Journal of Phytogeography and Taxonomy 54 :15-20, 2006 !The Society for the Study of Phytogeography and Taxonomy 2006

Yu Masuda, Lucien Faliniaina and Katsuhiko Kondo : Phylogenetic analysis of Malagasy Gastrorchis and Phaius （）based on internal transcribed spacer（ITS） sequence

Laboratory of Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, 1―4―3 Kagamiyama, Higashi-Hiroshima City 739―8526, Japan

Abstract The molecular phylogenetics among five species of Gastrorchis and Phaius pulchellus all endemic to Madagas- car and additional four species of non-Malagasy Phaius was studied on the basis of the sequence analysis of ITS region of rDNA. The species of Gastrorchis and those of Phaius studied constituted two respective clades, except- ing Malagasy P . pulchellus was placed in the clade of Gastrorchis. This fact suggests that Malagasy P . pulchell- us might be shared and originated from the common ancestor of Gastrorchis.

Key words : Gastrorchis, ITS, molecular phylogenetics, Phaius.

The terrestrial or epiphytic genera Gastrorchis had no spur and no fusion of the base of the lip Schlct. and Phaius Lour.（Orchidaceae）are tax- with the column（Hermans 1999）. onomically placed in the subfamily Epidendroi- Only a karyomorphological approach in Gas- deae, the tribe , sub-tribe Bletiinae trorchis and Phaius is available up to date in （Dressler 1982）. Gastrorchis endemic to Mada- most standard references（Faliniaina and Kondo gascar（Schlechter 1825）consists of six species 2003）: Four species and one variety of Gastror- according to Perrier de la Bathie（1939―1941）or chis and Phaius pulchellus and its variety had nine species and three varieties according to Du common karyomorphological characteristics and Puy et al.（1999）. Phaius consists of approxi- the chromosome number of 2n=40 and thus, they mately 50 species and is distributed from Africa, seem to closely related to each other. These east to Asia down to Polynesia（Perrier de la karyomorphological similarities support Dressler’s Bathie 1939―1941 ; Ohwi 1965）. However, Phaius hypothesis（1982）that Gastrorchis and Phaius pulchellus Kraenzl is only the species in the ge- seem to be closely related to each other on the nus found in Madagascar as well as French Re- basis of morphological characters. union（Bosser 1971 ; Du Puy et al. 1999）. The nucleotide sequences of the internal tran- Gastrorchis has been firstly considered to be scribed spacer 1 and 2（ITS 1 and ITS 2）from the placed as the subgenus in Phaius in the Orchi- nuclear ribosomal DNA（nrDNA）region are daceae（Blume 1858）and then, has been treated widely used in molecular phylogenetics and sys- as the distinct genus isolated from Phaius（Per- tematics at the species and genus levels（Bald- rier de la Bathie 1939―1941）. Moreover, Gastror- win et al. 1995）．The ITS regions have been well chis has been once treated as a section in -used in phylogenetic analysis in orchidaceous Phaius（Summerhayes 1964）. More recently, （Whitten et al. 2000 ; Pridgeon et al. Gastrorchis and Phaius have been taxonomically 2001 ; Gravendeel et al. 2001 ; Salazar et al. revised to be separate genera in the subtribe 2003 ; Tsai et al. 2004 ; Van Den Berg et al. Bletiinae by Dressler（1982）. Malagasy Gastror- 2005）. However, any molecular phylogeny has chis and Phaius were easily identified by some not yet been applied to Gastrorchis, Phaius and morphological characters ; e.g., the former genus their close relatives of sub-tribe Bletiinae.

