudinal sections of young plants of approximately the same age, and in this way a clear conception was obtained of the arrangement of the vascular system within the stem of the young sporophyte. THE 1)1 \ I I OPMl NT OF THE VASCULAR SYS I I M l\ DAN E V Especial attention has been given to the development of the vascular system in Datura and Kaulfussia, as these have received less attention than Mar atti a and Angiopteris, and moreover are probably more primitive forms whose structures have departed less from the ancestral type than those of either Marattia or Angiopteris. A longitudinal section of the young sporophyte of Danaa jamatcensis at the period when the root is just about to emerge is shown in fig. 121, A. The root in this species, as in D. simplicifolia, described by Brebner, is the first organ to appeal THE OLDER SPOROPHVTE 161 outside the prothallium; the cotyledon at this time is still very small and the lamina is scarcely developed. The root apex does not show in the section figured and all that can be seen is the upper portion of its stele, which joins the two young vascular bundles that extend respectively into the cotyledon and the second leaf, which can already be recognized. The greater part of the young sporophyte is made up of large thin-walled cells. Figure 121, B, shows a median section of the young cotyledon from this same embryo, which is strongly bent over the stem apex. It is possible that the square terminal cell, which in the drawing has the nucleus indicated, is the apical cell of the leaf, but this is not at all certain. The outer part of the cotyledon is made up of large thin-walled cells like those which compose the bulk of the sporophyte, but at its apex there is active cell division, and traversing the young cotyledon and placed somewhat toward its inner side, can be seen the primary vascular bundle, made up of elongated procambium cells. This bundle can be easily followed down into the body of the sporophyte, where, as we have seen, it is Fig. 145. Transverse ection "I i trer) voting sporophyte of Danaa jamaicensiz. Xtco. A shows cotyledon apex; C, apices ?f the stem and second leaf; G shows root apex. continued without interruption into the stele of the primary root. The stem apex, which does not show in the figure, is in a slight depression close to the base of the cotyledon and shows, as usual, a single deep apical cell. Nearly opposite the cotyledon is the beginning of the second leaf (/ !); this is shown in median section in fig. 1 2 1 , B. The group of narrow columnar cells that compose its apex does not show a recog- nizable single initial cell. A short distance below its apex, the procambium of its vascular bundle becomes differentiated and is continued downward until it joins the bundle from the cotyledon. The tissue arising above the junction of these two bundles and continuing upward into the apex of the stem is composed of absolutely undifferentiated parenchyma and there is no evidence at all of any stelar tissue in the stem above the junction of these two primary leaf traces; indeed, the stem apex lies decidedly on one side of the plane which traverses the vascular bundles of the two leaves. Fig. 145 shows a number of cross-sections from a series taken from a large embryo of Dancva jamaicensis, before the emergence of the cotyledon (//), 11 162 nil- MARAT] 1 M.l S whose apex is still undivided. ('. shows the apices of the stem and second leaf, the former being show n on a larger scale in (/'. The section <>t the cotyledonary bundle, lying to the right of the stem apex, is plainly evident in section (',. D shows the separated bundles of the cotyledon and very young second leaf, which lower down unite into a single strand. Whether we can properly speak of a stem at all in connection with the tissues composing the central region of the young sporophyte is questionable. It is per- fectly evident that this central region is composed partly of tissue belonging to the base of the cotyledon, which is in no sense to he looked upon as an adjunct of the Fig. 146. -Ten transverse sections of a young sporophyte of Danaa jamaicensis. A-C. Thi m apex, which is shown in B F-J. Show the condition of the vascular strand in the lower and more highly magnified in D. Fig. C is reversed in region of the sporophyte. position with reference to A. H, I. Are in the transition region. L. The central region of C. J. Shows the root stele. Xzoo. stem, and partly of the root base; the two structures, i. e., the bases of the root and the cotyledon, merge insensibly into each other. To this tissue, derived from the root and the cotyledon, must be added a large amount ol superficial tissue derived originally from the foot of the embryo. In speaking of the axis of the young sporo- phyte as a "stem" it must therefore be borne in mind that this stem is really a composite structure, partly leaf, partly root, and partly foot. Fig. 138, A and B, shows two longitudinal sections of an older stage after the emergence of the cotyledon. A passes through the stem apex and it is evident that there is no vasculai bundle extending into the stem. The tracheaiy tissue is now well developed in the leaf trace. B shows the trace from the second hat at its point of junction with the bundle from the cotyledon. In fig. 138, C is a similar THE OLDER SPOROPHYTE 163 stage to that shown in A , but cut at right angles to it, so as to show the continuity of the bundles of the cotyledon and primary root, which together constitute the prima- ry axial vascular bundle of the sporophyte, as they do in Ophioglossum moluccanum. The similarity of such a section as that figured to the similar stage in 0. moluccanum is sufficiently obvious. The stem apex is shown in fig. 140, A, and the rudiment of the second leaf is shown in fig. 140, B. Figure 146 shows ten transverse sections from a series made from a young sporo- phyte of D. jamaicensis of almost exactly the same age as that shown in fig. 138, B. A is cut above the level of the stem apex and passes through the cotyledon and apex of the second leaf. The arrangement of the cells at the apex of the latter suggests the possibility of a single apical cell. The base of the petiole of the cotyledon is much flattened and is made up of large, thin-walled parenchyma with no traces of tannin cells or mucilage ducts. The vascular bundle is oval in outline and decidedly collateral in structure. There is no recognizable endodermis, and I shall not attempt to say whether the cells immediately surrounding the bundle should be spoken of as endodermis or pericycle. Except for two small tracheids on the inner side of the bundle the vascular bundle is composed of delicate tissue, apparently parenchyma, but some of the larger elements undoubtedly represent sieve tubes. Brebner states that the sieve tubes in D. simplicifolia (Brebner 3, page 524) do not show clearly in sections mounted in Canada balsam, but when examined in other media they show thick, glistening walls and pitted areas. This point was not further examined in the species under consideration, but would probably agree with the observations made by Brebner upon D. simplicifolia. Section B passes through the stem apex, whose apical cell is shown on a larger scale in D. The cotyledon trace in this section shows three tracheids. In D the section is made a short distance below the stem apex and shows the second leaf trace bending in to join the trace from the cotyledon. The central part of the section is shown more enlarged in E, and it is perfectly clear that the region immediately below the apex and lying between two leaf bases shows nothing which can be interpreted as a cauline stele. F and G show sections taken still further down, illustrating the coalescence of the two leaf traces to form the single axial stele. In G there is seen the first tracheid belonging to the leaf trace from the second leaf, and in H the central bundle is complete and there are seen the two xylem masses corresponding respectively to the two leaf traces. One of these, belonging to the primary leaf trace, is more developed than the other one. The larger xylem mass in this case had five tracheids and the smaller one three. / and / are transitional stages between the bundle of the cotyledon and the root, and, proceeding down- ward, the bundle becomes somewhat smaller and there is a reduction in the number of tracheids, which, however, are plainly seen to be continuous in their development with the two xylem masses in the central bundle of the stem, formed by the junction of the two primary leaf traces, which maintain their identity until they merge into the two xylem masses of the diarch root. Within the root itself the endodermis surrounding the bundle is very conspicuous. The cells making up the endodermis are of large size and the characteristic markings upon their radial walls are very easily seen. The limits of the endodermis become much less clear in the transi- tional region, and higher up it is difficult to make out a clearly defined endodermis. Brebner figures a very irregular endodermis in the stem of D. simplicifolia, and perhaps we may assume that a proper endodermis is present also in the young stem of£>. jamaicensis, but it is certainly very inconspicuous, to say the least, in the central region of the young sporophyte, in strong contrast to the very conspicuous endo- dermis in the root. Farmer (Farmer 3) was able by treatment with sulphuric acid 164 THK MARATTIALKS to demonstrate an external endodermis in the stem bundles of Angiopteris,Marattia, and Kaulfussia, and presumably similar treatment would show its presence in the young s em of Daneea. It is evident that we can not speak of the young sporophyte in Daneea jamai- censis as possessing in its early stage a " protostele," or "haplostele," to use Brebner's terminology, unless we choose to call the single leaf trace of the cotyledon a protostele. |ust so soon as the two first leaf trans unite, the axial vascular bundle of the young sporophyte has two quite independent xylems. Figure 147 is a longitudinal section of a sporophyte of D. elliptica, slightly older than that of D.jamaicensi i shown in fig. 138. The second leaf(/2) had already begun to develop the lamina, whose apex was forked so that there wire two veins present. A longitudinal section of one of the two leaf loins is shown in D. The stem apex closely resembles that of D. jamatcensis, the apical cell having much the same form and size. The stem apex does not lie in the same plane as the leaf traces Fig. 147. Danaa tlliptica, A. Nearly median section of a young sporophyte, showing C. Median section of second leaf. X35. origin of second root, r2 . X35. D. Apex of second leaf. X150. B. Central region. ■' 1 ;o. 1. Stem apex. 150. and hence does not show in the figure, which is a section passing directly through the leaf traces. This plant showed very clearly the very earliest stage of the second root(fig. 147, y/, r ). The apical cell of the young root is cut out from one of the cells of the parenchyma below the stem apex, lying almost exactly half-way between the two primary leaf traces. Only a single segment had been cut off from the apical cell in this case and both the apical cell and this primary segment were easily dis- tinguished from the surrounding tissue by then denser contents. There is nothing to indicate that the cell which hail assumed the function of the root initial was in any way essentially different from the neighboring cells. If it is an endodermal cell there is no way of recognizing this, as the endodermis could not be recognized in this part of the sporophyte. Figure 147, B, shows an enlarged view of this young mot initial. King above the junction of the two primary leaf traces. Conspicuous reticulate tracheitis with pointed ends are present in the trace from the cotyledon, but as yet no permanent tissue has been developed in the bundle from the second leaf. THK OLDER SPOROPHVTE 165 A somewhat older sporophyte from D. jamaicensis is shown in fig. 148. In this plant the third leaf was plainly visible and the second root had developed into a short conical body which was pushing its way through the tissues between the . , >t Fig. 148. A, B, C. Three sections of a sporophyte of Daruta jamaicensis with three leaves D. Median section of the second leaf. In C is shown the second root, r . X20. insertion of the first and second leaves. By this time tracheary tissue has developed in the bundle from the second leaf, and now for the first time we note the presence of large tannin cells in the neighborhood of the vascular bundles. Three of these are shown in fig. 149, lying next the bundle of the second leaf. The irregular ring Fig. 14Q. A, the stem apex; B. apex of the second root of the sporophyte shown in fig. 148. X 150. The shaded cells in A are tannin sacs. of narrow cells shown in the figure and lying below the apical meristem marks the point of insertion of the second root. Figure 149, B, shows a nearly median section of the apex of this second mot. The apical cell is nearly oblong in form and the lateral segments are very large and periclinal segments which contribute to the root 166 THE MARATTIALF.S cap arc cut <>tf from them. I he outer cells of the root cap are very much enlarged and there is an evident space between the root and the surrounding tissues. The trace from the third leaf joins that of the second one close to its union with the bundle from the cotyledon (fig. 148, H). In fig. 150 are shown nine cross-sections from a series made from a plant of D. jamaicensis, in which four leaves win- evident. A microscopic examination of the stem apex showed, however, that a fifth leaf was also present, which stood nearly directly opposite the cotyledon. A shows sections only of leaves •; and 4, the latter having the stipules conspicuous, while these are absent or scarcely developed at all on the first three leaves, at least in the specimen in question. Between the leaves are seen sections of the numerous scales which beset the surface of the young leaves. B is taken somewhat lower down and includes a section of leaf 5. C passes through the stem apex and shows clearly the spiral arrangements of the first five leaves, each of which has in its petiole a single concentric bundle which becomes larger in I'iG. 150. IK jamaii ensi ■ a ,miiu' sporophyte, with three fully-developed leaves an.! two jrounger ones. Section C pa through the stem apex. Roman figures indical ?e leavi each successive leaf, with a corresponding increase in the development of the xvlem. As the sections are examined, farthei ami farther down in the stem ( /) to /■'), one can sir very clearly tin- way in which the single bundle in the lower part of the stem owes irsoii»in to the coalescence of the leaf traces. Proceeding downward th. traces of leaves 4 and ^ ate seen to approach gradually and finally to become completely coalescent; ami still further down ( /•' ) the leaf trace from 3 joins that from 4, ami a smidc bundle results, crescent-form in section, but showing clearly its compound nature. The three x\ leins never become entirely confluent. THE OLDER SPOROPHVTE 167 For a long time sections of the stem show this single central bundle of crescentic form, at first derived from the coalescence of the third, fourth, and fifth leaf traces, but continued upward in the same form and added to by the addition of the traces from the subsequent leaves. This crescentic stele, which, for convenience may be spoken of as the stele of the stem, is entirely of foliar origin. The crescent nevei becomes completely closed and its opening in the earlier stages of development can not be properly called a foliar gap. The parenchyma which is inclosed within its curve belongs from the first to the ground tissue and is not part of the stele. Some of the surrounding cells show traces of the typical endodermal markings and it is perhaps safe to say that the stele is bounded by an endodermis, as Farmer states is the case in Angwpteris and Brebner in D. simplicifolia. The limits of the endo- dermis, however, especially upon the concave side of the stele, are very vague. The stele, after the complete fusion of the three leaf traces, may perhaps best be described Fig. 151. Four longitudinal sections of a young sporophyte of Darurn elliptica. X 18. The fourth leaf, /4, has the stipules well developed; r3, sections of the third root, m, mucilage duct; it, stipules. as concentric in structure, with phloem developed all around the xylem, but there are probably traces of phloem also between the three xylems which represent the three confluent leaf traces. In the older portions of the stem the bundles become still more completely fused and the compound bundle is oval in outline, but still shows plainly the three xylems of its constituent leaf traces (fig. 150, G). In this region the endodermis is rather better developed than it is higher up, but its limits are still rather vague. At this level the traces of the leaves, I and 2, are still free, but have approached nearer to the central bundle than is the case higher up. Still lower down, the trace from the second leaf joins the bundle formed from the later leaf traces, which no longer clearly shows the separate xylems, but ap- proaches the condition which has been described as "protostelic," or "haplostelic," usintr Brebner's terminology. The xylem elements, however, do not form a solid 168 THE MAR VII I \I.I s core, but are more or less scattered, with thin-walled elements between. After fusion of the second leaf trace with this is complete, the section ot the stem shows only two bundles, representing the two first leaf traces, and finally (fig. 150,// and/) the section of the stem shows the condition already described for the sporophyte with but two leaves. These finally merge into a single primary axial bundle (/), which, followed downward, merges imperceptibly into the stele of the primary root. Near the middle of these lower sections can be seen a section of the second root, which pursues a downward course through the cortical tissue for a very long distance, but finally emerges and grows downward, side by side with the primary root. The third root (fig. 150, G, rJ) emerges much higher up and breaks through the cortex at about the level of the junction of the three youngest leaf traces. Fig. 153 shows the details of the central tissue from the same sporophyte as that just described. A passes through the stem apex and shows the apical cell cut somewhat obliquely, and near it the section of the trace from the tilth leaf. D is a section of the fourth leaf trace from the same level. This shows but two tracheids at a point near the inner limits of the bundle, while on the outer side there is a con- spicuous curved line of protophloem cells. B is a section taken somewhat lower down and shows the bending in of the leaf trace as it descends into the stem. This is still more marked in the fifth leaf trace, which, at this level, has a crescentic form. Fig. 152 A. Apex of fifth leaf from sporophyte shown in fig. 151. X150. B. Stem apex of same. C. Base of third root, showing triarch bundle and ring of sclercnchyma in cortex. X90. but no permanent elements yet developed. It is probable that a portion of this crescentic mass of procambium represents the sixth leaf trace. The fourth leaf trace at this point shows three tracheids which are decidedly larger than those higher up, and still lower down the tracheids increase still more in size and several tannin cells can be seen in contact with them. The section of the fourth leaf trace is connected with the large but undifferentiated trace of the fifth leaf by an isthmus of procambium cells, so that the sections of the two bundles form the crescent-shaped section, similar to that seen lower down ( fig. 153, 6). The tracheary elements of the fused portion of the fourth leaf trace are noticeably larger again than those in the free portion of the leaf trace. We now notice, for the first time, traces of the mucilage ducts which afterwards become so conspicuous in the stem. I Inst- first appear in section as rounded cells (m), and it is evident, as Farmer has shown in Angiopteris, that the earliest mucilage canals are of lysigenous origin, i. e., the) are formed by a THE OLDER SPOROPHYTE 169 fusion of cells and not by a splitting apart of the cells and the development of secreting cells about this intercellular space. In his earlier paper Brebner states that the mucilage ducts are schizogenous, but in his later paper he admits that they may be of lysigenous origin, which certainly is the case in the species of Dancea described here. The