Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

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Molecular Phylogenetics and Evolution

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Short Communication

Phylogeny of the island archipelago frog genus Sanguirana: Anotherendemic Philippine radiation that diversified ‘Out-of-Palawan’q

http://dx.doi.org/10.1016/j.ympev.2015.10.0101055-7903/� 2015 Elsevier Inc. All rights reserved.

q This paper was edited by the Associate Editor A. Larson.⇑ Corresponding author at: Biodiversity Institute, 1345 Jayhawk Blvd., Lawrence,

KS 66045, USA. Fax: +1 785 864 5335.E-mail addresses: rafe@ku.edu (R.M. Brown), ycsu@ku.edu (Y.-C. Su),

bkbwc3@mail.missouri.edu (B. Barger), camsiler@ou.edu (C.D. Siler), mbsanguila@urios.edu.ph (M.B. Sanguila), arvin.diesmos@gmail.com (A.C. Diesmos),dblackburn@flmnh.ufl.edu (D.C. Blackburn).

Please cite this article in press as: Brown, R.M., et al. Phylogeny of the island archipelago frog genus Sanguirana: Another endemic Philippine radiatidiversified ‘Out-of-Palawan’. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.10.010

Rafe M. Brown a,⇑, Yong-Chao Su a, Brenna Barger a, Cameron D. Siler b, Marites B. Sanguila c,Arvin C. Diesmos d, David C. Blackburn e

aBiodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045-7561, USAb Sam Noble Oklahoma Museum of Natural History and Department of Biology, University of Oklahoma, Norman, OK 73072-7029, USAcArts and Sciences Program, Saturnino Urios University, San Francisco St., 8600 Butuan City, PhilippinesdHerpetology Section, Zoology Division, Philippine National Museum, Burgos St., Ermita 1000, Manila, Philippinese Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA

a r t i c l e i n f o

Article history:Received 16 July 2015Revised 9 October 2015Accepted 10 October 2015Available online xxxx

Keywords:Cascade frogsGeographic radiationPalawan Arc HypothesisPleistocene Aggregate Island Complexes(PAICs)Stream frogs

a b s t r a c t

Recent higher-level frog phylogenetic analyses have included a few members of the endemic Philippinefrog genus Sanguirana. Although the monophyly of the group has never been disputed, the recentphylogenetically-supported inclusion of the Palawan Wood Frog (Sanguirana sanguinea) in this cladewas highly unexpected. In addition, species boundaries and relationships remain unclear and new speciescontinue to be discovered. We estimate the phylogeny for this endemic Philippine genus using twomitochondrial gene regions and six nuclear loci and complete sampling for all known species. We usea time-calibrated Bayesian estimate of phylogeny and model-testing approach to biogeographic inferenceto infer ancestral areas and probable means of diversification. These analyses identify Sanguirana as anadditional clade for which the biogeographic ‘Out-of-Palawan’ biogeographic scenario is unambiguouslypreferred. This study lends additional support to recent work suggesting that a substantial portion ofPhilippine vertebrate megadiversity originated via colonization of the archipelago from the Palawanmicrocontinent, with subsequent invasion of oceanic islands (e.g., range expansion over Huxley’sModification of Wallace’s Line), numerous instances of overwater dispersal, and geographic radiationacross the archipelago.

� 2015 Elsevier Inc. All rights reserved.

1. Introduction

Our limited understanding of the biogeographic patterns andevolutionary processes behind Southeast Asia’s exceptionally highlevels of vertebrate diversity is both a crippling limitation for con-servation biologists (Woodruff, 2010) and a remarkable opportu-nity for evolutionary biologists and biogeographers (Brown andDiesmos, 2009; Lohman et al., 2011; Brown et al., 2013). StudiesSoutheast Asian biodiversity have been hampered by the patchyand non-comprehensive nature of basic survey work in its forests(Lim et al., 2008), the deterioration, lack of financial support, andthreats to existing biodiversity collections (Kemp, 2015), the

lugubrious pace of taxonomic work (Joppa et al., 2011), andcountry-specific, idiosyncratic, and variable policies of naturalresource management (La Viña et al., 1997; Lim et al., 2008;Meijaard, 2011).

