Molecular Ecology (2007) 16,4789^807 doi:10.1111/j.l365-294X.2007.03524.x

The role of tropical dry forest as a long-term barrier to dispersal: a comparative phylogeographical analysis of dry forest tolerant and intolerant frogs

ANDREW J. CRAWFORD,* ELDREDGE BERMINGHAM* and CAROLINA POLANÍA S.t *Smithsonian Tropical Research Institute, Apartado 0840-0309, Balboa, Ancón, Panama, and -^Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Apartado 7495, Bogotá, Colombia

Abstract

We used a comparative phylogeographical approach to investigate the origins of the disjunct wet forest biota of the Golfo Dulce region along the Pacific slope of Costa Rica. This region is isolated by Pacific dry forests north and south and isolated from Caribbean wet forests by mountains. We studied three sympatric lowland frog species in the Craugastor fitzingeri species group that prefer wet forest but differ in their response to dry habitats. In dry forest, C. fitzingeri can survive along streams while C. crassidigitus and C. talamancae are entirely absent. We collected samples from across the ranges of all three species, and obtained mitochondrial DNA sequence data from the COI and cytochrome b genes. We observed significant phylogeographical structure in C. crassidigitus and C. talamancae, but much less in C. fitzingeri, demonstrating that mountain barriers and dry forest habitat have reduced mitochondrial gene flow in the strictly wet-forest species. Additionally, we discovered that the Golfo Dulce and Central Panama populations of C crassidigitus appear to have diverged in the Pliocene or earlier, suggesting that the dry forest separating these popula- tions is old. Our phylogenetic analysis of 12 of approximately 16 species of the C. fitzingeri species group suggests that the three lowland species are each other's closest relatives. Because of this shared phylogenetic history, we attribute the striking differences in phylo- geographical structure to the different ecologies of the frogs. In summary, we find that what appear to be minor differences in the natural history of these three closely related species may profoundly impact the potential for dispersal, range size, and cladogenesis. Keywords: Central America, dispersal, phylogeography, SOWH test, tropical dry forest, tropical wet forest Received 1 March 2007; revision received 4 June 2007; accepted 1 August 2007

and past conditions are often unknown. Furthermore, Introduction species ffiay be able to acclimate and survive harsh con- The primary goal of biogeographical studies is to character- ditions long enough to traverse apparent environmental ize the distribution patterns of organisms and determine barriers. Thus, we ivould like to knoiv which environmental the evolutionary forces, environmental conditions, and barriers have influenced organismal distributions over the historical events that have shaped these patterns (Loffiolino long term and w^hich are only recent or ephemeral phenomena et al. 2004). Comparing species ranges ivith habitat (e.g. Wüster et al. 2005). Here, we use the genealogical heterogeneity may allow one to evaluate the influence information provided by comparative phylogeographical of environmental conditions on the distribution of species analysis (e.g. Berminghaffi & Avise 1986; Avise 2000; (Graham et al. 2004). However, our ability to infer process Schäuble & Moritz 2001; Campbell et al. 2006) to from pattern is limited because environments also change investigate the relative roles of mountains and tropical dry forest habitat in shaping the geographical distributions of Correspondence: Andrew J. Crawford, Smithsonian Tropical Research related species of predoffiinantly wet forest leaf-litter frogs Institute, Naos Labs, Unit 0948, APO, AA 34002-0948, USA. Fax: on the Pacific and Caribbean versants of lower Central +507 212-8790; E-mail: [email protected]; [email protected] America (Fig. 1).

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4790 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S.

CENTRAL AMERICA

^* i ^ 1 SOUTH 1 AMERICA ^Mc.fi tzingeri and C. crassidigitus ^ zingeri ^ MMC.fi 9, ,300 •4^ rang e limits of C. talamancae kilometres

Bocas del Toro filter barrier

WWF simplified forest types M Carib. WET ^PM Pacific WET 3 Seasonal moist 4 DRY ^^M montane Gulfo Dulce region ^^M (fresh water) (KNMOPQR)

Fig. 1 Map of Isthmian Central America showing sampling sites for Craugastor fitzingeri group samples used in this study. Letters (A-Z) and numbers (1-5) in red circles denote areas where at least one of the following species was sampled: C. crassidigitus, C, fitzingeri, or C. talamancae. Red dots may combine neighbouring collecting localities into a single 'area', e.g. area 'W covers collecting localities 'Altos del Maria' and 'El Valle de Anton' (see Table 1). Inset map shows the geographical distributions of C. crassidigitus, C, fitzingeri, and C. talamancae on a political map of lower Central America, according to the Global Amphibian Assessment database (lUCN et al. 2004). Note, the geographical isolation of Golfo Dulce C. crassidigitus may be seen more clearly in Savage (2002). The geographical distribution of C. talamancae is limited to the Caribbean versant, with northern and eastern range limits indicated by grey arrows. Coloured regions in the main map represent simplified World Wildlife Fund ecoregions ('Caribbean WET' combines three WWF ecoregions: Central American Atlantic moist. Isthmian-Atlantic moist, and Chocó-Darién moist. 'DRY' combines two ecoregions: Central American and Panamanian dry forests. 'Montane' combines three WWF ecoregions: Central American, Talamancan and Eastern Panamanian montane forests). Note, the distributions of wet- vs. dry-forest amphibians in the GAA database would suggest that the eastern half of the 'Pacific Wet' WWF ecoregion indicated here may be more akin to dry forest. The Golfo Dulce region and the approximate location of the Bocas del Toro filter barrier (see Discussion) are indicated by orange arrows.

