Molecular Phylogeny of Phalaenopsis Blume (Orchidaceae) Based on the Internal Transcribed Spacer of the Nuclear Ribosomal DNA

Pl. Syst. Evol. 256: 1–16 (2006) DOI 10.1007/s00606-005-0356-y

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

C. C. Tsai1,2, S. C. Huang3, and C. H. Chou2,4

1Kaohsiung District Agricultural Improvement Station, Pingtung, Taiwan 2National Pingtung University of Science and Technology, Pingtung, Taiwan 3Floricultural Research Center, Taiwan Agricultural Research Institute, Taichung, Taiwan 4National Sun Yat-sen University, Kaohsiung, Taiwan

Received July 29, 2003; accepted May 21, 2005 Published online: October 20, 2005 Ó Springer-Verlag 2005

Abstract. The internal transcribed spacer (ITS1, information for elucidating the phylogeny of this 5.8S rDNA, and ITS2) region of nuclear ribosomal genus. DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity Key words: Phalaenopsis, phylogeny, rDNA, internal in the genus Phalaenopsis (Orchidaceae). A phy- transcribed spacer. logeny was developed for the genus based on the neighbor-joining and maximum parsimony analy- The genus Phalaenopsis Blume (Orchidaceae), ses of molecular data. Results of these analyses provided support for the monophyly of the genus a beautiful and popular orchid, comprises Phalaenopsis and concurred in that the genera approximately 66 species according to the Doritis and Kingidium should be treated as being latest classification of Christenson (2001), parts of the genus Phalaenopsis as suggested by who divided this genus into five subgenera, Christenson (2001). Within the genus Phalaenopsis, namely Proboscidioides, Aphyllae, Parishianae, neither subgenera Aphyllae nor Parishianae were Polychilos, and Phalaenopsis. Of these, subge- monophyletic, and they were highly clustered with nus Polychilos was subdivided into four sec- subgenus Proboscidioides plus sections Esmeralda tions, namely Polychilos, Fuscatae, Amboinen- and Deliciosae of subgenus Phalaenopsis based on ses, and Zebrinae. In addition, subgenus ITS data. Those species also have the same Phalaenopsis was also subdivided into four characters of morphology of four pollinia and sections, namely Phalaenopsis, Deliciosae, similar biogeographies. Furthermore, neither sub- Esmeralda, and Stauroglottis. genus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only Species of the genus Phalaenopsis are section Phalaenopsis was highly supported as being found throughout tropical Asia and the larger monophyletic. As for the subgenus Polychilos, only islands of the Pacific Ocean. The western section Polychilos was moderately supported as distribution of Phalaenopsis is in Sri Lanka being monophyletic. In conclusion, the present and South India. The eastern limit of the range molecular data obtained from the ITS sequence of is in Papua New Guinea. To the north, they nrDNA of the genus Phalaenopsis provide valuable are distributed in Yunnan Province (southern 2 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences

China) and Taiwan. The southern limit is in treated as section Parishianae of the genus northern Australia (Christenson 2001). Differ- Phalaenopsis by Sweet (1968). Christenson ent subgenera of Phalaenopsis have distinct (2001) agreed with Sweet’s treatment and geographic distributions. Subgenera Aphyllae, placed this complex in the subgenus Parishi- Parishianae, and Proboscidioides are distrib- anae of Phalaenopsis. Furthermore, the other uted in southern China and India extending to four-pollinia species, P. lowii, was also placed northern Vietnam, Myanmar, and Thailand, in the section Proboscidioides of the genus respectively. The subgenus Polychilos has a Phalaenopsis (Sweet 1980) and in the subgenus few species distributed as far west as north- Proboscidioides of the genus Phalaenopsis by eastern India, but it is primarily centered in Christenson (2001). Shim (1982), however, Indonesia and the Philippines (Christenson disagreed with Sweet’s concept (1980) and 2001). Subgenus Phalaenopsis is centered in separated sections Proboscidioides, Aphyllae, the Philippines with two species extending to Parishianae, Polychilos, Zebrinae, Fuscatae, Taiwan (P. aphrodite subsp. formosana and and Amboinenses as the genus Polychilos from P. equestris) and one wide-ranging species a narrowly defined Phalaenopsis. In addition, (P. amabilis) found from the Philippines and the genus Doritis was traditionally separated Indonesia to northern Australia (Christenson from Phalaenopsis because of its pollinium 2001). number, lip structure, and adaptations to a All Phalaenopsis species (with the exception terrestrial habitat (Sweet 1980, Seidenfaden of the natural tetraploid species, P. buyssoni- 1988a). This group was also treated as the ana) have 38 chromosomes (2n=38) (Woo- genus Phalaenopsis and placed in the section dard 1951, Shindo and Kamemoto 1963, Esmeralda of the subgenus Phalaenopsis Tanaka and Kamemoto 1984, Christenson (Christenson 2001). 2001). Furthermore, the chromosome number The internal transcribed spacer (ITS) of the subtribe Aeridinae (syn. Sarcanthinae) region of nuclear ribosomal DNA (nrDNA) (with only a few exceptions of tetraploid or has provided valuable information for resolv- hexaploid species) is consistent with that of ing phylogenetic relationships at different tax- Phalaenopsis (Christenson 2001). Crosses onomic levels (e.g. Baldwin 1992, 1993; Suh between Phalaenopsis species and other close et al. 1993; Sun et al. 1994; Bayer et al. 1996; genera, namely Aerides, Arachnis, Ascocen- Cox et al. 1997), particularly at the infragen- trum, Doritis, Neofinetia, Renanthera, Rhyn- eric level because of relatively rapid evolution- chostylis, Vanda, and Vandopsis, also show ary rates. In this study, we followed the various degrees of fertility (Sweet 1980). systematics proposed and references described Based on the number of pollinia, tradi- by Christenson (2001). In order to reconstruct tional species of Kingidium (which have four the phylogeny within the genus, the nucleotide pollinia) were segregated from the genus Pha- sequences of the ITS region of the nrDNA laenopsis (which has two pollinia) (Sweet 1980, from 53 taxa of Phalaenopsis plus 13 outgroup Seidenfaden 1988b). Christenson (2001) trea- species were analyzed to address the phylogeny ted the traditional genus Kingidium as Pha- of the genus. laenopsis and split it into different parts of Phalaenopsis, placing some species into the subgenus Aphyllae (P. braceana, P. minus, and Materials and methods P. taenialis) and some into the section Delicio- Taxonomy and nomenclature of Phalaenopsis sae (P. chibae and P. deliciosa) of the subgenus species in this study followed Sweet (1980) and Phalaenopsis. Another group, the P. parishii Christenson (2001). Plant materials used include complex, having four pollinia, was proposed as 53 taxa of the genus Phalaenopsis. In addition, a segregated genus Grafia, by Hawkes (see specimens of each of seven related genera, namely Christenson 2001). This complex was first Aerides, Ascocentrum, Neofinetia, Renanthera, C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 3

