Molecular Phylogenetics and Evolution 72 (2014) 35–41
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Molecular Phylogenetics and Evolution
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Short Communication Archipelago colonization by ecologically dissimilar amphibians: Evaluating the expectation of common evolutionary history of geographical diffusion in co-distributed rainforest tree frogs in islands of Southeast Asia
Paulette Gonzalez a, Yong-Chao Su a,b, Cameron D. Siler c, Anthony J. Barley a, Marites B. Sanguila d, ⇑ Arvin C. Diesmos a,e, Rafe M. Brown a,e, a Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045-7561, USA b Department of Biological Science, Tunghai University, Taichung, Taiwan c Sam Noble Museum and Department of Biology, University of Oklahoma, Norman, OK 73073-7029, USA d Father Saturnino Urios University, 8600 Butuan City, Philippines e Herpetology Section, Zoology Division, National Museum of the Philippines, Rizal Park, Padre Burgos Avenue, Manila, Philippines article info abstract
Article history: Widespread, co-distributed species with limited relative dispersal abilities represent compelling focal Received 5 July 2013 taxa for comparative phylogeography. Forest vertebrates in island archipelagos often exhibit pronounced Revised 19 December 2013 population structure resulting from limited dispersal abilities or capacity to overcome marine barriers to Accepted 21 December 2013 dispersal. The exceptionally diverse Old World tree frogs of the family Rhacophoridae have colonized the Available online 3 January 2014 forested island archipelagos of Southeast Asia on multiple occasions, entering the islands of Indonesia and the Philippines via a ‘‘stepping stone’’ mode of dispersal along elongate island chains, separated Keywords: by a series of marine channels. Here we evaluate the prediction that two tightly co-distributed Philippine Kurixalus appendiculatus rhacophorids colonized the archipelago during concomitant timescales and in the same, linear, ‘‘island- Old World tree frogs Mindanao tree frog hopping’’ progression. We use a new multilocus dataset, utilize dense genetic sampling from the eastern Philippines arc of the Philippines, and we take a model-based phylogeographic approach to examining the two Rhacophorus bimaculatus species for similar topological patterns of diversification, genetic structure, and timescales of diversifica- Frilled Tree Frog tion. Our results support some common mechanistic predictions (a general south-to-north polarity of colonization) but not others (timescale for colonization and manner and degree of lineage diversifica- tion), suggesting differing biogeographic scenarios of geographical diffusion through the archipelago and unique and idiosyncratic ecological capacities and evolutionary histories of each species. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction endemism (Brown and Diesmos, 2009) have fueled the search for common mechanisms of diversification, and piqued biogeogra- Understanding of the evolutionary and biogeographic processes phers’ interest in population substructuring, evolutionary parti- that have facilitated colonization of island archipelagos is of funda- tioning, and maintenance of biodiversity (Evans et al., 2003; mental interest to biodiversity specialists focused on the global Esselstyn and Brown, 2009; Oaks et al., 2013; Brown et al., 2013). conservation hotspots of Southeast Asia and the Pacific (Lohman Although the application of molecular data and rigorous statistical et al., 2011; Brown et al., 2013), where rates of forest destruction tools has greatly enhanced biogeographers’ ability to address ques- has been higher than anywhere else in the world (Whitmore and tions of diversification in a hypothesis-testing framework (Oaks Sayer, 1992; Brooks et al., 2002). In the Philippine biodiversity hot- et al., 2013; Brown et al., 2013), these efforts have been hampered spot, high levels of land vertebrate diversity and soaring rates of by a lack of comprehensive biodiversity surveys, logistical obsta- cles to field work, and a prevailing focus on fine-scale patterns of diversification associated with widespread microendemism ⇑ Corresponding author. Address: Biodiversity Institute, 1345 Jayhawk Blvd., (Brown and Diesmos, 2009; Brown et al., 2013). As a result, bioge- Lawrence, KS 66045, USA. Fax: +1 785 864 3403. ographers have only recently begun to identify more widespread, E-mail addresses: [email protected] (P. Gonzalez), [email protected] co-distributed groups of species, suitable for hypothesis-testing (Y.-C. Su), [email protected] (C.D. Siler), [email protected] (A.J. Barley), mbsanguila@ urios.edu.ph (M.B. Sanguila), [email protected] (A.C. Diesmos), [email protected] with a multi-taxon approach (Roberts, 2006; Esselstyn et al., (R.M. Brown). 2010; Siler et al., 2010; Brown et al., 2010; Oaks et al., 2013).