While most phylogeographical and biogeographical (Formicarius analis, Formicariidae), buff-throated foliage- studies of lower Central America have focused on the gleaner (Automolus ochrolaemus, Furnariidae), bicoloured biotic interchange between North and South America antbird {Gymnopithys leucaspis, Thamnophüidae), and wedge- (e.g. Simpson 1940; Vanzolini & Heyer 1985; Webb 1985; billed woodcreeper (Glyphoryiichus spirurus, Dendroco- Bermingham & Martin 1998; Zeh el at. 2003a, b), a smaller laptidae) (Ridgely et al. 2003). The Golfo Dulce disjunction but perhaps more enigmatic puzzle has been largely pattern is rare among lizards but common among snakes, ignored. Many of the plant and animal species found in including the eyelash viper {Bothrops schlegelii, Viperidae) the tropical moist and ivet forests (Holdridge 1967) of the and various colubrids (e.g. Erythrolnmprus mimus, RJiadi- Caribbean loivlands of lower Central America also have naea decoraia, Tantilla supracincta, and Urothecn guentheri) discontinuous populations in the wet forest on the Pacific (Köhler 2003). Frog species that show this disjunct pattern versant in the Golfo Dulce region of southwest Costa Rica include Bufo haematiticus (Bufonidae), Dendropsophus ebmc- (McDiarmid & Savage 2005). Geographical distributions catus and Smilisca phaeotn (Hylidae), as well as Craugastor are generally well characterized for vertebrates, and the crassidigitus (Brachycephalidae) (Savage 2002). disjunct pattern of the Golfo Dulce fauna has been docu- Wet-forest populations in the Pacific Golfo Dulce region mented for birds, snakes, and frogs. Specific examples from are isolated from conspecific populations elseivhere by birds of various families include black-faced antthrush dry forests and mountains. On the Pacific slope, the Golfo

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Dulce is bounded on the northivest and southeast sides by interaction of the C. fitzingeri group with the geography the tropical dry forests of the Guanacaste Province of Costa and environments of lower Central America provides an Rica and the Azuero Peninsula of Panama, respectively opportunity to analyse the relative contributions of bioge- (Holdridge 1967; Tosi 1969). Caribbean populations are ographical history and ecological preference in the distri- separated from the Golfo Dulce by the 2000-m high con- bution of wet-forest amphibians found in the Golfo Dulce tinental divide of the Talamanca Mountains (Savage 1982; region of Pacific Costa Rica. We use phylogenetic analyses McDiarmid & Savage 2005). The Pacific Coast dry forests of of mitochondrial DNA (mtDNA) sequence data to distin- Guanacaste and the Azuero Peninsula presumably result guish among three a priori biogeographical hypotheses from rain shadoivs caused by the Pilarán and Tabasará regarding the origin of the Golfo Dulce populations of C. mountains, respectively, which block the moist Caribbean crassidigitus and C. fitzingeri. As illusfrated in Fig. 2, these trade ivinds from the northeast. The Golfo Dulce region, in populations may have been connected historically to contrast, hosts a ivet forest habitat because the Talamanca Caribbean populations (Northern Route), to Cenfral Panama Mountains paralleling this area are high enough that a (Eastern Route) or may have been isolated by the rise of the zone of negative pressure is created over the landscape as cenfral Talamanca Mountains (vicariance). In turn, we the high -winds pass over the mountains. Moist air from the compare the phylogeographical structure of the three species Pacific Ocean is drawn into this loiv-pressure zone and in the C. fitzingeri group to determine if geographical range and supplies the region ivith rain (Coen 1983). habitat preference correlate with the degree of population We use a comparative phylogeographical analysis of subdivision and historical isolation in these frogs. three closely related and ividely sympatric frog species to investigate the origins of the Golfo Dulce fauna, and ascer- Materials and methods tain the role of dry forests and mountains in shaping the wet-forest communities of loivland Central America. By Sampling studying species ivith overlapping distributions, we aim to infer the geological and environmental processes that Twenty-nine Craugastor crassidigitus, 31 C. fitzingeri and 10 influenced the patterns of dispersal and isolation responsible C. talamancae samples were collected from across Costa for the ecological assemblages we observe today (Bermingham Rica (CR), Panama (PA) and Honduras (HN). Five of the & Avise 1986; Hickerson & Cunningham 2005; Kerdelhué C. fitzingeri samples were collected from four localities et al. 2006). Our focal species are Fitzinger's leaf-litter frog beyond the range of C. crassidigitus: marked localities A, B, Craugastor fitzmgeri, the slender-toed leaf-litter frog, C. J, and L in Fig. 1. To place our three focal species in their crassidigitus, and the Almirante robber frog, C. talamancae phylogenetic context, we also sampled six additional named (Anura: Brachycephalidae). All three are members of the species and three undescribed species of the 13-member C. fitzmgeri species group (Miyamoto 1986; Savage et al. 2004; C. fitzingeri group (Savage et al. 2004). Of these nine Crawford & Smith 2005). The geographical distribution additional C. fitzingeri group species, one is a lowland of C. talamancae is almost entirely nested within that of C. species restricted to eastern Panama and Colombia (C. crassidigitus, whose distribution in turn is nested entirely raniformis) and eight are montane frogs resfricted to Costa within that of C. fitzingeri (Fig. 1, inset). Because C. crassi- Rica and Panama west of the Canal. The montane species digitus is absent from the Pacific Coast dry forests, the included in this analysis were C. andi, C. cuáquero, C. long- Golfo Dulce populations are well isolated (Savage 2002). irostris, C. melanostictus, C. tabasarae, C. sp. cf. crassidigitus Craugastor fitzingeri covers the range of C. crassidigitus plus (Fortuna, PA), C. sp. cf. loitgirostris (near El Copé, PA), and it extends northward to eastern Honduras and southward C. sp. 'pn' (cenfral and eastern PA). Outgroups for phylo- into western Colombia, and is found in dry forest habitats genetic analyses were chosen based on a broad-scale study of northern Pacific Costa Rica and southern Panama of Craugastor (Crawford & Smith 2005) and included C. ranoides (Lynch & Myers 1983; Ibáñez et al. 2001; Ruíz-Carranza and C. fieischmanni (C. rugulosus species group) plus C. et al. 1996; Köhler 2001; McCranie & Wilson 2002; Savage megacephalus (C. biporcatus species group). 2002; Lynch & Suárez-Mayorga 2004). Craugastor talaman- Frogs were collected in the field, photographed, and cae occurs from 15 to 646-m elevation in Costa Rica (Savage euthanized with dilute chloretone (CH3)COHCCl3. Fresh 2002), but we have found this frog as high as 895 m in Cen- liver samples were stored in an NaCl-saturated buffer tral Panama (Table 1). Craugastor fitzingeri occurs from near containing 0.25 M EDTA and 20% dimethyl sulphoxide sea level to 1520 m (Savage 2002), while C. crassidigitus has (DMSO, Seutin et al. 1991). Corresponding voucher speci- been recorded from near sea level to a recorded high of mens were fixed in 10% formalin, stored in 70% ethanol 2000 m (Lynch & Myers 1983). (Pisani 1973), and deposited in biodiversity collections History and ecology both confribute to the evolution of at public research institutions. Additional samples were species ranges and the assembly of communities (Ricklefs generously provided by research museums. Detailed & Schlüter 1993; Schneider et al. 1998). The evolutionary saniple information is provided in Table 1.

