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The overall structure of the immune system is highly conserved across jawed vertebrates, but characterization and description of the immune system is heavily biased toward mammals. One arm of the vertebrate immune system, the adaptive immune system, mounts pathogen-specific responses that tend to be robust and effective at clearing pathogens. This system requires selection against self-recognition and modulation of the immune response. One of the mechanisms of immune modulation is the presence of regulatory T cells that suppress other effector immune cells. Regulatory T cells and their primary activator forkhead box protein P3 (FOXP3) have been well characterized in mammalian models but unexplored in most other vertebrate taxa. Amphibians are a good focal group for the characterization of FOXP3 due to their phylogenetic position on the vertebrate tree of life, and their susceptibility to emerging pathogens. In this study, we mined available transcriptomic and genomic data to confirm the presence of FOXP3 across the amphibian tree of life. We find that FOXP3 is present in all major clades of amphibians. We also test whether selection on FOXP3 shows signatures of intensification among the three main clades of amphibians, which may reflect shifts in the stringency of natural selection on this gene. Our findings provide insights into the evolutionary history of the vertebrate immune system and confirm the conservation of vertebrate immune genes within amphibians., Competing Interests: Declarations. Competing interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

TAS2Rs are a family of G protein-coupled receptors that function as bitter taste receptors in vertebrates. Mammalian TAS2Rs have historically garnered the most attention, leading to our understanding of their roles in taste perception relevant to human physiology and behaviors. However, the evolution and functional implications of TAS2Rs in other vertebrate lineages remain less explored. Here, we identify 9,291 TAS2Rs from 661 vertebrate genomes. Large-scale phylogenomic analyses reveal that frogs and salamanders contain unusually high TAS2R gene content, in stark contrast to other vertebrate lineages. In most species, TAS2R genes are found in clusters; compared to other vertebrates, amphibians have additional clusters and more genes per cluster. We find that vertebrate TAS2Rs have few one-to-one orthologs between closely related species, although total TAS2R count is stable in most lineages. Interestingly, TAS2R count is proportional to the receptors expressed solely in extra-oral tissues. In vitro receptor activity assays uncover that many amphibian TAS2Rs function as tissue-specific chemosensors to detect ecologically important xenobiotics., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: J.-K.W. is a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, Galixir and Inari Agriculture, which develop biotechnologies related to natural products, drug discovery, and agriculture. The remaining authors declare no other competing interests., (Copyright: © 2025 Higgins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Amphibians are the most threatened group of vertebrates and are in dire need of conservation intervention to ensure their continued survival. They exhibit unique features including a high diversity of reproductive strategies, permeable and specialized skin capable of producing toxins and antimicrobial compounds, multiple genetic mechanisms of sex determination and in some lineages, the ability to regenerate limbs and organs. Although genomic approaches would shed light on these unique traits and aid conservation, sequencing and assembly of amphibian genomes has lagged behind other taxa due to their comparatively large genome sizes. Fortunately, the development of long-read sequencing technologies and initiatives has led to a recent burst of new amphibian genome assemblies. Although growing, the field of amphibian genomics suffers from the lack of annotation resources, tools for working with challenging genomes and lack of high-quality assemblies in multiple clades of amphibians. Here, we analyse 51 publicly available amphibian genomes to evaluate their usefulness for functional genomics research. We report considerable variation in genome assembly quality and completeness and report some of the highest transposable element and repeat contents of any vertebrate. Additionally, we detected an association between transposable element content and climatic variables. Our analysis provides evidence of conserved genome synteny despite the long divergence times of this group, but we also highlight inconsistencies in chromosome naming and orientation across genome assemblies. We discuss sequencing gaps in the phylogeny and suggest key targets for future sequencing endeavours. Finally, we propose increased investment in amphibian genomics research to promote their conservation., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Limbs are a defining characteristic of tetrapods, yet numerous taxa, primarily among amphibians and reptiles, have independently lost limbs as an adaptation to new ecological niches. To elucidate the genetic factors contributing to this convergent limb loss, we present a 12 Gb chromosome-level assembly of the Banna caecilian (Ichthyophis bannanicus), a limbless amphibian. Our comparative analysis, which includes the reconstruction of amphibian karyotype evolution, reveals constrained gene length evolution in a subset of developmental genes across 3 large genomes. Investigation of limb development genes uncovered the loss of Grem1 in caecilians and Tulp3 in snakes. Interestingly, caecilians and snakes share a significantly larger number of convergent degenerated conserved noncoding elements than limbless lizards, which have a shorter evolutionary history of limb loss. These convergent degenerated conserved noncoding elements overlap significantly with active genomic regions during mouse limb development and are conserved in limbed species, suggesting their essential role in limb patterning in the tetrapod common ancestor. While most convergent degenerated conserved noncoding elements emerged in the jawed vertebrate ancestor, coinciding with the origin of paired appendage, more recent degenerated conserved noncoding elements also contribute to limb development, as demonstrated through functional experiments. Our study provides novel insights into the regulatory elements associated with limb development and loss, offering an evolutionary perspective on the genetic basis of morphological specialization., Competing Interests: Conflict of Interest Authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Green algae usually assigned to the genus Oophila are known to colonize egg capsules of amphibian egg masses across the Nearctic and Palearctic regions. We study the phylogenetic relationships of these algae using a phylotranscriptomic data set of 76 protein-coding single-copy nuclear genes. Our data set includes novel RNAseq data for six amphibian-associated and five free-living green algae, and draft genomes of two of the latter. Within the Oophila clade (nested within Moewusinia), we find samples from two European frogs (Rana dalmatina and R. temporaria) closely related to those of the North American frog R. aurora (Oophila subclade III). An isolate from the North American R. sylvatica (subclade IV) appears to be sister to the Japanese isolate from the salamander Hynobius nigrescens (subclade J1), and subclade I algae from Ambystoma maculatum are sister to all other lineages in the Oophila clade. Two free-living algae (Chlamydomonas nasuta and Cd. pseudogloeogama) are nested within the Oophila clade, and a strain of the type species of Chlorococcum (Cc. infusionum) is related to this assemblage. Our phylotranscriptomic tree suggests that recognition of different species within the Oophila clade ("clade B" of earlier studies) is warranted, and calls for a comprehensive taxonomic revision of Moewusinia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large-scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species-specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species-specific CRISPR-Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term 'ampliscanning', for 18 of the 22 amphibian species in Switzerland. We sub-selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR-Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR-Dx combined with universal primers for large-scale monitoring of multiple endangered species across landscapes to inform conservation measures., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium ) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to "leap" to the next level., (© 2024. The Author(s).)

