Art. XI.—The Prothallia of Three New Zealand Lycopods.
[Read before the Auckland Institute, 16th December, 1914.]
In no group of vascular cyptogams is so much variation shown in the gametophyte as in the genus Lycopodium. Up to the present time the species investigated include only tropical and European forms, though L. cernuum and L. Selago, two widely spread species, occur in New Zealand. The present investigation deals with the following New Zealand species: (1) L. volubile Forst., Prodr.; (2) L. scariosum Forst., Prodr.; (3) L. L. Billardieri Spring., Monog. Lycop.
Up to recent times the prothallia of Lycopodium were quite unknown. The process of the germination of the spore in Lycopodium was first described by De Bary in 1858. Then Fankhauser, in 1873, described the mature prothallium of L. annotinum. Between 1884 and 1890 Treub obtained important results. He described the prothallium of L. cernuum from the germination of the spore to the formation of the young sporophyte, and demonstrated the presence of a protocorm in this species. He also described the prothallia of the epiphytic species L. phlegmama, L. carinatum, L. nummularifolium, and L. Hippuris. In 1898–99 the mature prothallia of L. clavatum, L. annotinum, L. complanatum, and L. Selago were investigated by H. Bruchmann, and those of L. clavatum by Lang. Later, in 1910, Bruchmann added to his results.
All the investigators mentioned above deal with European or tropical species (except L. Selago and L. cernuum, very widely spread species). The only mention of New Zealand species is in a paper by Mr. J. Holloway, entitled “A Comparative Study of the Anatomy of Six New Zealand Species of Lycopodium,”* where he mentions the external features of the prothallia of L. cernuum, L. scariosum, L. laterale, and L. Billardieri.
Material and Methods.
Before going further it is advisable to say something of the material and methods employed in this investigation.
Several prothallia of L. volubile were found on the side of a bank on which a mature sporophyte and several young sporophytes were growing. Six of these prothallia were found attached to young sporophytes, but further search of the soil around revealed a single prothallium which gave no indication of bearing a sporophyte. Later this material was supplemented by abundant material collected some years ago by Mr. Holloway. The earliest stages of the prothallium, however, were not obtained; even the smallest examined already bore sexual organs.
The prothallia of L. scariosum was also gathered by Mr. Holloway, and in this case again all the younger stages were wanting, even the smallest one sectioned showing the foot of a young sporophyte.
In the case of the epiphytic L. Billardieri, part of the material was obtained from Professor Thomas, and some fresh material was also gathered by the writer. These prothallia were found growing on trees, in humus
[Footnote] * Trans. N.Z. Inst., vol. 42, p. 356, 1910.
an inch or two in thickness. They are filamentous, and ramify beneath the surface of the humus. Usually the young plants are found in colonies, and, by tracing them down from young to younger still, one can obtain those which are still attached to prothallia.
In the case of L. Billardieri, and also of L. Billardieri var. novaezelandicum Colenso, spores were sown in humus in which the mature sporophytes had been found growing. These spores were sown in April, but up till November showed no signs of germination.
The prothallia gathered by the writer were fixed in chromo-acetic acid, and serial sections cut.
The size and external form of the prothallium in L. volubile can be seen from figs. 1 and 2, which represent in natural size two of the prothallia examined. They are usually subterranean, being buried ¼–½ in. below the surface of the soil. They appear a dirty-white colour, lighter on the upper surface, which is destitute of the long rhizoids which clothe the rest of the prothallium; but the prothallium depicted in fig. 1 contained some chlorophyll in the ridge at the upper right-hand corner, where it had evidently projected above the surface of the soil.
Figs. 1, 2.—Prothallia of L. volubile. Natural size.
Figs. 1a, 1b.—Prothallium represented in fig. 1, seen from two different sides.
The prothallia varied somewhat in shape, but in most cases the younger ones examined were more or less conical below, increasing in size higher up. Probably in an earlier stage the prothallium forms a cylindrical body or primary tubercle, which later expands at right angles to its axis, so that in older prothallia the primary tubercle can be distinguished as a small projection from the lower surface of the mature prothallium.
The outer edge of the upper surface is, in most prothallia, surrounded by a ridge, on the inner surface of which the reproductive organs are produced. This ridge does not pass regularly round the whole margin, but is interrupted in places. Thus the upper surface is concave; and possibly this promotes the fertilization of the archegonia, as Lang suggests for the parallel case of L. clavatum.
