Molecular Phylogenetics and Evolution 92 (2015) 204–216 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier .com/locate /ympev Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences q http://dx.doi.org/10.1016/j.ympev.2015.06.011 1055-7903/� 2015 Elsevier Inc. All rights reserved. q This paper was edited by the Associate Editor A. Larson. ⇑ Corresponding author. E-mail address: bolsoni@unicamp.br (L.B. Lourenço). Luciana B. Lourenço a,⇑, Cíntia P. Targueta a, Diego Baldo b, Juliana Nascimento a, Paulo C.A. Garcia c, Gilda V. Andrade d, Célio F.B. Haddad e, Shirlei M. Recco-Pimentel a a Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, 13083-863 Campinas, São Paulo, Brazil b Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (CONICET-UNaM), Facultad de Ciencias Exactas Químicas y Naturales, Universidad Nacional de Misiones, Félix de Azara 1552, CPA N3300LQF, Posadas, Misiones, Argentina c Laboratório de Herpetologia, Departamento de Zoologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, 31270-901 Belo Horizonte, Minas Gerais, Brazil d Departamento de Biologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Maranhão-UFMA, 65080-040 São Luís, Maranhão, Brazil e Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista, C.P. 199, 13506-900 Rio Claro, São Paulo, Brazil a r t i c l e i n f o Article history: Received 8 December 2014 Revised 16 June 2015 Accepted 17 June 2015 Available online 2 July 2015 Keywords: Phylogeny Systematics Anura Physalaemus a b s t r a c t Although some species groups have been recognized in the leiuperine genus Physalaemus, no phylogenetic analysis has previously been performed. Here, we provide a phylogenetic study based on mitochondrial and nuclear DNA sequences from 41 of the 46 species of Physalaemus. We employed the parsimony criterion using the software TNT and POY and the Bayesian criterion using the software MrBayes. Two major clades were recovered inside the monophyletic Physalaemus: (i) the highly sup- ported Physalaemus signifer Clade, which included P. nattereri and the species previously placed in the P. deimaticus and P. signifer Groups; and (ii) the Physalaemus cuvieri Clade, which included the remaining species of Physalaemus. Five species groups were recognized in the P. cuvieri Clade: the P. biligonigerus Group, the P. cuvieri Group, the P. henselii Group, the P. gracilis Group and the P. olfersii Group. The P. gracilis Species Group was the same as that previously proposed by Nascimento et al. (2005). The P. henselii Group includes P. fernandezae and P. henselii, and was the sister group of a clade that comprised the remaining species of the P. cuvieri Clade. The P. olfersii Group included P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga. The P. biligonigerus Species Group was composed of P. biligonigerus, P. marmoratus, P. santafecinus and P. riograndensis. The P. cuvieri Group inferred here differed from that recognized by Nascimento et al. (2005) only by the inclusion of P. albifrons and the exclusion of P. cicada. The paraphyly of P. cuvieri with respect to P. ephippifer was inferred in all the analyses. Distinct genetic lineages were recognized among individuals currently identified as P. cuvieri and they were congruent with cytogenetic differences reported previously, supporting the hypothesis of occurrence of formally unnamed species. � 2015 Elsevier Inc. All rights reserved. 1. Introduction Physalaemus is one of the largest genera in Leptodactylidae, but its intergeneric and internal relationships remain unclear. Over the last decade, the taxonomy of leptodactylid anurans has changed as a result of a number of phylogenetic analyses (Frost et al., 2006; Grant et al., 2006; Pyron and Wiens, 2011; Faivovich et al., 2012; Fouquet et al., 2013; de Sá et al., 2014), and according to the latest studies Physalaemus is included in the subfamily Leiuperinae. Pyron and Wiens (2011) recognized the leptodactylid subfamily Leiuperinae to comprise the genera Edalorhina, Engystomops, Eupemphix [raised as a junior synonym of Physalaemus by Faivovich et al. (2012)], Physalaemus, Pleurodema (including Somuncuria) and Pseudopaludicola. Fouquet et al. (2013), in an extensive analysis of Leptodactylidae, also recovered a mono- phyletic Leiuperinae, whereas Faivovich et al. (2012), in a study designed to analyze the internal relationships in Pleurodema, did not recover Pseudopaludicola as belonging to the clade that includes all the remaining leiuperines. Although Pleurodema has been repeatedly recovered as the sister group of a clade composed of Edalorhina, Engystomops and Physalaemus (Frost et al., 2006; Grant et al., 2006; Lourenço et al., 2008; Pyron and Wiens, 2011; http://crossmark.crossref.org/dialog/?doi=10.1016/j.ympev.2015.06.011&domain=pdf http://dx.doi.org/10.1016/j.ympev.2015.06.011 mailto:bolsoni@unicamp.br http://dx.doi.org/10.1016/j.ympev.2015.06.011 http://www.sciencedirect.com/science/journal/10557903 http://www.elsevier.com/locate/ympev L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 205 Faivovich et al., 2012; Fouquet et al., 2013), it is not clear which is the sister group of Physalaemus. The internal phylogenetic relationships of Physalaemus are even less well studied than its intergeneric cladistic proximity. Currently, 46 named species of Physalaemus are known (listed in Frost, 2014), but even in the most comprehensive phylogenetic analyses of leiuperines conducted to date, only a few species of Physalaemus were included [in addition to P. nattereri, Pyron and Wiens (2011), Fouquet et al. (2013) and de Sá et al. (2014) analyzed eight species of Physalaemus, whereas Faivovich et al. (2012) ana- lyzed only five]. Tárano and Ryan (2002), in a study of the advertisement call of Physalaemus enesefae (currently a junior synonym of Physalaemus fischeri), presented in the introduction of their paper a preliminary phylogenetic inference of Physalaemus relationships performed by D. Cannatella, M. Holder, D. Hillis, A. S. Rand and M. J. Ryan. This inference included ten species of Physalaemus in addition to ‘‘P. enesefae’’ and, according to Tárano and Ryan (2002), it was based on morphological characters, allozymes and mitochondrial DNA sequences, but no further information about the phylogenetic methods used was provided. Tárano and Ryan (2002) noted two monophyletic groups in that cladogram, one of them composed of the species currently assigned to Engystomops and the other composed of the species of Physalaemus (Fig. 1), but the authors did not discuss the internal relationships of those groups. Despite the absence of a study specially designed to evaluate the phylogenetic relationships in Physalaemus, morphological groups of species have been recognized. Lynch (1970), who consid- ered Engystomops a synonym of Physalaemus, recognized four spe- cies groups in Physalaemus: the P. pustulosus Group (including species currently in the genus Engystomops), the P. cuvieri Group, Fig. 1. Phylogenetic relationships of Physalaemus species presented by Tárano and Ryan Hillis, A.S. Rand and M.J. Ryan. the P. biligonigerus Group and the P. signifer Group. Based on a phe- netic analysis of morphometric data, external morphology, color patterns and osteological characters, Nascimento et al. (2005) reviewed the proposal of Lynch (1970) and in addition to resurrect- ing the genera Engystomops and Eupemphix, rearranged the Physalaemus species into seven groups (P. albifrons, P. cuvieri, P. deimaticus, P. gracilis, P. henselii, P. olfersii and P. signifer Species Group). However, the monophyly of each of these species groups has not been tested, and recent studies have noted the lack of known synapomorphies for two of them, i.e., the P. henselii (Tomatis et al., 2009) and the P. albifrons (Vittorazzi et al., 2014) Groups. The need for phylogenetic analysis of Physalaemus is evident not only from a taxonomic point of view but also because such analysis will enable accurate evolutionary studies of several character sys- tems, including chromosomal data. Cytogenetic data are available for 