Functional diversification of lepidopteran opsins following gene duplication.

A comparative approach was taken for identifying amino acid substitutions that may be under positive Darwinian selection and are correlated with spectral shifts among orthologous and paralogous lepidopteran long wavelength-sensitive (LW) opsins. Four novel LW opsin fragments were isolated, cloned, and sequenced from eye-specific cDNAs from two butterflies, Vanessa cardui (Nymphalidae) and Precis coenia (Nymphalidae), and two moths, Spodoptera exigua (Noctuidae) and Galleria mellonella (Pyralidae). These opsins were sampled because they encode visual pigments having a naturally occurring range of lambda(max) values (510-530 nm), which in combination with previously characterized lepidopteran opsins, provide a complete range of known spectral sensitivities (510-575 nm) among lepidopteran LW opsins. Two recent opsin gene duplication events were found within the papilionid but not within the nymphalid butterfly families through neighbor-joining, maximum parsimony, and maximum likelihood phylogenetic analyses of 13 lepidopteran opsin sequences. An elevated rate of evolution was detected in the red-shifted Papilio Rh3 branch following gene duplication, because of an increase in the amino acid substitution rate in the transmembrane domain of the protein, a region that forms the chromophore-binding pocket of the visual pigment. A maximum likelihood approach was used to estimate omega, the ratio of nonsynonymous to synonymous substitutions per site. Branch-specific tests of selection (free-ratio) identified one branch with omega = 2.1044, but the small number of substitutions involved was not significantly different from the expected number of changes under the neutral expectation of omega = 1. Ancestral sequences were reconstructed with a high degree of certainty from these data. Reconstructed ancestral sequences revealed several instances of convergence to the same amino acid between butterfly and vertebrate cone pigments, and between independent branches of the butterfly opsin tree that are correlated with spectral shifts.     

Opsin evolution and expression in Arthropod compound Eyes and Ocelli: Insights from the cricket Gryllus bimaculatus

ABSTRACT: BACKGROUND: Opsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial to understand the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect. RESULTS: Phylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes. CONCLUSIONS: Our data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.     

小地老虎UV视蛋白基因的克隆及序列分析

视蛋白是感光物质的重要组成成分,是动物感知周围光环境的重要途径之一。本文以小地老虎(Agrotis ypsilon)3日龄成虫为材料,利用RT-PCR和RACE末端扩增技术克隆得到小地老虎UV视蛋白基因的cDNA序列。序列分析表明,小地老虎视蛋白基因的cDNA序列1 632 bp,包括一个1 140 bp的完整开放阅读框架,编码379个氨基酸,理论蛋白分子量(Mw)41.50 ku,等电点(pI)7.56。GenBank登录号为JN185654。UV视蛋白包括7个跨膜拓扑结构和一个G蛋白偶联受体家族,第107位赖氨酸与UV视蛋白的紫外敏感性有重要关系。同源性分析显示,小地老虎UV视蛋白基因与其他昆虫的UV视蛋白基因具有较高同源性。本研究对深入探究UV视蛋白在动物夜行生活中的作用具有重要意义。     

Comparative visual ecology of cephalopods from different habitats

Previous investigations of vision and visual pigment evolution in aquatic predators have focused on fish and crustaceans, generally ignoring the cephalopods. Since the first cephalopod opsin was sequenced in late 1980s, we now have data on over 50 cephalopod opsins, prompting this functional and phylogenetic examination. Much of this data does not specifically examine the visual pigment spectral absorbance position (λ_max) relative to environment or lifestyle, and cephalopod opsin functional adaptation and visual ecology remain largely unknown. Here we introduce a new protocol for photoreceptor microspectrophotometry (MSP) that overcomes the difficulty of bleaching the bistable visual pigment and that reveals eight coastal coleoid cephalopods to be monochromatic with λ_max varying from 484 to 505 nm. A combination of current MSP results, the λ_max values previously characterized using cephalopod retinal extracts (467–500 nm) and the corresponding opsin phylogenetic tree were used for systematic comparisons with an end goal of examining the adaptations of coleoid visual pigments to different light environments. Spectral tuning shifts are described in response to different modes of life and light conditions. A new spectral tuning model suggests that nine amino acid substitution sites may determine the direction and the magnitude of spectral shifts.     