Older prothalliia—e.g., that represented in fig. 3—seemed to form a rather flat plate, with a ridge running round the margin of the upper surface. This flattened form was particularly noticeable in some of the prothallia to which young sporophytes were attached.
The internal structure of the prothallium of L. volubile agrees in its main characters with that of L. clavatum or L. annotinum. A glance at fig. 4 shows that there is a differentiation into several layers of tissues which correspond in a general way to those of L. clavatum. The following tissues can be distinguished:—
(a.) On the lower surface a limiting layer of cells, elongated parallel to the surface and destitute of fungus hyphae, except certain cells which form the basal cells of rhizoids. These rhizoids apparently have no fungus hyphae in their cavities, the hyphae penetrating the wall of the basal cell and ramifying in the humus.
(b.) Next come several layers of equi - dimensional cells, thin-walled, whose cavities are densely packed with fungus hyphae, which stain deeply with haemalum. Nuclei are present in these cells, but are often somewhat obscured by the fungus. Those cells which border on layer (a) are often less densely filled with fungus.
(c.) Then follows a rather wide band of nucleated cells, elongated at right angles to the surface of the prothallium. The cell-walls are thick, and many of them contain fungus hyphae, which collect especially where two or more cell-walls meet. The presence of the fungus filaments in the walls renders it rather difficult to see the exact shape of the cells. This layer also has the fungus in the cell cavities.
The layers marked (b) and (c) extend not only parallel to the base but also parallel to the sides in the younger radial prothallia. In the older, more flattened, forms only a few rows of cells immediately below the reproductive organs are free from the fungus. From the layer (c) starch is absent.
Fig. 4.—Section of young prothallium of L.volubile. a, limiting layer; b, cells with intercellular fungus; c, cells with inter- and intra-cellular fungus; d, storage tissue; e, generative tissue; rh, rhizoid. x 82.
(d.) The whole of the central part of the prothallium is occupied by large thin-walled parenchymatous storage-cells, in which starch is stored in great abundance. The cells of this tissue bordering on (c) are often smaller and more densely filled with starch granules.
(e.) The layer (d) passes gradually into a rather delicate layer of small-celled tissue, which passes into more or less radial rows of small-celled
tissue from which the antheridia and archegonia arise on the upper surface of the prothallium.
In comparing the structure of this prothallium with that of L. clavatum and of L. annotinum one or two striking differences appear. First is the absence of a single layer of cells, elongated at right angles to the surface of the prothallium, lying between the layers marked (b) and (c), in L. volubile, and with the fungus intracellular. This layer of cells is represented by Bruchmann as extremely well marked in L. annotinum, and by Lang as quite distinct in L. clavatum. Secondly, the occurrence of the fungus in the cell-cavities as well as in the walls of layer (c). Thirdly, the absence of starch from layer (c) and its abundance in the cells of layer (d).
The formation of new tissue takes place all round the margin of the prothallium, just beneath the reproductive ridge. The meristematic cells are found on the lower surface of a bay or indentation just below the reproductive ridge (fig. 4, x). A section through this region shows a row of cells with large deeply staining nuclei. These cells divide both by periclinal and anticlinal walls. Towards the outer surface of the prothallium, cells formed by anticlinal divisions gradually pass into the elongated cells of the limiting layer; while, within this, meristematic cells pass gradually into the layers which contain the fungus hyphae. More internally, a layer of secondary meristem seems to be differentiated, which gives rise towards the upper surface of the prothallium to radial rows of small cells, and below gradually passes into the large-celled parenchymatous storage region.
Mention has already been made of the fact that certain cells of the prothallium harbour an endophyte. Not only does this fungus not injure the prothallium, but no mature prothallium is found without it. Moreover, the cells in which the fungus is present, are quite normal, and a nucleus is always present in them, though it may be almost hidden in the coils of the fungus. It seems quite clear that this is a case of symbiosis, though the exact way in which the endophyte and the prothallium benefit is not known. Possibly, in the first place the fungus attacked the prothallium of Lycopodium parasitically, but the Lycopodium prothallium was able to use the fungus to aid its nutrition. In any case, the fungus must obtain some advantage from the association or it would not so generally be found in the prothallial cells. Before the exact relation between the two organisms can be determined it is necessary to know more about the processes of nutrition in the fungus.
