Systematic Botany (2009), 34(3): pp. 521–529 © Copyright 2009 by the American Society of Taxonomists

More Miocene Dispersal Between and —the Case of ()

Yongquan Li,1,2,3 Stefan Dressler,4 Dianxiang Zhang 5,1 and Susanne S. Renner2 1 South 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 , including commercially important timber . 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 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 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 -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 /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 () 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 . B. Inflorescence of Bridelia affinis. C. Flies visiting male flowers of . 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 . 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 ). 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. (2005 ). To test the phylo- B. “retusa ” from Guangdong, B. tomentosa from Guangdong, B. tomentosa genetic placement of Bridelia and to assess the amount of genetic diversity from Singapore, B. affinis from Yunnan Mengzi, and B. balansae from Laos. 2009] LI ET AL.: BIOGEOGRAPHY OF BRIDELIA 523

As an additional plastid region, we sequenced the trnL-trnF IGS, using the sent the “typical” entity (i.e. the one to which the published Taberlet et al. (1991 ) primers e and f. As a nuclear marker, we employed name should be assigned). the ITS region, including ITS1, 5.8S rDNA gene, and ITS2, using the universal primers 17SE and 26SE (Sun et al. 1994). All ITS PCR prod- Of the 40 commonly accepted species of Bridelia , we sam- ucts were resolved on agarose gels as a single band and could be directly pled 25, but B. exaltata, B. glauca , and B. tulasneana were not sequenced (as found by Kawakita et al. 2004 and Kathriarachchi et al. included in all analyses because of incomplete sequences 2006 ). (Appendix 1 specifies the regions for which they were All reaction products were purified with Wizard SV gel and PCR clean- sequenced). Separate analyses of the chloroplast data (matK up kits (Promega, Madison, Wisconsin), and cycle sequencing was per- formed with BigDye Terminator v3.0 cycle sequencing kits (Applied gene and trnL-trnF IGS) and the nuclear (ITS) data yielded Biosystems, Foster City, California), using quarter-scale reaction mixtures. similar topologies that showed no statistically supported con- The dye terminators were removed by Sephadex G-50 Superfine gel fil- tradictions. We therefore combined all partitions into a sin- tration (Amersham Biosciences, Pittsburgh, Pennsylvania) on MultiScreen gle matrix of 3,177 aligned nucleotides sequenced for 50 plant TM-HV membrane plates (Millipore, Billerica, Massachusetts) according to the manufacturers’ protocols. Purified sequencing reactions were run accessions. on an ABI Prism 3100 Avant sequencer. The PCR primers were the same A matK gene for 114 accessions representing 51 gen- as those used for sequencing. Sequences were edited with Sequencher 4.7 era of Phyllanthaceae ( Fig. 2 ) has the topology also found by (Gene Codes, Ann Arbor, Michigan) and aligned by eye, using MacClade Kathriarachchi et al. (2005) and reveals the strikingly short 4.06 ( Maddison and Maddison 2003 ). branch lengths (genetic distances) within Bridelia compared to A total of 122 new sequences were generated for this study and have been deposited in GenBank (accession numbers see Appendix 1). other African/Asian clades. Bridelia is clearly monophyletic Phylogenetic Analysis—Maximum likelihood (ML) analyses under and sister to an Asian-Australian clade that comprises species an approximation of the GTR + G model of substitution were performed currently placed in the genus Cleistanthus . using RAxML (Stamatakis et al. 2008), with model parameters estimated Within Bridelia, the 3,177 nucleotides of nuclear and chloro- over the duration of specified runs. Bootstrap support values were esti- mated in RAxML with 100 replicate heuristic searches under the same plast DNA provide insufficient phylogenetic signal to resolve model as used in the searches. deeper relationships (Fig. 3), but do indicate that B. balansae Divergence Time Estimation—Estimation of divergence times relied and B. insulana are sister species (100% ML bootstrap support) on penalized likelihood (PL; Sanderson 2002 , 