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South African Journal of 89 (2013) 143–149

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

First molecular phylogeny of the pantropical genus : implications for infrageneric circumscription and biogeography

Mohammad Vatanparast a,⁎, Bente B. Klitgård b, Frits A.C.B. Adema c, R. Toby Pennington d, Tetsukazu Yahara e, Tadashi Kajita a

a Department of Biology, Graduate School of Science, Chiba University, 1-33 Yayoi, Inage, Chiba, Japan b Herbarium, Library, Art and Archives, Royal Botanic Gardens, Kew, Richmond, United Kingdom c NHN Section, Netherlands Centre for Biodiversity Naturalis, Leiden University, Leiden, The Netherlands d Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh, EH3 5LR, United Kingdom e Department of Biology, Kyushu University, Japan

article info abstract

Available online 21 July 2013 The genus Dalbergia with c. 250 has a pantropical distribution. In spite of the high economic and eco- logical value of the genus, it has not yet been the focus of a species level phylogenetic study. We utilized ITS Edited by JS Boatwright nuclear sequence data and included 64 Dalbergia species representative of its entire geographic range to pro- vide a first phylogenetic framework of the genus to evaluate previous infrageneric classifications based on Keywords: morphological data. The phylogenetic analyses performed suggest that Dalbergia is monophyletic and that Biodiversity it probably originated in the New World. Several clades corresponding to sections of these previous classifi- Biogeography Conservation cations are revealed. Taking into account that there is not a complete correlation between geography and – – Dalbergia phylogeny, and the estimation that the Dalbergia stem and crown clades are 40.4 43.3 mya and 3.8 12.7 Dispersal mya, respectively, it is plausible that several long distance dispersal events underlie the pantropical distribu- Phylogeny tion of the genus. Systematics © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Yahara et al., 2013). Within the GLDA, biodiversity assessments of multiple genera (Bauhinia L., Dalbergia L.f., Desmodium Desv., Understanding evolutionary history of species is a fundamental factor Mucuna Adans., and Vigna Savi.) have been prioritized (Yahara et al., for biodiversity assessment and setting conservation priorities (Purvis 2013) implementing Phylogenetic Diversity (PD; Faith, 1992), Function- and Hector, 2000), especially when taxa are economically and ecological- al Diversity (FD; Tilman, 2001) and ecological niche modeling methods. ly important (Brooks et al., 2006; Margules and Pressey, 2000). Among One of the genera chosen was Dalbergia, because of its species-richness, flowering , the Leguminosae () is the third largest family pantropical distribution, and economic importance. Robust species level with over 19,300 species found in most of the world's vegetation types phylogenies are the starting points for the above-mentioned analy- (Lewis et al., 2005). The economic and ecological importance of the le- ses, and here we present the first molecular phylogeny of Dalbergia gumes is exceptionally high because they are sources of food, fodder, (Leguminosae: ). , fuels, medicines, and through their ability to enrich soils. The genus Dalbergia is pantropical with around 250 species and Because various ecosystems and are under significant centers of diversity in Central and South America, Africa, Madagascar threat by human activities (Lee and Jetz, 2008; Rockstrom et al., and Asia (Klitgaard and Lavin, 2005). Several species of Dalbergia 2009), monitoring of biodiversity and ecosystem functioning is now produce fine timbers of high economic value. These are generally critical (Yahara et al., 2012). Because of its economic and ecological known as (e.g. D. cochinchinensis Pierre, D. latifolia Roxb., D. importance, the Leguminosae has been chosen as the focus of a Global melanoxylon Guill. & Perr., D. nigra (Vell.) Alleinao ex Benth., D. odorifera Diversity Assessment (Global Legume Diversity Assessment; GLDA; T.C.Chen and D. sissoo Roxb.,) and are, for example, used in fine carvings. More than 50 species of Dalbergia have been demonstrated to fix nitro- gen and to have aeschynomenoid type root nodules (Sprent, 2009). Several regional revisions, enumerations and flora treatments based exclusively on morphological characters exist for Dalbergia (Bentham, ⁎ Corresponding author at: Department of Biology, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. Fax: +81 43 290 2874. 1860; Carvalho, 1997; Chen et al., 2010; Niyomdham, 2002; Prain, E-mail address: [email protected] (M. Vatanparast). 1904; Sunarno and Ohashi, 1997; Thothathri, 1987). However, the only

