Ongoing declines for the world's amphibians in the face of emerging threats.

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

Evaluation of partial 12S rRNA, 16S rRNA, COI and Cytb gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae).

We evaluated the extent of intraspecific and interspecific genetic distances and the effectiveness of predefined threshold values using the main genes for estimates of biodiversity and specimens' identification in anurans. Partial sequences of the mitochondrial genes for small (12S) and large (16S) ribosomal subunits, cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb) of the family Hylidae were downloaded from GenBank and curated for length, coverage, and potential contaminations. We performed analyses for all sequences of each gene and the same species present in these datasets by distance and tree (monophyly)-based evaluations. We also evaluated the ability to identify specimens using these datasets applying "nearest neighbor" (NN), "best close match" (BCM) and "BOLD ID" tests. Genetic distance thresholds were generated by the function 'threshVal' and "localMinima" from SPIDER package and traditional threshold values (1%, 3%, 6% and 10%) were also evaluated. Coding genes, especially COI, had a better identification capacity than non-coding genes on barcoding gap and monophyly analysis and NN, BCM, BOLD ID tests. Considering the multiple factors involved in global DNA barcoding evaluations, we present a critical assessment of the use of these genes for biodiversity estimation and specimens' identification in anurans (e.g. hylids).

The dispersal between Amazonia and Atlantic Forest during the Early Neogene revealed by the biogeography of the treefrog tribe Sphaenorhynchini (Anura, Hylidae).

The Amazonia and the Atlantic Forest, separated by the diagonal of open formations, are two ecoregions that comprise the most diverse tropical forests in the world. The Sphaenorhynchini tribe is among the few tribes of anurans that occur in both rainforests, and their historical biogeographic have never been proposed. In this study, we infer a dated phylogeny for the species of the Sphaenorhynchini and we reconstructed the biogeographic history describing the diversification chronology, and possible patterns of dispersion and vicariance, providing information about how orogeny, forest dynamics and allopatric speciation affected their evolution in South America. We provided a dated phylogeny and biogeography study for the Sphaenorhynchini tribe using mitochondrial and nuclear genes. We analyzed 41 samples to estimate the ancestral areas using biogeographical analysis based on the estimated divergence times and the current geographical ranges of the species of Sphaenorhynchini. We recovered three characteristic clades that we recognize as groups of species (S. lacteus, S. planicola, and S. platycephalus groups), with S. carneus and G. pauloalvini being the sister taxa of all other species from the tribe. We found that the diversification of the tribe lineages coincided with the main climatic and geological factors that shaped the Neotropical landscape during the Cenozoic. The most recent common ancestor of the Sphaenorhynchini species emerged in the North of the Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. This is the first large-scale study to include an almost complete calibrated phylogeny of Sphaenorhynchini, presenting important information about the evolution and diversification of the tribe. Overall, we suggest that biogeographic historical of Sphaenorhynchini have resulted from a combination of repeated range expansion and contraction cycles concurrent with climate fluctuations and dispersal events between the Atlantic Forest and Amazonia.

DNA barcoding and phylogeography of the Hoplias malabaricus species complex.

Hoplias malabaricus (Bloch, 1794) is a carnivorous fish species widely distributed from northern to southern South America. This taxon is believed to be a good model for the investigation of biogeographic events that shape the ichthyofauna evolution in the Neotropical freshwater systems. However, many studies have revealed that H. malabaricus hides a species complex that hampers its taxonomic identity and limit its practical value for evolutionary and biogeographic studies. In this paper, we used the mitochondrial gene cytochrome c oxidase subunit I (COI) to delimit cryptic species and explore the phylogeography of H. malabaricus sensu stricto. We found genetic evidence for putative new species in the genus Hoplias and showed that H. malabaricus (Bloch, 1794) is a major clade assigned to barcode index number (BIN) BOLD:ABZ3047. This species is structured in six subpopulations differentiated by high Fst values and restricts gene flow. The subpopulations of the São Francisco/East Atlantic/Eastern Northeast Atlantic/Parnaíba/Itapecuru River basins and Tapajós River Basin were the most differentiated and showed demographic fluctuations. The present distributional pattern is most likely explained through a scenario from the Pleistocene.

Epidemiology of snake envenomation from Mato Grosso do Sul, Brazil.

Snake envenomation is considered a public health problem in tropical countries, where they occur in a high incidence. The present study reports the snake envenomation that occurred in Mato Grosso do Sul state (Brazil) between 2007 and 2017. Epidemiological data were obtained from the online platform of the Notification Disease Information System and were analyzed according to biome. A total of 5568 cases of snake envenomations were recorded during the study period, where the highest frequency was registered between October and April. The majority of envenomations occurred in working-age males (20 to 39 years), caused mainly by Bothrops snakes, and the duration of care after the envenomation in most cases took three hours. The municipalities that showed the highest snake envenomations case per 100,000 inhabitants presents low population density, and have their economy based on agricultural activity, which is a risk factor to snake envenomations. To the Mato Grosso do Sul state, the total number of snake envenomations had a positive relationship with the size of the municipality. Since this, larger areas usually have a mosaic of environments, which may harbor higher richness and abundance of snakes, and can cause more snake encounters with the population, resulting in more snake envenomations.

