Cytologia 48: 633-646, 1983

Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. The genus 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 ). 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 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 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.

H. intermedia Don.

Meiotic determination from PMC's revealed n=21 at M-I (Fig. 10) with some bivalents showing precocious disjunction. Secondary associations of bivalents were seen in some PMC's at M-I. Meiotic course was normal.

Somatic chromosome number 2n=42 (Fig. 11) determined from root-tips fur ther confirms the haploid count. The chromosomes belong to the medium-size category and range from 2.91-1.05ƒÊ. Total chromatin length is 75.49ƒÊ. The karyotype consists of 28m+6sm+8st chromosomes (Fig. 23). Six pairs (2nd,

5th, 10th, 11th, 12th and 17th) have faintly stained satellitic structures on their

short arms.

H. josephi Reichb. f.

PMC's revealed 20 loose bivalents of rather large size at diakinesis (Fig. 12).

The presence of a big nucleolus at this stage was striking. Although laggards were observed at A-I, the meiotic course was near normal.

H. latilabris Hook. f.

PMC's revealed 2n=42 at diakinesis with some of the bivalents showing early disjunction. Meiotic course was normal. Somatic chromosome number was fur ther confirmed from root-tip mitosis which showed 2n=42 chromosomes of small size (Fig. 13). The chromosome size ranges between 1.85-0.52ƒÊ and the total chro matin length is 54.56ƒÊ. The karyotype comprises 20 m+12sm+6st+4t chro mosomes.

H. lawn Hook. f.

This species has been investigated for the first time . Its somatic count from root-tip mitosis revealed 2n=42. The chromosomes fall under the big chromo some size category (Fig. 14). The chromosome size ranges between 4 .50-1.58ƒÊ. Total chromatin length is 116.55ƒÊ. The complement comprises 22m+20sm chromosomes (Fig. 24). Two pairs (15th and 16th) possess small stalellites on the short arm.

H. marginata Colebr.

Somatic chromosome number determination from root-tips revealed 2n=42.

Seven pairs were comparatively smaller than the rest . The chromosome size ranges between 2.38-1,05ƒÊ and the total chromatin length is 72 .84ƒÊ. The karyotype con sists 38m+4sm chromosomes.

H. pectinata Don.

Somatic studies from root-tip mitosis showed 2n=42 at metaphase (Fig . 15). 1983 Cytological Investigations on West-Himalayan Orchids. Tribe: Orchideae I. 641

The chromosomes measured between 2.11-0.92ƒÊ. The total chromatin length is

55.33ƒÊ. The karyotype consists of 30m+4sm+8st chromosomes.

Figs. 18-21. Habenaria susannae: 18, PMC with multiple association in the form of chains of upto 11 bivalents . 19, somatic cell with 2n=42 chromosomes. note stickiness. H. triflora: 20, somatic cell with 2n=42 chromosomes at early metaphase. H. edgeworthii: 21, morpho logical comparison of cytopypes with chromosome number 2n=42 and 2n=84 chromosomes. 642 S. K. Kashyapand P. N. Mehra Cytologia48

H. plantiginea Lindl. The chromosome number determination from PMC's revealed 2n=42 chro mosomes at M-I (Fig. 16). Multiple associations of different sorts were observed at this stage (cf. Table 3) suggesting reciprocal translocations. Mehra and Vij (1970, 1972) worked out this species from Darjeeling and discovered a hexaploid taxon with n=63 and 2n=126.

H. susannae R. Br. This species bearing white flowers with pleasing smell has horticultural possi bilities. The chromosome number determined from PMC's revealed n=21 at diakinesis (Fig. 17). Multivalent chains or rings involving upto 12 bivalents (Fig. 18) were seen in some PMC's at diakinesis. Anaphasic bridges and laggards were also frequent at A I and A II. This taxon which is diploid has obviously undergone reciprocal chromosomal translocations like the previous one. Somatic number 2n=42 (Fig. 19) is further confirmed from root-tip squashes. The chromosomes

Table3. Habenariaplantagenia (2n=42)

measured from 3.17-1.32ƒÊ and the total chromatin length is 85.03ƒÊ. The karyotype comprises 16m+12sm+14 st chromosomes.

