Cytological Investigations on West-Himalayan Orchids
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Cytologia 48: 633-646, 1983 Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. The genus Habenaria Willd. S. K. Kashyap and P. N. Mehra Departmentof Botany,Panjab University, Chandigarh-160014,India ReceivedDecember 23, 1981 Dressier and Dodson (1960) divided the tribe into four sub-tribes (Coryciinae, Epipogiinae, Orchidinae and Disinae). It includes 65 genera. Cytological data on sub-tribe Orchidinae are more than in the other sub-tribes but certainly not comprehensive. Out of 53 genera belonging to this sub-tribe, only 21 have been investigated. And only two genera of the sub-tribes Disinae and Epipogiinae, Satyriumand Epipogium, have been worked out (cf. Moore 1967-1971, Tanaka and Kamemoto 1972, 1974). The sub-tribe Coryciinae is untouched till date. The overall distribution of the members of this tribe is scanty in the Western Himalayas. Only the taxa belonging to the sub-tribe Orchidinae are well represent ed and occur at an elevation of 600m to 2500m. The members whcih are all ter restrialgrow either on open grasslands or in shade in thick forests. The main genera are Habenaria and Herminium with 30 and 6 species, respectively. Cytological contributionsto the members of this tribe in India have been made by Swamy (1944), Mehra and Pal (1961), Mehra and Bawa (1962, 1970), Arora (1968, 1971), Chat terji (1968a), Sharma and Roy (vide Chatterji, 1968b), Mehra and Vij (1970, 1972), Roy and Sharma (1972), Vij and Gupta (1975), Vij et al. (1976a, b) and Jorapur (1977). The present investigations have been divided into two parts. Part I deals with the genus Habenaria of which 16 species have been worked out, 12 of which have been studied karyotypically. Such detailed studies may throw some light on the cytogeneticmode of evolution of this genus. The remaining investigated genera will be presented in Part II. All the members have pollens in massulae which varyin number, size and shape in different taxa and their surface texture is mostly reticulate. Materials and methods All the members studied were collected from nature in the Western Himalayas (Table 1). Meiotic studies were made mostly from pollen mother cells (PMCs'). In a few taxa chromosomal studies were made from egg mother cells (EMC's) followingFeulgen staining technique. Buds were fixed at appropriate stages and antherswere squashed in aceto-carmine. In some cases somatic counts were also made from the root-tips after pre-treating with 8-hydroxyquinoline, followed by 634 S. K. Kashyap and P. N, Mehra Cytologia 48 Table 1. 1983 Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. 635 Table 1. (Continued) * New chromosome numbers/or B-chromosomes. * Species worked out for the first time . hydrolysis in IN HCl and stained with Feulgen staining technique. The de sired slides were made permanent in euparol. All the photomicrographs are at a uniform magnification of •~1510 except where otherwise mentioned. For karyo typic analysis cells at metaphase were enlarged to suitable magnification (•~4530) and chromosomes were cut, measured, pasted on the basis of arm-ratio (r-value) and chromosome length in descending size-order. Although the karyograms in 12 species have been studied, only representative samples of 3 size-class categories (small, medium, large) are presented in Figs. 22-24. For describing chromosome morphology, the terminology of Levan, Fredga and Sandberg (1964) is employed i.e. median region (m) with r-value 1-1.7 and d-value 0.0-2.5, submedian region (sin) with r-value 1.7-3 and d-value 2.5-5.0, subterminal region (st) with r-value 3.0-7.0 and d-value 5.0-7.5, terminal region (t) with r-value 7-a and d-value 7.5 -10.0. length of long arm/ r-value= length of short arm d-value=long arm-short arm Results HabenariaWilld. It is one of the largest genera of Orchidaceae comprising about 600 terrestrial speciesdistributed in the tropical and warm countries in the old and new world (AiryShaw 1973). The genus is represented in the Western Himalayas by 30 species, (Duthie1906). Many of the species of this polymorphic genus have been raised to genericranks on the basis of stigma structure (cf. Correll 1950). Present studies, as stated above, comprise cytological investigations on 16 species. 636 S. K. Kashyap and P. N. Mehra Cytologia 48 1983 Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. 