Rhynchostylis, Vanda,andVandopsis, which show sequences from the plant material in this study were various degrees of fertility with Phalaenopsis recovered using glassmilk (BIO 101, California). (Sweet 1980), Paraphalaenopsis, which was once These DNAs were directly sequenced following treated as Phalaenopsis (see Sweet 1980), as well the method of dideoxy chain-termination using an as of five other genera of the subtribe Aeridinae, ABI377 automated sequencer with the Ready namely Amesiella, Gastrochilus, Haraella, Thrix- Reaction Kit (PE Biosystems, California) of the spernum, and Tuberolabium, were selected as BigDyeä Terminator Cycle Sequencing. Sequenc- outgroup species (Table 1). All leaf material was ing primers were the same as those used for PCR. taken from living plants in the Kaohsiung Each sample was sequenced two or three times to District Agricultural Improvement Station (KDA- confirm the sequences. These reactions were per- IS) in Taiwan and from the collection of C. C. formed as recommended by the manufacturers. Tsai. The flower pictures of those species in this The sequence alignment was determined using study are available from the author (tsa- the program Clustal W multiple alignment in [email protected]). Using the cetyltrimethyl- BioEdit (Hall 1999). The alignment was then ammonium bromide (CTAB) method (Doyle and checked, and apparent alignment errors were cor- Doyle 1987), total DNA was extracted from fresh rected by hand. Genetic relationships were then etiolated leaves. Ethanol-precipitated DNA was determined using the program MEGA version 2.1 dissolved in TE (Tris-EDTA) buffer and stored at (Kumar et al. 2001). The genetic distance matrix )20°C. Qiagen (Valencia, CA, USA) columns was calculated by use of the two-parameter method were used to clean the DNA of samples which of Kimura (1980), and then it was used to construct was difficult to amplify by PCR. The approxi- phylogenetic trees using the Neighbor-joining (NJ) mate DNA yields were then determined using a method (Saitou and Nei 1987). Maximum parsi- spectrophotometer (model U-2001, Hitachi). mony (MP) analyses (Fitch 1971) were done using The ITS region, which includes the ITS1, 5.8S code modified from the Close-Neighbor-Inter- rDNA, and ITS2, was amplified by PCR with change (CNI) algorithm (Rzhetsky and Nei 1992) primers referenced to Tsai and Huang (2001). The in MEGA (Kumar et al. 1993). Bootstrapping (1025 protocols for the PCR were as follows: we used a replicates) was carried out to estimate the support 50-ll mixture containing 40 mM Tricine-KOH (pH for both NJ and MP topologies (Felsenstein 1985, 8.7), 15 mM KOAc, 3.5 mM Mg(OAc)2, 3.75 lg/ Hillis and Bull 1993). The strict consensus parsimo- ml BSA, 0.005% Tween 20, 0.005% Nonidet-P40, nious tree was then constructed using the program four dNTPs (0.2 mM each), primers (0.5 lM each), MEGA version 2.1 (Kumar et al. 2001). The aligned 2.5 of Advantage 2 DNA polymerase (Clontech), data matrix and tree files are available from the 10 ng genomic DNA, and a 50-ll volume of author ([email protected]). mineral oil. The PCR mixture for amplifying the ITS region included 10% dimethyl sulfoxide Results (DMSO) to reduce problems related to the second- ary structure and efficiency of PCR primer binding. Sequence alignment and ITS characteris- Amplification reactions were completed in a dry- tics. PCR products of each sample studied block with two-step thermal cycles (Biometra). In were directly sequenced except that of P. vir- the first step, the mixture was incubated at 94°C for idis. Since the ITS repeat sequences of 3 min, then it underwent 10 cycles of denaturation P. viridis were not homogeneous, the se- at 94°C for 45 s, annealing at 58°C for 45 s, and quences of this species were finally sequenced extension at 72°C for 1 min. The second step was after T-vector cloning. Three separate clones carried out by the following process: 30 cycles of were selected for sequencing. These three ITS denaturation at 94°C for 45 s, annealing at 54°C sequences were quite variable. Sequence var- for 45 s, and extension at 72°C for 1 min, with a final extension for 10 min at 72°C. These PCR iation among these three clones ranged from products were detected by agarose gel electropho- 2.8 to 6.7%. Furthermore, P. cochlearis resis (1.0%, w/v in TBE), stained with 0.5 lg/ml showed two separate PCR products for the ethidium bromide, and finally photographed under ITS region of PCR amplification. Their UV light exposure. The PCR products of ITS lengths were approximately 460 and 700 bp, 4 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences

Table 1. Names of specimens, geographical distribution, source, and GenBank accession numbers for the sequences of the internal transcribed spacer of nuclear ribosomal DNA Taxa and systematic Geographical Source GenBank classiﬁcation a distribution accession no. Genus Phalaenopsis Subgenus Proboscidioides (Rolfe) E. A. Christ. Phalaenopsis lowii Rchb.f. b Myanmar, and KDAIS KC-88 AY912236 adjacent western Thailand Subgenus Aphyllae (Sweet) E. A. Christ. Phalaenopsis wilsonii China (Sichuan, Yunnan, KDAIS KC-130 AY912257 Rolfe and eastern Tibet) Phalaenopsis minus endemic to Thailand KDAIS KC-227 AY912241 (Seidenf.) E. A. Christ. Phalaenopsis braceana Bhutan and China KDAIS KC-289 AY228495 (J. D. Hook.) E. A. Christ. Phalaenopsis honghenensis China (Yunnan) KDAIS KC-305 AY912229 F. Y. Liu Subgenus Parishianae (Sweet) E. A. Christ. Phalaenopsis gibbosa Sweet Vietnam and Laos KDAIS KC-52 AY912228 Phalaenopsis lobbii India, Bhutan, Myanmar, KDAIS KC-21 AY912235 (Rchb.f.) Sweet and Vietnam Phalaenopsis parishii eastern Himalayas, India, KDAIS KC-316 AY912245 Rchb.f. Myanmar, and Thailand Phalaenopsis appendiculata Endemic to Malaysia KDAIS KC-411 AY912218 C. E. Carr (Malay Peninsula) Subgenus Polychilos (Breda) E. A. Christ. Section Polychilos (Breda) Rchb.f. Phalaenopsis mannii northeast India, Nepal, KDAIS KC-22 AY912238 Rchb.f. and China to Vietnam Phalaenopsis cornu-cervi northeast India and the KDAIS KC-450 AY912222 (Breda) Bl. & Rchb.f. Nicobar Islands to Java and Borneo Phalaenopsis borneensis endemic to Borneo KDAIS KC-109 AF537024 Garay Phalaenopsis pantherina endemic to Borneo KDAIS KC-573 AY912244 Rchb.f. Phalaenopsis lamelligera endemic to Borneo KDAIS KC-263 AY912233 Sweet Section Fuscatae Sweet Phalaenopsis cochlearis Holtt. Malaysia (Malay KDAIS KC-484 AY912221 Peninsula) and Indonesia (Sarawak) Phalaenopsis viridis endemic to Indonesia KDAIS KC-127 J. J. Sm. (Sumatra) C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 5

