Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Molecular phylogeny of Neotropical rock frogs reveals a long history of vicariant diversification in the Atlantic forest Ariadne F. Sabbaga,⁎, Mariana L. Lyraa, Kelly R. Zamudiob, Célio F.B. Haddada, Renato N. Feioc, Felipe S.F. Leited, João Luiz Gasparinie,f, Cinthia A. Brasileirog aUniversidade Estadual Paulista, Instituto de Biociências, Departamento de Zoologia and Centro de Aquicultura (CAUNESP), 13506-900 Rio Claro, São Paulo, Brazil bDepartment of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA c Departamento de Biologia Animal, Museu de Zoologia João Moojen, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Viçosa, 36571-000 Viçosa, Minas Gerais, Brazil d Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Viçosa, 35690-000 Florestal, Minas Gerais, Brazil e Laboratório de Vertebrados Terrestre, Universidade Federal do Espírito Santo, 29932-540 São Mateus, Espírito Santo, Brazil fGrupo de História Natural de Vertebrados, Instituto de Biociências, Universidade Estadual de Campinas, 13083-970 Campinas, São Paulo, Brazil g Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Paulo, 09972-270 Diadema, São Paulo, Brazil A R T I C L E I N F O Keywords: Anurans Cycloramphidae Genetic diversity Molecular markers Thoropa A B S T R A C T The Brazilian Atlantic coastal forest is one of the most heterogeneous morphoclimatic domains on earth and is thus an excellent region in which to examine the role that habitat heterogeneity plays in shaping diversification of lineages and species. Here we present a molecular phylogeny of the rock frogs of the genus Thoropa Cope, 1865, native to the Atlantic forest and extending to adjacent campo rupestre of Brazil. The goal of this study is to reconstruct the evolutionary history of the genus using multilocus molecular phylogenetic analyses. Our to- pology reveals 12 highly supported lineages among the four nominal species included in the study. Species T. saxatilis and T. megatympanum are monophyletic. Thoropa taophora is also monophyletic, but nested within T. miliaris. Populations of T. miliaris cluster in five geographically distinct lineages, with low support for re- lationships among them. Although all 12 lineages are geographically structured, some T. miliaris lineages have syntopic distributions with others, likely reflecting a secondary contact zone between divergent lineages. We discuss a biogeographic scenario that best explains the order of divergence and the distribution of species in Atlantic forest and adjacent areas, and outline the implications of our findings for the taxonomy of Thoropa. 1. Introduction The Brazilian Atlantic forest is a highly complex and heterogeneous morphoclimatic and phytogeographic domain (Ab’Saber, 1977). It is a global biodiversity hotspot (Mittermier et al., 1998; Morellato and Haddad, 2000; Silva and Casteleti, 2003) and high habitat hetero- geneity, resulting from topographic complexity and large latitudinal range, is one of the main reasons proposed for its high biological di- versity (Ribeiro et al., 2009; Rodríguez et al., 2015). Therefore, the Atlantic forest is an excellent region to examine how heterogeneous habitats contribute to diversification (Rodríguez et al., 2015). Habitat heterogeneity has a potentially large effect on amphibian diversifica- tion, due to their low vagility and often specialized habitat preferences (Rodríguez et al., 2015). Indeed, the Atlantic forest is home to more than 500 frog species (Haddad et al., 2013; Toledo et al., 2014), ac- counting for 8.1% of the world’s known anuran diversity. More than 75% of Atlantic forest anurans are endemic to the domain (Haddad et al., 2013) and many of these endemic species inhabit montane en- vironments (Cruz and Feio, 2007; Haddad et al., 2013). Rock frogs in the genus Thoropa Cope, 1865, belong to the family Cycloramphidae (sensu Frost, 2017) and include the following six spe- cies endemic to Brazil: T. miliaris (Spix, 1824), T. petropolitana (Wandolleck, 1907), T. taophora (Miranda-Ribeiro, 1923), T. lutzi Cochran, 1938, T. megatympanum Caramaschi and Sazima, 1984, and T. saxatilis Cocroft and Heyer, 1988. All species of the genus, except T. megatympanum, inhabit rocky seashores, wet rocky outcrops, and boulders and waterfalls in montane rocky streams of the Atlantic forest (Bokermann, 1965; Cocroft and Heyer, 1988; Feio et al., 2006). Thoropa megatympanum inhabits the Atlantic forest-Cerrado and Atlantic forest- Caatinga ecotones (Caramaschi and Sazima, 1984) in the same kinds of habitats as its congeners. Thoropa species have specialized breeding requirements. Males are typically territorial and exhibit parental care of https://doi.org/10.1016/j.ympev.2018.01.017 Received 1 September 2017; Received in revised form 19 January 2018; Accepted 20 January 2018 ⁎ Corresponding author. E-mail address: ariadne.sabbag@gmail.com (A.F. Sabbag). Molecular Phylogenetics and Evolution 122 (2018) 142–156 Available online 02 February 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved. T http://www.sciencedirect.com/science/journal/10557903 https://www.elsevier.com/locate/ympev https://doi.org/10.1016/j.ympev.2018.01.017 https://doi.org/10.1016/j.ympev.2018.01.017 mailto:ariadne.sabbag@gmail.com https://doi.org/10.1016/j.ympev.2018.01.017 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ympev.2018.01.017&domain=pdf egg clutches (Giaretta and Facure, 2004; Muralidhar et al., 2014; Consolmagno et al., 2016), which are deposited on rocks in freshwater seeps or at the humid rocky margins of shallow rivulets (Barth, 1956; Bokermann, 1965; Rocha et al., 2002). Tadpoles are exotrophic and semiterrestrial, hatching and developing in the water seeps (Barth, 1956; Bokermann, 1965). A phylogeographic study of Thoropa miliaris and T. taophora showed that populations of the southern T. taophora are monophyletic, and nested within the northern T. miliaris, and inferred a north to south expansion and differentiation within these two species (Fitzpatrick et al., 2009). The relationships of these two species to the others in the genus and the mechanisms potentially contributing to their diversification are still unknown. In this study we reconstruct a multi- locus phylogeny for populations of all available species within the genus Thoropa. Our specific goals are to (1) describe the spatial distribution and genetic diversity of species and cryptic lineages within the genus; (2) infer the phylogenetic relationships of Thoropa species using mi- tochondrial and nuclear markers and date divergences among species and lineages; and (3) test for monophyly of the genus Thoropa and each of the nominal species within the genus. We discuss our results in the context of a biogeographic scenario that best explains the order of di- vergence and the current distribution of species in Atlantic forest and adjacent areas. Fig. 1. Collection localities for Thoropa samples included in this study. Black crosses labelled TL1-TL6 indicate type localities of T. saxatilis, T. megatympanum, T. miliaris, T. taophora, T. lutzi and T. petropolitana, respectively. Elevation is shown in gray scale ranging from white (0–50m above the sea level) to black (3900–3950m above sea level). A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 143 2. Material and methods 2.1. Taxon sampling and data matrix assembly The genus Thoropa is subdivided in two groups, based on morpho- logical characters: the T. petropolitana group, with T. petropolitana and T. lutzi, and the T. miliaris group, with T. miliaris, T. taophora, T. megatympanum, and T. saxatilis (Feio, 2002). The Thoropa petropolitana group is characterized by small adults (up to 30mm), males with nuptial spines only on finger II (numbers of fingers sensu Fabrezi and Alberch, 1996), and tadpoles with a large abdominal disc surpassing the lateral edge of the body and posterior portion of the abdomen (Feio, 2002). In contrast, species in the Thoropa miliaris group have medium to large (more than 35mm) adult body sizes, males with nuptial spines on fingers II, III, and IV (numbers of fingers sensu Fabrezi and Alberch, 1996), and tadpoles with reduced abdominal disc (Feio, 2002). Al- though these characteristics have been highlighted as important