In L. volubile the hyphae of the fungus pass into the basal cell of the rhizoids, penetrate the outer wall near the base of the rhizoid proper, and so come into contact with the humus in which the prothallium grows.
In order to examine the fungus and trace its distribution, hand-cut sections were soaked in caustic potash, washed in water, and stained with iodine. This treatment caused the fungus filaments to stain a red-brown colour. In different cells the fungus presented quite a different appearance. In many cells, particularly those of layer (b) (fig. 4), the hyphae formed a dense coil (fig. 5) or else a number of parallel threads. In others, again, especially in the basal cells of the rhizoids, it formed several thick densely staining threads. In other cells are seen what Lang terms “multinucleate vesicles” (figs. 6, 7, 8). These arise by the swelling-up of the hypha, and at first each contains only one nucleus (fig. 6), which stains deeply, though the vesicles themselves stain lightly. Several of these “multinucleate vesicles” may be present in a single cell. In other cells were dark spore-like bodies, but it was impossible to trace how they arose Possibly they arose by the bursting of multinucleate vesicles.
In L. volubile the antheridia and archegonia are formed on the same prothallium, and are both confined to the upper surface. Antheridia are usually first formed, the youngest being nearest to the meristematic zone; but as soon as a number have been formed archegonia begin to form nearest the growing-zone. Thus the central part of the upper surface of the prothallium often bears spent antheridia, while the marginal ridge bears mature archegonia. Apparently in some cases a fresh zone of antheridia may be formed, since on one of the prothallia which bore the foot of a young sporophyte all stages of antheridia from the single cell to the spent antheridium were found.
The antheridium arises from a single surface cell which is distinguished by its large nucleus. This cell divides into two, the outer of which forms the cover cells by anticlinal divisions only; while the inner divides repeatedly to form small-celled tissue, the spermatazoid mother cells. From the cells of the prothallium bordering on the mass of spermatazoid mother cells, narrow tabular cells are cut off so that the mature antheridium is surrounded by these cells. At first the cover cells are flush with the surface, but later they project a little in most cases. In one case an antheridium was found which appeared to project beyond the prothallium for fully half its length. Further examination, however, showed that only the six apical cells of the antheridium were cover cells, those lower down which are divided by periclinal walls being prothallial cells.
The archegonia also arise from a single superficial cell which contains a large deeply staining nucleus. This cell divides into a row of three cells, the outer of which forms the neck cells, the inner gives rise to the ovum, and the middle cell to the neck canal cells. The outer divides by a perpendicular wall, and these cells by further divisions form the neck, which in the mature archegonium projects considerably below the surface. A cross-section shows that the neck consists of four rows of cells. The mother cell of the neck canal cells divides. In a nearly mature archegonium only a single division wall had been formed in the neck canal mother cell; but nuclear division had continued so that in the outer cell there were six nuclei arranged in pairs, each pair surrounded by a little cytoplasm. A still later stage shows the ovum below containing a large deeply staining nucleus with a single
nucleolus, and, above, two cells each with a single nucleus; then a cell containing seven nuclei, the lowest four of which are arranged in pairs. The lower part of the archegonium is buried in the tissue of the prothallium, and a layer of tabular cells is cut off round the ovum from the neighbouring prothallial cells.
The Young Sporophyte.
Unfortunately, the writer was not able to trace the different stages in the development of the young sporophyte. The prothallia persist for a long time, and may be found still attached to a young plant which has reached a height of 1 ½ in. or 2 in. (fig. 9). It is quite common to find two, or even three, sporophytes attached to the same prothallium (figs. 10 and 11). As in all species of Lycopodium, the first root appears relatively late, some sporophytes an inch in height showing no trace of it.
Sections of two embryos were obtained which had not yet broken through the prothallium. The youngest was an oval parenchymatous mass, whose cells were very rich in starch (fig. 12). The suspensor consisted of one or two cells, and the stem and foot were quite undifferentiated. The other (fig. 13) was slightly older, and quite spherical, owing to the rapid growth of the cells of the foot segment. The cells of the stem tier were rather smaller, while the outer limiting layer of the foot consisted of large cells.
