1 Environmental versus geological barriers in the Great American Biotic Interchange: a

2 biogeographic analysis using frogs

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6 Carlos Alberto Jiménez Rivillas

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10 Andrew J. Crawford

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14 Catalina González Arango

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19 Universidad de los Andes

20 Departamento de Ciencias Biológicas

21 22 de Octubre de 2018

22 Bogotá D.C., Colombia 23

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26 He [the naturalist] looks upon every species of animal and plant now living as the

27 individual letters which go to make up one of the volumes of our earth's history; and, as a

28 few lost letters may make a sentence unintelligible, so the extinction of the numerous

29 forms of life which the progress of cultivation invariably entails will necessarily render

30 obscure this invaluable record of the past.

31 Alfred Russel Wallace (1863) On the physical geography of the Malay Archipelago. The

32 Journal of the Royal Geographical Society of London 33:217-234.

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36 I saw with regret, (and all scientific men have shared this feeling) that whilst the number of

37 accurate instruments was daily increasing, we were still ignorant

38 Alexander von Humboldt, Aimé Bonpland (1818) Personal Narrative of Travels to the

39 Equinoctial Regions of America, During the Year 1799-1804 - Volume 1.

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44 45 Environmental versus geological barriers in the Great American Biotic Interchange: a

46 biogeographic analysis using frogs

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48 Carlos Jiménez-Rivillas1,2, Paola Montoya3, Roberto Ibáñez4, Catalina Gonzalez-Arango5,

49 and Andrew J. Crawford2,4.

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51 1M.Sc. student in Biological Sciences, 2Biom|ics Lab, Biological Sciences Department,

52 Universidad de los Andes, Bogota D.C. – Colombia.

53 3Laboratorio de Evolución de Vertebrados (EvolVert), Biological Sciences Department,

54 Universidad de los Andes, Bogota D.C. – Colombia.

55 4Smithsonian Tropical Research Institute (STRI), Panama.

56 5Laboratorio de Paleoecología y Palinología (PaleoLab), Biological Sciences Department,

57 Universidad de los Andes, Bogota D.C. – Colombia.

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59 Abstract

60 The geological closure of the Isthmus of Panama (IP) precipitated one of the greatest

61 biogeographic events of the Cenozoic that indelibly changed composition of biotic

62 communities in South and North America. The precise timing of uplift and final closure of

63 the IP continues to be a topic of intense debate in geology and evolutionary biology. The

64 traditional or Young Isthmus model states that the definitive closure of the IP occurred

65 between 4 and 3 million years ago (Ma). The more recently proposed Old Isthmus model

66 states that the IP was completed during the middle Miocene (15 to 13 Ma). Regardless of 67 the closure data, the fossil record makes clear that at 2.7 Ma began the Great American

68 Biotic Interchange (GABI), a massive interchange of mammalian lineages, many affiliated

69 with dry and open environments. For the Old Isthmus hypothesis to be viable, one must

70 posit the existence of some non-oceanic barrier that delayed the interchange between

71 continents for some 10 million years. Here, we tested the hypothesis that an

72 environmental barrier in the form of a humid, closed-canopy forest was present on the IP

73 prior to the Pleistocene interchange and glacial cycling. Scant paleoenvironmental data

74 are available from the Neogene IP, so here we test our hypothesis indirectly using

75 comparative phylogeography of 69 species of anurans by reconstructing their

76 environmental affinities and estimating the timing of interchange between continents for

77 each lineage. We found that frog species with a preference for dry and open environments

78 all moved between continents after 3 Ma (n = 11 colonization events), while the mean

79 date of interchange for those associated with humid forests was 6.1 Ma (n = 24 events, SD

80 = 3.7 Ma), including some more recently than 3 Ma. Semi-arid species crossed

81 significantly later than humid forest species (randomization test, mean difference = 6.8

82 million years, P = 0.0075), as predicted by the hypothesis that a humid, closed-canopy

83 forest barrier existed during the Late Miocene and perhaps to a lesser extent in the

84 Pliocene. The Pleistocene was characterized by cooler and drier conditions which led to a

85 reduction in forest cover and this may have promoted directly the GABI across an IP

86 already 10 million years old.

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88 Keywords: Crown age, dispersal, humid-closed barrier, Stem age, dry-open corridor 89

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110 111 INTRODUCTION

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113 Barriers to dispersal are an important component of vicariance biogeography and

114 models of allopatric speciation (Platnick & Nelson, 1978; Ronquist, 1997). Physical

115 barriers are often invoked as isolating mechanisms driving allopatric speciation and

116 creating regions of endemism (Rosen, 1988). Examples include oceans, rivers, and

117 mountains (Jansson, 2003). Environmental heterogeneity can also create barriers to

118 dispersal, however, such as the warm desert of North America (Hafner & Riddle, 2011).

119 Wet forest can also be a barrier to dispersal for organisms that prefer open or xeric habitat,

120 e.g., the Amazonian rain forest separates the xeric and open habitats of the Dry Diagonal

121 in the South from the coastal xeric habitats of Venezuela and Colombia in the North

122 (Gutiérrez et al., 2014). Here we use comparative phylogeography to ask whether closed-

123 canopy wet forest could have been a barrier preventing animals from dispersing through

124 the Isthmus of Panama (IP) during the Pliocene and Miocene.

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126 Since the Early Cretaceous and throughout the Paleogene, South America lacked

127 connections with other major landmasses and was an island continent whose fauna and

128 flora remained in ‘splendid isolation’ (Simpson, 1980). Eventually the Central American

129 Seaway (CAS) closed via the formation of a complete and permanent land bridge known

130 as the Isthmus of Panama (IP) that joined South America with Central America, and thus

131 North America, separating the Caribbean Sea from the Pacific Ocean (Keigwin, 1978). The 132 date of the formation of the IP, however, is highly controversial and the scientific debate

133 itself has been termed the ‘Battle for the Americas’ (Stone, 2013).

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135 Positions regarding the geological formation and completion of the IP fall under

136 two basic models. The traditional or ‘Young Isthmus’ model states that the IP was not

137 completely formed until the Pliocene approximately 3 million years ago (Ma). Early

138 evidence came from biostratigraphy and isotope paleogeography (Keigwin, 1978).

139 Subsequent geological and paleontological studies, along with molecular phylogenetic

140 studies of marine geminate species, also dated the closure of the IP to between 4.0 to 3.0

141 Ma (e.g., Coates et al., 1992; O’Dea et al., 2016; Coppard & Lessios, 2017). These young

142 dates for the closure of the IP contrast sharply with the more recent ‘Old Isthmus’ model

143 that posits a closure around 15 to 13 Ma, based on analyses of petrogenesis of magmatic

144 rocks and paleo-volcanic activity (Montes et al., 2015).

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146 While controversy surrounds the estimated age of the closure of the IP, the date of

147 the Great American Biotic Interchange (GABI) is very well established. ‘GABI’ in the strict

148 sense refers to the massive, nearly simultaneous, and reciprocal colonization of North and

149 South America by diverse mammalian lineages at 2.7 Ma, a date clearly established by an

150 extensive fossil record involving species from 17 taxonomic families (Webb, 1976;

151 Marshall et al., 1982). The date of the GABI fits well with the Young Isthmus model,

152 whereas the Old Isthmus model posits a land bridge being completed at least 10 million

153 years before the GABI took place. To reconcile the well-dated GABI with the Old Isthmus 154 model, proponents of the latter would have to explain why a completed terrestrial corridor

155 was not used until 10 million years later.

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157 A hypothesis recently put forward to explain this 10 million year lag time invokes

158 an environmental barrier (Bacon et al., 2016). While a land bridge may have been in

159 place by the middle Miocene, successful dispersal would not take place if organisms

160 encounter conditions very different from their usual environment. The mammals

161 participating in GABI included a large proportion of species specializing in savannah

162 habitat (Stehli & Webb, 1985; Webb, 1991; Vrba, 1992; Woodburne, 2010). Thus, if a

163 complete, late Miocene Isthmus were covered predominately by closed-canopy wet

164 forests, such a landscape could have acted as an environmental, rather than physical,

165 barrier to dispersal between continents, delaying the GABI despite the existence of a

166 complete land bridge (Bacon et al., 2016).

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168 Unfortunately, details are lacking on the spatial and temporal distribution of paleo-

169 environments such as open and semi-arid versus closed and forested habitats during the

170 late Miocene, and the habitat preferences of many species that participated in the GABI

171 remain unconfirmed (Bacon et al., 2016). In this study, we proposed using comparative

172 phylogeography from non-mammalian groups to test for an association between habitat

173 preference and timing of continental interchange. Frogs may provide a valuable proxy for

174 the historical presence of open or wet versus closed or semi-arid habitats. As the only non-

175 amniote tetrapods, amphibians tend to be found close to source of water and most species 176 are poor dispersers (Cushman, 2006). These characteristics of frogs make them excellent

177 indicators of biogeographical processes (Ron, 2000; Paz et al., 2015). Amphibians tend to

178 retain ancestral niches and related ecological traits over time, thus displaying a high

179 degree of niche conservatism (Peterson et al., 1999; Wiens & Graham, 2005). Many

180 Neotropical lineages of frog appear to be constrained to one type of biome or other,

181 closed and mesic versus open and semi-arid environments (Rodríguez et al., 2015).

182 Further, we know that frogs crossed between continents over a wide temporal range,

183 including interchange well before and since the GABI (Weigt et al., 2005; Pinto-Sánchez

184 et al., 2012).

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186 We use multiple, independent lineages of frogs to test the hypothesis that a wet and

187 closed-canopy forest existed on the Isthmus prior to 3 Ma and this habitat acted as an

188 environmental barrier to dispersal. This hypothesis makes the following predictions that

189 we can test with molecular phylogenetics, spatial analyses, and comparative

190 phylogeographic analyses of modern frogs and their environmental preferences. (1) Any

191 lineages that crossed between continents prior to the GABI (i.e., before 2.7 Ma) should be

192 associated with wet and closed-canopy forest conditions, though young wet-forest species

193 could have crossed more recently, as well. (2) No species associated with semi-arid, open

194 environments could have crossed prior to 2.7 Ma. (3) Given the uncertainties around

195 divergence time estimation (Rutschmann, 2006; Zhu et al., 2015), we emphasize that

196 regardless of exact dates of interchange, those lineages associated with wet, closed-

197 canopy forests should show older dates relative to species associated with semi-arid or 198 open habitats. These predictions contrast with those made by the canonical ‘Young

199 Isthmus’ model in which all species are expected to have crossed the IP around 3 Ma

200 when the IP was completely formed, i.e., that (1) few species could have crossed before 3

201 Ma while those that did are considered ‘heralds’ (Webb, 1976), and (2) there should be no

202 co-variation between the age when taxa crossed the IP and their habitat preference.

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219 220 MATERIALS AND METHODS

221 Ethics statement

222 All field work and collecting in Colombia was conducted under the authority of the

223 Autoridad Nacional de Licencias Ambientales (ANLA) de Colombia (permiso de estudio

224 con fines de investigación científica en diversidad biológica No 27 del 22 de junio de

225 2012 and permiso de acceso a los recursos genéticos resolución No 0377 del 11 marzo de

226 2014 to A.J.C. plus permiso marco resolución No 1177 to the Universidad de los Andes).

227 Collections in Panama were conducted under permits Nos. SE/A-083-2001 and SE/A-37-

228 07 to R.I.D. and SE/AP-7-07 to E.B. by the Autoridad Nacional del Ambiente. Collection

229 and euthanasia followed institutional animal care and use protocols approved by

230 Institutional Committee on the Care and Use of Laboratory Animals (abbreviated CICUAL

231 in Spanish) of the Universidad de los Andes.

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233 Taxonomic and geographic sampling

234 We chose eight genera of Neotropical frogs whose geographic distribution

235 included both South America and Central America (Fig. 1), as follows.

236 Lithobates Fitzinger, 1843, is composed of 51 species (Frost, 2018) distributed from

237 North America to Bolivia and eastern Brazil in South America (Savage, 2002; Köhler,

238 2011; Frost, 2018). Most species exhibit mainly semi-aquatic habits and live close to

239 water bodies (Köhler, 2011).

240 Craugastor Cope, 1862, is a member of the unranked clade, Terraranae (New

241 World direct-developing frogs; Hedges et al., 2008), containing 117 species distributed 242 throughout the Neotropics from the southern-most United States (USA) South through

243 northwestern Ecuador (Crawford & Smith, 2005; Frost, 2015). Species of this genus are

244 generally cryptically colored inhabitants of the rainforest. A few species are diurnal leaf

245 litter inhabitants or nocturnal stream-dwellers (Köhler, 2011).

246 Leptodactylus Fitzinger, 1826, commonly referred to as “Neotropical grass frogs”, is

247 composed of 74 species (Frost, 2018). Grass frogs have a wide geographic distribution

248 from southern North America, across Central America and lowland South America, and

249 into the West Indies (Savage, 2002; Köhler, 2011; Sá et al., 2014a). They occupy a wide

250 range of environments from primary and secondary rainforest to open habitats over most

251 of subtropical and tropical lowland America (Sá et al., 2014a).

252 Agalychnis Cope, 1864, commonly known as red-eyed tree frogs, is an endemic

253 neotropical genus (Savage, 2002), composed of 13 species (Frost, 2018) with well-

254 resolved phylogenetic relationships (Faivovich et al., 2010; Gomez-Mestre et al., 2011;

255 Rivera-Correa et al., 2013). Red-eyed tree frogs are primarily denizens of the lowland

256 rainforest. They occupy a geographic range from northern coastal Mexico, throughout

257 Central America to northwestern Ecuador. Some species are found in the upper Amazon

258 of Colombia, Venezuela, Ecuador, and northern Peru (Savage, 2002; Frost, 2018).

259 Boana Gray, 1825, includes a group of rather large arboreal nocturnal treefrogs

260 termed “gladiator frogs” (Duellman, 1970; Köhler, 2011). This genus contains 99 species

261 distributed in tropical Central and South American from Nicaragua to Argentina, with

262 some species reaching in Trinidad and Tobago (Frost, 2018). Gladiator frogs can occupy a 263 wide variety of habitats from humid rainforests to dry tropical forest and other semi-arid

264 environments (Duellman, 1970, 1999; Savage, 2002).

265 Scinax Wagler, 1830, also known as “snouted treefrogs”, contains 71 species (Frost,

266 2018), distributed from southern Mexico to Argentina and Uruguay, and East to Trinidad

267 and Tobago and St. Lucia (Faivovich, 2002; Faivovich et al., 2005). All species are

268 nocturnal and arboreal, and many are found in a variety of habitats including dry, moist,

269 and wet forests (Duellman & Wiens, 1993; Köhler, 2011).

270 Dendropsophus Fitzinger, 1843, also known as “yellow tree frogs”, contains 105

271 species that range from northern Argentina and Uruguay, northward to tropical southern

272 Mexico (Frost, 2018). They are inhabitants of lowland moist, wet and dry forests, and

273 premontane wet forest and rainforests zones (Savage, 2002).

274 Smilisca Cope, 1865, contains only 9 species and is an essentially Middle

275 American genus that ranges through lowland and premontane areas from southern Texas

276 and Sonora, Mexico, to north-central Colombia and northwestern Ecuador (Savage, 2002).

277 Species are found in a variety of habitats such as dry, moist, and wet forests, including

278 secondary growth and pastures (Duellman, 1970; Köhler, 2011).

279 For each of eight genera of frogs we used molecular phylogenetic analyses to infer

280 the direction and timing of interchange between North America and South America. The

281 exact line separating the two continents is open to some debate. We therefore defined two

282 lines, one slightly more eastern and one slightly more western, and defined lineages as

283 ‘participants’ in the GABI if we inferred they had crossed both of these lines during the

284 evolution history of the genus (Fig. 2). The easternmost line we defined as the fault line 285 between the Panama microplate and the South American continent, which runs from the

286 western Caribbean Coast of Colombia (east of Panama) southward (parallel to the Atrato

287 River) along western edge of the Western Cordillera, continuing westward to the coast as

288 the Uramita fault (Duque-Caro, 1990; Montes et al., 2015; Fig. 2), Our second, more

289 western line, is defined by the structural boundary between the Chorotega and Choco

290 blocks of the Panama microplate that correspond to the Tertiary structural and

291 depositional basin called the Panama Canal basin (Coates et al., 2004; Kirby et al., 2008;

292 Fig. 2).

293 We used published species range maps (International Union for Conservation of

294 Nature [IUCN], 2019) to identify species or genera of frogs distributed on either side of the

295 two limits between continents. We then defined species as ‘participants’ in the GABI

296 based on previously published phylogenies that allowed us to estimate the geographic

297 origin of the taxonomic family or genus as either a northern lineage with one or more

298 representatives having dispersed from the Panamanian microplate to South American

299 continent, or as a southern lineage with one or more representatives having crossed

300 westward into what is today the country of Panama (Savage, 1966). To confirm the

301 direction of dispersal and estimate divergence times, we conducted molecular

302 phylogenetic analyses on a set of congeneric taxa selected as follows. We started with the

303 ‘participant’ species then used published phylogenies to identify each species’ most recent

304 common ancestor (MRCA) that has descendants on both continents. If this MRCA had only

305 a single taxonomic species as a descendant, we then included an additional species

306 identified as the sister lineage (i.e., descendant of the next most basal node to the MRCA 307 identified previously). The resulting taxonomic sampling can be thought of as our ingroup,

308 to which we added one outgroup identified by looking for a ‘non-participant’ species

309 having descended from the next basal-most node. A closely related non-participant is

310 defined as a species that occurs only on the continent where the more inclusive species-

311 group or genus originated. The resulting list of species analysed is found in Table 1.

312 For each ingroup species, we sought to obtain genetic samples from at least three

313 localities, or three localities per continent in the case of species that occurred on both

314 continents, whereas for the non-participant outgroup, one sample was considered

315 sufficient. Given the above requirements we included in this study a total of 35 ingroup

316 species (Table 1), divided among eight genera, as follows. For the genus of ranid frogs,

317 Lithobates Fitzinger, 1843 we selected 3 species as participants and 2 species as non-

318 participants (Table 1) by applying our selection criteria to the molecular phylogeny of

319 Yuan et al. (2016). For the terraranan genus, Craugastor Cope 1862, we identified 3

320 participant species and 3 non-participant species (Table 1) based on molecular

321 phylogenetic studies (Crawford et al., 2007; Pyron & Wiens, 2011). Finally, for

322 Leptodactylus Fitzinger, 1826 we use the molecular phylogenetic hypothesis proposed by

323 Sá et al. (2014) to select 6 participant species and 5 non-participant species (Table 1). For

324 the phyllomedusid genus Agalychnis Cope 1864 we selected 6 species as participants and

325 2 species as non-participants (Table 1) following the phylogeny by Rivera-Correa et al.,

326 2013 (Table 1). For the hylid genus, Boana Gray, 1825, we identified 5 species as

327 participants and 7 species as non-participants (Table 1; Faivovich et al., 2005). For the

328 hylid genus, Scinax Wagler, 1830, we use followed the phylogenies of Faivovich et al. 329 (2005) and Pyron & Wiens (2011) to choose 6 species as participants and 6 non-

330 participant species (Table 1). For the hylid Dendropsophus Fitzinger, 1843, we used the

331 molecular phylogeny by Gehara et al. (2014) to select 4 participant species and 7 non-

332 participant species (Table 1). For the sixth and final hylid genus, Smilisca Cope, 1865, we

333 use the phylogeny of Pyron & Wiens (2011) to select 2 participant species and 2 non-

334 participant species (Table 1). The complete data set for taxonomic and geographic

335 sampling for each species can be seen in Table S1.

336

337 Laboratory techniques

338 Because most published molecular phylogenetic studies of our target genera

339 included very poor geographic sampling from Panama and Colombia, we added

340 additional samples of all genera to published data sets. The new DNA sequence data were

341 collected as follows.

342 Mitochondrial genes are characterized by higher substitution rates than nuclear

343 genes, which allows one to resolve recent divergence events between populations and

344 species (Crawford, 2003a; Mueller, 2006), being the basis of most studies in comparative

345 phylogeography (Smith et al., 2014). The contrasting nuclear DNA sequence data helped

346 resolve deeper divergence events and provided a test of whether the mitochondrial gene

347 trees were in gross conflict with the inferred history of the nuclear genome.

348 We sequenced a fragment of the 16S rRNA (16S) gene which has a slower rate of

349 substitution compared to protein-coding genes in the mitochondrion (Patwardhan et al.,

350 2014), along with the 5’-end of the Cytochrome Oxidase subunit I (COI) gene, also known 351 as the animal ‘Barcoding of Life’ for its broad use in the identification of species (Hebert et

352 al., 2003; Patwardhan et al., 2014). Mitochondrial DNA (mtDNA) sequences were used to

353 create preliminary neighbor-joining trees (Saitou & Nei, 1987) assuming a HKY distance

354 correction (Hasegawa et al., 1985), which were then used to identify representative

355 samples of each major mtDNA lineage which were then used for PCR and sequencing of

356 two nuclear genes (ncDNA), the proopiomelanocortin gene (POMC) and a fragment of the

357 Tyrosinase precursor gene (TYR). Information on primers used in amplification and

358 sequencing is provided in Table 2.

359 We used the DNeasy Blood & Tissue Kit (Qiagen) to extract genomic DNA (gDNA)

360 from tissue samples of liver or thigh muscle stored at -80 C subsequent to being preserved

361 in 99% ethanol (EtOH), Nucleic Acid Preservation buffer (NAP; Camacho-Sanchez et al.,

362 2013), or 20% dimethyl sulfoxide (DMSO) in salt-saturated 0.25 EDTA (Seutin et al.,

363 1991). DNA sequences were amplified using the polymerase chain reaction (PCR) with a

364 total reaction volume of 30 μL. PCR experiments contained 15 μL of GoTaq Green Master

365 Mix (Promega), 2 μL each of the forward and reverse primers (Table 2) with a final

366 concentration of 10 μM, 9 μL of ddH2O, and 2 μL of gDNA with a concentration ≥ 30 ng

367 μL-1. For low-yield extractions with < 30 ng μL-1 of gDNA, we use a volume of 5 to 8 μL of

368 gDNA with an addition of bovine serum albumin (BSA) at 1% final concentration, or

369 glycerol anhydrous [ ≥99.5% (GC)] at final concentration of 5% by volume. A 1% agarose

370 gel electrophoresis in Tris/Borate/EDTA (TBE) buffer was conducted to confirm the

371 presence of sufficient mass of amplicons. PCR products were cleaned by adding 2.062 μL

372 of the following mix: 1.65 μL of fast AP buffer, 0.30 μL of Fast AP enzyme, and 0.112 μL 373 of Exo I enzyme (Werle et al., 1994), followed by incubation at 37°C for 60 minutes and

374 an inactivation step at 85°C for 15 minutes. Purified PCR products were Sanger-sequenced

375 (Sanger & Coulson, 1975), using the BigDye reaction mix, and following the cycle

376 sequencing profile of 95°C for 10 minutes, 35 cycles of 96°C for 3 seconds, 62°C for 15

377 seconds, 68°C for 30 minutes, and ending with 72°C for 2 minutes. All sequences were

378 quality-controlled, cleaned, assembled, and edited using Geneious v. R8 (Drummond et

379 al., 2015). New sequence data obtained for this project were deposited in BoLD (Table

380 S1).

381

382 Phylogenetic methods

383 The newly generated sequences (Table S1) were combined with sequences from

384 GenBank (Table S3). Prior to concatenated DNA sequence analyses we perform a multiple

385 sequence alignment for each gene fragment using MAFFT ( v7.388; Katoh & Standley,

386 2013) implemented in Geneious Prime (v2019.1.3) and applying the FFT-NS-2 algorithm

387 (Katoh, 2013). This algorithm incorporates an iterative refinement and consistency-based

388 scoring approach (here using the PAM200 matrix), using a guide tree based on all-

389 pairwise comparisons to obtain a more reliable alignment (Katoh et al., 2002). To

390 accelerate the initial calculation of the distance matrix, we selected a k-mer counting (k =

391 2), in which the number of k-mers shared by a pair of sequences is counted and regarded

392 as an approximation of the degree of similarity (Katoh & Standley, 2013). We chose an

393 open gap penalty of 2.0 for protein-coding gene fragments (COI, TYR, and POMC), and

394 1.0 for the ribosomal gene fragment 16S, and assumed a gap extension penalty of 0.1 and 395 offset value of 0.123 for all genes. Each dataset was inspected for significant incongruence

396 (Wiens, 1998) by comparing preliminary NJ trees.

397

398 Given the importance in phylogenetics analysis of accounting for variation in rates

399 and patterns of evolution in DNA sequences (Yang, 1996; Lanfear et al., 2012, 2016), we

400 used PartitionFinder2 (Lanfear et al., 2016) to select best-fit partitioning schemes and

401 models of molecular evolution for concatenated fragments. Potential partitions included

402 each codon position in each protein-coding gene, plus the 16S gene fragment.

403 Combinations of substitution models and partition schemes were evaluated using the

404 corrected Akaike Information Criterion (AICc). Recommended models were implemented

405 in BEAST2 while a single model was applied to all partitions in RAxML (see below).

406

407 Concatenated molecular phylogenetic analyses were performed using maximum

408 likelihood (ML). ML analysis were run in the Randomized Axelerated Maximum

409 Likelihood program (RAxML v.8.0) (Stamatakis, 2014) assuming the GTRCAT model,

410 which approximates the GTR + Γ model (Yang, 1994). Because our research question

411 required detailed phylogeographic analyses below the species level, we did not attempt

412 species-tree phylogenetic inference. Outgroup DNA sequences for each genus were

413 selected based on wider phylogenetic analyses (see above and Table S4). Statistical

414 support for nodes was assessed via 1000 bootstrap replicates (Felsenstein, 1985). ML trees

415 were visualized using FigTree v.1.4.3 (Rambaut, 2016).

416 417 Divergence time analysis

418 To estimate divergence times between species and populations we used the

419 Bayesian Evolutionary Analysis by Sampling Trees program, BEAST2 v.2.5.2, that uses

420 Markov chain Monte Carlo (MCMC) to integrate over parameter space (including tree

421 space) where the probability of each tree is estimated by its relative frequency in the

422 posterior sample (Bouckaert et al., 2014). All analyses were conducted independently for

423 each genus and assumed the partition scheme and molecular evolution models

424 recommended by PartitionFinder2 (see above). We chose a relaxed clock model that

425 allows substitution rates to vary among branches according to an autocorrelated

426 Lognormal distribution (Thorne et al., 1998). The prior mean of the rate of substitution

427 (ucld.Mean) was assumed to be 0.009825 per lineage per million years for mtDNA

428 (quantiles 2.5% 0.0074 and 97.5% 0.1225), and 0.0006135 for ncDNA (quantiles 2.5%

429 0.000462 and 97.5% 0.000765) (Crawford, 2003b) assuming a lognormal distribution

430 with. We assumed the calibrated Yule model as the prior on tree shape, such that the rate

431 of cladogenesis is assumed to be constant over time (Gernhard et al., 2008), allowing

432 direct specification of the marginal prior distribution of the calibrated divergence time

433 with the restriction of monophyly (Heled & Drummond, 2012). To improve our estimates

434 of absolute divergence times in the absence of a strict molecular clock, we assumed one

435 or two temporal constraints on each tree. For each tree we applied a secondary calibration

436 to the divergence time of the most recent common ancestor (MRCA) of the outgroup and

437 ingroup (stem age). Mean divergence times were taken from the most recent, global

438 analysis of divergence times in frogs based on numerous fossil calibrations and 100s of 439 genes (Feng et al., 2017; Table 3). When divergence time estimates were also available for

440 one pair of species within an ingroup, that secondary calibration was also applied, based

441 on the assumption that fossil-calibrated, genomic-scale analyses should be more reliable

442 than our 4-gene analyses of each genus with no possibility of fossil calibrations. Bayesian

443 MCMC analyses were performed using two independent runs of 50 million generations.

444 To analyze the results of the MCMC, we used Tracer v.1.7.1 (Rambaut, 2018) to visually

445 inspect that each parameter had reached stationarity, calculate the effective sample size

446 (ESS), and confirm that all parameters achieved an ESS of >200. We employed

447 TreeAnnotator v. 2.4.8 (Rambaut & Drummond, 2017) to summarizing the posterior

448 sample of trees after removing the first 10% of trees as burnin. We used FigTree to

449 visualize the resulting Bayesian consensus trees.

450

451 Inferring environmental preferences

452 The hypothesis of a closed-canopy (wet) barrier on the Isthmus predicts that species

453 from semi-arid open (dry) habitats will have crossed between continents more recently,

454 thus requiring that we estimate the environmental preference of our study species. To this

455 end, we used multivariate statistics to characterize the environmental preferences of each

456 frog species based on the climatic and edaphic characteristics of occurrence records

457 across its distributional range. Using the same parameters and method we then

458 summarized environmental variation at ecoregion a priori defined by the World Wildlife

459 Fund (WWF; Dinerstein et al., 2017) as distinctly wet or distinctly dry. We used QGIS

460 v.3.0 Girona (QGIS Team, 2009) to extract three layers corresponding to the biome area, 461 soil and bioclimatic from four locations representing lowland biomes defined as humid

462 environments: Chocó-Darién moist forests, Japurá-Solimões-Negro moist forests,

463 Southwest Amazonian moist forests, and Central-American-Atlantic moist forests (Fig. 3).

464 Similarly, we extracted environmental data from four sites corresponding to iconic dry

465 environments: the Panamanian dry forests, Guajira-Barranquilla xeric scrub, La Costa

466 xeric shrublands, and Brazilian-Atlantic dry forests (Fig. 3).

467

468 We generate two shapefiles: one containing the four wet biomes and another with

469 the four dry biomes. Over these layers, we added shape files representing the 19

470 WorldClim bioclimatic variables (v2.0 Fick & Hijmans, 2017) plus two soil variables

471 representing the percentage of clays in the soil and the cation exchange (cmol kg-1)

472 obtained from the Soil and Terrain Database for Latin America and the Caribbean

473 (SOTERLAC; Dijkshoorn et al., 2005). We used R (R Core Team, 2017) to generate 1000

474 random points over each layer with the package ‘raster’ (Hijmans, 2017), which were then

475 used to perform a Principal Component Analysis (PCA) and a Linear Discriminant Analysis

476 (LDA) (Venables & Ripley, 2002) to assess the degree to which the set of environmental

477 and soil variables could distinguish wet and dry environments. From VertNET (Constable

478 et al., 2010) and the Global Biodiversity Information Facility (GBIF.org, 2018), we

479 obtained georeferenced locality records for the frog species studied here, and filtered the

480 data against the IUCN RedList spatial data (ASG & IUCN, 2018). The value of each

481 environmental and soil variable was recorded from each georeferenced locality record for

482 each species, and these data were then added to the PCA analysis to visualize where each 483 species occurred in environmental space along a multidimensional continuum between

484 WWF-defined wet and dry environments. Each PC was also visualized as a (univariate)

485 histogram to visualize the potential overlap of each species with the wet and dry

486 categories defined by WWF.

487

488 Comparing times of interchange

489 To test the differences among wet- versus dry-habitat species in their age of

490 interchange between continents we perform a permutation test in R (R Core Team, 2017).

491 This technique creates a null distribution of mean difference in interchange time between

492 two groups of the same size as the original data but whose membership is created by

493 randomly re-assigning values to each of the two categories (Edgington, 1987; Manly,

494 1991). First, we calculated the difference in mean age of interchange from the true data.

495 The difference in means is our test statistic. Values were permuted, i.e., randomly re-

496 assigned to the two categories and the difference of the means is calculated again and the

497 value noted (Edgington, 1987). We performed this procedure 9,999 times to generate the

498 null distribution differences in mean interchange times (Manly, 1991). The test statistic

499 calculated from the original data was then compared to the null distribution, as with any

500 null hypothesis test, to determine the probability that a value equal to or more extreme

501 than the original value of the test statistic could be observed by chance (Edgington, 1987).

502 The permutation test assumed an of 0.05 on a one-tailed test, since we had a clear

503 prediction of the direction of the difference in mean exchange times under the scientific

504 hypothesis. 505

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526 527 RESULTS

528 Taxonomic and geographic sampling

529 For each of the eight genera under study here, we obtained the following numbers

530 of mitochondrial and nuclear sequences reported for the first time here: Lithobates (10

531 16S, 7 COI, 3 POMC, and 2 TYR), Craugastor (30 16S, 26 COI, 19 POMC, and 17 TYR),

532 Leptodactylus (30 16S, 21 COI, 11 POMC, and 8 TYR), Agalychnis (18 16S,17 COI, 5

533 POMC, and 5 TYR), Boana (54 16S, 50 COI, 14 POMC, and 14 TYR), Dendropsophus (49

534 16S, 51 COI, 15 POMC, and 10 TYR), Scinax (44 16S, 39 COI, 20 POMC, and 18 TYR),

535 and Smilisca (9 COI, 15 16S, 11 POMC, and 11 TYR). We complemented these new data

536 with additional sequences from GenBank, as follows: Lithobates (12 16S, 6 COI, 3 POMC,

537 and 5 TYR), Craugastor (3 16S, 1 COI, and 2 TYR), Leptodactylus (14 16S, 3 COI, 4

538 POMC, and 6 TYR), Agalychnis (12 16S, 3 COI, 1 POMC, and 2 TYR), Boana (24 16S, 19

539 COI, 6 POMC, and 7 TYR), Dendropsophus (23 16S, 20 COI, 6 POMC, and 4 TYR),

540 Scinax (33 16S, 12 COI, 2 POMC, and 3 TYR), and Smilisca (6 16S, 3 COI, 3 POMC, and

541 1 TYR). The number of DNA sequences obtained per gene and per genus, and the length

542 per gene of each aligned data matrix, are provided in Table 5. premature stop codons

543 were inferred in any of the found for the protein-coding genes (COI, POMC, and TYR). For

544 both analyses, maximum likelihood and divergence times by Bayesian inference, a

545 partition scheme was obtained by codon for the protein-coding genes. The 16S

546 mitochondrial fragment was analyzed in a block, is a gene coding only for messenger

547 RNA (mRNA). For each of the partitions we use the best evolutionary models based on the 548 Akaike corrected criteria (AICc) (Table 6) obtained from the partition analysis done in

549 PartitionFinder2 (Table 7; Lanfear et al., 2016).

550

551 The goal of the ncDNA data set was to test the validity of phylogenetic inferences

552 based solely on mtDNA gene trees. Because the nuclear data set included many fewer

553 individuals and contained many fewer parsimony-informative sites, phylogenetic analyses

554 were conducted separately for the concatenated mtDNA and the concatenated ncDNA.

555 Because we were interested in phylogenetic information below the level of species, we

556 did not conduct phylogenetic analyses using the multispecies coalescent.

557

558 Phylogenetic analyses

559 The separate ML phylogenetic analyses of the concatenated mtDNA versus ncDNA

560 yielded phylogenetically informative estimates about the evolutionary relationships

561 between the species for each of the eight genera evaluated. However, the degree of

562 resolution and branch support varied widely for each of the 8 genera and each of the two-

563 character matrices. We define as a high node support, bootstrap values greater than 85%.

564 Results based on the mtDNA yielded the highest proportion of well-supported branches in

565 Agalychnis (30 of 22 nodes; Fig. 7), followed by Craugator (32/59; Fig. 5), Scinax (38/70;

566 Fig. 10) Dendropsophus (43/99; Fig. 9), Leptodactylus (25/99; Fig. 6), Boana (38/90; Fig.

567 8), Lithobates (8/25; Fig. 4), and finally Smilisca (9/30; Fig. 11). Phylogenetic results based

568 on concatenated nuclear genes again showed that Agalychnis obtained the best nodal

569 support (4/9; Fig. 15), followed by Boana (9/18; Fig. 16), Dendropsophus (9/22; Fig. 17), 570 Leptodactylus (7/17; Fig. 14), Lithobates (1/6; Fig. 12), Scinax (3/18; Fig. 18), Craugastor

571 (2/10; Fig. 13) and Smilisca (2/1; Fig. 19).