dif- ferences between the two groups (Cocroft and Heyer, 1988), group monophyly and the relationships among all nominal species have not yet been established. Species of the Thoropa miliaris group are common in their preferred habitats throughout their range. Thoropa miliaris and T. taophora occur in the Atlantic forest of southeastern Brazil: T. miliaris has a broader distribution in the states of Bahia, Espírito Santo, Minas Gerais, and Rio de Janeiro (Frost, 2017), occurring from rocky seashores to 1500m above sea level, while T. taophora is restricted to eastern mountains and coastal areas in the state of São Paulo (Feio et al., 2006). Thoropa megatympanum occurs in the Serra do Espinhaço mountain range in the states of Minas Gerais and Bahia, where they are found in campo ru- pestre (rupestrian grasslands, sensu Silveira et al., 2015) at the ecotone between the Atlantic forest and Cerrado (Caramaschi and Sazima, 1984). Thoropa saxatilis is the species with the southernmost distribu- tion, occurring in the Serra Geral mountain range, in the states of Santa Catarina and Rio Grande do Sul (Cocroft and Heyer, 1988). Species of the Thoropa petropolitana group are far less common. Thoropa petropolitana was known from mountain sites (more than 700m above the sea level) in the Serra dos Órgãos, state of Rio de Janeiro, and T. lutzi was known to occur in the states of Rio de Janeiro and Espírito Santo (Bokermann, 1965). Thoropa petropolitana has not been recorded for more than 40 years and T. lutzi disappeared from the state of Rio de Janeiro, but can still be found in Parque Nacional do Caparaó and in the municipalities of Alegre and Muniz Freire, state of Espírito Santo (Feio, 2002; J.L. Gasparini pers. obs.). We sampled throughout the distributions of Thoropa saxatilis, T. megatympanum, T. miliaris, and T. taophora (Fig. 1), including topotypic specimens of T. miliaris (municipality of Rio de Janeiro, state of Rio de Janeiro), T. megatympanum (Serra do Cipó, municipality of Santana do Riacho, state of Minas Gerais), and T. taophora (Paranapiacaba, muni- cipality of Santo André, state of São Paulo). We were unable to find topotypic specimens of T. saxatilis (road from municipality of Bom Jardim da Serra to municipality of Lauro Müller, state of Santa Cat- arina), and the closest locality sampled was from the municipality of Timbé do Sul (state of Santa Catarina, around 60 km in straight line to the type locality). We also included one tissue sample of a small juve- nile stored directly in 100% ethanol, identified morphologically as T. lutzi, collected in municipality of Alegre, state of Espírito Santo (Fig. 1). We did not include topotypic specimens of T. lutzi (Recreio dos Ban- deirantes, municipality of Rio de Janeiro, state of Rio de Janeiro) or any specimens of T. petropolitana (municipality of Petrópolis, state of Rio de Janeiro) because no other tissue samples are available for those two rare species. We gathered tissue samples from herpetological collections and from our own field efforts, and supplemented those with sequences obtained from GenBank. During field trips, we collected liver, muscle tissue, or toe clips, stored in 100% ethanol at −20 °C. Voucher speci- mens for most of the samples are deposited in Brazilian collections (Appendix A.1). Tissue samples without voucher specimens are ar- chived in the tissue collection of Coleção de Anfíbios Célio F. B. Haddad, Universidade Estadual Paulista, Rio Claro, São Paulo state (CFBH-T) and at Cornell University (Ithaca, NY, USA). 2.2. Laboratory protocols We assembled two multilocus matrices for analyses at two scales, one to analyze genetic diversity and phylogenetic relationships between species within the genus Thoropa (herein called matrix A) and the other to test the monophyly of the genus (matrix B). Matrix A included five gene fragments for 46 individuals of the Thoropa miliaris group (T. megatympanum, T. miliaris, T. saxatilis, and T. taophora) and four Cycloramphidae species (Cycloramphus boraceiensis, C. dubius, C. eleu- therodactylus, and Zachaenus parvulus) plus Proceratophrys boiei as out- group taxa. We rooted this tree with P. boiei (Appendix A.1). In contrast, matrix B included ten gene fragments for 13 samples from the four species of T. miliaris group (T. megatympanum, T. miliaris, T. saxatilis, and T. taophora), the single sample of T. lutzi from the state of Espírito Santo, and 33 species as outgroups (Appendix A.2). Outgroups for both matrices were chosen based on phylogenies published by Frost et al. (2006), Grant et al. (2006), Pyron and Wiens (2011), Fouquet et al. (2013), Blotto et al. (2013), Faivovich et al. (2014), and Castroviejo- Fisher et al. (2015). We used the Qiagen DNeasy® Blood and Tissue kit (Qiagen Inc.) to extract total DNA, following the manufacturer’s protocol. Extracted DNA was used directly (or diluted to 0.5–1 ng/μl in Mili-Q® water) for amplifications. For matrix A, we PCR amplified three mitochondrial and two nuclear gene fragments: the 3′ fragment of the ribosomal gene encoding 16S rRNA amplified with primers 16Sar-L and 16Sbr-H (Palumbi et al., 1991) (16S-ARBR), a fragment of the gene encoding NADH dehydrogenase subunit 2 (ND2), the 5′ region of the gene en- coding cytochrome c oxidase subunit 1 (COI), a fragment of the intron 1 of the fibrinogen A alpha polypeptide gene (FGA), and a fragment of the recombination activating gene 1 (RAG1). For matrix B we used eight mitochondrial and three nuclear DNA fragments: 12S rRNA (12S), tRNA-Val, 16S rRNA (16S), tRNA-Leu1, NADH dehydrogenase the sub- unit 1 (ND1), tRNA-Ile, COI, RAG1, a fragment of the proopiomelano- cortin gene (POMC) and a fragment of exon 1 of the rhodopsin gene (RHO) (Supplementary Material SI.1). PCR reaction conditions are in Supplementary Material SI.2. We purified positive amplicons and se- quenced them in both directions using BigDye v.3.1® (Applied Biosys- tems) sequencing kits at Macrogen Inc. (Seoul, South Korea), Centro de Estudos de Insetos Sociais (UNESP, Rio Claro, SP, Brazil), or at the Cornell University Genomics Facility (Ithaca, NY, USA). We cleaned sequences, removed primer sequences, and built consensus sequences for each fragment and each individual using Sequencher v.4.5 (Gene Codes). We aligned sequences of each fragment separately with the software Muscle (Edgar, 2004) implemented in MEGA v.6 (Tamura et al., 2013), using default parameters (gap open penalty: −400; gap extend penalty: 0; maximum iterations: 8; clustering method: UPGMB). We translated the coding genes (ND1, ND2, COI, POMC, RAG1, and RHO) to verify the absence of frameshifts and premature stop codons. 2.3. Phylogenetic inferences and genetic diversity For phylogenetic inferences using matrix A, we conducted four different analyses: one concatenating mitochondrial and nuclear frag- ments (16S-ARBR, ND2, COI, FGA, and RAG1), a second including only the mitochondrial fragments (16S-ARBR, ND2, and COI), and one for each nuclear fragment (FGA and RAG1). For phylogenetic inferences using matrix B we conducted a single analysis with all gene fragments concatenated. Concatenated matrices were built in Mesquite v.3.03 (Maddison and Maddison, 2015). We estimated the best-fit nucleotide evolution model for each gene fragment (or codon position) and the best partition strategy using Bayesian Information Criterion (BIC; A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 144 Schwarz, 1978) in PartitionFinder v.1.1.1 (Lanfear et al., 2012). We inferred phylogenies using MrBayes v.3.1.2 (Ronquist and Huelsenbeck, 2003) with 100 million generations, two parallel runs, and eight Markov chains for each run. The priors were unlinked for all partitions, and as follows: Dirichlet distribution prior for state fre- quencies and substitution rates; uniform distribution prior for gamma shape and for the proportion of invariable sites; variable partition rates; and branch lengths unconstrained. Trees were sampled each 1000 generations and the first 25% of trees were discarded as burn-in. Ana- lyses were run at the CIPRES Science Gateway (Miller et al., 2010). To diagnose convergence of the runs, we used four criteria: the standard deviation of split frequencies (SDSF) lower than 0.01; the Potential Scale Reduction Factor (PSRF+) close to 1.0; a stable plot of the gen- erations versus the log likelihood values; and examination of the output log in Tracer v.1.6 (Rambaut et al., 2014; Ronquist et al., 2005). We inferred a 50% majority rule consensus tree and considered posterior clade probabilities (PP) higher than 0.95 as indicative of strong nodal support (Alfaro et al., 2003; Erixon et al., 2003). We visualized and edited trees in FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/ figtree). To identify significantly divergent lineages in our tree, we used geographic concordance, node support, branch lengths, and the tree topology. To calculate average genetic distances within and between species and within and between lineages for two fragments used in matrix A (16S-ARBR and COI), we used uncorrected p-distance with pairwise deletion and 1000 replicates of bootstrap in software MEGA v.6 (Tamura et al., 2013). 