2004 , implemented in r8s and that B. tomentosa must include B. harmandii and B. curtisii version 1.71) which combines the advantages of a relaxed clock approach to become monophyletic (98% ML bootstrap support). Most with computational speed; computation time was an issue because our matrix consisted of 94 matK sequences (after the exclusion of all near- of the remaining species represented by multiple accessions identical sequences of Bridelia ). The outgroup taxa Chaetocarpus castano- are monophyletic except for B. retusa, which divides into a carpus , madagascariensis, and zeylanica were pruned B. retusa s. s. clade from and a clade from Guangdong prior to analysis, and the optimal smoothing parameter was found via in China ( Fig. 3 ). the cross validation routines implemented in r8s. All runs used the trun- cated Newton algorithm with bound constraints. To obtain confidence Biogeography and Age of the Stem and Crown Groups intervals on divergence times, we ran r8s analyses on trees obtained from of Bridelia— A chronogram resulting from the PL clock a Bayesian Markov chain Monte Carlo (MCMC) analysis of the 94-taxon approach is shown in Fig. 4. The divergence between Asian matrix in MrBayes version 3.1.2 ( Ronquist and Huelsenbeck 2003 ) under “Cleistanthus ” clade and Bridelia (i.e. the Bridelia stem age) the GTR + G + I model. Default settings in MrBayes were used for all prior occurred about 33 ± 5 Ma, while the onset of radiation distributions, and the chain was run for 1 million generations and sam- pled every 100th generations. Convergence was checked following the (i.e. the crown group) of Bridelia is estimated as 10 ± 2 Ma. procedures described in the MrBayes manual. Only the last 100 MCMC Unexpectedly, there are two (not one) African Bridelia clades trees were used, and the mean and standard deviation on age estimates of (labeled I and II in Figs. 3 and 4 ). The precise relationships interest were obtained with the r8s “profile” command. between the African and Asian clades are not resolved with To translate genetic distances into absolute time the r8s analyses used two simultaneous constraints: (i) The divergence between Phyllanthaceae statistical confidence (Fig. 2). In the chronogram, the better- and Picrodendraceae, i.e. the root node in our tree, was fixed at 108 Ma supported African clade (which has 99% ML bootstrap sup- based on Davis et al. (2005). The latter study used multiple fossil con- port; Fig. 3 ) is dated to 1.85 ± 1 Ma. Together, the topologies straints in a relaxed clock to estimate family-level nodes in . and time estimates suggest long-distance dispersal from Asia The Picrodendraceae divergence was placed at 108 Ma, with a 95% confi- to Africa sometime between 10 and 1.85 Ma. Australia was dence interval (CI) of 114–106 Ma. Fixing the age of any node is clearly a drawback because it underestimates the uncertainty in fossil dating and reached at least twice (probably three times, but we did not assignment; however, one fixed node is needed in penalized likelihood include an Australian individual of B. tomentosa), both times (Sanderson 2004) as well as most other clock approaches. (ii) The mini- ca. 2 Ma ( Figs. 2–4 ). mal age of was set to 55 Ma based on pollen described from the Lower Eocene Woolwich bed in Kent, England ( Gruas-Cavagnetto and Köhler 1992). Although the pollen of Phyllanthus is rather heterogeneous, Discussion the Woolwich pollen appears to be correctly assigned (W. Punt, personal communication, July 2008). As an alternative minimal constraint, we African/Asian Long-Distance Dispersal in Bridelia— Based could have used fossil fruits that resemble those of Phyllanthus from the on our findings, Bridelia dispersed from tropical Asia to Africa Upper Cretaceous (Maastrichtian, 65–70 Ma ago) Whitemud Formation in at least once over the past 10 million years, possibly twice, southern Saskatchewan, Canada ( Nambudiri and Binda 1989 ). For abso- lute ages, we relied on the geologic time scale of Gradstein et al. (2004 ). depending on the precise relationships of the two African clades. Together with the results from other genus-level African/Asian clades (see Introduction), our data support Results Thorne’s (1973) hypothesis that diaspore exchange across the Phylogenetics of Bridelia— GenBank accession numbers must be frequent since we only detect those