Bot. Bull. Acad. Sin. (2003) 44: 345-351

Yukawa et al. — Reappraisal of Kitigorchis

Reappraisal of Kitigorchis (Orchidaceae)

Tomohisa Yukawa1,*, Shih-Wen Chung2, Yibo Luo3, Ching-I Peng4,*, Arata Momohara5, and Hiroaki Setoguchi6

1Tsukuba Botanical Garden, National Science Museum, Amakubo, Tsukuba, 305-0005, Japan

2Division of Forest Biology, Taiwan Forestry Research Institute, Taipei, Taiwan 100

3Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China

4Institute of Botany, Academia Sinica, Taipei, Taiwan 115

5Faculty of Horticulture, Chiba University, Matsudo, 271-8510, Japan

6Faculty of Integrated Human Studies, Kyoto University, Kyoto, 606-8501, Japan

(Received April 21, 2003; Accepted September 10, 2003)

Abstract. Kitigorchis, a monotypic genus supposed to be endemic in the central part of Japan, was recently found in the Central Mountain Range of Taiwan. Further investigation of herbarium specimens and descriptions revealed that Kitigorchis itoana is conspecific with Oreorchis indica, previously recorded from the western Himalaya to southwestern China. This pattern of geographic distribution represents another example of disjunct distribution between Japan, Taiwan and the Himalayan region within the Eastern Asiatic Kingdom. Phylogenetic analyses using DNA sequence data from the internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA, matK, trnT-L intergenic spacer, trnL intron, trnL-F intergenic spacer, and rpL 16 intron showed that the monotypic Kitigorchis makes Oreorchis paraphyletic.

Keywords: Disjunct distribution; Japan; Kitigorchis itoana; Orchidaceae; Oreorchis indica; Phylogeny; Taiwan; Taxonomy.


Maekawa (1971) established the orchid genus Kitigorchis on the basis of Kitigorchis itoana F. Maek. (Figure 1) together with Oreorchis foliosa (Lindl.) Lindl., Oreorchis erythrochrysa Hand.-Mazz., and Tainia shimadai Hayata. He separated Kitigorchis from Oreorchis by (1) the clustered, multi-branched rhizomes, (2) the distinct mentum, (3) the poorly developed calli on the lip, (4) the rigid leaf blade, and (5) the prominent nerves on the leaf. Subsequently, Hashimoto and Kanda (1981), Satomi (1982), Masamune (1984), Hashimoto (1987), Hashimoto et al. (1991), Imai (1997), Inoue and Ikegami (1997), and Environmental Agency of Japan (2000) followed Maekawa's treatment.

In their revisionary work of Oreorchis Lindl., Pearce and Cribb (1997) discussed the status of Kitigorchis and concluded that Kitigorchis is monotypic, comprising only K. itoana. They remarked that the clustered, multi-branched rhizomes of K. itoana are quite different from the subterranean organs of Oreorchis, but they did not recognize the other features used to separate these genera by Maekawa

(1971) as significant. They thus rejected Maekawa's transfer to Kitigorchis of the three taxa that do not form multi-branched rhizomes. They speculated that Kitigorchis is a genus intermediate between Corallorhiza R. Br. and Oreorchis. Since Maekawa's (1971) description of K. itoana was devoid of Latin diagnosis, it was not validly published. Throughout this paper, we provisionally use this invalid name because the development of the subject can be recognized correctly by use of this name and K. itoana has been used widely at least among literature in Japan.

In 2000 S.W. Chung and his associates found a terrestrial orchid in Taiwan that T. Yukawa confirmed to be Kitigorchis itoana (Figure 2). Previously, K. itoana was thought to be endemic in coniferous forests in a very narrow range in the central part of Japan on Mt. Yatsugatake, Mt. Komagatake, Mt. Karakitake, and Mt. Fujisan. This peculiar disjunct distribution led us to reexamine Kitigorchis.

