A revision of species diversity in the Neotropical genus Oreobates (Anura: Strabomantidae), with the description of three new species from the Amazonian slopes of the Andes

We revisit species diversity within Oreobates (Anura: Strabomantidae) by combining molecular phylogenetic analyses of the 16S rRNA amphibian barcode fragment with the study of the external morphology of living and preserved specimens. Molecular and morphological evidence support the existence of 23 species within Oreobates, and three additional candidate species (Oreobates sp. [Ca JF809995], Oreobates sp. [Ca EU368903], Oreobates cruralis [Ca EU192295]). We describe and name three new species from the Andean humid montane forests of Departamento Cusco, southern Peru: O. amarakaeri New Species from Rio Nusinuscato and Rio Mabe, at elevations ranging from 670 to 1000 m in the Andean foothills; O. machiguenga, new species, from Rio Kimbiri (1350 m), a small tributary of the Apurimac River, in the western versant of Cordillera Vilcabamba; and O. gemcare, new species, from the Kosnipata Valley at elevations ranging from 2400 to 2800 m. The three new species are readily distinguished from all other Oreobates by at least one qualitative morphological character. Three species are transferred to Oreobates from three genera of Strabomantidae: Hypodactylus lundbergi, Pristimantis crepitans, and Phrynopus ayacucho (for which the advertisement call, coloration in life, and male characteristics are described for first time). Oreobates simmonsi is transferred to the genus Lynchius. Hylodes verrucosus is considered a junior synonym of Hylodes philippi. In addition, H. philippi is removed from the synonymy of O. quixensis and considered a nomem dubium within Hypodactylus. The inclusion of Phrynopus ayacucho in Oreobates extends the ecological range of the genus to the cold Andean puna. Oreobates is thus distributed from the Amazonian lowlands in southern Colombia to northern Argentina, reaching the Brazilian Atlantic dry forests in eastern Brazil, across an altitudinal range from ca. 100 to 3850 m.

A new species of Trachycephalus (Amphibia, Anura, Hylidae) from the State of Goias, Brazil

A new species of Trachycephalus is described for the Cerrado biome of Goias, Brazil. Trachycephalus mambaiensis sp. nov. is distinguished from the other ten species of the genus by the skin co-ossified with the skull, heavy cranial ossification, frontoparietal that fails to articulate with squamosal, absence of a crista occipitalis and secreting glands of milky and viscous substances. The skull of the new species shows an intermediary condition between species of Trachycephalus with a well ossified skull (Casque-headed frogs) and those without cranial ossification.

A new species of Leptodactylus (Anura : Leptodactylidae) from the Atlantic forest of Sao Paulo State, Brazil

A new species of Leptodactylus from Bertioga municipality, Sao Paulo State, Brazil is described. Leptodactylus ajurauna sp. nov. differs from others species in the L. marmoratus group by its dark brown throat and the advertisement call. The taxonomy of other species in the group and the status of the population of Leptodactylus marmoratus sympatric with the new species are discussed. The advertisement call is also described.

A new dwarf species of Proceratophrys Miranda-Ribeiro, 1920 (Anura, Cycloramphidae) from the highlands of Chapada Diamantina, Bahia, Brazil

A new species of Proceratophrys is described from the highlands of northeastern Brazil. Molecular and morphological data suggests that Proceratophrys redacta sp. nov. is sister to P. minuta, and related to P. schirchi and P. cristiceps. The new species is diagnosed by its small size, absence of rostral and palpebral appendages, sagittal ridges interrupted, absence of postocular swellings, snout vertical in profile and dorsal coloration lacking distinct ocelli. The new species represents another example of endemism for the genus Proceratophrys in Chapada Diamantina region, and of another appendageless small-sized species associated with highlands. The phylogenetic results indicate that current morphological groupings in Proceratophrys may not represent natural groups.

A new species of Pseudopaludicola Miranda-Ribeiro (Leiuperinae: Leptodactylidae: Anura) from the Cerrado of southeastern Brazil with a distinctive advertisement call pattern

A new species of Pseudopaludicola is described from the Cerrado of southeastern Brazil. The new taxon is diagnosed from the P. pusilla species group by the absence of either T-shaped terminal phalanges or toe tips expanded, and promptly distinguished from all (13) recognized taxa currently assigned to Pseudopaludicola by possessing isolated (instead of regular call series), long (117-187 ms) and non-pulsed advertisement calls.

Two new species of marsupial tree-frogs genus Gastrotheca Fitzinger, 1843 (Anura, Hemiphractidae) from the Brazilian Atlantic Forest

Two new species of Gastrotheca are described from northeastern Minas Gerais and southern Bahia, in the Atlantic Forest of Brazil. Data on morphology, calls, mitochondrial, and nuclear DNA are provided. Allied to G. fissipes and G. megacephala, the new taxa provide evidence for a higher diversity of species of Gastrotheca than previously thought at the Atlantic Forest. The data also suggest that G. pulchra, another Atlantic Forest taxon, is more closely related to non-Atlantic Forest species than to the remaining analyzed Brazilian Gastrotheca species. This implies that the Gastrotheca at the Brazilian coastal forests have at least two independent origins.