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.07.001 144 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 attempt at a complete infrageneric classification of the genus is by (PCR) were performed in reaction volumes of 10 μL containing 0.2 units Bentham (1860). of ExTaq (TaKaRa) or 0.25 units of MightyAmp DNA Polymerase

Combined analyses of a broadly circumscribed monophyletic tribe (TaKaRa), and 0.2 mM dNTPs., 10× PCR buffer contains 1.5 mM MgCl2, Dalbergieae based on molecular and morphological data (Lavin et al., 0.5–1 μM of each primer pair, and 20 ng genomic DNA. The PCR condi- 2001), placed Dalbergia in the Dalbergia clade as sister to the genus tions were as follows: 2 min for initial denaturation at 95 °C, followed Pers. and L. subgen. Ochopodium. Using by 35 amplification cycles of 45 sec denaturation at 95 °C, 1 min trnL and nuclear ribosomal DNA (ITS/5.8S) sequence data, Ribeiro et al. annealing at 56 °C, 1 min extension at 72 °C, and a final 10 min exten- (2007) resolved Machaerium as more closely related to Aeschynomene sion at 72 °C. The PCR products were visualized by performing 0.8% subgen. Ochopodium Vogel. than to Dalbergia.Inthatstudyonly14spe- agarose gel electrophoresis. Amplified DNA was purified using illustra cies of exclusively New World Dalbergia were included, with the addition ExoStar Enzymatic PCR and Sequencing Clean-Up kit (GE Healthcare, of the Old World D. sisso from an accession cultivated in Bahia, UK) according to the manufacturer's instructions. (Ribeiro et al., 2007). Therefore, a comprehensive phylogenetic study of The cycle sequencing reactions were carried out using the ABI the genus is needed, which includes a representative sample of African, BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) Asian and New World species. and sequencing reaction products were purified by an ethanol precipi- The nuclear ITS region has proven useful in species level phyloge- tation method. All DNA sequences were determined in forward and re- netic studies of various legume genera by many authors (Käss and verse strands using an ABI 3500 DNA sequencer (Applied Biosystems). Wink, 1997; Lavin et al., 2000; Wojciechowski et al., 1999). Lavin For 10 accessions where direct sequencing yielded electropherograms et al. (2001) used ITS sequence data for their broad-scale study of gen- showing polymorphism at several sites, the SSCP (Single strand confor- era resolved in the Dalbergioid clade and including several Dalbergia mation polymorphism) method was used (Hayashi, 1991). In this species. This motivated us to use sequence data of the ITS region for method multiple copies of ITS sequences are separated by conformation this study. We employed a relatively wide taxonomic sampling using of their molecular structure and weight on an SSCP gel. Distinct single 64 Dalbergia species from its centers of diversity in the Americas, Africa bands of SCCP gel were used as PCR templates for sequencing. All for- and Asia. The objectives of this study are to: (1) provide a first species ward and reverse strands for each sequence were assembled, manually level phylogenetic framework for Dalbergia, (2) evaluate previous edited and aligned with MUSCLE (Edgar, 2004). BLAST search (http:// infrageneric classifications of the genus, and (3) infer the biogeograph- blast.ncbi.nlm.nih.gov/) was conducted for all sequences to check for ical history of the genus. possible problems of amplification of contaminant DNA. Given the possi- bility of paralogy and/or pseudogenes among ITS sequences, we did not 2. Materials and methods include the sequences from SSCP which appeared to be pseudogenes in the sequences based upon the presence of indels and high divergence 2.1. Taxon sampling of sequences (Bailey et al., 2003).

