SAJB-00970; No of Pages 7 South African Journal of Botany xxx (2013) xxx–xxx Contents lists available at SciVerse 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 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 Article history: The genus Dalbergia with c. 250 species has a pantropical distribution. In spite of the high economic and eco- Received 19 May 2013 logical value of the genus, it has not yet been the focus of a species level phylogenetic study. We utilized ITS Received in revised form 29 June 2013 nuclear sequence data and included 64 Dalbergia species representative of its entire geographic range to pro- Accepted 1 July 2013 vide a first phylogenetic framework of the genus to evaluate previous infrageneric classifications based on Available online xxxx morphological data. The phylogenetic analyses performed suggest that Dalbergia is monophyletic and that fi Edited by JS Boatwright it probably originated in the New World. Several clades corresponding to sections of these previous classi - cations are revealed. Taking into account that there is not a complete correlation between geography and Keywords: phylogeny, and the estimation that the Dalbergia stem and crown clades are 40.4–43.3 mya and 3.8–12.7 Biodiversity mya, respectively, it is plausible that several long distance dispersal events underlie the pantropical distribu- Biogeography tion of the genus. Conservation © 2013 SAAB. Published by Elsevier B.V. All rights reserved. Dalbergia Dispersal Phylogeny Systematics 1. Introduction multiple legume genera (Bauhinia L., Dalbergia L.f., Desmodium Desv., Mucuna Adans., and Vigna Savi.) have been prioritized (Yahara et al., Understanding evolutionary history of species is a fundamental factor 2013) implementing Phylogenetic Diversity (PD; Faith, 1992), Function- for biodiversity assessment and setting conservation priorities (Purvis al Diversity (FD; Tilman, 2001) and ecological niche modeling methods. and Hector, 2000), especially when taxa are economically and ecological- One of the genera chosen was Dalbergia, because of its species-richness, ly important (Brooks et al., 2006; Margules and Pressey, 2000). Among pantropical distribution, and economic importance. Robust species level flowering plants, the Leguminosae (Fabaceae) is the third largest family phylogenies are the starting points for the above-mentioned analy- with over 19,300 species found in most of the world's vegetation types ses, and here we present the first molecular phylogeny of Dalbergia (Lewis et al., 2005). The economic and ecological importance of the le- (Leguminosae: Dalbergieae). gumes is exceptionally high because they are sources of food, fodder, The genus Dalbergia is pantropical with around 250 species and woods, fuels, medicines, and through their ability to enrich soils. centers of diversity in Central and South America, Africa, Madagascar Because various ecosystems and habitats are under significant and Asia (Klitgaard and Lavin, 2005). Several species of Dalbergia threat by human activities (Lee and Jetz, 2008; Rockstrom et al., produce fine timbers of high economic value. These are generally 2009), monitoring of biodiversity and ecosystem functioning is now known as rosewoods (e.g. D. cochinchinensis Pierre, D. latifolia Roxb., D. critical (Yahara et al., 2012). Because of its economic and ecological melanoxylon Guill. & Perr., D. nigra (Vell.) Alleinao ex Benth., D. odorifera importance, the Leguminosae has been chosen as the focus of a Global T.C.Chen and D. sissoo Roxb.,) and are, for example, used in fine carvings. Diversity Assessment (Global Legume Diversity Assessment; GLDA; More than 50 species of Dalbergia have been demonstrated to fix nitro- Yahara et al., 2013). Within the GLDA, biodiversity assessments of 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 Please cite this article as: Vatanparast, M., et al., First molecular phylogeny of the pantropical genus Dalbergia: implications for infrageneric circumscription and biogeography, South African Journal of Botany (2013), http://dx.doi.org/10.1016/j.sajb.2013.07.001 2 M. Vatanparast et al. / South African Journal of Botany xxx (2013) xxx–xxx 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 Machaerium Pers. and Aeschynomene 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, Brazil 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