Fig. 9.—Prothallium of L. volubile, bearing a single sporophyte. s, stem; pr, prothallium; rt, root. Natural size. Figs. 10, 11.—Prothallia of L. volubile, bearing two and three young sporophytes respectively. Natural size.
Figs. 12, 13.—Sections of two young embryos of L. volubile. st, stem segment; ft foot segment. x 267.
A section of a basal part of a young sporophyte still attached to the prothallium revealed the presence of a massive foot, the outer cells of which form an absorptive layer (fig. 14). The cells of this absorptive layer have much-thickened outer and side walls, which stain very deeply with haemalum.
They have also relatively larger nuclei and more abundant cell-contents. There was no sign of a protocorm. The tissue of the prothallium below the foot consists of rather flattened cells, in which starch is present. Examination of several young sporophytes proved that the vascular bundle does not extend into the foot. It is quite clear that the embryo of L. volubile is of the L. clavatum-annotinum type.
The prothallia of L. scariosum are, according to Mr. Holloway, always subterranean, being deeply buried (4–6 cm.). Like L. volubile, the prothallium of L. scariosum resembles that of L. clavatum, but it is even larger than that of L. volubile. The largest specimen cut was 14 mm. by 9 mm. The prothallia were conical below, becoming cylindrical above (figs. 15 and 16). The upper surface of the prothallium is concave, lighter in colour, devoid of rhizoids, and with a ridge running round the margin, on the inner surface of which the sexual organs are found. In other cases—e.g., fig. 17—two ridges have been formed, one towards the centre of the prothallium and the other round the margin. The prothallium mentioned above as the largest was quite different in shape (fig. 15). It was obtuse-angled below, and the whole surface very much lobed and wrinkled.
The general arrangement of tissues in L. scariosum is like that of L. volubile, or still more that of L. clavatum; but a striking point in which L. scariosum differs from both is in the small proportion of its cells occupied by the fungus hyphae. A glance at figs. 18 and 19 will show that the tissue infected by the fungus occupies only something like a quarter or a fifth of the whole prothallium, while in L. volubile and also in L. clavatum it occupies half. Fig. 18 shows that the tissue infected by the fungus extends also, in parts, to the lobes of the upper surface.
Fig. 18.—Section of prothallium of L. scariosum, showing proportion of cells occupied by the fungus.
Fig. 19.—Section of lower part of prothallium of L. scariosum. a, limiting layer; b, c, and d, the layers occupied by the fungus; e, storage layer. x 80.
The tissues of the prothallium of L. scariosum are,—
(a.) A limiting layer of elongated cells, from some of which rhizoids spring. These rhizoids may be merely prolongations of an epidermal cell or may be cut off from a basal cell by a cell-wall. They show the presence of fungus hyphae inside their cavities.
(b.) Several layers of cells, the lower two or three elongated parallel to the surface, and containing only a few thickened filaments of the fungus; the upper equi-dimensional, and containing a dense skein of fungus filaments.
(c.) Then a single layer of cells elongated at right angles to the surface, with the endophyte intracellular. This is a fairly definite layer, and corresponds to the similar one described by Lang and Bruchmann in L. clavatum and by Bruchmann in L. annotinum.
(d.) Then several layers of cells elongated at right angles to the surface, thick-walled, especially at the angles, and with the fungus in their walls but not in the cell-cavities. In this it differs from L. volubile, but agrees with L. elavatum. When sections were tested with iodine to demonstrate the presence of starch the cell-walls of this layer showed a faint blue colour, though no starch grains could be detected. The question arises whether starch was present in small quantities in the filaments of the fungus which spread in the cell-walls.
(e.) Above (d) was the storage layer of parenchymatous cells as in L. volubile.
The meriatematic tissue and the fungus were exactly as in L. volubile.
The position of the reproductive organs is similar to that of L. volubile. Unfortunately, all the prothallia examined were rather old, even the smallest showing the presence of a foot of a young plant when cut.
It was not, therefore, possible to trace the development of the antheridium in detail, but the mature antheridium is quite sunk beneath the surface of the prothallium, and probably opens by a single cap cell. In one antheridium some mature spermatazoids were found, and these were of the usual Lycopodiaceous type.