H. triflora Don.

This species has horticultural potential due to its non-white flowers. Somatic chromosome number determined from root-tip mitosis here too revealed 2n=42

(Fig. 20) at early metaphase. Most of the chromosomes were metacentric. This species is investigated cytologically for the first time.

Discussion

The somatic chromosomes of the 12 species studied can be classified into 3 size categories, small (H. densa, H. edgeworthii, H . elisabethae, H. ensifolia, H. latilabris, and H. pectinata), medium (H. intermedia, H. marginata) and large (H. fallax, H. lawii, H. commelinifolia and H. susannae). The total chromatin length of these is presented in Table 4.

There is also gradation in size of chromosomes in each of these species but 1983 Cytological Investigations on West-Himalayan Orchids . Tribe: Orchideae I . 643

Figs. 22-24 . Habenaria edgeworthii: 22 , karyogram of figure 5. H. intermedia: 23, karyogram of figure 11. H. lawii: 24, karyogram of figure 14. All•~4530. 644 S. K. Kashyapand P. N. Mehra Cytologia48

broadly they can be seen to fall into 3 groups of 7 chromosomes each (cf. Figs. 22 -24) which suggests the original basic chromosome number of the genus to be x=7. Since majority of the species show the chromosome count of n=21 and 2n=42, obviously the stabilization of the complement has occurred at the hexaploid level. In a number of species clear secondary associations of bivalents are manifested at M I (cf. H. densa, H. edgeworthii) not unoften falling into 7 groups, which too points in the same direction. The occurrence of 2n=28 in H. sagittifera (Miduno 1939, 1940, Mutsuura and Nakahira 1958, Mutsuura 1959) further supports this view point. Other variant numbers known in the genus are n=16, 24, 2n=64 (cf. Duncan 1959, Chatterji 1968b, Mehra and Vij 1970, 1972). This suggests the derivation of a secondary basic chromosome number of x=8. Richardson (1935) also reported 2n=16 for H. ciliaris.

Table4.

Chromosome numbers of some other genera of Orchidinae are also directly or indirectly based on x=7. Even the split members of Habenaria sensu lato (Brachy corythis, Coeloglossum, , etc.) are also mostly based on x=7. Diploid and tetraploid cytotypes have been found in H. edgeworthii in which but for gigantism in size in the latter cytotype no appreciable qualitative morph logical differences were noticed. Aneuploid races with 2n=44, 48 in H. densa did not reveal any difference in their morphology either . Intra and inter-specific polyploidy as well as aneuploidy in Habenaria is common (see Duncan 1959, Mehra and Vij 1972, Tanaka and Kamemoto 1972, 1974).

Conclusions

The original basic chromosome number of the genus appears to be n=7 . The derived secondary basic numbers are x=8, 15, the latter probably representing com pounding of the two basic numbers x=8, 7. Aneuploidy is frequent in speciation of the genus which in other words means that the addition or deletion of one or more chromosomes does not materially 1983 CytologicalInvestigations on West-HimalayanOrchids. Tribe: Orchideae I. 645 affectthe physiological balance of the taxa for survival. Intraspecific polyploidy has been noticed in a few cases (H. edgeworthii: 2n= 42,84). Variation in chromatin length as well as in the karyotypic formulae (see Table 4) is indicative of structural chromosomal alterations in the form of repatterning, and chromatic deletions or additions in this dynamic genus which is in an active state of speciation. It looks as if the genes or gene blocks which determine the basicflower pattern characteristic of the genus remain intact and unaltered except for undergoing minor variations affecting only the flower size or the proportions of its various parts during speciation. That such structural changes are even at present in the offing is suggested to a certain extent by multivalent formations as a result of reciprocal interchanges in the otherwise diploidised taxa (H. plantagenia, H. susannae)built on the present day basic chromosome number n=21.

Summary

Sixteen species of Habenaria are studied cytologically. H. lawii (2n=48) and H. trjora (2n=42) are investigated for the first time. Karyotype analysis is made of 12 species. Three different size categories i.e. small, medium and large are observed in different species. Chromosomal races are seen in H. densa (2n=44, 48)and H. edgeworthii (2n=42, 84). It is concluded that the original basic number is x=7 and most of the species in the genus have been stabilised at the hexaploid levelwith n=21 chromosomes. Aneuploidy is not infrequent.

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