Home to numerous celebrated clades of co-distributedterrestrial vertebrates (Brown and Diesmos, 2009), the PhilippineArchipelago recently has been the focus of several integrative stud-ies of amphibian radiations (Setiadi et al., 2011; Blackburn et al.,2013; Brown and Siler, 2013; Brown et al., 2013, 2015). One mod-erately sized clade is the endemic frog genus Sanguirana (formerlythe Rana everetti Complex; Brown et al., 2000), which consists ofseven described and morphologically diagnosable species andone or two undescribed ‘‘cryptic” species (Brown et al., 2000;Fuiten et al., 2011). Additional new species most likely await dis-covery in unsurveyed portions of the archipelago (Brown et al.,2013; Brown, 2015).

In a recent review, Brown et al. (2013) distinguished betweenthe archipelago’s partially or fully characterized adaptiveradiations (e.g., Blackburn et al., 2013; Brown et al., 2015) and

on that

2 R.M. Brown et al. /Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

the possibly non-adaptive, ‘‘geographic” radiations (e.g., Setiadiet al., 2011; Brown and Siler, 2013). The latter, loosely defined cat-egory (Brown et al., 2013) includes clades with one or more repre-sentative species on each island bank, or Pleistocene AggregateIsland Complex (PAIC; Brown and Diesmos, 2009; Fig. 1). In theseclades, species have been characterized as ecologically similar withlittle to no evidence of a phenotype-environment correlation orwithin-island, ecologically associated diversification (Brownet al., 2000, 2013; Brown and Siler, 2013). Of particular interestare clades that show a mixture of diversification patterns, with sin-gle ecological generalist species on some islands, but evidence ofintra-island diversification, habitat specialization, and multiplesympatric species on other islands (Setiadi et al., 2011; Brownet al., 2013, 2015).

We estimate the phylogeny and biogeographic history of onesuch group: Philippine endemic stream frogs of the genusSanguirana (Brown et al., 2000; Fuiten et al., 2011). Aside frombeing one of only two amphibian genera endemic to the country(the other being Platymantis; Brown et al., 2015), Sanguirana isinteresting because of its pattern of species’ distributions. Thegenus consists of at least eight species (Fuiten et al., 2011), withall taxa restricted to individual PAICs (Brown et al., 2000). Severalof the species (S. albotuberculata, S. everetti, S. sanguinea, and S. sp.[undescribed]) possess exclusive, non-overlapping distributionsisolated to single PAICs or island subsets within PAICs, but theremainder (S. aurantipunctata, S. igorota, S. luzonensis, and

Fig. 1. Map of the Philippine archipelago (A) with sampling color coded by species (seecontour forming the major Pleistocene Aggregate Island Complexes (PAICs; Brown and Dconsensus tree resulting from the post-burnin posterior distribution from the Bayesian aP70% ML) are denoted with black dots) and moderately supported nodes (0.90–0.95 Baresulted in weakly-supported differences in branching order among species but resolvecoalescence as one possible explanation for incongruence between mtDNA and nDNA greader is referred to the web version of this article.)

Please cite this article in press as: Brown, R.M., et al. Phylogeny of the island arcdiversified ‘Out-of-Palawan’. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/1

S. tipanan) features some degree of sympatry in the mountains ofLuzon Island (Figs. 1 and 2; Brown et al., 2000).

Here we infer the biogeographic history of this group and askwhether any of the previously hypothesized, shared mechanismsof diversification for Philippine terrestrial vertebrates (Brownet al., 2013) apply to Sanguirana. Our multilocus phylogenetic esti-mate and ancestral inference of biogeography identifies the genusas yet another clade with a biogeographic history consistent withthe ‘Out-of-Palawan’ biogeographic scenario (Blackburn et al.,2010; Esselstyn et al., 2010; Siler et al., 2012; Brown et al.,2013). As such, the genus Sanguirana represents the first amphib-ian clade for which the Palawan Arc Hypothesis (Blackburn et al.,2010; Siler et al., 2012) is a tractable explanation for the initial iso-lation, biogeographic origin, and subsequent diversification of oneof the archipelago’s more spectacular and completely endemicradiations (Brown and Diesmos, 2009; Brown et al., 2013).