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Laboratory protocols

Genomic DNA ivas extracted using a standard phenol- chloroform protocol. A 717-bp fragment of the cytochrome b (cyt b) gene was amplified using primers CBl (5'- CCATCCAACATCTCAGCATGATGAAA-3') and CB3 (5'-GGCGAATAGGAAGTATCATTC-3') with an annealing temperature of 42 °C (aka primers 144 and 164, respectively, from Table 3 in Goebel et al. 1999). A 639-bp fragment of the COI gene was amplified using primers COIf (5'-CCTGCA- GGAGGAGGAGAYCC-3') and COIa (5'-AGTATAAGCG- TCTGGGTAGTC-3') with an annealing temperature of 57 °C Golfo Central Caribbean (aka primers 109 and 124, respectively, from Table 3 in Dulce Panama Costa Rica Goebel et al. 1999). Amplified fragments were sequenced using polymerase chain reaction (PCR) primers and BigDye 3.1 terminator reaction chemistry and analysed on either an ABI PRISM 377 or 3100 automated sequencer (Applied Biosystems Inc.). DNA sequences were aligned Eastern with SEQiiENCHER 4.2 (Gene Codes Corporation) and checked Route by eye. We inferred the amino acid sequences for all DNA sequences to check for the presence of premature stop codons or other nonsense mutations.

Caribbean Central Golfo Phylogenetic analysis Costa Rica Panama Dulce To evaluate potential phylogenetic heterogeneity between the cyt b and COI data sets, we used the incongruence length difference (ILD) permutation test (Farris et al. 1995) with 500 random partitions of the combined data set as implemented in PAUP* 4.0blO (Swofford 1998). This test may be used to investigate the possibility of incomplete Vicariance linkage among vertebrate mitochondrial genes (Ballard & Whitlock 2004) or to detect contamination among samples. The ILD test is conservative (Sullivan 1996) and a significance level of a = 0.01 or 0.001 may be most appropriate Golfo Caribbean Central (Cunningham 1997). Dulce Costa Rica Panama We tested the data for significant departure from this assumption of stationarity using a chi-squared test imple- mented in PAUP*. Fifty-six models of DNA sequence evolution were then evaluated using the Bayesian information criterion, as implemented in DT-MODSEL (version: 13-Aug-02) by Northern Minin et al. (2003). This method uses decision theory and Route incorporates branch length error as an additional consider- ation in choosing the most appropriate model. Maximum- likelihood (ML) models were evaluated and parameters Fig. 2 Upper panel shows a map of Costa Rica and Panama with calculated from neighbour-joining (NJ) trees (Saitou & Nei three labelled geographical regions. Double-headed dashed arrows 1987) obtained from a matrix of LogDet distances (e.g. represent possible dispersal routes that may have connected the Lockhart et al. 1994). Using this method, we sought the Golfo Dulce region genealogically to the other two regions. The longer single best-likelihood model of DNA sequence evolution arrow illustrates a possible Eastern Route, the shorter arrow applicable to all sites, as well as the most appropriate models represents a possible Northern Route. The three cladograms represent for each codon position alone. phylogenetic predictions based on three hypotheses tested here con- cerning the biogeographical origin of the Golfo IXilce Cmugastor fauna: ML and most parsimonious (MP) tiees were inferred using two possible dispersal routes. Eastern and Northern, plus a possible PAUP* by heuristic searches. We conducted ML searches using vicariance origin resulting from the rise of the Talamanca Mountains. fixed parameter values which were estimated iteratively:

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4796 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S.

the chosen model and preliminary parameter values were Divergence time estimation used to infer an ML tree, and this new tree topology was used to re-estimate paraffieter values for the final ML tree We employed a likelihood-ratio test (LRT) to evaluate w^hether searches. MP searches were conducted six times, each analysis the combined data set was significantly unlikely under used 5000 random addition sequence repKcates. Confidence the assumption of rate constancy of molecular evolution limits on MP trees were inferred using the nonparametric (Felsenstein 1981). To ensure the two models (with and bootstrap (Felsenstein 1985) in PAUP* and involved 2000 without clock) were nested hierarchically, the topology we bootstrap pseudoreplicates each of ivhich ivas analysed via inferred from the unconstrained ML tree search was used 30 replicate searches. For both MP and bootstrap analyses, as a consfraint tree during the searches under the enforced- MaxTrees was set to 100 000. clock model while branch lengths and parameter values In conducting a Bayesian Markov chain Monte Carlo were re-estimated. (MCMC) phylogenetic analysis (Rannala & Yang 1996; To estimate divergence times within and among our three Yang & Rannala 1997) using the computer program, MRBAYES focal species of frogs, we applied one molecular-clock method version 3.1.2 (Ronquist & Huelsenbeck 2003), one has the (utilizing only the genetic distance information) and one option of applying independent models to different parti- relaxed-clock approach (relying upon the phylogenetic tions of the data, thereby creating far more complex models tree). First, we applied a 1.91% rate of total divergence than the 10-parameter general time reversible model per million years for model-corrected amphibian mtDNA (lavaré 1986). Huelsenbeck & Rannala (2004) showed that sequence divergence (Crawford 2003a, b). This rate was phylogenetic accuracy is more vulnerable to model under- obtained by applying a model-based correction to the original parameterization than over-parameterization. While Baye- mtDNA data used in Macey et al. (1998) to estimate a sian MCMC deals efficiently with highly complex models, molecular clock in toads based on a 10-million-year-old striking the balance between phylogenetic accuracy and calibration point (see also Macey et al. 2001 for a comparison parameter identifiability is a nontrivial problem (Castoe of rates among vertebrates). This re-calibrated rate was then et al. 2004; Nylander ci al. 2004). Therefore, we increased applied to our model-corrected genetic distance in Table 2 model complexity by partitioning the data by the three (plus/minus two standard errors), because uncorrected codon positions, while avoiding over-parameterization by genetic distances may bias divergence time estimates towards selecting the appropriate model for each partition using the calibration point (Arbogast et ni. 2002). Second, we used the decision theory method, which tends to choose less nonparametric rate smoothing (NPRS) (Sanderson 1997) complex models (Minin et al. 2003). We conducted two par- to create an ultramefric free, which we then calibrated by allel runs of the MCMC algorithm for 6 million generations assuming that the divergence of the most recent common each, sampled one tiee per 1000 generations, discarded the ancestor (MRCA) of the C.fitzingeri species group occurred first 1001 saved trees as bum-in, and estimated the posterior between 20 and 37 million years ago (Ma), as estimated by probability distribution of topologies, branch lengths Crawford & Smith (2005). These authors used a Bayesian and parameter values from the combined 10 000 samples MCMC method (Thome et al. 1998; Thorne & Kishino 2002) collected. All runs employed four chains with Metropolis- and a biogeographical hypothesis for the origin of the genus coupled MCMC heating. For comparison, we also conducted Crnugastor to estimate divergence times of all species a nonpartitioned Bayesian MCMC analysis. groups within Crnugastor based on mitochondrial (ND2

