Cytological Studies on Some Representative Species of the Tribe Orchideae (Orchidaceae) from China

Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 20042004 1452 231238 Original Article

CYTOLOGY OF CHINESE ORCHIDEAE Y.-B. LUO

Botanical Journal of the Linnean Society, 2004, 145, 231–238. With 21 figures

Cytological studies on some representative species of the tribe Orchideae (Orchidaceae) from China

YI-BO LUO*

Laboratory of Systematic and Evolutionary Botany and Herbarium (PE), Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing 100093, China

Received July 2003; accepted for publication October 2003

Cytological studies were carried out on 14 taxa belonging to Amitostigma, Chusua, Galearis, Habenaria, Hemipilia, Hemipiliopsis, Herminium, Peristylus and Ponerochis, collected mostly from the south-eastern part of the Hengduan Mountain Region, south-west China. Cytological data on 11 of the taxa are reported for the ﬁrst time. The interphase nuclei were either of the simple chromocentre type or intermediate between simple and complex chromocentre types. The nuclear morphology of Hemipiliopsis at interphase supports the conclusion that it is related more closely to Chusua and Ponerochis than to Habenaria. At the whole tribe level, however, the results did not indicate a clear correlation between morphological features of the interphase nuclei and phylogeny. The somatic chromosome numbers were 2n = 42 in ten species and 2n = 32, 38, 40, 64 and 72 in four species. The chromosome counts of 2n = 32 and 64 in Habenaria aitchsonii are rare in the genus. It is proposed that the repeated change of chromosome number from x = 7 to x = 8 has played an important role in the evolution of the tribe Orchideae. This change has occurred mainly in the European subtribe Orchidinae, but also in the Asian subtribe Habenariinae. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 231–238.

ADDITIONAL KEYWORDS: Habenariinae – Orchidinae.

INTRODUCTION In China this tribe is represented by about 232 species (Lang, 1999). Some of the genera are endemic The tribe Orchideae (sensu Cribb, 2001) comprises 62 to China, or their species occur mostly in China. genera and approximately 1800 species distributed all These include Amitostigma, Diphylax, Hemipilia, over the world. Among the genera, the classification of Herminium, Neottianthe, Pecteilis, Smithorchis and Orchis has been a subject of considerable argument. Tsaiorchis. Until now, these Chinese species have Considering the feature of bursicles, the broad Lin- been considered largely in taxonomic and floristic naean concept of Orchis is supported by some authors accounts, but from a biosystematic point of view, cyto- (Tang, Wang & Lang, 1980; Lang, 1987, 1999); while logical evidence is also helpful to understand the phy- the more strict concept of Orchis, which is typified by logeny and evolution of Orchideae. There is a large O. militaris and comprises about 33 species distrib- amount of literature on the cytology of this tribe uted in Europe, temperate Asia and North Africa, is (D’Emerico, 2001), but the chromosome number of accepted by most students, based on a wide spectrum only one species of Chinese Orchideae has been of evidence (Vermeulen, 1947, 1972; Senghas, 1973; reported previously, Platanthera minor (Yang and Delforge, 1995; Bateman, Pridgeon & Chase, 1997; Zhu, 1984). This paper will report cytological observa- Pridgeon et al., 1997; Luo and Chen, 1999, 2000; Bate- tions in a further 14 species of the Chinese Orchideae, man et al., 2001, 2003; Cribb, 2001; Wood, 2001). In comprising nine genera. this study I adopted the strict concept of Orchis, and the generic limitations of Ponerochis, Chusua and Galearis followed Cribb (2001) and Wood (2001). MATERIAL AND METHODS Plant material was collected mainly from the Hengduan Mountain Region, south-west China *E-mail: [email protected] (Table 1). Voucher specimens are deposited in PE and

