View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Plant Syst. Evol. 247: 203–213 (2004) DOI 10.1007/s00606-004-0133-3 A phylogenetic analysis of Apostasioideae (Orchidaceae) based on ITS, trnL-F and matK sequences A. Kocyan1, Y.-L. Qiu1,2, P. K. Endress1, and E. Conti1 1University of Zurich, Institute of Systematic Botany, Zurich, Switzerland 2University of Michigan, Department of Ecology and Evolutionary Biology, Ann Arbor, USA Received May 23, 2003; accepted January 24, 2004 Published online: May 10, 2004 Ó Springer-Verlag 2004 Abstract. The orchid subfamily Apostasioideae Introduction consists of two genera, Apostasia and Neuwiedia. To study the position of Apostasioideae within The genera Apostasia and Neuwiedia form the Orchidaceae and their intra- and intergeneric rela- subfamily Apostasioideae (Orchidaceae), with tionships, a molecular phylogenetic analysis has a total of 15 species mainly occurring in been conducted on the nuclear ITS region and the Southeast Asia. In contrast to all other two plastid DNA regions trnL-F intron and matK. orchids, they have a very simple gynostemium The two genera traditionally ascribed to Aposta- and three stamens, one of which may be a sioideae are each monophyletic. In Apostasia, staminode in Apostasia (Kocyan and Endress A. nuda, with two stamens and no staminode, is 2001). Traditionally, the subfamily is placed as sister to a clade comprising three species charac- sister to the rest of Orchidaceae (see Stern et al. terised by two stamens and one staminode. Within 1993, Kocyan and Endress 2001, Rudall and Neuwiedia, maximum parsimony analyses place Bateman 2002, for recent reviews of their N. zollingeri as sister to the clade formed by N. borneensis and N. veratrifolia. A family-wide taxonomic history). This placement is also phylogenetic analysis of matK sequences represent- supported by recent molecular (rbcL, 18S, ing all proposed subfamilies of Orchidaceae pro- nad1 b-c; Cameron et al. 1999, Cameron and duced five moderately to well-supported clades. One Chase 2000, Freudenstein et al. 2000) and non- of these clades, Apostasioideae, is sister to the clade molecular analyses (Freudenstein and Ras- formed by Vanilloideae, Cypripedioideae, Orchidoi- mussen 1999). However, little information deae and Epidendroideae. High transition-transver- has been available on the systematic structure sion ratio and the absence of stop codons in the of these two genera. De Vogel (1969) proposed individual sequences suggest that matK is at the two different sections in Apostasia based on the transition from a possibly functional gene to a presence or absence of the staminode, which pseudogene in Apostasioideae, contrary to what is represents the vestige of the median abaxial found in some other groups of Orchidaceae. stamen: sect. Apostasia, with a staminode, and Key words: Molecular phylogenetics, plastid DNA, sect. Adactylus without a staminode. This nrDNA, Orchidaceae, pseudogene, Apostasia, division was also confirmed by a recent study Neuwiedia. on floral structures and developmental pat- 204 A. Kocyan et al.: Phylogenetic analysis of Apostasioideae (Orchidaceae) terns of Apostasioideae (Kocyan and Endress increase the species sampling we also tried to 2001). The relationships among Neuwiedia extract DNA from herbarium material without species still remain to be resolved, because success. Non-apostasioid orchids were sampled in morphological results are somewhat ambigu- various botanical gardens. Nomenclature of Apos- ous (de Vogel 1969, Kocyan and Endress tasia and Neuwiedia follows that of de Vogel 2001). In order to investigate inter- and (1969). Vouchers are deposited in the Zu¨rich herbarium [Z+ZT] or in the pickled collection of intrageneric relationships within Apostasioi- A. Kocyan. ITS and matK sequences of various deae, which are to date mainly based on orchid taxa were obtained from Genbank morphological observations, we have pro- (Table 1). duced sequences from three commonly used DNA extraction and sequencing. Leaf material DNA regions: 1) the internal transcribed was dried and preserved in silica gel. Total genomic spacer (ITS) of the nuclear ribosomal DNA DNA was extracted from dried material following (e.g. Baldwin 1992, Cox et al. 1997, Conti et al. the modified CTAB procedure of Doyle and Doyle 1999, von Balthazar et al. 2000, Whitten et al. (1987) or the SDS protocol after Eichenberger et al. 2000, Gravendeel et al. 2001); 2) the plastid (2000). Apostasia nipponica was extracted with the gene matK (e.g. Johnson and Soltis 1994, 1995; DNeasyä Plant Mini Kit (Qiagen, Basel, Switzer- Conti et al. 1999; Kores et al. 2000; Whitten land). The selected DNA regions were amplified with standard polymerase chain reactions (PCR). et al. 2000; Goldman