Table 2 Means and standard errors of pairwise genetic distances within and among three geographical regions (Fig. 2) for three species of Craugastor. Within each cell, the top value indicates C. crassidigitus, the middle value C. talamancae, and the bottom value indicates C. fitzingeri. Genetic diversity within regions based on average pairwise distances is shown along the diagonal. Below the diagonal are values for between-region total genetic distance. Values are Tamura-Nei+F model-corrected genetic distances (see text) based on samples with data for both genes (COI and cyt b). CR, Costa Rica. Note: C. talamancae does not occur in the Golfo Dulce region

Golfo Dulce Central Panama Caribbean CR

Golfo Dulce 0.0313 (0.0045) C. crassidigitus n/a C. talamancae 0.0082! ((0.0017) C.fitzingeri Central Panama 0.1388Î ((0.0154) 0.0658 (0.0076) n/a 0.1115 (0.0156) 0.0258Î ((0.0042) 0.0114 (0.0024) Caribbean CR 0.2620) ((0.0310) 0.3120 (0.0340) 0.0743 (0.0109) n/a 0.2444 (0.0272) 0.0597 (0.0080) 0.0662 (0.0098) 0.0662 (0.0094) 0.0140 (0.0026)

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd FROG PHYLOGEOGRAPHY AND TROPICAL FORESTS 4797 plus five tRNA genes) and nuclear (c-myc) gene sequences. (Prager & Wilson 1988), both implemented in PAUP*. AS (This same highly parametric Bayesian MCMC method is with the SH test, these tests compare, on a per-site basis, the not appropriate for the present data set because it would support provided by the data, but trees are compared in a confound pol5rmorphism with divergence.) pairwise fashion, ignoring the fact that one of the two trees may be the optimal tree (Felsenstein 2004). Finally, ive performed a parametric bootstrap analysis of Hypothesis testing each of the 11 null hypotheses via a SOWH-Kke test (Swofford To evaluate statistical support for our three a priori et al 1996; Goldman et al 2000). In the SO WH test, the prob- biogeographical hypotheses for the origin of the Golfo ability of the observed difference, S, in tree scores between Dulce populations, ive compared the scores of optimal the optimal tree (Hj) and the constrained tree (HQ) is evaluated trees without vs. with topological constraints enforced. We by comparison against a null distribution of differences enforced the three topological constraints on C. crassidigitus in tree scores obtained from simulated data sets that are and C. fitzingeri separately, for a total of six predictions generated using the ML model of evolution and the null based on our three biogeographical scenarios (Fig. 2). hypothesis topology (Huelsenbeck et al. 1996; Goldman We tested five additional null hypotheses concerning the et al. 2000). The SO WH test is more powerful than the SH evolution of the C. fitzingeri species group. We tested the test and performs better than all other topological tests (Shi monophyly of our three focal species (C. crassidigitus, C. et al. 2005), but may be sensitive to model misspecification fitzingeri, and C. talamancae), the monophyly of four lowland (Buckley 2002; Felsenstein 2004). Full or even partial ML species (the previous three species plus C. raniformis), and optimization of simulated data sets was computationally the monophyly of all five lowland species (the previous prohibitive for the present data set. Therefore, we evaluated four plus C. longirostris). We also tested two hypotheses of the difference between optimal ML and constrained ML Savage et al. (2004): the sister-relationship between C fitzingeri topologies using the MP criterion, making it a 'SOWH- and C. raniformis (two species with yellow spots on the like' test, but one commonly used (e.g. Pauly et al. 2004). posterior of the thighs) and the monophyly of Talamancan This modified test is potentially more conservative than highland species. In total, we evaluated 11 hypotheses of the standard likelihood method because the ML and MP relationships. trees may differ. When the ML tree topology is evaluated For testing the relative support of alternative topologies, under the parsimony criterion, the ML tree could well be we used five different statistical tests. First, we calculated longer than the most-parsimonious (MP) tree, in which the marginal posterior probability of the monophyletic case S may be reduced and the null hypothesis harder to group predicted by each of our 11 null hypotheses. This reject. method should provide a straightforward estimate of the Under the MP criterion, we calculated the length of each posterior probability of the clade or combination of clades of our 11 HQ trees and the Hj tree. Subtracting the tree in question, but its validity depends on using the correct length (TL) of Hj from the TL of each HQ gave us our 11 test model of evolution (Huelsenbeck & Rannala 2004). statistics (Ô). Next, 100 simulated data sets were generated Next, we performed one likelihood-based nonparametric using SEQ-GEN version 1.3.2 (Rambaut & Grassly 1997). For test, the paired-sites test (aka, SH test) of Shimodaira & each of the simulated data sets, paired unconstrained and Hasegawa (1999), as implemented in PAUP*. This test uses constrained MP trees were inferred by 300 replicate MP bootstrap resampling and corrects critical values for multiple searches. The probabiKty of rejecting a given null hypothesis comparisons, and is knoivn to be conservative (Buckley was obtained by comparing the observed S-value to the 2002; Shimodaira 2002). The significance of the difference distribution of ô obtained from the 100 simulated data sets. in the sum of sitewise log-likelihoods for all trees was We compared levels of mtDNA sequence polymorphism evaluated by bootstrap sampling of site scores (RELL sam- and divergence within three species (C. crassidigitus, C. fit- pling) with 2000 replicates (Kishino & Hasegawa 1989) and zingeri and C. talamancae) by calculating model-corrected calculating how far the observed differences are from the levels of genetic variation within and among the three geo- mean of the bootstrap replicates. The accuracy of this test graphical regions in question (Fig. 2). Means and standard is increased ivith the inclusion of all reasonable trees, in errors (based on 2000 bootstrap replicates) were calculated addition to the ML tree (Hj) and the constrained tree (HQ) using MEGA version 3.0 (Kumar et al. 2004). (Shimodaira & Hasegaiva 1999). Therefore, we included 12 arbitrarily chosen MP trees as ivell, and then obtained Results the one-tailed P values for each Hg tree relative to Hj and the 12 MP trees. We obtained DNA sequence from both the COI and cyt h We performed two parsimony-based paired-sites tests of genes from 67 frogs of the total sample of 89 Craugastor, topology. We used the Wilcoxon signed-ranks test, aka, the including 26 C. fitzingeri, 24 C. crassidigitus, 7 C. talamancae, Templeton test (Templeton 1983) and the winning-sites test and 8 samples representing five additional species in the C.