Rod Monochromacy and the Coevolution of Cetacean Retinal Opsins

Cetaceans have a long history of commitment to a fully aquatic lifestyle that extends back to the Eocene. Extant species have evolved a spectacular array of adaptations in conjunction with their deployment into a diverse array of aquatic habitats. Sensory systems are among those that have experienced radical transformations in the evolutionary history of this clade. In the case of vision, previous studies have demonstrated important changes in the genes encoding rod opsin (RH1), short-wavelength sensitive opsin 1 (SWS1), and long-wavelength sensitive opsin (LWS) in selected cetaceans, but have not examined the full complement of opsin genes across the complete range of cetacean families. Here, we report protein-coding sequences for RH1 and both color opsin genes (SWS1, LWS) from representatives of all extant cetacean families. We examine competing hypotheses pertaining to the timing of blue shifts in RH1 relative to SWS1 inactivation in the early history of Cetacea, and we test the hypothesis that some cetaceans are rod monochomats. Molecular evolutionary analyses contradict the “coastal” hypothesis, wherein SWS1 was pseudogenized in the common ancestor of Cetacea, and instead suggest that RH1 was blue-shifted in the common ancestor of Cetacea before SWS1 was independently knocked out in baleen whales (Mysticeti) and in toothed whales (Odontoceti). Further, molecular evidence implies that LWS was inactivated convergently on at least five occasions in Cetacea: (1) Balaenidae (bowhead and right whales), (2) Balaenopteroidea (rorquals plus gray whale), (3) Mesoplodon bidens (Sowerby''s beaked whale), (4) Physeter macrocephalus (giant sperm whale), and (5) Kogia breviceps (pygmy sperm whale). All of these cetaceans are known to dive to depths of at least 100 m where the underwater light field is dim and dominated by blue light. The knockout of both SWS1 and LWS in multiple cetacean lineages renders these taxa rod monochromats, a condition previously unknown among mammalian species.     

Evolution of opsin genes reveals a functional role of vision in the echolocating little brown bat (Myotis lucifugus)

Echolocating bats are able to orientate, navigate and forage without visual cues. To probe the role of vision in bats, we studied the visual opsin genes from the echolocating little brown bat (Myotis lucifugus). Short-wavelength sensitive (SWS1) opsin, middle/long-wavelength sensitive (M/LWS) opsin and rhodopsin cDNA sequences were identified from the Ensembl database and validated by the sequencing of genomic DNA. We retrieved the published orthologous genes from eleven additional representative species of mammals from GenBank and conducted an evolutionary analysis. We found that the M/LWS opsin and rhodopsin genes were both under strong purifying selection, whereas the SWS1 opsin gene has undergone positive selection at two amino acid sites and one lineage, though the main evolutionary force is still purifying selection. Two-ratio model of the SWS1 opsin gene revealed that the ω ratio for the little brown bat lineage was nearly three times lower than the background ratio, suggesting a much stronger functional constraint. Our relative rate tests show the little brown bat has a lower nonsynonymous substitution rate than those in other mammals (on average 32% lower) for the SWS1 opsin gene. However, no such significant differences were detected for the M/LWS opsin and rhodopsin genes. The results of the relative ratio tests are consistent with that of tests for selection, showing a history of purifying selection on the little brown bat opsin genes. These findings suggest a functional role of vision in the little brown bat despite being nocturnal and using echolocation. We speculate that this echolocating bat may be able to use visual cues to orientate, navigate and forage at night, to discriminate color under moonlight and starlight conditions, or to avoid predation by diurnal raptors.     

Parallel and Convergent Evolution of the Dim-Light Vision Gene RH1 in Bats (Order: Chiroptera)

Rhodopsin, encoded by the gene Rhodopsin (RH1), is extremely sensitive to light, and is responsible for dim-light vision. Bats are nocturnal mammals that inhabit poor light environments. Megabats (Old-World fruit bats) generally have well-developed eyes, while microbats (insectivorous bats) have developed echolocation and in general their eyes were degraded, however, dramatic differences in the eyes, and their reliance on vision, exist in this group. In this study, we examined the rod opsin gene (RH1), and compared its evolution to that of two cone opsin genes (SWS1 and M/LWS). While phylogenetic reconstruction with the cone opsin genes SWS1 and M/LWS generated a species tree in accord with expectations, the RH1 gene tree united Pteropodidae (Old-World fruit bats) and Yangochiroptera, with very high bootstrap values, suggesting the possibility of convergent evolution. The hypothesis of convergent evolution was further supported when nonsynonymous sites or amino acid sequences were used to construct phylogenies. Reconstructed RH1 sequences at internal nodes of the bat species phylogeny showed that: (1) Old-World fruit bats share an amino acid change (S270G) with the tomb bat; (2) Miniopterus share two amino acid changes (V104I, M183L) with Rhinolophoidea; (3) the amino acid replacement I123V occurred independently on four branches, and the replacements L99M, L266V and I286V occurred each on two branches. The multiple parallel amino acid replacements that occurred in the evolution of bat RH1 suggest the possibility of multiple convergences of their ecological specialization (i.e., various photic environments) during adaptation for the nocturnal lifestyle, and suggest that further attention is needed on the study of the ecology and behavior of bats.     