2. Materials and methods

2.1. Taxon sampling and data collection

Ingroup sampling included 161 individuals collected from 47localities, including all known species of Sanguirana (Fig. 1;Appendix A, Supplemental Material). We selected closely relatedoutgroup species Glandirana minima and Hylarana erythraea, on

key). Lighter blue shadings indicate shallow seas and the underwater bathymetriciesmos, 2009). Hypothesized relationships of Sanguirana species (B), depicted by thenalysis of the concatenated dataset. Strongly supported nodes (P0.95 Bayesian PP;yesian PP) are indicated with gray dots. Bayesian analysis of nuclear genes only (C)the paraphyly of S. luzonensis (with respect to S. igorota and S. tipanan); (D) deep

ene lineages. (For interpretation of the references to color in this figure legend, the

hipelago frog genus Sanguirana: Another endemic Philippine radiation that0.1016/j.ympev.2015.10.010

Fig. 2. Time-calibrated Bayesian topology from BEAST (see text for results from individual calibration schemes) with approximate geological reconstructions from Hall (1998)and the results of ancestral range estimation under the best-fit, 3-parameter DIVALIKE+J model of geographical range evolution (Table 2) implemented in the R packageBioGeoBEARS (Matzke, 2013). Color-coded boxes on nodes correspond to PAIC ancestral range estimates, indicated on right (colored vertical bars). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.)

R.M. Brown et al. /Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx 3

the basis of uncontroversial higher-level phylogenetic relation-ships of the family Ranidae (Wiens et al., 2009), and sequenced atotal of 6098 nucleotides from two mitochondrial gene regionsand six nuclear loci (Table 1). Primers, PCR methods, thermal pro-files, and sequencing protocols follow our recent phylogeneticstudies (Brown and Siler, 2013; Brown et al., 2015). We visualizedproducts on 1.5% agarose gels and used ABI BigDye Terminator v3.1sequencing chemistry (Life Technologies, Grand Island, NY) forcycle sequencing. PCR products and sequencing reactions werepurified using ExoSAP-IT and Sephadex cleanups, respectively(Amersham Biosciences, Piscataway, NJ). Purified products wereanalyzed with an ABI Prism 3730xl Genetic Analyzer, andsequences were assembled and edited using Geneious vR8.1.7(Kearse et al., 2012). All novel sequences were deposited inGenBank (Appendix A, Supplemental Material).

2.2. Sequence characteristics, alignment, and phylogenetic analyses

Initial alignments were produced in MAFFT v7.221 (Katoh andStandley, 2013) with slight manual adjustments. To assess genecongruence, we estimated gene trees with Bayesian and likelihood

Please cite this article in press as: Brown, R.M., et al. Phylogeny of the island arcdiversified ‘Out-of-Palawan’. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10

analyses and compared estimates from these independent parti-tions for the presence of strongly supported conflict between generegions. Finding none, we concatenated data for subsequent anal-yses. We then assembled two datasets: one with all genes included(but with missing nuclear data for many individuals [Table 1];Appendix A, Supplemental Material) and another reduced to sam-ples with near-complete nuclear gene sampling. Because explora-tory comparative analyses yielded the same strongly supportednodes, we felt justified in including all available data in subsequentphylogenetic analyses.

Partitioned Bayesian analyses were conducted inMrBayes v3.2.5(Ronquist and Huelsenbeck, 2003). We partitioned the ribosomalRNA mitochondrial sequences by region (12S, tRNAval, and 16S),nuclear DNA was partitioned by locus, with gene-specific protein-coding regions partitioned by codon position (Table 1). The AkaikeInformation Criterion (AIC), as implemented in jModelTest v2.1.7(Darriba et al., 2012, was used to select the best model of nucleotidesubstitution for each partition (Table 1). We ran four MCMC analy-ses, eachwith fourMetropolis-coupled chains, an incremental heat-ing of 0.02, and an exponential distribution with 25 as the rateparameter prior on branch lengths. All analyses were run for

hipelago frog genus Sanguirana: Another endemic Philippine radiation that.1016/j.ympev.2015.10.010

Table 1Loci, number of sequences (N), characteristics, partitioning, and results of model selection procedure.

