Microreserves are an important tool for amphibian conservation.

Initiatives to protect 30% of Earth by 2030 prompt evaluation of how to efficiently target shortcomings in the global protected area (PA) network. Focusing on amphibians, the most vulnerable vertebrate class, we illustrate the conservation value of microreserves, a term we employ here to refer to reserves of <10 km2. We report that the network continues to under-represent threatened amphibians and that, despite this clear shortcoming in land-based conservation, the creation of PAs protecting amphibians slowed after 2010. By proving something previously assumed-that amphibians generally have smaller ranges than other terrestrial vertebrates-we demonstrate that microreserves could protect a substantial portion of many amphibian ranges, particularly threatened species. We find existing microreserves are capable of hosting an amphibian species richness similar to PAs 1000-10,00X larger, and we show that amphibians' high beta diversity means that microreserves added to a growing PA network cover amphibian species 1.5-6x faster than larger size categories. We propose that stemming global biodiversity loss requires that we seriously consider the conservation potential of microreserves, using them to capture small-range endemics that may otherwise be omitted from the PA network entirely.

A phylogeny-informed characterisation of global tetrapod traits addresses data gaps and biases.

Tetrapods (amphibians, reptiles, birds, and mammals) are model systems for global biodiversity science, but continuing data gaps, limited data standardisation, and ongoing flux in taxonomic nomenclature constrain integrative research on this group and potentially cause biased inference. We combined and harmonised taxonomic, spatial, phylogenetic, and attribute data with phylogeny-based multiple imputation to provide a comprehensive data resource (TetrapodTraits 1.0.0) that includes values, predictions, and sources for body size, activity time, micro- and macrohabitat, ecosystem, threat status, biogeography, insularity, environmental preferences, and human influence, for all 33,281 tetrapod species covered in recent fully sampled phylogenies. We assess gaps and biases across taxa and space, finding that shared data missing in attribute values increased with taxon-level completeness and richness across clades. Prediction of missing attribute values using multiple imputation revealed substantial changes in estimated macroecological patterns. These results highlight biases incurred by nonrandom missingness and strategies to best address them. While there is an obvious need for further data collection and updates, our phylogeny-informed database of tetrapod traits can support a more comprehensive representation of tetrapod species and their attributes in ecology, evolution, and conservation research.

Carbon nitride dots do not impair the growth, development, and telomere length of tadpoles.

Carbon nanoparticles, or carbon dots, can have many beneficial uses. However, we must consider whether they may have any potential negative side effects on wildlife or the ecosystem when these particles end up in wastewater. Early development stages of amphibians are particularly sensitive to contaminants, and exposure to carbon dots could disrupt their development and cause morbidity or death. Past studies have investigated short-term exposure to certain types of nanoparticles, but if these particles get into wastewater exposure may not be short term. Therefore, we tested whether chronic exposure to different concentrations of carbon dots affects the growth, metamorphosis, and telomere length of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We exposed 12 groups of five tadpoles each to different concentrations of carbon dots and a control for three months and tracked survival, growth and metamorphosis. We used carbon nitride dots approximately 2 nm in size at concentrations of 0.01 mg/ml and 0.02 mg/ml, known to interrupt development in zebrafish embryos. After three months, we measured telomere length from tissue samples. We found no difference in tadpole survivorship, growth, development rate, or telomere length among any of the groups, suggesting that carbon dots at these concentrations do not disrupt tadpole development.

Salmonella enterica Infection of Synanthropic Non-native Geckos in Southern Florida.

Wild geckos are a significant source of human salmonellosis. We swabbed the cloacas of 37 non-native synanthropic geckos (Gekko gecko, n = 16; Phelsuma grandis, n = 21) from southern Florida, USA, and assayed swab DNA extracts using quantitative polymerase chain reaction of the invA gene. Salmonella enterica was detected in both species with a pooled prevalence of 13.5% (5/37; 95% CI 5.3-27.1%), indicating the potential for zoonotic transmission. Implications for human health in the region are discussed.