572

573 Bayesian MCMC phylogenetic inference of trees and divergence times made

574 independently for two concatenated data sets, mtDNA versus nuclear DNA, resulted in

575 well-supported phylogenies for most of the genera tested. Analyses of both data sets were

576 run until all parameters achieved an ESS > 200 for all eight genera. The nodes in the

577 Bayesian consensus trees obtained for each genome and each genus achieved, node

578 support based on greater than or equal to 0.945 (0.95) posterior probability for a majority

579 of nodes. For the mtDNA data set, Agalychnis achieved the highest proportion of nodes

580 with high support (26 of 35 nodes; Fig. 23), followed by Craugastor (36/62; Fig. 21),

581 Leptodactylus (42/73; Fig. 22), Smilisca (16/31; Fig. 27), Lithobates (17/27; Fig. 20), Scinax

582 (44/70; Fig. 26), Dendropsophus (54/103; Fig. 25) and Boana (39/96; Fig. 24). For the

583 concatenated nuclear DNA analysis, Lithobates achieved the highest proportion of nodes

584 with high support (7/8; Fig. 28), followed by Agalychnis (5/11; Fig. 31), Smilisca (6/13; Fig.

585 35), Craugastor (3/12; Fig. 29), Leptodactylus (4/18; Fig. 30), Dendropsophus (5/24; Fig.

586 33), Scinax (8/20; Fig. 34) and Boana (8/21; Fig. 32). See below for divergence time results

587 placed in their biogeographic context.

588

589 The differences between mtDNA versus ncDNA datasets might be influenced by

590 the limitless source of independent phylogenetic information (Thornton & Desalle, 2000).

591 Eukaryotic nuclear genomes are composed of multiple chromosomes that undergo 592 recombination, exist genes found on different chromosomes, or even on the same

593 chromosome. If the distance is sufficiently far apart, are effectively evolutionarily

594 independent of each other (Small et al., 2002). This complicated genetic architecture of

595 eukaryotic nuclear genomes carries out possible difficulties in isolating and identifying

596 orthologous genes. Also, the possibilities of concerted evolution and/or recombination

597 among paralogous sequences and the presence of intraspecific and intrapopulation

598 heterozygosity (Henikoff et al., 1997).

599

600 For the genus Lithobates, the results found in the phylogenetic analysis support the

601 position of the species in the phylogeny and rescue the evolutionary relationships between

602 sister species. In this way, our work supports the evolutionary relationships between L.

603 juliani and L. vaillanti as a sister group of the South American clade composed of L. bwana

604 and L. palmipes (Pyron & Wiens, 2011; Yuan et al., 2016).

605

606 In the genus Craugastor the evolutionary relationships within species, are consistent

607 in both analyses, ML and Bayesian MCMC phylogenetic inference. Under these

608 inferences, we found that C. longirostris are the sister species of the other species in the

609 phylogeny. Our results are comparable with the phylogenetic hypothesis for this group

610 provided by (Crawford & Smith, 2005). Our analyses also showed that C. raniformis is the

611 sister species of C. crassidigitus (Crawford & Smith, 2005; Pyron & Wiens, 2011).

612 613 The genus Leptodactylus the results indicate that L. knudseni is the sister species of

614 the rest of species within Leptodactylus. The evolutionary relationships between species L.

615 knudseni, L. pentadactylus and L. savagei, also, L. melanonotus, L. bolivianus, L. latrans, L.

616 colombiensis, and, L. wagneri, L. fragilis, L. longirsotris, L. poecilochilus, and L. fuscus.

617 These evolutionary relationships between species coincide with that found by de Sá et al.

618 (2014). Who used integrative taxonomy analysis, using mitochondrial and morphological

619 data.

620

621 For the genus Agalychnis the ML analysis showed, both in the mtDNA topology

622 and the ncDNA topology, a similar pattern with some differences between the ratio of the

623 closest taxa in the topology with nuclear data (Figs.7 & 15). The relationship between the

624 species is maintained in the topologies by Bayesian MCMC phylogenetic inference (Figs

625 23 & 31). Our results rescue the evolutionary relationships between Agalychnis callydrias

626 and A. terranova proposed by Rivera-Correa et al. (2013). We also rescue the relationship

627 between A. spurrelli and A. saltator (Pyron & Wiens, 2011; Faivovich et al., 2010; Rivera-

628 Correa et al., 2013). However, our results suggest that A. terranova, recently reported for

629 the Colombian Pacific coast (Palacios-Rodríguez et al., 2016), forms a cluster distinct from

630 the individuals of the Magdalena river valley, type locality for this species (Rivera-Correa

631 et al., 2013). What suggests a colonization to the isthmus of Panama, taking into account

632 that this area of South America corresponds to the westernmost part of the IP (Montes et

633 al., 2015).

634 635 For the Boana genus, the results of divergence times and ML are not the same in

636 both types of molecular data. Although the relations between species are maintained in

637 the two topologies, the position of the clades in the phylogeny changes between the

638 mtDNA and ncDNA (Figs. 8 & 16). The group composed of B. pellucens and B. rufitelus

639 are the sister group of the rest of the species of the genus (Pyron & Wiens, 2011; Faivovich

640 et al., 2005). While, in the nuclear phylogeny, this position is occupied by the group

641 composed of B. geographica and B. semilineata. We included two species that previously

642 had not been in previous works (Pyron & Wiens, 2011; Faivovich et al., 2005). One of

643 them is B. pugnax, species more closely related to the clade formed by B. xerophylla and

644 B. crepitans. The other species is B. xerophylla, the latter because it was recently separated

645 from the synonymy of B. crepitans (Orrico et al., 2017). Our results also confirm the

646 relationship between these two species as sister species.

647

648 The results obtained from the phylogenetic analysis of mitochondrial fragments as

649 nuclear for the genus Dendropsophus present consistency between the different

650 topologies. The internal relations between the different topologies suggest D. koechlini

651 and D. parviceps as the sister species of the rest of the members of the genus (Figs. 9 &

652 17). The evolutionary relationships between the species of the genus Dendropsophus, are

653 kept in agreement with the obtained by Pyron & Wiens (2011), we found that the relations

654 between D. koechlieni, D. parviceps, and D. brevifrons are supported by the proposed in

655 this study, as well as the relationship between D. rhodopeplus and D. microcephalus.

656 657 For the genus Scinax, the ML analysis showed differences in the topologies for

658 mitochondrial and nuclear genes. The results for mitochondrial genes locate the clade

659 composed of S. altae and S. staufferi as the sister group of the rest of the species of the

660 genus, while the topology made for nuclear genes infers two main clades, one composed

661 of S. rostratus and S. boulengeri and the other composed by S. cruentomma, S. ruber and

662 S. x-signatus. In turn of this clade diverges another composed by S. wandae, S. altae, S.

663 staufferi and S. elaeochroa (Pyron & Wiens, 2011; Duellman et al., 2016; Faivovich et al.,

664 2005).

665

666 For the genus Smilisca, the evolutionary relationships between the different species

667 remain consistent in the ML analyzes and Bayesian MCMC phylogenetic inference. The

668 different topologies show a pattern congruent with the formation of two large clades, the

669 first consisting of S. sordida and S. sila and the other by S. puma and S. phaeota. The

670 relationships and position of these species for the topologies found in this study support

671 the previously reported evolutionary relationships for the genus (Pyron & Wiens, 2011;

672 Duellman et al., 2016; Faivovich et al., 2005).

673

674 Environmental preferences

675 In the PCA conducted on random points sampled from each four "wet" and “dry”

676 ecosystems, PC1 was defined by the variables CLYPPT, BIO18, BIO12, BIO17, BIO14,

677 BIO19 and BIO3 (Table 4) and explained variation of 46% of the environmental variation

678 in wet ecosystems. These Bioclim variables are, of course, related to precipitation, while 679 CLYPPT indicates high percentage of clay in the soil that best describe the areas selected

680 as "wet" for this analysis (Fig. 36). On the other hand, PC1 also was defined by variables

681 BIO6, BIO9, BIO11, BIO1, BIO8, BIO10 related with temperature, and high levels of

682 CECSOL, describes the “dry” environments. The LDA showed how environmental

683 variables allowed identifying differences between each of the categories of "wet" and "dry"

684 environment (Fig. 37). In this way, the two types of environments analyzed in this study

685 present differences that allow them to be classified as two different environments based on

686 the analysis of the comparison of bioclimatic and soil variables. This allows us to

687 conclude that humid environments are environmentally different from dry environments.

688

689 Once wet and dry ecosystems were established in multivariate, we proceeded to

690 characterize the environmental preferences of each of the 69 species of interest. We used

691 georeferenced points from across the distribution of each species. These points were

692 assigned to each of the environments from the geographic distance they were, thus, the

693 points closest to the areas defined as "dry" environments are classified as species with a

694 preference for this type of environment, as well for "wet" environments. Of the 69 species

695 analyzed, we found that 11 species live in relatively dry environments, 45 species live in

696 humid environments, and the remaining 12 species appeared to be environmental

697 generalists and indeed they have very large geographic distributions (Table 8).

698

699

700 701 Divergence times

702 The genus Lithobates managed to diverge between Central and South America in a

703 single event, where the divergence of L. vaillanti with two species L. bwana and L.

704 palmipes. Results based mitochondrial and nuclear data indicate that L. juliani is the sister

705 group of the species participating in the interchange between continents, the divergence

706 (stem age) between this species and the participating clade, composed of L. vaillanti, L.

707 bwana and L. palmipes occurred between 9.0 Ma (with 95% credibility interval, CI: 0.3--

708 24.5 Ma, mtDNA) and 9.8 Ma (CI: 1.8--49.6 Ma, ncDNA). The diversification in South

709 America (crown age) by L. vaillanti occurred 6.2 Ma (CI: 0.0--15.9 Ma, mtDNA). The

710 origin of the South American clade (L. bwana and L. palmipes) occurred 4.4 Ma (CI: 0.0--

711 10.53 Ma, mtDNA). Finally, L. palmipes managed to radiate in South America between

712 1.91 Ma (CI: 0.1--10.0, mtDNA) according to mitochondrial data and 2.1 Ma (CI: 0.0--

713 20.3 Ma, ncDNA).

714

715 For the genus Craugastor we found five events of divergence, three of these include

716 to C. longirostris. The first occurred at 8.1 Ma (CI: 3.0--35.1 Ma, mtDNA), followed by

717 diversification in South America at 0.1 Ma (CI: 0.0-2.3 Ma, mtDNA), and then the

718 subsequent diversification of Central America at 3.6 Ma (CI: 0.0-9.7 Ma, mtDNA). The

719 second event shows the divergence between C. longirostris at 9.8 Ma (CI: 3.3--24.8 Ma,

720 mtDNA). Followed by diversification in South America at 1.0 Ma (CI: 0.2--5.0 Ma,

721 mtDNA). In the third event, C. longirostris diverged at 6.9 Ma (CI: 0.0--11.1 Ma, mtDNA)

722 with a subsequent diversification in South America at 2.5 Ma (CI: 0.4--12.3 Ma, mtDNA). 723 A fourth divergence event include the species C. raniformis, which showed a divergence

724 at 3.2 Ma (CI: 0.8-12.5 Ma, mtDNA), and 18.4 Ma (CI: 0.0-48.4 Ma, ncDNA). According

725 to ncDNA, the diversification in South America of C. raniformis occurred at 0.25 Ma (CI:

726 0.0-2.8 Ma, ncDNA). Finally, the fifth diversification event was the divergence of C.

727 fitzingeri with C. crassidigitus and C. talamancae. This event occurred at 21.9 Ma (CI:

728 15.1--29.3 Ma, mtDNA), and 8.1 Ma (CI: 0.0--25.49 Ma, ncDNA). The diversification of

729 C. fitzingeri in South America occurred at 11.7 Ma (CI: 7.2--16.8 Ma, mtDNA), and 5.39

730 Ma (CI: 0.0-11.1 Ma, ncDNA).

731

732 The genus Leptodactylus showed six divergence events between South America

733 and Central America (crown age). The first of these events consists of the divergence

734 between L. pentadactylus and L savagei between 4.3 Ma (CI: 0.0--12.6 Ma, mtDNA) and

735 3.1 Ma (CI: 0.0--3.5 Ma, ncDNA), with a subsequent diversification in Central America

736 (steam age) at 1.5 Ma (CI: 0.0--10.9 Ma, mtDNA). In the second event, the divergence

737 between L. latrans and L. melanonotus occurred between 8.7 Ma (CI: 0.7--23.7 Ma,

738 mtDNA) and 2.7 Ma (CI: 0.0-1.7 Ma, ncDNA). In the third event, the divergence between

739 L. colombiensis and L. bolivianus occurred at 5.2 Ma (CI: 0.6--18.8 Ma, mtDNA), and

740 diversification of L. bolivianus in Central America at 0.6 Ma (CI: 0.0--1.1 Ma).

741 Subsequently, occurred a new diversification of South America by L. bolivianus at 1.2 Ma

742 (CI: 0.5--1.9 Ma, mtDNA). The divergence between L. melanonotus and L. colombiensis at

743 2.6 Ma (0.0--3.8 Ma, mtDNA). Later the diversification of L. melanonotus in Central

744 America occurred at 2.0 Ma (CI: 0.0--4.1 Ma, mtDNA). A second divergence event 745 between L. melanonotus and L. wagneri occurred at 0.9 Ma (CI: 0.0--3.9 Ma, ncDNA).

746 The next event consists of the divergence between the clade composed of L. longirostris, L.

747 poecilochilus and L. fuscus with L. fragilis at 14.7 Ma (CI: 0.4--26.4 Ma, mtDNA). The

748 diversification of L. fragilis in Central America occurred at 0.3 Ma (CI: 0.8--3.7 Ma,

749 mtDNA). The last event consists in the divergency between L. poecilochilus and L.

750 longirostris at 9.1 Ma (CI: 5.6--13.0 Ma, mtDNA), the divergence between Central

751 America from South America by the L. poecilochilus occurred at 3.4 Ma (CI: 1.7--5.3 Ma,

752 mtDNA) and finally the diversification in South America occurred at 0.8 Ma (CI: 0.1-8.3

753 Ma, mtDNA). We found disparities between mitochondrial and nuclear data that suggest

754 L. poecilochilus is the sister species of L. fuscus and the divergence between the two

755 species occurred at 1.5 Ma (0.3--1.8 Ma, ncDNA), the diversification of L. poecilochilus in

756 Central America occurred at 0.7 Ma (CI: 0.0--1.1 Ma, ncDNA).

757

758 For the genus Agalychnis we found four divergence events. A first event shows the

759 divergence of A. buckleyi and A. hulli with A. lemur at 11.7 Ma (CI: 3.8--20.3 Ma,

760 mtDNA), and 21.9 Ma (CI: 6.8--33.7 Ma, nc DNA). The diversification of A. lemur in

761 Central America occurred at 2.9 Ma (CI: 0.9--10.3 Ma, mtDNA), and 4.4 Ma (CI: 0.09--

762 31.61 Ma, ncDNA). A second divergence event have occurred by A. spurrelli between

763 South America and Central America at 0.4 Ma (CI: 0.0--5.9 Ma, mtDNA) and 1.2 Ma (CI:

764 0.0--23.6 Ma, ncDNA). The diversification of this linage in Central America occurred at

765 0.2 Ma (CI: 0.0--1.9 Ma, mtDNA). The third event was the divergence between South

766 America and Central America, where A. annae diverged from the clade containing A. 767 terranova and A. callydrias at 7.7 Ma (CI: 0.0--6.48 Ma, mtDNA), and 12.3 Ma (CI: 0.0--

768 28.3 Ma, ncDNA), then later divergence between A. terranova and A. callydrias at 3.3 Ma

769 (CI: 0.2--5.8 Ma, mtDNA), and 4.45 Ma (CI: 0--23.48 Ma, ncDNA) originating the

770 diversification of A. callydrias in Central America 1.8 Ma ( CI: 0.2--6.6 Ma, mtDNA)

771 according to the mitochondrial estimation.

772

773 For the Boana the first event consists of the divergence between B. pellucens and B.

774 rufitela at 8.5 Ma (CI: 2.8--23.6 Ma, mtDNA), then the subsequent diversification of B.

775 rufitela in Central America at 7.4 Ma (CI: 0.2--16.8 Ma, mtDNA). The results obtained

776 from nuclear data indicate different divergence events for this clade, estimating an age of

777 10.1 Ma (CI: 2.9-41.5 Ma, ncDNA). The second event consists of the divergence between

778 B. boans and the clade composed of B. semilineata and B. geographica at 9.0 Ma (CI: 3.6-

779 -21.8 Ma, mtDNA), and 21.2 Ma (0.0--40.5 Ma, ncDNA). The radiation of B. boans in

780 Central America occurred at 2.0 Ma (CI: 1.6--17.8 Ma, mtDNA). we found a second

781 diversification event in South America by this species at 0.9 Ma (CI: 0.0--4.5 Ma, mtDNA).

782 For B. rosenbergi, we found that this species diverged at 16.5 Ma ago (CI: 7.4--32.8 Ma,

783 mtDNA), with a diversification date in Central America at 7.1 Ma (CI: 1.6--21.1 Ma,

784 mtDNA), and diversification in South America at 2.67 Ma (CI: 0.3--10.8 Ma, mtDNA), and

785 a second event of diversification in Central America at 1.9 Ma (CI: 0.0--8.2 Ma, mtDNA).

786 In the next event, the divergence of B. pugnax from the clade composed of B. crepitans

787 and B. xerophylla occurred at 14.0 Ma (CI: 6.2--26.9 Ma, mtDNA), followed by a

788 diversification in Central America at 2.9 Ma (CI: 0.3--12.0 Ma, mtDNA) and a 789 diversification in South America with the subsequent at 3.4 Ma (CI: 0.9--14.4 Ma,

790 mtDNA). The last event showed was the divergence between B. xerophylla and B.

791 crepitans at 11.3 Ma (CI: 0.0--14.6 Ma, mtDNA) and 14.7 Ma (CI: 0.0--20.7 Ma, ncDNA),

792 diversification of B. xerophylla in Central America at 2.0 Ma (CI: 0.3--6.6 Ma, mtDNA),

793 and 1.37 Ma (CI: 0.07--18.57 Ma, ncDNA) according to the nuclear analysis.

794

795 For the genus Dendropsophus we found three different divergence events between

796 Central America and South America. The first was the divergence between D. subocularis

797 and D. ebraccatus at 4.7 Ma (CI: 1.8--9.3 Ma, mtDNA), and 8.7 Ma (CI: 0.0--26.7 Ma,

798 ncDNA). Following this event, D. ebraccatus diversified in Central America at 2.9 Ma (CI:

799 1.2--6.2 Ma, mtDNA) and 4.7 Ma (CI: 0.0--18.2 Ma, ncDNA). Subsequently, the

800 diversification in South America occurred at 1.4 Ma (CI: 0.4--2.3 Ma, mtDNA) and 3.1 Ma

801 (CI: 0.0--11.3 Ma, ncDNA). The second event showed the divergence between D.

802 phlebodes and D. rhodopeplus at 7.6 Ma (CI: 5.2--10.1 Ma, mtDNA), followed by the

803 diversification in Central America of D. phlebodes at 1.4 Ma (CI: 0.0-2.5 Ma, mtDNA) and

804 4.1 Ma (CI: 0.0-29.3 Ma, ncDNA). The third event involves the divergence between D.

805 microcephalus and D. rhodopeplus at 7.6 Ma (CI: 5.2--10.1 Ma, mtDNA) and the

806 diversification in Central America at 3.0 Ma (CI: 1.8--4.2 Ma, mtDNA), and 5.3 Ma (CI:

807 0.0--17.8 Ma, ncDNA). Finally, we found that this species diversified in South America at

808 0.5 Ma (CI: 0.3--0.8 Ma, mtDNA).

809 810 For the genus Scinax the first divergence event was between S. nebulosus and S.

811 boulengeri at 13.1 Ma (CI: 0.7--57.7 Ma, mtDNa), and the diversification in Central

812 America at 9.2 Ma (CI: 0.1--30.8 Ma, mtDNA), and 29.4 Ma (0.8--45.0 Ma, ncDNA). S.

813 boulengeri diversified in Central America at 5.4 Ma (CI: 0.3--19.71 Ma, mtDNA), and 8.2

814 Ma (CI: 1.8-48.3 Ma, ncDNA). The second event was the divergence of S. elaeochroa at

815 9.7 Ma (CI: 0.1--19.8 Ma, mtDNA), and 14.8 Ma (CI: 0.0--26.8 Ma, ncDNA), followed by

816 the diversification in Central America between 3.8 Ma (CI: 0.3--16.3 Ma, mtDNA), and

817 3.1 Ma (CI: 0.3--30.5 Ma, ncDNA). The third event showed the origin in Central America

818 of S. staufferi and S. altae from the divergence with S. cruentomma and S. wandae from

819 South America. This event occurred at 33.7 Ma (CI: 0.3--35.6 Ma, mtDNA), and 17.8 Ma

820 (CI: 0.0-33.3 Ma, ncDNA). The divergence between S. staufferi and S. altae in Central

821 America occurred at 2.4 Ma (0.4--10.8 Ma, mtDNA) and 5.7 Ma (CI: 0.0--40.6 Ma,

822 ncDNA). The last colonization event found for this genus is the divergence of S. ruber in

823 Central America at 3.7 Ma (CI: 0.06-9.63 Ma, mtDNA), and the diversification in Central

824 America at 0.9 Ma (CI: 0.0--4.9 Ma, mtDNA), and 27.5 Ma (CI: 0.0-27.5 Ma, ncDNA).

825 The mitochondrial analysis shows an event of diversification event in South America

826 occurred at 2.1 Ma (CI: 0.1-6.9 Ma, mtDNA).

827

828 Finally, for the genus Smilisca, we found two divergence events between Central

829 America and south America. In the first event was the divergence between S. sordida and

830 S. sila occurred at 39.5 Ma (CI: 0.0-31.9 Ma, mtDNA), and 38.0 Ma (CI: 0.5-81.7 Ma,

831 ncDNA). While the diversification of S. sila in South America was estimated at 8.5 Ma (CI: 832 0.3-79.5 Ma, mtDNA), and 11.6 Ma (CI: 0.2--63.1 Ma, ncDNA). The second divergence

833 participated S. puma and S. phaeota, the divergence between the species occurred at 6.5

834 Ma (CI: 0.0-68.4 Ma, mtDNA), and 21.2 Ma (CI: 0.0-45.4 Ma, ncDNA). The

835 diversification of S. phaeota in South America occurred at 3.4 Ma (CI: 0.1-15.3 Ma,

836 mtDNA), and 10.9 Ma (CI: 0.0-55.3 Ma, ncDNA). The diversification in South America

837 occurred at 1.73 Ma (CI: 0-21.52 Ma, mtDNA), and 6.58 Ma (CI: 0.0-33.3 Ma, ncDNA).

838

839 Interchange time patterns

840 The results obtained from the analysis of the difference between the diversification

841 time means of the participating species that belonged to “wet” or “dry” environments, it

842 tells us that there are no significant differences between these two categories (Pvalue =

843 0.0075). This indicates that the times of diversification of the participating species

844 between continents follow a statistically significant temporal pattern. The environments to

845 which the species correspond to establish a barrier to the dispersion of the species. We

846 make the comparison of these two categories with the species without preference for one

847 of the two (“both”), we find that there are no differences between the times of

848 diversification of these species with those that show a clear preference for any of the

849 environments (“dry” vs “both”: Pvalue = 0.42, “wet” vs “both”: Pvalue = 0.84).

850

851

852

853 854 DISCUSSION

855 One of the goals of biogeography is understand how geographical and

856 environmental processes could affect diversification. Inside this, comparative

857 phylogeography allow us to identify the historical processes that may have influenced

858 populations and communities from ecological or demographic forces acting on single

859 evolutionary lineages or species (Bermingham & Moritz, 1998a; Crawford et al., 2007).

860 This study attempted to recover the biogeographic history of species that participated in

861 the faunal interchange between South and Central America once the closure of the IP

862 occurred. The patterns inferred from the phylogenetic and divergence time analyses in

863 eight different genera of frogs are strikingly consistent to determine the evolutionary

864 relationships within species, the age of the divergences and diversification events between

865 species from Central America and South America. Our results support different events of

866 interchange in a different period of time.

867

868 This study attempted to recover the phylogenetic history of species that participated

869 in the faunal exchange between South America and Central America once the closure of

870 the IP occurred. The patterns inferred from the phylogenetic and divergence time analyses

871 in eight different genera of frogs are strikingly consistent to determine the evolutionary

872 relationships within species and the age of the divergences and diversification events

873 between species from Central America and South America. Our results support different

874 events of interchange, in a different period of time, but these events seem to do not have a

875 relation with the hypothesis about the existence origin of a biological barrier for the 876 species with a strong dependence of environmental conditions. The effectiveness of

877 barriers to prevent dispersal depends not only on the nature of the environment but also

878 on the characteristics of the organisms themselves (Paz et al., 2015). These features vary

879 among taxonomic groups, so the barriers may not affect all members of biota equally

880 (Bernardo et al., 2007). Corridors are areas where members of the local biota can

881 circumvent barriers, allowing the movement of species from one region to another

882 (Panzacchi et al., 2016). Therefore, a corridor permits the formation of taxonomically

883 balanced assemblages of organisms between otherwise distinct regions. The difference

884 between biotas on either end the corridor results from ecological and evolutionary

885 responses to contrasting environments (Paz et al., 2015).

886

887 Mitochondrial versus nuclear

888 As genetic markers we chose mitochondrial and nuclear loci. With this

889 information, we inferred the stem ages and crown ages of lineages participating in the

890 GABI (broad sense). We use both types of genomic sequences to make separate analyzes

891 because mitochondrial protein-coding genes have much faster mutation rates than nuclear

892 protein-coding genes (Vawter & Brown, 1986; Crawford, 2003). Therefore, the use of

893 mitochondrial information for the reconstruction of the evolutionary history between

894 species has been more widely used from mitochondrial genetic information (Wiens, 1998;

895 Crawford et al., 2013). Nuclear genes are recommended for the reconstruction of

896 phylogenetic relationships between higher-level taxa since divergences between groups

897 are usually older, which is not possible to effectively trace using mitochondrial 898 information (Springer et al., 2001). A second difference is that the mitochondrial genome

899 is inherited as a single, haploid, non-recombinant binding unit. This haploid mode of

900 inheritance leads to a smaller effective population size compared to the nuclear genome

901 (Springer et al., 2001). In turn, a less effective population size results in a shorter expected

902 coalescence time for the mitochondrial loci compared to the nuclear loci and a higher

903 probability that the mitochondrial gene tree accurately reflects the species tree for the

904 nodes closely spaced compared to nuclear gene trees (Moore et al., 2005). However, we

905 decided to incorporate nuclear genetic information in order to find a common pattern in

906 the divergence between species from the comparison between the two types of analysis.

907 We do not perform a combined analysis between both types of genomic information

908 because this may sacrifice accuracy in the part of the tree affected by the different

909 genomic histories (Wiens, 1998).

910

911 Biogeographic history

912 Our hypothesis about the delayed in the GABI due the existence of a humid biome

913 that acted like environmental barrier, while it was restricting the pass for species of dry

914 biomes, but it was allowing the interchange between continents of species of humid

915 biomes. This hypothesis is supported with our phylogenetics and divergence time analysis.

916 Our results suggest that occurred 32 interchange events between South and North

917 America (Table 9). Due the uncertainty that surrounded the dates of nodes in Bayesian

918 trees, we interpreted both the stem age how the earliest time of migration, and the crown

919 age how the latest age of colonization (Cody et al., 2010). 24 of these events occurred in 920 South America direction to Central America. 8 events occurred in Central America

921 direction to South America. Pyron (2014) in his analysis of the reconstruction of ancestral

922 areas for tropical amphibians, estimated the area of origin of several of the genera

923 included in this study. According to the results of their analysis, we find that genera such

924 as Agalychnis, Boana, Dendropsophus, Leptodactylus, and Scinax have an origin in South

925 America, which corroborates our inference. Smilisca and Craugastor are genera that

926 originated in South America but colonized Central America through the Proto-Antilles

927 model between the Cretaceous-Paleocene. These groups subsequently recolonized South

928 America via the IP (Crawford & Smith, 2005; Pyron, 2014). So, we treat this event in

929 particular as a secondary colonization of South America. Lithobates seems to have

930 expanded to South America through the Isthmus of Panama, from the Nearctic (Pyron,

931 2014).

932

933 In general terms, the divergence times of the species participating in the GABI

934 support the Old Isthmus model (Montes et al., 2012). Where once formed the IP during

935 the Miocene, this formed a land bridge between Central America and South America that

936 allowed the colonization of these continents in both directions, contrary to the

937 reconstructions that preferred the hypothesis of dispersions on water (Heinicke et al.,

938 2007). The Old Isthmus model (Montes et al., 2012) has been supported in other works

939 based on the analysis of divergence times with plants (Bacon et al., 2013; Poelchau &

940 Hamrick, 2013), arthropods (Zeh et al., 2003; Winston et al., 2017), fishes (Bermingham &

941 Moritz, 1998b; Picq et al., 2014), snakes (Daza et al., 2009, 2010), freshwater turtles 942 (Iverson et al., 2013), birds (Weir et al., 2009; Smith & Klicka, 2010; Ornelas et al., 2014),

943 salamanders (Elmer et al., 2013), and others frogs of the genus Pristimantis (Pinto-Sánchez

944 et al., 2012).

945

946 Traditionally it has been estimated that the age of closure of the IP occurred at 4.0--

947 3.0 Ma, taking as the greatest source of evidence the GABI (Coates & Stallard, 2013).

948 However, as we have found in the results of this study in addition to the other sources of

949 evidence mentioned above, the colonization of Central America by organisms other than

950 the GABI mammals occurred before the date established by the New Isthmus model. In

951 this study we tested Bacon et al. (2016) hypothesis, who proposed that there was a delay

952 of about 10 Ma in GABI of mammals given the existence of a humid forest barrier for

953 these species that, according to previous studies, they lived in dry environments such as

954 the neotropical savannas. We evaluated the environmental preferences of different species

955 of dry and wet forest anurans, we found a pattern in which the GABI participating species

956 with a preference for dry environments managed to colonize Central America after the

957 date proposed by the Young Isthmus model of 3.5 Ma. However, although there is some

958 evidence of the existence of a tropical humid forest environment in the IP since the

959 Miocene (Jaramillo et al., 2014). It is not the same to support the origin of the dry forest in

960 Central America (Retallack & Kirby, 2007). This study gives an idea that there might have

961 been a dry forest 3 million years ago. So, it is important to analyze this type of variables in

962 other organisms to see if a common pattern can be rescued that allows reconstructing with

963 greater precision the environmental conditions in the Isthmus of Panama. 964

965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

981

982

983

984

985 986 Acknowledgements

987 We thank to Luis Mosquera, Pablo Palacio and Jonard Echavarria for help collecting

988 specimens. Specimens were collected under the project “Diversidad criptica de anfibios y

989 reptiles de Colombia” P.R.6.2015.2182, movilization permit number P02182S207_N0030

990 granted by Autoridad Nacional de Licencias Ambientales (ANLA). Financial support was

991 provided by the Departamento Administrativo de Ciencia, Tecnología e Innovación

992 (Colciencias), Programa Nacional en Ciencias Básicas, award 120456934310 to A.J.C.

993 and by a Proyecto Semilla grant from the Facultad de Ciencias, Universidad de los Andes.

994 To Juan M. Daza, Vivian P. Paez and Brian C. Bock from Grupo Herpetologico de

995 Antioquia (GHA), and Museo de Herpetologia de la Universidad de Antioquia (MHUA) in

996 the adquisition of frog tissues. Angelica Cajamarca for their help analyzing samples in

997 laboratory. To Andres Lopez-Rubio from Universidad de Antioquia in the analysis of

998 sequences. We thank to biom|ics lab members specially to Santiago Herrera-Alvarez,

999 Luisa Castellanos, Erika Rodriguez, Daniela Garcia, Valentina Muñoz, Juan Pablo

1000 Ramirez, Andrea Paz and Lucas Barrientos. From EvolVert lab to Laura Cespedes, Catalina

1001 Palacio, Maria Alejandra Meneses, from EcoFiv lab to Fabian Salgado, from CIMPAT to

1002 Juan Carlos Santos and David Andres Ayala-Usma for the invaluable help during the

1003 research process and their friendship.

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1381 Biology, 64, 267–280. 1382 1 Table 1. Taxonomic sampling of species participants and non-participants in the Great American Biotic Interchange (GABI).

2 The selection criteria of each species for this study was based in the phylogenetic relationships among species published that

3 includes species distributed from Central America to South America and their sister groups that does not cross between

4 continents.

Family Species Status Lineage References Ranidae Lithobates bwana (Hillis and de Sá, 1988) Participant Northern Yuan et al., 2016 Non- Ranidae Lithobates juliani (Hillis and de Sá, 1988) participant Northern Yuan et al., 2016 Ranidae Lithobates palmipes (Spix, 1824) Participant Northern Yuan et al., 2016 Ranidae Lithobates vaillanti (Brocchi, 1877) Participant Northern Yuan et al., 2016 Non- Ranidae Lithobates warszewitschii (Schmidt, 1857) participant Northern Yuan et al., 2016 Non- Craugastoridae Craugastor crassidigitus (Taylor, 1952) participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Craugastoridae Craugastor fitzingeri (Schmidt, 1857) Participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Craugastoridae Craugastor longirostris (Boulenger, 1898) Participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Craugastoridae Craugastor raniformis (Boulenger, 1896) Participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Non- Craugastoridae Craugastor tabasarae (Savage et al., 2004) participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Non- Craugastoridae Craugastor talamancae (Dunn, 1931) participant Northern Pyron & Wiens, 2014; Crawford & Smith, 2005 Phyllomedusidae Agalychnis annae Duellman (1963) Participant Southern Rivera-Correa et al., 2013 Non- Phyllomedusidae Agalychnis buckleyi (Boulenger, 1882) participant Southern This study Phyllomedusidae Agalychnis callidryas (Cope, 1862) Participant Southern Rivera-Correa et al., 2013 Non- Phyllomedusidae Agalychnis hulli (Duellman & Mendelson, 1995) participant Southern Rivera-Correa et al., 2013 Phyllomedusidae Agalychnis lemur (Boulenger, 1882) Participant Southern Rivera-Correa et al., 2013 Phyllomedusidae Agalychnis saltator Taylor, 1955 Participant Southern Rivera-Correa et al., 2013 Phyllomedusidae Agalychnis spurrelli Boulenger, 1913 Participant Southern Rivera-Correa et al., 2013 Phyllomedusidae Agalychnis terranova Rivera-Correa et al., 2013 Participant Southern Rivera-Correa et al., 2013 Hylidae Boana boans (Linnaeus, 1758) Participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana crepitans (Wied-Neuwied, 1824) participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana faber (Wied-Neuwied, 1821) participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana geographica (Spix, 1824) participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana lundii (Burmeister, 1856) participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana pardalis (Spix, 1824) participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana pellucens (Werner, 1901) participant Southern Pyron, 2011; Faivovich et al., 2005 Hylidae Boana pugnax (Schmidt, 1857) Participant Southern This study Hylidae Boana rosenbergi (Boulenger, 1898) Participant Southern Pyron, 2011; Faivovich et al., 2005 Hylidae Boana rufitela (Fouquette, 1961) Participant Southern Pyron, 2011; Faivovich et al., 2005 Non- Hylidae Boana semilineata (Spix, 1824) participant Southern Pyron, 2011; Faivovich et al., 2005 Hylidae Boana xerophylla (Duméril & Bibron, 1841) Participant Southern Pyron, 2011; Faivovich et al., 2005 Hylidae Scinax altae (Dunn, 1933) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Scinax boulengeri (Cope, 1887) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax cruentomma (Duellman, 1972) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Scinax elaeochrous (Cope, 1875) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax kennedyi (Pyburn, 1973) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax nebulosus (Spix, 1824) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Scinax rostratus (Peters, 1863) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Scinax ruber (Laurenti, 1768) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax squalirostris (Lutz, 1925) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Scinax staufferi (Cope, 1865) Participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax wandae (Pyburn & Fouquette, 1971) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Scinax x-signatus (Spix, 1824) participant Southern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Dendropsophus brevifrons (Duellman & Crump, 1974) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Hylidae Dendropsophus ebraccatus (Cope, 1874) Participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus koechlini (Duellman & Trueb, 1989) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus mathiassoni (Cochran & Goin, 1970) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Hylidae Dendropsophus microcephalus (Cope, 1886) Participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus parviceps (Boulenger, 1882) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Hylidae Dendropsophus phlebodes (Stejneger, 1906) Participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus rhodopeplus (Günther, 1858) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus sarayacuensis (Shreve, 1935) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Hylidae Dendropsophus subocularis (Dunn, 1934) Participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Non- Hylidae Dendropsophus triangulum (Günther, 1869) participant Southern Pyron & Wiens 2014; Wiens et al., 2010 Hylidae Smilisca phaeota (Cope, 1862) Participant Northern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Smilisca puma (Cope, 1885) participant Northern Pyron & Wiens 2014; Faivovich et al., 2005 Hylidae Smilisca sila Duellman and Trueb, 1966 Participant Northern Pyron & Wiens 2014; Faivovich et al., 2005 Non- Hylidae Smilisca sordida (Peters, 1863) participant Northern Pyron & Wiens 2014; Faivovich et al., 2005 Leptodactylidae Leptodactylus bolivianus Boulenger, 1898 Participant Southern de Sá et al., 2014 Leptodactylidae Leptodactylus fragilis (Brocchi, 1877) Participant Southern de Sá et al., 2014 Leptodactylidae Leptodactylus fuscus (Schneider, 1799) Participant Southern de Sá et al., 2014 Non- Leptodactylidae Leptodactylus knudseni Heyer, 1972 participant Southern de Sá et al., 2014 Non- Leptodactylidae Leptodactylus latrans (Steffen, 1815) participant Southern de Sá et al., 2014 Non- Leptodactylidae leptodactylus longirostris Boulenger, 1882 participant Southern de Sá et al., 2014 Leptodactylidae Leptodactylus melanonotus (Hallowell, 1861) Participant Southern de Sá et al., 2014 Non- Leptodactylidae Leptodactylus pentadactylus (Laurenti, 1768) participant Southern de Sá et al., 2014 Leptodactylidae Leptodactylus poecilochilus (Cope, 1862) Participant Southern de Sá et al., 2014 Leptodactylidae Leptodactylus savagei Heyer, 2005 Participant Southern de Sá et al., 2014 Non- Leptodactylidae Leptodactylus wagneri (Peters, 1862) participant Southern de Sá et al., 2014 5

6

7

8

9

10

11

12

13

14

15

16

17

18

19 20 Table 2. Secondary calibration dates with the 95% confidence intervals used in the calibration point with the outgroups for

21 analyses with mitochondrial and nuclear data.

