Bull. Natl. Mus. Nat. Sci., Ser. B, 37(1), pp. 9–22, February 22, 2011 Taxonomic Reconsideration and Phylogeographic Implication for Nertera yamashitae (Rubiaceae) Goro Kokubugata1,*, Koh Nakamura2, Yumiko Hirayama1, Ching-I Peng2 and Masatsugu Yokota3 1 Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305–0005 Japan 2 Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan 3 Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903–0213 Japan * E-mail: [email protected] (Received 16 November 2010; accepted 22 December 2010) Abstract Anatomical and morphological characters and phylogenetic position of Nertera ya- mashitae were investigated with those of related species in the family Rubiaceae. In the anatomy and morphology, disk morphology and number of ovules in an ovary locule of N. yamashitae differed from those of other Nertera species, and were more similar to those of Ophiorrhiza species. In the sequence similarity search of ITS in nrDNA, and atpB–rbcL and trnK/matK in cpDNA, N. yamashitae showed the highest similarity to Ophiorrhiza species. In the MP phyloge- netic analyses based on nrITS, atpB–rbcL, and trnK/matK, N. yamashitae was outside of a clade of the genus Nertera in tribe Anthospermeae, while it was included in a clade of the genus Ophior- rhiza in tribe Ophiorrhizeae. The present study, in taxonomy, suggests that N. yamashitae should be treated as a species in Ophiorrhiza as a new combination; and in phylogeography, implies that it could be a relict species lineage in Amami Island of the Ryukyu Archipelago. Key words : dwarfism, Nertera, Ophiorrhiza, Rubiaceae, Ryukyus. it has small leaves, four stamens, and a four- Introduction lobed corolla, showing the generic-representative The genus Nertera Banks et Sol. ex Gaertn. is morphologies for Nertera (e.g. Lo, 1999; Liu and composed of ca. 16 (or six in a strict sense) Yang, 1998). This species has been thought to be species found in Malesia, Oceania and Americas; an insular endemic species of Amami Island, and and is classified in tribe Anthospermeae (sub- only three populations have been found in two family Rubioideae), being well supported by river systems in this island (Hotta, 2004). There- morphological and molecular data (Bremekamp, fore, it is treated as critically endangered by the 1966; Bremer and Manen, 2000; Bremer, 2009). Japanese Ministry of the Environment (2007). Although Heads (1996) included Nertera species After Yamazaki’s description (1998), although a in the genus Coprosma, disagreeing with most few studies on cytology (Kokubugata et al., taxonomic treatments, he still recognized it as a 1998) and conservation biology (Hotta, 2004) taxon at section level, namely sect. Nertera. As have been carried out, further taxonomical recon- the first species of the genus Nertera discovered sideration based on anatomical, morphological, in Japan, Nertera yamashitae T. Yamazaki (Fig. and molecular data has never been applied to this 1) studied herein was described based on a type species. specimen collected from Amami Island in the Dwarfism, a phenomenon in which body size Ryukyu Archipelago (Yamazaki, 1998), because is strongly reduced, is well-known in alpine 10 Goro Kokubugata et al. Fig. 1. Nertera yamashitae. (A) Habit (photographed by H. Yamashita); (B) flower in plants (GK11029) culti- vated in Tsukuba Botanical Garden, National Museum of Nature and Science. Barϭ5 mm. ecosystems (e.g. Körner, 1999). In the Ryukyus, Materials and Methods some leaf dwarfisms are known at elevations over 1,000 m on Yakushima Island in the northern Plant materials Ryukyus, and phylogenetic and their evolution- Plants of Nertera yamashitae were collected ary biology has been examined in these plants from two localities in two river systems in (e.g. Yokoyama et al., 2003; Shinohara and Amami Island (Table 1). Also, plants of N. Murakami, 2006; Tsukaya et al., 2007). Also, granadense (Mutis ex L.f.) Druce following a certain dwarf leaf species are known at altitudes strict taxonomic treatment of Liu and Yang of less than 100 m on Amami Island, such as N. (1998) in four Asian countries, and two Ophior- yamashitae studied herein, Lysimachia liukiuen- rhiza species in two Asian countries were collect- sis Hatus. (Myrsinaceae), Oxalis exilis A. Cunn. ed and used in the present molecular phylogenet- (Oxalidaceae), Rubus amamianus Hatus. et Ohwi ic analyses (Table 1). Voucher specimens were (Rosaceae), and Sphenomeris minutula Sa. Kura- deposited in the herbarium of the National Muse- ta (Lindsaeaceae) (Hatusima, 1975). In contrast um of Nature and Science (TNS). with leaf dwarfism at high elevation on Yakushi- ma Island, however, few phylogenetic studies DNA extraction, polymerase chain reaction, and have examined for the