Diversity and molecular evolution of non-visual opsin genes across environmental, developmental, and morphological adaptations in frogs.

Non-visual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, non-visual opsins form photopigments involved in various non-visual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here we investigate the molecular evolution of non-visual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of non-visual opsins due to nocturnal ancestry and potential functional differences in non-visual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multi-tissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which non-visual 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 non-visual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 non-visual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on non-visual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many non-visual 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.

Circadian rhythm and surface activity in soil-dwelling caecilians (Amphibia: Gymnophiona).

The degree to which burrowing, soil-dwelling caecilian amphibians spend time on the surface is little studied, and circadian rhythm has not been investigated in multiple species of this order or by manipulating light-dark cycles. We studied surface-activity rhythm of the Indian caecilians Ichthyophis cf. longicephalus and Uraeotyphlus cf. oxyurus (Ichthyophiidae) and Gegeneophis tejaswini (Grandisoniidae), under LD, DD and DL cycles. We examined daily surface activity and the role of light-dark cycles as a zeitgeber. All three species were strictly nocturnal and G. tejaswini displayed the least surface activity. Four out of thirteen individuals, two I. cf. longicephalus, one G. tejaswini and one U. cf. oxyurus, displayed a more or less distinct surface-activity rhythm in all three cycles, and for the nine other animals the activity patterns were not evident. An approximately 24 h free-run period was observed in the three species. When the light-dark cycle was inverted, surface activity in the three species shifted to the dark phase. The findings of this study suggest that caecilians have a weak circadian surface-activity rhythm, and that the absence of light can act as a prominent zeitgeber in these burrowing, limbless amphibians.

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.

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.

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.

On the taxonomy of Uropeltis petersi (Beddome, 1878) (Serpentes: Uropeltidae) and description of a new, closely related species from the Western Ghats of India.

A new species of the uropeltid snake genus Uropeltis Cuvier, 1829 is described from the environs of Munnar in the Anamalai hils of the Western Ghats of peninsular India. Uropeltis tricuspida sp. nov. superficially resembles the poorly known and closely related U. petersi, but differs from that species in having more ventral and subcaudal scales, and a tri- rather than bicuspid posterior tip to the terminal scute.

Identity of the holotype and type locality of Rhabdophis leonardi (Wall, 1923) (Colubridae: Natricinae), with notes on the morphology and natural history of the species in southwestern China.

The original description of Natrix leonardi (currently Rhabdophis leonardi) by Frank Wall in 1923, based on a specimen from the "Upper Burma Hills," lacked important morphological details that have complicated the assignment of recently collected material. Furthermore, although the holotype was never lost, its location has been misreported in one important taxonomic reference, leading to further confusion. We report the correct repository of the holotype (Natural History Museum, London), together with its current catalog number. We also describe key features of that specimen that were omitted from the original description, and provide new details on the morphology of the species, including sexual dichromatism unusual for the genus, based upon specimens from southern Sichuan, China. Rhabdophis leonardi is distinguished from its congeners by the following characters: 15 or 17 DSR at midbody and 6 supralabials; distinct annulus around the neck, broad and red in males, and narrow and orange with a black border in females; dorsal ground color light green or olive; some lateral and dorsal scales possessing black edges, the frequency of black edges gradually increasing from anterior to posterior, forming irregular and ill-defined transverse black bands; eye with prominent green iris; black ventral spots with a red edge, most numerous at midbody but extending halfway down the length of the tail. In southwestern China, this species is frequently found at 1730-2230 m elevation. It has been documented to prey upon anuran amphibians, including toads. A recently published phylogenetic analysis showed this species to be deeply nested with the genus Rhabdophis, as a member of the R. nuchalis Group. That analysis also revealed the existence of two closely related but geographically distinct subclades in the molecular analysis, one of which may represent an unnamed taxon.

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.

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.

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.

Rediscovery and systematics of Rhinophis fergusonianus Boulenger, 1896 (Serpentes: Uropeltidae) from Indias Western Ghats.

The shieldtail snake Rhinophis fergusonianus Boulenger, 1896 was previously known only from the holotype specimen collected approximately 130 years ago from an imprecise Indian locality (Cardamom Hills). We report the rediscovery of this species from four localities, from low-elevation hills on both sides of the Palghat Gap in the southern part of the Western Ghats of peninsular India. We document new specimens of R. fergusonianus and provide a reassessment of the systematics of the species based on external morphology and DNA-sequence data. We also provide a revised key to the identification of Indian species of Rhinophis.

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.

The unexpected gecko: A new cryptic species within Urocotyledon inexpectata (Stejneger, 1893) from the northern granitic Seychelles.

Urocotyledon inexpectata, a small gecko endemic to the granitic islands of the Seychelles, has previously been demonstrated to comprise two highly distinct clades based on mitochondrial and nuclear DNA sequences, with one lineage exclusive to a northern group of islands, and the second lineage exclusive to the more southerly islands. Here we complement the genetic data with additional analyses to determine if the clades should be considered distinct species. We present and analyse new morphological data, including skull and jaw osteology, and supplement the available genetic data with DNA sequences for individuals from the previously unsampled island of Felicit, which cluster with the other northern island samples. Despite the high morphological similarity between Urocotyledon populations from northern and southern islands, diagnostic characters were identified, with the northern lineage having a more completely ossified skull, more tooth loci on maxilla and dentary, shorter hindlimbs, relatively smaller eye diameter and relatively wider head. We therefore describe the northern lineage as a distinct species, Urocotyledon norzilensis sp. nov., and discuss the biological and conservation management implications of this taxonomic action.

Rediscovery and systematics of the rarely encountered Blue-bellied kukri snake (Oligodon melaneus Wall, 1909) from Assam, India.

We report the rediscovery of Oligodon melaneus 112 years after its original description and document the third, and only non-type, specimen for the species. The new specimen was found 267 km east of the type locality (Tindharia, West Bengal state) from Assam state, India. We designate a lectotype for the species, and provide an extended description of a freshly collected male specimen. Phylogenetic analyses of 16s and cytb mitochondrial genes provide support for O. melaneus being closely related to the widespread South Asian endemic O. arnensis.

Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients.

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.

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.

A new pseudosuchian archosaur, Mambawakale ruhuhu gen. et sp. nov., from the Middle Triassic Manda Beds of Tanzania.

The Manda Beds of southwest Tanzania have yielded key insights into the early evolutionary radiation of archosaurian reptiles. Many key archosaur specimens were collected from the Manda Beds in the 1930s and 1960s, but until recently, few of these had been formally published. Here, we describe an archosaur specimen collected in 1963 which has previously been referred to informally as Pallisteria angustimentum. We recognize this specimen as the type of a new taxon, Mambawakale ruhuhu gen. et sp. nov. The holotype and only known specimen of M. ruhuhu comprises a partial skull of large size (greater than 75 cm inferred length), lower jaws and fragments of the postcranium, including three anterior cervical vertebrae and a nearly complete left manus. Mambawakale ruhuhu is characterized by several cranial autapomorphies that allow it to be distinguished with confidence from all other Manda Beds archosaurs, with the possible exception of Stagonosuchus nyassicus for which comparisons are highly constrained due to very limited overlapping material. Phylogenetic analysis suggests that M. ruhuhu is an early diverging pseudosuchian, but more precise resolution is hampered by missing data. Mambawakale ruhuhu is one of the largest known pseudosuchians recovered to date from the Middle Triassic.