2.4. Coalescent-based species tree We reconstructed a coalescent-based species tree using *Beast (Heled and Drummond, 2010), and estimated time to the most recent common ancestor (tMRCA), in the software BEAST v.2.3 (Bouckaert et al., 2014). We prepared the input file with the BEAUti utility, using only the 35 terminal taxa from matrix A that possessed all five gene fragments sequenced (Appendix A.1). We used the package bModelTest (Bouckaert, 2015) to find the best models of nucleotide evolution for each gene fragment, partitioned or not by codon positions, for each data set. BEAST v.2.3 runs the *Beast analysis concomitantly with nucleotide substitution model selection through the bModelTest package. Lineages were assigned as “species” in a mapping file (required by BEAUti) following the phylogenetic tree inferred using matrix A. We performed the analysis without outgroup, and including sites with missing data. The best partition scheme did not include partition by codon, and each fragment was set for an in- dependent nucleotide evolution model. We used strict clock for 16S- ARBR and ND2, and lognormal relaxed clock (Drummond et al., 2006) for COI, FGA, and RAG1. We also adopted the Yule speciation process and unchecked the function “fix mean substitution rates” for each gene partition. Rates of nucleotide evolution were set to “estimate”, except for ND2 fragment, to which we applied the instantaneous rate of 0.00957 base changes per lineage per million years (Crawford, 2003). The best-fitting parameters were established after preliminary searches including different datasets (with and without outgroups and sites with missing data), best model of molecular clock rate variation, best tree model prior, and best partition schemes. Performance and accuracy were checked in Tracer v.1.6 (Rambaut et al., 2014) and an analysis was considered reliable if all ESS values were higher than 200 (Rambaut et al., 2014). Final analyses included one run of 100 million generations, sampled each 10 000 generations. The final maximum clade credibility tree was generated with TreeAnnotator v.2.3 (Bouckaert et al., 2014), discarding 10% as burn-in and using median node heights. 3. Results 3.1. Phylogenetic inferences and genetic diversity For matrix A, including the outgroup, the mitochondrial con- catenated alignment consisted of 2288 bp, the alignment of FGA was 535 bp long, the alignment of RAG1 was 429 bp long, and the total concatenated alignment (mitochondrial and nuclear) consisted of 3252 bp. For matrix B, also including outgroups, the mitochondrial concatenated alignment consisted of 4330 bp and the total con- catenated alignment (mitochondrial and nuclear) consisted of 5576 bp. The models of nucleotide substitution found for the fragments through BIC (Schwarz, 1978) and the best partition scheme are shown in Sup- plementary Material SI.3, for both matrices. The number of partitions identified by PartitionFinder v.1.1.1 was eight both for matrix A and matrix B. 3.1.1. Diversity of the Thoropa miliaris group The Bayesian consensus tree inferred from the concatenated (mi- tochondrial + nuclear) dataset of matrix A showed that Thoropa species form a monophyletic group in relation to the outgroup species, with high PP values (Fig. 2). The other trees (mitochondrial, FGA, and RAG1) also recovered Thoropa as monophyletic (Supplementary Material SI.4, SI.5, and SI.6). The nuclear gene trees recovered only T. saxatilis as monophyletic. The species Thoropa megatympanum, T. saxatilis, and T. taophora were recovered as monophyletic in all concatenated phylo- genetic analyses (mitochondrial + nuclear, and only mitochondrial). However, Thoropa miliaris was recovered as paraphyletic in relation to T. taophora, and populations of this species clustered in five well sup- ported cryptic lineages (Fig. 2). Thoropa saxatilis included two well supported and distinct lineages, both distributed in the slopes of the Serra Geral: sax-1, a northern lineage from municipality of Timbé do Sul in the state of Santa Catarina; and sax-2, a southern lineage distributed throughout the states of Santa Catarina and Rio Grande do Sul (Fig. 3), approximately 40 km distant from each other. Thoropa megatympanum included two well supported lineages (meg-1 and meg-2; Fig. 2) distributed in the northern and southern portions of Serra do Espinhaço, respectively (Fig. 3). The topotype (from Santana do Riacho, Minas Gerais state) belongs to the southern meg-2 lineage. Thoropa miliaris is composed of five well-supported lineages, al- though paraphyletic in their relationship to T. taophora (Fig. 2): lineage mil-1, distributed in southern Espírito Santo state; lineage mil-2, dis- tributed in montane regions of the states of Bahia, Minas Gerais, Espírito Santo and Rio de Janeiro; lineage mil-3, distributed in central and southern regions of Espírito Santo; lineage mil-4, located in northern Serra dos Órgãos in the state of Rio de Janeiro; and lineage mil-5 distributed primarily in the state of Rio de Janeiro, including coastal populations and the topotype sample of T. miliaris (Fig. 3). Fi- nally, Thoropa taophora is composed of three lineages: tao-1, distributed in the northern coastal regions of the state of São Paulo; tao-2, dis- tributed in southern coastal regions of the state of São Paulo, and in- cluding the topotype specimen of T. taophora; and tao-3, from Estação Ecológica Juréia-Itatins, a coastal site in southern São Paulo state. We found five localities where lineages of T. miliaris were syntopic (Santa Maria Madalena, Rio de Janeiro state; Santa Teresa, Cachoeiro do Itapemirim, and Domingos Martins, Espírito Santo state; Carangola, Minas Gerais state) and one region of syntopy of lineages mil-5 of T. miliaris and tao-1 of T. taophora (Paraty, Rio de Janeiro state) (Fig. 2). According to our analyses, the deepest phylogenetic split separates Thoropa saxatilis from all other Thoropa, with T. megatympanum forming the sister lineage to five lineages of T. miliaris plus T. taophora (Fig. 2). Both nuclear fragments and the concatenated analyses recovered the same results. Thoropa miliaris and T. taophora form a complex of poorly resolved populations. Lineages mil-1 and mil-2 are sister lineages (PP=92), and combined form the sister taxon (PP=100) to the A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 145 http://tree.bio.ed.ac.uk/software/figtree http://tree.bio.ed.ac.uk/software/figtree remaining lineages (mil-3+mil-4+mil-5+ T. taophora). Mil-3, in turn, is the sister lineage of mil-4+mil-5+ T. taophora, again with low support (PP= 69, Fig. 2). Mil-4+mil-5+ T. taophora form a well- supported clade (PP= 100). The average genetic distances found between pairwise species ranged from 4.4 to 8.1% for 16S-ARBR and 13.5 to 17% for COI (Table 1). The intraspecific genetic distances ranged from 1.1 to 3.1% for 16S-ARBR and from 3.5 to 11.4% for COI. The genetic distances among lineages varied from 1.1 to 9.4% for 16S-ARBR and from 3.7 to 17.8% to COI (Table 1). Within lineages they varied from 0.0 to 2.0% for 16S-ARBR and from 0.2 to 9.7% to COI (Table 1). 