details of the older central bundles of the sections F and G, fig. 150, are shown in fig. 153, E, F; the shaded cells in C are tannin cells. Figure 151 shows longitudinal sections of a young sporophyte of D. ellipiica, of about the same age as the sporophyte which has just been described. This also shows three fully developed leaves, while the fourth and fifth leaves are well advanced. Fig. 1 J3. Details of the vascular system of sporophyte shown in fig. 150. A. Passes through the stem apex; B and C, lower down, showing the fourth and fifth leaf traces. X150. D. A single leaf trace. X 150. E, F. Vascular bundles from the lower part of the stem, m, m, young mucilage ducts. The central bundle of the stem formed by the junction of the three first leaf traces is now conspicuous and forms a stout central strand, continuing downward into the primary root (r1). A is a nearly median section, passing through the stem apex, whose large and conspicuous apical cell is shown in fig. 152, B. I he apex of the fifth leaf is seen just above this and a more enlarged figure of this is shown in fig. 152, A. The apex of the fifth leaf (which is here cut transversely) is occupied by a small group of large cells, of which the central one may perhaps represent a single initial cell. Below the stem apex, but separated from it by a considerable amount of tissue, can be seen the bases of the leaf trans from the fourth and fifth leaves respectively. These are joining the central bundle of the stem near the junction 17(1 I III MARATTIALKS of the third leaf trace, and at this point there may be seen the base of the third root (ra ). Between the fourth and fifth leaf traces is present a section of the first mucilage duct. B is a section some distance to one side of the apex, also passing through the primary mucilage canal and the fourth root, which arises between the fourth and fifth leaf traces. Figures C and /) are sections on opposite sides of the stem apex from H. The fourth leaf, with its conspicuous stipules (si), shows in these sections, and the third root can also he seen. Fig. 152, C, is a mote enlarged view of the third root shown in fig. 151, /J. The root is march and in the peripheral part of the cortex there is visible a row of very conspicuous cells whose walls stain very strongly with sat rani in- and in section closely resemble la lire tracheal y elements. A longitudinal section of these cells, which are also conspicuous in the later roots (fig. 155), shows that they are elongated sclerenchyma cells whose thick walls are conspicuously pitted. Farmer (Farmer 3) observed similar cells in the roots of Angiopteris. Fig. 154. Two sections of an older sporophyte of D.jenmani. X18. in, mucilage ducts; *r, stipules of young leaf; r, young root. Figures 1 54 and 155 show three longitudinal sections of a pretty well advanced young sporophyte of D. jenmani. The root (shown in the figure emerging at the base) is probably the second root. The stem has already begun to assume a dorsi- ventral form, and the basal part of the root, which is strongly curved, is cut away and SO does not show in these sections. The earliest leaves are no longer recognizable and it is impossible to determine just how many leaves have bun formed. The leaves first formed have the single trace characteristic of the fiist leaves in all of the species, but the youngest leaves show that the leaf trace is double. In this sec- tion (fig. [55, A) is shown a very young leaf, cut through parallel to its surface and showing clearly the stipules at its base and also the young vascular bundles. Within the leaf are two bundles, which are seen to join at its base, but separate again lower down, the two bundles thus forming two distinct traces in the stem. In fig. 154, II, the section passes through the youngest root, which is seen to have its stele joined to one of the leaf traces from the youngest leaf. Several conspicuous mucilage ducts are now present in the stun near the vascular bundles; these are THE OLDER SPOROPHYTE 171 formed of rows of large cells which become more or less fused together and their contents, composed of a dense granular mucilage, stain very strongly, either with safranine or with Bismarck brown, which were the principal stains used in preparing the sections. Numerous tannin cells are also present, scattered irregularly through the cortical tissue and also in the vascular bundles. Figure 155, B, is a median section through the base of one of the later roots, showing its connection with the corresponding bundle in the stem. The elongated cells (sc) are the cortical sclerenchyma cells already referred to. These are shown on a larger scale in C and D, where the conspicuous pitted walls can be seen. The shaded cells in the figure are tannin cells. figure 156 shows cross-sections taken at different heights from a sporophyte of D.jenmani, a plant of about the same age as that which was shown in the preceding series of longitudinal sections. Sections made near the apical region, A, B, and C, Fig. 155. A. Another section of specimen shown in fig. 154. IS. Hase of a root showing sclerenchyma, sc, in cortex. X40. C. Sclerenchyma cells, showing pitted walls. X;zo. D. Surface view, showing pits. show two pretty well advanced leaves and the first trace of a third, the apex of which is indicated in C and E. Unlike the earlier leaves with their single leaf trace, these later leaves, as we have seen, have the petiole traversed by two vascular bundles whose sections are plainly visible. These, as we have remarked from a study of the longitudinal sections, fuse into one near the base of the leaf (//), and two small bundles are also present on either side, belonging to the stipules. As the sections are followed downward, the main bundle is found to divide into the double leaf trace entering the stem, and the two small stipule bundles become united with these, so that within the stem, at about tin- level of the stem apex, two distinct bundles are seen, corresponding to each leaf. The leaves are now arranged in two series, indicating that the dorsiventral character of the stem in this species is established and the youngest bat lies almost opposite the next youngest one, whose leaf traces can be seen. /'. shows the stem apex with the apical cell, \, and the youngest leaf rudiment, /. 172 THE MARATTIALES Below the level of the stem apex, the stele of the axis closely resembles that already described from the somewhat younger plant and forms a somewhat irregular crescent, evidently composed of two portions which are joined on the side facing the two leaf traces from the oldest leaf which shows in this section. The opening of the crescent at this level is turned away from the leaf. No permanent elements are present in the central stele as yet. Following this downward, the crescent is seen to open on the side next the two oldest leaf traces and there is thus formed a. foliar gap, while on the opposite side the space becomes closed. In section /), which is taken lower down, the position of the section of the central stele is reversed. At Fig. 156. A-D. Four transverse sections of a young sporophyte of D. jenmani. X20. Section C shows stem apex. E. The apical meristcm with apical cell of stem, x, anil a leaf initial, /. X180. F-I. Four sections from the same series, but taken lower down. X20. Figs. F. < J , show Fusi in of double leaf trace with central stele, m, mucilage ducts. Between the mucilage ducts in G can be seen the small commissural strand. this point the section of the central stele forms a crescent of tissue, most of which has not passed beyond the procambial stage. This crescent-shaped mass is evi- dently composed of two parts and represents mainly the basal undivided bundles of the two youngest leaves. Trachearv tissue has begun to develop in the older of these two masses. ( )ccupying the space within this crescentic stele are three young mucilage canals, but none are yet developed in the cortical tissue of the stem. Within this central space there can also be seen the section of a small isolated "roup THE OLDER SPOROPHVTE 173 of procambial cells. This is the single axial stele or commissural strand which had not yet begun to develop in the plant with five leaves. The older leaf base, shown in this section, has a single large vascular bundle, crescent-shaped in outline and con- centric in structure, and there are also seen the two small concentric bundles belong- ing to the stipules. All the leaf traces in the older leaves are concentric in structure, with well- developed internal phloem, and thus differ from the collateral traces of the earliest leaves. This concentric structure of the bundle is already well marked in the fourth leaf, where, however, the protophloem is mainly developed upon the outer side. F shows the arrangement of the bundles in a section somewhat further down. The two strands from the oldest of the three leaves seen in the apex have joined the horns of the crescentic central bundle, which still shows clearly its dual nature. In the younger of the two portions of these sections the first tracheids have just appeared near the free end, while in the older half a line of tracheids extends nearly through the whole of the center. The mucilage ducts at this level are much larger and their cells have become confluent. Close to the large mucilage canal can be seen the *s o O ^ " 0 o'- i^-^m A Fig. 157. A. Section of an oldex stem of Danaa jenmani, showing cortical mucilage ducts, »»/, and central or commissural strand, c. //.leaf trace, r, root. X20. B. Shows a section higher up, with free leaf traces and commissural strand. section of the axial vascular strand, which somewhat higher up turns outward and becomes fused with the inner face of the crescentic central stele. The leaf traces soon become completely merged with the broad crescentic stele and their limits can no longer be recognized. The foliar gap gradually closes up and the two por- tions of the crescent separate at a point opposite so as to form a leal gap, nearly opposite the first one; but I have seen no cases where a section of the central stele appears as an unbroken ring, although it is possible that this might occur foi a brief period. Much the same condition of things is seen lower down and it is clear that in all cases, after the double leaf trace has been developed, one bundle fusts with an older and one with a younger leaf trace. The axial bundle shows a central mass of tracheary tissue, and in somewhat older stages an endodermis is pretty well developed. Finally this bundle can be traced downward to where it connects with the main stele, composed of the united leaf traces. The main stele gradually passes down into the base of the stem, where it shows the structure already described for the younger sporophyte. In older sporophytes the crescentic central stele becomes broken up permanendy into two elongated masses when seen in section (fig. 157), but the junction of the 174 THE M \K .VI I I All s leaf traces with these takes place exactly as in the youngei plant. Fig. 157, 5, shows the separate leal traces and the free axial strand, wink- in ./ the leaf traces are becoming fused with the broad central strands ami tin- axial strand is also anastomos- ing with one ol tin- latter. This lias taken place at tin- point of junction with a root. While 111 the younger sporoph) te the mucilage duets are confined to tin- central region, 111 the older plant they occur also in the peripheral region of the stem (hj>. 157, ///). 1 he details ol the vasculai bundles are shown in fi^s. 1 5 S ami 15c). n 0 Fig. i ^S. — Details ol v.iscular bundles From sections shown in fi^. 156. \ .in. I H .ire the ends of the crescentic bundle shown in fig. 156, l> ; '.'. is .1 leaf trace and I) pari <>t an older bundle. ■. 1 so. To recapitulate: The vascular system in the young sporophyte of Dancea begins as a single axial strand, which is continuous through the cotyledon ami mot. At a very early period a second vascular bundle or stele is formed in the second leaf connecting with the primary strand, and this is followed by a similar single strand or stele in each succeeding leaf, up to about the seventh. Up to this time, except for the steles of the secondary roots, the whole vasculai system is built up of united in.. 159. \, part of central stele; B, the commi ural bundle from a yoi D ijcnmani. leaf traces and there is no cauline bundle in the strict sense of tin- word, although we may speak of the bundle, or stele of the stem, as soon as there is a solid central strand formed below the junction of the earlier leaf traces. This primary stele never has the form of a true protostele, however, as tin- xylems belonging to the separate leaf nans can be recognized and the compound nature of this central bundle is unmistakable. THE OLDER SPOROPHYTE 175 At a later stage, probably about the time that the seventh leaf is formed, there arises the single axial (commissural) strand, which is cauline in its origin and which, as Brebner has stated for D. stmplicifolia, is the only strictly cauline portion of the vascular system of the stem. This can first be seen at about the same time that the first double leaf trace appears. The development of the vascular system was not traced further, as it has already been adequately described by Kuhn (Kuhn 2), and except for the increased number of leaf trace and commissural strands the vascular system is essentially the same in a young plant like that shown in fig. 157 as it is in the adult sporophyte. Brebner' s account of the development of the young sporophyte in Dancea simplicifolta agrees essentially with my own studies of D. elliptica, D. jenmani, and D. jamatcensis. He failed, however, to get successful sections of the transition region between the root and the cotyledon, or, as he puts it, "between the root and stem," and his statements that the central bundle begins as a "haplostele," i. e., a protostele, is not borne out by my studies on the other species. Moreover, his statement that "in this way the somewhat irregular adelosiphonic dictyostele of Datura is established now by a process which is, to all intents and purposes, a blanching of the haplostele, due to the departure of the leaf traces of closely set, spirally arranged leaves," might better be stated, "the dictyostele arises by a process which is essentially the fusion of the leaf traces of closely set, not necessarily spirally arranged leaves." He fully recognized the important part which the leaf traces play in the building up of the dictyostele of the stem, but he did not apparently recognize these as the sole factors in the development of the dictyostele of the older stem. He does, however, specifically state that the axial or commissural strand is the only part of the vascular system which is strictly of cauline origin. THE ADULT SPOROPHYTE OF DAN^EA. The anatomy of what was supposed to be the sporophyte of Dantea was first described by Holle (Holle 2), but the later investigations of Kuhn showed that the plant investigated by Holle could not have been a Dancea, nor could it even have belonged to the Marattiaceae, since Holle describes the rhizome as developing a sheath of sclerenchyma, a condition of things which has not been found to exist in any Marattiaceas. Kuhn's account is very far from complete and the description of the sporophyte given by Brebner (Brebner 3) for D. simplicifolta does not deal with the adult sporophyte. The account given here is based mainly upon a study of D. jamatcensis and D. elliptica, but an investigation of certain points was also made in D. jenmani. The rhizome in D. jamatcensis and D. jenmani is a markedly dorsiventral one. These two species are a good deal alike, but are readily dis- tinguished by the difference in the form of the leaves. In the former the leaves are rather larger and have more numerous pinna-, while the terminal pinna is developed. /). jenmani has somewhat smaller leaves with from seven to nine pairs of leaflets, and the leaves are abruptly pinnate, the terminal pinna not being developed (plate 9, A, fig. 1). The leaves are arranged very much as they are in Helmtnthostachys or in Ophtoglossum pendulum, the fleshy leaf base being provided with two verj conspic- uous stipules which are developed very early in the life of the sporophyte. The exact number of roots corresponding to a leaf is not easy to make out, but there are at least two, and it is quite possible that the number may sometimes be greater. I hese roots branch freely, especially toward the rips. D. elliptica (plate 10) is a good deal larger than the other species and the stout rhizome, which, including the persistent leaf bases, lias a diameter of 3 centimeters 176 THE MARATT1 \l I S or more, is upright, the leaves being spirally arranged as they are in Marattia and Angiopteris. The petioles in this species are characterized by curious enlargements or nodes, which may occur in the other species also, but are much less prominent. The leaves of I), elliptica reach a length of about 50 centimeters and the leaflets, which are much larger than those in the other species, are less numerous, there being usually about eleven. The conspicuous nodose swellings seen in the petiole are repeated on a smaller scale in the rachis at the base of each pair of leaflets. Sections of the stems were examined in /). jamaicensis and I), elliptica, which, except for a difference in size, agree closely in their structure. The ground tissue, as was lust pointed out by Kuhn, consists entirely of parenchyma, through which are scattered many conspicuous tannin sacs which, in preserved material, appear to the naked eye as numerous black specks. The section of the vascular cylinder looks very much like that of the younger sporophytes already described, but the number of bundles seen in the section is larger, and there is a group of several bundles instead of the single medullary strand seen in the section of the younger stem. In a section from a large specimen of D. elliptica in which the rhizome, including the old leaf bases, Fig. 160. A. Section "t petiole "I fourth leal of Danaa jamaicen is. 4". B. Leal trace. C. Bundle ol petiole. ■ iSo. was about •; centimeters in diameter, the outer ring of bundles contained thirteen, while within this was a smaller circle composed of five medullary bundles, three large ones and two smaller ones. In a similai section of a medium-sized section of D. jamaicensis, in which the rhizome was about half the size of that in /). elliptica, there were nine bundles in the outer ring and four in the middle, two large ones and two small ones. In this section the junction of a root was cut through and the root stele was seen to be applied by its broad base to one of the larger of the- medullary bundles (fig. 161, / The relation of this central group of bundles in tin- stem to the primary medullary strand was not investigated. Brebner has shown that in the- late 1 stages of the- young sporophyte in D. simplicifolia the original medullary strand which, as we have seen, is really the- only part of the- vascular system which is strictly of cauline origin fuses at certain points with the- bundles of the outer ring, and at this point of fusion there may be a branching so that a section above this shows two of these medullary bundles. Whether all of the central group of bundles seen in the- stem of the adult THE oldi:r sporophvtk 177 sporophyte in D. jamaicensis and D. elltpttca are due to further branching of the primary medullary strand, or whether some of them may be new bundles of cauline origin, remains to be investigated. The arrangement of the characteristic mucilage ducts is the same in the two species studied. There is a ring of these in the outer region of the cortex and a second group in the central region, these central ones usually being close to the vascular bundles. Fig. 161. A. Section of adult rhizome of Danaa jamaicensis. X2.25 B. Section of rhizome of D. elliptica. X1.5. The mucilage ducts are not shown. THE ANATOMY OF THE LEAF. A section of the petiole of the adult leaf appears very much like a similar section of the stem, except that there is developed in the cortical region, separated by several rows of cells from the epidermis, a band of sclerenchyma, yellowish or brownish in color, and very much indeed like that common in the typical ferns. Holle's statement that a similar mantle of sclerenchyma occurs in the stem was shown to be an error, the result of his having investi- gated a fern which was not a Dan&a. There certainly was no trace of scleren- chyma in any of the specimens I have examined. The distribution of the vascular bun- dles in the petiole is very similar to that in Hclmtnthostachys, except that inside the ring of bundles there is a single larger centra] one, instead of the two small ones which occur in Helminthostachys. All the bundles are continued into the stem as separate strands or there may be a certain amount of anastomosing among them, as there is in Ophioglossum pendulum, which Datura resembles more in the character of its leaf traces than it does Helmin- thostachys, where the bundles of the petiole are united into a single trace within the cortex of the stem. The distribution of the mucilage ducts in the petiole is very much like that of the stem. There is a definite ring of these inside the zone of sclerenchyma and several larger ones in the central region, near the vascular bundles. The form and venation of the leaf is essentially the same as in the younger sporophytes which have already been described, and stomata are developed only upon the lower side, the epidermal cells showing a strongly undulate outline, except for the series of narrow cells which 12 Fig. 162. — Apex oi small root of Danaa jenmani. 