The earliest stages of archegonia found showed a neck of two cells, while the central row of cells consisted of ovum and three canal cells. In one instance the end cell of the central series showed two nuclei. A later stage shows an ovum and six cells in the central series, in three of which the nuclei are paired. The difficulty of counting the neck canal nuclei is increased by the fact that the cells of the neck contain deeply staining nuclei, which often lie against the wall separating the neck cells from the axial row of cells. In the mature archegonium the cell-walls of the axial row of cells have disappeared, and there remain the ovum and nine nuclei, the six lowest of which are is pairs. The neck cells split apart, and the four rows of cells diverge.
In Lycopodium the number of cells in the axial row varies widely. In L. clavatum and L. annotinum there are 6–10 or more, especially in L. annotinum; L. phlegmaria has 3–5, according to Treub; whereas in L. cernuum and L. inundatum the number may be reduced to one. Further, it is very common for some of the nuclear divisions not to be followed by cell-division, so that the nuclei are associated in pairs.
The Young Sporophyte.
The fertilization of one archegonium of a prothallium of L. scariosum does not prevent the fertilization of others. There may be only one sporophyte to a prothallium (fig. 20), but it is quite common to find two young sporophytes attached to the same prothallium, and in one case
(fig. 21) three young sporophytes had reached practically the same height. Although they were all about 1½ in. high, the prothallium showed no signs of decay.
Unfortunately, it was not possible to obtain sufficient stages to trace the development of the embryo, but it is probably of the L. clavatum type. The foot is large and persistent, a fact which is in relation to the depth of the prothallium below the surface of the soil, and the late appearance of the first root. The foot may be recognized as a small projection at the base of a young sporophyte which has become detached from the prothallium. The first-formed leaves are scale-like.
A longitudinal section of the base of a young plant still attached to a prothallium passing through the foot shows essentially the same structure as in L. volubile. There is a limiting layer of cells with more abundant contents and large nuclei. The cells of the foot are large, and though there is no prolongation of the vascular tissue into the foot the central cells are elongated to assist conduction (fig. 22).
Figs. 20, 21.—Prothallia of L. scariosum, bearing one and three young sporophytes respectively. Natural size.
The early stages of development of the prothallium in L. Billardieri were not found. The mature prothallium is quite devoid of chlorophyll, and consists of filaments which branch rather sparingly, the formation of vegetative branches taking place in acropetal succession. The prothallium is thickly covered with rhizoids, inclined to the axis of the branch at an angle of 60 or 70 degrees (figs. 23–31). The branches are cylindrical in form, and growth is terminal; but the young branches are usually obovate, being thicker at the distal end. The extreme ends of the branches also appear lighter in colour, owing to the absence of the fungus hyphae from their cells.
Fig. 24.—Prothallium of L. Billardieri, surrounded by detached branches. x 6. Fig. 25.—Reproductive branch of L. Billardieri, bearing vegetative branches radiating out in all directions. x 6. Fig. 26.—Prothallium of L. Billardieri, bearing young sporophyte. Figs. 27, 28.—Reproductive branch of L. Billardieri, bearing young sporophyte. Natural size.
The vegetative branches may grow to some length, but some become thicker at their growing end and bear antheridia. After the formation of antheridia has continued some time the same branch may form archegonia. As soon as a branch begins to form sexual organs it gives off branches much
more freely, several branches often arising side by side, which never occurs in a vegetative branch. (Figs. 24, 25, and 26.)
The branches may die off behind and form new prothallia. The same would happen if a branch were accidentally detached. In some cases prothallia are found surrounded by numerous detached branches, whose rhizoids are interlaced with those of the parent prothallium. In this way prothallia are able to increase rapidly when circumstances are favourable, a power which is no doubt of great value, since the initial difficulty in the germination of the spore is so great.
Mirnute Structure of Vegetature Branches.
A longitudinal section of a vegetative branch shows a certain differentiation of tissues. A mature branch (fig. 32) shows,—
(a.) In the centre several rows of elongated cells with scanty contents.
(b.) Then two rows of cells, the region infected by the fungus. These cells are equi-dimensional and contain a distinct nucleus. In many of these cells the fungus hyphae form a dense skein, and in others there were several bodies which stain very deeply with haemalum. Further search showed that these dark bodies arose from the fungus, and are probably spores. The fungus was also intercellular.
(c.) A single row of elongated cells.