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In this paper, we attempt to determine genetic 109（TaKaRa Bio, Otsu, Japan） wasspreadonto and systematic relationship between Malagasy LB agar plate（LB medium, including 100 mg/ml Gastrorchis and Phaius pulchellus based on the ampicillin, 0.5 mM IPTG, 40 μg/ml X-Gal）for sequence analysis of ITS region. blue/white selection and incubated at 37℃ over- night. Between 5-10 white colonies of each spe- Materials and methods cies were picked up at random and checked pres- Plant materials : ence of ITS region by PCR described above. At Four species and one variety of Gastrorchis least four clones of each species with ITS region and one species and one variety of Phaius in were incubated overnight in LB medium includ- Madagascar were used in this study（Table 1）. ing 100 mg/ml ampicillin and plasmid DNA were For comparison, Calanthe sylvatica（Thouars） extracted and purified. The ITS regions were se- Lindl. in Madagascar was chosen and used as quenced by ABI 377 automated sequencer using the outgroup since it was taxonomically closely Big Dye Terminator kit（ABI, Foster city, CA, related to Gastrorchis and Phaius placed in the USA）following the manufacturer’s protocol. Se- subtribe Bletiinae and shared same geographical quencing primer was ITS 4. localities and habitats（Dressler 1982）.They were collected and cultivated in Laboratory of Data analysis : Plant Chromosome and Gene Stock, Graduate The sequences obtained were aligned with School of Science, Hiroshima University, Japan. CLUSTAL W program（Thompson et al. 1994）on Additional four species of Phaius in Indonesia, the DDBJ website（http : //www.ddbj.nig.ac.jp）. Japan, French New Caledonia and American Sa- The phylogenetic tree was constructed with a moa, respectively were collected and cultivated parsimony method using PAUP 4.0 b program in Hiroshima Botanical Garden. （Swofford 2000）. The heuristic search was con- ducted with 1,000 random addition replicates, DNA extraction, amplification, cloning and se- rearrangements limited to 100,000 per replicates, quencing : tree bisection-reconnection（TBR）and MulTree Total DNA of each of the studied taxa was ex- on. The bootstrap analysis was carried out on tracted from fresh leaves using CTAB method 1,000 replicates using the heuristic search option （Doyle and Doyle 1987）. The nucleotide se- in PAUP（Felsenstein 1985）．To quantify the di- quences of ITS 1, 5.8 S rDNA and ITS 2（ITS re- versity of ITS sequences, the number of polymor- gion）were amplified from total DNA using ITS phic nucleotide sites per nucleotide site was cal- 5（5’-GGAAGTAAAAGTCG-TAACAAGG-3’）and culated（Nei 1987）. ITS 4（5’-TCCTCCGCTTATTGATATGC-3’）PCR primers（White et al. 1990）. The PCR amplifica- Results and discussion tions were performed in a GeneAmp PCR system Nucleotide sequence analysis and comparison 2700（Perkin-Elmer, Boston, MA, USA）thermal Amplification of ITS regions using the primer cycler for an initial 1 min denaturation at 97℃, pair described above gave PCR products of ap- then 27 cycles of 1 min at 97℃,1minat52℃, proximately 750 bp in length. The sequences of and 2 min at 72℃ followed by an extention pe- them were comprised of a partial sequence of 18 riod of 7 min at 72℃. The ITS PCR products S rRNA gene, the ITS region, and a partial se- were purified by the High Pure PCR Product Pu- quence of 26S rRNA sequence. The boundaries of rification Kit（Roche, Indianapolis, IN, USA）and the ITS 1 and ITS2 and nuclear rDNA coding re- cloned by the pGEM-T easy vector system gions were determined by comparison with an （Promega, Madison, WI, USA）because the peaks almost complete sequence for the 18S―26S of direct sequencing data of ITS PCR products nrDNA cistron of Arabidopsis thaliana（Acces- were broad or overlapped and they were impossi- sion No. X 52320）. The nucleotide sequence data ble to be analyzed. Ligation and transformation reported in this paper will appear in the DDBJ, were prepared according to the manufacturer’s EMBL and GenBank databases under accession protocol. Transformed Escherichia coli strain JM numbers AB 222023 to 222034 and AB 239254 to

