Art. XXVII.—Studies in the New Zealand Species of the Genus Lycopodium: Part IV—The Structure of the Prothallus in Five Species.
[Read before the Philosophical Institute of Canterbury, 3rd December, 1919; received by Editor, 31st December, 1919; issued separately, 23rd June, 1920.]
In three previous papers (6, 7, 9) I have given general descriptions of the form and the manner of occurrence of the prothalli of eight New Zealand species of Lycopodium. In the last of these papers I also noted, without figures, certain details in the structure of the prothalli which bore upon the general subject of the great variability of the New Zealand species of Lycopodium. The eight species whose prothalli were described are L. Billardieri Spring, L. Billardieri var. gracile T. Kirk, L. varium R. Br. Prodr., L.cernuum Linn., L. laterale R. Br. Prodr., L. ramulosum T. Kirk, L. volubile Forst., L. fastigiatum R. Br. Prodr., L. scariosum Forst. These prothalli are representative of four out of the five known types. It is a striking fact that these eight species introduce no new types of prothallus beyond those which have become known through the researches-especially of Treub and Bruchmann, but they present some very interesting variations from those types. The prothallus of L. cernuum has been, of course, known to science since Treub's papers on several tropical species studied by him in Java were published, but the other seven mentioned above have only recently become known. Two other workers have published the results of their investigations on the prothalli of several of the New Zealand species—namely, Miss Edgerley (4) and Professor C. J. Chamberlain (3)—the former's paper dealing with L. volubile, L. scariosum, and L. Billardieri, and the latter's with L. volubile, L. scariosum, and L. laterale.
The present paper is on the structure of the prothallus of the five species L. Billardieri, L. Billardieri var. gracile, L. varium, L. cernuum, L. laterale, and L. ramulosum. I hope to publish a similar account with regard to L. volubile, L. fastigiatum, and L. scariosum in a fifth part.
The literature dealing with the various other species—European, Tropical, and American—which have been described is enumerated below at the beginning of the sections to which they respectively belong. Several of these papers I have not had access to, but have had to depend for my knowledge of them on brief summaries and figures copied from them in various standard books of reference. This is, of course, unfortunate, as it has made less possible for the purpose of this paper a full comparative study of the different types of Lycopodium prothalli.
I am glad to record my thanks to Dr. Charles Chilton, Professor of Biology at Canterbury College, for the interest he has always shown in my work, and for his kind permission to use the biological laboratory of Canterbury College from time to time. I desire also here to recall and acknowledge the guidance of Professor A. P. W. Thomas, who first suggested to me, when working under him in the biological laboratory at Auckland University College, the study of the New Zealand species of Lycopodium.
L. Billardieri Spring; L. Billardieri var. gracile T. Kirk; L. varium R. Br. Prodr.
The first papers published on the prothalli of this section were those of Treub (14, 15) dealing with the four tropical species L. Phlegmaria Linn., L. carinatum Desv., L. Hippuris Desv., and L. nummularifolium Blume. In a paper on the stem-anatomy of certain New Zealand species of Lycopodium I gave a very brief description of the prothallus of L. Billardieri, stating that it corresponded to the Phlegmaria type (6), and Miss Edgerley a few years later (4) described it more fully, with figures. In two papers of the present series (7, 9) I have given certain details concerning the external form and the structure of this same prothallus and of the two allied forms L. Billardieri var. gracile and L. varium. Literature dealing with two species of prothallus belonging to the closely related Selago section must also be mentioned—viz., Bruchmann's two accounts (1, 2) of the prothallus of L. Selago Linn., and a very brief description, with one figure, of that of L. lucidulum Michx. by Spessard (11).
General Form of the Prothallus.
The complete mature prothallus of all these three New Zealand forms, similarly to those of the four tropical epiphytic species described by Treub, consists essentially of a central body of tissue, which may be either bulky or more or less elongated, and a number of branches which arise adventitiously from this central body. In fig. 1 is shown such a complete mature prothallus of L. Billardieri var. gracile in external view, the natural size being also indicated in the illustration. The central body of the prothallus as here shown is somewhat slender and elongated, a condition which I have found generally to be the rule in this variety. It possesses two complete, and also two broken, thin vegetative branches; a young, stouter branch; and also, nearer the forward end, a short club-shaped “resting” process. The two complete branches on the left side of the figure have begun to put forth secondary branches, while one has also commenced to expand at its growing end preparatory to there bearing sexual organs. The oldest end of the prothallus is intact, and shows clearly the original cone form with which the prothallus of the epiphytic type always begins. At the apex of the cone the cell first formed from the spore still persists. The main prothallial body shows the presence of fungus in its internal tissues, this fungal inhabitant occupying the whole of the tissues in the dark basal cone-like region, but being more irregularly distributed farther forward. The fungus is also present in the vegetative branches, being there also somewhat irregularly distributed, and the single club-shaped resting process is very dark with it. The ends of the branches are all quite free of fungus and are translucent in appearance. The forward end of the main prothallial body is slightly more bulky than the rest, and is quite clear of the fungus. This is the main generative region of the prothallus, and bears paraphyses, archegonia, and also a young plant. The whole prothallus is covered with long rhizoids inclining forward towards the growing apices, except on the terminal bulky region, which is wholly devoid of them, and on the basal cone-like region, from which they have decayed away, leaving only short, stubby projections. The description of this particular prothallus in its external
appearance will suffice to illustrate the general features of the three New Zealand epiphytic species here dealt with.
L. Billardieri var. gracile grows abundantly throughout Westland on stems of the tree-fern Dicksonia squarrosa, and it has also been reported from various other parts of New Zealand in the same situation. The
Fig. 1.—L. Billardieri var. gracile. Complete mature prothallus in general view, showing basal cone intact, lateral branches, and plantlet. × 13. The small figure represents this prothallus at ¾ natural size.
prothalli and young plants occur more especially on the younger stems of the Dicksonia in between the bases of the stipites, which in this tree-fern run down the stem a considerable length before they begin to be overgrown by the mass of hard brittle aerial rootlets. It is in this more open part
of the tree-fern stem-surface that the prothalli and plantlets of both L. Billardieri var. gracile and of Tmesipteris occur. As the tree-fern grows in height the covering of aerial rootlets spreads up the stem, and so plantlets of increasing age have to be carefully dissected out from the mass of the brittle rootlets. The prothalli of this species of Lycopodium are often to be found adhering closely to the hard black surfaces of the stipites, and are there readily found by tearing away the humus and the debris of old tomentum which collects between the bases of the stipites. Their rather delicate, attenuated form is probably the result of this particular position of growth.
Fig. 6.—L. varium. Prothallus in general view, showing central body and branches, and a young plant. × 10.
Fig. 7.—L. varium. Prothallus in general view, showing basal end, and young embryo in the generative region. × 10.
Fig. 8.—L. varium. A branched “resting” process in general view. × 10.
The prothalli of L. Billardieri, on the other hand, occur for the most part in masses of humus on elevated positions in the forks of the forest trees and of their main branches. The central prothallial body is generally more bulky than that of L. Billiardieri var. gracile, but otherwise the prothallus is identical both in appearance and in structure. With regard to the tropical forms studied by him, Treub states that the prothalli of L. Hippuris are much larger and thicker than-those of L. Phlegmaria, while those of L. nummularifolium are exceedingly thin. These epiphytic prothalli have very much the appearance of a mass of root-ends, but a little
experience in dissecting soon leads one to detect the presence of a central region and so distinguish the prothalli from rootlets. Moreover, the root-ends and vegetable fibres so commonly to be met with in the humus are more dead-white or yellowish in appearance, the prothalli in their fungus-free regions being somewhat translucent. Figs. 2–5 show the central bulky region of four prothalli of L. Billardieri, from all of which the oldest basal region is absent. The distribution of the fungus is indicated in these figures by dark shading. It will be seen that the forward region of the central prothallial body is the most bulky, and is wholly free from fungus. It is also quite devoid of rhizoids. It bears on one surface—the upper—paraphyses in large numbers, and also sexual organs. The archegonia and antheridia are not intermingled, but occur in clearly defined zones arising immediately behind the growing apex of the prothallus. The surface appearance of an old antheridium is shown in fig. 3a, the triangular opercular cell being a very distinct feature. The under-surface of the generative portion of the prothallus is always quite naked and smooth.
Lycopodium varium is closely allied to L. Billardieri, but grows terrestrially and has a somewhat different habit of growth. Its prothalli are in every respect identical with those of L. Billardieri. Three prothalli are shown in figs. 6–8. That in fig. 6 bears a young plant; its basal end is not seen. That in fig. 7 shows the basal end dark and withered, and a very young embryo can be seen through the tissues of the forward generative region. In fig. 8 is shown a branched “resting” process. In none of these figures is the distribution of the fungus indicated.
Position in the Soil
The prothalli belonging to these three forms have apparently no regular position relative to the surface of the soil in which they grow. The branches of the prothallus extend in any direction. Frequently they are inclined forward in the same direction as that of the growth of the main body, but this is not always the case. Bruchmann has shown that in the case of L. Selago the more deeply growing, elongated, cylindrical forms of prothallus extend in a vertical direction towards the surface. This is not the case with the prothalli of the terrestrially-growing L. varium, which are quite similar both in their form and in their indefinite position of growth to those of the ordinary epiphytic species. The prothalli of L. Billardieri and of the two other allied New Zealand forms are, however, quite markedly dorsiventral in structure. The paraphyses and sexual organs are to be found only along the uppermost side of the prothallus, whether they occur on the central region or on the lateral branches.
Early Stages in the Development.
The youngest prothallus found by me belonged to the form L. Billardieri var. gracile, and is shown in fig. 9. This prothallus consisted of the first-formed, conical region, which was entirely infested with the fungus and was covered with the old bases of broken-off rhizoids, and a forward translucent region showing several young paraphyses and an antheridium immediately behind the apex. This younger portion of the prothallus also bore the usual long rhizoids. The fungus entirely occupied the main portion of the prothallus except in its epidermal cells. It also bore a young lateral process, on which rhizoids were beginning to arise by the outward growth
of epidermal cells. This lateral process was free of the fungus, except in certain isolated subepidermal cells which lay at the bases of the young rhizoids. From this latter fact it is apparent that, at any rate when the extension in length of a branch is rapid, infection may take place from without through the rhizoids, and that the distribution of fungus throughout the prothallus does not take place simply through its forward extension from the older regions. The original apex of the oldest conical region
Fig. 9.—L. Billardieri var. gracile. A young prothallus complete, in general view. × 60.
Fig. 10.—L. Billardieri var. gracile. Longitudinal section of the basal cone of the prothallus illustrated in fig. 1, showing remains of spore on the first-formed cell. × 170.
Fig. 11.—L. Billardieri var. gracile. Longitudinal section of central body of mature prothallus, showing general arrangement of tissues, and also foot of young plant. × 47.
was intact, and the first-formed cells were clearly to be traced. Most of the mature prothalli of these epiphytic species which I found were incomplete in their basal region, probably on account of the decaying-away of these oldest tissues through age; but I also found several mature prothalli both of L. Billardieri and of the variety gracile in which the original end was intact. This was the case with that shown in fig. 1. A highly
magnified view of the lower portion of the conical region of this particular prothallus is given in fig. 10, in which the remains of the original spore can be seen still attached to the basal cell of the cone. Whether or not a filament ever is formed from the germinating spore, as is known to occur in the prothallus of Tmesipteris, cannot be here stated. The presence of the fungus in these basal cells serves to keep the latter from collapsing for a considerable period, and if a filament is ever formed in the epiphytic Lycopodium prothalli one would expect that, as in the case of Tmesipteris, it would not easily be lost. It is clear, at any rate, from fig. 10 that in some cases at least the germinating spore gives rise immediately to the cone-shaped tissue body. In his description of the prothalli of L. Selago and L. complanatum Bruchmann figures a single cell at the basal point of the prothallus.
Main Body of the Prothallus.
The main body of the prothallus consists essentially of two regions, the hinder vegetative and the forward generative region. There is no such differentiation in structure in the fungus-bearing tissues of the central
Fig. 12.—L. Billardieri var. gracile. Transverse section of basal cone of mature prothallus, showing fungus throughout, also first branch in longitudinal section × 108.
