1958 291

Studies on the Cytology and Phylogeny of the VI. Observations on the

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, , and . 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 Ophioglossum is typical of the family and is perhaps the most ancient of all living . 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 mother cells and double these numbers in 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 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 and dense growth of fibrous . 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 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 and rhizome. While the rhizome in O. costatum is tuberous, that in O. Aitchisonii is elongated and the 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. 14 and Text-fig. 14a). While the former shows a single large nucleolus in most of the cells, the latter had two relativery small nucleoli in dividing cells. 4. O. nudicaule L. fil. (locality: Belgaum) Plants of this collection are almost similar in appearance to the Palghat materials (with pale green colour of leaves and stipes). Preparations of meiosis clearly showed the presence of 240 bivalents in spore mother cells (Fig. 12 and Text-fig. 12a). 5. O. nudicaule L. fil. (locality: Palai) This is much different from all the other plants of O. nudicaule des cribed above. The leaves which more or less resemble those of O. nudicaule var. tenerum have blunt apices unlike the acute leaf tips characteristic of the latter. Chromosome counts of this variety showed the presence of 240 bivalents in a spore mother cell (Fig. 8). There is only a single nucleolus in a cell. 6. O. nudicaule L. fil. (locality: Kumbanad) This variety was collected in the wild condition from Kumbanad, Central Travancore. The leaves of this are much smaller than those of all the other varieties of O. nudicaule so far studied and are closely appressed to the soil. The spikes which are about two centimetres long, are borne on long stalks. Chromosome counts showed the presence of 240 bivalents in spore mother cells (Fig. 13 and Text-fig. 13a). The bivalents are slightly fuzzy in appearance. They are decidedly smaller than those of all the other species of Ophioglossum investigated in the present study. O. lusitanicum L. ssp. lusitanicum This species was collected from the Christian College Campus, Tambaram, 296 C. A. Ninan Cytologia 23

Figs. 5-9. 5, meiosis in a spore mother cell of Helminthostachys zeylanica L. Hk. from

Trivandrum. n=94. •~750. 6, a spore mother cell of O. nudicaule L. fil. var. tenerum

(Mettenius) n. comb. from Bangalore. n=120. •~750. 7, meiosis in Botrychium lanuginosum Wall. from Kodaikanal. n=90. •~750. 8, meiosis in O. nudicaule L. fil. var. No. 5. n=240. •~750. 9, a spore mother cell from the Belgaum material of O.

costatum R. Br. n=120. •~750. 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 297

Madras, where plenty of them sprout after the first showers in July-August . Fresh collections of this species were brought to Trivandrum and planted in

Figs. 10-14. 10, meiotic metaphase in a spore mother cell of O . lusitanicum L. ssp. l usitanicum from Madras. n=240. •~750. 11, first meiotic metaphase in O. nudicaule L. fil. var. No. 3. n=240. •~750. 12 , a spore mother cell of O. nudicaule L. fil. var. N o. 4. n=240. •~750. 13, meiosis in O. nudicaule L. fil. var. No. 6. n=240. Note the small size of the bivalents compared to other varieties of O. nudicaule •~750. 14, meiosis in O. nudicaule L. fil. var. No. 3. (with reddish brown stipes). n=240. •~750. the garden where they thrived excellently for a few seasons. The prepara- 298 C. A. Ninan Cytologia 23 tion shown in Fig. 10 (see also Text-fig. 10a) was obtained from a spike which matured under cultivation. This shows clearly 240 bivalents. This number has been verified from quite a large number of preparations. All