Among tetrapod (terrestrial) vertebrates, amphibians remain more closely tied to an amphibious lifestyle than amniotes, and their visual opsin genes may be adapted to this lifestyle. Previous studies have discussed physiological, morphological, and molecular changes in the evolution of amphibian vision. We predicted the locations of the visual opsin genes, their neighboring genes, and the tuning sites of the visual opsins, in 39 amphibian genomes. We found that all of the examined genomes lacked the Rh2 gene. The caecilian genomes have further lost the SWS1 and SWS2 genes; only the Rh1 and LWS genes were retained. The loss of the SWS1 and SWS2 genes in caecilians may be correlated with their cryptic lifestyles. The opsin gene syntenies were predicted to be highly similar to those of other bony vertebrates. Moreover, dual syntenies were identified in allotetraploid Xenopus laevis and X. borealis. Tuning site analysis showed that only some Caudata species might have UV vision. In addition, the S164A that occurred several times in LWS evolution might either functionally compensate for the Rh2 gene loss or fine-tuning visual adaptation. Our study provides the first genomic evidence for a caecilian LWS gene and a genomic viewpoint of visual opsin genes by reviewing the gains and losses of visual opsin genes, the rearrangement of syntenies, and the alteration of spectral tuning in the course of amphibians' evolution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Unusually for oceanic islands, the granitic Seychelles host multiple lineages of endemic amphibians. This includes an ancient (likely ca. 60 million years) radiation of eight caecilian species, most of which occur on multiple islands.These caecilians have a complicated taxonomic history and their phylogenetic inter-species relationships have been difficult to resolve. Double-digest RAD sequencing (ddRADseq) has been applied extensively to phylogeography and increasingly to phylogenetics but its utility for resolving ancient divergences is less well established. To address this, we applied ddRADseq to generate a genome-wide SNP panel for phylogenomic analyses of the Seychelles caecilians, whose phylogeny has so far not been satisfactorily resolved with traditional DNA markers. Based on 129,154 SNPs, we resolved deep and shallow splits, with strong support. Our findings demonstrate the capability of genome-wide SNPs for evolutionary inference at multiple taxonomic levels and support the recently proposed synonymy of Grandisonia Taylor, 1968 with Hypogeophis Peters, 1879. We revealed three clades of Hypogeophis (large-, medium- and short-bodied) and identify a single origin of the diminutive, stocky-bodied and pointy-snouted phenotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural selection can drive organisms to strikingly similar adaptive solutions, but the underlying molecular mechanisms often remain unknown. Several amphibians have independently evolved highly adhesive skin secretions (glues) that support a highly effective antipredator defence mechanism. Here we demonstrate that the glue of the Madagascan tomato frog, Dyscophus guineti, relies on two interacting proteins: a highly derived member of a widespread glycoprotein family and a galectin. Identification of homologous proteins in other amphibians reveals that these proteins attained a function in skin long before glues evolved. Yet, major elevations in their expression, besides structural changes in the glycoprotein (increasing its structural disorder and glycosylation), caused the independent rise of glues in at least two frog lineages. Besides providing a model for the chemical functioning of animal adhesive secretions, our findings highlight how recruiting ancient molecular templates may facilitate the recurrent evolution of functional innovations., (© 2024. The Author(s).)