178 THE MAR All [ALES immediately surround the stoma. A section of the lamina presents an appearance very much like that of Helmtnthostachys. Below the upper epidermis is a well- developed palisade layer, while below this the spongy mesophyll has the characteristic intercellular spaces found in the leaves of most of the higher plants. Danaa tricho- manoides,3 very small species from Peru, has delicate membranaceous leaves, which probably would show a much simpler structure than that of the coriaceous leaves of the largei species. I), snitcnsis, a species from Porto Rico, which is in the herba- rium of the British Museum, has adventitious buds developed at the leaf tip. cot 1 10. 163. \. B. Two longitudinal sections of a young sporophyte of Kaulfussia. /' tnd ird lcavi ; r2, second f">t. X20. C. Stem apex. X180. D. Apex of third leaf. XiSo. THE VPICAL GROWTH OF THE ROOTS. I he earlier roots grow from a single initial cell, but this is later replaced by a group of similar initials which, in the large roots, are very much like those described tor the mot apex of Angiopteris (Koch I). Figure [62 shows a section of a root from the young sporophyte which is somewhat transitional in charactei between the form with a single definite initial cell and tin- largei root with its group of initials. I he tell v, which is very much like the apical cell of the primal) loot, m.iv perhaps still be considered as the single apical cell, but the ad jaunt segments are almost equal in size to this, and it is clear that they contribute to the plerome cylinder of the root as well as to the cortex and the root cap. In sections of large routs taken from the adult sporophyte, there is found at the growing point not a single initial cell, but a group of apparently similar initial cells somewhat wedge-shaped in longitudinal section and arranged in a radiating fashion. \o single one of these can he clearly recognized as a primary initial cell. These cells divide at intervals by longitudinal walls and from the bases additions are made THE OLDER SPOROPHYTE 179 to the plerome cylinder, while from the outer cells of the meristem group arise the new cells for the root cap. The outer segments cut off from the peripheral cells of the apical group of cells undergo more rapid periclinal divisions than those nearer the center and the cells thus formed contribute to the cortex of the root. Unlike the roots of most of the Ophioglossaceae, those of Daneea branch freely in a monopodial fashion. This is paralleled among the Ophioglossaceae by some of the larger species of Botrychium, Helmtnthostachys, and Ophioglossum pendulum. Kiihn's statement that the roots are unbranched is incorrect. The branching of the roots was perhaps somewhat more frequent in D. jamaicensis than in D. elliptica, but the latter species also shows numerous short lateral rootlets. The root hairs are not conspicuous on these older roots, but are readily seen upon the younger ones and are then found to be multicellular, as was first shown by Brebner for D. simplicifolia. Sections of the larger roots were examined in these two species and, while they agreed in the main, there were some slight differences. In a large root of D. elliptica, about 2 millimeters in diameter, the outer portion was occupied by about four rows of brownish thin-walled cells, very probably more or less suberized. Within this was a ring of brownish sclerenchyma cells about three cells in thickness, and between this and the exceedingly conspicuous endodermis was a broad zone of parenchyma, the cells containing much starch. A short distance outside of the endodermis was a ring of conspicuous mucilage ducts. There were twelve xylem rays alternating with as many rays of phloem. These did not extend to the middle of the stele, which was occupied by a pith of considerable size. The roots of D. jamaicensis were somewhat smaller and the main differences were: first, the thicker layer of tissue outside the ring of sclerenchyma, which was much less developed than in D. elliptica (in many places this ring was but one cell in thickness and very seldom more than two cells); second, the mucilage ducts were relatively very much larger; third, there were but nine xylem masses instead of the twelve in the root of D. elliptica. The endophyte, which is usually found in the primary roots of all the Marat- tiaceae is generally wanting from the larger roots of the adult sporophyte. THE SPOROPHYTE OE KAULFUSSIA. The development of the young sporophyte in Kaulfussia resembles that of Daneea in most respects, but from the first it is markedly dorsiventral, like the sporophyte of Helmtnthostachys. The cotyledon, while not provided with the char- acteristic free stipules of the later leaves, still has its base connected with a ridge which extends around the stem apex and youngest leaves, which thus lie in a cavity at the base of the cotyledon in much the same way that the stem apex is situated in the young sporophyte of Ophioglossum; but the sheath in Kaulfussia forms a shallow open cup, instead of the closed conical sheath found in Ophioglossum. This stipular sheath seems to be in all respects similar in its origin to that found in Ophioglossum. The second leaf lies nearly opposite the first and the third next to the hist, and so on, the distichous arrangement being evident from the beginning. We have already pointed out that in the very young sporophyte the vascular bundle from the cotyledon is continued directly into that of the root and that the bundle from the second leaf joins this common bundle, as shown in fig. 163. These primary bundlis run on one side of the apical meristem, which, as in Daneea, is of very limited extent and does not contribute at all to these bundles. The apical cell of the young sporophyte, up to tin oldest stages that were in- vestigated, is very much like that of the very young plant and is broader than that of 'Daneea, more resembling both in cross and longitudinal sections the apical cell 180 THE MARATTIALES ol Ophioglossum. The young leaves arise right and left in succession on eithei side of the stem apex, and from .1 very early period are strongly bent over, showing the characteristic circinate vernation of the Marattiaceae. Sections mack- through the apex of the young leaf show that ir has at first a definite apical cell a good deal like that which is found in the stem apex, and although no satisfactory cross-sections ol this apical cell were found, there seems to be no question that there is present a single definite initial cell which probably has much the same form as that of the stem apex. 1'lie young leaf, at this stage, has the dorsal region strongly convex and composed of large parenchyma cells. I In apex is bent over forward and downward. Fig. i(>4. Scries of transverse sections from a young spot ' bowing the coalescence of the two primary leaf traces. I", second leaf; .'J, third leaf; r", second root. X40. D. Stem apex more enlarged. F-H. Central region of the sections. The fusion is complete in H. The shaded area is a mucilage duet. The tissues of the apex and the adjacent region are composed of small, actively dividing cells. The young vascular bundle can be clearly seen extending nearly to the apex and continuing downward into the stem until it joins the bundle from the next older leaf. The second root arises at about the same rime that the third leaf is recognizable and apparently its origin is exactly the same as it is in Dance a. Its stele joins the central bundle of the stem neai its junction with the third leaf trace. The young leaves, like those in Dancea, are sparingly covered with hairs and scales, hut these scales do not have the peltate form found in Dancea, being usually short rows of stout cells; or the hair may have its base composed of an oblong mass of cells attached by a narrow stalk and having its apex drawn our into a slender filament composed of two or three elongated cells. The terminal cells of these hairs usually stain verj strongly, indicating that they contain tannin. 1 he young leaves are relatively stouter than in Dancea, but otherwise resemble them closely. In tin young sporophyte, up to tin time of the formation of the third leaf, there may be no trace of the mucilage duets, although these may be developed THE OLDER SPOROPHYTE 181 at an earlier period. The mucilage ducts throughout the life of the sporophyte are less conspicuous in Kaulfussia than in Dance a. A section taken at the level of the stem apex in the very young sporophyte (fig. 137) presents an appearance not unlike that found in the bud in Ophioglossum. The base of the cotyledon extends around the stem apex, so that in the section the stem apex and the second leaf are inclosed in this cavity formed by the stipular sheath of the cotyledon. Higher up, the base of the cotyledon becomes free from the second leaf, but this takes place earlier on one side than on the other. The development of the vascular system in the stem is exactly as it is in Dancea. The stem apex in the young sporophyte takes no part in the development of the vascular bundles, the stele in the stem being made up entirely of the united leaf traces. In the specimen figured (fig. 164), in which the second leaf was still quite young, the bundle from the cotyledon passes downward into the stem and continues its downward course until it joins the second leaf trace. The resulting solid stele at first shows the two separated xylems of the component leaf traces which lower down merge into a single thick band of xylem completely surrounded by the phloem Fk;. 1C5. — Three longitudinal sections of a young sporophyte of Kaulfussia, with three leaves. X25. (fig. 164, EH). This stage corresponds to Brebner's "haplostele" in Dancea sim- plicifoha. This condition merges insensibly into the primary root with its diarch bundle, the two xylems in the younger part of the root being quite separate, but forming a single band at the base of the transitional region, between the root and the bundle in the stem. The bundle of the root shows a conspicuous endodermis which becomes less and less evident in the transitional region, although it prob- ably never quite disappears. The primary root usually shows the presence of an endophytic fungus like that occurring in the prothallium and in the primary root of most of the Ophioglossaceae. Fig. 166 shows cross-sections of a young sporophyte in which the fifth leaf is still quite small, but with its trace showing the first tracheary tissue. This fifth leaf trace unites below with the trace from tin- fourth leaf and forms :i single bundle, with the xylems separate. The xylem from the fourth leaf above its junction with the fifth leaf trace forms at first a continuous band which divides into two parts, one of which unites with the xylem from the fifth leaf trace, while the other remains distinct. This separation of the xylem in the fourth leaf trace is the first indication of the for- mation of tlu- double leaf trace, such as we shall find occurs in the later leaves. 182 THE MARATTIALES The separated xylems become joined lower down into a single transverse plate, just before the junction of the strand with the trace from tin third leaf (hg. 166, /-/), with which it joins in the same way and is continued downward to the junction with the strand arising from the fusion of the second hat trace with tin- cotyledon. Above the junction of the hist and second leaves, the stile of flu second root, which grows vertically downward, joins the stele formed by the junction ol the three youngest leaf traces. Above the junction the section of the bundle is nearly circular in outline, the lar<*e tracheids forming a broken group in the center of the bundle, quite like a Fig. [66. A-C. Series of trail pte of Kaulfussia, of about i D. Stem apei. H-J. Three sections lower clown. K. Centr.il vascular cylinder ^t ill lower down. that shown in fig. 165. corresponding stage in Darteea. Some of tin- cells adjacent to the bundle show the typical thickenings on the radial walls, indicating that the) belong to the endodermis, but the limits of tin endodermis are extremely vague. In this plant two mucilage ducts had developed in tin fourth leaf, one f stem apex of .1 young sporophyte <>f Kaulfutiia ol about same age i tli.it shown in fij;. 167. C-J. Sections uikrn lower down from sanu- series. mt mucilage ducts, K. Central bundle of section ]. X140. I.. Part til tin- bundle *>l section Tl, more enlarged. G , V , I, |. show only tin- central region of tin- secti ins. Ophioglossum than it is like the other Marattiacea'. In the intermediate condition between the single solid stele found in the very young stem and the entirely separated leaf traces of the older rhizome, there is a transitional condition with a "siphono- stele," very much as in Helminthostat hys (fig. [68, A ). In one very young sporophyte there was found, in the cortex of the primary loot neat its base, a large lacuna which recalled the similar lacuna found in the first internode of Helminthostachys. THE OLDER SPOROPHYTE 185 The young sporophyte of Kaulfussia is almost completely destitute of the tannin cells which are so conspicuous a feature in Dancea, and these tannin cells are prac- tically entirely absent from the sporophyte throughout its whole existence. In this respect there is a marked resemblance to Ophtoglossum. I he mucilage ducts are also somewhat less developed than in the other Marattiacea.' and are mainly con- fined to the central part of the stem in proximity to the vascular bundles. At a late period they may also appear in the cortical region. Their lysigenous origin is less evident than in Dancea and it is not impossible that they may sometimes be of schizogenous origin, as Brebner states is often the case in Dancea. The structure of the vascular strands is very much like that of Dancea, as a reference to the figures will show. There are developed slender, spirally-marked protoxylem elements like those in Dancea and which we have already seen occur also among the Ophioglossacea?, in Helminthostachys. The stipules in the young plants of Kaulfussia are much less definite than they are in Dancea. A cross-section through the bases of the young leaves shows that instead of the bases appearing free, each with its distinct stipules, the leaf is confluent with the next one, one side being slightly extended and free. The leaf base thus incloses the next younger leaf completely on one side, but leaves it free on the Fig. 169. A. Bundle from intermediate region of a very young sporophyte of Kaulfussia. X150. B. Section of primary root. X150. C. Section of bundle of second root, en, endodermis. X150. other (fig. 168, B). In the later leaves the stipules become more conspicuous, but they are always connected by a very conspicuous commissure in front (fig. 171, com) so that a section of the young leaf base shows a space between the front of the petiole and the stipular sheath. In short, the structure is very similar to the open stipular sheath of Botrychium virginianum and apparently arises in much the same way. The structure of the adult sporophyte has been carefully investigated by Kuhn, who pointed out that it is the simplest of the Marattiacea; in the structure of the stem (fig. 171, B). As in the young sporophyte, the stem shows in section a single circle of vascular bundles, but there is in addition a single median strand within tin- circle of bundles belonging to the dictyostele, and this bundle is presumably <>t cauline origin, like the corresponding one in Dancea. The whole vascular skeleton of the stem, when removed by maceration, was found by Kuhn to be a hollow cylinder with large open meshes. I hese are more elongated on the ventral side and from these ventral strands alone, according to Kuhn, the roots are developed. In this respect Kaulfussia offers a certain analogy to Helminthostachys and perhaps to Ophioglossum pendulum. As in the other Marattiacea-, the ground tissue of the rhizome is composed ot simple parenchyma. Tin- outer layers have their walls brown in color and show the reaction of cork. Except for tin- presence of the central strand, a section of the 186 llll MARATTIAI.F.S rhizome of Kaulfussia presents almost the exact appearance of a similar section of Ophtogloi sum. In a rhizome having a diameter ol about a centimeter twelve bundles showed in a cross-section taken through the internode, one ot these being tin- medullary strand. The section of a petiole of a leaf taken from tin- same plant is shown in fig. 171. C, and tin- structure of tin- section closely resembles that of the rhizome. There were eight bundles arranged in a circle, within which were two medullary strands. The cortical region was composed of several rows of brownish cells, prob- ably similar to those in the outer part of the rhi/ome, and within these, separated Fig. 170. Centra] bundle from .1 young sporophyte of Kaul- fussia, showing two xylems. Xico. Fig. 171. A. kin. oi a large sporophyte of Kaulfussia. u, stip- ules; com, commissure. X;,. B. Section of rhizome. Xz. I . Set ti"n of petiole. X2. from them by three or four rows of cells, was a conspicuous band of collenchyma, a tissue characteristic of the leaves of most of the Marattiaceae. According to Kiihn, the bundles of the leaves are continued separately into the cortex of the stem ami do not unite into a sin are formed. I In- reduced numbei oi xylems in the root of Kaul- fussia, as compared with the other Marattiaceae, is anothei indication phioglossaceae, the sti ucture of the root being very similai indeed to that of H elminthostachys or Ophioglossum pendulum. I ill SPOROPHYTE 01 M \\< VTTIA. The published observations upon the young sporophyte of Marattia are far from complete. Kuhn ( Kuhn I ) has described the stem structure in young plants of M. fraxinea, hut it is evident that these plants were already too far advanced to show the early arrangement of the bundles, as the stems he described had reached a length of nearly i centimeters. Farmer ami Mill (Farmer 3) have also given some details as to the early stem structure in the same species. 1 hese young plants, especially those described by Kuhn, have the stem relatively longer than is tin- case either in M. tinuolasn, which I have studied somewhat in detail, or M . alata, or »\/. sambucina, which I have also examined. In all of these species the stem of the very young sporophyte is still quite short and very soon assumes the compact globular form with the crowded leaves that it has in the adult sporophyte. It may be said, however, that in M . alata the buds which develop upon the old leaf bases have the young stem somewhat more elongated, but not nearly so much as Kuhn's figures would indicate to be the case in M . fraxinea* In the youngest specimens examined by Kuhn the cross-section of the stem showed a ring of bundles corresponding to the leaf traces and a central medullary strand, the whole arrangement being very similai to that which is found in the adult rhizome of Kaulfussia. 