Calibration point Estimated time (My) 95% CI Source Outgroup Ingroup Phyllomedusa Agalychnis 24.5 20.0 - 29.7 Feng et al. (2017) Aplastodiscus Boana 25.2 19.6 - 31.0 Feng et al. (2017) Haddadus Craugastor 48.5 43.2 - 53.7 Feng et al. (2017) Pseudis Dendropsophus 35.3 30.6 - 40.2 Feng et al. (2017) Lithodytes Leptodactylus 36.2 29.6 - 42.5 Feng et al. (2017) Rana Lithobates 22.2 19.5 - 25.3 Feng et al. (2017) Scarthyla Scinax 58.5 55.5 - 66.1 Kumar et al. (2017) 22

23

24

25

26

27

28

29

30

31

32 33 Table 3. Bioclimatic variables using in the habitat preferences analyses.

Code Bioclimatic variable BIO1 Annual Mean Temperature BIO2 Mean Diurnal Range BIO3 Isothermality BIO4 Temperature Seasonality BIO5 Max Temperature of Warmest Month BIO6 Min Temperature of Coldest Month BIO7 Temperature Annual Range BIO8 Mean Temperature of Wettest Quarter BIO9 Mean Temperature of Driest Quarter BIO10 Mean Temperature of Warmest Quarter BIO11 Mean Temperature of Coldest Quarter BIO12 Annual Precipitation BIO13 Precipitation of Wettest Month BIO14 Precipitation of Driest Month BIO15 Precipitation Seasonality BIO16 Precipitation of Wettest Quarter BIO17 Precipitation of Driest Quarter BIO18 Precipitation of Warmest Quarter BIO19 Precipitation of Coldest Quarter 34

35

36

37

38 39 Table 4. Best partitions scheme of mitochondrial and nuclear for each genus.

Genus Scheme lnL AICc Parameters Sites (bp) Partitions Agalychnis Cope, 1864 Mitochondrial -5770.08 11763.77 102 1175 4 Nuclear -1546.44 3179.19 41 842 4 Boana Gray, 1825 Mitochondrial -10964.00 22478.76 224 1206 4 Nuclear -3076.33 6311.11 74 1138 4 Craugastor Cope, 1862 Mitochondrial -9343.62 19048.98 157 1197 4 Nuclear -1832.91 3767.06 48 949 4 Dendropsophus Fitzinger, 1843 Mitochondrial -12351.22 25296.64 240 1254 4 Nuclear -2378.72 4915.23 73 990 4 Leptodactylus Fitzinger, 1826 Mitochondrial -11072.39 22562.71 179 1255 4 Nuclear -2404.65 4948.31 65 1018 4 Lithobates Fitzinger, 1843 Mitochondrial -4875.84 9918.43 78 1226 4 Nuclear -1261.68 2596.90 35 748 4 Scinax Wagler, 1830 Mitochondrial -11739.46 23900.45 180 1240 4 Nuclear -2373.70 4901.90 71 890 5 Smilisca Cope, 1865 Mitochondrial -4639.82 9473.82 90 1246 4 Nuclear -1797.89 3699.55 49 899 4 40

41

42

43

44

45 46 Table 5. Best fit model selection in the partition analyses with PartitionFinder2. These models were used in the analyses of

47 divergence times by Bayesian inference in Beast2.

Genus Scheme Partition names Best-fit model Sites (bp) Agalychnis Cope, 1864 Mitochondrial 16S-1 TRN 522 COI-1 HKY + I 218 COI-2 TRN 218 COI-3 TRN 217 Nuclear POMC -1, 2, TYR -3 TRN 432 POMC-3 HKY 152 TYR-1 JC 129 TYR-2 HKY 129 Boana Gray, 1825 Mitochondrial 16S-1 GTR + I + � 546 COI-1 TRN + I + � 220 COI-2 HKY + I 220 COI-3 TRN + � 220 Nuclear POMC-1 TRN 202 POMC-2, 3 HKY 404 TYR-1 TRN 178 TYR-2, 3 TRN 354 Craugastor Cope, 1862 Mitochondrial 16S-1 TRN + � 575 COI-1 TRN + � 208 COI-2 HKY + I 207 COI-3 TRN + � 207 Nuclear POMC-1, TYR-3 HKY + I + � 316 POMC-2, TYR-1 HKY 317 POMC-3 TRN 165 TYR-2 TRN 151 Dendropsophus Fitzinger, 1843 Mitochondrial 16S-1 TRN 579 COI-1 TRN + I + � 225 COI-2 TRN 225 COI-3 TRN + I + � 225 Nuclear POMC-1 HKY + � 153 POMC-2, TYR-2 TRN 330 POMC-3, TYR-3 HKY + I 329 TYR-1 HKY + � 178 Leptodactylus Fitzinger, 1826 Mitochondrial 16S-1 TRN 597 COI-1 GTR 220 COI-2 TRN + I 219 COI-3 HKY + I 219 Nuclear POMC-1, TYR-1 TRN 340 POMC-2, TYR-2 HKY + � 339 POMC-3 TRN + � 160 TYR-3 HKY + � 179 Lithobates Fitzinger, 1843 Mitochondrial 16S-1 GTR + � 555 COI-1 TRN + I 224 COI-2 HKY 224 COI-3 TRN + � 223 Nuclear POMC-1 HKY 97 POMC-2, 3 HKY 194 TYR-1, 2 HKY + I 305 POMC-3 HKY 152 Scinax Wagler, 1830 Mitochondrial 16S-1 TRN 584 COI-1 TRN 219 COI-2 TRN + I + � 219 COI-3 TRN + � 218 Nuclear POMC-1 TRN 133 POMC-2, TYR-1 TRN 297 POMC-3 HKY 132 TYR-2 HKY + � 164 TYR-3 HKY 164 Smilisca Cope, 1865 Mitochondrial 16S-1 TRN 578 COI-1 TRN + I 223 COI-2 JC + I 223 COI-3 TRN 222 Nuclear POMC-1, TYR-1 HKY 300 POMC-2 HKY 144 POMC-3, TYR-3 TRN 299 TYR-2 HKY + � 156 48

49

50

51

52

53

54

55

56

57

58

59 60 Table 6. lengths of the different fragments of mitochondrial and nuclear DNA product of sequencing alignment using MAFFT.

Number of DNA fragments (bp) Number of DNA fragments (bp) Genus tips tips 16S COI POMC TYR Agalychnis Cope, 1864 37 522 652 12 456 385 Boana Gray, 1825 101 546 659 22 606 531 Craugastor Cope, 1862 63 575 621 13 497 451 Dendropsophus Fitzinger, 1843 103 579 674 25 548 531 Leptodactylus Fitzinger, 1826 75 597 657 20 481 536 Lithobates Fitzinger, 1843 28 555 670 9 291 456 Scinax Wagler, 1830 74 584 655 21 397 492 Smilisca Cope, 1865 32 578 667 14 432 466 61

62

63

64

65

66

67

68

69

70

71

72 73 Table 7. predictions of environmental preferences for each of the species included in this study.

Family Species Status Prediction dry Prediction wet Classification Ranidae Lithobates bwana (Hillis and de Sá, 1988) Participant 1.00 0.00 dry Ranidae Lithobates juliani (Hillis and de Sá, 1988) Non-participant 0.00 1.00 wet Ranidae Lithobates palmipes (Spix, 1824) Participant 0.14 0.86 wet Ranidae Lithobates vaillanti (Brocchi, 1877) Participant 0.19 0.81 wet Ranidae Lithobates warszewitschii (Schmidt, 1857) Non-participant 0.35 0.65 wet Craugastoridae Craugastor crassidigitus (Taylor, 1952) Non-participant 0.29 0.71 wet Craugastoridae Craugastor fitzingeri (Schmidt, 1857) Participant 0.14 0.86 wet Craugastoridae Craugastor longirostris (Boulenger, 1898) Participant 0.03 0.97 wet Craugastoridae Craugastor raniformis (Boulenger, 1896) Participant 0.05 0.95 wet Craugastoridae Craugastor tabasarae (Savage et al., 2004) Non-participant 1.00 0.00 dry Craugastoridae Craugastor talamancae (Dunn, 1931) Non-participant 0.04 0.96 wet Phyllomedusidae Agalychnis annae Duellman (1963) Participant 0.55 0.45 both Phyllomedusidae Agalychnis buckleyi (Boulenger, 1882) Non-participant 0.00 1.00 wet Phyllomedusidae Agalychnis callidryas (Cope, 1862) Participant 0.15 0.85 wet Phyllomedusidae Agalychnis hulli (Duellman & Mendelson, 1995) Non-participant 0.00 1.00 wet Phyllomedusidae Agalychnis lemur (Boulenger, 1882) Participant 0.50 0.50 both Phyllomedusidae Agalychnis saltator Taylor, 1955 Participant 0.05 0.95 wet Phyllomedusidae Agalychnis spurrelli Boulenger, 1913 Participant 0.08 0.92 wet Phyllomedusidae Agalychnis terranova Rivera-Correa et al., 2013 Participant 0.00 1.00 wet Hylidae Boana boans (Linnaeus, 1758) Participant 0.05 0.95 wet Hylidae Boana crepitans (Wied-Neuwied, 1824) Non-participant 0.66 0.34 dry Hylidae Boana faber (Wied-Neuwied, 1821) Non-participant 1.00 0.00 dry Hylidae Boana geographica (Spix, 1824) Non-participant 0.20 0.80 wet Hylidae Boana lundii (Burmeister, 1856) Non-participant 1.00 0.00 dry Hylidae Boana pardalis (Spix, 1824) Non-participant 1.00 0.00 dry Hylidae Boana pellucens (Werner, 1901) Non-participant 0.12 0.88 wet Hylidae Boana pugnax (Schmidt, 1857) Participant 0.38 0.62 wet Hylidae Boana rosenbergi (Boulenger, 1898) Participant 0.07 0.93 wet Hylidae Boana rufitela (Fouquette, 1961) Participant 0.29 0.71 both Hylidae Boana semilineata (Spix, 1824) Non-participant 0.00 1.00 wet Hylidae Boana xerophylla (Duméril & Bibron, 1841) Participant 0.67 0.33 dry Hylidae Scinax altae (Dunn, 1933) Participant 0.75 0.25 dry Hylidae Scinax boulengeri (Cope, 1887) Participant 0.06 0.94 wet Hylidae Scinax cruentomma (Duellman, 1972) Non-participant 0.00 1.00 wet Hylidae Scinax elaeochrous (Cope, 1875) Participant 0.08 0.92 wet Hylidae Scinax kennedyi (Pyburn, 1973) Non-participant 0.00 1.00 wet Hylidae Scinax nebulosus (Spix, 1824) Non-participant 0.06 0.94 wet Hylidae Scinax rostratus (Peters, 1863) Participant 0.20 0.80 wet Hylidae Scinax ruber (Laurenti, 1768) Participant 0.24 0.76 wet Hylidae Scinax squalirostris (Lutz, 1925) Non-participant 1.00 0.00 dry Hylidae Scinax staufferi (Cope, 1865) Participant 0.49 0.51 both Hylidae Scinax wandae (Pyburn & Fouquette, 1971) Non-participant 0.09 0.91 wet Hylidae Scinax x-signatus (Spix, 1824) Non-participant 0.51 0.49 both Hylidae Dendropsophus brevifrons (Duellman & Crump, 1974) Non-participant 0.00 1.00 wet Hylidae Dendropsophus ebraccatus (Cope, 1874) Participant 0.12 0.88 wet Hylidae Dendropsophus koechlini (Duellman & Trueb, 1989) Non-participant 0.00 1.00 wet Hylidae Dendropsophus mathiassoni (Cochran & Goin, 1970) Non-participant 0.08 0.92 wet Hylidae Dendropsophus microcephalus (Cope, 1886) Participant 0.26 0.74 both Hylidae Dendropsophus parviceps (Boulenger, 1882) Non-participant 0.02 0.98 wet Hylidae Dendropsophus phlebodes (Stejneger, 1906) Participant 0.00 1.00 wet Hylidae Dendropsophus rhodopeplus (Günther, 1858) Non-participant 0.03 0.97 wet Hylidae Dendropsophus sarayacuensis (Shreve, 1935) Non-participant 0.00 1.00 wet Hylidae Dendropsophus subocularis (Dunn, 1934) Participant 0.17 0.83 wet Hylidae Dendropsophus triangulum (Günther, 1869) Non-participant 0.00 1.00 wet Hylidae Smilisca phaeota (Cope, 1862) Participant 0.12 0.88 wet Hylidae Smilisca puma (Cope, 1885) Non-participant 0.00 1.00 wet Hylidae Smilisca sila Duellman and Trueb, 1966 Participant 0.47 0.53 both Hylidae Smilisca sordida (Peters, 1863) Non-participant 0.32 0.68 both Leptodactylidae Leptodactylus bolivianus Boulenger, 1898 Participant 0.33 0.67 both Leptodactylidae Leptodactylus fragilis (Brocchi, 1877) Participant 0.49 0.51 both Leptodactylidae Leptodactylus fuscus (Schneider, 1799) Participant 0.52 0.48 both Leptodactylidae Leptodactylus knudseni Heyer, 1972 Non-participant 0.06 0.94 wet Leptodactylidae Leptodactylus latrans (Steffen, 1815) Non-participant 0.77 0.23 dry Leptodactylidae leptodactylus longirostris Boulenger, 1882 Non-participant 0.00 1.00 wet Leptodactylidae Leptodactylus melanonotus (Hallowell, 1861) Participant 0.63 0.37 dry Leptodactylidae Leptodactylus pentadactylus (Laurenti, 1768) Non-participant 0.00 1.00 wet Leptodactylidae Leptodactylus poecilochilus (Cope, 1862) Participant 0.46 0.54 both Leptodactylidae Leptodactylus savagei Heyer, 2005 Participant 0.12 0.88 wet Leptodactylidae Leptodactylus wagneri (Peters, 1862) Non-participant 0.01 0.99 wet 74 A B C D

E F G H

1 2 3 Figure 1. Geographical distribution of genera Lithobates (A), Craugastor (B), Leptodactylus 4 (C), Agalychnis (D), Boana (E), Dendropsophus (F), Scinax (G), and Smilisca (H). In gray is 5 the geographical range of the species of each genus. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Figure 2. Spatial location of the proposed boundaries between Central and South America. 31 The suture more to the west is the Uramita fault and more to the east is the Panama Canal 32 basin. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

51 52 Figure 3. Ecoregions selected as wet closed-canopy and semi-arid open environments to 53 assignation of distributional points for each species to categorize each species in its 54 environment of preference. In yellow corresponds to dry biomes, and green to wet 55 biomes. 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 Rana_luteiventris

Pichi_354_Lithobates_CR_Cartago 100 AMNH_A_118801_Lithobates_palmipes_VE_Amazonas

AJC_1798_Lithobates_warszewit_PA_Panama

100 CH_6145_Lithobates_warszewit_PA_Bocas_Changuinola

89 KRL_1567_Lithobates_warszewit_PA_Cocle_Cope 100 AJC_1387_Lithobates_warszewit_PA_Panama_SanCarlos 96 TNHC_60324_Lithobates_juliani_BZ_Cayo_Vaqueros 64 Pichi_357_Lithobates_vaillanti_CR_Alajuela

CH_6554_Lithobates_vaillanti_PA_Bocas_Changuinola 41

AJC_3541_Lithobates_vaillanti_CO_Tolima_Mariquita 82 23 MHUA_A_4880_Lithobates_vaillanti_CO_Antioquia_Maceo

QCAZ_13964_Lithobates_bwana_EC_Loja_Zapotillo

57 MHNLS_17185_Lithobates_palmipes_VE_Zulia

CFBHT_10214_Lithobates_palmipes_BR_MatoGrosso_AltaFloresta 46

42 MNCN_ADN_50843_Lithobates_palmipes_BR_MatoGrosso_Juruena 31 56 TG_204_Lithobates_palmipes_BR_MatoGrosso_Paranaita

29 CFBHT_07835_Lithobates_palmipes_BR_Alagoas_CampoAlegre

90 90 CFBHT_12435_Lithobates_palmipes_BR_RioGrandeDoNorte_TibauDoSul 94 CFBHT_11134_Lithobates_palmipes_BR_Paraiba_JoaoPessoa

AJC_3266_Lithobates_palmipes_VE_DeltaAmacuro 60 AMNH_A_166454_Lithobates_palmipes_GY_PotaroSiparuni

18 AJC_2308_Lithobates_palmipes_CO_Casanare_Orocue

CFBHT_07601_Lithobates_palmipes_BR_Acre_CruzeiroDoSul28

39KU_202896_Lithobates_palmipes_EC_Napo_Misahualli

41 AJC_5473_Lithobates_palmipes_CO_Caqueta_Valparaiso 45 JMP_1615_Lithobates_palmipes_PE_Loreto

72 0.2 73 74 Figure 4. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 75 (mtDNA) for the species of the genus Lithobates. Numbers in nodes correspond to 76 bootstrap support values. In blue species from South America, in red species from North 77 America. 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 CFBHT_12711_Haddadus_binotatus_BR_EspiritoSanto_MimosoDoSol 100 CFBHT_13706_Haddadus_binotatus_BR_RioDeJaneiro_SantaMariaMadalena 76 CFBHT_12658_Haddadus_binotatus_BR_SantaCatarina CH_6513_Craugastor_longirostris_PA_Bocas_Changuinola 97 KRL_1399_Craugastor_aff_longirostris_PA_Cocle_Cope 92 AJC_1955_Craugastor_aff_longirostris_PA_Panama_Chagres 99 AJC_1939_Craugastor_aff_longirostris_PA_Panama_Chepo 98 AJC_2116_Craugastor_longirostris_CO_Choco_Tacarcuna 100 AJC_2125_Craugastor_longirostris_CO_Choco_Tacarcuna 88 CH_6390_Craugastor_longirostris_PA_Darien_Pinogana 63 CH_9040_Craugastor_longirostris_PA_Darien 100 CH_9068_Craugastor_longirostris_PA_Darien_Garachine AB_10_Craugastor_melanostictus_PA_Chiriqui_Naranjos 97 100 Pichi_393_Craugastor_melanostictus_CR_Limon_Talamanca 18 EVACC_024_Craugastor_tabasarae_PA_Panama_Chame 99 CH_6055_Craugastor_tabasarae_PA_Panama_Chepo 42 KRL_0706_Craugastor_tabasarae_PA_Cocle_Cope 67 EVACC_219_Craugastor_tabasarae_PA_Panama_Valle_RioMaria AJC_1201_Craugastor_crassidigitus_PA_Panama_Chame 93 22 AJC_1598_Craugastor_crassidigitus_PA_Darien_Chepigana AJC_1731_Craugastor_crassidigitus_PA_Bocas_Changuinola 30 MVZ_207248_Craugastor_crassidigitus_CR_Puntarenas_Monteverde AJC_1719_Craugastor_talamancae_PA_Bocas_Changuinola 100 AJC_1907_Craugastor_talamancae_PA_Panama_Chepo 98 73 KRL_1087_Craugastor_talamancae_PA_Cocle_Cope 100 KRL_1520_Craugastor_talamancae_PA_Cocle_Cope MHUA_A_4305_Craugastor_fitzingeri_CO_Choco_Nuqui 100 26 MHUA_A_4306_Craugastor_fitzingeri_CO_Choco_Nuqui 85 MHUA_A_4883_Craugastor_fitzingeri_CO_Choco_Nuqui 91 43 CHOCO019_15_Craugastor_fitzingeri_LAR_0005_CO_Choco 100AJC_7036_Craugastor_cf_fitzingeri_CO_Choco_UnPanam 77 100 LAR_0005_Craugastor_fitzingeri_CO_Choco_Quibdo CH_9012_Craugastor_fitzingeri_PA_Darien_Garachine KRL_0693_Craugastor_fitzingeri_PA_Cocle_Cope 75 100AJC_1774_Craugastor_fitzingeri_PA_Panama_Chilibre 14 Pichi_070_Craugastor_fitzingeri_CR_Puntarenas 58 45 CH_8438_Craugastor_fitzingeri_PA_Chiriqui Pichi_173_Craugastor_fitzingeri_CR_Puntarenas_Naranjito 26 40 CH_6152_Craugastor_fitzingeri_PA_Bocas_Changuinola 98Pichi_068_Craugastor_fitzingeri_CR_Alajuela_Gavilan 74 Pichi_067_Craugastor_fitzingeri_CR_Guanacaste_Murcielago AJC_2079_Craugastor_longirostris_CO_Valle_Buenaventura 59 AJC_4279_Craugastor_longirostris_CO_Narino_Tumaco 99 KU_177803_Craugastor_longirostris_EC_Pichincha_SantoDomingo 100 LAR_0010_Craugastor_longirostris_CO_Choco_Quibdo 92 AJC_2092_Craugastor_longirostris_CO_Choco_Arusi 82 AJC_1193_Craugastor_longirostris_CO_Choco_Arusi AJC_1199_Craugastor_aff_longirostris_CO_Tolima_Mariquita MHUA_A_7286_16S_Craugastor_raniformis_CO_Antioquia_Maceo32 71 7 MHUA_A_7259_16S_Craugastor_raniformis_CO_Antioquia_SanCarlos 100 AJC_1336_Craugastor_longirostris_CO_Antioquia_Maceo 50MHUA_A_7259_Craugastor_raniformis_CO_Antioquia_SanCarlos MHUA_A_7286_Craugastor_raniformis_CO_Antioquia_Maceo20 17 AJC_3885_Craugastor_longirostris_CO_Santander_Sabana 100 LAR_0007_Craugastor_raniformis_CO_Choco_Quibdo 100LAR_0006_Craugastor_fitzingeri_CO_Choco_Quibdo AJC_2080_Craugastor_raniformis_CO_Valle_Cali87 84 AJC_2095_Craugastor_raniformis_CO_Choco_Nuqui 99 AJC_1515_Craugastor_raniformis_PA_Panama_Chepo 87 AJC_1612_Craugastor_raniformis_PA_Darien_Chepigana 99 CH_6074_Craugastor_raniformis_PA_Panama_IslaCana 62MHUA_A_6769_Craugastor_longirostris_CO_Antioquia_Chigorodo 98 MHUA_A_6766_Craugastor_longirostris_CO_Antioquia_Chigorodo

94 0.3 95 96 Figure 5. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 97 (mtDNA) for the species of the genus Craugastor, numbers in nodes correspond to 98 bootstrap support values. In blue species from South America, in red species from North 99 America. 100 101 102 103 104 105 106 107 108 109 AJC_3451_Lithodytes_lineatus_CO_Meta_Arama

AJC_0953_Leptodactylus_melanonotus_CR_Alajuela_VolcanArenal

KRL_1114_Leptodactylus_insularum_PA_Cocle_Cope 57 AJC_3509_Leptodactylus_insularum_CO_Santander_SanVicente

AJC_4133_Leptodactylus_fuscus_CO_Cordoba_Montelibano32 83 51MUJ_2187_Leptodactylus_bolivianus_CO_Bolivar_Cartagena

81JDL_24887_Leptodactylus_bolivianus_CO_Sucre_SanMarcos 2563 ULABG_5112_Leptodactylus_bolivianus_VE_Merida_RioLimones

CH_4956_Leptodactylus_insularum_PA_Panama_Uveros

CFBHT_10542_Leptodactylus_latrans_BR_RiodeJaneiro_Niteroi 7 100 CFBHT_00832_Leptodactylus_latrans_BR_MinasGerais_Camanducaia

28 AJC_5035_Leptodactylus_colombiensis_CO_Atlantico_Tubara

62 AJC_3978_Leptodactylus_colombiensis_CO_Boyaca_Miraflores AJC_5784_Leptodactylus_melanonotus_CO_Boyaca_SantaMaria 69 LSUNZ_H_12885_Leptodactylus_wagneri_EC 28 100 USNM_320988_Leptodactylus_wagneri_EC_Pastaza_Coca

42 JSM_178_Leptodactylus_colombiensis_CO_Huila_Garzon

AJC_6198_Leptodactylus_melanonotus_CO_Antioquia_Anori 30 98 AJC_3848_Leptodactylus_colombiensis_CO_Santander_PteNal 18 USNM_535964_Leptodactylus_melanonotus_BZ_Cayo_SanJacinto

31 AJC_0858_Leptodactylus_melanonotus_PA_Cocle_Anton 77 AJC_1149_Leptodactylus_melanonotus_PA_Panama_SanCarlos

AMNH_20102_Leptodactylus_pentadactylus_BR_Roraima_Florencia 63 PK_1234_Leptodactylus_knudseni_GY_PotaroSiparuni 99 AJC_3429_Leptodactylus_knudseni_16S_CO_Casanare_Orocue 100 MHNLS_15590_Leptodactylus_knudseni_VE_Bolivar_Purumay

100 CFBH_39775_Leptodactylus_pentadactylus_BR_MatoGrosso_Paranaita

CORBIDI_0024_Leptodactylus_pentadactylus_EC_Sucumbios_GarzaCocha 100 AJC_4761_Leptodactylus_pentadactylus_CO_Amazonas_Leticia 57 83 CH_6585_Leptodactylus_savagei_PA_Bocas_Changuinola

94USNM_298079_Leptodactylus_savagei_PA_Bocas_IslaPopa 85 33 USNM_534219_Leptodactylus_savagei_HN_Colon_Machin

MHUA_A_7387_Leptodactylus_savagei_CO_Bolivar_Norosi

AJC_6581_Leptodactylus_savagei_CO_Magdalena_SantaMarta 74 98 AJC_6545_Leptodactylus_savagei_CO_Magdalena_SantaMarta 84 KRL_0838_Leptodactylus_savagei_PA_Cocle_Cope

97AJC_1502_Leptodactylus_savagei_PA_Panama_Chepo 46 82 AJC_1809_Leptodactylus_savagei_PA_Darien_Pinogana

AJC_2231_Leptodactylus_fuscus_16S_CO_Casanare_Palmarito

91AJC_2301_Leptodactylus_fuscus_16S_CO_Casanare_Orucue

62AJC_4078_Leptodactylus_fuscus_16S_CO_Casanare_Sabanalarga 56 95 AJC_3467_Leptodactylus_fuscus_16S_CO_Meta_Arama

JSM_169_Leptodactylus_fuscus_CO_Magdalena_Granada

70AJC_3892_Leptodactylus_fuscus_16S_CO_Santander_SabanaDeTorres

63AJC_4153_Leptodactylus_fuscus_CO_Cordoba_Montelibano

62JSM_206_Leptodactylus_fuscus_CO_Huila_Garzon 62 AJC_4619_Leptodactylus_fuscus_CO_Antioquia_Pto_Berrio

92 76bm_Leptodactylus_longirostris_FG_MontArawa 34 ROM_20591_Leptodactylus_longirostris_GY_PotaroSiparuni_Kaieteur 89 103AF_Leptodactylus_longirostris_SU_Para_Apura 96 199mc_Leptodactylus_longirostris_FG_GrandSanti

KRL_0678_Leptodactylus_fragilis_PA_Cocle_Cope

AJC_1523_Leptodactylus_fragilis_PA_Panama_Chepo 100 43 AJC_4684_Leptodactylus_fragilis_CO_Antioquia_Yondo 63 JSM_201_Leptodactylus_fragilis_CO_Huila_Garzon28 11 AJC_4476_Leptodactylus_fragilis_CO_Magdalena_Pinuelas 99 JSM_262_Leptodactylus_fragilis_CO_Bolivar_Baru 49 AJC_4664_Leptodactylus_fragilis_CO_Antioquia_Yondo 54 45 AJC_4628_Leptodactylus_fragilis_CO_Antioquia_PtoBerrio 53 AJC_2294_Leptodactylus_fragilis_CO_Tolima_Mariquita

PK_1507_Leptodactylus_longirostris_GY_PotaroSiparuni

100 531AF_Leptodactylus_longirostris_FG_Nouragues_Inselberg 64 MHNLS_17323_Leptodactylus_longirostris_VE_Bolivar_Uayaraca

77 JSM_0011_Leptodactylus_poecilochilus_CO_NorteSantander_Bochalema 93 AJC_4871_Leptodactylus_poecilochilus_CO_NorteSantander_Patios

CH_9077_Leptodactylus_poecilochilus_PA_Darien_Garachine 96 90 KRL_0886_Leptodactylus_poecilochilus_PA_Veraguas_SantaFe

86 JSM_227_Leptodactylus_poecilochilus_CO_Bolivar_Zambrano 94 JSM_172_Leptodactylus_poecilochilus_16S_CO_Magdalena_Granada 100 CH_6373_Leptodactylus_poecilochilus_PA_Darien_Pinogana 100 AJC_1813_Leptodactylus_poecilochilus_PA_Darien_Pinogana

110 0.3 111 112 Figure 6. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 113 (mtDNA) for the species of the genus Leptodactylus, numbers in nodes correspond to 114 bootstrap support values. In blue species from South America, in red species from North 115 America. 116 117 118 CFBHT_12869_Hylomantis_aspera_BR_Bahia_Ilheus

CFBHT00392_Hylomantis_granulosa_BR_Pernambuco_Jaqueira

Pichi_259_Agalychnis_saltator_CR_Puntarenas_Bandera 99 Pichi_258_Agalychnis_lemur_CR_Limon_Asuncion

100 CH_6779_Agalychnis_lemur_PA_Panama_Chilibre

95 KRL_0940_Agalychnis_lemur_PA_Cocle_Cope 76 toe_134_Agalychnis_lemur_PA_Cocle_Cope 65

LSB_135_Agalychnis_buckleyi_CO_Antioquia_Yarumal 100 LSB_134_Agalychnis_buckleyi_CO_Antioquia_Yarumal

86 MZUTI_313_Agalychnis_hulli_EC_Pastaza

100MZUTI_170_Agalychnis_hulli_EC_Pastaza

62 MZUTI_169_Agalychnis_hulli_EC_Pastaza

100 34 MZUTI_168_Agalychnis_hulli_EC_Pastaza

QCAZ_13217_Agalychnis_spurrelli_EC_Esmeraldas_Durango

97AJC_7022_Agalychnis_spurrelli_CO_Choco_UnPanam

73 Pichi_261_Agalychnis_spurrelli_CR_Limon_Guayacan 56 Pichi_262_Agalychnis_spurrelli_CR_SanJose_Carara

100 MVZ_203768_Agalychnis_saltator_CR_Heredia_Sarapiqui

100 Pichi_260_Agalychnis_saltator_CR_Alajuela_SanCarlos 87 JMR_609_Agalychnis_saltator_CR_Heredia_LaSelva

59 Pichi_251_Agalychnis_annae_CR_Heredia_Roble

MHUA_L_0198_2_Agalychnis_terranova_CO_Antioquia_Sonson 83 MHUA_A_7316_Agalychnis_terranova_CO_Antioquia_Sonson 82 100 AJC_3510_Agalychnis_terranova_CO_Santander_SanVicente 99 AJC_3515_Agalychnis_terranova_CO_Santander_SanVicente

100 AJC_2086_Agalychnis_CO_Choco_Nuqui

100 COLZOOCH_H_2679_Agalychnis_terranova_CO_Choco_UnPanam 73 AJC_2087_Agalychnis_CO_Choco_Nuqui

84 Pichi_253_Agalychnis_callidryas_CR_SanJose_Carara

RdS_537_Agalychnis_callidryas_BZ_StannCreek 6812 AJC_1735_Agalychnis_callidryas_PA_Bocas_Changuinola

48Pichi_254_Agalychnis_callidryas_CR_Limon_Talamanca

KRL_0917_Agalychnis_callidryas_PA_Cocle_Cope 32 89 AJC_1768_Agalychnis_callidryas_PA_Panama_Chilibre 83 AJC_1565_Agalychnis_callidryas_PA_Panama_Chepo 89 AJC_1595_Agalychnis_callidryas_PA_Darien_Chepigana

119 0.2 120 121 Figure 7. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 122 (mtDNA) for the species of the genus Agalychnis, numbers in nodes correspond to 123 bootstrap support values. In blue species from South America, in red species from North 124 America. 125 126 127 128 129 130 131 132 133 CFBHT_13076_Apl_leucopygius_BR_RioDeJaneiro_NovaFriburgo

CFBHT_14484_Apl_arildae_BR_RioDeJaneiro_Itatiaia

AJC_2553_Boana_nympha_CO_Amazonas_Leticia

23680_Boana_pellucens_EC_Guayas_Huaquillas 100 30594_Boana_pellucens_EC_ElOro_Avanzada

100 AJC_7043_Boana_rufitelus_CO_Choco_UnPanam 100 AJC_7040_Boana_rufitelus_CO_Choco_UnPanam 91 AJC_0877_Boana_rufitelus_PA_BocasDelToro_IslaPopa

96 AJC_1099_Boana_rufitelus_PA_Colon_Gatun 92 KRL_0798_Boana_rufitelus_PA_Cocle_Cope 65 51 AJC_1382_Boana_rufitelus_PA_Panama_Chame

11A_Boana_semilineatus_BR_EspiritoSanto_RioBananal

AJC_2476_Boana_boans_CO_Guainia_PtColombia 52 MHNLS_19727_Boana_geographicus_VE_Bolivar_Suapure 99 KM390786_1_Boana_semilineata_BR 52 99 97 MNKA_9497_Boana_geographicus_BO_StaCruz_Tucumancito

99 MZUSP_157060_Boana_geographicus_BR_Amazonas_Tefe

MZUSP_157090_Boana_geographicus_BR_Acre_SerraDoDivisor64 22 CORBIDI_5253_Boana_geographicus_PE_MadreDios_PtoMaldonado 60 73 MZUSP_157083_Boana_geographicus_BR_Amazonas_Purus

AJC_3443_Boana_boans_CO_Meta_SanJuan

MHNLS_19685_Boana_boans_VE_Bolivar_Suapure

87 88AMNH_20098_Boana_boans_BR_Roraima_Rorainopolis 37 AJC_3268_Boana_boans_VE_DeltaAmacuro 89 43 PK_1129_Boana_boans_GY_PotaroSiparuni

CORBIDI_5173_Boana_boans_PE_MadreDios_PtoMaldonado

AJC_2329_Boana_boans_CO_Vaupes_Mitu 33 90 AJC_2541_Boana_boans_CO_Amazonas_Leticia 40 45 MTR_ALCX69P42_Boana_boans_BR_Amazonas_NovoAripuana

