Long-wavelength sensitive visual pigments of the guppy (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>): six opsins expressed in a single individual

Background

The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns.

Results

We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages.

Conclusion

The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppy's behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.

     

Why UV vision and red vision are important for damselfish (Pomacentridae): structural and expression variation in opsin genes

Coral reefs belong to the most diverse ecosystems on our planet. The diversity in coloration and lifestyles of coral reef fishes makes them a particularly promising system to study the role of visual communication and adaptation. Here, we investigated the evolution of visual pigment genes (opsins) in damselfish (Pomacentridae) and examined whether structural and expression variation of opsins can be linked to ecology. Using DNA sequence data of a phylogenetically representative set of 31 damselfish species, we show that all but one visual opsin are evolving under positive selection. In addition, selection on opsin tuning sites, including cases of divergent, parallel, convergent and reversed evolution, has been strong throughout the radiation of damselfish, emphasizing the importance of visual tuning for this group. The highest functional variation in opsin protein sequences was observed in the short‐ followed by the long‐wavelength end of the visual spectrum. Comparative gene expression analyses of a subset of the same species revealed that with SWS1, RH2B and RH2A always being expressed, damselfish use an overall short‐wavelength shifted expression profile. Interestingly, not only did all species express SWS1 – a UV‐sensitive opsin – and possess UV‐transmitting lenses, most species also feature UV‐reflective body parts. This suggests that damsels might benefit from a close‐range UV‐based ‘private’ communication channel, which is likely to be hidden from ‘UV‐blind’ predators. Finally, we found that LWS expression is highly correlated to feeding strategy in damsels with herbivorous feeders having an increased LWS expression, possibly enhancing the detection of benthic algae.     

Visual pigment evolution in Characiformes: The dynamic interplay of teleost whole‐genome duplication,surviving opsins and spectral tuning

Vision represents an excellent model for studying adaptation, given the genotype‐to‐phenotype map that has been characterized in a number of taxa. Fish possess a diverse range of visual sensitivities and adaptations to underwater light, making them an excellent group to study visual system evolution. In particular, some speciose but understudied lineages can provide a unique opportunity to better understand aspects of visual system evolution such as opsin gene duplication and neofunctionalization. In this study, we showcase the visual system evolution of neotropical Characiformes and the spectral tuning mechanisms they exhibit to modulate their visual sensitivities. Such mechanisms include gene duplications and losses, gene conversion, opsin amino acid sequence and expression variation, and A₁/A₂‐chromophore shifts. The Characiforms we studied utilize three cone opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform's entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss (SWS1, RH2) and duplication (LWS, RH1). The LWS‐ and RH1‐duplicates originated from a teleost specific whole‐genome duplication as well as characiform‐specific duplication events. Both LWS‐opsins exhibit gene conversion and, through substitutions in key tuning sites, one of the LWS‐paralogues has acquired spectral sensitivity to green light. These sequence changes suggest reversion and parallel evolution of key tuning sites. Furthermore, characiforms' colour vision is based on the expression of both LWS‐paralogues and SWS2. Finally, we found interspecific and intraspecific variation in A₁/A₂‐chromophores proportions, correlating with the light environment. These multiple mechanisms may be a result of the diverse visual environments where Characiformes have evolved.     

Gene Duplication and Divergence of Long Wavelength-Sensitive Opsin Genes in the Guppy, Poecilia reticulata

Female preference for male orange coloration in the genus Poecilia suggests a role for duplicated long wavelength-sensitive (LWS) opsin genes in facilitating behaviors related to mate choice in these species. Previous work has shown that LWS gene duplication in this genus has resulted in expansion of long wavelength visual capacity as determined by microspectrophotometry (MSP). However, the relationship between LWS genomic repertoires and expression of LWS retinal cone classes within a given species is unclear. Our previous study in the related species, Xiphophorus helleri, was the first characterization of the complete LWS opsin genomic repertoire in conjunction with MSP expression data in the family Poeciliidae, and revealed the presence of four LWS loci and two distinct LWS cone classes. In this study we characterized the genomic organization of LWS opsin genes by BAC clone sequencing, and described the full range of cone cell types in the retina of the colorful Cumaná guppy, Poecilia reticulata. In contrast to X. helleri, MSP data from the Cumaná guppy revealed three LWS cone classes. Comparisons of LWS genomic organization described here for Cumaná to that of X. helleri indicate that gene divergence and not duplication was responsible for the evolution of a novel LWS haplotype in the Cumaná guppy. This lineage-specific divergence is likely responsible for a third additional retinal cone class not present in X. helleri, and may have facilitated the strong sexual selection driven by female preference for orange color patterns associated with the genus Poecilia.     

