South African Journal of Botany 89 (2013) 143–149 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb First molecular phylogeny of the pantropical genus Dalbergia: 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 Herbarium, Library, Art and Archives, Royal Botanic Gardens, Kew, Richmond, United Kingdom 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 b c a r t i c l e i n f o Available online 21 July 2013 Edited by JS Boatwright Keywords: Biodiversity Biogeography Conservation Dalbergia Dispersal Phylogeny Systematics a b s t r a c t The genus Dalbergia with c. 250 species has a pantropical distribution. In spite of the high economic and ecological value of the genus, it has not yet been the focus of a species level phylogenetic study. We utilized ITS nuclear sequence data and included 64 Dalbergia species representative of its entire geographic range to provide a first phylogenetic framework of the genus to evaluate previous infrageneric classifications based on morphological data. The phylogenetic analyses performed suggest that Dalbergia is monophyletic and that it probably originated in the New World. Several clades corresponding to sections of these previous classifications are revealed. Taking into account that there is not a complete correlation between geography and phylogeny, and the estimation that the Dalbergia stem and crown clades are 40.4–43.3 mya and 3.8–12.7 mya, respectively, it is plausible that several long distance dispersal events underlie the pantropical distribution of the genus. © 2013 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction Understanding evolutionary history of species is a fundamental factor for biodiversity assessment and setting conservation priorities (Purvis and Hector, 2000), especially when taxa are economically and ecologically important (Brooks et al., 2006; Margules and Pressey, 2000). Among flowering plants, the Leguminosae (Fabaceae) is the third largest family with over 19,300 species found in most of the world's vegetation types (Lewis et al., 2005). The economic and ecological importance of the legumes is exceptionally high because they are sources of food, fodder, woods, fuels, medicines, and through their ability to enrich soils. Because various ecosystems and habitats are under significant threat by human activities (Lee and Jetz, 2008; Rockstrom et al., 2009), monitoring of biodiversity and ecosystem functioning is now critical (Yahara et al., 2012). Because of its economic and ecological importance, the Leguminosae has been chosen as the focus of a Global Diversity Assessment (Global Legume Diversity Assessment; GLDA; ⁎ 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. E-mail address: vatanparast@gmail.com (M. Vatanparast). Yahara et al., 2013). Within the GLDA, biodiversity assessments of multiple legume genera (Bauhinia L., Dalbergia L.f., Desmodium Desv., Mucuna Adans., and Vigna Savi.) have been prioritized (Yahara et al., 2013) implementing Phylogenetic Diversity (PD; Faith, 1992), Functional Diversity (FD; Tilman, 2001) and ecological niche modeling methods. One of the genera chosen was Dalbergia, because of its species-richness, pantropical distribution, and economic importance. Robust species level phylogenies are the starting points for the above-mentioned analyses, and here we present the first molecular phylogeny of Dalbergia (Leguminosae: Dalbergieae). The genus Dalbergia is pantropical with around 250 species and centers of diversity in Central and South America, Africa, Madagascar and Asia (Klitgaard and Lavin, 2005). Several species of Dalbergia produce fine timbers of high economic value. These are generally known as rosewoods (e.g. D. cochinchinensis Pierre, D. latifolia Roxb., D. melanoxylon Guill. & Perr., D. nigra (Vell.) Alleinao ex Benth., D. odorifera 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 nitrogen 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, 1860; Carvalho, 1997; Chen et al., 2010; Niyomdham, 2002; Prain, 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 