Text-fiigs. lOa-14a. Explanatory diagrams of Figs. 10-14 reproduced at the same magni fication as the photographs. 10a, O. lusitanicum. L. ssp. lusitanicum. n=240. •~750. lla, O. nudicaule L. fil. var. No. 3. n=240. •~750. 12a, O. nudicaule L. fil. var. No. 4. n =240. •~750. 13a, O . nudicaule L. fil. var, No. 6. n=240. •~750. 14a, O. nudicaule L. fil. var. No. 3. (with reddish brown stipes). n=240. •~750. the spore mother cells studied showed a single nucleolus each. Another variety of O. lusitanicum was recently collected from Veli (sea-coast), near Trivandrum City. Cytologically this was found to be similar 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 299 to the Madras materials of O. lusitanicum. Spore mother cells clearly showed 240 bivalents. O. petiolatum Hk. This species resembles O. reticulatum L. in many of its characters. Clausen (1938) regards this as a separate species, quite distinct from O. reticulatum and O. pedunculosum with which it has been identified by some authors. He states: "O. petiolatum really is very closely related to O. reticulatum and in certain parts of the world, notably in India, China and Mexico, the two populations seem somewhat to intergrade. Usually, however, they appear distinct and are readily separable. O. petiolatum generally may be distinguished by its lanceovate blade which is acute at the apex, by the rather long slender fertile segment, and by the lax venation. Typically, also, it is somewhat smaller than O. reticulatum". Cytological study of O. petiolatum from materials collected from Ettappadappu in the Western Ghats showed the presence of 480 bivalents in spore mother cells (Figs. 15 and 15a). O. pendulum L. ssp. falcatum (Presl.) Clausen Fixed sporangia of this species were obtained by post from Ceylon. Due to the scarcity of material at the correct stage of meiotic division, only a few preparations of meiosis could be obtained. The spore mother cell shown in Fig. 16 (see also Fig. 16a) is the best one obtained and this shows approximately 480 bivalents. The meiotic chromosomes of this species (which represents the subgenus ) are in no way different from those of O. petiolatum or O. reticulatum (representing the subgenus Euophio glossum). Though fixed material of O. pendulum ssp. typicum was obtained from Malaya, chromosome counts at meiosis could not be made due to lack of mature sporangia. Preparations of archesporial mitosis showed that this is also a high numbered form, not very different from the Ceylon material. O. vulgatum L. Wild collections of this species were obtained from Munnar (5000 ft.) where they grow in marshy places along with Equisetum debile. Prepara tions of mciosis made from a spike matured under cultivation showed the presence of c. 570 bivalents in spore mother cells (Fig. 18 and 18a). The meiotic chromosomes of this species closely resemble those of O. petiolatum and O. pendulum. There are four nucleoli in most of the cells of which two are larger than the other two. O. reticulatum L. Of all the species of Ophioglossum investigated in the present study, this is the most interesting from a cytological point of view. Within the same species materials collected from different localities showed considerable variations in chromosome number. In view of the peculiar cytological interest of this species, it was collected from as many localities in India as possible and subjected to detailed cytological study. 300 C. A. Ninan Cytologia 23

The lowest chromosome number in this species was seen in mate rials collected from Ettappa dappu in the Western Ghats, which showed c. 435 bivalents in spore mother cells. Plants collected from Ponmudi (3500 ft.) sho wed c. 450 bivalents, while those obtained from Bonaccord Estate, a few miles from Ponmudi, showed 480 bivalents in spore mother cells (Fig. 17 and 17a). An other collec tion from Trivandrum showed 495 bivalents (Fig. 19 and 19a). Wild collec tions of O. reticulatum from Trivan drum showed Figs. 15 and 15a. 15, a spore mother cell of O. petiolatum Hk. showing 570 bivalents. 480 bivalents. •~750. 15a, explanatory diagram of Fig 15. •~750. Materials of 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 301 the same species collected from Mysore, Trichinopoly, Bengal and Dehra Dun also showed the same number of bivalents. Plants collected from the rain forests of Parampikulam in the Western Ghats showed n=c. 630 (631 bivalents and 10 fragments in a spore mother cell) (Fig. 20 and 20a). This indicates that the sporophy tic number for this is over 1260 which is by far the largest chromosome number yet reported in any species (Abraham and Ninan 1954). Root tip squashes of this species gave prepara tions showing approximately double the number of bivalents ob served at mei osis in spore mother cells of the same plant. A root tip prepara tion of the Figs. 16 and 16a. 16, a spore mother cell of O. pendulum L. (Ceylon Trivandrum material) showing 480 bivalents at meiotic metaphase. •~750. 16a, explanatory diagram of Fig. 16. •~750. material (with

n=c. 570) is illustrated in Fig. 21. Round about 1100 chromosomes were

counted from this. The somatic chromosomes vary from 1.5ƒÊ to 4.5ƒÊ and 2(V) C. A. Ninan Cytologia 23

the constric

tions are not

very clear.