1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.12.006 36 P. Gonzalez et al. / Molecular Phylogenetics and Evolution 72 (2014) 35–41
One group of amphibians intimately tied to the geographical two species (Brown and Alcala, 1994) along the eastern Philippine template as a result of their variable natural history and ecological island arc lays the foundation for a simple biogeographic predic- tolerances are the Old World tree frogs of the family Rhacophori- tion of concomitant pattern and history of colonization, which dae (Brown and Alcala, 1994; Li et al., 2013). Two such unrelated can be tested phylogeny. species, Kurixalus appendiculatus and Rhacophorus bimaculatus are Our surprising results are consistent with the prediction of sim- widely (but patchily) distributed throughout the eastern island ilar initial routes of colonization in both taxa (entryway into the arc of the Philippines (Brown and Alcala, 1970, 1994), with sepa- Southwestern portions of the archipelago, presumably via coloni- rate origins in the Philippines via presumed colonization from zation from the Sunda Shelf island of Borneo via the elongate sulu the islands of the Sunda Shelf (e.g., Borneo; Li et al., 2013; Yu Archipelago, and a general south-to-north diffusion into the et al., 2013). Kurixalus appendiculatus is a forest floor, stagnant remaining Philippines), emphasizing the linear nature of the Phil- ephemeral pool or swamp-breeding species, and R. bimaculatus is ippines’ eastern island arc, but include exceptions to the pure a streamside vegetation-inhabiting species (Inger, 1954; Brown expectation of stepwise colonization. Additionally, pronounced and Alcala, 1994) most commonly encountered perched in vegeta- phylogeographic and population-genetic differences among popu- tion above spray zones and high-gradient cascading waterfalls lations of both taxa suggest different colonization histories and (RMB, personal observation). Rhacophorus bimaculatus exhibits veg- disparate patterns of geographically based genetic variation, con- etation-suspended larval development via foam-nest construction; sistent with variable natural history and microhabitat preferences the reproductive biology and larval ontogeny of Philippine popula- of each respective species. tions of K. appendiculatus have not been well studied (Alcala and Brown, 1982; Brown and Alcala, 1994). Because of their differing ecological and life-history strategies and their partially co-distrib- 2. Materials and methods uted ranges (overlapping five or six major hypothesized barriers to dispersal; Fig. 1), we were motivated to evaluate the prediction 2.1. Taxon sampling and data collection that they share a common history of archipelago colonization and similar evolutionary consequences of dispersal and isolation, Sampling included individuals collected over the past two possibly within similar timescales for diversification. Although decades from localities throughout the eastern island arc of the other studies have shown species-specific individual colonization Philippines in an effort to target sampling from extreme histories (Evans et al., 2003; Brown et al., 2010; Brown and Siler, southwestern Mindanao (adjacent to Borneo) to the northern por- 2013) the near-perfect co-distributed known occurrences of these tions of the northern most island of Luzon (Fig. 1; Appendix 1). To
Fig. 1. Hypothesized relationships of rhacophorid frogs related to R. bimaculatus and K. appendiculatus, illustrated by the maximum clade credibility tree resulting from Bayesian analyses. Nodes supported by P0.95 Bayesian PP were considered highly supported. Terminals are labeled with taxonomic names of outgroups, and localities within the two focal species. The expected topology stepwise colonization across the eastern arc is depicted at lower left, and a color-coded key to general localities and tree tip labels is presented at upper right, with hypothesized barriers to dispersal indicated for reference; these include: (1) the Mid-Sierra Filter Zone, (2) the Bicol–Luzon Isthmus, (3) the San Bernardino Strait, (4) the Dinagat–Siargao Transition Zone, (5) the Zamboanga-Mindanao Isthmus, and (6) the Sulu Strait. P. Gonzalez et al. / Molecular Phylogenetics and Evolution 72 (2014) 35–41 37 assess the monophyly of each species, test biogeographic hypoth- assessed by the effective sample size (ESS) values of parameters eses, and investigate appropriate outgroup taxa, a broad sampling (>200), likelihood scores through time plots using TRACER v1.5 (19 taxa) from the family Rhacophoridae was included (Appendix (Rambaut and Drummond, 2009). 