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.     

Six Opsins from the Butterfly Papilio glaucus: Molecular Phylogenetic Evidence for Paralogous Origins of Red-Sensitive Visual Pigments in Insects

It has been hypothesized that the UV-, blue-, and green-sensitive visual pigments of insects were present in the common ancestor of crustaceans and insects, whereas red-sensitive visual pigments evolved later as a result of convergent evolution. This hypothesis is examined with respect to the placement of six opsins from the swallowtail butterfly Papilio glaucus (PglRh1–6) in relationship to 46 other insect, crustacean, and chelicerate opsin sequences. All basal relationships established with maximum parsimony analysis except two are present in the distance and maximum likelihood analyses. In all analyses, the six P. glaucus opsins fall into three well-supported clades, comprised, respectively, of ultraviolet (UV), blue, and long-wavelength (LW) pigments, which appear to predate the radiation of the insects. Lepidopteran green- and red-sensitive visual pigments form a monophyletic clade, which lends support to the hypothesis from comparative physiological studies that red-sensitive visual pigments in insects have paralogous origins. Polymorphic amino acid sites (180, 197, 277, 285, 308), which are essential for generating the spectral diversity among the vertebrate red- and green-sensitive pigments are notably invariant in the Papilio red- and green-sensitive pigments. Other major tuning sites must be sought to explain the spectral diversification among these and other insect visual pigments. Received: 6 December 1999 / Accepted: 3 April 2000     

Opsin gene sequence variation across phylogenetic and population histories in Mysis (Crustacea: Mysida) does not match current light environments or visual-pigment absorbance spectra

The hypothesis that selection on the opsin gene is efficient in tuning vision to the ambient light environment of an organism was assessed in 49 populations of 12 Mysis crustacean species, inhabiting arctic marine waters, coastal littoral habitats, freshwater lakes ('glacial relicts') and the deep Caspian Sea. Extensive sequence variation was found within and among taxa, but its patterns did not match expectations based on light environments, spectral sensitivity of the visual pigment measured by microspectrophotometry or the history of species and populations. The main split in the opsin gene tree was between lineages I and II, differing in six amino acids. Lineage I was present in marine and Caspian Sea species and in the North American freshwater Mysis diluviana, whereas lineage II was found in the European and circumarctic fresh- and brackish-water Mysis relicta, Mysis salemaai and Mysis segerstralei. Both lineages were present in some populations of M. salemaai and M. segerstralei. Absorbance spectra of the visual pigment in nine populations of the latter three species showed a dichotomy between lake (λ(max) =554-562 nm) and brackish-water (Baltic Sea) populations (λ(max) = 521-535 nm). Judged by the shape of spectra, this difference was not because of different chromophores (A2 vs. A1), but neither did it coincide with the split in the opsin tree (lineages I/II), species identity or current light environments. In all, adaptive evolution of the opsin gene in Mysis could not be demonstrated, but its sequence variation did not conform to a neutral expectation either, suggesting evolutionary constraints and/or unidentified mechanisms of spectral tuning.     

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.     

Salmonid Opsin Sequences Undergo Positive Selection and Indicate an Alternate Evolutionary Relationship in Oncorhynchus

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey–McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.     

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.     

Ant opsins: Sequences from the Saharan silver ant and the carpenter ant

cDNA clones encoding opsins from compound eyes of carpenter ant,Camponotus abdominalis, and Saharan silver ant,Cataglyphis bombycina, were isolated from cDNA libraries. The opsin cDNAs from each species code for deduced proteins with 378 amino acids which are 92% identical. Of the 30 amino acid differences between the two proteins, 13 are non-conservative. Eight of these non-conservative substitutions are within the membrane spanning domain. The presence of a potential Schiff-base counterion in helix III in both species suggests that these opsins are the protein moiety of the visible range pigments. When compared to all known opsins, these opsins are most similar to the opsin from preying mantis (76% identity at the amino acid level). Phyletic comparisons group the two ant opsins with the other arthropod long wavelength opsins.     