Our sampling strategy was to cover the total distribution range of 2.3. Phylogenetic inferences Dalbergia and to include representative species from various taxo- nomic ranks (subgenus, section and series). In total, 64 species of Maximum Parsimony (MP) and Bayesian Inference (BI) were used Dalbergia (represented by 66 accessions) were included: 31 species for the phylogenetic analyses. The MP analyses were conducted using from Asia, 21 species from Africa including Madagascar, and 12 spe- PAUP* version 4.0b10 (Swofford, 2002). Heuristic searches were cies from America (Table 1). material for DNA extraction was performed using the -bisection–reconnection (TBR) branch swap- obtained from recent field surveys in South East Asia by Tetsukazu ping algorithm and all gaps removed from analyses. All characters Yahara and his colleagues, and from E, L, MO and WAG (acronym were selected as unweighted and unordered. Branch support was eval- after Thiers, 2013). Twenty-one accessions of Dalbergia species were uated using the bootstrapping method of Felsenstein (1985) based on obtained from GenBank. 10000 replicates. In order to evaluate the monophyly of Dalbergia we included Bayesian inference was performed using MrBayes version 3.2.1 additional accessions from the Dalbergia clade: cristata (Ronquist et al., 2012). The GTR + I + G model was selected as the Wight & Arn., africana Endl., Bryaspis humulariodes Gledhill best-fit model based on the Akaike information criterion (Akaike, and Aeschynomene falcata (Poir.) DC. (Table 1). Designated outgroups 1974) using the program jModeltest (Darriba et al., 2012; Guindon and were selected based on their placement outside of the Dalbergia clade Gascuel, 2003). Two independent Markov Chain Monte Carlo (MCMC) in Lavin et al. (2001) and included parvifolia Phil., analyses with four simultaneous chains and 8 × 106 generations were acapulcensis Rose and correntina (Burkart) Krapov. & W.C.Greg. run. were sampled every 500 generations and the first 4000 Sequences of all outgroups were retrieved from GenBank. Voucher trees were discarded as burn-in. The convergence of MCMC chains specimens of the newly collected samples in this study were deposited was visualized with the Tracer program version 1.5 (Rambaut and in the herbarium of Kyushu University (FU). All newly generated DNA Drummond, 2009) and likelihood scores for the sampled trees were sequences in this study are submitted to the DNA Data Bank of Japan inspected. (DDBJ, AB828599–AB828668). 3. Results 2.2. DNA extraction, PCR, and sequencing The total sequence length of the ITS (ITS1, 5.8S, and ITS2) sequences Genomic DNA was extracted from silica gel dried or herbar- for 64 representatives of Dalbergia and four species from Dalbergia clade ium specimens using the DNeasy Mini Kit (Qiagen) following ranged from 634 to 737 nucleotides sites. When aligned, 362 sites were the manufacturer's instructions with a modified protocol for herbari- parsimony informative. um materials. The concentration of genomic DNA was measured with a GeneQuant 100 electrophotometer (GE Healthcare, Life Sciences). 3.1. Phylogenetic inferences To amplify complete sequence of ITS (ITS1, 5.8S and ITS2) primer pairs ITS5–ITS4 (White et al., 1990) were used. In the case of failed The maximum parsimony analysis resulted in 4420 most parsimoni- amplification, for most of the herbarium samples, internal primers ous trees with a length of 1405 steps, consistency index (CI; Kluge and ITS2 and ITS3 (Baldwin, 1992) were used to amplify the ITS region in Farris, 1969) of 0.429 and retention index (RI; Farris, 1989) of 0.793. two pieces with ITS5 and ITS4, respectively. Polymerase chain reactions The MP analysis was not well-resolved, with the strict consensus tree M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 145

Table 1 Species list and voucher information for Dalbergia species and four related genera from Dalbergia clade.