dwarf leaf species at low sequencing altitude on Amami Island without Lysimachia Fresh leaves were harvested from the plants liukiuensis (Kokubugata et al., 2010). cultivated in a greenhouse of the Tsukuba Botan- In the present study, we investigated the floral ical Garden for DNA extraction, which was con- anatomy and morphology of N. yamashitae; and ducted using the DNeasy Plant Mini Kit (Qiagen, its phylogenetic position among related species Valencia, CA) following the manufacturer’s pro- of subfamily Rubioideae based on three molecu- tocols. Total DNA samples isolated were deposit- lar loci to reconsider the taxonomic status, and to ed in the Molecular Biodiversity Research Center discuss the dwarfism of this species. of the National Museum of Nature and Science. Taxonomy and Phylogeography of Nertera yamashitae 11 In the present study, ITS1, 5.8S rDNA and ITS2 with part of the 18S rDNA (ITS) of nuclear matK / (type) ribosomal DNA (nrDNA), the intergenic spacer trnK of atpB and rbcL genes (atpB–rbcL), the matK / and maturaseK gene and adjacent trnK introns trnK (trnK/matK) of cpDNA were selected for the rbcL – molecular phylogenetic analyses. In the poly- b merase chain reaction (PCR), we amplified atpB (type) ITS using the primer set AB101 and AB102 rbcL (Douzery et al., 1999), atpB–rbcL using atpB2F – Accession no. and rbcL2R (Nakamura et al., 2006), trnK/matK atpB using trnK–3914(D)F (Johnson and Soltis, 1994) and psbA5ЈR (M. Nepokroeff, University of South Dakota, personal communication). The PCR amplifications were performed using Takara ITS (type) Ex Taq (Takara, Tokyo) with Ampdirect Plus (Shimadzu, Kyoto) on an iCycler (BioRad, Her- cules, CA). The PCR profile comprised 35 cycles a of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C for ITS, and 35 cycles of 30 s at 94°C, 30 s at 55°C, and 3 min at 72°C for atpB–rbcL and trnK/matK. After purification of PCR products using ExoSAP–IT (USB Corp., Cleveland, OH), direct sequencing was performed using the BigDye Terminator Cycle Sequencing Kit ver. 3 (ABI PRISM; Applied Biosystems, Foster City, CA), with the PCR primers AB101 and AB102, and two additional primers ITS2 and ITS3 (White et al., 1990) for the ITS; the PCR primers atpB2F and rbcL2R for atpB–rbcL; and the PCR investigated in the present study, and their localities, voucher numbers, and ITS, and their localities, voucher in the present study, investigated primers trnK–3914 (D) and psbA5ЈR and addi- tional primers of matK–300F (Yamashita and Tamura, 2000), trnK2621R (Liston and Kadereit, Ophiorrhiza 1995), matK–AF, matK–8R (Ooi et al., 1995), JAPAN, Ryukyus: Sumiyo, Amami Is., Kagoshima Sumiyo, Ryukyus: JAPAN, GK11029 Kaohsiung Taoyuan, TAIWAN: Kaohsiung Taoyuan, TAIWAN: AB559527 (A) LuzonPHILIPPINES: Bontoc, Mountain Province, GK8301 Benguet, Luzon Is.PHILIPPINES: Trinidad, AB559539 (a) Luzon Is.PHILIPPINES: Banaue, Ifuga, Borneo: Kinabaru, SabahMALAYSIA, AB559531 (D) Cibodas, Cianjur Java: INDONESIA, West AB559551(I) GK8254 GK8824 AB559543 (e) GK9647 GK10821 AB559532 (E) GK8559 GK7903 AB559528 (B) — AB559530 (C) AB559533 (E) AB559544 (f)and AB559535 (G) AB559534 (F) AB559541 (c) AB559542 (d)matK AB559545 (g) — AB559547 (i) AB559546 (h) — — –AR — — — (Kazempour Osaloo et al., 1999) and for matK/trnK. Automated sequencing was per- Sequences investigated here and registered in the DDBJ/EMBL/GenBank database; letters parentheses indicate locus type. here and registered Sequences investigated b formed on a 3130xl Genetic Analyzer (Applied Nertera Biosystems). The sets of sequence data were assembled using ATGC ver. 4.01 (GENETYX Blume Cibodas, Cianjur Java: INDONESIA, West Co., Tokyo). GK8503 AB559536Sequence AB559548 data of AB559552 ITS, atpB–rbcL, T. Yamaz.T. Naze, Amami Is., Kagoshima Ryukyus: JAPAN, GK9854 AB559526 (A) AB559538 (a) AB559550(I) and trnK/matK were deposited in the DDBJ/ Species Locality Voucher (Mutis ex L.f.) Druce(Mutis ex Nanao, Ilan TAIWAN: GK11028EMBL/GenBank AB559529 (B) AB559540 (b) — international DNA database sp. THAILAND: Ang Kang, Chiang Mai GK12391 AB559537 AB559549 AB559553 under the accession numbers shown in Table 1. GenBank accession numbers GK: G. Kokubugata (TNS). GK: G. Kokubugata a Table 1. Plant materials of Ophiorrhiza Nertera yamashitae Nertera granadense N. Ophiorrhiza longiflora 12 Goro Kokubugata et al. Sequence similarity searches and taxon sampling of sequences, with the ACCTRAN character in DNA database optimization, tree bisection–reconnection (TBR) Sequence similarity searches in the ITS, branch swapping, MULTREES, and STEEPEST atpB–rbcL, and matK/trnK regions of N. ya- DESCENT options on. Statistical support for mashitae were conducted with the program Basic each clade was assessed by bootstrap analysis