232 Y.-B. LUO

Table 1. Localities and chromosome numbers of the material studied

Interphase nucleus Species Locality Altitude Vouchers 2n morphology*

Hemipilia cruciata Finet Yunnan, Lijiang 2700 m Luo 55 42 Intermediate 2450 m Luo 93 42 2780 m Luo 87 42 2620 m Luo 90 42 2520 m Luo 91 42 Hemipilia ﬂabellata Bur. et Franch Yunnan, Zhongdian 2810 m Luo 65 42 Intermediate Yunnan, Heqin 2100 m Luo 39 42 Hemipilia calyophylla Par. & Rchb. f. Guangxi, Rongan 300 m – 42 Intermediate Amitostigma gracile (Bl.) Schltr. Hunan, Xinling 1300 m – 42 Intermediate Ponerorchis chusua (D. Don) Soó Yunnan, Lijiang 3400 m Luo 53 c. 98 Simple Yunnan, Zhongdian 2700 m Luo 66 42 3660 m Luo 73 63 Yunnan, Weixi 3300 m Luo 102 42 2630 m Luo 79 42 Chusua brevicalcarata (Finet) Yunnan, Zhongdian 3200 m Luo 161 42 Simple P. F. Hunt (9734) Galearis diantha (Schltr.) P. F. Hunt Yunnan, Zhongdian 3660 m Luo 74 42 Intermediate Hemipiliopsis purpureopunctata (K. Y. Xizang, Nyingchi 2550 m Luo 14 42 Simple Lang) Y. B. Luo and S. C. Chen Habenaria delavayi Finet Yunnan, Lijiang 2700 m Luo 56 42 Complex Habenaria mairei Schltr. Yunnan, Zhongdian 2540 m Luo 169 40 Complex (9742) Habenaria aitchisonii Rchb.f. Yunnan, Zhongdian 2550 m Luo 68 64 Complex Yunnan, Heqin 2300 m Luo 40 32 Peristylus coeloceras Finet Yunnan, Lijiang 2720 m Luo 52 42 Intermediate Herminium lanceum (Thunb.) Vujik Yunnan, Weixi 2630 m Luo 84 72 Complex Herminium monorchis (L.) R. Br. Yunnan, Zhongdian 3000 m Luo 71 38 Complex

*Classiﬁcation of Tanaka (1971).

K. Root tips were harvested and pretreated in 0.002 M counted in material of H. cruciata from five locali- 8-hydroxyquinoline for 5 h at room temperature ties, in H. flabellata from two localities and in before being fixed in modified Carnoy’s fluid (99% H. calyophylla from one locality (Figs 6–8). In this ethanol : chloroform : glacial acetic acid = 2 : 1 : 1). genus, only H. cordifolia has previously been observed They were macerated in a mixture of one part 45% cytologically, with Mehra and Bawa (1962, 1970) acetic acid and two parts 1 M hydrochloric acid at reporting 2n = 44 and Mehra (1983) reporting 2n = 42. 60 ∞C for about 20 s. Then they were stained and The interphase nuclei, mitotic prophase and squashed in 2% aceto-orcein or in carbol fuchsin. Clas- metaphase chromosomes of the three species were sification of interphase nuclei and mitotic prophase basically similar. The chromatin in the interphase chromosomes follows Tanaka (1971, 1987). nuclei was lightly stained and contained large, darkly stained chromocentres, which were round to rod shaped, numbering about the half of the chromosome RESULTS number (Fig. 3). This interphase nucleus is of a type Chromosome numbers and interphase nucleus types intermediate between the simplex and the complex are given in Table 1. Detailed descriptions are as chromocentre types of Tanaka (1971, 1987). In follows. prophase, early condensing segments were observed in the proximal region of the chromosomes (Fig. 4).

HEMIPILIA CRUCIATA FINET, H. CALYOPHYLLA PAR. ET RCHB. F. AND H. FLABELLATA BUR. ET FRANCH. AMITOSTIGMA GRACILE (BL.) SCHLTR. Chromosome numbers of these three species are Plants of this species from one locality in south- reported for the ﬁrst time. The number 2n = 42 was western Hunan, central China, had 2n = 42 (Fig. 9),

