Cytologia 49: 583-595, 1984

Cytological Observations on Some West Himalayan Orchids Tribe: Epidendreae I (Subtribes: Pogoniinae, Thuniinae, Bletiinae, , Epidendrinae)

P. N. Mehra and S. K. Kashyap

Department of Botany, Panjab University, Chandigarh,

Received October 20, 1982

Epidendreae is the largest tribe of the family with 29 subtribes covering about 465 terrestrial and epiphytic genera. A great majority of them belong to the subtribes Sarcanthineae, Oncidiinae and Epidendrinae. All the genera with few exceptions possess hard and waxy pollinia which is genrally accepted to be an advanced character in the family Orchidaceae. The systematics of some of the genera is highly debatable. Bulbophyllum Thou. and Dendrobium Sw. with about 1400 each belong to this category.

Most of the epiphytic as well as terrestrial members of this tribe have great horticultural value for their fascinating flowers. Cytological information is still quite meagre considering its great numerical strength. Important contributions on the subject have been made by many workers (cf. Moore 1973, Tanaka and Kame moto 1972, 1974).

Contributions on the Indian members of the tribe have been made by Sharma and Chatterji (1966), Vij and Mehra (1976), and Mehra and Sehgal (1974, 1975, 1976, 1978, 1980). Presently cytological investigations have been carried out on 56 West

Himalayan species belonging to 26 genera included in 10 subtribes. These results are presented in a series of three papers. The present paper includes 14 species under 8 genera. Discussion, conclusions, and phyletic relationships of the tribe Epidendreae as a whole is presented in the last paper of the series.

Material and methods

All the taxa studied were collected in nature. Procedures adopted for meiotic studies from PMC's and EMC's as well as mitotic studies from root-tip squashes, are the same as described earlier (Mehra and Kashyap, 1983). The same applies with regard to the description of the karyotypes.

All the photomicrographs were made at a uniform magnification of •~1510 except where otherwise stated.

Table I summarises the results of the present investigation. A few notes are presented below for individual taxon.

Results and discussion

Subtribe: Pogoniinae Pfitz. Table 1. * worked out for the first time . + New chromosome number added/B-chromosomes . 586 P. N. Mehra and S. K. Kashyap Cytologia49

Nervilia Comm. ex Gaud. The genus comprises a total 80 tropical and subtropical species and is confined to the old world. N. aragoana Gaud. is a terrestrial species which occurs between 1000-1500m in the Kumaon bills. It flowers earlier to the rainy season after which the leaves appear. Mitotic studies from root-tip revealed 2n=72 chromosomes. The chro mosomes in the complement have wide gradation in size. This count falls in line with the only previous record of 2n=72 by Chennave eraiah and Jorapur (1966). These authors reported variable numbers 2n=54, 72, 108 and 144 for different species of the genus and have suggested x=9 or 18 as its basic number. Undoubtedly polyploidy has played a considerable role in specia tion within the genus. Dressler and Dodson (1960) considered Pogoniinae close to Vanillinae and dis tantly placed from Sobraliinae, Arethusinae and Gastrodiinae. On the basis of occurrence of individual pollen grains in Vanilla and Pogonia, the two subtribes Vanillinae and Pogoniinae can further be considered as related to each other. They also seem primitive within the tribe Epidendreae where the general rule is the formation of hard and waxy pollinia.

Subtribe: Thuniinae Schltr. This subtribe is not well represented in the Western Himalayas since Thunia is the only genus with a single species reported from the area. T. alba Reichb. occurs in the Kumaon hills as an epiphyte or lithophyte between the elevations of 1000-1500m. Its flowers are charming white and fragrant. The species has ornamental value. Flowering period is May-June. Twenty bivalents were clearly counted in the PMC's at M-I (Fig. 1). Secon dary associations of bivalents were noticed in about 20% cells (Fig. 2). However, the number of groups formed varied from cell to cell. Majority of the cells showed equal segregation of 20:20 chromosomes at A-I. They were secondarily associated at this stage too. Meiosis was mostly normal although occasionally a few laggards were seen at A-I. Pollen grains were associated in tetrads forming soft pollinia. Forty small chromosomes were counted at root tip mitosis . The chromosomes possesssed median or submedian primary constriction. The present count of n=20, 2n=40 for T. alba confirms the previous reports (cf. Mehra and Vij 1970, Vij and Mehra 1976). Mehra and Sehgal (1978) found an asynaptic strain with 2n=c. 40. Variable chromosome numbers 2n=42 and 2n= 44 are also on record (Tanaka 1964, Roy and Sharma 1972). Vij and Mehra (1976) suggested x=20 as the basic number for the genus on the basis of chromosome counts of n-20 in T. alba and T. marshalliana. The presence of variable number 2n=38, 40, 42 and 44 as reported in literature may suggest the role of aneuploidy in evolution within the genus. In the great tribe Epidendreae, the subtribe Thuniinae is considered to be primitive by Dressler and Dodson (1960) and distantly related to the subtribes Epidendrinae, Coelogyninae and Bletiinae . The occurrence of relatively large chro mosomes in members of the subtribe and individual pollen grains in one of its 1984 Cytological Observations on Some West Himalayan Orchids 587

