Systematic Botany (2009), 34(3): pp. 521–529 © Copyright 2009 by the American Society of Plant Taxonomists More Miocene Dispersal Between Africa and Asia—the Case of Bridelia (Phyllanthaceae) Yongquan Li, 1,2,3 Stefan Dressler, 4 Dianxiang Zhang 5,1 and Susanne S. Renner 2 1 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China 2 Department of Biology, University of Munich, Germany 3 The Graduate University of the Chinese Academy of Science, Beijing, 100039 China 4 Forschungsinstitut Senckenberg, Frankfurt/Main, Germany 5 Author for correspondence ([email protected]) Communicating Editor: Thomas A. Ranker Abstract— Several hundred angiosperm genera range from Africa to Asia and Australia, among them Bridelia (Phyllanthaceae), with ca. 40 species, including commercially important timber trees. We here use nuclear and chloroplast DNA sequences from herbarium mate- rial, plus new collections from China, to test the monophyly of Bridelia and to infer the geologic times when it acquired its disjunct range. For the Southeast Asian mainland, within-species sampling, including material collected close to the type localities, allowed testing current spe- cies concepts. Based on a sample of 114 chloroplast matK sequences of Phyllanthaceae, Bridelia is monophyletic and sister to an Asian clade which requires resurrecting an older generic name to make the African Cleistanthus monophyletic. Within Bridelia , gene trees from the com- bined data (3,177 aligned nucleotides from 25 species of Bridelia plus outgroups) agree with most morphological species boundaries. Exceptions are that B. tomentosa must include B. harmandii and B. curtisii to become monophyletic and that B. fordii is distinct from B. retusa . The topol- ogy, together with relaxed clock divergence times, implies that Bridelia dispersed from tropical Asia to Africa once or twice between 10 and 1.85 million years ago (Ma). Australia was reached, probably from New Guinea, at least twice, both times ca. 2 Ma. Together with earlier stud- ies, there are now at least eight cases of Neogene long distance dispersal between Africa and Asia (followed by speciation), with no directional bias apparent so far. Keywords— Africa , Asia , Australia , biogeography , Bridelia , Cleistanthus , long distance dispersal , molecular clock. Several hundred genera of flowering plants are disjunctly Here we investigate the biogeography and time of radia- distributed between tropical Africa, Asia, and Australia tion of Bridelia (Phyllanthaceae), an African/Asian clade that ( Thorne 1973 ), but only a handful have been investigated attracted our attention because it is almost equally species- with molecular methods. Part of the reason is the cost of col- rich on both continents, making it intuitively difficult to infer lecting material from multiple continents. Another reason is the direction of possible dispersal: Bridelia has 18–20 species that workers may initially be unaware of the need to sam- in Africa and Madagascar (all endemic), 18 in tropical Asia ple in Africa, Asia, and Australia because species have been (a few of these shared with Australia), and five in Australia, allocated to different genera. This was the case in Cucumis , three of them endemic ( Dressler 1996 ; Forster 1999 ; Li and which until 2007 was thought to have some 30 species in Dressler 2008 ). The species are shrubs, treelets, or trees with Africa and two in Asia, but which turned out to instead minute unisexual flowers ( Fig. 1a-d ) that are produced in include 13 Asian and Australian species, all of which were successive male-female-male batches, a strategy known as hiding under different generic names ( Renner et al. 2007 ; duodichogamy ( Borges et al. 1997 : B. retusa ; Luo et al. 2007 : Renner and Schaefer 2008 ). B. tomentosa ). The fruits are indehiscent drupes, except in a Nevertheless, at least eight African/Asian genus-level few species in which the endocarp splits open (e.g. B. stip- clades have been investigated biogeographically. Of these, ularis ). A previous molecular-phylogenetic study sampled Cucumis apparently dispersed from Africa to Asia <10 mil- three species of Bridelia and found them to form the sister lion years ago (Ma; Renner et al. 2007 ); Adansonia (Malvaceae- clade to an Asian subgroup of Cleistanthus , a large poly- Bombacoideae) from Africa (one species) and Madagascar phyletic genus of ca. 140 species also distributed in Africa, (six species) to Australia (one species) 2–15 Ma ( Baum et al. Madagascar, and tropical Asia ( Kathriarachchi et al. 2005 ; 1998 ); Osbeckia (Melastomataceae) from Africa (five spe- see Results). cies) and Madagascar (10–13) to India/Sri Lanka (26) and The African and Asian species of Bridelia have been treated in Australia (two species) 7–16 Ma ( Renner and Meyer 2001 ; regional floras ( Léonard 1955 , 1962 ; Leandri 1958 ; Hutchinson Renner 2004 ); Gaertnera (Rubiaceae) from Africa (c. 30 species) and Dalziel 1958 ; Airy Shaw 1975 , 1978 , 1980 , 1981 ; Radcliffe- and Madagascar (25 species) to Southeast Asia (16 species) Smith 