Diversity and Molecular Evolution of Antimicrobial Peptides in Caecilian Amphibians.

Antimicrobial peptides (AMPs) are key molecules in the innate immune defence of vertebrates with rapid action, broad antimicrobial spectrum, and ability to evade pathogen resistance mechanisms. To date, amphibians are the major group of vertebrates from which most AMPs have been characterised, but most studies have focused on the bioactive skin secretions of anurans (frogs and toads). In this study, we have analysed the complete genomes and/or transcriptomes of eight species of caecilian amphibians (order Gymnophiona) and characterised the diversity, molecular evolution, and antimicrobial potential of the AMP repertoire of this order of amphibians. We have identified 477 candidate AMPs within the studied caecilian genome and transcriptome datasets. These candidates are grouped into 29 AMP families, with four corresponding to peptides primarily exhibiting antimicrobial activity and 25 potentially serving as AMPs in a secondary function, either in their entirety or after cleavage. In silico prediction methods were used to identify 62 of those AMPs as peptides with promising antimicrobial activity potential. Signatures of directional selection were detected for five candidate AMPs, which may indicate adaptation to the different selective pressures imposed by evolutionary arms races with specific pathogens. These findings provide encouraging support for the expectation that caecilians, being one of the least-studied groups of vertebrates, and with ~300 million years of separate evolution, are an underexplored resource of great pharmaceutical potential that could help to contest antibiotic resistance and contribute to biomedical advance.

Caecilian Genomes Reveal the Molecular Basis of Adaptation and Convergent Evolution of Limblessness in Snakes and Caecilians.

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.

Chytrid fungus in amphibians from the lowland Brazilian Amazon.

Infectious diseases are one of the main threats to biodiversity. The fungus Batrachochytrium dendrobatidis (Bd) is associated with several amphibian losses around the globe, and environmental conditions may dictate the success of pathogen spread. The Brazilian Amazon has been considered climatically unsuitable for chytrid fungus, but additional information on Bd dynamics in this ecoregion is still lacking. We sampled 462 amphibians (449 anurans, 4 caudatans and 9 caecilians), representing 57 species from the Brazilian Amazon, and quantified Bd infections using qPCR. We tested whether abiotic variables predicted the risk of Bd infections, and tested for relationships between biotic variables and Bd. Finally, we experimentally tested the effects of Bd strains CLFT 156 and CLFT 102 (from the southern and northern Atlantic Forest, respectively) on Atelopus manauensis. We detected higher Bd prevalence than those previously reported for the Brazilian Amazon, and positive individuals in all 3 orders of amphibians sampled. Both biotic and abiotic predictors were related to prevalence, and no variable explained infection load. Moreover, we detected higher Bd prevalence in forested than open areas, while the host's reproductive biology was not a factor. We detected higher mortality in the experimental group infected with CLFT 156, probably because this strain was isolated from a region characterized by discrepant climatic conditions (latitudinally more distant) when compared with the host's sampling site in Amazon. The lowland Brazilian Amazon is still underexplored and future studies targeting all amphibian orders are essential to better understand Bd infection dynamics in this region.

Morphology of the Cutaneous Poison and Mucous Glands in Amphibians with Particular Emphasis on Caecilians (Siphonops annulatus).

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

Research on helminths from Mexican amphibians: gaps, trends, and biases.

We present a taxonomic, spatial, and thematic overview of the current state of knowledge on helminth parasites of Mexican amphibians. Sixty-six host species have been studied so far, representing 17.5% of the amphibian species distributed in Mexico. A total of 139 nominal species of helminths - 68 platyhelminths, 62 nematodes, three acanthocephalans, three annelids (hirudineans), and three arthropods (pentastomids) - have been recorded parasitizing these hosts. Most taxa found in larval stages have not been identified at the species level. The gastrointestinal nematode Aplectana itzocanensis exhibits the broadest host range, while the bladder fluke Gorgoderina attenuata and A. itzocanensis show the widest geographic distribution. Our analysis of helminthological studies evidenced gaps and biases on research efforts that have been devoted to relatively few host species, regions, and approaches. Most helminthological records come from two species, the cane toad Rhinella marina and the Montezuma's frog Lithobates montezumae, and most studies have focused on describing the helminth fauna of a host species in a particular location or on the description of new helminth species. The highest proportion of records corresponds to the Veracruzan biogeographic province, and helminth richness is significantly correlated with host richness and with total amphibian richness by biogeographic province. Only three provinces (Yucatan Peninsula, Pacific Lowlands, and Baja Californian) have positive, yet still low helminth species discovery effort. Based on our findings, we recommend pursuing research approaches unexplored in Mexico and we provide guidelines to improve research on helminths parasitizing amphibians.