Most described species have not been explicitly included in phylogenetic trees-a problem named the Darwinian shortfall-owing to a lack of molecular and/or morphological data, thus hampering the explicit incorporation of evolution into large-scale biodiversity analyses. We investigate potential drivers of the Darwinian shortfall in tetrapods, a group in which at least one-third of described species still lack phylogenetic data, thus necessitating the imputation of their evolutionary relationships in fully sampled phylogenies. We show that the number of preserved specimens in scientific collections is the main driver of phylogenetic knowledge accumulation, highlighting the major role of biological collections in unveiling novel biodiversity data and the importance of continued sampling efforts to reduce knowledge gaps. Additionally, large-bodied and wide-ranged species, as well as terrestrial and aquatic amphibians and reptiles, are phylogenetically better known. Future efforts should prioritize phylogenetic research on organisms that are narrow-ranged, small-bodied and underrepresented in scientific collections, such as fossorial species. Addressing the Darwinian shortfall will be imperative for advancing our understanding of evolutionary drivers shaping biodiversity patterns and implementing comprehensive conservation strategies.

Most vertebrates develop distinct females and males, where sex is determined by repeatedly evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master sex-determining gene, the dmrt1-paralogue (dm-w) of female-heterogametic clawed frogs (Xenopus; ZW♀/ZZ♂), is known across >8740 species of amphibians. In this study, by combining chromosome-scale female and male genomes of a non-model amphibian, the European green toad, Bufo(tes) viridis, with ddRAD- and whole genome pool-sequencing, we reveal a candidate master locus, governing a male-heterogametic system (XX♀/XY♂). Targeted sequencing across multiple taxa uncovered structural X/Y-variation in the 5'-regulatory region of the gene bod1l, where a Y-specific non-coding RNA (ncRNA-Y), only expressed in males, suggests that this locus initiates sex-specific differentiation. Developmental transcriptomes and RNA in-situ hybridization show timely and spatially relevant sex-specific ncRNA-Y and bod1l-gene expression in primordial gonads. This coincided with differential H3K4me-methylation in pre-granulosa/pre-Sertoli cells, pointing to a specific mechanism of amphibian sex determination., (© 2024. The Author(s).)

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1-a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray-finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe-finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Effective population size (N e ) is a particularly useful metric for conservation as it affects genetic drift, inbreeding and adaptive potential within populations. Current guidelines recommend a minimum N e of 50 and 500 to avoid short-term inbreeding and to preserve long-term adaptive potential respectively. However, the extent to which wild populations reach these thresholds globally has not been investigated, nor has the relationship between N e and human activities. Through a quantitative review, we generated a dataset with 4610 georeferenced N e estimates from 3829 populations, extracted from 723 articles. These data show that certain taxonomic groups are less likely to meet 50/500 thresholds and are disproportionately impacted by human activities; plant, mammal and amphibian populations had a <54% probability of reaching N ̂ e = 50 and a <9% probability of reaching N ̂ e = 500. Populations listed as being of conservation concern according to the IUCN Red List had a smaller median N ̂ e than unlisted populations, and this was consistent across all taxonomic groups. N ̂ e was reduced in areas with a greater Global Human Footprint, especially for amphibians, birds and mammals, however relationships varied between taxa. We also highlight several considerations for future works, including the role that gene flow and subpopulation structure plays in the estimation of N ̂ e in wild populations, and the need for finer-scale taxonomic analyses. Our findings provide guidance for more specific thresholds based on N e and help prioritise assessment of populations from taxa most at risk of failing to meet conservation thresholds., (© 2024 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Applications of environmental DNA (eDNA) analysis methods for biomonitoring have grown exponentially over the last decade and provide a wealth of new information on the distribution of species. However, eDNA methods have limited application for estimating population-level metrics. Environmental RNA (eRNA) has the potential to address ecological questions by gathering population demographic information from environmental media but may be challenging to detect and analyze. We developed gene-specific eRNA assays targeting keratin-associated genes in two focal species, American bullfrogs (Lithobates catesbeianus) and tiger salamanders (Ambystoma mavortium) to answer an important question in amphibian management: whether species detections represent breeding populations versus transitory adults. We performed an extensive laboratory validation with amphibians housed across development stages, where we collected 95 and 127 environmental samples for bullfrogs and salamanders, respectively. Both assays were highly specific to the larval stage and amplified with high sensitivity (90% in bullfrog and 88.4% in tiger salamander samples). We then applied our validated assays to multiple natural systems. When larvae were present, we found 74.1% overall detection in bullfrog field samples and 70.8% and 48.5% overall detection in field samples from ponds with A. macrodactylum and A. californiense larvae, correlating with eDNA detection rates. When only adults were present, we did not detect larvae-specific eRNA in A. macrodactylum ponds, despite high eDNA detection rates. Although much work is ahead for optimizing assay design, sampling and filtering methods, we demonstrate that eRNA can successfully be used to discern life stages with direct application for ecology and conservation management., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species-think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the in vitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results., (© 2024 John Wiley & Sons Ltd.)