Based on the results of a global phylogenetic analysis of the tribe Calypsoeae Dressler using matK, the maturase-encoding gene located in an intron of the plastid gene trnK (Yukawa, unpublished), Kitigorchis forms a clade with Oreorchis and Corallorhiza. Cremastra Lindl. is the sister group to the three genera and can be used as the outgroup for further analyses. As mentioned above, the generic status of Kitigorchis was based primarily on the

*Corresponding authors: Tomohisa Yukawa, Fax: +81-29-853-8998; E-mail:; Ching-I Peng, Fax: +886-2-2789-1623; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

single character, the clustered, multi-branched rhizomes. This character, however, may represent a plesiomorphic character in tribe Calypsoeae because Corallorhiza and Cremastra also have it. On the other hand, we did not find any morphological synapomorphies to unite Oreorchis

and Corallorhiza. It was apparent that phylogenetic analyses using macromolecular characters would be necessary to determine the generic status of Kitigorchis. In this paper we report on both morphological and macromolecular characters of K. itoana and allied taxa to clarify their taxonomic status and the phylogenetic affinity of the species.

Materials and Methods

Materials collected for morphological and macromolecular studies are summarized in Table 1. Herbarium specimens studied are indicated in the taxonomic treatment. Fresh materials were preserved in 60% ethanol for morphological observations.

The molecular experimental methods are described in Yukawa et al. (1993, 1996). The internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA (including 5.8S ribosomal DNA and parts of 18S and 26S ribosomal DNA), matK of the plastid (including parts of trnK introns), trnT-L intergenic spacer of the plastid, trnL intron of the plastid, trnL-F intergenic spacer of the plastid, and rpL 16 intron of the plastid were amplified via the polymerase chain reaction (PCR) from a total DNA extract. We used the primers described in Douzery et al. (1999) for ITS; Yukawa et al. (1999) for matK; Taberlet et al. (1991) for trnT-L intergenic spacer, trnL intron, and trnL-F intergenic spacer; and Jordan et al. (1996) and Inoue and Yukawa (2002) for rpL16 intron. DNA sequences were aligned manually, and gaps were treated as missing characters. The aligned data file is available from the first author upon request.

We initially conducted molecular phylogenetic analyses using two data sets. One was derived from ITS which represents the nuclear DNA sequences; the other comprising the five plastid DNA regions as mentioned above. Furthermore, the two data sets were combined to conduct analyses with more phylogenetic signals. Parsimony and distance analyses were conducted with PAUP* Version 4.0b10 (Swofford, 2002). The branch and bound search option was used to perform Fitch parsimony analyses (Fitch, 1971). Branch lengths for trees were

Figure 1. Flowering plant of Kitigorchis itoana F. Maek. (identified as Oreorchis indica (Lindl.) Hook. f. in this study) from Mt. Yatsugatake, Honshu, Japan. Photograph taken by Hiroshi Nakayama.

Yukawa et al. — Reappraisal of Kitigorchis

Figure 2. Kitigorchis itoana F. Maek. (identified as Oreorchis indica (Lindl.) Hook. f. in this study). A, Habit; B, Flower, front view; C, Column and labellum, side view; D, Dorsal sepal; E, Petal; F, Lateral sepal; G, Labellum; H, Column and column foot. Drawn from Chung 3161 collected from Mt. Hohuanshan, Taiwan.

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

calculated using the DELTRAN optimization (Swofford and Maddison, 1987). Distance trees were obtained using the neighbor-joining method (Saitou and Nei, 1987) with a Kimura two-parameter correction (Kimura, 1980). To assess the relative robustness for branches, the bootstrap method (Felsenstein, 1985) was used with 10,000 replicates.

Results and Discussion


Comparison of Japanese and Taiwanese plants of K. itoana did not show significant differences except for larger vegetative parts (leaves: to 19 cm long × 2.7 cm wide in Japanese material, to 30 cm long × 2.5 cm wide in Taiwanese material; inflorescence: to 30 cm in Japanese material, to 50 cm long in Taiwanese material) and more distinct lateral lobes of the labellum in plants from Taiwan (Figure 2). We suspected that the discovery of this significant, disjunct population might indicate a wider distribution of the species. We therefore examined specimens and descriptions of other genera in tribe Calypsoeae and found that Oreorchis indica (Lindl.) Hook. f., a species reported from the western Himalaya to southwestern China, is conspecific with K. itoana. Descriptions and illustrations of O. indica [owing to misidentification, illustrated as Oreorchis foliosa (Lindl.) Lindl. in King and Pantling (1898), Benerji and Pradhan (1984), and Deva and Naitani (1986); see Pearce and Cribb (1997)] coincide with K. itoana from Japan and Taiwan. Consequently, the distribution pattern of K. itoana represents another example of disjunct distribution between Japan, Taiwan and the Himalayan region within the Eastern Asiatic Kingdom (Wu and Wu, 1998).