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AB 239295. The length of ITS region in our sam- Phylogenetic analysis ples varied from 229―244 bp for the ITS 1 region, Maximum parsimony analysis of the ITS data 167 bp for the 5.8S rRNA gene, and 255―262 bp produced 89186 equally parsimonious trees of for the ITS 2 region（Table 1）. Their intra- 282 steps with a consistency index including un- individual polymorphism was confirmed in all informative characters（CI）of 0.816 and a reten- species in this study : One to 11 polymorphic tion index（RI）of 0.965. Same topology was sites and 1―4 indels between respective clones yielded by MP analysis with indels or without were found in each species and some of their indels. Thus, the strict consensus tree with in- clones had same sequence（Table 1）.These dels was drawn in Fig. 1. The phylogram showed clones of the same sequences were treated here two major clades ; one consisted of the Malagasy as one common clone following the phylogenetic taxa of Phaius and Gastrorchis and the other analysis. consisted of non-Malagasy Phaius. The mono- ITS sequences of the five species of Gastror- phyly of Malagasy clade of Gastrorchis and chis（17 clones）, six species of Phaius（23 clones） Phaius pulchellus was supported by 100% boot- and two species of Calanthe（5clones）as the out- strap values（BS）, while the monophyly of non- group were aligned and yielded 670 characters. Malagasy Phaius was supported by 63.4% BS. Percentage of polymorphic sites of the ITS re- This result suggested that the genus Phaius was gion excluding the indels within 17 taxa of Gas- polyphyletic taxon, and Malagasy Phaius was trorchis was 8.3%, that between 23 taxa of Gas- differentiated from a part of the clade of Gas- trorchis and Malagasy Phaius was 9.6%, that trorchis. between 40 taxa of Gastrorchis, Malagasy In the Malagasy clade of Gastrorchis and Phaius and non-Malagasy Phaius was 25.3%, Phaius pulchellus, a subclade was inferred with that between 45 taxa of Gastrorchis, Phaius and 68.8% BS, which included G. humblotii（Rchb. f） Calanthe（outgroup）was 29.3%, respectively（Ta- Schltr. var. schlechteri（H. Perrier）Senghas ex ble 2）. J. Bosser et P.J. Cribb, G . lutea（Ursch et Toill.