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4798 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S. fitzingeri species group (Savage et al. 2004), as ivell as two model was selected. For second position sites, the outgroup taxa, C. fleischmanni and C. ranoides (Crawford & HKY+r+i model (Hasegawa et al. 1985) was selected. For Smith 2005; Puschendorf et al. 2005). The alignment was third position sites, the TIM+F model was selected. In unambiguous, and the inferred amino acid sequence MRBAYES, the K2P was implemented by fixing the priors on contained no stop codons. We obtained 639 bp of the COI all base frequencies at the constant value 0.25, while the gene and 714 bp of the cyt b gene, for a total of 1353 bp in TIM model was replaced by a full six-rate model. the final data set. Among ingroup taxa with data for both Parameter values for the nonpartitioned TrN93+r+i genes, we counted 528 parsimony-informative sites plus model were estimated iteratively with final values as 75 singletons. follows: frequencies of nucleotides A, C, G and T were The 22 samples from ivhich ive obtained DNA sequence 0.262952, 0.350508, 0.114824 and 0.271716, respectively; the from just one gene included an additional 5 C. fitzingeri A-G transition rate was 20.63020 and the C-T fransition samples, 5 C. crassidigitus, 3 C. talamancae, 1 sample each of rate was 7.64818, relative to the transversion rate of 1.0; a C. cuáquero, C. longirostris, and C. tabasarae, plus 5 samples was 1.118286 with an I parameter value of 0.489922. Using representing four additional species not in the two-gene this model and fixed parameter values, the ML tree data set: C. aiidi, C. melanostictus, C. sp. cf. crassidigitus, C. sp. inferred from the 69 frogs with complete two-gene data nov. 'pn', and an additional outgroup taxon (C. megaceplmlus). showed a support value of -12157.65888 log-Hkelihood units These 22 additional samples ivere used in phylogenetic in repeated searches. The first MP search recovered 864 reconstructions but ivere not used in model selection or shortest trees of 2476 steps. After repeating the MP search hypothesis testing. See Table 1. five more times, we recovered no new shortest trees. We also conducted MP, bootstrap and Bayesian analyses on the complete data set of 89 samples. The results of our Evolutionary models and phylogenetic results tri-partitioned Bayesian analysis are presented in Fig. 3. No Using a chi-squared test, we detected no departure from appreciable differences were observed between the tri- homogeneity of nucleotide frequencies among all samples, partitioned and nonpartitioned Bayesian consensus trees including the outgroup taxa (P = 0.9853). The ILD failed to (unlike in Castoe et al. 2004). We observed no significant reject the null hypothesis of homogeneity between the COI conflict between Bayesian and MP bootstrap support (bss), and cyt b data sets for the ingroup (P = 0.350) but repeating with one exception: while Bayesian analyses placed Carib- the test with the outgroup C. fleischmanni rejected homo- bean and Pacific CR samples of C. fitzingeri as sisters with geneity of the data partitions (P = 0.005). a marginal posterior probability (mpp) = 0.85 (Fig. 3), the The most appropriate ML model for the combined data MP bootstrap analysis placed Pacific CR with Central PA set was the Tamura & Nei (1993) model with the distribution samples with a significant bss = 98. of rates of evolution among sites described by the gamma Our sampling provided the following insights into inter- density shape parameter, a (Yang 1994), and a proportion specific relationships of the C. fitzingeri group. Craugastor of invariable sites, I (Hasegawa et al. 1987) or TrN93+r+í fleischmanni and other outgroup samples appeared far model. When using MEGA to calculate average pairwise removed from the rest of the ingroup samples (result not distances within and among the three geographical regions shown), while C. longirostris from eastern Panama formed of interest (Fig. 2), we adopted the TrN93+r model because the sister lineage to all other C. fitzingeri group samples MEGA does not implement the 1 parameter. To compensate, (Fig. 3). Craugastor talamancae and C. crassidigitus are sister we re-estimated a for this model (0.227184) and used this species with mpp = 1.0 and bss = 59. Some support was value in MEGA. found for a monophyletic group formed by our four low- When the data were partitioned by codon position, the land species: C. crassidigitus, C. fitzingeri, C. talamancae, and following models were recommended. For first position C. raniformis. These four species formed a clade with a non- sites the Kimura 2-parameter or K2P (Kimura 1980) +r+i significant mpp = 0.90, but this result was supported by the

Fig. 3 Phylogeny of all Craugastor fitzingeri group samples (Table 1) inferred from a Bayesian phylogenetic analysis of tri-partitioned mtDNA sequence data. Scale bar reflects the more conservative genetic distances estimated from the unpartitioned ML model. Outgroups and the distantly related C. fleischmanni are not shown. Statistical support for each node indicated by Bayesian marginal posterior probability (mpp) before the slash, and MP bootstrap scores (bss) following the slash. An asterisk (*) indicates maximal statistical support: a mpp value of 1.0 or a bss value of > 95%. A dot (•) indicates still significant support: a mpp of 0.99-0.95 or a bss value of 94-80%. Each mtDNA sample is represented by its sample code given in Table 1. For each sample code, the letter (A-Z) or number (1-5) preceding the period (.) refers to an area illustrated in Fig. 1, while the numbers following the period distinguish different frogs sampled within named areas. Sample codes with lower case letters (a-d) are not illustrated in Fig. 1, but are listed in Table 1. Sample codes in bold represent individuals from which data for both genes were obtained. The dagger (t) indicates that sample 1.2 (Table 1) actually comes from a collecting locality on the Pacific versant, 2 km from the continental divide (Fig. 1).

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd FROG PHYLOGEOGRAPHY AND TROPICAL FORESTS 4799

-T sJ } C. longirostris G.1 C. andi H.2 H.3 } C. cuáquero C.I C. melanostictus V.4 C. sp. cf. longirostris C 1J(| } C. tabasarae a.l C. sp. cf. crassidigitus \ w1i }C. sp. nov. 'pn'

Central Panama

}western PA Caribbean CR

Central Panama

Golfo Dulce region (Pacific CR)

Caribbean CR

Northern CR mountains

Panama

JDarien } Central Pacific ^

Pacific CR

}Nicoya Peninsula }western PA

Caribbean

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4800 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S.