1 his stage is also very similar to the condition found in the young sporophyte of Datura. Farmer and Hill describe the vascular skeleton of the very young plant of M. fraxinea as a "siphonostele" with much larger foliar gaps than those found in Angiopteris, and thus more nearly resembling Kaulfussia or Datura. The leaf traces are at first single, hut the later leaves have double leaf traces, such as we have described in Datura and Kaulfussia. The writer has already published some details in regard to the young sporo- phyte of Marattia douglash (Campbell 3, 4), and some additional facts are here added to those that have already been published; hut as the series of specimens available for study was not at all complete, further investigation is desirable to complete the history of the development of the vascular system in Marattia. The only material available for a study of the young sporophyte in M. douglash was a series of slides made a good many years ago from material collected on the island of Kauai in the Hawaiian Islands. This material was supplemented by a small number of very young plants of M. sambucina, collected in |ava. Material of older sporophytes of M. alata was collected in Jamaica in the summer of 1908. Un- fortunately, all the preparations of M. douglash, except the very youngest stages, were longitudinal sections, so that it was difficult to follow out satisfactorily the course of the vascular bundles in tin- later stages. Longitudinal sections of a young sporophyte before the cotyledon was com- pletely expanded are shown in fig. [33. The section was cut nearly in the plane of the cotyledon ami the bent-over apex of the latter was cut so as to show plainly the two lobes arising from the first dichotomy of its apex. The second leaf is ahead)' formed and differs in no essential particular from the corresponding leaf in kanl- ■■*. 1 iit.-ii a pap;r has ap| ■ ■ larli I i vascular system of the young ] >- ph) tc of M. tfttaa. The stele of the ver) young plant i le cribed a i protostele which passes abrupt 1) into a olenostele. THh" OLDER SPOROPHYTE 189 fussia or Danaa. It was not quite certain whether a single apical cell was present at this stage, but a triangular cell which could be seen at the apex was probably the apical cell of the young leaf. The root apex of this specimen had been injured so that the form of the apical cell could not be clearly made out, but it was probably the same as we have already described for the young sporophyte. The apical cell of the stem meristem was broader in outline than that of Danaa and truncate below. The vascular system at this stage, as we have already indicated earlier, consists of the common bundle of the root and the cotyledon, which is joined at a point quite close to the stem apex by the second leaf trace. The first tracheaiy tissue is visible at this point in the form of short, reticulately marked tracheids such as we have already seen in the young sporophyte of Daneea. At this stage no mucilage ducts or tannin cells had developed. About the base of the young leaves are short hairs Fig. 172. A, B. Two longitudinal sections of a young sporophyte of Marattia dougJasii. I2, second leaf; r", second root. Xiao. C. Part of primary root, showing cells in- fected by endophytic fungus. Fig 173. Section of a young sporophyte of Marattia douplasii. v the separated steles of the single leaf tracts, which are beginning to form the open dictyi isti le characteristic of the adult sporophyte. I he exact nature of its origin could not be followed in the material at my disposal, but it is presumably THE OLDER SPOROPHYTE 191 much the same as that described for Dana a, since the appearance of the longitudinal sections in Marattia is exactly like that of corresponding stages in Dancea. The apex of the stem, however, is much broader than in Dancea. There is in the middle of the apical region a cell which from its size and position may be pretty certainly denominated the apical cell (fig. 174, C). There is present a strand of procam- bium which ends abruptly a short distance below the stem apex. This strand probably represents the primary commissural strand, which, as in Dancea, is in all probability a truly cauline bundle and has no direct connection with the leaf traces. In the central region of the stem there are now several large mucilage ducts, but tannin cells are still absent. In the roots, however, the tannin cells are abundantly developed. Farmer and Hill's brief account of the development of Marattia fraxinea agrees with my own observations so far as they have gone, except for the interpretation of the vascular bundles. The "protostele" found in the lower part of the stem is undoubtedly the common bundle of the primary root and the cotyledon, and the open "siphonostele" is really made up of separate leaf traces, which anastomose at certain points to form the large meshes of the very open dictyostele. Farmer and Hill call attention to the fact that the "foliar gaps" are much wider than in Angi- opteris, and in consequence the separate strands, seen in section, form a circle of apparently quite separate bundles, evidently closely approximating the condition found in Dancea and Kaulfussia. Farmer and Hill do not make it quite clear that the commissures which they found developed later, connecting the strands of the dictyostele, were really parts of the central cauline strand, but they presumably assume that such was the case, as this is explicitly stated in the case of Angiopteris, which they also studied. The structure of the bundle in the latest stage described by Farmer and Hill agrees pretty closely with the condition described by Kuhn in the youngest specimens which he studied, where the stem was about a centimeter in diameter. 1 he early leaf traces, as in the other Marattiacea?, are single, but later on double traces are formed. According to Farmer and Hill, the two bundles of the leaf trace unite before joining the vascular cylinder of the stem. No material was available for a further study of the development of the bundle in M. douglasii, but it probably agrees with that of M. fraxinea. Several young plants of M. alata were examined for comparison with the young germ plants of M. douglasii. The specimens in question (fig. 175) had arisen as adventitious buds upon the old leaf bases which had become separated from the stem. This manner of formation of the young plants is very common in this species (see plate 12, A). In one of these young plants, in which the stem was about 4 centimeters long and 1.5 centimeters in diameter, exclusive of the leaf bases, tin oldest expanded leaf had a petiole of about 8 millimeters in diameter. In these young plants there is usually only one leaf expanded, so that they have a very different appearance from the fully developed sporophyte with its crowded circle of leaves, and these monophyllous plants remind one very much of sterile specimens of some of the larger species of Botrychium (plate 12, B). A section of the petiole of this specimen showed ten vascular bundles arranged in a circle, within which, on the ventral side, were two other bundles. The section resembled almost exactly that of the petiole of a fully developed leaf of Helminthostachys. At the base of the leaf are the two very conspicuous stipules, one of which overlaps the other. These stipules are connected by a commissure which joins tin two stipules near the base and is entirely concealed within the cavity formed bv the overlapping stipules, but can be seen on raising these, as a hood-like membrane, overarching the next V)2 I III MARAT! I.M.I S younger leaf. Fig. 175, E, shows .1 young leaf from anothei plant, in which one siilc has been cut away so as to show the relation of the stipules to the leaf base. This leal was still coiled up and its apex was quite concealed within the large overlapping stipules. Both as to its position and its relation to the stipules, the commissure exactly resembles the lip-like basal extension of the stipular sheath in Botrychium and Helminthostachys, and there is no reason to suppose that it is not exactly homologous with this. Partially inclosed by the stipular sheath of the expanded leaf is the next youngei leaf, which is cut in a plane nearly at 1 1 lc li t angles with that of the oldest leaf, so that the overlapping of the stipules, which entirely conceal the rest of the leaf, is very plainly seen (fig. ljv H, /-'). Within the stipular sheath of this leaf is a still younger one, which is entirely concealed from view from the outside, hut shows plainly in longitudinal sections, taken next to the center of the hud. The stem apex is entirely concealed within the sheath of the younger leaf. The resemblance of this section of Fig. 175. — Marattia alata Smith. A, B. Young plant developed us a bud upon an old leaf base. The outer tissue bas been cut away, and the two sides of the thick central section arc shown. XI. C. Section "t petiole of leaf. D. Three sections of a rhizome of a small plant. :■: i .; ; E. Youne. leaf. The lamina is coiled up within the large stipules, si. In (l) one of the stipules has been cut away to show the commissure, com. Xi.;; the young plant to a similar section of the hud in one of the larger species of Botry- chium or of Helminthostachys is sufficiently striking. I he bundles forming the ting in the petiole anastomose freely in the leaf base, as they do in Kaulfussta or Ophioglossum pendulum, so that the number of strands in the leaf trace is smaller than the number of bundles within the petiole. I he vascular bundles in the stem form a single very open mesh-work, with which the leaf tracts join and which is, with little question, composed entireh of these hat tracts, as it is m the earlier stage. Indeed, except tor the larger size of these plants, there is no essential difference between them and the young germ plants, described in .\/. douglasii. Several roots are usually developed before the first leaf of these young hud-plants unfolds. Probably one root develops for each leaf, hut it is not certain that this is always the case. ( >wing to the thickness of the cortical region which has to In- traversed by the root before it emerges, the roots have already reached a large size before tiny appear upon the outside. This deep- THE OLDER SPOROPHYTE 193 seated origin of the roots of the Marattiaceae has often been figured and described. The roots from the base of this plant showed the hexarch structure, the six xylem masses being united in the middle of the stele. About half-way between the stele and the epidermis of the root is a circle of large mucilage ducts, but these were quite absent from the outer cortex. No mycorrhiza could be found, except that in the outermost layers of cells, which constitute a rudimentary periderm, occasional fungus filaments can be detected, but these are very different in appearance from the typical mycorrhiza found in the primary root of the germ plant. No root hairs could be found, but occasionally short stumps were seen which looked as if a root hair had been broken oft. Fig. 175, D, shows three consecutive free-hand sections of a stem from a plant of about the same size as the one we have just described, but evidently much older, as there were the remains of many leaves and the whole caudex, except for its smaller size, was very much like that of the adult plant. The broad vascular strands made up of the confluent leaf traces formed an irregular circle which in fig. 175, D, 2, 3, shows the free central strand which lower down forms the com- missure across the central parenchyma. The arrangement of the bundles, therefore, in the young plant of Marattiaceae is the same as in the adult rhizome of Kaulfussia. In these sections two roots are shown, one of which is cut through the point of junction with a strand of the dictyostele (fig. 175, D, 1). As in the younger germ plants, tannin cells are quite absent from the stem, although in the roots they are conspicuous. A few large mucilage ducts, however, occur in the ground tissue of the stem. Farmer and Hill give one figure of a cross-section of the stele from the young stem of Marattia which shows that it has essentially the same structure as that of Datura. There is a fairly well-marked endodermis, within which lies a broad zone of phloem, entirely surrounding the central mass of xylem. According to Kuhn, there is in the older plant a second circle of bundles within the first, but no satisfactory account is given as to the relation of the bundles making up this second circle with those of the outer dictyostele. However, it is probably composed also of elements derived from the leaf traces, but it is possible that some of the strands may be of cauline origin. THE ADULT SPOROPHYTE OF MARATTIA. The genus Marattia includes about 25 extremely variable species, some of which, e. g., M. fraxinea, closely resemble Angiopteris in their general habit (see Bitter 1, page 441; Christensen 1). They occur in the moist, tropical regions of both the Old and New World, and one species, M. salicifolia, extends as far south as the Cape Region of South Africa. M. douglasii is a conspicuous fern of the Hawaiian Islands. This species I have studied somewhat in detail, as well as the West Indian M. alata, which is abundant in the mountain forests of Jamaica. These two species closely resemble each other in general habit and have the leaves very much more divided than is the case in M. fraxinea and its allies (see plate 12). The stem in the adult plant is an almost globular, upright caudex, a foot or more in diameter in large plants. The closely set, spirally arranged leaves have very stout petioles, 5 or 6 centimeters in diameter at their base, which is enlarged and provided with two very large wing-like fleshy stipules, which, with the base of the leaf, remain attached and completely cover the caudex after the petioles have fallen away. There is at the base of the leaf, as in the other Marattiace;e, a pulvinus- like enlargement, where the leaf breaks off, leaving a clean scar marked by the broken ends of the vascular bundles. The leaves may reach a length of 2 to 3 meters or 13 194 THE MARATTIALES even more. The thick, fleshy texture of the leaflets, much like that of Botrychium ternatum or />'. silaifolium, at once distinguishes Marattia from any of" the large leptosporangiate ferns with which it may be associated. The resemblance to the leaves of Botrychium is especially marked in the young plants growing from the stipular buds. These leaves show a marked triangulai outline, curiously similar to that of the larger species of Botrychium (see plate 12, B). The venation of the leaf- lets is also very like, resembling that of Botrychium more than it does that of Danaa or Angiopteris. In the species with large linear leaflets, e. »., M. fraxinea, the form of the leaf is quite similar to that of Angiopteris or Danesa, but the veins are not so closely approximated. The structure of the leaf was particularly studied in M. alata, which closelj resembles the Hawaiian M. douglasii. In well-grown specimens the leaves measure 2 or 3 meters in length, with a petiole which is 5 or 6 centimeters in diameter above the insertion of the stipules and is somewhat larger lower down. Where it joins the stem there is a marked constriction and the leaves often become broken off at this point, the fleshy leaf base remaining alive and often giving rise to adventitious buds ( pi. ire 12, A, 3). A section of the leaf base above the stipules shows the vascular Fig. 176. A Section -it ultimate rachis "I .1 leaflet of Marattia alata. The shaded area ij> collcnchytua; m, mucilage dui ti - li. Collcnchyma cells, more highly magnified. C. Pari of vasculax bundle. The shaded cells arc tannin sacs. bundles to be arranged in two large concentric circles, within which is a smaller third ring corresponding to the two inner bundles found in the section of tin younger leaf. Below this point there are numerous anastomoses of the bundles before they enter the cortex of the stem, and the leaf trace consists of a much smaller number of bundles, eight in the specimen examined, which are arranged in a single circle, open on the adaxial side. The distribution of the bundles to the stipules was not Studied, but it no doubt corresponds to that in other species, where the veins form .111 extensively branched system, connected with the bundles of the petiole. I he large leaves of M . alata, when lullv developed, are five-pinnate, the ultimate leaflets being about 2 centimeters long. From the midrib extend lateral veins which usually fork once but may remain undivided (plate 12, A, 1 ). The bundles of the rachis in the later divisions of the leaf diminish in number and show the horseshoe- like arrangement seen in the leaf trace after it leaves the petiole. In the final divisions the crescent of bundles seen in the larger rachis is always completely united and in section appears as a single horseshoe-shaped bundle (fig. 176, A). In the main stipe and secondary ami tertiary rachis there is a conspicuous hypodermal sheath of sclerenchyma. Ibis sclercnchvma passes into collcnchyma near the base of the THE OLDER SPOROPHYTE 195 stipe and finally disappears entirely before the stem is reached. The s.clerenchyma is also replaced by collenchyma in the rachis of the terminal divisions of the leaf. Tannin sacs are quite absent except from the immediate vicinity of the bundles, where they occur upon the inner concave side, either in direct contact with the bundle or actually within it. The leaf lamina is only about half as thick as that of the leath- ery leaf of Dancca elhptica, but shows much the same structure. The palisade cells are not quite so well developed and there is a complete absence of sclerenchyma, but otherwise the structure is very similar. The cells of the epidermis are undulate in outline and the stomata, like those in Danaa, are surrounded by a series of accessory cells. The large roots may attain a diameter of 6 to 7 millimeters. In the absence of the hypodermal sheath of sclerenchyma they more nearly resemble Kaulfussia than Danaa, but they are more like Dancea in the greater number of xylem rays, which number twelve in the largest roots examined. In these later roots the central part of the stele is occupied by the pith, the xylem rays not being united as they Fig. 177. A. Apex 'it .t small root oi Marattia douglasii, showing two initial cells. B. Transverse section of apex of .1 similar root. C. Longitudinal section of the apex of the second root. D. Apex of a large root of M. alala. x x, initial cells. X200. .in in the toots from the younger plant, but in some cases a few scattered tracheids appear in the central region. There is usually a ring of conspicuous mucilage ducts in the cortex, but these were sometimes not very well developed. Numerous tannin sacs are scattered through the cortex and also occur within the stele. THE SPOROPHYTE OF ANGIOPTERIS. Angtoptcrts is the largest and most specialized of the Marattiacc.i . As we have indicated before, there is much difference of opinion as to the number of species which should be recognized and many botanists consider that all of the forms bel<>n» to a single extremely variable species. Bitter, however, in his account of the Marat- tiaceae in the "Naturliche Pflanzenfamilien," thinks that from 20 to 30 species should be recognized, following in this respect the classification given by Presl and l)e Vries; while Christensen (Christensen I) recognizes 62. I have examined ma- terial from Australia furnished by Mr. J. H. Maiden, director of the Botanical Gardens in Sydney, as well as material collected by myself in Ceylon and Java. Some of the specimens growing in the Botanical Garden at Buitenzorg are the largest that I have seen. Leaves 6 meters and upwards in length were measured and the upright caudex was almost as big as a barrel. The specimens seen in Ceylon were somewhat smaller. 