60 CH_6332_Boana_boans_PA_Darien_Pinogana

93AJC_1051_Boana_boans_PA_Panama_Chepo 63 AJC_1375_Boana_boans_CO_Tolima_Falan 98 AJC_1320_Boana_boans_CO_Tolima_Mariquita

TG_368_Boana_faber_BR_SaoPaulo_PedroToledo

100 CFBHT_15407_Boana_faber_BR_SaoPaulo_Nazare 66 CFBHT_04381_Boana_faber_BR_RioDeJaneiro_Petropolis 100 CFBHT_06588_Boana_faber_BR_SaoPaulo_SaoLuis

85 CFBHT_09065_Boana_pardalis_BR_EspiritoSanto_SantaLeopoldina

CFBHT_04366_Boana_pardalis_BR_RioDeJaneiro_Petropolis96 98 CFBHT_14367_Boana_pardalis_BR_SaoPaulo_SaoJose 90 79 CFBHT_01279_Boana_pardalis_BR_SaoPaulo_SaoLuis

CFBHT_12635_Boana_lundii_BR_Brasilia_DF

100CFBHT_02106_Boana_lundii_BR_Goias_AltoParaiso 77 CFBHT_01902_Boana_lundii_BR_MinasGerais_SaoRoque 87 CFBHT_12573_Boana_lundii_BR_SaoPaulo_SaoCarlos

Pichi_281_Boana_rosenbergi_CR_Puntarenas_Quepos 100 69 BSUCR283_12_Boana_rosenbergi_Pichi_283_CR_Puntarenas_AguaBuena 100 MHUA_A_7797_Boana_rosenbergi_CO_Antioquia_Chigorodo

100 CH_9082_Boana_rosenbergi_PA_Darien_Garachine

97CH_6337_Boana_rosenbergi_PA_Darien_Pinogana 79 CH_8792_Boana_rosenbergi_PA_Colon_Gamboa 70 AJC_1509_Boana_rosenbergi_PA_Panama_Chepo

MHNLS_16827_Boana_pugnax_VE_Zulia_Derote

CH_8501_Boana_pugnax_PA_Veraguas 100 JMR_5959_Boana_pugnax_CO_Sucre_SanOnofre 85 96 AJC_4161_Boana_xerophylla_CO_Santander_Guapota 9981 AJC_4149_Boana_sp_CO_Cordoba_Montelibano 79 AJC_4135_Boana_sp_CO_Cordoba_Montelibano 78 AJC_5138_Boana_pugnax_CO_Cesar_Bosconia

AJC_3870_Boana_pugnax_CO_Santander

814 AJC_2345_Boana_xerophylla_CO_Guajira_Dibulla 78 AJC_2341_Boana_sp_CO_Guajira_Dibulla 75 67AJC_2344_Boana_sp_CO_Guajira_Dibulla

73 AJC_4627_Boana_pugnax_CO_Antioquia_PtoBerrio

47 MHUA_A_7285_Boana_xerophylla_CO_Antioquia_Maceo

AJC_3870_Boana_pugnax_CO_SAntioquiaander_SabanaTorres 53 89 AJC_4686_Boana_xerophylla_CO_Antioquia_Yondo 11 AJC_3501_Boana_pugnax_CO_Santander_SanVicente 17 AJC_4614_Boana_pugnax_CO_Antioquia_PtoBerrio

CFBHT_02360_Boana_crepitans_BR_Minas_GraoMogol 100 CFBHT_12841_Boana_crepitans_BR_Bahia_Camamu 78 CFBHT_07825_Boana_xerophylla_BR_Alagoas_CampoAlegre

98 INPA_H_31229_Boana_xerophylla_BR_Roraima_Rorainopolis AJC_2857_Boana_xerophylla_VE_Amazonas_PtoAyacucho

JSM_181_Boana_xerophylla_CO_Huila_Garzon 99 97JSM_183_Boana_xerophylla_CO_Huila_Garzon

52AJC_4341_Boana_xerophylla_CO_Cundinamarca_Nilo

AJC_2253_Boana_xerophylla_CO_Cundinamarca_Guaduas 71 8920 AJC_3765_Boana_pugnax_CO_Cundinamarca_Nilo 50 AJC_4341_Boana_crepitans_CO_Cundinamarca

48AJC_1323_Boana_crepitans_CO_Tolima_Mariquita 52 AJC_1323_Boana_crepitans_CO_Mariquita 95 34 AJC_1316_Boana_xerophylla_CO_Tolima_Mariquita

AJC_1738_Boana_xerophylla_CO_Meta_Arama

96AJC_4108_Boana_crepitans_CO_Casanare

AJC_4014_Boana_xerophylla_CO_Boyaca_Miraflores44

8AJC_4108_Boana_xerophylla_CO_Casanare_Sabanalarga

15AJC_4563_Boana_xerophylla_CO_Meta_Restrepo 9 89 AJC_4010_Boana_xerophylla_CO_Boyaca_Miraflores 26 AJC_4300_Boana_xerophylla_CO_Meta_SanMartin

MHNLS_17218_Boana_xerophylla_VE_Merida_AndresBello

AJC_1078_Boana_xerophylla_PA_Panama_Buenavista 49 100 AJC_1096_Boana_xerophylla_PA_Panama_Pacora 76 AJC_1123_Boana_xerophylla_PA_Panama_Cumbres 77 AJC_3843_Boana_xerophylla_CO_Santander_PtNal

100AJC_3505_Boana_xerophylla_CO_Santander_SanVicente 83 AJC_3394_Boana_cf_xerophylla_CO_Santander_PuenteNal 75 AAV_151_Boana_xerophylla_CO_Santander_Piedecuesta

134 0.2 135 136 Figure 8. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 137 (mtDNA) for the species of the genus Boana, numbers in nodes correspond to bootstrap 138 support values. In blue species from South America, in red species from North America. 139 140 141 142 AJC_2382_Pseudis_paradoxa CORBIDI_5217_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado 100 CORBIDI_5235_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado MHUA_A_6585_Dendropsophus_subocularis_CO_Antioquia_SanRoque 92 MJH_7101_Dendropsophus_brevifrons_PE_Huanuco_PtoInca 41 76 APL_16416_Dendropsophus_brevifrons_BR_Rondonia_PortoVelho 57 QCAZA_18174_Dendropsophus_brevifrons_EC_Napo_JatunSacha 88 QCAZA_55848_Dendropsophus_brevifrons_EC_Sucumbios_Lorocachi 52 84 QCAZA_17826_Dendropsophus_brevifrons_EC_Orellana_Yasuni 99 QCAZA_28273_Dendropsophus_brevifrons_EC_Sucumbios_Cuyabeno LSB_331_Dendropsophus_subocularis_CO_Antioquia_SanRafael

37 APL_16749_Dendropsophus_parviceps_BR_Rondonia_PortoVelho QCAZ_52752_Dendropsophus_parviceps_EC_Pastaza_Sarayacu 100 100 QCAZ_52026_Dendropsophus_parviceps_EC_Tungurahua_RioNegro 92 QCAZ_51195_Dendropsophus_parviceps_EC_Orellana_Yasuni 89QCAZ_48919_Dendropsophus_parviceps_EC_Napo_Sumaco 70 QCAZ_39515_Dendropsophus_parviceps_EC_Pastaza_Coconaco QCAZA_32637_Dendropsophus_sarayacuensis_EC_MoronaSantiago_NueveOctubre 99 QCAZA_36697_Dendropsophus_sarayacuensis_EC_Napo_Toachi 100 QCAZA_17429_Dendropsophus_sarayacuensis_EC_Pastaza_Fatima 100 QCAZA_23030_Dendropsophus_sarayacuensis_EC_Tungurahua_Banos 99 QCAZA_44826_Dendropsophus_triangulum_EC_Orellana_RioNapo 100 CORBIDI_12194_Dendropsophus_triangulum_PE_Loreto_Requena 65 SCF_012_Dendropsophus_triangulum_PE_Ucayali 100 ZONIAAF_MTR19029_Dendropsophus_triangulum_BR_Amazonas 100 98 WED_54094_Dendropsophus_triangulum_EC AJC_3837_Dendropsophus_subocularis_CO_Santander_PteNal 100 AJC_3845_Dendropsophus_subocularis_CO_Santander_PteNacional Pichi_267_Dendropsophus_ebraccatus_CR_Puntarenas_Golfito 99 100 CH_6627_Dendropsophus_ebraccatus_PA_Bocas_Changuinola 78 AJC_0406_Dendropsophus_ebraccatus_CR_Limon_Pacurita 99 AJC_0951_Dendropsophus_ebraccatus_CR_Alajuela_Arenal 92 CH_9321_Dendropsophus_ebraccatus_PA_Darien_RioChucunaque 93AJC_1790_Dendropsophus_ebraccatus_PA_Panama_Chilibre CH_6644_Dendropsophus_ebraccatus_PA_Panama_Chilibre62 52 91 AJC_1118_Dendropsophus_ebraccatus_PA_Colon_Gamboa CH_6335_Dendropsophus_ebraccatus_PA_Darien_Pinogana 97 LSB_323_Dendropsophus_ebraccatus_CO_Antiquia_SanCarlos 52 93 AJC_1338_Dendropsophus_ebraccatus_CO_Antioquia_Maceo 100 AJC_3504_Dendropsophus_ebraccatus_CO_Santander_SanVicente 99 AJC_3502_Dendropsophus_ebraccatus_CO_Santander_SanVicente AJC_2883_Dendropsophus_minusculus_VE_Amazonas_PtoAyacucho 100 AJC_2322_Dendropsophus_mathiassoni_CO_Casanare_Orocue 81 AJC_1740_Dendropsophus_mathiassoni_CO_Meta_Arama 33AJC_4052_Dendropsophus_mathiassoni_CO_Casanare_Sabanalarga 55 LSB_363_Dendropsophus_mathiassoni_CO_Meta_Villavicencio AJC_2728_Dendropsophus_rhodopeplus_PE_SanMartin_Tarapoto APL_16847_Dendropsophus_rhodopeplus_BR_Rondonia_PortoVelho 100 89 67 IDLAR_4665_Dendropsophus_rhodopeplus_PE_Cuzco_SanLorenzo 79 KST_0542_Dendropsophus_rhodopeplus_PE_MadreDeDios_Manu 89 AJC_3571_Dendropsophus_rhodopeplus_CO_Amazonas_Tanimboca 100 JMP_2003_Dendropsophus_rhodopeplus_CO_Amazonas_Leticia 100 QCAZA_44584_Dendropsophus_rhodopeplus_EC_Orellana_Huiririma QCAZA_44328_Dendropsophus_rhodopeplus_EC_Orellana_Chiroisla100 62 94 QCAZA_44329_Dendropsophus_rhodopeplus_EC_Orellana_Chiroisla COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_UnPanam 100 COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_UnPanam__reversed_ 87 AJC_1788_Dendropsophus_phlebodes_PA_Panama_Chilibre 94 CH_6806_Dendropsophus_phlebodes_PA_Panama_Chilibre MHNLS_17186_Dendropsophus_microcephalus_VE_Zulia_Tocuco MHNLS_17186_Dendropsophus_microcephalus_VE 88 100 76 MHNLS_18951_Dendropsophus_microcephalus_VE_Yaracuy_Nirgua 61MNCN_ADN_59473_Dendropsophus_microcephalus_VE_Anzoategui_Independencia EBEF_4_Dendropsophus_microcephalus_VE_Apure_ElFrio61 60MHNLS_17736_Dendropsophus_microcephalus_VE_DeltaAmacuro 76 AJC_3177_Dendropsophus_microcephalus_VE_Bolivar_PedroChien 100 AJC_0954_Dendropsophus_microcephalus_CR_Alajuela_Arenal 100 ENS_10627_Dendropsophus_microcephalus_HN_Paraiso_Chichicaste 100 Pichi_273_Dendropsophus_microcephalus_NG_RioFrio_Corra Pichi_272_Dendropsophus_microcephalus_CR_Puntarenas_AguaBuena 92 CH_5683_Dendropsophus_microcephalus_PA_Chiriqui_David 80 AJC_1507_Dendropsophus_microcephalus_PA_Panama_Chepo AJC_1514_Dendropsophus_microcephalus_PA_Panama_Chepo 9224CH_9066_Dendropsophus_phlebodes_PA_Darien_Garachine 69 100 AJC_3804_Dendropsophus_phlebodes_PA_Darien_Garachine 59 54AJC_3805_Dendropsophus_phlebodes_PA_Darien_Garachine AJC_0994_Dendropsophus_microcephalus_PA_Panama_LagoBayano 26 AJC_1791_Dendropsophus_microcephalus_PA_Panama_Chilibre 56 AJC_0899_Dendropsophus_microcephalus_PA_Colon_Galeta 96 AJC_1098_Dendropsophus_microcephalus_PA_Panama_Naranjal49 71 AJC_1117_Dendropsophus_microcephalus_PA_Cocle_Cope AJC_1049_Dendropsophus_microcephalus_CO_Antioquia_Maceo 38 AJC_3561_Dendropsophus_microcephalus_CO_Tolima_Mariquita AJC_4838_Dendropsophus_microcephalus_CO_Atlantico_Tubara 56 81AJC_5766_Dendropsophus_microcephalus_CO_Sucre_SanMarcos 38 AJC_5224_Dendropsophus_microcephalus_CO_Tolima_Armero 28AJC_5354_Dendropsophus_microcephalus_CO_Huila_Neiva 51 36AJC_5337_Dendropsophus_microcephalus_CO_Tolima_Coello MHUA_A_7174_Dendropsophus_microcephalus_CO_Antioquia_GomezPlata 4 AJC_4613a_Dendropsophus_microcephalus_CO_Antioquia_PtoBerrio 1AJC_4633_Dendropsophus_microcephalus_CO_Antioquia_PtoBerrio AJC_5746_Dendropsophus_microcephalus_CO_Santander_Barrancabermeja 2 AJC_4433_Dendropsophus_microcephalus_CO_Magdalena_Pinuelas 74AJC_5741_Dendropsophus_microcephalus_CO_Antioquia_Yondo 9 JMR_5987_Dendropsophus_microcephalus_CO_Bolivar_MariaLaBaja 21 5AJC_2352_Dendropsophus_microcephalus_CO_Guajira_Dibulla AJC_2254_Dendropsophus_microcephalus_CO_Cundinamarca_Guaduas 10AJC_4035_Dendropsophus_microcephalus_CO_Santander_SanVicente 5AJC_3869_Dendropsophus_microcephalus_CO_Santander_Sabana 10 AJC_2349_Dendropsophus_microcephalus_CO_Guajira_Dibulla

143 0.2 144 145 Figure 9. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 146 (mtDNA) for the species of the genus Dendropsophus, numbers in nodes correspond to 147 bootstrap support values. In blue species from South America, in red species from North 148 America. 149 150 151 152 153 154 QULC_2340_Sca_goinorum_BR CFBHT_16443_Sca_vigilans_VE CFBHT_10951_Scinax_nebulosus_BR_BaixaGrande_Piaui 100 AJC_3274_Scinax_nebulosus_VE_DeltaAmacuro 89 AMNH_20118_Scinax_nebulosus_BR_Roraima 38 MTKD_48004_Scinax_nebulosus_GY_Iwokrama

86 MVZ_207215_Scinax_boulengeri_CR 100 AJC_1115_Scinax_boulengeri_PA_Colon_Cumbres 97AJC_0577_Scinax_boulengeri_PA_Darien 81 CH_6346_Scinax_boulengeri_PA_Darien_Pinogana 45 MHNLS_20261_Scinax_rostratus_VE_Anzoategui AJC_4964_Scinax_rostratus_CO_NteSantander_Patios JSM_171_Scinax_rostratus_CO_Magdalena_Granada 100 96 95 AJC_4142_Scinax_rostratus_CO_Cordoba_Montelibano 98 AJC_4648_Scinax_rostratus_CO_Antioquia_PtBerrio 98 72 AJC_3422_Scinax_rostratus_CO_Santander_SanVicente AJC_1747_Scinax_cf_kennedyi_CO_Meta_Arama 94 100 AJC_1741_Scinax_cf_kennedyi_CO_Meta_Arama 99 AJC_4058_Scinax_boulengeri_CO_Casanare_Sabanalarga AJC_4051_Scinax_cf_kennedyi_CO_Casanare_Sabanalarga75 96 AJC_4061_Scinax_cf_kennedyi_CO_Casanare_Sabanalarga CFBHT_08180_Scinax_squalirostris_BR_RioGrandeDoSul_BomJesus 98 CFBHT_15638_Scinax_squalirostris_BR_SaoPaulo_SaoJoseDoBarreiro 100 CFBHT_00502_Scinax_squalirostris_BR_SaoPaulo_Itirapina AJC_0872_Scinax_elaeochrous_PA_Bocas_Colon 100 MVZ_203919_Scinax_elaeochrous_CR_Heredia_Selva 100 AJC_0872_Scinax_elaeochrous_PA 70 COLZOOCH_H_2676_Scinax_elaeochrous_CO_Choco_UnPanam 73 COLZOOCH_H_2676_16S_Scinax_elaeochrous_CO_Choco_UnPanam68 38AJC_1817_Scinax_sp_PA_Darien_Pinogana 311825_AJC_Scinax_sp_PA_Darien_Pinogana AJC_1827_Scinax_sp_PA_Darien_Pinogana99 32 AJC_1821_Scinax_sp_PA_Darien_Pinogana JMP_1847_Scinax_cruentommus_CO_Amazonas 74 AJC_3089_Scinax_wandae_VE_Amazonas 36 100 AJC_4105_Scinax_wandae_CO_Casanare_Sabanalarga 93 AJC_3461_Scinax_wandae_CO_Meta_PtoGaitan 100 AJC_3464_Scinax_wandae_CO_Meta_Arama 43 JMP_1574_Scinax_cruentommus_PE_Loreto UTA_A_50749_Scinax_staufferi_GT_Zacapa_Teculatan 82 67 ENS_10717_Scinax_staufferi_HN_Corocito 100 AJC_1093_Scinax_altae_PA_Panama_Naranjal 100AJC_1094_Scinax_altae_PA_Panama_Naranjal 19 AJC_1125_Scinax_altae_PA_Panama_Cumbres62 64 CH_5684_Scinax_altae_PA_Chiriqui_Guarumal CFBHT_05375_Scinax_x_signatus_BR_Ceara_Ubajara 100CFBHT_05377_Scinax_cf_x_signatus_BR_Ceara_Ubajara 67CFBHT_08860_Scinax_x_signatus_BR_Pernambuco_FernandodeNoronha 37 CFBHT_03433_Scinax_x_signatus_BR_Bahia_Itabuna AJC_3884_Scinax_ruber_CO_Santander_SabanaDeTorres 82 AJC_3562_Scinax_ruber_CO_Tolima_Mariquita 10034 AJC_4146_Scinax_elaeochrous_CO_Cordoba_Montelibano 23 AJC_2324_Scinax_ruber_CO_Casanare_Orocue 41 AJC_4609_Scinax_x_signatus_CO_Antioquia_PtBerrio 98 AJC_3569_Scinax_ruber_CO_Amazonas_Tanimboca AJC_4053_Scinax_ruber_CO_Casanare_Sabanalarga 99 AJC_3378_Scinax_ruber_CO_Casanare_Sabanalarga 96 AJC_2273_Scinax_ruber_CO_Vaupes 99 68AJC_3446_Scinax_ruber_CO_Meta_Arama 98 AJC_3952_Scinax_ruber_CO_Meta_Arama AJC_1119_Scinax_ruber_PA_Colon_Gamboa 79 93 AJC_1120_Scinax_ruber_PA_Colon_Gamboa 100AJC_1584_Scinax_ruber_PA_Panama_Chepo AJC_1583_Scinax_ruber_PA_Panama_Chepo95 97CH_9157_Scinax_ruber_PA_Darien_Cana 100 100 CH_9319_Scinax_ruber_PA_Darien_Chucunaque AJC_3524_Scinax_ruber_CO_Santander_SanVicente AJC_4131_Scinax_elaeochrous_CO_Cordoba_Montelibano 99100 AJC_4160_Scinax_ruber_CO_Cordoba_Montelibano 59 AJC_3751_Scinax_ruber_CO_Cordoba_Monteria 73JSM_234_Scinax_boulengeri_CO_Bolivar_Zambrano 67 AJC_3532_Scinax_ruber_CO_Santander_SanVicente

155 0.3 156 157 Figure 10. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 158 (mtDNA) for the species of the genus Scinax, numbers in nodes correspond to bootstrap support 159 values. In blue species from South America, in red species from North America. 160 161 162 163 164 165 166 167 168 169 170 171 Pseudacris_maculata

Pichi_327_Smilisca_sordida_CR_Puntarenas

99 Pichi_329_Smilisca_sordida_CR_Alajuela 100 Pichi_331_Smilisca_sordida_CR_Limon

MHUA_A_7773_Smilisca_sila_CO_Antioquia_Chigorodo 80 100 MHUA_A_4744_Smilisca_sila_CO_Antioquia_Sabanalarga 76 MHUA_A_7289_Smilisca_sila_CO_Antioquia_Maceo

100 TCWC_84597_Smilisca_sila_CR_SanJose_Carara

50 AJC_1626_Smilisca_sila_PA_Darien_Chepigana 100 AJC_1618_Smilisca_sila_PA_Darien 99

KRL_1641_Smilisca_sila_PA_Cocle_Cope

81FROG_1000_Smilisca_sila_PA_Cocle_Cope

52 AJC_1978_Smilisca_sila_PA_Panama_Chilibre 90 AJC_1350_Smilisca_sila_PA_Panama_Chame

KRL_0865_Smilisca_phaeota_PA_Cocle_Cope

VCR_179_Smilisca_puma_CR_Heredia 99 Pichi_322_Smilisca_puma_CR_Alajuela

28 32 Pichi_319_Smilisca_phaeota_CR_Puntarenas

78 Pichi_320_Smilisca_phaeota_CR_SanJose 78 Pichi_311_Smilisca_phaeota_CR_Alajuela 39

CH_6316_Smilisca_phaeota_PA_Darien_Pinogana

AJC_1185_Smilisca_phaeota_CO_Choco_Arusi

59 100MHUA_A_7798_Smilisca_phaeota_CO_Antioquia_Chigorodo

38 MHUA_A_7083_Smilisca_phaeota_CO_Choco_BahiaSolano 46 40LAR_0001_Smilisca_phaeota_CO_Choco_Quibdo

AJC_4280_Smilisca_phaeota_CO_Narino_Tumaco

KU_217770_Smilisca_phaeota_EC_Manabi 36 18 AJC_3696_Smilisca_phaeota_CO_Valle_Cali

7 AJC_1048_Smilisca_phaeota_CO_Antioquia_Maceo

63 AJC_1517_Smilisca_phaeota_PA_Panama_Chepo 73 AJC_3525_Smilisca_phaeota_CO_Santander_SanVicente

172 0.2 173 174 Figure 11. Maximum likelihood (ML) phylogenetic tree of mitochondrial DNA sequences 175 (mtDNA) for the species of the genus Smilisca, numbers in nodes correspond to bootstrap 176 support values. In blue species from South America, in red species from North America. 177 178 179 180 181 182 183 184 185 186 187 188 189 190 MVZ_225749_Rana_luteiventris

KRL_823_Lithobates_warszewitschii_PA_Cocle_Cope

94 AJC_1387_Lithobates_warszewitschii_PA_Panama_Carlos

66 CH_6145_Lithobates_warszewitschii_PA_Bocas_Changuinola

56

JSF_1127_Lithobates_warszewitschii_PA

KU_195299_Lithobates_vaillanti_MX_Oaxaca_Tapanatepec

85

TNHC_60324_Lithobates_juliani_BZ_Cayo_LittleVaquerosCreek

78

AMNH_A_166454_Lithobates_palmipes_GY_PotaroSiparuni

75 AJC_3541_Lithobates_vaillanti_CO_Tolima_Mariquita

66 MHUA_A_4880_Lithobates_vaillanti_CO_Antioquia_Maceo

38

CH_6554_Lithobates_vaillanti_PA_Bocas_Changuinola

191 0.004 192 193 Figure 12. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 194 (ncDNA) for the species of the genus Lithobates, numbers in nodes correspond to 195 bootstrap support values. In blue species from South America, in red species from North 196 America. 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 ICN_55762_Pristimantis_parectatus_CO_Antioquia_Sonson

CH_9068_Craugastor_longirostris_PA_Darien_Garachine 86 CH_6513_Craugastor_longirostris_PA_Bocas_Changuinola 64 AJC_1955_Craugastor_longirostris_PA_Panama_Chagres

KRL_1387_Craugastor_tabasarae_PA_Cocle_Cope 94 CH_6055_Craugastor_tabasarae_PA_Panama_Chepo

AJC_1719_Craugastor_talamancae_PA_Bocas_Changuinola 87 AJC_1907_Craugastor_talamancae_PA_Panama_Chepo 70 AJC_0207_Craugastor_talamancae_PA_GunaYala_Warsobtugua

AJC_1201_Craugastor_crassidigitus_PA_Panama_Chame 35 49 82 AJC_0209_Craugastor_crassidigitus_PA_KunaYala_Nusgandi 57 AJC_1731_Craugastor_crassidigitus_PA_Bocas_Changuinola 63 AJC_1720_Craugastor_crassidigitus_PA_Bocas_Changuinola 33

AJC_7036_Craugastor_cf_fitzingeri_CO_Choco_Panamericana

27 CH_6152_Craugastor_fitzingeri_PA_Bocas_Changuinola 46 63 CH_9012_Craugastor_fitzingeri_PA_Darien_Garachine 78 AJC_1774_Craugastor_fitzingeri_PA_Panama_Chilibre

AJC_4279_Craugastor_longirostris_CO_Narino_Tumaco 68 KU_177803_Craugastor_longirostris_EC_Pichincha_SantoDomingo

AJC_1336_Craugastor_longirostris_CO_Antioquia_Maceo 48 92 MHUA_A_7259_Craugastor_raniformis_CO_Antioquia_Carlos 93 MHUA_A_7286_Craugastor_raniformis_CO_Antioquia_Maceo 45 MHUA_A_7800_Craugastor_raniformis_CO_Antioquia_Chigorodo

52MHUA_A_6769_Craugastor_longirostris_CO_Antioquia_Chigorodo

27 AJC_2095_Craugastor_raniformis_CO_Choco_Nuqui

51CH_6074_Craugastor_raniformis_PA_Panama_Canas 50 AJC_1612_Craugastor_raniformis_PA_Darien_Chepigana 97 AJC_1515_Craugastor_raniformis_PA_Panama_Chepo

215 0.02 216 217 Figure 13. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 218 (ncDNA) for the species of the genus Craugastor, numbers in nodes correspond to 219 bootstrap support values. In blue species from South America, in red species from North 220 America. 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 55MC_Lithodytes_lineatus_FG_GrandSanti

QCAZ_16621_Lithodytes_lineatus_EC_MoronaSantiago_Mendez

MHUA_A_7387_Leptodactylus_savagei_CO_Bolivar_Norosi

JSM_206_Leptodactylus_fuscus_CO_Huila_Garzon

JSM_227_Leptodactylus_poecilochilus_CO_Bolivar_Zambrano

94 94 AJC_0350_Leptodactylus_poecilochilus_PA_Colon_Gamboa 72 AJC_1813_Leptodactylus_poecilochilus_PA_Darien_Pinogana 100 63 AJC_0535_Leptodactylus_fragilis_PA 55 KRL_0678_Leptodactylus_fragilis_PA_Cocle_Cope 100 AJC_4664_Leptodactylus_fragilis_CO_Antioquia_Choco

8 AJC_1523_Leptodactylus_fragilis_PA_Panama_Chepo 47 12 CH_8503_Leptodactylus_fragilis_PA_Veraguas_Montijo

109MC_Leptodactylus_pentadactylus_FG_Kaw

AJC_1502_Leptodactylus_savagei_PA_Panama_Chepo 10 88 AJC_1809_Leptodactylus_savagei_PA_Darien_Pinogana 17 CH_6585_Leptodactylus_savagei_PA_Bocas_Changuinola 1 396MC_Leptodactylus_knudseni_FG_Lucifer 4 531AF_Leptodactylus_longirostris_FG_Nouragues_Inselberg 51 ULABG_4591_Leptodactylus_fuscus_VE_Bolivar_Canaima

AJC_0858_Leptodactylus_melanonotus_PA_Cocle_Anton 26 73 MVZ_207294_Leptodactylus_melanonotus_CR_Guanacaste_SantaRosa 99 AJC_0953_Leptodactylus_melanonotus_CR_Alajuela_Arenal 69

J_AF_2012_978163_Leptodactylus_cf_wagneri_BR_MatoGrosso_VilaRica

27 MD_2279_Leptodactylus_latrans_BR_Bahia_Una 91 MACN_38648_Leptodactylus_latrans_AR_BuenosAires_Escobar

239 0.02 240 241 Figure 14. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 242 (ncDNA) for the species of the genus Leptodactylus, numbers in nodes correspond to 243 bootstrap support values. In blue species from South America, in red species from North 244 America. 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 ZUFRJ_7926_Hylomantis_granulosa

SMS_Agalychnis_hulli_EC_Napo_SelvaViva

CH_6779_Agalychnis_lemur_PA_Panama_Chilibre

97

KRL_955_Agalychnis_lemur_PA_Cocle_Cope

HenryVilaZoo_Agalychnis_annae_CR_SanJose

63 54 AJC_7022_Agalychnis_spurrelli_CO_Choco_Panamericana

86

KU_217507_Agalychnis_spurrelli_EC_Pichincha_VicenteMaldonado

95 IGM_12_Agalychnis_saltator_CR_Limon_AltoColorado

3

COLZOOCH_H_2679_Agalychnis_aff_callidryas_CO_Choco_Panamericana

32 IGM_10_Agalychnis_annae_CR_SanJose_ZooSimonBolivar

33 AJC_1768_Agalychnis_callidryas_PA_Panama_Chilibre

46 RdS_537_Agalychnis_callidryas_BZ_StannCreek

2 AJC_1735_Agalychnis_callidryas_PA_Bocas_Changuiola

15

IGM_11_Agalychnis_callidryas_CR_Limon_Colorado

264 0.006 265 266 Figure 15. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 267 (ncDNA) for the species of the genus Agalychnis, numbers in nodes correspond to 268 bootstrap support values. In blue species from South America, in red species from North 269 America. 270 271 272 273 274 275 276 277 278 279 280 281 282 SIUC_H_06926_Hyloscirtus_colymba

SIUC_H_06924_Hyloscirtus_palmeri

33mc_Boana_geographicus_GY_GrandSanti 100 CFBH_5424_Boana_semilineatus_BR_RioDeJaneiro_DuqueDeCaixas

99 99bm_Boana_boans_GY_MontBakra 31 RWM_17746_Boana_boans_VE_Amazonas_AguaBlanca

95 CH_7040_Boana_boans_PA

63 CH_5307_Boana_boans_PA 78 99 CH_6332_Boana_boans_PA_Darien_Pinogana

KU_215191_Boana_boans_PE_MadreDeDios_PtoMaldonado

ROM_44089_Boana_xerophylla_GY_Parish_Hill

AJC_7040_Boana_rufitela_CO_Choco_Panamericana

55 88 AJC_7043_Boana_rufitela_CO_Choco_Panamericana

89 30594_Boana_pelluces_EC_ElOro_Avanzada 64

96 23680_Boana_pelluces_EC_Guayas_Huaquillas

12 CH_10337_Boana_rufitela_PA

AJC_1180_Boana_rufitela_PA 41 21 AJC_1389_Boana_rufitela_PA_Panama_Chame

5 CH_10046_Boana_rufitela_PA

37AJC_1099_Boana_rufitela_PA_Colon_Gatun

14 CH_10014_Boana_rufitela_PA 34 15 KRL_0798_Boana_rufitelus_PA_Cocle_Cope

CH_8501_Boana_pugax_PA_Veraguas_Catival 100 CH_8505_Boana_pugax_PA_Veraguas_Montijo

69 MHUA_A_7797_Boana_rosenbergi_CO_Antioquia_Chigorodo

89 CH_9082_Boana_rosebergi_PA_Darien_Garachine 98 AJC_1509_Boana_rosebergi_PA_Panama_Chepo 99

CFBH_4000_Boana_lundii_BR_SaoPaulo_RioClaro 96 USNM_303046_Boana_pardalis_BR_SaoPaulo_Salesopolis

56 CFBH_2966_Boana_crepitans_BR_Alagoas_Piranhas

ROM_20560_Boana_xerophylla_GY_Kurupukari 46 40 ROM_28436_Boana_xerophylla_GY_Paramakatoi

59 AJC_1323_Boana_xerophylla_CO_Tolima_Mariquita

83 AJC_1078_Boana_xerophylla_PA_Panama_BuenaVista 89 AJC_1124_Boana_xerophylla_PA_Panama_Ancon

283 0.02 284 285 Figure 16. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 286 (ncDNA) for the species of the genus Boana, numbers in nodes correspond to bootstrap 287 support values. In blue species from South America, in red species from North America. 288 289 290 291 292 293 294 295 296 297 298 299 Pseudis_paradoxa

LSB_331_Dendropsophus_subocularis_CO_Antioquia_Rafael 88 KU_215248_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado

COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_Panamericana

88AJC_0876_Dendropsophus_phlebodes_PA_Bocas_Colon

68 AJC_1788_Dendropsophus_phlebodes_PA_Panama_Chilibre 70 CH_9066_Dendropsophus_phlebodes_PA_Darien_Garachine 97

AJC_2352_Dendropsophus_microcephalus_CO_Guajira_Dibulla

AJC_0899_Dendropsophus_microcephalus_PA_Colon_Galeta 8974 CH_5683_Dendropsophus_microcephalus_PA_Chiriqui_David

20 AJC_4838_Dendropsophus_microcephalus_CO_Atlantico_Tubara 96 AJC_4035_Dendropsophus_microcephalus_CO_Santander_Vicente

73 35 AJC_0954_Dendropsophus_microcephalus_CR_Alajuela_Arenal

92 ENS_10627_Dendropsophus_microcephalus_HN_Paraiso_Chichicaste 50 UTA_50632_Dendropsophus_microcephalus_HN_Atlantida_RioViejo

28mc_Dendropsophus_brevifrons_FG_Cayenne_MtKaw 99 AMNH_A_139315_Dendropsophus_parviceps_BR_Acre_RioBranco

QCAZA_44539_Dendropsophus_triangulum_EC_Orellana_Huiririma 30 99 QCAZA_44457_Dendropsophus_triangulum_EC_Orellana_Chiroisla 64 QCAZA_44826_Dendropsophus_triangulum_EC_Orellana_RioNapo 17

MJH_3844_Dendropsophus_triangulum_BR_Acre

50 AJC_3837_Dendropsophus_subocularis_CO_Santander_Nacional

59 CH_6627_Dendropsophus_ebraccatus_PA_Bocas_Changuinola

33AJC_0406_Dendropsophus_ebraccatus_CR_Limon_Pacurita

33 UTA_51789_Dendropsophus_ebraccatus_NG_Matagalpa_PenasBlancas

22RdS_790_Dendropsophus_ebraccatus_BZ_StannCreek

40CH_6335_Dendropsophus_ebraccatus_PA_Darien_Pinogana

38AJC_3502_Dendropsophus_ebraccatus_CO_Santander_Vicente

54 AJC_1338_Dendropsophus_ebraccatus_CO_Antioquia_Maceo 52 LSB_323_Dendropsophus_ebraccatus_CO_Antioquia_Carlos

300 0.02 301 302 Figure 17. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 303 (ncDNA) for the species of the genus Dendropsophus, numbers in nodes correspond to 304 bootstrap support values. In blue species from South America, in red species from North 305 America. 306 307 308 309 310 311 312 313 314 315 316 317 318 AMNH_A_141040_Hypsiboas_multifasciatus

MVZ_207215_Scinax_boulengeri_CR

89 AJC_0577_Scinax_boulengeri_PA_Darien_PNNDarien 95 AJC_1115_Scinax_boulengeri_PA_Colon_Cumbres 89 AJC_4142_Scinax_rostratus_CO_Cordoba_Montelibano

99 247mc_Scinax_rostratus_FG

25 AJC_1518_Scinax_rostratus_PA_Panama_Chepo 71 JSM_171_Scinax_rostratus_CO_Magdalena_Granada

320MC_Scinax_cf_ruber_x_signatus_FG_Bourda

71 AJC_4146_Scinax_elaeochroa_CO_Cordoba_Montelibano 85 AJC_4609_Scinax_x_signatus_CO_Antioquia_Berrio 69 2131VOGT_Scinax_ruber_BR_Amazonas_Cachoeirinha