ML and strict consensus MP trees based on the two-gene Divergence time estimation data set (not shown). The recently described C. tabasarae (Savage et al. 2004) was the sister species of an undescribed We applied the LRT to the subset of 67 samples with data taxon, C. sp. cf. longirostris with strong statistical support for both genes and rejected the molecular clock (P < 0.001). (Fig. 3). Craugastor cuáquero appeared to be the sister to a We applied the NPRS (relaxed clock) method to the phylo- taxon tentatively identified as C. andi (Fig. 3). Most other geny based on the complete data set (Fig. 3) and obtained basal relationships among members of the C. fitzingeri the following six divergence time intervals of particular group were poorly resolved. Of principal importance to interest (for comparison, the molecular clock results are this study mtDNA haplotypes representing conspecific given in parentheses). Comparing the Golfo Dulce vs. individuals of C. crassidigitus, C. fitzingeri, and C. talamancae Central Panama populations, we estimated that in C. were recovered as monophyletic with very high mpp and fitzingeri, these populations diverged in the Pliocene, 2.6- bss values, although C. crassidigitus monophyly received a 4.7 Ma (0.9-1.8 Ma). In C. crassidigitus, the Golfo Dulce marginal bss = 69 (Fig. 3). and Central Panama populations last shared a common ancestor as long ago as the Late Miocene, 4.2-8.1 Ma (5.8- 9.3 Ma). Craugastor fitzingeri samples last shared a common Intraspecific relationships ancestor in the Late Miocene, 5.3-9.8 Ma (2.6-4.5 Ma). The Within C. crassidigitus, C. fitzingeri, and C. talamancae, we MRCA of C. talmanacae diverged in the Late Miocene, 5.5- uncovered striking differences among species in branch 11 Ma (10-17 Ma). The MRCA of C. crassidigitus diverged lengths and in degree of support for intraspecific relation- in the Mid- to Late Miocene, 8.0-15 Ma (13-20 Ma). All ships. Although C. crassidigitus and C. talamancae have the more three species shared a common ancestor in the Early to restricted distributions, their mtDNA haplotypes showed Mid-Miocene, 12-23 Ma (19-36 Ma). larger genetic distances among geographical regions and well-resolved basal relationships (Fig. 3). Golfo Dulce and Phylogenetic hypothesis testing Central Panama samples of C. crassidigitus were reciprocally monophyletic and together formed the sister lineage to the All five topology tests rejected the 'northern route' and Caribbean CR + northern mountain CR samples (Fig. 3). 'vicariance' hypotheses for C. crassidigitus (Fig. 2). All Within regions, ive observed long branch-lengths and tests failed to reject any biogeographical hypothesis for significant phylogenetic structure in both C. crassidigitus C. fitzingeri (Table 3). Regarding our statistical tests of and C. talamancae (Table 2). interspecific relationships, the three nonparametric tests The phylogeography of C fitzingeri differed from C. crassi- (SH and parsimony-based) failed to reject any hypothesis. digitus and C. talamancae most notably in the low intraspecific All tests failed to reject the monophyly of our three focal genetic diversity (Table 2) and poorly supported relation- lowland species (C. crassidigitus, C. fitzingeri, C. talamancae), ships at basal nodes (Fig. 3). Genetic diversity within any the monophyly of the preceding three + C. raniformis (also of the three geographical regions ivas 3.8-fold to 9.8-fold lowland), or the monophyly of C. fitzingeri + C. raniformis. lower in C. fitzingeri than in the other two species, while However, Bayesian mpp and the SOWH test rejected the between any given pair of regions, C. fitzingeri showed 3.7- monophyly of C. longirostris + the other four lowland fold to 5.4-fold lower total genetic divergence (Table 2). species, while the SOWH test also rejected the monophyly Craugastor fitzingeri samples from outside the range of both of of highland taxa (Table 3). the other two species (areas A, B, J, and L in Fig. 1) represented very little additional genetic divergence (haplotypes A.l, Discussion B.l, B.2, J.l and L.l in Fig. 3) relative to other haplotypes. The phylogeographical data from C. crassidigitus, C fitz- Comparative phylogeography permits us to separate ingeri, and C. talamancae shared some features in common. historical processes that may have influenced whole None of these three species rejected our initial assumption communities of organisms from ecological or demographic that Central Panama samples represent a single clade, forces acting on single evolutionary lineages or species despite being drawn from two coasts and two mountain (Bermingham & Avise 1986; Campbell et al. 2006). In the ranges (Fig. 2). In C. fitzingeri, basal relationships are unclear present study, we are interested in the age and degree of (Fig. 3). In both C. crassidigitus and C fitzingeri, Caribbean geographical isolation of the Golfo Dulce community of CR appeared genetically isolated relative to the other two Craugastor frogs, which we place in a broader geographical regions (Table 2 and Fig. 3). In both C. crassidigitus and context in order to also analyse the influence of ecology on C. talamancae, the deep intraspecific root node clearly separ- the phylogeography of these frogs. Our focal species form ated Caribbean CR from Central Panama samples, despite a monophyletic clade (Fig. 3), thus, minimizing differences the continuous lowland wet forest habitat connecting these that may owe purely to phylogeny (e.g. grossly different two regions along the Caribbean coast. mechanics of nucleotide substitution), and are broadly