Sex determination systems have greatly diversified between amphibians and reptiles, with such as the different sex chromosome compositions within a single species and transition between temperature-dependent sex determination (TSD) and genetic sex determination (GSD). In most sex chromosome studies on amphibians and reptiles, the whole-genome sequence of Xenopous tropicalis and chicken have been used as references to compare the chromosome homology of sex chromosomes among each of these taxonomic groups, respectively. In the present study, we reviewed existing reports on sex chromosomes, including karyotypes, in amphibians and reptiles. Furthermore, we compared the identified genetic linkages of sex chromosomes in amphibians and reptiles with the chicken genome as a reference, which is believed to resemble the ancestral tetrapod karyotype. Our findings revealed that sex chromosomes in amphibians are derived from genetic linkages homologous to various chicken chromosomes, even among several frogs within single families, such as Ranidae and Pipidae. In contrast, sex chromosomes in reptiles exhibit conserved genetic linkages with chicken chromosomes, not only across most species within a single family, but also within closely related families. The diversity of sex chromosomes in amphibians and reptiles may be attributed to the flexibility of their sex determination systems, including the ease of sex reversal in these animals., (© 2023 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In recent years, environmental DNA (eDNA) has received attention from biologists due to its sensitivity, convenience, labor and material efficiency, and lack of damage to organisms. The extensive application of eDNA has opened avenues for the monitoring and biodiversity assessment of amphibians, which are frequently small and difficult to observe in the field, in areas such as biodiversity survey assessment and detection of specific, rare and threatened, or alien invasive species. However, the accuracy of eDNA can be influenced by factors such as ambient temperature, pH, and false positives or false negatives, which makes eDNA an adjunctive tool rather than a replacement for traditional surveys. This review provides a concise overview of the eDNA method and its workflow, summarizes the differences between applying eDNA for detecting amphibians and other organisms, reviews the research progress in eDNA technology for amphibian monitoring, identifies factors influencing detection efficiency, and discusses the challenges and prospects of eDNA. It aims to serve as a reference for future research on the application of eDNA in amphibian detection., (© 2023 Wiley Periodicals LLC.)

Background: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management., Results: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA., Conclusions: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg., (© 2023. The Author(s).)

Microbiomes play an important role in determining the ecology and behaviour of their hosts. However, questions remain pertaining to how host genetics shape microbiomes, and how microbiome composition influences host fitness. We explored the effects of geography, evolutionary history and host genetics on the skin microbiome diversity and structure in a widespread amphibian. More specifically, we examined the association between bacterial diversity and composition and the major histocompatibility complex class II exon 2 diversity in 12 moor frog (Rana arvalis) populations belonging to two geographical clusters that show signatures of past and ongoing differential selection. We found that while bacterial alpha diversity did not differ between the two clusters, MHC alleles/supertypes and genetic diversity varied considerably depending on geography and evolutionary history. Bacterial alpha diversity was positively correlated with expected MHC heterozygosity and negatively with MHC nucleotide diversity. Furthermore, bacterial community composition showed significant variation between the two geographical clusters and between specific MHC alleles/supertypes. Our findings emphasize the importance of historical demographic events on hologenomic variation and provide new insights into how immunogenetic host variability and microbial diversity may jointly influence host fitness with consequences for disease susceptibility and population persistence., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Since Ficetola et al. (2008) alerted ecologists and conservation biologists to the existence of environmental DNA (eDNA), the number of studies using eDNA has exploded, with a rapidly increasing diversity of research, monitoring, and management objectives. Initial applications focused on amphibians and fishes while today's taxonomic targets span the phylogenetic tree. The environmental media that are sampled have expanded from freshwater to saltwater to soils, and, most recently, to air. In this issue of Molecular Ecology Resources, Lynggaard et al. (Molecular Ecology Resources, 2023) use eDNA captured on air filters to census vertebrate biodiversity in a forest. With a three day, six sample period, 143 sample effort in a nature park in a rural area of Zealand, Denmark, their wild species detections comprised about 25% of the terrestrial vertebrates that are known to occur in the area, including about 33% of the mammal, 17% of the bird, and 60% of the amphibian species. This study demonstrates that air sampling for eDNA has the potential to become a powerful standard method for terrestrial biodiversity assessment that is complementary to traditional methods (e.g., trapping, visual and acoustic observation, collection of scat and hair)., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Invasive wild pigs (Sus scrofa) are one of the most widespread, destructive vertebrate species globally. Their success can largely be attributed to their generalist diets, which are dominated by plant material but also include diverse animal taxa. Wild pigs are demonstrated nest predators of ground-nesting birds and reptiles, and likely pose a threat to amphibians given their extensive overlap in wetland use. DNA metabarcoding of fecal samples from 222 adult wild pigs culled monthly from 2017 to 2018 revealed a diverse diet dominated by plant material, with 166 plant genera from 56 families and 18 vertebrate species identified. Diet composition varied seasonally with availability for plants and was consistent between sexes. Amphibians were the most frequent vertebrate group consumed and represented the majority of vertebrate species detected, suggesting amphibians are potentially vulnerable to predation by wild pigs in our study region. Mammal, reptile, and bird species were also detected in pig diets, but infrequently. Our results highlight the need for research on the impacts of wild pigs on amphibians to better inform management and conservation of imperiled species., (© 2023. The Author(s).)

