Multiple Ecological Axes Drive Molecular Evolution of Cone Opsins in Beloniform Fishes.

Ecological and evolutionary transitions offer an excellent opportunity to examine the molecular basis of adaptation. Fishes of the order Beloniformes include needlefishes, flyingfishes, halfbeaks, and allies, and comprise over 200 species occupying a wide array of habitats-from the marine epipelagic zone to tropical rainforest rivers. These fishes also exhibit a diversity of diets, including piscivory, herbivory, and zooplanktivory. We investigated how diet and habitat affected the molecular evolution of cone opsins, which play a key role in bright light and colour vision and are tightly linked to ecology and life history. We analyzed a targeted-capture dataset to reconstruct the evolutionary history of beloniforms and assemble cone opsin sequences. We implemented codon-based clade models of evolution to examine how molecular evolution was affected by habitat and diet. We found high levels of positive selection in medium- and long-wavelength beloniform opsins, with piscivores showing increased positive selection in medium-wavelength opsins and zooplanktivores showing increased positive selection in long-wavelength opsins. In contrast, short-wavelength opsins showed purifying selection. While marine/freshwater habitat transitions have an effect on opsin molecular evolution, we found that diet plays a more important role. Our study suggests that evolutionary transitions along ecological axes produce complex adaptive interactions that affect patterns of selection on genes that underlie vision.

Ecologically mediated differences in electric organ discharge drive evolution in a sodium channel gene in South American electric fishes.

Active electroreception-the ability to detect objects and communicate with conspecifics via the detection and generation of electric organ discharges (EODs)-has evolved convergently in several fish lineages. South American electric fishes (Gymnotiformes) are a highly species-rich group, possibly in part due to evolution of an electric organ (EO) that can produce diverse EODs. Neofunctionalization of a voltage-gated sodium channel gene accompanied the evolution of electrogenic tissue from muscle and resulted in a novel gene (scn4aa) uniquely expressed in the EO. Here, we investigate the link between variation in scn4aa and differences in EOD waveform. We combine gymnotiform scn4aa sequences encoding the C-terminus of the Nav1.4a protein, with biogeographic data and EOD recordings to test whether physiological transitions among EOD types accompany differential selection pressures on scn4aa. We found positive selection on scn4aa coincided with shifts in EOD types. Species that evolved in the absence of predators, which likely selected for reduced EOD complexity, exhibited increased scn4aa evolutionary rates. We model mutations in the protein that may underlie changes in protein function and discuss our findings in the context of gymnotiform signalling ecology. Together, this work sheds light on the selective forces underpinning major evolutionary transitions in electric signal production.

Convergent evolution of dim light vision in owls and deep-diving whales.

Animals with enhanced dim-light sensitivity are at higher risk of light-induced retinal degeneration when exposed to bright light conditions.1,2,3,4 This trade-off is mediated by the rod photoreceptor sensory protein, rhodopsin (RHO), and its toxic vitamin A chromophore by-product, all-trans retinal.5,6,7,8 Rod arrestin (Arr-1) binds to RHO and promotes sequestration of excess all-trans retinal,9,10 which has recently been suggested as a protective mechanism against photoreceptor cell death.2,11 We investigated Arr-1 evolution in animals at high risk of retinal damage due to periodic bright-light exposure of rod-dominated retinas. Here, we find the convergent evolution of enhanced Arr-1/RHO all-trans-retinal sequestration in owls and deep-diving whales. Statistical analyses reveal a parallel acceleration of Arr-1 evolutionary rates in these lineages, which is associated with the introduction of a rare Arr-1 mutation (Q69R) into the RHO-Arr-1 binding interface. Using in vitro assays, we find that this single mutation significantly enhances RHO-all-trans-retinal sequestration by ∼30%. This functional convergence across 300 million years of evolutionary divergence suggests that Arr-1 and RHO may play an underappreciated role in the photoprotection of the eye, with potentially vast clinical significance.

Origins and evolution of biological novelty.

Understanding the origins and impacts of novel traits has been a perennial interest in many realms of ecology and evolutionary biology. Here, we build on previous evolutionary and philosophical treatments of this subject to encompass novelties across biological scales and eco-evolutionary perspectives. By defining novelties as new features at one biological scale that have emergent effects at other biological scales, we incorporate many forms of novelty that have previously been treated in isolation (such as novelty from genetic mutations, new developmental pathways, new morphological features, and new species). Our perspective is based on the fundamental idea that the emergence of a novelty, at any biological scale, depends on its environmental and genetic context. Through this lens, we outline a broad array of generative mechanisms underlying novelty and highlight how genomic tools are transforming our understanding of the origins of novelty. Lastly, we present several case studies to illustrate how novelties across biological scales and systems can be understood based on common mechanisms of change and their environmental and genetic contexts. Specifically, we highlight how gene duplication contributes to the evolution of new complex structures in visual systems; how genetic exchange in symbiosis alters functions of both host and symbiont, resulting in a novel organism; and how hybridisation between species can generate new species with new niches.

Adaptation of Antarctic Icefish Vision to Extreme Environments.