Table 1. (Continued) Taxa and systematic Geographical Source GenBank classification a distribution accession no. ‘‘clone 1’’ AY912254 ‘‘clone 2’’ AY912255 ‘‘clone 3’’ AY912256 Phalaenopsis kunstleri Myanmar and Malay KDAIS KC-540 AY912232 J. D. Hook. Peninsula Section Amboinenses Sweet Phalaenopsis pulchra endemic to the Philippines KDAIS KC-579 AY912248 (Rchb.f.) Sweet (Luzon and Leyte) Phalaenopsis bellina Malaysia (Malay Peninsula) KDAIS KC-107 AY900290 (Rchb.f.) E. A. Christ. and East Malaysia (Sarawak) Phalaenopsis violacea Indonesia (Sumatra) and KDAIS KC-153 AY390229 Witte Malaysia (Malay Peninsula) Phalaenopsis micholitzii Philippines (Mindanao) KDAIS KC-382 AY912240 Rolfe Phalaenopsis fimbriata Indonesia (Java, Sarawak, KDAIS KC-62 AF537013 J. J. Sm. and Sumatra) Phalaenopsis floresensis endemic to the island KDAIS KC-241 AY912227 Fowlie of Flores Phalaenopsis fasciata endemic to the Philippines KDAIS KC-191 AY912226 Rchb.f. (Luzon, Bohol, and Mindanao) Phalaenopsis doweryensis east Malaysia, Sabah, without KDAIS KC-516 AY912224 Garay & E. A. Christ. a precise locality Phalaenopsis modesta endemic to the island of Borneo KDAIS KC-353 AY912242 J. J. Sm. in East Malaysia (Sabah) and Indonesia (Kalimantan) Phalaenopsis maculata Malaysia (Pahang), East KDAIS KC-49 AF537008 Rchb.f. Malaysia (Sabah and Sarawak), and Indonesia (Kalimantan Timur) Phalaenopsis javanica endemic to Indonesia (Java) KDAIS KC-39 AY912231 J. J. Sm. Phalaenopsis mariae endemic to the Philippines KDAIS KC-11 AY912239 Burb. ex Warn. & B. S. Wms. and Indonesia (Kalimantan and Borneo) Phalaenopsis amboinensis Indonesia (Molucca Archipelago KDAIS KC-157 AY912217 J. J. Sm. and Sulawesi) Phalaenopsis lueddemanniana endemic to the Philippines KDAIS KC-262 AY912237 Rchb.f. Phalaenopsis venosa endemic to Indonesia (Sulawesi) KDAIS KC-220 AY912253 Shim & Fowlie Phalaenopsis pallens endemic to the Philippines KDAIS KC-258 AY912243 (Lindl.) Rchb.f. Phalaenopsis bastianii endemic to the Philippines KDAIS KC-245 AY912219 Gruss & Rollke Phalaenopsis hieroglyphica endemic to the Philippines KDAIS KC-33 AF537000 (Rchb.f.) Sweet 6 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences

Table 1. (Continued) Taxa and systematic Geographical Source GenBank classiﬁcation a distribution accession no. Phalaenopsis reichenbachiana endemic to the Philippines KDAIS KC-389 AY912249 Rchb.f. & Sander Section Zebrinae Pﬁtz. Phalaenopsis inscriptiosinensis endemic to Indonesia (Sumatra) KDAIS KC-298 AY912230 Fowlie Phalaenopsis tetraspis Rchb.f. India (Andaman and Nicobar KDAIS KC-531 AY912252 Islands) and Indonesia (Sumatra) Phalaenopsis corningiana Rchb.f. Borneo (Sarawak and elsewhere KDAIS KC-346 AY390247 on the island) Phalaenopsis sumatrana widespread from Myanmar, KDAIS KC-161 AY390241 Korth. & Rchb.f. Thailand, Vietnam, to Indonesia (Java and Sumatra), Malaysia (Perak and Johore), East Malaysia (Sabah), and the Philippines (Palauan) Phalaenopsis zebrina Witte KDAIS KC-257 AY390251 Subgenus Phalaenopsis Section Phalaenopsis Benth. Phalaenopsis philippinensis endemic to the Philippines KDAIS KC-534 AY912246 Golamco ex Fowlie & Tang Phalaenopsis amabilis widespread from Sumatra KDAIS KC-327 AY391524 (L.) Blume and Java to the southern Philippines, and east to New Guinea and Queensland, Australia Phalaenopsis aphrodite Rchb.f. northern Philippines and KDAIS KC-173 AY391537 southeastern Taiwan Phalaenopsis sanderiana Rchb.f. endemic to the Philippines KDAIS KC-175 AY391552 Phalaenopsis schilleriana Rchb.f. endemic to the Philippines KDAIS KC-429 AY912250 Phalaenopsis stuartiana Rchb.f. endemic to the island of KDAIS KC-528 AY912251 Mindanao in the southern Philippines Section Deliciosae E. A. Christ. Phalaenopsis chibae Yukawa endemic to Vietnam KDAIS KC-488 AY912220 Phalaenopsis deliciosa Rchb.f. widespread from Sri Lanka KDAIS KC-255 AY912223 and India to the Philippines and Sulawesi Section Esmeralda Rchb.f. Phalaenopsis pulcherrima widespread from northeast KDAIS KC-256 AY912247 (Lindl.) J. J. Sm. India and southern China throughout Indochina to Malaysia (Malay Peninsula), Indonesia (Sumatra), and East Malaysia (Sabah) C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 7