Bentham (1860). Combined analyses of a broadly circumscribed monophyletic tribe Dalbergieae based on molecular and morphological data (Lavin et al., 2001), placed Dalbergia in the Dalbergia clade as sister to the genus Machaerium Pers. and Aeschynomene L. subgen. Ochopodium. Using trnL and nuclear ribosomal DNA (ITS/5.8S) sequence data, Ribeiro et al. (2007) resolved Machaerium as more closely related to Aeschynomene subgen. Ochopodium Vogel. than to Dalbergia. In that study only 14 species of exclusively New World Dalbergia were included, with the addition of the Old World D. sisso from an accession cultivated in Bahia, Brazil (Ribeiro et al., 2007). Therefore, a comprehensive phylogenetic study of the genus is needed, which includes a representative sample of African, Asian and New World species. The nuclear ITS region has proven useful in species level phylogenetic studies of various legume genera by many authors (Käss and Wink, 1997; Lavin et al., 2000; Wojciechowski et al., 1999). Lavin et al. (2001) used ITS sequence data for their broad-scale study of genera resolved in the Dalbergioid clade and including several Dalbergia species. This motivated us to use sequence data of the ITS region for this study. We employed a relatively wide taxonomic sampling using 64 Dalbergia species from its centers of diversity in the Americas, Africa and Asia. The objectives of this study are to: (1) provide a first species level phylogenetic framework for Dalbergia, (2) evaluate previous infrageneric classifications of the genus, and (3) infer the biogeographical history of the genus. 2. Materials and methods 2.1. Taxon sampling Our sampling strategy was to cover the total distribution range of Dalbergia and to include representative species from various taxonomic ranks (subgenus, section and series). In total, 64 species of Dalbergia (represented by 66 accessions) were included: 31 species from Asia, 21 species from Africa including Madagascar, and 12 species from America (Table 1). Leaf material for DNA extraction was obtained from recent field surveys in South East Asia by Tetsukazu Yahara and his colleagues, and from E, L, MO and WAG (acronym after Thiers, 2013). Twenty-one accessions of Dalbergia species were obtained from GenBank. In order to evaluate the monophyly of Dalbergia we included additional accessions from the Dalbergia clade: Geissaspis cristata Wight & Arn., Kotschya africana Endl., Bryaspis humulariodes Gledhill and Aeschynomene falcata (Poir.) DC. (Table 1). Designated outgroups were selected based on their placement outside of the Dalbergia clade in Lavin et al. (2001) and included Adesmia parvifolia Phil., Pterocarpus acapulcensis Rose and Arachis correntina (Burkart) Krapov. & W.C.Greg. Sequences of all outgroups were retrieved from GenBank. Voucher specimens of the newly collected samples in this study were deposited in the herbarium of Kyushu University (FU). All newly generated DNA sequences in this study are submitted to the DNA Data Bank of Japan (DDBJ, AB828599–AB828668). (PCR) were performed in reaction volumes of 10 μL containing 0.2 units of ExTaq (TaKaRa) or 0.25 units of MightyAmp DNA Polymerase (TaKaRa), and 0.2 mM dNTPs., 10× PCR buffer contains 1.5 mM MgCl2, 0.5–1 μM of each primer pair, and 20 ng genomic DNA. The PCR conditions were as follows: 2 min for initial denaturation at 95 °C, followed by 35 amplification cycles of 45 sec denaturation at 95 °C, 1 min annealing at 56 °C, 1 min extension at 72 °C, and a final 10 min extension at 72 °C. The PCR products were visualized by performing 0.8% agarose gel electrophoresis. Amplified DNA was purified using illustra ExoStar Enzymatic PCR and Sequencing Clean-Up kit (GE Healthcare, UK) according to the manufacturer's instructions. The cycle sequencing reactions were carried out using the ABI BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) and sequencing reaction products were purified by an ethanol precipitation method. All DNA sequences were determined in forward