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© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4802 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S. sympatric (Fig. 1) thus minimizing differences in popu- colonization by an immigrant, w^hereas in the second case the lation structure resulting from different environmental immigrant would have only a 1 /N probability of replacing histories. a conspecific resident's mtDNA type in the population. The present study highlights two serious difficulties fac- In all three species, the deepest phylogeographical split ing all researchers interested in the evolution of amphibian separates northwestern from southeastern populations communities. First, many amphibian species remain un- along the Caribbean versant. While the species' ranges discovered or undescribed, and many named species harbour appear to correspond ivell with habitat heterogeneity lineages that have apparently been separated for signifi- (or perhaps with lack of collecting effort in the case of cant periods of time (e.g. Ron et al. 2006; Stuart et al. 2006). C. talamancae in eastern Panama), this Caribbean phylo- This study alone included three undescribed highland geographical break is someivhat surprising given the exist- species and revealed substantial cryptic phylogeographical ence of a continuous belt of wet forest linking Costa Rica diversity in two named lowland species. Second, amphibians •with Central Panama along the Caribbean coast (Fig. 1). are declining at an alarming rate (Stuart et al. 2004). One of This regional phylogeographical discontinuity has been our three undescribed highland taxa, Craugastor sp. cf. lon- observed in a number of freshwater fish (Bermingham & girostris, may well be extinct already (see Lips et al. 2006). Martin 1998; Perdices et al. 2002; Reeves & Bermingham Some of the known highland species may be gone as well. 2006), manakins (Brumfield & Braun 2001), dirt frogs Craugastor cuáquero is known only from the type locality, (Crawford 2003a) and the tree, Symphonia globulifera (Dick the site of drastic amphibian decline (Pounds & Crump 1994; et al. 2003). Across species, the phylogeographical breaks Pounds et al. 1997) while Craugastor andi and C.fleischmanni correspond to the Bocas del Toro Province near the border are both listed as 'Critically Endangered' and 'possibly of Panama and Costa Rica, but do not match perfectly in Extinct' by the Global Amphibian Assessment (lUCN location. Taken together, the multispecies data establish the et al. 2004). If the Museum of Vertebrate Zoology had not existence of a filter barrier in the Bocas del Toro region preserved these samples before the amphibian declines, (Fig. 1). A filter barrier reduces but does not eliminate we would not have had these highland forms for our phylo- the probability of dispersal between geographical areas genetic analyses and our conclusions regarding the (Remington 1968). monophyly of our focal species ivould have been called The effect of a filter barrier on different types of organisms into question. should vary with the dispersal ability of the species (e.g. Notwithstanding the differences among the phylogeo- Schäuble & Moritz 2001). In the case of C. fitzingeri, C. graphical patterns of our three focal frog species, the species crassidigitus, and C. talamancae, mean intraspecific genetic share tivo striking phylogeographical traits ivith one distances across the Bocas del Toro barrier varied threefold; another and with a variety of other species that have been uncorrected genetic distances were 5%, 14%, and 13%, studied in Central America. First, populations of Craugastor respectively, suggesting that the barrier has impeded crassidigitus, C. talamnncne, and to a lesser extent C.fitzingeri, C.fitzingeri less than its congeners, or that C.fitzingeri expanded are genetically discontinuous across the narroiv regional its geographical range more recently. The intraspecific landscape, thus providing additional evidence that many divergences observed for Craugastor species across the conspecific populations inhabiting loiver Central America Bocas del Toro filter barrier are similar to interspecific have been evolutionarily independent for long periods of divergences estimated for other Neotiopical frog genera. time. Similar patterns of regional phylogeographical struc- Although these studies are not based on the same combi- ture have been documented for primary freshwater fishes nation of mitochondrial genes, we note that related but (Bermingham & Martin 1998; Perdices et al. 2002; Reeves nonsister Epipedobates species (Dendrobatidae) from Peru are & Bermingham 2006), birds (Brumfield & Braun 2001; only 7-8% diverged in mtDNA sequence (Roberts et al. 2006), González et al. 2003), frogs (Crawford 2003a; Weigt et al. while Guiana Atelopus (Bufonidae) are 7-9% different from 2005) and a ividespread tree species (Dick et al. 2003). their closest relatives found in Peru and Brazil (Noonan & Second, C. crassidigitus and C. talamancae certainly occupied Gaucher 2005). much of their current geographical range relatively early Although the Bocas del Toro region is geologically very in their evolutionary history suggesting that the physical dynamic (Coates et al. 2003), the geological aspect apparent ability to disperse, in and of itself, is not limiting contem- today that potentially accounts for the filter barrier is porary gene floiv between populations. Reeves & Bermingham simply the narrowness of the lo^wland "wet forest. In the short (2006), commenting on a similar pattern among freshivater south-to-north distance from the Talamanca Mountains fish in the region, hypothesized that dispersal across a to the Caribbean Ocean, the elevation drops off sharply, landscape devoid of competitors vs. one populated with leaving a very narro'w corridor for dispersal along the conspecifics could leave significantly different mtDNA Caribbean versant (Fig. 1). There is no evidence to suggest traces. In the first case, suitable environment may be all that this region previously lacked continuous wet forest habitat that is needed to yield a very high probability of successful (Piperno & Pearsall 1998).

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd FROG PHYLOGEOGRAPHY AND TROPICAL FORESTS 4803