36 135PG_Scinax_cruentomma_FG

43 190MC_Scinax_x_signatus_FG

23 AJC_1120_Scinax_ruber_PA_Colon_Gamboa

91 AJC_1584_Scinax_ruber_PA_Panama_Chepo 63 96 AJC_1119_Scinax_ruber_PA_Colon_Gamboa

AJC_0872_Scinax_elaeochroa_PA_Bocas_Colon 37 MVZ_203919_Scinax_elaeochroa_CR_Heredia_LaSelva 61 COLZOOCH_H_2676_Scinax_elaeochroa_CO_Choco_Panamericana

94 AJC_1827_Scinax_sp_PA_Darien_Pinogana 87 54 AJC_1817_Scinax_sp_PA_Darien_Pinogana

AJC_3461_Scinax_wandae_CO_Meta_Gaitan

CH_5684_Scinax_altae_PA_Chiriqui_Guarumal 54 70 AJC_1125_Scinax_altae_PA_Panama_Cumbres 90 UTA_A_50749_Scinax_staufferi_GT_Zacapa_Teculatan

50 UTA_A_50749_Scinax_staufferi_GU_Zacapa 62 ENS_10717_Scinax_staufferi_HN_Olancho_Corocito

319 0.02 320 321 Figure 18. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 322 (ncDNA) for the species of the genus Scinax, numbers in nodes correspond to bootstrap 323 support values. In blue species from South America, in red species from North America. 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 JFBM_14310_Pseudacris_maculata

CH_6543_Smilisca_sordida_PA_Bocas_Changuinola

94CH_6621_Smilisca_sordida_PA_Bocas_Changuinola

10 MF4720_Smilisca_sordida_CR_SanJose 1 CH_6537_Smilisca_sordida_PA_Bocas_Changuinola

AJC_1350_Smilisca_sila_PA_Panama_Chame 88 AJC_0855_Smilisca_sila_PA_Cocle_Anton

60 CH_9358_Smilisca_sila_PA_Darien_Garachine

29 MHUA_A_4744_Smilisca_sila_CO_Antioquia_Sabanalarga

93 MHUA_A_7289_Smilisca_sila_CO_Antioquia_Maceo 32 30 MHUA_A_7773_Smilisca_sila_CO_Antioquia_Chigorodo

CH_6130_Smilisca_phaeota_PA_Bocas_Changuinola 76 AJC_0933_Smilisca_phaeota_CR_Limon_Siquirres

30 MFF4410_Smilisca_puma_CR_Heredia

48 VCR_179_Smilisca_puma_CR_Heredia

39 KU_217770_Smilisca_phaeota_EC_Manabi

59AJC_4280_Smilisca_phaeota_CO_Narino_Tumaco

64AJC_3525_Smilisca_phaeota_CO_Santander_Vicente

91 CH_6316_Smilisca_phaeota_PA_Darien_Pinogana 25 LAR_0001_Smilisca_phaeota_CO_Choco_Quibdo

343 0.02 344 345 Figure 19. Maximum likelihood (ML) phylogenetic tree of nuclear DNA sequences 346 (ncDNA) for the species of the genus Smilisca, numbers in nodes correspond to bootstrap 347 support values. In blue species from South America, in red species from North America. 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 Rana_luteiventris

AMNH_A_118801_Lithobates_palmipes_VE_Amazonas

Pichi_354_Lithobates_CR_Cartago

AJC_1798_Lithobates_warszewit_PA_Panama

CH_6145_Lithobates_warszewit_PA_Bocas_Changuinola

AJC_1387_Lithobates_warszewit_PA_Panama_SanCarlos

KRL_1567_Lithobates_warszewit_PA_Cocle_Cope

Pichi_357_Lithobates_vaillanti_CR_Alajuela

TNHC_60324_Lithobates_juliani_BZ_Cayo_Vaqueros

CH_6554_Lithobates_vaillanti_PA_Bocas_Changuinola

AJC_3541_Lithobates_vaillanti_CO_Tolima_Mariquita

MHUA_A_4880_Lithobates_vaillanti_CO_Antioquia_Maceo

QCAZ_13964_Lithobates_bwana_EC_Loja_Zapotillo

MHNLS_17185_Lithobates_palmipes_VE_Zulia

CFBHT_10214_Lithobates_palmipes_BR_MatoGrosso_AltaFloresta

MNCN_ADN_50843_Lithobates_palmipes_BR_MatoGrosso_Juruena

TG_204_Lithobates_palmipes_BR_MatoGrosso_Paranaita

CFBHT_07835_Lithobates_palmipes_BR_Alagoas_CampoAlegre

CFBHT_11134_Lithobates_palmipes_BR_Paraiba_JoaoPessoa

CFBHT_12435_Lithobates_palmipes_BR_RioGrandeDoNorte_TibauDoSul

AJC_3266_Lithobates_palmipes_VE_DeltaAmacuro

AMNH_A_166454_Lithobates_palmipes_GY_PotaroSiparuni

AJC_2308_Lithobates_palmipes_CO_Casanare_Orocue

CFBHT_07601_Lithobates_palmipes_BR_Acre_CruzeiroDoSul

KU_202896_Lithobates_palmipes_EC_Napo_Misahualli

AJC_5473_Lithobates_palmipes_CO_Caqueta_Valparaiso

JMP_1615_Lithobates_palmipes_PE_Loreto

367 40 35 30 25 20 15 10 5 0 368 369 Figure 20. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 370 Lithobates. Purple bars show confidence intervals for the node age. Country names are 371 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 372 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 373 from South America, in red species from North America. 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 CFBHT_12711_Haddadus_binotatus_BR_EspiritoSanto_MimosoDoSol CFBHT_12658_Haddadus_binotatus_BR_SantaCatarina CFBHT_13706_Haddadus_binotatus_BR_RioDeJaneiro_SantaMariaMadalena AJC_1939_Craugastor_aff_longirostris_PA_Panama_Chepo AJC_1955_Craugastor_aff_longirostris_PA_Panama_Chagres CH_6513_Craugastor_longirostris_PA_Bocas_Changuinola KRL_1399_Craugastor_aff_longirostris_PA_Cocle_Cope AJC_2116_Craugastor_longirostris_CO_Choco_Tacarcuna AJC_2125_Craugastor_longirostris_CO_Choco_Tacarcuna CH_6390_Craugastor_longirostris_PA_Darien_Pinogana CH_9040_Craugastor_longirostris_PA_Darien CH_9068_Craugastor_longirostris_PA_Darien_Garachine AB_10_Craugastor_melanostictus_PA_Chiriqui_Naranjos Pichi_393_Craugastor_melanostictus_CR_Limon_Talamanca CH_6055_Craugastor_tabasarae_PA_Panama_Chepo EVACC_024_Craugastor_tabasarae_PA_Panama_Chame EVACC_219_Craugastor_tabasarae_PA_Panama_Valle_RioMaria KRL_0706_Craugastor_tabasarae_PA_Cocle_Cope LAR_0010_Craugastor_longirostris_CO_Choco_Quibdo AJC_1193_Craugastor_longirostris_CO_Choco_Arusi AJC_2092_Craugastor_longirostris_CO_Choco_Arusi AJC_2079_Craugastor_longirostris_CO_Valle_Buenaventura AJC_4279_Craugastor_longirostris_CO_Narino_Tumaco KU_177803_Craugastor_longirostris_EC_Pichincha_SantoDomingo AJC_1336_Craugastor_longirostris_CO_Antioquia_Maceo MHUA_A_7286_Craugastor_raniformis_CO_Antioquia_Maceo AJC_3885_Craugastor_longirostris_CO_Santander_Sabana MHUA_A_7259_Craugastor_raniformis_CO_Antioquia_SanCarlos AJC_1199_Craugastor_aff_longirostris_CO_Tolima_Mariquita MHUA_A_7259_16S_Craugastor_raniformis_CO_Antioquia_SanCarlos MHUA_A_7286_16S_Craugastor_raniformis_CO_Antioquia_Maceo LAR_0007_Craugastor_raniformis_CO_Choco_Quibdo LAR_0006_Craugastor_fitzingeri_CO_Choco_Quibdo AJC_2080_Craugastor_raniformis_CO_Valle_Cali AJC_2095_Craugastor_raniformis_CO_Choco_Nuqui MHUA_A_6766_Craugastor_longirostris_CO_Antioquia_Chigorodo MHUA_A_6769_Craugastor_longirostris_CO_Antioquia_Chigorodo CH_6074_Craugastor_raniformis_PA_Panama_IslaCana AJC_1515_Craugastor_raniformis_PA_Panama_Chepo AJC_1612_Craugastor_raniformis_PA_Darien_Chepigana AJC_1201_Craugastor_crassidigitus_PA_Panama_Chame AJC_1598_Craugastor_crassidigitus_PA_Darien_Chepigana AJC_1731_Craugastor_crassidigitus_PA_Bocas_Changuinola MVZ_207248_Craugastor_crassidigitus_CR_Puntarenas_Monteverde AJC_1719_Craugastor_talamancae_PA_Bocas_Changuinola AJC_1907_Craugastor_talamancae_PA_Panama_Chepo KRL_1087_Craugastor_talamancae_PA_Cocle_Cope KRL_1520_Craugastor_talamancae_PA_Cocle_Cope CHOCO019_15_Craugastor_fitzingeri_LAR_0005_CO_Choco AJC_7036_Craugastor_cf_fitzingeri_CO_Choco_UnPanam LAR_0005_Craugastor_fitzingeri_CO_Choco_Quibdo MHUA_A_4305_Craugastor_fitzingeri_CO_Choco_Nuqui MHUA_A_4306_Craugastor_fitzingeri_CO_Choco_Nuqui MHUA_A_4883_Craugastor_fitzingeri_CO_Choco_Nuqui CH_6152_Craugastor_fitzingeri_PA_Bocas_Changuinola Pichi_067_Craugastor_fitzingeri_CR_Guanacaste_Murcielago Pichi_068_Craugastor_fitzingeri_CR_Alajuela_Gavilan CH_8438_Craugastor_fitzingeri_PA_Chiriqui Pichi_070_Craugastor_fitzingeri_CR_Puntarenas Pichi_173_Craugastor_fitzingeri_CR_Puntarenas_Naranjito CH_9012_Craugastor_fitzingeri_PA_Darien_Garachine AJC_1774_Craugastor_fitzingeri_PA_Panama_Chilibre KRL_0693_Craugastor_fitzingeri_PA_Cocle_Cope

392 70 60 50 40 30 20 10 0 393 394 Figure 21. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 395 Craugastor. Purple bars show confidence intervals for the node age. Country names are 396 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 397 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 398 from South America, in red species from North America. 399 400 401 402 403 404 405 406 407 408 AJC_3451_Lithodytes_lineatus_CO_Meta_Arama AJC_0953_Leptodactylus_melanonotus_CR_Alajuela_VolcanArenal CFBHT_00832_Leptodactylus_latrans_BR_MinasGerais_Camanducaia CFBHT_10542_Leptodactylus_latrans_BR_RiodeJaneiro_Niteroi AJC_5035_Leptodactylus_colombiensis_CO_Atlantico_Tubara CH_4956_Leptodactylus_insularum_PA_Panama_Uveros KRL_1114_Leptodactylus_insularum_PA_Cocle_Cope AJC_3509_Leptodactylus_insularum_CO_Santander_SanVicente AJC_4133_Leptodactylus_fuscus_CO_Cordoba_Montelibano ULABG_5112_Leptodactylus_bolivianus_VE_Merida_RioLimones JDL_24887_Leptodactylus_bolivianus_CO_Sucre_SanMarcos MUJ_2187_Leptodactylus_bolivianus_CO_Bolivar_Cartagena AJC_3978_Leptodactylus_colombiensis_CO_Boyaca_Miraflores AJC_5784_Leptodactylus_melanonotus_CO_Boyaca_SantaMaria LSUNZ_H_12885_Leptodactylus_wagneri_EC USNM_320988_Leptodactylus_wagneri_EC_Pastaza_Coca JSM_178_Leptodactylus_colombiensis_CO_Huila_Garzon AJC_0858_Leptodactylus_melanonotus_PA_Cocle_Anton AJC_1149_Leptodactylus_melanonotus_PA_Panama_SanCarlos USNM_535964_Leptodactylus_melanonotus_BZ_Cayo_SanJacinto AJC_3848_Leptodactylus_colombiensis_CO_Santander_PteNal AJC_6198_Leptodactylus_melanonotus_CO_Antioquia_Anori AJC_3429_Leptodactylus_knudseni_16S_CO_Casanare_Orocue MHNLS_15590_Leptodactylus_knudseni_VE_Bolivar_Purumay AMNH_20102_Leptodactylus_pentadactylus_BR_Roraima_Florencia PK_1234_Leptodactylus_knudseni_GY_PotaroSiparuni AJC_4761_Leptodactylus_pentadactylus_CO_Amazonas_Leticia CORBIDI_0024_Leptodactylus_pentadactylus_EC_Sucumbios_GarzaCocha CH_6585_Leptodactylus_savagei_PA_Bocas_Changuinola USNM_298079_Leptodactylus_savagei_PA_Bocas_IslaPopa USNM_534219_Leptodactylus_savagei_HN_Colon_Machin CFBH_39775_Leptodactylus_pentadactylus_BR_MatoGrosso_Paranaita MHUA_A_7387_Leptodactylus_savagei_CO_Bolivar_Norosi AJC_6545_Leptodactylus_savagei_CO_Magdalena_SantaMarta AJC_6581_Leptodactylus_savagei_CO_Magdalena_SantaMarta AJC_1502_Leptodactylus_savagei_PA_Panama_Chepo AJC_1809_Leptodactylus_savagei_PA_Darien_Pinogana KRL_0838_Leptodactylus_savagei_PA_Cocle_Cope AJC_2231_Leptodactylus_fuscus_16S_CO_Casanare_Palmarito AJC_2301_Leptodactylus_fuscus_16S_CO_Casanare_Orucue AJC_3467_Leptodactylus_fuscus_16S_CO_Meta_Arama AJC_4078_Leptodactylus_fuscus_16S_CO_Casanare_Sabanalarga JSM_169_Leptodactylus_fuscus_CO_Magdalena_Granada AJC_3892_Leptodactylus_fuscus_16S_CO_Santander_SabanaDeTorres AJC_4153_Leptodactylus_fuscus_CO_Cordoba_Montelibano AJC_4619_Leptodactylus_fuscus_CO_Antioquia_Pto_Berrio JSM_206_Leptodactylus_fuscus_CO_Huila_Garzon AJC_1523_Leptodactylus_fragilis_PA_Panama_Chepo KRL_0678_Leptodactylus_fragilis_PA_Cocle_Cope AJC_4684_Leptodactylus_fragilis_CO_Antioquia_Yondo JSM_201_Leptodactylus_fragilis_CO_Huila_Garzon AJC_4664_Leptodactylus_fragilis_CO_Antioquia_Yondo JSM_262_Leptodactylus_fragilis_CO_Bolivar_Baru AJC_4476_Leptodactylus_fragilis_CO_Magdalena_Pinuelas AJC_2294_Leptodactylus_fragilis_CO_Tolima_Mariquita AJC_4628_Leptodactylus_fragilis_CO_Antioquia_PtoBerrio PK_1507_Leptodactylus_longirostris_GY_PotaroSiparuni ROM_20591_Leptodactylus_longirostris_GY_PotaroSiparuni_Kaieteur 76bm_Leptodactylus_longirostris_FG_MontArawa 103AF_Leptodactylus_longirostris_SU_Para_Apura 199mc_Leptodactylus_longirostris_FG_GrandSanti 531AF_Leptodactylus_longirostris_FG_Nouragues_Inselberg MHNLS_17323_Leptodactylus_longirostris_VE_Bolivar_Uayaraca AJC_4871_Leptodactylus_poecilochilus_CO_NorteSantander_Patios JSM_0011_Leptodactylus_poecilochilus_CO_NorteSantander_Bochalema CH_9077_Leptodactylus_poecilochilus_PA_Darien_Garachine KRL_0886_Leptodactylus_poecilochilus_PA_Veraguas_SantaFe AJC_1813_Leptodactylus_poecilochilus_PA_Darien_Pinogana CH_6373_Leptodactylus_poecilochilus_PA_Darien_Pinogana JSM_172_Leptodactylus_poecilochilus_16S_CO_Magdalena_Granada JSM_227_Leptodactylus_poecilochilus_CO_Bolivar_Zambrano

40960 50 40 30 20 10 0 410 411 Figure 22. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 412 Leptodactylus. Purple bars show confidence intervals for the node age. Country names are 413 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 414 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 415 from South America, in red species from North America. 416 CFBHT00392_Hylomantis_granulosa_BR_Pernambuco_Jaqueira

CFBHT_12869_Hylomantis_aspera_BR_Bahia_Ilheus

Pichi_258_Agalychnis_lemur_CR_Limon_Asuncion

Pichi_259_Agalychnis_saltator_CR_Puntarenas_Bandera

CH_6779_Agalychnis_lemur_PA_Panama_Chilibre

KRL_0940_Agalychnis_lemur_PA_Cocle_Cope

toe_134_Agalychnis_lemur_PA_Cocle_Cope

LSB_134_Agalychnis_buckleyi_CO_Antioquia_Yarumal

LSB_135_Agalychnis_buckleyi_CO_Antioquia_Yarumal

MZUTI_313_Agalychnis_hulli_EC_Pastaza

MZUTI_170_Agalychnis_hulli_EC_Pastaza

MZUTI_168_Agalychnis_hulli_EC_Pastaza

MZUTI_169_Agalychnis_hulli_EC_Pastaza

QCAZ_13217_Agalychnis_spurrelli_EC_Esmeraldas_Durango

AJC_7022_Agalychnis_spurrelli_CO_Choco_UnPanam

Pichi_261_Agalychnis_spurrelli_CR_Limon_Guayacan

Pichi_262_Agalychnis_spurrelli_CR_SanJose_Carara

Pichi_260_Agalychnis_saltator_CR_Alajuela_SanCarlos

JMR_609_Agalychnis_saltator_CR_Heredia_LaSelva

MVZ_203768_Agalychnis_saltator_CR_Heredia_Sarapiqui

Pichi_251_Agalychnis_annae_CR_Heredia_Roble

AJC_3510_Agalychnis_terranova_CO_Santander_SanVicente

AJC_3515_Agalychnis_terranova_CO_Santander_SanVicente

MHUA_A_7316_Agalychnis_terranova_CO_Antioquia_Sonson

MHUA_L_0198_2_Agalychnis_terranova_CO_Antioquia_Sonson

AJC_2086_Agalychnis_CO_Choco_Nuqui

AJC_2087_Agalychnis_CO_Choco_Nuqui

COLZOOCH_H_2679_Agalychnis_terranova_CO_Choco_UnPanam

AJC_1565_Agalychnis_callidryas_PA_Panama_Chepo

AJC_1595_Agalychnis_callidryas_PA_Darien_Chepigana

AJC_1768_Agalychnis_callidryas_PA_Panama_Chilibre

KRL_0917_Agalychnis_callidryas_PA_Cocle_Cope

AJC_1735_Agalychnis_callidryas_PA_Bocas_Changuinola

Pichi_254_Agalychnis_callidryas_CR_Limon_Talamanca

Pichi_253_Agalychnis_callidryas_CR_SanJose_Carara

RdS_537_Agalychnis_callidryas_BZ_StannCreek

417 40 35 30 25 20 15 10 5 0 418 419 Figure 23. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 420 Agalychnis. Purple bars show confidence intervals for the node age. Country names are 421 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 422 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 423 from South America, in red species from North America. 424 425 426 427 428 429 430 431 432 433 434 435 436 CFBHT_13076_Apl_leucopygius_BR_RioDeJaneiro_NovaFriburgo CFBHT_14484_Apl_arildae_BR_RioDeJaneiro_Itatiaia AJC_2553_Boana_nympha_CO_Amazonas_Leticia 23680_Boana_pellucens_EC_Guayas_Huaquillas 30594_Boana_pellucens_EC_ElOro_Avanzada AJC_7040_Boana_rufitelus_CO_Choco_UnPanam AJC_7043_Boana_rufitelus_CO_Choco_UnPanam AJC_0877_Boana_rufitelus_PA_BocasDelToro_IslaPopa AJC_1099_Boana_rufitelus_PA_Colon_Gatun AJC_1382_Boana_rufitelus_PA_Panama_Chame KRL_0798_Boana_rufitelus_PA_Cocle_Cope AJC_2476_Boana_boans_CO_Guainia_PtColombia KM390786_1_Boana_semilineata_BR MHNLS_19727_Boana_geographicus_VE_Bolivar_Suapure 11A_Boana_semilineatus_BR_EspiritoSanto_RioBananal MNKA_9497_Boana_geographicus_BO_StaCruz_Tucumancito MZUSP_157060_Boana_geographicus_BR_Amazonas_Tefe MZUSP_157083_Boana_geographicus_BR_Amazonas_Purus CORBIDI_5253_Boana_geographicus_PE_MadreDios_PtoMaldonado MZUSP_157090_Boana_geographicus_BR_Acre_SerraDoDivisor AJC_3443_Boana_boans_CO_Meta_SanJuan AJC_3268_Boana_boans_VE_DeltaAmacuro PK_1129_Boana_boans_GY_PotaroSiparuni AMNH_20098_Boana_boans_BR_Roraima_Rorainopolis MHNLS_19685_Boana_boans_VE_Bolivar_Suapure CORBIDI_5173_Boana_boans_PE_MadreDios_PtoMaldonado AJC_2329_Boana_boans_CO_Vaupes_Mitu AJC_2541_Boana_boans_CO_Amazonas_Leticia MTR_ALCX69P42_Boana_boans_BR_Amazonas_NovoAripuana CH_6332_Boana_boans_PA_Darien_Pinogana AJC_1051_Boana_boans_PA_Panama_Chepo AJC_1320_Boana_boans_CO_Tolima_Mariquita AJC_1375_Boana_boans_CO_Tolima_Falan TG_368_Boana_faber_BR_SaoPaulo_PedroToledo CFBHT_15407_Boana_faber_BR_SaoPaulo_Nazare CFBHT_04381_Boana_faber_BR_RioDeJaneiro_Petropolis CFBHT_06588_Boana_faber_BR_SaoPaulo_SaoLuis CFBHT_09065_Boana_pardalis_BR_EspiritoSanto_SantaLeopoldina CFBHT_04366_Boana_pardalis_BR_RioDeJaneiro_Petropolis CFBHT_01279_Boana_pardalis_BR_SaoPaulo_SaoLuis CFBHT_14367_Boana_pardalis_BR_SaoPaulo_SaoJose CFBHT_01902_Boana_lundii_BR_MinasGerais_SaoRoque CFBHT_12573_Boana_lundii_BR_SaoPaulo_SaoCarlos CFBHT_02106_Boana_lundii_BR_Goias_AltoParaiso CFBHT_12635_Boana_lundii_BR_Brasilia_DF BSUCR283_12_Boana_rosenbergi_Pichi_283_CR_Puntarenas_AguaBuena Pichi_281_Boana_rosenbergi_CR_Puntarenas_Quepos MHUA_A_7797_Boana_rosenbergi_CO_Antioquia_Chigorodo CH_9082_Boana_rosenbergi_PA_Darien_Garachine CH_6337_Boana_rosenbergi_PA_Darien_Pinogana AJC_1509_Boana_rosenbergi_PA_Panama_Chepo CH_8792_Boana_rosenbergi_PA_Colon_Gamboa MHNLS_16827_Boana_pugnax_VE_Zulia_Derote AJC_4135_Boana_sp_CO_Cordoba_Montelibano AJC_4149_Boana_sp_CO_Cordoba_Montelibano AJC_4161_Boana_xerophylla_CO_Santander_Guapota JMR_5959_Boana_pugnax_CO_Sucre_SanOnofre CH_8501_Boana_pugnax_PA_Veraguas AJC_5138_Boana_pugnax_CO_Cesar_Bosconia AJC_2345_Boana_xerophylla_CO_Guajira_Dibulla AJC_2341_Boana_sp_CO_Guajira_Dibulla AJC_2344_Boana_sp_CO_Guajira_Dibulla AJC_4614_Boana_pugnax_CO_Antioquia_PtoBerrio AJC_4627_Boana_pugnax_CO_Antioquia_PtoBerrio AJC_3870_Boana_pugnax_CO_Santander MHUA_A_7285_Boana_xerophylla_CO_Antioquia_Maceo AJC_3501_Boana_pugnax_CO_Santander_SanVicente AJC_3870_Boana_pugnax_CO_SAntioquiaander_SabanaTorres AJC_4686_Boana_xerophylla_CO_Antioquia_Yondo CFBHT_02360_Boana_crepitans_BR_Minas_GraoMogol CFBHT_07825_Boana_xerophylla_BR_Alagoas_CampoAlegre CFBHT_12841_Boana_crepitans_BR_Bahia_Camamu INPA_H_31229_Boana_xerophylla_BR_Roraima_Rorainopolis AJC_2857_Boana_xerophylla_VE_Amazonas_PtoAyacucho JSM_181_Boana_xerophylla_CO_Huila_Garzon JSM_183_Boana_xerophylla_CO_Huila_Garzon AJC_1316_Boana_xerophylla_CO_Tolima_Mariquita AJC_1323_Boana_crepitans_CO_Mariquita AJC_1323_Boana_crepitans_CO_Tolima_Mariquita AJC_4341_Boana_xerophylla_CO_Cundinamarca_Nilo AJC_4341_Boana_crepitans_CO_Cundinamarca AJC_2253_Boana_xerophylla_CO_Cundinamarca_Guaduas AJC_3765_Boana_pugnax_CO_Cundinamarca_Nilo AJC_4010_Boana_xerophylla_CO_Boyaca_Miraflores AJC_4108_Boana_xerophylla_CO_Casanare_Sabanalarga AJC_1738_Boana_xerophylla_CO_Meta_Arama AJC_4563_Boana_xerophylla_CO_Meta_Restrepo AJC_4300_Boana_xerophylla_CO_Meta_SanMartin AJC_4014_Boana_xerophylla_CO_Boyaca_Miraflores AJC_4108_Boana_crepitans_CO_Casanare MHNLS_17218_Boana_xerophylla_VE_Merida_AndresBello AJC_1123_Boana_xerophylla_PA_Panama_Cumbres AJC_1078_Boana_xerophylla_PA_Panama_Buenavista AJC_1096_Boana_xerophylla_PA_Panama_Pacora AJC_3505_Boana_xerophylla_CO_Santander_SanVicente AJC_3843_Boana_xerophylla_CO_Santander_PtNal AAV_151_Boana_xerophylla_CO_Santander_Piedecuesta AJC_3394_Boana_cf_xerophylla_CO_Santander_PuenteNal

50 437 40 30 20 10 0 438 439 Figure 24. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 440 Boana. Purple bars show confidence intervals for the node age. Country names are coded 441 as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 442 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 443 from South America, in red species from North America. 444 445 446 447 AJC_2382_Pseudis_paradoxa CORBIDI_5217_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado CORBIDI_5235_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado APL_16416_Dendropsophus_brevifrons_BR_Rondonia_PortoVelho QCAZA_18174_Dendropsophus_brevifrons_EC_Napo_JatunSacha QCAZA_55848_Dendropsophus_brevifrons_EC_Sucumbios_Lorocachi QCAZA_17826_Dendropsophus_brevifrons_EC_Orellana_Yasuni QCAZA_28273_Dendropsophus_brevifrons_EC_Sucumbios_Cuyabeno MHUA_A_6585_Dendropsophus_subocularis_CO_Antioquia_SanRoque LSB_331_Dendropsophus_subocularis_CO_Antioquia_SanRafael MJH_7101_Dendropsophus_brevifrons_PE_Huanuco_PtoInca APL_16749_Dendropsophus_parviceps_BR_Rondonia_PortoVelho QCAZ_52026_Dendropsophus_parviceps_EC_Tungurahua_RioNegro QCAZ_52752_Dendropsophus_parviceps_EC_Pastaza_Sarayacu QCAZ_51195_Dendropsophus_parviceps_EC_Orellana_Yasuni QCAZ_39515_Dendropsophus_parviceps_EC_Pastaza_Coconaco QCAZ_48919_Dendropsophus_parviceps_EC_Napo_Sumaco QCAZA_17429_Dendropsophus_sarayacuensis_EC_Pastaza_Fatima QCAZA_23030_Dendropsophus_sarayacuensis_EC_Tungurahua_Banos QCAZA_32637_Dendropsophus_sarayacuensis_EC_MoronaSantiago_NueveOctubre QCAZA_36697_Dendropsophus_sarayacuensis_EC_Napo_Toachi CORBIDI_12194_Dendropsophus_triangulum_PE_Loreto_Requena QCAZA_44826_Dendropsophus_triangulum_EC_Orellana_RioNapo SCF_012_Dendropsophus_triangulum_PE_Ucayali WED_54094_Dendropsophus_triangulum_EC ZONIAAF_MTR19029_Dendropsophus_triangulum_BR_Amazonas AJC_3837_Dendropsophus_subocularis_CO_Santander_PteNal AJC_3845_Dendropsophus_subocularis_CO_Santander_PteNacional Pichi_267_Dendropsophus_ebraccatus_CR_Puntarenas_Golfito CH_6627_Dendropsophus_ebraccatus_PA_Bocas_Changuinola AJC_0406_Dendropsophus_ebraccatus_CR_Limon_Pacurita AJC_0951_Dendropsophus_ebraccatus_CR_Alajuela_Arenal CH_9321_Dendropsophus_ebraccatus_PA_Darien_RioChucunaque AJC_1118_Dendropsophus_ebraccatus_PA_Colon_Gamboa AJC_1790_Dendropsophus_ebraccatus_PA_Panama_Chilibre CH_6644_Dendropsophus_ebraccatus_PA_Panama_Chilibre CH_6335_Dendropsophus_ebraccatus_PA_Darien_Pinogana AJC_1338_Dendropsophus_ebraccatus_CO_Antioquia_Maceo LSB_323_Dendropsophus_ebraccatus_CO_Antiquia_SanCarlos AJC_3502_Dendropsophus_ebraccatus_CO_Santander_SanVicente AJC_3504_Dendropsophus_ebraccatus_CO_Santander_SanVicente AJC_2883_Dendropsophus_minusculus_VE_Amazonas_PtoAyacucho AJC_1740_Dendropsophus_mathiassoni_CO_Meta_Arama AJC_2322_Dendropsophus_mathiassoni_CO_Casanare_Orocue AJC_4052_Dendropsophus_mathiassoni_CO_Casanare_Sabanalarga LSB_363_Dendropsophus_mathiassoni_CO_Meta_Villavicencio AJC_2728_Dendropsophus_rhodopeplus_PE_SanMartin_Tarapoto APL_16847_Dendropsophus_rhodopeplus_BR_Rondonia_PortoVelho IDLAR_4665_Dendropsophus_rhodopeplus_PE_Cuzco_SanLorenzo KST_0542_Dendropsophus_rhodopeplus_PE_MadreDeDios_Manu AJC_3571_Dendropsophus_rhodopeplus_CO_Amazonas_Tanimboca JMP_2003_Dendropsophus_rhodopeplus_CO_Amazonas_Leticia QCAZA_44329_Dendropsophus_rhodopeplus_EC_Orellana_Chiroisla QCAZA_44328_Dendropsophus_rhodopeplus_EC_Orellana_Chiroisla QCAZA_44584_Dendropsophus_rhodopeplus_EC_Orellana_Huiririma COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_UnPanam COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_UnPanam__reversed_ AJC_1788_Dendropsophus_phlebodes_PA_Panama_Chilibre CH_6806_Dendropsophus_phlebodes_PA_Panama_Chilibre MHNLS_18951_Dendropsophus_microcephalus_VE_Yaracuy_Nirgua MHNLS_17186_Dendropsophus_microcephalus_VE MHNLS_17186_Dendropsophus_microcephalus_VE_Zulia_Tocuco AJC_3177_Dendropsophus_microcephalus_VE_Bolivar_PedroChien MHNLS_17736_Dendropsophus_microcephalus_VE_DeltaAmacuro EBEF_4_Dendropsophus_microcephalus_VE_Apure_ElFrio MNCN_ADN_59473_Dendropsophus_microcephalus_VE_Anzoategui_Independencia AJC_0954_Dendropsophus_microcephalus_CR_Alajuela_Arenal ENS_10627_Dendropsophus_microcephalus_HN_Paraiso_Chichicaste Pichi_273_Dendropsophus_microcephalus_NG_RioFrio_Corra CH_5683_Dendropsophus_microcephalus_PA_Chiriqui_David Pichi_272_Dendropsophus_microcephalus_CR_Puntarenas_AguaBuena AJC_1507_Dendropsophus_microcephalus_PA_Panama_Chepo AJC_1514_Dendropsophus_microcephalus_PA_Panama_Chepo CH_9066_Dendropsophus_phlebodes_PA_Darien_Garachine AJC_3804_Dendropsophus_phlebodes_PA_Darien_Garachine AJC_3805_Dendropsophus_phlebodes_PA_Darien_Garachine AJC_0899_Dendropsophus_microcephalus_PA_Colon_Galeta AJC_1791_Dendropsophus_microcephalus_PA_Panama_Chilibre AJC_0994_Dendropsophus_microcephalus_PA_Panama_LagoBayano AJC_1098_Dendropsophus_microcephalus_PA_Panama_Naranjal AJC_1117_Dendropsophus_microcephalus_PA_Cocle_Cope AJC_4613a_Dendropsophus_microcephalus_CO_Antioquia_PtoBerrio AJC_4633_Dendropsophus_microcephalus_CO_Antioquia_PtoBerrio AJC_5224_Dendropsophus_microcephalus_CO_Tolima_Armero AJC_4838_Dendropsophus_microcephalus_CO_Atlantico_Tubara AJC_5766_Dendropsophus_microcephalus_CO_Sucre_SanMarcos AJC_5337_Dendropsophus_microcephalus_CO_Tolima_Coello AJC_5354_Dendropsophus_microcephalus_CO_Huila_Neiva AJC_4433_Dendropsophus_microcephalus_CO_Magdalena_Pinuelas AJC_5746_Dendropsophus_microcephalus_CO_Santander_Barrancabermeja AJC_1049_Dendropsophus_microcephalus_CO_Antioquia_Maceo AJC_3561_Dendropsophus_microcephalus_CO_Tolima_Mariquita AJC_5741_Dendropsophus_microcephalus_CO_Antioquia_Yondo JMR_5987_Dendropsophus_microcephalus_CO_Bolivar_MariaLaBaja AJC_2349_Dendropsophus_microcephalus_CO_Guajira_Dibulla AJC_2352_Dendropsophus_microcephalus_CO_Guajira_Dibulla AJC_2254_Dendropsophus_microcephalus_CO_Cundinamarca_Guaduas AJC_4035_Dendropsophus_microcephalus_CO_Santander_SanVicente AJC_3869_Dendropsophus_microcephalus_CO_Santander_Sabana MHUA_A_7174_Dendropsophus_microcephalus_CO_Antioquia_GomezPlata

