Search

Pupil constriction has important functional consequences for animal vision, yet the evolutionary mechanisms underlying diverse pupil sizes and shapes are poorly understood. We aimed to quantify the diversity and evolution of pupil shapes among amphibians and to test for potential correlations to ecology based on functional hypotheses. Using photographs, we surveyed pupil shape across adults of 1294 amphibian species, 74 families and three orders, and additionally for larval stages for all families of frogs and salamanders with a biphasic ontogeny. For amphibians with a biphasic life history, pupil shape changed in many species that occupy distinct habitats before and after metamorphosis. In addition, non-elongated (circular or diamond) constricted pupils were associated with species inhabiting aquatic or underground environments, and elongated pupils (with vertical or horizontal long axes) 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.

Made available in DSpace on 2022-04-29T08:39:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 The spectral characteristics of vertebrate ocular lenses affect the image of the world that is projected onto the retina, and thus help shape diverse visual capabilities. Here, we tested whether amphibian lens transmission is driven by adaptation to diurnal activity (bright light) and/or scansorial habits (complex visual environments). Spectral transmission through the lenses of 79 species of frogs and six species of salamanders was measured, and data for 29 additional frog species compiled from published literature. Phylogenetic comparative methods were used to test ecological explanations of variation in lens transmission and to test for selection across traits. Lenses of diurnal (day-active) and scansorial (climbing) frogs transmitted significantly less shortwave light than those of non-diurnal or non-scansorial amphibians, and evolutionary modelling suggested that these differences have resulted from differential selection. The presence of shortwave-transparent lenses was common among the sampled amphibians, which implies that many are sensitive to shortwave light to some degree even in the absence of visual pigments maximally sensitive in the UV. This suggests that shortwave light, including UV, could play an important role in amphibian behaviour and ecology. Shortwave-absorbing lens pigments likely provide higher visual acuity to diurnally active frogs of multiple ecologies and to nocturnally active scansorial frogs. This new mechanistic understanding of amphibian visual systems suggests that shortwave-filtering lenses are adaptive not only in daylight conditions but also in those scotopic conditions where high acuity is advantageous. Read the free Plain Language Summary for this article on the Journal blog. Department of Life Sciences The Natural History Museum Department of Herpetology California Academy of Sciences Department of Vertebrate Zoology National Museum of Natural History Smithsonian Institution Department of Biology Amphibian and Reptile Diversity Research Center The University of Texas at Arlington Department of Biology York University Ecology Evolution and Development Group Department of Wetland Ecology Estación Biológica de Doñana (CSIC) Departamento de Biodiversidade and Centro de Aquicultura (CAUNESP) I.B. Universidade Estadual Paulista Department of Biology The Pennsylvania State University Department of Biological Sciences Institute for the Environment Florida International University Programa de Pós-graduação em Conservação da Fauna Universidade Federal de São Carlos Division of Optometry & Visual Science School of Health Sciences City University of London Departamento de Biodiversidade and Centro de Aquicultura (CAUNESP) I.B. Universidade Estadual Paulista

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.

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.

Animals with biphasic lifecycles often inhabit different visual environments across ontogeny. Many frogs and toads (Amphibia: Anura) have free-living aquatic larvae (tadpoles) that metamorphose into adults that inhabit a range of aquatic and terrestrial environments. Ecological differences influence eye size across species, but these relationships have not yet been explored across life stages in an ontogenetic allometric context. We examined eye-body size scaling in a species with aquatic larvae and terrestrial adults, the common frog Rana temporaria, using a well-sampled developmental series. We found a shift in ontogenetic allometric trajectory near metamorphosis indicating prioritized growth in tadpole eyes. To explore the effects of different tadpole and adult ecologies on eye-body scaling, we expanded our taxonomic sampling to include developmental series of eleven additional anuran species. Intraspecific eye-body scaling was variable among species, with 8/12 species exhibiting a significant change in allometric slope between tadpoles and adults. Traits categorizing both tadpole ecology (microhabitat, eye position, mouth position) and adult ecology (habitat, activity pattern) across species had significant effects on allometric slopes among tadpoles, but only tadpole eye position had a significant effect among adults. Our study suggests that relative eye growth in the preliminary stages of biphasic anuran ontogenies is somewhat decoupled and may be shaped by both immediate ecological need (i.e. tadpole visual requirements) and what will be advantageous during later adult stages.