dis- and voucher information for all sequences used in this study persal events that were followed by successful establishment. are listed in Appendix 1. All data matrices were submitted Steppingstones between Africa and Asia are the Seychelles, to TreeBASE (study number S2259). The appendix also lists the Comores, and the Chagos archipelago, about halfway the type locality for many of the species because the discov- between Africa and Indonesia, and the Miocene collision of ery of polyphyletic genera and species made it necessary to the Afro-Arabian plate with Asia opened another channel of decide which species or population was more likely to repre- migration between the continents. Bridelia fruits are red to 524 SYSTEMATIC BOTANY [Volume 34

Fig. 2. Maximum likelihood (ML) phylogeny for Phyllanthaceae based on matK sequences (1,990 aligned nucleotides); rooting and clade labeling are based on Kathriarachchi et al. (2005) . Values at nodes indicate ML bootstrap support from 100 replicates. An asterisk marks the two nodes used for calibration in the molecular clock analyses. 2009] LI ET AL.: BIOGEOGRAPHY OF BRIDELIA 525

Fig. 3. Maximum likelihood phylogeny of Bridelia based on combined nuclear ITS and plastid matK and trnL-trnF sequences (3,177 aligned nucle- otides); rooting is based on the results shown in Fig. 2 . Values at nodes indicate ML bootstrap support from 100 replicates. The Guangdong population of B. fordii (collections Li 127 and 91) is considered as belonging to B. retusa in the Flora of China (Li and Dressler 2008), but based on the sequences here is genetically distinct from that species. black drupes, 0.7–3 cm in diameter ( Dressler 1996 ) that are germinate readily, as shown for the timber species B. cathartica dispersed by birds as documented for the Asian B. retusa and B. micrantha ( Akinnifesi et al. 2006 ; World Agroforestry and B. tomentosa ( Ridley 1930 ; Corlett 1992 ) and the African Centre 2009). The roots of some species, for instance B. fer- B. micrantha ( Snow 1981 ). The seeds are small (the dry seed ruginea, can persist and sprout for many years (Abbadie et al. mass of B. ferruginea is 47.8 ± 4.9 mg; Lahoreau et al. 2006) and 2006). There are no bird migratory flyways between Asia 526 SYSTEMATIC BOTANY [Volume 34

Fig. 4. Chronogram obtained from a relaxed clock applied to 94 matK sequences of Phyllanthaceae, rooted as in Fig. 2. The two nodes that were constrained to obtain absolute times are indicated by the letters A and B; the arrow marks the crown group of Bridelia . 2009] LI ET AL.: BIOGEOGRAPHY OF BRIDELIA 527 and Africa, but the monsoon trade winds and correspond- (e.g. B. ferruginea ), occur among species of subgenus Bridelia ing ocean currents may be transporting torn-off floating plant with bilocular fruits (e.g. B. scleroneura), implying that reduc- parts from southwestern Indonesia across the great expanse tion in locule number occurred several times. The clade of the Indian Ocean to Africa (the same route followed by formed by the Asian one-seeded B . balansae and B . insulana , Austronesian merchant ships for at least two thousand years). however, corresponds to Gerhmann’s section Cleistanthoideae , Thousands of sightings of floating debris and entire floating also characterized by brochidodromous leaf venation, and islands have been made from ships traveling the world’s his section Micranthae corresponds to our African clade I oceans ( Van Duzer 2004 , 2006 ), and small seeds could easily ( Fig. 3 ). In the absence of any obvious morphological species have been transported in this way. Sri Lanka and southern groups and given the still incomplete species sampling, a new India are less likely source areas since they harbor only four intrageneric classification would be premature. or five species of Bridelia, including the widespread B. retusa and B. stipularis . Pollination of Bridelia is by nonspecialized Acknowledgments. We thank the National Science Foundation of China (Grant No. 30870367 to DZ) and the Chinese Academy of Science flies (Luo et al. 2007), and the combination of all these vegeta- (Grant No. KSCX2-YW-Z-027 to DZ) for funding, H.