A new species of Phrynopus from the northeastern Andes of Peru, its phylogenetic position, and notes on the relationships of Holoadeninae (Anura: Craugastoridae).

We report the discovery of a geographically disjunct and morphologically distinctive species of direct-developing frog of the genus Phrynopus (Phrynopus mariellaleo sp. nov.) that changes considerably our understanding of the distribution of species in this Andean genus. The type locality lies on a subcordillera (Cerro de Campanario area) of the extreme northeastern portion of the Cordillera Central of Peru, on the headwaters of the Mayo River, Amazonas department, at 2575 m asl (6°6'42.9''S, 77°26'24''W). This area is situated 170 km to the NE from the northernmost record of Phrynopus known so far. Molecular phylogenetic analyses of a supermatrix (13269 aligned positions of gene sequences of four mitochondrial and ten nuclear genes) of 105 terminals (representing 93 named and 9 unnamed species of Holoadeninae) recover this new species as the sister to Phrynopus auriculatus, a species occurring more than 500 km south of the type locality of the new species. Both Phrynopus auriculatus and the new species occur at moderate elevations on the easternmost stretches of the Andean subcordilleras; their sister relationship point to a potentially broader distribution of species of Phrynopus along the poorly sampled intervening areas of the eastern hills of the Andes. The new species has a conspicuous and visibly large tympanic membrane (a trait rare in the clade), outlined by a marked bold black supratympanic fold and a black facial mask, and exhibits conspicuous dorsolateral, scapular, and middorsal Y-shaped folds. Specimens were found on the forest floor-a rocky substrate covered by a thick layer of leaf litter, moss and roots-of a primary humid montane forest (Yungas ecoregion) with scattered patches of bamboo (Chusquea spp.). Our phylogenetic analyses corroborate the monophyly of all Holoadeninae genera, including Euparkerella and Psychrophrynella, genera for which tests of monophyly were pending, and corroborates Hypodactylus nigrovittatus as part of Hypodactylus and sister to a clade that includes H. brunneus, H. elassodiscus and H. peraccai.

The advertisement call of the treefrog Nyctimantis rugiceps Boulenger, 1882 (Anura: Hylidae), with notes on its natural history and toxicity.

Nyctimantis rugiceps Boulenger, 1882 (Fig. 1A) is a Neotropical treefrog (Duellman Trueb 1976; Faivovich et al. 2005) known only from disjunct localities in Amazonian Colombia, Ecuador, and Peru (Pérez-Villota et al. 2009). This species has the skin of the skull co-ossified and reproduces-including calling behavior, egg deposition and tadpole development-in water-filled tree or bamboo cavities (Duellman Trueb 1976; Duellman 1978). Given its secretive behavior, this is a poorly known species and, as noted by Duellman (1978: 169), "the major clue to the life history of Nyctimantis is the calling behavior of the males". Unfortunately, the only quantitative description of the advertisement call of N. rugiceps is a brief passage in Duellman (1978) based on four specimens from Santa Cecilia, Ecuador, where important variables are missing (e.g., call duration). More importantly, graphs illustrating the waveform and spectrogram are missing. Considering these limitations and the importance of advertisement calls to the study of anurans (Köhler et al. 2017), we provide a quantitative description using a call recording obtained in Leticia, Amazonas, Colombia.

A new morphologically cryptic species of Phyllomedusa (Anura: Phyllomedusidae) from Amazonian forests of northern Peru revealed by DNA sequences.

We describe and name Phyllomedusa chaparroi sp. nov., a medium-sized species (snout-vent length in adult males 67.9-77.5 mm) of monkey frog from Amazonian rainforests of northern Peru. Although morphologically most similar to P. boliviana and P. camba (indistinguishable from the latter in external qualitative and quantitative traits), phylogenetic analysis of combined mitochondrial and nuclear markers place the new species sister to a clade containing P. neildi, P. tarsius, and P. trinitatis. Phyllomedusa chaparroi can be readily differentiated from these species by having a dark reddish-brown iris with indistinct tiny orange spots versus an orange iris with marked dark reticulation found in P. neildi, P. tarsius, and P. trinitatis. Furthermore, genetic distances for a 532 bp sequence of the 16S gene between the new species and its sister species are 2.8-4.1 %, whereas distances are 4.5-5.5 % to the morphologically cryptic P. camba. We briefly discuss the importance of DNA sequences in revealing morphologically cryptic species and modify the content of the P. tarsius species group based on phylogenetic analyses and observations on iris coloration.

A new species of poison-dart frog (Anura: Dendrobatidae) from Manu province, Amazon region of southeastern Peru, with notes on its natural history, bioacoustics, phylogenetics, and recommended conservation status.