Opsin expression predicts male nuptial color in threespine stickleback

Theoretical models of sexual selection suggest that male courtship signals can evolve through the build‐up of genetic correlations between the male signal and female preference. When preference is mediated via increased sensitivity of the signal characteristics, correlations between male signal and perception/sensitivity are expected. When signal expression is limited to males, we would expect to find signal‐sensitivity correlations in males. Here, we document such a correlation within a breeding population of threespine stickleback mediated by differences in opsin expression. Males with redder nuptial coloration express more long‐wavelength‐sensitive (LWS) opsin, making them more sensitive to orange and red. This correlation is not an artifact of shared tuning to the optical microhabitat. Such correlations are an essential feature of many models of sexual selection, and our results highlight the potential importance of opsin expression variation as a substrate for signal‐preference evolution. Finally, these results suggest a potential sensory mechanism that could drive negative frequency‐dependent selection via male–male competition and thus maintain variation in male nuptial color.     

Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations

Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.     

Evolutionary ecology of opsin gene sequence,expression and repertoire

Linking molecular evolution to biological function is a long‐standing challenge in evolutionary biology. Some of the best examples of this involve opsins, the genes that encode the molecular basis of light reception. In this issue of Molecular Ecology, three studies examine opsin gene sequence, expression and repertoire to determine how natural selection has shaped the visual system. First, Escobar‐Camacho et al. ( 2017 ) use opsin repertoire and expression in three Amazonian cichlid species to show that a shift in sensitivity towards longer wavelengths is coincident with the long‐wavelength‐dominated Amazon basin. Second, Stieb et al. ( 2017 ) explore opsin sequence and expression in reef‐dwelling damselfish and find that UV‐ and long‐wavelength vision are both important, but likely for different ecological functions. Lastly, Suvorov et al. ( 2017 ) study an expansive opsin repertoire in the insect order Odonata and find evidence that copy number expansion is consistent with the permanent heterozygote model of gene duplication. Together these studies emphasize the utility of opsin genes for studying both the local adaptation of sensory systems and, more generally, gene family evolution.     

Color vision diversity and significance in primates inferred from genetic and field studies

Color provides a reliable cue for object detection and identification during various behaviors such as foraging, mate choice, predator avoidance and navigation. The total number of colors that a visual system can discriminate is largely dependent on the number of different spectral types of cone opsins present in the retina and the spectral separations among them. Thus, opsins provide an excellent model system to study evolutionary interconnections at the genetic, phenotypic and behavioral levels. Primates have evolved a unique ability for three-dimensional color vision (trichromacy) from the two-dimensional color vision (dichromacy) present in the majority of other mammals. This was accomplished via allelic differentiation (e.g. most New World monkeys) or gene duplication (e.g. Old World primates) of the middle to long-wavelength sensitive (M/LWS, or red–green) opsin gene. However, questions remain regarding the behavioral adaptations of primate trichromacy. Allelic differentiation of the M/LWS opsins results in extensive color vision variability in New World monkeys, where trichromats and dichromats are found in the same breeding population, enabling us to directly compare visual performances among different color vision phenotypes. Thus, New World monkeys can serve as an excellent model to understand and evaluate the adaptive significance of primate trichromacy in a behavioral context. I shall summarize recent findings on color vision evolution in primates and introduce our genetic and behavioral study of vision-behavior interrelationships in free-ranging sympatric capuchin and spider monkey populations in Costa Rica.     

Relative LWS cone opsin expression determines optomotor thresholds in Malawi cichlid fish

Associating quantitative genetic traits with quantitative behaviors is a relatively unexplored region of sensory neurobiology. The visual system is an ideal place to test models associating these levels of sensory perception. In this study, we reared cichlid fish from Lake Malawi in different ambient light environments. We then tested the visual sensitivities of these fish using the optomotor response (OMR) behavioral paradigm and measured the relative expression of cone opsin genes. We found that the light environment experienced by fish during development can alter gene expression, particularly as it applies to the long wavelength-sensitive (LWS) opsin gene. Also, fish from different rearing conditions exhibited different behavioral sensitivities. We combined these data with predictions of opsin pigment absorption by the different OMR stimuli to determine which cone types are most likely to influence the OMR behavior. While we hypothesized that this behavior would be controlled by a random-wiring model reflecting the expression of both medium wavelength-sensitive (MWS) and LWS opsins, our models suggest that only the LWS pigment is required to predict behavior. Furthermore, analyses show that LWS expression variation accounts for ~20% of the observed behavioral variance. This work confirms that sensory gene expression influences behavior in a predictable fashion. It also suggests that the neural wiring of basal visual pathways in cichlid fish may differ from that observed in mammals and zebrafish, but is similar to that described in goldfish. This finding has important implications for the evolution of the magnocellular neural pathway in teleosts.     