29 of the 46 species of Physalaemus (Beçak, 1968; Beçak et al., 1970; Denaro, 1972; de Lucca et al., 1974; Silva et al., 1999, 2000; Amaral et al., 2000; Lourenço et al., 2006; Ananias et al., 2007; Quinderé et al., 2009; Tomatis et al., 2009; Milani et al., 2010; Nascimento et al., 2010; Provete et al., 2012; Vittorazzi et al., 2014), and this set of data has already been explored by Tomatis et al. (2009) and Vittorazzi et al. (2014) in an attempt to evaluate the arrangement of the P. henselii and P. albifrons Groups as proposed by Nascimento et al. (2005). Despite the noto- rious interspecific variation in the location of nucleolus organizer regions (NORs) in Physalaemus, the steps of karyotypic evolution in this genus and the recognition of chromosomal synapomorphies for species groups remain largely unknown (see comments in Tomatis et al., 2009 and Vittorazzi et al., 2014). One critical limita- tion to the cytogenetic studies of Physalaemus has been the scarcity (2002), based on unpublished analysis performed by D. Cannatella, M. Holder, D. 206 L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 of chromosomal markers, which prevents a reliable recognition of interspecific chromosome homeology, especially with regards to the smallest of the 22 chromosomes of the diploid complement (i.e., chromosome pairs 8–11) (see Vittorazzi et al., 2014). It is also noteworthy that the cytogenetic data, especially the NOR locations, have revealed cryptic diversity in Physalaemus, particularly with respect to P. cuvieri, which some have hypothesized indicates the presence of unnamed species (Quinderé et al., 2009). In this con- text, a phylogenetic hypothesis for the relationships of the species of Physalaemus, which should include a deeper analysis of the genetic diversity of P. cuvieri, would help elucidate the evolution- ary variation of the chromosomal data. In this study, we aim to investigate the interspecific relationships in Physalaemus, to test the monophyly of the recog- nized species groups and to provide a revision of the chromosomal data available for this genus in the light of a phylogenetic hypothesis. 2. Materials and methods 2.1. Taxon sampling We analyzed 41 of the 46 currently known species of Physalaemus (Appendix A), including all the nominal species of the P. albifrons, P. cuvieri, P. deimaticus, P. gracilis and P. henselii Groups; all but one species of the P. olfersii Group; and all but three species of the P. signifer Group. For ten of the analyzed spe- cies, topotypical specimens or individuals from the vicinities of the type localities were sampled. Because high diversity in P. cuvieri was revealed by cytogenetic analyses (Quinderé et al., 2009), which raises the hypothesis of existence of unnamed spe- cies, we used a large sample of this taxon by analyzing speci- mens from 19 localities (Appendix A), which included most of the sites sampled by Quinderé et al. (2009). We also included exemplars of all the other Leiuperinae genera (i.e., Edalorhina, Engystomops, Pleurodema and Pseudopaludicola) and of the two other subfamilies of Leptodactylidae (i.e., Leptodactylinae and Paratelmatobiinae) (Appendix A). Because the phylogenetic relationships of the family Leptodactylidae are still controversial (see Frost et al., 2006; Grant et al., 2006; Pyron and Wiens, 2011), we included representatives of the following families of Hyloidea: Alsodidae, Centrolenidae, Ceratophryidae, Cycloramphidae, Hylodidae, Odontophrynidae, Telmatobiidae and Hylidae, which was inferred by Frost et al. (2006) and Pyron and Wiens (2011) as the sister group of a clade that included Leptoctylidae and all the other aforementioned hyloid families, among others (Appendix A). The DNA sequences reported here and those recovered from GenBank are all indicated in Appendix A. 2.2. Mitochondrial and nuclear gene sequencing Genomic DNA was extracted from tissue samples obtained from scientific collections (see Appendix A). Tissue samples were immersed in a TNES buffer solution (50 mM Tris, pH 7.5, 400 mM NaCl, 20 mM EDTA, 0.5% SDS). The solution was subsequently supplemented with proteinase K (to a final concentration of 100 lg/mL) and the samples were incubated for 5 h at 55 �C. Then, 1/3 volume of 5 M NaCl was added, and the samples were centrifuged. DNA was precipitated from the supernatant with iso- propyl alcohol, washed with ethanol (70%), resuspended in TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and stored at �20 �C. The mitochondrial 12S and 16S ribosomal genes and the inter- vening tRNA-val region were PCR amplified using the primer pairs MVZ59(L) (Graybeal, 1997)/TitusI(H) (Titus, 1992) and 12L13(L) (Feller and Hedges, 1998)/16Sbr(H) (Palumbi et al., 1991). For PCR amplification of a segment of the nuclear gene RAG-1, the pri- mers RAG-1F and RAG-1R (Faivovich et al., 2005) were used. The PCR products were purified with GFX PCR and Gel Band DNA purifi- cation kits (GE Healthcare, England) and directly sequenced using BigDye Terminator kits (Applied Biosystems, Foster City, CA, USA) in an automatic DNA ABI/Prism sequencer (Applied Biosystems, Foster City, CA, USA). The mitochondrial genes were sequenced using the primers MVZ59(L) (Graybeal, 1997), MVZ50(H) (Graybeal, 1997), 12L13 (Feller and Hedges, 1998), TitusI(H) (Titus, 1992), Hedges16L2a (Hedges, 1994), Hedges16H10 (Hedges, 1994), 16Sar(L) (Palumbi et al., 1991) and 16Sbr(H) (Palumbi et al., 1991), whereas the RAG-1 segment was sequenced with the same primers used for PCR amplification. DNA sequences were edited using Bioedit version 7.0.1 (http://www.mbio.ncsu. edu/BioEdit/bioedit.html). 2.3. Phylogenetic inferences Homologous 165 segments with approximately 2.5 kb com- posed a mitochondrial DNA matrix, and these sequences together with 76 RAG-1 sequences of 410 bp generated a concatenated matrix. Because missing data may compromise phylogenetic infer- ences (e.g., Agnarsson and May-Collado, 2008; Simmons, 2012), we inferred phylogenetic relationships separately from the mitochon- drial data matrix and from the concatenated data matrix. Each matrix was analyzed using both maximum-parsimony and Bayesian criteria. When using the maximum-parsimony criterion, we inferred the phylogenetic relationships based on both dynamic-homology and static-homology hypotheses. The phylogenetic analysis based on direct optimization of una- ligned characters was implemented with the software POY v.5.1.1 (Varón et al., 2010). The phylogenetic searches performed with POY included tree building (of at least 18 Wagner trees), tree bisec- tion–reconnection (TBR) swapping, perturbation using a parsi- mony ratchet and tree fusing. The analyses were run with a maximum execution time of 5 days and cost 1 for gap opening, gap extension and nucleotide substitution. To obtain an implied alignment from the POY analysis, the characters were transformed into static characters and the generated matrix was exported using the command ‘‘phastwinclad.’’ The exported matrix was loaded with TNT v.1.1 to calculate bootstrap support based on 1000 pseu- doreplicates, using traditional search. For the analysis using the static homology hypothesis, the mtDNA sequences were aligned with Muscle (Edgar, 2004). The alignment obtained under default parameters was improved twice with the ‘‘refine’’ command, rendering a matrix with 2528 charac- ters. The alignment of the RAG-1 sequences was unambiguous because this data matrix had no indel and the final concatenated data matrix included 2938 characters. The phylogenetic analyses of the aligned mitochondrial and concatenated data matrices under the maximum-parsimony criterion were performed using the soft- ware TNT v.1.1 (Goloboff et al., 2003). Gaps were considered a fifth character state. The most parsimonious trees were inferred through heuristic searches performed using the new technology search, including combined sectorial searches, the ratchet, tree drifting and tree fusing. The trees were obtained from driven searches, and the best length was hit 100 times. The bootstrap val- ues of the branches inferred in these analyses were calculated with 1000 pseudoreplicates, using traditional search on TNT. For the Bayesian analyses of the aligned data matrices, the GTR+I+G model of DNA evolution was inferred for the mitochon- drial data, and the models JC+G, K80+G and SYM+G were inferred, respectively, for the first, second and third codon positions of the RAG-1 sequences, using the software MrModeltest v.2.3 (Nylander, 2004). The phylogenetic analyses were implemented in the software MrBayes v.3.2 (Ronquist et al., 2011). Two http://www.mbio.ncsu.edu/BioEdit/bioedit.html http://www.mbio.ncsu.edu/BioEdit/bioedit.html L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 207 simultaneous analyses were run, each with four chains (three heated and one cold). In each analysis, 20 million generations were run. One tree was sampled every 100 generations. Consensus topology and posterior probabilities were produced after discard- ing the first 25% of the trees generated. The ASDSF (Average Standard Deviation of Split Frequencies) value was below 0.01 and the PSRF (Potential Scale Reduction Factor) values were approximately 1.000. The stabilization of posterior probabilities was checked using Tracer v. 1.5 (Rambaut and Drummond, 2007). 2.4. Genetic distances among lineages related to Physalaemus cuvieri, P. ephippifer and P. fischeri The genetic distances among Physalaemus ephippifer, P. fischeri and the lineages of P. cuvieri recognized in the phylogenetic analy- ses were estimated by pairwise comparisons of �500 bp sequences of the 16S mitochondrial rRNA gene using the software Mega v.6.0 (Tamura et al., 2013). Sequences of P. cuvieri specimens from Bolivia studied by Jansen et al. (2011) (JF789851-JF789858) were also included. Additionally, pairwise comparisons were provided for the other species of the P. cuvieri Species Group. 2.5. Cytogenetic analysis Because the presence of a small telocentric chromosome becomes an important feature to be discussed in the present study and because no species of the Physalaemus deimaticus group had been karyotyped to date, we described the karyotype of the ZUEC 21193 specimen of P. deimaticus collected from Diamantina-MG, Brazil. Metaphase chromosome spreads were obtained from cell suspensions of intestine after an in vivo treat- ment with colchicine using a protocol adapted from King and Rofe (1976). Prior to the removal of the intestine, the animal was deeply anesthetized with a 2% lidocaine gel. Chromosomes were conventionally stained with 10% Giemsa and sequentially Fig. 2. Intergeneric relationships of Physalaemus. Overall relationships recovered in one mitochondrial and Rag-1 sequences. Branch lengths are proportional to inferred amount consensus tree. The interspecific relationships in the Physalaemus signifer Clade and Physa bootstrap values (P50%). Asterisks indicate bootstrap value of 100%. Numbers to the ri same data matrix (see Appendix F). C-banded (King, 1980) and silver stained by the Ag-NOR method (Howell and Black, 1980). 3. Results 3.1. Phylogenetic inferences In all our analyses, Physalaemus was monophyletic, Pleurodema was inferred as the sister group of a clade that included Physalaemus, Edolorhina and Engystomops, and Pseudopaludicola was the sister group of a clade composed of all the remaining leiu- perines. In contrast, the relationships among Physalaemus, Edalorhina and Engystomops differed among the cladograms gener- ated. Whereas Edalorhina was the sister group of the clade com- posed of Physalaemus and Engystomops in the Bayesian analyses (Appendices F and G), in all of the most parsimonious trees achieved by TNT and POY, Edalorhina was more closely related to Engystomops (Fig. 2; Appendices B–E). The relationships inferred for the species of Physalaemus in all of the analyses were quite congruent (Figs. 3–5; Appendices B–G). Physalaemus nattereri (Eupemphix nattereri according to Nascimento et al., 2005) was nested within the Physalaemus clade as the sister taxon of a clade composed of all the representatives of the P. signifer and P. deimaticus Groups (sensu Nascimento et al., 2005) (Fig. 3; Appendices B–G). The monophyly of the P. deimaticus Group as defined by Nascimento et al. (2005) was inferred in all the analyses, with high statistical support (Fig. 3; Appendices B–G). A highly supported clade composed of the representatives of the P. signifer Group (sensu Nascimento et al., 2005), except P. maculiven- tris, was also recovered in all the analyses (Fig. 3; Appendices B–G). A clade with all the species of the P. signifer Group as recognized by Nascimento et al. (2005), including P. maculiventris, however, was not achieved in our analyses, since P. maculiventris was inferred as the sister taxon of a clade that included the remaining species of the P. signifer Group and all the species of the P. deimaticus Group (Fig. 3; Appendices B–G). The clade composed of P. nattereri, of the 32 most parsimonious trees inferred in the TNT analysis of the concatenated s of sequence evolution. All of the nodes shown here were also present in the strict laemus cuvieri Clade are shown in Figs. 3 and 4. Numbers to the left of branches are ght of branches are posterior probabilities achieved in the Bayesian analysis of the Fig. 3. Interspecific relationships in the Physalaemus signifer Clade. Partial view of one of the 32 most parsimonious trees inferred in the TNT analysis of the concatenated mitochondrial and Rag-1 sequences. Branch lengths are proportional to inferred amounts of sequence evolution. All of the nodes were also present in the strict consensus tree. Numbers to the left of branches are bootstrap values (P50%). Asterisks indicate bootstrap value of 100%. Numbers to the right of branches are posterior probabilities achieved in the Bayesian analysis of the same data matrix (see Appendix F). Branches lacking posterior probability were not recovered in the Bayesian analysis. Fig. 4. Interspecific relationships in the Physalaemus cuvieri Clade. Partial view of one of the 32 most parsimonious trees inferred in the TNT analysis of the concatenated mitochondrial and Rag-1 sequences. Branch lengths are proportional to inferred amounts of sequence evolution. All of the nodes shown here were also present in the strict consensus tree. The interspecific relationships in the Physalaemus cuvieri Species Group are shown in Fig. 5. Numbers to the left of branches are bootstrap values (P50%). Asterisks indicate bootstrap value of 100%. Numbers to the right of branches are posterior probabilities achieved in the Bayesian analysis of the same data matrix (see Appendix F). Branches lacking posterior probability were not recovered in the Bayesian analysis. 208 L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 the P. deimaticus Group and all the species included in the P. signifer Group by Nascimento et al. (2005) was highly supported by boot- strap or posterior probability in our analyses (Fig. 3; Appendices B–G) and is hereafter called P. signifer Clade. The species of Physalaemus not included in the P. signifer Clade were nested in a distinct group (Figs. 4 and 5), which we call the P. cuvieri Clade (Fig. 3; Appendices B–G). In this major clade, a mono- phyletic group with all the five species assigned to the P. gracilis Group (sensu Nascimento et al., 2005) was identified in all the inferences (Fig. 4; Appendices B–G). In contrast, we could not sup- port the P. albifrons, P. cuvieri, P. henselii and P. olfersii phenetic groups, as they were recognized by Nascimento et al. (2005). Because Physalaemus albifrons was nested within the P. cuvieri Group instead of being closely related to P. biligonigerus, P. mar- moratus and P. santafecinus, the P. albifrons and P. cuvieri Groups proposed by Nascimento et al. (2005) were not corroborated. In addition, our analyses did not support inclusion of P. cicada in the P. cuvieri Group (Figs. 4 and 5; Appendices B–G). The Physalaemus henselii Group of Nascimento et al. (2005) was not monophyletic because P. riograndensis was not closely related to