1% THE MARATTIALES Forms of Angiopteris occur from Polynesia to Madagascai and northward as far as the Himalayas. The genus also occurs in northern Australia. The anatomy of the adult plant has been investigated more thoroughly than that of any other member of the Marattiacese. I he mosl recent account of the anatonn is thai given by Miss Shove (Shove I), while Farmer and I fill have care- fully investigated the vascular system of the young sporophyte. A number of prep- arations wire made by the writer for the purposes of comparison with the othei genera, hut no attempt was made to follow oul in detail the extremely complicated vascular skeleton of the adult sporophyte. Farmer and llill (Farmer 3) have given a detailed account of the development of the vascular system in the young sporo- phyte of Angiopteris, so that it was not thought necessary to make a large number of preparations of this species, the material of which was collected at the same place where Farmer obtained his plants. However, as these authors did not trace the development of the young bundles from the apical meristem, it seemed worth while to examine this point for the purpose of supplementing their account based upon the study of the tullv developed vascular skeleton. Fui. 178. — Four longitudinal sections of a young sporophyte of An^iopttris. The vascular system begins, as was recognized by Farmer in an earlier papei (Farmer I), as a single strand connecting the root and cotyledon, exactly as is the case in the other Marattiace;e. In the further study of the development of the vascular skel- eton Farmer and Hill employed the method of constructing a model of the vascular system, built up of superimposed sheets of wax, corresponding with the outline of the vascular bundles seen in the serial sections. The figures which they give, drawn from such models, show very clearly the relation of the vascular strands which make up the complicated skeleton in Angiopteris. The assumption is made that the single stele found in the young plant is really a cauline structure, the leaf traces being subsidiary. The earlv history of the vascular system of the young sporophyte is given bv them as follows: "The vascular skeleton in the young plant of Angiopteris consists of an axile rod of tissue, from which strands are given off to the roots and leaves respectively. The first lateral root is given off at a point not quite opposite the formation of the first leaf trace. It is separated from it by about 1 300. The regular relation be- tween the leaf and the corresponding root is, however, soon lost. I he gaps produced by the early leaf traces are very small and are immediately made good above. The THE OLDER SPOROPHYTE 197 first deeply depressed aperture or gap occurs at about the sixth or seventh leaf. The leaf traces still continue to issue from the stele as single strands till a varying number have been formed, but they begin to bifurcate while still within the cortex of the stem. "As the stem increases the leaf traces become more numerous and crowded and they take away a larger portion of the vascular tissue from the axile strand. The result is that the leaf gaps become less rapidly repaired. The stele is already hollow in this region, that is, it consists of a cylindrical vascular mass with perforations corresponding to foliar gaps and inclosing a core of parenchyma. Sooner or later, the gap above one leaf fails to be repaired until after the exit of the trace of the next leaf and then the original vascular cylinder becomes broken up and assumes a condition, in transverse section, conforming with that of polystely or dialystely." There is thus a gradual transition from the solid stele found in the earliest stages to a hollow cylinder or siphonostele with a core of pith, and by the formation of larger and larger leaf gaps there is a transition to. an open dictyostele like that found in the other genera. Finally there appears the commissural strand which forms a thick A-D. Four transverse sections of a verv \oune, sporopln h- of Irigiopter, the Fusion of tin- first three leaf traces. E. Centra) region of C. X200. F. Central region of D. X200. A. traverses stem apex. B, C, show sti'nd traversing the pith and undoubtedly of cauline origin. This central strand becomes more and rnore important as the plant develops and from its central position might be mistaken for the original axial strand of the young sporophyte. The vascular system at this stage is described as follows: "The leaf traces become more complex and anastomoses take place at irregular intervals with the strands which can still be recognized as the relics of the original siphonostele, as well as with one another. Irregularities also commence to become apparent as to the relative heights at which the two members ot the leal traces be- come freed from the plexus of tissue, and a stage is thus reached at which the vascular skeleton appears to consist of a stout axile strand, surrounded by upwardly diverging zones of steles, which ultimately pass out above to the leaves." It is evident that at this stage there is a condition which is very much like that figured by Kuhn for Kaulfussia, and it is pretty clear that the whole of the dictyostele, exclusive of the axial or commissural strand, is made up of the "upwardly diverging zones of steles which pass out above into the leaves," i. e., in other words, the dictyostele is composed of a union of multiple leaf traces. 198 THE MARATTIALES Transverse sections of a very young sporophyte of Angiopteris show the same relation of the parts as in the other genera. The junction of the traces of the Hist. second, and third leaves takes place at a very short distance below the stem apex, which, as Farmer pointed out, shows an unmistakable apical cell ot sonuw hat irregu- lar form. Compared with the other genera, the primary leaf trace in Angiopteris has the xylem better developed, and composed of about half a do/en traeheids, where the trace enters the stem. This primary leaf trace is concentric, as it is in the later leaves, and not collateral, as it is in Danica (fig. 179). Near the point of the junction of the three primary traces, there may be seen in the young traces from the second and third leaves the first tracheary tissue, consisting of one or two tracheids. As the sections are examined lower down the tracheary tissue of the second and third leaf traces increases in amount until it forms a solid band, separated from the corresponding xylem of the primary leaf trace by a band of parenchyma. The three leaf traces are now completely fused, but the sections of the two xylems are perfectly evident, and this band of tissue between Fig. 180. A-D. Four sections of an older sporophyte than that shown in ftp,. 179. X40. E. Stem apex. ■ 150. I - Central bundle from transitional region. G. Buniile of primary root. the two xylems (which really belongs to the ground tissue of the stem and not to the stele itself) corresponds to the "pith" figured by Farmer and Hill for the transi- tional region between the root and stem (fig. 180, C). Sometimes, in still lowei sections, the two xylems are connected, but I have found no cases where the pith was entirely surrounded by the xylem, and the two xylems of this axial strand merge gradually into the two xylems of the diarch primary root (fig 179, G). I he endo- dermis is clearly evident in the intermediate region and is recognizable also at higher levels, but is less easy to distinguish. In a series of sections, from a plant in which the fifth leaf was just recognizable, a section at the level of the stem apex shows the apical meristem to he composed nt several large cells, one of which is probably the single apical cell, but this is not easy to determine. The base of the fifth leaf is close to the apical group and the section of its stele is indicated by a group of small cells close to the stem apex. I he stele of the third leaf is clearly seen, separated from the fourth leaf by about one- third the circumference of the stem. Below the stem apex these two leaf traits THE OLDER SPOROPHYTE 199 approach, but are still separated by a considerable mass of tissue, lying directly under the stem apex. The two bundles finally join and between them there seems to be a sort of connective tissue which may perhaps belong to the stem itself, so that this central stele of the stem may possibly have a certain amount of cauline tissue in addition to that derived from the leaf traces; but it is quite as likely that this connective tissue between the bundles is nothing more than a lateral extension of the leaf trace itself, very much as is the case in Helmtnthostachys and Botrychium. Fig. 1X1. -Series "I transverse sections from a sporophyte of Angiopteris, showing 5 leav< s. r2, r3, second and third roots. X25. B, traverses the stem apex. When the fusion is complete, the section of the stele appears circular in outline and the whole of the tissue is apparently quite uniform, with the exception of a single tracheid marking the position of the xylem of the older leaf trace. There follows almost immediately the older leaf trace which joins the solid stele, formed by the fusion of the traces from the third and fourth leaf, without causing any break. At this level the first tracheary tissue can be seen in the fourth leaf trace also. 1 he structure of the stem below the junction of the second and third leaf traces is the same as that already described for the younger sporophyte. A very young root was 200 THE MARATTIALES formed just opposite where the second leaf trace joined the stele. 1 he apical cell, which had apparently developed from an endodermal cell, had only undergone the first division. This root was probably not the first lateral root, or if it was, it had formed very much later than is usual. The base of the plant had been somewhat injured and there was what looked like the remains of another root, which was probably the first lateral root. The oldest specimen of which sections were made had four fully developed leaves and the rudiments of the fifth and sixth. An examination of the lower series of sections showed the remains of oik- or two other leaves and it was probable that eight leaves all together had been formed. At the level of the stem apex (fig. 1 8 1 , H ) six separate leaf traces could be seen and the relation of the younger ones to the apex was exactly the same as in the younger sporophyte. Below the apex, the central stele of the stem shows a broken ring of procambium inclosing larger parenchyma, representing the pith. The procambium ring is made up evidently of two portions, one of which is certainly referable to the sixth leaf trace, while the opposite one perhaps represents the beginning of the next leaf trace, although the leaf to which it belongs is not yet evident above (fig. 181, D). The junction of the leaf traces so close to the stem apex makes this point very difhcult to decide. The fusion of the fifth and fourth traces follows quickly and the resulting Stele shows plainly the three separate xylems of its component bundles. The older portion of the stele shows that it is still solid, with no evident leaf gaps (fig. 181, E G), but with several groups of tracheids probably corresponding to as many leaf traces, though less distinct than in the younger plant. This is complicated by the fusion of the root traces, which disturbs the arrangement of the xylems from the leaf traces, hut the xylem is less compact than is figured by Farmer and llill and the "pith" much less definite; indeed, one can hardly speak of a pith in this connec- tion. No trace could be seen of the central commissural strand which later makes its appearance and which presumably arises in the same way that it does in Datura. In the early stages Angioptcm appears to agree closely with the other IYlarat- tiaceae in the development of its vascular system, hut the single central stele without leaf gaps is retained much longer than in the other genera and it also becomes much larger and has a better-developed xylem, and the open dictyostele, formed from the anastomosing of the early single leaf traces, characteristic of Daruea ami Marattia, is not present. It is not quite clear whether tin- "siphonostele" with its small leaf gaps, which is the next stage in the development, is made up entirely of leaf traces, and it is possible that the stelar tissue, connecting the adjacent leaf traces, may be composed in part of cauline tissue. On the other hand, it is quite as likely that the connection of the leaf traces is brought about merely by a broadening at the point of contact, such as occurs in Helminthostachys. Indeed, at this stage, the stele of Angiopteris is more like that of Helminthostachys than like that of the other Mafat- tiaceae. THE ADULT SPOROPHYTE or ANGIOPTERIS. In habit Angiopteris closely resembles the larger species of Marattia. The enormous leaves are arranged spirally about the thick upright caudex, which is covered completely by the persistent bases of the old leaves. I he leaves are usually twice-pinnate; the linear leaflets have more or less conspicuously serrate margins with a venation very much like that of Dance a, the veins being more closely set than in Marattia. In addition to the ordinary subterranean roots, large aerial roots are not infrequent, but it is doubtful if there is any essential difference between the two kinds of roots. THE OLDER SPOROPHYTE 201 The anatomy of the stem closely resembles that of Marattia, but the bundles seen in section are much more numerous and instead of being arranged in two circles, exclusive of the central commissural strand, are usually arranged in four or five. The first complete study of the arrangement of the bundles in the stem was made by Mettenius (Mettenius 2). A summary of his results is given in the paper by Miss Shove. Briefly stated, Mettenius describes the distribution of the bundles as follows (Shove 1, p. 498): "The vascular bundles form funnel-shaped zones, with their lower ends in the axis of the stem and their upper portions continued out into the leaves as leaf traces. It is the transverse sections of these concentrically arranged funnels which appear as the rings of separate bundles in the section of the stem. Segments from the outer zone pass into the leaves as the leaf traces and the gaps thus left are filled up by corresponding segments from the next inner zone." The detailed account made by Miss Shove was based upon a moderate-sized specimen from Ceylon. This stem is described as being somewhat dorsiventral in structure, which is certainly not usual in Angiopteris and may perhaps be explained by the conditions under which the plant was growing. I found Angiopteris growing frequently upon steep banks and it is quite possible that plants growing in such a position might be obliged to bend upward, in which case a somewhat dorsiventral structure would be developed. Plants growing upon level ground, so far as my observations go, are always strictly radial in structure. Miss Shove found essentially the same structure as that described by Mettenius, except that she states that the leaf traces are developed exclusively from the strands of the outer zone, while Mettenius states that strands are also contributed to the leaf trace from the second zone. The following is taken from her paper (Shove 1): "The general scheme of the arrangement of the vascular tissue in Angiopteris is almost clearly conceived by considering it in connection with the insertion of the leaves. The leaf bases, which are set in a rough spiral on the stem, show in their lower parts a meshed segment of vascular tissue having the form of part of the sur- face of a cylinder. This segment passes from the leaf base into the outermost zone of the stem, uniting right and left with the strands of this zone. Then, continuing in an obliquely descending direction, it passes on into the second zone and so on until it reaches the longitudinal axis of the stem, where it unites with other leaf- trace bundles and loses all individuality." Miss Shove found that the steles in the stem were both mesarch and endarch in structure. The protoxylem is found in groups of two or more spiral tracheitis, some of them at the periphery and some in the center of the stele. The number of protoxylems varies with the size of the stele, the larger ones usually containing five or six such groups. The earliest protoxylem appears at the periphery of the stele. Protophloem is developed upon the outer side of the stele in the form of discontinu- ous, small, thick-walled elements, which Miss Shove regards as sieve tubes. This protophloem does not occur upon the inner side of the xylem. A remarkable pecu- liarity of the bundle is the fact that the secondary sieve tubes are formed outside the protophloem instead of within it. The phloem is of greater breadth on the outer side of the stele than on the inner. No endodermis could be detected about the steles in the stem. The usual absence of sclerenchyma was noted for the stem. Miss Shove was unable to determine the nature of the apical meristem and the question still remains open, whether the stem grows from a single apical cell or from a group, such as is found in the larger roots. It appears from the account of both the older and more recent investigators that the complicated system of concentric meshed zones in the adult stem of Angiop- 202 THF MARATTIALES teris is really built up ol leal traces, the so-called "compensating segment" being nothing more than the lower part of ;i leaf trace which higher up emerges as a meshed segment from the outer zone and passes into tbe base of the leaf. The structure of the petiole (fig. 182, .7) is like that ol Marattia, except that the vascular bundles are more numerous and are arranged in several circles, inclos- ing one or two small bundles in tin center. A cross-section ol a leaflet ( fig. 182, II) shows the vascular bundle which traverses the midrib to be horseshoe-shape in section, having a central mass of tracheids, with reticulate or scalariform markings, and surrounded by the phloem, largely made up of large sieve tubes, but having also protophloem cells and bast. The ground tissue is composed largely of paren- chyma, but on both sides below the epidermis is a conspicuous band of collenchyma. In the larger divisions of the leaf the collenchyma, as in Marattia, is replaced by sclerenchyma. The structure of the lamina is very much like that of Dancea, but the palisade parenchyma is even better developed. A comparison was made of the leaves of two forms (species?) of Angiopteris, one from Australia, the other from Ceylon. These showed several notable differ- ences. The leaflets of the Ceylonese specimens were thinner and sharply serrate, /' t'lo. 182. -Atigiopteris. A. Section of petiole from an adult sporophyte, somewhat reduced. B. Section of a leaflet, col, collenchyma: pt palisade tissue. X14. C. Part of sporophyll, showing the sori. X4. while in the Australian specimens the serrations were almost wanting, except at the tapering apex of the leaflet. There were also marked anatomical differences. In the Australian form the palisade cells are very much elongated and are separated hum the epidermis by a layer of colorless hypodermal cells, and the spongy meso- phyll of the lower part of the leaf is decidedly more compact than in the form from Ceylon. In the latter, the palisade cells are noticeablj shorter and abut directly upon the epidermis. Undoubtedly the differences in the anatomy of the two forms are associated with the difference between the moist, tropical climate of Ceylon and the drier and cooler climate of Australia, and perhaps do not necessarily imply that the two species are distinct, although it is highly probable that such is the case. The roots originate, for the most parr, in the inner zones, but a few may arise in connection with tin bundles of the outer /.ones and must necessarily traverse a very large amount of tissue before they finally emerge. Russow (Russow I) described two sorts of roots — earth roots which were branched and had but five xvlem rays completely lignified to the center of the bun- dles, and aerial roots which wire much larger and unbranched and had twelve to THF OLDER SPOROPHYTE 203 twenty xylem rays, only the outer tracheids being lignified. In the plant which was examined by Miss Shove only the earth roots were found, and these, instead of having only five xylems as Russow asserts, had from ten to thirteen and the xylem elements were entirely lignified. The probabilities are that there is no sharp line to be drawn between the aerial roots and the earth roots. Figure 183 shows a sec- tion of a root from the base of a small plant from Australia, in which there were fourteen xylem masses. As will be seen, there is the same circle of large mucilage ducts and the tannin cells that are found in the root of Mar atti a, which it very closely resembles. Root hairs are nearly or quite absent, in which respect these resemble the older roots of other Marattiaceae. In two cases Miss Shove found a dichotomous branching of the root. This, while probably anomalous, is interesting, as it recalls the method of branching in the roots of Ophioglossum. ARCHANGIOPTERIS. The genus Archangiopteris, with a single species, A . henryi Christ and Giesen- hagen, is at present known only from a single locality in southern China. Arch- angiopteris in habit resembles a large Dancca. The leaves, which reach a length of Fig. 183. — Angiopteris. A. Section of a large root. mt mucilage ducts. X14. B. Part of the central cylinder of the root, en, endodermis. X70. about a meter, have from seven to twelve leaflets, much like those oiDantea elliptica, both in form and venation. The stalk of each leaflet is swollen in a manner that suggests the nodular swellings in the petiole of Datura elliptica (fig. 184). The only account of the structure of the plant is that of Gwynne-Vaughn (Gwynne- Vaughn 2), but as he had only a fragment of a stem he was not able to make a complete study of the vascular system. To judge from the fragment of the stem which he examined, tin leaves seem to be arranged spirally and the stem is probably radial in structure. The general structure of the leaf base seems to be most like that of Kaulfussia. The leaf trace consists of only two vascular strands, which divide up later into several (eight or nine) separate strands, arranged at the base of the leaf in a horseshoe curve, quite like that of Kaulfussia. 