Fig. 13.—L. Billardieri var. gracile. Transverse section of mature prothallus above basal cone, showing initial development of the central strand. × 108.
body of this prothallus as is found in those of the clavatum and complanatum types. In these latter prothalli the fungal tissues are clearly marked off into cortical and palisade zones, and in the case of the New Zealand species L. volubile and L. fastigiatum there is also a wide and very characteristic zone in which the fungal hyphae are also intercellular. The particular type of structure found in these deeply-growing terrestrial types can be put in connection with the general habit of growth of the prothallus. This is the opinion expressed by Bruchmann in his description of the prothalli of L. clavatum, L. annotinum, and L. complanatum (1, pp. 18, 58). As will be seen below, even the prothalli of the Cernua section have attained to a certain degree of differentiation in their fungus-bearing regions. The fungus-infested cells in the main prothallial body of the three New Zealand species which belong to the Phlegmaria type are all practically similar to one another in their form and contents. In longitudinal section it is apparent that transverse divisions have taken place in them so that
they are more equidimensional than are the adjacent cells (fig. 11). It is rather the centrally-placed, fungus-free cells which have become changed in form. They are always considerably elongated, and are somewhat narrower than the cells of the fungal zones (fig. 11). This is, of course, quite in accordance with the function of translocation which they are called upon to perform in these elongated prothalli. Certain of the epidermal cells grow out as rhizoids, a transverse wall separating the rhizoid proper from its parent cell. The width of the fungal zone in the main prothallus body varies in different parts, but, generally speaking, it diminishes towards the growing region. However, if lateral branches are borne well forward on the prothallus the fungus will be found well forward also. The fungus is always massed around the base of a branch, but in those portions of the main body which lie in between the branches
Fig. 14.—L. Billardieri. Transverse section of the prothallus shown in fig. 3, at the point c. × 60.
Fig. 15.—L. Billardieri. Transverse section of the prothallus shown in fig. 5, at the point a. Compare also fig. 2, c. × 60.
Fig. 16.—L. Billardieri. Transverse section of the prothallus shown in fig. 3, at the point a. × 135.
the fungal zone may be no more than one or two cells in width. In the basal cone-like region the cells, which are all equidimensional, are all infested with the fungus, there being here no centrally-placed conducting strand. At the actual base of the prothallus even the epidermal cells show the presence of the fungus (fig. 10). Fig. 12 is a transverse section of the basal cone, showing the first-formed lateral branch in longitudinal section. It will be seen that in the central region in this figure the fungus is inter-cellular as well as within the cell-cavities, and the cells here consequently appear roundish in shape. Farther forward still the centrally-placed cells are free from fungus and represent the beginning of the conducting-strand (fig. 13). Fig. 14 is a transverse section of the prothallus which is shown in fig. 3 taken at the point marked c. In this region of the main body the fungus has extended forward so that it underlies the old antheridia. The rhizoids are here ventrally borne, and the fungus is aggregated along the ventral side so that the prothallus-shows a dorsiventral structure. Fig. 15
is a transverse section of the prothallus shown in fig. 5 at aa, and represents the most forward position occupied by the fungus in this prothallus. It will be seen that the fungus in the lateral branch which is also included in this figure is not in connection with that in the main body. The same figure would also represent in transverse section the appearance of the prothallus shown in fig. 2 at cc.
The generative portion of the central prothallial body is always the most bulky, and shows no differentiation of its tissues whatever Immediately behind the growing apex of the prothalli shown in figs. 2, 3, and 5 young antheridia are being produced. Fig. 16 represents a transverse
Fig. 17.—L. Billardieri. Transverse section of the prothallus shown in fig. 3 at the point b. × 135.
Fig. 18.—L. Billardieri. Transverse section of the dorsal side of the prothallus shown in fig. 2 at the point b. × 135.
Fig. 19.—L. Billardieri. Oblique section through the prothallus shown in fig. 2 at the point a. × 135.
section of the prothallus shown in fig. 3 at a. Fig. 17 is a transverse section through an older antheridial zone of the same prothallus at b, while fig. 18 shows the grouping of the archegonia in amongst the paraphyses on the prothallus illustrated in fig. 2 at bb. The sexual organs occur in large numbers in distinct zones. The close grouping of the antheridia, for example, is shown in fig. 19, which is an oblique section taken through the point of the prothallus illustrated in fig. 2 at aa. The generative region in transverse section bears evidence of repeated cell-divisions, and the cell nuclei and contents are very prominent, as if there had been extensive translocation of food material to this region of the prothallus. A longitudinal section of the generative region shows that the extensive development of sexual organs and paraphyses along the dorsal surface brings it about that
the actual apical meristem is somewhat ventrally placed. Fig. 20 shows this forward region of a prothallus in longitudinal section, but the older vegetative region is not included, as the prothallus was curved in shape. The generative region in longitudinal section is also shown in figs. 11 and 34, both of which prothalli bear a young, developing plant.
The Lateral Branches.
The lateral branches or vegetative processes arise from the main central body when the vegetative region of the latter is ceasing to cope with its main function of nourishing the growing generative region. The examination of such prothalli as those given in figs. 1, 9, and 10 shows both that the rhizoids on the older portion of the main body have decayed away and that the fungal coils in the cells of this older region have given place to the fungal “spores.” The cells are full of these spores, which in all the old prothalli that I have examined do not seem to disintegrate further. The lateral branches are thickly covered with rhizoids, and show the presence of a fungus distributed in the cells more or less along their entire length.
The tissues of the branch are similar to those in the vegetative region of the main body, except, of course, that in accordance with the smaller girth of the branch both the fungal zone and the centrally-placed con-ducting-strand are there correspondingly less in width. Fig. 21 shows a well-developed branch in transverse section, and fig. 22 in longitudinal section. In transverse section there is seen to be a central group of six fungus-free cells, surrounded by a single layer of larger fungus-bearing
cells, some of which have divided. Outside this again is another single layer of somewhat smaller cells which may or may not contain the fungus, bounded peripherally by the epidermal layer, whose external walls are cuticularized. These same layers can also be distinguished in fig. 22 in longitudinal section. Thinner branches show less cell-multiplication in the fungal zone. In longitudinal section it is apparent that the fungus-containing cells have divided transversely, so that they are more nearly equidimensional than those either of the central strand or of the epidermis. The fungus is present in the form of hyphal coils or clusters of oval dark-staining “spores.” The fungus also extends between the cells, so that the latter are frequently roundish in shape.
Sooner or later a branch will show a thickening of its tissues at the apex, and on this swollen region paraphyses and sexual organs will arise (figs. 6, 23, 24). Such a swollen region will be free from the fungus, but the hyphal coils are often aggregated very thickly in that portion of the branch which lies immediately behind it (fig. 24). It is always antheridia which are first formed in such branches, the archegonia occurring only on the main central region of a prothallus. However, a branch may thicken and develop to such an extent that it practically becomes a new prothallus. Probably not a few mature prothalli have arisen not from the germination of a spore, but by a branch having become detached from an older prothallus and having gone on growing in thickness. Such a branch will put forth lateral branches, and will eventually bear archegonia as well as antheridia on its main body. This will account for the fact that most of the mature prothalli found by me did not show the typical cone-like basal region. They are in fact prothalli which have arisen adventitiously. Fig. 27 shows in external view the terminal region of a stoutish branch of L. Billardieri var. gracile which has begun to develop antheridia and paraphyses. Some of the detached branches which are dissected out of the humus are imperfect at both extremities, a fact which indicates that they may persist in the humus and remain self-nourishing for a considerable time.
In dissecting out prothalli of these species of Lycopodium from the substratum in which they lie, one frequently comes across detached prothallial branches which have probably arisen by the decaying-away of the parent prothallus. Three such branches of L. Billardieri var. gracile are shown in figs. 28–30. In this particular species these branches are frequently very long and thin, and they may show the presence of paraphyses along extended portions of their length. Lateral branches frequently bear short club-shaped processes, sometimes in great numbers (fig. 30), which are quite dark with the fungal inhabitant. These processes are to be regarded as “resting” processes, and they may occur also on mature prothalli of the ordinary kind (figs. 1, 11). They are frequently met with detached in the humus, and are either of a simple nature or are branched (figs. 8, 25). The actual apex of the resting process is white and clear of fungus, but the rest is very dark, being thickly infested with it. Fig. 31 represents such a resting process in transverse section, from which it will be seen that the central cells contain the fungus as well as those more medianly situated, and that the fungus is intercellular as well as intracellular. The apex of one of the branches of the stout resting process shown in fig. 25 is given in longitudinal section in fig. 32. Immediately behind the apex the fungus is hyphal only and does not penetrate between the cells. Farther back, however, the hyphal coils have to a large extent been replaced by the “spores,” and the fungus is also intercellular. Instances may be met with
Fig. 21.—L Billardieri. Transverse section of lateral vegetative branch. × 170
Fig. 22.—L. Billardieri var. gracile. Longitudinal section of a lateral vegetative branch. × 170.
Figs. 23, 24.—L. Billardieri var. gracile. Swollen ends of lateral branches, bearing antheridia and paraphyses, in general view. × 22.
Fig. 25.—L. Billardieri var. gracile. Branched detached “resting” process, in general view. × 22.
Fig. 26.—L. Billardieri var. gracile. Detached “resting “process in general view, showing initiation of renewed growth. × 22.
Fig. 27.—L. Billardieri var. gracile. Growing end of lateral branch in general view, showing apical meristem, young antheridia, and paraphyses. × 80.
Figs. 28–30. —L. Billardieri var. gracile. Detached branches in general view, bearing paraphyses and club-shaped “resting” processes, and showing disposition of fungus. × 7
in which a resting process is resuming its extension in length. Generally it proceeds immediately to form paraphyses and antheridia (fig. 26), and probably develops ultimately into a large prothallus. Thus these species are able to propagate themselves vegetatively. In this connection it will be remembered that Treub has described a rather different mode of vegetative propagation in the prothallus of L. Phlegmaria.
Fig. 31.—L. Billardieri var. gracile. Transverse section of a “resting” process. × 135.
Fig. 32.—L. Billardieri var. gracile. Longitudinal section of one of the apices of the “resting” process shown in fig. 25. × 135.
Fig. 33.—L. Billardieri. Longitudinal section of a very young lateral branch. × 135.
Fig. 34.—L. Billardieri var. gracile. Longitudinal section of the generative region of the main prothallial body, showing suspensor, foot, and first root of plantlet in longitudinal section. × 42.
Position of the Meristem.
In the young prothallus of L. Billardieri var. gracile shown in fig. 9 it is apparent that the meristem is confined to the actual apex, and that more than one of the apical cells are meristematic. This position and form of the meristem holds throughout the life of the prothallus of this particular type. In fig. 27 are shown the growing tips of lateral prothallial branches. However irregular in shape the central body of the prothallus may be, there is always a forward growing end to be found where the meristem is localized. In the prothalli shown in figs. 2–5 the growing apex is easily to be distinguished, but it is not always so apparent. As I have mentioned above, the rapid formation of sexual organs behind the growing apex generally brings it about that the meristematic cells are somewhat displaced from the actual apex of the prothallus and lie slightly towards the underside, this being apparent in the longitudinal section shown in fig. 20. Again, I must add
that in this particular figure the hinder region of the prothallus is not cut longitudinally, so that although the fungal tissue in the figure seems to bear some relation to the meristem this is really not the case. In the branches in which the fungus is found almost throughout the entire length it is absent from the whole of the tip of the branch, even in the case of the “resting” processes which are so packed with fungus (fig. 32). Thus the growing tips always appear clear and translucent. A lateral branch arises adventitiously on the central prothallial body by one or more of the externally-placed cells of the latter setting up a local meristem. This can be seen from fig. 33, which shows in longitudinal section a very young lateral branch being formed alongside some old paraphyses. At first the process extends in length by the activity of a single triangular apical cell (fig. 33), but probably very early the apex broadens and more than one cell becomes meristematic. Miss Edgerley states that at the tips of the branches she found two initials (4, p. 105).
Relation of the Young Plant to the Prothallus.