Molecular Evolution of Arthropod Color Vision Deduced from Multiple Opsin Genes of Jumping Spiders

Among terrestrial animals, only vertebrates and arthropods possess wavelength-discrimination ability, so-called “color vision”. For color vision to exist, multiple opsins which encode visual pigments sensitive to different wavelengths of light are required. While the molecular evolution of opsins in vertebrates has been well investigated, that in arthropods remains to be elucidated. This is mainly due to poor information about the opsin genes of non-insect arthropods. To obtain an overview of the evolution of color vision in Arthropoda, we isolated three kinds of opsins, Rh1, Rh2, and Rh3, from two jumping spider species, Hasarius adansoni and Plexippus paykulli. These spiders belong to Chelicerata, one of the most distant groups from Hexapoda (insects), and have color vision as do insects. Phylogenetic analyses of jumping spider opsins revealed a birth and death process of color vision evolution in the arthropod lineage. Phylogenetic positions of jumping spider opsins revealed that at least three opsins had already existed before the Chelicerata-Pancrustacea split. In addition, sequence comparison between jumping spider Rh3 and the shorter wavelength-sensitive opsins of insects predicted that an opsin of the ancestral arthropod had the lysine residue responsible for UV sensitivity. These results strongly suggest that the ancestral arthropod had at least trichromatic vision with a UV pigment and two visible pigments. Thereafter, in each pancrustacean and chelicerate lineage, the opsin repertoire was reconstructed by gene losses, gene duplications, and function-altering amino acid substitutions, leading to evolution of color vision. Mitsumasa Koyanagi and Takashi Nagata contributed equally to this work. Sequence data from this article have been deposited with the DDBJ under accession nos. AB251846–AB251851.     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

Molecular characterization of visual pigments in Branchiopoda and the evolution of opsins in Arthropoda

Studies on color vision in invertebrates have focused primarily on insect visual pigments, with little attention given to crustacean visual pigments. None of the blue-green-, blue-, or ultraviolet (UV)-sensitive-opsins have been identified in crustaceans. In addition, the discussion of visual pigments has been limited to long-wavelength-sensitive opsins in Pancrustacea. Here, we focused on Branchiopoda (Crustacea), which is a sister group of Hexapoda including insects. In the tadpole shrimp Triops granarius, the visual pigment chromophore was retinal. Multiple opsins were isolated from each of three branchiopod species, T. granarius, Triops longicaudatus, and the fairy shrimp Branchinella kugenumaensis (five, five, and four opsins from these species, respectively). Phylogenetic analyses and the presence of a lysine residue corresponding to position 90 in bovine rhodopsin suggested that three of the branchiopod opsins comprise UV-sensitive pigments. In addition, the phylogenetic relationships between insect and branchiopod UV-sensitive opsins revealed that the divergence of blue- and UV-sensitive pigments predates the Branchiopoda and Insecta divergence. The other branchiopod opsins show distant relationships to other known insect opsins and form novel clusters. The present results strongly suggest that the ancestral arthropod of the Chelicerata-Pancrustacea lineages possessed at least four types of opsins. The ancestors of Pancrustacea and the Insecta-Branchiopoda lineages possessed at least five and six types of opsins, respectively. Our results suggest that in the evolutionary process associated with each lineage, several opsins appeared and diversified with repeated gene duplication, of which some have been lost in some taxa.     

Genetic variability of the sws1 cone opsin gene among New World monkeys

Vision is a major sense for Primates and the ability to perceive colors has great importance for the species ecology and behavior. Visual processing begins with the activation of the visual opsins in the retina, and the spectral absorption peaks are highly variable among species. In most Primates, LWS/MWS opsins are responsible for sensitivity to long/middle wavelengths within the visible light spectrum, and SWS1 opsins provide sensitivity to short wavelengths, in the violet region of the spectrum. In this study, we aimed to investigate the genetic variation on the sws1 opsin gene of New World monkeys (NWM) and search for amino acid substitutions that might be associated with the different color vision phenotypes described for a few species. We sequenced the exon 1 of the sws1 opsin gene of seven species from the families Callitrichidae, Cebidae, and Atelidae, and searched for variation at the spectral tuning sites 46, 49, 52, 86, 90, 93, 114, 116, and 118. Among the known spectral tuning sites, only residue 114 was variable. To investigate whether other residues have a functional role in the SWS1 absorption peak, we performed computational modeling of wild-type SWS1 and mutants A50I and A50V, found naturally among the species investigated. Although in silico analysis did not show any visible effect caused by these substitutions, it is possible that interactions of residue 50 with other sites might have some effect in the spectral shifts in the order of ~14 nm, found among the NWM. We also performed phylogenetic reconstruction of the sws1 gene, which partially recovered the species phylogeny. Further studies will be important to uncover the mutations responsible for the phenotypic variability of the SWS1 of NWM, and how spectral tuning may be associated with specific ecological features such as preferred food items and habitat use.     