Figs. 1-8. Thunia alba: 1 and 2, PMC showing 20 bivalents at M-I. Note secondary associations in Fig. 2. 3-4: Calanthe puberula. 3, somatic cell with 2n=40+1B (at arrow). 4, a polysomatic cell (2n=80+2B's at arrow) •~1160. C. mannii: 5, a somatic cell with 40 chromosomes. C. trivarinata: 6, haploid complement (n=20) at pollen mitotic metaphase. Pachystoma senile: 7, 8,

PMC and EMC with 20 bivalents at diakinesis respectively. Note smaller size of bivalents in EMC. 588 P. N. Mehra and S. K. Kashyap Cytologia49 members, Arundina (cf. Mehra and Sehgal, unpublished), also suggest its primiti veness.

Subtribe Bletiinae Bentham

Calanthe R. Br.

It is a genus of perennial ornamental comprising nearly 120 species met in warm climates (Airy Shaw 1973) with a wide range of distribution from South

Africa and the Islands of the Indian Ocean through India to China and Japan and further south and eastwards from to Australia and Tahiti (Santapau and Kapadia 1966). Duthie (1960) recorded 7 species from the Western Himalayas.

Many species of this genus are exploited horticulturally.

C. puberula Lindl. grows in moist situations on rotting leaf-mold in thick forests at an elevation of about 2200 m. Mitotic studies from root-tips revealed the chro mosome number to be 2n=40+1B (Fig. 3, B-chromosome at arrow). The chromo somes showed consistent size gradation measuring from 3.44-1.45ƒÊ. Total chro mosome length is 93.24ƒÊ. Karyotype formula is 14m+18 sm+8 st. The B-chro mosome was observed in all the dividing cells. A few polyploid cells were observed with double the chromosome number and 2B's (Fig. 4, B's at arrows). Gametic chromosome number n=20 was reported by Arora (1968) and Mehra and Vij (1970) for this species but the occurrence of a B-chromosome is reported for the first time.

C. manii Hook. f. is met in humus rich soil under thick shady forests at an altitude of about 2100m in the Kumaon hills. Somatic studies from root tips reve aled 2n=40 chromosomes at metaphase (Fig. 5). The chromosomes showed mild gradation in size and appear to possess median or submedian primary constriction, The present count of 2n=40 falls in line with the earlier reports of n=20 by Mehra and Sehgal (1974) and Vij et al. (1976).

C. tricarinata Lindl, grows in shady places in forests at an altitude of about 2500m in the Western Himalayas. Its flowering period is June-July. Twenty chro mosomes were clearly counted at pollen mitotic metaphase (Fig. 6). The chromo somes measured from 3.70-2.11 p. Total chromosome length of the haploid com plement is 59.33ƒÊ. Karyotype is symmetrical with 15m+5sm chromosomes (Fig. 17). Pollen grains form soft pollinia.

The present count of n=20 is in accord with the earlier records (cf. Tanaka and

Kamemoto 1972, 1974). Ito and Mutsuura (1958) reported 2n=40, 60 for this species.

Darlington and Wylie (1955) suggested x=20 as the basic number for the genus. Majority of species of the genus possess n=20 or 2n=40. Triploid taxon with 2n=60 was found only in C. tricarinata (Ito and Mutsuura 1958). Other numbers reported in the genus are 2n=38, 42, 44 and 58 (cf. Tanaka and Kamemto

1972, 1974, Tanaka et al. 1981).

Pachystoma senile Reichb. f. A terrestrial species distributed in the plains and foothills of Northern India from Garhwal to Sikkim, Khasi hills and Manipur , Presently it was collected from the plains on sandy soil among Saccharum bushes. Its flowering period is March

April. 1984 CytologicalObservations on SomeWest HimalayanOrchids 589

Meiotic studies revealed 20 bivalents at diakinesis (Fig. 7). The bivalents were ring or rod-shaped. Secondary associations among some of them were seen in a few PMC's at M-I. Laggards were common at A-I and sometimes a fragment was also noticed. Bridges and laggards were seen even at T-II. Pollen grains form soft

Figs. 9-16: Coelogyne cristata . 9, somatic cell with 2n=40 at metaphase. C. stricta: 10, somatic cell with 2n=40 at metaphase . C. ovalis: 11, somatic cell with 2n=40 at metaphase. Otochilus POrrecta: 12, somatic cell with 2n=40 at metaphase. Pholidota articulata: 13, somatic cell with 2n=40 at metaphase . 14, PMC with n=19+1B at diakinesis. B-chromosome is at arrow. P. imbricata: 15 , somatic cell with 2n=40 at metaphase. Eria spicata: 16, somatic cell with 2n=40 at metaphase. 590 P. N. Mehra and S. K. Kashyap Cytologia 49

pollinia. Meiotic studies from EMC's confirmed n=20 as the gametic chromosome num

ber for the species but the bivalents here were distinctly smaller than those in the

PMC's (Fig. 8). Root-tip mitosis showed 2n=40 chromosomes. They were almost of the same

size with median or submedian primary constriction. The species is investigated for the first time. Since there is no earlier record for the genus, x=20 is possibly

its basic number.