Locus N Total characters Pars-inform char’s Un-informative char’s Variable char’s Mean p-distance Substitution model

12S 161 485 129 67 196 1.32972 TrN+CtRNA-Val 154 70 25 8 33 0.97069 K80+C16S 151 1457 428 258 686 1.83154 GTR+I+CCLT-1st 28 228 12 7 19 0.13111 TPM1uf+ICLT-2nd 28 228 8 3 11 0.06915 TrN+ICLT-3rd 28 227 28 10 38 0.31727 TPM1uf+I+CCPT-1st 21 249 1 8 9 0.03614 HKYCPT-2nd 21 249 3 7 10 0.04217 HKYCPT-3rd 21 248 14 23 37 0.18975 K80+ICXCR-1st 43 197 4 1 5 0.04158 JCCXCR-2nd 43 197 1 0 1 0.01204 TrNCXCR-3rd 43 196 23 7 30 0.34387 TPM1uf+I+CRAG1-1st 23 202 3 1 4 0.08108 F81RAG1-2nd 23 203 11 23 34 0.2354 HKY+CRAG1-3rd 23 202 7 6 13 0.13289 HKY+ITyro-1st 40 181 5 8 13 0.09734 TPM1uf+CTyro-2nd 40 181 21 31 52 0.4392 HKY+CTyro-3rd 40 181 4 13 17 0.18681 HKYDNAH3-1st 26 306 5 5 10 0.03268 F81DNAH3-2nd 26 306 2 4 6 0.01961 HKYDNAH3-3rd 26 306 16 9 25 0.09818 HKY

4 R.M. Brown et al. /Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

20 � 107 generations, with parameters and topologies sampledevery 1000 generations. We assessed stationarity with Tracer v1.6(Rambaut and Drummond, 2007) and convergence with AWTY(Wilgenbusch et al., 2004). Stationarity was achieved after 5 � 106

generations (first 25% samples), which we discarded as burn-in.Partitioned maximum likelihood (ML) analyses were conducted

in RAxMLHPC v8.0.0 (Stamatakis, 2014) on the concatenated data-set using the same partitions (Table 1) but with the GTR+ C modelfor each. One hundred replicate inferences were performed, eachinitiated with a random starting tree. Nodal support was assessedwith 1000 bootstrap pseudoreplicates. Alignments and resultingtopologies were deposited in Dryad (doi: 10.5061/dryad.6394c).

2.3. Divergence time estimation and biogeographic inference

We used BEAST v1.8.2 (Drummond et al., 2012) to estimatedivergence times. We unlinked clock models and original substitu-tion rates per gene/partition, and employed the following priors fortwo calibration strategies: (1) we applied a range of empiricallyobserved amphibian divergence rates (1.4% Mya�1, 95%HPD = 0.8–1.9%) following Sanguila et al. (2011) as the rate calibra-tion for the mitochondrial genes in our data set; (2) for compar-ison, we applied the time estimate of the approach of the oceanicPhilippines to the Palawan microcontinent block (8 Mya, 95%HPD = 10–6; Hall, 1998) to provide a crude estimate of the originof the oceanic Philippine lineage. We then combined four separateruns for each calibration (25% burn-in for each) and consideredeffective sample sizes adequate if >200. We diagnosed convergencewith Tracer v1.6 and used TreeAnnotator v1.8.2 to generate ourfinal maximum clade credibility topology.

We used BioGeoBEARS (Matzke, 2013) to gain insight intopotential speciation modes and to estimate ancestral geographicalranges. We considered a total of six models (Matzke, 2013): Dispersal-Extinction-Cladogenesis (DEC; Ree and Smith, 2008), Ronquist’s(1997) Dispersal-Vicariance Analysis (DIVA; Ronquist, 1997), andthe BayArea model (Landis et al., 2013); and an additional parame-ter j that was considered for each model to evaluate founder-eventspeciation, which could be associated with long distance dispersaland subsequent divergence of colonizing lineages (Matzke, 2013).We then estimated biogeographic ancestral ranges and transitionsthereof under the best-fit model (selected with AIC).