Opsin Genes and Visual Ecology in a Nocturnal Folivorous Lemur

Primate color vision has traditionally been examined in the context of diurnal activity, but recent genetic and ecological studies suggest that color vision plays a role in nocturnal primate behavior and ecology as well. In this study, we united molecular analyses of cone visual pigment (opsin) genes with visual modeling analyses of food items to explore the evolution of color vision in the folivorous woolly lemur (genus Avahi). Previous studies have shown that leaf quality, e.g., protein content, leaf toughness, and protein/toughness ratio, is significantly correlated with green-red and blue-yellow chromatic differences, suggesting a potential role of color in leaf discrimination in Avahi, and, consequently, a potential adaptive advantage to color vision in this taxon. Phylogenetic selection tests determined that the strength of selection on the SWS1 opsin gene to retain blue-sensitive SWS cones did not significantly differ in Avahi compared to day-active primates. Genotyping of the M/LWS opsin gene in 60 individuals from nine species found that the 558-nm-sensitive (red-sensitive) allele is conserved across all Avahi. Finally, we measured spectral reflectance from five species of young leaves consumed by Avahi and background foliage in Ranomafana National Park and modeled performance of possible S and M/L pigment pairs for detecting these food items under different nocturnal illuminations (e.g. twilight, moonlight). We found that the observed cone pigment pair in Avahi was optimally tuned for color-based detection of young green leaves in all nocturnal light environments, suggesting a potential adaptive role of nocturnal color vision in selection for dichromacy in this genus.     

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.     

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.     

Allelic Variation in Malawi Cichlid Opsins: A Tale of Two Genera

The role of sequence variation in the spectral tuning of color vision is well established in many systems. This includes the cichlids of Lake Victoria where sequence variation has been linked to environmental light gradients and speciation. The cichlids of Lake Malawi are a similar model for visual evolution, but the role of gene sequence variation in visual tuning between closely related species is unknown. This work describes such variation in multiple species of two rock-dwelling genera: Metriaclima and Labidochromis. Genomic DNA for seven cone opsin genes was sequenced and the structure of the opsin proteins was inferred. Retinal binding pocket polymorphisms were identified and compared to available data regarding spectral absorbance shifts. Sequence variation with known or potential effects on absorbance spectra were found in four genes: SWS1 (UV sensitive), SWS2B (violet sensitive), RH2Aβ (green sensitive), and LWS (red sensitive). Functional variation was distributed such that each genus had both a variable short-wavelength and long-wavelength sensitive opsin. This suggests spectral tuning is important at the margins of the cichlid visual spectrum. Further, there are two SWS1 opsin alleles that differ in sensitivity by 10 nm and are >2 MY divergent. One of these occurs in a haplotype block >1 kb. Potential haplotype blocks were found around the RH2 opsin loci. These data suggest that molecular diversification has resulted in functionally unique alleles and changes to the visual system. These data also suggest that opsin sequence variation tunes spectral sensitivities between closely related species and that the specific regions of spectral tuning are genus-specific.     

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.     

Cone monochromacy and visual pigment spectral tuning in wobbegong sharks

Much is known regarding the evolution of colour vision in nearly every vertebrate class, with the notable exception of the elasmobranchs. While multiple spectrally distinct cone types are found in some rays, sharks appear to possess only a single class of cone and, therefore, may be colour blind. In this study, the visual opsin genes of two wobbegong species, Orectolobus maculatus and Orectolobus ornatus, were isolated to verify the molecular basis of their monochromacy. In both species, only two opsin genes are present, RH1 (rod) and LWS (cone), which provide further evidence to support the concept that sharks possess only a single cone type. Examination of the coding sequences revealed substitutions that account for interspecific variation in the photopigment absorbance spectra, which may reflect the difference in visual ecology between these species.     