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.

A new species of Andean frog of the genus Phrynopus (Anura: Strabomantidae) from southeastern Peru.

The Cordillera de los Andes is one of the most important regions for biodiversity. Among amphibians, many endemic species of terrestrial-breeding frogs have recently been discovered. Herein we describe Phrynopus sancristobali from the Andes of southeastern Peru based on molecular and morphological data. The new species is known from the ecotone between humid puna and montane forest at 3910 m a.s.l. on the left side of the Apurímac Valley in the Department of Ayacucho. The new species differs from congeners by having dorsum bearing pustules and light brown with dark brown reticulations surrounding the areolas, and coloration consisting of flanks golden brown with gray or dark brown marks, palms pale orange, soles deep orange, toes IV and V dark brown in dorsal and ventral view, belly cream to yellow with gray to light brown marks, and groin and throat deep orange. The snout-vent length (SVL) is 20.7 and 22.2 in two females, and 19.5 mm in one male. According to our phylogeny inferred using Maximum Likelihood with a concatenated dataset of three mitochondrial and two nuclear genes, P. sancristobali is sister taxon of P. apumantarum, recently described from Department Huancavelica. Our description extends the known geographic range of Phrynopus 73 km to the south, and P. sancristobali is the only species in the genus known to occur south of the Mantaro River, whose deep valley is hypothesized to be a biogeographic barrier for high-Andean organisms. The discovery of P. sancristobali confirms the high levels of endemism and beta diversity of Phrynopus in the moist puna grasslands and montane forests of the high Andes of Peru, and suggests that further work will reveal the presence of additional species in southern Peru.

First evidence of ranavirus in native and invasive amphibians in Colombia.

Ranaviruses can cause mass mortality events in amphibians, thereby becoming a threat to populations that are already facing dramatic declines. Ranaviruses affect all life stages and persist in multiple amphibian hosts. The detrimental effects of ranavirus infections to amphibian populations have already been observed in the UK and in North America. In Central and South America, the virus has been reported in several countries, but the presence of the genus Ranavirus (Rv) in Colombia is unknown. To help fill this knowledge gap, we surveyed for Rv in 60 species of frogs (including one invasive species) in Colombia. We also tested for co-infection with Batrachochytrium dendrobatidis (Bd) in a subset of individuals. For Rv, we sampled 274 vouchered liver tissue samples collected between 2014 and 2019 from 41 localities covering lowlands to mountaintop páramo habitat across the country. Using quantitative polymerase chain reaction (qPCR) and end-point PCR, we detected Rv in 14 individuals from 8 localities, representing 6 species, including 5 native frogs of the genera Osornophryne, Pristimantis and Leptodactylus, and the invasive American bullfrog Rana catesbeiana. Bd was detected in 7 of 140 individuals, with 1 co-infection of Rv and Bd in an R. catesbeiana specimen collected in 2018. This constitutes the first report of ranavirus in Colombia and should set off alarms about this new emerging threat to amphibian populations in the country. Our findings provide some preliminary clues about how and when Rv may have spread and contribute to understanding how the pathogen is distributed globally.

A new cryptic species of terrestrial breeding frog of the Pristimantisdanae Group (Anura, Strabomantidae) from montane forests in Ayacucho, Peru.

Based on morphological and molecular characters, we describe a new species of terrestrial breeding frog of the Pristimantisdanae Group from montane forests of La Mar Province, Ayacucho Department in southern Peru, at elevations from 1200 to 2000 m a.s.l. The phylogenetic analysis, based on concatenated sequences of gene fragments of 16S rRNA, RAG1, COI and TYR suggests that the new species is a sister taxon of a clade that includes one undescribed species of Pristimantis from Cusco, Pristimantispharangobates and Pristimantisrhabdolaemus. The new species is most similar to P.rhabdolaemus, which differs by lacking scapular tubercules and by its smaller size (17.0-18.6 mm in males [n = 5], 20.8-25.2 mm in females [n = 5] in the new species vs. 22.8-26.3 mm in males [n = 19], 26.0-31.9 mm in females [n = 30] of P.rhabdolaemus). Additionally, we report the prevalence of Batrachochytriumdendrobatidis (Bd) in this species.