Please cite this article in press as: Brown, R.M., et al. Phylogeny of the island arcdiversified ‘Out-of-Palawan’. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/1

3. Results

3.1. Phylogeny

Bayesian and likelihood analyses (Figs. 1 and 2) of the completedataset strongly support a monophyletic Philippine Sanguirana(MrBayes posterior probabilities [PP] = 1.00, Maximum Likelihoodbootstrap support [MLBS] = 100). Sanguirana sanguinea fromPalawan Island is the sister taxon to the remaining clade of oceanicPhilippine lineages (PP = 0.99, MLBS = 80; Fig. 1). Within this cladeof oceanic Philippine species are three strongly supported taxawhose relationships to each other are not resolved with strong sup-port: (1) the Luzon endemic S. aurantipunctata, (2) the Mindanaoclade of S. albotuberculata + S. everetti (PP = 1.00, MLBS = 100), and(3) a clade in which an undescribed species from the West Visayanislands (Figs. 1 and 2; PP = 0.99,MLBS = 93) is sister to a clade of spe-cies from Luzon (S. igorota, S. luzonensis, and S. tipanan; PP = 0.73,MLBS = 88). The relationships among S. igorota, S. luzonensis, andS. tipanan remain poorly resolved (Fig. 1). Weakly supported differ-ences between themitochondrial gene tree (not shown; identical tothe concatenated tree in Fig. 1B) and a separate nuclear topology(Fig. 1C), involves a possibly non-monophyletic S. luzonensis withrespect to S. igorota and S. tipanan (Fig. 1).

3.2. Biogeographic inference and time frame for diversification

We subsampled 49 individuals (at least two samples/clade fromthe 161-individual dataset) with near-complete mitochondrial andnuclear sequences to estimate the divergence times among themajor clades. When our amphibian mitochondrial molecular clock(1.4% divergence between species Mya�1) was applied, weestimated the oldest ingroup node as 12.1 Mya (95% HPD = 16.9–7.7), which approximates the divergence (14.1 Mya, 95%HPD = 18.3–10.2) inferred when using our geological calibration(10–6 Mya, Hall, 1998). The estimated divergence times for theoceanic clade were nearly identical (molecular clock: 7.8 Mya,95% HPD = 10.6–5.2; geology: 7.9 Mya, 95% HPD = 9.5–6.4).Divergence times within the oceanic Philippines were similarlycoincident, and dated during the late Miocene to Pliocene (Fig. 2).

The best-fit biogeographic model selected by AIC weights wasMatzke’s (2013) likelihood implementation of the DIVA model(i.e., two-parameter model; dispersal allowed but penalized

hipelago frog genus Sanguirana: Another endemic Philippine radiation that0.1016/j.ympev.2015.10.010

Table 2Results of biogeographic model selection procedure via Akaike Information Criterion assessment of model fit (Matzke, 2013).

Model LnL Numparams d e j AIC Delta-AIC AIC Wt.

DIVALIKE+J �8.098662 3 1.00E�12 1.00E�12 0.05097011 22.197324 �0.870998 0.58DEC �9.534161 2 1.00E�12 1.00E�12 0 23.068322 0 0.37DEC+J �8.927847 3 1.00E�12 2.36E�09 0.04630856 23.855694 0.787372 0.25DIVALIKE �11.064518 2 7.87E�03 1.00E�12 0 26.129036 3.060714 0.08BAYAREALIKE+J �10.410781 3 1.00E�07 1.00E�07 0.07690819 26.821562 3.75324 0.06BAYAREALIKE �16.305238 2 9.80E�03 1.05E�01 0 36.610476 13.542154 0.00

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relative to vicariance; Ronquist, 1997), which incorporates disper-sal to a geographic area not observed in the parental lineages(DIVALIKE+J; Table 2). The ancestral range estimate based on thismodel inferred a process of vicariance for diversification betweenthe ephemerally accreted (or, at least, adjacent) Palawan micro-continent + proto-Luzon block (Hall, 1998; Brown and Diesmos,2009), which then separated as the latter slipped farther northalong the Philippine trench (Fig. 2; Hall, 1998; Brown et al.,2013). Subsequently, because the oceanic Philippines arrived atits current configuration within the last eight million years (Hall,1998; Brown and Diesmos, 2009), we infer that speciation duringthis period was associated with repeated bouts of overwaterdispersal and colonization between isolated PAICs (Luzon toMindanao �7.3 Mya; Luzon to Western Visayas �5.9 Mya; Fig. 2).