When com

pared to those of ancient

genera like , in

which the so

matic chro

mosomes vary

from 4.5ƒÊ to

18ƒÊ in length,

the chromo

somes of Op

hioglossum

are much

smaller. Un

like the situa

tion in most

of the angio

sperms, the

growth of the root tips in Op

hioglossum is

very slow and

mitotic figures

are very rare

ly met with.

Botrychium

lanuginosum

(Wall.) Hk. et Grev. var.

typicum

The only

species of the

genus Botry chium so far

obtained from

South India is

B. lanugino

sum. This

Figs. 17 and 17a. 17, meiotic metaphase in a spore mother cell of O. was collected reticulatum L. n=480. •~750. 17a, explanatory diagram of Fig. 17. •~ 750. from three 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 303

localities, Oot acamund (7500 ft.), Ko daikanal (7000 ft.) and Mun nar area (5000 ft.). Though fresh materi als were col lected and planted in the garden several times, none of them thrived well under green house conditions at Trivandrum. Cytological examination of the Kodai kanal material fixed in the field showed the presence of 90 bivalents in spore mo ther cells (Fig. 7). Prepara tions of arch esporial mito sis of the Ootacamund material and root tip squ ashes of the Munnar ma terial showed 180 chromo somes each .

For B. lanugi Figs. 18 and 18a. 18, meiosis in a spore mother cell of O. vulgatum nosum there L. showing ca. 570 bivalents at metaphase. •~750. 18a, explanatory fore, the ha- diagram of Fig. 18. •~750.

Cytologia 23, 1958 20 304 C. A. Ninan Cytologia 23

ploid chro

mosome number is n=90. This species is a tetraploid on the num ber n=45 found in B. lunaria (Manton 1950). Helmintho stachys zeylanica (L.) Hk. This monotypic genus occurs at Poojappura and Thiru vallam in the suburbs of Trivand rum City. It is also reported to occur at Calcutta (Majumdar 1930). Pre parations of meiosis made from materials collected from the two localiti

Figs. 19 and 19a. 19, meiosis in a spore mother cell of O. reticulatum es near Tri

L. from Trivandrum. 495 bivalents are present. •~750. 19a, explanatory vandrum diagram of Fig. 19. •~750. showed the presence of 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 305

94 bivalents in spore mother cells (Fig. 5) and archespori al mitosis showed the diploid number to be 188.

Discussion Phylogeny and interre lationships of the Ophioglos saceae Though chromoso me studies on the Ophioglos saceae have appeared a few times in current cytological literature (Manton 1950, 1953; Britton 1953; Man ton and Sledge 1954; Abra ham and Ninan 1954; Wagner and Lord 1956 and Verma Figs. 20 and 20a. 20, meiosis in a spore mother cell of O. reticula tum L. from Parampikulam showing 631 bivalents and 10 fragments. 1956), our •~ 750. 20a, explanatory diagram of Fig. 20. •~750.

20 * 306 C. A. Ninan Cytologia 23

knowledge regarding the cytolo gy of this family is rather in complete. Excepting for the genus Bo trychium, even the correct ha ploid num bers are not clearly es tablished. Manton (1950) has recorded n=c. 128 in O. lusi tanicum L. while Ver ma (1956) reported n=116-117 in O. Ait chisonii (Clarke) d'Almeida. The present study has shown very clearly that

Figs. 21-22. 21, a root tip squash preparation of O. reticulatum L. the lowest from Trivandrum showing over 1000 chromosomes in a cell. •~750. haploid 22, a group of spore mother cells from a smear preparation of O. number in nudicaule L. fil. var. tenerum photographed at a lower magnification. •~ 400. Ophioglos sum is n= 120. Botrychium has the haploid number of n=45 (Manton 1950, Britton 1953, Wagner and Lord 1956), while Helminthostachys has n=94 (Ninan 1956a). The above data clearly show that haploid numbers like n=120 in Ophioglos- 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 307