and reverse strands using an ABI 3500 DNA sequencer (Applied Biosystems). For 10 accessions where direct sequencing yielded electropherograms showing polymorphism at several sites, the SSCP (Single strand conformation polymorphism) method was used (Hayashi, 1991). In this method multiple copies of ITS sequences are separated by conformation of their molecular structure and weight on an SSCP gel. Distinct single bands of SCCP gel were used as PCR templates for sequencing. All forward and reverse strands for each sequence were assembled, manually edited and aligned with MUSCLE (Edgar, 2004). BLAST search (http:// blast.ncbi.nlm.nih.gov/) was conducted for all sequences to check for possible problems of amplification of contaminant DNA. Given the possibility of paralogy and/or pseudogenes among ITS sequences, we did not include the sequences from SSCP which appeared to be pseudogenes in the sequences based upon the presence of indels and high divergence of sequences (Bailey et al., 2003). 2.3. Phylogenetic inferences Maximum Parsimony (MP) and Bayesian Inference (BI) were used for the phylogenetic analyses. The MP analyses were conducted using PAUP* version 4.0b10 (Swofford, 2002). Heuristic searches were performed using the tree-bisection–reconnection (TBR) branch swapping algorithm and all gaps removed from analyses. All characters were selected as unweighted and unordered. Branch support was evaluated using the bootstrapping method of Felsenstein (1985) based on 10000 replicates. Bayesian inference was performed using MrBayes version 3.2.1 (Ronquist et al., 2012). The GTR + I + G model was selected as the best-fit model based on the Akaike information criterion (Akaike, 1974) using the program jModeltest (Darriba et al., 2012; Guindon and Gascuel, 2003). Two independent Markov Chain Monte Carlo (MCMC) analyses with four simultaneous chains and 8 × 106 generations were run. Trees were sampled every 500 generations and the first 4000 trees were discarded as burn-in. The convergence of MCMC chains was visualized with the Tracer program version 1.5 (Rambaut and Drummond, 2009) and likelihood scores for the sampled trees were inspected. 3. Results 2.2. DNA extraction, PCR, and sequencing Genomic DNA was extracted from silica gel dried leaves or herbarium specimens using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions with a modified protocol for herbarium materials. The concentration of genomic DNA was measured with a GeneQuant 100 electrophotometer (GE Healthcare, Life Sciences). 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 amplification, for most of the herbarium samples, internal primers ITS2 and ITS3 (Baldwin, 1992) were used to amplify the ITS region in two pieces with ITS5 and ITS4, respectively. Polymerase chain reactions The total sequence length of the ITS (ITS1, 5.8S, and ITS2) sequences for 64 representatives of Dalbergia and four species from Dalbergia clade ranged from 634 to 737 nucleotides sites. When aligned, 362 sites were parsimony informative. 3.1. Phylogenetic inferences The maximum parsimony analysis resulted in 4420 most parsimonious trees with a length of 1405 steps, consistency index (CI; Kluge and Farris, 1969) of 0.429 and retention index (RI; Farris, 1989) of 0.793. The MP analysis was not well-resolved, with the strict consensus tree 145 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 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 D. abrahamii D. acariiantha D. adamii D. afzeliana D. arbutifolia D. armata D. assamica D. aurea D. balansae D. baronii D. benthamii D. bignonae D. bintuluensis D. boehmii D. bojeri D. bracteolata D. candenatensis D. candenatensis D. canescens D. capuronii D. cearensis D. cochinchinensis D. cochinchinensis D. cultrata D. dongnaiensis D. ecastophyllum D. falcata D. floribunda D. frutescens var. frutescens D. frutescens var. tomentosa D. glomerata D. godefroyi D. greveana D. hancei D. havilandii D. horrida D. hostilis D. humbertii D. inundata D. junghuhnii D. kurzii D. lactea D. lakhonensis