1958 291 Studies on the Cytology and Phylogeny of the Pteridophytes VI. Observations on the Ophioglossaceae C. A. Ninan Department of Botany, University College , Trivandrum, India Received January 24, 1958 Introduction The Ophioglossaceae is a "very distinctive and circumscribed family" of primitive megaphyllous Pteridophytes consisting of three living genera, Ophioglossum, Botrychium and Helminthostachys. Without any known fossil record and with very distinctive features, the three genera constitute a natural family, their common character being the possession of the fertile spike. This family enjoys world-wide distribution and consists of one hundred species (Carl Christensen 1905-1934), the monotypic Helmin thostachys being restricted to the Australian and Indo-Malayan regions. Bower (1926) recognizes 78 species in this family while Clausen (1938) reduces them to 50. The three genera are typically of the eusporangiate type and combine several points of interest in cytology, phylogeny and evolution. The genus Ophioglossum is typical of the family and is perhaps the most ancient of all living ferns. It consists of 56 species according to Christensen's index (Bower and Clausen recognize only 43 and 26 species respectively). Most of them are ground growing forms while two species, O. pendulum and O. palmatum are epiphytic. Over a dozen species are indigenous to India and are found distributed in a variety of habitats. Botrychium is represented by 43 species (Christensen 1905-1934). Bower and Clausen recognize 34 and 23 species respectively for this genus. In the tropics this genus is usually confined to higher elevations. The only species of the genus Helminthostachys (H. zeylanica) is usually found to occur in low lands or river sides which get inundated. During the course of an extensive investigation on the cytology of tropical Pteridophytes with special reference to those native to the southern part of India, materials of all the three genera became available for study. The present paper embodies the cytological study of nine species of Ophio glossum (including several varieties, cytological and geographic races), and one species each of Botrychium and Helminthostachys. Material and methods The materials used in this study were collected from different places and grown in the Botanical Garden of the Kerala University. A very wide 292 C. A. Ninan Cytologia 23 collection of all the Indian species of Opioglossum was made from as many localities as possible. Botrychium was obtained from hill-stations like Munna, Kodaikanal and Ootacamund. Helminthostachys was collected from Trivandrum where it grows in the wild condition. In addition, two varieties of O. pendulum were obtained from Malaya and Ceylon. When compared to the Lycopods or most of the leptosporangiate ferns, the Ophioglossaceae are fairly easy cytological materials and respond well to simple aceto-carmine squash technique. The fixative used was a modified proportion of absolute alcohol, glacial acetic acid and chloroform (Ninan 1955). Spikes were fixed for nearly a week. In certain cases excellent preparations were obtained even after a fixation time of about three months in 3:1 Carnoy. Botrychium, Helminthostachys and the low numbered species of Ophioglossum (n=120), are easy to handle. Species like O. vulgatum, O. Pendulum, O. petiolatum and O. reticulatum with very large number of chromosomes in a cell (ranging from 436 to c. 630 bivalents in spore mother cells and double these numbers in root tip cells), present great difficulties and it is not easy to get preparations showing individual chromo somes sufficiently spaced out, to permit clear analysis. However, by suitable modification of the technique, clear preparations of meiosis were obtained in most of the high chromosome numbered forms. Maturation of spores in Ophioglossum begins at the apex of the spike and proceeds downwards. Even if the upper half has matured spores, all stages of division could be obtained from the lower region. Meiosis normally sets in, when the spikes are about an inch long. In Botrychium meiosis commences very early, even before the unfurling of spikes, which at the time of meiosis are not more than five millimetres in length. In Helmin thostachys meiosis starts when the spikes are about two inches long. Like Botrychium it is also an easy cytological material. Excepting the high chromosome numbered species of Ophioglossum like O. retieulatum, O. vulgatum and O. petiolatum, preparations of all the other species used in this study were photographed at a standard magnifica tion of •~750, using planochromatic objective •~100 and photo eye-piece •~ 6. But most of the preparations of the high numbered species had to be photographed at a lower magnification (•~500) to get all the chromosomes in focus in one plane. Explanatory diagrams were made on enlarged photographic prints and reduced to the desired size in reproduction, Exceptionally clear preparations were photographed in fresh acetocarmine and made permanent after com pleting the necessary observations. McClintock's (1929) method with slight modifications was adopted for making the smears permanent. 