In the epiphytic species, whereas the antheridia are frequently borne on certain parts of the lateral branches as well as on the central body of the prothallus, the archegonia are borne on the latter only. At the same time, it will be remembered that this central body may not have grown directly from the germinating spore, but may have originated by the development of a detached branch. This central body of the prothallus, as has been described, consists of a hinder vegetative region and a forward more bulky generative region, and it is to the latter that the young plant is attached (figs. 1, 6, 7, 11, 34, 36, 37) Not infrequently more than one developing plantlet is attached to the same prothallus (fig. 37).
The prothallus, being placed well below the surface of the humus, is called upon to nourish the young plant altogether until the latter can reach the surface and produce its first leaves. The naked stems of the developing plantlets are sometimes as much as ½ in. long before they reach the light. In accordance with this considerable degree of dependence of the young plant upon its parent prothallus, that part of the base of the plant which is in contact with the prothallial tissues functions as an absorbing “foot.” This foot is a well-marked feature in the plantlets of the epiphytic species, although it does not there assume so large a size as it does in the plantlets
which are borne on the clavatum and complanatum types of prothallus. In these latter species the prothallus is still more deeply buried, and the leaves on the young stem are also no more than scales, so that the prothallus functions as the source of food-supply for a lengthy period. The size of the foot in the New Zealand epiphytic species can be seen from figs. 11 and 34, the foot being in transverse-section in the former figure and in longitudinal section in the latter. There is a well-defined epithelial cell-layer around the periphery of the foot where the latter is in contact with the prothallus, and the outer walls of these epithelial cells are strongly defined, staining darkly. The adjoining prothallial tissue is small-celled and contains abundant protoplasm and darkly-staining nuclei, extensive cell-division having taken place here contemporaneously with the development of the plant. The central cells in the foot are large, with their long axes directed towards the stem-apex. All of these features, of course, point to the fact that there is a well-established translocation of food material from the prothallus into the developing plantlet, and that the epithelial layer functions as an absorbing tissue.
The first root develops comparatively late. It may be recognized as a conical outgrowth at the base of the stem on the side which lies away from the prothallus even before the stem-apex has reached the surface of the soil, but it never seems to develop farther until the first leaves are being produced. Fig. 34 shows the first root in longitudinal section, it being apparent from this figure that the main vascular tissues of the stem lead down bodily into the root, while just a few narrow conducting-elements connect the former with the central tissue of the foot. Fig. 34, which is a drawing of the young plant of L. Billardieri var. gracile is strikingly similar to the figure which Bruchmann gives of the young plant of L. Selago (1, pl. 7, fig. 43). I have observed a number of young plants of the New Zealand epiphytic species in this condition, so that it may be taken as representing characteristically this stage in the development of the young plant and its mode of dependence upon the prothallus in both the Selago and Phlegmaria sections.
Treub stated that in the young plant of L. Phlegmaria he had found an indication of a swelling which he regarded as a rudimentary protocorm. Bower, however, has questioned this, and Treub's statement has never been established. I have found no indication of a protocorm in any of the three New Zealand species which belong to this section of the genus. In his Origin of a Land Flora Bower says that he regards the Selago and Phlegmaria type of embryo plant as being the primitive type for the genus, as it certainly is the most simple. The clavatum and complanatum type of embryo, he says, is not very dissimilar to it, but has become more modified through the large development of the foot consequent on the deeply subterranean habit of the prothallus. The cernuum-inundatum type of embryo stands rather by itself. The intraprothallial swelling called the “foot” is here practically absent, but instead there is the extra-prothallial swelling which Treub called the “protocorm.” Bower and others hold strongly that the protocorm is not a primitive character, as Treub had supposed, but merely a physiological modification.
Details of the Fungal Symbiont.
A symbiotic fungus occurs very commonly throughout the Pteridophyta in the subterranean forms of prothallus, and it has been carefully studied in most of those species of Lycopodium of which the prothalli are known.
In the three New Zealand species dealt with above it is present at the actual base of the prothallus, having entered probably very early in its development. It occupies the whole of the tissues in the basal cone except at the meristematic apex, being found even in the epidermal cells at the basal point (figs. 1, 9). At first the fungus is in the form of hyphal coils which are in actual connection with the mycelium in the outside soil by means of hyphae which are to be found running through the rhizoids. As these rhizoids die off from the older parts of the prothallus, each being cut off at its base by a strongly thickened transverse wall, the fungus in these older regions becomes isolated from that in the soil, and probably ceases to function. The fungal coils soon disappear in many of the cells, their place being taken by clusters of darkly-staining oval “spores” (fig. 10). I do not know whether or not the fungus is at all used up by the growing prothallus, but I should say not, as in even mature prothalli the cells in the basal cone are still occupied either by the coils or by the “spores.”
A little forward from the basal cone the fungus has begun to penetrate between the cells as well as occupying their cavities, so that the cells appear roundish in section. It is especially present in this intercellular position at the centre of the prothallus, and produces its spores there also (fig. 12). A little higher up the prothallial body the fungus becomes more localized, avoiding the central core of cells which has begun to function in the translocation of food material. The cells of this fungal zone throughout the vegetative portion of the main prothallial body preserve their ordinary form and are in no way altered by the presence of the fungus. Their nuclei also appear large and healthy, showing that the fungus has exercised no harmful effect. I have not been able to distinguish in these prothalli the multinucleate vesicles which have been described as occurring in the fungal zone in the prothalli of the clavatum and complanatum types. All that are here apparent are the dense hyphal coils and the clusters of spores. As the prothallus grows, the fungus pushes forward, always occupying its particular zone, except that at length when the sexual organs and paraphyses are initiated it is confined to the ventral side of the prothallus body (fig. 14). In the forward, bulky, generative region the fungus is altogether absent. Its most forward position is shown in (fig. 15), and it will be observed that even here the coils have begun to be transformed into the spores.
The fungus is also present in the lateral branches, being confined there also to a particular zone, avoiding the centrally-placed conducting-cells and the epidermis. Here, too, it is intercellular as well as intracellular, and the clusters of spores are a well-marked feature (figs. 20–22). The distribution of the fungus in some of the long thin branches is discontinuous (fig. 1), from which it would appear that fresh infection can take place from without through the rhizoids (see also fig. 9). In those stouter branches which have begun to form sexual organs and paraphyses the fungus is only present along the opposite side of the branch (figs. 28–30), the branches thus being bilateral. Sometimes it is to be noticed that when a branch is beginning to swell at the apex preparatory to the formation of antheridia the fungus is thickly aggregated immediately behind this point, there occupying the whole of the tissues of the branch (fig. 24). The “resting” club-shaped branches are always very densely infected, there being no long narrow central conducting-cells (figs. 1, 25, 26, 30). The actual apex of the resting process is of course, clear of the fungus, but the latter reaches right up behind the apex, being found there as dense
fungal coils (figs. 31, 32). Throughout the greater portion of these resting processes the coils have given place to the clusters of spores, as takes place also in the other parts of the prothallus.
I have not been able to trace how the spores are formed, or even what is their exact nature. It seems unlikely that they are used up by the developing prothallus as food, for they are more thickly present in the oldest parts of the prothallus than elsewhere. Nor have I observed the presence of oil globules in the fungal tissues, which is so well known a feature in the prothalli of the clavatum and complanatum types. Probably the prothallus benefits from the fungus only when the latter is in the form of hyphal coils, these coils being in direct connection with the external mycelium. The rapid growth in length of the prothallus and its branches accounts for there being no storing-up of food material in any of its vegetative parts. Even in the bulky generative region I have not been able to discover the presence of starch, although it is possible that it may there be present at certain stages in the development of the sexual organs or young plant, but there is abundant protoplasm and the nuclei are very large in the cells around the growing apex. The younger regions of the lateral branches are probably self-nourishing, in this matter being quite independent of the older parts of the prothallus. In fact, isolated branches or portion of branches are capable of continued growth, this being a very common method of propagation in these epiphytic species. The central conducting-strand in the branches functions in the translocation of food material from the younger regions of the branches down into the main prothallial body, the food being there needed in the forward generative region.
Miss Edgerley figures the clusters of spore-like bodies, stating that she found them very commonly in L. Billardieri, but that she observed no nucleated vesicles (4, p. 109).
Comparison of L. Selago and L. Billardieri (& c.).
In his description of the prothalli of L. Selago, Bruchmann (1, pp. 87 et seq.) shows that the more compact surface-growing forms which are found in this species possess a ring-like marginal meristem. This bears a close resemblance to that which is found in the prothallus of the clavatum and complanatum types. The elongated prothalli of L. Selago are found in deeper soil, and possess, according to Bruchmann an erect position of growth, as if striving to reach the surface. Bruchmann derives the peculiar growth of the latter from the ring-like marginal growth of the former, stating that the apparently apical meristem of the elongated forms represents a part of the margin which has grown forward. It will be necessary to compare this manner of elongated growth with that which takes place in the prothalli belonging to the Phlegmaria section.
The prothallus of both L. Selago and L. Billardieri (& c.) starts with the cone form, the cone being from the first richly occupied by the fungus. Judging from the arrangement of cells at the lowest part of the cone, growth at first takes place from a single apical cell, but this probably soon gives place to a group of apical meristematic cells. The girth of the cone is also probably increased by cell-divisions taking place in all the body-cells stimulated by the presence of the fungus. From this point onwards the further growth of the prothallus could either result in the extension of the cone form or the prothallus could pass out of the cone form into an elongated, cylindrical form. The first of these methods seems to
be the normal rule in L. Selago—at any rate, in its surface-growing forms— and the latter in the prothallus of the Phlegmaria section. It is significant to note that both these methods of growth are represented in those New Zealand species which belong to the Cernua section, and which are described later in this paper, although in these species they are not so clearly differentiated as in the former. The prothalli of the Phlegmaria section represent the extreme attained within the genus by the elongated, cylindrical type of growth, and the prothalli of the clavatum and complanatum sections represent, on the other hand, the extreme attained by the continued cone-like manner of growth. The prothalli of L. Selago, as Lang has pointed out (10, p. 305), show that these two main types are not fundamentally dissimilar, for they both here occur in the same species. In saying this, however, it must clearly be remembered that, according to Bruchmann's account, the elongated, cylindrical forms of the L. Selago prothallus do not altogether correspond to the elongated prothalli of the Phlegmaria section, for growth in them is not truly apical, but is rather a one-sided marginal extension from the cone. The elongated prothallus of L. ramulosum, as will be shown, is really more comparable to the prothallus of L. Phlegmaria, & c., although, even here, the comparison is not an exact one. The elongated bilateral extensions from the basal cone in L. Selago are better compared with the flattened extension from the margin which Bruchmann described and figured in L. annotinum (1), being of the same nature but much more pronounced.
The more compact surface-growing form of the L. Selago prothallus is nearer to a self-nourishing, chlorophyllous type of prothallus, which must certainly be regarded as more primitive than a wholly saprophytic one. The very young prothallus, as has been said, begins with the cone form, and next has to set apart a central conducting-core which reaches up behind the actual growing apex. The apex, of course, will be more or less conical in shape, and at this stage is certainly not broad. If the prothallus does not proceed to elongate rapidly, the stimulation to increase in size will be largely confined to increasing its girth, and this will take place not only by a broadening of the apical mersitem, but also by divisions taking place in the body-cells generally. At the same time the fungus-free central core will extend in width as the apex broadens and the prothallus slowly extends in length, until it assumes above the form of a cushion of tissue. If chloropyhll is developed in it, this upper cushion will contain a general distribution of starch. In proceeding to explain how the original apical meristem could become transposed to a marginal ring it seems not unnatural to suppose that as the upper fungus-free tissue becomes more bulky, and so loses the tendency to a localized concentration of food material, the stimulation exerted by the supply of food is felt more in those parts of the prothallus where the fungus is youngest and therefore in its fullest functioning power. This will naturally be along the upper margin of the enveloping fungal zone which extends as a ring around the prothallus. Here, then, the meristematic activity of the prothallus will be localized, As the prothallus still continues slowly to increase in size the fungus will push forward bit by bit into the more newly formed cells, the prothallus still retaining the original cone form.
