1968 401

Cytological Studies in

K. Ramachandran

Department of Botany, University of Kerala , Trivandrum,

Received November 8, 1967

Introduction The family Dioscoreaceae consists of ten genera and about 650 species (Lawrence 1960), dominated by the Dioscorea which accounts for about 620 species. The family is distributed in all tropical regions and a few species are also found in temperate Europe and North America . The genus Dioscorea is of great economic importance. A few species , the true yams, are cultivated for edible tubers in many tropical countries. The genus is also of interest as tubers of some wild species yield precursors of cortisone and related steroid drugs. The dioecious species of Dioscorea received the attention of cytologists studying the sex chromosome mechanism in (Meurman 1925, Nakajima 1933, 1934, 1936, 1937, 1942, Smith 1937). Recently, since the discovery of cortisone precursors in tubers of some wild species, a great deal of work on the cytology and experimental breeding of Dioscoreas has been done by Martin and co-workers (Martin 1966, Martin and Cabanillas 1963, 1966, 1967, Martin and Ortiz 1963, 1966, Martinet al. 1963). These studies have shed valuable light on the pattern of evolution in the family. Cytological studies in the family have been summarised by Martin and Ortiz (1963). Previously, Indian species of Dioscorea were studied by Sundara Raghavan (1958, 1959). A list of chromosome numbers of 9 species and a few varieties are given in his earlier paper, but no figures of chromosomes have been published. Chromosome numbers of two more species were reported later (Sundara Raghavan 1959). Sharma and De (1956) and Sharma and Sharma (1957) studied two species of Dioscorea. The present study was undertaken as part of a larger programme on the breeding of the yams. A preliminary report of this work was published earlier (Ramachandran 1962) in which chromosome numbers and sex determi nation mechanism in Dioscorea were discussed. The present paper gives a detailed account of the cytology of all the South Indian species studied.

Materials and methods

A large collection of cultivated and wild species of Dioscorea and Tri chopus zeylanicus was made from the different parts of Kerala State, South India. Three clones of D. bulbifera were also obtained from Poona, Western 402 K. Ramachandran Cytologia 33 1968 Cytological Studies in Dioscoreaceae 403

India. The plants were grown in the botanical garden of the Kerala Uni

versity, Trivandrum. Cytological studies were made from acetocarmine smears . Root tips for somatic chromosome studies were obtained from yam heads , aerial tubers or, in a few cases, leaf-bud cuttings planted in pots . Root tips were kept at 6•K-10•Ž for an hour and fixed in Carney's fluid (alcohol , acetic acid and chloroform in the proportion of 3:1:1) mordanted with a trace of

iron-acetate. For meiotic studies male spikes were fixed in alcohol-acetic acid

(3:1). Anthers were dissected out from buds and smeared in acetocarmine .

Observations

Dioscorea Plim. ex L.

The genus consists of about 620 species , distributed in the tropics, with a few species in temperate Europe and North America . A few species are cultivated in most tropical countries. The four species commonly cultivated

in South India are D. alata, D. esculenta, D. sativa and D. spinosa. All

these have been investigated in the present study.

1. D. alata L. Known variously as 'white yam' , 'water yam' or ' winged yam', this is the most important of the yams, being very widely

cultivated in Asiatic tropics, Africa, the Pacific Islands and tropical America.

There are a large number of varieties of this species under cultivation. Twenty

six clones of this species have been assembled in our collections. They differ

chiefly in their growth habit, shape and size of leaves, and shape, size and

coloration of skin and flesh of the yams. Other differences relate to the

presence or absence of spines, colouration of stem and petioles and shape and size of bulbils.

Twenty three out of the twenty six clones of D. alata had 40 chromo

somes, two had 60 and the other 80 chromosomes in root tip cells. Fig. 1

shows the somatic chromosomes of a male having 40 chromosomes.

They are small ranging between 0.6ƒÊ and 2.1ƒÊ in length. One of the chro

mosomes is appreciably larger than the others (indicated by arrow in figure).

Meiosis was normal and 20 bivalents have been observed at metaphase I

(Fig. 2). No irregularities have been found in the few cells examined.

Two clones (both female plants) of D. alata in our collection had 60 chromosomes in root tip cells. Fig. 3 shows the chromosome complement of one of these. The chromosomes are between 0.6 and 1.7ƒÊ in length.

