Phylogenetic relationships of the Boana pulchella Group (Anura: Hylidae).

In this paper we present a phylogenetic analysis of the treefrogs of the Boana pulchella Group with the goals of (1) providing a rigorous test of its monophyly; (2) providing a test of relationships supported in previous studies; and (3) exploring the relationships of the several species not included in previous analyses. The analyses included>300 specimens of 37 of the 38 species currently included in the group, plus 36 outgroups, exemplars of the diversity of Boana and the other genera of the hylid tribe Cophomantini. The dataset included eight mitochondrial genes (12S, 16S, CytB, COI, ND1, tRNAIle, tRNALeu, and tRNAVal) and five nuclear genes (RHO, TYR, RAG-1, CXCR4, SIAH1). The phylogenetic analyses recover the monophyly of the B. pulchella Group with lower support than previous studies, as a result of the inclusion of the B. claresignata Group, which is recovered as its sister taxon. Within the B. pulchella Group, the inclusion of almost all species of the group had little impact on previous notions of its phylogeny, except for the rejection of the hypothesized B. polytaenia Clade (B. goiana and B. phaeopleura are nested in the clade here called the B. prasina Clade), which is redefined. Phylogenetic support is strong for five major clades, which collectively include all but three of the species sampled: the B. balzani Clade (B. aguilari, B. balzani, B. gladiator, B. melanopleura, B. palaestes), the redefined B. polytaenia Clade (B. botumirim, B. buriti, B. cipoensis, B. jaguariaivensis, B. leptolineata, B. polytaenia, B. stenocephala, and two undescribed species), the B. prasina Clade (B. bischoffi, B. caingua, B. cordobae, B. goiana, B. guentheri, B. marginata, B. phaeopleura, B. prasina, B. pulchella, and one undescribed species), the B. riojana Clade (B. callipleura, B. marianitae, B. riojana), and the B. semiguttata Clade (B. caipora, B. curupi, B. joaquini, B. poaju, B. semiguttata, B. stellae, and two undescribed species). The monophyly of the B. prasina + B. riojana Clades, and that of the B. polytaenia + B. semiguttata Clades are well-supported. The relationships among these two clades, the B. balzani Clade, B. ericae + B. freicanecae, and B. cambui (representing the deepest phylogenetic splits within the B. pulchella Group) are recovered with weak support. We discuss the phenotypic evidence supporting the monophyly of the B. pulchella Group, and the taxonomy of several species, identifying three new synonyms of Boana polytaenia, one new synonym of Boana goiana, and one new synonym of B. riojana.

Integrative taxonomy reveals a new but common Neotropical treefrog, hidden under the name Boana xerophylla.

Boana xerophylla is a common treefrog widely distributed in northern Brazil, Colombia, French Guiana, Guyana, Panama, Suriname, Trinidad and Tobago, and Venezuela. A recent study found molecular, acoustic, and morphometric differences between the populations located on opposite sides of the Orinoco River. Here, we carry out an updated molecular phylogenetic analysis, including new samples from all the countries along the distribution area, and analyzed additional call recordings from Brazil, Colombia, French Guiana, Panama, Trinidad and Tobago, and Venezuela. Our phylogenetic inference reveals three geographically restricted lineages: one in the eastern Guiana Shield (corresponding to B. xerophylla sensu stricto), another in the western Guiana Shield, and a third one north of the Orinoco River. Morphological and acoustic data agree with the differentiation between the populations north of the Orinoco River and the eastern Guiana Shield despite the low genetic p-distances observed (16S rRNA: 0.72.2 %). We argue that the populations north of the Orinoco River correspond to a new species, sister of B. xerophylla. We name and describe Boana platanera sp. nov. from the southern versant of the Cordillera de Mérida (08º48'26'' N, 70º30'46'' W, WGS 84; 947 m asl), Venezuela, and refer all the populations north of the Orinoco River currently identified as B. xerophylla to this species. The new species can be readily diagnosed from B. xerophylla (characters of the latter in parentheses) by a pale orange-yellow or light brown dorsal coloration (dark brown to green), palpebral membrane with dark pigments (pigments absent); pericloacal region dark brown (cream), advertisement call with shorter first note length than B. xerophylla. This study represents an empirical example regarding false negatives behind genetic thresholds for species discovery, appraising the use of integrative taxonomic approaches.

Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica.

Taxonomic changes have frequently occurred in the anuran genus Pseudopaludicola as a consequence of high morphological similarity among its species. The present work reports karyotypic analysis of three Pseudopaludicola species sampled in their type locality and four Pseudopaludicola populations from distinct localities, aiming at contributing to the systematics of this genus. Chromosomes were stained with Giemsa or submitted to the silver staining (Ag-NOR) and C-banding techniques. The karyotype was 2n=22 in P. mineira, Pseudopaludicola sp. and two populations of P. saltica. The chromosome pair 8 was heteromorphic in P. saltica, characterizing a XX/XY sex-determination system with telocentric X and submetacentric Y. Highly similar karyotypes with 2n=18 chromosomes were observed in P. canga, P. aff. canga from Barreirinhas, State of Maranhão, Uberlândia, State of Minas Gerais and Icém, State São Paulo. The high similarity among the karyotypes 2n=18 suggested that the populations of P. aff. canga belong to the group 'pusilla', the same group of P. canga. The data demonstrated also that P. aff. canga from Barreirinhas (northeast region) is cytogenetically identical to P. canga with regarding the NOR site position in pair 3 and the presence of a heterochromatic block in the pair 2, whereas P. aff. canga from Uberlândia and Icém (southeast) had the NOR in the pair 9. Moreover, the cytogenetic data discriminated P. mineira and Pseudopaludicola sp. from the previously analyzed species with 22 chromosomes, and suggested that Pseudopaludicola sp. is an undescribed species. Sexual heteromorphic chromosomes are firstly reported in Pseudopaludicola and the data indicated the need of an extensive taxonomic review in this genus.

Unraveling the species diversity and relationships in the Leptodactylus mystaceus complex (Anura: Leptodactylidae), with the description of three new Brazilian species.

Members of the Leptodactylus mystaceus species complex are widely distributed in forests and open formations of South America east of the Andes. Species of the complex are morphologically similar or indistinguishable among each other, but acoustic data have been the cornerstone for species discrimination across their geographic ranges. In this paper, we re-examine the monophyly, species diversity, and relationships in the L. mystaceus complex on the basis of morphology, coloration, acoustics, and DNA sequences. Morphological and color patterns originally used to the allocation of species to the L. mystaceus complex are also reassessed. Our results revealed three new species from the Brazilian Atlantic Forest and Cerrado, which are named and described herein, based mainly on acoustic and molecular data. Populations assigned to the lineage widely distributed across the South American Dry Diagonal (DD), reported in this study as L. cf. mystaceus, is likely paraphyletic with respect to the nominal species (Amazonian lineage), but additional data are still needed to address the taxonomic status of the DD lineage.

Contrasting genetic, acoustic, and morphological differentiation in two closely related gladiator frogs (Hylidae: Boana) across a common Neotropical landscape.

Mountain chains and rivers are often found to represent barriers promoting vicariant differentiation in terrestrial vertebrates. Previous studies have supported the idea that the Cordillera de Mérida (CM), the easternmost branch of the Northern Andes, represents a geographic barrier for vertebrates, including frogs. Previous studies have also suggested that the Orinoco River (OR), the biggest river in Venezuela, also represents a geographic barrier for terrestrial vertebrates. Boana pugnax and B. xerophylla are two Neotropical hylids, members of the B. faber species group, that are distributed on either side of the CM, and whose ranges extend up to 605 and 2450 m in elevation, respectively. In addition, B. xerophylla occurs on either side of the OR. Herein, we assess the genetic, acoustic, and morphological differentiation within B. pugnax and within B. xerophylla across the CM and within B. xerophylla across the OR, and test if genetic differentiation is correlated with geographic distance. We also evaluated the acoustic differentiation between the recently recognized B. xerophylla and its sister species, B. crepitans, and found marked differences between advertisement calls, corroborating their status as distinct species. Genetic and morphometric analyses of populations from opposite sides of the CM revealed differentiation in B. pugnax but not in B. xerophylla. Within the latter species, we found molecular, acoustic, and morphometric differentiation among samples of B. xerophylla from western Venezuela versus the Guiana Shield. Genetic variation within B. pugnax and within B. xerophylla was not explained by geographic distance. Thus, our data show conspecific population structure across the CM in B. pugnax, plus the possible existence of two species within what today is considered B. xerophylla, yet the CM apparently is not involved in this divergence. These results suggest that even for closely related species with shared ecology and distribution, genetic and phenotypic differentiation respond differently to common ecological or historical factors.

