Bull. Natl. Mus. Nat. Sci., Ser. B, 37(1), pp. 9–22, February 22, 2011

Taxonomic Reconsideration and Phylogeographic Implication for yamashitae ()

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, 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 in the 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 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 Nertera in tribe , while it was included in a clade of the genus Ophior- rhiza in tribe . The present study, in , 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 ), 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 , 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 (GK11029) culti- vated in Tsukuba Botanical Garden, National Museum of Nature and Science. Bar5 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 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 psbA5R (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 psbA5R 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.: Trinidad, AB559539 (a) Luzon Is.PHILIPPINES: Banaue, Ifuga, Borneo: Kinabaru, SabahMALAYSIA, AB559531 (D) Cibodas, Cianjur Java: , 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 Local Alignment Search Tool (BLAST) ver. (Felsenstein, 1985). One thousand replicates of 2.2.18 (Altschul et al., 1997) in the DDBJ/ heuristic searches, with the TBR branch swap- EMBL/GenBank database (http://blast.ddbj.nig. ping and MULTREES options off, were per- ac.jp/top-j.html) on 23 February 2010. Following formed to calculate bootstrap values. Strict con- the BLAST results, we selected tribe members sensus trees of the output tree files from PAUP that showed high similarity to N. yamashitae in were generated by TREEVIEW (Page, 1996). the examined three regions, and their sequence data deposited in the DNA database were added Floral anatomical and morphological observa- to the present phylogenetic analyses under the tions in Nertera yamashitae accession numbers shown in Table 2. Corolla morphology of N. yamashitae was observed based on living plants and herbarium Phylogenetic analysis specimens (GK 9854 and 11029 in TNS). Num- DNA sequences were aligned using the pro- ber of ovules was counted on a mature flower gram ClustalX 1.8 (Thompson et al., 1994) and (GK 11029) fixed in FAA solution after cultiva- then manually adjusted. Phylogenetic analyses tion in the Tsukuba Botanical Garden of National were conducted based on a maximum parsimony Museum of Nature and Science. The ovary was (MP) criterion using PAUP* version 4.0b10 torn using a pair of tweezers, the number of (Swofford, 2002). First, to verify the phylogenetic ovules counted, and their position observed and position of N. yamashitae at the tribe level, MP photographed under a light microscope. phylogenetic analyses based on ITS and atpB–rbcL were independently conducted with Results members of tribes Anthospermeae including Nertera species, Ophiorrhizeae including Ophi- Sequence similarity searches of ITS, atpB–rbcL, orrhiza, and Urophylleae, which is thought to be and matK/trnK a sister group of tribe Ophiorrhizeae (Rydin et In two plants of Nertera yamashitae collected al., 