1 he arrange- ment of the vascular system of the stipules is probably the same as in the other Marattiaceae. The structure of the stem is also apparently very much like that of Kaulfussia, a section showing only one circle of bundles with a single central strand. The root, which is like that of the other Marattiaceae, has from seven to ten xylem rays. 204 THE MARATTIALES MACROGLOSSUM. This represents a new genus of Marattiaceae, the one species of which, M. alidce Copeland, has recently been described (Copeland I). It is a large fern from Borneo, and is evidently related to Angiopteris, from which it differs mainly in its simply pinnate leaves, which may reach a length of about 3 meters (see Copeland 1, plate V). TISSUES OF THE MARATTIACE E. There are often present upon the leaf bases of the Marattiaceae peculiar lenticel- like structures to which German writers have given the name "Staubgrubchen." These arise beneath the stomata, and form small cavities whose peripheral cells become detached and dried up, forming a dust-like powder. Rod-shaped bodies, mainly composed of cal- cium pectate, are of common occurrence in the intercellular spaces of the tissues of the Marat- tiaceae. Siliceous deposits and crystals of calcium oxalate have also been observed in Angiopteris (see Bitter I). The elements of the vascular bundles are much like those in the bundles of the ordinary ferns. 1 he tracheal v tissue is composed mainly of scalariform elements, and the sieve tubes have numerous lateral sieve plates, like those of the othei ferns. Ililland Farmer have noted a slight secondary formation of wood in Angiopteris, somewhat similar to but much less marked than that in the stem of Botryi h- ium virginianum (see Mill I, Farmer 3). THE SPOROPHYLL OF Till Al \RATTJ UT E. The Marattiaceae differ most strikingly from the ( Jphioglossaceae in the character of the sporo- phylls. In all of the living Marattiaceae the sporan- gia Or Synangia are borne upon the lower Surface F>c \l\.—Archmgioptcrit henryi (aftei Chrisl nt the leaves, which usually are not at all different from the sterile ones. In Danaa (fig. 189) the sporophylls are decidedly contracted and the very large synangia almost completely cover the lower surface of the leaflets, but in the other genera the sporophyll is not at all contracted and much the greater part of the leaf surface is free (figs. 182, 184, 185, 188). Kaulfussia (fig. 188) differs most from the other genera, the very peculiar cir- cular or rarely oval synangia being scattered apparently without any definite order over the whole lower surface of the leaf. In Dancea the elongated synangia, which lie over the veins, extending from the midrib nearly to the margin of the leaflet, are crowded so that they leave very little of the surface free. In Marattia and Angiop- teris the synangia lie over the veins as they do in Dnna-a, but they are very much shorter and are formed near the margin of the leaflet, within which they form a single line which occupies only a very small part of the leaf. Archangiopteris, except that the synangia are very much longer, agrees closely with Angiopteris. \D proper indusium is present in the Marattiaceae, although very commonly about the base of the synangium there are found a few hairs or scales which have sometimes been considered to represent an indusium. In Dancea there is a growth of tissue between the elongated synangia, which grows up into a sort of ridge, the THE OLDER Sl'OROl'HYTE 205 top of which is expanded so that it overarches the synangia. This ridge in section appears T-shape. Whether this growth of leaf tissue between the synangia in Dana-a is to be considered as an indusium may be questioned. It appears to be very similar in structure to the oblong cavity or fovea, within which is sunk the not very dissimilar synangium of Isoetes (fig. 189, B). THE SPORANGIUM OF THE MARATTIAC 1 I In Angiopterts and Archangiopteris there are formed separate sporangia not very unlike those found in Botrychium or Helminthostachys, but in all the othei genera the sporangia are fused into a synangium which might be compared with the spike of Ophtoglossum. The synangium in Dun, in simplicifolia may reach a length of over 3 centimeters and contain upward of 100 loculi. The first studv of the development was made upon Marattia by Luerssen (Luerssen 3). According to his statement, the differenti- ation of the synangium begins while the leaf is still very small and rolled up between the stip- ules. The tissue about a vein begins to develop into an elevated cushion following the vein. Upon this receptacle there are devel- oped two parallel ridges of tissue, separated by a cleft. These two ridges later grow upward and meet above, so that their edges completely close up the cleft, which no longer shows from the outside. In each half of this syn- angium, very much as is the case in the sporangial spike of Ophto- glossum, there are developed sep- arate archesporial groups corre- sponding to the separate cham- bers that are found in the fully developed synangium. Luerssen states that the whole process takes about six months for its completion (fig. 186). In Angiopteris (fig. 187) the development of the sporangium begins at a much later period, when the leaf is almost completely developed. The sporangia here are arranged in an oblong group or sorus, which corresponds to the synangium in Marat- tia. The formation of the sorus begins, according to Goebel, as an oblong depression above a young vein, and about this depression there is formed a circle of short hairs which are sometimes supposed to represent an indusium. Two ridges corresponding to those found in the young synangium of" Marattia are formed, but upon these the young sporangia develop separately, very much as they do in Botrychium. Goebel states that the archesporium in Angiopteris can be traced back to a single hypodermal cell. This cell divides repeatedly but apparently without any definite order, and there is finally formed a large mass of sporogenous tissue, each cell of which gives rise in the usual way to four spores. The cells about the arche- sporium develop the tapetum. Goebel states that these tapetal cells are destroyed before the division of the spore mother cells, but I have found that this is not the Fig. iS$.—Maraiiia douglasii. A. Leaf from a young sporophyte. st, stipules. B. Leaflet with synangia. X4. C. Horizontal section of a synangium. Xio. X2. 206 THE MARATTIALES case and that the tapetal cells persist until the division of the spores is complete. Bower has confirmed this statement and found that the same condition of things obtains in the other genera. The most complete account of the development of the sporangium is that of Bower (Bower 6), who has studied the development in all of the genera except Archangiopteris. Except for the difference in form, there is no essential difference in the development of the sporangia in Kaulfussia and Daneea from that found in Marattia. In Daneea, however, the individuality of the loculi of the synangium is less clear than in the other genera. Very often Bower found that the arche- sporium became divided more or less completely by parts of the sterile tissue, some- what in the same fashion that the so-called "trabecular" are formed in Isoetes. In Daneea, moreover, the synangium from the first is solid, and the cleft which is present in the young synangium of Marattia is absent. In Kaulfussia the develop- ment of the synangium differs in that a single circular ridge is formed instead of the Fio. tS<>. A. Section "I -i young synangium "l Marattia fraxinca. ■ iz^. B, Locului from an older synangium. x, the tapctum (after Bower). two parallel oiks found in Marattia, and the loculi or chambers of the synangium are thus arranged in a circle around a central pit-like depression. Bower states that the sporogenous tissue of each loculus in all the forms he examined can usually be traced to a single mother cell. He also found that the tapetum always arises from the cells adjacent to the a i chcsporium, and that normally all of the sporogenous tissue develops into spores. In these respects the Marattiaceae closely resemble Helminthostachys and Botrychium. In Daneea and Kanlfus.ua there is no mechanical tissue representing the annulus found in the more specialized terns. The dehiscence of the sporangium in these forms is brought about merely by a shrinking of the cells on either side of the slit by means of which each loculus opens. This slit is very short in Daneea and may finally appear as a circular pore, but it is not essentially different from the more elongated slit found in Kaulfussia and Marattia. In the latter then- is developed, in the outer tissues of the synangium, mechanical tissue which causes the two hakes of the synangium to separate, very much like the two covers of a book, and the elongated slit from each loculus opens into this space between the widely separated halves of the synangium. THE OLDER SPOROPHYTE 207 The dehiscence of the individual loculi is affected by the contraction of the thinner-walled cells surrounded by firmer tissue. The sporangium of An giopteris is undoubtedly a more specialized structure than the synangium of the other Maratti- aceae. In Angiopteris each individual sporangium has the wall on the outer side much thicker than that on the inner one, and the superficial cells have their walls much thickened. The inner wall is sometimes composed of but one layer of cells outside of the tapetum, but more commonly there are one or two layers of cells between the tapetum and the epidermis. Near the top of the sporangium on its outer side there is a transverse band of cells with thicker walls, and these constitute a rudimentary annulus very much like that found in the Osmundaceae. By the contraction of this thickened annulus the longitudinal slit on the inner face of the sporangium is made to open widely at maturity. The number of spores produced in each loculus, according to Bower, is approximately 1,750 for DancEa, 7,500 for kiiuljits.ua, 2,500 for Marattia, and 1,450 for Angiopteris. Which type of sporangium in the Marattiacea? is the more primitive is very difficult to say, as both the free sporangium like that of Angiopteris and the compact synangium like that of Marattia and Dancea are of about equal antiquity, so far as the geological record goes. It must be remem- bered that the living Marattiaceaj are almost certainly merely a few isolated fragments of a once large group, and it is by no means necessary to assume that the spo- rangia of the living forms must necessarily all conform to a com- mon primitive type. It seems quite as likely that the free spo- rangia, like those of Angiopteris, may have originated directly from some ancient prototype which resembled, perhaps, forms like Botrychtum or Helminthostach ys , while the genera with the solid synangium like Dancea may have come from forms with completely united spo- rangia like Opkiogldssum. It appears from a study of the most ancient ferns that these had dimorphic leaves, the sporophyll probably resembling the fertile spikes of Botrychtum or the fertile leaf segments of Osmund a. I low the modern Marattiaceae originated from forms of this type is not by any means clear. The development in Helminthosta, hys of sterile leaf-like lobes associated with the sporangia may perhaps afford a clew to the method of sterilization by which the sporangiophores of some type allied to tin ( )phioglossaceae may gradually have developed sterile green hat segments, bearing upon their lower surface sporangia or synangia like those of the modern Marattiaceae. These sterile segments found in Helminthostachys are occasionally strikingly leaf- like, and as the groups of sporangia in Helminthostachys an- sometimes united into small synangia, the development of similar synangia upon the lower surface of such green sterile segments of the sporangiophore is quite conceivable. Another explanation might be the coalescence of the fertile and the sterile pa its of a sporophyll like that found in the Ophioglossaceae, hut the fact that the spo- Fxc 187. — Angiopteris. A, B, sections of young sporangia (after Goebel). C, an older sporangium. r, the annulus; /, the persistent tapetum. X75. 20S THE MARATTIALliS rangiophore in all of the living Ophioglossaceae is adaxial, while in the Marattiaceae the synangia are abaxial, is a serious objection to such a hypothesis; and the most plausible explanation at present seems to be that of a progressive sterilization ot part of the sporophyll itself and the expansion ot the sterile tissue into the broad lamina of the leaf bearing the separated sporangia or synangia upon its lower surface. It must he admitted that the difference between the sporophylls of the existing Marattiaceae ami those of the Ophioglossaceae is very great, and the leaves of the former, superficially at least, are much more like those of the leptosporangiate ferns; but the sporangia themselves are very similar in then development to those of the 0 *&~ A. Lower surface of a sporophyll of Kaulfussia, showing circular synangia. X i .5. B. Median section of a synangium. ■ '-■ C, I). Cross-sections of a synangium* C. Near top, showing openings of loctili. D. Near middle. X12. Ophioglossaceae, and offer no obstacle to the assumption of a fairly close relationship between the Ophioglossaceae and the Marattiaceae, which is indicated by the history of the development of the gametophyte, and the vegetative organs and tissues of the sporophyte. Perhaps the most marked difference between the tissues of the Marat- tiaceae and the Ophioglossaceae is the prevalence of the mucilage ducts in the tissues of Marattiaceae, but these arise rather late in the history of the sporophyte, and it is Fig. 189. — Dantta jamaicensis. A. Base of a fertile leaflet, showing synangia, sp. Xz. B. Transverse sections of three synangia. X12. C. Horizontal section of a synangium. significant that they are less developed in the presumably more primitive Kaulfussia than they are in the more specialized types like Angiopteris. Another difference is the development of sclerenchyma, which is <]uite absent from the ( Ophioglossaceae; but this again is also quite absent from Kaulfussia, which, on the whole, must be considered to be the most primitive of the living Marattiaceae and also the form which most closely approaches the Ophioglossaceae. Part III. THE ORIGIN AND RELATIONSHIPS OF THE EUSPORANGIATE. The eusporangiate ferns as they now exist represent but a few isolated fragments of what in earlier geologic time was presumably a very much larger and more con- nected group. As the geological history of these forms is very far from clear (this being especially true of the Ophioglossaceae), we are, perforce, dependent mainly upon a comparative study of the few existing types for information concerning their relationships. In the foregoing pages an endeavor has been made to trace the de- velopment of these forms, both in their gametophytic and early sporophytic stages, and the result of these studies has been to confirm the belief that a real genetic- relationship exists between the Marattiaceae and the Ophioglossaceae. While the three genera of the Ophioglossaceae differ in certain particulars from each other, there is no question as to their being comparatively closely related, and the same is true of the different genera of the Marattiaceae, although perhaps the differences here are somewhat greater than in the Ophioglossaceae. It has been assumed in these studies that the sporophyte of the ferns is the result of a progressive specialization of the sporogonium of some form allied to the Bryophytes, though it is highly improbable that any of the existing Bryophytes are directly related to this progenitor of the primitive ferns. No attempt will be made here to discuss the reasons for accepting the "antithetic" theory of the alternation of generations, rather than the homologous theory. These reasons have been set forth at length elsewhere. It is hopeless to expect that any satisfactory fossil traces will be found of these predecessors of the true ferns. The fern type is exceedingly ancient, but it must have been preceded by simpler forms connecting it with some bryophytic type. These forms, as well as the earlier true Pteridophytes, were almost certainly plants of small size and delicate texture, probably not very unlike some of the small and delicate species of Ophioglossum. Such plants, composed entirely of soft, perish- able tissues, could hardly be expected to leave recognizable fossil traces, and their absence from the ancient Paleozoic rocks is not to be wondered at. There still exist, however, among the Bryophytes, certain forms which, if they are not directly related to the eusporangiate ferns, nevertheless show many striking similarities in structure, which help to explain at least what may very well have been the character of the liverwort-like ancestors from which the ferns are descended. Among the living Bryophytes, as is well known, an interesting series of types may be traced, showing the gradual increase in the importance of the neutral generation — the sporophyte — starting as little more than a mass of spores, finally by a progressive sterilization of what was originally sporogenous tissue, and an accompanying special- ization of the sterile tissues thus formed, attaining a condition of almost complete independence. The importance of this process of sterilization of potentially sporo- genous tissue, has been especially clearly expounded in the works of Professor Bower. In two classes, the true mosses or Musci and the horned liverworts or Antho- cerotes, the sporophyte continues its growth for several months and develops an elaborate system of green, assimilative tissue, quite comparable to that found m the vascular plants. The spore-producing function is correspondingly subordinated and the spore formation is delayed until a late period in the life of the sporophyte. 14 2oy 210 ORIGIN AM) RELATIONSHIPS of nil I I M'oK WGIATAE I lu large, green sporophyte, however (fig, 190, A, .«/>), never attains complete inde- pendence, as no proper root is developed and it is dependent for its water supply upon tin gametophyte. In all of the Eusporangiates, the- embryo is much latei in developing its organs than in tin- Leptosporangiates, and thus resembles the Bryophytes; moreover, the embryo reaches a very much larger size before it attains its independence. Even after the young sporophyte has developed several roots and leaves, it may still Fig. 110. A. Gametophyte of Anthoctroi sp. with the large porophyte, sp, attached to it. Uppei part .