1 2 3 4

5 6 7 8 9

10 11 12

Figures 1–12. Figs 1–4. Morphology of interphase nucleus and prophase nucleus. Fig. 1. Habenaria delavayi, complex type of interphase nucleus. Fig. 2. Ponerorchis chusua, simple type of interphase nucleus. Fig. 3. Hemipilia calyophylla, interphase nucleus intermediate between the simplex and the complex types. Fig. 4. Hemipilia cruciata, early condensing segments of prophase nucleus. Figs 5–12. Metaphase chromosomes. Fig. 5. Hemipilia cruciata, 2n = 42. Fig. 6. Hemipilia calyophylla, 2n = 42. Fig. 7. Hemipilia ﬂabellata, 2n = 42. Fig. 8. Amitostigma gracile, 2n = 42. Fig. 9. Ponerorchis chusua, 2n = 42. Fig. 10. Ponerorchis chusua, 2n = 63. Fig. 11. Ponerorchis chusua, 2n = c. 98. Fig. 12. Chusua brevicalcarata, 2n = 42. Scale bar = 10 mm.

© 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 231–238 234 Y.-B. LUO agreeing with a previous report based on material The interphase nuclei are of the simple chromocen- from Japan (Yokota, 1987, 1990). tre type. At mitotic prophase, most chromosomes had The interphase nuclei and mitotic prophase chromo- early condensing segments at the proximal region of somes were similar to those of Hemipilia described one or both arms. above (Figs 3, 4). Yokota (1990) considered that the interphase nuclei of this species were more similar to the complex chromocentre type than to the simple HABENARIA AITCHISONII RCHB. F., H. DELAVAYI chromocentre one. However, he still referred to it as FINET AND H. MAIREI SCHLTR. the intermediate type between the simple and the Chromosome numbers of H. delavayi and H. mairei complex chromocentre types. are reported here for the first time, with 2n = 42 counted in H. delavayi from one locality and 2n = 40 in H. mairei from one locality (Figs 16, 17). Mehra & Vij PONERORCHIS CHUSUA (D. DON) SOÓ (1970, 1972; see Mehra, 1983) twice reported n = 21 The chromosome number of this species is reported for H. aitchisoni. My counts of 2n = 32 and 64 in this here for the first time. The chromosome numbers species from two localities differ from the previous showed a great variation in material from five locali- reports (Figs 18, 19). ties. Plants from three localities of relatively lower ele- The interphase nuclei of Habenaria aitchisoni, H. delavayi and H. mairei were similar to each other, vations (Table 1) had 2n = 42 (Fig. 10), whereas those from two localities at relatively higher elevations had and of the complex chromocentre type of Tanaka (1971) (Fig. 1). At mitotic prophase in all the three spe- 2n = 63, a triploid, and 2n = c. 98, an aneuploid (Figs 11, 12). cies, most chromosomes had early condensing seg- All of them showed the same type of interphase ments at the proximal region of one or both arms. nucleus and mitotic prophase, which is referable to the simple chromocentre type of Tanaka (1971) (Fig. 2). PERISTYLUS COELOCERAS FINET The number 2n = 42 was counted in plants from one locality of from Lijiang, north-west Yunnan (Fig. 20), CHUSUA BREVICALCARATA (FINET) P. F. HUNT and this is the first report of its chromosome number. Material from one locality had 2n = 42, the first report Chromosome numbers of seven species in this genus for this species (Fig. 13). have been reported previously, with reports of The interphase nuclei and mitotic prophase were of 2n = 28, 36, 38, 42, 44, 46 and 88 (Tanaka and Kame- the simple chromocentre type of Tanaka (1971). moto, 1984; Yokota, 1987; Sheikh, Kondo & Tanaka, 1993). The interphase nuclei were similar to that of Hemi- GALEARIS DIANTHA (SCHLTR.) P. F. HUNT pilia species described above. At mitotic prophase, most chromosomes had early condensing segments at The chromosome number of this species, reported here the proximal region of one or both arms. for the first time, is 2n = 42 based on material from one locality from Zhongdian, north-west Yunnan (Fig. 14). Two other species of this genus, G. cyclochila HERMINIUM LANCEUM (THUNB.) VUJIK AND and G. spectabilis, have been reported to have 2n = 42 H. MONORCHIS (L.) R. BR. (Humphrey, 1932a, b; Löve & Simon, 1968; Yokota, The chromosomes H. lanceum were counted as 1987, 1990). See Tanaka & Kamemoto (1984). 2n = 72, based on material from Weixi, north-western The interphase nuclei are intermediate between the Yunnan (Fig. 21). This number is different from previ- simple and the complex chromocentre types of Tanaka ous reports, which vary from n = 19, 20, 38 or 2n = 38, (1971). At mitotic prophase, most of the chromosomes 40, 76 (Yokota, 1990). Within this species, x = 19 has had early condensed segments at the proximal region been reported eight times and x = 20 only once of one or both arms. (Yokota, 1990). Overall, chromosome numbers of 11 species of Hemininium have been reported (D’Emer- ico, 2001), with only one species, H. gramineum, hav- HEMIPILIOPSIS PURPUREOPUNCTATA (K. Y. LANG) ing 2n = 36, x = 18 (Mehra & Kashyap, 1978). In Y. B. LUO ET S. C. CHEN H. monorchis, 2n = 38 was counted in material from The chromosome number of this species is reported one locality at Zhongdian, north-western Yunnan here for the first time. It was counted as 2n = 42, based (Fig. 21). In the Asian area, this species has been on material from one locality from north-west Xizhang reported twice to have n = 20 and once n = 19 (Mehra, (Tibet) (Fig. 15). 1983). In the European region, it was reported to have