2009a, 2009b). Second, to elucidate the phy- from two different river systems on Amami Is- logenetic position of N. yamashitae among phy- land, no intraspecific sequence variation was de- logenetically closely related species, MP phylo- tected in the three sequences of ITS, atpB–rbcL, genetic analysis was conducted based on com- and trnK/matK. The results of the sequence simi- bined data of ITS, atpB–rbcL, and trnK/matK. larity searches using BLAST are summarized in The combinability of the two cpDNA regions Table 3. The BLAST analyses indicated that ITS was assessed using the incongruence length dif- (684 bp) of N. yamashitae had highest similarity ference (ILD) test (Farris et al., 1994), with the to 18 accessions of six Ophiorrhiza species in partition homogeneity test implemented in Japan and Taiwan. An accession of N. dichondri- PAUP* version 4.0b10 (Swofford, 2002). Indels folia Hook. f. (DQ501279; Wright et al., 2006) were treated as missing data. Characters were was 91st in the BLAST list. The atpB–rbcL (1149 treated as unordered, and character transforma- bp) of N. yamashitae had the highest similarity to tions were weighted equally. The branch collapse 17 accessions of six Ophiorrhiza species in option was set to collapse at a minimum length Japan and Taiwan. An accession for atpB–rbcL of zero. A heuristic parsimony search was per- of Nertera sp. (DQ131755; J. F. Manen, unpubl. formed with 200 replicates of random additions data) was 78th in the BLAST list. The trnK/matK Taxonomy and Phylogeography of Nertera yamashitae 13 (type) (II) (II) (II) (II) (II) (II) (II) (II) (III) (IV) (III) (V) (V) (V) (V) (V) (VI) (VI) (VII) (VI) (VI) (VII) (VI) (IX) (X) (XI) (XII) (XII) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 matK / trnK — — — b (j)(j) AB270601 (j) AB270601 (j) AB270601 (k) AB270601 (k) AB270601 (j) AB270601 (j) AB270601 (l) AB270601 (l) AB247245 (l) AB247121 (m) AB247245 (m) AB257122 (m) AB257122 (m) AB257122 (m) AB257122 (n) AB257122 (n) AB247246 (n) AB247246 (n) AB247247 (n) AB247246 (o) AB247246 (p) AB247248 (l) AB247246 (l) AB247250 (q) AB247249 (q) AB247252 (q) AB247253 AB247253 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 (type) 1 2 2 rbcL – Accession no. AJ234028 —— —— AJ233997 —— AJ233995 —— —— atpB (H)(H)(H) AB269877 (H) AB269877 (H) AB269877 (H) AB269877 (I) AB269878 (I) AB269878 (J) AB269877 (K) AB269877 (K) AB247238 (K) AB247238 (K) AB247238 (K) AB257115 (K) AB257115 (K) AB257115 (K) AB257115 (K) AB257115 (K) AB247239 (L) AB247239 (L) AB247239 (K) AB247239 (K) AB247239 (M) AB247240 (N) AB247241 (O) AB247238 (P) AB247238 (P) AB247243 AB247243 AB247243 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 4 4 ITS (type) sequences according to the DDBJ/EMBL/GenBank database matK / trnK and rbcL – NKC004(RYU) AB247339 NK061456(RYU)NKT0128(RYU)NKT0129(RYU)NK061458(RYU)NKT0127(RYU)NK061457(RYU) AB269875 NKT0104(RYU) AB269875 NKT0107(RYU) AB269875 NKT0111(RYU) AB269875 NK061419(RYU) AB269876 NK050501(RYU) AB269876 NKT0113(RYU)NK050790(RYU) AB269874 NKT0118(RYU) AB269874 NKT0120(RYU) AB269874 NKT0119(RYU) AB269874 NKT0121(RYU) AB269874 NKT0122(RYU) AB269874 