D. lanceolaria D. lateriflora D. latifolia D. maritima D. martinii D. melanocardium D. melanoxylon D. miscolobium D. monetaria D. oliveri D. parviflora D. pinnata D. revoluta D. rimosa var. foliacea D. rostrata D. sandakanensis D. sericea D. sissoo D. spruceana D. stipulacea D. thorelii D. trichocarpa Aeschynomene falcata Bryaspis humularioides Geissaspis cristata Kotschya africana Craib Bosser & R. Rabev. Harms Berhaut G.Don Baker E.Mey. Benth. Bosser & R. Rabev. Prain Baker Prain Berhaut Sunarno & Ohashi Taub. Drake Baker (Dennst.) Prain (Dennst.) Prain (Elmer) Merr. Bosser & R. Rabev. Ducke Pierre Pierre Pittier Pierre (L.) Taub. Prain Craib (Vell.) Britton (Vogel) Benth. Hemsl. Prain Baill. Benth. Prain (Dennst.) Mabb. Benth. R.Vig. Benth. Benth. Prain Vatke Gagnep. L.f. Benth. Roxb. R.Vig. F.White Pittier Guill. & Perr. Benth. L.f. Prain Roxb. (Lour.) Prain Ducke (Benth.) Thoth. Hassk. Sunarno & Ohashi G.Don DC. (Benth.) Benth. Roxb. Gagnep. Baker (Poir.) DC. Gledhill Wight & Arn. Endl. Kessler, P.J.A. Forest Sally Bidgood & R Abdallah & K Vollesen Jongkind, C.C.H. Wieringa, J.J. Kayombo, C.J. Groenendijk, E.M.C. Ninh, T.N. Capuron, R.P.R. Hu, S.Y. Rabevohitra, R. Hu, S.Y. Ptsultaf Sidiyasa, K. Kayombo, C.J. Randrianaivo, R. Wohlhauser, S. Maxwell, J.F. Wightman, G.M. Soejarto, D.D. Capuron, R.P.R. Jardim, J. Maxwell, J.F. Nanthavong, K. Maxwell, J.F. Maxwell, J.F. Croat, T.B. Ambriansyah Maxwell, J.F. Wasum, R. Solomon Sousa, M. Maxwell, J.F. Randrianaivo, R. Hu, S.Y. Ambriansyah Reddi, B.V. Jongkind, C.C.H. Meyers Meireles, J. E. Gardette, E. Maxwell, J.F. De Wilde Maxwell, J.F. Maxwell, J.F. Silva Ambriansyah Faliniaina Brumitt Wendt, T. I FIRIIS Sano Acevedo Parakosonh, K.P. Niyomdham, C. Huq, A.M. s.n. FRDU Polak, A.M. Kessler, P.J.A. Huq, A.M. Shetty, B.V. Smith Chin, S.C. Toyama, Tagane & Yahara Service Forestier de Madagascar Antezana Valera C. John Thomas Hiep, N.T. Roy E. Gereau 2022 5659 1348 8088 3844 1026 241 11472 3068 22805 3875 21989 7983 2375 6861 694 60139 166 2701 7479 27807 1775 1342 643 196 849 23287 448 159 1585 9409 9349 65 441 21333 2599 25899 7790 229 367 2177 286 6120 444 921 1057 963 10 16910 3889 6876 14516 7608 161 811 10430 12 1160 1386 10336 1278 13743 3498 1033 21658 1218 10097 10736 2498 Asia Africa Africa Africa Africa Africa Africa Asia Africa Asia Africa Asia Africa Asia Africa Africa Africa Asia Asia Asia Africa America Asia Asia Asia Asia America Asia Asia America America America Asia Africa Asia Asia Asia Africa Africa America Asia Asia Africa Asia Asia America Asia Africa Africa America Africa America America Asia Asia Asia America Asia Asia Asia Asia Asia America Asia Asia Africa America Africa Asia Africa Indonesia Madagascar Tanzania Guinea Gabon Tanzania Mozambique Vietnam Madagascar China Madagascar China Benin Indonesia Tanzania Madagascar Madagascar Singapore Australia Philippines Madagascar Brazil Thailand Laos Thailand Thailand Belize Indonesia Thailand Brazil Bolivia Mexico Thailand Madagascar China Indonesia India Guinea Madagascar Brazil Malaysia Thailand Ethiopia Thailand Thailand Brazil Indonesia Madagascar Zambia Mexico Ethiopia Brazil Ecuador Laos Thailand Bangladesh Peru Thailand Indonesia Indonesia Bangladesh India Bolivia Indonesia Cambodia Madagascar Bolivia Liberia Vietnam Tanzania L L L WAG WAG L WAG L WAG L WAG L WAG L WAG WAG WAG L L L WAG MO L L L L L L L L L L L L L L L WAG L MO L L WAG L L MO L L L L WAG MO U L L L MO L L L L L U L FU L MO MO MO MO 146 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 Adesmia parvifolia JN579628 Pterocarpus acapulcensis AF269175 0.76 1/98 0.99 Arachis correntina AF203554 Aeschynomene martii EF451088 Aeschynomene histrix FM242599 Aeschynomene falcata FM242597 Aeschynomene falcata Bolivia Outgroups Genus Aeschynomene sect. Ochopodium 0.96 Machaerium scleroxylon EF451085 1 1/84 0.99/63 Machaerium opacum EF451097 Machaerium oblongifolium EF451096 1/97 0.68/67 Genus Machaerium Pictetia marginata AF068177 Diphysa ormocarpoides AF068167 Ormocarpum muricatum