Extreme environments, such as Antarctic habitats, present major challenges for many biological processes. Antarctic icefishes (Crynotothenioidea) represent a compelling system to investigate the molecular basis of adaptation to cold temperatures. Here, we explore how the sub-zero habitats of Antarctic icefishes have impacted rhodopsin (RH1) function, the temperature-sensitive dim-light visual pigment found in rod photoreceptors. Using likelihood models and ancestral reconstruction, we find that accelerated evolutionary rates in icefish RH1 underlie unique amino acid mutations absent from other deep-dwelling fishes, introduced before (S160A) and during (V259M) the onset of modern polar conditions. Functional assays reveal that these mutations red-shift rhodopsin spectral absorbance, consistent with spectral irradiance under sea ice. These mutations also lower the activation energy associated with retinal release of the light-activated RH1, and accelerate its return to the dark state, likely compensating for a cold-induced decrease in kinetic rates. These are adaptations in key properties of rhodopsin that mediate rod sensitivity and visual performance in the cold dark seas of the Antarctic.

Evolution, inactivation and loss of short wavelength-sensitive opsin genes during the diversification of Neotropical cichlids.

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.

Genetic structure of endangered lake chubsucker Erimyzon sucetta in Canada reveals a differentiated population in a precarious habitat.

We used mitochondrial DNA to assess the genetic structure of endangered lake chubsucker Erimyzon sucetta across its Canadian range. We found unique mitochondrial haplotypes in Lyons Creek, a tributary of the Niagara River that faces a strong potential for habitat deterioration. Lyons Creek may therefore serve as a reservoir of unique genetic diversity. The sensitivity of Lyons Creek, combined with the genetic uniqueness of its E. sucetta population, call for further investigation into whether this population should be considered a separate designatable unit for conservation purposes.

Convergent selection pressures drive the evolution of rhodopsin kinetics at high altitudes via nonparallel mechanisms.

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.

Insights into visual pigment adaptation and diversity from model ecological and evolutionary systems.

Sensory systems provide valuable insight into the evolution of molecular mechanisms underlying organismal anatomy, physiology, and behaviour. Visual pigments, which mediate the first step in visual transduction, offer a unique window into the relationship between molecular variation and visual performance, and enhance our understanding of how ecology, life history, and physiology may shape genetic variation across a variety of organisms. Here we review recent work investigating vertebrate visual pigments from a number of perspectives. Opsin gene duplication, loss, differential expression, structural variation, and the physiological context in which they operate, have profoundly shaped the visual capabilities of vertebrates adapting to novel environments. We note the importance of conceptual frameworks in investigating visual pigment diversity in vertebrates, highlighting key examples including evolutionary transitions between different photic environments, major shifts in life history evolution and ecology, evolutionary innovations in visual system anatomy and physiology, as well as shifts in visually mediated behaviours and behavioural ecology. We emphasize the utility of studying visual pigment evolution in the context of these different perspectives, and demonstrate how the integrative approaches discussed in this review contribute to a better understanding of the underlying molecular processes mediating adaptation in sensory systems, and the contexts in which they occur.

Accelerated Evolution and Functional Divergence of the Dim Light Visual Pigment Accompanies Cichlid Colonization of Central America.

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.

Evolution of nonspectral rhodopsin function at high altitudes.

High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.

An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease.

Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.

A comparative study of rhodopsin function in the great bowerbird (Ptilonorhynchus nuchalis): Spectral tuning and light-activated kinetics.

Rhodopsin is the visual pigment responsible for initiating the phototransduction cascade in vertebrate rod photoreceptors. Although well-characterized in a few model systems, comparative studies of rhodopsin function, particularly for nonmammalian vertebrates are comparatively lacking. Bowerbirds are rare among passerines in possessing a key substitution, D83N, at a site that is otherwise highly conserved among G protein-coupled receptors. While this substitution is present in some dim-light adapted vertebrates, often accompanying another unusual substitution, A292S, its functional relevance in birds is uncertain. To investigate functional effects associated with these two substitutions, we use the rhodopsin gene from the great bowerbird (Ptilonorhynchus nuchalis) as a background for site-directed mutagenesis, in vitro expression and functional characterization. We also mutated these sites in two additional rhodopsins that do not naturally possess N83, chicken and bovine, for comparison. Both sites were found to contribute to spectral blue-shifts, but had opposing effects on kinetic rates. Substitutions at site 83 were found to primarily affect the kinetics of light-activated rhodopsin, while substitutions at site 292 had a larger impact on spectral tuning. The contribution of substitutions at site 83 to spectral tuning in particular depended on genetic background, but overall, the effects of substitutions were otherwise surprisingly additive, and the magnitudes of functional shifts were roughly similar across all three genetic backgrounds. By employing a comparative approach with multiple species, our study provides new insight into the joint impact of sites 83 and 292 on rhodopsin structure-function as well as their evolutionary significance for dim-light vision across vertebrates.

Comparative sequence analyses of rhodopsin and RPE65 reveal patterns of selective constraint across hereditary retinal disease mutations.

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 (d N/d S) 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.

Spectral tuning in vertebrate short wavelength-sensitive 1 (SWS1) visual pigments: can wavelength sensitivity be inferred from sequence data?