We describe and name a new species of poison-dart frog from the Amazonian slopes of the Andes in Manu Province, Madre de Dios Department, Peru; specifically within the Amarakaeri Communal Reserve and the buffer zone of Manu National Park. Ameerega shihuemoy sp. nov. is supported by a unique combination of characters: black dorsum with cream to light orange dorsolateral lines, blue belly reticulated with black, and the lack of axillary, thigh and calf flash marks. Within Ameerega, it shares the general appearance of A. altamazonica, A. boliviana, A. hahneli, A. ignipedis, A. petersi, A. picta, A. pongoensis, A. pulchripecta, A. simulans, A. smaragdina, and A. yungicola; each possessing a granular black to brown dorsum, a light labial bar, a conspicuous dorsolateral line running from the snout to the groin, and a metallic blue belly and underside of arms and hind limbs. From most of these species it can be distinguished by lacking flash marks on the axillae, thighs, and calves (absent in only A. boliviana and A. smaragdina, most A. petersi, and some A. pongoensis), by having bright cream to orange dorsolateral stripes (white, intense yellow, or green in all other species, with the exception of A. picta), and by its blue belly reticulated with black (bluish white and black in A. boliviana, green and blue with black marbling in A. petersi, and green and blue lacking black marbling in A. smaragdina). Its mating call also shows clear differences to morphologically similar species, with a lower note repetition rate, longer space between calls, and higher fundamental and dominant frequencies. Phylogenetic analyses based on the 16S mitochondrial rRNA fragment also support the distinctiveness of the new species and suggest that A. shihuemoy is most closely related to Ameerega macero, A. altamazonica, A. rubriventris, and two undescribed species (Ameerega sp. from Porto Walter, Acre, Brazil, and Ameerega sp. from Ivochote, Cusco, Peru). Genetically, the new species is most similar to the sympatric A. macero, from which it clearly differs in characteristics of its advertisement call and coloration. The new species is found near rocky streams during the dry season and near temporary water bodies during the rainy season. Tadpoles are found in lentic water along streams, or in shallow, slow-moving streams. Given its small geographic range, we recommend that A. shihuemoy should be considered 'Near threatened' (NT) according to IUCN Red List criteria.

A new species of Andean frog of the genus Bryophryne from southern Peru Anura: Craugastoridae) and its phylogenetic position, with notes on the diversity of the genus.

We describe a new species of terrestrial frog of the genus Bryophryne (Anura: Craugastoridae) from the wet puna and elfin forests of the Amazonian versant of the Andes. The new species seems to be restricted to high altitude environments at elevations between 3506-3651 m in the area now protected by Megantoni National Sanctuary and Manu National Park (Distrito de Echarate, Provincia La Convención, Departamento Cusco, Peru). The new species is characterized by lacking vomerine processes of vomers, by having tympanic annulus and tympanic membrane not evident through the skin, smooth dorsal skin with scattered warts, conspicuous dorsolateral, middorsal, and occipital folds, warty flanks, areolate skin on ventral surfaces of the body, and by lacking finger and toe fringes and basal web on feet. In life, specimens have bright and highly variable dorsal coloration that ranges from olive-green to red with variable combinations of red or orange marks (red or orange in the green form and olive-green in the red form). Molecular phylogenetic analyses of mitochondrial and nuclear DNA place the new species within the genus Bryophryne and as sister group of B. cophites. Bryophryne bustamantei, also sequenced for this study, is found as the sister group of the clade formed by B. cophites and the new species. Bryophryne is found as sister group of Psychrophrynella in maximum likelihood analyses and as the sister group of a large clade of holoadenines in parsimony analyses. The genus Bryophryne now contains nine species, all of them distributed along the Cordillera Oriental of the Peruvian Andes, southeast of the Apurimac River valley.

Molecular phylogenetics, species diversity, and biogeography of the Andean lizards of the genus Proctoporus (Squamata: Gymnophthalmidae).

The family Gymnophthalmidae comprises ca. 220 described species of Neotropical lizards distributed from southern Mexico to Argentina. It includes 36 genera, among them Proctoporus, which contains six currently recognized species occurring across the yungas forests and wet montane grasslands of the Amazonian versant of the Andes from central Peru to central Bolivia. Here, we investigate the phylogenetic relationships and species limits of Proctoporus and closely related taxa by analyzing 2121 base pairs of mitochondrial (12S, 16S, and ND4) and nuclear (c-mos) genes. Our taxon sampling of 92 terminals includes all currently recognized species of Proctoporus and 15 additional species representing the most closely related groups to the genus. Maximum parsimony, maximum likelihood and Bayesian phylogenetic analyses recovered a congruent, fully resolved, and strongly supported hypothesis of relationships that challenges previous phylogenetic hypotheses and classifications, and biogeographic scenarios. Our main results are: (i) discovery of a strongly supported clade that includes all species of Proctoporus and within which are nested the monotypic Opipeuter xestus (a genus that we consider a junior synonym of Proctoporus), and two species of Euspondylus, that are therefore transferred to Proctoporus; (ii) the paraphyly of Proctoporus bolivianus with respect to P. subsolanus, which is proposed as a junior synonym of P. bolivianus; (iii) the detection of seven divergent and reciprocally monophyletic lineages (five of them previously assigned to P. bolivianus) that are considered confirmed candidate species, which implies that more candidate species are awaiting formal description and naming than currently recognized species in the genus; (iv) rejection of the hypothesis that Proctoporus diversified following a south to north pattern parallel to the elevation of the Andes; (v) species diversity in Proctoporus is the result of in situ diversification through vicariance in the grasslands of the high Andes, with at least five dispersals contributing to montane forest species.

From Amazonia to the Atlantic forest: molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges.

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today.

Tests of biogeographic hypotheses for diversification in the Amazonian forest frog, Physalaemus petersi.

Several hypotheses have been proposed to explain the biogeographic processes that generate the high species richness of the Amazon basin. We tested two of them in a terra firme (upland) forest frog species, Physalaemus petersi: (1) the riverine barrier hypothesis; and (2) the elevational gradient hypothesis. Mitochondrial DNA sequence data (2.4 kb) from the 12S, 16S, and intervening valine tRNA genes were obtained from 65 P. petersi individuals and 4 outgroup taxa and analyzed with a combination of phylogenetic and population genetic approaches. Moderate support for the riverine barrier hypothesis was found for one of the three rivers examined, but little evidence was found for the elevational gradient hypothesis. Phylogenetic analyses revealed that high levels of sequence divergence (an average of 4.57-4.79%) separate three well-supported clades from the northwestern, southwestern, and eastern Amazon. Strong evidence for recent population expansion in P. petersi in the southwestern region of the Amazon basin was also uncovered.