637 H, arietina Hook. f. Somatic determination from root-tips revealed 2n=42 at metaphase. Mehra and Bawa (1970) also reported n=21 from Silma hills. Other numbers, n=23 and 2n=48, were reported by Chatterji (1968b) and Roy and Sharma (1972) respec tively, which, if correct, may suggest occurrence of aneuploid races. H. commelinifolia Wall. Somatic chromosome number 2n=42 was determined from root-tip mitotis (Fig. 1). Chromosomes are large but occur in a graded series measuring between 3.17-1.05ƒÊ. Total chromatin length is 103.57ƒÊ. Chromosome morphology was not clear in 3-4 chromosomes but karyotypic analysis seems to consist of 16m+ 12sm+14st chromosomes. An interesting feature is the presence of a secondary constriction in many of the chromosomes. H. densa Wall. Presently two races were discovered with 2n=44 and 48 chromosomes. a) Cytotype with 2n=44: Twenty two bivalents were observed at M-I (Fig. 2) which exhibited secondary associations. In a few cells lagging chromosomes were also seen at A-I. Further meiotic course was almost normal. b) Cytotype with 2n=48: Root-tips revealed 2n=48 (Fig. 3) at mitosis. The species belongs to the category of small-sized chromosomes. There is gradual gradation in size of the chromosomes too within the complement. They measured from 2.11-0.66ƒÊ. Total chromtain length is 58.27ƒÊ. The karyotypic formula is 36m+4sm+6st+2t. The presently studied cytological races are in conformity with the earlier records of n=22, 2n=44 (Chatterji 1968b) and n=24 (Mehra and Vij 1972). The two cytotypes did not reveal any significant morphological differences in their phenotypes. H. edgeworthii Hook. f. Present cytological studies revealed diploid and tetraploid races with chromo some number n=21, 2n=42 and n=42, 2n=84 respectively. a) Cytotype with n=21, 2n=42: Meiotic determination revealed 21 bivaltnts at M-I (Fig. 4). Secondary assocaitions among the bivalents were frequent but such groups varied from cell to cell. Somatic determination from root-tip mitosis showed 2n=42 (Fig. 5). Seven chromosome pairs were somewhat larger than the rest (Fig. 22). The chromosome size ranges between 1.72-0.66ƒÊ. Total chromarin length is 47.15ƒÊ. The karyo type comprised 28m+10sm+4st chromosomes (Fig. 22). The 9th pair appears Figs. 1-9. Habenaria commelinifolia: 1, somatic cell with 42 chromosomes at metaphase. H. densa: 2, PMC with 2n=44 at M-I. Note secondary associations. 3, somatic cells with 2n= 48. H. edgeworthii: cytotype 'a'. 4, 21 bivalents at M-I showing secondary associations. 5, somatic cells with 2n=42 chromosomes; cytotype 'b'. 6, somatic cell with 2n=84 chromosomes at metaphase. H. ensifolia: 7 , M-I with n=31+1B at arrow below and two lightly stained nucleoli at arrow above. 8, somatic cell with 2n=42 chromosomes. H. fallax: 9, somatic cell with 2n=30 chromosomes at metaphase. 638 S. K. Kashyapand P. N. Mehra Cytologia48 to bear a terminal satellite. b) Cytotype with n=42, 2n=84: Meiotic determination revealed 2n=84 at diakinesis. Most of the bivalents showed early disjunction. Multiple associations were seen at M-I. The chromosome number was furhter confirmed by somatic analysis of root-tips which revealed 2n=84 (Fig. 6). Morphological characters of these two cytotypes are compared in Table 2. Polyploidy has led to gigantism (Fig. 21). The present finding of 2n=84 falls in line with the earlier reports by different authors (see Table 1). H. elisabethae Duthie Somatic chromosome number determination from root-tip mitosis revealed 2n=42. Seven pairs were comparatively smaller than the rest. Chromosome Table2. Habenariaedgeworthii size measured from 2.11-0.79ƒÊ within the complement. Total chromatin length is 57.21ƒÊ. The karyotype comprises 32m+10sm chromosomes. H. ensifolia Lindl. PMC's clearly revealed n=21+1B at M-I (Fig. 7). At diakinesis some of the bivalents showed precocious disjunction. The chromosome number was further confirmed from somatic root-tip mitosis which revealed 2n=42 chromosomes of small size (Fig. 8). They measured from 1.98-0.52ƒÊ within the complement. Total chromatin length is 49.53ƒÊ. Karyotype comprises 24m+8sm+4t chromosomes. H. fallax K. and P. Somatic chromosome number determination from root-mitosis showed 2n=30 (Fig. 9). The chromosomes are large in size ranging from 3.97-1.72ƒÊ within the complement. Total chromatin length is 88.21 u . Karyotype consists of 14 m+ 1983 Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. 639 Figs. 10-17. H. intermedia: 10, M-I with 21 bivalents. note precocious disjunction of some bivalents. 11, somatic cell with 2n=42 chromosomes. H. josephi: 12, PMC with 20 bivalents at diakineiss. 13, somatic cell with 2n=40 chromosomes. H. lawii: 14, somatic cell with 2n=42 chromosomes . H. pectinata: 15, somatic cell with 2n=42 chromosomes. H. plantaginea: 16, PMC with 2n=42 at M-I . note multiple associations (11I+8II+1III+lv+lvII). H. susannae: 17, PMC with 21 bivalents at diakinesis. 640 S. K. Kashyap and P. N. Mehra Cytologia 48 12sm+4st chromosomes. The present count of 2n=30 differs from the only existing report of n=21 by Mehra and Vij (1970). It seems that the present taxon may be aneuploid at tetraploid level on the base number x=7.