13 14 15

16 17

18 19 20 21

Figures 13–21. Metaphase chromosomes. Fig. 13. Galearis diantha, 2n = 42. Fig. 14. Hemipiliopsis purpureopunctata, 2n = 42. Fig. 15. Habenaria delavayi, 2n = 42. Fig. 16. Habenaria mairei, 2n = 40. Fig. 17. Habenaria aitchisonii, 2n = 32. Fig. 18. Habenaria aitchisonii, 2n = 64. Fig. 19. Peristylus coeloceras, 2n = 42. Fig. 20. Herminium lanceum, 2n = 72. Fig. 21. Herminium monorchis, 2n = 38. Scale bar = 10 mm.

2n = 40 and 42 (Delforge, 1995). This study conﬁrms gested that within the subtribe Orchidinae, polyploidy the previous count of n = 19. is conﬁned largely to the Dactylorhiza–Coeloglossum Both species studied here show the same type of and Gymnadenia–Nigritella clades that are distrib- interphase nuclei, which are of the complex chro- uted widely in Europe. Among the 43 taxa of the sub- mocentre type of Tanaka (1971). tribe Orchidinae observed cytologically in this study and previous works (Yokota, 1990), which account for nearly one-third of the total taxa in this subtribe from DISCUSSION Asia, only two taxa, Platanthera hyperborea and Pon- Tanaka (1971, 1987) suggested that in Orchidaceae erorchis chusua, appear to be polyploid. This fact the morphological features of chromosome at inter- seems to support the conclusion reached by Pridgeon phase are closely correlated with the phylogenetic et al. (1997). tree. Yokota (1990) also considered that karyomorpho- D’Emerico (2001) suggested that there are four logical studies are useful for understanding the inter- basic chromosome numbers in Orchideae (sensu Cribb, and intrageneric relationships of the tribe Orchideae, 2001), x = 16, 18, 20 and 21. Up to the present, there basing his comments on chromosomal studies on 48 were no reports of x = 16 or 18 in the Asian members of taxa from Asia. In my study, three types of interphase the subtribe Orchidinae; by contrast, these two num- nucleus were found in 14 species of nine genera, but bers have been reported frequently in the European the results could not clearly elucidate the species’ rela- subtribe Orchidinae, such as in Ophrys, Serapias and tionships. For example, three species of Hemipilia Anacamptis s.l. (Bateman et al., 1997; Pridgeon et al., shared similar interphase nucleus features with Gale- 1997; D’Emerico, 2001). The chromosome number of aris diantha, but the genus Hemipilia is not consid- the genera that are endemic or nearly endemic to Asia, ered to be phylogenetically related to the genus such as Ponerorchis, Chusua, Galearis and Hemipili- Galearis on the basis of molecular evidence (Bateman opsis, is generally x = 21, based on available data. The et al., 2001, 2003). By contrast, Hemipiliopsis pur- numbers x = 20 and 21, however, were often found in pureopunctata is a recently raised genus based on the genera distributed both in Asia and in Europe, Habenaria purpureopunctata (Luo & Chen, 2003). It is such as Dactylorhiza, Orchis (s.s.), Gymnadenia and morphologically and phylogenetically different from Coeloglossum. Therefore, it seems that the chromo- other species of Habenaria (Bateman et al., 2001, some numbers x = 16 and 18 may represent derived 2003; Luo & Chen, 2003). It has 2n = 42, which is the states, whereas x = 21 in the Asian region is a primi- most common chromosome number in the tribe tive basic number in the subtribe Orchidinae. Orchideae. The morphology of its interphase nucleus, The remaining six species studied here belong to the the simple type, is similar to that of Chusua brevical- subtribe Habenariinae, comprising about 930 species carata and Ponerorchis chusua rather than Habenaria in 23 genera, of which the largest genus is Habenaria aitchisonii, H. delavayi and H. mairei. This result with about 600 species (Dressler, 1993). The chromo- seems to support the close phylogenetic relationships some number of the other genera in the subtribe based on molecular evidence (Bateman et al., 2001, shows less variation than that in Habenaria (D’Emer- 2003). Perhaps a thorough cytological study on the ico, 2001). In Habenaria, the somatic chromosome tribe Orchideae would be helpful in clarifying their number varies from 2n = 28 to 168, but 84% of the 43 karyomorphological evolution. species with available chromosomal data have 2n = 42 Eight of the 14 species studied in this paper belong (D’Emerico, 2001), and such species are distributed to the subtribe Orchidinae, which is distributed in North America (Felix and Guerra, 1998), India mainly in the North Temperate region (Cribb, 2001), (Mehra, 1983) and Japan (Yokota, 1990). In this work, and all have 2n = 42. Only at two localities of Poner- the chromosome number 2n = 42 is found in one of orchis chusua were some polyploid individuals with the three species studied. Considering that there are 2n = 63 and c. 98 found. Whether those two popula- nearly 70 species of Habenaria in China, the species tions could be regarded as entirely polyploid, however, cytologically studied in this region are obviously too still needs further study, especially in the case of the few to allow any conclusion to be reached. triploid, as triploids cannot produce stable, sexually Two chromosome numbers, 2n = 32 and 64, were reproducing populations because of their meiotic found in Habenaria aitchisonii. The number 2n = 32 irregularity and subsequent sterility. Yokota (1990) has been found only in one other species of Habenaria, studied the chromosomes of 48 taxa of the tribe H. dentata (Sw.) Schltr. (Yokota, 1987, 1990). The Orchideae (sensu Dressler, 1993) from Japan, of which interphase nuclei of both species are basically similar, 34 are in subtribe Orchidinae. The number 2n = 42 the complex type of Tanaka (1971, 1987). The chromo- was counted in most of the taxa, except Platanthera some number 2n = 32 has also been reported in the hyperborea (2n = 84), Gymnadenia conopsea and Coel- genus Pecteilis, a small satellite genus of Habenaria oglossum viride (2n = 40). Pridgeon et al. (1997) sug- with only about eight species (Cribb, 2001), P. radiata

