Chromosome Botany (2007) 2: 127-132 © Copyright 2007 by the International Society of Chromosome Botany

Molecular phylogeny of sensu lato () based on ITS sequences data: An evidence for the inclusion of Neisosperma

Hendrian1 and Katsuhiko Kondo1・2

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

2Author for correspondence: ([email protected]) Received October 3, 2006; accepted October 17, 2007

ABSTRACT. Molecular phylogeny of Ochrosia sensu lato (Ochrosia sensu stricto and Neisosperma) was investigated by maximum parsimony analysis of sequence data from the internal transcribed spacer (ITS) of nuclear ribosomal DNA. Twenty-one species including two varieties (25 accessions), were defined as the ingroup. Three outgroup species were selected from tribe Vinceae (Kopsia arborea, K. flavida, and Rauvolfia serpentina). The ITS sequence data showed that Ochrosia s.l. is a highly defined monophyletic group (supported with 100% bootstrap value). As shown in the strict consensus tree, all Neisosperma species were placed at the basal part of the tree with N. glomerata and N. nakaiana as the basalmost taxa. All species belong to Ochrosia s.str. were grouped into one clade and nested within Neisosperma resulting in monophyletic Ochrosia s.str. and paraphyletic Neisosperma. This suggested that Neisosperma should be included into Ochrosia, and the two groups are treated as a single genus. The analysis also showed that subdivision of Ochrosia s.l. into sections based on fruit morphology (fibrous vs. massive and thick endocarp) is not supported.

KEYWORDS: Internal Transcribed Spacer, Molecular phylogeny, Neisosperma, Ochrosia sensu lato, Ochrosia sensu stricto.

Ochrosia sensu lato comprises about 40 species (Hendrian Rauvolfia and Vinca. 2004). The species are widely spread from the Mascarene In opposition to them, Leeuwenberg (1987, 1994), and Seychelles in the west throughout south Asia, Indo- Middleton (1999), and Hendrian (2004) put cavity- China, Malesia, northern , and Pacific Islands as fruited and fibrous-fruited species together into one far east as the Marquesas and Hawaiian Islands. They genus, Ochrosia Juss. In his paper, Hendrian mentioned occur mostly in coastal areas and lowland forests. that despite the diversity in fruit type the species are all Ochrosia was proposed by Jussieu in his Genera very similar in flower and vegetative characters. Plantarum (1789). Within the genus there is a striking Other clear characters found are more useful for delim- diversity in fruit morphology, which has been the basis iting species rather than genera or sections. However, it for recognition of two sections: Ochrosia section Lactaria should be noted that it was based on morphological with a massive, thick endocarp containing two lateral characters only. cavities filled with spongy tissue, and Ochrosia section Until now no phylogenetic approach has been attempted Echinocaryon with an endocarp split into diverging fibers on genus Ochrosia s.l. exclusively, and the relationship (Mueller 1871). between Ochrosia s.str. and Neisosperma is still unclear. Since then, there has been a long and on-going debate In a paper on phylogenetic relationships within Apocy- regarding the relationship between these two groups. naceae s.l. by Potgieter and Albert (2001), Neisosperma Subdivision proposed by Muller was followed by nakaiana and came out as sister taxa Valeton (1895) - as subgenus, and Pichon (1947), who (unfortunately, in this study, each of the two ‘groups’ was added the third section - Ochrosia section Phragmochro- represented with one species only, so there was not much sia Pichon - which characterized by having both numerous to be concluded). fibers and rudimentary lateral cavities. Molecular systematics studies have led to a new and Conversely, Fosberg and Sachet (1972, 1974, 1977) better-supported framework of angiosperm phylogeny resurrected the name Neisosperma for the fibrous-fruited (Endress, Baas & Gregory 2000). The greatest advantage species, and restricted the name Ochrosia Juss. only to of molecular data in systematic appears to be the large the portion containing the type (the cavity fruited species). number of observable characters available for analysis. Thus, they treated Neisosperma and Ochrosia as two Another advantage of molecular data is the wide range of different genera. This application was then followed by substitution rates that exist across nucleotide sites. Markgraf (1979), Boiteau and Allorge (1981), and Molecular data also have the advantage that characters Forster (1993). In the most recent classification of can be selected and defined in a relatively objective Apocynaceae, Endress and Bruyns (2000) also treated manner (Hillis & Wiens 2000). Neisosperma separately from Ochrosia. The two genera Three of the eukaryotic nuclear ribosomal RNA genes were placed in tribe Vinceae, subfamily , are organized in a cluster that includes a small subunit together with Amsonia, Catharanthus, Kopsia, Petchia, gene (16S to 18S), a large subunit gene (26S to 28S), and