Protocols used to extract mRNA from frog retinas, create cDNA libraries, and amplify opsins by PCR for sequencing at the UT core facility under their standard protocols. These protocols were used to obtain the opsin sequences in the paper: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection.

A correction to this paper has been published: https://doi.org/10.1007/s10682-021-10109-w

Most vertebrates use a combination of rod and cone photoreceptors to enable vision in conditions ranging from starlight to direct sunlight. Nocturnal geckos, however, have simplex retinas that contain only rods in terms of morphology and physiology, but these rods are thought to be derived from cones through an evolutionary process known as photoreceptor transmutation. To investigate this, we generated eye transcriptomes and analyzed patterns of phototransduction gene evolution in geckos in comparison to other reptiles. We confirm that geckos have lost several major components of the rod phototransduction pathway, including rod opsin (RH1), which we identified as a pseudogene in multiple genomes. We also identified a partial rod transducin transcript, but found no evidence of the protein in retinal sections. However, we find that geckos express several complete rod phototransduction transcripts in the eye, which may contribute to the rod-like physiology of nocturnal gecko photoreceptors. Finally, we found surprising evidence that even though photoreceptor transmutation evolved independently in geckos and snakes, they have experienced parallel shifts in selective constraint on phototransduction genes. These results implicate adaptive change in the underlying molecular machinery of visual transduction, in addition to the convergent changes in cellular morphology, during photoreceptor transmutation.

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.

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.

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.

Natural variation in the number, expression and function of sensory genes in an organism's genome is often tightly linked to different ecological and evolutionary forces. Opsin genes, which code for the first step in visual transduction, are ideal models for testing how ecological factors such as light environment may influence visual system adaptation. Neotropical cichlid fishes are a highly ecologically diverse group that evolved in a variety of aquatic habitats, including black (stained), white (opaque) and clear waters. We used cross-species exon capture to sequence Neotropical cichlid short wavelength-sensitive (SWS) opsins, which mediate ultraviolet (UV) to blue visual sensitivity. Neotropical cichlid SWS1 opsin (UV-sensitive) underwent a relaxation of selective constraint during the early phases of cichlid diversification in South America, leading to pseudogenization and loss. Conversely, SWS2a (blue-sensitive) experienced a burst of episodic positive selection at the base of the South American cichlid radiation. This burst coincides with SWS1 relaxation and loss, and is consistent with findings in ecomorphological studies characterizing a period of extensive ecological divergence in Neotropical cichlids. We use ancestral sequence reconstruction and protein modelling to investigate mutations along this ancestral branch that probably modified SWS2a function. Together, our results suggest that variable light environments played a prominent early role in shaping SWS opsin diversity during the Neotropical cichlid radiation. Our results also illustrate that long-term evolution under light-limited conditions in South America may have reduced visual system plasticity; specifically, early losses of UV sensitivity may have constrained the evolutionary trajectory of Neotropical cichlid vision.

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.

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.

Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via non-parallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems. This article is protected by copyright. All rights reserved

Ngatiwai Trust Board. et al., The tuatara (Sphenodon punctatus)—the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2—is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which—at approximately 5 Gb—is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing., N.J.G. acknowledges the support of Ngatiwai iwi, Allan Wilson Centre, University of Otago, New Zealand Department of Conservation, New Zealand Genomics and Illumina. J.I.A. was supported by CONICYT National Doctoral Scholarship No. 21130515. M.W. was supported by NIH grant R35 GM124827. Ensembl annotation was supported by the Wellcome Trust (WT108749/Z/15/Z) and the European Molecular Biology Laboratory.

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.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana

Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.