sum and n=45 in Botrychium are referable to a basic number of 15. The author (Ninan 1956 a) has shown that though the haploid number in Helminthostachys (n=94) is slightly higher than an exact multiple of 15, it is also traceable in ultimate origin to the same basic number . This indicates a remote common ancestry of the three genera, and lends support to the suggestion that the three genera of this group , though unlike in appearance, are closely related (Eames 1936). The relationship of the Ophioglossaceae to other families of ferns has been found to be very difficult to trace. Bower (1926) suggests some degree of affinity of the Ophioglossaceae to the Marattiaceae and Osmundaceae among living ferns, while Campbell (1904, 1920) presents an alternative suggestion of affinity to the Lycopods. Cytological evidence however does not seem to support any of these suggestions. The Marattiaceae and the Osmundaceae are shown to have evolved from ancestral types with 13 and 11 respectively as the basic chromosome numbers (Ninan 1956 b, 1956 d) while numbers like 9, 10, 11, 12, 13 and 17 are characteristic of the modern Lycopods (Manton 1950, Delay 1953; Abraham and Ninan 1958; Ninan 1958 a; Mehra and Verma 1957). The Ophioglossaceae with the basic number 15 clearly stands apart from the above groups. Manton and Sledge (1954) have suggested that chromosome numbers like 29 and 30 seen in most of the Pteroid, Gymnogrammoid and Adiantoid ferns might be very ancient numbers retained in these genera through some direct ancestor in the Schizaeaceae in close relation to the genus Lygodium, which shows in the different species studied, basic numbers like 29 and 30 (Manton and Sledge 1954 and Ninan, unpublished). It is quite probable that 30 might be the original number of which 29 might be a later aneuploid derivative. The number 30 in turn might have evolved from 15, which it is shown, is also the basic number of the Ophioglossaceae. This at once suggests probable remote relationship of the Ophioglossaceae to Lygodium, the most primitive genus of the Schizaeaceae. In this connection, it is worthy of observation that other lines of evidences also seem to indicate possible relationship between these two families. Chrysler (1941) suggests that "the 'monangial sorus' in the Schizaeaceae may be indication of affinity with the Ophioglossales, while the pronounced dichotomy in Schizaea marks the family as a primitive one to which we may reasonably turn while seeking a parallel to the evolutionary history of the Ophioglossaceae". Both Ophioglossum and Schizaea are very ancient genera and probably represent relics of a pre-glacial flora. The existence of very high chromo some numbers in these two genera further indicates possible parallelism in their evolutionary history. The three genera of the Ophioglossaceae are sometimes considered as forming a rough series in reduction and simplification (Eames 1936). In such a series, Botryclzium with open venation and relatively much divided 308 C. A. Ninan Cytologia 23

leaf is interpreted as the most primitive genus, Ophioglossum with reticulate venation and little divided leaf as most advanced, and Helminthostachys intermediate between the two. On a consideration of the characters it will be seen that Ophioglossum is primitive while Botrychium is the most advanced, Helminthostachys again being intermediate. The distribution of the various species of the three genera if regarded as an indication of possible phylogeny (Willis 1922, 1923) would suggest that Ophioglossum with the greatest range of distribution is primitive while Botrychium less primitive and Helminthostachys with very restricted range of distribution, the least primitive. From a careful comparative study of the three genera, Bower (1926) considers Helminthostachys as the most primitive and Ophioglossum as the most advanced. Nishida (1952) regards Helminthostachys as the most advanced. There is thus very little agreement among competent investigators as to which of these genera is primitive and which is advanced. Cytological study of the three genera indicates that Ophioglossum with very high chromosome numbers like n=120, 240, 480, c. 570, c. 630 etc. is extremely specialised while Helminthostachys with n=94 is relatively primitive. Botrychium with the lowest haploid number of n=45 is decidedly the most primitive of the three genera.