-lit into v. lives which allow the escape of spores. B, C. Two gametophytes of Marattia ilouglasit. with attached sporophyte. Sporophyte emerges upon upper side of gametophyte, very much as in Jnthoceros, but a root, r, is developed which penetrates lower side of gametophyte and connects the young sporophyte with the earth. g, gametophyte; sp, sporophyte; /, first leaf; r, primary root. maintain its connection with the gametophyte and he to a certain extent dependent upon it in a way which forcibly recalls the relation of gametophyte and sporophyte in the mosses. This condition, which furnishes a very strong argument in favor of the primitive nature of the Eusporangiates when compared with the Leptosporan- giates, is especially pronounced among the Ophioglossaceae, where it may even go so far that spores are developed before the connection of gametophyte and sporophyte is finally sundered. In the true mosses the sporophyte is exceedingly specialized and shows but a very remote resemblance to that of the Ptei idophytes. In Anthoccros, however, the Fig. 91. Diagrams which show the similarity in form of the very youni: sporophyte in hithocrros, A, and Ophioplossum , B. In the former the upper portion becomes an elong- ated capsule within which spores are produced. The shaded portion shows layer of sporogenous tissue sur- rounding central sterile tissue or columella. The zone, m, between capsule and foot, /, is composed of mcristematic tissue. In Ophio^lossutn the upper part of the young sporophyte forms the primary leaf, or rledon, cor, which is not sporogenous. The colum- ella of Jnthoceros is replaced by the axial vascular bundle, v.b. At the junction of the cotyledon and foot arises the primary root, r, which finally penetrate! the foot and enters the ground. most highly developed of the horned liverworts, then are a number of very significant structural details that are very strongly reminiscent of the young sporophyte ol Ophi'jglossum moluccanum. In both of these forms tin young embryo has a very large toot and a conical upper region 1 tig. [91). This upper portion in Ophioglossum develops into the cotyledon; in Anth . into tin spore-bearing part of the sporo- phyte. Above the toot in Anthoceros there is developed a zone of meristematic tissue by means of which new cells are added to the base of the growing sporo- ORIGIN AND RELATIONSHIPS OF THE EUSFORANGIATAE 211 phyte, and in Ophioglossum much the same thing occurs in the cotyledon, although at first this grows from a single apical cell. It is probable that in the ancestors of the Ophioglossaceae this upper conical portion of the embryo was developed directly into a spore-bearing organ. There is, however, a marked difference which soon appears, viz, the development of a true root in Ophioglossum. This root, pushing down through the foot, penetrates the lower side of the gametophyte and places the young sporophyte directly into communication with the water supply from the earth, and thus the latter becomes entirely independent. Were the large sporophyte of Anthoceros to develop a similar root from the basal meristem, it also would become entirely self-supporting. Indeed, so marked are the resemblances in the early stage of development that they make the inference almost irresistible that the Ophioglossaceae must have descended from some simpler forms whose sporophyte bore a strong resemblance to Anthoceros. This " pio-Ophioglossum," if we may call it so, pre- sumably produced spores upon the first leaf, instead of its being a sterile cotyle- don as in the existing forms. From a study of the development of the sporophyte Fig. 192. — Comparison of sexual organs in Anthocerotaccu: and Marattia. A. Longitudinal section of the thallus of Anthoceros pcarsoni Howe, showing an antheridium mother cell, with the superimposed cover cell (d). B. An older antheridium within a cavity covered by a double layer of cover cells. C. Young archegonium of Megaceros tjibodensis Campbell, a form closely related to Anthoceros. D. Sections of young anthcridia of Marattia douglasii Baker. The inner cell divides at once into the sperm cells, and the cover remains single-layered. J, the cover. E. Young archegonia of Marattia douglasii. b, the basal cells of the archegonium; d, neck. in O. moluccanum, it may be inferred that this ancestral form had no stem, but consisted simply of this spore-bearing leaf and a root. The sporangia were presum- ably simpler than in the existing forms of Ophioglossum, and perhaps intermediate in character between such sporangia as those of the smaller species of Ophioglossum and {the imperfectly segregated spore groups which occur in the Anthocerotes. Not only does the sporophyte of Anthoceros show important resemblances to that of Ophioglossum, but the development of the sexual organs also shows striking analogies. The archegonium, unlike that of other Bryophytes, is sunk below the surface in much the same fashion as that of the eusporangiate ferns, and the short neck of the archegonium in the latter is probably to be compared to the four terminal neck cells which may occur in the Anthocerotes (fig. 192). The endogenous anther- idia of the Anthocerotes also may perhaps be compared to the sunken antheridia of the eusporangiate ferns, and in their early stages the resemblances are very close. 212 ORIGIN VND RELATIONSHIPS OF THE EUSPORANGIATA1 The most marked difference in the character of the reproductive organs is the spermato/.okl. The spcrmato/oids in the Eusporangiates, especially in Ophio- glossum, are very large and possess numerous cilia, while in Anthoceros they are minute and have but two cilia, like the other Bryoph) tes. I his is perhaps the strong- est reason for assuming that there is not a direct connection between tin \nthoce- rotes and the Ophioglossaceae; hut both gametophyte and sporophyte have so many points in common that it may be pretty safel) assumed that the progenitors of the ( Iphioglossaceae were not very different, in appearance at least, from the living An- thoceros. Whether the differences in the spermatozoid are secondary remains to be seen, hut in view of the extraordinary constancy of the form of the spermatozoids in all of the main groups of the Archegoniates, one would certainty expect that large multiciliate spermatozoids would be found in the ancestors of the Eusporangiates, losing these should ever be discovered. There is no question that the subterranean prothallium of the < )phioglossaceae is a secondary condition, derived from some green gametophyte, probably very much like that of the Marattiaceae. Not only has the chlorophyll been lost, but in Qphio- glossum and Helminthostachys the dorsiventral form of the gametophyte has been replaced by a radially symmetrical thallus. While this may be partially explained as the result of the absence of light, it must be remembered that the equally sub- terranean prothallium of Botrychium is dorsiventral, although the reproductive organs are borne upon the dorsal surface and not upon the ventral one, as they are in most terns. Whether this position of the reproductive organs is the result of the conditions of growth or whether it indicates that the green gametophyte from which this saprophytic underground form is descended also bore the reproductive organs dorsally, as most liverworts do, can only be conjectured. In the Marattiaceae antheridia are not uncommon upon the dorsal surface, and according to Jonkmann archegonia also may occur dorsally. Whether this is normal or is the result of unusual light conditions was apparently not in\ estigated. I he formation of archegonia in the ordinary ferns may be induced upon the dorsal side of the prothallium, provided it is illuminated from below. If the illumination is equal on both sides, archegonia will develop both on the ventraiPand dorsal surface. Of the investigated species of Ophioglossum, 0. moluccanum and the nearly related and perhaps identical 0. pendunculosum probably represent the most primi- tive type. In these species there may be a feeble development of chlorophyll in the gametophyte under certain conditions and the saprophytic habit is much less pro- nounced than it is in the other species of Ophioglossum and Botrychium. The development of an endotropic mycorrhiza in the prothallium of the Marattiaceae is an interesting suggestion as to the probable beginning of the sapro- phytic habit which characterizes the 'gametophyte of the Ophioglossaceae. There seems to be good reason to suppose' that the peculiar type of symbiosis, which is developed so highly in the Ophioglossaceae, began by the development of a fungus associate in some green gametophyte like that of the Marattiaceae. The reproductive organs of the Ophioglossaceae and Marattiaceae are very similar indeed. It is probable that the short-necked archegonium found in the Marattiaceae and-lin Ophioglossum^s more primitive than the long-necked arche- gonium of Botrychium or Helminthostachys. This conclusion is based upon the assumption that the four-necked archegonium of the ferns is a development of the four terminal cells in the neck of the Bryophyte archegonium, Anthoceros most nearlv approaching this hypothetical ancestral type. This being the case, the forms with the shortest neck would most nearly resemble this assumed ancestral type. ORIGIN AND RELATIONSHIPS OF THE EUSI'ORANGIATAE 213 The question of the canal cells is a puzzling one. In the Marattiacea?, with the exception of Dancea, two neck canal cells, or at any ran two nuclei, are present and the ventral canal cell is conspicuous. In Dancea, however, the canal cells are very much less perfectly developed, the ventral canal cells especially being exceedingly difficult to demonstrate. In the latter respect Dancea shows a remarkable resem- blance to the ( )phioglossace.e, especially to Ophtoglossuni, where the ventral canal cell is equally difficult to demonstrate. This apparent degeneration of the ventral canal cell is probably secondary, as all of the Bryophytes and the other Pteridophytes have the ventral canal cell conspicuous. The antheridium in all of the Marattiaceae is almost exactly like that ofOphio- glossum, while Botrychium and Helminthostachys differ in having the outer wall of the antheridium more or less completely two-layered. If the antheridium is derived from a type like that of Anthoceros (fig. 192), where the antheridial cavity is covered by a double layer of cells, it would seem that Botrychium and Helminthostachys are more primitive than Op/uoglossum or the Marattiaceae in this respect. ( )f the Lusporangiates, there is no question that the type of embryo found^'in Ophioglossum moluccanum comes the nearest to the assumed ancestral form. This hypothetical pro-fern may be assumed to have developed simply a spore-bearing organ or sporangiophore, perhaps more or less leaf-like, and a root developed from the inner part of the massive foot in the same way that the root actually does develop in the embryo of the Marattiace;e and Ophioglossum moluccanum. Except that the cotyledon of 0. moluccanum is sterile, the young sporophyte in this species is actually like this hypothetical type, viz, it is composed simply of root and leaf. It is probable that the fertile spike of Ophioglossum is not unlike the primitive sporangiophore, and the ancestral form presumably developed such a spike-like sporangiophore from the first, the sterile lamina being a secondary structure arising perhaps from a basal meristem, like that found in the sporophyte of Anthoceros. The early development of the spores in the ()phioglossace:e is a further indication of their primitive nature. The monophyllous condition which prevails among the Ophioglossaceae must also be regarded not as a secondary condition but as a primitive one, and a study of the development of the sporophyte lends no support to the theory that we have to do with a reduced strobilus. Of the Marattiacea?, Kaulfussia normally approaches the monophyllous condition more closely than any other genus. The older plants, as a rule, have only two or three leaves expanded at one time and there may fre- quently be but one. In strong contrast with this is the dense crown of leaves found in Marattia and Angiopteris, although even in these forms the number of leaves is less than it is in the majority of leptosporangiate ferns. The tendency to the mono- phyllous condition is shown in the younger plants of Marattia, developed from the buds on the old leaf bases, where, as a rule, only one leaf is expanded at a time. The young sporophyte of 0. moluccanum has no stem. Without exception, for a considerable time at least, the vascular system of the sporophyte is composed exclusively of tissue belonging to the leaves and roots, the stem apex playing no part in the building up of the vascular skeleton. The theory that there is a special stele belonging to the stem, of which the leaf traces and the root traces are sub- sidiary structures, is not borne out by a detailed study of the evolution of the vascular system in either the Marattiacea- or Ophioglossaceae. In all of these that I have examined the vascular system begins as a single strand common to the primary leaf (or cotyledon) and the primary root, and the more complicated vascular system of the older stem for a very considerable period is built up exclusively by additions of new leaf tracts or root steles. This condition is permanent in the Ophioglos- 214 ORIGIN AND RELATIONSHIPS OF THE EUSPORANGIATAE sacea?. In the Marattiacea? small cauline bundles (commissural bundles) may be developed at later stages, but these arc insignificant compared with the complex of united leaf traces. The antithetic theory of alternation implies that the sporophvlls arc older structures than the sterile leaves, and those ferns in which the sporogenous function of the sporophylls is most pronounced ma)' be assumed, other things being equal, to be the most primitive. In this respect, as well as in the usually monophyllous condition, the Ophioglossacea? are distinctly more primitive than the Marattiaceae. The sporangiophore or fertile leaf segment is a practically independent structure, being differentiated at an extremely early period in the development of the leaf. In the Marattiacea? the sporangia are subordinate and the leaves upon which they are borne usually differ in no respect from the sterile leaves. The most marked exception to this is Datura, in which the sporophylls differ decidedly from the sterile leaves and the synangia are much larger than those of the other genera, although the leaves themselves are usuallv smaller. Bower thinks that the circular synangium of Kaulfussia is the most primitive tvpe among the existing Marattiacea?, but there is some reason to doubt the sound- ness of this conclusion; at any rate, on theoretical grounds, the large synangia and contracted sporophylls of Dana- a must be assumed to be more primitive than the type of leaf found in Kaulfussia. In regard to the character of the sporophylls, tnerefore, none of the living Marattiacea? can be regarded as being very primitive. All of the oldest fern fossils show the sporangia to be borne upon special leaves or leaf segments, in which the lamina is nearly or quite absent (Scott 1, Bower 9). In this respect, therefore, these ancient fossil ferns were more like the ( )phiogloss- aceae than like the Marattiace;e, although their sporangia came nearer to the type of Marattiacea?. It is by no means impossible that these oldest ferns, e.g., the Botryopterideae, were related to the Ophioglossaceae. The single apical cell found in the stem apex throughout the life of the sporo- phyte in Ophioglossum is probably a more primitive condition than the group of cells found in Angiopteris. It still remains to be seen whether the single apical cell found in the other Marattiace;e in their early stages is persistent throughout life or is replaced, as in the roots, by a group of initial cells. The single axial strand of collateral structure throughout cotyledon and root may be taken as the starting point for two types of vascular skeleton which have arisen from it. The first is that of Ophioglossum, where, with the development of the new kaves, there is built up the wide-meshed cylindrical network or dictyostele, cjmposed of single collateral leaf traces. A further development of the same type results in the much more complicated dictyosteles of the Marattiacea?, where, more- over, the collateral bundles are replaced by concentric ones. This greater com- plexity is due primarily to the much larger leaves of the Marattiacea?, in which the leaf traces are compound. In the earlier stages of these forms, however, we have seen that the young leaf traces are single anil tin structure of the vascular skeleton exactly as it is in Ophioglossum. Of the Marattiacea', Kaulfussia more nearly retains this primitive condition than the other genera and next to this comes Datura. while further removed from the primitive type is the exceedingly complex skeleton found in the massive stem of Angiopteris. The concentric bundles, characteristic of flu- Marattiaceae, are presumably of secondary origin and we find that in the early bundles, especially of Datura, a true collateral structure is present. The second type of skeleton is that found in Botrychium and Helminthostachys, This is a solid, hollow cylinder with inconspicuous leaf <>ts <>t tin Marattiaceae, its form is more like that of the Marattiaceae than it is like that of Botrychium. The endophyte which occurs in the older roots of the < )phioglossaceae, as well as in the primary one, I have not found in the older roots of the Marattiaceae, at least not as a regular thing, although it seems to be always present in the primary root. 216 ORIGIN AND RELATIONSHIPS OF THE EUSPORANGIATAE The Ophioglossaceae and Marattiaceae are alike in the absence of any mechan- ical tissues in the cortex of the sum. Sclerenchyma, which is well developed in the leaves of most of the Marattiaceae, seems to In- entirely wanting in all of the Ophioglossaceae, but the collenchyma which replaces sclerenchyma in certain organs of the Marattiaceae, and is the only form ot mechanical tissue in Kaulfussia, is occasionally found in the ( Ophioglossaceae. A notable case of this is the broad and very conspicuous zone of collenchyma surrounding the root bundle in Ophioglossum palmatum. The mucilage ducts so characteristic of Marattiaceae are apparently quite wanting in all of tin < Ophioglossaceae, and the tannin sacs which are a nearly constant feature in the Marattiaceae are absent in Botrychium and Ophioglossum, but occur in Helminthostachys. In the general structure of the vascular bundles there are important resem- blances. In both families the first-formed tracheids in the young sporophyte are all of the short, reticulate form, and this is retained permanently in Ophioglossum, but is replaced later by pitted tracheids in Helminthostachys and Botrychium. Helminthostachys alone among the Ophioglossaceae shows spiral protoxylem ele- ments like those found in the later bundles of the Marattiaceae. It is probable that the collateral bundles of Ophioglossum represent the prim- itive type from which the concentric type found in the Marattiaceae has been derived. In Botrychium and Helminthostachys, while the collateral type of bundle is found in the stem, in the petioles the bundles are concentric, as they are in the Marattiaceae. The collateral type of bundle reappears again in the early stages of the sporophyte in Datnvu. Angiopteris, which is probably the most specialized of the living Marattiaceae, has concentric bundles only. CONCLUSION. While the more specialized Marattiacea?, like Marattia and Angiopteris, apparently differ very much from the Ophioglossacea?, it appears from a study of the development of these forms, as well as that of the simpler and presumably more primitive genera Damza and Kaulfussta, that the most conspicuous differences are of secondary rather than of primary importance, and the conclusion is justified that the two families of the kusporangiata? really arose from the same primitive stock. Of the Ophioglossacea?, Helminthostachys is undoubtedly the form which, on the whole, comes nearest to the Marattiacea?, although Ophioglossum is more like them in the character of the reproductive organs and in the vascular skeleton of the sporophyte. Ophioglossum also recalls Kaulfussta in the form and venation of its leaves. While the green gametophyte in the Marattiaceae is undoubtedly a more primitive structure than the saprophytic prothallium of the Ophioglossacea?, the sporophyte of the latter is certainly a more primitive one than that of the Marat- tiaceae. The monophyllous condition, the early development of the sporangia, and the fact that the whole of the sporangiophore is spore-bearing, all point to this. The way in which the sporophyll of the Marattiacea? has been derived from a type like that of the Ophioglossacea? is not clear, and it is by no means certain that all of the Marattiacea? have been developed in the same way. The occurrence of fossil forms with sporangia of the marattiaceous type grouped together — much like the sporangia of Botrychium or Osmunda— suggests that such a condition might have been preliminary to the separation of the sporangia by the development of sterile green tissue in the sporangiophore, such as sometimes occurs in Helminthostachys. 