Taxon Author Collector Collection number Continent Country Source

Dalbergia L.f. D. abbreviata Craib Kessler, P.J.A. 2022 Asia L D. abrahamii Bosser & R. Rabev. Forest 5659 Africa Madagascar L D. acariiantha Harms Sally Bidgood & R Abdallah & K Vollesen 1348 Africa Tanzania L D. adamii Berhaut Jongkind, C.C.H. 8088 Africa Guinea WAG D. afzeliana G.Don Wieringa, J.J. 3844 Africa Gabon WAG D. arbutifolia Baker Kayombo, C.J. 1026 Africa Tanzania L D. armata E.Mey. Groenendijk, E.M.C. 241 Africa Mozambique WAG D. assamica Benth. Ninh, T.N. 11472 Asia L D. aurea Bosser & R. Rabev. Capuron, R.P.R. 3068 Africa Madagascar WAG D. balansae Prain Hu, S.Y. 22805 Asia China L D. baronii Baker Rabevohitra, R. 3875 Africa Madagascar WAG D. benthamii Prain Hu, S.Y. 21989 Asia China L D. bignonae Berhaut Ptsultaf 7983 Africa Benin WAG D. bintuluensis Sunarno & Ohashi Sidiyasa, K. 2375 Asia Indonesia L D. boehmii Taub. Kayombo, C.J. 6861 Africa Tanzania WAG D. bojeri Drake Randrianaivo, R. 694 Africa Madagascar WAG D. bracteolata Baker Wohlhauser, S. 60139 Africa Madagascar WAG D. candenatensis (Dennst.) Prain Maxwell, J.F. 166 Asia Singapore L D. candenatensis (Dennst.) Prain Wightman, G.M. 2701 Asia Australia L D. canescens (Elmer) Merr. Soejarto, D.D. 7479 Asia Philippines L D. capuronii Bosser & R. Rabev. Capuron, R.P.R. 27807 Africa Madagascar WAG D. cearensis Ducke Jardim, J. 1775 America Brazil MO D. cochinchinensis Pierre Maxwell, J.F. 1342 Asia L D. cochinchinensis Pierre Nanthavong, K. 643 Asia L D. cultrata Pittier Maxwell, J.F. 196 Asia Thailand L D. dongnaiensis Pierre Maxwell, J.F. 849 Asia Thailand L D. ecastophyllum (L.) Taub. Croat, T.B. 23287 America Belize L D. falcata Prain Ambriansyah 448 Asia Indonesia L D. floribunda Craib Maxwell, J.F. 159 Asia Thailand L D. frutescens var. frutescens (Vell.) Britton Wasum, R. 1585 America Brazil L D. frutescens var. tomentosa (Vogel) Benth. Solomon 9409 America Bolivia L D. glomerata Hemsl. Sousa, M. 9349 America Mexico L D. godefroyi Prain Maxwell, J.F. 65 Asia Thailand L D. greveana Baill. Randrianaivo, R. 441 Africa Madagascar L D. hancei Benth. Hu, S.Y. 21333 Asia China L D. havilandii Prain Ambriansyah 2599 Asia Indonesia L D. horrida (Dennst.) Mabb. Reddi, B.V. 25899 Asia L D. hostilis Benth. Jongkind, C.C.H. 7790 Africa Guinea WAG D. humbertii R.Vig. Meyers 229 Africa Madagascar L D. inundata Benth. Meireles, J. E. 367 America Brazil MO D. junghuhnii Benth. Gardette, E. 2177 Asia L D. kurzii Prain Maxwell, J.F. 286 Asia Thailand L D. lactea Vatke De Wilde 6120 Africa Ethiopia WAG D. lakhonensis Gagnep. Maxwell, J.F. 444 Asia Thailand L D. lanceolaria L.f. Maxwell, J.F. 921 Asia Thailand L D. lateriflora Benth. Silva 1057 America Brazil MO D. latifolia Roxb. Ambriansyah 963 Asia Indonesia L D. maritima R.Vig. Faliniaina 10 Africa Madagascar L D. martinii F.White Brumitt 16910 Africa Zambia L D. melanocardium Pittier Wendt, T. 3889 America Mexico L D. melanoxylon Guill. & Perr. I FIRIIS 6876 Africa Ethiopia WAG D. miscolobium Benth. Sano 14516 America Brazil MO D. monetaria L.f. Acevedo 7608 America Ecuador U D. oliveri Prain Parakosonh, K.P. 161 Asia Laos L D. parviflora Roxb. Niyomdham, C. 811 Asia Thailand L D. pinnata (Lour.) Prain Huq, A.M. 10430 Asia L D. revoluta Ducke s.n. - America Peru MO D. rimosa var. foliacea (Benth.) Thoth. FRDU 12 Asia Thailand L D. rostrata Hassk. Polak, A.M. 1160 Asia Indonesia L D. sandakanensis Sunarno & Ohashi Kessler, P.J.A. 1386 Asia Indonesia L D. sericea G.Don Huq, A.M. 10336 Asia Bangladesh L D. sissoo DC. Shetty, B.V. 1278 Asia India L D. spruceana (Benth.) Benth. Smith 13743 America Bolivia U D. stipulacea Roxb. Chin, S.C. 3498 Asia Indonesia L D. thorelii Gagnep. Toyama, Tagane & Yahara 1033 Asia FU D. trichocarpa Baker Service Forestier de Madagascar 21658 Africa Madagascar L Aeschynomene falcata (Poir.) DC. Antezana Valera C. 1218 America Bolivia MO Bryaspis humularioides Gledhill John Thomas 10097 Africa Liberia MO Geissaspis cristata Wight & Arn. Hiep, N.T. 10736 Asia Vietnam MO Kotschya africana Endl. Roy E. Gereau 2498 Africa Tanzania MO 146 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149