Table 1. Chromosome numbers in the genus Botrychium

Based on characters afforded by vernation, division of the leaf and , the genus Botrychium has been divided into three subgenera, Eubotiychium, and Osmundopteris. There is however, no 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 309 unanimity of opinion as to which of these subgenera is primitive and which is advanced (Bower 1926, Clausen 1938, 1954; Chrysler 1945; Nishida 1952). Lyon (1905) raises the subgenus Sceptridium to generic rank, while Nishida (1952) elevates all the three subgenera to generic status. It will be seen from the table below that the section Osmundopteris contains species with the haploid number of n=90 while the number n=45 predominates in the other two sections, Sceptridium and Eubotrychium, which marks them as relatively less specialised in a cytological sense. It is also seen from the table that there is no cytological distinctness among the three subgenera which would justify their elevation to the status of separate genera as done by some authors. The genus Ophioglossum has likewise been divided into four subgenera, Euophioglossum, Ophioderma, and Rhizoglossum, based on the nature and division of the blade, habit and condition of the fertile segment. Nishida (1952), Clausen (1954) and Wagner (1952) consider Euophioglossum as the most primitive. Cytological study shows that out of the two subgenera Euophioglossum and Ophioderma, the former alone has retained the haploid number n=120, which is the lowest recorded for the genus, while the latter exhibits numbers like n=c. 370 and n=480 which are most probably derived from n=120 (see table below). This observation further supports the suggestion that the epiphytic habit (characteristic of species included in Ophioderma) is specialised (Clausen 1954). As far as these two subgenera are concerned, it is obvious that there is no distinct cytological discontinuity which would justify their separation into two sub genera. Cytological evidence does not support their being raised to generic status as suggested by Nishida (1952). As Clausen (1938) has remarked, "the arguments for merging them under Ophioglossum seem much stronger than the arguments in favour of segregation". Polyploidy in the genus Ophioglossum The genus Ophioglossum with haploid numbers like n=120, 240, c. 370, 480, c. 570, c. 630 etc, in the different species represents the highest grade of polyploidy so far known in any species in the plant kingdom. Even the lowest haploid number for this genus (n=120) is very high, compared with those of the other genera of Pteridophytes or with numbers like 9, 10, 11, 12, 13 etc. commonly met with in the gymnosperms and angiosperms (Darlington and Wylie 1955). A comparative study of chromosome numbers of species of Ophioglossum from very distinct climatic conditions shows that the tropical forms exhibit much higher levels of polyploidy than those from temperate areas. Abraham and Ninan (1954) tentatively suggested that within the same species, materials from the older strata of the earth's crust, which have longest escaped great geological upheavals, would show higher chromosome numbers, compared with materials of the same from relatively more recent formations. Further knowledge of chromosome numbers from 310 C. A. Ninan Cytologia 23