D. alata var. purpurea Roxb. Female plants of this variety collected from different localities showed

Figs. 1-9. Chromosomes of Dioseorea species. •~1250. 1, D. alata L., 2n=40. Note one larger chromosome (possibly Y) indicated by arrow. 2, D. alata L. Diakinesis in a

pollen mother cell of a tetraploid plant showing 20 bivalents. 3, D. alata L., 2n=60. 4, D. alata var. purpurea Roxb., 2n=80. 5, D. bulbifera L., clone l from Maharashtra, 2n=80. 6, D. bulbifera L., clone from Ponmudi (Kerala), 2n=80. 7, D. bulbifera L., clone 2 from Maharashtra, 2n=80. 8, D. bulbifera L., clone 3 from Maharashtra, 2n=80. 9, D. bulbifera L., clone from Karthicappally (Kerala), 2n=80. 404 K. Ramachandran Cytologia 33 1968 Cytological Studies in Dioscoreaceae 405

2n=80 chromosomes (Fig. 4). Male plants have not been collected so far .

2. D. bulbifera L. Five different clones of D . bulbifera, 2 from Kerala State and 3 from Maharashtra State possessed 80 somatic chromosomes . Another plant from Kozhencherry (Kerala) showed 2n=100 chromosomes .

The somatic chromosomes of the six clones of D . bulbifera are shown in Figs. 5-10.

Fig. 11 shows a group of pollen mother cells from a male plant of clone

2. Meiotic chromosomes at metaphase I are seen clumped together . No

irregularities have been found in subsequent stages of meiosis . Ninety per

cent of pollen grains appeared to be normal , as shown by staining with acetocarmine.

3. D. esculenta Burkill. This is a widely cultivated species , charac terised by slender round stems with small prickles , cordate leaves and several underground tubers in a cluster. There are two easily recognisable cultivated

varieties of this species. One variety ('Nanakizhengu') has larger leaves and

tubers than the other ('Cherukizhengu'). The former variety had 90 somatic

chromosomes (Fig. 12), while the later showed 100 chromosomes (Fig. 13).

4. D. hispida Dennst. This is a common wild species of the Western

Ghats. Plants of this species showed 40 chromosomes in root tip cells (Fig.

14). Chromosomes are between 0.9 and 2.7ƒÊ in length.

5. D. oppositifolia L. var. linnaei Pr. and Burk. The chromosome

complement of this variety (2n=40) is illustrated in Fig. 15. Chromosomes

range between 1ƒÊ and 2ƒÊ in length.

Another clone of D. oppositifolia collected from Kannikatty (Madras

State) also showed 40 somatic chromosomes (Fig. 16).

6. D. pentaphylla L. Two varieties of D. pentaphylla have been ex

amined cytologically in the present study.

D. pentaphylla var. linnaei Pr. and Burk.

The chromosome number in male plants of this variety collected from

Kallar (Kerala State) is 2n=40 (Fig. 17). One chromosome of the com

plement is larger than the others being 2ƒÊ long. The others are between 1.2ƒÊ and 1.8ƒÊ in length.

D. pentaphylla var. rheedei Pr. and Burk.

This is found cultivated in some places in Kerala and produces smooth

white cylindrical tubers and elongated bulbils. A female plant of this variety

has been examined cytologically. It possessed 40 chromosomes (Fig. 18).

Chromosome length ranges between 1.2 and 1.8ƒÊ.

Figs. 10-16. Chromosomes of Dioscorea species. All figures except Fig. 11. •~1250. 10,

D. bulbifera L., clone from Kozhencherry (Kerala), 2n=100. 11, a group of pollen mother cells from the clone of D. bulbifera from Ponmudi. •~750. 12, D. esculenta Burkill,

2n=90. 13, D. esculenta Burkill, 2n=100. 14, D. hispida Dennst., 2n=40. 15, D. op

positifolia L. var. linnaei Pr. and Burk., 2n=40. 16, D. oppositifolia L. from Kannikatty

(Madras State), 2n=40. 406 K. Ramachandran Cytologia 33 1968 Cytological Studies in Dioscoreaceae 407

7. D. sativa Thun. (The Potato yam). This species is cultivated

widely in tropical Asia, West Indies and Africa for its edible aerial tubers.

Female plants of this species were cytologically studied.

There are clearly 80 chromosomes in root tip cells (Fig . 19). 8. D. spinosa Roxb. Santapau (1951) considers this as synonymous

with D. esculenta. But it is quite distinct with very large tubers (30-40cm

long and 15-25cm in diameter) and numerous large spines at the base. The

tubers of this species are also edible. It is cultivated in many parts of Kerala

and has also been reported as occurring wild in some parts of India.

Male plants of this species showed 2n=90 chromosomes (Fig. 20). One

chromosome, 2.4ƒÊ long, is appreciably larger than the others, which range

between 0.75ƒÊ and 1.8ƒÊ in length.