60 448 50 40 30 20 10 0 449 450 Figure 25. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 451 Dendropsophus. Purple bars show confidence intervals for the node age. Country names 452 are coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador 453 (EC), Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue 454 species from South America, in red species from North America. 455 456 457 CFBHT_16443_Sca_vigilans_VE QULC_2340_Sca_goinorum_BR MVZ_207215_Scinax_boulengeri_CR AJC_1115_Scinax_boulengeri_PA_Colon_Cumbres AJC_0577_Scinax_boulengeri_PA_Darien CH_6346_Scinax_boulengeri_PA_Darien_Pinogana CFBHT_10951_Scinax_nebulosus_BR_BaixaGrande_Piaui AMNH_20118_Scinax_nebulosus_BR_Roraima AJC_3274_Scinax_nebulosus_VE_DeltaAmacuro MTKD_48004_Scinax_nebulosus_GY_Iwokrama MHNLS_20261_Scinax_rostratus_VE_Anzoategui AJC_4051_Scinax_cf_kennedyi_CO_Casanare_Sabanalarga AJC_4061_Scinax_cf_kennedyi_CO_Casanare_Sabanalarga AJC_4058_Scinax_boulengeri_CO_Casanare_Sabanalarga AJC_1741_Scinax_cf_kennedyi_CO_Meta_Arama AJC_1747_Scinax_cf_kennedyi_CO_Meta_Arama AJC_4964_Scinax_rostratus_CO_NteSantander_Patios AJC_3422_Scinax_rostratus_CO_Santander_SanVicente AJC_4648_Scinax_rostratus_CO_Antioquia_PtBerrio AJC_4142_Scinax_rostratus_CO_Cordoba_Montelibano JSM_171_Scinax_rostratus_CO_Magdalena_Granada CFBHT_08180_Scinax_squalirostris_BR_RioGrandeDoSul_BomJesus CFBHT_00502_Scinax_squalirostris_BR_SaoPaulo_Itirapina CFBHT_15638_Scinax_squalirostris_BR_SaoPaulo_SaoJoseDoBarreiro AJC_0872_Scinax_elaeochrous_PA AJC_0872_Scinax_elaeochrous_PA_Bocas_Colon MVZ_203919_Scinax_elaeochrous_CR_Heredia_Selva COLZOOCH_H_2676_Scinax_elaeochrous_CO_Choco_UnPanam AJC_1817_Scinax_sp_PA_Darien_Pinogana COLZOOCH_H_2676_16S_Scinax_elaeochrous_CO_Choco_UnPanam 1825_AJC_Scinax_sp_PA_Darien_Pinogana AJC_1821_Scinax_sp_PA_Darien_Pinogana AJC_1827_Scinax_sp_PA_Darien_Pinogana AJC_3461_Scinax_wandae_CO_Meta_PtoGaitan AJC_3464_Scinax_wandae_CO_Meta_Arama JMP_1847_Scinax_cruentommus_CO_Amazonas AJC_3089_Scinax_wandae_VE_Amazonas AJC_4105_Scinax_wandae_CO_Casanare_Sabanalarga JMP_1574_Scinax_cruentommus_PE_Loreto ENS_10717_Scinax_staufferi_HN_Corocito UTA_A_50749_Scinax_staufferi_GT_Zacapa_Teculatan AJC_1093_Scinax_altae_PA_Panama_Naranjal AJC_1125_Scinax_altae_PA_Panama_Cumbres AJC_1094_Scinax_altae_PA_Panama_Naranjal CH_5684_Scinax_altae_PA_Chiriqui_Guarumal CFBHT_05375_Scinax_x_signatus_BR_Ceara_Ubajara CFBHT_05377_Scinax_cf_x_signatus_BR_Ceara_Ubajara CFBHT_03433_Scinax_x_signatus_BR_Bahia_Itabuna CFBHT_08860_Scinax_x_signatus_BR_Pernambuco_FernandodeNoronha AJC_2324_Scinax_ruber_CO_Casanare_Orocue AJC_3562_Scinax_ruber_CO_Tolima_Mariquita AJC_4609_Scinax_x_signatus_CO_Antioquia_PtBerrio AJC_3884_Scinax_ruber_CO_Santander_SabanaDeTorres AJC_4146_Scinax_elaeochrous_CO_Cordoba_Montelibano AJC_3569_Scinax_ruber_CO_Amazonas_Tanimboca AJC_2273_Scinax_ruber_CO_Vaupes AJC_3378_Scinax_ruber_CO_Casanare_Sabanalarga AJC_4053_Scinax_ruber_CO_Casanare_Sabanalarga AJC_3446_Scinax_ruber_CO_Meta_Arama AJC_3952_Scinax_ruber_CO_Meta_Arama AJC_1119_Scinax_ruber_PA_Colon_Gamboa AJC_1120_Scinax_ruber_PA_Colon_Gamboa CH_9157_Scinax_ruber_PA_Darien_Cana AJC_1584_Scinax_ruber_PA_Panama_Chepo AJC_1583_Scinax_ruber_PA_Panama_Chepo CH_9319_Scinax_ruber_PA_Darien_Chucunaque AJC_3524_Scinax_ruber_CO_Santander_SanVicente AJC_4131_Scinax_elaeochrous_CO_Cordoba_Montelibano AJC_4160_Scinax_ruber_CO_Cordoba_Montelibano AJC_3751_Scinax_ruber_CO_Cordoba_Monteria AJC_3532_Scinax_ruber_CO_Santander_SanVicente JSM_234_Scinax_boulengeri_CO_Bolivar_Zambrano

458100 90 80 70 60 50 40 30 20 10 0 459 460 Figure 26. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 461 Scinax. Purple bars show confidence intervals for the node age. Country names are coded 462 as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 463 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 464 from South America, in red species from North America. 465 466 Pseudacris_maculata

MHUA_A_7773_Smilisca_sila_CO_Antioquia_Chigorodo

MHUA_A_4744_Smilisca_sila_CO_Antioquia_Sabanalarga

MHUA_A_7289_Smilisca_sila_CO_Antioquia_Maceo

TCWC_84597_Smilisca_sila_CR_SanJose_Carara

AJC_1618_Smilisca_sila_PA_Darien

AJC_1626_Smilisca_sila_PA_Darien_Chepigana

AJC_1350_Smilisca_sila_PA_Panama_Chame

AJC_1978_Smilisca_sila_PA_Panama_Chilibre

FROG_1000_Smilisca_sila_PA_Cocle_Cope

KRL_1641_Smilisca_sila_PA_Cocle_Cope

Pichi_327_Smilisca_sordida_CR_Puntarenas

Pichi_329_Smilisca_sordida_CR_Alajuela

Pichi_331_Smilisca_sordida_CR_Limon

KRL_0865_Smilisca_phaeota_PA_Cocle_Cope

Pichi_322_Smilisca_puma_CR_Alajuela

VCR_179_Smilisca_puma_CR_Heredia

Pichi_319_Smilisca_phaeota_CR_Puntarenas

Pichi_311_Smilisca_phaeota_CR_Alajuela

Pichi_320_Smilisca_phaeota_CR_SanJose

AJC_1048_Smilisca_phaeota_CO_Antioquia_Maceo

AJC_1517_Smilisca_phaeota_PA_Panama_Chepo

AJC_3525_Smilisca_phaeota_CO_Santander_SanVicente

KU_217770_Smilisca_phaeota_EC_Manabi

AJC_3696_Smilisca_phaeota_CO_Valle_Cali

AJC_4280_Smilisca_phaeota_CO_Narino_Tumaco

CH_6316_Smilisca_phaeota_PA_Darien_Pinogana

AJC_1185_Smilisca_phaeota_CO_Choco_Arusi

LAR_0001_Smilisca_phaeota_CO_Choco_Quibdo

MHUA_A_7083_Smilisca_phaeota_CO_Choco_BahiaSolano

MHUA_A_7798_Smilisca_phaeota_CO_Antioquia_Chigorodo

46760 50 40 30 20 10 0 468 469 Figure 27. Bayesian MCMC divergence times analysis with mtDNA samples for the genus 470 Smilisca. Purple bars show confidence intervals for the node age. Country names are 471 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 472 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 473 from South America, in red species from North America. 474 475 476 477 478 479 480 481 482 483 484 485 MVZ_225749_Rana_luteiventris

KRL_823_Lithobates_warszewitschii_PA_Cocle_Cope

AJC_1387_Lithobates_warszewitschii_PA_Panama_Carlos

CH_6145_Lithobates_warszewitschii_PA_Bocas_Changuinola

JSF_1127_Lithobates_warszewitschii_PA

KU_195299_Lithobates_vaillanti_MX_Oaxaca_Tapanatepec

TNHC_60324_Lithobates_juliani_BZ_Cayo_LittleVaquerosCreek

AMNH_A_166454_Lithobates_palmipes_GY_PotaroSiparuni

AJC_3541_Lithobates_vaillanti_CO_Tolima_Mariquita

CH_6554_Lithobates_vaillanti_PA_Bocas_Changuinola

MHUA_A_4880_Lithobates_vaillanti_CO_Antioquia_Maceo

486 40 35 30 25 20 15 10 5 0 487 488 Figure 28. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 489 Lithobates. Purple bars show confidence intervals for the node age. Country names are 490 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 491 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 492 from South America, in red species from North America. 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 ICN_55762_Pristimantis_parectatus_CO_Antioquia_Sonson

CH_9068_Craugastor_longirostris_PA_Darien_Garachine

AJC_1955_Craugastor_longirostris_PA_Panama_Chagres

CH_6513_Craugastor_longirostris_PA_Bocas_Changuinola

CH_6055_Craugastor_tabasarae_PA_Panama_Chepo

KRL_1387_Craugastor_tabasarae_PA_Cocle_Cope

AJC_1719_Craugastor_talamancae_PA_Bocas_Changuinola

AJC_0207_Craugastor_talamancae_PA_GunaYala_Warsobtugua

AJC_1907_Craugastor_talamancae_PA_Panama_Chepo

AJC_0209_Craugastor_crassidigitus_PA_KunaYala_Nusgandi

AJC_1201_Craugastor_crassidigitus_PA_Panama_Chame

AJC_1720_Craugastor_crassidigitus_PA_Bocas_Changuinola

AJC_1731_Craugastor_crassidigitus_PA_Bocas_Changuinola

AJC_7036_Craugastor_cf_fitzingeri_CO_Choco_Panamericana

CH_6152_Craugastor_fitzingeri_PA_Bocas_Changuinola

AJC_1774_Craugastor_fitzingeri_PA_Panama_Chilibre

CH_9012_Craugastor_fitzingeri_PA_Darien_Garachine

AJC_4279_Craugastor_longirostris_CO_Narino_Tumaco

KU_177803_Craugastor_longirostris_EC_Pichincha_SantoDomingo

AJC_1336_Craugastor_longirostris_CO_Antioquia_Maceo

MHUA_A_7259_Craugastor_raniformis_CO_Antioquia_Carlos

MHUA_A_7286_Craugastor_raniformis_CO_Antioquia_Maceo

MHUA_A_6769_Craugastor_longirostris_CO_Antioquia_Chigorodo

MHUA_A_7800_Craugastor_raniformis_CO_Antioquia_Chigorodo

AJC_2095_Craugastor_raniformis_CO_Choco_Nuqui

AJC_1612_Craugastor_raniformis_PA_Darien_Chepigana

AJC_1515_Craugastor_raniformis_PA_Panama_Chepo

CH_6074_Craugastor_raniformis_PA_Panama_Canas

70 510 60 50 40 30 20 10 0 511 512 Figure 29. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 513 Craugastor. Purple bars show confidence intervals for the node age. Country names are 514 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 515 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 516 from South America, in red species from North America. 517 518 519 520 521 522 523 524 525 526 527 528 529 530 55MC_Lithodytes_lineatus_FG_GrandSanti

QCAZ_16621_Lithodytes_lineatus_EC_MoronaSantiago_Mendez

JSM_206_Leptodactylus_fuscus_CO_Huila_Garzon

JSM_227_Leptodactylus_poecilochilus_CO_Bolivar_Zambrano

AJC_0350_Leptodactylus_poecilochilus_PA_Colon_Gamboa

AJC_1813_Leptodactylus_poecilochilus_PA_Darien_Pinogana

AJC_0535_Leptodactylus_fragilis_PA

AJC_1523_Leptodactylus_fragilis_PA_Panama_Chepo

AJC_4664_Leptodactylus_fragilis_CO_Antioquia_Choco

CH_8503_Leptodactylus_fragilis_PA_Veraguas_Montijo

KRL_0678_Leptodactylus_fragilis_PA_Cocle_Cope

109MC_Leptodactylus_pentadactylus_FG_Kaw

396MC_Leptodactylus_knudseni_FG_Lucifer

MHUA_A_7387_Leptodactylus_savagei_CO_Bolivar_Norosi

CH_6585_Leptodactylus_savagei_PA_Bocas_Changuinola

AJC_1502_Leptodactylus_savagei_PA_Panama_Chepo

AJC_1809_Leptodactylus_savagei_PA_Darien_Pinogana

531AF_Leptodactylus_longirostris_FG_Nouragues_Inselberg

ULABG_4591_Leptodactylus_fuscus_VE_Bolivar_Canaima

AJC_0858_Leptodactylus_melanonotus_PA_Cocle_Anton

AJC_0953_Leptodactylus_melanonotus_CR_Alajuela_Arenal

MVZ_207294_Leptodactylus_melanonotus_CR_Guanacaste_SantaRosa

J_AF_2012_978163_Leptodactylus_cf_wagneri_BR_MatoGrosso_VilaRica

MACN_38648_Leptodactylus_latrans_AR_BuenosAires_Escobar

MD_2279_Leptodactylus_latrans_BR_Bahia_Una

531 60 50 40 30 20 10 0 532 533 Figure 30. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 534 Leptodactylus. Purple bars show confidence intervals for the node age. Country names are 535 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 536 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 537 from South America, in red species from North America. 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 ZUFRJ_7926_Hylomantis_granulosa

SMS_Agalychnis_hulli_EC_Napo_SelvaViva

CH_6779_Agalychnis_lemur_PA_Panama_Chilibre

KRL_955_Agalychnis_lemur_PA_Cocle_Cope

HenryVilaZoo_Agalychnis_annae_CR_SanJose

AJC_7022_Agalychnis_spurrelli_CO_Choco_Panamericana

KU_217507_Agalychnis_spurrelli_EC_Pichincha_VicenteMaldonado

IGM_12_Agalychnis_saltator_CR_Limon_AltoColorado

COLZOOCH_H_2679_Agalychnis_aff_callidryas_CO_Choco_Panamericana

RdS_537_Agalychnis_callidryas_BZ_StannCreek

IGM_10_Agalychnis_annae_CR_SanJose_ZooSimonBolivar

AJC_1768_Agalychnis_callidryas_PA_Panama_Chilibre

AJC_1735_Agalychnis_callidryas_PA_Bocas_Changuiola

IGM_11_Agalychnis_callidryas_CR_Limon_Colorado

555 50 40 30 20 10 0 556 557 Figure 31. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 558 Agalychnis. Purple bars show confidence intervals for the node age. Country names are 559 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 560 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 561 from South America, in red species from North America. 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 SIUC_H_06924_Hyloscirtus_palmeri

SIUC_H_06926_Hyloscirtus_colymba

33mc_Boana_geographicus_GY_GrandSanti

CFBH_5424_Boana_semilineatus_BR_RioDeJaneiro_DuqueDeCaixas

KU_215191_Boana_boans_PE_MadreDeDios_PtoMaldonado

99bm_Boana_boans_GY_MontBakra

RWM_17746_Boana_boans_VE_Amazonas_AguaBlanca

CH_5307_Boana_boans_PA

CH_6332_Boana_boans_PA_Darien_Pinogana

CH_7040_Boana_boans_PA

ROM_44089_Boana_xerophylla_GY_Parish_Hill

AJC_7040_Boana_rufitela_CO_Choco_Panamericana

30594_Boana_pelluces_EC_ElOro_Avanzada

23680_Boana_pelluces_EC_Guayas_Huaquillas

AJC_7043_Boana_rufitela_CO_Choco_Panamericana

AJC_1389_Boana_rufitela_PA_Panama_Chame

CH_10337_Boana_rufitela_PA

AJC_1180_Boana_rufitela_PA

KRL_0798_Boana_rufitelus_PA_Cocle_Cope

CH_10046_Boana_rufitela_PA

AJC_1099_Boana_rufitela_PA_Colon_Gatun

CH_10014_Boana_rufitela_PA

CH_8501_Boana_pugax_PA_Veraguas_Catival

CH_8505_Boana_pugax_PA_Veraguas_Montijo

MHUA_A_7797_Boana_rosenbergi_CO_Antioquia_Chigorodo

AJC_1509_Boana_rosebergi_PA_Panama_Chepo

CH_9082_Boana_rosebergi_PA_Darien_Garachine

CFBH_4000_Boana_lundii_BR_SaoPaulo_RioClaro

USNM_303046_Boana_pardalis_BR_SaoPaulo_Salesopolis

AJC_1323_Boana_xerophylla_CO_Tolima_Mariquita

AJC_1078_Boana_xerophylla_PA_Panama_BuenaVista

AJC_1124_Boana_xerophylla_PA_Panama_Ancon

ROM_28436_Boana_xerophylla_GY_Paramakatoi

CFBH_2966_Boana_crepitans_BR_Alagoas_Piranhas

ROM_20560_Boana_xerophylla_GY_Kurupukari

577 80 70 60 50 40 30 20 10 0 578 579 Figure 32. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 580 Boana. Purple bars show confidence intervals for the node age. Country names are coded 581 as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 582 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 583 from South America, in red species from North America. 584 585 586 587 588 589 590 Pseudis_paradoxa

28mc_Dendropsophus_brevifrons_FG_Cayenne_MtKaw

KU_215248_Dendropsophus_koechlini_PE_MadreDeDios_PtoMaldonado

AMNH_A_139315_Dendropsophus_parviceps_BR_Acre_RioBranco

LSB_331_Dendropsophus_subocularis_CO_Antioquia_Rafael

COLZOOCH_H_2551_Dendropsophus_phlebodes_CO_Choco_Panamericana

AJC_0876_Dendropsophus_phlebodes_PA_Bocas_Colon

AJC_1788_Dendropsophus_phlebodes_PA_Panama_Chilibre

CH_9066_Dendropsophus_phlebodes_PA_Darien_Garachine

AJC_2352_Dendropsophus_microcephalus_CO_Guajira_Dibulla

AJC_0899_Dendropsophus_microcephalus_PA_Colon_Galeta

CH_5683_Dendropsophus_microcephalus_PA_Chiriqui_David

AJC_4035_Dendropsophus_microcephalus_CO_Santander_Vicente

AJC_4838_Dendropsophus_microcephalus_CO_Atlantico_Tubara

AJC_0954_Dendropsophus_microcephalus_CR_Alajuela_Arenal

ENS_10627_Dendropsophus_microcephalus_HN_Paraiso_Chichicaste

UTA_50632_Dendropsophus_microcephalus_HN_Atlantida_RioViejo

MJH_3844_Dendropsophus_triangulum_BR_Acre

QCAZA_44826_Dendropsophus_triangulum_EC_Orellana_RioNapo

QCAZA_44457_Dendropsophus_triangulum_EC_Orellana_Chiroisla

QCAZA_44539_Dendropsophus_triangulum_EC_Orellana_Huiririma

AJC_3837_Dendropsophus_subocularis_CO_Santander_Nacional

CH_6627_Dendropsophus_ebraccatus_PA_Bocas_Changuinola

AJC_0406_Dendropsophus_ebraccatus_CR_Limon_Pacurita

UTA_51789_Dendropsophus_ebraccatus_NG_Matagalpa_PenasBlancas

RdS_790_Dendropsophus_ebraccatus_BZ_StannCreek

CH_6335_Dendropsophus_ebraccatus_PA_Darien_Pinogana

AJC_1338_Dendropsophus_ebraccatus_CO_Antioquia_Maceo

AJC_3502_Dendropsophus_ebraccatus_CO_Santander_Vicente

LSB_323_Dendropsophus_ebraccatus_CO_Antioquia_Carlos

591 60 50 40 30 20 10 0 592 593 Figure 33. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 594 Dendropsophus. Purple bars show confidence intervals for the node age. Country names 595 are coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador 596 (EC), Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue 597 species from South America, in red species from North America. 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 AMNH_A_141040_Hypsiboas_multifasciatus

MVZ_207215_Scinax_boulengeri_CR

AJC_0577_Scinax_boulengeri_PA_Darien_PNNDarien

AJC_1115_Scinax_boulengeri_PA_Colon_Cumbres

247mc_Scinax_rostratus_FG

AJC_4142_Scinax_rostratus_CO_Cordoba_Montelibano

AJC_1518_Scinax_rostratus_PA_Panama_Chepo

JSM_171_Scinax_rostratus_CO_Magdalena_Granada

135PG_Scinax_cruentomma_FG

320MC_Scinax_cf_ruber_x_signatus_FG_Bourda

AJC_4146_Scinax_elaeochroa_CO_Cordoba_Montelibano

AJC_4609_Scinax_x_signatus_CO_Antioquia_Berrio

2131VOGT_Scinax_ruber_BR_Amazonas_Cachoeirinha

190MC_Scinax_x_signatus_FG

AJC_1120_Scinax_ruber_PA_Colon_Gamboa

AJC_1119_Scinax_ruber_PA_Colon_Gamboa

AJC_1584_Scinax_ruber_PA_Panama_Chepo

AJC_0872_Scinax_elaeochroa_PA_Bocas_Colon

MVZ_203919_Scinax_elaeochroa_CR_Heredia_LaSelva

COLZOOCH_H_2676_Scinax_elaeochroa_CO_Choco_Panamericana

AJC_1817_Scinax_sp_PA_Darien_Pinogana

AJC_1827_Scinax_sp_PA_Darien_Pinogana

AJC_3461_Scinax_wandae_CO_Meta_Gaitan

AJC_1125_Scinax_altae_PA_Panama_Cumbres

CH_5684_Scinax_altae_PA_Chiriqui_Guarumal

UTA_A_50749_Scinax_staufferi_GU_Zacapa

ENS_10717_Scinax_staufferi_HN_Olancho_Corocito

UTA_A_50749_Scinax_staufferi_GT_Zacapa_Teculatan

125 613 100 75 50 25 0 614 615 Figure 34. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 616 Scinax. Purple bars show confidence intervals for the node age. Country names are coded 617 as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 618 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 619 from South America, in red species from North America. 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 JFBM_14310_Pseudacris_maculata

MF4720_Smilisca_sordida_CR_SanJose

CH_6543_Smilisca_sordida_PA_Bocas_Changuinola

CH_6537_Smilisca_sordida_PA_Bocas_Changuinola

CH_6621_Smilisca_sordida_PA_Bocas_Changuinola

CH_9358_Smilisca_sila_PA_Darien_Garachine

AJC_0855_Smilisca_sila_PA_Cocle_Anton

AJC_1350_Smilisca_sila_PA_Panama_Chame

MHUA_A_7289_Smilisca_sila_CO_Antioquia_Maceo

MHUA_A_4744_Smilisca_sila_CO_Antioquia_Sabanalarga

MHUA_A_7773_Smilisca_sila_CO_Antioquia_Chigorodo

AJC_0933_Smilisca_phaeota_CR_Limon_Siquirres

CH_6130_Smilisca_phaeota_PA_Bocas_Changuinola

MFF4410_Smilisca_puma_CR_Heredia

VCR_179_Smilisca_puma_CR_Heredia

KU_217770_Smilisca_phaeota_EC_Manabi

AJC_4280_Smilisca_phaeota_CO_Narino_Tumaco

AJC_3525_Smilisca_phaeota_CO_Santander_Vicente

CH_6316_Smilisca_phaeota_PA_Darien_Pinogana

LAR_0001_Smilisca_phaeota_CO_Choco_Quibdo

70 635 60 50 40 30 20 10 0 636 637 Figure 35. Bayesian MCMC divergence times analysis with ncDNA samples for the genus 638 Smilisca. Purple bars show confidence intervals for the node age. Country names are 639 coded as follows: Belize (BZ), Brazil (BR), Colombia (CO), Costa Rica (CR), Ecuador (EC), 640 Guyana (GY), Mexico (MX), Panama (PA), Peru (PE), and Venezuela (VE). In blue species 641 from South America, in red species from North America. 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 dry wet

5

bio_18

bio_12 bio_14bio_17 bio_16bio_13 CLYPPT_M_sl7_1km_ll bio_19

bio_4 0 bio_7bio_2 bio_3

PC2 (26.4% explained var.) explained (26.4% PC2 CECSOL_M_sl7_1km_llbio_6

bio_15 bio_9 bio_11 bio_8bio_1

bio_5 bio_10

-5

-5 0 5 10 PC1 (46.2% explained var.) 658 659 660 Figure 36. Principal Component Analysis (PCA) for the bioclimatic and soil variables. PC1 661 explain the 46.2% of the variation with the variables of precipitation that describes the 662 “wet” environments, and temperature variables that describes the “dry” environments. 663 664 665 666 667 668 669 670 0.4 0.2 0.0

-4 -2 0 2 4 6

group dry 0.4 0.2 0.0

-4 -2 0 2 4 6

group wet 671 672 673 Figure 37. Histogram for the predictors of the Linear Discriminant Analysis (LDA). The 674 analysis shows two groups of variables that describes the “wet” and “dry” environments. 675 1 Table S1. Data set of new sequences of mitochondrial and nuclear sequences for taxonomic and geographic sampling for

2 each species

Mitochondrial Institutional/Field Deparmen Municipality Longitu Family Species Country Latitude Elevation genes Nuclear genes collection number t/Province /District de 16S COI POMC TYR Phyllomedusida AJC 1768 e Agalychnis callidryas Panama Panama Chilibre 9.23 -79.40 675 - - 555 531 Phyllomedusida AJC 2086 e Agalychnis callidryas Colombia Choco Nuqui 5.57 -77.50 20 689 - - - Union Phyllomedusida Panamerica AJC 7022 e Agalychnis spurrelli Colombia Choco na 5.32 -76.62 138 689 614 555 531 Phyllomedusida CH 6779 e Agalychnis lemur Panama Panama Chilibre 9.23 -79.40 800 - - 555 531 Union COLZOOCH-H Phyllomedusida Panamerica 2679 e Agalychnis terranova Colombia Choco na 5.31 -76.61 151 689 614 555 531 Phyllomedusida AJC 1595 e Agalychnis callidryas Panama Darien Chepigana 8.79 -78.45 797 - - 555 531 Bocas Del AJC 0877 Hylidae Boana rufitelus Panama Toro Isla Popa 9.22 -82.11 20 - - 555 531 AJC 1051 Hylidae Boana boans Panama Panama Chepo 9.00 -78.75 130 - - 555 531 AJC 1078 Hylidae Boana xerophylla Panama Panama Buena Vista 9.17 -79.41 640 - - 555 531 AJC 1099 Hylidae Boana rufitelus Panama Colon Gatun 9.25 -79.96 140 - - 555 531 AJC 1124 Hylidae Boana xerophylla Panama Panama Ancon 9.07 -79.65 89 - - 555 531 AJC 1323 Hylidae Boana xerophylla Colombia Tolima Mariquita 5.31 -74.91 516 - - 555 531 AJC 1375 Hylidae Boana boans Colombia Tolima Falan 5.12 -74.97 580 - 614 - - AJC 1509 Hylidae Boana rosenbergi Panama Panama Chepo 8.96 -78.46 120 - - 555 531 AJC 2345 Hylidae Boana xerophylla Colombia La Guajira Dibulla 11.25 -73.56 25 689 614 - - AJC 2553 Hylidae Boana nympha Colombia Amazonas Leticia -4.14 -69.92 89 689 - - - Cundinam AJC 3765 Hylidae Boana pugnax Colombia arca Nilo 4.36 -74.56 700 689 - - - Puente AJC 3843 Hylidae Boana xerophylla Colombia Santander Nacional 5.90 -73.70 1720 689 614 - - AJC 4883 Hylidae Boana pugnax Colombia Atlantico Tubara 10.89 -75.00 203 689 - - - Union Panamerica AJC 7040 Hylidae Boana rufitelus Colombia Choco na 5.33 -76.63 81 689 614 555 531 Union Panamerica AJC 7043 Hylidae Boana rufitelus Colombia Choco na 5.32 -76.62 110 689 614 555 531 CH 6332 Hylidae Boana boans Panama Darien Pinogana 7.72 -77.66 449 - - 555 531 CH 8501 Hylidae Boana pugnax Panama ------555 531 CH 8505 Hylidae Boana pugnax Panama ------555 531 CH 9082 Hylidae Boana rosenbergi Panama Darien Garachine 8.07 -78.36 6 - - 555 531 MHUA-A 7797 Hylidae Boana rosenbergi Colombia Antioquia Chigorodo 7.53 -76.59 94 689 614 555 531 Craugastor Kuna Yala, AJC 0207 Craugastoridae talamancae Panama San Blas Nusgandi 9.32 -78.98 400 689 - 555 531 AJC 1515 Craugastoridae Craugastor raniformis Panama Panama Chepo 8.96 -78.47 130 - - 555 531 AJC 1612 Craugastoridae Craugastor raniformis Panama Darien Chepigana 8.79 -78.45 797 - - 555 531 Craugastor Bocas Del AJC 1719 Craugastoridae talamancae Panama Toro Changuinola 9.07 -82.47 245 - - 555 531 AJC 1774 Craugastoridae Craugastor fitzingeri Panama Panama Chilibre 9.23 -79.40 675 - - 555 531 AJC 1955 Craugastoridae Craugastor longirostris Panama Panama San Miguel 9.32 -79.29 800 - - 555 531 AJC 2095 Craugastoridae Craugastor raniformis Colombia Choco Nuqui 5.57 -77.50 20 - - 555 531 AJC 4279 Craugastoridae Craugastor longirostris Colombia Narino Tumaco 1.42 -78.37 251 - - 555 531 Union Craugastor cf. Panamerica AJC 7036 Craugastoridae fitzingeri Colombia Choco na 5.33 -76.63 109 689 614 555 531 CH 6055 Craugastoridae Craugastor tabasarae Panama Panama Chepo - - - - - 555 531 CH 6074 Craugastoridae Craugastor raniformis Panama Panama Isla Canas 8.37 -78.82 2 - - 555 531 Bocas Del CH 6152 Craugastoridae Craugastor fitzingeri Panama Toro Changuinola 9.21 -82.48 120 - - 555 531 Bocas Del CH 6513 Craugastoridae Craugastor longirostris Panama Toro Changuinola 9.14 -82.50 117 - - 555 531 CH 9012 Craugastoridae Craugastor fitzingeri Panama Darien Garachine 8.00 -78.35 250 - - 555 531 CH 9068 Craugastoridae Craugastor longirostris Panama Darien Garachine 7.98 -78.36 697 - - 555 531 MHUA-A 6769 Craugastoridae Craugastor longirostris Colombia Antioquia Chigorodo 7.52 -76.59 56 689 614 555 - MHUA-A 7259 Craugastoridae Craugastor raniformis Colombia Antioquia San Carlos -74.87 859.00 859 689 614 555 531 MHUA-A 7286 Craugastoridae Craugastor raniformis Colombia Antioquia Maceo -74.64 508.00 508 689 614 555 531 MHUA-A 7800 Craugastoridae Craugastor raniformis Colombia Antioquia Chigorodo 7.53 -76.59 94 689 614 555 - Dendropsophus AJC 0899 Hylidae microcephalus Panama Colon Galeta 9.40 -79.85 20 - - 555 531 Dendropsophus AJC 1338 Hylidae ebraccatus Colombia Antioquia Maceo 6.54 -74.64 504 - - 555 - Dendropsophus AJC 1788 Hylidae phlebodes Panama Panama Chilibre 9.17 -79.41 630 - - 555 531 Dendropsophus AJC 2352 Hylidae microcephalus Colombia La Guajira Dibulla 11.25 -73.56 25 - - 555 - Dendropsophus San Vicente AJC 3502 Hylidae ebraccatus Colombia Santander de Chucuri 7.08 -73.55 478 - - 555 531 Dendropsophus Puente AJC 3837 Hylidae subocularis Colombia Santander Nacional 5.90 -73.70 1720 - - 555 531 Dendropsophus San Juan de AJC 3924 Hylidae mathiassoni Colombia Meta Arama 3.23 -73.85 325 689 - - - Dendropsophus San Vicente AJC 4035 Hylidae microcephalus Colombia Santander de Chucuri 7.08 -73.55 155 - - 555 - Dendropsophus AJC 4366 Hylidae microcephalus Colombia Cordoba Planeta Rica 8.40 -75.58 52 689 - - - Dendropsophus AJC 4838 Hylidae microcephalus Colombia Atlantico Tubara 10.89 -75.00 64 689 - 555 - Dendropsophus CH 5683 Hylidae microcephalus Panama Chiriqui David 8.36 -82.52 10 - - 555 531 Dendropsophus CH 6335 Hylidae ebraccatus Panama Darien Pinogana 7.72 -77.66 462 - - 555 531 Dendropsophus Bocas Del CH 6627 Hylidae ebraccatus Panama Toro Changuinola 9.23 -82.50 84 - - 555 531 Dendropsophus CH 9066 Hylidae phlebodes Panama Darien Garachine 8.07 -78.36 6 - - 555 531 COLZOOCH-H Dendropsophus Union 2551 Hylidae phlebodes Colombia Choco Panamerica 5.33 -76.63 111 689 614 555 - Dendropsophus ENS 10627 Hylidae microcephalus Honduras Paraiso Chichicaste 14.11 -86.30 550 - - 555 531 Dendropsophus LSB 323 Hylidae ebraccatus Colombia Antioquia San Carlos 6.23 -74.85 832 - - 555 531 Leptodactylus AJC 0350 Leptodactylidae poecilochilus Panama Colon Gamboa 9.12 -79.70 30 - - 555 531 Leptodactylus AJC 0858 Leptodactylidae melanonotus Panama Cocle Anton 8.60 -80.14 612 - - 555 531 Leptodactylus Costa AJC 0953 Leptodactylidae melanonotus Rica Alajuela San Carlos 10.47 -84.74 587 - - 555 531 AJC 1502 Leptodactylidae Leptodactylus savagei Panama Panama Chepo 8.96 -78.47 130 - - 555 531 AJC 1523 Leptodactylidae Leptodactylus fragilis Panama Panama Chepo 8.96 -78.47 130 - - 555 531 AJC 1809 Leptodactylidae Leptodactylus savagei Panama Darien Pinogana 7.76 -77.68 525 - - 555 531 Leptodactylus AJC 1813 Leptodactylidae poecilochilus Panama Darien Pinogana 7.76 -77.68 550 - - 555 531 AJC 4133 Leptodactylidae Leptodactylus fuscus Colombia Cordoba Montelibano 7.97 -75.59 64 689 - - - AJC 4476 Leptodactylidae Leptodactylus fragilis Colombia Magdalena Pinuelas 10.45 -74.41 40 689 - - - Puerto AJC 4619 Leptodactylidae Leptodactylus fuscus Colombia Antioquia Berrio 6.49 -74.41 111 689 - - - Bocas de AJC 4664 Leptodactylidae Leptodactylus fragilis Colombia Antioquia Barbacoas 6.73 -74.42 102 - - 555 - AJC 4684 Leptodactylidae Leptodactylus fragilis Colombia Santander Santanderes 6.73 -74.21 122 689 - - - Leptodactylus AJC 4761 Leptodactylidae pentadactylus Colombia Amazonas Leticia -3.86 -70.21 90 689 - - - Leptodactylus Norte de AJC 4871 Leptodactylidae poecilochilus Colombia Santander Santanderes 7.75 -72.53 410 689 - - - Leptodactylus AJC 5784 Leptodactylidae melanonotus Colombia Boyaca Santa Maria 4.78 -73.28 520 689 - - - Leptodactylus AJC 6198 Leptodactylidae melanonotus Colombia Antioquia Anori 6.93 -75.16 860 689 - - - AJC 6545 Leptodactylidae Leptodactylus savagei Colombia Magdalena Santa Marta 11.23 -74.13 57 689 - - - Bocas Del CH 6585 Leptodactylidae Leptodactylus savagei Panama Toro Changuinola 9.07 -82.47 173 - - 555 531 JSM 206 Leptodactylidae Leptodactylus fuscus Colombia Huila Garzon 2.21 -75.64 811 - - 555 - Leptodactylus JSM 227 Leptodactylidae poecilochilus Colombia Bolivar Zambrano 9.75 -74.83 16 - - 555 - KRL 0678 Leptodactylidae Leptodactylus fragilis Panama Cocle El Cope 8.67 -80.59 750 - - 555 531 AJC 2308 Ranidae Lithobates palmipes Colombia Casanare Orucue 4.91 -71.43 138 689 - - - AJC 3541 Ranidae Lithobates vaillanti Colombia Tolima Mariquita 5.14 -74.90 382 - - 555 531 AJC 5473 Ranidae Lithobates palmipes Colombia Caqueta Valparaiso 1.09 -75.57 191 689 - - - Bocas Del CH 6554 Ranidae Lithobates vaillanti Panama Toro Changuinola 9.07 -82.47 173 - - 555 531 MHUA-A 4880 Ranidae Lithobates vaillanti Colombia Antioquia Maceo 6.55 -74.64 499 689 614 555 - AJC 0577 Hylidae Scinax boulengeri Panama Darien Pinogana 7.76 -77.68 500 - - 555 531 Bocas Del AJC 0872 Hylidae Scinax elaeochrous Panama Toro Isla Colon 9.35 -82.23 5 - - 555 531 AJC 1115 Hylidae Scinax boulengeri Panama Colon Las Cumbres 9.10 -79.68 80 - - 555 531 AJC 1119 Hylidae Scinax ruber Panama Colon Gamboa 9.11 -79.70 50 - - 555 531 AJC 1125 Hylidae Scinax altae Panama Panama Las Cumbres 9.07 -79.65 89 - - 555 531 AJC 1518 Hylidae Scinax rostratus Panama Panama Chepo 8.96 -78.46 120 - - 555 531 AJC 1583 Hylidae Scinax ruber Panama Panama Chepo 8.96 -78.47 130 - - 555 531 AJC 1817 Hylidae Scinax sp. Panama Darien Pinogana 7.76 -77.68 550 - - 555 531 AJC 1827 Hylidae Scinax sp. Panama Darien Pinogana 7.76 -77.68 525 - - 555 531 Puerto AJC 3461 Hylidae Scinax wandae Colombia Meta Gaitan 3.23 -73.86 308 - - 555 531 San Vicente AJC 3524 Hylidae Scinax ruber Colombia Santander de Chucuri 7.08 -73.55 155 689 - - - AJC 3562 Hylidae Scinax ruber Colombia Tolima Mariquita 5.26 -74.89 490 - 614 555 - AJC 3751 Hylidae Scinax ruber Colombia Cordoba Monteria 8.79 -75.86 60 689 - - - AJC 4058 Hylidae Scinax boulengeri Colombia Casanare Sabanalarga 4.77 -73.04 315 689 - - - AJC 4142 Hylidae Scinax rostratus Colombia Cordoba Montelibano 7.97 -75.59 64 - - 555 531 AJC 4146 Hylidae Scinax elaeochrous Colombia Cordoba Montelibano 7.97 -75.59 64 - 614 555 531 Puerto AJC 4609 Hylidae Scinax x-signatus Colombia Antioquia Berrio 6.49 -74.42 111 689 614 555 531 Puerto AJC 4648 Hylidae Scinax rostratus Colombia Antioquia Berrio 6.49 -74.42 111 689 - - - Norte de AJC 4964 Hylidae Scinax rostratus Colombia Santander Santanderes 7.75 -72.53 410 689 - - - CH 5684 Hylidae Scinax altae Panama Chiriqui Guarumal 8.36 -82.52 10 - - 555 531 COLZOOCH-H Union 2676 Hylidae Scinax elaeochrous Colombia Choco Panamerica 5.22 -76.67 90 689 614 555 531 ENS 10717 Hylidae Scinax staufferi Honduras Olancho Corocito 14.98 -86.10 879 - - 555 531 JSM 171 Hylidae Scinax rostratus Colombia Magdalena Granada 10.98 -74.79 40 - - 555 531 JSM 234 Hylidae Scinax boulengeri Colombia Bolivar Zambrano 9.75 -74.83 16 - 614 555 - AJC 0855 Hylidae Smilisca sila Panama Cocle Anton 8.60 -80.14 612 689 - 555 531 Costa AJC 0933 Hylidae Smilisca phaeota Rica Limon Siquirres 10.06 -83.55 600 - - 555 531 AJC 1350 Hylidae Smilisca sila Panama Panama Chame 8.64 -80.07 920 - - 555 531 San Vicente AJC 3525 Hylidae Smilisca phaeota Colombia Santander de Chucuri 7.08 -73.55 155 - - 555 531 AJC 4280 Hylidae Smilisca phaeota Colombia Narino Tumaco 1.42 -78.37 270 - - 555 - CH 6316 Hylidae Smilisca phaeota Panama Darien Pinogana 7.76 -77.68 525 - - 555 531 Bocas Del CH 6546 Hylidae Smilisca phaeota Panama Toro Changuinola 9.14 -82.50 387 - - 555 531 CH 9358 Hylidae Smilisca sila Panama Darien Garachine 8.01 -78.35 215 - - 555 531 LAR 0001 Hylidae Smilisca phaeota Colombia Choco Quibdo 5.68 -76.59 72 - - 555 531 MHUA-A 4744 Hylidae Smilisca sila Colombia Antioquia Sabanalarga 6.85 -75.82 790 689 614 555 531 Bahia MHUA-A 7083 Hylidae Smilisca phaeota Colombia Choco Solano 6.11 -77.44 100 689 - - - MHUA-A 7289 Hylidae Smilisca sila Colombia Antioquia Maceo 6.54 -74.64 521 689 - - 531 MHUA-A 7773 Hylidae Smilisca sila Colombia Antioquia Chigorodo 7.53 -76.59 94 689 614 555 531 MHUA-A 7798 Hylidae Smilisca phaeota Colombia Antioquia Chigorodo 7.53 -76.59 94 689 - - - 3

4

5

6

7

8

9 10 Table S2. Samples obtained from the Barcode of Life Data system (BoLD) for mitochondrial fragments.