An extraordinary new species of Melanophryniscus (Anura, Bufonidae) from southeastern Brazil

We describe a new species of bufonid from a lowland, sandy soil, restinga habitat in the state of Espirito Santo, southeastern Brazil. Based on the shared occurrence of putative morphological synapomorphies of Melanophryniscus and the results of a phylogenetic analysis of DNA sequences of a broad sample of bufonids, and other anurans, we assign the new species to Melanophryniscus. The new species possesses several peculiar character states that distinguish it from all other Melanophryniscus including, but not limited to: fingers II, III, and V much reduced; nuptial pad with few enlarged, brown-colored spines on medial margin of finger II; seven presacral vertebrae, the last fused with the sacrum; and ventral humeral crest prominent, forming a spinelike projection.

A relict new species of Oreobates (Anura, Strabomantidae) from the Seasonally Dry Tropical Forests of Minas Gerais, Brazil, and its implication to the biogeography of the genus and that of South American Dry Forests

A new species of Oreobates is described from Cavernas do Peruacu National Park, Januaria, Minas Gerais state, in the Atlantic Dry Forests of Brazil. The new species is distinguished from all other Oreobates by having the following combination of characters: large tympanum, discs broadly enlarged and truncate on Fingers III and IV, smooth dorsal skin, nuptial pads absent, snout subacuminate, and a very short pulsatile (2-3 pulses) single-noted advertisement call with dominant frequency of about 3150 Hz, and no harmonic structure. Molecular phylogenetic analyses using partial sequences of the mitochondrial genes cytochrome b (cyt b) and 16S using multiple outgroups recovered the new species within Oreobates and sister to O. heterodactylus. The latter species inhabits the Dry Forests of Mato Grosso (Cerrado) and Bolivia (Chiquitano forests), and is strictly associated to these habitats, which suggests a preterit connection between Chiquitano and Atlantic Dry Forests. The discovery of a new Oreobates in the Atlantic Dry Forest is of great importance for the conservation of these dry forests, as it is known only from this type of habitat.

Multiple Quaternary Refugia in the Eastern Guiana Shield Revealed by Comparative Phylogeography of 12 Frog Species

The Guiana Shield (GS) is one of the most pristine regions of Amazonia and biologically one of the richest areas on Earth. How and when this massive diversity arose remains the subject of considerable debate. The prevailing hypothesis of Quaternary glacial refugia suggests that a part of the eastern GS, among other areas in Amazonia, served as stable forested refugia during periods of aridity. However, the recently proposed disturbance-vicariance hypothesis proposes that fluctuations in temperature on orbital timescales, with some associated aridity, have driven Neotropical diversification. The expectations of the temporal and spatial organization of biodiversity differ between these two hypotheses. Here, we compare the genetic structure of 12 leaf-litter inhabiting frog species from the GS lowlands using a combination of mitochondrial and nuclear sequences in an integrative analytical approach that includes phylogenetic reconstructions, molecular dating, and Geographic Information System methods. This comparative and integrated approach overcomes the well-known limitations of phylogeographic inference based on single species and single loci. All of the focal species exhibit distinct phylogeographic patterns highlighting taxon-specific historical distributions, ecological tolerances to climatic disturbance, and dispersal abilities. Nevertheless, all but one species exhibit a history of fragmentation/isolation within the eastern GS during the Quaternary with spatial and temporal concordance among species. The signature of isolation in northern French Guiana (FG) during the early Pleistocene is particularly clear. Approximate Bayesian Computation supports the synchrony of the divergence between northern FG and other GS lineages. Substructure observed throughout the GS suggests further Quaternary fragmentation and a role for rivers. Our findings support fragmentation of moist tropical forest in the eastern GS during this period when the refuge hypothesis would have the region serving as a contiguous wet-forest refuge.

HELMINTH PARASITES OF HYPSIBOAS PRASINUS (ANURA: HYLIDAE) FROM TWO ATLANTIC FOREST FRAGMENTS, SAO PAULO STATE, BRAZIL

Seventy-seven males of Hypsiboas prasinus from 2 Atlantic forest fragments in the municipalities of Botucatu and Jundiai, sao Paulo State, Brazil, were examined for endoparasites. The frogs were captured in summer (January until March) and winter (July/August) of 2008 and 2009. Thirty-three males (75%) from Botucatu were infected with Rhabdias cf. fuelleborni, cosmocerciid nematodes, and Cylindrotaenia americana. Twenty-five tree frogs (78.5%) from Jundiai were infected by Rhabdias cf. fuelleborni, Physaloptera sp., and cosmocerciid nematodes. Only cosmocerciid nematodes presented a statistically significance difference in prevalence (z = 4.345; P < 0.001) and mean abundance (t = 562.0; P < 0.001) between Botucatu and Jundiai during the winter. Also, the cosmocerciids exhibited higher mean abundance (t = 196.0; P = 0.034) in winter when compared with summer at the Jundiai site. Moreover, to our knowledge, this is the first report of C. americana in the Brazilian Hylidae. This study presents 4 new records of nematodes in H. prasinus.