Morphology of the cutaneous poison and mucous glands in amphibians with particular emphasis on caecilians (Siphonops annulatus)

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

Amphibians of Serra das Torres Natural Monument: a reservoir of biodiversity in the Atlantic Forest of southeastern Brazil / Anfíbios do Monumento Natural Serra das Torres: um reservatório da biodiversidade da Mata Atlântica no sudeste do Brasil

Abstract The Brazilian Atlantic Forest holds a major part of the country's amphibian species richness and high rates of endemism. In this study, we conducted surveys using the Rapid Assessment (RA) method to sample the amphibian fauna of the Serra das Torres Natural Monument (MONAST), an Atlantic Forest remnant in southeastern Brazil. We sampled actively with a 6-10-person team to collect standard samples from 09:00 to 12:00 hours for the daytime period, and from 18:00 to 22:00 hours for the crepuscular/nighttime period, with a total of approximately 1,320 hours of sampling effort. We supplemented these data with 720 hours of passive sampling, using pitfall traps with drift fences (30 bucket-days). We recorded 54 amphibian species (two gymnophionans and 52 anurans), and the species richness estimated by the Bootstrap method indicates that a slightly larger number of species (n = 60) may occur in the study area. The most speciose family was Hylidae (n = 21), followed by Brachycephalidae (n = 8). Overall, 25% of the species (n = 13) were recorded only once (singletons) and 15% (n = 8) only twice (doubletons). Most amphibians recorded in this study (71%, n = 37 species) were restricted to the Atlantic Forest biome, two species (Euparkerella robusta and Luetkenotyphlus fredi) are endemic to the Espírito Santo state, and one of them, the leaf litter species E. robusta, is endemic to the MONAST. Euparkerella robusta is currently listed as Vulnerable by the IUCN and is classified as Critically Endangered in the Espírito Santo State red list, while L. fredi has yet to be evaluated due to its recent description. Thoropa lutzi is currently listed as Endangered (EN) by both the IUCN and in the State list. Nine species are listed as Data Deficient (DD) and populations of 13 species are considered to be declining by the IUCN. We extend the geographical distribution of two anuran species (Hylodes babax and Phasmahyla lisbella) and fill an important gap in the distribution of Siphonops hardyi. Amphibians associated with the forest floor represented 42% of the species richness from MONAST, and 43% of these species inhabit the leaf litter exclusively. Our study revealed that Serra das Torres preserves a considerable diversity of Atlantic Forest amphibians, which reinforces the need for the conservation of this forest remnant.

Resumo A Mata Atlântica brasileira guarda importante porção da riqueza de anfíbios e altas taxas de endemismos. Neste estudo, nós realizamos pesquisas usando o Método de Avaliação Rápida (RA) com o objetivo de inventariar a fauna de anfíbios de um remanescente da Mata Atlântica no sudeste do Brasil, o Monumento Natural Serra das Torres (MONAST). Amostramos ativamente com uma equipe de 6 a 10 pessoas para coletar amostras padronizadas entre 09:00 e 12:00 horas durante o peíodo diurno e entre 18:00 e 22:00 duranto período crepuscular/noturno, totalizando aproximadamente 1320 horas de esforço amostral. Complementamos estes dados com 720 horas de amostragem passiva usando armadilhas de queda com cercas-guia (30 dias de balde). Registramos 54 espécies de anfíbios (dois gimnofionos e 52 anuros) e a riqueza de espécies estimada pelo Bootstrap indicou um número relativamente maior de espécies (n = 60). A família mais especiosa foi Hylidae (n = 21), seguida por Brachycephalidae (n = 8). No geral, 25% das espécies (n = 13) foram registradas apenas uma vez - singletons e 15% (n = 8) apenas duas vezes (doubletons). A maioria dos anfíbios registrados neste estudo (71%, n = 37 espécies) esteve restrita ao bioma Mata Atlântica, duas espécies (Euparkerella robusta e Luetkenotyphlus fredi) são endêmicas do estado do Espírito Santo, sendo uma delas, a espécie de serapilheira E. robusta, endêmica do MONAST. Euparkerella robusta está atualmente listada como Vulnerável pela IUCN e classificada como Criticamente Ameaçada na lista vermelha do estado do Espírito Santo, enquanto L. fredi ainda não foi avaliada devido a sua descrição ser muito recente. Thoropa lutzi está atualmente listada como Ameaçada (EN) pela IUCN e na lista estadual. Nove espécies estão listadas como Deficiente de Dados (DD) e as populações de 13 espécies são consideradas em declínio pela IUCN. Estendemos a distribuição geográfica de duas espécies de anuros (Hylodes babax e Phasmahyla lisbella) e preenchemos uma importante lacuna na distribuição de Siphonops hardyi. Os anfíbios associados ao chão da floresta representaram 42% das espécies do MONAST e 43% destas espécies habitavam exclusivamente a serapilheira. Nosso estudo revelou que a Serra das Torres preserva diversidade considerável de anfíbios da Mata Atlântica, o que reforça a necessidade de conservação desse remanescente florestal.