Table 2. Chromosome numbers in the genus Ophioglossum

the tropics has only served to strengthen this suggestion. It is also pertinent to note in this connection that both and Phylloglossum, restricted in distribution to Australia and New Zealand (which represent very old geological formations), also show very high chromosome numbers like 2n =408-420 in Tmesipteris (Barber 1955) and 2n=502-510 in Phylloglossum (Blackwood 1953). The regular bivalent formation observed in all species of Ophioglossum investigated in the present study indicates that in this genus we might be dealing with an allopolyploid series. However, the absence of multivalents 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 311 in the surviving species does not in any way preclude the possibility of their being autopolyploids at some stage in their long history. It is quite probable that in the course of evolution, the multivalent pairing may be replaced by bivalent pairing consequent on the accumulation of genie or chromosome mutations. Establishment of homozygous types involving mutations of any magnitude is facilitated by a sexual mode of reproduction. When a polyploid has through a combination of mutations established sufficient differences among originally identical chromosomes, it may behave like diploids during meiosis. The interpolation of a vegetative mode of reproduction would perpetuate any otherwise viable type, irrespective of the nature of chromosomal pairing at meiosis, as the products of meiosis are of no importance in the life-history in such a case. What we are currently dealing with most probably represent upper members of a series whose bases have been lost, and as such we cannot easily decide whether auto or allopolyploidy or a combination of the two might have been operative at different stages in their long history. Evidences from artificially produced autopolyploids indicate that autoployploidy is rarely, if ever, successful at higher levels than tetraploidy (Manton 1950, Stebbins 1950). Investigations on the genus Psilotum, which represents a natural autopolyploid series in a , have also shown that the highest level of polyploidy attained is the tetraploid stage and that a large majority of the natural populations are tetraploids (Ninan 1956c). The regular bivalent formation associated with degrees of polyploidy far beyond the tetraploid level in Ophioglossum may be interpreted as indicating an allopoly ploid origin. It is well recognized that within very closely related plants, like different species in a genus, where the chromosome number varies from species to species, the size of chromosomes decreases with increase in number. Obser vation on the genus Ophioglossum has shown that this correlation between small size and large number is of no general validity. In O. reticulatum with 630 bivalents in spore mother cells, the chromosomes are as large as those in species like O. costatum with 120 bivalents in spore mother cells. In other ancient genera like Psilotum, Angiopteris etc. it is seen that large size of chromosomes continues to be associated with large numbers. It seems that the incidence of high levels of polyploidy has not in any way affected chromosome size in these genera. It is worthwhile to consider whether there is any relationship between polyploidy and vegetative propagation, so frequently met with in the Ophio glossaceae. It must be admitted that the prevalence of vegetative propagation has promoted the survival of different strains with high polyploid constitution and of some with aneuploid numbers. Muntzing (1936) and Gustafsson (1947, 1948) hold that accessory methods of vegetative reproduction are direct consequences of polyploidy. Stebbins (1950) considers that polyploidy can reinforce the action of genes favouring vegetative reproduction. Propagation 312 C. A. Ninan Cytologia 23 through root buds is characteristic of most of the ground growing species of Ophioglossum, and it is very prevalent in O. reticulatum which shows very high chromosome numbers coupled with aneuploid constitution in many populations. It must be admitted that there is some correlation between high numbers and the frequency of vegetative propagation in Ophioglossum. Coming to purely cytological considerations, it is seen that the course of meiosis in all the species investigated in this study is perfectly normal, leading to the formation of well filled and seemingly viable spores. The bivalents have quite normal shapes and represent the usual X, Y, 0 and V types. The large majority of them at metaphase of meiosis show a single interstitial chiasma while the others show 2-3 chiasmata. The nucleolus appears to persist till late metaphase of heterotypic division. Though there seems to be no consistent correlation between polyploidy and the number of nucleoli in Ophioglossum, it is seen that forms with n=120 (diploids?) have a single large nucleolus while the higher numbered forms show a varying number of nucleoli (1-4) in a cell. of the genus Ophioglossum The taxonomic status of O. reticulatum, O. vulgatum and O. petiolatum has been much disputed. Clausen (1938) considers O. reticulatum and O. petiolatum as perhaps representing forms of the same species, but treats them as distinct species in his taxonomic system. Similarly, O. reticulatum and O. vulgatum have been regarded as forms of the same species (d' Almeida 1922), connected by a number of transitional types. However, Clausen (1938) recognizes them as distinct species. Cytological situation in these three species shows that they are all high polyploid strains with haploid numbers like n=c.435, c.450, 480, 495, c.570, c.630 etc. in O. reticulatum, n=340-46, 385-90, 410, 480, 515-520 and 570 in O. vulgatum and n=480 and 510 515 in O. petiolatum. It is clear that there is no cytological discontinuity which would justify their treatment as separate species. O. nudicaule likewise presents several difficulties to the taxonomist. Due to the extreme variability among the different populations, certain authors consider some of these variants as distinct species. O. nudicaule L. fil. var. typicum and O. nudicaule var. tenerum have thus been regarded by some authors as separate species, though Clausen (1938) includes them in the synonymy of O. nudicaule L. fil. He considers these differences as normal variations in any local population. Regarding O. nudicaule Clausen (1938) remarks: "O. nudicaule is an exceedingly variable form from which many spurious species have been segregated. Since the several extremes of develop ment within this species population freely intergrade, and since these vari ations cannot be satisfactorily correlated with range, it has seemed impossible to consider this here either as species or as subspecies". Four out of the six varieties of O. nudicaule investigated in this study show the haploid number of n=240, while the other two (O. nudicaule var. typicum and 1958 Studies on the Cytology and Phylogeny of the Pteridophytes VI 313

O. nudicaule var. tenerum), show n=120. On cytological grounds therefore it seems reasonable that these two varieties should be raised as two distinct species (this aspect is dealt with in another paper) . Again, minor variations within the same species in which no gross cytological difference can be detected, must be regarded as the result of evolutionary changes other than polyploidy-most probably gene mutations . The cumulative effects of such changes may lead to the establishment of new forms which may be ranked as separate species. This may account for the morphological differences between species with the same number of chromosomes.