-J. Esser and two tive and reproductive characters probably facilitates success- anonymous reviewers for comments on the manuscript, P. Sebastian and ful establishment of Bridelia following dispersal. N. Holstein for help in the lab, I. Telford for material from Australia, and The five Australian species of Bridelia ( Forster 1999 ), of C. Heibl for help with illustrations. which B. exaltata, B. finalis, and B . leichhardtii are endemic, are not monophyletic and go back to at least two arrivals in Literature Cited Australia about 1–2 Ma ( Figs. 2–4 ). At 1–2 Ma, most islands between Borneo and New Guinea emerged from the sea and Abbadie, L., J. Gignoux, X. Le Roux, and M. Lepage . 2006 . Lamto: Structure, functioning, and dynamics of a savanna ecosystem series: Ecological stud- started to provide steppingstones for plant dispersal (e.g. ies . New York : Springer Verlag . Hall 2002 ). Bridelia probably dispersed to New Guinea (or the Akinnifesi, F. K., F. Kwesiga, J. Mhango, T. Chilanga, A. Mkonda, C. A. C. Lesser Sunda Islands) first and then reached Australia from Kadu, I. Kadzere, D. Mithofer, J. D. K. Saka, G. Sileshi, T. Ramadhani, there (however, we have not sampled from New Guinea). and P. Dhliwayo . 2006 . Towards the development of Miombo fruit trees as commercial tree crops in southern Africa. Forests. Trees and Generic Boundaries of Bridelia and Cleistanthus, and Livelihoods 16 : 103 – 112 . Species Concepts in Tropical Asian Bridelia— Based on our Airy Shaw, H. K. 1975 . The Euphorbiaceae of Borneo. Kew Bulletin Add. sampling of 11 of the 18 Asian species of Bridelia , three of the Ser. 4 . Kew : Royal Botanic Gardens . five Australian species, and 12 of the 18–20 African and Airy Shaw, H. K. 1978 . Notes on Malesian and other Asiatic Euphorbiaceae . Malagasy species, Bridelia is monophyletic. By contrast, Kew Bulletin 33 : 25 – 77 . Airy Shaw, H. K. 1980 . The Euphorbiaceae of New Guinea. Kew Bulletin Cleistanthus is polyphyletic and will need taxonomic readjust- Add. Ser. 8 . Kew : Royal Botanic Gardens . ment. The type species of Cleistanthus , C. polystachyus from Airy Shaw, H. K. 1981 . The Euphorbiaceae of Sumatra . Kew Bulletin 36 : , clusters with other African species including the 239 – 374 . small genera Pseudolachnostylis and possibly Pentabrachion (the Baum, D. A., R. L. Small, and J. F. Wendel . 1998 . Biogeography and floral evolution of baobabs (Adansonia , Bombacaceae) as inferred from mul- position of which is not statistically supported). The Asian tiple data sets . Systematic Biology 47 : 181 – 207 . “Cleistanthus” species therefore require a new name. Because Borges, R. M., H. Somnanthan, and S. Mali . 1997 . Alternations of sexes in our sampling does not include C. myrianthum (Hassk.) Kurz a deciduous tree: temporal dioecy in Bridelia retusa . Current Science from Java, the type species of Nanopetalum Hassk., nor C. fer- 72 : 940 – 944 . ruginea (Baill.) Müll. Arg. from Sri Lanka, the type of Lebidiera Corlett, R. T. 1992 . Plants attractive to frugivorous birds in Hong Kong . Memoirs of the Hong Kong Natural History Society 19 : 115 – 116 . Baill., we cannot decide whether any of these, or both, Davis, C. C., C. O. Webb, K. J. Wurdack, C. A. Jaramillo, and M. J. Donoghue . would group near C. sumatranus (Miq.) Müll. Arg., the type 2005 . Explosive radiation of Malpighiales supports a mid-Creta- of Leiopyxis Miq., which is included in our study. Depending ceous origin of modern tropical rain forests. American Naturalist 165 : on the topology, the oldest of these three names would be an E36 – E65 . Dressler, S. 1996 . The genus Bridelia Willd. (Euphorbiaceae) in Malesia and appropriate genus name for the Asian “Cleistanthus .” Indochina: a regional revision . Blumea 41 : 263 – 331 . We included multiple accessions of most Asian species Dressler, S. and P. C. van Welzen . 2005 . Bridelia . Pp. 141 – 152 , figs. 31, 32, ( Fig. 3 ) to test current species concepts for the Indochinese plate V: 3, 4 in Flora of Thailand 8 , eds. K. Chayamarit and P. C. van species of Bridelia (Dressler 1996; Li and Dressler 2008). For Welzen . Bangkok : The Forest Herbarium . most species, the gene tree obtained here agrees with mor- Forster, P. I. 1999 . A taxonomic revision of Bridelia Willd. (Euphorbiaceae) in Australia. Austrobaileya 5 : 405 – 419 . phological species boundaries. Exceptions are that B. har- Gehrmann, K. 1908 . Vorarbeiten zu einer Monographie der Gattung mandii and B. curtisii will need to be included in B. tomentosa Bridelia mit besonderer Berücksichtigung der afrikanischen if that species is to become monophyletic ( Fig. 3 ) and that Arten . Botanische Jahrbücher für Systematik, Pflanzengeschichte und B. retusa will need to be circumscribed more narrowly because Pflanzengeographie 41. Beibl . 