This group includes the Lycopodiales, Sphenophyllales, Psilotales, Pseudoborniales, Equisetales, and Filicales. The Sphenophyllales and Pseudoborniales occur only as fossils and the Psilotales are confined to tropical and subtropical regions. The Lycopodiales are commonly called club mosses or ground pines, the Equisetales are called horsetail rushes or scouring rushes, and the Filicales are the common ferns. The Ophioglossaceae, a family of the Filicales, is often treated as an order. Two of its genera, Ophioglossum and Botrychium, are widely distributed and well known. Material, except in the Psilotales, is abundant and so easily recognized that anyone who pays a little attention to collecting can, in a single season, get a fine supply for a study of the group. Some desirable forms may not be present in all localities, but these will be few, and can be secured at a reasonable price from those who make a business of collecting.
Nothing but impressions has yet been found in Pseudoborniales. Gametophytes of Psilotales have been found only recently. Their young sporangia cut easily, but older stages are very refractory and should receive extreme care in dehydrating, clearing, and infiltration.
Lycopodium.-The genus is evergreen, and consequently some stage in development can be secured at any season. In general, the tropical species are easier to cut than the temperate. Without any regard to taxonomic sequence, we shall consider the vegetative structure, the strobili, and the prothallia.
Vegetative structure.-Formalin alcohol, with or without the addition of acetic acid, is an excellent fixing agent and, quite contrary to prevalent notions, the staining capacity of material seems to improve with several months' immersion. We prefer the following formula: formalin 10 c.c., acetic acid 5 c.c., 50 per cent alcohol 100 c.c.
The growing points of stems and roots cut easily in paraffin, and when the material becomes too hard to cut in paraffin it can be cut without any imbedding. It is easier to get good sections of L. lucidulum and L. inundatum than of drier species, like L. obscurum and L. clavatum, Safranin and Delafield's haematoxylin is a reliable stain. Safranin with anilin blue or light green is also good, and the light green gives particularly clear views of the phloem.
If young sporelings are available they afford a beautiful example of a very primitive type of stele, in transverse section showing an exarch protostele with 4 or 5 radiating arms of metaxylem, each tipped with a comparatively large group of protoxylem cells. In most species, this simple radial stele of the sporeling passes into a complicated, banded stele in the adult plant. Even in the adult plant the protoxylem and metaxylem are easily distinguished in sections near the growing point of the stem or root (Fig. 78). Not only in Lycopodium, but in any vascular plant, sections at this age are useful in pointing out the protoxylem.
Sections of the stem and root of Lycopodium complanatum, mounted on the same slide, show an interesting parallelism of structures. Transverse sections of the stem of Lycopodium pithyoides, a Mexican species, show not only the stem structures but excellent transverse sections of roots which grow down through the cortex.
The strobilus - For longitudinal sections, cut a slab from each side of the strobilus to insure fixing and infiltration. If a strobilus, or similar organ, is simply halved, both pieces are likely to curve. Among north temperature species, Lycopodium inundatum is the most easily cut. A young strobilus 1 cm. in length may show all stages from the archesporium to the spore mother-cell. Iron-haematoxylin is the best stain for differentiating the archesporial cells. The divisions in the spore mother-cell stain intensely, so that care must be taken not to overstain.
Strobili of Lycopodium dendroideum or L. obscurum 6 or 7 mm. in length show a beautiful series in the development of the sporangium from the earliest stages up to tetrads. These young stages fix well in chromo-acetic acid, with or without a little osmic acid.
The gametophyte - In most species the gametophyte, or prothallium, is subterranean, tuberous, and has no chlorophyll; in other species the prothallium is partly subterranean and partly aerial, the aerial portion being green and bearing the archegonia and antheridia.
The prothallia of Lycopodium inundatum, the only species in the United States known to have aerial green prothallia, have not yet been discovered in this country, although they have been found in Europe.
It would seem natural to get the prothallia by germinating the spores, but here again no one has had any notable success, except Bruchmann. In some species, the spores do not germinate for several years, but when the prothallia are once developed they continue to bear archegonia and antheridia for several years. The spores of L. Selago germinate in 3 to 5 years after shedding; those of L. clavatum and L. annotinum in 6 to 7 years. In L. clavatum and L. annotinum archegonia and antheridia develop in 12 to 15 years after the spores are shed. L. inundatum germinates more promptly-in 10 days to 6 months-but no one has succeeded in keeping a culture up to the archegonium stage.