(d.) The peripheral layer, usually a single row of cells, elongated parallel to the axis of the branch, and covered with a thin cuticle. At intervals are cells rather shorter, which give rise to rhizoids. These cells are the only ones of the peripheral layer to contain fungus filaments. A branch treated with caustic potash and stained with iodine showed the presence of numerous pits on the cell-walls lying between any two of the cells of the peripheral layer. The external wall and the wall opposite to it were not pitted. Careful search failed to reveal similar pits in the transverse walls of the central tissue which Treub describes as present in L. phlegmaria.
A longitudinal section of the growing end of a branch revealed the presence of two initial cells (fig. 33) which are characterized by large
nuclei. Apparently the mode of growth is exactly the same as in L. phlegmaria. Both transverse and longitudinal divisions are formed so that the cells at the apex of a branch are equi-dimensional. A short distance behind the apex differentiation of the tissues takes place. Few transverse divisions take place in the central cells, so that elongated cells are formed. Those outside, the central cells divide by transverse walls and form equi-dimensional cells, which soon become infected with the fungus. Some of the cells of the peripheral layer divide by a periclinal wall (fig. 33, x), and the outer wall of the external cell forms a thick cuticle. The external cell grows out and forms a rhizoid, which may grow out to a great length. The wall which separates the rhizoid from the basal cell is extremely thickened and cuticularized.
Fig. 32.—Longitudinal section of vegetative branch of L. Billardieri. a, central cells; b, fungus-infected cells; c, single row of cells; d, peripheral layer; b.c, basal cell of rhizoid; nc, nucleus. x 267. Fig. 33.—Longitudinal section of apex of vegetative branch. a, central cells; b, fungus-infected cells; c, single row of cells; d, peripheral layer; nc, nucleus; i, iritial ell. x 367.
The development of young branches was not followed in detail, but a young stage showed a few projecting cells of the peripheral layer, and below them a few small parenchymatous cells.
Repeated attempts were made to demonstrate the presence of starch in the cells of the branch, but only the slightest traces of it were found.
The sexual branches of L. Billardieri are always dorsiventral, the sexual organs being borne on the upper surface. The presence of sexual organs on any part of the thallus can readily be detected by means of the paraphyses which are always present on the reproductive region (fig. 34). The
reproductive branches differ from the ordinary vegetative branches also in being thicker. Antheridia and archegonia are developed on the same branch, but the antheridia are first formed. A branch about to bear antheridia becomes club-shaped, and the antheridia are formed near the apex, though the apical cell is not used up in their formation (fig. 35). Owing to the rapid division of cells, the antheridia come to lie on the upper surface. The formation of antheridia may continue for some time, but eventually archegonia are formed at the apical end. In the antheridial branch the lower vegetative portion is infected with the fungus, only a few cells immediately below the antheridia being free from it. From the cells of the lower surface rhizoids spring, as in the ordinary vegetative branches.
The paraphyses in L. Billardieri consist of three cells, each containing a nucleus and rather scanty protoplasm. The basal cell is often smaller than the others, and the apical cell usually becomes narrower at its distal end. Each arises by the division of a surface cell into two, the outer of which divides again. In only one paraphysis was a tendency to branching seen, where a small protuberance had been formed on one side of the lowest cell. In the small number of cells of which the paraphyses are composed, and in their infrequent branching, they differ strikingly from L. phlegmaria and L. Selago, in both of which branching of paraphyses is common, and the number of cells may reach nine or twelve.
Antheridia arise, as in other species of Lycopodium, from single cells which divide into an outer and inner cell; the outer cell gives rise to the cover cell, and the inner to the mass of spematazoid mother cells.
Fig. 34.—Young reproductive branch of L. Billardieri, bearing antheridia and young vegetative branches. v.br, vegetative branch; an, antheridia; p, paraphyses. x 82.
Development is the same as in other species, and the mature antheridium does not project above the surface. The antheridia may arise so close to one another that they are separated only by a single plate of tabular cells. There is no doubt that in this case the spermatazoids escape by the disintegration of a single cap cell, since when the spermatazoids have escaped the remaining portion of the wall of the cap cell becomes yellow. The development of the spermatazoids was not followed in detail.
Archegonia apparently only develop on branches which have already borne antheridia. As is the case in the formation of antheridia, the meristematic region of the branch is towards the lower surface. The cells of the growing region are distinguished by their large deeply staining nuclei.