Table 1. (Continued) Taxa and systematic Geographical Source GenBank classification a distribution accession no. Section Stauroglottis (Schauer) Benth. Phalaenopsis equestris Philippines and Taiwan KDAIS KC-75 AY912225 (Schauer) Rchb.f. Phalaenopsis celebensis endemic to Indonesia KDAIS KC-482 AF537014 Sweet (Sulawesi) Phalaenopsis lindenii Loher endemic to the Philippines KDAIS KC-119 AY912234 Outgroups Aerides multiflora Roxbury widespread from Nepal to KDAIS Van-10 AY912258 Indochina Amesiella philippinensis endemic to the Philippines KDAIS KC-46 AY912259 (Ames) Garay Ascocentrum ampullaceum endemic to Nepal and KDAIS Van-24 AY912260 (Lindl.) Schltr. Thailand Gastrochilus japonicus endemic to Taiwan and KDAIS KC-69 AY228503 (Makino) Schltr. Japan Haraella retrocalla endemic to Taiwan KDAIS KC-70 AY912261 (Hayata) Kudo Neofinetia falcata from Japan and Korea KDAIS Aer-36 AY912262 (Thunb.) H.H. Hu to the Ryukyu Is. (Jpn.) Paraphalaenopsis labukensis endemic to Borneo KDAIS KC-121 AF537028 (P.S. Shim) A. Lamb & C.L. Chan. Renanthera matutina widespread from Thailand, KDAIS Van-33 AY912263 (Blume)Lindl. Sumatra, Java and Malaysia Rhynchostylis gigantea Indochinese peninsula KDAIS KC-45 AY912264 (Lindl.) Ridley Thrixspermum formosanum endemic to Taiwan KDAIS KC-78 AY912265 (Hayata) Schltr. Tuberolabium kotoense Yamam. Taiwan and the Philippines KDAIS KC-68 AF537003 Vanda coerulescens Griffith China, Burma, and Thailand KDAIS Van-29 AY912266 Vandopsis lissochiloides Thailand to Indonesia KDAIS Van-7 AY912267 (Gaudich) Pfitzer and the Philippines a The systematics of Phalaenopsis are based on Christenson (2001). b Plant materials were cultivated at the Kaohsiung District Agricultural Improvement Station, Taiwan. respectively. The sequence of the long PCR deleted fragment included the ITS1 region, product was unique from other Phalaenopsis and parts of it included 5.8S rDNA. The species as well as the 13 outgroup species boundaries of the internal transcribed spacers studied after phylogenetic analyses based on (ITS1 and ITS2) and nrDNA coding regions both the distance method and maximum in the 66 taxa studied were determined by parsimony method (data not shown). There- comparison to several published sequences fore, the short PCR product of P. cochlearis obtained from a range of angiosperms was selected for analysis in the following (Takaiwa et al. 1984, 1985; Kiss et al. study. The short ITS sequence of P. cochle- 1989a; Kiss et al. 1989b; Baldwin 1992). aris showed a 237-bp deletion. Parts of the The accession numbers of the 53 species of 8 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 9 the genus Phalaenopsis plus 13 outgroup the MP strict consensus tree constructed from species are shown in Table 1. The ITS ITS data were generally congruent. Based on lengths obtained from Phalaenopsis species the phylogenetic trees (Figs. 1, 3), none of the and outgroup species were similar to those outgroup species was nested within the genus reported from a broad sample of Phalaenopsis. This results support that the angiosperms (Baldwin et al. 1995). Sequences genus Phalaenopsis as described by Christen- from the 53 Phalaenopsis species plus 13 son (2001) is a monophylum. Within the genus outgroup species of related genera were Phalaenopsis, two major clades were shown. aligned and resulted in 719 characters which The first clade comprised the subgenus Poly- were subdivided as follows: the ITS1 spacer chilos and sections Phalaenopsis and Stauro- region comprised 262 characters among glottis of the subgenus Phalaenopsis (with which 165 were variable sites and 113 were bootstrap values of 63% in the NJ tree and potentially parsimony informative sites, the 59% in the MP strict consensus tree); and the ITS2 spacer region comprised 294 characters second clade consisted of sections Esmeralda among which 154 were variable sites and 109 and Deliciosae of the subgenus Phalaenopsis, were potentially parsimony informative sites, as well as subgenera Proboscidioides, Parishi- and 5.8S rDNA comprised 163 characters anae, and Aphyllae (Figs. 1, 3) (with bootstrap among which 16 were variable sites and 8 values of 83% in the NJ tree and 81% in the were potentially parsimony informative sites. MP strict consensus tree). The genetic distances among the 53 Phalaen- The monophyly of the subgenus Polychilos opsis species were in the range of 0.00 was not supported, since parts of the subgenus between P. bellina and P. violacea to 0.137 Phalaenopsis, namely sections Phalaenopsis and between P. aphrodite and P. viridis (clone 3) Stauroglottis, were nested within the subgenus with an average of 0.073 using the 2-param- Polychilos. Within the subgenus Polychilos, eter method of Kimura (1980) (data not only the section Polychilos showed monophyly shown). supported with bootstrap values of 80% in the ITS phylogeny. The phylogenetic tree of NJ tree and 61% in the MP strict consensus tree. regions of the ITS1, 5.8S rDNA, and ITS2 Parts of section Amboinenses, namely P. hiero- used character state changes which were glyphica, P. reichenbachiana, P. bastianii, equally weighted. The phylogenetic tree con- P. pallens, P. lueddemanniana, P. fasciata, and structed following the NJ method is shown in P. pulchra, formed a clade supported with Fig. 1. Based on the MP method, the analysis bootstrap values of 99% in both NJ and MP yielded 118 equally parsimonious trees with a strict consensus trees. In addition, parts of the length of 911 steps, a consistency index (CI) of section Amboinenses, namely P. doweryensis 0.50, and a retention index (RI) of 0.76. One of and P. maculata, formed a clade with the the 118 most parsimonious trees and the strict section Fuscatae supported by bootstrap values consensus tree are shown in Figs. 2 and 3, of 90% in the NJ tree and 91% in the MP strict respectively. Bootstrap values higher than 50% consensus tree (Figs. 1, 3). are shown below/above the supported Furthermore, the monophyly of the subge- branches for both the NJ and MP trees. nus Phalaenopsis was also not supported in Among Phalaenopsis species, the NJ tree and this study. Within the subgenus Phalaenopsis, b Fig. 1. Neighbor-joining tree of 53 Phalaenopsis species plus 13 outgroups obtained from sequence comparisons of the ITS region of rDNA. Numbers above the internodes indicate bootstrap values from 1025 replicates. Bootstrap values > 50% are shown on each branch. A solid circle (d) on the tree indicates that this species was traditionally treated as the genus Doritis. Solid squares (j) on the tree indicate that these species were traditionally treated as the genus Kingidium 10 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 11 the monophyly of the section Phalaenopsis was section Deliciosae of the subgenus Phalaenop- supported with bootstrap values of 100% in sis and parts (P. minus, P. braceana, and the NJ tree and 99% in MP strict consensus P. taenialis) of the subgenus Aphyllae were tree. Section Stauroglottis was shown not to be treated as parts of the genus Kingidium (Sei- monophyletic. Parts of the section Stauroglot- denfaden 1988b). Recently, Christenson (2001) tis, namely P. equestris and P. lindenii, showed presented a generic revision of Phalaenopsis a sister group relationship to the section and treated Doritis as a synonym of the genus Phalaenopsis supported by bootstrap values Phalaenopsis based on the high hybrid fertility of 88% in the NJ tree and 67% in the MP and similar morphology of microspores be- strict consensus tree. Furthermore, section tween P. pulcherrima (syn. Doritis pulcherrima) Deliciosae was not monophyletic (Figs. 1, 3). and parts (P. lobbii or P. parishii)ofPhalaen- Sections Deliciosae and Esmeralda of the opsis (Aoyama et al. 1994, Christenson 2001). subgenus Phalaenopsis were both separated In addition, the genus Kingidium was also from most species of the subgenus Phalaenop- treated as a synonym of the genus Phalaenopsis sis, namely sections Phalaenopsis and Stauro- based on the fact that it shares small subsac- glottis. Instead they were to be shown closely cate lip bases with parts of Phalaenopsis, related to the subgenera Parishianae, Aphyllae, namely the subgenus Aphyllae, as well as it and Proboscidioides. has four pollinia as have parts of Phalaenopsis, In addition, subgenus Parishianae was not namely the subgenera Proboscidioides, Aphyl- shown to be monophyletic either since the lae, and Parishianae (Christenson 2001). bootstrap value in the NJ tree was low Molecular data from the ITSs of nrDNA (<50%) (Fig. 1), and its members did not showed that outgroup species were not nested form a clade in the MP strict consensus tree within the genus Phalaenopsis. In addition, (Fig. 3). Furthermore, there was conflict be- traditional Kingidium and Doritis species were tween the phylogenies of the subgenus Aphyl- not separated from Phalaenopsis species. lae depicted by the NJ and MP strict consensus Therefore, the monophyly of the genus Pha- trees based on ITS data. The NJ tree showed it laenopsis as described by Christenson (2001) to be monophyletic supported with a moderate was supported in the present study. bootstrap value (of 68%), but its members did Infrageneric relationships within Phalaen- not form a clade in the MP strict consensus opsis tree (Figs. 1, 3). As for the monotypic subge- (1) Subgenera Proboscidioides, Aphyllae, and nus Proboscidioides, P. lowii had a sister rela- Parishianae tionship to the subgenus Aphyllae in NJ tree, Molecular data from ITS sequences showed and was nested within the subgenus Aphyllae that the monotypic subgenus Proboscidioides, in the MP strict consensus tree (Figs. 1, 3). namely P. lowii, formed a clade with the subgenera Aphyllae and Parishianae plus the sections Esmeralda and Deliciosae of subgenus Discussion Phalaenopsis. The result is in agreement with Monophyly of the genus Phalaenopsis the morphological characteristics of this clade, Phalaenopsis pulcherrima was previously bearing four separate pollinia, as well as with treated as a member of the genus Doritis the fact that they are geographically distrib- (Seidenfaden 1988a), while members of the uted in close proximity to one another (Fig. 4, b Fig. 2. One of 118 most parsimonious trees of 53 Phalaenopsis species plus 13 outgroups obtained from sequence comparisons of the ITS region of rDNA. Bootstrap values > 50% are shown on each branch. A solid circle (d) on the tree indicates that this species was traditionally treated as the genus Doritis. Solid squares (j) on the tree indicate that these species were traditionally treated as the genus Kingidium 12 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 13