1 he converse is not tenable unless we accept the view that the sterile leaf is an older structure than the sporangiophore, a view which we believe is not warranted by the facts. It has been suggested in a previous chapter that the sporophyll of the Marattiacea? might be accounted for by a theory of concrescence of the sporangiophore and sterile lamina in the Ophioglossacea?. Ophioglossum palmatum, with the two series of small sporangiophores and broad leaf lamina, is the form which comes nearest to such a condition. A very serious objection to this view was mentioned, however — the fact, that the sporangiophore is an adaxial structure, whereas the synangia of the Marattiacea? are always abaxial in their position. Comparing the eusporangiate ferns with the leptosporangiate, it is generally conceded that the Osmundacea? are the ferns which most closely approach the F.usporangiata?. The Osmundacea? show points of resemblance to both the Ophio- glossacea? and Marattiacea?, this being true both of the vegetative tissues and the sporangia. In Osmunda the arrangement of the sporangia suggests Botrvi hium, while in I odea the sporangia are borne upon the backs of the leaves, much as they are in the Marattiacea?. Bower has suggested that the Gleicheniaceae, which are, however, certainly allied to the Osmundacea? also, show certain suggestions of a marattiaceous affinity, especially in the arrangement of the sporangia. We may then briefly summarize our conclusion as follows: From some form, allied to the simpler existing species of Ophioglossum, the whole fern series is descended. In this whole series the leaf is the predominant organ, the stem at first bring quite subordinate in importance. 1 his ancestral fern was monophyllous and the leaf at first was a sporophyll, perhaps without any definite sterile segment. 217 218 CONCLUSION From this central type presumably several lines diverged, of which only a few frag- ments persist. One of these is seen in the different forms of Hot r\c In urn, to which Helminthostachys is probably not very remotely allied. The whole of this series is characterized by a subterranean gametophyte, and a more or less saprophytic habit of the sporophyte is indicated by the development of a mycorrhiza in the roots. The Marattiaceae, as they now exist, probably do not represent a single un- broken line of descent, and show strong evidences of a possible multiple derivation from the earlier stock. The point of contact with the Ophioglossaceae is probably in the neighborhood of Helminthostachys, which, on the whole, is more like the Marattiaceae than are the other Ophioglossaceae; but it is improbable that the solid synangium, such as characterizes most of the Marattiaceae, was derived from a group of separated sporangia like those in Botrychium or Helminthostachys, and it is more likely that they arose from a structure which resembled the spike of Ophiu- glossum. Angiopteris is, with little question, the most specialized of the living Marattiaceae and has probably departed further from the ancestral type than any of the other forms, while Kaulfussia, on the other hand, is probably the most primitive. On the whole the Marattiaceae are nearer to the leptosporangiate ferns than the Ophioglossaceae are, and it is likely that the Leptosporangiates are directly descended from some ancient fern forms, allied to the Marattiacea?, but differing from any of the existing types. BIBLIOGRAPHY. Arber, E. A. Newell. i. On the Past History of the Ferns. Ann. Hot., 1906, 20, 215. Atkinson, G. F. 1. Symbiosis in the Ophioglossaceae. Bull. Torrey Bot. Club, 1893, 20, 356. Bary, A. de 1. Comparative Anatomy of the Vegetative Organs of the Phanerogams and Ferns. Oxford, 1884. Beer, R. I. On the Development of the Spores of II 'elminthostachys zeylanica. Ann. Bot., 1906, 20, 177. Belajeff, W. 1. Ueber Bau und Entwickelung der Spermatozoiden der Gefasskryptogamen. Ber. d. deutsch. bot. Gesell., 1888, 7, 122. 2. Ueber Bau und Entwickelung der Spermatozoiden der Pflanzen. Flora, 1894, 79, Erganzsb., 1-48. 3. Ueber den Nebenkern in spermatogenen Zellen und die Spermatogenese bei den Farnkrauter. Ber. d. deutsch. bot. Gesell., 1897, 15, 339. 4. Ueber die Cilienbildner in den spermatogenen Zellen. Ber. d. deutsch. bot. Gesell., 1898, 16, 140. 5. Ueber die Centrosomen in den spermatogenen Zellen. Ber. d. deutsch. bot. Gesell., 1899, 17, 199-205. Bitter, G. 1. Marattiaceae. Engler and Prantl. Die naturlichen Pflanzenfamilien, 1900, Th. I, Abt. iv, 422-444. 2. Ophioglossaceae. Ibid., 449-472. Bower, F. O. 1. Comparative Morphology of the Leaf of the Vascular Cryptogams and Gymnosperms. Proc. Roy. Soc, 1884, xxxvn, 61; Phil. Trans. Royal Soc, 1884, 175, 565. 2. The Comparative Examination of the Meristems of Ferns as a Phylogenetic Study. Ann. Bot., 1889, 3, 305. 3. Is the Eusporangiate or the Leptosporangiate the more Primitive Type of Fern? Ann. Bot., 1891, s, 109. 4. Studies in the Morphology of the Spore-producing Members: Equisetineae and Lycopodineae. Phil. Trans. Roy. Soc, 1894, sen B, 185, 473-572. 5. Studies in the Morphology of Spore-producing Members, 11: Ophioglossaceae. London, 1896. 6. Studies in the Morphology of Spore-producing Members, m: Marattiaceae. Phil. Trans. Roy. Soc, 1897, ser. B, 189, 35-81. 7. Studies in the Morphology of Spore-producing Members, v. General Comparisons and Con- clusions. Phil. Trans. Roy. Soc, 1903, ser. B, 196, 191-257. 8. Ophioglossum simplex Ridley. Ann. Bot., 1904, 18, 205. 9. The Origin of a Land Flora. London, 1908. 10. Note on Ophioglossum palmatum. Report Brit. Assoc. Adv. Sci., Sheffield, Sept. 1910. Boodle, L. A. 1. On some Points in the Anatomy of the Ophioglossaceae. Ann. Bot., 1899, 13, 377-394. Brebner G. 1. On the Mucilage Canals of the Marattiaceae. Journ. Linn. Soc, London, 1895, 33, 444. 2. On the Prothallus and Embryo of Danaa simplicifolia Rudge. Ann. Bot., i8i;6, 10, 109. 3. On the Anatomy of Datura and other Marattiaceae. Ann. Bot., 1902, 16, 517-552. Britton, E. G. 1. A Revision of the North American Species of Ophioglossum. Bull. Torrey Hot. Club, 1897, 24. S4S-S59- Bruchmann, H. 1. Ueber das Prothallium und die Keimpflanze von Ophioglossum vulgatum L. Bot. Zeif., 1904, 62, 227-247. 2. Ueber das Prothallium und die Sporenpflanze von Botrychium lunaria L. Flora, 1906, 96, 203. BURLINGAME, L. L. I. The Sporangium of the Ophioglossales. Bot. Gaz., 1907, 44, 34. 219 220 lill'.LIOGRAPHY Campbell, 1). II. i. On the Affinities of the Filicineae. Bot. Gaz. 1S90, 15, 1. 2. On the Relationships of the Archegoniatae. Mot. (!az., 1 Sn the Structure and Affinities of Helminthostachys leylanica. Ann. Bot., Sept. 1899, 13. 3. (and T. G. Mill). On the Arrangement and Structure of the Vasculai Strands in Angiopteris evecta and some other Maratriacca'. Ann. Bot., Iq02, 16, ^1-402. ( !oi BEL, K. i. Beitrage zur vergleichenden Entwickelungsgeschichte der Sporangien. Bot. Zeit., 1880, 545 : 1881,681. 2. Vergleichende Entwickelungsgeschichte der Pflanzenorgane. Schenk. Handbuch der Botanik, 1884, 3. 3. Outlines of Classification and Special Morphology. I I ranslation of the German edition.) Oxford, Clarendon Press, 1887. 4. Organographie der Pflanzen. 4 pts. Jena, 1898-1901. ( Irim 1. icil V 1 1. R. 1. Flora of the British West Indian Islands. London, 1864. ( iw ynm -V \i 1,1m, D. T. 1 <)n an Unexplained Point in the Anatomy of Helminthostachys zeylanica. Ann. Bot., 1902. 16, 170. 2. On the Anatomy of Archangwptens henryi and othei Marattiaceae. Ann. Bot., i>>o^ 19, 411. II \NNIG, I'.. 1 I (her die Staubgrubchen an den Stammen und Blattstielen
l sterile leaf; 4, fertile leaf; s- sterile leaf of D. iamaicensis Underwood. B. 1, young plant ol Dantea clliptica Sin". (?); 2, sporophyll of I), jamaicensis; 3, 4, very young plants of /). jamaicensii (?). All specimens collected in Jamaica. I'm 1 10. Adult sporophyte i>i Dancea elliptica. [amaica. Plate 11. K aulfussia test ulifolia Bl. 1. Young plant showing the rhizome. 1. Sporophyll. Specimens collected neai Buitenzorg, [ava. Plate 1 1. A. Marattia alata Swz. 1. Part of fertile leaf. 2. A single fertile pinna. 3. Fallen leaf-base with adventitious buds, k. B. Marattia alata. 1. Leaf from young plant developed from an adventitious bud. 2, }. Young sporophytes arising from fertilization. All specimens from Jamaica. Plate [3. Angioptens evecta (Forst.) Hoffni. 1-3. Specimens from Australia; 4, 5, specimens from Peradeniya, Ceylon. 1. caudexofa small plant; 2, part of a sporophyll; 3, cross-section of the petioleofa full-grown leaf; 4, young sporophyte arising from the prothallium; 5, single leaf from a similar sporo- plivte. CAMPBELL - 4 1. Two spores of O. moluccanum, showing range in size. X 500. 2. Two-celled gametophyte of O. moluccanum. 3. A three-celled stage, sp, ruptured spore membrane. 4. A four-celled stage, seen from above. 5. Two transverse optical sections of a four-celled gametophyte. 6. Three-celled gametophyte of O. intermedium Hk. 7-9. Young gametophytes of O. pendulum L., showing mycorrhi/a! infection, m, mycorrhizal filaments. 10. Adult gametophyte of O. moluccanum. X 10. /, basal tuber; J , anthcridia. 11-14. Gametophytes of O. pendulum. X 3. b, adventitious buds; sp, young sporophytc. 15. Upper part of ripe antheridium of O. pendulum. X 275. o, opercular cell. 16-20. Development of the spermatozoid in O. moluccanum. X 950. bt, blepharoplasts. CAMPBELL W&* bl 21 bl .;*...- '■**.•. 23 A£- 25 22 bl 28 2G 27 ' bl 3 " 29 3 ■ V • -_ 30 .V 21-29. Spermatogenesis in Ophioglossum pendulum. X 950; 21 shows 34-37 spermatocyte before final nuclear division, with two blepharoplasts, bl; 22, 23, nuclear spindle from side and pole; 24-29, development of spermatozoid ; n, nucleus of spermatozoid ; c, cilia; u, protoplasmic vesicle. 30-32. Three stages in development of archegonium of O. pendulum. X 275. b, basal cell; nl, n-, neck canal cells. 33. Venter of archegonium showing apparently a ventral canal cell, 0. n, one of the nuclei of the neck canal cell. X 650. 44. 45, 46-48 Spermatogenesis in B. virginianum; 38-41, in Kaul/ussia; 42, 43, in Danaea. All X 950. Nearly ripe spermatozoids of Angiopteris. X 950. n, nucleus ; bl, blepharoplast; c, cilia; u, vesicle. Nearly ripe archegonium of Danaa, showing apparent ventral canal cell, v. Recently fertilized egg-cells of Danaa. sp, spermatozoid (?) within the egg-nucleus. PLATE 3 H s Ji E E o i E £ -P T3 E CAMPBELL PLATE 4 A i H A. Six plants of Ophinglossum (Ophioderma) intermedium Hk., reduced one-half. Collected at Buitenzorg, Java. B. Ophioglossum (Ophhderma) pendulum L. Collected in the Hanwella Reserve Forest. Ceylon. I, 2. Full-grown sporophytes of O. pendulum, much reduced. In 2 the sporangidl spike, sp, is forked. 3-6. Details of the sporophyll and sporangiophorc. Ophioglossum (Chciroglossa) palmatum L. Collected in Jamaica. I. The whole plant. 2, 3. Sporophylls; in 3 there is a single median spike. CAMPBELL PLATE 6 s =3 03 3 Q- E E .a -5 u 5> CAMPBELL PLATE 8 a U 5 • < 5 to 1 ° 3" g cu < u. CAMPBELL PLATE 13 ft* w - IT! 1 CL u INDEX Adder-tongue fern. See Ophioglossum. Adventitious buds. See Buds. Alternation of generations, 209 Aneura, 120 Angiopteris, 4, 117, 118, 120, 121, 126, 139, 140, 141, 146, 150, 152, 153, 154, 155, 158, 164, 196, 198, 199, 201, 202, 203, 204, 205, 207 Figs. 88, 89, 95, 101, 109, no, in, 112, 124, 130, 134, 180, 181, 182, 183, 187 Plate 13 Angiopteris, species of, 196 Angiopteris evecta (Forst.) Hoffm., n Marattia, 1 ;*, 147. '5°. 'S1 Ophioglossum, 1 ;. 42 Ci \ stals. Sit- Calcium oxalate. (\ >.u!s, 30 Danaea, 17, 53. s-' 84. "7. "8, 124, 125, 126, i:S. [35, 142, 151, 154, 160, 163, 164. 203. 212 Danaea elliptica Smith, 1:4, 125, 1:6, [32, 136, 14:. 14S. 154 Figs. 92,93, 103, 105, 117. 125, i;i, 14;,. 147. 151, 1,2, 161. Plates 9, 10 1). jamaicensis I nderwood, 124, 1:6, 136, 14;, 144, 146, 14s, 154, 155, 156 Figs. 'd. 93, 98, 99, 100, 104, 114, 115, ll6, Il8, 119, I20, 121. 12,. I2C). I 58, 140. I43, 144. 14;. I46, I4S, 141. 1 50, 1 ;;. 160, 161, 189 Plate 9 I), jenmani I (nderwood, 1 24 Figs. 91, [54, 155, 156, 157, 162 Plateg D. simplicifolia Rudge, 124, 127, 136, 142, 154, 160, 175, 176, 206 D. sintensis, 178 I), trichomanoides Spruce, 178 Dehiscence "I — Antheridium, 24, 25, 129 Sporangium, 109 Synangium <>f Marattiaceae, 207 Dichotomy of — Prothallium in Marattiac -ex, 121 Root in Ophioglossum, 93 Dicotyledons, 65 Dictvostele in — Vngiopteris, 197 Danaea, 17; Kaulfussia. 185 Marattia, 192 Drosera, 32 Embryo of — Vngiopteris, 1 jg Botrychium. 46, 47, 48, 51, 136 Danaea, 1 1*. 136, 142 Helminthostachys, 54, 67 Kaulfussia, I 41 Marattia, 1;;. 136, 137 Marattiaceae, 118, 135 Ophioglossum, 34, 35, 36, 37, 38, 43 I ndodermis: \ngioptt us, id |, 201 Danaea, i<>;. 167 Helminthostachys, 72 Kaulfussia. 164, 182, IS; Marattia. 164. 193 Ophioglossum, >>i Endarch bundles in Helminthostachys, 77 Endophyte. See Mycoi rhiza. Entomophthoreae, 22 Equisetum, Equisetineae, 2(>, 27. ;•). 1 ; -. [39 Eubotrychium (see also Botrychium), 100 Euophioglossum (see also Ophioglossum), s;, 86,89,91, 93 Eusporangiatae, 3 Comparison with Bryophytes, 209. 210, 212 Nature of vascular system, 214 Relation to Leptosporangiatae, 217 Fegatella, ;; Fei ns. origin of, 209 Fertilization: Botrychium, 52 Marattiaceae, 134 Ophioglossum, i 1 Fibro-vasculai system is., also Leaf-trace, Stele): Angiopteris, 196, [98, [99, 201 Botrychium, 60, 62, 66 Danaea, 160, 162, i(>;, 104, 166, 172, 174, 176 Helminthostach) s. 7;. 76, 77, 78, 106 Kaulfussia, [81, 1X2. 184, is; Marattia. 189, 19!. [93 I'nssils: Bryophytes, 209 Ferns, 3 Marattiaceae, 1 17 Gametophyte: \ngiopteris, 121 Botrychium, 16, 17, 18 Danaea, 124, 12; Helminthostachys, 20, 21. 22 Kaulfussia, 122, 123 Marattia, 1 19, 121, 122, 210 Ophioglossum, 6, 10, 11, 12, 13, 14. 15, 211 Germination of spores: Marattiaceae, 1 19, 120 ( )phioglossaceae, 7, 8, 9 Gleichenia, mycorrhiza in. ,i Gymnogramme, 26 Hairs: Marattiaceae, 1 50 Ophioglossum palmatum, 99 Helminthostachys, ;. 8, 10. 19. 20, 21, 2,. 51, ;:, 54, 67, 68. 69, 7°, 7>. 72. 73. 74V 75. 76. 77, 78, 79, 80, 81, 82, 84, 104, 10,, 106, 107, 108, 112, 115, 116, 213, 214 Figs. 10,11,45, 46. 47,48,49. ;°. 5ii ^:» ;;• s4. 78, 79. 80, 86 Plate 8 INDEX 227 Horned liverworts (see also Anthocerotes), Indusium, 204 Isoetes, 205, 206 Kaulfussia, 1, 82, 84, 1 17, 1 18. » J9, 122, 123, 141, 147, H9- 152, !53, 154. 157, 158, 164, 178, 179, 180, 182, 183, 185, 186, 187, 206, Figs. 90, 96, 102, 113, 123, 128, 141, 163, 164, 166, 167, 168, 171. 188 Plate 11 Lacunae: Internodal in Helminthostachys, 75 Internodal in Kaulfussia, 184 Leaf: Angiopteris, 201 Botrychium, 61, 101 Dana-a, 176, 178 Helminthostachys, 67, 70, 74 Kaulfussia, 185 Marattia, 194 Marattiaceae, 118, 157, IS9. -'4 ( (phioglossum, 57, 84, 87, 88 Leaf, anatomy: Angiopteris, 202 Botrychium, 102 Danzea, 160 Helminthostacry s, 107 Kaulfussia, 187 Marattia, 194, 195 Ophioglossum, 93, 96 Leaf, fertile (see also Sporophyll), 5, 85, 109 213 Leaf, gaps, 78, 173 Leaf, trace, 66, 71, 8o, 173, 186, 191, 197 Leptosporangiatae, 3, 217 Liverworts, mycorrhiza in, 33 Lycopodium, 9, 10, 24 L. cernuum, 9, 10 Mantle cells (of antlieridium), 24, 129 Marattia, 117, »9> I2°, I22' I26' I28' I32' 136, 137, 147. x49, 152, 'S3. 156, 164, 188, 189, 192, 193, 195, 204, 206 M. alata Sw.irtz, 138, 188, 191, 192, 193- Figs. 175, 176. 177- Plate 12 M. cicutaefolia Klf., 119, 132, 136 M. douglasii (Pr.) Baker, 119, 120, 122, 126, 136, 137, 147, 149. JSi, 153, 188, 189, 190, 205,210 Figs. 87, 88, 94, 106, 107, 108, 122, 132, 133, 141, 172. 173- '74- 185, 190, 191 M. fraxinea Smith, 120, 188, 191, 193 Figs, 87, 186 M. salicifolia Schrad., 193 M. sambucina Blume, 120, 188 Fig. 87 M. weinmannia-folia Liehm., 136 209 132, 155, 181, 208 136, 170, 204, 135. iSS. 194, 194 135. 156, 127. [77. Marattiaceae, 3, 35- 39. 48, 53. 61, 83, 84, 117, 129, 130, 134. 135. 209, 212, 214, 215 Macroglossum, 117, 204 M. alidae Copeland, 204 Mesarch vascular bundles in Helminthostach) s, 78 Monotropa, 33 Mosses, 209 Mucilage cells, 168, 170, 173, 177. 182, 185, 187, 190 Mucilage ducts, 168, 170, 173. l77- 182, 185, 187, 190 Mycorrhiza, 6, 9, "> IS. l8, '9- 20, 21, 22, 32, 33, 127, 179. 181, 212 Operculum (of antlieridium), 24, 25, 128, 129 Ophioderma (see also Ophioglossum). 5, 87, 94 Ophioglossacea-, 3, 5, 6, 7, 34. 82, 86, 108, 109, 208, 213, 214 Ophioglossum, 5, 6, 7, 9. 10, II, 12, 13, 14. •?. 22, 24, 26, 27, 28. 35, 39, 42. 43. 56. 60, 82. 83, 85. 86, 87, 89, 91. 207, 209, 210,213,214,215 O. bergianum Schlecht, 5, 87, 92 O. capense Sw., 92 O. californicum Prantl., 5 (). ellipticum Hk., 92 O. intermedium Hooker, 88, 97, Fig. 69 Plate 4 ( ). lusitanicum L., 86, 87 (>. moluccanum Schlecht., 5, 6, 7. »> l-< 26, 28, 34- 35. 36. 38, 39, 4°. 42. 45. 55. 56, 57, 59, 86, 90,94, 112, 210, 212 Figs. 2, 4, 12, 18, 22, 23, 24, 25, 26, 27, 37, 38, 39, 55, 56. 57- 58. 59. 6°- 61, 62, 81, 83, 192 Plates 1, 3 O. palmatum L., 88, 89, 98, 99 Fig. 70 Plate 5 O. pedunculosum Desv., 10, 11, 13, 34, 3°, 39, 4°, 42, 57 Fig- 3 , o 0. pendulum L., 5, 6, 7, 9, l°> !4> 18, 26, 28, 29, 36, 37, 38, 40, 42, 44, 56, 88, 95, 96, 109, 112 Figs. 1, 3, 4, 13, 16, 20, 21, 63, 64, 65, 66, 67, 68, 82, 84 Plates 1, 2, 4 (). reticulatum L., 6, 40, 90 (). simplex Ridley, 88. 98. 109 Fig. 71 0. vulgatum L., 3, 5, °, 10, 13, 15, 27, 28, 29, 34, 43, 44, 57, 58, 59, 86, 87, 92 Figs. 3, 5. !9 Orchids as humus saprophytes, 33 Osmunda, Osmundaceae, 121, 123, 207 Osmundopteris. See Botrychium virginianum. Palisade tissue, 107, 178, [95, 202 228 INDEX Pcllia, 122 Periderm, 66, 8o, 193 Peronosporeae, 22 Phyllotrichium (sec also Botrychium), 100, 102 Pith, in Helminthostachys, 76, 79 Polypodium quercifolium, 10 Prothallium. See Gametophyte, Pro-ophioglossum, 21 1 Protocorm, S3 Protophloem in Angiopteris, 201 Protostele, 17;, 188 Protoxylem: Angiopteris, 201 Helminthostachys, 79, 82 Kaulfussia, 185 Pteris cretica, 53 Pythium, 22 Rhizoids: Botrychium, 18 Danxa, 127 Helminthostachys, 20 Kaulfussia, 1 23 Ophioglossum, 12 Rhizome (see also Stem) : Botrychium, 101 Danxa, 175, 176, 177 Helminthostachys, 106 Kaulfussia, 186 Ophioglossum, 91, 94 Root: Angiopteris, 140, 202, 203 Botrychium, 47, 51, 64, 103, 104 Danxa, 157, 164, 177, 179 Helminthostachys, 70, 80, 81, 105, 107, 108 Kaulfussia, 187 Marattia, 138, 156, 192, 195 Ophioglossum, 35, 39, 43, 56, 83, 87, 89, 93, 97.98 Root apex: Botrychium, 49 Danxa, 178 Helminthostachys, 72, 80 Marattia, 138, l$6, [95 Ophioglossum, 94 Root hairs, multicellular, of Kaulfussia, 183, 187 Scales, epidermal: Danxa, 151 Helminthostachys, 74, 75 Kaulfussia, 180 Marattiacex, 150 Sceptridium. See Botrychium obliquum. Sclerenchyma, 170, 171, 194 Secondary wood: Angiopteris, 204 Botrychium, 65, 82 Helminthostachys, 78 Sex-organs. See Antheridium, Archegonium. Sieve rubes, 151, 163, 201, 202 Siliceous deposits in Marattiacex, 204 Siphonostele: Angiopteris, 197 Kaulfussia, 184 Marattia, 188 Spermatogenesis: Botrychium, 28 Marattiaceae, i2<>, 130 Ophioglossum, 26, 27 Spermatozoids : Botrychium, 2s Marattiaceae, 130 Ophioglossum, 26, 27 Spike of Ophioglossaceae. See Sporangiophore. Sporangium (see also Synangium): Angiopteris, 205 Botrychium, 109, m, 114, 11^ Danxa, 205, 206, 207 Helminthostachys, ill, 116 Kaulfussia, 207 Marattia, 20; Ophioglossum, 109, 111, 113 Sporangiophore: Botrychium, 109, no Helminthostachys, 105, 106 Ophioglossacex, 5, 85, 108, 109, 208 Ophioglossum, 109, 1 10 Spore: Marattiacex, 208 Ophioglossacex, 6, 7 Spore division, 1 14 Sporophyll: Angiopteris, 204, 206 Archangiopteris, 204 Botrychium, 100, 102 Danxa, 204 Helminthostachys, in, 116 Kaulfussia, 204, 208 Marattia, 204 Marattiacex, 204, 208, 214 Ophioglossum, 85, 88, 93, 109, no, 112, 214 Sporophvte (see also Embryo): Angiopteris, 196, 198, 199, 201 Botrychium, 51, 59, 60, 62, 63, 99 Danxa, 163, 178 Helminthostachys, $4. 67, 68, 77, 104, 105 Kaulfussia. I 79. 1 80, l86 Marattia, 188, 189 Ophioglossaceae, comparison of young spor- ophyte, 82 ( )phioglossum, 38, 39, 44, 45, 55 Staubgriibchen of Marattiaceae, 204 Stele: Botrychium, 82 Helminthostachys, 52, 75, 82 Stem, stem apex: Angiopteris, 153, 198, 199 Botrychium, 49, 53, 101, 103 Danxa, 118, 136, 156, 170 Helminthostachys, 70, 71, 74 Kaulfussia, 185, 187 INDEX 229 Stem, stem apex: Marattia, 192 Marattiaceae, 154, 162 Ophioglossum, 42, 50, 56, 89, 91, 94, 97, 99 Sterilization of sporogenous tissue, 209 Stipule, stipular sheath: Botrychium, 61, 101 Daruea, 1 70 Helminthostachys, 71, 73, 105 Kaulfussia, 185, 187 Marattia, 195 Marattiaceae, 118 Ophioglossaceae, 83, 84 Ophioglossum, 91 Stomata: Botrychium, 101 Damea, 151 Helminthostachys, 106 Kaulfussia, 1 52, 187 Marattia, 195 Ophioglossum, 96 Suspensor: Botrychium, 47, 48, 53, 136 Dana?a, 118, 136 Synangium, 204, 205, 206, 207, 208 Tannin cells: Angiopteris, 146 Danaea, 165, 169, 171 Helminthostachys, 79, 84 Kaulfussia, 185 Marattia, 189 Marattiaceae, 150 Tapetum, ill, 116, 206 Trabecule?, 207 Tracheary tissue: Angiopteris, 201 Botrychium, 66 Helminthostachys, 68, 79, 82 Kaulfussia, 185 Marattia, 189 Ventral canal-cell, 30, 31, 133. 134, 176, 187, 194 201, 213 Vestigial leaves: Botrychium lunaria, 51 Helminthostachys, 69, 81 Ophioglossum vulgatum, 35 MBL WHOI LIBRARY hi H IflJI 1 3*5"l M ■ H agj ■SH He