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

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

Diversity and Molecular Evolution of Nonvisual Opsin Genes across Environmental, Developmental, and Morphological Adaptations in Frogs.

Nonvisual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, nonvisual opsins form photopigments involved in various nonvisual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here, we investigate the molecular evolution of nonvisual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of nonvisual opsins due to nocturnal ancestry and potential functional differences in nonvisual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multitissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which nonvisual opsins are present in anuran genomes and those that are also expressed in the eyes, compare selective constraint among genes, and test for potential adaptive evolution by comparing selection between discrete ecological classes. At the genomic level, we recovered all 18 ancestral vertebrate nonvisual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 nonvisual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on nonvisual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many nonvisual opsins remain poorly understood, these findings provide insight into the diversity and evolution of these genes across anurans, filling an important gap in our understanding of vertebrate opsins and setting the stage for future research on their functional evolution across taxa.

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Genomic SNPs resolve the phylogeny of an ancient amphibian island radiation from the Seychelles.

Unusually for oceanic islands, the granitic Seychelles host multiple lineages of endemic amphibians. This includes an ancient (>63 million years old) 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.

Comparative morphology of oral glands in snakes of the family Homalopsidae reveals substantial variation and additional independent origins of salt glands within Serpentes.

Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we examined the morphology of the oral glands of 12 species of the family Homalopsidae. Snakes of this family exhibit substantial interspecific morphological variation in their oral glands. Particular variables are the venom glands, ranging from large (e.g., Subsessor bocourti) to small (e.g., Erpeton tentaculatum). The supra- and infralabial glands are more uniform in morphology, being the second most developed in almost all the sampled species. Premaxillary glands distinct from the supralabial glands were observed in five species (Myron richardsonii, Bitia hydroides, Cantoria violacea, Fordonia leucobalia, and Gerarda prevostiana), in addition to Cerberus rynchops, the only species in which this condition was previously documented associated with the excretion of salt. In the three species of the saltwater group of homalopsids (C. violacea, F. leucobalia, and G. prevostiana), the premaxillary glands also extend posteriorly, occupying a large area above the supralabial gland, a condition not observed in any other species of snake studied thus far. Character evolution analyses indicate that premaxillary glands differentiated from the supralabial gland and evolved independently three or four times in the family, always in lineages that invaded marine habitats. Our results suggest that the differentiated premaxillary glands are likely salt glands, as is the case in C. rynchops. If corroborated, this increases to six or seven the number of independent evolutionary origins of salt glands in snakes that have undergone an evolutionary transition to marine life.

The systematics and evolution of the Sri Lankan rainforest land snail Corilla: New insights from RADseq-based phylogenetics.

The stylommatophoran land-snail genus Corilla is endemic to Sri Lanka and India's Western Ghats. On the basis of habitat distribution and shell morphology, the 10 extant Sri Lankan species fall into two distinct groups, lowland and montane. Here, we use phylogenetic analyses of restriction-site-associated DNA sequencing (RADseq) data and ancestral-state reconstructions of habitat association and shell morphology to clarify the systematics and evolution of Sri Lankan Corilla. Our dataset consists of 9 species of Corilla. Phylogenetic analyses were based on 88 assemblies (9,604-4,132,850 bp) generated by the RADseq assembler ipyrad, using four parameter combinations and different levels of missing data. Trees were inferred using a maximum likelihood (ML) approach. Ancestral states were reconstructed using maximum parsimony (MP) and ML approaches, with 1 binary state character analysed for habitat association (lowland vs montane) and 6 binary state characters analysed for shell morphology (shape, colour, lip width, length of upper palatal folds, orientation of upper palatal folds and collabral sculpture). Over a wide range of missing data (40-87 % missing individuals per locus) and assembly sizes (62,279-4,132,850 bp), nearly all trees conformed to one of two topologies (A and B), most relationships were strongly supported and total branch support approached the maximal value. Apart from the position of Corilla odontophora 'south', topologies A and B showed similar, well-resolved relationships at and above the species level. Our study agrees with the shell-based taxonomy of C. adamsi, C. beddomeae, C. carabinata, C. colletti and C. humberti (all maximally supported as monophyletic species). It shows that C. erronea and C. fryae constitute a single relatively widespread species (for which the valid name is C. erronea) and that the names C. gudei and C. odontophora each apply to at least two distinct, yet conchologically-cryptic species. The MP and ML ancestral-state reconstructions yielded broadly similar results and provide firm evidence that diversification in Sri Lankan Corilla has involved evolutionary convergence in the shell morphology of lowland lineages, with a pale shell and wide lip having evolved on at least two separate occasions (in C. carabinata and C. colletti) from montane ancestors having a dark, narrow-lipped shell.