ResumenDescribimos una nueva especie de rana terrestre de desarrollo directo del grupo Pristimantisdanae de bosques montanos procedentes de la provincia de La Mar, departamento de Ayacucho al sur de Perú con rango de distribución altitudinal entre los 1200­2000 msnm, en base a caracteres morfológicos y moleculares. El análisis filogenético basado en las secuencias concatenadas de los fragmentos de genes ARNr 16S, COI, RAG1 y TYR sugiere que la nueva especie es un taxón hermano del clado que incluye a una especie de Pristimantis no descrita de Cusco, Pristimantispharangobates y Pristimantisrhabdolaemus. La nueva especie se asemeja más a P.rhabdolaemus; de la cual difiere por la ausencia de tubérculos escapulares y su menor tamaño corporal (17.0­18.6 mm en machos [n=5], 20.8­25.2 mm en hembras [n=5] en la nueva especie vs 22.8­26.3 mm en machos [n=19], 26.0­31.9 mm en hembras [n=30] de P.rhabdolaemus). Adicionalmente, reportamos la prevalencia de Batrachochytriumdendrobatidis (Bd) en esta especie de Terrarana.

An insight into embryogenesis interruption by carbon nitride dots: can they be nucleobase analogs?

The carbon nitride dot (CND) is an emerging carbon-based nanomaterial. It possesses rich surface functional moieties and a carbon nitride core. Spectroscopic data have demonstrated the analogy between CNDs and cytosine/uracil. Recently, it was found that CNDs could interrupt the normal embryogenesis of zebrafish. Modifying CNDs with various nucleobases, especially cytosine, further decreased embryo viability and increased deformities. Physicochemical property characterization demonstrated that adenine- and cytosine-incorporated CNDs are similar but different from guanine-, thymine- and uracil-incorporated CNDs in many properties, morphology, and structure. To investigate the embryogenesis interruption at the cellular level, bare and different nucleobase-incorporated CNDs were applied to normal and cancerous cell lines. A dose-dependent decline was observed in the viability of normal and cancerous cells incubated with cytosine-incorporated CNDs, which matched results from the zebrafish embryogenesis experiment. In addition, nucleobase-incorporated CNDs were observed to enter cell nuclei, demonstrating a possibility of CND-DNA interactions. CNDs modified by complementary nucleobases could bind each other via hydrogen bonds, which suggests nucleobase-incorporated CNDs can potentially bind the complementary nucleobases in a DNA double helix. Nonetheless, neither bare nor nucleobase-incorporated CNDs were observed to intervene in the amplification of the zebrafish polymerase-alpha 1 gene in quantitative polymerase chain reactions. Thus, in conclusion, the embryogenesis interruption by bare and nucleobase-incorporated CNDs might not be a consequence of CND-DNA interactions during DNA replication. Instead, CND-Ca2+ interactions offer a plausible mechanism that hindered cell proliferation and zebrafish embryogenesis originating from disturbed Ca2+ homeostasis by CNDs. Eventually, the hypothesis that raw or nucleobase-incorporated CNDs can be nucleobase analogs proved to be invalid.

Pristimantis achupalla sp. n., a new minute species of direct-developing frog (Amphibia, Anura, Strabomantidae) inhabiting bromeliads of the montane forest of the Amazonian Andes of Puno, Peru.