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

We present genome sequences for the caecilians Geotrypetes seraphini (3.8 Gb) and Microcaecilia unicolor (4.7 Gb), representatives of a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. More than 69% of both genomes are composed of repeats, with retrotransposons being the most abundant. We identify 1,150 orthogroups that are unique to caecilians and enriched for functions in olfaction and detection of chemical signals. There are 379 orthogroups with signatures of positive selection on caecilian lineages with roles in organ development and morphogenesis, sensory perception, and immunity amongst others. We discover that caecilian genomes are missing the zone of polarizing activity regulatorysequence (ZRS) enhancer of Sonic Hedgehog which is also mutated in snakes. In vivo deletions have shown ZRS is required for limb development in mice, thus, revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

How does amphibian MHC diversity fit in the landscape of vertebrate evolution? Mimnias et al. (2022) address this gap in the field of MHC evolution by focusing on the lesser described MHC class I in salamanders. These findings contribute to understanding MHC diversity and the susceptibility of amphibians to pathogens, which could lead to future research on a major threat to amphibian biodiversity, chytrid fungi., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Studies on the globin family are continuously revealing insights into the mechanisms of gene and protein evolution. The rise of a new globin gene type in Pelobatoidea and Neobatrachia (Amphibia:Anura) from an α-globin precursor provides the opportunity to investigate the genetic and physical mechanisms underlying the origin of new protein structural and functional properties. This amphibian-specific globin (globin A/GbA) discovered in the heart of Rana catesbeiana is a monomer. As the ancestral oligomeric state of α-globins is a homodimer, we inferred that the ancestral state was lost somewhere in the GbA lineage. Here, we combined computational molecular evolution with structural bioinformatics to determine the extent to which the loss of the homodimeric state is pervasive in the GbA clade. We also characterized the loci of GbA genes in Bufo bufo. We found two GbA clades in Neobatrachia. One was deleted in Ranidae, but retained and expanded to yield a new globin cluster in Bufonidae species. Loss of the ancestral oligomeric state seems to be pervasive in the GbA clade. However, a taxonomic sampling that includes more Pelobatoidea, as well as early Neobatrachia, lineages would be necessary to determine the oligomeric state of the last common ancestor of all GbA. The evidence presented here points out a possible loss of oligomerization in Pelobatoidea GbA as a result of amino acid substitutions that weaken the homodimeric state. In contrast, the loss of oligomerization in both Neobatrachia GbA clades was linked to independent deletions that disrupted many packing contacts at the homodimer interface., Competing Interests: Declaration of competing interest The authors declare that there is not any conflict of interest in this manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl - ) cotransporters. Na + /K + /2Cl - (Nkcc) and Na + /Cl - cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na + /Cl - cotransporter ( ncc gene: slc12a3 ), whereas teleost fishes possess multiple ncc genes, slc12a3 ( ncc1 ) and slc12a10 ( ncc2 ), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 ( ncc1 ) and ncc3 . However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 ( ncc2 ) in mammals has not been clarified. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10 , and slc12a10 is present in most ray-finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray-finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as compared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing variants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional constraints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates. NEW & NOTEWORTHY slc12a10 is not a fish-specific gene and is present in a few mammals (e.g., platypus and horse), non-avian reptiles, amphibians, but was pseudogenized or deleted in most mammals (e.g., human, mouse, cat, cow, and rhinoceros), birds, and some ray-finned fishes (pufferfishes).

Background: Reptiles and amphibians provide untapped potential for discovering how a diversity of genetic pathways and environmental conditions are incorporated into developmental processes that can lead to similar functional outcomes. These groups display a multitude of reproductive strategies, and whereas many attributes are conserved within groups and even across vertebrates, several aspects of sexual development show considerable variation., Summary: In this review, we focus our attention on the development of the reptilian and amphibian ovary. First, we review and describe the events leading to ovarian development, including sex determination and ovarian maturation, through a comparative lens. We then describe how these events are influenced by environmental factors, focusing on temperature and exposure to anthropogenic chemicals. Lastly, we identify critical knowledge gaps and future research directions that will be crucial to moving forward in our understanding of ovarian development and the influences of the environment in reptiles and amphibians., Key Messages: Reptiles and amphibians provide excellent models for understanding the diversity of sex determination strategies and reproductive development. However, a greater understanding of the basic biology of these systems is necessary for deciphering the adaptive and potentially disruptive implications of embryo-by-environment interactions in a rapidly changing world., (© 2022 S. Karger AG, Basel.)

In the amphibian intestine during metamorphosis, most of the larval epithelial cells undergo apoptosis, while a small number of the epithelial cells dedifferentiate into stem cells (SCs). The SCs actively proliferate and then newly generate the adult epithelium analogous to the mammalian counterpart, which is continuously renewed from the SCs throughout adulthood. This larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) through interacting with the surrounding connective tissue that develops as the stem cell niche. Thus, the amphibian intestine provides us a valuable opportunity to study how the SCs and their niche are formed during development. To clarify the TH-induced and evolutionally conserved mechanism of SC development at the molecular level, numerous TH response genes have been identified in the Xenopus laevis intestine over the last three decades and extensively analyzed for their expression and function by using wild-type and transgenic Xenopus tadpoles. Interestingly, accumulating evidence indicates that thyroid hormone receptor (TR) epigenetically regulates the expression of TH response genes involved in the remodeling. In this review, we highlight recent progress in the understanding of SC development, focusing on epigenetic gene regulation by TH/TR signaling in the X. laevis intestine. We here propose that two subtypes of TRs, TRα and TRβ, play distinct roles in the intestinal SC development via different histone modifications in different cell types., (Copyright © 2023 Elsevier Inc. All rights reserved.)