AF068157 Ormocarpum clade 1 Geissaspis cristata Vietnam Kotschya africana Tanzania 1 0.65/54 0.65/57 0.87 0.68 Asia Africa America 0.51 Aeschynomene aspera Sri Lanka FM242623 Aeschynomene uniflora FM242635 Aeschynomene pfundii FM242629 Aeschynomene Bryaspis humularioides Liberia Bryaspis lupulina AF204234 1/99 Soemmeringia semperflorens AF189027 Aeschynomene scabra FM242632 Aeschynomene rudis FM242631 Aeschynomene virginica FM242592 1/85 1 Aeschynomene denticulata FM242626 0.77 Aeschynomene indica AIU59892 1 clade 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 which have bootstrap support values greater than 50%. These clades are consistent with those found in the Bayesian analyses (Figs. 1 and 2), and these have higher posterior probability (PP) than the parsimony bootstrap values (though this is usual; Alfaro et al., 2003). The 50% majority rule consensus tree of the Bayesian tree revealed better resolution of taxa with higher PP support (Figs. 1 and 2). Within the Dalbergia clade the West African genus Bryaspis P. A. Duvign. is nested within the Aeschynomene clade (Fig. 1). Geissaspis cristata and Kotschya africana are also nested within the Aeschynomene clade, though their relationships are weakly resolved. Dalbergia is monophyletic with two Neotropical species (D. miscolobium Benth. and D. spruceana (Benth.) Benth., Clade I) resolved sister to the remaining species, though with relatively weak support (0.61 PP). A second New World Clade II consisting of six species (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 neither node is resolved in the parsimony strict consensus tree. The species in clades I and II belong to sections Dalbergia and Selenolobium Benth. sensu Carvalho (1997), and our results show that these sections are non-monophyletic. Clade III contains three subclades (III-a, III-b and III-c). The well-supported subclade III-a contains D. adamii Berhaut from Africa as sister to four members of the Neotropical section Ecastaphyllum (P. Browne) Ducke sensu Carvalho (1997). Subclades III-b and III-c are exclusively Asian. These three subclades are well supported, but relationships among them are unresolved. Although Clade IV has low PP support (PP = 0.54), it brings D. afzeliana G. Don, sampled from Gabon, as sister to two subclades (IV-a and IV-b) resolved with PP support of 1 and 0.8, respectively. Subclade IV-a includes an African-Madagascan clade (M1) as sister to an entirely Asian clade. Subclade IV-b, resolved with relatively high support (PP = 0.8), reveals a similar pattern to subclade IV-a, namely a sister relationship of an African-Madagascan clade with a clade consisting entirely of Asian species. Species sampled from section Dalbergaria sensu Prain (1904) are in this subclade, though with low support (PP = 0.54). Clade V, resolved with high PP support (PP = 0.96), contains three major subclades (V-a, V-b and V-c) which correspond geographically to the New World (V-a), Africa (V-b) and Asia (V-c), respectively, with the exception of D. acariiantha Harms from Tanzania which is nested within the Asian subclade V-c (Fig. 2). Subclade V-a contains New World species from sect. Triptolemea A (Mart. Ex Benth.) Benth. Basally branching to the three well-supported subclades in Clade V is a grade consisting of a mixture of New and Old World species. 4. Discussion 4.1. Phylogenetic relationships within the Dalbergia Clade Dalbergioid legumes were recently circumscribed as a pantropical group of papilionoids based on molecular and morphological data, and initially comprised 44 genera with ca. 1100 species (Lavin et al., 2001, 2000). Recently, however, three additional genera were placed in the Dalbergioid clade: Acosmium Schott s.s. (Cardoso et al., 2012), Maraniona C.E. Hughes, G.P. Lewis, Daza & Reynel. (Hughes et al., 2004), Steinbachiella Harms (Lewis et al., 2012), taking the total to 47. Nearly all dalbergioids occur in the Neotropics and Africa. In contrast, only about 100 species belonging to six of these 47 genera are distributed in tropical Asia. Most of these Asian species belong to Dalbergia (Klitgaard and Lavin, 2005; Lavin et al., 2001). In this study we included ITS sequence data of four species from Aeschynomene s.l. (Table 1) along with Dalbergia clade accessions from GenBank. However, relationships among Aeschynomene and segregate genera such as Bryaspis, Geissaspis Wight & Arn. and Kotschya Endl. are weakly resolved. Extensive sampling within Aeschynomene s.s. is required for a better understanding 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. 