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 cor- relation 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 © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 231–238 231 232 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), © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 231–238 CYTOLOGY OF CHINESE ORCHIDEAE 233 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.