95 : 1 – 42 . Gradstein, F. M., J. G. Ogg, A. G. Smith, W. Bleeker, and L. J. Lourens . 2004 . sequences from its Chinese populations do not cluster with A new Geologic Time Scale, with special reference to Precambrian the Thai populations ( Fig. 3 ). The type specimen of B. retusa and Neogene . Episodes 27 : 83 – 100 . comes from Sri Lanka, making it likely that the name applies to Gruas-Cavagnetto, C. and E. Köhler . 1992 . Pollens fossils d’Euphorbiacées the Thai populations rather than the Chinese ones, for which de l’Eocene français . Grana 31 : 291 – 304 . an available name is B. fordii Hemsley, which was based on Hall, R. 2002 . Cenozoic geological and plate tectonic evolution of SE Asia and the SW Pacific: computer-based reconstructions, model and ani- plants from Guangdong ( Li 1994 ). The remaining Asian spe- mations . Journal of Asian Earth Sciences 20 : 353 – 431 . cies of Bridelia could all be reliably bar-coded for easy identi- Hutchinson, J. and J. M. Dalziel . 1958 . Euphorbiaceae . Pp. 364 – 423 in Flora fication using just the trnL-trnF IGS. of West Tropical Africa Ed. 2 , 1 ( 2 ), ed. R. W. J. Keay . London : Crown In terms of intrageneric taxonomic groups within Bridelia , Agents for Overseas Governments and Administrations . Jablonsky, E. 1915 . Euphorbiaceae - Phyllanthoideae - Bridelieae . In: A. our data do not support Gehrmann’s (1908) subgenera (still Engler (ed.), Das Pflanzenreich. Heft 65 . Leipzig : Wilhelm Engelmann . accepted by Jablonsky [1915 ] and Dressler [1996 ]) since spe- Kathriarachchi, H., P. Hoffmann, R. Samuel, K. J. Wurdack, and M. W. cies of subgenus Gentilia, characterized by unilocular fruits Chase . 2005 . Molecular phylogenetics of Phyllanthaceae inferred 528 SYSTEMATIC BOTANY [Volume 34

from five genes (plastid atpB, matK , 3’ ndhF, rbcL and nuclear PHYC ) . Sanderson, M. J. 2002 . Estimating absolute rates of molecular evolution Molecular Phylogenetics and Evolution 36 : 112 – 134 . and divergence times: a penalized likelihood approach. Molecular Kathriarachchi, H., R. Samuel, P. Hoffmann, J. Mlinarec, K. J. Wurdack, Biology and Evolution 19 : 101 – 109 . H. Ralimanana, T. F. Stuessy, and M. W. Chase . 2006 . Phylogenetics Sanderson, M. J. 2004 . R8s, version 1.70. User’s manual . http://ginger. of the (Phyllanthaceae; Euphorbiaceae sensu ucdavis.edu/r8s/ r8s1.7.manual.pdf . lato) based on nrITS and plastid matK DNA sequence data . American Snow, D. W. 1981 . Tropical frugivorous birds and their food plants: a Journal of Botany 93 : 637 – 655 . world survey . Biotropica 13 : 1 – 14 . Kawakita, A., A. Takimura, T. Terachi, T. Sota, and M. Kato . 2004 . Stamatakis, A., P. Hoover, and J. Rougemont . 2008 . A rapid bootstrap algo- Cospeciation analysis of an obligate pollination mutualism: have rithm for the RAxML web-servers . Systematic Biology 75 : 758 – 771 . Glochidion trees (Euphorbiaceae) and pollinating Epicephala moths Sun, Y., D. Z. Skinner, G. H. Liang, and S. H. Hulbert . 1994 . Phylogenetic (Gracillariidae) diversified in parallel? Evolution 58 : 2201 – 2214 . analysis of sorghum and related taxa using internal transcribed Kulju, K. K. M., S. E. C. Sierra, S. G. A. Draisma, R. Samuel, and P. C. spacer of nuclear ribosomal DNA. Theoretical and Applied Genetics 89 : van Welzen . 2007 . Molecular phylogeny of Macaranga, Mallotus , and 26 – 32 . related genera (Euphorbiaceae S.S.): insights from plastid and nuclear Taberlet, P., L. Gielly, G. Pautou, and J. Bouvet . 1991 . Universal primers for DNA sequence data . American Journal of Botany 94 : 1726 – 1743 . amplification of three non-coding regions of chloroplast DNA. Plant Lahoreau, G., S. Barot, W. A. Hoffmann, S. A. Setterfield, and P. R. Williams . Molecular Biology 17 : 1105 – 1109 . 