147 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 Fig. 1 Dalbergia spruceana Bolivia 1/82 Dalbergia miscolobium Brazil Dalbergia miscolobium EF451069 Dalbergia elegans EF451066 Dalbergia lateriflora Brazil Dalbergia inundata Brazil 1/68 0.65 Dalbergia revoluta Peru Dalbergia foliolosa EF451067 Dalbergia villosa EF451068 1/97 Dalbergia cuiabensis EF451065 Clade I 1/90 0.99 0.99/51 0.94/85 Clade II Dalbergia acuta EF451064 Dalbergia adamii Guinea Dalbergia monetaria Ecuador 1/95 Dalbergia ecastophyllum Belize 1 Dalbergia monetaria EF451073 0.79 0.97 Dalbergia ecastaphyllum EF451071 1 Dalbergia havilandii Indonesia Dalbergia sericea Bangladesh 1 Dalbergia abbreviata Indonesia 1/95 Dalbergia rostrata Indonesia Dalbergia pinnata Bangladesh 0.57 Dalbergia candenatensis Australia 1 Dalbergia candenatensis Singapore 1 Dalbergia afzeliana Gabon Dalbergia boehmii Tanzania Dalbergia capuronii Madagascar 1 Dalbergia maritima Madagascar 1 1 Dalbergia floribunda Thailand 0.54 Dalbergia latifolia Indonesia 0.98 1 Dalbergia latifolia JX856442 Dalbergia cochinchinensis FR854123 0.72 Dalbergia cochinchinensis Thailand 1 1 Dalbergia cochinchinensis Laos 0.58 Dalbergia bignonae Benin 0.93 Genus Dalbergia sect. Ecastaphyllum Carvalho 1997 III-a Clade III III-b 0.61 0.81 1 1 1 0.8 1 0.92 0.79 0.54 0.81 0.55 1/53 0.74 1/94 0.74 1/59 0.96 1/93 0.97 III-c M1 Dalbergia aurea Madagascar M2 Dalbergia lactea Ethiopia Dalbergia falcata Indonesia Dalbergia kurzii Thailand Dalbergia dongnaiensis Thailand Dalbergia lakhonensis Thailand Dalbergia oliveri Laos IV-a Clade IV sect. Dalbergaria Prain 1905 1 0.98 Dalbergia oliveri FR854137 Dalbergia stipulacea Indonesia Dalbergia godefroyi Thailand Dalbergia lanceolaria Thailand Dalbergia lanceolaria subsp. paniculata FR854132 Dalbergia lanceolaria JX856440 Dalbergia balansae China Dalbergia assamica Vietnam Dalbergia assamica FR854119 IV-b Dalbergia nigra EF451075 Dalbergia bintuluensis Indonesia Dalbergia sandakanensis Indonesia Dalbergia hostilis Guinea Dalbergia armata Mozambique Dalbergia horrida India Dalbergia junghuhnii Malaysia 1 Dalbergia thorelii Cambodia Dalbergia bracteolata Madagascar Dalbergia hancei China Dalbergia melanoxylon Ethiopia 0.63 Asia Africa America 0.1 1/95 Dalbergia arbutifolia Tanzania Dalbergia benthamii China 0.73 Dalbergia canescens Philippines Dalbergia decipularis EF451077 Dalbergia frutescens EF451078 1 Dalbergia congestiflora AF068140 Dalbergia glomerata Mexico 1 1 0.53 0.98 Dalbergia melanocardium Mexico 0.82 Dalbergia frutescens var. tomentosa Bolivia sect. Triptolemea Dalbergia brasiliensis EF451076 1/62 Dalbergia frutescens var. frutescens Brazil Carvalho 1997 1 Dalbergia cearensis Brazil 0.75 Dalbergia maritinii Zambia 0.93 0.78 Dalbergia bojeri Madagascar Dalbergia baronii Madagascar 0.52 Dalbergia humbertii Madagascar M3 Dalbergia abrahamii Madagascar 0.95 Dalbergia greveana Madagascar 0.99 0.97 1 Dalbergia trichocarpa Madagascar Dalbergia acariiantha Tanzania 1 Dalbergia cultrata Thailand Dalbergia sissoo India 1 1/94 Dalbergia sissoo EF451079 Dalbergia parviflora Thailand 0.97 Dalbergia tonkinensis FR854138 0.93 V-b V-c 0.96 Dalbergia rimosa var. foliacea Thailand V-a Clade V 148 M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 4.2. Infrageneric classification of Dalbergia In 1860, Bentham (1860) divided the 64 species of Dalbergia known at that time into six series (Triptolemea Americanae, Triptolemea, Sissoae Americanae, Sissoae Gerontogee, Dalbergariae and