Insights into the skin of caecilian amphibians from gene expression profiles.

BACKGROUND: Gene expression profiles can provide insights into the molecular machinery behind tissue functions and, in turn, can further our understanding of environmental responses, and developmental and evolutionary processes. During vertebrate evolution, the skin has played a crucial role, displaying a wide diversity of essential functions. To unravel the molecular basis of skin specialisations and adaptations, we compared gene expression in the skin with eight other tissues in a phylogenetically and ecologically diverse species sample of one of the most neglected vertebrate groups, the caecilian amphibians (order Gymnophiona). RESULTS: The skin of the five studied caecilian species showed a distinct gene expression profile reflecting its developmental origin and showing similarities to other epithelial tissues. We identified 59 sequences with conserved enhanced expression in the skin that might be associated with caecilian dermal specialisations. Some of the up-regulated genes shared expression patterns with human skin and potentially are involved in skin functions across vertebrates. Variation trends in gene expression were detected between mid and posterior body skin suggesting different functions between body regions. Several candidate biologically active peptides were also annotated. CONCLUSIONS: Our study provides the first atlas of differentially expressed sequences in caecilian tissues and a baseline to explore the molecular basis of the skin functions in caecilian amphibians, and more broadly in vertebrates.

Functional traits explain amphibian distribution in the Brazilian Atlantic Forest.

AIM: Species distributions are one of the most important ways to understand how communities interact through macroecological relationships. The functional abilities of a species, such as its plasticity in various environments, can determine its distribution, species richness and beta diversity patterns. In this study, we evaluate how functional traits influence the distribution of amphibians, and hypothesize which functional traits explain the current pattern of amphibian species composition. LOCATION: Atlantic Forest, Brazil. TAXON: Amphibia (Anura and Gymnophiona). METHODS: Using potential distributions of Brazilian amphibians from Atlantic Forest based on their functional traits, we analysed the influence of biotic and abiotic factors on species richness, endemism (with permutation multivariate analysis) and beta diversity components (i.e. total, turnover and nestedness dissimilarities). RESULTS: Environmental variables explained 59.5% of species richness, whereas functional traits explained 15.8% of species distribution (geographical species range) for Anuran and 88.8% for Gymnophiona. Body size had the strongest correlation with species distribution. Results showed that species with medium to large body size, and species that are adapted to living in open areas tended to disperse from west to east direction. Current forest changes directly affected beta diversity patterns (i.e. most species adapted to novel environments increase their ranges). Beta diversity partitioning between humid and dry forests showed decreased nestedness and increased turnover by increasing altitude in the south-eastern region of the Atlantic Forest. MAIN CONCLUSIONS: Our study shows that functional traits directly influence the ability of the species to disperse. With the alterations of the natural environment, species more apt to these alterations have dispersed or increased their distribution, which consequently changes community structure. As a result, there are nested species distribution patterns and homogenization of amphibian species composition throughout the Brazilian Atlantic Forest.

What lies beneath? Molecular evolution during the radiation of caecilian amphibians.