© 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 231–238 CYTOLOGY OF CHINESE ORCHIDEAE 237 as 2n = 32, 48 (Miduno, 1939, 1940; Yokota, 1987). Bateman RM, Hollingsworth PM, Preston J, Luo YB, Similarly to Habenaria, chromosome numbers of this Pridgeon AM, Chase MW. 2003. Molecular phylogenetics small genus also showed variation, because 2n = 42 of the Orchidinae and selected Habenariinae (Orchidaceae). was counted in P. latilabris (Lind.) Mitra and in Botanical Journal of the Linnean Society 142: 1–40. P. sagarikii Seidenfaden (Arora, 1971; Yokota, 1987). Bateman RM, Pridgeon AM, Chase MW. 1997. Phylogenet- Considering that the original basic chromosome num- ics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based ber of the genus Habenaria and its allies might be on nuclear ITS sequences. 2. Infrageneric relationships and reclassification to achieve monophyly of Orchis sensu stricto. x = 7 (Mehra and Bawa, 1970; Yokota, 1990; Felix and Lindleyana 12: 113–141. Guerra, 1998), then x = 8 might be regarded as an Cribb PJ. 2001. Distribution of Orchideae, Ponerochis. In: important variation of the chromosome number in Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN, eds. Habenaria. Yokota (1987) even suggested the chromo- Genera Orchidacearum, Vol. 2, part 1: Orchidioideae. New some number 2n 32 should be regarded as a phylo- = York: Oxford University Press, 215; 354. genetic relic. Another interesting point is that the D’Emerico S. 2001. Cytogenetics. In: Pridgeon AM, Cribb PJ, species in the subtribe Habenariinae with chromo- Chase MW, Rasmussen FN, eds. Genera Orchidacearum, some numbers 2n = 32 are distributed in Asia. Vol. 2, part 1: Orchidioideae. New York: Oxford University The repeated occurrence of chromosomal number Press, 216–224. change from x = 7 to x = 8 has thus played an impor- Delforge P. 1995. Orchids of Britain and Europe. London: tant role in the evolution of the tribe Orchideae. In the Harper Collins. subtribe Orchidinae this change has occurred mainly Dressler RL. 1993. Phylogeny and classification of the orchid in Europe, but in the subtribe Habenariinae it has family. Portland, OR: Timber Press. occurred in Asia. D’Emerico (2001) stated that there is Felix LP, Guerra M. 1998. Cytogenetic studies on species of some correlation between the degree of karyotype Habenaria (Orchidoideae: Orchidaceae) occurring in the asymmetry and heterochromatin among some Euro- Northeast of Brazil. Lindleyana 13: 224–230. pean groups of Orchidinae with chromosome numbers Humphrey LM. 1932a. Somatic chromosomes in certain Min- x = 16 and 18, such as Anacamptis, Serapias, Ophrys, nesota orchids. American Naturalist 66: 471–474. Barlia and Himantoglossum. The present study and Humphrey