NKT0123(RYU) AB269874 NKT0124(RYU) AB269874 GK3989(TNS) AB269874 NKG001(RYU) AB269874 AB247258 NKC002(RYU) AB247258 AB269874 AB269874 AB247259 AB247260 AB247339 atpB L.f. Bremer 3039(UPS) EU145355 Hook.f. Munzinger 532(MO) AF257930 (Hatus.) Koh Nakam.,(Hatus.) Koh NK061455(RYU) AB269875 Benth. ex ThwaitesBenth. ex Thor 601(S) EU145376 Hook. B & Bremer 937(S) EU145375 Masam. NKC001(RYU) AB247336 Bl. NKT01015(RYU) AB247257 L. Bremer 2201(UPS) EU145377 (Mutis ex L.f.) Druce(Mutis ex — AF257927 Merr. 233(AAU) Kjeldsen & Poulsen EU145378 Species Voucher Valeton Axelius 343(S)EU145374 Axelius Valeton Denda, Kameshima et Yokota NKT0126(RYU) AB269875 Nertera championii Ophiorrhiza elmeri neocaledonica bracteata Lerchea zeylanicus Neurocalyx Ophiorrhiza mungos Ophiorrhiza amamiana Ophiorrhiza japonica Ophiorrhiza hayatana herbaceum a Table 2. classification of cited species and their ITS, Tribal Tribe Ophiorrhizeae Anthospermeae 14 Goro Kokubugata et al. (type) (XIII) (XIV) (XV) (XV) (XVI) (XVII) (XVII) (XVII) (XVII) 5 5 5 5 5 5 5 5 5 matK / (2009a, 2009b); trnK — — — — — — — — et al. b (q)(q) AB247254 (r) AB247255 (r) AB247256 (s) AB247256 (t) AB247251 (t) AB247150 (t) AB247150 (t) AB247150 AB247150 3 5 5 5 5 5 5 5 5 5 1 1 1 (type) 2 2 2 2 Rydin 1 (2006). : rbcL – et al. Accession no. —— EU145567 AJ233996 EU145337 —— AJ233998 EU145338 —— AJ234001 AJ234002 atpB Nakamura (Q)(R)(S) AB247243 (T) AB247243 (U) AB247244 (V) AB247244 (V) AB247242 (W) AB247147 (X) AB247147 AB247147 AB247147 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 ITS (type) (GenBank unpublished data); (GenBank unpublished et al. NKC005(RYU)NKT0142(RYU)NKH002(RYU)NKT0137(RYU)GK3979(TNS) AB247338 AB247342 AB247273 AB247274 AB247272 Table 2. (Continued) CL Anderson 4 (Blume) Bremek. Ridsdale 2471(L) EU145384 Ridl. Ridsdale 2473(L) EU145380 (S. Moore) Bremek. Bremer 2090(UPS) EU145386 (Masam.) H.S. Lo NKW001(RYU) AB247340 (Valeton) Axelius(Valeton) 316(S) Axelius EU145381 (Hiern) Bremek. Lantz 123(UPS) EU145385 Bremek. Beaman Beaman 7950(S) EU145388 (Wight) Thwaites(Wight) Lundqvist 11085(UPS) EU145389 (Reinw. ex Blume) Korth. ex (Reinw. Boeea 7887(S) — DQ131793 Masam. NKC003(RYU) AB247337 Ohwi NKT0141(RYU) AB247341 (Standl.) Standl. 736(UPS) Clark & Watt EU145383 (Merr.) Bremek.(Merr.) — EU145387 Champ. ex. Benth.Champ. ex. NKH001(RYU) AB247273 Species Voucher Kurz 55(S) J.B.H. EU145379 Manen (GenBank unpublished data); Manen (GenBank unpublished 3 capitellatum Xanthophytum corymbosus Maschalocorymbus symplocoides Pauridiantha suberosa Praravinia arboreum Urophyllum Urophyllum ellipticum Urophyllum Xanthophytum borneense Xanthophytum paucinervis Pauridiantha leucocarpa Pravinaria Ophiorrhiza kuroiwae Ophiorrhiza michelloides Ophiorrhiza pumila bifida Spiradiclis Ophiorrhiza hayatana Amphidasya ambigua a Tribe Bremer and Manen (2000); Sequences in the DDBJ/EMBL/GenBank database; letters parentheses indicate types of three loci; references are as follows 2 Taxonomic treatment follows Bremer and Manen (2000). treatment follows Taxonomic a b Ophiorrhizeae Urophylleae Taxonomy and Phylogeography of Nertera yamashitae 15