3.1.2. Monophyly of the genus Thoropa The Bayesian consensus tree from the concatenated (mitochondrial and nuclear) analysis of matrix B showed that the genus Thoropa is not monophyletic, as T. lutzi was recovered as the sister lineage of Cycloramphus+ Zachaenus with high PP support (Fig. 4). The Thoropa miliaris group is monophyletic, and the sister taxon of this clade is composed of Cycloramphus+ Zachaenus+ T. lutzi. Within the T. miliaris group, most of the relationships were recovered with PP higher than in the analyses of matrix A. The only node with low PP was the node uniting mil-5 and T. taophora (78%). The families Aromobatidae, Ba- trachylidae, Bufonidae, Centrolenidae, Ceratophryidae, Cyclor- amphidae, Dendrobatidae, Hylidae, Hylodidae, Leptodactylidae, Odontophrynidae and Rhinodermatidae were each recovered as monophyletic. Alsodidae is the only family not recovered as mono- phyletic, because Limnomedusa macroglossa was recovered as the sister taxon of Odontophrynidae. 3.2. Coalescent-based species tree The *Beast species tree showed a similar topology to the matrix A phylogeny based on concatenated data, but with differences in PP (Fig. 5). The best-fit models of nucleotide evolution inferred by bMo- delTest are in SI.7. The topology recovered the two lineages of Thoropa saxatilis (sax-1+ sax-2) form the sister group to the remaining species; two lineages of T. megatympanum (meg-1+meg-2) form the sister group to T. miliaris+ T. taophora; mil-3 as the sister lineage to the other four lineages of T. miliaris+ T. taophora; mil-1 as the sister lineage of mil-2, mil-5, mil-4, and T. taophora; mil-2 as the sister lineage of mil-5; mil-2+mil-5 form the sister group to mil-4 and T. taophora; tao-1 as the sister lineage to tao-2; and tao-1+ tao-2 as the sister group to tao-3. Estimates of times to the most common ancestor (tMRCA) showed that divergences within Thoropa span a large timeframe, ranging from the Oligocene to the Pleistocene (Fig. 5). The common ancestor of the four species dates to the Oligocene. The common ancestor between the two lineages of Thoropa megatympanum and the common ancestor among the three lineages of T. taophora date to the Pliocene-Pleisto- cene, and the common ancestor of tao-1 and tao-2 dates to the Pleis- tocene. 4. Discussion This is the broadest phylogenetic study of species in the genus Thoropa published to date. Additionally, the inclusion of the single available sample of T. lutzi raised doubts about the monophyly of the genus (see discussion below). Combined, the trees derived from mi- tochondrial and nuclear markers resolve relationships within Thoropa at different levels of divergence. The mitochondrial tree showed the Fig. 2. Phylogeny of Thoropa species based on concatenated (mitochondrial + nuclear) dataset (matrix A) and Bayesian Inference (50% majority rule consensus). Numbers at each node represent posterior probabilities. Terminals in bold represent topotype specimens for T. megatympanum, T. miliaris, and T. taophora. The symbol // indicates branch lengths that are not to scale. Samples CFBH 30,616 and CFBH 31,105 were identified as Thoropa miliaris because of their collection locality (Paraty, Rio de Janeiro), but belong to T. taophora, thus we renamed them. The codes BA, MG, ES, RJ, SP, SC, RS refer to the states of Bahia, Minas Gerais, Espírito Santo, Rio de Janeiro, São Paulo, Santa Catarina, and Rio Grande do Sul, respectively. A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 146 monophyly of T. saxatilis, T. megatympanum, T. taophora, and also showed a paraphyletic T. miliaris with respect to T. taophora. The nu- clear markers alone revealed well supported deeper divergences within the genus, especially the early split of T. saxatilis and T. megatympanum from other Thoropa. 4.1. Biogeography of diversification in Thoropa Our data indicate that diversification of Thoropa began with a north- south split (between T. saxatilis and the other species). The distribu- tional gap between T. saxatilis and the most southern locality of T. taophora (Peruíbe and Iguape, state of São Paulo) as well as its ancient separation from other species in the group corroborate this observation. This split was followed by a west-east division, with the separation between T. megatympanum and the complex T. miliaris+ T. taophora. This pattern of diversification corroborates the hypothesis of allopatric speciation of Thoropa species from a widespread ancestor proposed by Cocroft and Heyer (1988), although the speciation events were older than they postulated. Subsequently, the diversification of T. mili- aris+ T. taophora extended to the coast, in a process that likely in- cluded both vicariance and dispersal. Despite the long process of di- versification of lineages, the distribution of Thoropa species is still Fig. 3. Distribution of Thoropa samples, in different Brazilian states, identified to species (symbols) and to lineage (colors). Symbols labelled TL1-TL6 indicate type localities of T. saxatilis, T. megatympanum, T. miliaris, T. taophora, T. lutzi and T. petropolitana, respectively. Black crosses represent type localities not sampled in this study. Localities where lineages occur in syntopy are represented by squares and multiple colors. Elevation is shown in gray scale ranging from white (0–50m above the sea level) to black (3900–3950m above sea level). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 147 highly associated with rocky seashores or wet rocky outcrops near mountain formations, which indicates that this habitat preference is probably the ancestral state for the genus. The deep diversification times found for Thoropa species are similar to those found in many other South American amphibians, beginning in late Oligocene and continuing through the early Pliocene (Rull, 2008). Similar temporal patterns were found in Adenomera (Fouquet et al., 2014) and Lepto- dactylus (Fouquet et al., 2013). The disjunct distribution of Thoropa saxatilis in relation to other species in this genus may be explained by paleogeographic changes to the landscape during the Miocene and Pliocene (Rull, 2008). Thoropa saxatilis and T. megatympanum have remained largely geographically isolated from all other species, except for a small contact zone between T. megatympanum and T. miliaris (mil-2), in Catas Altas, Minas Gerais state, and other known localities in the Espinhaço mountain range in Minas Gerais state (Feio et al., 2006). These cases of syntopy seem to represent secondary contact of T. miliaris and T. megatympanum following the west-east vicariance. Populations of Thoropa miliaris and T. taophora show a more com- plex scenario of diversification. Given the largely allopatric geographic distributions of T. miliaris lineages and T. taophora, and the paraphyly of T. miliaris, we also consider vicariance for the split between the two forms, followed by secondary contact of lineages in some localities where the species are syntopic. This posterior contact may have oc- curred naturally, following past expansion and retraction hypothesized for Atlantic forest due to climatic oscillations (Carnaval and Moritz, 2008). Another possibility for syntopic distributions of T. miliaris lineages is anthropogenic mediated migration, but this latter hypothesis can only be ruled out after we have a better understanding of the geographic distributions of lineages. The split between Thoropa miliaris lineages and T. taophora occurred sometime between 15.5 and 4.0 million years in the late Miocene or early Pliocene (Fig. 5). However, this split was not accompanied by morphological change, as T. miliaris and T. taophora do not have any diagnostic morphological phenotypes Table 1 Average uncorrected p-distances (percentage, with standard errors in parentheses), within species (diagonal) and between species (below diagonal), and within lineages (diagonal) and between lineages (below diagonal), calculated from 16S-ARBR and COI fragments. (a) Within and between species for 16S-ARBR fragment. (b) Within and between species for COI fragment. (c) Within and between lineages for 16S fragment. (d) Within and between lineages for COI fragment. (a) 16S-ARBR fragment, by species Species N T. saxatilis T. megatympanum T. miliaris T. taophora T. saxatilis 4 2.0 (0.4) T. megatympanum 6 8.1 (1.0) 1.2 (0.3) T. miliaris 27 6.7 (0.9) 6.4 (0.8) 3.1 (0.5) T. taophora 9 7.4 (1.0) 7.4 (0.9) 4.4 (0.7) 1.1 (0.3) (b) COI fragment, by species Species N T. saxatilis T. megatympanum T. miliaris T. taophora T. saxatilis 4 6.6 (0.8) T. megatympanum 6 14.6 (1.2) 3.9 (0.5) T. miliaris 24 16.3 (1.2) 16.0 (1.1) 11.4 (0.9) T. taophora 7 17.0 (1.2) 15.9 (1.3) 13.5 (1.0) 3.5 (0.5) (c) 16S-ARBR fragment, by lineage Lineage N sax-1 sax-2 meg-1 meg-2 mil-1 mill-2 mil-3 mil-4 mil-5 tao-1 tao-2 tao-3 sax-1 1 – sax-2 3 3.7 (0.9) 0.3 (0.2) meg-1 2 9.4 (1.4) 7.2 (1.0) 1.1 (0.4) meg-2 4 9.4 (1.4) 7.9 (1.1) 1.7 (0.5) 0.7 (0.3) mil-1 4 7.8 (1.2) 7.8 (1.1) 7.3 (1.0) 7.1 (1.0) 2.0 (0.4) mil-2 10 7.0 (1.2) 6.6 (1.1) 6.0 (0.9) 6.6 (1.0) 3.6 (0.6) 1.3 (0.3) mil-3 3 8.0 (1.3) 6.5 (1.1) 5.5 (0.8) 5.9 (0.9) 4.4 (0.8) 3.3 (0.6) 1.9 (0.4) mil-4 2 6.7 (1.2) 5.9 (1.0) 6.0 (1.0) 6.3 (1.0) 3.9 (0.8) 3.0 (0.6) 3.1 (0.7) 0.2 (0.2) mil-5 8 7.1 (1.1) 6.0 (0.9) 6.0 (0.9) 6.3 (0.9) 4.0 (0.7) 3.8 (0.6) 4.0 (0.7) 2.5 (0.6) 1.6 (0.4) tao-1 2 8.2 (1.2) 7.1 (1.1) 6.8 (0.9) 7.3 (1.0) 5.6 (0.9) 4.4 (0.8) 4.2 (0.8) 3.4 (0.8) 4.6 (0.8) 0.0 (0.0) tao-2 5 7.5 (1.2) 7.4 (1.1) 7.1 (1.0) 7.4 (1.0) 5.6 (0.9) 4.3 (0.7) 4.6 (0.8) 3.4 (0.7) 4.4 (0.8) 1.1 (0.4) 0.5 (0.2) tao-3 2 7.2 (1.1) 7.6 (1.1) 7.5 (1.1) 8.1 (1.1) 5.0 (0.8) 4.0 (0.7) 4.8 (0.8) 3.2 (0.7) 4.2 (0.8) 2.0 (0.6) 1.7 (0.5) 0.0 (0.0) (d) COI fragment, by lineage Lineage N sax-1 sax-2 meg-1 meg-2 mil-1 mil-2 mil-3 mil-4 mil-5 tao-1 tao-2 tao-3 sax-1 1 – sax-2 3 10.7 (1.1) 2.5 (0.5) meg-1 2 15.2 (1.5) 14.7 (1.3) 4.4 (0.8) meg-2 4 15.0 (1.5) 14.3 (1.4) 5.4 (0.7) 1.8 (0.4) mil-1 3 16.4 (1.4) 16.3 (1.4) 16.9 (1.5) 16.8 (1.5) 5.7 (0.7) mil-2 9 17.0 (1.4) 16.5 (1.3) 15.5 (1.3) 15.8 (1.4) 11.5 (1.1) 6.8 (0.6) mil-3 2 15.3 (1.4) 17.3 (1.4) 15.9 (1.2) 16.4 (1.3) 13.2 (1.0) 14.1 (1.1) 9.7 (1.2) mil-4 2 15.7 (1.5) 16.6 (1.3) 15.7 (1.3) 16.8 (1.4) 12.9 (1.2) 13.9 (1.3) 13.0 (1.2) 0.5 (0.3) mil-5 8 15.3 (1.3) 15.8 (1.3) 16.3 (1.3) 15.5 (1.3) 13.2 (1.1) 13.4 (1.1) 13.3 (1.1) 12.3 (1.1) 5.3 (0.6) tao-1 2 16.2 (1.5) 17.8 (1.3) 17.4 (1.5) 17.4 (1.5) 15.4 (1.3) 14.2 (1.2) 13.2 (1.2) 12.0 (1.3) 11.9 (1.1) 0.2 (0.2) tao-2 4 16.7 (1.5) 17.2 (1.4) 15.5 (1.4) 15.8 (1.5) 15.1 (1.3) 14.7 (1.2) 13.4 (1.1) 12.4 (1.2) 12.6 (1.1) 3.7 (0.7) 1.0 (0.3) tao-3 1 14.3 (1.4) 16.2 (1.5) 13.8 (1.3) 13.9 (1.3) 15.2 (1.5) 13.3 (1.2) 11.8 (1.2) 10.7 (1.4) 11.4 (1.1) 6.8 (0.9) 6.3 (1.0) – A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 148 (Feio et al., 2006). The more recently derived lineages of Thoropa miliaris+ T. taophora complex (mil-5, tao-1, tao-2, and tao-3) include populations that reach the coast of Brazil and inhabit rocky seashores. Seashores are an odd environment for an amphibian and few species are known to tolerate high-salinity habitats (Abe and Bicudo, 1991; Gordon et al., 1961; Romspert and McClanahan, 1981). Nonetheless, the two lineages have evolved to occupy saline microhabitats and although these populations live in hostile environments, they are locally abundant (Bokermann, 1965; Brasileiro et al., 2010). Additionally, Thoropa saxatilis, T. megatympanum, and T. taophora may also be experiencing ongoing diversification as our topology showed geographical structure within these species (sax-1 and sax-2; meg-1 and meg-2; tao-1, tao-2, and tao-3). The north-south divergence in T. taophora was identified by Fitzpatrick et al. (2009), with the possibility of a southern São Paulo refugium forming the third lineage (“Juréia”=tao-3; Carnaval et al., 2009). Our data including the four Thoropa species corroborate that the distribution of habitats may play an important role in genetic connectivity or discontinuity of lineages and species (Fitzpatrick et al., 2009). 4.2. On the monophyly of Thoropa Although Cycloramphidae was recovered as a monophyletic family, its relationships with the other families remain poorly resolved, as found in previous studies (Grant et al., 2006; Pyron and Wiens, 2011; Fouquet et al., 2013; Blotto et al., 2013; and Faivovich et al., 2014). Clearly, the higher-level systematics of Athesphatanura needs further study. The inclusion of a sample of T. lutzi resulted in the potential para- phyly of the genus Thoropa (Figs. 2 and 4), with the single T. lutzi sample more closely related to Cycloramphus plus Zachaenus than to other members of the genus Thoropa. The position of this sample in the phylogeny raises interesting questions about the phylogenetic re- lationships of these three genera. For example, an externally non-visible tympanum is a synapomorphy for Cycloramphus+ Zachaenus (Verdade, 2005), and in our sample of T. lutzi, the tympanum is visible. This in- dicates that this feature might have two independent origins in Cy- cloramphidae and is thus not a synapomorphy for Cycloramphus, or that the Cycloramphidae ancestor had a visible tympanic membrane but it was lost in Cycloramphus+ Zachaenus. We cannot discard the possibility that our sample of Thoropa lutzi is not T. lutzi sensu stricto, because we did not analyze samples from T. lutzi Fig. 4. Phylogeny based on concatenated (mitochondrial + nuclear) dataset (matrix B) and Bayesian Inference (50% majority rule consensus) for Thoropa species and outgroups. Numbers at each node represent clade posterior probabilities. The branches leading to Stefania evansi are not to scale. Sample CFBH 31105 was identified as Thoropa miliaris because of its collection locality (Paraty, Rio de Janeiro), but it has been renamed T. taophora. The codes MG, ES, RJ, SP, SC, RS refer to the states of Minas Gerais, Espírito Santo, Rio de Janeiro, São Paulo, Santa Catarina, and Rio Grande do Sul, respectively. Color coded lineages of Thoropa are as shown in Fig. 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 149 type locality. Feio (2002) studied the morphology of a population from municipality of Muniz Freire (state of Espírito Santo), near the Alegre population we sampled, and found some morphological differences from the type series of T. lutzi. To test these relationships, we need more extensive sampling, with systematic analyses of molecular and mor- phological features, to infer the probable synapomorphies of Thoropa. Meanwhile, we recommend that this sample be designated as Thoropa cf. lutzi, until we can further clarify its taxonomic position. 4.3. Paraphyly and taxonomic considerations in Thoropa Previous studies on the underestimation of diversity of frog species revealed thresholds of uncorrected p-distances that could be used to recognize “candidate species”, based on the most commonly used bar- code gene fragments for amphibians (16S-ARBR and COI) (e.g., Vences et al., 2005a; Vences et al., 2005b; Fouquet et al., 2007). For 16S-ARBR, a threshold of 5% (Vences et al., 2005a) or 3% (Fouquet et al., 2007) of divergence have been used, while for COI the proposed threshold is 10% (Vences et al., 2005b). These levels of sequence divergence be- tween lineages might correspond to different species and not merely intraspecific genetic variation. If we analyze the four recognized species in our study, every value calculated for 16S-ARBR is higher than 3% (threshold sensu Fouquet et al., 2007), and only one value (4.4%, between Thoropa miliaris and T. taophora) is slightly smaller than 5% (threshold sensu Vences et al., 2005a). Thus, in general, the four nominal species are well supported, although T. miliaris and T. taophora might be considered synonyms if the threshold for 16S-ARBR is considered strictly. Our data show some evidence of deep intraspecific divergences within species. When com- paring lineages within each species, they all differ genetically by less than 3% (sensu Fouquet et al., 2007) and less than 5% (sensu Vences et al., 2005a) for 16S-ARBR, and less than 10% for COI, with one ex- ception. The genetic distance of COI among lineages within T. miliaris is slightly higher at 11.4%, which could indicate that T. miliaris contains high internal diversity and represents a species complex. With respect to genetic distances between species estimated from the 16S-ARBR fragment, our values (4.4–8.1) are similar to those found in studies of Proceratophrys (1–11%, Dias et al., 2013), Osteocephalus (0.6–6.1%, Jungfer et al., 2013), Physalaemus (1.2–11.7%, Lourenço et al., 2015), Ceratophryidae (0.4–7.2%, Faivovich et al., 2014), and Oreobates (2.8–11.7%, Padial et al., 2008). For the COI fragment, our between-species values (13.5–17.0) are higher than the values pub- lished for species of Alsodes and Eupsophus (Blotto