Table 1. The materials used in this study and intra-individual variation in ITS region of Gastrorchis, Phaius and Calanthe species 㪘㪹 㫍㪼㫉㪹 㫀 㫋㪸 㫀㫆㫅 㫀㪺㪼㫇㪪 㪼㫊 㫆㪭 㫀㪺㪼㫇㫊㫉㪼㪿㪺㫌 㪸㫅㪼㫄 㪻㫅 㪠㪼㪻㫀㫋㫆㪼㫃㪺㫌㪥㫊㪼㫅㫆㫃㪺㪽㫆㪅㫆㪥 㫅㫆㫀㫊㫊㪼㪺㪺㪸㪼㫊㪸㪹㪸㫋㪸㪛㪿㫋㪾㫅㪼㫃㪪㪫㪠㫃㪼㪻㫅 㪼㪺㫉㫌㫆㫊 㫆㪺 㫌 㫉㫋㫅 㫐 㫅㫆㫀㫋㫌㫋㫀㫋㫊㪹㫌㫊 㪀㫇㪹㩿 㪞㪅 㫅㪸㫉㪽 㫊㪸㪞 㫉㫋 㪸㫉㪽㫊㫀㪿㪺㫉㫆 㫅 㫀㫊㫀㫆㪺 㫀 㪺㪪 㫃㪿 㫋 㪅㫉 㪣㪧 㪇㪎㪄㪪㪞㪚 㪑㪌 㪸㪾㪸㪻㪸㪤 㫉㪸㪺㫊 㪊 㪊 㪇 㪌㪌㪄㪋㪌㪉㪐㪊㪉㪙㪘㪃㪊㪉㪇㪉㪉㪉㪙㪘㪉㪍㪍 㪅㪞 㪹㫄㫌㪿 㪞 㫊㪸 㫀㫀㫋㫆㫃㪹㫄㫌㪿㫊㫀㪿㪺㫉㫆㫉㫋 㪩㩿 㪺 㪹㪿 㪽㪅 㪀㪅 㪿㪺㪪 㫃㫋 㪅㫉 㪣 㪎㪄㪪㪞㪚㪧 㪑㪍㪇 㪸㪻㪸㪤 㪾㪸㫊 㫉㪸㪺 㪊 㪉 㪇 㪐㪌㪄㪏㪌㪉㪐㪊㪉㪙㪘㪃㪋㪉㪇㪉㪉㪉㪙㪘㪇㪍㪍 㪞㪅 㪿 㪹㫄㫌 㪅 㫊㪺㪿 㪞 㫊㪸 㪺㫉㫆㫉㫋 㪿㫀 㪿㫊 㪹㫄㫌 㫃㫆㫋 㫀㫀 㫉㪸㫍 㪅 㫊㪺㪿㫃㪼 㫋㪿㪺 㪼㫉㫀 㪪㪞㪚㪧㪣 㪄 㪸㪤㪑㪎㪇㪎 㫉㪸㪺㫊㪸㪾㪸㪻 㪌 㪎 㪈 㪌㪍㪄㪉㪍㪉㪐㪊㪉㪙㪘㪃㪌㪉㪇㪉㪉㪉㪙㪘㪈㪎㪍㪄㪇㪎㪍 㪧㪅㪟㩿 㪼 㫉㫉 㫀㪼㫉 㫊㫆㪙㫏㪼㫊㪸㪿㪾㫅㪼㪪㪀 㫊㪼㫉 㪹㪹㫀㫉㪚㪅㪡㪅㪧㪻㫅㪸 㪞 㫋㪅 㫌㪹㪼 㪸㫊㫆㫃㫌㪺㫉㪼㪹㫌㫋㫊㫀㪿㪺㫉㫆㫉㫋㫊㪸㪞 㩿 㫆㪫 㫉㪸㫌 㪀㫊 㪪 㫃㪿㪺 㫋 㪅㫉 㪣㪧 㫉㪸㪺㫊㪸㪾㪸㪻㪸㪤㪑㪏㪇㪎㪄㪪㪞㪚 㪉 㪌 㪇 㪎㪍㪉㪐㪊㪉㪙㪘㪃㪍㪉㪇㪉㪉㪉㪙㪘㪐㪍㪍 㪼㫋㫌㫃㪅㪞 㪞 㫊㪸 㫉㫆㫉㫋 㪺 㫋㫌㫃㫊㫀㪿 㪼㪸 㪅㫅㪼㪞㪅㫃㫃㫀㫆㪫㪻㫅㪸㪿㪺㫊㫉㪬㩿 㫉㪸㪺㫊㪸㪾㪸㪻㪸㪤㪑㪐㪇㪎㪄㪪㪞㪚㪧㪣 㪋 㪊 㪇 㪈㪎㪄㪐㪍㪉㪐㪊㪉㪙㪘㪃㪎㪉㪇㪉㪉㪉㪙㪘㪐㪍㪍 㫏㪼 㪙㫆 㫊㫊 㪼㫉㪀 㫇㪅㪧 㪅㫃㫌 㫇 㫃㫌 㪸㪿㪧 㫀 㫊㫌 㫇㫌㫃 㪼㪿㪺 