4. Discussion

4.1. Phylogeny

Our phylogenetic estimate identifies weakly supported differ-ences between mitochondrial and nuclear dataset partitions. Thesediscrepancies involve the first-branching lineages within theapparently rapidly-radiating oceanic Philippines clade(Fig. 1B and C), and also the non-monophyly of S. luzonensis withrespect to S. igorota and S. tipanan (Fig. 1D). The latter conceivablycould be explained by the presence of a cryptic species (SierraMadre and Zambales mountains ranges of Luzon and possiblyMarinduque Island; Fig. 1B), maintenance of ancestral polymor-phism, deep coalescence of mitochondrial gene lineages, and/orthe stochastic nature of lineage sorting in the coalescent(Fig. 1D). Distinguishing between these hypotheses is beyond thescope of the current dataset given the lack of support at somenodes and limited number of loci available.

4.2. ‘Out-of-Palawan’ biogeography in Philippine stream frogs

The analysis presented here identifies another Philippine radia-tion for which the Palawan Ark Hypothesis (Blackburn et al., 2010;Siler et al., 2012; Brown et al., 2013) best fits the available data.Because of low support for the clade ((S. everetti + S. albotubercu-lata) (S. sp. Negros-Panay (S. luzonensis (S. igorota + S. tipanan + S.luzonensis))))), we are unable to infer the sequence of island colo-nization following the initial invasion of the oceanic portions ofthe archipelago from Palawan (Fig. 1D). We expect that animproved, fine-scale biogeographic inference may be possible withinformation from additional loci. Despite the lack of support for thesequence of island colonization (and order of first branching withinthe oceanic Philippine clade; Fig. 1B and C), the genus Sanguiranaclearly diversified within the Philippines after an initial invasionfrom the Palawan microcontinent. It has not escaped our attentionthat if current phylogenetic estimates are correct, and Glandirana isin fact Sanguirana’s closest mainland relative (Wiens et al., 2009),then biogeography of Sanguirana is similar to other‘Out-of-Palawan’ taxa in that its closest relatives apparently arenot present on landmasses of the Sunda Shelf, but instead are more

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diverse and widespread on the Asian mainland (Blackburn et al.,2010; Siler et al., 2012; Brown et al., 2013).

4.3. Common mechanisms of diversification for land vertebrates ofSoutheast Asian island archipelagos?

Much attention has been paid to hypotheses of commonmecha-nisms of diversification in terrestrial vertebrates of the Philippines,an iconic Southeast Asian island archipelago (Woodruff, 2010;Lohman et al., 2011; Brown et al., 2013). In the Philippines, adisproportionate amount of this attention has focused on the‘PAIC-paradigm,’ its impact in unrelated taxa, deviations from themodel, and other recent (Pleistocene) explanations (reviews:Brown and Diesmos, 2009; Brown et al., 2013; Oaks et al., 2013).However, more recent inferences from increasingly robust multilo-cus molecular phylogenies (Esselstyn et al., 2010; Setiadi et al.,2011; Brown et al., 2015) suggest that alternate explanations areplausible, and clearly have contributed to establishing Philippinevertebratemegadiversity (BrownandDiesmos, 2009). These includewithin-island habitat connectivity/discontinuity, ecological diver-gence along altitudinal gradients, contingency in processes of insu-lar community assembly, and the possibility paleo-transport ofancient isolated lineages on continental land fragments during theassembly of the archipelago (Hall, 1998; Blackburn et al., 2010;Brown et al., 2013). The Palawan Ark Hypothesis is one such expla-nation that may characterize the evolutionary history of more ter-restrial biodiversity in the oceanic Philippines than has generallybeen appreciated (Esselstyn et al., 2010). Moving beyond the PAICparadigm toward the acceptance of a plurality of shared mecha-nisms and a greater appreciation of overseas dispersal has been aninevitable and welcomed consequence of the accumulation newdata from recent biodiversity surveys throughout the archipelago(Brown et al., 2013).