Craugastor crassidigitus, C. lalamancae, and C.fitzingeri all compatible with the data from C. crassidigitus (Table 3). Our share sufficient aspects of their evolutionary history to analyses indicate that Panama's Pacific slope served as the warrant consideration of their ecology in any attempt most recent link between the Golfo Dulce and the rest of to reconcile the observed phylogeographical differences C. crassidigitus. Independent of the direction of this hypo- among species. These three focal species prefer wet forest thesized dispersal event, two scenarios may explain these habitats, but C.fitzingeri appears to have a higher tolerance results. The Golfo Dulce C. crassidigitus may have arisen by for dry forests. It persists in dry habitat along sheltered vicariance with the Caribbean CR populations (8.0-15 Ma), riparian gallery forests (Savage 2002) and has been with subsequent dispersal out of the Golfo Dulce and recorded froffi drier sites along the Pacific Coast (Lynch & into Cential Panama via the Pacific Coast (4.2-8.1 Ma). Myers 1983; Köhler 2001; Savage 2002). All three species Alternatively the first divergence event may have separated overlap extensively in elevation, but C. talamancae does not Caribbean CR from Central Panama at the Bocas del Toro extend as high as the other two. AU three are found in both barrier, with subsequent dispersal into the Golfo Dulce forest edges and forest interiors, but C.fitzingeri appears to along the Pacific Coast. predominate in marginal habitats. Lynch & Myers (1983, Under either of the above scenarios, we may draw two p. 534) report that C.fitzingeri is often abundant 'in disturbed conclusions regarding the history of the region and environ- or edge situations.' Craugastor crassidigitus occurs 'in both mental influences on C. crassidigitus diversification: (i) the dry mature and second-growth forest, and also in forest-edge forests of the Pacific Coast of Panama have been less of a barrier situations' but 'it generally is more abundant in upland than the dry forests or mountains of northern CR (Fig. 1), but forest' (L5mch & Myers 1983, pp. 526-527). (ii) the Pacific Panania dry forest have served as a barrier to The modest differences in habitat association affiong C. crassidigitus since 4.2 Ma or prior (genetic divergence was species would favour dispersal by C. fitzingeri relative to 14%; Table 2). Using comparative phylogeography, we argue the other two species. The phylogeography of C. fitzingeri that the dry forest specifically is what separates Golfo Dulce demonstrates that this species has either higher levels of and Central Panama populations of C. crassidigitus. Other contemporary gene flow or has recently expanded across the historical or geological factors would have affected the landscape. Because our three focal species share a common intiaspecific divergence within C. crassidigitus and C.fitzingeri environmental distribution and common phylogenetic similarly, while in fact we found that the Golfo Dulce history until their separation in the Miocene, we conclude haplotypes from C.fitzingeri showed only 2.6% mean genetic that the slight differences in habitat preference provides distance rather than the 14% observed in C. crassidigitus. C. fitzingeri ivith a greater dispersal potential ivhich in turn explains the significantly reduced phylogeographical Craugastor as a model for the origin of the Golfo Dulce fauna stiucturing relative to C. crassidigitus and C. talamancae. The greater dispersal potential of C.fitzingeri is also sup- The 'Eastern Route' connecting Golfo Dulce with Central ported by the phylogeographical pattern of the samples Panama via the dry Pacific versant seemed a priori the least collected from the extreme ends of the species' geographical likely of the three possible results because (i) Central range, beyond the regions of sympatry The C.fitzingeri samples Panama is connected with Caribbean CRby the continuous from beyond the geographical range of the other two species wet forest of the Caribbean versant (across the Bocas del (areas A, B, J, and L in Fig. 1) ivere not genetically distinct Toro filter barrier), (ii) the 'Northern Route' required only (haplotypes A.l, B.l, B.2, J.l, and L.l in Fig. 3), demonstiat- traversing the very short distance between Caribbean and ing they have been recently connected by migration ivith Pacific CR (Fig. 2), and (iii) there is no evidence to suggest more central localities. This result could not have been that the dry forests of Isthmian Cenfral America were recently predicted a priori, because more widespread species some- wet. The Pacific dry forests of today were savannah habitat times show the greater phylogeographical structure (e.g. during Pleistocene glacial maxima (Pipemo & Pearsall 1998) Roberts 2006). Thus, we find that Craugastor ecology and would have been even more inhospitable to forest- predicts both the degree of phylogeographical stiucturing dwelling frogs during these climatic drying and cooling and the extent of species' ranges. events. However, our biogeographical conclusions corre- spond well with the environmental history of the region. Dry forest habitat in Panama substantially predates the The origin of the Golfo Dulce populations Pleistocene, going back > 4 million years (Graham & Dilcher Only the phylogenetic data representing C. crassidigitus 1995), a date that agrees well with our minimum divergence permitted us to distinguish among our three a priori hypo- time estimate (4.2 Ma) for C. crassidigitus populations theses (Fig. 2) for the origin of the Golfo Dulce populations separated by this dry forest. Thus, the palaeontological, of C. fitzingeri and C. crassidigitus. The n:\tDNA data for C. geological, and phylogeographical data together support fitzingeri were unable to reject any of the three a priori the old age of the spatial pattern of wet and dry forests of hypotheses, whereas 'Eastern Route' is the only hypothesis lower Central America.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 4804 A. J. CRAWFORD, E. BERMINGHAM andC. POLANÍA S.

While our genealogical information alone does not the Project for the Management of the Wildlands of Krma Yala demonstrate the direction of dispersal, we hypothesize that (PEMASKY). In Costa Rica permits were obtained with the C. crassidigitus colonized the Golfo Dulce from the Pacific support of Javier Guevara at the Ministerio de Ambiente y Energía (MINAE) and Federico Bolaños at the UCR. We thank the follow- based on three observations: (i) this species potentially pre- ing landowners and authorities for permission to collect samples dated the Golfo Dulce wet forest, (ii) the Caribbean coast in Costa Rica: Luis Diego Gomez and Sergio Jimenez (OTS), Brian hosts all three lowland species, ivhereas the Pacific Coast Kubicki (CR Amphibian Research Center), Bert Kohlmann (Univer- lacks C. talamancae (Fig. 1), and (iii) the Golfo Dulce shows sidad EARTH), Osa Pulchra Women's Collective, Reserva Biológica less ivithin-region genetic diversity than the other two Quebradas, Frans van Harén and Gerardo Budowski (Universidad regions (Table 2). If so, colonization of Golfo Dulce ivould para la Paz), and Ulises Jimenez (Reserva Biológica Quebradas). have taken place from the east, via Central Panama Likewise in Panama, we thank Señor Bodero (Urbanización Altos de Cerro Azul), TropicStar Lodge, ANCÓN Expeditions, and Iñaki (Table 3). This pattern of colonization ivas also suggested Ruiz Osaba (Burbayar Lodge). We also thank the following cura- for Phyllohates (Maxson & Myers 1985), a genus of frogs of tors and collectors for sharing tissue samples: Federico Bolaños, likely South American origin. Cachi Chaves and Robert Puschendorf (UCR), Karen Lips and As a further test of the age of wet forest habitat, we can Figa Brenes (SIUC), Frank Solis, César Jaramillo and Roberto compare phylogeographical data from open-habitat spe- Ibáñez (Círculo Herpetológico de Panamá and Universidad de cies found on either side of the wet forest (e.g. Wüster et ni. Panamá), Edgardo Griffith, Gwen Keller, Carla Cicero and David 2005). If the Golfo Dulce wet forest has been isolated by dry Wake (MVZ), Randy McCranie, George Zug and Steve Gott (USNM), and Mahmood Sasa (OTS). For help in the laboratory, we forest or savannah since > 4 (Ma), as we contend, then any would especially like to thank Catalina Perdomo and Armando dry-forest or open-habitat species occurring on either side Castillo, plus Maribel Gonzalez, Carlos Vergara, Grettehun Grethel of the Golfo Dulce should show a complementary genetic Grajales, Nimiadina Gomez, and Oris Sanjur. This work was sup- break of similar age. This prediction was borne out in a recent ported by a Smithsonian Institution Postdoctoral Fellowship in analysis of the fungara frog, which is found in the open and Molecular Evolution to A.J.C. and an STRI Internship to C.P.S. We dry forest habitats on either side of the Golfo Dulce region. thank Wolfgang Wüster and two anonymous reviewers for their This Golfo Dulce 'break' was dated to 4-16 Ma using help in substantially improving this manuscript. either of two South American calibration points and a clock-independent temporal analysis (Weigt et ni. 2005). 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