㫃 㫊㫌㫃 㪸㫍 㫉㪅 㫌㫃㫃㪼㪿㪺㫃㫌㫇 㫊 㪢㫉㪸㪼㫅㫑㫃㪅 㪣 㪪㪞㪚㪧 㪄 㪈㪎 㪇 㪸㪤㪑 㫉㪸㪺㫊㪸㪾㪸㪻 㪋 㪋 㪋 㪍㪍 㪋 㪍㪍㪄 㪏 㪉㪙㪘 㪉 㪉㪇㪉 㪏 㪉㪙㪘㪃 㪊 㪎㪉㪐 㪊㪄㪎㪌 㪧㪅 㫇 㫃㫌 㪅 㫅㪸㫊 㪻 㫊㫌㫃㫃㪼㪿㪺㫃㫌㫇㫊㫌㫀㪸㪿㪧 㪸㫍 㫉㪅 㫊 㫅㪸 㪻 㫅㪸㫉 㪾 㫊㫀㫊㫅㪼㫋㪸 㫆㪙 㫊㫊 㪼㫉 㪣 㪑㪈㪈㪎㪄㪪㪞㪚㪧 㪤 㪻㪸 㪾㪸 㪸㫊 㫉㪸㪺 㪉 㪋 㪇 㪍㪎㪉㪐㪊㪉㪙㪘㪃㪐㪉㪇㪉㪉㪉㪙㪘㪌㪍㪍 㪅㪧 㪸㫄㪹㫆 㪧㪿㪸 㫌㫀 㫊 㫄㪸 㪹㫆㫀 㪼㫅 㫅㫊㫀㫊 㪙 㫌㫃 㫄㪼 㪏㪌 㪐㪐 㪈㪄 㪠㪑 㫅 㫆㪻 㫅 㫊㪼 㫀㪸 㪌 㪈㪈 㪈 㪙㪘㪋㪌㪍 㪉㪉 㪉㪙㪘㪃㪇㪊㪇㪉 㪉㪏㪄㪐㪎㪉㪐㪊 㫄㪅㪧 㫀㫅㫆 㫀㪸㪿㪧 㫌 㫉㫆㫅㫀㫄㫊 㪙㫃 㪼㫄㫌 㪏㪋 㪐㪎 㪸㪡㪑 㫅㪸㫇 㪊 㪊 㪇 㪌㪍 㪉㪉㪉㪙㪘㪊 㪙㪘㪃㪈㪊㪇 㪊㪏㪉㪐㪊㪉 㪋㪏㪄 㪅㪧 㫋 㪺㫅㪸 㪿㪧 㪸 㪼㪸㪼㫃㫃㫀㫍㫉㪸㪺㫅㪸㫋㫊㫌㫀 㩿㪙㪸㫅㫂㫊 㪼㫏 㩾㪣 㪀㪅㫉㪼㪟 㫃㪙 㫌㫄 㪎㪼 㪌 㪄㪎㪏 㪈 㪺㫅㪼㫉㪝㪑 㪿 㪥㪼 㫃㪸㪚㫎 㪼㪻㫆 㪌㪸㫀㫅 㪍 㪊 㪍㪌㪍 㪐㪌㪍㪄 㪙㪘 㪉㪉 㪃㪉㪊㪇㪉 㪙㪘 㪉㪊 㪐㪏㪄㪍㪏㪉㪐 㪧 㪾㪅 㫉㪸㪼 㫀㪼㪽㪽㪼㪸㫉㪾㫊㫌㫀㪸㪿㪧 㪩㪺 㪅㪹㪿 㪽㪅 㪈㪇㪈 㪌 㪸㫆㫄㪸㪪㫅㪸㪺㫀㫉㪼㫄㪘㪑㪏 㪊 㪉 㪇 㪈㪐㪄㪇㪐㪉㪐㪊㪉㪙㪘㪃㪊㪊㪇㪉㪉㪉㪙㪘㪍㪌㪍 㪚㪅 㫊 㫃㫐 㫍 㫃㪸㪚 㪸㫅 㪿㫋 㫐㫊㪼 㫃 㫀㫋㪸㫍 㪺㪸 㪸㫌㫆㪫㩿 㫉 㪻㫅㫀㪣㪀㫊 㪅㫃 㪣㪧 㪞㪚 㪄㪪 㪎㪈 㪻㪸㪤㪑㪉 㪸 㪺㫊㪸㪾 㪸㫉 㪋 㪈 㪉 㪍㪌㪍㪄㪍㪌 㪙㪘㪎 㪉㪉㪉㪇㪊 㪙㪘㪃㪋 㪉㪊㪐㪉㪐 㪌㪐㪄㪊 㫄㪅㪧 㫀㫅 㪋㪶㫆 㫀㪸㪿㪧 㫌㫊 㫀㫄 㫆㫅 㫉 㪼㫄㫌㫃㪙 㪛㫅㪸㪭 㪼 㪙㫅 㪼㫉 㪼㪾 㫋 㫃㪸 㪅 㪉㪇㪇㪌 㪍 㪌㪝㪘㪎㪌 㪈㪌㪇㪈㪉 㪚 㪸㪅 㫃 㫊㫀 㪸㫃㪸㪚 㫅 㪼㪿㫋 㪸㫃㫀 㫄㫊 㪸 㪸㫀㫃㫆㪽㪼 㫅㫀㪣 㪻㫃㪅 㪣 㪼㫆㪸㫀 㫋 㪅㫃㪸 㪉㪇㪇㪌 㪌㪈㪍㪉㪏㪏㪰㪘㪋㪌㪍