While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens., (© 2022 John Wiley & Sons Ltd.)

Temporal changes and transmission patterns in host-associated microbial communities have important implications for host health. The diversity of amphibian skin microbial communities is associated with disease outcome in amphibians exposed to the fungal pathogen Batrachochytrium dendrobatidis (Bd). To successfully develop conservation strategies against Bd, we need a comprehensive understanding of how skin microbes are maintained and transmitted over time within populations. We used 16S rRNA sequence analysis to compare Epipedobates anthonyi frogs housed with one conspecific to frogs housed singly at four time points over the course of 1 year. We found that both α and β diversity of frog skin bacterial communities changed significantly over the course of the experiment. Specifically, we found that bacterial communities of cohabitating frogs became more similar over time. We also observed that some bacterial taxa were differentially abundant between frogs housed singly and frogs housed with a conspecific. These results suggest that conspecific contact may play a role in mediating amphibian skin microbial diversity and that turnover of skin microbial communities can occur across time. Our findings provide rationale for future studies exploring horizontal transmission as a potential mechanism of host-associated microbial maintenance in amphibians.

The epidermal differentiation complex (EDC) is a cluster of genes encoding components of the skin barrier in terrestrial vertebrates. EDC genes can be categorized as S100 fused-type protein (SFTP) genes such as filaggrin, which contain two coding exons, and single-coding-exon EDC (SEDC) genes such as loricrin. SFTPs are known to be present in amniotes (mammals, reptiles and birds) and amphibians, whereas SEDCs have not yet been reported in amphibians. Here, we show that caecilians (Amphibia: Gymnophiona) have both SFTP and SEDC genes. Two to four SEDC genes were identified in the genomes of Rhinatrema bivittatum, Microcaecilia unicolor and Geotrypetes seraphini. Comparative analysis of tissue transcriptomes indicated predominant expression of SEDC genes in the skin of caecilians. The proteins encoded by caecilian SEDC genes resemble human SEDC proteins, such as involucrin and small proline-rich proteins, with regard to low sequence complexity and high contents of proline, glutamine and lysine. Our data reveal diversification of EDC genes in amphibians and suggest that SEDC-type skin barrier genes have originated either in a common ancestor of tetrapods followed by loss in Batrachia (frogs and salamanders) or, by convergent evolution, in caecilians and amniotes., (© 2022. The Author(s).)

The evolutionary position and lifestyle of amphibians highlights the important roles of the immune system in adaptive radiation and their adaptation to a complex pathogenic environment. Toll-like receptors (TLRs) are membrane-like sensors that recognize and bind conserved molecular motifs in pathogens to initiate downstream immune responses. To understand the evolutionary patterns of TLRs in amphibians, we analyzed TLR genes from the genomes and transcriptomes of 102 amphibian species. Phylogenetic results showed that 578 intact amphibian TLR sequences belonged to 16 TLR genes and were divided into seven subfamilies. The TLR4 subfamily was only identified in the Anura. Purification selection plays a leading role in amphibian TLR evolution and mean ω (dN/dS) values ranged from 0.252 for TLR7 to 0.381 for TLR19. Furthermore, the ω values of different domains were significantly different. We found positive selection patterns for 141 of 12,690 codons (1.1%) in all amphibian TLRs, most of which were located in leucine-rich repeats (LRRs). We also observed low to moderate levels of single-nucleotide polymorphisms (SNPs) in Pelophylax nigromaculatus and Bombina orientalis. This study provided critical primers, meaningful information regarding TLR gene family evolution in amphibians, and insights into the complex evolutionary patterns and implications of TLR polymorphisms., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The genus Pipa is a species-poor clade of Neotropical frogs and one of the most bizarre-looking due to many highly derived anatomical traits related to their fully aquatic lifestyle. With their African relatives, they form the Pipidae family, which has attracted much attention, especially regarding its anatomy, reproductive biology, paleontology and biogeography. However, the actual diversity and phylogenetic relationships within Pipa remain poorly understood, and thus so do their historical biogeography and the evolution of striking features, such as the absence of teeth and endotrophy in some species. Using short mtDNA sequences across the distribution of the genus, we identified 15 main lineages (Operational Taxonomic Units - OTUs). This more than doubles the number of the currently seven valid nominal species. Several closely related OTUs do not share nuDNA alleles, confirming species divergence. Time-calibrated phylogenies obtained from mitogenomes and from 10 nuclear loci provide highly similar topologies but strikingly distinct node ages for Pipa. High dN/dS ratios and the variation of substitution rates across the trees suggest a strong effect of saturation on fast evolving positions of mtDNA, producing a substantially shorter stem branch of Pipa. Focusing on the nuDNA topology, we inferred an early Neogene Amazonian origin of the diversification of Pipa, with an initial split between the Guiana-Brazilian Shields and Western Amazonia, a pattern observed in many other co-distributed groups. All the western species are edentate, suggesting a single loss in the genus. Each of these groups diversified further out of Amazonia, toward the Atlantic Forest and toward trans-Andean forests, respectively. These events are concomitant with paleogeographic changes and match patterns observed in other co-distributed taxonomic groups. The two Amazonian lineages have probably independently acquired endotrophic larval development., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen., (© 2021 European Peptide Society and John Wiley & Sons, Ltd.)