Bats represent one of the largest and most striking nocturnal mammalian radiations, exhibiting many visual system specializations for performance in light-limited environments. Despite representing the greatest ecological diversity and species richness in Chiroptera, Neotropical lineages have been undersampled in molecular studies, limiting the potential for identifying signatures of selection on visual genes associated with differences in bat ecology. Here, we investigated how diverse ecological pressures mediate long-term shifts in selection upon long-wavelength (Lws) and short-wavelength (Sws1) opsins, photosensitive cone pigments that form the basis of colour vision in most mammals, including bats. We used codon-based likelihood clade models to test whether ecological variables associated with reliance on visual information (e.g. echolocation ability and diet) or exposure to varying light environments (e.g. roosting behaviour and foraging habitat) mediated shifts in evolutionary rates in bat cone opsin genes. Using additional cone opsin sequences from newly sequenced eye transcriptomes of six Neotropical bat species, we found significant evidence for different ecological pressures influencing the evolution of the cone opsins. WhileLwsis evolving under significantly lower constraint in highly specialized high-duty cycle echolocating lineages, which have enhanced sonar ability to detect and track targets, variation inSws1constraint was significantly associated with foraging habitat, exhibiting elevated rates of evolution in species that forage among vegetation. This suggests that increased reliance on echolocation as well as the spectral environment experienced by foraging bats may differentially influence the evolution of different cone opsins. Our study demonstrates that different ecological variables may underlie contrasting evolutionary patterns in bat visual opsins, and highlights the suitability of clade models for testing ecological hypotheses of visual evolution.

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin--the vertebrate visual pigment mediating the first step in phototransduction at low-light levels--via codon-based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well-studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.

Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein invitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats.

Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here, we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via nonparallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems.

Three complete mitochondrial genomes of South American electric fishes (Gymnotiformes), derived from high-throughput RNA sequencing (RNA-Seq), are reported herein. We report the complete mitochondrial genome of the bluntnose knifefish Brachyhypopomus n.sp. VERD, determined from newly sequenced data. We also provide the complete mitochondrial genomes for Sternopygus arenatus and the electric eel Electrophorus electricus, assembled from previously published transcriptome data. The mitochondrial genomes of Brachyhypopomus n.sp. VERD, Sternopygus arenatus and Electrophorus electricus have 13 protein-coding genes, 1 D-loop, 2 ribosomal RNAs and 22 transfer RNAs, and are 16,547, 16,667 and 16,906 bp in length, respectively. Phylogenetic analysis of the eight available mitochondrial genomes of gymnotiform fishes shows Apteronotus to be the sister lineage of other gymnotiformes, contradicting the “Sinusoidea” hypothesis that Apteronotidae and Sternopygidae are sister taxa.

Studies of cichlid evolution have highlighted the importance of visual pigment genes in the spectacular radiation of the African rift lake cichlids. Recent work, however, has also provided strong evidence for adaptive diversification of riverine cichlids in the Neotropics, which inhabit environments of markedly different spectral properties from the African rift lakes. These ecological and/or biogeographic differences may have imposed divergent selective pressures on the evolution of the cichlid visual system. To test these hypotheses, we investigated the molecular evolution of the dim-light visual pigment, rhodopsin. We sequenced rhodopsin from Neotropical and African riverine cichlids and combined these data with published sequences from African cichlids. We found significant evidence for positive selection using random sites codon models in all cichlid groups, with the highest levels in African lake cichlids. Tests using branch-site and clade models that partitioned the data along ecological (lake, river) and/or biogeographic (African, Neotropical) boundaries found significant evidence of divergent selective pressures among cichlid groups. However, statistical comparisons among these models suggest that ecological, rather than biogeographic, factors may be responsible for divergent selective pressures that have shaped the evolution of the visual system in cichlids. We found that branch-site models did not perform as well as clade models for our data set, in which there was evidence for positive selection in the background. One of our most intriguing results is that the amino acid sites found to be under positive selection in Neotropical and African lake cichlids were largely nonoverlapping, despite falling into the same three functional categories: spectral tuning, retinal uptake/release, and rhodopsin dimerization. Taken together, these results would imply divergent selection across cichlid clades, but targeting similar functions. This study highlights the importance of molecular investigations of ecologically important groups and the flexibility of clade models in explicitly testing ecological hypotheses.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

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.