Table2.Percentageofpolymorphisms*amongGastrorchis, Malagasy Phaius and non-Malagasy Phaius Group ITS 1（%） 5.8 S（%） ITS 2（%） Total（%） within Gastrorchis 14.0 1.8 7.4 8.3 Gastrorchis and Malagasy Phaius 15.4 3.6 8.1 9.6 Gastrorchis, Malagasy Phaius and non-Malagasy Phaius 36.1 9.7 25.6 25.3 Gastrorchis, Phaius and Calanthe（outgroup） 40.9 9.7 32.0 29.3 * The percentages were calculated by Nei（1987）

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G.fran_2 G.fran_3 G.fran_1 G.humb_1 G.humb_3 G.humb_2 P.pul.pul_3 100 P.pul.pul_2 P.pul.pul_4 60.2 P.pul.pul_1 68.8 P.pul.sand 1 90.8 P.pul.sand_2 G.humb.sch_1 Malagasy G.humb.sch_3 G.humb.sch_5 100 G.humb.sch_2 G.humb.sch_4 G.lutea_2 92.1 70.7 G.lutea_4 G.lutea_1 G.lutea_3 100 99.7 G.tube_1 90.0 G.tube_2 76.6 P.ambo_1 P.ambo_3 85.5 P.ambo_4 91.2 P.ambo_2 59.9 P.ambo_5 P.tanc_2 100 P.tanc_3 P.tanc_4 non- 99.3 P.tanc_5 98.8 P.tanc_1 Malagasy P.grae_2 P.grae_3 63.4 100 P.grae_1 100 P.mino_4 P.mino_1 P.mino_3 93.9 P.mino_2 C.alis C.sylv_1 C.sylv_2 Outgroup 85.4 C.sylv_3 C.sylv_4

Fig. 1. The strict consensus tree of 89186 most parsimonious trees based on the internal transcribed spacer（ITS） sequence data（Tree length=282, CI=0.816. RI=0.965）. Numbers listed along the branch show bootstrap values. See Table 1 for explanation of the abbreviations.