1). These included representative taxa of the following genera: Partitioned maximum likelihood (ML) analyses were conducted Kurixalus (5 species), Rhacophorus (9 species), Nyctixalus (2 spe- in RAxML-VI-HPC v7.0 (Stamatakis, 2006) on the concatenated cies), and Theloderma (3 species). dataset using the same partitioning strategy as for Bayesian analy- Genomic DNA was extracted from liver tissues stored in 95% eth- sis. The more complex model (GTR + I + C) was used for all subsets anol or RNALater. Two mitochondrial gene fragments, 12S ribo- (Table 1), and 100 replicate ML inferences were performed for each somal RNA gene (12S; 723 bp) and Cytochrome b (Cytb; 569 bp), analysis. Each inference was initiated with a random starting tree and the nuclear gene Rhodopsin (Rhod; 323 bp) were sequenced and nodal support was assessed with 100 bootstrap pseudorepli- using the primers and protocols of Brown et al. (2010) and Li et al. cates (Stamatakis et al., 2008). (2013; and citations therein). Amplified products were visualized on 1.5% agarose gels, and PCR products were purified with 1 lLof 2.3. Estimating a relative temporal framework for divergence a 20% dilution of ExoSAP-IT (US78201, Amersham Biosciences, Pis- cataway, NJ). Cycle sequencing reactions were run using ABI Prism Lineage divergence times were estimated in BEAST version 1.7.5 BigDye Terminator chemistry (Ver. 3.1; Applied Biosystems, Foster (Drummond et al., 2012). Recent empirical studies of amphibians City, CA), and purified with Sephadex (NC9406038, Amersham Bio- (using a variety of calibration strategies, taxa, and mtDNA gene sciences, Piscataway, NJ) in Centri-Sep 96 spin plates (CS-961, fragments) have inferred model-corrected mitochondrial sequence Princeton Separations, Princeton, NJ). Purified products were ana- divergence rates between 0.8% and 1.9% total divergence per mil- lyzed with an ABI Prism 3130xl Genetic Analyzer (Applied Biosys- lion years (for review, see Sanguila et al., 2011, and citations there- tems). Continuous gene sequences were assembled and edited in) for various gene regions of the mitochondrial genome. We used using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI). All novel this range, and a relaxed clock approach, as implemented in BEAST, sequences are deposited in GenBank (Appendix 1). allowing branch lengths to vary according to an uncorrelated log- normal distribution, employing a Yule process tree prior and all 2.2. Sequence alignment and phylogenetic analyses remaining priors left as default. We selected a rate prior with a normal distribution and a mean of 1.4% (CI, 0.008–0.019) and simultaneously estimated divergence dates and chronogram topol- Initial alignments were produced in Muscle (Edgar, 2004), 9 followed by manual refinements. Alignments of protein-coding ogy. Four independent MCMC analyses were run for 1 Â 10 gener- regions (Cytb, Rhod) were optimized using translated amino acid ations, sampling every 1000 generations. We discarded the first sequences. To assess phylogenetic congruence between the mito- 25% of samples as burn-in. Three independent runs were per- chondrial and nuclear data, we inferred the phylogeny for each formed and convergence was determined as outlined above (see gene independently using likelihood and Bayesian analyses and as- Table 2). sessed all strongly supported nodes for differences in relationships between gene trees. Following the observation of no statistically 2.4. Ancestral range reconstruction and the eastern arc significant incongruence between datasets (no strong nodal sup- biogeographical diffusion scenario port for conflicting topologies), we concatenated the data for anal- ysis. Exploratory analyses of the combined dataset of 92 We used the Bayesian stochastic search variable selection, individuals (including all taxa, some of which were missing data BSSVS (Lemey et al., 2009), of the discrete phylogeographic model for one of the three sampled genes; Appendix 1) and a reduced dataset of individuals with no missing data exhibited identical Table 2 relationships; we therefore chose to include all available data for Uncorrected mitochondrial sequence divergence (%) among populations of Kurixalus appendiculatus and Rhacophorus bimaculatus distributed along the eastern island arc subsequent analyses of the concatenated dataset. of the Philippines (calculated in MEGA5; Tamura et al., 2011). Partitioned Bayesian analyses were conducted in MrBayes v3.2.1 (Ronquist and Huelsenbeck, 2003), with protein-coding 1234 genes partitioned by codon position; we treated the 12S sequence Kurixalus appediculatus data as a single partition. The Akaike Information Criterion (AIC), as 1. Zamboanga 2. Mindanao Island 0.073 implemented in jModeltest v2.1.3 (Posada, 2008) was used to 3. Bohol, Leyte, Samar 0.074 0.046 select the best model of nucleotide substitution for each partition 4. Luzon Island 0.063 0.049 0.044 (Table 1). For Bayesian analyses, two independent Markov Chain Rhacophorus bimaculatus 8 Monte Carlo (MCMC) chains were run for 2 Â 10 generations with 1. Zamboanga Markov chains being sampled every 1000 generations. The first 2. E. Mindanao Island 0.046 25% of the sampled trees was discarded as burn-in after the aver- 3. S. Mindanao Island 0.039 0.019 age standard deviation of split frequencies of the two runs falling 4. Leyte Island 0.046 0.014 0.018 5. Luzon Island 0.053 0.020 0.025 0.022 below the value of 0.01. The convergence of runs was further
Table 1 Substitution models for each partition selected by the Akaike information criterion (AIC).