Table 3. The top five accessions with high similarity to the sequences of Nertera yamashitae using BLAST and their localities

Species Locality Locusa Accession Score Identities no.b (bits)

ITS

Ophiorrhiza amamiana JAPAN (Okinawa Is.) ITS(I) AB269876 1144 659/684 (96%) Ophiorrhiza amamiana JAPAN (Amami, Tokunoshima, ITS(H) AB269875 1136 658/684 & Okinawa Isls. in the Ryukyus) (96%) Ophiorrhiza japonica TAIWAN ITS(N) AB247260 1128 657/684 (96%) Ophiorrhiza japonica JAPAN (Shikoku, Kyushu & Amami, ITS(K) AB269874 1120 656/684 Tokunoshima, Okinawa, Ishigaki (95%) & Iriomote Isls. in the Ryukyus) Ophiorrhiza japonica TAIWAN ITS(M) AB247259 1112 655/684 (95%) atpB–rbcL

Ophiorrhiza japonica JAPAN (Tokunoshima & atpB–rbcL(m) AB257115 1132 597/608 Okinawa Isls.) (98%) Ophiorrhiza hayatana TAIWAN atpB–rbcL(q) AB247243 1132 597/608 (98%) Ophiorrhiza michelloides TAIWAN atpB–rbcL(s) AB247242 1132 597/608 (98%) Ophiorrhiza japonica JAPAN (Iriomote Is.); Taiwan atpB–rbcL(p, l) AB247241, 1132 597/608 AB247238 (98%) Ophiorrhiza amamiana JAPAN (Amami, Tokunoshima, atpB–rbcL(j) AB269877 1124 596/608 & Okinawa Isls. in the Ryukyus) (98%) matK/trnK

Ophiorrhiza michelloides TAIWAN matK/trnK(XVI) AB247251 4220 2276/2335 (97%) Ophiorrhiza japonica JAPAN (Ishigaki & Iriomote Isls. matK/trnK(VI) AB247246 4209 2274/2334 in the Ryukyus) (97%) Ophiorrhiza hayatana TAIWAN matK/trnK AB247254, 4205 2274/2335 (XIII, XIV) AB247255 (97%) Ophiorrhiza japonica JAPAN (Iriomote Is. in the Ryukyus) matK/trnK(VII) AB247248 4201 2273/2334 (97%) Ophiorrhiza hayatana TAIWAN matK/trnK(XI) AB247252 4197 2273/2335 (97%)

a Letters in parentheses indicate locus type in Table 2. b Sequences in the DDBJ/EMBL/GenBank database. locus (2328 bp) of N. yamashitae had highest ITS sequence (Fig. 2), 233 of the 313 variable similarity to 19 accessions of six Ophiorrhiza characters were parsimony-informative, and 305 species in Japan and Taiwan. No accession for equally most parsimonious trees of 726 steps trnK/matK of Nertera species was found in the were obtained, with a consistency index (CI) DDBJ/EMBL/GenBank database. 0.65, a retention index (RI)0.87, and a rescaled consistency index (RC)0.56. In the MP analy- Phylogenetic position of Nertera yamashitae at ses at tribe level based on the atpB–rbcL se- the tribe level quences (Fig. 3), 110 of the 228 variable charac- In the MP analyses at tribe level based on the ters were parsimony-informative, and 216 equal- 16 Goro Kokubugata et al.

Fig. 2. Unrooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera yamashitae in tribes Anthospermeae, Ophiorrhizeae, and Urophylleae based on the ITS locus. Species with underlines and asterisks are members of tribes Ophiorrhizeae and Anthospermeae, respectively (Bremer and Manen, 2000). Numerals above branches indicate bootstrap percentages (50%). Letters in parentheses indicate loci types in N. yamashitae, other Nertera species, and Ophiorrhiza species (Tables 1 and 2). ly most parsimonious trees of 288 steps were ob- (Hatus.) Koh Nakam., Denda, Kameshima et tained (CI0.89, RI0.95, and RC0.85). Both Yokota, O. japonica Bl., O. hayatana Masam. of the unrooted strict consensus trees based on and O. michelloides (Masam.) H.S. Lo. the ITS and atpB–rbcL loci indicated that N. ya- mashitae was not positioned in a clade with other Phylogenetic relationship of Nertera yamashitae Nertera species nor in a clade of Anthospermeae to the other Ophiorrhiza species (Figs. 2 and 3). On the other hand, N. yamashitae To analyze the phylogenetic relationship with- was included in a clade with Ophiorrhiza species in the subclade consisting of N. yamashitae and and Spiradiclis bifida (tribe Ophiorrhizeae) based four Ophiorrhiza species (Figs. 2 and 3), the MP on analysis of the ITS sequence (BP99.9%; analysis was conducted based on the full set of Fig. 2). Based on the atpB–rbcL sequence (Fig. ITS, atpB–rbcL, and matK/trnK loci with other 3), a clade consisting of N. yamashitae and Ophi- four Ophiorrhiza species, namely O. kuroiwae orrhiza species had high bootstrap support Ohwi, O. pumila Champ. ex Benth., O. longiflora (97.2%). In both of the unrooted strict consensus Blume and an unknown Ophiorrhiza species as trees, N. yamashitae included in a subclade with an outgroup following the two independent four Ophiorrhiza species of O. amamiana analyses based on ITS (Fig. 2) and atpB–rbcL Taxonomy and Phylogeography of Nertera yamashitae 17