The nomenclatural confusion between O. indica and O. foliosa was described in detail by Pearce and Cribb (1997) who treated the two taxa as varieties of a single species, O. foliosa var. foliosa and O. foliosa var. indica (Lindl.) N. Pearce and P. J. Cribb. The two taxa are easily distinguished by the presence or absence of a mentum and the raised lamellae between the lateral lobes of the labellum. Since these variations are not continuous and reflect different reproductive strategies, we consider the two taxa to be different species.

Tainia gokanzanensis Masamune, another species similar to K. itoana, is incompletely known (Turner, 1992) because the type specimen could not be located. Interestingly, the type was collected from Mt. Hohuanshan, the same mountain where K. itoana was found in Taiwan. When we examined the original description of T. gokanzanensis meticulously, we concluded that T. gokanzanensis from Taiwan is conspecific with K. itoana.

Reinvestigation of morphological characters of K. itoana supported Pearce and Cribb's (1997) view in which they recognized the clustered, multi-branched rhizomes as the sole diagnostic character of the genus. We also did not find synapomorphic characters for K. itoana, Oreorchis foliosa, Oreorchis erythrochrysa, and Tainia shimadai to validate Maekawa's (1971) original circumscription of Kitigorchis.

DNA Analyses

Initially, we determined the genetic divergence between the Japanese and Taiwanese plants of K. itoana using ITS sequences. Since the two sequences were identical, the Japanese material was used for phylogenetic analyses.

The results of the phylogenetic analyses derived from the nuclear and plastid data sets were consistent (results not shown). In such cases, analyses of combined data sets provide more resolution and internal support for relationships than do the individual data sets (e. g. Olmstead and Sweere, 1995). A combined data set was therefore used for further analyses.

The parsimony analysis using the combined data set resulted in a single most parsimonious (MP) tree of 483 steps (Figure 3), which showed a sister group relationship between K. itoana and O. erythrochrysea (100% bootstrap support). This clade further formed a clade with Corallorhiza and the remaining species of Oreorchis, albeit with moderate bootstrap support (68%). The tree had a consistency index (CI) of 0.94 (0.86 excluding uninformative characters) and a retention index (RI) of 0.89. The

Figure 3. A single most-parsimonious Fitch tree based upon ITS, matK, trnT-L intergenic spacer, trnL intron, trnL-F intergenic spacer, and rpL 16 intron sequences: length=483, consistency index=0.94 (0.86 excluding uninformative characters), retention index of 0.89. Numbers above internodes correspond to branch length (DELTRAN optimisation). Numbers below internodes indicate bootstrap values from 10,000 replicates of Fitch parsimony analysis.

Yukawa et al. — Reappraisal of Kitigorchis

neighbor-joining (NJ) topology (Figure 4) was identical for the relationship between K. itoana and O. erythrochrysea (100% bootstrap support). In the NJ tree, this clade further made a clade with the rest of the species of Oreorchis (52% bootstrap support). Sixteen percent of the MP bootstrap replications showed this relationship.

Both the MP and NJ results definitely showed a sister group relationship between K. itoana and O. erythrochrysea (100% bootstrap support in both analyses). The relationships between K. itoana-O. erythrochrysea clade and the remaining species of Oreorchis and Corallorhiza are not clear. Scattered sampling in Oreorchis and Corallorhiza and long branches in Corallorhiza probably resulted in the low resolution. Ambiguous relationships among the 3 clades, namely, K. itoana-O. erythrochrysea clade, O. patens-O. fargesii clade, and C. trifida-C. wisteriana clade, did not provide a conclusive taxonomic arrangement of these genera. At the least, the current circumscription of Kitigorchis makes Oreorchis paraphyletic. If O. erythrochrysea is transferred to Kitigorchis, all three genera, Kitigorchis, Oreorchis, and Corallorhiza, exhibit monophyly. We did not, however, find any synapomorphic morphological characters found only in K. itoana and O. erythrochrysea. Another option is to unite the three genera into the single genus Corallorhiza, the earliest name among the three genera. Inclusion of more taxa from both Oreorchis and Corallorhiza in further phylogenetic analyses is desirable to determine the taxonomy of this group. Our provisional conclusion, based on our data, is to treat K. itoana as conspecific with Oreorchis indica, since doing otherwise would make Oreorchis paraphyletic.

