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Non-visual opsins are transmembrane proteins expressed in the eyes, skin, and brain of many animals. When paired with a light-sensitive chromophore, non-visual opsins form photopigment systems involved in various non-visual, light-detection functions, including circadian rhythm regulation, light-seeking behavior, and detection of seasonality. Previous research has primarily explored the diversity and function of non-visual opsins in model organisms, with few studies investigating their molecular evolution in non-model species. Here we explored molecular evolution of non-visual opsin genes in anuran amphibians (frogs and toads). With diverse lifestyles ranging from fossorial to aquatic, anurans inhabit a diverse array of light environments, which makes them a compelling system for studying the evolution of light detection mechanisms. Using whole-eye transcriptomes from 79 anuran species, as well as genomes and multi-tissue transcriptomes from an additional 15 species, we 1) identify which non-visual opsin genes are expressed in the eyes of anurans; 2) compare selective constraint (ω, or dN/dS) among non-visual opsin genes; and 3) test for potential adaptive evolution by comparing selection between discrete ecological classes in anurans (e.g. diurnal versus non-diurnal). We consistently recovered 14 non-visual opsin genes from anuran eye transcriptomes, compared to 18 genes that we recovered genome wide, and detected positive selection in a subset of these genes. We also found variation in selective constraint between discrete ecological and life-history classes, which may reflect functional adaptation in non-visual opsin genes. Although non-visual opsins remain poorly understood, these findings provide insight into their molecular evolution and set the stage for future research on their potential function across taxa with diverse environmental, developmental, and morphological adaptations.

Among major vertebrate groups, anurans (frogs and toads) are understudied with regards to their visual systems and little is known about variation among species that differ in ecology. We sampled North American anurans representing diverse evolutionary and life histories that likely possess visual systems adapted to meet different ecological needs. Using standard molecular techniques, visual opsin genes, which encode the protein component of visual pigments, were obtained from anuran retinas. Additionally, we extracted the visual opsins from publicly available genome and transcriptome assemblies, further increasing the phylogenetic and ecological diversity of our dataset. We found that anurans consistently express four visual opsin genes (RH1, LWS, SWS1, and SWS2, but not RH2) even though reported photoreceptor complements vary widely among species. We found the first evidence of visual opsin duplication in an amphibian with the duplication of the LWS gene in the African bullfrog, which had distinct LWS copies on the sex chromosomes. The proteins encoded by these genes showed considerable sequence variation among species, including at sites known to shift the spectral sensitivity of visual pigments in other vertebrates and thus mediate dim-light and color vision. Using molecular evolutionary analyses of selection (dN/dS) we found significant evidence for positive selection at a subset of sites in the dim-light rod opsin gene RH1 and the long wavelength sensitive cone opsin gene LWS. The function of sites inferred to be under positive selection are largely unknown, but a few are likely to affect spectral sensitivity and other visual pigment functions based on proximity to previously identified sites in other vertebrates. The observed variation cannot fully be explained by evolutionary relationships among species alone. Taken together, our results suggest that other ecological factors, such as habitat and life history, as well as behaviour, may be driving changes to anuran visual systems.

Pupil constriction has important functional consequences for animal vision, yet the evolutionary mechanisms underlying diverse pupil sizes and shapes, often among animals that occupy optically similar environments, are poorly understood. We aimed to quantify the diversity and evolution of pupil shapes among amphibians and test for potential correlations to ecology based on functional hypotheses. Using photographs, we surveyed pupil shape and the orientation of the constricted pupil across adults of 1293 amphibian species, 72 families, and 3 orders, and additionally for larval life stages for all families of frogs and salamanders with a biphasic ontogeny. Pupil shape is exceptionally diverse in amphibians with evolutionary transitions throughout the amphibian tree of life. For amphibians with a biphasic life history, we found that pupils change in many species that occupy distinct habitats before and after metamorphosis. Finally, we found that non-elongated (round or diamond) constricted pupils were correlated with species inhabiting consistently dim light environments (burrowing and aquatic species) and that elongated pupils (vertical and horizontal) were more common in species with larger absolute eye sizes. We propose that amphibians provide a valuable group within which to explore the anatomical, physiological, optical, and ecological mechanisms underlying the evolution of pupil shape.

Many animals have complex life cycles where larval and adult forms have distinct ecologies and habitats that impose different demands on their sensory systems. While the adaptive decoupling hypothesis predicts reduced genetic correlations between life stages, how sensory systems adapt across life stages at the molecular level is not well understood. Frogs are a compelling system to study this question in because most species rely on vision as both aquatic tadpoles and terrestrial adults, but these habitats present vastly different light environments. Here we used whole eye transcriptome sequencing to investigate differential expression between aquatic tadpoles and terrestrial juveniles of the southern leopard frog (Lithobates sphenocephalus). Because visual physiology changes with light levels, we also tested how constant light or dark exposure affected gene expression. We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles, versus 5% for light/dark exposure. Analyses targeting a curated set of visual genes revealed significant differential expression between life stages in genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Light/dark exposure had a significant effect on a smaller set of visual genes. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Overall, we identified extensive expression-level differences in the eyes of tadpole and juvenile frogs related to observed morphological and physiological changes through metamorphosis, and corresponding adaptive shifts to optimize vision in aquatic versus terrestrial environments.