et al. 2001; Gravendeel Newly designed primers (von Balthazar et al. 2000) et al. 2001; Scho¨nenberger and Conti 2003); were used to amplify the entire ITS1, 5.8S rDNA and 3) the non coding trnL-F intergenic spacer and ITS2 region in a single reaction (Baldwin of the plastid genome (e.g. Taberlet et al. 1991, 1992). Primers c and f of Taberlet et al. (1991) were Pfosser and Speta 1999, Whitten et al. 2000). used to amplify the trnL(UAA)5¢ exon and Furthermore, a matK-data matrix representing trnF(GAA) of the chloroplast genome in a single all major orchid clades has been produced to reaction. The matK gene was amplified in two test the monophyly and the phylogenetic overlapping parts using the -19F forward position of Apostasioideae within Orchi- (CGTTCTGACCATATTGCACTATG; Kores daceae. et al. 2000, Molvray et al. 2000) and 834R reverse (AAAGACTCCARAAGATRTTG) primers, and the 580F (ACTAATACCCYATCCCATMC) and Materials and methods R1(CATTTTTCATTGCACACGRC) primers. Primer R1 anneals slightly upstream of the often Taxon sampling. Our study comprises four (out of used trnK-2R primer. Primers 580F, 834R and R1 seven) Apostasia species, three (out of eight) were newly designed for this study. The PCR Neuwiedia species, 15 representatives of other protocol used to amplify ITS, trnL-F intron and major orchid clades, as well as Blandfordia punicea matK was as follows: 35 cycles of 30 sec at 95 °C (Blandfordiaceae) and Curculigo capitulata (Hyp- denaturation, 1 min annealing (50°C for ITS, 55 °C oxidaceae) as outgroups (Table 1). Blandfordiaceae for trnL-F, 49 °C for matK), 1 min 40 sec and Hypoxidaceae have been identified as close elongation at 72 °C. Three species appeared to relatives of Orchidaceae in broad analyses of contain multiple copies of ITS, and PCR products Asparagales (Rudall et al. 1997, Chase et al. 2000, were cloned with the TOPO TA cloningÒ kit Fay et al. 2000) (Table 1). With the exception of (Invitrogen). Several clones were sequenced and Apostasia nipponica (kindly offered by T. Yukawa, subjected to phylogenetic analyses. A putative Tsukuba Botanical Garden, Amakubo, Japan) the ortholog was identified based on branch length, material was collected during field trips to Sabah/ sequence characteristic, and phylogenetic place- Malaysia and Tasmania/Australia by the first ment, and was then used in final analyses. Prior to author. The sampling that represents less than 50 % cycle sequencing, PCR products were purified with of the known species diversity may be critizised. the Qiaquickä PCR Purification Kit (Qiagen, However, all Apostasioideae species are rare, none Basel, Switzerland). For cycle sequencing BigDye is available in cultivation, and some are known Terminator Ready Reaction Kit (Applied Biosys- only from very few (old) herbarium collections. To tems) was used. Additional primers were used for A. Kocyan et al.: Phylogenetic analysis of Apostasioideae (Orchidaceae) 205 Table 1. Species analysed in this study. Subfamily names according to Pridgeon et al. (1999). Taxa Voucher number / GenBank Accession Number DNA number (AK = A. Kocyan) matK trnL-F ITS Ingroup Apostasioideae Apostasia nipponica Masam. Yukawa 99-92 / AK92 AY557215 AY557222 AY557231 Apostasia nuda R.Br. AK971126-3-01 / AK44 AY557214 AY557221 AY557230 Apostasia odorata Blume AK971109-1-01 / AK15 AY557213 AY557220 AY557229 Apostasia wallichii R.Br AK981023-1-02 / AK14 AY557212 AY557219 AY557228 Neuwiedia borneensis de Vogel AK971114-1-01 / AK19 AY557209 AY557216 AY557225 Neuwiedia veratrifolia Blume AK971115-1-01 / AK21 AY557211 AY557218 AY557227 Neuwiedia zollingeri Reichenb.f. AK971114-1-04 / AK20 AY557210 AY557217 AY557226 var. javanica (J.J.Sm.) de Vogel Cypripedioideae Cypripedium calceolus L. AK990519-1-02 / AK56 AY557208 AY557224 AY557232 Paphiopedilum glaucophyllum AK990525-1-25 / AK75 AY557205 J. J. Smith Phragmipedium longifolium Rolfe AK990525-1-02 / AK71 AY557204 Vanilloideae Cleistes rosea Lindl. AJ310006* Pogonia ophioglossoides (L.) Jussieu AJ310055* Vanilla planifolia Andr. s.n. / AK49 AJ310079* AY557223 U66819* Orchidoideae Disa glandulosa Burch. ex Lindl. AF263654* Ophrys apifera Hudson AJ310049* Lower Epidendroideae Corymborkis veratrifolia AK981020-1-01 / AK06 AY557203 (Reinw.) Blume Nervilia cf. aragoana Gaud. AJ310048* Palmorchis trilobata L.O. PTR310052* Williams in Woodson & Schery Higher Epidendroideae Aerides multiflora Roxb. 920432 # / AP02 AY557201 Calanthe vestita Lindl. AF263634* Collabium simplex Rchb.f. AK991017-1-04 / AK95a AY557200 Rhynchostylis gigantea 913013 # / AP07 AY557202 (Lindl.) Ridl. Outgroup Blandfordia punicea Sweet AK981013-1-01 / AK01 AY557206 (Blandfordiaceae) Curculigo capitulata Kuntze AK981228-1-01 / AK05 AY557207 (Hypoxidaceae) * Sequence taken from GenBank