Institutional/Field Latitud Longitud Elevatio collection Family Species Country State/Province Municipality Processes 16S COI e e n number Phyllomedusida Costa 567[0n Pichi 251 e Agalychnis annae Rica Heredia Roble 10.07 -84.16 1400 BSUCR251-12 ] 657[0n] Phyllomedusida 568[0n LSB 134 e Agalychnis buckleyi Colombia Antioquia Yarumal 7.09 -75.47 1776 BSAMS271-12 ] 658[0n] Phyllomedusida 568[0n LSB 135 e Agalychnis buckleyi Colombia Antioquia Yarumal 7.09 -75.47 1776 BSAMS272-12 ] 658[0n] Phyllomedusida 566[0n AJC 1565 e Agalychnis callidryas Panama Panama Chepo 8.96 -78.46 120 BSUCR260-9 ] 658[0n] Phyllomedusida 566[0n AJC 1735 e Agalychnis callidryas Panama Bocas Del Toro Changuinola 9.07 -82.47 245 BSUCR260-6 ] 658[0n] Phyllomedusida Costa Pacifico Pichi 252 e Agalychnis callidryas Rica Puntarenas Central 9.51 -84.38 10 BSUCR252-12 N/A 635[5n] Phyllomedusida Costa 568[0n Pichi 253 e Agalychnis callidryas Rica San Jose Carara 9.74 -84.53 300 BSUCR253-12 ] 657[0n] Phyllomedusida toe 1 Acal e Agalychnis callidryas Panama Cocle El Cope 8.67 -80.59 750 BSAMP352-07 N/A 648[0n] Phyllomedusida Agalychnis cf. 569[0n AJC 2087 e callidryas Colombia Choco Nuqui 5.57 -77.50 20 BSAMS042-12 ] 658[0n] Comunidad Phyllomedusida Simon Bolivar ZONIA5323- 804[0n MZUTI 168 e Agalychnis hulli Ecuador Pastaza Sacha Yacu -1.41 -77.70 914 18 ] 613[0n] Comunidad Phyllomedusida Simon Bolivar ZONIA5324- 806[0n MZUTI 169 e Agalychnis hulli Ecuador Pastaza Sacha Yacu -1.41 -77.70 914 18 ] 588[0n] Comunidad Phyllomedusida Simon Bolivar ZONIA5325- 804[0n MZUTI 170 e Agalychnis hulli Ecuador Pastaza Sacha Yacu -1.41 -77.70 914 18 ] 597[0n] Comunidad Phyllomedusida Simon Bolivar ZONIA5326- 765[0n MZUTI 185 e Agalychnis hulli Ecuador Pastaza Sacha Yacu -1.41 -77.70 878 18 ] 617[0n] Comunidad Phyllomedusida Simon Bolivar ZONIA5327- 818[0n MZUTI 313 e Agalychnis hulli Ecuador Pastaza Sacha Yacu -1.25 -77.42 838 18 ] 612[0n] Phyllomedusida 602[0n toe 134 Hlem e Agalychnis lemur Panama Cocle El Cope 8.67 -80.59 750 BSAMP356-07 ] N/A Phyllomedusida Costa 561[0n Pichi 258 e Agalychnis lemur Rica Limon Asuncion 9.93 -83.19 400 BSUCR258-12 ] 656[0n] Phyllomedusida Costa Pacifico 540[0n Pichi 262 e Agalychnis spurrelli Rica San Jose Central 9.74 -84.53 300 BSUCR262-12 ] N/A 560[0n AJC 1320 Hylidae Boana boans Colombia Tolima Mariquita 5.31 -74.91 516 BSAMS020-12 ] 658[0n] 555[0n AJC 2329 Hylidae Boana boans Colombia Vaupes Mitu 1.21 -70.62 - ZONIF110-12 ] 655[0n] Puerto 554[0n AJC 2476 Hylidae Boana boans Colombia Guainia Colombia 2.27 -68.27 - ZONIF188-12 ] 655[0n] 556[0n AJC 2541 Hylidae Boana boans Colombia Amazonas Leticia -4.14 -69.92 - ZONIF232-12 ] 655[0n] Venezuel 558[0n AJC 3268 Hylidae Boana boans a Delta Amacuro - 8.34 -61.69 - ZONIA222-12 ] 661[0n] San Juan de 559[0n AJC 3443 Hylidae Boana boans Colombia Meta Arama 3.23 -73.86 - BSECO220-11 ] 658[0n] 555[0n AMHN 20098 Hylidae Boana boans Brazil Roraima Rorainopolis 0.20 -60.65 - ZONIG370-13 ] 585[0n] Puerto 556[0n CORBIDI 5173 Hylidae Boana boans Peru Madre de Dios Maldonado -12.84 -69.30 BSAPE404-12 ] 658[0n] Venezuel 558[0n MHNLS 19685 Hylidae Boana boans a Bolivar Suapure 7.12 -65.23 - AMTMP214-15 ] 658[0n] Novo 556[0n MTR ALCX69P42 Hylidae Boana boans Brazil Amazonas Aripuana -7.62 -60.67 - ABRAM047-13 ] 464[0n] 555[0n PK 1129 Hylidae Boana boans Guiana Potaro-Siparuni - 5.17 -59.48 - ZONIE382-12 ] 655[0n] San Vicente 585[0n AJC 3501 Hylidae Boana pugnax Colombia Santander de Chucuri 7.08 -73.55 478 BSAMS110-12 ] 584[0n] Sabana de 570[0n AJC 3870 Hylidae Boana pugnax Colombia Santander Torres 7.40 -73.50 - BSECO299-11 ] 658[0n] 588[0n AJC 5138 Hylidae Boana pugnax Colombia Cesar Boscania 9.98 -73.89 - DRYCO035-15 ] 658[0n] 474[0n JMR 5959 Hylidae Boana pugnax Colombia Sucre San Onofre 10.02 -75.52 - DRYCO079-15 ] 658[0n] 472[0n AJC 4627 Hylidae Boana pugnax Colombia Antioquia Puerto Berrio 6.49 -74.42 - DRYCO015-15 ] 625[0n] Venezuel 569[0n MHNLS 16827 Hylidae Boana pugnax a Zulia El Derote 10.26 -72.08 - AMTMP308-15 ] 642[0n] BSAMP1025- 565[0n CH 6337 Hylidae Boana rosenbergi Panama Darien Pinogana 7.72 -77.66 - 09 ] 658[0n] 574[0n CH 8792 Hylidae Boana rosenbergi Panama Colon Gamboa - - - BSAMQ591-13 ] 658[0n] Costa 567[0n Pichi 281 Hylidae Boana rosenbergi Rica Puntarenas Quepos 9.41 -84.16 148 BSUCR281-12 ] 657[0n] Costa Pacifico Pichi 282 Hylidae Boana rosenbergi Rica San Jose Central 9.74 -84.53 300 BSUCR282-12 N/A 587[5n] 568[0n AJC 1382 Hylidae Boana rufitelus Panama Panama Chame 8.64 -80.07 920 BSAMM044-09 ] 517[0n] 598[0n KRL 0798 Hylidae Boana rufitelus Panama Cocle El Cope 8.67 -80.59 750 BSAMP060-07 ] 648[0n] 570[0n AJC 2341 Hylidae Boana sp. Colombia La Guajira Dibulla 11.25 -73.56 - BSAMS091-12 ] 658[0n] 570[0n AJC 2344 Hylidae Boana sp. Colombia La Guajira Dibulla 11.25 -73.56 - BSAMS092-12 ] 658[0n] 570[0n AJC 4135 Hylidae Boana sp. Colombia Cordoba Montelibano 7.97 -75.59 - BSAMS188-12 ] 658[0n] 583[0n AJC 4149 Hylidae Boana sp. Colombia Cordoba Montelibano 7.97 -75.59 - BSAMS192-12 ] 654[0n] 570[0n AAV 151 Hylidae Boana xerophylla Colombia Santander Piedecuesta 6.78 -73.02 - BSAMS001-12 ] 658[0n] 568[0n AJC 1096 Hylidae Boana xerophylla Panama Panama Pacora 9.16 -79.21 65 BSAMQ272-09 ] 658[0n] 568[0n AJC 1123 Hylidae Boana xerophylla Panama Panama Las Cumbres 9.07 -79.65 - BSAMM010-09 ] 658[1n] San Juan de 601[0n AJC 1738 Hylidae Boana xerophylla Colombia Meta Arama 3.23 -73.85 - BSECO171-11 ] 658[0n] 564[0n AJC 2253 Hylidae Boana xerophylla Colombia Cundinamarca Guaduas 5.06 -74.60 - ZONIF087-12 ] 658[0n] Venezuel Puerto 567[0n AJC 2857 Hylidae Boana xerophylla a Amazonas Ayacucho 5.69 -67.61 - ZONIA003-12 ] 660[0n] Puente 570[0n AJC 3394 Hylidae Boana xerophylla Colombia Santander Nacional 5.88 -73.68 1623 BSECO102-11 ] 658[0n] San Vicente 570[0n AJC 3505 Hylidae Boana xerophylla Colombia Santander de Chucuri 7.08 -73.55 478 BSAMS114-12 ] 658[0n] 569[0n AJC 4014 Hylidae Boana xerophylla Colombia Boyaca Miraflores 5.21 -73.14 - BSECO163-11 ] 658[0n] 569[0n AJC 4108 Hylidae Boana xerophylla Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO058-11 ] 658[0n] 551[1n AJC 4300 Hylidae Boana xerophylla Colombia Meta San Martin 3.70 -73.70 405 DRYCO006-15 ] 608[0n] 567[1n AJC 4341 Hylidae Boana xerophylla Colombia Cundinamarca Nilo 4.36 -74.57 700 DRYCO010-15 ] 608[0n] 562[0n AJC 4563 Hylidae Boana xerophylla Colombia Meta Restrepo 4.26 -73.56 570 DRYCO018-15 ] N/A 563[1n AJC 4686 Hylidae Boana xerophylla Colombia Antioquia Yondo 6.73 -74.21 122 DRYCO033-15 ] N/A 568[0n JSM 183 Hylidae Boana xerophylla Colombia Huila Garzon 2.21 -75.64 811 BSAMS251-12 ] 658[0n] Venezuel 564[0n MHNLS 17218 Hylidae Boana xerophylla a Merida Andres Bello 8.68 -71.48 - ZONIF354-12 ] 655[0n] 564[0n INPA-H 31229 Hylidae Boana xerophylla Brazil Roraima - 0.20 -60.65 - ZONIG361-13 ] 655[0n] 570[0n JSM 181 Hylidae Boana xerophylla Colombia Huila Garzon 2.21 -75.64 - BSAMS237-12 ] 658[0n] MHUAA042- 491[0n MHUA-A 7285 Hylidae Boana xerophylla Colombia Antioquia Maceo - - - 13 ] 586[0n AJC 4161 Hylidae Boana xerophylla Colombia Santander Guapota 6.35 -73.31 - DRYCO347-16 ] 654[0n] Craugastor aff. 563[0n AJC 1939 Craugastoridae longirostris Panama Panama Chepo 9.32 -79.29 - BSAMP709-09 ] 658[0n] Craugastor aff. 602[0n KRL 1399 Craugastoridae longirostris Panama Cocle El Cope 8.67 -80.59 - BSAMP237-07 ] 648[0n] Craugastor 555[0n AJC 1201 Craugastoridae crassidigitus Panama Panama - 8.64 -80.07 - BSAMM033-09 ] 639[1n] Craugastor 538[0n AJC 1598 Craugastoridae crassidigitus Panama Darien Chepigana 8.79 -78.45 - BSAMP467-08 ] 658[0n] Craugastor BSAMP1208- 508[0n AJC 1731 Craugastoridae crassidigitus Panama Bocas Del Toro Changuinola 9.07 -82.47 - 09 ] 658[0n] 602[0n KRL 0693 Craugastoridae Craugastor fitzingeri Panama Cocle El Cope 8.67 -80.59 750 BSAMP021-07 ] 648[0n] CHOCO064- MHUA-A 4305 Craugastoridae Craugastor fitzingeri Colombia Choco Nuqui 5.59 -77.35 10 15 N/A 658[0n] Costa Sector 473[0n Pichi 067 Craugastoridae Craugastor fitzingeri Rica Guanacaste Murcielago 10.90 -85.73 100 BSUCR067-12 ] N/A Costa 472[0n Pichi 068 Craugastoridae Craugastor fitzingeri Rica Alajuela El Gavilan 10.87 -84.35 170 BSUCR068-12 ] N/A Costa 485[0n Pichi 070 Craugastoridae Craugastor fitzingeri Rica Puntarenas Pacifico Sur 8.18 -82.93 - BSUCR070-12 ] 479[0n CH 8438 Craugastoridae Craugastor fitzingeri Panama Chiriqui - - - - BSAMQ558-13 ] CHOCO019- 568[0n LAR 0005 Craugastoridae Craugastor fitzingeri Colombia Choco Quibdo 5.68 -76.59 - 15 ] 658[0n] Craugastor CHOCO060- 571[0n AJC 2092 Craugastoridae longirostris Colombia Choco Arusi 5.57 -77.50 20 15 ] N/A Craugastor 561[0n CH 6390 Craugastoridae longirostris Panama Darien Pinogana 7.77 -77.73 1597 BSAMP790-09 ] N/A Craugastor CHOCO024- LAR 0010 Craugastoridae longirostris Colombia Choco Quibdo 5.69 -76.59 - 15 N/A 615[0n] Craugastor CHOCO069- MHUA-A 6766 Craugastoridae longirostris Colombia Antioquia Chigorodo 7.58 -76.63 49 15 N/A 658[0n] Craugastor 563[0n CH 9040 Craugastoridae longirostris Panama Darien - 7.98 -78.36 - BSAMQ618-13 ] 658[0n] Craugastor 563[0n AJC 2116 Craugastoridae longirostris Colombia Choco Tacarcuna 8.16 -77.24 - BSAMS057-12 ] 658[0n] Craugastor 563[0n AJC 2125 Craugastoridae longirostris Colombia Choco Tacarcuna 8.16 -77.24 - BSAMS066-12 ] 658[0n] Craugastor Valle del 565[0n AJC 2079 Craugastoridae longirostris Colombia Cauca Buenaventura - - - BSAMS038-12 ] 658[0n] Craugastor 514[0n AJC 1193 Craugastoridae longirostris Colombia Choco Arusi 5.57 -77.50 65 BSATE135-11 ] 612[0n] Valle del 561[0n AJC 2080 Craugastoridae Craugastor raniformis Colombia Cauca Cali 3.55 -76.88 565 BSAMS039-12 ] 609[0n] CHOCO021- 565[0n LAR 0007 Craugastoridae Craugastor raniformis Colombia Choco Quibdo 5.69 -76.60 30 15 ] 658[0n] 658[0n EVACC_219 Craugastoridae Craugastor tabasarae Panama El Valle Rio Maria 8.63 -80.08 900 EVACC048-12 ] 561[0n] 648[0n KRL 0706 Craugastoridae Craugastor tabasarae Panama Cocle El Cope 8.67 -80.59 750 BSAMP026-07 ] 602[0n] Craugastor 495[0n AJC 1907 Craugastoridae talamancae Panama Panama Chepo 9.28 -79.34 810 BSAMP677-09 ] 564[1n] Craugastor 602[0n KRL 1520 Craugastoridae talamancae Panama Cocle El Cope 8.67 -80.59 - BSAMP291-07 ] 648[0n] Dendropsophus Costa 568[0n AJC 0406 Hylidae ebraccatus Rica Limon Pacurita 10.01 -83.54 530 BSAMQ216-09 ] 602[0n] Dendropsophus Costa 568[0n AJC 0951 Hylidae ebraccatus Rica Alajuela Volcan Arenal 10.47 -84.74 587 BSAMQ265-09 ] 658[0n] Dendropsophus 568[0n AJC 1118 Hylidae ebraccatus Panama Colon Gamboa 9.11 -79.70 50 BSAMM007-09 ] 658[0n] Dendropsophus 567[0n CH 6644 Hylidae ebraccatus Panama Panama Chilibre 9.17 -79.41 675 BSAMP604-08 ] N/A Dendropsophus 567[0n AJC 1790 Hylidae ebraccatus Panama Panama Chilibre 9.17 -79.41 - BSAMP597-08 ] 658[1n] Dendropsophus San Vicente 569[0n AJC 3504 Hylidae ebraccatus Colombia Santander de Chucuri 7.08 -73.55 - BSAMS113-12 ] 658[0n] Dendropsophus 504[0n CH 9321 Hylidae ebraccatus Panama Darien - - - - BSAMQ688-13 ] 658[0n] Dendropsophus San Juan de 568[0n AJC 2110 Hylidae mathiassoni Colombia Meta Arama 3.23 -73.85 325 BSECO216-11 ] 534[0n] Dendropsophus 568[0n AJC 2322 Hylidae mathiassoni Colombia Casanare Orucue 4.91 -71.43 138 BSAMS086-12 ] 609[0n] Dendropsophus 568[0n AJC 4052 Hylidae mathiassoni Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO013-11 ] 658[0n] Dendropsophus 568[0n LSB 363 Hylidae mathiassoni Colombia Meta Villavicencio 4.15 -73.63 538 BSAMS282-12 ] 658[0n] Dendropsophus San Juan de 568[0n AJC 1740 Hylidae mathiassoni Colombia Meta Arama 3.23 -73.85 - BSECO173-11 ] 611[0n] Dendropsophus 568[0n AJC 2322 Hylidae mathiassoni Colombia Casanare Orucue 4.91 -71.43 - BSAMS086-12 ] 609[0n] Dendropsophus 564[0n AJC 0994 Hylidae microcephalus Panama Panama Lago Bayano 9.03 -78.76 96 BSAMQ286-09 ] N/A Dendropsophus 562[0n AJC 1117 Hylidae microcephalus Panama Cocle El Cope 8.66 -80.59 460 BSAMQ288-09 ] 658[0n] Dendropsophus AJC 1514 Hylidae microcephalus Panama Panama Chepo 8.96 -78.47 130 BSAMP384-08 N/A 658[0n] Dendropsophus 561[0n AJC 1791 Hylidae microcephalus Panama Panama Chilibre 9.17 -79.41 630 BSAMP598-08 ] 658[0n] Dendropsophus 563[0n AJC 2349 Hylidae microcephalus Colombia Antioquia Maceo 6.54 -74.64 504 BSAMS094-12 ] 658[0n] Dendropsophus Sabana de 563[0n AJC 3869 Hylidae microcephalus Colombia Santander Torres 7.40 -73.50 - BSECO298-11 ] 658[0n] Dendropsophus AJC 4433 Hylidae microcephalus Colombia Magdalena Pinuelas 10.45 -74.41 40 DRYCO239-16 N/A 611[0n] Dendropsophus 520[0n AJC 4613a Hylidae microcephalus Colombia Antioquia Puerto Berrio 6.49 -74.42 111 DRYCO032-15 ] N/A Dendropsophus Armero AJC 5224 Hylidae microcephalus Colombia Tolima Guayabal 5.01 -74.91 300 DRYCO268-16 N/A 611[0n] Dendropsophus AJC 5337 Hylidae microcephalus Colombia Tolima Coello 4.26 -74.92 400 DRYCO279-16 N/A 611[0n] Dendropsophus AJC 5354 Hylidae microcephalus Colombia Huila Neiva 2.84 -75.33 400 DRYCO285-16 N/A 611[0n] Dendropsophus Barrancaberm AJC 5746 Hylidae microcephalus Colombia Santander eja 7.04 -73.86 68 DRYCO310-16 N/A 611[0n] Dendropsophus AJC 5766 Hylidae microcephalus Colombia Sucre San Marcos 8.66 -75.14 22 DRYCO318-16 N/A 611[0n] Dendropsophus JMR 5987 Hylidae microcephalus Colombia Bolivar Maria la baja 9.98 -75.30 14 DRYCO070-15 N/A 656[1n] Dendropsophus Costa 563[0n Pichi 272 Hylidae microcephalus Rica Puntarenas Agua Buena 8.41 -83.34 100 BSUCR272-12 ] 657[0n] Dendropsophus 561[0n AJC 1507 Hylidae microcephalus Panama Panama Chepo 8.96 -78.47 - BSAMP377-08 ] 658[0n] Dendropsophus 562[0n AJC 1098 Hylidae microcephalus Panama Panama - 9.16 -79.21 - BSAMQ278-09 ] 658[0n] Dendropsophus 562[0n AJC 1117 Hylidae microcephalus Panama Cocle El Cope 8.66 -80.59 - BSAMQ288-09 ] 658[0n] Dendropsophus 563[0n AJC 1049 Hylidae microcephalus Colombia Antioquia Maceo 6.54 -74.64 - BSAMS010-12 ] 658[0n] Dendropsophus Palomino, 563[0n AJC 2349 Hylidae microcephalus Colombia La Guajira Dibulla 11.25 -73.56 - BSAMS094-12 ] 658[0n] Dendropsophus 563[0n AJC 3561 Hylidae microcephalus Colombia Tolima Mariquita 5.26 -74.89 - BSAMS134-12 ] 658[0n] Dendropsophus San Vicente 563[0n AJC 4035 Hylidae microcephalus Colombia Santander de Chucuri 7.08 -73.55 44 BSAMS182-12 ] 658[0n] Dendropsophus 559[0n AJC 2254 Hylidae microcephalus Colombia Cundinamarca Guaduas 5.06 -74.60 35 ZONIF088-12 ] 655[0n] Dendropsophus 545[0n AJC 4633 Hylidae microcephalus Colombia Antioquia Puerto Berrio 6.49 -74.42 - DRYCO023-15 ] 561[5n] Dendropsophus Venezuel 561[0n AJC 3177 Hylidae microcephalus a Bolivar - 8.05 -61.84 - ZONIA165-12 ] 658[0n] Dendropsophus Venezuel 562[0n MHNLS 17736 Hylidae microcephalus a Delta Amacuro - 9.81 -61.59 - AMTMP372-15 ] 657[0n] Dendropsophus Venezuel 559[0n EBEF 4 Hylidae microcephalus a Apure - - - - ZONIF310-12 ] 655[0n] Dendropsophus Venezuel 562[0n MNHLS 18951 Hylidae microcephalus a Yaracuy - 10.30 -68.66 - ZONIJ413-15 ] 645[0n] Dendropsophus Venezuel 559[0n MNHLS 17186 Hylidae microcephalus a Zulia - - - - ZONIF496-12 ] 655[0n] MNCN- Dendropsophus Venezuel 561[0n ADN59473 Hylidae microcephalus a Anzoategui - 8.30 -62.88 - AMTMP196-15 ] 657[0n] Dendropsophus Costa 563[0n AJC 0954 Hylidae microcephalus Rica Alajuela - 10.47 -84.74 - BSAMQ267-09 ] 658[0n] Dendropsophus Venezuel 567[0n AJC 2883 Hylidae minusculus a Amazonas - 5.69 -67.61 - ZONIA017-12 ] 660[0n] Dendropsophus 491[0n AJC 3804 Hylidae phlebodes Panama Darien - 8.07 -78.36 - BSAMQ420-13 ] Dendropsophus 562[0n AJC 1788 Hylidae phlebodes Panama Panama Chilibre 9.17 -79.41 - BSAMP595-08 ] 658[0n] Dendropsophus 563[0n CH 6806 Hylidae phlebodes Panama Panama Chilibre 9.23 -79.41 - BSAMP666-09 ] 658[0n] Dendropsophus Costa 564[0n Pichi 275 Hylidae phlebodes Rica Alajuela Finca Palmira 10.95 -84.73 - BSUCR275-12 ] 657[0n] Dendropsophus Reserva 569[0n AJC 3571 Hylidae rhodopeplus Colombia Amazonas Tanimboca -4.11 -69.95 - BSAMS140-12 ] 658[0n] Dendropsophus LSB 331 Hylidae subocularis Colombia Antioquia San Rafael 6.39 -75.01 - BSAMS279-12 N/A 658[0n] Dendropsophus Puente 570[0n AJC 3845 Hylidae subocularis Colombia Santander Nacional 5.90 -73.70 - BSECO284-11 ] 658[0n] Dendropsophus 566[0n SCF 012 Hylidae triangulum Peru Ucayali - -9.77 -70.71 - ZONIJ485-15 ] 658[0n] ZONIAAF-MTR Dendropsophus ZONIA2565- 745[0n 19029 Hylidae triangulum Brazil Amazonas - -4.72 -62.13 - 16 ] 571[0n AJC 2294 Leptodactylidae Leptodactylus fragilis Colombia Tolima Mariquita 5.26 -74.90 568 BSAMS078-12 ] 658[0n] 552[0n AJC 4628 Leptodactylidae Leptodactylus fragilis Colombia Antioquia Puerto Berrio 6.49 -74.42 111 DRYCO021-15 ] 564[0n] 551[1n AJC 4682 Leptodactylidae Leptodactylus fragilis Colombia Antioquia Yondo 6.73 -74.21 122 DRYCO026-15 ] 575[0n] 571[0n JSM 201 Leptodactylidae Leptodactylus fragilis Colombia Huila Garzon 2.21 -75.64 811 BSAMS247-12 ] 658[0n] 571[0n JSM 262 Leptodactylidae Leptodactylus fragilis Colombia Bolivar Baru 10.15 -75.68 17 BSAMS266-12 ] 658[0n] MHUAA004- 470[0n MHUA-A 3995 Leptodactylidae Leptodactylus fragilis Colombia Antioquia Maceo 6.55 -74.64 500 13 ] 591[0n] Reserva Natural 567[0n AJC 2231 Leptodactylidae Leptodactylus fuscus Colombia Casanare Palmarito 4.83 -71.66 145 BSAMS071-12 ] 658[0n] 569[0n AJC 2301 Leptodactylidae Leptodactylus fuscus Colombia Casanare Orucue 4.91 -71.43 138 BSAMS081-12 ] 658[0n] San Juan de 569[0n AJC 3467 Leptodactylidae Leptodactylus fuscus Colombia Meta Arama 3.23 -73.86 308 BSECO243-11 ] 658[0n] Sabana de 569[0n AJC 3892 Leptodactylidae Leptodactylus fuscus Colombia Santander Torres 7.40 -73.50 - BSECO319-11 ] 658[0n] 569[0n AJC 4078 Leptodactylidae Leptodactylus fuscus Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO029-11 ] 658[0n] 569[0n AJC 4153 Leptodactylidae Leptodactylus fuscus Colombia Cordoba Montelibano 7.97 -75.59 64 BSAMS193-12 ] 627[0n] 570[0n JSM 169 Leptodactylidae Leptodactylus fuscus Colombia Magdalena GLithobatesda 10.98 -74.79 40 BSAMS227-12 ] 658[0n] Leptodactylus Venezuel 568[0n MHNLS 15590 Leptodactylidae knudseni a Bolivar - 5.91 -62.74 64 AMTMP230-15 ] 623[0n] Leptodactylus Kaieteur 567[0n PK 1234 Leptodactylidae knudseni Guiana Potaro-Siparuni National Park 5.17 -59.50 - ZONIE397-12 ] 655[0n] leptodactylus Venezuel 565[0n MHNLS 17323 Leptodactylidae longirostris a Bolivar - 5.69 -62.53 111 ZONIF500-12 ] 655[0n] leptodactylus Kaieteur 565[0n PK 1507 Leptodactylidae longirostris Guiana Potaro-Siparuni National Park 5.17 -59.48 - ZONIE447-12 ] 655[0n] Leptodactylus 539[0n 450[63n AJC 1149 Leptodactylidae melanonotus Panama Panama San Carlos 8.63 -80.08 940 BSAMM031-09 ] ] Leptodactylus 566[0n AMNH 20102 Leptodactylidae pentadactylus Brazil Roraima Floresta 0.20 -60.65 - ZONIG374-13 ] 655[0n] Leptodactylus 569[0n CORBIDI 0024 Leptodactylidae pentadactylus Ecuador Sucumbios Sucumbios -0.48 -75.34 811 BSAPE510-12 ] 639[1n] Leptodactylus Norte de 572[0n JSM 0011 Leptodactylidae poecilochilus Colombia Santander Bochalema 7.58 -72.69 2364 BSAMS225-12 ] 658[0n] Venezuel Delta 564[0n AJC 3266 Ranidae Lithobates palmipes a - Amacuro 8.34 -61.69 312 ZONIA220-12 ] 661[0n] Venezuel 552[0n MHNLS 17185 Ranidae Lithobates palmipes a Zulia El Tocuco 9.84 -72.82 70 ZONIF495-12 ] 544[0n] MNCN- 549[0n ADN50843 Ranidae Lithobates palmipes Brazil Mato Grosso Juruena -10.33 -58.49 239 ZONIE707-12 ] 655[0n] Costa Llanuras del 551[0n Pichi 357 Ranidae Lithobates vaillanti Rica Alajuela Norte 10.95 -84.73 47 BSUCR357-12 ] N/A Lithobates 552[0n AJC 1387 Ranidae warszewitschii Panama Panama San Carlos 8.64 -80.07 - BSAMM055-09 ] 516[2n] Lithobates BSAMP1240- 553[0n CH 6145 Ranidae warszewitschii Panama Bocas Del Toro Changuinola 9.07 -82.47 245 09 ] 658[0n] Lithobates 551[0n AJC 1798 Ranidae warszewitschii Panama Panama Chepo 9.32 -79.29 50 BSAMP668-09 ] 658[0n] Kaieteur 565[0n PK 1239 Leptodactylidae Lithodytes lineatus Guiana Potaro-Siparuni National Park 5.13 -59.42 - ZONIE400-12 ] 644[0n] 574[0n AJC 1093 Hylidae Scinax altae Panama Panama Naranjal 9.16 -79.21 - BSAMQ268-09 ] 657[0n] 574[0n AJC 1094 Hylidae Scinax altae Panama Panama Naranjal 9.16 -79.21 - BSAMQ271-09 ] 658[0n] BSAMP1034- 575[0n CH 6346 Hylidae Scinax boulengeri Panama Darien Pinogana 7.72 -77.66 80 09 ] 658[0n] Costa Llanuras del 577[0n Pichi 297 Hylidae Scinax boulengeri Rica Alajuela Norte 10.88 -84.33 98 BSUCR297-12 ] 657[0n] 573[0n AJC 4061 Hylidae Scinax cf. kennedyi Colombia Casanare Sabanalarga 4.77 -73.04 206 BSECO009-11 ] 658[0n] 603[0n AJC 4051 Hylidae Scinax cf. kennedyi Colombia Casanare Sabanalarga 4.77 -73.04 855 BSECO012-11 ] 655[0n] San Juan de 573[0n AJC 1741 Hylidae Scinax cf. kennedyi Colombia Meta Arama 3.23 -73.85 74 BSECO174-11 ] 658[0n] San Juan de 573[0n AJC 1747 Hylidae Scinax cf. kennedyi Colombia Meta Arama 3.23 -73.85 325 BSECO180-11 ] 658[0n] 576[0n JMP 1847 Hylidae Scinax cruentommus Colombia Amazonas Leticia -4.12 -69.95 - ZONIA463-12 ] 654[0n] 574[0n JMP 1574 Hylidae Scinax cruentommus Peru Loreto Pucaurquillo -3.33 -71.92 - ZONIC640-12 ] 655[0n] 594[0n AJC 4131 Hylidae Scinax elaeochrous Colombia Cordoba Montelibano 7.97 -75.59 192 BSAMS187-12 ] 658[0n] Venezuel Delta 574[0n AJC 3274 Hylidae Scinax nebulosus a - Amacuro 8.32 -61.74 138 ZONIA228-12 ] 661[0n] 571[0n MTKD 48004 Hylidae Scinax nebulosus Guiana - - 4.67 -58.69 208 ZONIE553-12 ] 655[0n] 559[0n AMNH 20118 Hylidae Scinax nebulosus Brazil Roraima - 1.19 -60.18 240 ZONIG385-13 ] 655[0n] 571[0n AJC 1503 Hylidae Scinax rostratus Panama Panama Chepo 8.96 -78.46 120 BSAMP373-08 ] N/A San Vicente 590[0n AJC 3422 Hylidae Scinax rostratus Colombia Santander de Chucuri 7.08 -73.55 155 BSAMS108-12 ] 613[0n] MHUAA038- 492[0n MHUA-A 7171 Hylidae Scinax rostratus Colombia Antioquia Gomez Plata 6.59 -75.20 1085 13 ] N/A Venezuel Independenci 571[0n MHNLS 20261 Hylidae Scinax rostratus a Anzoategui a 8.30 -62.88 206 AMTMP199-15 ] 636[0n] 574[0n AJC 2324 Hylidae Scinax ruber Colombia Casanare Orucue 4.91 -71.43 138 BSAMS088-12 ] Reserva 576[0n AJC 3569 Hylidae Scinax ruber Colombia Amazonas Tanimboca -4.12 -69.95 88 BSAMS138-12 ] 658[0n] Sabana de 574[0n AJC 3884 Hylidae Scinax ruber Colombia Santander Torres 7.40 -73.50 - BSECO312-11 ] 658[0n] San Juan de 321[0n AJC 3952 Hylidae Scinax ruber Colombia Meta Arama 3.23 -73.85 325 BSECO215-11 ] 658[0n] 578[0n AJC 4053 Hylidae Scinax ruber Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO014-11 ] 607[0n] 575[0n AJC 4160 Hylidae Scinax ruber Colombia Cordoba Montelibano 7.97 -75.59 64 BSAMS195-12 ] 658[0n] 578[0n AJC 3378 Hylidae Scinax ruber Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO095-11 ] 658[0n] San Juan de 578[5n AJC 3446 Hylidae Scinax ruber Colombia Meta Arama 3.23 -73.85 - BSECO223-11 ] 658[0n] Comunidad 574[0n AJC 2273 Hylidae Scinax ruber Colombia Vaupes Pirasemo 1.32 -70.39 - ZONIF106-12 ] 612[0n] San Vicente 575[0n AJC 3532 Hylidae Scinax ruber Colombia Santander de Chucuri 7.08 -73.55 - BSAMS125-12 ] 658[0n] 574[0n AJC 1120 Hylidae Scinax ruber Panama Colon Gamboa 9.11 -79.70 204 BSAMM009-09 ] 658[0n] 573[0n AJC 1584 Hylidae Scinax ruber Panama Panama Chepo 8.96 -78.47 44 BSAMP453-08 ] 658[0n] 494[0n CH 9319 Hylidae Scinax ruber Panama Darien - - - 810 BSAMQ686-13 ] 658[0n] 499[0n CH 9157 Hylidae Scinax ruber Panama Darien Cana - - 750 BSAMQ671-13 ] 658[0n] 576[0n AJC 1825 Hylidae Scinax sp. Panama Darien Pinogana 7.76 -77.68 525 BSAMP925-09 ] 658[0n] 576[0n AJC 1821 Hylidae Scinax sp. Panama Darien Pinogana 7.76 -77.68 229 BSAMP921-09 ] 658[1n] 580[0n AJC 4105 Hylidae Scinax wandae Colombia Casanare Sabanalarga 4.77 -73.04 - BSECO056-11 ] 658[0n] Venezuel 565[0n AJC 3089 Hylidae Scinax wandae a Amazonas Atures 5.55 -67.45 114 ZONIA120-12 ] 620[0n] San Juan de 542[0n AJC 3464 Hylidae Scinax wandae Colombia Meta Arama 3.23 -73.85 - BSECO240-11 ] 658[0n] 286[0n AJC 1048 Hylidae Smilisca phaeota Colombia Antioquia Maceo 6.54 -74.64 504 BSAMS009-12 ] 658[0n] CHOCO010- 573[0n AJC 1185 Hylidae Smilisca phaeota Colombia Choco Arusi 5.57 -77.50 30 15 ] 658[4n] 569[0n AJC 1517 Hylidae Smilisca phaeota Panama Panama Chepo 8.96 -78.47 130 BSAMP387-08 ] 656[1n] Valle del CHOCO002- 573[0n AJC 3696 Hylidae Smilisca phaeota Colombia Cauca Cali 3.56 -76.88 597 15 ] 602[2n] Costa 568[0n Pichi 311 Hylidae Smilisca phaeota Rica Alajuela - 10.87 -84.35 90 BSUCR311-12 ] N/A Costa 568[0n Pichi 319 Hylidae Smilisca phaeota Rica Puntarenas - 9.46 -84.06 200 BSUCR319-12 ] N/A 658[0n CH 6190 Hylidae Smilisca sordida Panama Bocas Del Toro Changuinola 9.21 -82.48 120 BSAMQ043-09 ] 568[0n] 543[3n CH 6539 Hylidae Smilisca sordida Panama Bocas Del Toro Changuinola 9.14 -82.50 184 BSAMQ140-09 ] 568[0n] 658[0n CH 6621 Hylidae Smilisca sordida Panama Bocas Del Toro Changuinola 9.23 -82.50 84 BSAMQ187-09 ] 567[0n] 11

12

13

14

15

16

17

18

19

20

21

22

23

24 25 Table S3. Data set for mitochondrial and nuclear sequences obtained from the GenBank database.