Taxonomic Treatment

Oreorchis indica (Lindl.) Hook. f., Fl. Brit. Ind. 5: 709. 1890. Corallorhiza indica Lindl., J. Linn. Soc. Bot. 3: 26. 1859. Oreorchis foliosa (Lindl.) Lindl. var. indica (Lindl.) N. Pearce & P. J. Cribb, Edinburgh J. Bot. 54: 307. 1997.—TYPE: INDIA. Western Himalaya, Simla, on Hattu, 5 August 1847, T. Thomson 1724 (holotype: K-LINDL).

Tainia gokanzanensis Masam. in Humbert, Notul. Syst. (Paris) 6: 38. 1937, syn. nov.—TYPE: TAIWAN. Taityusyu, Mt. Gôkanzan (Hohuanshan), alt. ca. 3,300m, G. Masamune s. n. (holotype: TAI, not located).

Kitigorchis itoana F. Maek., The Wild Orchids of Japan in Colour. 300. 1971, nom. nud.

Habitat and Ecology. Terrestrial in coniferous forest and open meadows. Elevation 1,950-4,500 m. Flowering observed from June to August.

Distribution. Himalayan India, Nepal, Bhutan, China (Xizang, Yunnan, and Sichuan), Taiwan, Japan (central part of Honshu).

Specimens examined. JAPAN: Honshu, Mt. Yatsugatake, alt. 1,950 m, 24 Jun 2001, T. Yukawa 01-1 (TNS); Honshu, Mt. Fujisan, 2,000 m, 19 Jul 1984, F. Konta 15375 (TNS). TAIWAN: Hualien Hsien, Hsiulin Hsiang,

Figure 4. Neighbor joining (NJ) tree based upon ITS, matK, trnT-L intergenic spacer, trnL intron, trnL-F intergenic spacer, and rpL 16 intron sequences. Numbers below internodes indicate bootstrap values from 10,000 replicates of NJ analysis

Mt. Hohuanshan, behind High Altitude Experimental Station, Taiwan Endemic Species Research Institute, Abies forest, along a dry brook, ca. 3,000 m, N 24° 29' 42" E 121° 16' 37", 8 Jun 2000, flowering, S. W. Chung 3161 (TAIF); same loc., 1 Dec 2000, sterile, C. I. Peng 18199 (HAST). CHINA: Tibet, 3,400 m, 10 Jun 1956, Unknown collector 308 (PE). BHUTAN: Chamsa-Yabu Thang, 3,200-3,700 m, 15 May 1967, H. Kanai et al. s. n. (TI): Nala-Tzatogang, 3,200-3,400 m, 26 May 1967, H. Kanai et al. s. n. (TI).

Acknowledgement. The authors thank Alexandra Berkutenko, Barbara Carlsward, Kouichi Kita, Yuji Kurashige, Hiroshi Nakayama, Norio Tanaka, and Takahiro Yagame for collecting the plant material studied; Tomoko Fujimoto and Hideaki Shimizu for technical assistance; Hiroshi Nakayama for taking the photograph (Figure 1) of Oreorchis indica in its natural habitat in Japan; Chien-Yu Ke for assistance with the line drawing of O. indica (Figure 2); Kuo-Hsiang Lai and Yau-De Tzeng for assistance/accompanying in the field trip in Taiwan. Directors and curators of TAI, TI, TNS, PE, and HAST kindly permitted us to study herbarium specimens. This study was supported in part by Grants-in-Aid to Scientific Research from the Japan Society for the Promotion of Science (13640708 to Tomohisa Yukawa; 15405014 to Hiroaki Setoguchi), grants from Institute of Botany, Academia Sinica to Ching-I Peng, and grants from Taiwan Forestry Research Institute to Shih-Wen Chung.

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

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