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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

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.

Among major vertebrate groups, anurans (frogs and toads) are understudied with regard 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 to 33 species in total. We found that anurans consistently express four visual opsin genes (

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.

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.

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.

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

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.

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.

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.

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.

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.