Background: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths., Results: Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content., Conclusions: Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost., (© 2022. The Author(s).)

Host-associated microbial biofilms can provide protection against pathogen establishment. In many host-microbe symbioses (including, but not limited to humans, plants, insects, and amphibians), there is a correlation between host-associated microbial diversity and pathogen infection risk. Diversity may prevent infection by pathogens through sampling effects and niche complementarity, but an alternative hypothesis may be that microbial biomass is confounded with diversity and that host-associated biofilms are deterring pathogen establishment through space preemption. In this study, we use the amphibian system as a model for host-microbe-pathogen interactions to ask two questions: (i) is bacterial richness confounded with biofilm thickness or cell density, and (ii) to what extent do biofilm thickness, cell density, and bacterial richness each deter the establishment of the amphibian fungal pathogen Batrachochytrium dendrobatidis? To answer these questions, we built a custom biofilm microcosm that mimics the host-environment interface by allowing nutrients to diffuse out of a fine-pore biofilm scaffolding. This created a competitive environment in which bacteria and the fungal pathogen compete for colonization space. We then challenged bacterial biofilms ranging in community richness, biofilm thickness, bacterial cell density, and B. dendrobatidis (also known as Bd )-inhibitory metabolite production with live B. dendrobatidis zoospores to determine how B. dendrobatidis establishment success on membranes varies. We found that biofilm thickness and B. dendrobatidis-inhibitory isolate richness work in complement to reduce B. dendrobatidis establishment success. This work underscores that physical aspects of biofilm communities can play a large role in pathogen inhibition, and in many studies, these traits are not studied. IMPORTANCE Our finding highlights the fact that diversity, as measured through 16S rRNA gene sequencing, may obscure the true mechanisms behind microbe-mediated pathogen defense and that physical space occupation by biofilm-forming symbionts may significantly contribute to pathogen protection. These findings have implications across a wide range of host-microbe systems since 16S rRNA gene sequencing is a standard tool used across many microbial systems. Further, our results are potentially relevant to many host-pathogen systems since host-associated bacterial biofilms are ubiquitous.

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m -1 ). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m -1 ) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture-recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)

The growing availability of genetic data sets, in combination with machine learning frameworks, offers great potential to answer long-standing questions in ecology and evolution. One such question has intrigued population geneticists, biogeographers, and conservation biologists: What factors determine intraspecific genetic diversity? This question is challenging to answer because many factors may influence genetic variation, including life history traits, historical influences, and geography, and the relative importance of these factors varies across taxonomic and geographic scales. Furthermore, interpreting the influence of numerous, potentially correlated variables is difficult with traditional statistical approaches. To address these challenges, we analysed repurposed data using machine learning and investigated predictors of genetic diversity, focusing on Nearctic amphibians as a case study. We aggregated species traits, range characteristics, and >42,000 genetic sequences for 299 species using open-access scripts and various databases. After identifying important predictors of nucleotide diversity with random forest regression, we conducted follow-up analyses to examine the roles of phylogenetic history, geography, and demographic processes on intraspecific diversity. Although life history traits were not important predictors for this data set, we found significant phylogenetic signal in genetic diversity within amphibians. We also found that salamander species at northern latitudes contained low genetic diversity. Data repurposing and machine learning provide valuable tools for detecting patterns with relevance for conservation, but concerted efforts are needed to compile meaningful data sets with greater utility for understanding global biodiversity., (© 2020 John Wiley & Sons Ltd.)

Human land transformation is one of the leading causes of vertebrate population declines. These declines are thought to be partly due to decreased connectivity and habitat loss reducing animal population sizes in disturbed habitats. With time, this can lead to declines in effective population size and genetic diversity which restrict the ability of wildlife to efficiently cope with environmental change through genetic adaptation. However, it is not well understood whether these effects generally hold across taxa. We address this question by repurposing and synthesizing raw microsatellite data from online repositories for 19 amphibian species sampled at 554 georeferenced sites in North America. For each site, we estimated gene diversity, allelic richness, effective population size, and population differentiation. Using binary urban-rural census designations, and continuous measures of human population density, the Human Footprint Index, and impervious surface cover, we tested for generalizable effects of human land use on amphibian genetic diversity. We found minimal evidence, either positive or negative, for relationships between genetic metrics and urbanization. Together with previous work on focal species that also found varying effects of urbanization on genetic composition, it seems likely that the consequences of urbanization are not easily generalizable within or across amphibian species. Questions about the genetic consequences of urbanization for amphibians should be addressed on a case-by-case basis. This contrasts with general negative effects of urbanization in mammals and consistent, but species-specific, positive and negative effects in birds., (© 2021 John Wiley & Sons Ltd.)

Background: Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified., Results: Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana's inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog's prestin was functionally different from Rana., Conclusions: We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin., (© 2021. The Author(s).)