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 293 Cytological observations . costatum R. Br. (=O. fibrosunz Schum.) This species is characterised by the presence of a relatively massive rhizome and dense growth of fibrous roots. It was collected in the wild condition from Baroda, Palghat and Belgaum. They grow in moist sandy areas in association with grasses and other species of Ophioglossum like O. nudicaule, O. gramineum etc. The Palghat material was fixed in the wild condition in July 1954 and brought to Trivandrum along with several live plants which were cultivated in the garden. This species like O. reticulatum thrives well under green house conditions for a long period of time. Several clear preparations of meiosis were obtained of which two are illustrated in Figs. 1 and 2. One hundred and twenty bivalents are clearly seen in both the preparations. This number has also been verified from a large number of spore mother cells. The Belgaum material showed the same number of bivalents in spore mother cells (Fig. 9). There is only a single large nucleolus which persists till metaphase of the first meiotic divi sion. Examination of a large number of spore mother cells showed that 3-4 bivalents are always associated with the nucleolus. O. Aitchisonii (Clarke) d'Almeida This species is almost similar to O. costatum from which it differs mainly in the shape and size of the leaf and rhizome. While the rhizome in O. costatum is tuberous, that in O. Aitchisonii is elongated and the leaves narrower. This species occurs in the wild condition in Poona (Bombay State). Live plants along with young spikes fixed in Carnoy were brought to Trivandrum by Prof. Abraham in December 1954. Preparations of meiosis showed the presence of 120 bivalents in spore mother cells (Fig. 3). Meiosis in this species is also perfectly normal. The meiotic chromosomes are slightly larger than those of O. costatum. O. gramineum Willd. Wild collections of this species were obtained from Palghat and Baroda. Usually this is found to grow in association with O. costatum and O. nudicaule. Due to the scarcity of meiotic material at the correct stage of development, very clear preparations of meiosis could not be obtained. An approximate count of 120 bivalents has been made from a spore mother cell. Details of the correct haploid number remain to be verified.O . nudicaule L. fil. This is a highly polymorphic species. Several races of this species were collected from different localities and cytologically investigated. The cytology of some of the important varieties studied is given below. 1. O. nudicaule L. fil. var. typicum This is very closely similar to O. parvifolium Greville and Hooker, which Clausen (1938) considers to be synonymous with O. nudicaule L. O 294 C. A. Ninan Cytologia 23 fil. Wild collections of this variety were made by Prof. Abraham from the campus of the Central College, Bangalore. Several spore mother cells showing meiosis were studied and very clear counts of n=120 were made. Figs. 1-4. 1, first meiotic metaphase in a spore mother cell of Ophioglossum costatum R. Br. 120 bivalents are clearly seen. •~750. 2, another spore mother cell of the same species showing 120 bivalents. •~750. 3, O. Aitchisonii (Clarke) d'Almeida. n=120. •~ 750, 4, a spore mother cell of O. nudicaule L. fil. var. typicum. n=120. •~750. One such spore mother cell with 120 bivalents is shown in Fig. 4. The meiotic chromosomes of this species are larger than those of O, costatum 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 295 and O. Aitchisonii. Only one nucleolus is present in each nucleus in all the cells examined. 2. O. nudicaule L. fil. var. tenerum (Mettenius) n. comb. This variety closely resembles O. ypanernense Martius and O. mononeuron St. John. both of which Clausen (1938) includes in the synonymy of O. nudicaule L. fil. Wild collections of this variety were made from Bangalore and Palghat. Preparations of meiosis showed the presence of 120 bivalents in spore mother cells (Fig. 6). A group of spore mother cells of this variety photographed at a lower magnification is shown in Fig. 22. 3. O. nudicaule L. fil. (locality: Palghat) Wild collections of this were obtained from Palghat where they grow in plenty, practically covering the whole of a hillock, near the Head Works of the Malampuzha Dam. This variety is distinguished by the pale green colour of the leaves and stipes. Preparations of meiosis showed the presence of 240 bivalents in spore mother cells (Fig. 11 and Text-fig. 1la). Inter mingled with these were also found similar plants with reddish brown tinge on the stipes and the leaves. Cytologically this was found to be identical with the previous form in that the chromosome number for this is also n=240 (Fig.