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.

Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Factors Influencing Bacterial and Fungal Skin Communities of Montane Salamanders of Central Mexico.

Host microbial communities are increasingly seen as an important component of host health. In amphibians, the first land vertebrates that are threatened by a fungal skin disease globally, our understanding of the factors influencing the microbiome of amphibian skin remains incomplete because recent studies have focused almost exclusively on bacteria, and little information exists on fungal communities associated with wild amphibian species. In this study, we describe the effects of host phylogeny, climate, geographic distance, and infection with a fungal pathogen on the composition and structure of bacterial and fungal communities in seven tropical salamander species that occur in the Trans-Mexican Volcanic Belt of Central Mexico. We find that host phylogenetic relatedness is correlated with bacterial community composition while a composite climatic variable of temperature seasonality and precipitation is significantly associated with fungal community composition. We also estimated co-occurrence networks for bacterial and fungal taxa and found differences in the degree of connectivity and the distribution of negative associations between the two networks. Our results suggest that different factors may be responsible for structuring the bacterial and fungal communities of amphibian skin and that the inclusion of fungi in future studies could shed light on important functional interactions within the microbiome.

Genetic variation of Batrachochytrium dendrobatidis is linked to skin bacterial diversity in the Pacific treefrog Hyliola regilla (hypochondriaca).

Symbiotic bacterial communities are crucial to combating infections and contribute to host health. The amphibian skin microbiome plays an important role in protecting their hosts against pathogens such as Batrachochytrium dendrobatidis (Bd), one of the causative agents of chytridiomycosis, which is responsible for dramatic amphibian population declines worldwide. Although symbiotic skin bacteria are known to inhibit Bd growth, an understanding of the relationship between Bd genetic variability, environmental conditions, and skin bacterial communities is limited. Therefore, we examined the associations between Bd infection load, Bd genetic diversity and skin bacterial communities in five populations of Hyliola regilla (hypochondriaca) from environmentally contrasting sites in Baja California, Mexico. We observed differences in Bd genetics and infection load among sites and environments. Genetic analysis of Bd isolates revealed patterns of spatial structure corresponding to the five sites sampled. Amphibian skin bacterial diversity and community structure differed among environments and sites. Bacterial community composition was correlated with Bd genetic differences and infection load, with specific bacterial taxa enriched on infected and un-infected frogs. Our results indicate that skin-associated bacteria and Bd strains likely interact on the host skin, with consequences for microbial community structure and Bd infection intensity.

Comparative Analysis of Skin Bacterial Diversity and Its Potential Antifungal Function Between Desert and Pine Forest Populations of Boreal Toads Anaxyrus boreas.

The skin microbiome in amphibians has gained a lot of attention as some of its members play a protective role against pathogens such as the fungus Batrachochytrium dendrobatidis (Bd). The composition of skin bacterial communities has been suggested as one of the factors explaining differences in susceptibility to Bd among amphibian species and populations. The boreal toad Anaxyrus boreas is known to be susceptible to Bd, and severe population declines in its southeastern range have been documented. However, throughout A. boreas distribution, populations present differences in susceptibility to Bd infections which may be associated with differences in skin microbial diversity. This study compared the skin bacterial diversity and Bd infection levels of A. boreas in one desert population and one pine forest population from Baja California, Mexico. We found that desert and pine forest toad populations exhibit differences in skin bacterial community structure but show similar Bd infection levels. Using a predictive method, we found that the abundance of bacteria with potential Bd-inhibitory properties differed between uninfected and infected individuals but not between populations. Our data suggest that several bacteria in the skin community may be offering protection from Bd infections in these A. boreas populations. This study provides foundational evidence for future studies seeking to understand the skin-microbial variation among boreal toads' populations and its relation with Bd susceptibility.

Early presence of Batrachochytrium dendrobatidis in Mexico with a contemporary dominance of the global panzootic lineage.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a devastating infectious disease of amphibians. Retrospective studies using museum vouchers and genetic samples supported the hypothesis that Bd colonized Mexico from North America and then continued to spread into Central and South America, where it led to dramatic losses in tropical amphibian biodiversity (the epizootic wave hypothesis). While these studies suggest that Bd has been in Mexico since the 1970s, information regarding the historical and contemporary occurrence of different pathogen genetic lineages across the country is limited. In the current study, we investigated the historical and contemporary patterns of Bd in Mexico. We combined the swabbing of historical museum vouchers and sampling of wild amphibians with a custom Bd genotyping assay to assess the presence, prevalence, and genetic diversity of Bd over time in Mexico. We found Bd-positive museum specimens from the late 1800s, far earlier than previous records and well before recent amphibian declines. With Bd genotypes from samples collected between 1975-2019, we observed a contemporary dominance of the global panzootic lineage in Mexico and report four genetic subpopulations and potential for admixture among these populations. The observed genetic variation did not have a clear geographic signature or provide clear support for the epizootic wave hypothesis. These results provide a framework for testing new questions regarding Bd invasions and their temporal relationship to observed amphibian declines in the Americas.