Table 1) (Sweet 1980, Christenson 2001). This Phalaenopsis, the rest of subgenus Phalaenop- four-pollinia clade, namely subgenera Aphyllae sis, namely sections Phalaenopsis and Stauro- and Parishianae, plus the sections Esmeralda glottis, formed a monophyletic group and Deliciosae of subgenus Phalaenopsis,is supported by moderate bootstrap values based separate from two-pollinia clade, namely sub- on the ITS sequences. The close relationship genus Polychilos and sections Phalaenopsis and between sections Phalaenopsis and Stauroglot- Stauroglottis of subgenus Phalaenopsis based tis was also supported by morphological on phylogenetic tree derived from ITS data. characters of sharing similar geographic Based on the biogeography of Phalaenopsis, ranges, small chromosome sizes, and flowers the four-pollinia clade is separated from the lacking transversely barred patterns (Christen- two-pollinia clade by oceans/straits. As for son 2001), RAPD analyses (Chen et al. 1995), Phalaenopsis species of the Malay Peninsula, and the IGS sequence of 5S rDNA (Kao 2001). the border between the two-pollinia clade and Within subgenus Phalaenopsis, the mono- the four-pollinia clade is approximately lo- phyly of section Phalaenopsis was supported in cated at the Isthmus of Kra. This region is a the study. The section Phalaenopsis bears famous phytogeographical break between SE flowers with broad petals, which are much Asia and Malaysia (Van Steenis 1950). broader than the sepals. Additionally, they Within the four-pollinia clade, neither bear prominent, erect, somewhat glossy calli subgenus Aphyllae nor Parishianae were (Christenson 2001). Furthermore, based on monophyletic based on the ITS sequences. geographical distributions (Sweet 1980, Chris- Furthermore, the monotypic subgenus Probos- tenson 2001), and molecular data of the cidioides, namely P. lowii, had a close relation- intergenic spacer (IGS) of 5S nrDNA (Kao ship to the subgenus Aphyllae. Therefore, the 2001), the monophyly of section Phalaenopsis unique characters in P. lowii, namely the long was supported as well. Monophyly of section beak-like rostellum and lateral lobes of the lip Stauroglottis was not supported in this study, in the form of recurved hooks, might have since P. celebensis was unique from other been overemphasized in the systematics of members of the section Stauroglottis. Accord- Phalaenopsis proposed by Christenson (2001). ing to the geographical distribution of this In addition, section Deliciosae of the subgenus section, P. celebensis (distributed in Sulawesi, Phalaenopsis was not monophyletic either. Indonesia) was also separated from other (2) Subgenus Phalaenopsis species of the section Stauroglottis (distributed This subgenus is subdivided into four sections, in the Philippines) (Christenson 2001). Fur- namely sections Phalaenopsis, Deliciosae, thermore, section Deliciosae was not shown to Esmeralda, and Stauroglottis, based on the be monophyletic based on ITS sequence either. Christenson’s systematics of Phalaenopsis. (3) Subgenus Polychilos This subgenus is characterized by having a This large subgenus is subdivided into the four single callus (with the exception of the three sections Polychilos, Fuscatae, Amboinenses, and species of the section Deliciosae) and smooth Zebrinae based on the systematics of Christen- lateral lobes of the lip (Christenson 2001). son (2001). The subgenus is characterized by Monophyly of the subgenus Phalaenopsis was having fleshy, long-lasting flowers with two not supported in this study. Excluding sections pairs of calli on the lip (biseriate), the lateral Deliciosae and Esmeralda of the subgenus lobes of the lip producing a raised tooth along b Fig. 3. The strict consensus parsimonious tree of 53 Phalaenopsis species plus 13 outgroups obtained from sequence comparisons of the ITS region of rDNA. Bootstrap values > 50% are shown on each branch. A solid circle (d) on the tree indicates that this species was traditionally treated as the genus Doritis. Solid squares (j) on the tree indicate that these species were traditionally treated as the genus Kingidium 14 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences

Fig. 4. Correlation between the biogeography and phylogenetic clades among five subgenera of Phalaenopsis the leading edge, and by two pollinia (Christen- and P. maculata, formed a unique clade and son 2001). In the present study, the monophyly were separated from other members of the of subgenus Polychilos was not supported, since subgenus Polychilos. Furthermore, ITS data parts of the subgenus Phalaenopsis, namely also revealed no differentiation between sec- sections Phalaenopsis and Stauroglottis, were tions Amboinenses and Zebrinae. In addition, nested within subgenus Polychilos. Within sub- within section Amboinenses, seven species, genus Polychilos, section Polychilos was mono- namely P. bastianii, P. pallens, P. hieroglyph- phyletic. The result is in agreement with the ica, P. reichenbachiana, P. lueddemanniana, morphological characters of this section, which P. fasciata, and P. pulchra, distributed in the features a fleshy flattened rachis (with the Philippines, formed a subclade and were sepa- exception of P. mannii), non-fragrant flowers, rated from other members of the clade. The petals narrower than sepals, a triseriate callus, a result is in agreement with the geographical slightly saccate lip base, a transversely lunate distribution of those species which are distrib- mid-lobe of the lip, a lip base continuous with uted in the Philippines (Sweet 1980, Christenson the column foot, and a pair of knee-like 2001) as well as the traditional classification of projections at the base of the column (Christen- seven species (with the exception of P. bastianii, son 2001). However, other sections of subgenus a newly discovered species) which were treated Polychilos, namely Amboinenses, Zebrinae, and as one species, P. lueddemanniana (see Chris- Fuscatae, were not monophyletic. Based on ITS tenson 2001). Until Sweet (1968, 1969), this data, the section Fuscatae and two species of the highly variable species was fully resolved and section Amboinenses, namely P. doweryensis treated as different species. C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences 15