Adesmia parvifolia JN579628 Pterocarpus acapulcensis AF269175 Outgroups AF203554

Aeschynomene martii EF451088 0.76 Aeschynomene histrix FM242599 Genus Aeschynomene 1/98 Aeschynomene falcata FM242597 sect. Ochopodium 0.99 Aeschynomene falcata Bolivia 0.96 1 Machaerium scleroxylon EF451085 Genus Machaerium opacum EF451097 1/84 0.99/63 Machaerium oblongifolium EF451096 Machaerium

Pictetia marginata AF068177 ormocarpoides AF068167 1/97 clade 0.68/67 Ormocarpum muricatum AF068157 1 Geissaspis cristata Vietnam Kotschya africana Tanzania 1 FM242623 Aeschynomene uniflora FM242635 0.65/54 0.65/57 0.87 Aeschynomene pfundii FM242629 0.68 Bryaspis humularioides Liberia Aeschynomene clade Asia 1/99 Bryaspis lupulina AF204234 semperflorens AF189027 Africa Aeschynomene scabra FM242632 0.51 America FM242631 1/85 1 FM242592 0.77 Aeschynomene denticulata FM242626 1 Aeschynomene indica AIU59892 0.1

0.94/85 Genus Dalbergia Fig. 2

Fig. 1. The 50% majority rule consensus tree resulting from Bayesian analysis of the ITS for the members of Dalbergia clade. Numbers along branches are posterior probabilities and bootstrap values from Bayesian analysis and Maximum parsimony, respectively. Values b50% were not shown. Bold lines represent clades and subclades with PP N90%. Sequences for OTUs with accession number obtained from GenBank. showing a basal polytomy. However, major clades are resolved, some of section Dalbergaria sensu Prain (1904) are in this subclade, though which have bootstrap support values greater than 50%. These clades are with low support (PP = 0.54). Clade V, resolved with high PP sup- consistent with those found in the Bayesian analyses (Figs. 1 and 2), and port (PP = 0.96), contains three major subclades (V-a, V-b and these have higher posterior probability (PP) than the parsimony boot- V-c) which correspond geographically to the New World (V-a), Africa strap values (though this is usual; Alfaro et al., 2003). The 50% majority (V-b) and Asia (V-c), respectively, with the exception of D. acariiantha rule consensus tree of the Bayesian tree revealed better resolution of Harms from Tanzania which is nested within the Asian subclade V-c taxa with higher PP support (Figs. 1 and 2). (Fig. 2). Subclade V-a contains New World species from sect. Triptolemea Within the Dalbergia clade the West African genus Bryaspis P. A. A (Mart. Ex Benth.) Benth. Basally branching to the three well-supported Duvign. is nested within the Aeschynomene clade (Fig. 1). Geissaspis subclades in Clade V is a grade consisting of a mixture of New and Old cristata and Kotschya africana are also nested within the Aeschynomene World species. clade, though their relationships are weakly resolved. Dalbergia is monophyletic with two Neotropical species (D. miscolobium Benth. and D. spruceana (Benth.) Benth., Clade I) re- 4. Discussion solved sister to the remaining species, though with relatively weak sup- port (0.61 PP). A second New World Clade II consisting of six species 4.1. Phylogenetic relationships within the Dalbergia Clade (Fig. 2) is then sister to all other Dalbergia species (Fig. 2), though again, support for this relationship is not exceptionally high (0.88 PP), and nei- Dalbergioid were recently circumscribed as a pantropical ther node is resolved in the parsimony strict consensus tree. The species group of papilionoids based on molecular and morphological data, in clades I and II belong to sections Dalbergia and Selenolobium Benth. and initially comprised 44 genera with ca. 1100 species (Lavin et al., sensu Carvalho (1997), and our results show that these sections are 2001, 2000). Recently, however, three additional genera were placed non-monophyletic. Clade III contains three subclades (III-a, III-b and in the Dalbergioid clade: Schott s.s. (Cardoso et al., 2012), III-c). The well-supported subclade III-a contains D. adamii Berhaut from C.E. Hughes, G.P. Lewis, Daza & Reynel. (Hughes et al., Africa as sister to four members of the Neotropical section Ecastaphyllum 2004), Steinbachiella Harms (Lewis et al., 2012), taking the total to 47. (P. Browne) Ducke sensu Carvalho (1997). Subclades III-b and III-c are Nearly all dalbergioids occur in the Neotropics and Africa. In contrast, exclusively Asian. These three subclades are well supported, but relation- only about 100 species belonging to six of these 47 genera are distribut- ships among them are unresolved. Although Clade IV has low PP support ed in tropical Asia. Most of these Asian species belong to Dalbergia (PP = 0.54), it brings D. afzeliana G. Don, sampled from Gabon, as sister (Klitgaard and Lavin, 2005; Lavin et al., 2001). In this study we included to two subclades (IV-a and IV-b) resolved with PP support of 1 and 0.8, ITS sequence data of four species from Aeschynomene s.l. (Table 1)along respectively. Subclade IV-a includes an African-Madagascan clade (M1) with Dalbergia clade accessions from GenBank. However, relationships as sister to an entirely Asian clade. Subclade IV-b, resolved with relative- among Aeschynomene and segregate genera such as Bryaspis, Geissaspis ly high support (PP = 0.8), reveals a similar pattern to subclade IV-a, Wight & Arn. and Kotschya Endl. are weakly resolved. Extensive sam- namely a sister relationship of an African-Madagascan clade with pling within Aeschynomene s.s. is required for a better understanding a clade consisting entirely of Asian species. Species sampled from of relationships among these genera.