Botanists in Lycopodium localities should look for prothallia. Since the subterranean forms are perennial and as large as a grain of wheat; some reaching a length of 1.5 cm., it would seem as if they should be found wherever Lycopodium grows.
Selaginella - Material of Selaginella, in all phases of the lifehistory, is easy to secure, but not so easy to handle after it is obtained. As many as 340 species, mostly tropical, have been described, only 3 of which are common in the range of Gray's Manual. Of these 3, Selaginella apus is best for sections. It is found in moist or wet situations on the borders of ponds, along ditches, or on moist meadows. While the plant is very small, it has large spores. Several of the tropical species are common in greenhouses, and they fruit abundantly.
Vegetative structure - Growing points and root-tips are easily cut in paraffin. In most species, the older parts of the stem are too hard and brittle to cut in paraffin and are too small to cut well freehand. It might be worth while to try hydrofluoric acid or the cellulose acetate method when that method becomes developed. At present, patience and a sharp knife seem to be the only reliance. Some of the tropical species, like Selaginella Wildenovii, have stems nearly as large as a lead pencil, with polystelic structure, and are not hard to cut. The vascular cylinder is an exarch protostele or, when polystelic, each bundle is an exarch protostele. It is exceptionally easy to get a brilliant, differentiated stain when once the sections are cut.
The strobilus - Very young strobili cut easily in paraffin, but after the megaspore coats begin to harden, there are few objects which make more trouble than the strobili of Selaginella. For stages up to the young megaspores, fix in chromo-acetic acid, with or without the addition of a little osmic acid; but for later stages use hot corrosive sublimate acetic acid in 50 per cent alcohol. If this is not available, use formalin, acetic alcohol. Sometimes sections of the difficult later stages stay on the slide with Mayor's albumen fixative.
The strobili of most species are square in transverse section. To get longitudinal sections showing the relations of sporangia, sporophylls and axis, cut diagonally, from corner to corner, never parallel to the flat side. For archesporial cells, use iron-haematoxylin; for young megaspores and the development of spore coats, use safranin, gentian-violet, orange; for later stages, use safranin and light green.
The gametophytes - In most cases the spores germinate while still within the sporangium and, in some cases, like Selaginella apus, the female gametophyte develops up to the archegonium initial stage before shedding.
The various stages of the female gametophyte and embryo are not hard to stain; but the walls throughout the development of the male gametophyte are very thin and extremely hard to stain. Safranin and light green is a good combination. Light green in clove oil may prove more satisfactory than the alcoholic solution.
Isoetes - This peculiar genus is widely distributed and 16 of its 64 species occur within the United States. It looks so much like a sedge that it is easily overlooked, even when rather abundant. As a genus, it is hydrophytic, growing in wet places or even under water.
Vegetative structure.-The short, thick stem, even in old plants, cuts easily in paraffin. Fix in formalin alcohol and stain in safranin and light green. Sporelings with stems about 2 mm. in diameter and young plants with stems up to 5 mm. in diameter are best for a study of the peculiar vascular system of this plant. These young stages fix well in chromo-acetic acid and are not hard to cut.
Sporangia - All the sporangia of the plant may be said to constitute a single strobilus of the Selago type. Both longitudinal and transverse sections should be cut. The stem is so short that, in a plant of medium size, a longitudinal section may include the stem, the sporangium, and the sporophyll, up to the top of the ligule. Such sections, 10 to 15(, or even 20( in thickness, are best for demonstration. Transverse sections through the whole cluster of sporophylls show the arrangement of megasporophylls and microsporophylls and also the relations of the sporangia to sporophylls.
The gametophytes - The spores are shed in the uninucleate stage, and consequently it is not so easy to find the germination as in the case of Selaginella. When the large megasporangium begins to decay, let the megaspores dry naturally. They retain their power of germination for a year at least. Simply wet them with tap water and the earlier stages are easily secured, quite clean and ready for cutting. There must be soil in the dish for later stages. Try a similar method for microspores. Also, look at the top of the stem of old plants for stages developing naturally. The cell walls of the male gametophyte, as in the case of Selaginella, are rather hard to differentiate. Use anilin blue or light green.
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