P. henselii and P. fernandezae. In all our analyses, P. riograndensis was recovered as the sister species of a clade composed of P. biligonigerus, P. marmoratus and P. santafecinus (Fig. 4; Appendices B–G). Fig. 5. Interspecific relationships in the Physalaemus cuvieri Species Group. Partial view of one of the 32 most parsimonious trees inferred in the TNT analysis of the concatenated mitochondrial and Rag-1 sequences. Branch lengths are proportional to inferred amounts of sequence evolution. Circles indicate nodes that collapsed in the strict consensus tree (see Appendix B). Numbers to the left of branches are bootstrap values (P50%). Asterisks indicate bootstrap value of 100%. Numbers to the right of branches are posterior probabilities achieved in the Bayesian analysis of the same data matrix (see Appendix F). Branches lacking posterior probability were not recovered in the Bayesian analysis. The lineages of ‘‘P. cuvieri’’, P. ephippifer and P. fischeri are shown in different colors and their sampling localities are indicated in the inset map. L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 209 The Physalaemus olfersii Group of Nascimento et al. (2005) was polyphyletic because P. aguirrei did not form a clade with P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga (the two latter spe- cies were described after the proposal of the P. olfersii Group and were assigned to this group by Cassini et al., 2010) (Fig. 4; Appendices B–G). In all the analyses, P. aguirrei was the sister group of a clade composed of the P. gracilis Group, the clade (P. olfersii, P. soaresi, P. maximus, P. feioi, P. lateristriga) and the clade (P. riograndensis, P. biligonigerus, P. marmoratus P. santafecinus), but the relationships between these two latter clades and the P. gracilis Group varied among the analyses. In the TNT analyses, the clade (P. riograndensis, P. biligonigerus, P. marmoratus, P. santafecinus) was the sister group of a clade composed of the P. gra- cilis Group and the clade (P. olfersii, P. soaresi, P. maximus, P. feioi, P. lateristriga) (Fig. 4; Appendices B–C). In the POY analyses (Appendices D–E) and in the Bayesian analysis of the mitochon- drial matrix (Appendix G), the clade (P. riograndensis, P. biligo- nigerus, P. marmoratus, P. santafecinus) was the sister group of the P. gracilis Group. In the Bayesian analysis of the concatenated matrix (Appendix F), the relationships between P. gracilis Group, the clade (P. olfersii, P. soaresi, P. maximus, P. feioi, P. lateristriga) and the clade (P. riograndensis, P. biligonigerus, P. marmoratus, P. santafecinus) remain unresolved. 3.1.1. Relationships of Physalaemus cuqui and P. albonotatus We notice that Physalaemus cuqui composed with P. albonotatus a clade that was the sister group of a clade that included individu- als collected from Paraguay and Argentina [here named Physalaemus sp. (aff. albonotatus)], which are morphologically sim- ilar to P. albonotatus but distinguished from it by the advertisement call (Fig. 5; Appendices B–G). In all the analyses, we also inferred paraphyly of P. albonotatus with regard to P. cuqui because the specimen of P. cuqui analyzed here was nested among the exem- plars of P. albonotatus (Fig. 5; Appendices B–G). The genetic dis- tance, estimated from 16S partial sequences, between P. cuqui and the group composed of these exemplars of P. albonotatus was only 0.3%. In contrast, a high uncorrected p-distance (7.3%) was observed between the clade (P. albonotatus, P. cuqui) and Table 1 Uncorrected p-distances between 16S partial genes of individuals of species/lineages of the Physalaemus cuvieri Species Group. In gray, the uncorrected p-distances found within each lineage or species. –: data not estimated because only one sequence was available. 210 L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 its sister group [Physalaemus sp. (aff. albonotatus) clade] (Table 1). 3.1.2. Relationships of Physalaemus cuvieri, P. ephippifer and P. fischeri The paraphyly of Physalaemus cuvieri with respect to P. ephip- pifer was inferred in all our analyses. The specimens first identified as Physalaemus cuvieri, collected from 19 localities within the wide geographical distribution of this species, clustered in distinct clades. One of the clades comprised the specimens distributed from Central to Southern Brazil and Argentina (Vitória da Conquista-BA, Palmeiras-BA, Chapada dos Guimarães-MT, Uberlândia-MG, Nova Itapirema-SP, Palestina-SP, Vitória Brasil-SP, Embu-SP, Rio Claro-SP, Passo Fundo-RG, Puerto Iguazú-MI,) (Fig. 5) and was highly supported in all our inferences (Fig. 5; Appendices B–G). Another highly supported clade clustered the specimens from Porto Nacional-TO (Fig. 5; Appendices B–G). The remaining specimens of P. cuvieri, which were distributed in Northern/Northeastern Brazil, were clustered into two clades, one of them composed of the specimens from Caruaru-PE and Alagoinha-BA, and the other composed of the individuals from Crateús–CE, Balsas–MA, São Luís–MA, Urbano Santos–MA and Araruna-PB (Fig. 5; Appendices B–G). In the maximum -parsimony analyses, both of these clades together composed the sister group of P. ephippifer (Fig. 5; Appendices B–E). In the Bayesian analysis of the concatenated matrix, the clade with the individuals from Caruaru-PE and Alagoinha-BA was recovered as the sister group of P. ephippifer, despite with very low support (0.54) (Appendix F). In the Bayesian analysis of the mitochondrial matrix, the clade that grouped the specimens from Caruaru-PE and Alagoinha-BA was recovered in a polytomy together with the P. ephippifer clade and the clade that clustered the remaining spec- imens from Northern/Northeastern Brazil (Appendix G). The clade composed of the two clades that comprised the exemplars of P. cuvieri from Northern/Northeastern Brazil and the P. ephippifer clade was highly supported in all the analyses; this clade was the sister group of the clade that comprised the specimens from Central to Southern Brazil and Argentina (Fig. 5; Appendices B–G). Therefore, based on the phylogenetic inferences, four different groups, distributed over distinct geographic areas, may be recog- nized for P. cuvieri, diagnosing putatively independent evolution- ary lineages, hereafter called Lineage 1A (Caruaru-PE, Alagoinha-BA), Lineage 1B (Crateús-CE, Urbano Santos-MA, Araruna-PB, Balsas-MA, São Luís-MA), Lineage 2 (Vitória da Conquista-BA, Palmeiras-BA, Chapada dos Guimarães-MT, Uberlândia-MG, Nova Itapirema-SP, Palestina-SP, Vitória Brasil-SP, Embu-SP, Rio Claro-SP, Passo Fundo-RG, Puerto Iguazú-MI) and Lineage 3 (Porto Nacional-TO) of ‘‘P. cuvieri’’. The relationships of Physalaemus fischeri and the Lineage 3 of ‘‘P. cuvieri’’ with the clade that includes P. ephippifer and the remaining lineages recognized among the individuals first identified as P. cuvieri remain unclear. In the Bayesian inferences (Appendices F–G) and POY analyses (Appendices D–E) P. fischeri was the sister group of a clade that includes P. ephippifer and all the individuals first identified as P. cuvieri, whereas in the TNT analyses (Fig. 5; Appendices B–C) the Lineage 3 of ‘‘P. cuvieri’’ was the sister group of a clade composed of all the other lineages of ‘‘P. cuvieri’’, P. ephip- pifer and P. fischeri, rendering P. cuvieri paraphyletic also with respect to P. fischeri. Neither of these two arrangements was highly supported by bootstrap or posterior probability, whereas the clade that includes all the lineages of ‘‘P. cuvieri’’, P. ephippifer and P. fischeri was (Fig. 5; Appendices B–G). 