A near-complete species-level phylogeny of uropeltid snakes harnessing historical museum collections as a DNA source.

Uropeltidae is a clade of small fossorial snakes (ca. 64 extant species) endemic to peninsular India and Sri Lanka. Uropeltid taxonomy has been confusing, and the status of some species has not been revised for over a century. Attempts to revise uropeltid systematics and undertake evolutionary studies have been hampered by incompletely sampled and incompletely resolved phylogenies. To address this issue, we take advantage of historical museum collections, including type specimens, and apply genome-wide shotgun (GWS) sequencing, along with recent field sampling (using Sanger sequencing) to establish a near-complete multilocus species-level phylogeny (ca. 87% complete at species level). This results in a phylogeny that supports the monophyly of all genera (if Brachyophidium is considered a junior synonym of Teretrurus), and provides a firm platform for future taxonomic revision. Sri Lankan uropeltids are probably monophyletic, indicating a single colonisation event of this island from Indian ancestors. However, the position of Rhinophis goweri (endemic to Eastern Ghats, southern India) is unclear and warrants further investigation, and evidence that it may nest within the Sri Lankan radiation indicates a possible recolonisation event. DNA sequence data and morphology suggest that currently recognised uropeltid species diversity is substantially underestimated. Our study highlights the benefits of integrating museum collections in molecular genetic analyses and their role in understanding the systematics and evolutionary history of understudied organismal groups.

Diet and habit explain head-shape convergences in natricine snakes.

The concept of ecomorphs, whereby species with similar ecologies have similar phenotypes regardless of their phylogenetic relatedness, is often central to discussions regarding the relationship between ecology and phenotype. However, some aspects of the concept have been questioned, and sometimes species have been grouped as ecomorphs based on phenotypic similarity without demonstrating ecological similarity. Within snakes, similar head shapes have convergently evolved in species living in comparable environments and/or with similar diets. Therefore, ecomorphs could exist in some snake lineages, but this assertion has rarely been tested for a wide-ranging group within a single framework. Natricine snakes (Natricinae) are ecomorphologically diverse and currently distributed in Asia, Africa, Europe and north-central America. They are primarily semiaquatic or ground-dwelling terrestrial snakes, but some are aquatic, burrowing or aquatic and burrowing in habit and may be generalist or specialist in diet. Thus, natricines present an interesting system to test whether snakes from different major habit categories represent ecomorphs. We quantify morphological similarity and disparity in head shape among 191 of the ca. 250 currently recognized natricine species and apply phylogenetic comparative methods to test for convergence. Natricine head shape is largely correlated with habit, but in some burrowers is better explained by dietary specialism. Convergence in head shape is especially strong for aquatic burrowing, semiaquatic and terrestrial ecomorphs and less strong for aquatic and burrowing ecomorphs. The ecomorph concept is useful for understanding natricine diversity and evolution, though would benefit from further refinement, especially for aquatic and burrowing taxa.

Transcriptomic evidence for visual adaptation during the aquatic to terrestrial metamorphosis in leopard frogs.

BACKGROUND: Differences in morphology, ecology, and behavior through ontogeny can result in opposing selective pressures at different life stages. Most animals, however, transition through two or more distinct phenotypic phases, which is hypothesized to allow each life stage to adapt more freely to its ecological niche. How this applies to sensory systems, and in particular how sensory systems adapt across life stages at the molecular level, is not well understood. Here, we used whole-eye transcriptomes to investigate differences in gene expression between tadpole and juvenile southern leopard frogs (Lithobates sphenocephalus), which rely on vision in aquatic and terrestrial light environments, respectively. Because visual physiology changes with light levels, we also tested the effect of light and dark exposure. RESULTS: We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles and 5% for light/dark exposure. Analyses targeting a curated subset of visual genes revealed significant differential expression of genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. CONCLUSIONS: Overall, we identified extensive expression-level differences in the eyes of tadpoles and juveniles related to observed morphological and physiological changes through metamorphosis and corresponding adaptive shifts to improve vision in the distinct aquatic and terrestrial light environments these frogs inhabit during their life cycle. More broadly, these results suggest that decoupling of gene expression can mediate the opposing selection pressures experienced by organisms with complex life cycles that inhabit different environmental conditions throughout ontogeny.