As in other species, the archegonia can each be traced to a single cell which divides into three, the outer giving rise to the neck, the middle to the neck canal cells, and the innermost to the ovum. The nucleus of the ovum stains deeply, and often shows two deeply staining nucleoli. In the central row of cells only two walls apparently are formed. In a mature archegonium ready for fertilization there appeared a large ovum with a small nucleus close to it, then a large nucleus and three pair of smaller nuclei separated from the ovum by a wall.
Fig. 35.—Longitudinal section of a young antheridial branch, showing initial cell (i) pushed to the lower surface; an, antheridum; rh, rhizoid. x 267.
Fig. 36.—Longitudinal section of foot of young plant of L. Billardieri, showing suspensor (susp). ar, remains of archegonium; l.l, limiting layer x 267.
Fig. 37.—Prothallium of L. Billardieri, bearing young sporophyte, showing only one leaf, l. ft, foot; s, stem; par, paraphysis; veg.br, vegetative branch. x 82.
The Young Sporophyte.
A glance at figs. 23 to 31 will show that the young sporophyte may remain attached to the prothallium for a long time. Thus sporophytes with four or five leaves and a young root may still be attached to a prothallium. The first-formed leaves are never scaly, since they are produced above the surface of the humus. Even after the young plant has become detached from the prothallium the foot may be distinguished on it (fig. 29). In no case was more than one sporophyte developed on a single branch of the prothallium.
A section through the basal part of a young sporophyte still attached to a prothallium shows a fairly large foot composed of parenchymatous cells, with its outer row of cells characterized by thick walls and more abundant contents. In fig. 36 the suspensor is seen, which consists of only a few cells. No trace of a protocorm was found. Fig. 37 shows a young sporophyte with only a single leaf developed.
Apparently similar to that of L. volubile, but no nucleated vesicles were observed. On the other hand, cells with dark spore-like bodies were very common (fig. 32).
The results obtained in this investigation may be summarized as follows:—
(1.) The spores of L. Billardieri and L. novae-zelandicum Colenso, like those of many other species of Lycopodium, do not germinate readily. After a period of five months there was no sign of germination.
(2.) The prothallia of L. volubile and L. scariosum resemble one another externally, but that of L. scariosum is larger. Both are usually colourless and saprophytic, but prothallia of L. volubile may come above the surface.
The prothallia of L. Billardieri, like those of other epiphytic species, closely resemble those of L. phlegmaria.
(3.) Internal Form of Prothallia.—In a general way, both L. volubile and L. scariosum resemble L. clavatum, but L. scariosum differs from both L. volubile and L. clavatum in the small proportion of its cells in which the fungus is found, the infected tissue being only about a sixth of the whole prothallium in L. scariosum, while about a half in L. volubile and L. clavatum. L. scariosum resembles both L. clavatum and L. annotinum in having a single row of cells elongated at right angles to the surface on the prothallium, and having the fungus intracellular. This layer is absent in L. volubile. Again, in the layer of cells marked (c) in L. volubile and (d) in L. scariosum the fungus is intercellular only in L. scariosum, and both inter- and intra-cellular in L. volubile.
The internal structure of L. Billardieri closely resembles that of L. phlegmaria, but in L. Billardieri no pits were seen in the walls of the elongated central cells as described by Treub in L. phlegmaria, though the pits in the walls of the peripheral layer were very conspicuous.
(4.) Starch was abundant in both L. volubile and L. scariosum in the parenchymatous storage layer. In L. volubile it was quite absent from the layer below this; but in L. scariosum the walls of the cells showed a light-blue colour when treated with iodine, showing possibly that the fungus filaments in the wall contain starch.
(5.) Fungus was present in all three species. In L. volubile the fungus does not enter the rhizoids, but pierces the wall of the basal cell of the
rhizoid, and so comes into contact with the humus; while in L. scariosum the filaments of the fungus are found also in the cavities of the rhizoids.
(6.) Reproductive Organs.—Antheridia in all three species were of the usual Lycopodiaceous type, those in L. volubile projecting a little above the surface, while those in L. scariosum and L. Billardieri were completely sunk in the prothallium.
The development of the archegonia is the same as in other species of Lycopodium, the only point of variation being in the neck canal cells, which number four to seven in L. volubile, six in L. scariosum, and five in L. Billardieri.