3.2. Genetic comparisons among the lineages related to Physalaemus cuvieri, P. ephippifer and P. fischeri Low genetic distance was observed between the Lineages 1A and 1B of ‘‘P. cuvieri’’ (1.4%) as well as between them and P. ephip- pifer (1.5% and 1.2%) (Table 1). In contrast, high values of uncor- rected p-distance were observed when Lineages 2 and 3 of ‘‘P. cuvieri’’ were compared with each other (6.0%) or with P. ephippifer and the Lineages 1A and 1B of ‘‘P. cuvieri’’ (ranging from 4.0% to 7.5%) (Table 1). By comparing 16S gene fragments of specimens from two Bolivian lineages of Physalaemus cuvieri recognized by Jansen et al. (2011) with the equivalent gene fragments obtained from individuals nested in the four population-level lineages of P. cuvieri recognized here, high values of uncorrected p-distance were observed (3.4% to 7.1%), as was the distance between the Bolivian lineages (3.1%) (Table 1). Several of the aforementioned values were higher than that which emerged from the comparison of samples from P. erikae and P. kroyeri (3.6%), two valid species also assigned to the P. cuvieri Group (Table 1). The uncorrected p-dis- tances estimated between P. fischeri and all of those P. cuvieri lin- eages were also high (ranging from 7.9% to 10.1%), as was that calculated between P. fischeri and P. ephippifer (7.8%) (Table 1). 3.3. The karyotype of Physalaemus deimaticus All the 25 metaphases of the SMRP 497.2 specimen of Physalaemus deimaticus showed 22 chromosomes, including 10 pairs of metacentric or submetacentric chromosomes and one pair (chromosome pair 11) of telocentric chromosomes (Fig. 6A). Large amounts of C-banded heterochromatin were detected in the cen- tromeric/pericentromeric regions of all the chromosomes (Fig. 6A). The nucleolus organizer region (NOR) was detected in Fig. 6. A. Karyotype of Physalaemus deimaticus stained with Giemsa (top) and C-banded (bottom). Note the telocentric chromosome pair 11. In B, the same NOR-bearing chromosome 1 C-banded in A after staining with Giemsa (top) or submitted to the Ag-NOR method. The arrows in B indicate the secondary constrictions of the NORs in the Giemsa stained chromosome and the silver stained NORs. L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 211 the long arm of the large metacentric chromosomes classified as number 1, and it coincided with a C-band (Fig. 6B). 4. Discussion 4.1. Intergeneric relationships of Physalaemus The close relationship of Pleurodema with a clade composed of Physalaemus, Edalorhina and Engystomops, previously inferred by Pyron and Wiens (2011), Faivovich et al. (2012) and Fouquet et al. (2013), was also recovered in all our phylogenetic inferences. Although with low statistical support, our inferences also provide additional evidence of the close relationship between Pseudopaludicola and the remaining Leiuperinae, corroborating Fouquet et al. (2013). In contrast, our analyses were not conclusive with regard to the closer intergeneric relationships of Physalaemus. Although the Bayesian inferences yielded Physalaemus as the sister genus of Engystomops, as also recovered by the Bayesian analyses of Pyron and Wiens (2011) and Fouquet et al. (2013), the maximum-parsimony analyses yielded a closer relationship between Edalorhina and Engystomops, as previously inferred by Frost et al. (2006), Grant et al. (2006), Lourenço et al. (2008) and Faivovich et al. (2012). 4.2. Interspecific relationships in Physalaemus We increased from one to three the number of exemplars (from different localities) of Physalaemus nattereri and from eight to 41 the number of species of Physalaemus included in phylogenetic analyses, providing a reliable test of the monophyly of this genus. In all our phylogenetic inferences, P. nattereri was recovered inside the Physalaemus clade, corroborating that Eupemphix should be considered a junior synonym of Physalaemus, as previously stated by Faivovich et al. (2012). The topologies recovered in all our phylogenetic inferences sug- gest two major clades in Physalaemus: (i) the Physalaemus signifer Clade, composed of P. nattereri, and the species previously placed in the P. deimaticus and P. signifer Groups; and (ii) the Physalaemus cuvieri Clade, with the remaining species of Physalaemus. This inference is congruent with the topologies obtained by previous authors (Pyron and Wiens, 2011; Faivovich et al., 2012). Among leiuperines, two morphological characters have been used for supra-specific arrangements: the maxillary and premaxil- lary dentition (e.g., Cochran, 1955; Bokermann, 1962, 1966; Cannatella et al., 1998); and the tarsal tubercle (the inner tarsal tubercle in some papers) (e.g., Cochran, 1955; Cannatella and Duellman, 1984; Cannatella et al., 1998; Funk et al., 2008). Lynch (1970) considered this variation too discordant to reveal species relationships. However, more recently, Nascimento et al. (2005) noted the absence of premaxillary and maxillary dentition for P. nattereri (as Eupemphix) and for all species they assigned to the P. signifer and P. deimaticus Groups, which correspond to the P. sig- nifer Clade inferred in our analyses. In contrast, they noted the presence of premaxillary and maxillary teeth in the remaining spe- cies groups (i.e., the P. albifrons, P. cuvieri, P. gracilis, P. henselii and P. olfersii Groups). Nevertheless, as shown by several authors (Cardoso and Haddad, 1985; Heyer, 1985; Heyer and Wolf, 1989; Lobo, 1996; Pombal and Madureira, 1997; Haddad and Sazima, 2004; Pimenta et al., 2005; Weber et al., 2005; Cruz et al., 2007), most species of the P. signifer Clade have maxillary and premaxil- lary teeth (in some papers indicated as ‘‘teeth not visible but dis- cernible by probe’’). In addition, all species of Edalorhina, Pleurodema and Pseudopaludicola, and the species of Engystomops of the Duovox clade (i.e., E. coloradorum, E. guayaco, E. montubio, E. pustulatus, E. puyango and E. randi) have maxillary teeth (Lynch, 1970; Lobo, 1995; Ron et al., 2006). The maxillary teeth are absent in species of Engystomops of the E. edentulus clade (E. freibergi, E. petersi and E. pustulosus), and this character state opti- mizes as a synapomorphy of this clade, as previously indicated (Cannatella et al., 1998; Ron et al., 2005; Ron et al., 2006; Ron et al., 2010). Therefore, the presence of premaxillary and maxillary teeth is plesiomorphic in Physalaemus, and the absence versus presence of premaxillary and maxillary teeth must be carefully considered in this genus. In contrast, the tarsal tubercle clearly allows characterizing the species of Physalaemus as belonging to two major clades. All species of the P. signifer Clade lack tarsal tubercles, whereas all species of the P. cuvieri Clade have a tarsal tubercle with vari- able development (polymorphic in P. lateristriga, see Cassini et al., 2010). Analogously, this character was used for characterizing the two major groups in Engystomops, and the absence of the tarsal tubercle was suggested as a synapomorphy of the Duovox clade of Engystomops (Cannatella et al., 1998; Ron et al., 2005, 2006). In the remaining leiuperines, this character also shows variation; all species of Pseudopaludicola and most Pleurodema lack the tar- sal tubercle (except for P. allium and P. diplolister, which do have tarsal tubercles; Maciel and Nunes, 2010), whereas in the two species of Edalorhina, tubercles are present (Heyer, 1975). Thus, although in both Physalaemus and Engystomops these characters discriminate major groups, their optimization in leiuperines is ambiguous. 