-Gen. ex Bosser）Senghas and G . tuberculosa might be needed. （Thouars）Schltr, P . pulchellus var. pulchellus In spite of the Malagasy Phaius pulchellus and P . pulchellus var. sandrangatensis J. Bosser, having common flower characters with the non- and the others included G . francoisii Schltr. and Malagasy Phaius, the present results suggested G . humblotii. In the subclade, G . humblotii var. that the Malagasy P . pulchellus was placed in schlechteri, G . lutea and G . tuberculosa made a the Gastrorchis cluster（100% BS ; Fig. 1）.The clade supported by 92.1% BS. This fact sug- possibility for the incongruence between mor- gested that the species of G . humblotii was not phology and ITS sequence data could be conver- monophyletic, and both G . humblotii and G . gent evolution or gene transfer through intro- humblotii var. schlechteri were originated from gressive hybridization because Gastrorchis and different ancestor, respectively. Thus, revision of Phaius could easily make hybrid（Dressler 1982）. the taxonomic position of the G . humblotii Although the data in this paper were not enough

－ 18 － October 2006 J. Phytogeogr. Taxon. Vol. 54. No. 1 to solve this problem, yet they indicated at least 2001. Molecular phylogeny of Coelogyne Phaius pulchellus was not voyaged from some- （ ; Orchidaceae）based on plas- where else but evolved in Madagascar. tid RFLPS, matK and nuclear ribosomal ITS Many more molecular analyses using not only sequences : evidence for polyphyly. Am. Journ. genomic DNA but also plastid DNA as much as Bot. 88 : 1915―1927. chloroplast DNA and/or mitochondria DNA se- Hermans J. 1999. Phaius pulchellus var. san- quences should be made for clarification, justifi- drangatensis orchidanceae. Curtis’s Botanical cation and revision of the relationship between Magazine. 16, 4 : 262―266. Gastrorchis, Malagasy Phaius and non-Malagasy Liao,F.S.,Do,Y.Y.,Cheng,C.F.,Lee,S.J., Phaius. Cheng, S. H. and Huang, P. L. 2005. Molecu- lar phylogeny of related species in Calanthe This paper was contributed from Laboratory of based on analysis of internal transcribed Plant Chromosome and Gene Stock, Graduate spacer and large-subunit regions of ribosomal School of Science, Hiroshima University（Contri- DNA. Unpublished（on DDBJ database）. bution No. 83）. Nei, M. 1987. Molecular evolutionary genetics. 512 pp. Columbia Univ. Press, New York. References Ohwi, J. 1965. Flora of Japan. 1067 pp. Smitho- Baldwin,B.G.,Sanderson,M.J.,Porter,J.M., nian Inst., Washington, D.C. Wojciechowski, M. F., Campbell, C. S. and Perrier de la Bathie 1939―1941. Orchidees, 49 e Donoghue, M. J. 1995. The ITS region of nu- famille, in Hembert, H., Flore de Madagascar, clear ribosomal DNA : A valuable source of vol. 2 : 299―307, Tananarive : Imprierie Offi- evidence on Angiosperm phylogeny. Ann. Mis- cielle. souri Bot. Gard. 82 : 247―277. Pridgeon,A.M.,Solano,R.andChase,M.W. Blume, C. L. 1858. Collection des orchidees les 2001. Phylogenetic relationships in Pleurothal- plus remarquables de l’Archipel Indien et du linae（Orchidaceae）: Combined evidence from Japon. 1. G.G.Sulpke, Amsterdam. nuclear and plastid DNA sequences. Am. Bosser J. 1971. Contribution a l’etude des orchi- Journ. Bot. 88 : 2286―2308. daceae de Madagascar, XV!. Revision du Salazar,G.A.,Chase,M.W.,SotoArenas,M.A. genre Phaius Lour., Adansonia, ser. 2, 11 : and Ingrouille, M. 2003. Phylogenetics of 519―543. Cranichideae with emphasis on Spiranthinae Doyle, J. J. and Doyle, J. L. 1987. A rapid DNA （Orchidaceae, Orchidoideae）:evidence from isolation procedure for small quantities of plastid and nuclear DNA sequences. Am. fresh leaf tissue. Phytochem. Bull. Bot. Soc. Journ. Bot. 90 : 777―795. Am. 19 :11―15. Schlecter, R. 1825. Die Orchideen. 836 pp., Ver- Dressler, R. L. 1982. The orchids, Natural his- lagsbuchhandlung Paul Parey, Berlin. tory and classification. 332 pp., Harvard Univ. Summerhayes, V. S. 1964. African orchids XXIX. Press, Cambridge. Kew Bull. 17 : 511―560. DuPuy,D.,Cribb,P.,Bosser,J.,Hermans,J. Swofford, D. L. 2000. PAUP*. Phylogenetic and Hermans, C. 1999. The orchids of Mada- Analysis Using Parsimony（*and Other Meth- gascar. p.376. Roy. Bot. Gard., Kew. ods）. Version 4. Sinauer Associates, Sunder- Faliniaina, L. and Kondo, K. 2003. Karyomor- land, Massachusetts. phological studies of Malagasy Gastrorchis Thompson,J.D.,Higgins,D.G.andGibson,T.J. and Phaius（Orchidaceae）.Chrom.Sci.7 :49 1994. CLUSTAL W : improving the sensitivity ―53. of progressive multiple alignment through se- Felsenstein, J. 1985. Confidence limits on phylo- quence weighting, position specific gap penal- genies : an approach using the bootstrap. Evo- ties and weight matrix choice. Nucleic Acid lution 6 : 227―242. Res. 22 : 4673―4680. Gravendeel, B., Chase, M. W., De Vogel, E. F., Tsai,C.C.,Peng,C.I.,Huang,S.C.,Huang,P. Roos, M. C., Mes, T. H. M and Bachmann, K. L. and Chou, C. H. 2004. Determination of the