Dressler and Dodson (1960) considered the subtribe Bletiinae to be primitive

amongst the Epidendreae on the basis of floral and vegetative characters. Large size of chromosomes, terrestrial habitat and presence of individual pollen grains in

Acanthephippium striatum and Anthogonium gracile (Mehra and Sehgal: unpublish

ed) further support its primitiveness. Bletiinae seems to be close to Thuniinae.

Subtribe: Coelogyninae Bentham Coelogyne Lindl.

It is a large genus comprising 200 mostly epiphytic or lithophytic species dis

tributed in West China, Indomalayan and Pacific regions (Airy Shaw 1973). Many species are of horticultural importance. Three species have been investigated.

C. cristate Lindl. was collected from Kumaon hills at an elevation of 1300m.

Forty small chromosomes were clearly seen at root-tip mitosis (Fig. 9). The chromo somes showed gradual size gradation and measured from 1.98-0.92ƒÊ. Total chro

mosome length is 56.15y. Karyotype consists of 28 m+6 sm+6 st chromosomes

(Fig. 18). The present count of 2n=40 confirms the previous reports for this species of n=20 by Mehra and Vij (1970), 2n=40 by Tanaka (1964) and Vij et al. (1976). Vij and Mehra (1976) discovered a tetraploid race with n=40 from the Eastern Him alayas.

C. stricta (Don.) Schltr. grows on trunks and branches of angiospermic trees in thick forests of Kumaon hills between 1400-1600m. Its somatic number from root tip mitosis was found to be 2n=40 (Fig. 10). The chromosomes here too showed size gradation and measured from 2.64-1.05ƒÊ. Total chromosome length is 61.98ƒÊ.

The somatic chromosome complement comprises 32 m+8 sm chromosomes.

The previous records for the species (under C. elata) are n=20 (Arora 1968, Mehra and Vij 1970, Vij and Mehra 1976) and 2n=44 (Roy and Sharma 1972 , Sharma and Sarkar 1971). A very irrelevant number n=12 was given by Arora

(1971) which seems erroneous. C. ovalis Lindl. grows as an epiphyte on branches of trees in the thick forests of Kumaon hills at an elevation of 1350m . Its flowering period is September

October. Twenty bivalents were observed at diakinesis . Ten of them - appeared to be somewhat bigger than the rest. Early disjunction of some of the smaller bivalents was also noticed. Meiotic course was normal . Pollen grains form hard pollinia.

Root tip mitosis revealed 2n=40 chromosomes (Fig . 11). Chromosome size ranges from 2.25-0.92ƒÊ. Total chromosome length is 63 .04ƒÊ. Karyotype con 1984 Cytological Observations on Some West Himalayan Orchids 591

Figs. 17-19. 17, karyogram of figure 6 (Calanthe tricarinata). 18, karyogram of figure 9 (Co

elogyne cristata). 19, karyogram of figure 13 (Pholidata articulata). All •~4530. 592 P. N. Mehra and S. K. Kashyap Cytologia 49

sists of 20 m+12 sm+8 st.

The present finding of n=20, 2n=40 falls in line with the reports by Mehra and Vij (1970) and Vij and Mehra (1976).

A perusal of literature reveals that the majority of the species of the genus in vestigated so far are based on x=20. However, 2n=38 has been recorded for C. corymbosa, C. nitida, and C. occulata (Mehra and Sehgal 1974, 1976, 1980) which therefore, are aneuploids. Polyploidy associated with gigantism was noticed in C. cristata (n=20, 40) by Vij and Mehra (1976) from the populations growing in Eastern

Himalayas. Darlington and Wylie (1955) suggested x=20 as the basic number for the genus.

Otochilus porrecta Lindl. This is the only species of the genus Otochilus met in the Kumaon hills as an epiphyte in thick and moist forests at about 1500m. Forty small chromosomes were noticed at metaphase in root-tip mitosis (Fig. 12). The chromosomes possessed median or submedian primary constriction. The only re port for this species is n=20 by Mehra and Vij (1970). To date all the chromosomally known species of the genus are found to be uni formly built on x=20. A discordant report of 2n=36 for O. alba by Chatterji (1966) needs confirmation.