127 128 Hendrian and Kondo the 5.8S gene. In addition, two internal transcribed MATERIAL AND METHODS spacers (ITS1 and ITS2) lie between these genes and Plant materials Twenty-one species (25 accessions), there is an external transcribed spacer (ETS) at the 5’ end comprising ten Neisosperma species. and 11 Ochrosia s.str. of the transcribed RNA (Palumbi 1996). species. (including two varieties of Ochrosia mariannensis: The ITS region, defined as the unit containing the var. mariannensis and var. crassicarpa) were defined as ITS1, 5.8S gene, and ITS2, has proven to be valuable for the ingroup in this study. Outgroup taxa were chosen phylogenetic reconstruction in angiosperms. It has become from Rauvolfia (R. serpentina) and Kopsia (K. arborea one of the most popular sequences for phylogenetic and K. flavida), both genera belong to tribe Vinceae. inference at the generic and infrageneric levels in . Taxon names, voucher information, and GenBank According to Alvarez and Wendel (2003), during the last accessions numbers were given in Table 1 below. five years, of 244 phylogenetic papers published in the most prominent systematics and evolution journals, two- DNA isolation Leaf material was obtained from living thirds (66%) included ITS sequence data, and more than collections in botanical gardens and arboretum. Samples of one third (34%) were based exclusively on ITS sequences. 11 species were obtained from herbarium specimens. Three The favorable properties of ITS region for phylogenetic and two accessions were sequenced for Neisosperma analysis of angiosperms was discussed in several papers oppositifolia and Neisosperma nakaiana respectively. (Baldwin et al. 1995, Soltis and Soltis 1998, Alvarez and Wendel 2003) Amplification and sequencing Total genomic DNA was Further study on the Ochrosia s.l. complex is much isolated from fresh leaf tissues, silica-dried leaf material, needed. The present study aims to resolve this long- or from herbarium specimens using DNeasy Plant Mini standing question and reconstruct species relationships Kit (Qiagen) following the manufacturer’s protocol. The within Ochrosia s.l. internal transcribed spacer (ITS) region was amplified using universal ITS5 forward primer (5’-GGAAGTAAA

Table 1. List of the species studied

Species Vouchers Locality DDBJ acc. no. O. coccinea IV.A.26 (Bogor Bot. Garden) Moluccas, Indonesia AB331864 O. compta 721228 (NTBG) Oahu, Hawaii AB331873 O. kauaiensis 030324 (NTBG) , Hawaii AB331875 O. mariannensis var. mariannensis 75s25 (Waimea Arboretum) Guam AB331871 O. mariannensis var. crassicarpa 76s12 (Waimea Arboretum) Guam AB331872 Ochrosia sp. 95s123 (Waimea Arboretum) Tonga AB331870 O. mulsanti MacKee 22097 (L) AB331877 O. silvatica Sevenet 399 (L) New Caledonia AB331878 O. vitiensis Smith 9478 (L) Fiji AB331869 O. sandwicensis Spence 7 (L) Hawaii AB331874 O. grandiflora Veillon 7950 (P) New Caledonia AB331879 O. elliptica 5557 (PTBG) Kauai, Hawaii AB331876 N. oppositifolia1 IV.A.176 (Bogor Bot. Garden) Sumatra, Indonesia AB331858 N. oppositifolia2 82s318 (Waimea Arboretum) Saipan AB331859 N. oppositifolia3 970511.001 (NTBG) Tonga AB331860 N. nakaiana1 740323.001 (NTBG) Ogasawara, Japan AB331862 N. nakaiana2 75s2230 (Waimea Arboretum) Ogasawara, Japan AB331861 N. glomerata VIII.G.191(Bogor Bot. Garden) Moluccas, Indonesia AB331863 N. miana Jaffre 3273 (P) New Caledonia AB331881 N. sevenetti MacKee 31150 (L) New Caledonia AB331883 N. thiollierei MacKee 26988 (P) New Caledonia AB331882 N. kilneri Forster PIF 29925 (BRI) Queensland AB331868 N. poweri Forster PIF 26594 (BRI) Queensland AB331867 N. citrodora IV.A.200 (Bogor Bot. Garden) Papua, Indonesia AB331866 N. acuminata Leeuwenberg et al. 14542 (A) Sulawesi, Indonesia AB331865 Rauvolfia serpentina XXIV.A.XXI.10 Java, Indonesia AB331857 Kopsia arborea IV.A.52 (Bogor Bot. Garden) Java, Indonesia AB331855 Kopsia flavida IV.A.43 (Bogor Bot. Garden) Seram, Indonesia AB331856 ITS sequences in Ochrosia sensu lato (Apocynaceae) 129