Molecular Evolution of the Genomic RNA of Apple Stem Grooving Capillovirus

The complete genome of the German isolate AC of Apple stem grooving virus (ASGV) was sequenced. It encodes two overlapping open reading frames (ORFs), similarly to previously described ASGV isolates. Two regions of high variability were detected between the ASGV isolates, variable region 1 (V1, from amino acids (aa) 532 to 570), and variable region 2 (V2, from aa 1,583 to 1,868). The phylogenetic analysis of the V1 and V2 regions suggested that the ASGV diversity was structured by host plant species rather than geographical origin. The dN/dS ratio between nonsynonymous and synonymous nucleotide substitution rates varied greatly along the ASGV genome. Most of ORF1 showed predominant negative selection except for the two regions V1 and V2. V1 showed an elevated dN and an average dS when compared to the ORF1 background but no significant positive selection was detected. The V2 region of ORF1 showed an elevated dN and a low dS when compared to the ORF1 background with an average dN/dS????3.0 indicative of positive selection. However, the V2 area includes overlapping ORFs, making the dN/dS estimate biased. Joint estimates of the selection intensity in the different ORFs by a recent method indicated that this region of ORF1 was in fact evolving close to neutrality. This was convergent with previous results showing that introduction of stop codons in this region of ORF1 did not impair plant infection. These data suggest that the elimination of a stop codon caused the overprinting of a novel coding region over the ancestral ORF.     

The Population Genetics of dN/dS

Evolutionary pressures on proteins are often quantified by the ratio of substitution rates at non-synonymous and synonymous sites. The dN/dS ratio was originally developed for application to distantly diverged sequences, the differences among which represent substitutions that have fixed along independent lineages. Nevertheless, the dN/dS measure is often applied to sequences sampled from a single population, the differences among which represent segregating polymorphisms. Here, we study the expected dN/dS ratio for samples drawn from a single population under selection, and we find that in this context, dN/dS is relatively insensitive to the selection coefficient. Moreover, the hallmark signature of positive selection over divergent lineages, dN/dS>1, is violated within a population. For population samples, the relationship between selection and dN/dS does not follow a monotonic function, and so it may be impossible to infer selection pressures from dN/dS. These results have significant implications for the interpretation of dN/dS measurements among population-genetic samples.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.     

Evolution and mechanism of spectral tuning of blue-absorbing visual pigments in butterflies

The eyes of flower-visiting butterflies are often spectrally highly complex with multiple opsin genes generated by gene duplication, providing an interesting system for a comparative study of color vision. The Small White butterfly, Pieris rapae, has duplicated blue opsins, PrB and PrV, which are expressed in the blue (λ _max = 453 nm) and violet receptors (λ _max = 425 nm), respectively. To reveal accurate absorption profiles and the molecular basis of the spectral tuning of these visual pigments, we successfully modified our honeybee opsin expression system based on HEK293s cells, and expressed PrB and PrV, the first lepidopteran opsins ever expressed in cultured cells. We reconstituted the expressed visual pigments in vitro, and analysed them spectroscopically. Both reconstituted visual pigments had two photointerconvertible states, rhodopsin and metarhodopsin, with absorption peak wavelengths 450 nm and 485 nm for PrB and 420 nm and 482 nm for PrV. We furthermore introduced site-directed mutations to the opsins and found that two amino acid substitutions, at positions 116 and 177, were crucial for the spectral tuning. This tuning mechanism appears to be specific for invertebrates and is partially shared by other pierid and lycaenid butterfly species.