Fig. 2. The 50% majority rule consensus tree resulting from Bayesian analysis of the ITS for Dalbergia species. Numbers along branches are posterior probabilities and bootstrap values from Bayesian analysis and maximum parsimony, respectively. Values b50% were not shown. Bold lines represent clades and subclades with PP N90%. Sequences for OTUs with accession number obtained from GenBank. M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 147

Fig. 1

Dalbergia spruceana Bolivia

1/82 Dalbergia miscolobium Brazil Clade I 1/90 Dalbergia miscolobium EF451069 Dalbergia elegans EF451066 Dalbergia lateriflora Brazil 0.99 1/68 Dalbergia inundata Brazil 0.65 Dalbergia revoluta Peru Clade II 0.99/51 Dalbergia foliolosa EF451067 0.94/85 Dalbergia villosa EF451068 1/97 Dalbergia cuiabensis EF451065 0.93 Dalbergia acuta EF451064 Dalbergia adamii Guinea Genus Dalbergia monetaria Ecuador 1/95 Dalbergia ecastophyllum Belize sect. Ecastaphyllum III-a 1 Dalbergia 0.79 Dalbergia monetaria EF451073 Carvalho 1997 0.97 Dalbergia ecastaphyllum EF451071 1 Dalbergia havilandii Indonesia Clade III 0.61 1 Dalbergia sericea Bangladesh III-b 1/95 Dalbergia abbreviata Indonesia Dalbergia rostrata Indonesia Dalbergia pinnata Bangladesh 0.57 1 Australia III-c 1 Dalbergia candenatensis Singapore Dalbergia afzeliana Gabon Dalbergia boehmii Tanzania 1 Dalbergia capuronii Madagascar M1 1 1 Madagascar 0.54 Dalbergia floribunda Thailand Dalbergia latifolia Indonesia IV-a 0.98 0.81 1 Dalbergia latifolia JX856442 0.72 Dalbergia cochinchinensis FR854123 1 Dalbergia cochinchinensis Thailand 1 Dalbergia cochinchinensis Laos 0.58 Dalbergia bignonae Benin Dalbergia aurea Madagascar 1 M2 1 Dalbergia lactea Ethiopia Clade IV Dalbergia falcata Indonesia 1 Dalbergia kurzii Thailand 0.8 1 Dalbergia dongnaiensis Thailand Dalbergia lakhonensis Thailand 0.92 Dalbergia oliveri Laos sect. Dalbergaria 1 IV-b 0.79 0.98 Dalbergia oliveri FR854137 0.54 Indonesia Prain 1905 Dalbergia godefroyi Thailand Thailand 0.81 Dalbergia lanceolaria subsp. paniculata FR854132 0.55 Dalbergia lanceolaria JX856440 Dalbergia balansae China Dalbergia assamica Vietnam 1/53 Dalbergia assamica FR854119 EF451075 0.74 Dalbergia bintuluensis Indonesia 1/94 Dalbergia sandakanensis Indonesia 0.74 Dalbergia hostilis Guinea 1/59 Dalbergia armata Mozambique India 0.96 1/93 Dalbergia junghuhnii Malaysia 1 Dalbergia thorelii Cambodia Dalbergia bracteolata Madagascar 0.97 China Ethiopia 0.63 1/95 Dalbergia arbutifolia Tanzania Dalbergia benthamii China 0.73 Dalbergia canescens Philippines Dalbergia decipularis EF451077 1 Dalbergia frutescens EF451078 Dalbergia congestiflora AF068140 1 1 Dalbergia glomerata Mexico Clade V 0.53 0.98 Dalbergia melanocardium Mexico V-a 0.82 Dalbergia frutescens var. tomentosa Bolivia Dalbergia brasiliensis EF451076 sect. Triptolemea 1/62 Dalbergia frutescens var. frutescens Brazil Carvalho 1997 1 Dalbergia cearensis Brazil Asia 0.75 Dalbergia maritinii Zambia 0.93 0.78 Dalbergia bojeri Madagascar Madagascar 0.52 Dalbergia humbertii Madagascar Africa M3 V-b 0.95 Dalbergia abrahamii Madagascar 0.99 Dalbergia greveana Madagascar America 0.97 1 Dalbergia trichocarpa Madagascar Dalbergia acariiantha Tanzania 1 Dalbergia cultrata Thailand 0.1 Dalbergia sissoo India 1 1/94 Dalbergia sissoo EF451079 V-c 0.97 Thailand 0.93 Dalbergia tonkinensis FR854138 0.96 var. foliacea Thailand 148 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149