AGTCGTAACAAGG-3’) and ITS4 reverse primer (5’-T were obtained by stepwise addition, with 1000 random CCTCCGCTTATTGATATGC-3’) (White et al. 1990). addition sequence replicates. Branch support was Polymerase chain reaction was performed in 20 µl, using evaluated through 1000 replicates of bootstrap analysis Promega PCR Core System I (Promega) or TaKaRa Ex (Felsenstein 1985, Kitching et al. 1998, Soltis and Soltis Taq™ (Takara Bio Inc.). Bovine Serum Albumin (BioLabs, 2003) Inc., USA) was added to the reactions. Amplification was ® carried out using a GeneAmp PCR System 2700 (Applied RESULTS AND DISCUSSION Biosystem). Amplification of some accessions was con- ITS characteristics are shown in Table 2. The ITS 1 ducted using Takara PCR Thermal Cycler Dice version region ranged from 224 base pairs (bp) in Neisosperma III, TP 600 (Takara Bio, Inc.). Cycling was performed miana to 230 bp in Neisosperma poweri, with an aligned with an initial denaturation step of 2 min at 95°C, length of 237 bp. Variable sites found were 108 (45.57%), followed by 30 cycles of 1 min denaturation at 95°C, 1 with 83 (35.02%) informative base substitution characters minute annealing at 55°C, and 2 min extension at 72°C, and 7 informative indels, ranging from 1 bp (at base with 5 min final extension at 72°C. PCR products were position: 88, 137, 155, and 212) to 4 bp (at base position: then run in 1.5% agarose gel, stained with ethidium 35-38). The G+C content was 66.08%, ranging from bromide, and purified with the QIAquick PCR Purifica- 62.99% in Ochrosia mariannensis var. crassicarpa to tion Kit (Qiagen). 72.25% in Kopsia flavida. Cycle sequencing reactions were performed using The 5.8S rRNA gene is highly conserved, with 173 BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied (89.18%) constant sites, providing only 10 (5.15%) Biosystem), according to the manufacturer’s protocol. informative base substitution characters. Only 1 informa- The same primers described above were used for these tive indel found in this region (1 bp, at base 261). In seed reactions. Templates were purified using sodium acetate/ plant sequences generally the proportion of potentially EDTA/ethanol precipitation method. The products were informative sites of 5.8 gene is about 14% (Soltis and then sent to the Center for Gene Science, Hiroshima Soltis 1998). The gene is invariant in length. All sequences University, Japan (sequenced with Automated Sequencer (except those of Kopsia flavida and Ochrosia mariannen- 3130x/Genetic Analyzer - ABI Prism). For some sis var. crassicarpa) have a length of 189 bp. The aligned accessions, products were sent to Microgen, Inc., Korea length was 194 bp. The G+C content was 56.64% - lower (sequenced with ABI 3730xl) than for the spacer regions - ranging from 56.08 in Ochrosia coccinea to 58.20 in Kopsia arborea. The Alignment and phylogenetic analysis Sequences were conserved 14 bp motif (5’-GAATTGCAGAATCC-3’) aligned using ClustalW (Thompson et al. 1994) and that can be used to differentiate flowering plants from adjusted manually by eye. Regions of ambiguous bryophytes, and several orders of algae and fungi (Jobes alignment were excluded from analysis. The sequences and Thien 1997) were found in all sequences examined. then were compared with Alstonia scholaris (GenBank The length of ITS 2 region was shorter than that of Accession no. DQ 358880) to determine boundaries of ITS 1. It ranged from 210 bp in Rauvolfia serpentina to ITS1 and ITS2. 214 bp in Ochrosia coccinea, with an aligned length of Maximum Parsimony analysis was performed using 220 bp. The G+C content was 66.06% - a little bit lower PAUP* ver. 4.0b10 (Swofford 2002). Informative charac- than that of ITS 1 - ranging from 62.15 in Ochrosia coc- ters were unordered and equally weighted. Uninformative cinea to 73.24 in Kopsia arborea. Of the 102 (46.36%) characters were excluded. A heuristic search was used to variable sites recorded there were 61 (27.73%) informa- obtain most parsimonious trees (MPT). Starting trees tive base substitution characters. Six informative indels