Ocular lens morphology is influenced by ecology and metamorphosis in frogs and toads.

The shape and relative size of an ocular lens affect the focal length of the eye, with consequences for visual acuity and sensitivity. Lenses are typically spherical in aquatic animals with camera-type eyes and axially flattened in terrestrial species to facilitate vision in optical media with different refractive indices. Frogs and toads (Amphibia: Anura) are ecologically diverse, with many species shifting from aquatic to terrestrial ecologies during metamorphosis. We quantified lens shape and relative size using 179 micro X-ray computed tomography scans of 126 biphasic anuran species and tested for correlations with life stage, environmental transitions, adult habits and adult activity patterns. Across broad phylogenetic diversity, tadpole lenses are more spherical than those of adults. Biphasic species with aquatic larvae and terrestrial adults typically undergo ontogenetic changes in lens shape, whereas species that remain aquatic as adults tend to retain more spherical lenses after metamorphosis. Further, adult lens shape is influenced by adult habit; notably, fossorial adults tend to retain spherical lenses following metamorphosis. Finally, lens size relative to eye size is smaller in aquatic and semiaquatic species than other adult ecologies. Our study demonstrates how ecology shapes visual systems, and the power of non-invasive imaging of museum specimens for studying sensory evolution.

Molecular phylogenetics of sub-Saharan African natricine snakes, and the biogeographic origins of the Seychelles endemic Lycognathophis seychellensis.

Phylogenetic relationships of sub-Saharan African natricine snakes are understudied and poorly understood, which in turn has precluded analyses of the historical biogeography of the Seychelles endemic Lycognathophis seychellensis. We inferred the phylogenetic relationships of Seychelles and mainland sub-Saharan natricines by analysing a multilocus DNA sequence dataset for three mitochondrial (mt) and four nuclear (nu) genes. The mainland sub-Saharan natricines and L. seychellensis comprise a well-supported clade. Two maximally supported sets of relationships within this clade are (Limnophis,Natriciteres) and (Afronatrix,(Hydraethiops,Helophis)). The relationships of L. seychellensis with respect to these two lineages are not clearly resolved by analysing concatenated mt and nu data. Analysed separately, nu data best support a sister relationship of L. seychellensis with (Afronatrix,(Hydraethiops,Helophis)) and mt data best support a sister relationship with all mainland sub-Saharan natricines. Methods designed to cope with incomplete lineage sorting strongly favour the former hypothesis. Genetic variation among up to 33 L. seychellensis from five Seychelles islands is low. Fossil calibrated divergence time estimates support an overseas dispersal of the L. seychellensis lineage to the Seychelles from mainland Africa ca. 43-25 million years before present (Ma), rather than this taxon being a Gondwanan relic.

The roles of vicariance and isolation by distance in shaping biotic diversification across an ancient archipelago: evidence from a Seychelles caecilian amphibian.

BACKGROUND: Island systems offer excellent opportunities for studying the evolutionary histories of species by virtue of their restricted size and easily identifiable barriers to gene flow. However, most studies investigating evolutionary patterns and processes shaping biotic diversification have focused on more recent (emergent) rather than ancient oceanic archipelagos. Here, we focus on the granitic islands of the Seychelles, which are unusual among island systems because they have been isolated for a long time and are home to a monophyletic radiation of caecilian amphibians that has been separated from its extant sister lineage for ca. 65-62 Ma. We selected the most widespread Seychelles caecilian species, Hypogeophis rostratus, to investigate intraspecific morphological and genetic (mitochondrial and nuclear) variation across the archipelago (782 samples from nine islands) to identify patterns and test processes that shaped their evolutionary history within the Seychelles. RESULTS: Overall a signal of strong geographic structuring with distinct northern- and southern-island clusters were identified across all datasets. We suggest that these distinct groups have been isolated for ca. 1.26 Ma years without subsequent migration between them. Populations from the somewhat geographically isolated island of Frégate showed contrasting relationships to other islands based on genetic and morphological data, clustering alternatively with northern-island (genetic) and southern-island (morphological) populations. CONCLUSIONS: Although variation in H. rostratus across the Seychelles is explained more by isolation-by-distance than by adaptation, the genetic-morphological incongruence for affinities of Frégate H. rostratus might be caused by local adaptation over-riding the signal from their vicariant history. Our findings highlight the need of integrative approaches to investigate fine-scale geographic structuring to uncover underlying diversity and to better understand evolutionary processes on ancient, continental islands.