1987 ; Dressler and van Welzen 2005 ; Li and Dressler 5–6 Ma ( Malcomber 2002 ); and Exacum (Gentianaceae) from 2008 ), but never in a comprehensive manner. Diagnostically Madagascar (38 species) to Sri Lanka/India (17), Southeast important characters in Bridelia , such as leaf venation, shape, Asia, and Australia < 35 Ma ( Yuan et al. 2005 ). In the other and indumentum, can be difficult to employ, and secure iden- direction, Uvaria (Annonaceae) appears to have dispersed tification of Bridelia specimens therefore requires access to from Southeast Asia (70 species) and Australia (3 species) an herbarium with authenticated material for comparison. to Africa (70 species) 12–15 Ma ( Richardson et al. 2004 ); and The DNA sequences used here mostly come from specimens Macaranga and Mallotus (Euphorbiaceae) from Asia (where housed in the herbaria of Edinburgh, Leiden, Munich, Paris, they have several hundred species) to Africa and Madagascar and St. Louis, and from the first author’s collections from (together ca. 40 species) < 27 Ma ( Kulju et al. 2007 ). Of course, China. Sequencing revealed many doubtfully or wrongly these estimates are rough, and more detailed species sampling named collections (c. 15%), necessitating the sampling of is needed, but nevertheless it is clear that diaspore dispersal, multiple accessions per species and material collected close followed by successful speciation, has occurred in both direc- to the type localities. Morphological species concepts in tions, east to west and west to east. Bridelia are relatively broad ( Dressler 1996 ), with widespread 521 522 SYSTEMATIC BOTANY [Volume 34 Fig. 1. Habits and flowers of selected species of Bridelia in Guangdong, China. A. Habit of Bridelia retusa . B. Inflorescence of Bridelia affinis . C. Flies visiting male flowers of Bridelia stipularis . D. Fruits of Bridelia tomentosa. species correspondingly variable. This is the case in B. balansae, between African and Asian Bridelia relative to that among other African/ B. retusa , B. stipularis , and B. tomentosa . Our analysis includes Asian sister taxa, we created a matK matrix of 114 accessions represent- ing 51 genera of Phyllanthaceae (the majority from Kathriarachchi et al. up to four accessions of these species to represent their geo- 2005 ), rooted on Picrodendraceae, Euphorbiaceae, and Putranjivaceae. graphic ranges. The GenBank matK sequence of Sauropus androgynus was renamed S. gar- After rigidly testing the monophyly and closest relatives of rettii based on Pruesapan et al. (2008 ). Bridelia , we address two questions: (1) At what time(s) and DNA Isolation, Amplification, and Sequencing— Total genomic by which likely means did Bridelia acquire its African/Asian/ DNA was isolated from silica-dried leaves or herbarium material using DNeasy plant mini kits (QIAGEN, Valencia, California) or NucleoSpin- Australian range; and (2) are the current broad morphologi- Plant kits (Macherey-Nagel, Düren, Germany). The PCR protocols used cal species concepts in tropical Asian Bridelia supported by were as follows: Initial denaturation at 95°C for 5 min, followed by 35 molecular data? cycles of 30 sec at 95°C for denaturation, 1 min for primer annealing at 48°C and 2 min 40 sec at 72°C for DNA elongation. Reactions were per- formed with 10 mM of primers, 25 μM MgCl , 1.25 μM of each dNTP, 2.5 μ × 2 Materials and Methods M of 10 PCR buffer, 10% of BSA, 0.5 units of Taq DNA polymerase, and 10–50 ng of template DNA per 25 μl reaction volume. When ampli- Taxon Sampling— We sampled 11 of the 18 Asian species of Bridelia , fication failed, we used the more reactive Phusion polymerase (Phusion three of the five Australian species (including two endemics), and 12 of TM High Fidelity PCR Kit, Finnzymes, Espoo, Fiinland) according to the the 18–20 African and Malagasy species. Of the seven species listed in the manufacturer’s protocol. Flora of China ( Li and Dressler 2008 ), we lack B. parvifolia Kuntze (which The plastid maturase K ( matK ) gene and part of its flanking trnK occurs on the island of Hainan) and Chinese material of B. glauca (suppos- intron were amplified using the primers designed by Samuel et al. edly in Guangdong, Guangxi, and Yunnan). Chinese B. “glauca” specimens (2005 ). We used their primers 570, 80F, 800F, 1200F, 190R, 950R, that we sequenced turned out to represent one of the other Chinese spe- and 1710R. In addition, we designed two new primers, LY-390F cies, and in the case of rare B. parvifolia, originally described from Vietnam, (5’-CGATCAATTCATTCAATATTTC-3’) and LY-1300R (5’-CGAAGTA- we were unable to obtain a loan of the Hainan material assigned to this TATATTTGATTCGATACA-3’) by modifying Samuel et al.’s primers species. As outgroups, we included 10 species of Cleistanthu s (including 390F and 1300R. For quality control, we re-extracted and resequenced the type species of that genus) and one species each of Pentabrachion and the first section of the matK gene (ca. 1,000 bps) from single collections of Pseudolachnostylis, based on Kathriarachchi et al.