DNA barcoding for identification of anuran species in the central region of South America.

The use of COI barcodes for specimen identification and species discovery has been a useful molecular approach for the study of Anura. Here, we establish a comprehensive amphibian barcode reference database in a central area of South America, in particular for specimens collected in Mato Grosso do Sul state (Brazil), and to evaluate the applicability of the COI gene for species-level identification. Both distance- and tree-based methods were applied for assessing species boundaries and the accuracy of specimen identification was evaluated. A total of 204 mitochondrial COI barcode sequences were evaluated from 22 genera and 59 species (19 newly barcoded species). Our results indicate that morphological and molecular identifications converge for most species, however, some species may present cryptic species due to high intraspecific variation, and there is a high efficiency of specimen identification. Thus, we show that COI sequencing can be used to identify anuran species present in this region.

Correction: Four new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Craugastoridae) in the eastern Amazon.

[This corrects the article DOI: 10.1371/journal.pone.0229971.].

A new tiny toad species of Amazophrynella (Anura: Bufonidae) from east of the Guiana Shield in Amazonia, Brazil.

The combination of different approaches has successfully delimited new species within many Neotropical species complexes traditionally classified as a single nominal organism. Recent studies have shown that the Amazonian endemic genus Amazophrynella, currently composed of 12 small-sized species, could harbor several additional species. Based on morphology and molecular data, we describe a new species of Amazophrynella from east of the Guiana Shield, in Pará state, Brazil. The new species is characterized by having one of the biggest size of the genus (SVL of males 16.0-17.8 mm and females 22.9-24.4 mm), presence of a large palmar tubercle (occupying 2/4 of the palmar surface), 5.6-8.1% uncorrected p-distance from its sister clade (including A. teko, A. sp.1, and A. manaos) for the 16S mitochondrial gene, and 8.8% for the COI. The new species described here represents a newly discovered lineage. Of the 12 Amazophrynella species currently recognized, two were describe in the last century (A. bokermanni and A. minuta) and the remaining species were recently discovered and described (in the last six years), which underscores the degree to which species richness of Amazophrynella is underestimated.

Four new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Craugastoridae) in the eastern Amazon.

The Pristimantis genus (Anura: Craugastoridae) is the most diverse among all vertebrates with 531 described species. The highest diversity occurs in Ecuador (215 species), followed by Colombia (202), Peru (139), Venezuela (60), Brazil (30), Bolivia (17), Guyana (6) Suriname and French Guiana (5). The genus is divided into 11 species groups. Of these, the P. conspicillatus group (containing 34 species), distributed in extreme southeastern Costa Rica, Isla Taboga (Panama), northern South America (from Colombia to eastern Guyana), south Bolivia, and is the best represented in Brazil (16 species). The main characteristics of this group are the tympanic membrane and tympanic annulus distinct (except in P. johannesdei); dorsum smooth or shagreen; dorsal lateral fold present or absent; usually smooth belly, but may be weakly granular in some species; toe V slightly larger than the toe III. Most of the taxonomic inconsistencies in species of Pristimantis could be due to its much conserved morphology and the lack of comprehensive taxonomic evaluations. Thus, an ongoing challenge for taxonomists dealing with the Pristimantis genus is the ubiquitous abundance of cryptic species. In this context, accurate species delimitation should integrate evidences of morphological, molecular, bioacoustics and ecological data, among others. Based on an integrative taxonomy perspective, we utilize morphological, molecular (mtDNA) and bioacoustic evidence to describe four new species of the Pristimantis conspicillatus group from the eastern Amazon basin. Pristimantis giorgii sp. nov. is known from the Xingu/Tocantins interfluve and can be distinguished from the other Pristimantis species of the region by presenting discoidal fold, dorsolateral fold absent, vocalization composed of three to four notes and genetic distance of 7.7% (16S) and 14.8% (COI) from P. latro, the sister and sympatric species with respect P. giorgii sp. nov.. Pristimantis pictus sp. nov. is known to the northern Mato Grosso state, Brazil, and can be distinguished from the other species of Pristimantis by presenting the posterior surface of the thigh with light yellow patches on a brown background, also extending to the inguinal region, vocalization consisting of four to five notes and a genetic distance of 11.6% (16S) and 19.7% (COI) from P. pluvian sp. nov., which occurs in sympatry. Pristimantis pluvian sp. nov. is known to the northern Mato Grosso state, Brazil, and may be distinguished from the other Pristimantis species by having a posterior surface of the thigh reddish and vocalization composed of two notes. Pristimantis moa sp. nov. is known to the northern Tocantins state and southwestern Maranhão state. This species can be distinguished from the other Pristimantis species by possessing slightly perceptible canthal stripe, external thigh surface with dark yellow spots on brown background, vocalization consisting of three to five notes and genetic distance of 2.3-11.7 (16S) and 10.5-23.1 (COI) for the new Pristimantis species of this study.