et al., 2013). Thus, species delimitation in Thoropa is well defined, even considering the T. miliaris+ T. taophora complex. Lineages of Thoropa miliaris and T. taophora may represent an on- going diversification in this species complex, with T. miliaris being a “paraphyletic species” (see Hörandl and Stuessy, 2010; Schmidt- Lebuhn, 2012; Stuessy and Hörandl, 2014 for discussion). In a review of the occurrence of paraphyly and polyphyly in animal phylogenies of mitochondrial DNA, Funk and Omland (2003) found 21.3% of amphi- bian topologies exhibiting paraphyly or polyphyly. Similarly, 22% of Amazonia-Guianas amphibians exhibit a paraphyletic topology in phylogenies (Fouquet et al., 2007). Even with high genetic distances, the existence of syntopy between lineages of Thoropa miliaris and T. taophora makes it difficult to infer species limits. Moreover, the T. miliaris+ T. taophora complex has high phenotypic variation (Feio et al., 2006), precluding unambiguous diagnosis of species lineages, because of extensive morphometric and morphological overlap found in the OTUs (Feio et al., 2006). Thoropa miliaris and T. taophora surely represent a species complex, and this lineage needs further study, with a focus on systematics and population genetics. Combined, our analyses underscore the high genetic diversity of lineages endemic to the Atlantic forest and adjacent regions. The high degree of geographic isolation among species and lineages and the re- latively old age of these divergence events indicate that high habitat heterogeneity and a dynamic history of climatic change have structured diversification in this genus. Our study shows the importance of 3.0 mil-3 meg-2 mil-4 mil-1 tao-1 meg-1 tao-2 tao-3 mil-2 mil-5 90 100 52 37 70 58 99 95 100 23.9523 13.177 11.6256 7.1887 3.0467 10.8462 9.3991 0.8772 3.3005 05101520253035 PleistocenePlioceneMioceneOligoceneEocene My sax-1 sax-2 100 100 28.4907 9.023 Fig. 5. Topology inferred from coalescent species tree analysis treating each lineage of Thoropa as distinct population, with respective tMRCA for each node. Numbers above nodes indicate posterior probabilities, and numbers below nodes indicate average tMRCA (in millions of years – “My”). Blue bars indicate standard deviations of tMRCA. A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 150 inferring these spatial processes to clarify taxonomy and species di- versity, and the processes that might threaten species conservation in tropical biomes. Acknowledgements We thank G. Pontes (MCP), P. C. A. Garcia (UFMG), H. E.-D. Zaher, T. Grant and C. Mello (MZUSP), J. P. Pombal Jr., and N. Gonzaga (MNRJ), M. Solé and E. Silva (MZUESC) for facilitating tissue dona- tions. We thank P. Colombo, N. C. Pupin, A. T. de Carvalho e Silva, K. Ceron, H. Morrinho, M. Jordani, A. Mendes, L. A. Fusinatto, F. Centro, M. W. Faria, C. S. Cassini, V. G. D. Orrico, T. H. Condez, A. N. Figueiredo, J. P. de Côrtes, B. v. M. Berneck, J. A. Passamani, A. Diogo, G. Sobrinho (IBAMA – ES), A. P. L. S. Almeida, W. Santos (Fazenda Recanto da Mata), R. Kautsky, S. L. Mendes, M. Musso, F. Musso, J. E. Simon, P. L. V. Peloso, R. Zorzal, J. Aragon and B. Becacici for help with field trips. We thank F. Brusquetti, M. T. C. Thomé, C. T. O. Brunes, B. Blotto, F. R. do Amaral, K. Pellegrino, V. G. D. Orrico, C. P. A. Prado, B. v. M. Berneck, C. M. Lopes, and D. Baêta for help with analyses and constructive review of the manuscript, P. Lemes for map editions, and L. C. Irber Jr. for computational help. This research benefitted from re- sources of the Center for Scientific Computing (NCC/GridUNESP) of the São Paulo State University (UNESP) and the Centro de Estudos de Insetos Sociais (CEIS, UNESP, Rio Claro, SP, Brazil). Field collections license provided by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio-SISBIO number 30181-1). Fundings This research was supported by the São Paulo Research Foundation (FAPESP), São Paulo, Brazil [grants #08/50928-1 and #13/50741-7]; by FAPESP/Fundação Grupo Boticário de Proteção à Natureza [grant #2014/50342-8]; by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil; and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil [grants #564955/2010-8 and #302518/2013-4]. Appendix A Appendix A.1. Samples of Thoropa and outgroup species used in this study (matrix A), with accession numbers, collection localities (“simplified locality, municipality, state” or “municipality, state”), coordinates, GenBank accession numbers, and lineage membership. Samples in bold are topotype specimens. Asterisks indicate samples used in Fitzpatrick et al. (2009). Crosses indicate samples used also in matrix B, and full circles indicate samples used in coalescent species tree analysis. Samples originally recorded as T. miliaris, but considered as T. taophora because of their topology position, are shown with superscript “a”. Voucher specimens are deposited in Coleção de Anfíbios Célio F. B. Haddad, Universidade Estadual Paulista, Rio Claro, São Paulo (CFBH); Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro (MNRJ); Museu de Zoologia da Universidade de São Paulo, São Paulo (MZUSP); Museu de Zoologia João Moojen de Oliveira, Universidade Federal de Viçosa, Viçosa, Minas Gerais (MZUFV); Coleção Herpetológica da Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais (UFMG); Museu de Ciências Nat- urais, Pontifícia Universidade Católica de Minas Gerais, Belo Horizonte, Minas Gerais (MCNAM); and Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul (MCP). Collection abbreviations follow Sabaj Pérez (2014).Appendix A.2. Samples of Thoropa and outgroup species used in this study (matrix B) sorted by family, with collection numbers and GenBank accession numbers. Samples in bold are Thoropa topotype specimens. Samples originally recorded as T. miliaris, but considered as T. taophora because of their topology position, are shown with superscript “a”. Voucher specimens are deposited in Coleção de Anfíbios Célio F. B. Haddad, Universidade Estadual Paulista, Rio Claro, São Paulo (CFBH); Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro (MNRJ); Museu de Zoologia da Universidade de São Paulo, São Paulo (MZUSP); and Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul (MCP). Collection abbreviations follow Sabaj Pérez (2014). A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 151 Sp ec ie s A cc es si on nu m be r C ol le ct io n lo ca lit y La ti tu de Lo ng it ud e G en Ba nk A cc es si on N um be rs C la de 16 S- A R BR N D 2 C O I FG A R A G 1 T. m eg at ym pa nu m ● U FM G -A 58 57 A lt o da Se rr a Se te Q ue da s, Li cí ni o de A lm ei da ,B A − 14 .5 24 36 7 − 42 .5 39 99 6 M G 79 95 89 M G 79 97 12 M G 79 96 24 M G 79 96 66 M G 79 97 54 m eg -1 T. m eg at ym pa nu m ● U FM G -A 11 51 2 Se rr a de Bo tu m ir im ,B ot um ir im ,M G − 16 .8 48 18 6 − 43 .0 42 21 7 M G 79 95 88 M G 79 97 11 M G 79 96 23 M G 79 96 65 M G 79 97 53 m eg -1 T. m eg at ym pa nu m ● U FM G -A 11 54 2 Pa rq ue N ac io na l da s Se m pr e V iv as , D ia m an ti na , M G − 17 .7 29 29 0 − 43 .7 77 30 0 M G 79 95 92 M G 79 97 15 M G 79 96 29 M G 79 96 70 M G 79 97 59 m eg -2 T. m eg at ym pa nu m ● U FM G -A 94 41 Fe ch ad os , Sa nt an a do Pi ra pa m a, M G − 18 .7 86 48 3 − 43 .8 75 64 9 M G 79 95 90 M G 79 97 14 M G 79 96 27 M G 79 96 68 M G 79 97 57 m eg -2 T. m eg at ym pa nu m † ● M C N A M 20 72 Pa rq ue N ac io na l da Se rr a do C ip ó, Sa nt an a do R ia ch o, M G − 19 .3 01 72 5 − 43 .6 04 12 2 M G 79 95 75 M G 79 97 13 M G 79 96 26 M G 79 96 67 M G 79 97 56 m eg - 2 T. m eg at ym pa nu m U FM G -A 11 04 9 C ac ho ei ra da O st ra ,S er ra do R ol a M oç a, Br um ad in ho ,M G − 20 .0 99 47 2 − 44 .1 91 08 9 M G 79 95 91 – M G 79 96 28 M G 79 96 69 M G 79 97 58 m eg -2 T. m ili ar is ● M ZU FV 40 48 Fa ze nd a A lt o C ar ir i, Sa lt o da D iv is a, M G − 16 .3 00 00 0 − 39 .9 83 33 3 M G 79 96 03 M G 79 97 27 M G 79 96 41 M G 79 96 82 M G 79 97 70 m il- 2 T. m ili ar is M ZU FV 41 64 Fa ze nd a R am ai an a, Jo aí m a, M G − 16 .7 00 00 0 − 40 .8 16 66 7 M G 79 96 04 – M G 79 96 42 – M G 79 97 71 m il- 2 T. m ili ar is C FB H -T 19 80 0 Se rr a G ra nd e, ao la do da Pe dr a do M on te do Pe sc oç o, It am ar aj u, BA − 16 .9 93 25 0 − 39 .5 94 50 0 M G 79 96 05 M G 79 97 18 – – – m il- 2 