The tuatara (Sphenodon punctatus), the only living member of the archaic reptilian order Rhynchocephalia (Sphenodontia) once widespread across Gondwana, is an iconic and enigmatic terrestrial vertebrate endemic to New Zealand. A key link to the now extinct stem reptiles from which dinosaurs, modern reptiles, birds and mammals evolved, the tuatara provides exclusive insights into the ancestral amniotes. The tuatara genome, at ∼5 Gbp, is among the largest vertebrate genomes assembled. Analysis of this genome and comparisons to other vertebrates reinforces the uniqueness of the tuatara. Phylogenetic analyses indicate tuatara diverged from the snakes and lizards ∼250 MYA. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Genome sequence analysis identifies expansions of protein, non-protein-coding RNA families, and repeat elements, the latter of which show an extraordinary amalgam of reptilian and mammalian features. Sequencing of this genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. It also provides important insights into both the technical challenges and the cultural obligations associated with genome sequencing.

Colubridae is the largest and most diverse family of snakes, with visual systems that reflect this diversity, encompassing a variety of retinal photoreceptor organizations. The transmutation theory proposed by Walls postulates that photoreceptors could evolutionarily transition between cell types in squamates, but few studies have tested this theory. Recently, evidence for transmutation and rod-like machinery in an all cone retina has been identified in a diurnal garter snake (Thamnophis), and it appears that the rhodopsin gene at least may be widespread among colubrid snakes. However, functional evidence supporting transmutation beyond the existence of the rhodopsin gene remains rare. We examined the all cone retina of another diurnal colubrid,Pituophis melanoleucus, distantly related toThamnophis. We found thatP. melanoleucusexpresses two cone opsins (SWS1, LWS) and rhodopsin (RH1) within the eye. Immunohistochemistry localized rhodopsin to the outer segment of photoreceptors in the all-cone retina of the snake and all opsin genes produced functional visual pigments when expressedin vitro. Consistent with other studies, we found thatP. melanoleucusrhodopsin is extremely blue-shifted. Surprisingly,P. melanoleucusrhodopsin reacted with hydroxylamine, a typical cone opsin characteristic. These results support the idea that the rhodopsin-containing photoreceptors ofP. melanoleucusare the products of evolutionary transmutation from rod ancestors, and suggests that this phenomenon may be widespread in colubrid snakes. We hypothesize that transmutation may be an adaptation for diurnal, brighter-light vision, which could result in increased spectral sensitivity and chromatic discrimination with the potential for colour vision.Summary StatementThe all cone retina of the colubrid snake,Pituophis melanoleucuscontains a blue-shifted rhodopsin with cone opsin-like properties, which may have been adaptive in diurnal snakes.

Despite continued advances in sequencing technologies, there is a need for methods that can efficiently sequence large numbers of genes from diverse species. One approach to accomplish this is targeted capture (hybrid enrichment). While these methods are well established for genome resequencing projects, cross-species capture strategies are still being developed and generally focus on the capture of conserved regions, rather than complete coding regions from specific genes of interest. The resulting data is thus useful for phylogenetic studies, but the wealth of comparative data that could be used for evolutionary and functional studies is lost. Here we design and implement a targeted capture method that enables recovery of complete coding regions across broad taxonomic scales. Capture probes were designed from multiple reference species and extensively tiled in order to facilitate cross-species capture. Using novel bioinformatics pipelines we were able to recover nearly all of the targeted genes with high completeness from species that were up to 200 myr divergent. Increased probe diversity and tiling for a subset of genes had a large positive effect on both recovery and completeness. The resulting data produced an accurate species tree, but importantly this same data can also be applied to studies of molecular evolution and function that will allow researchers to ask larger questions in broader phylogenetic contexts. Our method demonstrates the utility of cross-species approaches for the capture of full length coding sequences, and will substantially improve the ability for researchers to conduct large-scale comparative studies of molecular evolution and function.

SummaryWe present BlastPhyMe (BLAST, Phylogenies, and Molecular Evolution) a new application to facilitate the fast and easy generation and analysis of protein-coding sequence datasets. The application uses a portable database framework to manage and organize sequences along with a graphical user interface (GUI) that makes the application extremely easy to use. BlastPhyMe utilizes several existing services and applications in a unique way that save researchers considerable time when building and analyzing protein-coding datasets. The application consists of two modules that can be used separately or together. The first module enables the assembly of coding sequence datasets. BLAST searches can be used to obtain all related sequences of interest from NCBI. Full GenBank records are saved within the database and coding sequences are automatically extracted. A feature of particular note is that sequences can be sorted based on NCBI taxonomic hierarchy before export for visualization using existing tools, such as fast. The application provides GUIs for automatic alignment of sequences with the popular tools MUSCLE and PRANK, as well as for reconstructing phylogenetic trees using PhyML. The second module incorporates selection analyses using codon-based likelihood methods. The alignments and phylogenetic trees generated with the dataset module, or those generated elsewhere, can be used to run the models implemented in the codeml PAML package. A GUI allows easy selection of models and parameters. Importantly, replicate analyses with different parameter starting values can be automatically performed in order to ensure selection of the best-fitting model. Multiple analyses can be run simultaneously based on the number of processor cores available, while additional analyses will be run iteratively until completed. Results are saved within the database and can be exported to publication-ready Excel tables, which further automatically compute the appropriate likelihood ratio test between models in order to determine statistical significance. Future updates will add additional options for phylogenetic reconstruction (eg, MrBayes) and selection analyses (eg, HYPHY). BlastPhyMe saves researches of all bioinformatics experience levels considerable time by automating the numerous tasks required for the generation and analysis of protein-coding sequence datasets using a straightforward graphical interface.AvailabilityInstallation package and source code available from: https://github.com/ryankschott/BlastPhyMeContactschottr@si.edu