9. D. tomentosa Koenig. Male plants of this species have been cyto

logically examined in the present study. They showed 40 chromosomes in

root tip cells (Fig. 21). One chromosome is remarkably larger than the others

of the complement.

At metaphase I of meiosis twenty bivalents have been observed in pollen

mother cells (Fig. 22). The larger chromosome found in somatic plates is

found paired with one of the medium sized chromosomes to form an asym

metrical bivalent. No irregularities have been found in the later stages of

division.

10. D. wallichii Hook. f. A male clone of this species has been

studied. Forty chromosomes are found in root tip cells (Fig. 23). Their size

ranges between 0.9 and 1.7ƒÊ.

11. Gaertn. This is a monotypic genus of the Dioscoreaceae

represented by the single species T. zeylanicus.

. zeylanicus Gaertn.

This plant is found at elevations of over 3000 ft. in the Western Ghats.

There are 28 chromosomes in root tip cells (Fig. 24). Chromosomes

range in length between 1.5 and 2.7ƒÊ.

Meiosis is regular. Fourteen bivalents were observed at metaphase I in

pollen mother cells (Fig. 25). The plant shows good seed setting.

Discussion

The cytology of ten species of Dioscorea and the monotypic genus

Figs. 17-25. Chromosomes of Dioscorea species. •~1250. 17, D. pentaphylla var.•@ linnaei

Pr. and Burk., 2n=40. Note one larger chromosome (possibly Y) indicated by arrow. 18,

D. pentaphylla var. rheedei Pr. and Burk., 2n=40. 19, D. sativa Thun., 2n=80. 20, D.

spinosa Roxb., 2n=90. Note one larger chromosome (possibly Y)•@ indicated by arrow.

21, D. tomentosa Koenig., 2n=40. Note the larger chromosome (possibly Y) indicated by

arrow. 22, metaphase I in a pollen mother cell of D. tomentosa showing 20 bivalents.

An asymmetrical bivalent is indicated by arrow. 23. D. wallichii Hook. f., 2n=40. 24,

Trichopus zeylanicus Gaertn., 2n=28. 25, metaphase I in a pollen mother cell of Trichopus

zeylanicus showing 14 bivalents.

T 408 K. Ramachandran Cytologia 33

Trichopus from South India has been investigated in the present study. The chromosome numbers observed during this study and those reported earlier for these species are given in Table 1. As the data in the table show, in Dioscorea the chromosome numbers are in multiples of the basic number 10 (40, 60, 80, 90, 100). has a chromosome number 2n=28, suggesting a basic number 14. Table 1. Chromosome numbers of South Indian species of Dioscorea and Trichopus

Previous investigators have also found 10 as the basic number of Asiatic species of Dioscorea (Smith 1937, Sundara Raghavan 1958, 1959, Martin and Ortiz 1963). A basic number 9 has been found to be characteristic of New World species (Martin and Ortiz 1963, 1966). As some African species showed both 9 and 10 as basic numbers (Miege 1954), an African centre of origin for the genus was suggested by Martin and Ortiz (1963) with the 9 chromosomed races spreading West and 10 chromosomed races migrating East 1968 Cytological Studies in Dioscoreaceae 409

and North. Burkill (1960), however, considers eastern continental Asia as the centre of origin of the genus on morphological grounds . Two species of Dioscorea from Pyrenees suggest a basic number 12 (Miege 1954). Heslot (1953), however, has suggested that these two species should be included in a separate genus (Borderea) . Recent hybridization studies of Martin and Cabanillas (1966) have shed light on the evolutionary pattern and relationships of species in Dioscorea . Their data show that strong reproductive barriers exist between Old and New World species of Dioscorea. Crosses between New and Old World species failed entirely. Barriers between New World species were less strong , and even distant crosses between Dioscorea and Rajania produced seeds . Fertility studies of F1 hybrids showed that in speciation gross structural changes of chromosomes or polyploidy were of little importance. The status of Trichopus has always presented problems to taxonomists . Knuth (1924) included the genus under the tribe Stenomeridae. Hutchinson (1959) raised it to familial status within , assigning Trichopus and Averta to Trichopodaceae. Burkill (1960), however, has again reduced it to generic status. Ayensu (1966) has supported Hutchinson's classification on anatomical evidence. He has suggested that the family Trichopodaceae should be retained for Trichopus and that Averta should be excluded from this family. Cytologically Trichopus is distinct from other genera of the Dioscoreaceae so far known in that the basic number 14 is not found in any other genera. This seems to indicate a separate line of descent for Trichopus. Cytological data lend support to the creation of a separate family Trichopodaceae as done by earlier authors on anatomical and morphological evidence.