Table 2. Sequence characteristics for the ITS Sequences characters ITS 1 5.8S ITS 2 Combined Aligned length (bp) 237 194 220 651 Unaligned length, mean (bp) 227.36 189.21 211.5 628.07 Unaligned length, range (bp) 224 - 230 189 - 194 210 - 214 624 - 632 G+C content, range (%) 62.99 - 72.25 56.08 - 58.20 62.15 - 73.24 61.42 - 68.25 G+C content, mean (%) 66.08 56.64 66.06 63.22 Constant sites 129 (54.43%) 173 (89.18%) 118 (53.64%) 420 (64.52%) Variable sites 108 (45.57%) 21 (10.82%) 102 (46.36%) 231 (35.48%) Informative base substitutions 83 (35.02%) 10 (5.15%) 61 (27.73%) 154 (23.66%) Number of informative indels 7 1 6 14 Indel size, range (bp) 1 - 4 1 1 - 5 1 - 5 Total informative characters 90 11 67 168 130 Hendrian and Kondo were found, ranging from 1 bp (at base position: 480 and supported with 100% bootstrap value, and clearly 596) to 5 bp (at base position: 641-645). There was also separated from the outgroup species Kopsia arborea, 1 autapomorphic indel at base position 637-638 (for Kopsia flavida and Rauvolfia serpentina. All Neisosperma Ochrosia citrodora). species were found to be placed at the basal part of the In total, the aligned length of the region was 651 bp. tree with Neisosperma glomerata and N. nakaiana as the There were 168 informative characters obtained (consisted basalmost taxa. All species belong to Ochrosia s.str. were of 154 informative base substitutions and 14 informative grouped into 1 clade (clade B), supported with 95% indels). Parsimony analysis yielded 2 most parsimonious bootstrap value. This Ochrosia clade is nested within trees (MPT) of length 396. Ensemble consistency (CI) Neisosperma, resulting in monophyletic Ochrosia and and retention (RI) indices were 0.7803 and 0.8346 respec- paraphyletic Neisosperma. Consequently, this ITS tively. The strict consensus tree has 23 resolved nodes sequence data convincingly suggests that Neisosperma with 19 nodes supported by bootstrap values > 50% should be sunk into Ochrosia, and the two ‘groups’ (Fig.1). should be treated as one genus. All ingroup species (Ochrosia s.str. spp. and Neisosper- The clade formed by the basalmost taxa - N. glomerata ma spp.) were grouped into 1 clade (clade A) strongly and N. nakaiana - was supported with 99% bootstrap