2006 . Positive effect of seed size on seedling survival in fire-prone Thorne, R. F. 1973 . Floristic relationships between tropical Africa and trop- savannas of Australia, Brazil and West Africa . Journal of Tropical ical America . Pp. 27 – 47 in Tropical forest ecosystems in Africa and South Ecology 22 : 719 – 722 . America: a comparative review , eds. B. J. Meggers and W. D. Duckworth . Leandri, J. 1958 . Euphorbiacées-Bridelia . Pp. 192 – 197 in Flore de Madagascar Washington : Smithsonian Institution Press . et des Comores 111e famille , ed. H. Humbert . Paris : Firmin-Didot . Van Duzer, C. 2004 . Floating islands: A global bibliography . Los Altos Hills, Léonard, J. 1955 . Observations sur divers Bridelia africains . Bulletin du California : Cantor Press . Jardin botanique de l’Etat à Bruxelles 25 : 359 – 374 . Van Duzer, C. 2006 . Addenda to Floating islands: a global bibliography . Los Léonard, J. 1962 . Euphorbiaceae-Bridelieae . Pp. 5 – 50 in Flore du Congo et du Altos Hills, California : Cantor Press . Rwanda- 8 (1) . Brussels : INEAC . World Agroforestry Centre. 2009 . http://www.worldagroforestry.org/sea/ Li, P. T. 1994 . Flora reipublicae popularis sinicae . Beijing : Science Press . products/afdbases/af/asp/SpeciesInfo.asp?SpID=374 (last accessed Li, P. T. and S. Dressler . 2008 . Euphorbiaceae . Pp. 172 – 177 in Flora of China May 2009). vol. 11 , ed. Z. Y. Wu, P. H. Raven, and D. Y. Hong . Beijing : Science Yuan, Y. M., S. Wohlhauser, M. Möller, J. Klackenberg, M. W. Callmander, Press / St Louis: Missouri Botanical Garden Press . and P. Küpfer . 2005 . Phylogeny and biogeography of Exacum Luo, S., D. Zhang, and S. S. Renner . 2007 . Duodichogamy and androdi- (Gentianaceae): a disjunctive distribution in the Indian Ocean basin oecy in the Chinese Phyllanthaceae Bridelia tomentosa . American resulted from long distance dispersal and extensive radiation. Journal of Botany 94 : 260 – 265 . Systematic Biology 54 : 21 – 34 . Maddison, W. P. and D. K. Maddison . 2003 . MacClade: analysis of phy- logeny and character evolution, version 4.06 . Sunderland : Sinauer Appendix 1. Species and nuclear regions sequenced, their GenBank Associates . accession numbers (ITS , matK , trnL-trnF , — = sequence not obtained), Malcomber, S. T. 2002 . Phylogeny of Gaertnera Lam. (Rubiaceae) based vouchers, and geographic localities. Type locations are indicated for the on multiple DNA markers: Evidence of a rapid radiation in a wide- Asian species (based on Dressler 1996) for which we tried to obtain intra- spread, morphologically diverse genus . Evolution 56 : 42 – 57 . specific accessions from throughout their ranges. To facilitate data loca- Nambudiri, E. M. V. and P. L. Binda . 1989 . Dicotyledonous fruits asso- tion, taxa are listed in alphabetical order by genus and species. ciated with coprolites from the Upper Cretaceous (Maastrichtian) Whitemud Formation, southern Saskatchewan, Canada . Review of Bridelia affinis Craib (Type locality: Thailand, Chiang Mai, Doi Palaeobotany and Palynology 59 : 57 – 66 . Suthep); FJ439898, FJ439935, FJ439979; Li Heng et al. 10572 (E); China, Pruesapan, K., I. R. H. Telford, J. J. Bruhl, S. G. A. Draisma, and P. C. van Yunnan, Gaoligong Shan Region, Baoshan, Mangkuan, 1,000 m, 24°57’N, Welzen . 2008 . Delimitation of Sauropus (Phyllanthaceae) based on 98°18’E. Bridelia affinis Craib; FJ439899, FJ439936, FJ439980; Y. Li lyq107 plastid matK and nuclear ribosomal ITS DNA sequence data. Annals (IBSC, M); China, Yunnan, Mengzi, Panzhihua, 1, 362 m, 23°23’N, of Botany 102 : 1007 – 1018 . 103°26’E. Bridelia angolensis Müll. Arg.; FJ439924, FJ439964, FJ440005; Radcliffe-Smith, A. 1987 . Euphorbiaceae (Part 1) . Flora of Tropical East G. Boss s. n. (M); , Sendi, 14°36’S, 14°15’E. Bridelia atroviridis Müll. Africa , ed. R. M. Polhill . Rotterdam : Balkema . Arg.; FJ439921, FJ439961, FJ440002; M. A. Mwangoka 1371 (M); Tanzania, Renner, S. S. 2004 . Multiple Miocene Melastomataceae dispersal between Tanga, Muheza District, Kwamngumi Forest Reserve, 100 m, 04°56’00’S, Madagascar, Africa, and India . Philosophical Transactions of the Royal 38°44’E. Bridelia balansae Tutcher (Type locality: Hongkong Bot. Garden, Society of London. Series B 359 : 1485 – 1494 . without information on wild provenience); FJ439904, FJ439942, FJ439985; Renner, S. S. and K. Meyer . 2001 . Melastomataceae come full circle: GaoligongShan Expedition 1997#9907 (E); China, Yunnan, Gaoligong Shan Biogeographic reconstruction and molecular clock dating. Evolution Region, Nujiang Lisu Aut. Pref., Lushi Co., ca. 5 km SW from Shanjiang, 55 : 1315 – 1324 . 1,000–1,150 m, 25°31’N, 98°08’E. Bridelia balansae Tutcher; FJ439903, Renner, S. S. and H. Schaefer . 