The paraphyses of L. Billardieri are composed of fewer cells than in L. Selago and L. phlegmaria, and are usually unbranched.
(7.) Young Sporophyte.—Though the embryology was not traced in detail, the embryos of L. volubile and L. scariosum are undoubtedly of the L. clavatum type. There is a large persistent foot and no sign of a protocorm. In L. Billardieri the foot is fairly large and persistent.
In the gametophyte of Lycopodium there is much greater variation than in any other vascular cyptogams. Thus, H. Bruchmann describes five different types of prothallia—(1) L. clavatum, (2) L. complanatum, (3) L. Selago, (4) L. inundatum, and (5) L. phlegmaria. After carefully examining these different types he comes to the following conclusion: “It follows from the above facts that the groups of Lycopodium characterized especially by means of their sexual generation do not stand in close relation to one another; especially not such as one would expect in species of plants which have found their position together in one genus. The knowledge leads to a separation of the Lycopodiums into groups, or, still better, into genera, to which it would be quite in place to give new names. There arise as many groups as the sexual generation allows to be distinguished.” Thus, Bruchmann regards the species of Lycopodium as derived from several stocks of Lycopodiaceous plants. This is essentially the same conclusion as that arrived at by Dr. Treub in 1886, when, after remarking on the great difference between the prothallia, embryos, and young plants of L. cernuum and L. phlegmaria, he proceeds to say, “This is one of the reasons why I believe I may offer the opinion that the profound differences between the prothallia of the Lycopods are of very ancient date, and that they are not the result of very recent adaptations.”
But another view of the variations in the prothallia of different species of Lycopodium has been given by Lang in 1899, and by Goebel in his “Organography of Plants” in 1905, and it is this view that the facts obtained in the present investigation seem to support. Lang's view is that many of the differences between the different types of Lycopodium prothallia are physiologically adaptive in character, and so are not of generic value; and similarly Goebel sees no valid ground for regarding the gap between the several forms of the prothallia of the Lycopodia to be so great as Treub and Bruchmann will have it.
With regard to external form, the differences are not so great as they appear, for L. Selago may be regarded as intermediate between the radial green prothallia of L. cernuum and the filamentous saprophytic L. phlegmaria. Again, in regard to internal structure Lang points out that the differences are in the tissues infected by the fungus, and this has been seen also to be the case in L. volubile and L. scariosum. Thus they are due to
the physiological relation between the fungus and the cells it inhabits and so cannot be used for classification.
In the same way, the differences between the embryo and young plants are closely related to the position of the prothallium, whether at the surface of the soil or some distance below the surface. Again, the appearance of chlorophyll in the tissues is not constant. Thus, in L. Selago, when the prothallium grows at the surface of the soil, chlorophyll appears in its subaerial part; and this is also the case in L. volubile.
In other characters which are not adaptive the different species of Lycopodium agree closely. Thus all species of Lycopodium usually show biciliate spermatazoids, archegonia with no basal cell, and a suspensor in the embryo. In all but the epiphytic forms of prothallia growth is essentially intercalary, and in the epiphytic forms the apical growth is in relation to the branched filamentous form.
Thus it appears that in Lycopodium the gametophyte and the young stages of the sporophyte are peculiarly adaptive. The experience of botanists proves that the mature sporophyte of plants form a better basis for the interpretation of affinities than does the more plastic embryonal stages, and this is especially the case in Lycopodium.
In conclusion, I wish to record my grateful thanks to Professor A. P. W. Thomas for his ever-ready interest in my work, and also to Mr. J. Holloway for permitting me to use material gathered by him.
1. De Bary. “Sur la germination des Lycopodes.” Ann. de Sci. nat., ser. 4, vol. ix, p. 30, 1858.
2. Fankhauser. “Ueber der Vorkeim von Lycopodium.” Botanische Zeitune, 1873.
3. Treub. “Etudes sur les Lycopodiacées.” Annales du Jardin botanique de Buitenzorg, vols. iv, v, vii, and viii.
4. Bruchmann. “Ueber die Prothallien und die Keimpflanze von mehrerer Europaischer Lycopodien.” 1898.
5. Bruchmann. “Prothallium of L. complanatum.” Bot. Zeitung, vol. lxvi, pp. 168–81.
6. Lang. “Prothallus of Lycopodium clavatum.” Annals of Botany, 1899.