BACKGROUND: Evolution leaves an imprint in species through genetic change. At the molecular level, evolutionary changes can be explored by studying ratios of nucleotide substitutions. The interplay among molecular evolution, derived phenotypes, and ecological ranges can provide insights into adaptive radiations. Caecilians (order Gymnophiona), probably the least known of the major lineages of vertebrates, are limbless tropical amphibians, with adults of most species burrowing in soils (fossoriality). This enigmatic order of amphibians are very distinct phenotypically from other extant amphibians and likely from the ancestor of Lissamphibia, but little to nothing is known about the molecular changes underpinning their radiation. We hypothesised that colonization of various depths of tropical soils and of freshwater habitats presented new ecological opportunities to caecilians. RESULTS: A total of 8540 candidate groups of orthologous genes from transcriptomic data of five species of caecilian amphibians and the genome of the frog Xenopus tropicalis were analysed in order to investigate the genetic machinery behind caecilian diversification. We found a total of 168 protein-coding genes with signatures of positive selection at different evolutionary times during the radiation of caecilians. The majority of these genes were related to functional elements of the cell membrane and extracellular matrix with expression in several different tissues. The first colonization of the tropical soils was connected to the largest number of protein-coding genes under positive selection in our analysis. From the results of our study, we highlighted molecular changes in genes involved in perception, reduction-oxidation processes, and aging that likely were involved in the adaptation to different soil strata. CONCLUSIONS: The genes inferred to have been under positive selection provide valuable insights into caecilian evolution, potentially underpin adaptations of caecilians to their extreme environments, and contribute to a better understanding of fossorial adaptations and molecular evolution in vertebrates.

Phylogenetic and Trait-Based Prediction of Extinction Risk for Data-Deficient Amphibians.

Amphibians are among the most highly threatened lineages, with at least 2,000 species estimated to be in danger of extinction [1, 2]. Alarmingly, another ∼2,200 species (∼25% of all ∼7,900 known species) are data deficient or not evaluated (hereinafter termed data deficient) by the International Union for Conservation of Nature (IUCN) [1]. Without an estimate of their status, data-deficient species are usually overlooked in conservation planning and resource allocation [3]. Amphibians have the highest proportion of data-deficient species of any vertebrate group [1, 4], which highlights the need to estimate their threat status considering potentially imminent extinctions. We apply a trait-based spatio-phylogenetic statistical framework [5] to predict threat status for data-deficient species. Because ecological, geographical, and evolutionary attributes increase extinction risk [6, 7], we used geographic distribution data [1, 8], phylogenetically imputed ecological traits, and an amphibian phylogeny [9] to provide initial baseline predictions. We estimate that half of the ∼2,200 data-deficient species are threatened with extinction (vulnerable, endangered, or critically endangered), primarily in the Neotropics and Southeast Asia. This increases the number of amphibian species estimated to be threatened with extinction by ∼50%. Of these, we predict that ∼500 species are endangered or critically endangered, and three may be extinct already. We highlight families that are most at risk and suggest where urgent conservation is needed to avert their loss. We show that some of the most vulnerable species may also be the most poorly known and offer an analytical framework for preliminary analysis of their threat status in the face of deficient empirical data.

Morphological evolution and modularity of the caecilian skull.

BACKGROUND: Caecilians (Gymnophiona) are the least speciose extant lissamphibian order, yet living forms capture approximately 250 million years of evolution since their earliest divergences. This long history is reflected in the broad range of skull morphologies exhibited by this largely fossorial, but developmentally diverse, clade. However, this diversity of form makes quantification of caecilian cranial morphology challenging, with highly variable presence or absence of many structures. Consequently, few studies have examined morphological evolution across caecilians. This extensive variation also raises the question of degree of conservation of cranial modules (semi-autonomous subsets of highly-integrated traits) within this clade, allowing us to assess the importance of modular organisation in shaping morphological evolution. We used an intensive surface geometric morphometric approach to quantify cranial morphological variation across all 32 extant caecilian genera. We defined 16 cranial regions using 53 landmarks and 687 curve and 729 surface sliding semilandmarks. With these unprecedented high-dimensional data, we analysed cranial shape and modularity across caecilians assessing phylogenetic, allometric and ecological influences on cranial evolution, as well as investigating the relationships among integration, evolutionary rate, and morphological disparity. RESULTS: We found highest support for a ten-module model, with greater integration of the posterior skull. Phylogenetic signal was significant (Kmult = 0.87, p < 0.01), but stronger in anterior modules, while allometric influences were also significant (R2 = 0.16, p < 0.01), but stronger posteriorly. Reproductive strategy and degree of fossoriality were small but significant influences on cranial morphology (R2 = 0.03-0.05), after phylogenetic (p < 0.03) and multiple-test (p < 0.05) corrections. The quadrate-squamosal 'cheek' module was the fastest evolving module, perhaps due to its pivotal role in the unique dual jaw-closing mechanism of caecilians. Highly integrated modules exhibited both high and low disparities, and no relationship was evident between integration and evolutionary rate. CONCLUSIONS: Our high-dimensional approach robustly characterises caecilian cranial evolution and demonstrates that caecilian crania are highly modular and that cranial modules are shaped by differential phylogenetic, allometric, and ecological effects. More broadly, and in contrast to recent studies, this work suggests that there is no simple relationship between integration and evolutionary rate or disparity.