Opsin gene duplication and divergence in ray-finned fish

Opsin gene sequences were first reported in the 1980s. The goal of that research was to test the hypothesis that human opsins were members of a single gene family and that variation in human color vision was mediated by mutations in these genes. While the new data supported both hypotheses, the greatest contribution of this work was, arguably, that it provided the data necessary for PCR-based surveys in a diversity of other species. Such studies, and recent whole genome sequencing projects, have uncovered exceptionally large opsin gene repertoires in ray-finned fishes (taxon, Actinopterygii). Guppies and zebrafish, for example, have 10 visual opsin genes each. Here we review the duplication and divergence events that have generated these gene collections. Phylogenetic analyses revealed that large opsin gene repertories in fish have been generated by gene duplication and divergence events that span the age of the ray-finned fishes. Data from whole genome sequencing projects and from large-insert clones show that tandem duplication is the primary mode of opsin gene family expansion in fishes. In some instances gene conversion between tandem duplicates has obscured evolutionary relationships among genes and generated unique key-site haplotypes. We mapped amino acid substitutions at so-called key-sites onto phylogenies and this exposed many examples of convergence. We found that dN/dS values were higher on the branches of our trees that followed gene duplication than on branches that followed speciation events, suggesting that duplication relaxes constraints on opsin sequence evolution. Though the focus of the review is opsin sequence evolution, we also note that there are few clear connections between opsin gene repertoires and variation in spectral environment, morphological traits, or life history traits.     

The molecular basis of color vision in colorful fish: Four Long Wave-Sensitive (LWS) opsins in guppies (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>) are defined by amino acid substitutions at key functional sites

Background  

Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae.     

Molecular characterization of crustacean visual pigments and the evolution of pancrustacean opsins

Investigations of opsin evolution outside of vertebrate systems have long been focused on insect visual pigments, whereas other groups have received little attention. Furthermore, few studies have explicitly investigated the selective influences across all the currently characterized arthropod opsins. In this study, we contribute to the knowledge of crustacean opsins by sequencing 1 opsin gene each from 6 previously uncharacterized crustacean species (Euphausia superba, Homarus gammarus, Archaeomysis grebnitzkii, Holmesimysis costata, Mysis diluviana, and Neomysis americana). Visual pigment spectral absorbances were measured using microspectrophotometry for species not previously characterized (A. grebnitzkii=496 nm, H. costata=512 nm, M. diluviana=501 nm, and N. americana=520 nm). These novel crustacean opsin sequences were included in a phylogenetic analysis with previously characterized arthropod opsin sequences to determine the evolutionary placement relative to the well-established insect spectral clades (long-/middle-/short-wavelength sensitive). Phylogenetic analyses indicate these novel crustacean opsins form a monophyletic clade with previously characterized crayfish opsin sequences and form a sister group to insect middle-/long-wavelength-sensitive opsins. The reconstructed opsin phylogeny and the corresponding spectral data for each sequence were used to investigate selective influences within arthropod, and mainly "pancrustacean," opsin evolution using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a historical (i.e., phylogenetic) context. Although the conservative dN/dS methods did not detect any selection, 4 amino acid properties (coil tendencies, compressibility, power to be at the middle of an alpha-helix, and refractive index) were found to be influenced by destabilizing positive selection. Ten amino acid sites relating to these properties were found to face the binding pocket, within 4 A of the chromophore and thus have the potential to affect spectral tuning.     

A Fish Eye Out of Water: Ten Visual Opsins in the Four-Eyed Fish,Anableps anableps

The “four-eyed” fish Anableps anableps has numerous morphological adaptations that enable above and below-water vision. Here, as the first step in our efforts to identify molecular adaptations for aerial and aquatic vision in this species, we describe the A. anableps visual opsin repertoire. We used PCR, cloning, and sequencing to survey cDNA using unique primers designed to amplify eight sequences from five visual opsin gene subfamilies, SWS1, SWS2, RH1, RH2, and LWS. We also used Southern blotting to count opsin loci in genomic DNA digested with EcoR1 and BamH1. Phylogenetic analyses confirmed the identity of all opsin sequences and allowed us to map gene duplication and divergence events onto a tree of teleost fish. Each of the gene-specific primer sets produced an amplicon from cDNA, indicating that A. anableps possessed and expressed at least eight opsin genes. A second PCR-based survey of genomic and cDNA uncovered two additional LWS genes. Thus, A. anableps has at least ten visual opsins and all but one were expressed in the eyes of the single adult surveyed. Among these ten visual opsins, two have key site haplotypes not found in other fish. Of particular interest is the A. anableps-specific opsin in the LWS subfamily, S180γ, with a SHYAA five key site haplotype. Although A. anableps has a visual opsin gene repertoire similar to that found in other fishes in the suborder Cyprinodontoidei, the LWS opsin subfamily has two loci not found in close relatives, including one with a key site haplotype not found in any other fish species. A. anableps opsin sequence data will be used to design in situ probes allowing us to test the hypothesis that opsin gene expression differs in the distinct ventral and dorsal retinas found in this species.     