Inhibition of Batrachochytrium dendrobatidis Infection by Skin Bacterial Communities in Wild Amphibian Populations.

Skin-associated bacteria are known to inhibit infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd) in amphibians. It has also been postulated that skin-associated bacterial community is related to Bd infection intensity. However, our understanding of host microbial dynamics and their importance in regulating Bd intensity is limited. We analyzed Bd infection and skin-associated bacteria from two amphibian species, the salamander Ambystoma rivulare and the frog Lithobates spectabilis that co-occurred in a tropical high-altitude site in central Mexico. Sixty-three percent of sampled salamander individuals and 80% of frog individuals tested positive for Bd. Overall, we registered 622 skin-associated bacterial genera, from which 73 are known to have Bd inhibitory effects. These inhibitory taxa represented a relative abundance of 50% in relation to total relative bacterial abundance. Our results indicated that, although sharing some bacterial taxa, bacterial community from the skin of both species was different in taxonomic composition and in relative abundance. Pseudomonas spp. and Stenotrophomonas spp. were among the five most abundant bacterial taxa of both species. Both bacterial taxa inhibit Bd infection. We detected that bacterial richness and relative abundance of inhibitory Bd bacteria were negatively related to intensity of Bd infection independent of species and seasons. Despite the high Bd prevalence in both host species, no dead or sick individuals were registered during field surveys. The relatively low levels of Bd load apparently do not compromise survival of host species. Therefore, our results suggested that individuals analyzed were able to survive and thrive under a dynamic relation with enzootic infections of Bd and their microbiota.

The Influence of Habitat and Phylogeny on the Skin Microbiome of Amphibians in Guatemala and Mexico.

Microbial symbionts are increasingly recognized as playing a critical role in organismal health across a wide range of hosts. Amphibians are unique hosts in that their skin helps to regulate the exchange of water, ions, and gases, and it plays an active role in defense against pathogens through the synthesis of anti-microbial peptides. The microbiome of amphibian skin includes a diverse community of bacteria known to defend against pathogens, including the global pandemic lineage of Batrachochytrium dendrobatidis associated with mass amphibian die-offs. The relative influence of host phylogeny and environment in determining the composition of the amphibian skin microbiome remains poorly understood. We collected skin swabs from montane amphibians in Mexico and Guatemala, focusing on two genera of plethodontid salamanders and one genus of frogs. We used high throughput sequencing to characterize the skin bacterial microbiome and tested the impact of phylogeny and habitat on bacterial diversity. Our results show that phylogenetic history strongly influences the diversity and community structure of the total bacterial microbiome at higher taxonomic levels (between orders), but on lower scales (within genera and species), the effect of habitat predominates. These results add to a growing consensus that habitat exerts a strong effect on microbiome structure and composition, particularly at shallow phylogenetic scales.

Phylogenomic insights into the diversification of salamanders in the Isthmura bellii group across the Mexican highlands.

Mountain formation in Mexico has played an important role in the diversification of many Mexican taxa. The Trans-Mexican Volcanic Belt in particular has served as both a cradle of diversification and conduit for dispersal. We investigated the evolutionary history of the Isthmura bellii group of salamanders, a widespread amphibian across the Mexican highlands, using sequence capture of ultraconserved elements. Results suggest that the I. bellii group probably originated in southeastern Mexico in the late Miocene and later dispersed across the Trans-Mexican Volcanic Belt and into the Sierra Madre Occidental. Pre-Pleistocene uplift of the Trans-Volcanic Belt likely promoted early diversification by serving as a mesic land-bridge across central Mexico. These findings highlight the importance of the Trans-Volcanic Belt in generating Mexico's rich biodiversity.

Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

Positive selection drives the evolution of a major histocompatibility complex gene in an endangered Mexican salamander species complex.