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genetic relationship of Dendrobium species （Received July 15, 2005 ; accepted April 20, （Ochidaceae）in Taiwan based on the sequence 2006） of the internal transcribed spacer of ribosor- malDNA.Sci.Hort.101 : 315―325. 増田 優・ファリニアイナ ルシエン・近藤勝彦： Van Den Berg, C., Goldman, D. H., Freudenstein, マダガスカル産ラン科 Gastrorchis と Phaius の J. V. Pridgeon, A. M., Cameron, K. M. and 分子系統学的関係 Chase, M. W. 2005. An overview of the phylo- マダガスカル固有の Gastrorchis 5 種と Phaius genetic relationships within Epidendroideae pulchellus とその 1 変種，マダガスカル以外に分 inferred from multiple DNA regions and recir- 布する Phaius 4 種，そして outgroup として両属 cumscription of and Arethuseae に近縁な Calanthe sylvatica を用いて，系統学的 （Orchidaceae）. Am. Journ. Bot. 92 : 613―624. 関係の分析を行った。本研究では，それぞれの核リ White, T. J., Bruns, T., Lee, S. and Taylor, J. ボソーマルDNA の ITS 領域をPAUP ver. 4.0 プ 1990. Amplification and direct sequencing of ログラムを用いて解析した。その結果，作成した分 fungal ribosomal RNA genes for phylogenetics. 子系統樹から，マダガスカル固有の Gastrorchis と In PCR protocols: A guide to method and ap- Phaius pulchellus は同じクレードに属し，マダガ plications.（M. A. Innis, D. H. Gelfand, J. J. スカル以外に分布する Phaius は，異なるクレード Sninsky and T. J. White, Eds）pp. 315―322. に属することが明らかとなった。このことは，マダ Academic Press, San Diego, California. ガスカルに固有の Phaius pulchellus が Gastror- Whitten, W. M., Williams, N. H. and Chase, M. chis との共通の祖先から派生したこと，マダガス W. 2000. Subtribal and generic relationships カル以外に分布する Phaius とは別系統であること of Maxillarieae（Orchidaceae）with emphasis を示唆している。 on Stanhopeinae : combined molecular evi- （〒739―8526 東広島市鏡山 1―4―3 広島大学大学 dence. Am. Journ. Bot. 87 : 1842―1856. 院理学研究科附属植物遺伝子保管実験施設）

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