Evolutionary renovation of L/M opsin polymorphism confers a fruit discrimination advantage to ateline New World monkeys

New World monkeys exhibit prominent colour vision variation due to allelic polymorphism of the long‐to‐middle wavelength (L/M) opsin gene. The known spectral variation of L/M opsins in primates is broadly determined by amino acid composition at three sites: 180, 277 and 285 (the ‘three‐sites’ rule). However, two L/M opsin alleles found in the black‐handed spider monkeys (Ateles geoffroyi) are known exceptions, presumably due to novel mutations. The spectral separation of the two L/M photopigments is 1.5 times greater than expected based on the ‘three‐sites’ rule. Yet the consequence of this for the visual ecology of the species is unknown, as is the evolutionary mechanism by which spectral shift was achieved. In this study, we first examine L/M opsins of two other Atelinae species, the long‐haired spider monkeys (A. belzebuth) and the common woolly monkeys (Lagothrix lagotricha). By a series of site‐directed mutagenesis, we show that a mutation Y213D (tyrosine to aspartic acid at site 213) in the ancestral opsin of the two alleles enabled N294K, which occurred in one allele of the ateline ancestor and increased the spectral separation between the two alleles. Second, by modelling the chromaticity of dietary fruits and background leaves in a natural habitat of spider monkeys, we demonstrate that chromatic discrimination of fruit from leaves is significantly enhanced by these mutations. This evolutionary renovation of L/M opsin polymorphism in atelines illustrates a previously unappreciated dynamism of opsin genes in shaping primate colour vision.     

Opsin gene duplication and diversification in the guppy, a model for sexual selection

Identification of genes that control variation in adaptive characters is a prerequisite for understanding the processes that drive sexual and natural selection. Male coloration and female colour perception play important roles in mate choice in the guppy (Poecilia reticulata), a model organism for studies of natural and sexual selection. We examined a potential source for the known variation in colour perception, by analysing genomic and complementary DNA sequences of genes that code for visual pigment proteins. We find high sequence variability, both within and between populations, and expanded copy number for long-wave sensitive (LWS) opsin genes. Alleles with non-synonymous changes that suggest dissimilar spectral tuning properties occur in the same population and even in the same individual, and the high frequency of non-synonymous substitutions argues for diversifying selection acting on these proteins. Therefore, variability in tuning amino acids is partitioned within individuals and populations of the guppy, in contrast to variability for LWS at higher taxonomic levels in cichlids, a second model system for differentiation owing to sexual selection. Since opsin variability parallels the extreme male colour polymorphism within guppy populations, we suggest that mate choice has been a major factor driving the coevolution of opsins and male ornaments in this species.     

A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra-retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod-opsins from the retina of these species. By PCR, we have determined that the novel rod-like opsin is not expressed in the salmon retina, and the retinal rod-opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod-like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod-like opsins, their extra-retinal sites of expression, and phylogenetic position we have termed these novel opsins 'extra-retinal rod-like opsins' (ERrod-like opsins). We speculate that the differences between retinal rod-opsins and ERrod-like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.     

Molecular Evidence that Only Two Opsin Subfamilies,the Blue Light- (SWS2) and Green Light-Sensitive (RH2), Drive Color Vision in Atlantic Cod (Gadus morhua)

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.     

Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and d_N/d_S ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.     

Opsin Evolution in Damselfish: Convergence, Reversal, and Parallel Evolution Across Tuning Sites

The visual system plays a role in nearly every aspect of an organism??s life history, and there is a direct link between visual pigment phenotypes and opsin genotypes. In previous studies of African cichlid fishes, we found evidence for positive selection among some opsins, with sequence variation greatest for opsins producing the shortest and longest wavelength visual pigments. In this study, we examined opsin evolution in the closely related damselfish family (Pomacentridae), a group of reef fishes that are distributed widely and have a documented fossil record of at least 50?million years (MY). We found increased functional variation in the protein sequences of opsins at the short- and long-wavelength ends of the visual spectrum, in agreement with the African cichlids, despite an order of magnitude difference in the ages of the two radiations. We also reconstructed amino acid substitutions across opsin tuning sites. These reconstructions indicated multiple instances of parallel evolution, at least one definitive case of convergent evolution, and one evolutionary reversal. Our findings show that the amino acids at spectral tuning sites are labile evolutionarily, and that the same codons evolve repeatedly. These findings emphasize that the aquatic light environment can shape opsin sequence evolution. They further show that phylogenetic approaches can provide important insights into the mechanisms by which natural selection ??tinkers?? with phenotypes.