T. m ili ar is * ● C FB H 18 01 3 Es ta çã o Bi ol óg ic a Sa nt a Lú ci a, Sa nt a Te re sa ,E S − 19 .9 65 07 4 − 40 .5 40 46 6 G Q 17 45 78 G Q 17 49 87 M G 79 96 33 M G 79 96 74 M G 79 97 63 m il- 1 T. m ili ar is † ● C FB H 30 70 7 R es er va Bi ol óg ic a A ug us to R us ch ii, N ov a Lo m ba rd ia ,S an ta Te re sa ,E S − 19 .9 11 94 4 − 40 .5 49 44 4 M G 79 95 79 M G 79 97 28 M G 79 96 43 M G 79 96 83 M G 79 97 72 m il- 2 T. m ili ar is ● M N R J 72 83 2 C as ca to na , R PP N Sa nt uá ri o do C ar aç a, C at as A lt as ,M G − 20 .0 70 50 0 − 43 .4 89 11 1 M G 79 96 02 M G 79 97 26 M G 79 96 40 M G 79 96 81 M G 79 97 69 m il- 2 T. m ili ar is * ● C FB H 18 44 6 M or ro da G am el a, V it ór ia ,E S − 20 .3 02 08 3 − 40 .2 18 33 3 G Q 17 46 17 G Q 17 50 17 M G 79 96 34 M G 79 96 75 M G 79 97 64 m il- 1 T. m ili ar is ● C FB H 18 43 1 Pe dr a A zu l, D om in go s M ar ti ns ,E S − 20 .4 35 33 3 − 41 .0 21 50 0 M G 79 95 98 M G 79 97 22 M G 79 96 36 M G 79 96 77 M G 79 97 66 m il- 2 T. m ili ar is † C FB H 29 31 7 Pa ne la s, D om in go s M ar ti ns ,E S − 20 .3 35 55 6 − 40 .6 28 05 6 M G 79 95 77 – M G 79 96 32 M G 79 96 73 M G 79 97 62 m il- 1 T. m ili ar is ● C FB H -T 10 80 6 Sí ti o R ec an to da M at a, M un iz Fr ei re ,E S − 20 .5 82 22 2 − 41 .4 73 61 1 M G 79 95 93 M G 79 97 16 M G 79 96 30 M G 79 96 71 M G 79 97 60 m il- 3 T. m ili ar is ● C FB H 27 29 0 C ac ho ei ra pr óx im a à Pe dr a D ou ra da , C am in ho da Lu z, C ar an go la , M G − 20 .7 96 00 0 − 42 .1 05 63 3 M G 79 95 99 M G 79 97 23 M G 79 96 37 M G 79 96 78 M G 79 97 67 m il- 2 T. m ili ar is ● C FB H 27 29 2 C ac ho ei ra pr óx im a à Pe dr a D ou ra da , C am in ho da Lu z, C ar an go la , M G − 20 .7 96 00 0 − 42 .1 05 63 3 M G 79 96 06 M G 79 97 29 M G 79 96 44 M G 79 96 84 M G 79 97 73 m il- 5 T. m ili ar is M N R J 25 98 4 Pa rq ue M un ic ip al Pi co do It ab ir a, C ac ho ei ro de It ap em ir im ,E S − 20 .8 48 90 6 − 41 .0 67 80 0 M G 79 95 95 – – – – m il- 1 T. m ili ar is M N R J 25 98 5 Pa rq ue M un ic ip al Pi co do It ab ir a, C ac ho ei ro de It ap em ir im ,E S − 20 .8 48 90 6 − 41 .0 67 80 0 M G 79 95 94 – – – – m il- 3 T. m ili ar is ● M N R J 25 98 9 Pa rq ue M un ic ip al Pi co do It ab ir a, C ac ho ei ro de It ap em ir im ,E S − 20 .8 48 90 6 − 41 .0 67 80 0 M G 79 96 10 M G 79 97 33 M G 79 96 49 M G 79 96 88 M G 79 97 78 m il- 5 T. m ili ar is † ● C FB H 25 47 3 Ba rr a Po nt õe s, 65 0 m de al ti tu de , M im os o do Su l, ES − 20 .9 46 19 7 − 41 .5 31 44 1 M G 79 95 76 M G 79 97 17 M G 79 96 31 M G 79 96 72 M G 79 97 61 m il- 3 T. m ili ar is ● M N R J 43 66 3 M ar ge m es qu er da do R io Pa ra íb a do Su l, V ol ta G ra nd e, M G − 21 .8 05 88 1 − 42 .5 08 00 8 M G 79 96 08 M G 79 97 31 M G 79 96 47 M G 79 96 86 M G 79 97 76 m il- 5 T. m ili ar is † ● C FB H 27 35 0 Pa rq ue Es ta du al do D es en ga no , M or um be ca , Sa nt a M ar ia M ad al en a, R J − 21 .8 79 25 0 − 41 .9 18 61 7 M G 79 95 82 M G 79 97 36 M G 79 96 52 M G 79 96 91 M G 79 97 81 m il- 4 T. m ili ar is ● C FB H 27 36 6 Pa rq ue Es ta du al do D es en ga no ,E st ra da pa ra o So ss eg o do Im bé ,S an ta M ar ia M ad al en a, R J − 21 .9 30 46 7 − 41 .7 92 50 0 M G 79 96 07 M G 79 97 30 M G 79 96 45 M G 79 96 85 M G 79 97 74 m il- 5 T. m ili ar is C FB H 28 05 4 Pa rq ue Es ta du al do D es en ga no ,t ri lh a pa ra o pi co do D es en ga no ,S an ta M ar ia M ad al en a, R J − 21 .8 80 31 0 − 41 .9 16 91 0 M G 79 96 00 M G 79 97 24 M G 79 96 38 M G 79 96 79 – m il- 2 A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 152 T. m ili ar is ● C FB H -T 21 27 6 R io A iu ru oc a, A iu ru oc a, M G − 21 .9 75 39 0 − 44 .6 03 54 0 M G 79 96 01 M G 79 97 25 M G 79 96 39 M G 79 96 80 M G 79 97 68 m il- 2 T. m ili ar is ● M N R J 70 70 1 Pa rq ue N at ur al M un ic ip al Fa ze nd a A ta la ia , M ac aé ,R J − 22 .3 04 60 0 − 41 .9 99 99 7 M G 79 96 12 M G 79 97 37 M G 79 96 53 M G 79 96 92 M G 79 97 82 m il- 4 T. m ili ar is † ● C FB H 10 12 5 Es tr ad a R io de Ja ne ir o- Pe tr óp ol is , km 86 ,7 18 m ,P et ró po lis ,R J − 22 .5 33 35 0 − 43 .2 33 84 0 M G 79 95 78 M G 79 97 21 M G 79 96 35 M G 79 96 76 M G 79 97 65 m il- 2 T. m ili ar is † M N R J 40 64 2 Pi st a C lá ud io C ou ti nh o, Pr ai a V er m el ha , R io de Ja ne ir o, R J − 22 .9 52 93 1 − 43 .1 57 71 9 M G 79 95 80 M G 79 97 19 M G 79 96 46 – M G 79 97 75 m il -5 T. m ili ar is ● M N R J 74 65 8 Pe dr a de It ac oa ti ar a, It ac oa ti ar a, N it er ói , R J − 22 .9 73 53 9 − 43 .0 25 10 0 M G 79 96 09 M G 79 97 32 M G 79 96 48 M G 79 96 87 M G 79 97 77 m il- 5 T. m ili ar is ● C FB H -T 15 49 5 Pa re dã o de pe dr a na R od ov ia R io -S an to s (B R -1 01 ), km 48 7, A ng ra do s R ei s, R J − 22 .9 81 30 2 − 44 .4 36 65 4 M G 79 96 11 M G 79 97 35 M G 79 96 51 M G 79 96 90 M G 79 97 80 m il- 5 T. m ili ar is † ● C FB H 31 10 5 C os tã o ro ch os o da pr ai a da s La ra nj ei ra s, Tr in da de ,P ar at y, R J − 23 .3 34 41 6 − 44 .6 76 56 3 M G 79 95 81 M G 79 97 34 M G 79 96 50 M G 79 96 89 M G 79 97 79 m il- 5 T. sa xa til is † ● M C P 11 91 8 C as ca ta da C or ti na ,S er ra da R oc in ha , Se rr a V el ha ,T im bé do Su l, SC − 28 .8 28 69 0 − 49 .9 15 58 5 M G 79 95 73 M G 79 97 07 M G 79 96 19 M G 79 96 61 M G 79 97 49 sa x- 1 T. sa xa til is ● C FB H 32 21 1 C ac ho ei ra da O nç a, Pr ai a G ra nd e, SC − 29 .1 60 00 0 − 49 .9 88 33 3 M G 79 95 87 M G 79 97 10 M G 79 96 22 M G 79 96 64 M G 79 97 52 sa x- 2 T. sa xa til is † ● C FB H 30 38 1 C as ca ta da Fo rq ue ta ,D is tr it o de Ba rr a do O ur o, M aq ui né ,R S − 29 .5 34 36 4 − 50 .2 03 17 1 M G 79 95 74 M G 79 97 09 M G 79 96 21 M G 79 96 63 M G 79 97 51 sa x- 2 T. sa xa til is ● C FB H 30 31 1 R es er va da Fa m íli a Li m a, en tr e a 3a e a 4a ca ch oe ir as ,S ap ir an ga ,R S − 29 .5 52 86 9 − 51 .0 16 90 0 M G 79 95 86 M G 79 97 08 M G 79 96 20 M G 79 96 62 M G 79 97 50 sa x- 2 T. ta op ho ra a C FB H 30 61 6 Pa re dã o de pe dr a na R od ov ia R io -S an to s (B R -1 01 ), pr óx im o a Ta ri tu ba ,P ar at y, R J − 23 .0 47 22 2 − 44 .5 77 02 8 M G 79 96 16 – – – – ta o- 2 T. ta op ho ra † a ● C FB H 31 10 2 Tr ilh a pa ra a pr ai a da s La ra nj ei ra s, Tr in da de ,P ar at y, R J − 23 .3 32 24 7 − 44 .6 79 72 8 M G 79 95 84 M G 79 97 40 M G 79 96 55 M G 79 96 95 M G 79 97 85 ta o- 1 T. ta op ho ra * ● C FB H 10 47 2 Il ha R ed on da ,P ra ia A lm ad a, U ba tu ba , SP − 23 .3 51 94 4 − 44 .9 04 44 4 G Q 17 45 53 G Q 17 49 60 M G 79 96 56 M G 79 96 96 M G 79 97 86 ta o- 1 T. ta op ho ra ● C FB H 19 92 0 Il ha A nc hi et a, U ba tu ba ,S P − 23 .5 41 36 7 − 45 .0 74 64 7 M G 79 96 15 M G 79 97 41 M G 79 96 57 M G 79 96 97 M G 79 97 87 ta o- 2 T. ta op ho ra † M ZU SP -A 14 37 13 C ac ho ei ra da Pe dr a Li sa ,P ar qu e N .M . N as ce nt es de Pa ra na pi ac ab a, Sa nt o A nd ré , SP − 23 .8 04 94 4 − 46 .3 01 00 0 M G 79 95 85 M G 79 97 20 M G 79 96 58 M G 79 96 98 – ta o- 2 T. ta op ho ra * ● C FB H 15 32 5 Il ha de Sã o Se ba st iã o, Pa rq ue Es ta du al de Il ha be la ,I lh ab el a, SP − 23 .8 31 11 1 − 45 .3 52 77 8 G Q 17 45 90 G Q 17 49 90 M G 79 96 60 M G 79 97 00 M G 79 97 89 ta o- 2 T. ta op ho ra ● C FB H 15 70 0 Il ha A s Il ha s, Sã o Se ba st iã o, SP − 23 .7 89 44 4 − 45 .7 10 31 4 M G 79 96 17 M G 79 97 42 M G 79 96 59 M G 79 96 99 M G 79 97 88 ta o- 2 T. ta op ho ra † ● C FB H 29 71 1 Es ta çã o Ec ol óg ic a Ju ré ia -I ta ti ns , N úc le o A rp oa do r, Pe ru íb e, SP − 24 .3 82 88 6 − 47 .0 18 24 7 M G 79 95 83 M G 79 97 38 M G 79 96 54 M G 79 96 93 M G 79 97 83 ta o- 3 T. ta op ho ra C FB H 34 89 8 C ac ho ei ra do Po ci nh o, Ig ua pe ,S P − 24 .5 71 93 3 − 47 .2 48 51 9 M G 79 96 13 M G 79 97 39 – M G 79 96 94 M G 79 97 84 ta o- 3 C .b or ac ei en si s – – – – K J9 61 57 0 – K J9 61 55 0 M G 79 97 03 K J9 61 59 0 – C .e le ut he ro da ct yl us – – – – K U 49 51 90 – M G 79 96 25 M G 79 97 04 M G 79 97 55 – C .d ub iu s – – – – G Q 17 45 85 M G 79 97 43 – – – – P. bo ie i – – – – K U 49 54 68 EU 01 77 81 K U 49 46 75 M G 79 97 02 M G 79 97 90 – Z. pa rv ul us – – – – K U 49 56 19 – K U 49 48 26 M G 79 97 01 M G 79 97 91 – A.F. Sabbag et al. Molecular Phylogenetics and Evolution 122 (2018) 142–156 153 Sp ec ie s Fa m ily G en Ba nk A cc es si on nu m be rs 12 S tR N A -V al 16 S tR N A -L eu N D 1 tR N A -I le