Pholidota Lindl.

An epiphytic genus with 55 species, distributed in China and from India to

Australia and Polynesia (Airy Shaw 1973). Only 2 species are found in the Western Himalayas (Duthie 1906).

P. articulata Lindl. occurs abundantly in Kumaon hills as an epiphyte at an altitude of 1400 m. Its flowering period is in April. One of the two different popu lations studied showed the occurrence of B-chromosomes.

Root tip mitosis revealed 2n=40 chromosomes (Fig. 13). The chromosomes are small ranging in size from 1.72-0.66ƒÊ. Total chromosome length is 45.69ƒÊ. Karyotype is symmetrical with all median (m) chromosomes (Fig. 19). A few aneu somatic cells with 2n=38 were also seen.

Meiotic studies in the other population showed n=20+0-3B's at diakinesis

(Fig. 14). Bivalents were both ring and rod-shaped. The presence of B's did not have any appreciable effect on the morphology of plants.

The present findings (n=20, 2n=40) are in agreement with the previous reports

(Arora 1968, Mehra and Vij 1970, Vij et al. 1976, Vij and Mehra 1976). Mehra and Pal (1961), however, reported n=21 which may be an aneuploid.

P. imbricata Lindl. occurs in Kumaon hills at lower elevations of about 1000 m as an epiphyte on trunks and branches of trees. Diploid chromosome number from root tip mitosis was observed to be 2n=40 (Fig. 15). Chromosomes showed gradu al size gradation and measured 2.38-0.66,u. Total chromosome length is 48.21 ,u.

Karyotype comprises 22 m+8 sm+10 st chromosomes. The present count is in conformity with the earlier report 2n=40 (Pancho

1965a). Mehra and Vij (1970) reported n=20 for this species. Pholidota appears to be uniformly built on x=20 (cf. Tanaka and Kamemoto 1972, 1974). All the presently studied members of the subtribe Coelogyninae consistently showed n=20 chromosomes. The basic chromosome number suggested for all 1984 CytologicalObservations on Some West HimalayanOrchids 593 these genera is x=20. Chromosomes are small showing gradation in size. According to Dressler and Dodson (1960) Coelogyninae is closely related to Epidendrinae but its relationship with Thuniinae and Bletiinae is remote. Our observations on the disparity in the size of the chromosomes in the former two sub tribes compared to the latter ones support this conclusion (cf. Figs. 17-19).

Subtribe: Epidendrinae D. and Dod. Only the genus Eria with about 4 species is distributed in the Western Hima layas of which two species have presently been investigated. Eria confusa Hook. f. grows as an epiphyte in thick forests of Kumaon hills at 1100m. Its flowering period is in June. Haploid set of 18 chromosomes was observed at pollen mitosis. Chromosomes are small in size and comparable to those of Coelogyninae. E. spicata (D. Don) Handel-Mazz. also grows as an epiphyte in the thick forests of Kumaon hills at an elevation of about 1400m. Thirty eight small chromosomes were seen at root-tip mitosis (Fig. 16). Mostly the chromosomes appeared to pos sess median or submedian primary constriction. The present record 2n=38 confirms the earlier report of n=19 by Chardard (1963) but differs from the number n=18, 2n=36 given by Chatterji (1965). An other number n=20 is reported by Mehra and Vij (1970) and Mehra and Sehgal (1975). A perusal of literature reveals variable gametic chromosome numbers in the genus viz. n=18, 19, 20, 21, 22 and 33 (cf. Moore 1973, Tanaka and Kamemoto 1972, 1974). It is, therefore, difficult to decide about the correct original basic num ber of this polymorphic genus. The subtribe Epidendrinae is undoubtedly a close relative of Coelogyninae as stated earlier.

Concluding remarks

It is to be noted that the bivalents at male meiosis are larger than those at female meiosis in Pachystoma senile. Furthermore, the bivalents in general apparently give the appearance of the chromosomes being larger at meiosis than at somatic mitosis for the same species. Consequently to be precise, if comparison has to be made of the chromosome size in various subtribes it has to be done consistently at the mitotic level. Such a comparison leaves us in no doubt that the chromosomes in Thuniinae are larger than in Coelogyninae or Epidendrinae.

Summary This paper deals with the cytology of 14 species comprising 8 genera of Epiden dreae. The genus Pachystoma (P. senile: n=20) has been investigated for the first time. B-chromosomes are reported in Calanthe puberula (2n=40+1B) and Pholi dota articulata (n=20+0-3B). An aneuploid race of Eria confusa (n=18) is dis covered. The chromosomes in Pogoniinae, Thuniinae and Bletiinae are larger in 594 P. N. Mehra and S. K. Kashyap Cytologia 49 size compared to those of Coelogyninae and Epidendrinae which are relatively much smaller.

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