Molecular Phylogenetics and Evolution 44 (2007) 474–482 www.elsevier.com/locate/ympev Short communication Contrasting patterns of genetic differentiation between endemic and widespread species of fruit bats (Chiroptera: Pteropodidae) in Sulawesi, Indonesia Polly Campbell a,b,*, Andrea S. Putnam c, Caitlin Bonney a,d, Rasit Bilgin e, Juan Carlos Morales f, Thomas H. Kunz a, Luis A. Ruedas g a Department of Biology, Boston University, Boston, MA 02215, USA b Department of Zoology, University of Florida, P.O. Box 118525, Gainesville, FL 32611, USA c Section of Ecology, Behavior, and Evolution, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA d Harvard School of Public Health, Boston, MA 02115, USA e Bog˘azic¸i University, Institute of Environmental Sciences, Bebek 34342, Istanbul, Turkey f Division of Environmental Biology, National Science Foundation, Arlington, VA 22230, USA g Museum of Vertebrate Biology and Department of Biology, Portland State University, Portland, OR 97207, USA Received 14 July 2006; revised 25 January 2007; accepted 4 February 2007 Available online 22 February 2007 1. Introduction Bridle et al., 2001) implicates evolutionarily recent adaptive radiation. The Indonesian island of Sulawesi is remarkable for its Although, the diversity of Old World fruit bats (Pteropo- complex geological history and high biodiversity. Lying didae) is higher on Sulawesi than on the islands of the Sunda directly East of Wallace’s original zoogeographic boundary shelf and the Malay peninsula combined (Corbet and Hill, between the Oriental and Australian Regions (Wallace, 1992), phylogenetic relationships among pteropodid bats 1876), Sulawesi (Fig. 1) constitutes the largest terrestrial in Wallacea are not well-defined at the species level, and nei- habitat in Wallacea, a biogeographical region prioritized ther broad-scale phylogeographic nor population-level as a global conservation hotspot (Myers et al., 2000). Oce- genetic structure have been examined in any species of bat anic isolation has played a significant role in the evolution from Sulawesi. Here, we evaluate the phylogenetic status of Wallacea’s highly endemic fauna (Whitten et al., 1997); of Cynopterus brachyotis on Sulawesi, and compare the Sulawesi’s considerable size (186,145 km2), complex topog- within-island genetic structure of this widespread fruit bat raphy and diversity of microclimates and habitats have to that of a forest-restricted endemic, Thoopterus nigrescens. provided ample opportunity for diversification and in situ Thoopterus nigrescens (67–99 g) is the sole member of a speciation (Bridle et al., 2001). Molecular studies of Sulaw- genus that is endemic to Sulawesi and parts of the Moluc- esi’s biota have found evidence for both historic vicariance can Island chain (Fig. 1), and is strongly associated with and ecological selection as key factors driving diversifica- primary forest (Bergmans and Rozendaal, 1988). Cynopte- tion (Evans et al., 2004; Bridle et al., 2004). Geographically rus brachyotis (36–41 g) is common in disturbed habitats on concordant patterns of genetic differentiation across dispa- Sulawesi (Bergmans and Rozendaal, 1988) and is the only rate taxa (amphibians and monkeys, Evans et al., 2003a,b) Wallacean representative of a genus whose diversity in pen- support vicariance hypotheses, while low genetic diver- insular Malaysia and the Greater Sunda Islands suggests a gence among ecologically differentiated congeners (shrews, Sunda shelf center of origin (Campbell et al., 2006). Ruedi, 1995, 1998; grasshoppers, Walton et al., 1997; Although the range of C. brachyotis extends from South- west India to Sulawesi (Corbet and Hill, 1992), recent phy- logenetic analysis demonstrates that it comprises a complex of evolutionarily distinct lineages, including allopatric lin- * Corresponding author. Address: Department of Zoology, University of Florida, P.O. Box 118525, Gainesville, FL 32611, USA. Fax: +1 352 eages from Sulawesi and the Philippines and two sympatric 392 3704. lineages from the Sunda shelf (C. brachyotis Forest and E-mail address: [email protected]fl.edu (P. Campbell). Sunda; Campbell et al., 2004). 