C O I PO M C R A G 1 R H O A llo ph ry ne ru th ve ni A llo ph ry ni da e A Y 84 35 64 A Y 84 35 64 A Y 84 35 64 A Y 81 94 58 A Y 81 94 58 – – A Y 81 90 77 EU 66 34 32 A Y 84 45 38 A ls od es ne uq ue ns is A ls od id ae A Y 84 35 65 A Y 84 35 65 A Y 84 35 65 JX 20 40 17 JX 20 40 17 JX 20 40 17 JX 20 38 91 K P2 95 56 1 A Y 84 43 62 A Y 84 45 39 Eu ps op hu s ro se us / E. ca lc ar at us A ls od id ae A Y 84 35 87 A Y 84 35 87 A Y 84 35 87 JX 20 40 54 JX 20 40 54 JX 20 40 54 D Q 50 28 52 K C 60 40 74 – A Y 84 45 60 Li m no m ed us a m ac ro gl os sa A ls od id ae A Y 84 36 89 A Y 84 36 89 A Y 84 36 89 – – – K C 59 33 45 K P2 95 57 9 A Y 84 44 71 A Y 84 46 82 A llo ba te s fe m or al is A ro m ob at id ae A Y 36 45 43 A Y 36 45 43 A Y 36 45 43 H Q 29 09 51 H Q 29 09 51 H Q 29 09 51 D Q 50 28 11 H Q 29 08 31 D Q 50 33 27 D Q 50 32 15 A te lo gn at hu s pa ta go ni cu s Ba tr ac hy lid ae A Y 84 35 71 A Y 84 35 71 A Y 84 35 71 – – – – K P2 95 56 2 A Y 84 43 68 A Y 84 45 45 Ba tr ac hy la le pt op us Ba tr ac hy lid ae A Y 84 35 72 A Y 84 35 72 A Y 84 35 72 JX 20 40 29 JX 20 40 29 JX 20 40 29 JX 20 39 10 K P2 95 56 3 A Y 84 43 69 A Y 84 45 46 H yl or in a sy lv at ic a Ba tr ac hy lid ae JX 20 42 22 JX 20 42 22 JX 20 42 22 – – – – – – – D ut ta ph ry nu s m el an os tic tu s Bu fo ni da e A Y 45 85 92 A Y 45 85 92 A Y 45 85 92 A Y 45 85 92 A Y 45 85 92 A Y 45 85 92 A Y 45 85 92 D Q 15 83 17 EU 71 28 21 D Q 28 39 67 Es pa da ra na pr os ob le po n C en tr ol en id ae JX 56 48 57 JX 56 48 57 JX 56 48 57 JX 56 48 57 JX 56 48 57 JX 56 48 57 FJ 76 65 91 A Y 81 90 85 EU 66 34 53 A Y 36 44 04 C er at op hr ys co rn ut a C er at op hr yi da e K P2 95 60 8 K P2 95 60 8 K P2 95 60 8 K P2 95 54 2 K P2 95 54 2 K P2 95 54 2 K P2 95 68 8 K P2 95 56 7 K P2 95 58 9 K P2 95 70 8 C ha co ph ry s pi er ot tii C er at op hr yi da e K P2 95 62 1 K P2 95 62 1 K P2 95 62 1 K P2 95 55 1 K P2 95 55 1 K P2 95 55 1 – K P2 95 57 3 K P2 95 59 7 K P2 95 71 6 Le pi do ba tr ac hu s la ev is C er at op hr yi da e K P2 95 63 1 K P2 95 63 1 K P2 95 63 1 K P2 95 55 6 K P2 95 55 6 K P2 95 55 6 K P2 95 69 9 K P2 95 57 6 K P2 95 59 9 K P2 95 72 0 C yc lo ra m ph us ac an ga ta n C yc lo ra m ph id ae H Q 63 41 62 – FJ 68 56 83 – – – – – FJ 68 51 03 K F2 14 19 8 C yc lo ra m ph us ba nd ei re ns is C yc lo ra m ph id ae H Q 63 41 61 – H Q 63 41 66 – – – – – – – C yc lo ra m ph us bo ra ce ie ns is C yc lo ra m ph id ae D Q 28 30 97 D Q 28 30 97 D Q 28 30 97 – – – D Q 50 28 56 – D Q 50 33 57 D Q 28 38 13 C yc lo ra m ph us du bi us C yc lo ra m ph id ae – – G Q 17 45 85 – – – – – – – C yc lo ra m ph us el eu th er od ac ty lu s C yc lo ra m ph id ae H Q 63 41 60 – K U 49 51 90 – – – M G 79 96 25 – M G 79 97 55 – C yc lo ra m ph us fu lig in os us C yc lo ra m ph id ae H Q 63 41 58 – H Q 63 41 63 – – – – – – – C yc lo ra m ph us or ga ne ns is C yc lo ra m ph id ae H Q 63 41 59 – H Q 63 41 64 – – – – – – – Th or op a cf .l ut zi (C FB H -T 10 ,5 62 ) C yc lo ra m ph id ae M G 79 97 06 M G 79 95 72 M G 79 95 72 M G 79 95 72 M G 79 95 72 – – – M G 79 97 48 M G 79 98 02 Th or op a m eg at ym pa nu m (M C N A M 20 72 ) C yc lo ra m ph id ae M G 79 95 75 M G 79 95 75 M G 79 95 75 M G 79 95 75 M G 79 95 75 M G 79 95 75 M G 79 96 26 – M G 79 97 56 M G 79 97 93 Th or op a m ili ar is (C FB H 29 ,3 17 ) C yc lo ra m ph id ae M G 79 95 77 M G 79 95 77 M G 79 95 77 M G 79 95 77 M G 79 95 77 M G 79 95 77 M G 79 96 32 M G 79 97 45 M G 79 97 62 M G 79 97 95 Th or op a m ili ar is (C FB H 30 ,7 07 ) C yc lo ra m ph id ae M G 79 95 79 – M G 79 95 79 M G 79 95 79 M G 79 95 79 M G 79 95 79 M G 79 96 43 – M G 79 97 72 M G 79 97 97 Th or op a m ili ar is (C FB H 25 ,4 73 ) C yc lo ra m ph id ae M G 79 95 76 M G 79 95 76 M G 79 95 76 M G 79 95 76 M G 79 95 76 M G 79 95 76 M G 79 96 31 – M G 79 97 61 M G 79 97 94 Th or op a m ili ar is (C FB H 27 ,3 50 ) C yc lo ra m ph id ae M G 79 95 82 M G 79 95 82 M G 79 95 82 M G 79 95 82 M G 79 95 82 M G 79 95 82 M G 79 96 52 M G 79 97 47 M G 79 97 81 M G 79 97 99 Th or op a m ili ar is (C FB H 10 ,1 25 ) C yc lo ra m ph id ae M G 79 97 05 M G 79 95 78 M G 79 95 78 M G 79 95 78 M G 79 95 78 M G 79 95 78 M G 79 96 35 M G 79 97 46 M G 79 97 65 M G 79 97 96 Th or op a m ili ar is (M N R J 40 ,6 42 ) C yc lo ra m ph id ae M G 79 95 80 M G 79 95 80 M G 79 95 80 – – – M G 79 96 46 – M G 79 97 75 – Th or op a m ili ar is (C FB H 31 ,1 05 ) C yc lo ra m ph id ae M G 79 95 81 M G 79 95 81 M G 79 95 81 M G 79 95 81 M G 79 95 81 M G 79 95 81 M G 79 96 50 – M G 79 97 79 M G 79 97 98 Th or op a sa xa til is (M C P 11 ,9 18 ) C yc lo ra m ph id ae M G 79 95 73 M G 79 95 73 M G 79 95 73 M G 79 95 73 M G 79 95 73 M G 79 95 73 M G 79 96 19 M G 79 97 44 M G 79 97 49 – Th or op a sa xa til is (C FB H 30 ,3 81 ) C yc lo ra m ph id ae M G 79 95 74 M G 79 95 74 M G 79 95 74 M G 79 95 74 M G 79 95 74 M G 79 95 74 M G 79 96 21 – M G 79 97 51 M G 79 97 92 Th or op a ta op ho ra (C FB H 31 ,1 02 ) a C yc lo ra m ph id ae M G 79 95 84 M G 79 95 84 M G 79 95 84 M G 79 95 84 M G 79 95 84 M G 79 95 84 M G 79 96 55 – M G 79 97 85 M G 79 98 01 Th or op a ta op ho ra (M ZU SP -A 14 3, 71 3) C yc lo ra m ph id ae M G 79 95 85 M G 79 95 85 M G 79 95 85 – – – M G 79 96 58 – – – Th or op a ta op ho ra (C FB H 29 ,7 11 ) C yc lo ra m ph id ae M G 79 95 83 M G 79 95 83 M G 79 95 83 – – – M G 79 96 54 – M G 79 97 83 M G 79 98 00 Za ch ae nu s pa rv ul us C yc lo ra m ph id ae K C 59 33 62 K C 59 33 62 K C 59 33 62 – – – – – – – C ol os te th us pr at ti D en dr ob at id ae H Q 29 09 69 H Q 29 09 69 H Q 29 09 69 H Q 29 09 69 H Q 29 09 69 H Q 29 09 69 D Q 50 28 65 H Q 29 08 47 D Q 50 33 61 K P2 95 70 8 St ef an ia ev an si H em ip hr ac ti da e A Y 84 37 67 A Y 84 37 67 A Y 84 37 67 A Y 81 94 90 A Y 81 94 90 – – A Y 81 91 08 D Q 67 93 07 A Y 84 47 55 H yp si bo as m ul tif as ci at us H yl id ae A Y 84 36 48 A Y 84 36 48 A Y 84 36 48 G Q 36 62 99 G Q 36 62 99 G Q 36 62 99 JN 97 07 62 G Q 36 60 36 A Y 84 44 36 A Y 84 46 33 C ro ss od ac ty lu s sc hm id ti H yl od id ae A Y 84 35 79 A Y 84 35 79 A Y 84 35 79 JX 20 40 31 JX 20 40 31 JX 20 40 31 D Q 50 27 38 H Q 29 08 28 D Q 50 32 98 A Y 84 45 52 H yl od es ph yl lo de s H yl od id ae D Q 28 30 96 D Q 28 30 96 D Q 28 30 96 – – – D Q 50 28 73 – D Q 50 33 67 D Q 50 32 53 A de no m er a an dr ea e Le pt od ac ty lid ae K C 52 06 83 K C 52 06 83 K C 52 06 83 H Q 29 09 44 H Q 29 09 44 H Q 29 09 44 K C 52 06 89 K C 60 40 61 K C 60 40 37 K C 60 40 94 M ac ro ge ni og lo ttu s al ip io i O do nt op hr yn id ae K C 59 33 60 K C 59 33 60 K C 59 33 60 K C 59 33 53 K C 59 33 53 K C 59 33 53 – – K C 59 33 55 K C 59 33 57 O do nt op hr yn us am er ic an us O do nt op hr yn id ae A Y 84 37 04 A Y 84 37 04 A Y 84 37 04 JX 20 40 59 JX 20 40 59 JX 20 40 59 JX 20 39 39 JX 29 81 41 FJ 68 57 06 A Y 84 46 95 A.F. 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http://refhub.elsevier.com/S1055-7903(17)30623-1/h0460 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0470 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0470 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0470 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0480 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0480 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0490 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0490 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0495 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0495 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0495 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0500 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0500 http://refhub.elsevier.com/S1055-7903(17)30623-1/h0500 Molecular phylogeny of Neotropical rock frogs reveals a long history of vicariant diversification in the Atlantic forest Introduction Material and methods Taxon sampling and data matrix assembly Laboratory protocols Phylogenetic inferences and genetic diversity Coalescent-based species tree Results Phylogenetic inferences and genetic diversity Diversity of the Thoropa miliaris group Monophyly of the genus Thoropa Coalescent-based species tree Discussion Biogeography of diversification in Thoropa On the monophyly of Thoropa Paraphyly and taxonomic considerations in Thoropa Acknowledgements Fundings Appendix A Supplementary material References