Acknowledgments

We thank the Biodiversity Monitoring Bureau (BMB) of thePhilippine government for facilitating permits, M. Diesmos and A.C. Alcala for collaboration and logistical support, and the U.S.National Science Foundation (DEB 073199, 0334952, 0743491,0804115, and 1418895) for supporting our research.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2015.10.010.

References

Blackburn, D.C., Bickford, D.P., Diesmos, A.C., Iskandar, D.T., Brown, R.M., 2010. Anancient origin for the enigmatic Flat-Headed Frogs (Bombinatoridae:Barbourula) from the islands of Southeast Asia. PLoS One 5 (8), e12090.

Blackburn, D.C., Siler, C.D., Diesmos, A.C., McGuire, J.A., Cannatella, D.C., Brown, R.M., 2013. An adaptive radiation of frogs in a Southeast Asian island archipelago.Evolution 67, 2631–2646.

hipelago frog genus Sanguirana: Another endemic Philippine radiation that.1016/j.ympev.2015.10.010

6 R.M. Brown et al. /Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

Brown, R.M., 2015. A new species of stream frog (genus Hylarana) from themountains of southern Mindanao Island, Philippines. Herpetologica 71,223–233.

Brown, R.M., Diesmos, A.C., 2009. Philippines, Biology. In: Gillespie, R., Clague, D.(Eds.), Encyclopedia of Islands. University of California Press, Berkeley,pp. 723–732.

Brown, R.M., Siler, C.D., 2013. Spotted stream frog diversification at the Australasianfaunal zone interface, mainland versus island comparisons, and a test of thePhilippine ‘dual-umbilicus’ hypothesis. J. Biogeogr. 41, 182–195.

Brown, R.M., McGuire, J.A., Diesmos, A.C., 2000. Status of some Philippine frogsrelated to Rana everetti (Anura: Ranidae), description of a new species, andresurrection of Rana igorota Taylor 1922. Herpetologica 56, 81–104.

Brown, R.M., Siler, C.D., Oliveros, C.H., Esselstyn, J.A., Diesmos, A.C., Hosner, P.A.,Linkem, C.W., Barley, A.J., Oaks, J.R., Sanguila, M.B., Welton, L.J., Blackburn, D.S.,Moyle, R.G., Peterson, A.T., Alcala, A.C., 2013. Evolutionary processes ofdiversification in a model island archipelago. Annu. Rev. Ecol. Evol. Syst. 44,411–435.

Brown, R.M., Siler, C.D., Richards, S., Diesmos, A.C., Cannatella, D.C., 2015. Multilocusphylogeny and a new classification for Southeast Asian and Melanesian forestfrogs (family Ceratobatrachidae). Zool. J. Linn. Soc. 174, 130–168.

Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. JModelTest 2: more models,new heuristics and parallel computing. Nat. Methods 9, 772.

Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogeneticswith BEAUti and the BEAST 1.7. Molec. Biol. Evol. 29, 1969–1973.

Esselstyn, J.A., Oliveros, C.H., Moyle, R.G., Peterson, A.T., McGuire, J.A., Brown, R.M.,2010. Integrating phylogenetic and taxonomic evidence illuminates complexbiogeographic patterns along Huxley’s modification of Wallace’s Line. J.Biogeogr. 37, 2054–2066.

Fuiten, A., Diesmos, A.C., Welton, L.J., Bartley, A., Oberheide, B., Rico, E.L.B., Brown, R.M., 2011. New species of stream frog from the mountains of Luzon Island,Philippines. Herpetologica 67, 89–103.

Hall, R., 1998. The plate tectonics of Cenozoic SE Asia and the distribution of landand sea. In: Hall, R., Holloway, J.D. (Eds.), Biogeography and GeologicalEvolution of SE Asia. Backhuys Publications, Lieden, pp. 99–131.

Joppa, L.N., Roberts, D.L., Pimm, S.L., 2011. The population ecology and socialbehaviour of taxonomists. Trends Ecol. Evol. 26, 551–553.

Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment softwareversion 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780.

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S.,Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P.,Drummond, A., 2012. Geneious Basic: an integrated and extendable desktopsoftware platform for the organization and analysis of sequence data.Bioinformatics 28, 1647–1649.