2008 . Phylogenetics of Cucumis FJ439940, FJ439984; M. Furuse 3697 (MO); Japan, Ryukyu Province, Mt. (Cucurbitaceae) as understood in 2008. Pp. 53 – 58 in Cucurbitaceae Omoto Is. Ishigaki, 80 m, 24°22’N, 124°10’E; FJ439902, FJ439939, FJ439983; 2008. Proceedings of the IXth EUCARPIA meeting on genetics and M. Newman et al. LAO497 (E); Laos, Khammouan, along path to Ban Silia, breeding of Cucurbitaceae, Avignon (France), May 21–24th, 2008, 560 m, 18°00’N, 105°30’E; —, FJ439941, —; S.-C. Wu 842 (E); China, , ed. M. Pitrat . Available at https://w3.avignon.inra.fr/dspace/ Taipei City, Taipei Bot. Gard, 200 m, 25°03’N, 121°31’E; FJ439901, FJ439938, handle/2174/236. FJ439982; Y. Li lyq110 (IBSC, M); China, Guangdong, Meizhou, Yinnashan Renner, S. S., H. Schaefer, and A. Kocyan . 2007 . Phylogenetics of Cucumis Nature Reserve, 416 m, 24°24’N, 116°23’E. Bridelia brideliifolia (Pax) (Cucurbitaceae): Cucumber (C. sativus ) belongs in an Asian/ Fedde; FJ439922, FJ439962, FJ440003; M. A. Mwangoka 759 (M); Tanzania, Australian clade far from melon (C. melo ) . BMC Evolutionary Biology Iringa, Mufindi District, Lulanda Village, N of Fufu, 1,575 m, 08°36’S, 7 : 58 . 35°37’E. Bridelia cathartica Bertol. f.; FJ439929, FJ439970, FJ440011; Richardson, J. E., L. W. Chatrou, J. B. Mols, R. H. J. Erkens, and M. D. Pirie . J. A. Mlangwa et al. 1430 (M); Tanzania, Kilimanjaro, Gonja-Ntenga Ngara, 2004 . Historical biogeography of two cosmopolitan families of flow- Ndori village, 04°14’S, 38°02’E. Bridelia curtisii Hook.f. (Type local- ering plants: Annonaceae and Rhamnaceae. Philosophical Transactions ity: Malaysia, Penang, at Tulloh Bahang.); FJ439914, FJ439954, FJ439995; of the Royal Society of London. Series B 359 : 1495 – 1508 . H.-J. Esser 98-106 (M); Thailand, Sa Kaeo Province, RFD Centennial Ridley, H. N. 1930 . The dispersal of plants throughout the world . Ashford, Botanical Garden, circa 12°35’N, 102°4’E. F. Muell.; —, Kent : L. Reeve . FJ439978, —; P. I. Forster PIF30448 (NE); Australia, Pullen Creek, Council Ronquist, F. and J. P. Huelsenbeck . 2003 . MrBayes 3: Bayesian phyloge- Reserve, D’Aguilar Range, 16 km WSW of Brisbane City Centre, 60 netic inference under mixed models . Bioinformatics 19 : 1572 – 1574 . m, 27°1’S, 152°2’E. Bridelia ferruginea Benth.; —, FJ439966, FJ440007; Samuel, R., H. Kathriarachchi, P. Hoffmann, M. H. J. Barfuss, K. J. M. Schmidt 862 (FR); Burkina Faso, Houet, Moami, 22 km W of Bobo, 457 m, Wurdack, C. C. Davis, and M. W. Chase . 2005 . Molecular phylogenet- 11°7’N, 4°27’W. Bridelia glauca Blume (Type locality: Indonesia, Java); —, ics of Phyllanthaceae: evidence from plastid matK and nuclear PHYC FJ439977, —; S. Reksodihaadjo 186 (L); Indonesia, Java, Bogor, Tjipajung, sequences . American Journal of Botany 92 : 132 – 141 . Megamendung Tea Estate, 800 m, 6°35’S, 106°47’E. Bridelia harmandii 2009] LI ET AL.: BIOGEOGRAPHY OF BRIDELIA 529

Gagnep. (Type locality: Cambodia, Stung-streng); FJ439900, FJ439937, M. Newman et al. LAO120 (E); Laos, Khammouan, vicinity of Ban Mak FJ439981; W. Fischer 365 (FR); Thailand, Nakhon Ratchasima, Sakaerat, Phueang, 572 m, 17°58’N, 105°25’E; FJ439913, FJ439953, FJ439994; Li Pak Tong Chai, 14°31’N, 101°55’E. Bridelia insulana Hance (Type local- Heng et al. 10576 (E); China, Yunnan, Gaoligong Shan Region, Baoshan, ity: South Vietnam); —, AY830263, —; P.I. Forster 27626 (L); Australia, Mangkuan, Laomiancheng, S of Mangkuan on the E side of Gaoligong Queensland. Cook District: Silver Plains, 13°48’30”S, 143°28’30”E. Bridelia Shan, 1,000 m, 24°57’N, 98°18’E; —, FJ439951, —; I. McDonald 4982 insulana Hance; FJ439906, FJ439944, FJ439987; D. J. Middleton et al. 1545 (E); Indonesia, Bali, Tabanan, vicinity of Pura Ulu Watu, 50 m, 8°32’S, (M); Thailand, Phetchaburi, Amphoe Kaeng Krachan, Kaeng Krachan 115°7’E. Bridelia tenuifolia Müll. Arg.; FJ439923, FJ439963, FJ440004; National Park, 550 m, 12°50’N, 99°19’E. Bridelia leichhardtii Baill. ex T. Leyens & W. Lobin 206 (M); Angola, Bengo Province, Quissama National Müll. Arg.; FJ439905, FJ439943, FJ439986; J. Bruhl 802 (NE); Australia, Park, near Kulemba, 270 m, 06°38’S, 31°47’E. Bridelia tomentosa Blume Nathan Gorge, 23 km SW of Cracow, Cabbagetree Creek, 160 m, 25°7’S, (Type locality: Indonesia, Java); FJ439915, FJ439955, FJ439996; Y. Li lyq125 150°0’E. (Hochst.) Baill.; FJ439920, FJ439960, FJ440001; (IBSC, M); China, Guangdong, Guangzhou, South