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.

Eye size and investment in frogs and toads correlate with adult habitat, activity pattern and breeding ecology.

Frogs and toads (Amphibia: Anura) display diverse ecologies and behaviours, which are often correlated with visual capacity in other vertebrates. Additionally, anurans exhibit a broad range of relative eye sizes, which have not previously been linked to ecological factors in this group. We measured relative investment in eye size and corneal size for 220 species of anurans representing all 55 currently recognized families and tested whether they were correlated with six natural history traits hypothesized to be associated with the evolution of eye size. Anuran eye size was significantly correlated with habitat, with notable decreases in eye investment among fossorial, subfossorial and aquatic species. Relative eye size was also associated with mating habitat and activity pattern. Compared to other vertebrates, anurans have relatively large eyes for their body size, indicating that vision is probably of high importance. Our study reveals the role that ecology and behaviour may have played in the evolution of anuran visual systems and highlights the usefulness of museum specimens, and importance of broad taxonomic sampling, for interpreting macroecological patterns.

POSTMORTEM FINDINGS IN EIGHT SPECIES OF CAPTIVE CAECILIAN (AMPHIBIA: GYMNOPHIONA) OVER A TEN-YEAR PERIOD.

Between July 2007 and June 2017 there were 86 deaths in the populations of eight caecilian species at the Zoological Society of London (ZSL) London Zoo. The mortality rate (deaths per animal-year at risk) ranged from 0.03 in the Congo caecilian (Herpele squalostoma) to 0.85 in Kaup's caecilian (Potomotyphlus kaupii). Among the 73 individuals examined post mortem, no cause of death or primary diagnosis could be established in 35 cases, but of the others the most common cause of death was dermatitis (22 cases). When all significant pathological findings were considered, skin lesions of varying types were again the commonest (56 cases), particularly among the aquatic species: Typhlonectes compressicauda (18 out of 21 cases), T. natans (8/10) and P. kaupii (12/14). Other common findings were poor gut-fill (35 cases), kidney and gastrointestinal lesions (10 cases each), generalized congestion (8 cases) and poor body condition (6 cases). This review adds to the growing body of knowledge regarding the presentations and causes of disease in captive caecilians.

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.

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.

Phototactic tails: Evolution and molecular basis of a novel sensory trait in sea snakes.

Dermal phototaxis has been reported in a few aquatic vertebrate lineages spanning fish, amphibians and reptiles. These taxa respond to light on the skin of their elongate hind-bodies and tails by withdrawing under cover to avoid detection by predators. Here, we investigated tail phototaxis in sea snakes (Hydrophiinae), the only reptiles reported to exhibit this sensory behaviour. We conducted behavioural tests in 17 wild-caught sea snakes of eight species by illuminating the dorsal surface of the tail and midbody skin using cold white, violet, blue, green and red light. Our results confirmed phototactic tail withdrawal in the previously studied Aipysurus laevis, revealed this trait for the first time in A. duboisii and A. tenuis, and suggested that tail photoreceptors have peak spectral sensitivities between blue and green light (457-514 nm). Based on these results, and an absence of photoresponses in five Aipysurus and Hydrophis species, we tentatively infer that tail phototaxis evolved in the ancestor of a clade of six Aipysurus species (comprising 10% of all sea snakes). Quantifying tail damage, we found that the probability of sustaining tail injuries was not influenced by tail phototactic ability in snakes. Gene profiling showed that transcriptomes of both tail skin and body skin lacked visual opsins but contained melanopsin (opn4x) in addition to key genes of the retinal regeneration and phototransduction cascades. This work suggests that a nonvisual photoreceptor (e.g., Gq rhabdomeric) signalling pathway underlies tail phototaxis, and provides candidate gene targets for future studies of this unusual sensory innovation in reptiles.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.