We describe a new species of bromeliad-dwelling Pristimantis from primary montane forest (2,225 m a.s.l.) in southern Peru. The type locality is near Thiuni, in the Department of Puno (province of Carabaya) in the upper watershed of a tributary of the Inambari River. Pristimantis achupalla sp. n. is characterized by a snout-vent length of 10.0-12.8 mm in adult males (n = 4), unknown in adult females, and is compared morphologically and genetically with species in the Pristimantis lacrimosus group, and with other similar species of Pristimantis. The new species is characterized by having skin on dorsum and flanks rugose, green brownish color, distinctive scapular folds, subacuminate or acuminate snout profile, upper eyelid bearing two or three subconical tubercles and some rounded tubercles, rostral papilla, flanks light brown to brown, with irregular dark brown marks.

Applied science facilitates the large-scale expansion of protected areas in an Amazonian hot spot.

Meeting international commitments to protect 17% of terrestrial ecosystems worldwide will require >3 million square kilometers of new protected areas and strategies to create those areas in a way that respects local communities and land use. In 2000-2016, biological and social scientists worked to increase the protected proportion of Peru's largest department via 14 interdisciplinary inventories covering >9 million hectares of this megadiverse corner of the Amazon basin. In each landscape, the strategy was the same: convene diverse partners, identify biological and sociocultural assets, document residents' use of natural resources, and tailor the findings to the needs of decision-makers. Nine of the 14 landscapes have since been protected (5.7 million hectares of new protected areas), contributing to a quadrupling of conservation coverage in Loreto (from 6 to 23%). We outline the methods and enabling conditions most crucial for successfully applying similar campaigns elsewhere on Earth.

A new species of Atelopus (Anura: Bufonidae) from southern Peru.

We describe a new harlequin frog (genus Atelopus) from the cloud forest near Anchihuay (Anco District, Ayacucho Department) from 2000 to 2150 m elevation in southern Peru, representing the first record for the genus in the Department of Ayacucho. The new species has a maximum snout-vent length of 21.5 mm in females and 21.6 mm in males, and resembles A. erythropus in general appearance, small size, and dorsal coloration. The new species can be distinguished from A. erythropus by its unique pattern of ventral coloration, dorsal skin texture, and snout shape. We detected the presence of the pathogenic fungus Batrachochytrium dendrobatidis in individuals of the new species. This pathogen is threatening the survival of harlequin frogs throughout the Neotropics. In addition to chytridiomycosis, habitat loss further threatens the single locality where the new species is known to occur.

Transcriptomics reveal immune downregulation of newts overwhelmed by chytrid co-infection.

What happens when two emergent diseases infect the same host? In a From the Cover article in this issue of Molecular Ecology, McDonald et al. (2020) compare transcriptomic responses to co-infection by the two chytrid fungi in the skin, liver and spleen of Eastern newts (Notophthalmus viridescens). Novel molecular tools, such as high-throughput DNA sequencing for genome discovery and transcriptomics, have revolutionized our understanding of host-pathogen interactions and disease ecology (Güimil et al. 2005; Rosenblum et al. 2012). For example, epidemiologists are using genomic data to track the spread of the emergent SARS-CoV-2 in real time, both locally and globally. RNA sequencing (RNA-Seq) is routinely employed to study response to disease in humans, improving disease diagnostics, profiling and development of intervention strategies. Transcriptomic profiles may be particularly informative for emergent diseases, whose pathologies and effect on host phenotype are poorly known. Fungal pathogens increasingly threaten a variety of wild and domesticated organisms (Fisher et al. 2012), and two chytrid fungi attacking amphibians are causing one of the worst losses of vertebrate biodiversity ever recorded (Scheele et al. 2019).

A new species of frog (Terrarana, Strabomantidae, Phrynopus) from the Peruvian Andean grasslands.