In the surface-growing prothallus of L. Selago this is what happens, and the sexual stage is fairly early initiated, the antheridia and later the archegonia arising from the meristem. A comparative study of the prothalli of the different species of Lycopodium shows that the cone type of
structure exhibits modifications in the different sections of the genus which are quite in accord with the conditions under which the prothalli of those sections live. There is no need for the surface-growing prothalli of L. Selago to increase in length, for they soon reach the light. Nor is there any great storing-up of food in their tissues, for the young plant when formed can quickly attain independance. Again, it is probably the inherited tendency towards the chlorophyllous, self-nourishing condition, which is strong in the prothallus of this species, which causes the more deeply buried individuals to adopt the peculiar and extensive prolongation of the upper margin of the cone into the cylindrical, bilateral, erect branches in the effort to reach the light. In the altogether subterranean humusloving prothalli of the Phlegmaria section the chlorophyllous habit has been completely lost, and the direction of growth of the prothallus bears no relation to thè surface of the soil. These prothalli are greatly elongated and branched, and the extensive development of long rhizoids, as in the case of the humus-growing prothalli of Tmesipteris and Psilotum, shows that it is necessary for the fungus in the prothallus to be brought into intimate contact with as large an area as possible of the humus for its nourishment. The method of elongation is here altogether different from that adopted by the underground prothalli of L. Selago. Starting from the cone form, the prothallus quickly passes into the cylindrical form without increasing its girth, the meristem continuing in the original apical position throughout the life of the prothallus. In this way the main prothallus body is formed, that of L. Billardieri var. gracile being more elongated than that of the other species examined. The somewhat bulky nature of the forward generative portion of this body of tissue goes hand in hand with the need for the prothallus to support the young plant for a longer period than in the case of the prothallus of L. Selago, owing to the correspondingly greater depth at which the former is buried. The-lateral branches arise adventitiously from any part of the main prothallial body and grow from an apical meristem. They are primarily vegetative, the sexual organs being borne normally on the central body. The latter in its generative region possesses a bilateral structure which is always dorsiventral, the sexual organs being borne only on the upper side. Here again this is not strictly comparable with the bilateral structure of the processes of the L. Selago prothallus, for in the latter it is, according to Bruchmann, simply carried over from the margin of the original basal cone and always bears evidence of this. Thus the modification of the original cone form in the epiphytic prothalli, as in those of L. Selago, is best explained in the light of the conditions under which the prothalli live.
The structure of the prothallus in the New Zealand species belonging to the Cernua section, and the modifications which there occur, will be considered in the next section of this paper, and a general comparative survey, including also the structure of the prothallus of the clavatum and complanatum types, will be instituted in the concluding remarks.
L. cernuum Linn.; L. laterale R. Br. Prodr.; L. ramulosum T. Kirk.
In 1884 Treub (13) published his account of the prothallus of L. cernuum. Goebel's (5) account of the prothallus of L. inundatum followed next in 1887, and in the following year Treub (15) described his laboratory cultures of
the prothallus of L. salakense. In 1902 Thomas (12) published a preliminary account of the prothallus of Phylloglossum. In 1910 (6) I noted the fact that I had found the prothalli of both L. cernuum and L. laterale in New Zealand, and stated that the latter was of the cernuum, type. In Part I of the present series of papers (7) I described and figured the external form of the prothalli and the structure of the young plant of
Fig. 38.—L. cernuum. Prothallus showing in general view lower part of shaft with two fungal regions. × 32.
Fig. 39.—L. cernuum. Longitudinal section of prothallus shown in fig. 38. × 32.
Fig. 40.—L. cernuum. Longitudinal section of prothallus, showing double basal tubercle and thick, short shaft. × 50.
Fig. 41.—L. cernuum. Longitudinal section of upper fungal region of a prothallus, with lateral group of lobes. × 50.
both these species, and also of L. ramulosum, noting especially the variations in form to be found in the prothallus of the latter. In 1917 Chamberlain (3) gave a brief description, with figures, of the prothallus and young plant of L. laterale from material which he had obtained in New Zealand. Lastly, in a third Part (9) I have enumerated the main results of my study of the three New Zealand species which belong to the Cernua section in connection with the plasticity to be observed in the genus as a whole.
General Form of the Prothallus.
L. cernuum.—The typical prothallus of L. cernuum consists, according to Treub, of a basal “primary tubercle,” a longer or shorter shaft, and a crown of lobes, immediately beneath which lies the meristem and the sexual organs. The prothallus is radial in build, erect in growth, and situated at the surface of the soil, being green in its lobes and in the upper portion of the shaft. It is the most delicate and the smallest of all the types of Lycopodium prothalli, the allied L. salakense possessing a still more
Fig. 42.—L. cernuum. Longitudinal section of prothallus of intermediate length, × 50.
Fig. 43.—L. cernuum. Longitudinal section of basal fungal region of prothallus, showing lateral extension. × 50.
Fig. 44.—L. cernuum. Longitudinal section of prothallus of thick, short form. × 32.
Fig. 45.—L. cernuum. Longitudinal section of meristematic region of prothallus, with a fertilized archegonium. × 50.
filamentous prothallus. Some of the prothalli of this species which I have found are comparatively long (7, fig. 17, and fig. 38 in the present paper). That shown in the latter of these two illustrations was broken at its upper extremity, so that I do not know its complete length. In others, again, the shaft is short and thick, and the general appearance of the prothallus more massive (7, fig. 20, and figs. 40 and 44 in the present paper). Forms of intermediate length are shown in a previous paper (7, figs, 18, 19, and 21) and in fig. 42 of the present paper. I have also found a considerable amount of variation in the distribution of the fungus, and, since the fungal regions are always more or less swollen, there is a corresponding variation in the
general form of the prothallus. There is sometimes a single basal tubercle of rounded form (7, figs. 17–20); or this basal fungal region may be extended laterally for a distance up one side of the shaft, thus giving to the prothallus a somewhat lop-sided appearance (7, fig. 21, and figs. 42–44 in the present paper). Again, there may be a second fungal region quite unconnected with the first and situated higher up the shaft (figs. 38, 39, 41). Lastly, there may be two distinct fungal regions situated side by side, the two constituting the basal portion of the prothallus and giving it a markedly thickset appearance (fig. 40). I have sectioned several prothalli which showed this latter form. Rhizoids are borne on both fungal swellings in those cases in which two are present. The lobes which are borne at the crown of the prothallus are flattened extensions (7, figs. 17–21), being generally not more than one to three cells thick in transverse section (figs. 40–42). In one or two instances I noticed from my serial sections that there was a group of lobes situated laterally on the shaft of the prothallus. In fig. 41, this is seen to be associated with the uppermost fungal region, as if the formation of this second fungal zone had initiated a meristematic activity in the adjoining regions of the shaft. The thickest part of the prothallus is always that situated at the base of the crown of lobes. Here is the meristem (figs. 42, 45) from which the lobes have arisen, and by whose continued activity the shaft of the prothallus can be extended in length and its upper region in width. Here, too, the sexual organs are to be found and the young plantlets (figs. 40, 44, 45). I have never found the chlorophyll extending much more than half-way down the shaft. Sometimes the lobes are bright green, or they may show a very slight tinge of colour or be quite colourless, this variation possibly depending simply upon the age of the prothallus.
L. laterale.—The prothallus of this species corresponds very closely with that of L. cernuum. I have not found that there is such a marked variation in the length of the shaft as is to be found in the latter species, the majority of the prothalli of L. laterale being more of the intermediate length (7, figs. 13–16, and pl. 17, fig. 3). The longest prothallus I have found is that shown in fig. 46. One prothallus of a very large size is shown in fig. 48. It was exceedingly massive in form, and of irregular growth. Its greatest length was in a horizontal direction, and it possessed a crown of irregular short lobes all along the top. It was almost colourless, though perhaps this was the result of its age. It bore a young plant which was of a bright-green colour, the foot of the plant also being bright green and showing very conspicuously through the prothallial tissues. Chamberlain (3, p. 57) states that in the prothalli of this species examined by him he found no sharply differentiated primary tubercle like that described by Treub in L. cernuum. From his figures it would appear that this was because his prothalli are rather of the short, thickset form. The basal tubercle is always more distinct when the shaft is well developed. I have found that, as in L. cernuum, the basal, fungal region may extend laterally for a short distance up one side of the shaft, thus giving a lop-sided appearance to the prothallus as a whole (7, fig. 13). Also, as in the other species, there is sometimes a second fungal region higher up the shaft and quite distinct from the basal tubercle (fig. 47). The leafy expansions on the crown of the prothallus of L. laterale are more filamentous and less lobe-like than those of L. cernuum (7, figs. 13–16, and pl. 17, fig. 3, and figs. 46 and 47 in the present paper). This appears also from Chamberlain's figures. A large proportion of the prothalli examined by me showed
Fig. 46.—L. laterale. Longitudinal section of prothallus and young plant. × 42.
Fig. 47.—L. laterale. Longitudinal section of prothallus, showing lobes and shaft, with an upper fungal region. × 42.
Fig. 48.—L. laterale. Large massive prothallus with young plant in general view. Also a very young prothallus entangled in the rhizoids of the former. × 16.
Fig. 49.—L. laterale. The very young prothallus shown in fig. 48. × 70.
a lateral group of lobes borne on the shaft. I previously thought that this was a distinguishing feature between the two species L. cernuum and L. laterale, but have since found it also in the prothalli of the former. In one of the figures given in Part I of this series of papers (7, fig. 16) I showed that a long, thin, club-shaped process is sometimes to be found attached to the basal tubercle of the prothallus of L. laterale, and I there expressed the opinion that this was the first-formed part of the prothallus, as it so often is also in the prothallus of L. ramulosum. My serial sections of this prothallus unfortunately do not make this satisfactorily clear, but none the less I feel confident that this is the right interpretation. The base of this first - formed process (7, fig. 16) shows the presence of the fungus, and it may be compared with the corresponding parts of L. ramulosum illustrated in the present paper in figs. 52–54. As in L. cernuum, the prothallus of L. laterale is normally short-lived, having decayed away by the time that its plantlet has developed two or three protophylls. Probably the large size of the prothallus in fig. 48 was due to the fact that for a considerable time no embryo was formed on it. Moreover, in this case, on account of its large size, the prothallus would persist attached to the young plant for a much longer time than usual.
Fig. 50.—L. ramulosum. Complete mature prothallus of elongated form in general view, showing several swollen fungal regions. × 30.
L. ramulosum.—The prothalli of this species vary very remarkably, both in form and structure. I have discovered these prothalli in several different localities and in considerable numbers, and so am able to give a fairly complete account of their typical form and of the variations of it. The prothalli, as in the other species belonging to the cernuum type, are more or less green in their upper regions, being situated at the surface of the ground. Some of the prothalli of this species are very similar to those of L. cernuum, while others show on the one Hand the elongated and on the other hand the compact, massive habit which I have described as occurring both in L. cernuum and in L. laterale, but here to a much more marked extent. Figs. 50–55 illustrate prothalli of the elongated form
and figs. 56–60 prothalli of the compact, massive build. Some of these figures were given in Part I of the present series of papers, but are here reproduced in greater detail. Photos of three elongated prothalli of this species in general view are also given in Plates XII, XIII, and XIV, and
Fig. 51.—L. ramulosum. Complete mature prothallus of elongated form in general view, showing several fungal regions and a young plantlet. × 30.
a photo of a more massive prothallus with its young plantlet is given in longitudinal section in Plate XV. The prothallus shown in Plate XII is the same as that given in fig. 50.