4.2.1. The Physalaemus signifer Clade A remarkable morphological characteristic of the species of the Physalaemus signifer Clade is the presence of a dark arrowhead-shaped mark on the dorsum (e.g., Bokermann 1966; Cardoso and Haddad, 1985; Pimenta et al., 2005; Caramaschi 212 L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 et al., 1991; Caramaschi et al., 2003). The presence of this trait in P. nattereri was noted by Ananias et al. (2007) and Kolenc et al. (2011), and it was previously proposed as a synapomorphy of the P. signifer Group (sensu Nascimento et al., 2005) shared with P. nattereri (Ananias et al., 2007). In our phylogenetic hypotheses, this character optimized as a synapomorphy of the P. signifer Clade. Physalaemus angrensis, P. caete and P. irroratus, not included in our analyses, were originally included in the P. signifer Group (sensu Nascimento et al., 2005) by Weber et al. (2005), Nascimento et al. (2005) and Cruz et al. (2007), respectively. These species lack the tarsal tubercles and have a dark arrowhead-shaped mark on the dorsum. Therefore, we tentatively assign them to the P. signifer Clade. Another synapomorphy for the Physalaemus signifer Clade emerges from the cytogenetic analysis. The five species of the P. signifer Group already karyotyped (i.e., P. atlanticus, P. crombiei, P. moreirae, P. spiniger and P. signifer) have a telocentric chromo- some pair as the smallest pair (chromosome pair 11) of the com- plement (de Lucca et al., 1974; Silva et al., 2000; Ananias et al., 2007), as do P. nattereri (Beçak, 1968; Lourenço et al., 2006; Ananias et al., 2007) and P. deimaticus (present work), which is the only species of the P. deimaticus Group karyotyped to date. In contrast, the remaining 23 species of Physalaemus studied cytogenetically have a biarmed chromosome pair 11 (Beçak et al., 1970; Denaro, 1972; de Lucca et al., 1974; Silva et al., 1999, 2000; Amaral et al., 2000; Quinderé et al., 2009; Tomatis et al., 2009; Milani et al., 2010; Nascimento et al., 2010; Provete et al., 2012; Vittorazzi et al., 2014), except for P. fernan- dezae (Tomatis et al., 2009). The phylogenetic relationships inferred herein provide strong evidence for the independent ori- gin of the telocentric chromosome 11 of P. fernandezae and the chromosome 11 of species previously allocated to the P. signifer Group, corroborating the hypothesis discussed by Tomatis et al. (2009). Our analyses also strongly suggest that the telocentric chromosome 11 of P. nattereri is homeologous to the chromo- some 11 found in the species of the P. signifer Group, constituting a synapomorphy for the P. signifer Clade (Fig. 5). Inside the Physalaemus signifer Clade, a monophyletic group that includes all the species of the P. deimaticus Group previously pro- posed by Nascimento et al. (2005) could be recognized and was strongly supported by our analyses. Physalaemus nattereri, the P. deimaticus Species Group (i.e., P. deimaticus, P. erythros and P. rupestris) and the remaining species of the P. signifer Clade (i.e., P. atlanticus, P. bokermanni, P. camacan, P. crombiei, P. nanus, P. obtectus, P. signifer, P. spiniger, P. maculiventris and P. moreirae) occur in different biomes. Physalaemus nattereri, which is the sister taxon to a clade comprising the remainder of the P. signifer Clade, is widespread in open areas from east of Paraguay and Bolivia to cen- tral and southeastern Brazil (IUCN, 2013.2), whereas the species of the P. deimaticus Group are restricted to high elevations of the Espinhaço Mountain Range (Nascimento et al., 2005; Frost, 2014), localized in the Brazilian state of Minas Gerais between the Cerrado and Atlantic rainforest biomes, and the remaining spe- cies of the P. signifer Clade occur in the Atlantic rainforest (Nascimento et al., 2005). The Atlantic rainforest and the Espinhaço Mountain Range are areas of high endemism and biodi- versity (Costa et al., 2000; Nogueira et al., 2011; Freitas et al., 2012). 4.2.2. The Physalaemus cuvieri Clade This major clade, which comprised all the Physalaemus species not included in the P. signifer Clade, was not strongly supported in our analyses, and further studies are still necessary to confirm it. However, five species groups may be recognized in the P. cuvieri Clade (the P. cuvieri Group, the P. biligonigerus Group, the P. gracilis Group, the P. henselii Group and the P. olfersii Group) (discussed below) in addition to P. cicada and P. aguirrei. Despite the P. henselii Group being the sister taxon to the remainder of the P. cuvieri Clade and P. aguirrei being the sister taxon of a clade composed of the P. biligonigerus, P. olfersii and P. gracilis Groups in all our inferences, the relationships between these three latter species groups remain to be elucidated. The larval oral disc in Physalaemus can have five different con- figurations based on the combination of three characters: ventral gap, ventrolateral gaps and number of lower tooth rows (see Vera Candioti et al., 2011). The common character that defines the oral disc configurations C4 and C5 (sensu Vera Candioti et al., 2011) is the presence of a ventral gap in the lower marginal papil- lae. This character state is unique among non-bufonid Leptodactyliformes and is shared by the P. henselii Group, P. cicada and all species of the P. cuvieri Group (as here defined). It is also present in a few Pseudopaludicola species (see Vera Candioti et al., 2011), but is not present in species of the P. olfersii, P. biligo- nigerus or P. gracilis Groups, or in P. fischeri (P. cuvieri Group) or in P. aguirrei. Optimization of this character on the phylogenetic hypotheses inferred here reveals that the presence of a ventral gap is a synapomorphy of the P. cuvieri Clade with reversions in P. fischeri and in the clade (P. aguirrei, P. biligonigerus Group, P. gracilis Group, P. olfersii Group). 4.2.2.1. The Physalaemus biligonigerus Species Group. All our analy- ses recovered a highly supported clade consisting of Physalaemus biligonigerus, P. marmoratus and P. santafecinus, species that share some morphological similarities previously reported by Lynch (1970) and Nascimento et al. (2005). These species were not closely related to P. nattereri as proposed by Lynch (1970) or to P. albifrons as stated by Nascimento et al. (2005), leaving both the P. nattereri Group by Lynch and the P. albifrons Group as defined by Nascimento and colleagues polyphyletic. Our phylogenetic inferences, therefore, corroborate that the large heterochromatic band present in the short arm of chromo- some 3 of Physalaemus biligonigerus, P. marmoratus and P. santafeci- nus is a synapomorphy of this clade, and that this C-band had an independent origin from that found in the short arm of chromosome 3 of P. nattereri, as hypothesized by Vittorazzi et al. (2014). In all our inferences, the clade composed of Physalaemus biligonigerus, P. marmoratus and P. santafecinus was the sister group of P. riograndensis, despite with low support. These four species share a similar tadpole oral disc morphogenetic pattern, characterized by two lower labial rows and marginal papillae developing without a ventral gap (C1 and C2 configurations sensu Vera Candioti et al., 2011). Whereas P. riograndensis maintains a configuration with ventrolateral gaps, tadpoles of the remaining three species have complete marginal papillae (Vera Candioti et al., 2011). The presence of two lower labial tooth rows in Physalaemus is known only in these four species and, among other leiuperines, in three species of Pleurodema (P. guayapae, P. nebulosum and P. tucumanum; Cei, 1980), although the developmental processes involved differ (Vera Candioti et al., 2011). The presence of two lower labial tooth rows may represent, therefore, a synapomorphy of these species of Physalaemus and defines an oral disc that trun- cates its development with regard to the plesiomorphic larval labial tooth-row formula 2/3. Based on this analysis of tadpole oral disc and on our phyloge- netic inferences, we recognize a Physalaemus biligonigerus Species Group composed of P. biligonigerus, P. marmoratus, P. santafecinus and P. riograndensis. L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 213 4.2.2.2. The Physalaemus henselii Species Group. This group is com- posed of Physalaemus henselii and P. fernandezae, which were recovered as sister species in all our analyses. The species of the P. henselii Group have the most southerly distribution among the Physalaemus species, inhabiting open areas in the Pampa and Uruguayan Savanna Ecoregions, and their reproduction occurs in the winter (Barrio, 1964; Kolenc et al., 2006; Maneyro et al., 2008). These species have been considered closely related based on similar adult and larval external morphology, ecology and geo- graphic distribution (Barrio, 1964; Barrio, 1965). Lobo (1996) sug- gested that Physalaemus fernandezae and P. henselii are sister taxa based on the presence in both species of a nonbifurcated sternal style and an open frontoparietal fontanelle. The very incomplete osteological data for Physalaemus limits the interpretation of this character in the phylogenetic context. Physalaemus henselii and P. fernandezae were formerly included in the Physalaemus cuvieri Group by Lynch (1970) or in the P. hen- selii Group (with P. riograndensis) by Nascimento et al. (2005). However, several works indicated that the larval configuration conflicts with the inclusion of P. riograndensis in these groups (Alcalde et al., 2006; Kolenc et al., 2006; Vera Candioti et al., 2011). Physalaemus henselii and P. fernandezae share with P. cicada the larval oral disc configuration that displays three lower labial tooth rows and marginal papillae with a ventral gap (C5 sensu Vera Candioti et al., 2011). This character combination of the oral disc is particular and, among the other leiuperines, was observed only in some specimens of Pseudopaludicola falcipes (revised by Vera Candioti et al., 2011). The presence of a third lower labial tooth row is a plesiomorphic character state observed in most leiu- perines (except in P. biligonigerus Group and some species of Pleurodema, see Vera Candioti et al., 2011). 