Partition Model Nst Substitution rates 12S TIM2 + C 6 6.3197, 10.2567, 6.3197, 1.0000, 46.7128, 1.0000 CytB, 1st codon position TPM3uf + C 6 0.2462, 4.8736, 1.0000, 0.2462, 4.8736, 1.0000 CytB, 2nd codon position HKY + C 2 ti/tv = 5.5194 CytB, 3rd codon position TrN + C 6 0.1959, 0.3043, 0.1187, 0.3811 Rhod, 1st codon position TIM1ef + I 6 1.0000, 18.9720, 1.0000, 1.0000, 11.2115, 1.0000 Rhod, 2nd codon position F81 1 Rhod, 3rd codon position HKY + I + C 2 ti/tv = 4.4152 38 P. Gonzalez et al. / Molecular Phylogenetics and Evolution 72 (2014) 35–41 implemented in BEAST 1.7.5 to reconstruct the ancestral ranges of information). Finally, to assess both species for evidence of popula- the lineages within K. appendiculatus and R. bimaculatus. We set up tion structure versus recent expansion or population panmixia, we the phylogeographic traits via the present-day Philippine island calculated mismatch distributions in Arlequin 3.5.1.3 (Excoffier boundaries for the ingroups. The distributional ranges of outgroup et al., 2005; Supplemental information). taxa were set as Sundaland. The prior settings, MCMC chain length, and sampling strategy were the same as in divergence-time esti- mation with additional specification of symmetric discrete trait 3. Results substitution model. The most likely ancestral range (the range with highest posterior probability) of each lineage is reported in the 3.1. Taxon sampling, data collection, and sequence alignment final tree reconstructed in BEAST (Fig. 2). To explore support within both focal clades for diversification The complete, aligned matrix contains 44 samples of Kurixalus patterns consistent with the geographic-dispersion hypothesis, appendiculatus from Borneo and the Philippines and 27 samples we estimated the probability of stepwise diversification along the of Rhacophorus bimaculatus from the Philippines. Nineteen addi- Eastern Philippine Arc within a Bayesian framework (Supplemental tional rhacophorid samples available on GenBank were also
Fig. 2. The 50% majority-rule consensus tree resulting from analyses in BEAST using the BSSVS method of ancestral-range estimation. Branch color indicates inferred ancestral range, with posterior probabilities of ancestral range provided at focal nodes. All nodes recovered with moderate to high support (0.51–1.0 Bayesian PP) unless indicated. Nodes supported by 60.50 Bayesian PP were considered weakly supported. See key (lower left) for color-coded geographical ancestral areas. Labeled nodes referenced in Sections 3 and 4. P. Gonzalez et al. / Molecular Phylogenetics and Evolution 72 (2014) 35–41 39 included (Fig. 1, Appendix 1). Following initial unrooted analyses, (Zamboanga) of the island of Mindanao (Fig. 2). Interestingly, and other recent rhacophorid phylogenetic analyses (Pyron and although only moderately supported in some cases, biogeographic Wiens, 2011; Li et al., 2013; Appendix 1) we rooted the tree using reconstructions do not support a stepwise dispersion model from a representative sample of Nyctixalus spinosus. Variable characters the Mindanao to Luzon Pleistocene Aggregate Island Complexes are: 354 of 723 (12S); 303 of 569 (Cytb); 39 of 300 (Rhod). (PAICs; Brown and Diesmos, 2009; Fig. 2). Instead, within both clades, analyses support multiple dispersal events out of Mindanao 3.2. Phylogenetic analyses and the expectation of linear, stepwise Island, producing the currently widespread distribution of both archipelago colonization species across the southeastern portions of the archipelago (Fig. 2). Topology tests reinforce this observation, with no observed Analyses yield topologies with high Bayesian posterior proba- support [posterior probability (pp) approaching zero] for the step- bilities among many species and divergent populations sampled wise dispersal model (Fig. 1) along the eastern island arc of the in this study (Fig. 1). Some of the recovered relationships among Philippines. outgroup species included in this study are congruent with several recently published studies involving rhacophorid frogs (Li et al., 4. Discussion 2012; Yu et al., 2013; Fig. 1). Deeply divergent populations are recovered with strong support within both focal species, R. bima- 4.1. Phylogenetic and biogeographic origins of Philippine tree frogs culatus, and K. appendiculatus); however, analyses reveal greater inter-population genetic diversity within K. appendiculatus, with Both of our focal lineages clearly entered the Philippine