In the elongated forms the fungal regions may be swollen so as to present the form of rounded tubercles, or, again, they may be scarcely swollen at all. Each fungal region invariably bears a group of rhizoids. In the massive forms the prothallus may be compact and show a certain
similarity in general appearance to the surface-growing forms of the L. Selago prothallus, or they may be so irregularly extended in length that their build is difficult to distinguish. In the elongated forms, along with the development of a number of fungal areas, there goes a corresponding development of several distinct generative regions bearing sexual organs and filamentous or lobe-like processes. Frequently, in this latter form of prothallus, the first-formed generative regions are brown and withered, suggesting that the prothallus had resumed growth on one or more occasions at the close of a dry period or on account of there being no embryo developed. I have always found that a young plant when present was borne on the last-formed region of the prothallus, so that growth in length is apparently not continued after the development of a young plant. The prothalli sometimes persist attached to the developing plant for a much longer time than in L. cernuum. I have found several instances of a healthy prothallus still attached to a plantlet which showed as many as seven or eight protophylls along an extended protocorm. The lateral processes are sometimes filamentous, and at others short and lobe-like. Owing to the fact that the prothallus of L. ramulosum is so variable in form, it is impossible to give many details in a general description of it. I will now proceed to enter more into detail in connection with the particular individuals figured in this paper.
The prothallus shown in fig. 50 and in Plate XII possessed five fungal areas, quite distinct from one another, each being swollen and rounded in form and bearing a group of rhizoids. There are two generative regions showing the presence of archegonia and bearing filamentous processes. The basal fungal swelling bore no evidence of any original filamentous stage, and was probably formed immediately on germination of the spore. As will be described later, a first-formed filamentous stage is frequently to be observed in the prothalli of this species. The uppermost fungal region is, of course, the youngest. It occurs at the base of the meristematic apex of the prothallus, and it is evident that both this growing apex and its fungal region have just begun to form. Both from the figure and from the photo it will be seen that here, curiously enough, the fungus lies internally to the epidermal cells. There is no special apical cell or group of cells, the whole apical region of the prothallus being meristematic. The succession of five swollen fungal regions shows very clearly that it is the fungus which causes the swelling in the tissue of the prothallus, and that, at any rate in this prothallus, the primary tubercle is in no wise different from any of the later-formed tubercles.
The prothallus shown in fig. 51 illustrates some interesting points in comparison with that just described. There are six distinct fungal areas, and the four lowest are closely approximated and are scarcely, if at all, swollen. There is a well-developed filament at the base of the prothallus, and the prothallus as a whole increases quite gradually in girth from this upwards. Even the first-formed fungal region is practically unswollen. I should judge that the extension in length of the prothallus in these early stages had been comparatively rapid, there being no cessation in growth in length consequent on the formation of a first generative region or of the storing of food material. This state of things can bear close comparison with what takes place normally in the cylindrical much-elongated epiphytic prothalli, in which growth is continuous and rapid and there is an entire absence of swellings in the fungal regions. The adoption, or rather the preservation, of the definite apical meristem in the epiphytic type is probably only the result of this habit of growth. The prothallus
of L. ramulosum shown in fig. 51 may best be described in its entirety as a gradually widening filament. The young plantlet borne at its upper extremity was peculiarly diminutive in size. There were no antheridia
Figs. 52–54.—L. ramulosum. Complete mature prothalli of elongated form in general view. a is the basal fungal region; a′ and a″, secondary fungal regions; b′ and b″ are generative regions bearing lobe-like outgrowths. In fig. 52 there is a lateral outgrowth (b) associated with the basal fungal region. The shading on b′ and b″ represents browning due to withering. × 20.
Fig 55.—L. ramulosum. Complete mature prothallus of elongated form in general view, showing branching above the second fungal region. × 20.
either on this prothallus or on that in fig. 50. The photo of the prothallus given in Plate XIII also illustrates the nature of the elongated manner of growth with the formation of several fungal regions.
The four prothalli outlined in figs. 52–55 show at their base a distinct fungal tubercle, that in fig. 53 bearing evidence of an original filament. The prothallus in Plate XIV shows a lower fungal region, which was, however, not the first-formed portion. The shaft is long, and bears a lateral process and also a terminal broad generative region which is associated with a second fungal area. In figs. 52 and 54 there is a fungal swelling located behind the growing end, it being evident also that in both these prothalli the apex is at the point of renewing its growth. A large number of prothalli of this species which I found were of the peculiar step-like form shown in figs. 52–54. Here the generative regions are sometimes of considerable extent, and, relatively to the long axis of the prothallus, lie horizontally. The position of these elongated prothalli in the soil is always vertical. The older generative regions are frequently brown and withered, either as the result of a dry spell in the weather or because they have ceased to be supplied with food and have begun to decay. These generative regions are frequently fairly bulky. It is probable that the irregularly massive forms of prothallus described next have acquired their form through an exceptionally bulky development of one or more of these successive generative regions, the intermediate shaft-like stages of growth having been suppressed. Sometimes these massive prothalli show the remains of the lowest shaft and first-formed tubercle still attached to them. Those prothalli of L. laterale which bear club-like processes attached to the primary tubercle, as described in a previous paper (7, fig. 16), probably come under this category. Fig. 55 shows a prothallus which has branched in its middle region, the two branches being borne on a particularly large fungal swelling. These two branches are clearly not to be regarded as processes of the kind usually borne on the generative regions. One of them showed a fungal region with rhizoids near its apex. Both are browned at the tip. It is certainly interesting to find that along with its power of cylindrical elongation the prothallus of this species is able also to branch, these variations suggesting that the Lycopodium prothallus is very plastic in character and that the several main types are by no means so fixed as has been supposed.
I pass now to the description of the massive and compact prothalli. Some of these show a very regular build (figs. 56, 57, 60, and Plate XV), being comparable with the surface-growing forms of L. Selago (see 1, pl. 6, fig. 37). They do not, however, show the same internal structure as the latter. The prothallus illustrated in fig. 56 possesses one fungal region only, which occupies the whole of the base. This leads up into a short, massive shaft and a wide, upper, generative region. The whole of the uppermost region is meristematic, there being no localized marginal, ring-like meristem as is the case in the compact prothallus of L. Selago. At the base of this prothallus there is a filament consisting of one long cell bent in the middle (fig. 56a) which does not show the presence of fungus. It leads up into a swollen region which can be described as the basal tubercle, this latter passing gradually again into a higher and more swollen region. The prothalli shown in figs. 57 and 60 and Plate XV are also of this same form, there being but one large basal fungal region. That in Plate XV is in longitudinal section, but the section does not pass medianly through either the base or the shaft, nor does it show the full width of the prothallus. This massive form of the prothallus of L. ramulosum is similar to that sometimes adopted by the prothallus of L. cernuum and L. laterale, howbeit in a more marked degree.
Fig. 56.—L. ramulosum. Complete mature prothallus of compact, massive form in general view, bearing young plant. × 27.
Fig. 56a.—L. ramulosum. Original end of prothallus shown in fig. 56 in general view. × 73.
Fig. 57.—L. ramulosum. Complete half-grown prothallus of massive form, in general view. × 35.
Figs. 58–60.—L. ramulosum. Mature prothalli of massive form, in general view. Those shown in figs. 58 and 59 have grown irregularly. × 14.
Another type of massive prothallus is that shown in figs. 58 and 59, where the continued bulky growth has resulted in a very irregular form. The oldest part of the prothallus can be recognized in both these figures, although whether or not this part represents the actual original basal end of the prothallus cannot be said. In fig. 58 there are two distinct generative regions, the uppermost of which is divided by a deep constriction. The foot of a young plant has been torn from one portion of this upper region. This particular prothallus is similar in its continued growth to those shown in figs. 52–54, except that the great bulkiness has rather obscured the typical cylindrical radial build. In fig. 59 the prothallus has grown still more irregularly, and there are apparently two growing regions, one of which is broken. The sexual organs are distributed along the whole of the top of the prothallus, and are in this case archegonia only. This particular prothallus is best compared with the basal portion and first generative region only of those shown in figs. 53 and 54. These massive, irregularly-grown prothalli usually show abundant chlorophyll.
Position in the Soil.
All the prothalli in these three species are situated at the surface of the soil and possess more or less chlorophyll. Their position in the soil is always erect. The chlorophyll is never present in the basal regions, but always in the lobes, and generally also in the upper part of the shaft. Consequent on this vertical position of growth, the prothalli never show such an elongated habit as do those of the epiphytic, subterranean type, but certain individuals, especially in L. ramulosum, show a marked tendency in this direction. I cannot say whether or not the more elongated forms in this species are more deeply buried in their basal part than the massive forms. They were all dissected out of humus consisting for the most part of a decaying short moss. In any case, the total length of even the most elongated forms is too small to have much significance in this respect. Probably the function of continued apical growth is due simply to the postponement of the formation of the embryo, the food-supply being maintained by the fungus through the infection of newer regions of the prothallus. The typical radial build is maintained by all these three species, although in some prothalli, more particularly in L. ramulosum, this is almost obscured by the adoption of a secondary, irregular manner of growth.
Early Stages in the Development.
I have been fortunate to secure several very young prothalli-of the two species L. laterale and L. ramulosum. No doubt the only perfectly satisfactory way of obtaining the youngest stages of growth in any prothallus is by the experimental germination of the spores in the laboratory under close observation. Even this method, however, is not above suspicion, for one can never be sure to what extent the artificial conditions influence the form of the young prothallus. Very young prothalli dissected out of soil collected in the field are open to the criticism that they may belong to some plant other than the one under consideration. I can only say that with regard to the young prothalli now to be described I am quite satisfied that they belong to the particular species of Lycopodium to which I have ascribed them, and the reasons will be stated.
In the case of L. cernuum, I have found no young stages. Treub has, however, described the germination of the spore in this species, having successfully carried this out in the laboratory. His figures, which I have
seen only in certain standard books of reference, show that a tubercle is at once initiated, which is apparently at first devoid of fungus. This tubercle was called by Treub the “primary tubercle,” and he supposed that it was a characteristic feature of the Lycopodium prothallus. He was also successful in experimentally cultivating the prothalli of L. salakense. This also begins with a globular body, the “primary tubercle,” from which several thin filamentous branches arise, one of the latter eventually thickening and producing the sexual organs. This prothallus remains altogether free from a symbiotic fungus. Treub also germinated the spores of L. curvatum Sw., from which he obtained “primary tubercles,” but he was unable to induce them to develop farther. All the mature prothalli of L. cernuum which I have found show the rounded basal tubercle, although in some instances this is somewhat pointed below (see 7, figs. 17–21, and figs. 38–44 in the present paper).
I have dissected out a number of young prothalli of the species L. ramulosum along with the more mature forms, and some of these are shown in figs. 61–65. I judge these to belong to this species since they are of the typical Lycopodium form and no other species of Lycopodium were present in the two localities from which they came. No other kind of prothallus was ever found by me in the turves from which I dissected out those of L. ramulosum, nor was any species of fern present in the near neighbourhood, except Gleichenia dicarpa. Finally, the individuals which are shown in figs. 61–65 form a series which leads on to the more mature prothalli which undoubtedly belong to L. ramulosum. The youngest prothallus was that shown in fig. 61. In it there was no basal swelling, nor was there any indication of the presence of a fungus. The lower half consisted of a single linear row of cells, whilst in the upper half there was a gradual increase in the number of cells and in the girth of the filament towards the apex. This upper half was not a flat expansion of cells as in the usual fern prothallus, but was radial in build. The cells at the apex were smaller than those farther back, and evidently functioned as the meristem. The whole filament contained chlorophyll corpuscles. The original spore-case was still attached to the basal cell. In fig. 62 is shown a prothallus in which the first-formed filamentous stage was very short, passing almost immediately and suddenly into a globular mass of cells which could be called the “primary tubercle.” This prothallus also was quite free of fungus, being green throughout. The apical region consisted of small-celled tissue, and a young sexual organ was developing near by. A group of rhizoids was borne on the basal tubercle. In the formation of a fungusless primary tubercle this young prothallus corresponds very closely to those early stages in L. cernuum, L. salakense, and L. curvatum described by Treub. With regard to the mature prothallus of L. ramulosum, the conclusion I arrived at was that any swelling in the fungal regions was primarily due to the localized presence of the fungus. From these other three species, however, it is apparent that there may be at first a primary tubercle quite apart from the presence of the fungus, and this also appears in such a young prothallus of L. ramulosum as that shown in fig. 62. However, this is not always the case, as in the particular prothallus shown in fig. 61. The three young prothalli shown in figs. 63–65 all possessed a primary tubercle, which was infested by fungus.