China Botanical J. A. Mlangwa & T. Kaniki 1022 (M); Tanzania, Kilimanjaro, Mbaga ward, Garden, 41 m, 23°10’N, 113°21’E. Bridelia tomentosa Blume; FJ439916, Chabara Dispensary, 1,600 m, 04°12’S, 37°57’E. Hochst.; FJ439956, FJ439997; C-C. Liao et al. 1643 (E); China, Taiwan, Chiayi Hsien, FJ439925, FJ439965, FJ440006; H. Merxmueller 250 (M); South Africa, Tapu Hsiang, Tsengwen Reservoir, along Provincial Road #3, 300–400 m, Gauteng Province, Pretoria, Rustenburg, Magaliesberge bei Bergheim, 23°28′N, 120°27′E; —, FJ439959, —; M. Newman et al. LAO565 (E); 25°43’S, 28°13’E. Bridelia ovata Decne. (Type locality: Timor); FJ439908, Laos, Khammouan, Wang Jang Reservoir, 570 m, 17°58’N, 105°25’E; FJ439946, FJ439989; D. J. Middleton et al. 1337 (M); Thailand, Prachuap FJ439917, FJ439957, FJ439998; P. Leong SING-MS-03 (FR); Singapore, Cluny Khiri Khan, Amphoe Thap Sakae, Huay Kang National Park, 150 m, Road, near the corner to Dalvy Road junction, 01°24’N, 103°59’E; FJ439918, 11°40’N, 99°35’E. Bridelia pervilleana Baill.; —, —, FJ440012; D. J. M. FJ439958, FJ439999; K. Williams, 1375 (M); Thailand, Trang, Tham Wang Du Puy 622 (P); Madagascar, Fianarantsoa Province, W of Ambositra, Phraya Songkham, 60 m, 8°00’N, 99°45’E. Bridelia tulasneana Baill.; —, 22°07’S, 46°51’E. Bridelia retusa (L.) A. Juss. (Type locality: Sri Lanka); —, FJ440019; P. J. Rakotomalaza et al. 1169 (P); Madagascar. —, FJ439947, —; H. -J. Esser, 99-7 (M); Thailand, Kamphaeng Phet Cleistanthus perrieri Leandri; —, AY552425, —; P. Hoffmann et al. 273 Province, Khlong Lan District, Mae Wong National Park, circa 16°33’N, (K); Madagascar. Cleistanthus suarezensis Leandri; —, AY830265, —; 99°30’E. Bridelia retusa (L.) A. Juss. (det. Dressler 29 Feb. 2008); FJ439907, P. Hoffmann et al. 423 (K); Madagascar. Cleistanthus concinnus Croizat; FJ439945, FJ439988; W. Fischer 672 (FR); Thailand, Chayaphum, Tat FJ439930, FJ439972, FJ440014; Y. Li lyq89 (IBSC, M); China, Hainan, Ton National Park, 16°00’N, 101°44’E. Bridelia retusa (L.) A. Juss. (det. Changjiang, Bawangling Nature Reserve, 140 m, 19°06’N, 109°04’E. as B. fordii by P.T. Li, 2008); FJ439909, FJ439948, FJ439990; Y. Li lyq91 Cleistanthus cunninghamii Müll. Arg.; —, AY830264, —; P. I. Forster 9176 (IBSC, M); China, Guangdong, Fengkai, Heishiding, 23°27’N, 111°53’E. (K); Australia, Queensland. Cleistanthus oblongifolius Müll. Arg.; —, Bridelia retusa (L.) A. Juss. (det. as B. fordii by P.T. Li, 2008); FJ439910, AY552424, —; M. W. Chase 1257 (K); Cult. Indonesia, Bogor Botanic Garden, FJ439949, FJ439991; Y. Li lyq127 (IBSC); China, Guangdong, Fengkai, as Cleistanthus chartaceus Müll. Arg., 6°35’S, 106°47’E. Cleistanthus pete- Heerkou, Xiaoguilin, 23°27’N, 111°53’E. Bridelia scleroneura Müll. Arg.; lotii Merrill ex Croizat; FJ439931, FJ439973, FJ440015; Y. Li lyq100 (IBSC, FJ439926, FJ439967, FJ440008; A. Wieckhorst 22 (FR); Benin, Atacora, Dept. M); China, Guangxi, Longgang Nature Reserve, 65 m, 22°28’N, 106°56’E. Pehunco, Pehunco, 370 m, 10°13’N, 1°59’E; FJ439927, FJ439968, FJ440009; Cleistanthus polystachyus Hook. f. ex Planchon; —, FJ439971, FJ440013; K. Küppers 596-1 (FR); Burkina Faso, Tapoa, Chaîne de Gobnangou, rd. L. Festo et al. 457 (MO); Tanzania, Kagera, Bukoba Rural, 1,140 m, 01°05’07’’S, Tansarga to Kodjari, Zollstation Tansarga, 11°52’N, 1°52’E; FJ439928, 031°26’13’’E. Cleistanthus sumatranus (Miq.) Müll. Arg.; FJ439933, FJ439969, FJ440010; N. A. Mwangulango 1233 (MO); Tanzania, Mpanda FJ439975, FJ440017; Y. Li lyq58 (IBSC, M); China, Hainan, Xinglong, District, Mlele Beekeeping zone, 1,560 m, 09°44’S, 13°44’E. Bridelia sp.; Xinglong Tropical Garden, 76 m, 18°41’N, 110°12’E; FJ439934, FJ439976, FJ439919, AY830262, FJ440000; G. Walters et al. 907 (MO); Gabon, Haut- FJ440018; M. Newman et al. 1149 (E); Thailand, Phrachuap Khiri Khan, Ogooué, 02°07’S, 14°04’E. Bridelia stipularis Blume (Type locality: India, Amphoe Pran Buri, Sam Roi Yot Natl. Park, trail over Khao Daeng, 200 m, Kattuko-kelang); FJ439911, FJ439950, FJ439992; Y. Li lyq126 (IBSC, M); 12°08’N, 99°57’E. Cleistanthus tomentosus Hance; FJ439932, FJ439974, China, Hainan, Changjiang, Bawangling Nature Reserve, 140 m, 19°06’N, FJ440016; Y. Li lyq92 (IBSC, M); China, Hainan, Sanya, Ganzhaling Nature 109°04’E. Bridelia stipularis Blume; FJ439912, FJ439952, FJ439993; Reserve, 289 m, 18°23’N, 109°39’E.