Immune gene evolution can be critical to species survival in the face of infectious disease. In particular, polymorphism in the genes of the major histocompatibility complex (MHC) helps vertebrates combat novel and diverse pathogens by increasing the number of pathogen-derived proteins that can initiate the host's acquired immune response. In this study, we used a combination of presumably adaptive and neutral markers to investigate MHC evolution in populations of five salamander species within the Ambystoma velasci complex, a group consisting of 15 recently diverged species, several of which are endangered. We isolated 31 unique MHC class II ß alleles from 75 total individuals from five species in this complex. MHC heterozygosity was significantly lower than expected for all five species, and we found no clear relationship between number of MHC alleles and species range, life history, or level of heterozygosity. We inferred a phylogeny representing the evolutionary history of Ambystoma MHC, with which we found signatures of positive selection on the overall gene, putative peptide-binding residues, and allelic lineages. We identified several instances of trans-species polymorphism, a hallmark of balancing selection observed in other groups of closely related species. In contrast, we did not detect comparable allelic diversity or signatures of selection on neutral loci. Additionally, we identified 17 supertypes among the 44 unique Ambystoma alleles, indicating that these sequences may encode functionally distinct MHC variants. We therefore have strong evidence that positive selection is a major evolutionary force driving patterns of MHC polymorphism in this recently radiated species complex.

Presence and prevalence of Batrachochytrium dendrobatidis in commercial amphibians in Mexico City.

In Mexico City, native and exotic amphibians are commonly sold through the pet trade. This study investigates the presence of Batrachochytrium dendrobatidis (Bd) in native amphibians being sold at two commercial markets and at a herpetarium in Mexico City. A total of 238 individuals (6 genera and 12 species) were tested for Bd using real-time polymerase chain reaction (PCR) analysis. There were 197 Bd-positive individuals (prevalence 82%) from five species of amphibians. Hyla eximia from the markets had very high Bd prevalence (100%; 76/76 and 99%; 88/89) but those from the herpetarium were Bd negative (0/12). Ambystoma mexicanum from the herpetarium also had a high Bd-positive prevalence (80%; 28/35). Though A. mexicanum is nearly extinct in the wild, a commercial market continues to flourish through the pet trade. Now that captive colonies of A. mexicanum are currently used for reintroduction programs, the authors recommend quarantine to reduce spread of Bd via movement of infected animals in the trade and between colonies and via disposal of wastewater from captive collections.

Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

Environment rather than character displacement explains call evolution in glassfrogs.

The acoustic adaptation hypothesis (AAH) and ecological character displacement (ECD) are two potential mechanisms shaping call evolution that can predict opposite trends for the differentiation of signals. Under AAH, signals evolve to minimize environmental degradation and maximize detection against background noise, predicting call homogenization in similar habitats due to environmental constraints on signals. In contrast, ECD predicts greater differences in call traits of closely related taxa in sympatry because of selection against acoustic interference. We used comparative phylogenetic analyses to test the strength of these two selective mechanisms on the evolution of advertisement calls in glassfrogs, a highly diverse family of neotropical anurans. We found that, overall, acoustic adaptation to the environment may outweigh effects of species interactions. As expected under the AAH, temporal call parameters are correlated with vegetation density, but spectral call parameters had an unexpected inverse correlation with vegetation density, as well as an unexpected correlation with temperature. We detected call convergence among co-occurring species and also across multiple populations from the same species in different glassfrogs communities. Our results indicate that call convergence is common in glassfrogs, likely due to habitat filtering, while character displacement is relatively rare, suggesting that costs of signal similarity among related species may not drive divergent selection in all systems.

Deep divergences and extensive phylogeographic structure in a clade of lowland tropical salamanders.

BACKGROUND: The complex geological history of Mesoamerica provides the opportunity to study the impact of multiple biogeographic barriers on population differentiation. We examine phylogeographic patterns in a clade of lowland salamanders (Bolitoglossa subgenus Nanotriton) using two mitochondrial genes and one nuclear gene. We use several phylogeographic analyses to infer the history of this clade and test hypotheses regarding the geographic origin of species and location of genetic breaks within species. We compare our results to those for other taxa to determine if historical events impacted different species in a similar manner. RESULTS: Deep genetic divergence between species indicates that they are relatively old, and two of the three widespread species show strong phylogeographic structure. Comparison of mtDNA and nuclear gene trees shows no evidence of hybridization or introgression between species. Isolated populations of Bolitoglossa rufescens from Los Tuxtlas region constitute a separate lineage based on molecular data and morphology, and divergence between Los Tuxtlas and other areas appears to predate the arrival of B. rufescens in other areas west of the Isthmus of Tehuantepec. The Isthmus appears responsible for Pliocene vicariance within B. rufescens, as has been shown for other taxa. The Motagua-Polochic fault system does not appear to have caused population vicariance, unlike in other systems. CONCLUSIONS: Species of Nanotriton have responded to some major geological events in the same manner as other taxa, particularly in the case of the Isthmus of Tehuantepec. The deep divergence of the Los Tuxtlas populations of B. rufescens from other populations highlights the contribution of this volcanic system to patterns of regional endemism, and morphological differences observed in the Los Tuxtlas populations suggests that they may represent an undescribed species of Bolitoglossa. The absence of phylogeographic structure in B. nympha, in contrast to the other widespread species in the subgenus, may be due to historical forest contraction and more recent range expansion in the region. Phylogeographic data provide substantial insight into the evolutionary history of these morphologically similar species of salamanders, and contribute to our understanding of factors that have generated the high biodiversity of Mesoamerica.