4.2. Infrageneric classification of Dalbergia (L.) Taub. are adapted to water dispersal (Carvalho, 1997; Croat, 1978). Additionally, Dalbergia species grow in diverse habitats in- In 1860, Bentham (1860) divided the 64 species of Dalbergia known cluding moist and dry topical forests, savannas, costal dunes and at that time into six series (Triptolemea Americanae, Triptolemea, Sissoae rocky outcrops (Carvalho, 1997; Du Puy and Moat, 2002; Mabberley, Americanae, Sissoae Gerontogee, Dalbergariae and Selenolobia). In the 2008) and contain a variety of woody life forms including , tree Neotropics, the Brazilian species of Dalbergia were studied intensively and climbing . This diversity of ecology and life forms may have by Carvalho (1989, 1997). He divided the 41 Brazilian Dalbergia species contributed to the ability of Dalbergia species to successfully colonize into five sections based on inflorescence and types. In Asia, Prain and thereby to expand their distribution range. (1904) classified the 86 South East Asian species of Dalbergia into two subgenera, five sections and 24 series. Finally, Thothathri (1987) catego- 4.4. Colonization of Madagascar rized the 46 Dalbergia species present in the Indian subcontinent into four sections and seven series based on androecium and fruit types. In Madagascar is one of the biodiversity hotspots in the world due to general Dalbergia species are morphologically variable, and they possess its high levels of diversity, endemism and its highly threatened biota a wide range of preferences which previously made it difficult to (Yoder and Nowak, 2006). Gondwanan vicariance and/or Cenozoic classify the New and Old World species into natural groups (Bentham, dispersal has been thought to be the two most probable mechanisms 1860; Carvalho, 1989; Prain, 1904). Although our ITS analyses only in- responsible for the diversity of Madagascar's biota (Queiroz, 2005). Of cluded 64 of the total 250 species in the genus, it contains representa- approximately 10,000 native species of higher plants in Madagascar tives from nearly all of the various subgenera, sections and series of about 80% are endemic. Legumes, with ca. 600 endemic species, have previous infrageneric classifications. Taking the Bayesian tree (Fig. 2) a major role in Madagascan biodiversity (Puy, 2002). The Malagasy into account, our results revealed that sect. Triptolemea, with cymose Dalbergia species have diverse distribution patterns and grow in differ- inflorescences and thin samaroid pods and sect. Ecastaphyllum, with ent habitats such as evergreen humid forests, coastal forests and rocky racemose or paniculate inflorescences and orbicular to suborbicular or outcrops (Du Puy and Moat, 2002). Based on the taxonomic revisions reniform , are potentially monophyletic. In contrast, sects. Dalbergia of Malagasy Dalbergia (Bosser and Rabevohitra, 1996, 2002, 2005), 47 and Selenolobium are non-monophyletic (Fig. 2). These results are con- species are endemic to Madagascar. Despite the diversity of gruent with the results of Ribeiro et al. (2007) based on ITS and trnLse- species in Madagascar, they are under high risk of extinction due to quences, and it suggests that inflorescence and fruit types may serve as heavy habitat loss by logging, mining and other human activities sources of synapomorphies for classifications of Dalbergia as also previ- (Barrett et al., 2010). In this study we included nine endemic species ously noted by Carvalho (1997).AmongtheAsianDalbergia species (D. abrahamii Bosser & R. Rabev., D. aurea Bosser & R.Rabev., D. baronii only members of sect. Dalbergaria (Prain, 1904) group monophyletically, Baker, D. bojeri Drake, D. capuronii Bosser & R.Rabev., D. greveana Baill., though with low support. Members of this section, with reflexed stan- D. humbertii R.Vig., D. maritima R.Vig. and D. trichocarpa Baker,) as dard petals and stamens usually in two bundles of five, are distributed well as D. bracteolata Baker, also native in mainland Africa (Table 1). throughout Southeast Asia. The Malagasy Dalbergia are resolved in three different clades along with other species from Africa (Fig. 2). The