Multi-tissue transcriptomes of caecilian amphibians highlight incomplete knowledge of vertebrate gene families.

RNA sequencing (RNA-seq) has become one of the most powerful tools to unravel the genomic basis of biological adaptation and diversity. Although challenging, RNA-seq is particularly promising for research on non-model, secretive species that cannot be observed in nature easily and therefore remain comparatively understudied. Among such animals, the caecilians (order Gymnophiona) likely constitute the least known group of vertebrates, despite being an old and remarkably distinct lineage of amphibians. Here, we characterize multi-tissue transcriptomes for five species of caecilians that represent a broad level of diversity across the order. We identified vertebrate homologous elements of caecilian functional genes of varying tissue specificity that reveal a great number of unclassified gene families, especially for the skin. We annotated several protein domains for those unknown candidate gene families to investigate their function. We also conducted supertree analyses of a phylogenomic dataset of 1,955 candidate orthologous genes among five caecilian species and other major lineages of vertebrates, with the inferred tree being in agreement with current views of vertebrate evolution and systematics. Our study provides insights into the evolution of vertebrate protein-coding genes, and a basis for future research on the molecular elements underlying the particular biology and adaptations of caecilian amphibians.

Study of the hydromineral regulation of Typhlonectes compressicauda according to the seasonal variation.

INTRODUCTION: Typhlonectes compressicauda is a viviparous gymnophionan amphibian living in tropical areas of South America. This lengthened amphibian is submitted to seasonal variations characterized by the rainy season (from January to June) and the dry season (from July to December). The mineral homeostasis in amphibians is partly ensured by the neurohormones arginine-vasotocin (AVT), and mesotocin (MST). These two hormones were localized in the hypothalamus, and their receptors, mesotocin receptors (MTR) and vasotocin receptors (VTR2) in the kidney. The aim of the study was to better understand the physiology of the hydromineral regulation of the studied species. MATERIAL AND METHODS: The specimens of T. compressicauda male and female adult were divided into 6 groups: males in the rainy season, males in the dry season, females pregnant in the rainy season, females pregnant in the dry season, females not pregnant in the rainy season, females not pregnant in the dry season. We studied the expression of hormones (AVT, MST) and their receptors (MTR, VTR2) in the hypothalamus and the kidney, respectively, by immunohistochemical and histological techniques. We also studied the expression of aquaporin-2 (AQP2), a water-channel protein in the kidney. RESULTS: We found that the MST (diuretic hormone) and its receptor were more intensively expressed during the rainy season, whereas the period of maximal AVT (anti-diuretic hormone) and VTR2 expression was the dry season. A quantitative analysis showed significant differences in the number of labeled cells in the hypothalamus depending on the seasonal variation. The expression of AQP2 was observed in renal tubules during both seasons with an increased intensity during the dry season. CONCLUSION: The expression of the MST/AVT in brain, their receptors MTRs/VTR2, and AQP2 in kidney changed in T. compressicauda according to the seasonal variations. A direct relationship between the seasonal cycle and reproduction cycle was demonstrated in this species.

Filling the BINs of life: Report of an amphibian and reptile survey of the Tanintharyi (Tenasserim) Region of Myanmar, with DNA barcode data.