LM. 1932b. The somatic chromosomes of eight previous work, however, have not shown similar rela- species of Orchidaceae. Proceedings of Iowa Academy of tionship among the Asian groups of Habenariinae Science 39: 137. with chromosome numbers 2n = 32 or 64. Perhaps fur- Lang Kai-yung. 1987. Orchis L., Hemipilia Lindl., Amitostigma ther investigation covering more species might reveal Schltr., Neottianthe Schltr., Gymnadenia R. Br. & Habenaria the evolutionary trends in Habenariinae. Meanwhile, Willd. In: Wu C-y, ed. Flora Xizangica, Vol. 5. Bejing: Science Press, 677–688, 710–720, 725–736 (in Chinese). the small size of the chromosomes in Habenariinae Lang Kai-yung. 1999. Flora Reipublicae Popularis Sinicae has also hindered our understanding of their karyo- Tomus 17. Beijing: Science Press (in Chinese). type structure, and thus further work using molecular Löve A, Simon W. 1968. Cytotaxonomical notes on some cytogenetics techniques is needed in this study. American orchids. Southwest Naturalist 13: 335–342. Luo YB, Chen SC. 1999. Observations on putative pollinators ACKNOWLEDGEMENTS of Hemipilia flabellata Bur. et Franch. (Orchidaceae) in north-west Yunnan Province, China. Botanical Journal of the The completion of this study has been possible Linnean Society 131: 45–64. through grants from the Chinese Academy of Sciences Luo YB, Chen SC. 2000. The floral morphology and ontog- (Key Innovation Plan KSCX2-SW-121) and the Zurich eny of some Chinese representatives of orchid subtribe Foundation for Orchid Conservation. I thank Dr Orchidinae. Botanical Journal of the Linnean Society 134: Phillip Cribb, Royal Botanic Gardens, Kew, for his 529–548. constant encouragement and supervision, and Dr Luo YB, Chen SC. 2003. Hemipiliopsis, a new genus of Orchi- Peter Brandham, Royal Botanic Gardens, Kew, for his daceae. Novon 13: 450–453. suggestions. Mehra PN. 1983. Cytology of orchids of Khasi and Jaintia Hills. New Delhi: Rajbandhu Industrial Company. REFERENCES Mehra PN, Bawa KS. 1962. Chromosome studies in Orchi- daceae. In: Abstracts of the 49th Session, Indian Science Con- Arora CM. 1971. In IOPB Chromosome number reports, 34. gress, Calcutta, 326–327. Taxon 20: 785–797. Mehra PN, Bawa KS. 1970. Cytological observations on some Bateman RM, Hollingsworth PM, Preston J, Luo YB, Northwest Himalayan orchids. Caryologia 23: 273–282. Pridgeon AM, Chase MW. 2001. Phylogenetics. In: Prid- Mehra PN, Kashyap SK. 1978. In: Löve A, ed. IOPB chromo- geon AM, Cribb PJ, Chase MW, Rasmussen FN, eds. Genera some number reports LX. Taxon 27: 226. Orchidacearum, Vol. 2 part 1: Orchidioideae. New York: Mehra PN, Vij SP. 1970. In: Löve, A, ed. IOPB chromosome Oxford University Press, 224–232. number reports XXV. Taxon 19: 106–111.