Are we to consider that a primary tubercle is a fundamental feature in the structure plan of the Lycopodium prothallus, or is it to be regarded as an added feature? Possibly we are to regard the radially-built filament
as being the original typical condition, this filament being drawn out, or short and bulky, according to whether growth has taken place rapidly or slowly. The basal tubercle of the mature prothallus when present would, according to this view, be the result largely of secondary cell-divisions
Fig. 61.—L. ramulosum. Very young prothallus, in general view, with spore still attached. × 75.
Fig. 62.—L. ramulosum. Young prothallus, in general view, showing basal tubercle with no fungus. × 45.
Figs. 63, 64.—L. ramulosum. Young prothalli, in general view, showing basal tubercle with fungus. × 30.
Fig. 65.—L. ramulosum. Young prothallus, in general view, showing first-formed lobes and the initiation of a second fungal region. × 45.
which had taken place in the first-formed basal cells owing to the stimulus excited by the storing of food material. Prothalli cultivated under artificial conditions seem generally to develop slowly, and this may explain why it is that none of Treub's prothalli showed a first-formed filamentous
stage. There can be no doubt that the symbiotic association of a fungus with the Lycopodium prothallus is a further added feature, the mature form of the Lycopodium prothalli being determined mainly by the nature of this association.
I found one very young prothallus of L. laterale which was entangled in the rhizoids and lobes of the large prothallus shown in fig. 48. This young prothallus is shown in fig. 49. It consists of a filament of cells which is green throughout, the filament being one cell in width in its lower half and two cells in width in its upper. At the apex there is a single small cell which is clearly functioning as the apical cell. In this species also, as in L. ramulosum, the primary tubercle is thus not invariably present.
Main Body of the Prothallus.
L. cernuum.—The main body of the prothallus of this species consists, as has been described above, of one or more basal fungal regions, a longer or shorter shaft, and a crown of lobes, at the base of which lies the meristem and the generative region. Sometimes there is present also a lateral group of lobes. As I have not seen Treub's original papers, I do not know whether or not he has given in his description any indication of a differentiation of structure in the fungus-bearing tissue, but, judging from the short summaries of his results in various standard books of reference, this does not seem to be the case. In the prothallus of this species as it occurs in New Zealand. I have observed that there is in this tissue a well-marked differentiation. The fungal coils are invariably confined, so far as their intracellular position is concerned, to the epidermal cells in the fungal area, but the fungus always extends also in between the cells of the adjoining tissue. This will be apparent from figs. 39–44, which show the fungal area in longitudinal section, and from figs. 66 and 67, which represent the basal tubercle in transverse section. Moreover, this layer of cells which shows the presence of the fungus in an intercellular position is always modified in structure, the cells being very narrow, with their long axes arranged at right angles to the peripheral fungal layer. A portion of the fungal region shown in fig. 41 is given in greater magnification in fig. 68. In describing the “palisade” fungal zone in the prothalli of L. clavatum and L. annotinum, Bruchmann (1, p. 18) suggests that its main function is to serve as a brace to the prothallus. This would seem to be the case also in L. cernuum. In this latter species its peculiar structure is more particularly developed in those fungal regions which extend up the shaft, and where the need of a strengthening-tissue is felt. In those prothalli in which a well-formed, rounded primary tubercle is present the whole of the internal tissue of the tubercle shows the intercellular fungus, but its cells are not so markedly differentiated (figs. 38, 39). In the elongated prothalli of L. ramulosum, in those cases in which the fungal areas are not much swollen, the tissue in which the fungus is intercellular does not show much modification in form. The fungus is chiefly apparent at the angles between the cells, but it also seems to enwrap the cells generally (figs. 66–68). I have not observed the presence of starch, as a rule; in the palisade cells, but it is frequently thickly accumulated in the adjoining part of the shaft. In the primary tubercle the cells are small and numerous (figs. 38, 39), and the rounded form of the tubercle is probably due mainly to the extensive cell-divisions, induced by the fungus, which there have taken place. In this species the intercellular fungus never spreads up the shaft to any great extent: As has been described above, in this species there
is sometimes a second fungal region quite distinct from the first, which may be situated either higher up the shaft or at the base alongside the primary tubercle. The characteristic difference in structure between the basal tubercle and an upper fungal area is clearly shown in fig. 39. The details in the basal structure of the other form are illustrated in fig. 40, the right-hand tubercle here being cut not quite medianly.
The shaft is built up of large cells whose long axes are directed towards the growing apex. In some prothalli this shaft is long and thin (figs. 38, 42), but in others it is short and thick (figs. 40, 44). An instance in which a lateral group of lobes is borne on the shaft is given in fig. 41, these lobes
Figs. 66, 67.—L. cernuum. Transverse sections of the basal tubercle of prothallus, showing distribution of fungus. × 42.
Fig. 68.—L. cernuum. Longitudinal section of portion of fungal area shown in fig. 41. × 135.
Fig. 69.—L. ramulosum. Median longitudinal section of basal tubercle of massive prothallus shown in Plate XV. × 42.
Fig. 70.—L. ramulosum. a and b, longitudinal section of an elongated prothallus with three fungal regions, the basal tubercle being shown at b. × 48. c, longitudinal section of apical head of the prothallus shown in fig. 70a. × 75.
Fig. 71.—L. ramulosum. Longitudinal section of two adjoining fungal regions in a large elongated prothallus. × 42.
here being in conjunction with a second fungal zone. At the top of the shaft is the crown of lobes, these being in transverse, section from two to four cells in thickness (figs. 40–42, 44). In amongst the bases of these lobes are situated the sexual organs (figs. 40, 44). Some of the prothalli bear organs of one kind only, that shown in fig. 40 bearing numerous antheridia but no archegonia. Others, again, bear both, as in the case of the massive prothallus shown in fig. 44.
L. laterale.—The structure of this prothallus corresponds more or less closely with that of L. cernuum. In the fungal regions, whether at the base of the prothallus or higher up the shaft, the hyphal coils are confined to the epidermal cells, and the hyphae penetrate the adjoining tissues in an
intercellular position (figs. 46, 47). In this species the cells which immediately adjoin the epidermal layer are not so clearly differentiated as a “palisade” layer as they are in the prothalli of L. cernuum. In the prothallus illustrated in fig. 47 the intercellular fungus which belonged to the upper fungal zone occupied a considerable extent of the tissues of the shaft and had spread well up towards the meristem. I have not observed anything approaching this condition in any of the prothalli of L. cernuum. The terminal processes are filamentous rather than lobe-like, and are to be seen in longitudinal section in figs. 46 and 47. Their characteristic appearance, however, is best seen in general view (7, figs: 13–16, and pl. 17, fig. 3). A group of lateral lobes is shown in fig. 46, there being an archegonium present at their base.
L. ramulosum.—The internal structure here shows nothing differing at all markedly from that of the other two species described above, except that, as in L. laterale, the” palisade “tissue is not so well developed as in L. cernuum. A prothallus of the massive type is shown in longitudinal section in Plate XV. A true median longitudinal section of the basal tubercle of this same prothallus is given in fig. 69. It will be apparent that this figure corresponds very closely to fig. 39, which shows the basal tubercle in L. cernuum in longitudinal section. The rest of the prothallus is free from fungus, and consists mainly of compact small-celled tissue. The crown of filaments in this prothallus is poorly developed. The structure of the elongated prothallus is shown in fig. 70, a and b. The cell-layer in which the fungus is in an intercellular position is here very limited in extent, and is also little differentiated as a tissue. The lateral generative region is brown, and the filaments are here missing. The apical head of the prothallus bears numerous antheridia (fig. 70, c). Fig. 71 shows in longitudinal section two adjoining fungal areas in an elongated prothallus of much larger build than the one last mentioned, and here it will be seen that the intercellular fungus spreads through a correspondingly greater extent of tissue, in which the cells are certainly arranged more palisade-like. The shaft portion of these elongated prothalli consists of large, elongated cells, but the various generative regions are of small-celled tissue. This is clearly seen in the prothallus shown in fig. 70, this prothallus bearing antheridia only.
Position of the Meristem.
In the cernuum type of prothallus the meristem is of a more indefinite, unlocalized nature than in either the epiphytic, cylindrical type or the subterranean, cone-like type. In his figures of very young stages in the development of the prothalli of L. cernuum, Treub shows that, in his laboratory specimens, immediately on germination of the spore, a triangular. apical cell is set. apart, by whose all-round segmentation a globular mass of cells is formed—the” primary tubercle.” However, this method of growth immediately gives place to a filamentous stage in which the filament is only one cell wide, new segments being formed by transverse walls appearing in the terminal cells of the filament. The filament next thickens by longitudinal walls appearing in all its cells. This also is what happens in L. salakense; but there several such filaments are formed from the primary tubercle, although only one proceeds to thicken.
From my material of the three New Zealand species which belong to this type of prothallus I have certainly found instances in which the spore seems to have given rise at once to a globular mass of cells, but others also in which there is an original filamentous stage. This filament extends at
first by transverse walls arising in the terminal cell (fig. 61), but later it would seem from fig. 49 that oblique walls in the terminal cell cut off segments alternately on one side and on the other, so that the filament becomes two cells in width. All indications that I have noticed point to the fact that this first-formed part of the filament does not subsequently thicken. It is to be found in this thin form still attached at the base of older prothalli (figs. 51, 53, 56), so that it has nothing to do with the “shaft” of the prothallus. Presumably the tubercle is formed by the activity of a single triangular apical cell as in Treub's material, though, of course, my material is not young enough to show this. The young prothalli of L. ramulosum given in figs. 63–65 have grown on from the primary tubercle not in a filament one cell wide, but in a shaft of the same width as the tubercle. This shaft becomes progressively wider as it grows on. If a tubercle shows subsequently a distinct rounded form it must have been due to secondary cellmultiplication having taken place in it owing to the presence of the fungus. In the prothalli of L. ramulosum shown in figs. 62, 63, and 64 the whole of the small-celled terminal portion is clearly meristematic, so that here, possibly after a period of rest, the whole of the forward end of the tubercle has become meristematic. In the prothallus shown in fig. 61 the original filament gradually thickens as it grows on, apparently by longitudinal walls appearing in the various cells, and the whole of the apex has taken on the function of growth.
In L. cernuum the apex of the shaft grows on and eventually gives rise to a crown of lobes. Some of Treub's figures seem to indicate that the lobes are initiated when the prothallus is not more than of middle age. The meristematic zone is then largely localized at the base of the lobes, and by its continued activity the lobes are extended in length, and also the length of the shaft is added to. At the same time the shaft can increase slowly in width by the division of its cells in a longitudinal direction. Under certain conditions, perhaps inducing quickness of growth, the shaft becomes somewhat elongated, and the lobes also are well developed (fig. 42). The opposite conditions bring about a thickening of the shaft unaccompanied by any great increase in its length (fig. 44). Thus in the prothallus practically the whole of the main body is more or less meristematic, and the localization of this function at the growing apex is not so definite as in the prothalli of the epiphytic or of the terrestrial subterranean types. Meristematic activity at the base of the lobes becomes most pronounced when the sexual organs are being formed, so that they are embedded in a small-celled tissue (figs. 40, 45).