We describe a new, medium-sized species of terrestrial frog of the genus Phrynopus from a single locality in the central Andes of Peru (Departamento de Huánuco) at 3,730 meters of elevation. Phylogenetic analyses supported Phrynopus remotum sp. nov. as an independent lineage, sister to most of its congeners. The new species is morphologically distinguishable by the presence of small tubercles on upper eyelids and heels, an areolate venter, and the absence of dorsolateral folds or ridges. This species inhabits the highlands adjacent to the Marañón Dry valley. The only sympatric amphibian species recorded is the marsupial frog Gastrotheca peruana.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Integrative taxonomy reveals first country record of Hyalinobatrachium mondolfii Señaris and Ayarzagüena 2001, and distribution range extensions for Cochranella nola Harvey 1996, and Rulyrana spiculata Duellman 1976 (Anura: Centrolenidae) in Peru.

We used an integrative taxonomy approach to investigate the taxonomic identity of several populations of glassfrogs from Peru, which are notoriously challenging to identify due to their overall similarity in morphology and coloration. We relied on comparisons of morphology, bioacoustics, and partial fragments of 16S rRNA DNA sequences. We report for the first time the presence of Hyalinobatrachium mondolfii in Peru, being this the southernmost locality known for the species. Likewise, we update and extend the distribution ranges of Rulyrana spiculata and Cochranella nola in the Andes of Peru, provide a 16S sequence of a topotype of R. spiculata, and confirm its presence in Bolivia. For all three species, we increase the current knowledge on their geographic distribution and genetic and phenotypic variation.

Arcanumophis, a new genus and generic allocation for Erythrolamprus problematicus (Myers 1986), Xenodontinae (Colubridae) from the Cordillera de Carabaya, southern Peru.

The systematics of South American Xenodontinae snakes has experienced large changes and improvements as a result of recent studies employing molecular data. Herein we assess the status of the rare Peruvian snake species, Erythrolamprus problematicus (Dipsadidae, Xenodontinae, Xenodontini), previously known from a single specimen collected in 1950. Based on new morphological and molecular data from a second specimen that we collected, we confirmed the presence of a crease in the rostral scale, mentioned in the original description (a unique trait among the Xenodontini), and recovered E. problematicus as the sister-taxon of all other Xenodontini, instead of nested among the current Erythrolamprus. Therefore, our phylogenetic results justify the erection of a new genus to accommodate the species.

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Thermal physiological traits in tropical lowland amphibians: Vulnerability to climate warming and cooling.

Climate change is affecting biodiversity and ecosystem function worldwide, and the lowland tropics are of special concern because organisms living in this region experience temperatures that are close to their upper thermal limits. However, it remains unclear how and whether tropical lowland species will be able to cope with the predicted pace of climate warming. Additionally, there is growing interest in examining how quickly thermal physiological traits have evolved across taxa, and whether thermal physiological traits are evolutionarily conserved or labile. We measured critical thermal maximum (CTmax) and minimum (CTmin) in 56 species of lowland Amazonian frogs to determine the extent of phylogenetic conservatism in tolerance to heat and cold, and to predict species' vulnerability to climate change. The species we studied live in sympatry and represent ~65% of the known alpha diversity at our study site. Given that critical thermal limits may have evolved differently in response to different temperature constraints, we tested whether CTmax and CTmin exhibit different rates of evolutionary change. Measuring both critical thermal traits allowed us to estimate species' thermal breadth and infer their potential to respond to abrupt changes in temperature (warming and cooling). Additionally, we assessed the contribution of life history traits and found that both critical thermal traits were correlated with species' body size and microhabitat use. Specifically, small direct-developing frogs in the Strabomantidae family appear to be at highest risk of thermal stress while tree frogs (Hylidae) and narrow mouthed frogs (Microhylidae) tolerate higher temperatures. While CTmax and CTmin had considerable variation within and among families, both critical thermal traits exhibited similar rates of evolutionary change. Our results suggest that 4% of lowland rainforest frogs assessed will experience temperatures exceeding their CTmax, 25% might be moderately affected and 70% are unlikely to experience pronounced heat stress under a hypothetical 3°C temperature increase.