Kemp, C., 2015. The endangered dead. Nature 518, 292–294.Landis, M.J., Matzke, N.J., Moore, B.R., Huelsenbeck, J.P., 2013. Bayesian analysis of

biogeography when the number of areas is large. Syst. Biol. 62, 789–804.Lim, B.K., Edger, J.L., Peterson, A.T., Robbins, M.B., Clayton, D.H., Bush, S.E., Brown, R.

M., 2008. Biodiversity in China: lost in the masses? Harvard Asia Quart. 11, 12–23.

Please cite this article in press as: Brown, R.M., et al. Phylogeny of the island arcdiversified ‘Out-of-Palawan’. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/1

Lohman, D.J., de Bruyn, M., Page, T., von Rintelen, K., Hall, R., Ng, P.K.L., Shih, H.-T.,Carvalho, G.C., von Rintelen, T., 2011. Biogeography of the Indo-Australianarchipelago. Annu. Rev. Ecol. Evol. Syst. 42, 205–226.

Matzke, N.J., 2013. BioGeoBEARS: Biogeography with Bayesian (and likelihood)Evolutionary Analysis in R Scripts. CRAN: The Comprehensive R ArchiveNetwork.

Meijaard, E., 2011. Indonesia has its share of scientists, so where’s the science?Jakarta Globe (23 March 2011).

Oaks, J.R., Sukumaran, J., Esselstyn, J.A., Linkem, C.W., Siler, C.D., Holder, M.T.,Brown, R.M., 2013. Evidence for Pleistocene-driven diversification? A cautionfor interpreting ABC inferences of simultaneous historical events. Evolution 67,991–1010.

Rambaut, A., Drummond, A.J., 2007. Tracer v1.4. <http://beast.bio.ed.ac.uk/Tracer>.Ree, R.H., Smith, S.A., 2008. Maximum likelihood inference of geographic range

evolution by dispersal, local extinction, and cladogenesis. Syst. Biol. 57, 4–14.Ronquist, F., 1997. Dispersal-vicariance analysis: a new approach to the

quantification of historical biogeography. Syst. Biol. 46, 195–203.Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference

under mixed models. Bioinformatics 19, 1572–1574.Sanguila, M.B., Siler, C.D., Diesmos, A.C., Nuñeza, O., Brown, R.M., 2011.

Phylogeography and conservation implications of geographic structure ofgenetic variation and potential species boundaries in Philippine slender toads.Mol. Phylogenet. Evol. 61, 333–350.

Setiadi, M.I., McGuire, J.A., Brown, R.M., Zubairi, M., Iskandar, D.T., Andayani, N.,Supriatna, J., Evans, B.J., 2011. Adaptive radiation and ecological opportunity inSulawesi and Philippine fanged frog (Limnonectes) communities. Am. Nat. 178,221–240.

Siler, C.D., Oaks, J.R., Welton, L.J., Linkem, C.W., Swab, J., Diesmos, A.C., Brown, R.M.,2012. Did geckos ride the Palawan raft to the Philippines? J. Biogeogr. 39, 1217–1234.

Stamatakis, A., 2014. RAxML Version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. http://dx.doi.org/10.1093/bioinformatics/btu033.

La Viña, A.G.M., Caleda, M.J.A., Baylon, M.L.L., 1997. Regulating Access to Biologicaland Genetic Resources in the Philippines: A Manual on the Implementation ofExecutive Order No. 247. Foundation for the Philippine Environment and WorldResources Institute, Philippines.

Wiens, J.J., Sukumaran, J.S., Pyron, R.A., Brown, R.M., 2009. Evolutionary andbiogeographic origins of high tropical diversity in old world frogs (Ranidae).Evolution 64, 1217–1231.

Wilgenbusch, J.C., Warren, D.L., Swofford, D.L., 2004. AWTY: A System for GraphicalExploration of MCMC Convergence in Bayesian Phylogenetic Inference. <http://ceb.csit.fsu.edu/awty>.

Woodruff, D.S., 2010. Biogeography and conservation in Southeast Asia: how 2.7million years of repeated environmental fluctuations affect today’s patterns andthe future of the remaining refugial-phase biodiversity. Biodiv. Conserv. 19,919–941.

hipelago frog genus Sanguirana: Another endemic Philippine radiation that0.1016/j.ympev.2015.10.010