archi- populations from Borneo recovered to be quite genetically distinct pelago through the Zamboanga Peninsula, a distinct and ancient from sampled populations in the Philippines (Fig. 1). The species R. geological component of the complex island of Mindanao (Yumul cyanopunctatus, previously considered conspecific with R. bimacul- et al., 2004). Now considered endemic to the Philippines (Brown atus, was not supported to be closely related to this species but and Alcala, 1994; data presented here), the Rhacophorus bimacula- instead fell into a an unresolved polytomy with mainland and Sun- tus lineage may have diverged from its closest relatives subsequent dalanx taxa (Figs. 2 and S1). to dispersal through the Sulu Archipelago into Zamboanga. Kurixa- Within both focal species, populations from the Zamboanga lus appendiculatus extends from approximately the Isthmus of Kra Peninsula of Mindanao Island (Southeast Mindanao, Fig. 1), form (Myanmar, Thailand), throughout the Malaysian Peninsula and the sister group to all other sampled Philippine populations (Figs. 1 Singapore, and into the Sundaic islands of Malaysia, Indonesia and 2). Notably, some phylogenetic patterns are observed in both (Borneo, Sumatra) and the Philippines (Brown and Alcala, 1994). focal species: (1) the Zamboanga Peninsula is inferred as both spe- Whether K. appendiculatus will continue to be recognized as a sin- cies’ entryway into the archipelago and (2) a general south-to- gle species (morphological and acoustic differences between some north progression is inferred in both species. Differences in order Philippine populations are apparent; RMB and CDS, personal obser- of island colonization and timing of diversification suggest differ- vations), is immaterial to the clear interpretation that it, too, ing biogeographic histories between the focal taxa (below). entered the Philippines through the Zamboanga Peninsula. This eastern-arc colonization route has been inferred for numerous 3.3. Timing of diversification other vertebrate lineages (Inger, 1954; Brown and Alcala, 1970; Evans et al., 2003; Brown and Diesmos, 2009; Sanguila et al., The four separate BEAST analyses converged on the same 2011; Brown et al., 2013). parameter space and were combined for further analysis using LogCombiner (BEAST 1.6.1; Drummond and Rambaut, 2007). With no internal calibrations available for this study, the confidence 4.2. Relative timing of diversification intervals for many node dates deeper in the chronogram are broad (Fig. 2); however, the results still provide interesting insights into Minimal overlap between the highest posterior-density inter- the timing of diversification among the two focal co-distributed vals around the divergence date estimated for the basal node for radiations of these two clades of tree frogs in the Philippines. Rela- each species (marking the inferred earliest possible date for a dis- tionships supported in the majority-rule consensus chronogram persal from Sundaland or the Sulu Archipelago to the Zamboanga) resulting from BEAST analyses mirror the ML and Bayesian topolo- refute simultaneous occurence (e.g., Oaks et al., 2013; Brown et al., gies (Figs. 1 and 2). Our results indicate that both radiations of rha- 2013) for the early divergence of our two focal species (Fig. 2). cophorid species in the Philippines represent relatively young Rather, it appears that the K. appendiculatus lineage entered the clades, having diversified in the archipelago within the last 3.9– Philippines via Zamboanga significantly earlier than the R. bimacul- 5.9 and 6.1–8.8 Ma (95% HPD; Fig. 2, Nodes A, B, respectively). atus lineage did. Differences in timing of early diversification allow Early diversification within the two clades does not appear to a possible biogeographic explanation (below) that differs from past have occurred concomitantly. Instead, early divergences in K. scenarios of stepwise colonization along the eastern arc (Brown appendiculatus pre-date those associated with R. bimaculatus and Siler, 2013; Brown et al., 2013). (Fig. 2, Nodes A, B; note minimally overlapping HPDs). Early diver- gence dates among the major clades of K. appendiculatus appear to 4.3. The eastern arc biogeographic diffusion scenario and an alternate pre-date the Pleistocene, possibly having occurred during the Plio- explanation cene and Miocene (Fig. 2, Nodes B, F, G), whereas the inferred divergences among major clades of R. bimaculatus may have Our Bayesian biogeographic