In L. ramulosum the prothallus is capable of much more extensive growth, probably on account of its continued infection by the fungus. At a fairly early stage the apex gives rise to the lobe-like outgrowths, the initiation of which is shown in figs. 63 and 64. In the young prothallus in fig. 65 two outgrowths are well formed and a sexual organ is developing at their base. The lobes are browned and slightly withered, as if they had been touched by a dry spell in the weather. At the same time the shaft of the prothallus is proceeding to extend on past the lobes so that these latter will eventually come to occupy a lateral position. Immediately behind the developing shaft-apex three of the epidermal cells have become infected with the fungus and one epidermal cell has grown out as a rhizoid. It certainly seems as if this fresh infection by the fungus had been the cause of the renewed activity of the prothallus. The study of the prothallus given in fig. 50 suggests that the apical meristem has alternately
functioned in the extension in length of the prothallus and in the production of sexual organs and processes, the growth in length of the prothallus being renewed after the formation of each generative area. If an archegonium had been fertilized and an embryo formed, all the food-supply of the prothallus would have been concentrated around the growing embryo, and hence the prothallus would have ceased to elongate. In the prothallus shown in fig. 51 there has probably been no intermission in the functioning of the apical meristem, with the result that this prothallus approximates more to the cylindrical form of the epiphytic type. In the prothalli shown in figs. 52–54 there has been an exceptionally extensive development of generative tissue, giving to these prothalli the curious step-like form. From these elongated prothalli and from the irregularly-grown, more massive forms shown in figs. 58 and 59 it would appear that the repeated fresh infection of the prothallus by the fungus has enabled it to grow on far beyond its original cernuum-like form. In such a prothallus as that shown in fig. 51 the position and appearance of the meristem are similar to the ordinary cernuum type, although it has been able to function for a much longer period than in L. cernuum. In the other elongated prothalli shown in figs. 50 and 52–54 the position and form of the meristem is somewhat modified on account of the repeated development of the generative tissue, so that the actual growing apex is small and somewhat displaced. There is never a definite group of apical meristematic cells present, the whole of the growing head functioning in this respect as in L. cernuum. The very compact cone-like prothallus given in fig. 56 also shows the same type of meristem, there being no tendency to a localized, marginal, ring-like meristem as there is in the compact surface-growing forms of L. Selago. Thus, although the prothallus of L. ramulosum in its two forms may be compared on the one hand with the compact cone-like forms of L. Selago (and so also of L. clavatum and L. complanatum), and on the other hand with the elongated cylindrical form of L. Phlegmaria, & c., this comparison is by no means a close one, for the position of the meristem shows that L. ramulosum belongs always to the cernuum type. It is significant to note that these main types of Lycopodium prothalli are in a plastic condition, and the variations which they show make it quite permissible to conjecture how the different methods of growth could have originated.
The mature prothallus of L. laterale also conforms to the cernuum type in the position of its meristem, although here too the normal form of the main prothallial body is sometimes considerably modified by its continued growth.
Relation of the Young Plant to the Prothallus.
Since the prothallus of these species is always situated at the surface of the ground, the young plant possesses chlorophyll from a very early stage. As soon as it emerges from the tissues of the prothallus it proceeds to form the characteristic tubercle which Treub called the “protocorm,” on which abundant rhizoids are produced. At the same time the first “protophyll” arises on the tubercle, showing numerous stomata. Thus the developing plantlet early becomes independent of the prothallus in the matter of food-supply. A protocorm is formed in the young plant of all three New Zealand species which belong to the Cernua section. Young plants attached to their parent prothalli are shown in fig. 72 (L. cernuum), in figs. 46 and 48. (L. laterale), and in figs. 51, 56, 58, 60, and Plate XV (L. ramulosum). Other figures are also given in Part I of this series of papers.
The development of an embryo seems to bring all further growth of the prothallus to an end. The prothallus of this species is generally small and delicate, and does not long persist attached to its young plant. It has in most cases decayed away by the time that the plant has developed two or three protophylls. However, I have several times found prothalli of L. ramulosum still persisting, in one case healthily green in colour, attached to plantlets of seven or eight protophylls.
The intraprothallial portion of the embryo plant is developed as an absorbing-organ, but only to a slight extent as compared with the same organ in the young plant of the Selago and Phlegmaria types. Still less is it comparable in importance with the large “foot” of the clavatum and complanatum types. This progressive importance of the foot in the different types, taken in the order in which they are mentioned, goes hand
Fig. 72.—L. cernuum. Longitudinal section of prothallus and young plant showing foot. × 30.
Fig. 73.—L. cernuum. Transverse section of suspensor and foot of young plant. × 42.
Fig. 74.—L. cernuum. Longitudinal section of foot and part of protocorm of young plant. × 42.
Fig. 75.—L. ramulosum. Oblique section of portion of basal fungal region showing three large “spore” capsules of undetermined nature. × 250.
in hand with the increase in the size of the prothalli and of the extent of the dependence of the young plant upon its parent prothallus. From the figures mentioned above it will be seen that the surface cells of the foot in the three species of the cernuum type are only slightly developed as an epithelial layer. They are best illustrated in fig. 73, which is a transverse section of the foot and suspensor of the young plant of L. cernuum, and fig. 74, which shows the foot in longitudinal section.
In Part II of these papers (8, p. 92) I stated that I had never observed the presence of fungal hyphae in any of the protocorms which I had examined. This statement I must correct. The fungus is certainly never present as
intracellular hyphal coils, as it is in the prothalli, but it seems to be always present in an intercellular position in the central tissues of the protocorm. These centrally-placed cells, which show abundant protoplasmic contents, are always roundish in form and are separated from one another, the intercellular spaces staining darkly with haematoxylin. The appearance of the fungus in the protocorm is thus in marked contrast to the definite epidermal fungal tissue of the prothallus, in which it occurs in hyphal coils. The hyphae can clearly be traced entering the protocorm through the rhizoids. They seem to cross the outer layers of cells mainly, if not entirely, through spaces between the cells and more especially at their angles, and they are not at all apparent in this zone. But when once one has learned to look for them, both the hyphae and also their “spores” can be seen in the spaces between the cells throughout the whole of the central region of the protocorm (figs. 46, 74, and Plate XV; see also 7, fig. 61, and pl. 17, fig. 2, and pl. 18, figs. 1, 2). The fungal hyphae can best be studied outside the protocorm, around the base of which they form an open mat. They are branched and unseptate. Here, too, the spores are abundant, and it can be frequently and clearly seen that they originate by a length of hyphal thread dividing up into a number of the spores. This also can be seen in the intercellular spaces at the centre of the protocorm. It is certainly a striking fact that the fungus never occupies the cell-cavities in the protocorm. The fungus undoubtedly assists the young plant to become self-nourishing.
Treub and others have supposed the protocorm to be an organ of high phylogenetic importance. Bower, however, believed it to be a physiological modification, just as is the intraprothallial swelling called the “foot.” My study of this organ as it occurs in the three New Zealand species certainly inclines me to accept Bower's suggestion. The Lycopodium plant in all its organs is in such a plastic condition that it is unlikely that any highly primitive structures should have been retained— at any rate, in its vegetative parts.* The different sections into which the modern genus is classified are strikingly in accord with its biological divisions, as Lang, with regard to the prothallus, has pointed out. It might be added that this holds also for the other main organs of the Lycopodium plant. Each type of prothallus is correlated with a certain type of embryo plant. The protocorm is always associated with the cernuum type of prothallus, and apparently with no other. The comparative study of the different types of prothallial structure certainly leads one to the conclusion that the cernuum type is the least modified of all, and its chlorophyllous habit especially points to this; but it does not necessarily follow that the cernuum embryo is also the least modified. The presence of the symbiotic fungus seems to have been the dominant factor in determining the form of the different Lycopodium prothalli, and it is a significant fact that the protocorm is also infested with it.
[Footnote] * There has quite recently been described by Kidston and Lang (Trans. Roy. Soc. Edin., vol. 52, pt. 3, pp. 603–27, 1920) a small plant from the Devonian of Scotland, which the authors have named Hornia Lignieri, whose basal portion consists of a protocormous rhizome. This plant the authors compare with the young plants of Lycopodium cernuum, L. laterale, and L. ramulosum, and with Phylloglossum. As they suggest, the demonstrated presence of the “protocorm” in archaic vascular cryptogams introduces quite a new argument into the discussion concerning the nature and origin of this peculiar organ. However, they also briefly point out (ibid., note at bottom of p. 612) that fungal hyphae occur in an intercellular position in the tissues of the rhizome of Hornea.
Details of the Fungus.
There is not very much to say here with regard to the details of the symbiotic fungus in these three species of prothalli. The hyphal coils are present only in the epidermal cells of the fungal region, although the hyphae penetrate farther into the adjoining tissue in an intercellular position. I have seen no formation of clusters of “spores” as occurs in the epiphytic prothalli. Spores occur, however, in the interior tissue of the protocorm of the young plant along with hyphae in between the cells.
One prothallus of L. ramulosum showed in its lower basal tubercle a number of large, spherical, dark-staining bodies, three of which are illustrated in fig. 75. These bodies possessed thick walls, and in section were seen to be full of numerous very small oval lightly-staining “spores.” My serial transverse sections of this tubercle showed that there were no fewer than twelve of these large bodies present. They lie within the cell-cavities in the region in which the fungus is intercellular. Each is large enough to completely fill a cell and bulge out its walls. In each of two other prothalli of this species I observed a single spore body of this nature. I was not able to determine their nature, but judge that they bear some relation to the symbiotic fungus.
A special interest attaches itself to the study of the prothalli of the different species of Lycopodium, arising mainly from the fact that several types of prothallial structure are represented in the genus, and also that each of these types goes hand in hand with certain characteristic features in other organs of the plant. So different are these types of prothalli one from another that both Treub and Bruchmann have expressed the opinion that these differences date from a very ancient period. Treub states his belief that they can be explained in a small degree only by the different conditions under which the prothalli live. Bruchmann would divide the genus into as many new genera as there are distinct prothallial types. As opposed to this view, Lang has suggested that the different prothalli show a similarity in ground plan, and that they are all more or less profound modifications of an original cernuum-like form. As further species of prothalli are found this interest is deepened, for it becomes clear that although the main types are all now probably known to us, yet there are many significant modifications of them, and also that further study of the genus will probably reveal more such. Instead of assuming that the different prothallial types are practically unrelated, it will probably be more productive of results to try to discover from the development of the individual prothalli and from their variations whether there is a fundamental plan of structure, and what has been the reason for its modification. Such a study will, at all events, help us to recognize which characters are of most importance.
The complete mature prothallus of all the sections of the genus except the Inundata and Cernua shows consistently at its basal, original end the form of an inverted cone, the apex of the cone being occupied by a single cell, to which the remains of the spore can sometimes be seen attached. It is, of course, possible that in some cases a brief filamentous stage may arise first from the spore before the cone form is attained, and that this drops away later and so becomes lost, but apparently it seems that the cone form is at once initiated. In the much-branched prothalli of the Phlegmaria type, which are radial in build but not upright in growth, the cone quickly
passes over into the elongated cylindrical main body which bears sexual organs on the dorsal side, so becoming bilateral. The prothalli of L. Selago grow upright in the soil. They either preserve the cone form throughout their life—being, however, much smaller than the prothalli of the clavatum or complanatum types—or they give rise to elongated bilateral extensions of portions of the upper margin of the cone. In the upright prothalli of the clavatum and complanatum types there is practically no disposition towards elongation, the cone form being preserved throughout the life of the comparatively massive prothallus, any extra meristematic activity showing itself merely in irregular lobing of the upper margin or in adventitious budding. The angle of divergence of the cone from its apex upwards is different in these two types, being greater in the former than in the latter, so that the mature forms of the two prothalli are somewhat different. The prothalli of the species which belong to the Cernua and Inundata sections are also upright in growth, and have a radial build, so that in transverse section the main body is circular in outline, but the cone form is not always present. Treub's description of the prothalli of L. cernuum suggested that a short cone-like stage always followed immediately upon the germination of the spore, and the” primary tubercle,” as it was called by him, was thus looked upon as a characteristic feature in this type of prothallus. In all the species of this type whose prothalli are known a primary tubercle is, indeed, often present, but not invariably so, as appears, for example, in the prothalli, both young and old, of L. ramulosum.
The cone form adopted by the Lycopodium prothallus must be considered along with the presence in its tissues of the symbiotic fungus. It is, of course, well known that this fungus is one of its most characteristic features, just as it is also of other subterranean pteridophytic prothalli. Treub states that the prothallus of L. salakense shows no fungus, and it is quite possible that other species belonging to either the Cernua or Inundata sections may prove to be without it. From what we know of the young prothallus of L. laterale and L. ramulosum it would seem that in the sections Cernua and Inundata the infection may be postponed considerably, for the fungus is frequently absent altogether from young prothalli of quite fair size, and also not infrequently mature prothalli show signs of the same initial fungusless region at their base.