4.2.2.3. The Physalaemus gracilis Species Group. Our phylogenetic inferences corroborated the Physalaemus gracilis Group as previ- ously recognized by Nascimento et al. (2005), which is composed of P. barrioi, P. evangelistai, P. gracilis, P. jordanensis and P. lisei. Additionally, an undescribed species from Argentina and Brazil (traditionally assigned to P. gracilis, Barrio, 1965) was recovered nested within this clade. No morphological synapomorphy sup- ports the composition of this group, and the cytogenetic informa- tion is very fragmented, with chromosome data available only for P. barrioi (Provete et al., 2012) and P. gracilis (Brum-Zorrilla and Sáez, 1968). Most species of this clade are distributed in high regions of the Rain Atlantic Forest, although some species also inhabit the Uruguayan savannas ecoregion (P. gracilis), or transi- tional areas of Atlantic Forest, Cerrado and Campos Rupestres mon- tane savannas in Serra do Cipó (P. evangelistai). Described tadpoles of this group share an oral disc configuration with three lower labial rows and complete marginal papillae (C3, sensu Vera Candioti et al., 2011). This condition is plesiomorphic and shared by the species included in P. signifier Clade, P. olfersi Species Group and several other Leiuperinaes. 4.2.2.4. The Physalaemus olfersii Species Group. The Physalaemus olfersii Group, as defined by Nascimento et al. (2005), was poly- phyletic in all our analyses because P. aguirrei is not closely related to the clade composed of P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga; therefore, P. aguirrei should be excluded from the P. olfersii Species Group. Most of the species of the P. olfersii Group share a similar advertisement call, pulsed, without har- monic structure and without frequency modulation (see Cassini et al., 2010; Giaretta et al., 2009), whereas P. aguirrei have an unpulsed call with harmonic and frequency modulation like most of the species of the P. cuvieri and P. gracilis Groups (Bokermann, 1966). Physalemus insperatus and P. orophilus were assigned to the P. olfersii Group (sensu Nascimento et al., 2005) in the original descriptions [Cruz et al. (2008) and Cassini et al. (2010), respec- tively]. Both species lack the dark arrowhead-shaped blotch in the dorsum, are morphologically very similar to other species of the group, and like all of them inhabit the Atlantic rainforest. In addition, P. insperatus have the tarsal tubercle weakly developed. In turn, P. orophilus lacks a tarsal tubercle, but adult males have an advertisement call consisting of only one pulsed note (with sub- pulses) like all the other members of P. olfersii Group (Giaretta et al., 2009; Cassini et al., 2010 and references therein). Therefore, we tentatively include both species in the P. olfersii Group. 4.2.2.5. The Physalaemus cuvieri Species Group. All our phylogenetic inferences show a clade that includes Physalaemus albifrons, P. albonotatus, P. centralis, P. cuqui, P. cuvieri, P. ephippifer, P. erikae, P. fischeri and P. kroyeri. This clade differs from the P. cuvieri Group recognized by Nascimento et al. (2005) only by the inclusion of P. albifrons and the exclusion of P. cicada. Our phylogenetic anal- yses conducted with POY and TNT did not group P. cicada with the species of the P. cuvieri Group, and in the Bayesian analyses (of the concatenated and mitochondrial matrices), the relationships of P. cicada in the P. cuvieri Clade remain unclear. Therefore, we avoided recognizing this species as a member of the P. cuvieri Group until further analyses are made. According to Nascimento et al. (2005), Physalaemus albifrons was grouped together with P. biligonigerus, P. marmoratus and P. santafecinus. The close relationship of P. albifrons with P. biligo- nigerus, P. marmoratus and P. santafecinus was previously ques- tioned by Vittorazzi et al. (2014) based on chromosomal data and tadpole morphology. Vittorazzi and colleagues noted that (i) the P. albifrons karyotype shows an interstitial heterochromatic band in the short arm of the metacentric chromosome 5, which is a characteristic shared by all the species of P. cuvieri group already karyotyped; and (ii) the P. albifrons karyotype does not have the large heterochromatic band in the short arm of chromo- some 3 that is found in P. biligonigerus, P. marmoratus and P. santafecinus (discussed in section 4.2.2.1). The phylogenetic inferences shown herein allow us to interpret the interstitial heterochromatic band of the metacentric chromosome 5 as a synapomorphy of the P. cuvieri Group (including P. albifrons) as suggested by Vittorazzi et al. (2014). With regard to tadpole morphological characters, the phyloge- netic relationships recovered herein validate the hypothesis of Vittorazzi et al. (2014; based on data described by Vera Candioti et al., 2011), who proposed that the persistence of ventrolateral gaps in larval stages is a synapomorphy of the Physalaemus cuvieri Group (including P. albifrons). Our phylogenetic analysis raised important taxonomic ques- tions about some of the species included in the Physalaemus cuvieri Group. One question refers to P. cuqui and P. albonotatus. Physalaemus cuqui was described by Lobo (1993), who distin- guished it from P. albonotatus by size, external morphology and osteological characters. Subsequently, Ferrari and Vaira (2001), based on exemplars from Parque Nacional Calilegua, Argentina, described the advertisement call of P. cuqui, which consisted of a long trilled whine substantially different from the unpulsed adver- tisement call of P. albonotatus described by Barrio (1965). In our study, the paraphyly of P. albonotatus with respect to the single specimen of P. cuqui was inferred, and a high level of genetic sim- ilarity between P. cuqui and P. albonotatus from Paraguay and Brazil was estimated, suggesting that a better study, including a morpho- logical and bioacoustical revision and a better sampling of P. cuqui, is necessary for evaluating the possibility that P. cuqui Lobo, 1993 is a junior synonym of Leiuperus albonotatus Steindachner, 1864. In 214 L.B. Lourenço et al. / Molecular Phylogenetics and Evolution 92 (2015) 204–216 contrast, a high value of genetic distance was found between the clade (P. cuqui, P. albonotatus from Paraguay and Brazil) and the clade composed of exemplars from Paraguay and Argentina that were not nested among the representatives of P. albonotatus (despite their morphological resemblance to this species). Because of these findings and because the exemplars from Paraguay and Argentina not nested inside the P. albonotatus clade differ greatly from P. albonotatus in advertisement call (Baldo et al., unpublished data), it is likely that these exemplars from Paraguay and Argentina represent a new species. Other taxonomic questions arose from the inference of para- phyly of Physalaemus cuvieri with respect to P. ephippifer and pos- sibly P. fischeri, which led to the recognition