Mehra PN, Vij SP. 1972. Cytological studies in the East Tanaka R, Kamemoto H. 1984. Chromosomes in orchids: Himalayan Orchidaceae-2: Orchideae. Caryologia 25: 335– counting and numbers. In: Arditii J, ed. Orchid biology: 351. reviews and perspectives III. Ithaca, NY: Cornell University Miduno T. 1939. Chromosomenstudien on Orchidazeen, II. Press, 325–328. Somatischen Chromosomenzahlen einiger Orchideen. Cyto- Tang Tsin, Wang Fa-tsuan, Lang Kai-yung. 1980. Materiae logia 9: 447–451. ad genus Ochidem L. sinicam. Acta Phytotaxonomica Sinica Miduno T. 1940. Chromosomenstudien on Orchidazeen, IV. 18: 408–419. Somatischen Chromosomenzahlen einiger Arten und Bas- Vermeulen P. 1947. Studies on dactylorchids. Utrecht, Neth- tarde bei Orchideen. Cytologia 11: 179–185. erlands: Schotanus & Jens. Pridgeon AM, Bateman RM, Cox AV, Hapeman JR, Vermeulen P. 1972. Übersicht zur Systematik und Taxon- Chase MW. 1997. Phylogenetics of subtribe Orchidinae omie der Gattung Orchis s. str. Jahresberichte des naturwiss- (Orchidoideae, Orchidaceae) based on Nuclear ITS chaftlichen Vereins in Wuppertal 25: 22–36. sequences. 1. Intergeneric relationships and polyphyly of Wood J. 2001. Glearis, Orchis. In: Pridgeon AM, Cribb PJ, Orchis sensu lato. Lindleyana 12: 89–109. Chase MW, Rasmussen FN, eds. Genera Orchidacearum, Senghas K. 1973. Unterfamilies: Orchidoideae. In: Brieger Vol. 2, part 1: Orchidioideae. New York: Oxford University FG, Maatsch R, Senghas K, eds. Die Orchideen. Schlechter R. Press, 290–292, 333–339. Berlin: Paul Parey Verlag, 199–284. Yang DQ, Zhu XF. 1984. The chromosome numbers of 12 Sheikh SA, Kondo K, Tanaka R. 1993. A chromosome study species in Orchidaceae from China. Acta Phytotaxonomica of eight orchid species in Bangladesh. La Kromosomo II Sinica 22: 252–255. 71–72: 2456–2464. Yokota M. 1987. Karyotypes and phylogeny in Orchidinae and Tanaka R. 1971. Types of interphase nuclei of Orchidaceae. allied subtribes. In: Saito K, Tanaka R, eds. Proceedings of Botanical Magazine Tokyo 84: 118–122. the 12th World Orchid Conference. Tokyo: 12th World Orchid Tanaka R. 1987. The karyotype theory and wide crossing as Conference Organizing Committee, 70–79. an example in Orchidaceae. In: Hong DY, ed. Plant chromo- Yokota M. 1990. Karyomorphological studies on Habenaria, some research 1987. Hiroshima: The Organizing Committee Orchidaceae and allied genera from Japan. Journal of of the Sino-Japanese Symposium on Plant Chromosomes, Science of the Hiroshima University Series. B, Division 2 1–15. (Botany) 23: 53–161.