Fig. 3. Unrooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera yamashitae in tribes Anthospermeae, Ophiorrhizeae, and Urophylleae based on the atpB–rbcL locus. Detail explanation given in Fig. 2.

(Fig. 3) loci. In the MP analysis, 124 of 188 Discussion variable characters were parsimony-informative, and six most parsimonious trees of 239 steps Taxonomic reconsideration of Nertera yama- were obtained, with CI0.86, RI0.94, and shitae RC0.81 (Fig. 4). Following previous taxonomic studies, the The MP analysis (Fig. 4) indicated that N. genera Nertera and Ophiorrhiza commonly have yamashitae was connected to a basal position of a two-celled ovary, but the former has a single a subclade consisting of O. amamiana, O. japoni- ovule in a locule, while the latter has many ca, O. hayatana, and O. michelloides with high ovules in a locule (e.g. Verdcourt, 1958; Liu and probability (BP99.4%). Another dwarf leaf Yang, 1998; Bremer and Manen, 2000). Further- species of O. michelloides in Taiwan was not sis- more, the former genus has an annular disk and a ter to N. yamashitae. style with stigma bilobed at the base, or no disk (Puff, 1986), a four-lobed corolla, and four sta- Floral anatomy and morphology of Nertera mens; while the latter genus has a thick disk yamashitae (Fukuoka, 1979), a style with stigma bilobed at of N. yamashitae had a four-lobed and the top, a five- or six-lobed corolla, and five or whitish corolla, a style with stigma bilobed at the six stamens (Chen and Zhu, unpubl. data). Al- top (Fig. 1) and four stamens (not shown). The though N. yamashitae was similar to other Nert- ovary of N. yamashitae was inferior, bilocular, era species in the numbers of corolla lobes and and had ca. 25 ovules in a locule, and the disk on stamens, it was similar to Ophiorrhiza species in the ovary was thick (Fig. 5). the number of ovules, the morphologies of disk and stigma. Out of these anatomical and morpho- 18 Goro Kokubugata et al.

Fig. 4. Rooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera yamashitae within the genus Ophiorrhiza based on the full dataset of ITS, atpB–rbcL, and trnK/matK loci. Numerals above branches indicate bootstrap percentages (50%). Letters in parentheses indicate the types of three loci in N. yamashitae and Ophiorrhiza species (Tables 1 and 2). logical characters, the number of ovules in an Previous phylogenetic studies at tribal or ovary locule was thought to be an important generic levels in the family Rubiaceae commonly character for delimitations at the tribes (Bremer verified that tribe Anthospermeae including Nert- and Manen, 2000) and generic level (e.g. Hooker, era spp., and tribe Ophiorrhizeae including Ophi- 1873; Bremekamp, 1966) in the family Rubi- orrhiza spp., branched at a basal position in a aceae. In terms of anatomy and morphology, N. clade of subfamily Rubioideae and that they were yamashitae should not be treated as a species of obviously not sister groups (Bremer and Manen, the genus Nertera. 2000; Bremer, 2009; Rydin et al., 2009a, 2009b). Taxonomy and Phylogeography of Nertera yamashitae 19

Fig. 5. Anatomical and morphological observations of the disk and ovules in Nertera yamashitae. Bar500 mm.