Conclusions oghue M. J. (1995) The ITS region of nuclear ribosomal DNA: a valuable source of evidence In conclusion, our molecular data offer new on angiosperm phylogeny. Ann. Missouri Bot. perspectives on the phylogeny among species of Gard. 82: 247–277. Phalaenopsis. The ITS region of nrDNA Bayer R. J., Soltis D. E., Soltis P. S. (1996) Phylo- appears to be adequate for resolving infragen- genetic inferences in Antennaria (Asteraceae: eric relationships in this group. The results from Gnaphalieae: Cassiniinae) based on sequences this study concurred in that the genera Doritis from nuclear ribosomal DNA internal transcribed and Kingidium can be treated as part of the spacers (ITS). Amer. J. Bot. 83: 516–527. genus Phalaenopsis. Neither subgenus Aphyllae Chen W. H., Fu Y. M., Hsieh R. M., Tsai W. T., nor Parishianae were monophyletic, and they Chyou M. S., Wu C. C., Lin Y. S. (1995) clustered with subgenus Proboscidioides plus Application of DNA amplification fingerprint- ing in the breeding of Phalaenopsis orchid. In: sections Esmeralda and Deliciosae of subgenus Terzi M., Cella R., Falavigna A. (eds.) Current Phalaenopsis. This clade has the same characters issues in plant molecular and cellular biology. of morphology with four pollinia and similar Kluwer Academic Publishers, Dordrecht, Bos- biogeographies. In addition, neither subgenus ton, and London, pp. 341–346. Phalaenopsis nor Polychilos were monophyletic Christenson E. A. (2001) Phalaenopsis. Timber based on this study. Within subgenus Phalaen- Press, Portland, OR. opsis, only section Phalaenopsis was highly Cox A. V., Pridgeon A. M., Albert V. A., Chase M. supported as being monophyletic. As for the W. (1997) Phylogenetics of the slipper orchids subgenus Polychilos, only section Polychilos (Cypripedioideae, Orchidaceae): nuclear rDNA was moderately supported as being monophy- ITS sequences. Pl. Syst. Evol. 208: 197–223. letic. In short, based on ITS data we support the Doyle J., Doyle J. (1987) A rapid DNA isolation systematics of the genus Phalaenopsis on the procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15. generic level but not on the subgeneric or Felsenstein J. (1985) Confidence limits on phylog- sectional levels for most groups as described enies: an approach using the bootstrap. Evolu- by Christenson (2001). tion 39: 783–791. This research was supported by funding from Fitch W. M. (1971) Towards defining the course of the National Science Council, Executive Yuan, evolution: minimum change for a specific tree R.O.C. We are grateful to Director F. H. Lin for topology. Syst. Zool. 20: 406–416. his valuable comments and helpful discussion in the Hall T. A. (1999) BioEdit: a user-friendly biological course of the study. sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41: 95–98. References Hillis D. M., Bull J. J. (1993) An empirical test of Aoyama M., Kojima K., Kobayashi M. (1994) bootstrapping as a method for assessing confi- Morphology of microspores in Phalaenopsis dence in phylogenetic analysis. Syst. Biol. 42: hybrids. Kinki Chugoku Agr. Res. 88: 49–53. 182–192. Baldwin B. G. (1992) Phylogenetic utility of the Kao Y. H. (2001) Phylogeny of Phalaenopsis internal transcribed sequences of nuclear ribo- species based on 5S rDNA intergenic sequences. somal DNA in plants: an example from the Institute of Botany National Taiwan University, Compositae. Molec. Phylogenet. Evol. 1: 3–16. Taipei, Taiwan, Master’s thesis. (in Chinese). Baldwin B. G. (1993) Molecular phylogenetics of Kimura M. (1980) A simple method for estimat- Calycadenia (Compositae) based on ITS se- ing evolutionary rates of base substitution quences of nuclear ribosomal DNA: chromo- through comparative studies of nucleotide somal and morphological evolution reexamined. sequences. Molec. Evol. 16: 111–120. Amer. J. Bot. 80: 222–238. Kiss T., Kis M., Solymosy F. (1989a) Nucleotide Baldwin B. G., Sanderson M. J., Porter J. M., sequence of a 25S rRNA gene from tomato. Wojciechowski M. F., Campbell C.S., Don- Nucl. Acids Res. 17: 796. 16 C. C. Tsai et al.: Molecular phylogeny of Phalaenopsis based on ITS sequences

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