of distinct lineages among the Brazilian individuals first identified as P. cuvieri. The low values of genetic distance observed between the Brazilian Lineages 1A and 1B of ‘‘P. cuvieri’’ and P. ephippifer, which together constitute a monophyletic group, raise the hypothesis that these lineages may in fact represent P. ephippifer population-level groups. However, a significant karyotypic divergence was observed between P. ephippifer (Nascimento et al., 2010) and the specimens in Lineage B already studied cytogenetically (i.e., specimens from Crateús-CE and Urbano Santos-MA) (Quinderé et al., 2009). The karyotype of the topotypes of P. ephippifer (including the two spec- imens used in the phylogenetic inferences presented herein) (Nascimento et al., 2010) differs from those found in the Lineage 1B of ‘‘P. cuvieri’’ (Quinderé et al., 2009) by presenting heteromor- phic sex chromosomes Z and W. In the P. ephippifer karyotype, the NORs were restricted to the Z and W chromosomes (Nascimento et al., 2010), whereas the karyotypes of specimens in the Lineage 1B show the NORs in chromosomes 8 and 9, in a highly polymor- phic condition (Quinderé et al., 2009). Therefore, further studies including the analysis of other genes and populations are needed to evaluate this taxonomic question as well as the role of the sex chromosome heteromorphism in the evolution of this group. It is noticeable that the Brazilian Lineages 2 and 3 of ‘‘P. cuvieri’’, which showed high uncorrected p-distances in the 16S partial gene when compared with each other and with the Lineages 1A and 1B of ‘‘P. cuvieri’’ and P. ephippifer, correspond to distinct karyotypic groups that differ especially in the location of nucleolus organizer regions (NORs) (Silva et al., 1999; Quinderé et al., 2009). In the karyotypes of specimens in the Lineage 2 of ‘‘P. cuvieri’’, the princi- pal NOR could be found in chromosome 8 or chromosome 11 [see Quinderé et al. (2009) for details], whereas in the karyotypes of the individuals from Porto Nacional (Lineage 3 of ‘‘P. cuvieri’’), multiple NORs were present and could be found in chromosomes 1, 3, 4, 5 and 10 (Quinderé et al., 2009), in contrast to the karyotypes of the Lineages 1B and 2. Taking these chromosomal data together with the phylogenetic inferences and the p-distance data, we sug- gest that each of the Lineages 2 and 3 may represent a valid spe- cies. Based on the genetic divergences estimated from 16S partial gene, we also infer that these putative species do not correspond to either of those lineages of ‘‘P. cuvieri’’ found in Bolivian localities by Jansen et al. (2011). However, because of the wide geographical distribution of Physalaemus cuvieri and the uncertainty of its type locality, a deep review is still necessary to resolve the taxonomy of this putative species complex. 4.3. The nucleolus organizer regions of Physalaemus The nucleolus organizer regions, in addition to the C-bands, are the most common traits used for the study of the karyotypes of anurans because techniques frequently employed for cytogenetic studies of other vertebrates, including G-banding, fail to provide good results in anuran chromosomes. In some of the anuran spe- cies karyotyped, a single pair of NORs was found in the diploid complement, and this character is apparently fixed in the sampled populations (e.g., Schmid, 1978a,b; Lourenço et al., 2000; Veiga-Menoncello et al., 2003; Cardozo et al., 2011; Rodrigues et al., 2011). In contrast, multiple NORs as well as interpopulational and/or intrapopulational variations in the numbers or locations of NORs were reported for a number of species of anurans (e.g., Wiley et al., 1989; Foote et al., 1991; Schmid et al., 1995; Kaiser et al., 1996; Bruschi et al., 2014). Such variation causes the inference of homology between interspecific NOR-bearing chromosomes as well as the evolutionary analysis of the NOR to be viewed with caution. In the case of Physalaemus, some inferences may be made with regard to the character ‘‘location of fixed NORs’’ after the analysis of the phylogenetic relationships inferred here. The NOR-bearing chromosomes 8 found in P. cuvieri, P. albonotatus and P. albifrons (Silva et al., 2000; Quinderé et al., 2009; Vittorazzi et al., 2014) are very similar in morphology. The chromosomal morphology and the position of the NOR in the NOR-bearing chromosome 8 of Pleurodema diplolister (Lourenço et al., 2006) and, to a lesser extent, the chromosome 8 of Edalorhina perezi (Lourenço et al., 2000) indicate that these could be homeologous chromosomes. Therefore, the condition ‘‘presence of an interstitial NOR in 8q’’, as found in all of these species, may be plesiomorphic in relation to the other patterns of NOR occurrence found in species of Physalaemus, among which we note the following: the presence of a pericentromeric NOR in 9q (found in P. centralis; see Vittorazzi et al., 2014); the presence of NOR in 8q and in the meta- centric chromosome 9 (found in P. albonotatus, see Vittorazzi et al., 2014); and the polymorphic condition found in exemplars from Porto Nacional identified here as Lineage 3 of ‘‘P. cuvieri’’) (Quinderé et al., 2009). It is equally remarkable that the homoplastic telocentric chro- mosomes 11 of Physalaemus fernandezae and P. nattereri also show a terminal NOR, providing an illustrative example of the impor- tance of considering this cytogenetic trait carefully. At last, we note that the NOR location is a valuable trait in the cytogenetics of Physalaemus because this character may vary among species (e.g., Vittorazzi et al., 2014), being helpful in detect- ing differences even among lineages morphologically undistin- guishable as those of ‘‘P. cuvieri’’. Acknowledgments The authors thank Daniel P. Bruschi, Diego Barrasso, Claudio Borteiro, Francisco Brusquetti, Dario Cardozo, Francisco Kolenc, Flavia Netto, Martín Pereyra and William P. Costa for their help in collecting specimens; Miguel Trefaut Rodrigues, for the loan of tissues; and Karin R. Seger and Alessandra Ferreira for their techni- cal assistance. This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, proc. 620163/2008-9) and São Paulo Research Foundation (FAPESP) #2008/50928-1, #2011/09239-0 and #2013/50741-7. DB acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and ANPCyT for their financial support: PIP 1112008010-2422, PIP 112201101-00875, PIP 11220110100889; PICT 2007-2202, 2011-1524, 2011-1895, 2012-2687 and 404-2013. Specimens were collected from Brazilian localities under a permit issued by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA) (proc. 10678-2 and 32483-1). Appendices A–G. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2015.06. 011. http://dx.doi.org/10.1016/j.ympev.2015.06.011 http://dx.doi.org/10.1016/j.ympev.2015.06.011 L.B. 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http://refhub.elsevier.com/S1055-7903(15)00181-5/h0530 Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences 1 Introduction 2 Materials and methods 2.1 Taxon sampling 2.2 Mitochondrial and nuclear gene sequencing 2.3 Phylogenetic inferences 2.4 Genetic distances among lineages related to Physalaemus cuvieri, P. ephippifer and P. fischeri 2.5 Cytogenetic analysis 3 Results 3.1 Phylogenetic inferences 3.1.1 Relationships of Physalaemus cuqui and P. albonotatus 3.1.2 Relationships of Physalaemus cuvieri, P. ephippifer and P. fischeri 3.2 Genetic comparisons among the lineages related to Physalaemus cuvieri, P. ephippifer and P. fischeri 3.3 The karyotype of Physalaemus deimaticus 4 Discussion 4.1 Intergeneric relationships of Physalaemus 4.2 Interspecific relationships in Physalaemus 4.2.1 The Physalaemus signifer Clade 4.2.2 The Physalaemus cuvieri Clade 4.2.2.1 The Physalaemus biligonigerus Species Group 4.2.2.2 The Physalaemus henselii Species Group 4.2.2.3 The Physalaemus gracilis Species Group 4.2.2.4 The Physalaemus olfersii Species Group 4.2.2.5 The Physalaemus cuvieri Species Group 4.3 The nucleolus organizer regions of Physalaemus Acknowledgments Appendices A–G Supplementary material References