To effectively monitor, manage and protect aquatic species and understand their interactions, knowledge of their spatiotemporal distribution is needed. In this study, we used a fine-scale spatiotemporal water sampling design, followed by environmental DNA (eDNA) 12S metabarcoding, to investigate occupancy patterns of a natural community of fish and amphibian species in a lentic system. In the same system, we experimentally estimated the spatial and temporal dispersion of eDNA by placing a community of different fish and amphibian species in cages at one side of the pond, creating a controlled point of eDNA emission. Analyses of this cage community revealed a sharp spatial decline in detection rates and relative eDNA quantities at a distance of 5-10 m from the source, depending on the species and its abundance. In addition, none of the caged species could be detected 1 week after removal from the system. This indicates high eDNA decay rates and limited spatial eDNA dispersal, facilitating high local resolution for monitoring spatial occupancy patterns of aquatic species. Remarkably, for seven of the nine cage species, the presence of a single individual could be detected by pooling water of subsamples taken across the whole water body, illustrating the high sensitivity of the eDNA sampling and detection method applied. Finally, our work demonstrated that a fine-scale sampling design in combination with eDNA metabarcoding can cover total biodiversity very precisely and allows the construction of consistent spatiotemporal patterns of relative abundance and local distribution of free-living fish and amphibian species in a lentic ecosystem., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

A universal phenomenon of using synonymous codons unequally in coding sequences known as codon usage bias (CUB) is observed in all forms of life. Mutation and natural selection drive CUB in many species but the relative role of evolutionary forces varies across species, genes and genomes. We studied the CUB in mitochondrial (mt) CO genes from three orders of Amphibia using bioinformatics approach as no work was reported yet. We observed that CUB of mt CO genes of Amphibians was weak across different orders. Order Caudata had higher CUB followed by Gymnophiona and Anura for all genes and CUB also varied across genes. Nucleotide composition analysis showed that CO genes were AT-rich. The AT content in Caudata was higher than that in Gymnophiona while Anura showed the least content. Multiple investigations namely nucleotide composition, correspondence analysis, parity plot analysis showed that the interplay of mutation pressure and natural selection caused CUB in these genes. Neutrality plot suggested the involvement of natural selection was more than the mutation pressure. The contribution of natural selection was higher in Anura than Gymnophiona and the lowest in Caudata. The codons CGA, TGA, AAA were found to be highly favoured by nature across all genes and orders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sex is determined genetically in amphibians; however, little is known about the sex chromosomes, testis-determining genes, and the genes involved in testis differentiation in this class. Certain inherent characteristics of the species of this group, like the homomorphic sex chromosomes, the high diversity of the sex-determining mechanisms, or the existence of polyploids, may hinder the design of experiments when studying how the gonads can differentiate. Even so, other features, like their external development or the possibility of inducing sex reversal by external treatments, can be helpful. This review summarizes the current knowledge on amphibian sex determination, gonadal development, and testis differentiation. The analysis of this information, compared with the information available for other vertebrate groups, allows us to identify the evolutionarily conserved and divergent pathways involved in testis differentiation. Overall, the data confirm the previous observations in other vertebrates-the morphology of the adult testis is similar across different groups; however, the male-determining signal and the genetic networks involved in testis differentiation are not evolutionarily conserved.

In this study, we used a bioinformatics approach to analyze the nucleotide composition and pattern of synonymous codon usage in mitochondrial ND genes in three amphibian groups, that is, orders Anura, Caudata, and Gymnophiona to identify the commonality and the differences of codon usage as no research work was reported yet. The high value of the effective number of codons revealed that the codon usage bias (CUB) was low in mitochondrial ND genes among the orders. Nucleotide composition analysis suggested that for each gene, the compositional features differed among Anura, Caudata, and Gymnophiona and the GC content was lower than AT content. Furthermore, a highly significant difference (p < .05) for GC content was found in each gene among the orders. The heat map showed contrasting patterns of codon usage among different ND genes. The regression of GC12 on GC3 suggested a narrow range of GC3 distribution and some points were located in the diagonal, indicating both mutation pressure and natural selection might influence the CUB. Moreover, the slope of the regression line was less than 0.5 in all ND genes among orders, indicating natural selection might have played the dominant role whereas mutation pressure had played a minor role in shaping CUB of ND genes across orders., (© 2020 Wiley Periodicals LLC.)

Until recently, different families of urodele amphibians were thought to express distinct subsets of immunoglobulin (Ig) isotypes. In this study, we explored cDNAs encoding Ig heavy-chains (H-chains) in three species of urodele amphibians. We found that Cynops pyrrhogaster, Pleurodeles waltl, and Ambystoma mexicanum each carry genes encoding four Ig H-chain isotypes, including IgM, IgY, IgD, and IgX, similar to those found in anuran amphibians. We also found that urodele IgDs have a long constant region similar to those found in anuran, reptiles, and bony fishes. We also found several putative IgD splice variants. Our findings indicated that P. waltl IgP is not a novel isotype but an IgD splice variant. Altogether, our findings indicate that IgD splice variants may be universally expressed among amphibian species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)