Summary

Cytological studies of ten species and a few varieties of Dioscorea and the monotypic genus Trichopus occurring in South India have been made. The chromosome numbers of South Indian Dioscorea species are seen to occur in multiples of the basic number 10 (40, 60, 80, 90, 100), as in the case of other Asiatic species reported previously. Trichopus zeylanicus (n= 14, 2n=28) shows a basic number of 14 not found in any other genus of the Dioscoreaceae so far studied. Cytological data support morphological and anatomical evidence suggesting creation of a separate family for the genus Trichopus.

Acknowledgments

Grateful acknowledgment is made of the financial support given by the Indian Council of Agricultural Research, New Delhi for a scheme under which this work was carried out. The author is indebted to Prof. A. Abraham, Head of the Department of Botany and Dean, Faculty of Science, University 410 K. Ramachandran Cytologia 33 of Kerala, under whose supervision this work was carried out for guidance and encouragement.

Literature cited

Ayensu, E. S. 1966. Taxonomic status of Trichopus: anatomical evidence. J. Linn. Soc. (Bot.) 59: 425-430. Burkill, I. H. 1960. The organography and the evolution of Dioscoreaceae, the family of the yams. J. Linn. Soc. (Bot.) 56: 319-412. Cox, D. K., Corzo, A. H., Matuda, E. and Duran, J. G. G. 1958. Estudio de las Dioscoreas Mexicanas. I. Dioscorea spiculiflora Hemsl. Bol. Soc. Bot. Mexico 22: 1-16. Heslot, H. 1953. Le nombre chromosomique des Dioscoreacees pyreneens et leur rattachement au genre Borderea Miegev. C. R. Acad. Sci. 237: 433-434. Hutchinson, J. 1959. The families of flowering plants. II. . 2nd ed. Oxford. Knuth, R. 1924. Dioscoreaceae. In Engler, Das Pflanzenreich. 87 (IV. 43): 1-387. Lawrence, G. H. M. 1960. of vascular plants. The Macmillan Co., New York. Martin, F. W. 1966. Sex ratio and sex determination in Dioscorea. Jour. Hered. 57: 95 -99.- and Cabanillas, E. 1963. A wild hybrid of sapogenin-bearing Dioscorea species. Bull. Torn Bot. Club. 90: 232-237. - and - 1966. The F, hybrids of some sapogenin-bearing Dioscorea species. Amer. J. Bot. 53: 350-358.- and - 1967. Heritability of yields in Dioscorea floribunda. Trop. Agriculture, Trin. 44: 45-51. - and Ortiz, S. 1963. Chromosome numbers and behavior in some species of Dioscorea. Cytologia 28: 96-101. - and - 1966. New chromosome numbers in some Dioscorea species. Cytologia 31: 105 -107. - , Delfel, N. E. and Cruzado, H. J. 1963. Dioscorea friedrichsthalii, another sapogenin bearing species. Turrialba 13: 159-163. Meurman, O. 1925. The chromosomal behaviour of some dioecious plants and their re latives, with special reference to the sex chromosomes. Soc. Sci. Fenn. Comm. Biol. 2: 1-105 Miege, J. 1954. Nombres chromosomiques et repartition geographique de quelque plantes tropicales et equatoriales. Rev. Cytol. Paris 15: 312-348. Nakajima, G. 1933. Chromosome numbers in some angiosperms. Jap. Jour. Gen. 9. (Not seen in original).- 1934. Chromosome numbers in some angiosperms. Jap. Jour. Gen. 10: 1-5. - 1936. Jap. Jour. Gen. 12: 211. (Not seen in original).- 1937. Cytological studies in some dioecious plants. Cytologia, Fugii Jub. vol.: 282-292. - 1942. Cytogenetic studies. in some flowering dioecious plants, with special reference to the sex chromosome. Cytologia 12: 212-270. Ramachandran, K. 1962. Studies on the cytology and sex determination of the Dioscore - aceae. Jour. Indian Bot. Soc. 41: 93-98. Sharma, A. K. and De, D. N. 1956. Polyploidy in Dioscorea. Genetica 28: 112-120. - and Sharma, A. 1957. Investigations leading to a new theory of differentiation in plant cells. Genet. Iber. 9: 143-162. Simmonds, N. W. 1954. Chromosome behavior in some tropical plants. Heredity 8: 139 -146. Smith, B. W. 1937. Notes on the cytology and distribution of the Dioscoreaceae. Bull. Torr. Bot. Club 64: 189-197. Sundara Raghavan, R. 1958. A chromosome survey of Indian Dioscoreas. Proc. Indian Acad. Sci. Sect. B. 48: 59-63.- 1959. A note on some South Indian species of the genus Dioscorea. Curr. Sci. 28: 337 - 338.