In the prothallus of Tmesipteris the fungus is found even in the basal filament, when that is present; but whether the infection is initiated from the very first or subsequently spreads back into the extreme lower parts of the prothallus is not known. I have found many prothalli of Tmesipteris, both young and also mature, which showed a short filament of cells at their base, and also, on the other hand, complete prothalli in which no filament was present, in the latter case the remains of the original spore being sometimes clearly apparent on the cell at the actual apex of the basal cone. Sometimes the Tmesipteris prothallus takes the form of a steadily tapering cone, and at others in its lowest regions it increases in girth by a progressive series of gentle swellings. One cannot avoid the suggestion that the dominating factor in the Tmesipteris prothallus is the presence of the fungus, the nature of the swelling being due to the consistency or intermittency, as the case may be. of its activity, and the omission of a filamentous stage being due to its very early entry into the germinating spore. When we turn to the prothalli of the Lycopodiaceae the same conclusion seems to be forced upon us. Large mature prothalli of L. ramulosum may show no primary tubercle at all, and the cone form may be altogether absent, so that the general form of this prothallus can then
best be described as a radially-built filament. The immediate effect of the presence of the fungus also can be clearly seen in the prothalli of this species. It almost invariably causes a swelling of the tissues of the prothallus at the infection spot, this swelling being due to the greater or less development of the internal fungal tissue in which the hyphae are intercellular. Sometimes the fungus is restricted to one place in the prothallus, this being towards or at its base, so that there is present a basal swelling. But in all three New Zealand species which belong to the cernuum type, and more especially in L. ramulosum, more than one distinct fungal area is frequently formed. Another form sometimes adopted by the prothallus of L. ramulosum, and to be seen also in those of L. laterale and L. cernuum, is that in which the usual shaft is practically absent, the crown of lobes immediately surmounting the basal fungal region. The latter is then more bulky than usual, the whole mature prothallus bearing more truly the cone form than is normally the case in the prothalli of these sections. That this particular massive form is not a fixed character appears from the fact that only infrequently are these bulky prothalli of regular shape and growth. However, just as the long-drawn-out, more filamentous form may bear some comparison with the epiphytic, elongated type, so may the massive form be compared with the deep-growing clavatum and complanatum types, and the prothallus of L. ramulosum is thus seen to possess as great significance as that of L. Selago in bearing witness to the plasticity of the Lycopodium prothallus.
When we turn to the epiphytic type of prothallus we find that there also the form of the prothallus must be considered along with the nature of the fungus-distribution in its tissues. The whole of the basal cone constitutes, as it were a primary turbercle which contains the fungal element throughout its tissues, being comparable to the first-formed tubercle in L. cernuum, when that is there present. The elongated, cylindrical regions of the epiphytic prothalli, have completely passed out of the cone form, nor do they show any localized swellings induced by the presence of the fungus. The fungus is distributed throughout the branches and the vegetative region of the central body, and the absence of swelling is probably due to the rapid elongation of the prothallus in all its parts. Even the discontinuous infection which is to be seen often in the branches does not result in any swelling of the tissues as it does in L. ramulosum. Thus, although the elongated prothalli of L. ramulosum may be compared with the epiphytic type, yet they never attain to the characteristic form or structure of the latter. It will suffice here to allude quite briefly to the statement made by certain other writers, notably Lang, that this difference is probably merely the expression of the fact that in the Cernua and Inundata types fungal assimilation has not assumed the same degree of importance in the life of the prothallus as it has in the epiphytic types, for the prothallus is surface-growing and never gives up its chlorophyllous, self-nourishing habit. The epiphytic prothalli, however, live in soil of a different nature, and have adopted a wholly subterranean mode of life, in which they depend altogether upon the activities of the fungus for their nourishment. This has been the immediate cause of their extensive ramifications and their character of continuous cylindrical growth. In this respect, therefore, they are to be regarded as more specialized than the chlorophyllous type.
Although I have not in the present paper given any description of the prothalli of the species L. volubile, L. fastigiatum, and L. scariosum, which belong to the clavatum and complanatum types, yet it is appropriate here to compare briefly their form and structure with that of the other New
Zealand species already dealt with. In these two types the cone form is at once initiated and is continued, throughout the life of the prothallus. There is no extension in length comparable even to that of the Cernua and Inundata types, although, as I mentioned in a former paper (7, p. 273) and hope to describe more fully in a later one, the young prothallus of L fastigiatum branches after it has passed through the initial cone stage. It assumes the shape of the letter “Y,” the arms of the prothallus being true branches, as indicated by the fact that in each of them there is a central core of fungus-free cells completely surrounded by the fungal zone, the meristem being located near, but not actually at, the apex of each arm. Although the mature prothallus in this species has the saucer form, yet it frequently bears evidence of this early branching. The prothalli of these two types have departed altogether from the self-nourishing, chlorophyllous habit, and have become wholly dependent upon the symbiotic fungus, although the prothallus of L. volubile is able to develop chlorophyll if it happens to grow at the surface of the ground. We find that the external tissues in the whole of the vegetative portion of the prothallus have been given over to the fungal inhabitant, so that in form this vegetative region is one large regularly-shaped cone. The prothalli of the clavatum and complanatum types, owing to their terrestrial habit, occur in soil of a nature different from that of the humus-growing, epiphytic prothalli. There seems to be no need for the prothallus to elongate or branch in order to come in contact with a sufficient supply of food, although, judging from the branching by which the young prothallus of L. fastigiatum assumes the saucer form, it could do so if necessary. The fact that these types of prothalli are so much larger in size than the surface-growing form of L. Selago is obviously due to the much greater degree of dependence upon them of the young plant than in the latter species. They function largely as storehouses, the large quantities of starch and oil which they contain having been noted by all who have described them.
The compact, massive form of the prothallus in L. ramulosum, L. laterale, and L. cernuum must not be compared too closely with that of L. Selago or of the terrestrial subterranean types, for the position of the meristem in the latter is. altogether different from that in the former. Nor can the elongated form of L. ramulosum be compared exactly, with that of L. Billardieri. It would seem, however, that the difference between the marginal meristem of the Selago prothallus and the apical meristem of the epiphytic types is not so great. The prothalli of both these types in variably begin with the cone form, and the subsequent manner of growth of each, and the form which the meristem takes, can be explained quite naturally (as I have attempted to do earlier in this paper) as resulting simply from the position of the prothallus in the soil. That there is a somewhat close relationship between these two types appears also from the fact of their similarity in the embryo plant and in the presence of paraphyses. The clavatum and complanatum types of prothallus also seem to be allied to that of the Selago type, possessing the same kind of marginal meristem, although they lack paraphyses. Moreover, the embryo plant of these deeply subterranean forms differs from that of L. Selago only in the abnormal development of the “foot,” a character which has quite obviously resulted from their position in the soil.
The prothalli of the Cernua and Inundata sections seem to stand rather apart from the rest of the genus. The meristem is of a quite different nature, the fungal habit has not been adopted to the same extent, the filamentous manner of growth is largely present, and the form of the
young plant is very peculiar. Any similarities in detailed structure between this type and any of the others are perhaps best regarded as instances of parallel development, such as the fungal “palisade” tissue in the prothallus of L. cernuum and of L. clavatum and L. complanatum, and also the elongated manner of growth in L. ramulosum and L. Billardieri. The varieties in form and structure which occur in the prothalli of the cernuum type are chiefly interesting as evidences of the great plasticity of the Lycopodium gametophyte. It is in the Cernua and Inundata sections that we meet with the least evidence of a permanently fixed type of prothallus. These prothalli have also proceeded the least of all to a saprophytic, mode of life, and generally show the least extent of specialization. The epiphytic prothalli have become thoroughly specialized to the epiphytic habit, and the clavatum and complanatum types to the subterranean terrestrial habit. We are justified, therefore, in regarding the Cernua and Inundata types as showing more nearly the original structure plan of the Lycopodium prothallus than the others.
Thus we may conclude from a comparative study of the general form and structure of the different Lycopodium prothalli that they are all more or less modified from some primitive type of structure, and that the chief factor in this modification has been the presence in them of the symbiotic fungus. This primitive type of structure was probably a more or less bulky filament of radial build, situated at the surface of the ground, and possessing chlorophyll. The adoption of a fungal habit opened the door to possibilities of modification of this simple type of structure, and gave to the Lycopodium prothallus its quality of plasticity. It was able to establish itself in new positions and soils, the different types of habitat resulting in different types of modification of the original structure. When the fungal habit was thoroughly adopted the early filamentous stage became lost, but in all its forms the Lycopodium prothallus has never departed from the radial build.
It seems clear from a comparative survey of all the main characters of the Lycopodium sporophyte and gametophyte that the different sections of the genus are natural ones, and that their characters are all interdependent. It is possible that the varied aspect of the genus as it exists to-day has been due largely to the phenomenon of symbiotic association with a fungus exhibited by its gametophyte generation, the varied structure of the sporophyte as regards the form adopted by the mature plant, the form of its spore-bearing regions, and its stem-anatomy following as a natural consequence from the spread of the gametophyte to different stations and soils.
1. Bruchmann, H., Über die Prothallien und die Keimpflanzen mehrerer europäischer Lycopodien, pp. 1–119, pl. 1–8, Gotha, 1898.
2.—— Die Keimung der Sporen und die Entwickelung der Prothallien von Lycopodium clavatum, L. annotinum, und L. Selago, Flora, vol. 101, pp. 220–67 (35 figs.), 1910.
3. Chamberlain, C. J., Prothallia and Sporelings of Three New Zealand Species of Lycopodium, Bot. Gaz., vol. 63, pp. 51–64, pl. 2 and 3, 1917.
4.Edgerley, Miss K. V., The Prothallia of Three New Zealand Lycopods, Trans. N.Z. Inst., vol. 47, pp. 94–111 (37 figs.), 1915.
5. Goebel, K., Über Prothallien und Keimpflanzen von Lycopodium Inundatum, Bot. Zeit., vol. 45, pp. 161–68, pl. 2, 1887.
6.Holloway, J. E., A Comparative Study of the Anatomy of Six New Zealand Species of Lycopodium, Trans. N.Z. Inst., vol. 42, pp. 356–70, pl. 31–34, 1910.
7.—— Studies in the New Zealand Species of the Genus Lycopodium, Part I, Trans. N.Z. Inst., vol. 48, pp. 253–303 (102 figs., pl. 17, 18), 1916.
8.Holloway, J. E., Studies in the New Zealand Species of the Genus Lycopodium: Part II, Methods of Vegetative Propagation, Trans. N.Z. Inst., vol. 40 pp. 80–93 (24 figs., pl. 8, 9), 1917.
9.—— Studies in the New Zealand Species of the Genus Lycopodium: Part III, The Plasticity of the Species, Trans. N.Z. Inst., vol. 51, pp. 161–216 (16 figs., pl. 9–14), 1919.
10.Lang, W. H., The Prothallus of Lycopodium clavatum, Ann. of Bot., vol. 13, pp. 279–317, pl. 16, 17, 1899.
11. Spessard, E. A., Prothallia of Lycopodium in America, Bot. Gaz., vol. 63, pp. 66–76 (19 figs.), 1917.
12. Thomas, A. P. W., Preliminary Account of the Prothallium of Phylloglossum, Proc. Roy. Soc. Land., vol. 69, pp. 285–91, 1902.
13.Treub, M., Études sur les Lycopodiacées: I, Le prothalle du Lycopodium cernuum L., Ann. Jard. Buitenzorg., vol. 4, pp. 107–35, pl. 9–17, 1884.
14.—— Études sur les Lycopodiacées II, Le prothalle du Lycopodium Phlegmaria L., ibid., vol. 5, pp. 87–115, pl. 11–22, 1886; III, Le développement de I'embryon chez L. Phlegmaria, ibid., pp. 115–39, pl. 23–31, 1886.
15.—— Études sur les Lycopodiacées: IV, Le Prothalle du Lycopodium salakense, ibid., vol. 7, pp. 141–46, pl. 16–18, 1888; V, Les Prothalle des Lycopodium carinatum, L. nummulariaefolium, et L. Hippuris, ibid., pp. 146–49, pl. 19, 1888.
16.—— Études sur les Lycopodiacées: VI, L'Embryon et la plantule du Lycopodium cernuum L., ibid., vol. 8, pp. 1–15, pl. 1–5, 1890; VIII, Considerations théoretiques, ibid., pp. 23–37, 1890.