Vocalizations, tadpole, and natural history of Crossodactylus werneri Pimenta, Cruz Caramaschi, 2014 (Anura: Hylodidae), with comments on distribution and intraspecific variation.

Crossodactylus werneri was described based on specimens collected in the 1970's at Parque Nacional do Itatiaia, being also reported for nearby localities. We collected specimens that we assigned to C. werneri, and recorded calls of the species during fieldworks at Serra das Cabras (Campinas, state of São Paulo). In this paper, we describe for the first time the vocalizations, tadpole, coloration in life, and comment on aspects of the natural history of C. werneri. Besides, the examination of specimens in zoological collections allowed us to extend the geographic range for this species. We also make remarks on morphological/chromatic variation and provide 16S rDNA sequences for the species. Adults were found along a slow-flowing streamlet with sandy/muddy bottom within a small fragment of secondary forest. Males called between sunset and first hours of the night. Advertisement call consisted of series of pulsed notes. Call duration lasted around 3 s, emitted at the highest rate of 17 calls per minute and six notes per second. Note duration lasted around 18 ms. Notes had poorly defined pulses (irregular and/or weak amplitude modulations along the note). The dominant frequency was about 3380 Hz. Territorial call had a long, well-defined pulsed portion followed by a higher-amplitude "squeak". The dominant frequency was around 3400 Hz. Tadpoles were essentially similar to those of other Crossodactylus species, except by not having nostril ornamentation. Our record of C. werneri in Serra das Cabras might be regarded a rediscovery of this species since C. werneri had not been recorded for more than 30 years until our first record of C. werneri in the field from 2011 and subsequent years. Our record is approximately 100 km west, and Mococa 200 km northwest, from Santo Antônio do Pinhal, the westernmost previous record for C. werneri up to date. Gene sequences (16S rRNA) give insights into the genetic divergence between C. werneri and some congeners.

The first species of Aplastodiscus endemic to the Brazilian Cerrado (Anura, Hylidae).

The genus Aplastodiscus includes 14 nominal species in four monophyletic groups with occurrence in the Atlantic Forest and Brazilian Cerrado (Brazilian Savanna) of South America. A recent study reviewed the taxonomy and phylogenetic relationships of the genus and suggested a third species for the Aplastodiscus perviridis Group. Herein, on the basis of morphology and advertisement call, we describe this species and test its monophyly. The new species is the only Aplastodiscus with endemic occurrence in the Cerrado Biome. In addition, its geographical distribution and conservation status are discussed.

The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species.

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species.

Using delta13C stable isotopes to quantify individual-level diet variation.

Individual-level diet variation can be easily quantified by gut-content analysis. However, because gut contents are a 'snapshot' of individuals' feeding habits, such cross-sectional data can be subject to sampling error and lead one to overestimate levels of diet variation. In contrast, stable isotopes reflect an individual's long-term diet, so isotope variation among individuals can be interpreted as diet variation. Nevertheless, population isotope variances alone cannot be directly compared among populations, because they depend on both the level of diet variation and the variance of prey isotope ratios. We developed a method to convert population isotope variances into a standardized index of individual specialization (WIC/TNW) that can be compared among populations, or to gut-content variation. We applied this method to diet and carbon isotope data of four species of frogs of the Brazilian savannah. Isotopes showed that gut contents provided a reliable measure of diet variation in three populations, but greatly overestimated diet variation in another population. Our method is sensitive to incomplete sampling of the prey and to among-individual variance in fractionation. Therefore, thorough sampling of prey and estimates of fractionation variance are desirable. Otherwise, the method is straightforward and provides a new tool for quantifying individual-level diet variation in natural populations that combines both gut-content and isotope data.

The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species.