A new species of spotted leaf frog, genus Phasmahyla (Amphibia, Phyllomedusidae) from Southeast Brazil.

Based on concordant differences in male advertisement call, tadpole morphology, and absence of haplotype sharing in the barcoding 16S mitochondrial DNA, we describe here a new species of spotted leaf frog of the genus Phasmahyla from Atlantic Forest, State of Rio de Janeiro, Southeast Brazil. The new species is most similar to P. cochranae (type locality) and P. spectabilis (type locality). It differs from these species by the size of the calcar, moderate-sized body (snout-vent length 30.4-34.4 mm in adult eight males), and in the advertisement call. The tadpoles of Phasmahyla lisbella sp. nov. differ from P. exilis, P. spectabilis, P. timbo, P. guttata and P. jandaia because they do not have row of teeth in the anterior part; differ from P. cruzi by the shape of the anterior end of the oral disc. Through genetic data (phylogenetic distance and haplotype genealogy) we diagnosed the new species where the genetic divergences among its congeners is about 3-6% in a fragment of the 16S rRNA gene, which is above the threshold typically characterizing distinct species of anurans. However, the new species can be distinguished from other congeneric species based on an integrative approach (molecular, bioacoustics, larval, and adult morphology).

The tadpole of Physalaemus nanus (Boulenger, 1888) (Anura, Leptodactylidae) from Southern Brazil.

The genus Physalaemus Fitzinger, 1826 currently comprises 47 described species distributed from Central America (Guianas) to Uruguay (Frost 2016). The genus has been recently organized in two main clades based on molecular data, Physalaemus signifer Clade and Physalaemus cuvieri Clade (Lourenço et al. 2015). The P. signifer Clade, with 15 species, is composed by the P. deimaticus and P. signifier groups, plus P. nattereri (Steindachner, 1863) and P. maculiventris (Lutz, 1925) (Nascimento et al. 2005; Lourenço et al. 2015).

Anurans from the Middle Jaguaribe River Region, Ceará State, Northeastern Brazil / Anuros da região do Médio Rio Jaguaribe, estado do Ceará, Nordeste do Brasil

The Middle Jaguaribe River region is a poorly sampled area in the Caatingas domain, located in the eastern Ceará State, northeastern Brazil. The Brazilian government agencies considered this region as a priority area for conservation and inventories. In order to fill this biodiversity knowledge gap, we conducted a ten-day rapid inventory of the anuran fauna from April 11 to 20, 2014, surveying different physiognomies of the region. We recorded 19 anuran species belonging to five families: Bufonidae (2), Hylidae (5), Leptodactylidae (9), Microhylidae (2) and Odontophrynidae (1). Most inventories of Caatinga amphibians published to date have been conducted inside protected areas and/or of high altitude (mesic areas known as "brejos de altitude"). The list we present herein contributes to the knowledge of lowland Caatinga sites outside protected areas. We discuss our results in light of taxonomic and geographic features of the anurans sampled.

A Região do Médio Rio Jaguaribe é uma área pobremente amostrada no domínio das Caatingas, localizada no leste do Estado do Ceará, Nordeste do Brasil. O Ministério do Meio Ambiente considera esta região como uma área prioritária para conservação e realização de inventários. Com o objetivo de preencher esta lacuna do conhecimento sobre a biodiversidade, nós conduzimos um inventário rápido da anurofauna de dez dias de 11 a 20 de Abril de 2014, amostrando diferentes fisionomias da região. Nós registramos 19 espécies de anfíbios anuros pertencentes a cinco famílias: Bufonidae (2), Hylidae (5), Leptodactylidae (9), Microhylidae (2) e Odontophrynidae (1). A maioria dos inventários de anfíbios na Caatinga publicados até o presente momento foram conduzidos em áreas de proteção e/ou de áreas de altitude (áreas mésicas conhecidas como “Brejos de Altitude”). A presente lista contribui para o conhecimento de áreas de baixada da Caatinga fora das áreas protegidas. Nós discutimos os resultados è luz de características taxonômicas e biogeográficas dos anuros amostrados.

A new and possibly critically endangered species of casque-headed tree frog Aparasphenodon Miranda-Ribeiro, 1920 (Anura, Hylidae) from southeastern Brazil.

A new species of casque-headed tree frog of the genus Aparasphenodon is described from the municipality of Cataguases (21°20'S, 42°45'W; 288 m a.s.l.) in the Atlantic Rain Forest of Minas Gerais State, southeastern Brazil. Aparasphenodon pomba sp. nov. is characterized by medium size (males, snout-vent length, SVL 51.6-60.5 mm; females, SVL 58.7-62.1 mm); snout almost round in dorsal view; dorsum and limbs with cream-colored reticulation on dark-brown background; spots on ventral surface cream-colored; lips white; cream-colored dorsolateral stripe originating on the snout, crossing the upper eyelid and extending posteriorly to the axilla level; and red iris.