Fig.1 A strict consensus tree of 2 most parsimonious trees (length 396; CI 0.78; RI 0.83). Numbers at the nodes are bootstrap value. Bold letters in brackets are clades highlighted in the paper. ITS sequences in Ochrosia sensu lato (Apocynaceae) 131 value. The next most basal taxon after the clade was species representing Ochrosia s.str. and Neisosperma is Neisosperma oppositifolia, the most widespread species required to get more accurate picture of relationships in the genus. N. acuminata was found to be the sister within Ochrosia s.l. and to make the result much more taxon to the Ochrosia s.str. species. Morphologically, N. convincing. acuminata clearly has characteristics of ‘Neisosperma type’. Its mesocarps are round in tranverse section, with ACKNOWLEDGMENTS. The authors have been fully sup- endocarps split into diverging fibres. No lateral cavities ported by Dissertation PhD Program for 2006 LIPI 10617, the Japan Society for the Promotion of Science. We wish to thank Drs. were found. The species has only been known from Mary Endress, University of Zurich, Switzerland, Tatyana Sulawesi, Indonesia. Livshultz, University of Nebraska, Omaha, U.S.A., David J. The Ochrosia clade (clade B) then was split into 2 Middleton, Royal Botanic Garden, Edinburgh, UK, and Siti Roosita Ariati, Bogor Botanical Garden, Indonesia for discussion subclades. The first subclade consisted of Ochrosia and valuable advices, S. Koyano, Subtropical Agriculture Center coccinea, O. mariannensis var. mariannensis, O. marian- Ogasawara, Tokyo, Japan, Erin Fooley, NTBG, Hawaii, U.S.A., nensis var. crassicarpa, O. elliptica, O. mulsanti, O. David Orr, Waimea Arboretum, Hawaii, U.S.A. and Curators of A, silvatica, and O. grandiflora. Ochrosia coccinea was BISH, L and P, for providing material. Drs. Goro Kokubugata (Tsukuba Botanical Garden, National Science Museum, Japan and found to be the basalmost taxon of this subclade. The Joko Ridho Witono, Bogor Botanical Garden, Indonesia for their relationship of 3 New Caledonian Ochrosia, O. mulsanti, help. O. silvatica, and O. grandiflora is supported with 81% bootstrap value. The three species having similar LITERATURE CITED morphological character: the endocarps have many small Alvarez, I. and J. F. Wendel. 2003. Ribosomal ITS sequences and obscure lateral cavities (whilst the endocarps of O. and plant phylogenetic inference. Molecular Phylogenetic elliptica - another species that also found in New and Evolution 29: 417-434. Caledonia - are massive and have very clear two lateral Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campbell and M.J. Donoghue. cavities). Ochrosia elliptica is found to be placed with O. 1995. The ITS region of nuclear ribosomal DNA: a mariannensis var. mariannensis and O. mariannensis var. valuable source of evidence on angiosperm phylogeny. crassicarpa, supported with 100% bootstrap value. O. Annals of the Missouri Botanical Garden 82: 247-277. mariannensis is endemic to Mariana Islands, in the north- Boiteau, P. and L. Allorge. 1981. Flore de la Nouvelle Cale- donie 10. Apocynacees. Museum National D’Histoire western Pacific Ocean. Morphologically,O. mariannensis Naturelle, Paris. var. crassicarpa differs from O. mariannensis var. De Jussieu, A. L. 1789. Genera Plantarum. mariannensis in having very thick fruit, yellow rather De Mueller, F. 1871. Fragmenta Phytographiae Australiae, Vol. VII. Melbourne. than red when ripe, and narrowly elliptic than obovate or Endress, P. K., P. Baas & M. Gregory. 2000. Systematic Plant oblanceolate leaves. Morphology and Anatomy - 50 Years of Progress. Taxon The second subclade was formed by Ochrosia vitiensis, 49: 401-434. Ochrosia sp., O. compta, O. sandwicensis, and O. kauai- Endress, M. E. and P. V. Bruyns. 2000. A Revised Classifica- tion of the Apocynaceae s.l. The Botanical Review 66 ensis. The first two are from Fiji and Tonga respectively, (1): 1-56. and the last three were endemic to Hawaiian Islands. Felsenstein, J. 1985. Confidence limits on phylogenies: an St.John (1978) in his paper on the Ochrosia of Hawaiian approach using the bootstrap. Evolution, vol.39 no.4: 783-791. Islands recognized 11 species. Later in 1990, Wagner et al. Fosberg, F. R. and M. H. Sachet. 1972. Plants of Southeastern reduced them into only 4 species - O. compta, O. haleaka- Polynesia. 2. Micronesica, Vol. 8, No. 1-2: 43-50. Agana, lae, O. kauaiensis, and O. kilaueaensis. They treated O. Guam. sandwicensis as a synonym of O. compta. Fosberg, F. R. and M. H. Sachet. 1974. Plants of Southeastern Polynesia. 3. Micronesica, Vol. 10, No. 2: 251-256. Agana, The analysis of ITS sequence data discussed above Guam showed that Neisosperma is paraphyletic, and Ochrosia Fosberg, F. R. and M. H. Sachet. 1977. Nomenclature of the s.str. is monophyletic. The two groups formed a Ochrosiinae (Apocynaceae): 1. Application of the Names Neisosperma Raf. and Calpicarpum G. Don. Adansonia, monophyletic Ochrosia s.l. clade that highly supported Vol 17, No. 1: 19-22. with 100% bootstrap value. Therefore, it is suggested Forster, P. I. 1993. A Taxonomic Revision of Neisosperma that Ochrosia s.str. and Neisosperma are best united into Raf. (Apocynaceae) in Australia, Together with A Key to a single genus. This finding should be reflected in Australian Genera of Apocynaceae. Austrobaileya, Vol. 4, No. 1: 13-20. taxonomic treatments. The analysis also showed that Hendrian. 2004. Revision of Ochrosia (Apocynaceae) in subdivision of Ochrosia s.l. into subgenus or sections - as Malesia. Blumea 49/1: 101-128. proposed by Mueller (1871), Valeton (1895) and Hendrian Hillis, D. M. & J. J. Wiens. 2000. Molecules Versus Morphol- ogy in Systematics. pp. 1-19. in Wiens, J. J. (ed.). Phylo- (2004) - based on fruit morphology (fibrous vs. massive genetic. 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