Assessing the use of genomic DNA as a predictor of the maximum absorbance wavelength of avian SWS1 opsin visual pigments

Recently, in vitro mutation studies have made it possible to predict the wavelengths of maximum absorbance (λ_max) of avian UV/violet sensitive visual pigments (SWS1) from the identity of a few key amino acid residues in the opsin gene. Given that the absorbance spectrum of a cone’s visual pigment and of its pigmented oil droplet can be predicted from just the λ_max, it may become possible to predict the entire spectral sensitivity of a bird using genetic samples from live birds or museum specimens. However, whilst this concept is attractive, it must be validated to assess the reliability of the predictions of λ_max from opsin amino acid sequences. In this paper, we have obtained partial sequences covering three of the known spectral tuning sites in the SWS1 opsin and predicted λ_max of all bird species for which the spectral absorbance has been measured using microspectrophotometry. Our results validate the use of molecular data from genomic DNA to predict the gross differences in λ_max between the violet- and ultraviolet-sensitive subtypes of SWS1 opsin. Additionally, we demonstrate that a bird, the bobolink Dolichonyx oryzivorus L., can have more than one SWS1 visual pigment in its retina.     

Color vision and niche partitioning in a diverse neotropical primate community in lowland Amazonian Ecuador

A recent focus in community ecology has been on how within‐species variability shapes interspecific niche partitioning. Primate color vision offers a rich system in which to explore this issue. Most neotropical primates exhibit intraspecific variation in color vision due to allelic variation at the middle‐to‐long‐wavelength opsin gene on the X chromosome. Studies of opsin polymorphisms have typically sampled primates from different sites, limiting the ability to relate this genetic diversity to niche partitioning. We surveyed genetic variation in color vision of five primate species, belonging to all three families of the primate infraorder Platyrrhini, found in the Yasuní Biosphere Reserve in Ecuador. The frugivorous spider monkeys and woolly monkeys (Ateles belzebuth and Lagothrix lagotricha poeppigii, family Atelidae) each had two opsin alleles, and more than 75% of individuals carried the longest‐wavelength (553–556 nm) allele. Among the other species, Saimiri sciureus macrodon (family Cebidae) and Pithecia aequatorialis (family Pitheciidae) had three alleles, while Plecturocebus discolor (family Pitheciidae) had four alleles—the largest number yet identified in a wild population of titi monkeys. For all three non‐atelid species, the middle‐wavelength (545 nm) allele was the most common. Overall, we identified genetic evidence of fourteen different visual phenotypes—seven types of dichromats and seven trichromats—among the five sympatric taxa. The differences we found suggest that interspecific competition among primates may influence intraspecific frequencies of opsin alleles. The diversity we describe invites detailed study of foraging behavior of different vision phenotypes to learn how they may contribute to niche partitioning.     

Mix and match color vision: tuning spectral sensitivity by differential opsin gene expression in Lake Malawi cichlids

Cichlid fish of the East African Rift Lakes are renowned for their diversity and offer a unique opportunity to study adaptive changes in the visual system in rapidly evolving species flocks. Since color plays a significant role in mate choice, differences in visual sensitivities could greatly influence and even drive speciation of cichlids. Lake Malawi cichlids inhabiting rock and sand habitats have significantly different cone spectral sensitivities. By combining microspectrophotometry (MSP) of isolated cones, sequencing of opsin genes, and spectral analysis of recombinant pigments, we have established the cone complements of four species of Malawi cichlids. MSP demonstrated that each of these species predominately expresses three cone pigments, although these differ between species to give three spectrally different cone complements. In addition, rare populations of spectrally distinct cones were found. In total, seven spectral classes were identified. This was confirmed by opsin gene sequencing, expression, and in vitro reconstitution. The genes represent the four major classes of cone opsin genes that diverged early in vertebrate evolution. All four species possess a long-wave-sensitive (LWS), three spectrally distinct green-sensitive (RH2), a blue-sensitive (SWS2A), a violet-sensitive (SWS2B), and an ultraviolet-sensitive (SWS1) opsin. However, African cichlids determine their spectral sensitivity by differential expression of primarily only three of the seven available cone opsin genes. Phylogenetic analysis suggests that all percomorph fish have similar potential.     