first is a clade comprising 4.3. Biogeography D. capuronii and D. maritima as sister to D. boehmii Taub. from Tanzania (M1 clade, Fig. 2). The second is D. aurea which is sister to D. lactea The placement of two Neotropical clades as sister to the remaining Vatke from Ethiopia (M2 clade, Fig. 2). The third is a clade of six species species in the rest of the genus is suggestive of the New World as the of Malagasy Dalbergia to which D. martinii F. White from Zambia is the area of origin for Dalbergia. However, the low support for these rela- sister group with high support (PP = 0.93) (M3 clade, Fig. 2). The tionships (Fig. 2) and the fact that they are not resolved in the parsimo- occurrence of the Malagasy Dalbergia species in three distinct clades ny strict consensus tree implies that this hypothesis should remain (M1-3, Fig. 2) suggests at least three independent migrations to the preliminary. island during the Cenozoic. The inference of African species as sister to Long distance dispersal (LDD) and vicariance are two processes each Madagascan clade suggests that migration may have occurred which have been suggested to explain pantropical distributions of vari- from Africa to Madagascar, but sampling of more African and Malagasy ous taxa (Bartish et al., 2011). Within the dalbergioid legumes (Lavin species is needed to confirm this. et al., 2001) the genus Dalbergia has a pantropical distribution with high species diversity in Asia (ca. 100 species) as well as in the Neotrop- 5. Conclusions ics and Africa. Using the penalized likelihood method (Sanderson, 2002) based on matK sequences, the estimated age of the stem of Dalbergia Our preliminary molecular systematic analyses of Dalbergia provide (MRCA of Machaerium copote Dugand and Dalbergia congestifolia Pittier) a first phylogenetic framework for future research and informative re- and the crown of Dalbergia (D. sissoo and D. congestifolia)were40.4–43.0 sults regarding previous infrageneric classifications. They also give a mya and 3.8–12.7 mya, respectively, (Matt Lavin, Department of Plant first biogeographic account of the genus. The results show that the ITS Sciences and Plant Pathology, Montana State University, Bozeman, region provides sufficient phylogenetic signal among Dalbergia species Montana; personal communication, see also Lavin et al. (2005, 2004)). to detect some agreement with classifications based on morphology. The Dalbergia stem clade is old because of the age constraint imposed Additional molecular markers from chloroplast and other nuclear re- on the Machaerium stem clade by a fossil record. The two species of gions and more exhaustive taxon sampling are needed to be able to elu- crown Dalbergia (D. sissoo and D. congestifolia) in our phylogenetic tree cidate evolutionary patterns among Dalbergia species and its related are in Clade V (Fig. 2), so the crown age is likely to be an underestimate. genera with greater confidence. Moreover, with a more comprehen- However it can give a broad time-frame for the age of transoceanic sively sampled phylogeny, PD methods can be implemented to crown clades within the Dalbergia clade. Dalbergia species are likely to provide assessments of the phylogenetic distinctiveness of Dalbergia be younger than any of the supposed inter-continental land connections species that are threatened or at risk of extinction. or stepping stones (e.g., the North Atlantic; Pennington and Dick, 2004) and suggest that multiple migration by LDD across oceans occurred to Acknowledgments achieve pantropical distribution of Dalbergia.IngeneralDalbergia species have samaroid pods which are adapted to wind dispersal (Van der We thank curators of Herbaria of E, L, MO, and WAG to giving DNA Pijl, 1982). Some species, particularly in section Ecastaphyllum, materials. Our special gratitude to Niels Raes, Gerard Thijsse, James such as D. brownei (Jacq.) Urb., D. monetaria L.f., and D. ecastaphyllum Solomon, Jan Wieringa and Shuichiro Tagane for arrangements of our M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 149 visit and generously providing materials. 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