ancestral ranges suggest that the occurred during the Pleistocene (Fig. 2, Nodes A, C–E). two species have a similar biogeographic history of geographic dif- fusion through the Philippines, with a general south-to-north pro- 3.4. Biogeographic reconstructions and eastern arc biogeographic gression (Figs. 2 and S1). However, both species’ reconstructions diffusion scenario suggest multiple, separate dispersals out of Mindanao, reaching Dinagat–Siargao separately from dispersal to Samar–Leyte and Bayesian biogeographic reconstructions provide strong support Bohol in R. bimaculatus and the Luzon PAIC prior to—or concomi- for Philippine radiations of both focal species originating in Sunda- tant with—separate dispersals to Dinagat–Siargao and to Samar– land and first colonizing the Philippines on the southwestern tip Leyte in K. appendiculatus (Fig. 2). A strict, linear sequence of 40 P. Gonzalez et al. / Molecular Phylogenetics and Evolution 72 (2014) 35–41 stepping-stone dispersal was not inferred in either case, and was assessment of each distribution, available habitat, and regional significantly rejected in Bayesian tests of topologies (PP approach- threats to continued survival. ing zero) in both species, suggesting that idiosyncratic, lineage- specific factors may have influenced the geographical diffusion of Acknowledgments each lineage through the archipelago. One possible explanation re- lates to difference between the two species’ timing of dispersal We thank the Philippine Department of Environment and Nat- northwards. At 6–9 mya (when K. appendiculatus presumably en- ural Resources for facilitating collecting and export permits. Field- tered the archipelago), Mindanao and Luzon were in much closer work was made possible with support from the University of proximity than they are today (Yumul et al., 2009; Hall, 2012), Kansas Biodiversity Institute, NSF DEB 0073199, DEB 0743491 and were also fragmented into smaller landmasses, possibly facil- and EF-0334952 to RMB, and DEB 0804115, Fulbright and Ful- itating early dispersal from the Zamboanga Block to Luzon, fol- bright-Hayes Fellowships to CDS. ACD Thanks the National Mu- lowed by subsequent or simultaneous dispersal to the Mindanao seum of the Philippines, J. Barnes, and Fauna and Flora faunal region. In contrast, by the relatively later time of the R. International for support of his fieldwork and MBS acknowledges bimaculatus dispersal through the Philippines ( 3 mya), the con- the FSUU Research office for funding and the VP Administrative figuration of the archipelago had changed by continued movement and Student Affairs Office for logistical assistance. During the prep- of the Philippine Mobile Belt (Yumul et al., 2009), separating these aration of this manuscript Y.-C. Su was supported by Tunghai Uni- two land masses and possibly requiring dispersal through the versity, Taiwan. We thank D. Bickford for use of a K. appendiculatus intervening eastern arc islands of Dinagat–Siargao, and Samar– genetic sample from Borneo, and L. Grimser for sharing genetic Leyte, and into Luzon via the Bicol Peninsula (Figs. 1 and 2). We samples of R. cyanopunctatus from peninsular Malaysia. An anony- note that at this time, the mobile nature of the archipelago had ac- mous reviewer provided helpful comments on an earlier draft of creted several previously isolated landmasses of Luzon and Minda- this manuscript. nao, possibly facilitating overland dispersal and south-to-north colonization (Yumul et al., 2009; Fig. S1). In support of this scenario Appendix A. Supplementary material is the observation that R. bimaculatus is common in the forests of the Bicol Peninsula (RMB and CDS, personal observations), but K. Supplementary data associated with this article can be found, appendiculatus has yet to be recorded on this geological component in the online version, at http://dx.doi.org/10.1016/j.ympev.2013. of Luzon (Brown and Alcala, 1994; see sampling, Fig. S1). The con- 12.006. spicuous absence of K. appendiculatus in the Bicol faunal region, despite numerous recent biodiversity survey efforts (RMB, CDS, References and ACD, unpublished data) supports a possible Bicol-mediated dis- persal to Luzon in R. bimaculatus but not in K. appendiculatus Alcala, A.C., Brown, W.C., 1982. Reproductive biology of some species of Philautus (Fig. S1). (Rhacophoridae) and other Philippine anurans. Philippine J. Biol. 11, 203–226. 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