The molecular mechanisms underlying the enormous diversity of visual pigment wavelength sensitivities found in nature have been the focus of many molecular evolutionary studies, with particular attention to the short wavelength-sensitive 1 (SWS1) visual pigments that mediate vision in the ultraviolet to violet range of the electromagnetic spectrum. Over a decade of study has revealed that the remarkable extension of SWS1 absorption maxima (λ max ) into the ultraviolet occurs through a deprotonation of the Schiff base linkage of the retinal chromophore, a mechanism unique to this visual pigment type. In studies of visual ecology, there has been mounting interest in inferring visual sensitivity at short wavelengths, given the importance of UV signaling in courtship displays and other behaviors. Since experimentally determining spectral sensitivities can be both challenging and time-consuming, alternative strategies such as estimating λ max based on amino acids at sites known to affect spectral tuning are becoming increasingly common. However, these estimates should be made with knowledge of the limitations inherent in these approaches. Here, we provide an overview of the current literature on SWS1 site-directed mutagenesis spectral tuning studies, and discuss methodological caveats specific to the SWS1-type pigments. We focus particular attention on contrasting avian and mammalian SWS1 spectral tuning mechanisms, which are the best studied among vertebrates. We find that avian SWS1 visual pigment spectral tuning mechanisms are fairly consistent, and therefore more predictable in terms of wavelength absorption maxima, whereas mammalian pigments are not well suited to predictions of λ max from sequence data alone.

Divergent positive selection in rhodopsin from lake and riverine cichlid fishes.

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.

Retinal region of polarization sensitivity switches during ontogeny of rainbow trout.

Polarization sensitivity (PS) in vertebrate vision is controversial, perhaps because its underlying mechanism has remained obscure. An issue that might have added to the controversy is that rainbow trout (Oncorhynchus mykiss), which have served as the primary model system for polarization-based orientation, lose their ability to orient relative to celestial polarized-light patterns when parr (fry) transform into migratory smolts (juveniles), which would benefit most from polarization-based orientation. Here we addressed two key questions: (1) what is the mechanism underling PS?, and (2) how can the paradoxical loss of PS in trout smolts be reconciled? We assessed PS from optic nerve recordings in parr and smolts and found that the retinal region with enhanced PS shifted from the ventral retina in parr to the dorsal retina in smolts. This adaptation may allow fish to use the most reliable polarization field encountered at each life stage, the celestial polarization field in the shallow-swimming parr and the depth-insensitive underwater polarization field in the deep-swimming smolts. In addition, we assessed spectral sensitivity across the retina and during ontogeny and fit a cascade retinal model to PS data. We found that differential contribution of two cone detectors with orthogonal PS could drive the variation in PS and that feedback from horizontal cells to cones could explain the differential amplification of PS. This elegant arrangement, in which weak PS of cones is amplified and tuned by retinal networks, allows for PS without interfering with sampling of other visual information and illustrates how sensory systems may simultaneously process disparate aspects of physical environments.

Geophagus crocatus, a new species of geophagine cichlid from the Berbice River, Guyana, South America (Teleostei: Cichlidae).

We describe a new Geophagus from the Berbice River of Guyana, bringing the total number of described species in the genus to 19, and of Guianese species to six.. Geophagus crocatus, new species, is distinguished from all species of Geophagus outside of the G. surinamensis group by the presence of an incomplete suborbital stripe (vs. complete), and the presence of six lateral bars, with bars 2 and 3 slightly sloping toward each other and fusing dorsally at the base of the dorsal fin. Geophagus crocatus is the only Geophagus species known from the Berbice River, and it is present above and below the Itabru Falls.

Ontogeny in the visual system of Nile tilapia.

Retinal neurogenesis in fish facilitates cellular rearrangement throughout ontogeny, potentially allowing for optimization of the visual system to shifts in habitat and behaviour. To test this possibility, we studied the developmental trajectory of the photopic visual process in the Nile tilapia. We examined ontogenetic changes in lens transmission, photoreceptor sensitivity and post-receptoral sensitivity, and used these to estimate changes in cone pigment frequency and retinal circuitry. We observed an ontogenetic decrease in ultraviolet (UV) photoreceptor sensitivity, which resulted from a reduction in the SWS1 cone pigment frequency, and was associated with a reduction in lens transmission at UV wavelengths. Additionally, post-receptoral sensitivity to both UV and long wavelengths decreased with age, probably reflecting changes in photoreceptor sensitivity and retinal circuitry. This novel remodelling of retinal circuitry occurred following maturation of the visual system but prior to reaching adulthood, and thus may facilitate optimization of the visual system to the changing sensory demands. Interestingly, the changes in post-receptoral sensitivity to long wavelengths could not be predicted by the changes observed in lens transmission, cone pigment frequency or photoreceptor sensitivity. This finding emphasizes the importance of considering knowledge of visual sensitivity and retinal processing when studying visual adaptations and attempting to relate visual function to the natural environment. This study advances our understanding of ontogeny in visual systems and demonstrates that the association between different elements of the visual process can be explored effectively by examining visual function throughout ontogeny.