Summary 1. A critical cytological investigation of nine species of Ophioglossum (including several cytological and geographic races), one species of Botrychium and the monotypic Helminthostachys has been made. 2. The lowest haploid number for Ophioglossum is n=120 and for Helminthostachys, n=94. 3. Almost all the species of Ophioglossum investigated in the present study (excepting the aneuploid series in O. reticulatum and perhaps O . vulgatum) are found to form a polyploid series based on the haploid number n=120. 4. O. reticulatum L. is shown to have the highest chromosome number yet recorded in any species (n=c. 630, 2n=c. 1260). 5. The basic chromosome number of the Ophioglossaceae is postulated to be 15. 6. Evidences of chromosome numbers obtained from this study show the probability that there is some parallelism in the cytological evolution of the Ophioglossaceae and the Schizaeaceae. Both Ophioglossum and Schizaea exhibit very high chromosome numbers. The basic number 15, characteristic of the Ophioglossaceae, is also shared by Lygodium, the most primitive genus of the Schizaeaceae. 7. Cytological evidence is in favour of regarding Botrychium as the most primitive of the three genera while Helminthostachys is intermediate and Ophioglossum the most specialised. 8. There is no cytological support for the segregation of species of Botrychium and Ophioglossum into subgenera or for raising these subgenera to generic status. 9. It is suggested that the greater percentage of polyploids as well as higher grades of polyploidy encountered in South Indian races of Ophioglossum may be related to the fact that Peninsular India is geologically one of the oldest land masses with a continued vegetation cover for a very long geo logical period. This suggestion is incidentally supported by evidences of chromosome numbers from ancient genera like Tmesipteris and Phylloglossum 314 C. A. Ninan Cytologia 23 from Australia which also represents a very old geological formation. 10. The taxonomic delimitation in species like O. reticulatum, O. petiolatum and O. vulgatum is seen to be very artificially drawn. 11. Taxonomic revision in the light of cytology is necessary for O. nudicaule.

Acknowledgement

This investigation was carried out under the supervision of Prof. A. Abraham, Head of the Department of Botany, University College, Trivandrum. The materials used in this study were assembled by Prof. Abraham through the kind co-operation of many Botanists. I am deeply indebted to Prof. Abraham for generously placing the materials at my disposal, for his un flagging interest in the progress of this investigation, for the valuable guidance and advice throughout. To Prof. R. T. Clausen, New York State College of Agriculture, Ithaca, New York, and the Director, Royal Botanic Gardens, Kew, I am grateful for the identification of a few species of Ophioglossum included in this study. For financial help through the award of a Senior Research Scholarship, I am thankful to the Ministry of Education, Govern ment of India. To the National Institute of Sciences of India, I am grateful for the award of a Research Fellowship which enabled me to complete this investigation. My thanks are also due to the University of Kerala for the excellent research facilities.