Despite threats of species extinctions, taxonomic crises, and technological advances in genomics and natural history database informatics, we are still distant from cataloguing all of the species of life on earth. Amphibians and reptiles are no exceptions; in fact new species are described nearly every day and many species face possible extinction. The number of described species continues to climb as new areas of the world are explored and as species complexes are examined more thoroughly. The use of DNA barcoding provides a mechanism for rapidly estimating the number of species at a given site and has the potential to record all of the species of life on Earth. Though DNA barcoding has its caveats, it can be useful to estimate the number of species in a more systematic and efficient manner, to be followed in combination with more traditional, morphology-based identifications and species descriptions. Herein, we report the results of a voucher-based herpetological expedition to the Tanintharyi (Tenasserim) Region of Myanmar, enhanced with DNA barcode data. Our main surveys took place in the currently proposed Tanintharyi National Park. We combine our results with photographs and observational data from the Chaung-nauk-pyan forest reserve. Additionally, we provide the first checklist of amphibians and reptiles of the region, with species based on the literature and museum. Amphibians, anurans in particular, are one of the most poorly known groups of vertebrates in terms of taxonomy and the number of known species, particularly in Southeast Asia. Our rapid-assessment program combined with DNA barcoding and use of Barcode Index Numbers (BINs) of voucher specimens reveals the depth of taxonomic diversity in the southern Tanintharyi herpetofauna even though only a third of the potential amphibians and reptiles were seen. A total of 51 putative species (one caecilian, 25 frogs, 13 lizards, 10 snakes, and two turtles) were detected, several of which represent potentially undescribed species. Several of these species were detected by DNA barcode data alone. Furthermore, five species were recorded for the first time in Myanmar, two amphibians (Ichthyophis cf. kohtaoensis and Chalcorana eschatia) and three snakes (Ahaetulla mycterizans, Boiga dendrophila, and Boiga drapiezii).

Amphibians of the Reserva Ecológica Michelin: a high diversity site in the lowland Atlantic Forest of southern Bahia, Brazil.

An inventory of the amphibians of the Reserva Ecológica Michelin - REM in southern Bahia, Brazil is presented. Sixty-nine species were recorded during a ten-year sampling period. Amphibians were distributed in two orders (Gymnophiona and Anura), belonging to twelve families [Aromobatidae (1), Bufonidae (3), Centrolenidae (1), Craugastoridae (5), Eleutherodactylidae (3), Hemiphractidae (2), Hylidae (34), Phyllomedusidae (5) Leptodactylidae (7), Microhylidae (4), Odontophrynidae (3) and Caeciliidae (1)]. Fifty per cent of the reproductive modes known for Atlantic forest anurans were recorded. While no threatened species were found at REM, six species are classified as data deficient (DD) by the Brazilian Red List of threatened species and deserve additional attention. Phasmahyla timbo and Vitreorana eurygnatha are listed as endangered in Bahia according to the list of threatened species of the state. Despite a higher diversity of amphibians in the Atlantic forest having been reported for mountainous regions, our results revealed that amphibian richness for lowland forests is also high.

Stem caecilian from the Triassic of Colorado sheds light on the origins of Lissamphibia.

The origin of the limbless caecilians remains a lasting question in vertebrate evolution. Molecular phylogenies and morphology support that caecilians are the sister taxon of batrachians (frogs and salamanders), from which they diverged no later than the early Permian. Although recent efforts have discovered new, early members of the batrachian lineage, the record of pre-Cretaceous caecilians is limited to a single species, Eocaecilia micropodia The position of Eocaecilia within tetrapod phylogeny is controversial, as it already acquired the specialized morphology that characterizes modern caecilians by the Jurassic. Here, we report on a small amphibian from the Upper Triassic of Colorado, United States, with a mélange of caecilian synapomorphies and general lissamphibian plesiomorphies. We evaluated its relationships by designing an inclusive phylogenetic analysis that broadly incorporates definitive members of the modern lissamphibian orders and a diversity of extinct temnospondyl amphibians, including stereospondyls. Our results place the taxon confidently within lissamphibians but demonstrate that the diversity of Permian and Triassic stereospondyls also falls within this group. This hypothesis of caecilian origins closes a substantial morphologic and temporal gap and explains the appeal of morphology-based polyphyly hypotheses for the origins of Lissamphibia while reconciling molecular support for the group's monophyly. Stem caecilian morphology reveals a previously unrecognized stepwise acquisition of typical caecilian cranial apomorphies during the Triassic. A major implication is that many Paleozoic total group lissamphibians (i.e., higher temnospondyls, including the stereospondyl subclade) fall within crown Lissamphibia, which must have originated before 315 million years ago.