Studies of large-scale diversity changes and patterns of evolution can be adversely affected by a lack of understanding of alpha-taxonomy and systematics, and pachycephalosaur dinosaurs have significantly contributed to this problem. This is primarily a result of the relatively incomplete nature of the pachycephalosaur fossil record and the lack of understanding of specific and ontogenetic variation within the group. The taxon previously known as ‘Prenocephale’ brevis has been particularly problematic and has been placed in several different genera in addition to being synonymized with the species Stegoceras validum multiple times. Here we evaluate the validity of this taxon based on old and new material and using multiple, independent lines of evidence. We identify and describe the first peripheral skull elements, as well as the first juvenile and adult specimens, assignable to this taxon. These provide information on key parts of the morphology that have previously been lacking. Comparative anatomy, bivariate and multivariate morphometrics, osteohistology, and systematic analyses were used to test the hypothesis that this material is representative of a distinct taxon and to evaluate the differences between the other pachycephalosaur species of the closely related Belly River Group assemblage. No support was found for the assignment of ‘P.’ brevis to Stegoceras or Prenocephale, and thus we have erected a new genus, Foraminacephale gen. nov., to contain this species. Our data support the conclusion that the Foraminacephale brevis material represents an ontogenetic growth series of a single species distinct from St. validum. Evaluation of the two other members of the Belly River Group assemblage (Hanssuesia sternbergi and Colepiocephale lambei) was hindered by the small number of known specimens, but our results suggest that specimens currently referred to H. sternbergi may represent at least two species. We find that both ontogeny and osteohistology can vary greatly amongst even closely related species. It is therefore critical that multiple, independent lines of evidence be used to establish the ontogenetic trajectories of species prior to delineation, and that results of these analyses are used to produce robust and phylogenetically informative characters for use in phylogenetic analyses.

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

The pachycephalosaurian squamosal is one of the most diagnostic bones in this enigmatic group of dinosaurs, but little is known about variation in its morphology. Despite this, features of squamosal morphology are often used in diagnoses and phylogenetic studies. The recently proposed hypothesis of an ontogenetic transition from Dracorex to Pachycephalosaurus implies a large amount of ontogenetic variation in squamosal ornamentation that has not been well documented due to the small number of squamosals for these taxa. Stegoceras validum provides an important model for examining variation in squamosal morphology due to the large number of squamosals available. Here we examine the morphology of 14 S. validum squamosals both qualitatively and quantitatively. We find that although both node number and shape vary considerably, there is no clear ontogenetic pattern, which suggests a high degree of individual variation. The ornamental pattern, however, is maintained throughout the sample. Increase in n...

Colepiocephale lambei from the Foremost Formation of Alberta is a problematic pachycephalosaurid that has recently been hypothesized as (1) synonymous with Stegoceras validum, (2) a distinct species within the Stegoceras clade, and (3) an unusual taxon more derived than Stegoceras. Conflict among these hypotheses arises from different interpretations of the parietosquamosal region of the dome. Re-evaluation of the morphology of C. lambei based on all known material from Alberta indicates that the posteromedial extension of the parietal is broken off at its base in every specimen, and what was interpreted as the posterior margin of the dome actually is the anterior margins of large supratemporal fenestrae. The squamosals are not preserved in any specimen, but the form of the parietal suggests that they were present in the typical pachycephalosaurid configuration. Colepiocephale lambei is considered to be a distinct taxon based on several autapomorphies and stratigraphic segregation from S. validum...

Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (dN/dS) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Rhodopsin (rh1) is the visual pigment expressed in rod photoreceptors of vertebrates that is responsible for initiating the critical first step of dim-light vision. Rhodopsin is usually a single copy gene, however, we previously discovered a novel rhodopsin-like gene expressed in the zebrafish retina, rh1-2, which we identified as a functional photosensitive pigment that binds 11-cis retinal and activates in response to light. Here, we localize expression of rh1-2 in the zebrafish retina to a subset of peripheral photoreceptor cells, which indicates a partially overlapping expression pattern with rh1. We also express, purify, and characterize Rh1-2, including investigations of the stability of the biologically active intermediate. Using fluorescence spectroscopy, we find the half-life of the rate of retinal release of Rh1-2 following photoactivation to be more similar to the visual pigment rhodopsin than to the non-visual pigment exo-rhodopsin (exorh), which releases retinal around 5 times faster. Phylogenetic and molecular evolutionary analyses show that rh1-2 has ancient origins within teleost fishes, is under similar selective pressures to rh1, and likely experienced a burst of positive selection following its duplication and divergence from rh1. These findings indicate that rh1-2 is another functional visual rhodopsin gene, which contradicts the prevailing notion that visual rhodopsin is primarily found as a single copy gene within ray-finned fishes. The reasons for retention of this duplicate gene, as well as possible functional consequences for the visual system, are discussed.

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin--the vertebrate visual pigment mediating the first step in phototransduction at low-light levels--via codon-based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well-studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.

Taphonomic biases dictate how organisms are represented in the fossil record, but their effect on studies of vertebrate diversity dynamics is poorly studied. In contrast to the high diversity and abundance of small-bodied animals in extant ecosystems, small-bodied dinosaurs are less common than their large-bodied counterparts, but it is unclear whether this reflects unique properties of dinosaurian ecosystems or relates to taphonomic biases. A new, fully domed pachycephalosaurid dinosaur, Acrotholus audeti, from the Santonian of Alberta predates incompletely domed taxa, and provides important new information on pachycephalosaur evolution and the completeness of the ornithischian fossil record. Here we provide the first empirical evidence that the diversity of small-bodied ornithischian dinosaurs is strongly underestimated based on ghost lineages and the high proportion of robust and diagnostic frontoparietal domes compared with other pachycephalosaur fossils. This suggests preservational biases have a confounding role in attempts to decipher vertebrate palaeoecology and diversity dynamics through the Mesozoic.

Historically, studies of pachycephalosaurs have recognized plesiomorphically flat-headed taxa and apomorphically domed taxa. More recently, it has been suggested that the expression of the frontoparietal dome is ontogenetic and derived from a flat-headed juvenile morphology. However, strong evidence to support this hypothesis has been lacking. Here we test this hypothesis in a large, stratigraphically constrained sample of specimens assigned to Stegoceras validum, the best known pachycephalosaur, using multiple independent lines of evidence including conserved morphology of ornamentation, landmark-based allometric analyses of frontoparietal shape, and cranial bone histology. New specimens show that the diagnostic ornamentation of the parietosquamosal bar is conserved throughout the size range of the sample, which links flat-headed specimens to domed S. validum. High-resolution CT scans of three frontoparietals reveal that vascularity decreases with size and document a pattern that is consistent with previously proposed histological changes during growth. Furthermore, aspects of dome shape and size are strongly correlated and indicative of ontogenetic growth. These results are complementary and strongly support the hypothesis that the sample represents a growth series of a single taxon. Cranial dome growth is positively allometric, proceeds from a flat-headed to a domed state, and confirms the synonymy of Ornatotholus browni as a juvenile Stegoceras. This dataset serves as the first detailed model of growth and variation in a pachycephalosaur. Flat-headed juveniles possess three characters (externally open cranial sutures, tuberculate dorsal surface texture, and open supratemporal fenestrae) that are reduced or eliminated during ontogeny. These characters also occur in putative flat-headed taxa, suggesting that they may also represent juveniles of domed taxa. However, open cranial sutures and supratemporal fenestrae are plesiomorphic within Ornithischia, and thus should be expected in the adult stage of a primitive pachycephalosaur. Additional lines of evidence will be needed to resolve the taxonomic validity of flat-headed pachycephalosaur taxa.