Selenolobia). In the Neotropics, the Brazilian species of Dalbergia were studied intensively by Carvalho (1989, 1997). He divided the 41 Brazilian Dalbergia species into five sections based on inflorescence and fruit types. In Asia, Prain (1904) classified the 86 South East Asian species of Dalbergia into two subgenera, five sections and 24 series. Finally, Thothathri (1987) categorized the 46 Dalbergia species present in the Indian subcontinent into four sections and seven series based on androecium and fruit types. In general Dalbergia species are morphologically variable, and they possess a wide range of habitat preferences which previously made it difficult to classify the New and Old World species into natural groups (Bentham, 1860; Carvalho, 1989; Prain, 1904). Although our ITS analyses only included 64 of the total 250 species in the genus, it contains representatives from nearly all of the various subgenera, sections and series of previous infrageneric classifications. Taking the Bayesian tree (Fig. 2) into account, our results revealed that sect. Triptolemea, with cymose inflorescences and thin samaroid pods and sect. Ecastaphyllum, with racemose or paniculate inflorescences and orbicular to suborbicular or reniform fruits, are potentially monophyletic. In contrast, sects. Dalbergia and Selenolobium are non-monophyletic (Fig. 2). These results are congruent with the results of Ribeiro et al. (2007) based on ITS and trnL sequences, and it suggests that inflorescence and fruit types may serve as sources of synapomorphies for classifications of Dalbergia as also previously noted by Carvalho (1997). Among the Asian Dalbergia species only members of sect. Dalbergaria (Prain, 1904) group monophyletically, though with low support. Members of this section, with reflexed standard petals and stamens usually in two bundles of five, are distributed throughout Southeast Asia. 4.3. Biogeography The placement of two Neotropical clades as sister to the remaining species in the rest of the genus is suggestive of the New World as the area of origin for Dalbergia. However, the low support for these relationships (Fig. 2) and the fact that they are not resolved in the parsimony strict consensus tree implies that this hypothesis should remain preliminary. Long distance dispersal (LDD) and vicariance are two processes which have been suggested to explain pantropical distributions of various taxa (Bartish et al., 2011). Within the dalbergioid legumes (Lavin et al., 2001) the genus Dalbergia has a pantropical distribution with high species diversity in Asia (ca. 100 species) as well as in the Neotropics and Africa. Using the penalized likelihood method (Sanderson, 2002) based on matK sequences, the estimated age of the stem of Dalbergia (MRCA of Machaerium copote Dugand and Dalbergia congestifolia Pittier) and the crown of Dalbergia (D. sissoo and D. congestifolia) were 40.4–43.0 mya and 3.8–12.7 mya, respectively, (Matt Lavin, Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana; personal communication, see also Lavin et al. (2005, 2004)). The Dalbergia stem clade is old because of the age constraint imposed on the Machaerium stem clade by a fossil record. The two species of crown Dalbergia (D. sissoo and D. congestifolia) in our phylogenetic tree are in Clade V (Fig. 2), so the crown age is likely to be an underestimate. However it can give a broad time-frame for the age of transoceanic crown clades within the Dalbergia clade. Dalbergia species are likely to be younger than any of the supposed inter-continental land connections or stepping stones (e.g., the North Atlantic; Pennington and Dick, 2004) and suggest that multiple migration by LDD across oceans occurred to achieve pantropical distribution of Dalbergia. In general Dalbergia species have samaroid pods which are adapted to wind dispersal (Van der Pijl, 1982). Some species, particularly in section Ecastaphyllum, such as D. brownei (Jacq.) Urb., D. monetaria L.f., and D. ecastaphyllum (L.) Taub. are adapted to water dispersal (Carvalho, 1997; Croat, 1978). Additionally, Dalbergia species grow in diverse habitats including moist and dry topical forests, savannas, costal dunes and rocky outcrops (Carvalho, 1997; Du Puy and Moat, 2002; Mabberley, 2008) and contain a variety of woody life forms including shrubs, tree and climbing lianas. This diversity of ecology and life forms may have contributed to the ability of Dalbergia species to successfully colonize and thereby to expand their distribution range. 