The present molecular analyses reveal that N. vation could be useful to investigate the yamashitae is apparently included in the Ophior- morphology of O. yamashitae. rhiza clade in tribe Ophiorrhizeae but is distantly related to the Nertera clade in tribe Anthosper- Phylogeography of Ophiorrhiza yamashitae meae. In the present phylogenetic trees based on ITS, For taxonomy based on the anatomical, mor- atpB–rbcL, and trnK/matK (Fig. 4) O. yama- phological and molecular results, we propose to shitae, being an insular-endemic to Amami treat N. yamashitae as a species of the genus Island of the Ryukyus, was connected to a basal Ophiorrhiza. position of a clade consisting of four Ophior- rhiza species, namely O. amamiana being en- Ophiorrhiza yamashitae (T. Yamaz.) Kokubu- demic to the central Ryukyus (Nakamura et al., gata, Koh Nakam. et Yokota, comb. nov. Ba- 2007); O. japonica widely distributing in China, sionym: Nertera yamashitae T. Yamazaki, J. Jpn. Japan, Taiwan, and Vietnam (Yamazaki, 1993); Bot. 73: 89–91, Figs. 1 and 2, 1998. and O. hayatana and O. michelloides being en- demic to Taiwan (Liu and Yang, 1998; Nakamura Fruit morphology is also a very valuable char- et al., 2006). Of four Ophiorrhiza species, O. acter to delineate the genera Nertera and Ophior- amamiana and O. japonica presently occur on rhiza; the former has succulent (drupe) and the Amami Island (Hatusima and Amano, 1994). latter has dry (dehiscent) (e.g. Liu and However, our phylogenetic analyses suggest that Yang 1998; Chen and Zhu, unpubl. data). How- O. yamashitae is not derived from O. amamiana ever, no fruit of O. yamashitae has been observed nor O. japonica. These results imply that the an- in wild in spite of long-term efforts to find it cestral species of O. yamashitae has been extinct, (Yamazaki, 1998; Hotta, 2004), and thus this and O. yamashitae could survived as a relict species is assumed to have lost ability to repro- species lineage in Amami Island. duce by means of intrapopulation crossing Previously, some species lineages assumed to (Hotta, 2004). Artificial hand-pollination be- be relicts are reported in the central part of the tween plants from different populations in culti- Ryukyu Archipelago, including Amami Group 20 Goro Kokubugata et al. and Okinawa Group for terrestrial plants (Hatu- chemical investigations of these dwarf leaf sima, 1975; Hotta, 2004), amphibians and rep- species on Amami Island and in Taiwan are nec- tiles (Hikida et al., 1989; Ota, 1998). Hikida and essary to address these hypotheses. Ota (1997) suggested that these relict species lin- eages of amphibians and reptiles migrated to the Acknowledgments central Ryukyus when the area was connected to surrounding landmasses by a landbridge via the We thank Hiroshi Yamashita (Amami, Japan), northern and southern Ryukyus. After partial Hendrian (LIPI, Indonesia), Cheng-I Huang submergence of the landbridge during the (HAST, Taiwan), Piyakaset Suksathan (Queen Pliocene, the lineages were isolated in the central Sirikit Bot. Gard., Thailand), and Monica Ryukyus and have been extinct in other areas of Suleiman (University of , Sabah) for as- this archipelago (Hikida and Ota, 1997). There is sisting on field trips. This study was conducted in a possibility that O. yamashitae might have a connection with the National Museum of Nature similar phylogeographic background to the relict and Science projects on “Integration of System- species lineages of the terrestrial amphibians and atics and Molecular Phylogenetics in all Groups reptiles in the central part of the Ryukyu Archi- of Organisms” and “Taxonomic and Conserva- pelago. tion Studies on Endangered Plant Species in Japan”, and supported in part by Grants-in-Aid Parallel evolution and ecological aspects of for Scientific Research, Program C (JSPS; No. dwarf leaf species 20510220; GK) and the 21st Century COE pro- In the Ophiorrhiza species in Japan and Tai- gram of the University of the Ryukyus (MY). wan, O. michelloides being endemic to Taiwan was regarded as a dwarf leaf species (Masamune, References 1932; Liu and Yang, 1998; Nakamura et al., 2006). Although O. yamashitae and O. michel- Altschul, S. F., Madden, T. L., Schaffer, A. 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