Spatial distribution of Batrachochytrium dendrobatidis in South American caecilians.

The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) is linked to population declines in anurans and salamanders globally. To date, however, few studies have attempted to screen Bd in live caecilians; Bd-positive caecilians have only been reported in Africa and French Guiana. Here, we performed a retrospective survey of museum preserved specimens to (1) describe spatial patterns of Bd infection in Gymnophiona across South America and (2) test whether areas of low climatic suitability for Bd in anurans predict Bd spatial epidemiology in caecilians. We used quantitative PCR to detect Bd in preserved caecilians collected over a 109 yr period, and performed autologistic regressions to test the effect of bioclimatic metrics of temperature and precipitation, vegetation density, and elevation on the likelihood of Bd occurrence. We detected an overall Bd prevalence of 12.4%, with positive samples spanning the Uruguayan savanna, Brazil's Atlantic Forest, and the Amazon basin. Our autologistic models detected a strong effect of macroclimate, a weaker effect of vegetation density, and no effect of elevation on the likelihood of Bd occurrence. Although most of our Bd-positive records overlapped with reported areas of high climatic suitability for the fungus in the Neotropics, many of our new Bd-positive samples extend far into areas of poor suitability for Bd in anurans. Our results highlight an important gap in the study of amphibian chytridiomycosis: the potential negative impact of Bd on Neotropical caecilians and the hypothetical role of caecilians as Bd reservoirs.

Patterns of Apoptosis and Proliferation throughout the Biennial Reproductive Cycle of Viviparous Female Typhlonectes compressicauda (Amphibia, Gymnophiona).

Typhlonectes compressicauda is an aquatic gymnophionan amphibian living in South America. Its breeding cycle is linked to seasons, characterized by a regular alternation of rainy and dry seasons. During a complex biennial cycle, the female genital tract undergoes a series of alternations of increasing and decreasing, governed by equilibrium of proliferation and apoptotic phenomena. Immunohistochemical methods were used to visualize cell proliferation with the detection of Ki67 antibody, a protein present in proliferative cells; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Apostain were performed to detect apoptotic cells on sections of ovaries and oviducts. In ovaries, both phenomena affect the germinal nests and follicles according to the cycle period. In the oviduct, the balance was in favor of proliferation during preparation for reproduction, and in favor of apoptosis when genital ducts regress. Apoptosis and proliferation are narrowly implicated in the remodeling of the genital tract and they are accompanied by the differentiation of tissues according to the phase of the breeding cycle. These variations permit the capture of oocytes at ovulation, always at the same period, and the parturition after 6-7 months of gestation, at a period in which the newborns live with their mother, protected in burrows in the mud. During the intervening year of sexual inactivity, the female reconstitutes body reserves.

Myxozoan infections of caecilians demonstrate broad host specificity and indicate a link with human activity.

Myxozoans are parasitic cnidarians that infect a wide variety of hosts. Vertebrates typically serve as intermediate hosts whereas definitive hosts are invertebrates, including annelids and bryozoans. Myxozoans are known to exploit species in two of the three extant amphibian orders (Anura: frogs and toads; Caudata: newts and salamanders). Here we use museum collections to determine, to our knowledge for the first time, whether myxozoans also exploit the third amphibian order (Gymnophiona: caecilians). Caecilians are a poorly known group of limbless amphibians, the ecologies of which range from aquatic to fully terrestrial. We examined 12 caecilian species in seven families (148 individuals total) characterised by a diversity of ecologies and life histories. Using morphological and molecular surveys, we discovered the presence of the myxozoan Cystodiscus axonis in two South American species (one of seven examined families) of aquatic caecilians - Typhlonectes natans and Typhlonectes compressicauda. All infected caecilians had been maintained in captivity in the United Kingdom prior to their preservation. Cystodiscus axonis is known from several Australian frog species and its presence in caecilians indicates a capacity for infecting highly divergent amphibian hosts. This first known report of myxozoan infections in caecilians provides evidence of a broad geographic and host range. However, the source of these infections remains unknown and could be related to exposure in South America, the U.K. or to conditions in captivity.