New records of the harlequin frog Atelopus nahumae RUIZ-CARRANZA, ARDILA-ROBAYO & HERNANDEZ-CAMACHO, 1994, in the Sierra Nevada de Santa Marta (Colombia), with notes of its distribution (Anura: Bufonidae)

The known distribution of the harlequin frog, Atelopus nahumae RUIZ-CARRANZA, ARDILA-ROBAYO & HERNANDEZ-CAMACHO, 1994, is reported along with three new locality records in the northwestern Sierra Nevada de Santa Marta (Colombia). In the new localities, observations were made on the frog's mating behavior. This report adds the Cordoba River Basin, another high valley of the Sierra Nevada de Santa Marta, to the known range of A. nahumae. Nevertheless, the general distribution of the species in this region is largely unknown.

New species of the Rhinella crucifer group (Anura, Bufonidae) from the Brazilian Cerrado

A new species of Rhinella of Central Brazil from the Rhinella crucifer group is described. Rhinella inopina sp. nov. is restricted to the disjunct Seasonal Tropical Dry Forests enclaves in the western Cerrado biome. The new species is characterized mainly by head wider than long, shape of parotoid gland, and oblique arrangement of the parotoid gland. Data on natural history and distribution are also presented.

The occurrence of defensive alkaloids in non-integumentary tissues of the Brazilian red-belly toad Melanophryniscus simplex (Bufonidae)

The red-belly toads (Melanophryniscus) of southern South America secrete defensive alkaloids from dermal granular glands. To date, all information on Melanophryniscus alkaloids has been obtained by extraction from either skins or whole organisms; however, in other amphibians, tetrodotoxins, samandarines, and bufadienolides have been detected in both skin and other organs, which raise the possibility that lipophilic alkaloids may occur in non-integumentary tissues in Melanophryniscus as well. To test this hypothesis, we studied the distribution of alkaloids in the skin, skeletal muscle, liver, and mature oocytes of the red-belly toad M. simplex from three localities in southern Brazil. Gas chromatography and mass spectrometry of skin extracts from 11 individuals of M. simplex resulted in the detection of 47 alkaloids (including isomers), 9 unclassified and 38 from 12 known structural classes. Each alkaloid that was present in the skin of an individual was also present in the same relative proportion in that individual's skeletal muscle, liver, and oocytes. The most abundant and widely distributed alkaloids were the pumiliotoxins 251D, 267C, and 323A, 5,8-disubstituted indolizidines 207A and 223D, 5,6,8-trisubstituted indolizidine 231B, 3,5-disubstituted pyrrolizidines cis-223B and cis- and trans-251K, and izidine 211C. We report the first record of piperidines in Melanophryniscus, bringing the total number of alkaloid classes detected in this genus to 16. Alkaloid composition differed significantly among the three study sites. The functional significance of defensive chemicals in non-integumentary tissues is unknown.

A new species of the Bokermannohyla circumdata group (Anura: Hylidae) from southeastern Brazil, with bioacoustic data on seven species of the genus

We describe a new species of the Bokermannohyla circumdata group from the Estacao de Pesquisa e Desenvolvimento Ambiental Galheiro (EPDA-Galheiro) (19 degrees 12'S; 47 degrees 08'W), Municipality of Perdizes, State of Minas Gerais, a mid-altitudinal (similar or equal to 850 m above sea level) riparian forest environment in the Cerrado of southeastern Brazil. Bokermannohyla napolii sp. nov. is allied to the large-sized species of the group, diagnosed on the basis of adult morphology/morphometrics, and mainly vocalizations. Adult specimens of the new species are most closely related to those of B. luctuosa and B. circumdata, but can be differentiated from the former by having distal subarticular tubercle of finger III bifid/divided in males, and finger IV bifid/divided in males and females; and from both B. luctuosa and B. circumdata by a distinctive advertisement call structure. We also provide bioacoustic data on seven other species of the genus, including previously unknown advertisement calls of B. circumdata and B. carvalhoi, and re-description of the advertisement calls of B. luctuosa, B. ibitiguara, B. nanuzae, B. sazimai, and B. hylax.

Antimicrobial capacity of plasma from anurans of the Atlantic Forest

The viability and interpretation of techniques for the evaluation of immunocompetence of animals in their natural environment has been largely debated. One of these methods is based on testing the antimicrobial capacity of the blood and/or plasma in vitro, which could rapidly and effectively assess the immunological conditions of natural populations. We tested the applicability of the antimicrobial capacity of plasma (ACP) assay in anuran amphibians from the Atlantic Forest. The assay was performed by measuring both the turbidity (in a spectrophotometer) and the colony forming units (CFU) of the remaining bacteria (Escherichia coli) following exposure to amphibian plasma. Although both assays were correlated, the ACP assay by spectrophotometry showed 10 times lower intra-assay variation. We also found interspecific variation in ACP, as well as the maintenance of ACP values in males from the same population, collected in different breeding seasons. Thus, the estimation of ACP by spectrophotometry provides a convenient and accurate method for evaluating innate immunocompetence in comparative and ecophysiological studies of anuran amphibians.