Molecular systematics of Batrachoseps (Caudata, Plethodontidae) in southern California and Baja California: mitochondrial-nuclear DNA discordance and the evolutionary history of B. major.

Inferences about species boundaries and evolutionary history are often complicated by discordance between datasets. In recent times, considerable effort has been devoted to understanding the causes of discordance between the patterns of genetic variation and structure shown by different unlinked molecular markers. The genus Batrachoseps (Caudata, Plethodontidae), the most diverse group of salamanders in western North America, is characterized by limited morphological variation and discordance between molecular datasets, making it a challenging group for taxonomists but also a good model to test newly developed analytical methods to sort out possible sources of discordance. In this study, we present a comprehensive assessment of the evolutionary history of B. major, one of the most widespread species in the genus, based on extensive sampling and phylogenetic and coalescent analyses of data from mitochondrial and nuclear markers. We found non-monophyly of mtDNA in B. major, with two lineages (northern and southern) that are more closely related to other species in the genus than to each other, but this division was not apparent in nuclear DNA. Despite non-monophyly in gene trees, species tree analyses recovered a sister group relationship between the two lineages of B. major, and coalescent simulations suggested that there is no need to invoke gene flow to account for the discordance across gene trees. The possibility that these two lineages represent sister, cryptic taxa, is discussed in the context of Bayesian methods of species/lineage delineation. Contrary to prior expectations, B. major has experienced extensive diversification on the Baja California Peninsula, where four endemic lineages have persisted for at least 4 million years.

Interaction between breeding habitat and elevation affects prevalence but not infection intensity of Batrachochytrium dendrobatidis in Brazilian anuran assemblages.

Chytridiomycosis, an infectious disease of amphibians, is caused by the fungus Batrachochytrium dendrobatidis (Bd) and has been linked to declining amphibian populations worldwide. The susceptibility of amphibians to chytridiomycosis-induced population declines is potentially influenced by many factors, including environmental characteristics, differences among host species and the growth of the pathogen itself. We investigated the effects of elevation and breeding habitat on Bd prevalence and individual infection intensity (zoospore loads) in 3 anuran assemblages of the Atlantic Coastal Forest of Brazil. Bd infection intensity was strongly influenced by elevation and breeding habitat, but we found no evidence of an interaction between those 2 variables in explaining the number of zoospores sampled from individual frogs. In contrast, Bd infection odds were predicted by elevation and by an interaction between elevation and breeding habitat, such that frogs had a higher probability of Bd infection in lotic habitats at low elevations. Our results indicate that Bd persists across a wide variety of habitats and elevations in the Atlantic Coastal Forest. Prevalence and infection intensity of Bd are highest at high elevations where overall environmental conditions for Bd are most favorable. In addition, at low elevations amphibian host habitat choice is also an important determinant of infection. Our study highlights the need to investigate interacting variables of host ecology and the environment simultaneously.

Dramatic declines in neotropical salamander populations are an important part of the global amphibian crisis.

We document major declines of many species of salamanders at several sites in Central America and Mexico, with emphasis on the San Marcos region of Guatemala, one of the best studied and most diverse salamander communities in the Neotropics. Profound declines of several formerly abundant species, including 2 apparent extinctions, are revealed. Terrestrial microhabitat specialists at mid- to high elevations have declined more than microhabitat generalists. These terrestrial microhabitat specialists have largely disappeared from multiple sites in western Guatemala, including in well-protected areas, suggesting that the phenomenon cannot be explained solely by localized habitat destruction. Major declines in southern Mexican plethodontid salamanders occurred in the late 1970s to early 1980s, concurrent with or preceding many reported frog declines. The species in decline comprise several major evolutionary lineages of tropical salamanders, underscoring that significant portions of the phylogenetic diversity of Neotropical salamanders are at risk. Our results highlight the urgent need to document and understand Neotropical salamander declines as part of the larger effort to conserve global amphibian diversity.