Institutional Mitochondrial genes Nuclear genes /Field State/ Latitud Longitud Family Species Country Municipality Elevation collection Province e e 16S COI POMC Tyr number Phyllomedusi Agalychnis San Vicente KP14918 AJC 3510 dae terranova Colombia Santander de Chucuri 7.08 -73.55 478 KP149386 3 N/A N/A Phyllomedusi Agalychnis San Vicente KP14914 AJC 3515 dae terranova Colombia Santander de Chucuri 7.08 -73.55 478 KP149341 4 N/A N/A Phyllomedusi FJ76671 KRL 0940 dae Agalychnis lemur Panama Cocle El Cope 8.67 -80.59 750 FJ784445 9 N/A N/A Phyllomedusi FJ76657 KRL 0917 dae Agalychnis callidryas Panama Cocle El Cope 8.67 -80.59 750 FJ784436 0 N/A N/A Phyllomedusi GQ366291. GQ3661 - dae Agalychnis annae Costa Rica San Jose - - - - 1 N/A N/A 98.1 Phyllomedusi Guanacas cab964 dae Agalychnis callidryas Costa Rica te - - - - FJ489263.1 N/A N/A N/A La Selva Phyllomedusi biological lse662 dae Agalychnis callidryas Costa Rica Heredia station - - - FJ489296.1 N/A N/A N/A Phyllomedusi gam2350 dae Agalychnis callidryas Panama Gamboa - - - - FJ489290.1 N/A N/A N/A Phyllomedusi sfe2127 dae Agalychnis callidryas Panama Veraguas Santa Fe - - - FJ489316.1 N/A N/A N/A Phyllomedusi DQ2830 RdS 537 dae Agalychnis callidryas Belize - - - - - AY843563.1 N/A N/A 18.1 MVZ Phyllomedusi 210463 dae Agalychnis lemur Panama Chiriqui - 8.70 -82.28 - AY819534.1 N/A N/A N/A MVZ Phyllomedusi 203768 dae Agalychnis saltator Costa Rica Heredia Sarapiqui 10.44 -84.01 - AY326044.1 N/A N/A N/A Phyllomedusi Alto EF158398 IGM 12 dae Agalychnis saltator Costa Rica Limon Colorado - - - EF396334.1 N/A .1 N/A Phyllomedusi GQ366228. JMR 609 dae Agalychnis saltator Costa Rica Heredia - - - - 1 N/A N/A N/A QCAZ Phyllomedusi Esmeralda 13217 dae Agalychnis spurrelli Ecuador s Durango 1.07 -78.65 - AY326043.1 N/A N/A N/A Phyllomedusi Vicente AY81915 KU 217507 dae Agalychnis spurrelli Ecuador Pichincha Maldonado - - - AY819532.1 N/A 1.1 N/A MHUA-A Phyllomedusi Agalychnis 7316 dae terranova Colombia Antioquia Sonson 5.88 -74.85 - KC589394.1 N/A N/A N/A MHUA-L Phyllomedusi Agalychnis 0198-2 dae terranova Colombia Antioquia Sonson 5.88 -74.85 - KC589395.1 N/A N/A N/A Phyllomedusi GQ36603 SMNS dae Agalychnis hulli Ecuador Napo - - - - N/A N/A 3.1 N/A Phyllomedusi EF158394 IGM 10 dae Agalychnis annae Costa Rica San Jose - - - 1150 N/A N/A .1 N/A Phyllomedusi GQ36603 KRL 0955 dae Agalychnis lemur Panama Cocle El Cope - - - N/A N/A 4.1 N/A Phyllomedusi EF158395 IGM 11 dae Agalychnis callidryas Costa Rica Limon Colorado - - 710 N/A N/A .1 N/A AJC 4010 Hylidae Boana xerophylla Colombia Boyaca Miraflores 5.21 -73.14 - KP149317 N/A N/A N/A KM390786_ - Hylidae Boana semilineata Brazil - - - - - 1 N/A N/A N/A MNKA Santa Tucumancit 9497 Hylidae Boana geographica Bolivia Cruz o 16.36 62.00 530 KF723060.1 N/A N/A N/A MZUSP Amazona 157060 Hylidae Boana geographica Brazil s Tefe -2.89 -68.36 64 KU168903.1 N/A N/A N/A MZUSP Amazona 157083 Hylidae Boana geographica Brazil s Purus -4.31 -61.81 49 KU168897.1 N/A N/A N/A MZUSP Serra do 157090 Hylidae Boana geographica Brazil Acre Divisor -7.43 -73.66 263 KU168898.1 N/A N/A N/A CFBHT Campo KU4944 07825 Hylidae Boana crepitans Brazil Alagoas Alegre 9.78 36.33 - KU495263.1 70.1 N/A N/A CFBHT KU4944 12841 Hylidae Boana crepitans Brazil Bahia Camamu 13.94 39.10 - KU495262.1 69.1 N/A N/A CFBHT Minas KU4944 02360 Hylidae Boana crepitans Brazil Gerais Grao Mogol 16.55 42.91 - KU495261.1 68.1 N/A N/A CFBHT Rio de KU4944 04381 Hylidae Boana faber Brazil Janeiro Petropolis -22.48 -43.13 - KU495265.1 72.1 N/A N/A CFBHT Sao Luis do KU4944 06588 Hylidae Boana faber Brazil Sao Paulo Paraitinga -23.35 -45.14 - KU495267.1 74.1 N/A N/A CFBHT Nazare KU4944 15407 Hylidae Boana faber Brazil Sao Paulo Paulista -23.25 -46.43 - KU495264.1 71.1 N/A N/A Pedro de KU4944 TG 368 Hylidae Boana faber Brazil Sao Paulo Toledo -24.33 -47.25 - KU495266.1 73.1 N/A N/A CFBHT Rio de KU4944 04366 Hylidae Boana pardalis Brazil Janeiro Petropolis -22.48 -43.13 - KU495275.1 82.1 N/A N/A CFBHT Sao Jose do KU4944 14367 Hylidae Boana pardalis Brazil Sao Paulo Barreiro -22.72 -44.62 - KU495276.1 83.1 N/A N/A CFBHT KU4944 12573 Hylidae Boana lundii Brazil Sao Paulo Sao Carlos -21.99 -47.88 - KU495271.1 78.1 N/A N/A CFBHT Distrito KU4944 12635 Hylidae Boana lundii Brazil Brasilia Federal -15.74 -47.89 - KU495268.1 75.1 N/A N/A CFBHT Minas Sao Roque KU4944 01902 Hylidae Boana lundii Brazil Gerais de Minas -20.19 -46.52 - KU495270.1 77.1 N/A N/A CFBHT Alto Paraiso KU4944 02106 Hylidae Boana lundii Brazil Goias de Goias -14.03 -47.68 - KU495269.1 76.1 N/A N/A KU 202734 Hylidae Boana pellucens Ecuador Pichincha San Juan -0.21 -78.51 - AY326058.1 N/A N/A N/A JN970902 23680 Hylidae Boana pellucens Ecuador Guayas Huaquillas -2.14 -79.90 - JN970652.1 N/A .1 N/A JN970901 30594 Hylidae Boana pellucens Ecuador El Oro Avanzada -3.54 -79.96 - JN970651.1 N/A .1 N/A Espirito KT23626 11A Hylidae Boana semilineatus Brazil Santo Rio Bananal -19.26 -40.28 - N/A 4.1 N/A N/A CFBHT Sao Luis do KU4944 01279 Hylidae Boana pardalis Brazil Sao Paulo Paraitinga -23.35 -45.14 - KU495274.1 81.1 N/A N/A QCAZ KM0243 16809 Hylidae Boana geographica Ecuador Orellana Yasuni - - - N/A 41.1 N/A N/A QCAZ Estero KM0243 23186 Hylidae Boana rosenbergi Ecuador Manabi Ancho - - - N/A 61.1 N/A N/A Santa KM0243 CFBH 8459 Hylidae Boana semilineata Brazil Catarina Itapema - - - N/A 64.1 N/A N/A Madre de Puerto AY81911 KU 215191 Hylidae Boana boans Peru Dios Maldonado - - - N/A N/A 4.1 N/A ROM Parish JN970905 44089 Hylidae Boana xerophylla Guiana Hill - - - - N/A N/A .1 N/A ROM Paramaka JN970904 28436 Hylidae Boana xerophylla Guiana toi - - - - N/A N/A .1 N/A ROM Kurupuka JN970903 20560 Hylidae Boana xerophylla Guiana ri - - - - N/A N/A .1 N/A Mont EF37612 99bm Hylidae Boana boans Guiana Bakra - - - - N/A N/A N/A 3.1 RWM Amazona AY84405 17746 Hylidae Boana boans Venezuela s Agua Blanca - - - N/A N/A N/A 5.1 AY84406 CFBH 2966 Hylidae Boana crepitans Brazil Alagoas Piranhas - - - N/A N/A N/A 7.1 Grand EF37612 33mc Hylidae Boana geographica Guiana Santi - - - - N/A N/A N/A 2.1 Rio de Duque de AY84410 CFBH 5424 Hylidae Boana semilineata Brazil Janeiro Caixas - - - N/A N/A N/A 8.1 USNM AY84409 303046 Hylidae Boana pardalis Brazil Sao Paulo Salesopolis - - - N/A N/A N/A 6.1 AY84408 CFBH 4000 Hylidae Boana lundii Brazil Sao Paulo Rio Claro - - - N/A N/A N/A 5.1 CFBHT Espirito Santa KU4944 09065 Hylidae Boana pardalis Brazil Santo Leopoldina -20.09 -40.54 - KU495272.1 79.1 N/A N/A Craugastorida Craugastor JN99155 AJC 1336 e longirostris Colombia Antioquia Maceo 6.54 -74.64 504 N/A N/A N/A 0.1 Craugastorida Craugastor Santo EF49348 KU 177803 e longirostris Ecuador Pichincha Domingo -0.05 -79.09 517 EF493395.1 N/A N/A 2.1 Dendropsophus WED 54094 Hylidae triangulum Ecuador Napo Misahualli -1.03 -77.67 - AY326053.1 N/A N/A N/A CORBIDI Dendropsophus KY40666 12194 Hylidae triangulum Peru Loreto Requena -5.06 -73.85 - KY406441.1 6.1 N/A N/A QCAZA Dendropsophus KY40666 KY40670 44826 Hylidae triangulum Ecuador Orellana Rio Napo -0.93 -75.40 - KY406438.1 4.1 6.1 N/A QCAZA Dendropsophus Union del KY40667 36697 Hylidae sarayacuensis Ecuador Napo Toachi -0.32 -78.95 - KY406445.1 0.1 N/A N/A QCAZA Dendropsophus Morona Nueve de KY40666 32637 Hylidae sarayacuensis Ecuador Santiago Octubre -2.30 -78.12 - KY406443.1 8.1 N/A N/A QCAZA Dendropsophus Tungurah KY40666 23030 Hylidae sarayacuensis Ecuador ua Banos -1.39 -78.43 - KY406444.1 9.1 N/A N/A Fatima Centro QCAZA Dendropsophus Experimenta KY40666 17429 Hylidae sarayacuensis Ecuador Pastaza l -1.49 -78.00 - KY406442.1 7.1 N/A N/A QCAZA Dendropsophus KY40669 44584 Hylidae rhodopeplus Ecuador Orellana Huiririma -0.71 -76.35 - KY406466.1 1.1 N/A N/A QCAZA Dendropsophus KY40668 44328 Hylidae rhodopeplus Ecuador Orellana Chiroisla -0.61 -75.88 - KY406464.1 9.1 N/A N/A QCAZA Dendropsophus KY40669 44329 Hylidae rhodopeplus Ecuador Orellana Chiroisla -0.61 -75.88 - KY406465.1 0.1 N/A N/A Dendropsophus MJH 7101 Hylidae brevifrons Peru Huanuco Puerto Inca -9.63 -74.97 - AY843611.1 N/A N/A N/A QCAZA Dendropsophus Sucumbio Playas de KY40668 28273 Hylidae brevifrons Ecuador s Cuyabeno -0.27 -75.75 - KY406459.1 4.1 N/A N/A Estacion QCAZA Dendropsophus Cientifica KY40668 17826 Hylidae brevifrons Ecuador Orellana Yasuni -0.67 -76.40 - KY406457.1 2.1 N/A N/A Estacion QCAZA Dendropsophus biologica KY40668 18174 Hylidae brevifrons Ecuador Napo Jatun Sacha -1.07 -77.62 - KY406458.1 3.1 N/A N/A QCAZA Dendropsophus Sucumbio 55848 Hylidae brevifrons Ecuador s Lorocachi -0.16 -75.99 - KT721787.1 N/A N/A N/A Sarayacu, QCAZ Dendropsophus Rio MG041935. MG0418 52752 Hylidae parviceps Ecuador Pastaza Palandayaku -1.73 -77.48 402 1 75.1 N/A N/A QCAZ Dendropsophus Tungurah MG041926. MG0418 52026 Hylidae parviceps Ecuador ua Rio Negro -1.41 -78.19 1218 1 65.1 N/A N/A QCAZ Dendropsophus MG041943. MG0418 39515 Hylidae parviceps Ecuador Pastaza Cononaco -1.56 -75.59 195 1 83.1 N/A N/A Parque QCAZ Dendropsophus Nacional MG041918. MG0418 51195 Hylidae parviceps Ecuador Orellana Yasuni -1.10 -75.81 229 1 56.1 N/A N/A QCAZ Dendropsophus MG041924. MG0418 48919 Hylidae parviceps Ecuador Napo Sumaco -0.47 -77.60 2033 1 62.1 N/A N/A QCAZA Dendropsophus Sucumbio KY40665 35504 Hylidae triangulum Ecuador s Garzacocha -0.50 -76.37 229 KY406426.1 2.1 N/A N/A CORBIDI Dendropsophus Madre de Puerto MG041949. MG0418 5217 Hylidae koechlini Peru Dios Maldonado -12.81 -69.32 - 1 89.1 N/A N/A CORBIDI Dendropsophus Madre de Puerto MG041950. MG0418 5235 Hylidae koechlini Peru Dios Maldonado -12.85 -69.44 - 1 90.1 N/A N/A Guiana Dendropsophus French mountain of EF37612 28mc Hylidae brevifrons Guiana Cayenne Kaw 3 4.62 -52.11 4 N/A N/A N/A 8.1 Dendropsophus Matagalp Peñas AY81911 UTA 51789 Hylidae ebraccatus Nicaragua a Blancas 13.27 -85.67 1309 N/A N/A 7.1 N/A Cokscomb Basin Dendropsophus Stann Wildlife AY84407 RdS 790 Hylidae ebraccatus Belize creek Sanctuary 16.79 -88.53 112 N/A N/A N/A 0.1 Dendropsophus Madre de Puerto AY81911 KU 215248 Hylidae koechlini Peru Dios Maldonado -12.58 -69.20 209 N/A N/A 9.1 N/A Dendropsophus AY81912 UTA 50632 Hylidae microcephalus Honduras Atlantida Rio Viejo 15.67 -86.67 288 N/A N/A 1.1 N/A AMNH A- Dendropsophus AY84409 139315 Hylidae parviceps Brazil Acre Rio branco -10.07 -67.63 228 N/A N/A N/A 7.1 Dendropsophus Lago AY84412 MJH 3844 Hylidae triangulum Brazil Acre Catalao - - - N/A N/A N/A 2.1 QCAZA Dendropsophus KY40670 44539 Hylidae triangulum Ecuador Orellana Huiririma -0.72 -75.57 194 N/A N/A 8.1 N/A QCAZA Dendropsophus KY40670 44457 Hylidae triangulum Ecuador Orellana Chiroisla -0.61 -75.88 206 N/A N/A 7.1 N/A Leptodactylid Leptodactylus Road to 103AF ae longirostris Suriname Para Apura 5.18 -55.65 77 EU201121.1 N/A N/A N/A Leptodactylid Leptodactylus French Grand 199mc ae longirostris Guiana Santi - 4.33 -54.25 100 EU201120.1 N/A N/A N/A Leptodactylid Leptodactylus French Nourague KC60399 KC60405 KC60408 531AF ae longirostris Guiana s Inselberg 4.08 -52.68 145 N/A 1.1 7.1 4.1 Leptodactylid Leptodactylus French Mont 76bm ae longirostris Guiana Arawa - 2.82 -53.37 215 EU201119.1 N/A N/A N/A CFBHT Leptodactylid Leptodactylus Minas Camanducai 00832 ae latrans Brazil Gerais a -22.89 -46.05 - KU495335.1 N/A N/A N/A CFBHT Leptodactylid Leptodactylus Rio de 10542 ae latrans Brazil Janeiro Niteroi -22.97 -43.02 - KU495336.1 N/A N/A N/A Leptodactylid Leptodactylus HQ232845. JDL 24887 ae bolivianus Colombia Sucre San Marcos 8.60 -75.15 - 1 N/A N/A N/A Kaieteur ROM Leptodactylid Leptodactylus Potaro- National KM091596. 20591 ae longirostris Guiana Siparuni Park 5.22 -59.44 97 1 N/A N/A N/A ULABG Leptodactylid Leptodactylus HQ232841. 5112 ae bolivianus Venezuela Merida Rio Limones 8.57 -71.18 1363 1 N/A N/A N/A USNM Leptodactylid Leptodactylus Bocas del 298079 ae savagei Panama Toro Isla Popa 9.16 -82.12 38 AY947866.1 N/A N/A N/A USNM Leptodactylid Leptodactylus 320988 ae wagneri Ecuador Pastaza Coca -1.12 -76.95 - EF632054.1 N/A N/A N/A USNM Leptodactylid Leptodactylus Quebrada 534219 ae savagei Honduras Colon Machin 15.32 -85.29 - AY947862.1 N/A N/A N/A USNM Leptodactylid Leptodactylus 535964 ae melanonotus Belize Cayo San Jacinto 17.17 -89.08 - AY943237.1 N/A N/A N/A Parque MVZ Guanacas Nacional AY84417 207215 Hylidae Scinax boulengeri Costa Rica te Santa Rosa 10.84 -85.71 227 AY843755.1 N/A N/A 7.1 CFBHT Baixa KJ00428 10951 Hylidae Scinax nebulosus Brazil Grande Piaui -8.49 -45.15 218 KJ004190.1 2.1 N/A N/A Estación MVZ Biologica la AY84417 203919 Hylidae Scinax elaeochrous Costa Rica Heredia Selva 10.43 -84.01 419 AY843757.1 N/A N/A 8.1 UTA-A AY84418 50749 Hylidae Scinax staufferi Guatemala Zacapa Teculutan 14.93 -89.72 456 AY843761.1 N/A N/A 3.1 CFBHT KU4947 00502 Hylidae Scinax squalirostris Brazil Sao Paulo Itirapina -22.24 -47.91 - KU495567.1 74.1 N/A N/A CFBHT Sao Jose do KU4947 15638 Hylidae Scinax squalirostris Brazil Sao Paulo Barreiro -22.72 -44.62 - KU495565.1 72.1 N/A N/A Rio CFBHT Grande KU4947 08180 Hylidae Scinax squalirostris Brazil do Sul Bom Jesus -28.66 -50.44 - KU495566.1 73.1 N/A N/A CFBHT KU4947 05377 Hylidae Scinax cf. x-signatus Brazil Ceara Ubajara -3.84 -40.90 650 KU495579.1 86.1 N/A N/A CFBHT Pernambu Fernando de KU4947 08860 Hylidae Scinax x-signatus Brazil co Noronha -3.86 -32.42 1100 KU495578.1 85.1 N/A N/A CFBHT KU4947 05375 Hylidae Scinax x-signatus Brazil Ceara Ubajara -3.84 -40.90 1100 KU495574.1 81.1 N/A N/A CFBHT KU4947 03433 Hylidae Scinax x-signatus Brazil Bahia Itabuna -14.79 -39.27 - KU495577.1 84.1 N/A N/A FJ76683 KRL 0865 Hylidae Smilisca phaeota Panama Cocle El Cope 8.67 -80.59 750 FJ784413.1 5.1 N/A N/A FJ76683 KRL 1641 Hylidae Smilisca sila Panama Cocle El Cope 8.67 -80.59 750 FJ784578.1 7.1 N/A N/A MVZ Craugastorida Craugastor Puntarena 207248 e crassidigitus Costa Rica s Monteverde 10.30 -84.82 1360 EU186715.1 N/A N/A N/A Craugastorida Craugastor EVACC 024 e tabasarae Panama Panama - 9.32 -79.29 848 KC014806.1 N/A N/A N/A MVUP Craugastorida Craugastor EF62942 2039 e tabasarae Panama Panama Chame 8.64 -80.07 895 N/A 7.1 N/A N/A Leptodactylid Leptodactylus MG030714. AJC 5035 ae colombiensis Colombia Atlantico Tubara 10.89 75.00 - 1 N/A N/A N/A Leptodactylid Leptodactylus HQ232842. MUJ 2187 ae bolivianus Colombia Bolivar Cartagena - - - 1 N/A N/A N/A UTA-A Leptodactylid Leptodactylus San AY27309 53817 ae melanonotus Guatemala Marcos San Marcos 14.96 -91.81 - N/A 9.1 N/A N/A CFBH Leptodactylid Leptodactylus Mato KX03261 39775 ae pentadactylus Brazil Grosso Paranaita -9.67 -56.47 276 N/A 3.1 N/A N/A Leptodactylid Leptodactylus KC60405 KC60408 MD 2279 ae latrans Brazil Bahia Una -15.30 -39.08 9 N/A N/A 5.1 2.1 Leptodactylid Leptodactylus French Massif JX298144 396MC ae knudseni Guyana Lucifer - 4.77 -53.94 388 N/A N/A .1 N/A MACN Leptodactylid Leptodactylus Buenos KP29557 38648 ae latrans Argentina Aires Escobar -35.25 -58.34 20 N/A N/A 8.1 N/A ULABG Leptodactylid AY34176 4591 ae Leptodactylus fuscus Venezuela Bolivar Canaima 6.24 -62.85 392 N/A N/A N/A 0.1 J AF-2012 Leptodactylid Leptodactylus cf. Mato JN69190 978163 ae wagneri Brazil Grosso Vila Rica -10.02 -51.12 245 N/A N/A N/A 2.1 Leptodactylid Leptodactylus French Mount JX29824 109MC ae pentadactylus Guyana Kaw - 4.51 -52.07 142 N/A N/A N/A 3.1 MVZ Leptodactylid Leptodactylus Guanacas DQ3471 207294 ae melanonotus Costa Rica te - 10.87 -85.75 39 N/A N/A N/A 93.1 UTA A- GQ36607 50749 Hylidae Scinax staufferi Guatemala Zacapa - - - - N/A N/A 1.1 N/A French JN69212 320MC Hylidae Scinax cf. ruber Guiana Bourda - - - - N/A N/A N/A 1.1 Amazona Cachoeirinh JN69212 2131VOGT Hylidae Scinax ruber Brazil s a - - - N/A N/A N/A 2.1 French EF36420 135PG Hylidae Scinax cruentommus Guiana - - - - - N/A N/A N/A 6.1 French EF36415 190MC Hylidae Scinax x-signatus Guiana - - - - - N/A N/A N/A 4.1 French EF37614 247mc Hylidae Scinax rostratus Guiana - - - - - N/A N/A N/A 5.1 FJ76683 FROG 1000 Hylidae Smilisca sila Panama Cocle El Cope 8.67 -80.59 - N/A 8.1 N/A N/A DQ830821. DQ05581 KU 217770 Hylidae Smilisca phaeota Ecuador Manabi - - - - 1 N/A 1.1 N/A DQ830876. DQ38872 MFF4410 Hylidae Smilisca puma Costa Rica Heredia - - - - 1 N/A 1.1 N/A AY84418 VCR 179 Hylidae Smilisca puma Costa Rica Heredia Chilamate - - - AY843765.1 N/A N/A 6.1 TCWC DQ830822. 84597 Hylidae Smilisca sila Costa Rica San Jose - - - - 1 N/A N/A N/A Parque Nacional DQ38872 MF4720 Hylidae Smilisca sordida Costa Rica San Jose Carara - - - N/A N/A 3.1 N/A 26

27 28 Table S4. Outgroups sequences used in the phylogenetic and divergence time analyses.

Institutional/ Mitochondrial genes Nuclear genes Field Countr State/Provinc Latitud Longitud Family Species Municipality Elevation collection y e e e 16S COI POMC Tyr number Leptodactylida Adenomera CFBHT 01797 e marmorata Brazil Sao Paulo Cubatao 23.86 46.44 - KU495118.1 KU494325.1 N/A N/A Leptodactylida Adenomera CFBHT 13834 e martinezi Brazil Tocatins Guarai 8.77 48.28 - KU495119.1 KU494326.1 N/A N/A Aplastodiscus Nova CFBHT 13076 Hylidae leucopygius Brazil Rio de Janeiro Friburgo -22.38 -42.56 - KU495141.1 KU494348.1 N/A N/A Aplastodiscus CFBHT 14484 Hylidae arildae Brazil Rio de Janeiro Itatiaia -22.47 -44.58 - KU495138.1 KU494345.1 N/A N/A Haddadus Governador CFBHT 12658 Craugastoridae binotatus Brazil Santa Catarina Celso Ramos -27.34 -48.57 491 KU495246.1 KU494453.1 N/A N/A Phyllomedusid Hylomantis CFBHT 12869 ae aspera Brazil Bahia Ilheus -14.66 -39.22 42 KU495254.1 KU494461.1 N/A N/A Phyllomedusid Hylomantis CFBHT 00392 ae granulosa Brazil Pernambuco Jaqueira -8.74 -35.80 202 KU495255.1 KU494462.1 N/A N/A Phyllomedusid Hylomantis GQ366 AY844127 ZUFRJ 7926 ae granulosa Brazil Pernambuco Jaqueira -8.74 -35.80 202 N/A N/A 032.1 .1 MTR Jequitinhonh ALCX197P6 Hylidae Pseudis fusca Brazil Minas Gerais a -17.16 -41.23 113 KU495490.1 KU494697.1 N/A N/A Orocue, Pseudis Colom Reserva AJC 2382 Hylidae paradoxa bia Casanare Wisirare 4.91 -71.43 - KP149190.1 KP149394.1 N/A N/A Argenti Islas del GQ366339. MACN 37786 Hylidae Pseudis minuta na Entre Rios Ibicuy -33.66 -58.83 2 1 N/A N/A N/A Pseudis Bom Jardin CFBHT 02346 Hylidae cardosoi Brazil Santa Catarina da Serra -28.39 -49.55 - KU495488.1 KU494695.1 N/A N/A Pseudis Argenti AY844167 MACN 38642 Hylidae paradoxa na Corrientes Bellavista -28.51 -59.04 - N/A N/A N/A .1 Trinida Pseudis d y AY819 USNM 306122 Hylidae paradoxa Tobago Mayaro Nariva 10.43 -61.06 2 AY819483.1 N/A 102.1 N/A Igarape Scarthyla Nova QULC 2340 Hylidae goinorum Brazil Amazonas Empresa -8.75 -63.85 40 AY843752.1 N/A N/A N/A Samoel Scarthyla Venezu Dario CFBHT 16443 Hylidae vigilans ela Tachira Maldonado -8.53 -71.91 - KU495532.1 KU494739.1 N/A N/A Haddadus JX267685. CFBH 19570 Craugastoridae binotatus Brazil Sao Paulo Iguape - - - N/A N/A N/A 1 Haddadus JX267684. MTR 13438 Craugastoridae binotatus Brazil Bahia Trancoso - - - N/A N/A N/A 1 Haddadus GQ345 USNM 303077 Craugastoridae binotatus Brazil Sao Paulo Salesopolis -22.19 -48.78 485 N/A N/A 259.1 N/A Aplastodiscus KU184263 CFBH 32533 Hylidae ibirapitanga Brazil Bahia Ibirapitanga - - - N/A N/A N/A .1 Aplastodiscus KU184236 MNRJ 51863 Hylidae ibirapitanga Brazil Bahia Amargosa - - - N/A N/A N/A .1 Hyloscirtus Colom Valle del AY819 KU 181167 Hylidae simmonsi bia Cauca Calima 3.92 -76.51 1759 N/A N/A 159.1 N/A Leptodactylida Lithodytes Colom San Juan de AJC 3451 e lineatus bia Meta Arama 3.23 73.85 - N/A KP149226.1 N/A N/A Leptodactylida Lithodytes French KC604 KC604087 55MC e lineatus Guiana Grand Santi - - - N/A N/A 060.1 .1 Leptodactylida Lithodytes Ecuado Morona HQ290 HQ29088 QCAZ 16621 e lineatus r Santiago Mendez -2.72 -78.32 521 N/A N/A 829.1 9.1 AMNH-A Leptodactylida Lithodytes Guyan AY844129 166426 e lineatus a Kwakwani - - - N/A N/A N/A .1 Haddadus Mimoso do CFBHT 12711 Craugastoridae binotatus Brazil Espirito Santo Sol 21.07 41.37 - KU495245.1 KU494452.1 N/A N/A Haddadus Santa Maria CFBHT 13706 Craugastoridae binotatus Brazil Rio de Janeiro Madalena 21.88 41.92 - KU495244.1 KU494451.1 N/A N/A Pseudis DCC 3284 Hylidae paradoxa Brazil Sao Paulo - - - AY326032.1 N/A N/A N/A Pseudis Barima- UTA 53104 Hylidae paradoxa Guiana Mabaruma Waini - - - EF153012.1 N/A N/A N/A Little Lithobates Vaqueros KX269 KX269748 TNHC 60324 Ranidae juliani Belize Cayo Creek - - - N/A N/A 445.1 .1 Lithobates Panam KX269 KX269771 JSF 1127 Ranidae warszewitschii a - - - N/A N/A 468.1 .1 Lithobates Panam DQ28295 KRL 0823 Ranidae warszewitschii a Cocle El Cope - - - N/A N/A N/A 8.1 Rana United KX269 KX269804 MVZ 225749 Ranidae luteiventris States Washington - - - KX269213.1 N/A 502.1 .1 Rana United MVZ 191016 Ranidae luteiventris States Montana Lincoln 48.16 -115.87 - AY779194.1 N/A N/A N/A Rana United MVZ 137417 Ranidae luteiventris States Montana Missoula 46.68 -114.22 - N/A KU985757.1 N/A N/A Scarthyla Madre de AY819 KU 215423 Hylidae goinorum Peru Dios - - - AY819521.1 N/A 139.1 N/A BIOUG- Pseudacris EAC004 Hylidae maculata Canada Manitoba Churchill 58.47 -94.04 - N/A KU986064.1 N/A N/A BIOUG- Pseudacris EAC005 Hylidae maculata Canada Manitoba Churchill 58.47 -94.04 - N/A KU985712.1 N/A N/A Pseudacris KM669712. U12-45 Hylidae maculata Canada Quebec - - - 1 N/A N/A N/A Pseudacris KM669711. U12-42 Hylidae maculata Canada Quebec - - - 1 N/A N/A N/A Pseudacris United EF9882 EF988332. JFBM14310 Hylidae maculata States Minnesota - - - N/A N/A 70.1 1 Hyloscirtus Ecuado AY844121 QCAZ 16704 Hylidae tapichalaca r Zamora - - - N/A N/A N/A .1 AMNH A- Hyloscirtus AY844050 165163 Hylidae armatus Bolivia Santa Cruz Caballero - - - N/A N/A N/A .1 Pristimantis Colom KY494 KY494220 ICN 55762 Craugastoridae parectatus bia Antioquia Sonson - - - N/A N/A 200.1 .1 29