References

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Allen and Unwinn. Delay, C. 1953. Rev. De Cyt. et De Biol. Veg. 4: 59. Eames, A. J. 1936. Morphology of vascular plants (lower groups). McGraw Hill Co., New York pp. 433. Gopal-lyengar. 1956. Origin and behaviour of chiasmata in Botrychium. Proc. Indian Sci. Cong. Gustafsson, A. 1947. Apomixis in higher plants III. Biotype and species formation Lunds. Univ. Arsskr. 44: 183-370.- 1948. Polyploidy, life-form, and vegetative reproduction. Hereditas 34: 1-22. Lyon, H. L. 1905. A new genus of Ophioglossaceae. Bot. Gaz. 40: 455-458. Majumdar, G. P. 1930. Report on the occurrence of Helminthostachys zeylaniea (L.) Hk. and Equisetu.m debile Roxb. in the suburbs of Calcutta. Proc. Indian Sci. Cong. (Allahabad). Manton, I. 1950. Problems of Cytology and Evolution in the Pteridophyta. Cambridge University Press, pp. 316.- 1953. The cytological evolution of the flora of Ceylon. Symp. Soc. expt. Biol. no. 7 Evolution 174-185. Manton, I. and Sledge, W. A. 1954. Observations on the cytology and taxonomy of the Pteridophyte flora of Ceylon. Phil. Trans. 238B: 127-185. McClintock, B. 1929. A method for making aceto-carmine smears permanent. Stain Tech. 4: 53-56. Mehra, P. N. and Verma, S. C. 1957. Cytology of Lycopodium. Curr. Sci. 26: 55-56. Muntzing, A. 1936. The evolutionary significance of autopolyploidy. Hereditas 21: 263-378. Ninan, C. A. 1955. Cytology of Equisetum debile Roxb. J. Indian hot. Soc. 34: 112-114.- 1956a. Cytology of the Ophioglossaceae. Curr. Sci. 25: 161-162.- 1956b. Studies on the cytology and phylogeny of the Pteridophytes. I. Observations on the Marattiaceae. J. Indian bot. Soc. 35: 233-239.- 1956c. Cytology of L. Cellule 57: 305-318.- 1956d. Studies on the cytology and phylogeny of the Pteridophytes. III. Observations on Osmunda regalis. J. Indian hot. Soc. 35: 248-251.- 1958a. Studies on the cytology and phylogeny of the Pteridophytes. II. Observations on the genus Lycopodium. Proc. Nat. Inst. Sci. India 24B: 54-66.- 1958b. Studies on the cytology and phylogeny of the Pteridophytes. V. Observations on the Isoetaceae. J. Indian Bot. Soc. 37: 93-103. Nishida, M. 1952a. Studies on the systematic position and constitution of the Pteridophyta. (1) On the dichotomy of the vascular system in the stalk of Ophioglossum. Jour. Coll. Arts and Sci., Chiba. Univ. 1: 32-38.- 1952b. Dichotomy of vascular system in the stalk of Ophioglossaceae. Jour. Jap. Bot. 27: 165-171.- 1952c. A new system of Ophioglossales. Jour. Jap. Bot. 27: 271-278. Stebbins, G. L. 1950. Variation and Evolution in Plants, Columbia University Press, New York. Verma, S. C. 1956. Cytology of Ophioglossum. Curr. Sci. 25: 398-399. Wagner, W. H. Jr. 1952. Types of foliar dichotomy in living ferns. Amer. J. Bot. 39: 578-592.- Lord, L. P. 1956. The morphological and cytological distinctness of and B. lunaria in Michigan. Bull. Torrey hot. Cl. 83: 261-280. Willis, J. C. 1922. Age and area, a study in geographical distribution and the origin of species. Cambridge 1-258.- 1923. Age and area, a reply to critcism with further evidence. Ann. Bot. 37: 193-215. 316 C. A. Ninan Cytologia 23

Note added in proof. In a recent paper in this Journal Verma (1957) reported n=120 and n=122 in Ophioglossum polyphyllum. In an earlier note (Verma 1956) he reported n=116-117 in the same species, which, following Chakravarty (1951) and Pichi-Sermolii (1954) he treats as syno nymous with O. Aitchisonii (Clarke) d'Almeida and O. capense Sw. In the taxonomic treatment I have followed Clausen's (1938) very careful study of the genus and find the above synonymy unacceptable. O. Aitchisonii is a very distinct species, not likely to be confused with any other validly described species in the genus, and careful examination of materials fixed in the field shows clearly 120 bivalents (fig. 3). In three species reported in the present study and based on examination of hundreds of spore mother cells, the haploid number was found to be indisputably 120 and in no case have I observed a lower number in this genus. While a certain range of chromosome numbers is met with in species having numbers above n=240, this is not observed in species with n=120 and n= 240. In view of the later admission by Verma of n=120 in his O. polyphyllum I am inclined to believe that this is the correct number for this species also. An exami nation of the photograph in his earlier paper (Verma 1956) shows that the observation is based on inadequate preparation. The danger of basing con clusions on basic numbers in a genus without study of adequate preparations in a number of species of the genus need not be stressed. C. A. N.

References

Chakravarty, H. L. 1951. Bull. Bot. Soc., Bengal 3: 1. Pichi-Sermolii, R. 1954. Webbia 9: 623. Verma, S. C. 1957. Cytologia 22: 393.