4.4. Colonization of Madagascar Madagascar is one of the biodiversity hotspots in the world due to its high levels of diversity, endemism and its highly threatened biota (Yoder and Nowak, 2006). Gondwanan vicariance and/or Cenozoic dispersal has been thought to be the two most probable mechanisms responsible for the diversity of Madagascar's biota (Queiroz, 2005). Of approximately 10,000 native species of higher plants in Madagascar about 80% are endemic. Legumes, with ca. 600 endemic species, have a major role in Madagascan biodiversity (Puy, 2002). The Malagasy Dalbergia species have diverse distribution patterns and grow in different habitats such as evergreen humid forests, coastal forests and rocky outcrops (Du Puy and Moat, 2002). Based on the taxonomic revisions of Malagasy Dalbergia (Bosser and Rabevohitra, 1996, 2002, 2005), 47 species are endemic to Madagascar. Despite the diversity of rosewood species in Madagascar, they are under high risk of extinction due to heavy habitat loss by logging, mining and other human activities (Barrett et al., 2010). In this study we included nine endemic species (D. abrahamii Bosser & R. Rabev., D. aurea Bosser & R.Rabev., D. baronii Baker, D. bojeri Drake, D. capuronii Bosser & R.Rabev., D. greveana Baill., D. humbertii R.Vig., D. maritima R.Vig. and D. trichocarpa Baker,) as well as D. bracteolata Baker, also native in mainland Africa (Table 1). The Malagasy Dalbergia are resolved in three different clades along with other species from Africa (Fig. 2). The first is a clade comprising 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 Vatke from Ethiopia (M2 clade, Fig. 2). The third is a clade of six species of Malagasy Dalbergia to which D. martinii F. White from Zambia is the sister group with high support (PP = 0.93) (M3 clade, Fig. 2). The occurrence of the Malagasy Dalbergia species in three distinct clades (M1-3, Fig. 2) suggests at least three independent migrations to the island during the Cenozoic. The inference of African species as sister to each Madagascan clade suggests that migration may have occurred from Africa to Madagascar, but sampling of more African and Malagasy species is needed to confirm this. 5. Conclusions Our preliminary molecular systematic analyses of Dalbergia provide a first phylogenetic framework for future research and informative results regarding previous infrageneric classifications. They also give a first biogeographic account of the genus. The results show that the ITS region provides sufficient phylogenetic signal among Dalbergia species to detect some agreement with classifications based on morphology. Additional molecular markers from chloroplast and other nuclear regions and more exhaustive taxon sampling are needed to be able to elucidate evolutionary patterns among Dalbergia species and its related genera with greater confidence. Moreover, with a more comprehensively sampled phylogeny, PD methods can be implemented to provide assessments of the phylogenetic distinctiveness of Dalbergia species that are threatened or at risk of extinction. Acknowledgments We thank curators of Herbaria of E, L, MO, and WAG to giving DNA materials. Our special gratitude to Niels Raes, Gerard Thijsse, James Solomon, Jan Wieringa and Shuichiro Tagane for arrangements of our M. Vatanparast et al. / South African Journal of Botany 89 (2013) 143–149 visit and generously providing materials. We thank Matt Lavin for estimated age and useful suggestions on the manuscript. 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