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.02.013 P. Campbell et al. / Molecular Phylogenetics and Evolution 44 (2007) 474–482 475 Fig. 1. Map of Sulawesi. Although currently a single island, Sulawesi was formed by accretion of a number of geologically distinct fragments. Major suture zones between fragments are shown in white. Scale bar on main map 100 km. Numbers refer to collecting localities in Table1: 1, Siuna; 2, Poso; 3, Lore Lindu; 4, Mangole Isl. (indicated on inset); 5, Tana Toraja; 6, Buton Isl.; 7, Kabaena Isl.; 8, G. Lompobatang. Inset map localizes Sulawesi (dark gray) and the Moluccas (black) in Southeast Asia. The Sunda shelf (light grey outline) defines the approximate area of additional terrestrial habitat exposed when sea levels dropped to 6120 m below present levels during Pleistocene glacial cycles. PM, peninsular Malaysia; S, Sumatra; B, Borneo; P, Philippines. Nias Island indicated by star. Scale bar on inset 1000 km. Within Sulawesi, the taxonomic status of C. brachyotis In the present study, we use mitochondrial DNA remains uncertain; variation in body size has been inter- (mtDNA) sequence data and nuclear microsatellites to preted as evidence that two allopatric populations (Berg- address three main questions. (1) Was Sulawesi colonized mans and Rozendaal, 1988) or two sympatric species by C. brachyotis from different geographic regions, as sug- (Kitchener and Maharadatunkamsi, 1991) occur on the gested by Kitchener and Maharadatunkamsi’s taxonomic island. In a morphometric evaluation of Indonesian Cyn- revision? Support for this scenario would include two opterus, Kitchener and Maharadatunkamsi (1991) pro- Sulawesi lineages, with sister relationships to either posed that C. brachyotis is replaced on Sulawesi by Philippine, or Sunda shelf C. brachyotis clades. (2) Is there C. luzoniensis and C. minutus, both considered synonyms evidence that two evolutionarily distinct lineages or species of C. brachyotis (Corbet and Hill, 1992; Simmons, 2005), occur currently on the island? Geographic overlap between taxa respectively described from the Philippines and Nias reciprocally monophyletic lineages would provide strong Island, West Sumatra (see Fig. 1). This assessment has support for this scenario. (3) Do T. nigrescens and not been universally accepted (Corbet and Hill, 1992; C. brachyotis exhibit comparable or different levels of Koopman, 1993, 1994, but see Simmons, 2005). While genetic structure within Sulawesi? Based on the endemicity phylogenetic analysis of Cynopterus recovered a single and higher habitat-specificity of T. nigrescens, we expected mitochondrial lineage from Sulawesi (Campbell et al., to find stronger population structure in this species relative 2004) sampling was insufficient to evaluate the Kitchener to the geographically widespread habitat generalist, and Maharadatunkamsi hypothesis. C. brachyotis. 476 P. Campbell et al. / Molecular Phylogenetics and Evolution 44 (2007) 474–482 2. Materials and methods 2.2. Analyses—mitochondrial DNA 2.1. Sampling, PCR, sequencing and fragment analysis Intraspecific phylogenies were reconstructed using max- imum parsimony (MP) and Bayesian criteria, implemented Cynopterus brachyotis was sampled from three main- in PAUP* (Version 4.0b10; Swofford, 2002) and MRBAYES land sites on Sulawesi and two offshore islands; Thoopte- (Version 3.1.2; Huelsenbeck and Ronquist, 2001). MP rus nigrescens was sampled from four mainland sites and analyses were run using a full heuristic search with tree- one island (Fig. 1 and Table 1). Total genomic DNA bisection-reconnection (TBR) branch-swapping and ran- was isolated from tissue samples (wing or liver) using Qia- dom stepwise addition (100 replicates). A ca. Seventy-seven gen (Valencia, CA) DNeasy extraction kits. Approxi- base pairs deletion common to all C. brachyotis Sunda mately 640 base pairs (bp) of the 50 end of the haplotypes (see below) was coded as a single synapomor- mitochondrial (mtDNA) control region were sequenced phic character. All other gaps were treated as a fifth char- for C. brachyotis (n = 21; see Table 1 for Genbank Acces- acter state; all characters were weighted equally. Nodal sion Nos.). Primers, PCR conditions and sequencing reac- support was evaluated with 1000 bootstrap replicates, each tion protocols are reported in Campbell et al. (2004); eight with five replicates of random taxon addition, a full heuris- haplotypes from this earlier study were included in the tic search and TBR branch-swapping. present analysis. The C. brachyotis tree was rooted with Megaerops ecaud- For T. nigrescens (n = 37), complete control region was atus, a putative sister genus to Cynopterus (Jones et al., 2002; initially amplified using universal primers (Kocher et al., Giannini and Simmons, 2003). Previously published haplo- 1989). The sequences obtained were used to design internal types from the Philippine-restricted C. brachyotis lineage, primers, ThoopIntF: 50-CCTGAAGTAAGAACCAGAT the Malaysian C. brachyotis Forest lineage, and the wide- G-30 and ThoopIntR: 50-ACGGCATCTGGTTCTTTC-30, spread C. brachyotis Sunda lineage (sensu Campbell et al., which amplified approximately 657 bp at the 50 end of 2004) were included to evaluate the geographic origin of the control region. Amplifications were carried out in a vol- Sulawesi C. brachyotis