Photoreceptors and visual pigments in three species of newts

Photoreceptor composition and retinal visual pigments in three newt (Caudata, Salamandridae, Pleurodelinae) species (Pleurodeles waltl, Lissotriton (Triturus) vulgaris, and Cynops orientalis) were studied by light microscopy and single-cell microspectrophotometry. Retinas of all three species contain “red” (rhodopsin/porphyropsin) rods, large and small single cones, and double cones. Large single cones and both components of double cones contain red-sensitive (presumably LWS) visual pigment whose absorption spectrum peaks between 593 and 611 nm. Small single cones are either blue- (SWS2, maximum absorption between 470 and 489 nm) or UV-sensitive (SWS1, maximum absorption between 340 and 359 nm). Chromophore composition of visual pigments (A₁ vs. A₂) was assessed both from template fitting of absorption spectra and by the method of selective bleaching. All pigments contained a mixture of A₁ (11-cis retinal) and A₂ (11-cis-3,4-dehydroretinal) chromophore in the proportion depending on the species and cell type. In all cases, A₂ was dominant. However, in C. orientalis rods the fraction of A₁ could reach 45%, while in P. waltl and L. vulgaris cones it did not exceed 5%. Remarkably, the absorption of the newt blue-sensitive visual pigment was shifted by up to 45 nm toward the longer wavelength, as compared with all other amphibian SWS2-pigments. We found no “green” rods typical of retinas of Anura and some Caudata (ambystomas) in the three newt species studied.     

Green‐shifting of SWS2A opsin sensitivity and loss of function of RH2‐A opsin in flounders,genus Verasper

We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2‐a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2‐a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λ_max) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue‐sensitive, whereas in V. variegatus (485.4 nm), it was green‐sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green‐sensitive, while its overall structure remains conserved as a blue‐sensitive opsin. Further, the rh2‐a function seems to have been reduced during the evolution of flounders. λ_max values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue‐sensitive. Thus, the green‐sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2‐A.     

Rapid light-induced shifts in opsin expression: finding new opsins, discerning mechanisms of change, and implications for visual sensitivity

Light-induced shifts in cone frequency and opsin expression occur in many aquatic species. Yet little is known about how quickly animals can alter opsin expression and, thereby, track their visual environments. Similarly, little is known about whether adult animals can alter opsin expression or whether shifts in opsin expression are limited to critical developmental windows. We took adult wild-caught bluefin killifish (Lucania goodei) from three different lighting environments (spring, swamp and variable), placed them under two different lighting treatments (clear vs. tea-stained water) and monitored opsin expression over 4 weeks. We measured opsin expression for five previously described opsins (SWS1, SWS2B, SWS2A, RH2-1 and LWS) as well as RH2-2 which we discovered via 454 sequencing. We used two different metrics of opsin expression. We measured expression of each opsin relative to a housekeeping gene and the proportional expression of each opsin relative to the total pool of opsins. Population and lighting environment had large effects on opsin expression which were present at the earliest time points indicating rapid shifts in expression. The two measures of expression produced radically different patterns. Proportional measures indicated large effects of light on SWS1 expression, whereas relative measures indicated no such effect. Instead, light had large effects on the relative expression of SWS2B, RH2-2, RH2-1 and LWS. We suggest that proportional measures of opsin expression are best for making inferences about colour vision, but that measures relative to a housekeeping gene are better for making conclusions about which opsins are differentially regulated.     

Spatial and seasonal patterns of fish assemblages in mountain streams of the Ren River,southwest China

The spatial–temporal patterns of fish assemblages in lotic systems can provide useful information in developing effective conservation measures. This study aimed to explore the spatial and seasonal changes in fish assemblages and their association with environmental factors in mountain streams of the Ren River, southwest China. Field investigations were conducted at 18 sites during the rainy and dry seasons in 2017. A total of 1,330 individuals, belonging to three orders, eight families, 19 genera, and 21 species, were collected. Analysis of similarities (ANOSIM) showed that the structure of fish assemblages varied significantly at the spatial scale, but not at the seasonal scale. In low‐order sites, fish assemblages were mainly dominated by cold‐water and rheophilic species (e.g., Rhynchocypris oxycephalus, Scaphesthes macrolepis, Metahomaloptera omeiensis, and Gnathopogon herzensteini), while those in high‐order sites were predominated by warm‐water and eurytopic or stagnophilic species (e.g., Squalidus argentatus, Hemiculter leucisculus, and Zacco platypus). Canonical correspondence analysis (CCA) showed that the fish assemblages were structured by a combination of large‐scale landscape factors (e.g., altitude and C‐link) and small‐scale habitat features (e.g., channel width, water temperature, and depth). Among these factors, landscape had the greatest influence on fish assemblages, while local habitat variables were less important or were only significant in certain seasons.