A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.     

Ultraviolet-sensitive vision in long-lived birds

Long-term exposure to ultraviolet (UV) light generates substantial damage, and in mammals, visual sensitivity to UV is restricted to short-lived diurnal rodents and certain marsupials. In humans, the cornea and lens absorb all UV-A and most of the terrestrial UV-B radiation, preventing the reactive and damaging shorter wavelengths from reaching the retina. This is not the case in certain species of long-lived diurnal birds, which possess UV-sensitive (UVS) visual pigments, maximally sensitive below 400 nm. The Order Psittaciformes contains some of the longest lived bird species, and the two species examined so far have been shown to possess UVS pigments. The objective of this study was to investigate the prevalence of UVS pigments across long-lived parrots, macaws and cockatoos, and therefore assess whether they need to cope with the accumulated effects of exposure to UV-A and UV-B over a long period of time. Sequences from the SWS1 opsin gene revealed that all 14 species investigated possess a key substitution that has been shown to determine a UVS pigment. Furthermore, in vitro regeneration data, and lens transparency, corroborate the molecular findings of UV sensitivity. Our findings thus support the claim that the Psittaciformes are the only avian Order in which UVS pigments are ubiquitous, and indicate that these long-lived birds have UV sensitivity, despite the risks of photodamage.     

Spectral tuning and evolution of primate short-wavelength-sensitive visual pigments

The peak sensitivities (λ(max)) of the short-wavelength-sensitive-1 (SWS1) pigments in mammals range from the ultraviolet (UV) (360-400 nm) to the violet (400-450 nm) regions of the spectrum. In most cases, a UV or violet peak is determined by the residue present at site 86, with Phe conferring UV sensitivity (UVS) and either Ser, Tyr or Val causing a shift to violet wavelengths. In primates, however, the tuning mechanism of violet-sensitive (VS) pigments would appear to differ. In this study, we examine the tuning mechanisms of prosimian SWS1 pigments. One species, the aye-aye, possesses a pigment with Phe86 but in vitro spectral analysis reveals a VS rather than a UVS pigment. Other residues (Cys, Ser and Val) at site 86 in prosimians also gave VS pigments. Substitution at site 86 is not, therefore, the primary mechanism for the tuning of VS pigments in primates, and phylogenetic analysis indicates that substitutions at site 86 have occurred at least five times in primate evolution. The sole potential tuning site that is conserved in all primate VS pigments is Pro93, which when substituted by Thr (as found in mammalian UVS pigments) in the aye-aye pigment shifted the peak absorbance into the UV region with a λ(max) value at 371 nm. We, therefore, conclude that the tuning of VS pigments in primates depends on Pro93, not Tyr86 as in other mammals. However, it remains uncertain whether the initial event that gave rise to the VS pigment in the ancestral primate was achieved by a Thr93Pro or a Phe86Tyr substitution.     

Why Aye‐Ayes See Blue

The capacity for cone‐mediated color vision varies among nocturnal primates. Some species are colorblind, having lost the functionality of their short‐wavelength‐sensitive‐1 (SWS1) opsin pigment gene. In other species, such as the aye‐aye (Daubentonia madagascariensis), the SWS1 gene remains intact. Recent studies focused on aye‐ayes indicate that this gene has been maintained by natural selection and that the pigment has a peak sensitivity (λ_max) of 406 nm, which is ～20 nm closer to the ultraviolet region of the spectrum than in most primates. The functional significance behind the retention and unusual λ_max of this opsin pigment is unknown, and it is perplexing given that all mammals are presumed to be colorblind in the dark. Here we comment on this puzzle and discuss recent findings on the color vision intensity thresholds of terrestrial vertebrates with comparable optics to aye‐ayes. We draw attention to the twilight activities of aye‐ayes and report that twilight is enriched in short‐wavelength (bluish) light. We also show that the intensity of twilight and full moonlight is probably sufficient to support cone‐mediated color vision. We speculate that the intact SWS1 opsin pigment gene of aye‐ayes is a crepuscular adaptation and we report on the blueness of potential visual targets, such as scent marks and the brilliant blue arils of Ravenala madagascariensis.     

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.     

The molecular evolution of avian ultraviolet- and violet-sensitive visual pigments

The shortwave-sensitive SWS1 class of vertebrate visual pigments range in lambda(max) from the violet (385-445 nm) to the ultraviolet (UV) (365-355 nm), with UV-sensitivity almost certainly ancestral. In birds, however, the UV-sensitive pigments present in a number of species have evolved secondarily from an avian violet-sensitive (VS) pigment. All avian VS pigments expressed in vitro to date encode Ser86 whereas Phe86 is present in all non-avian ultraviolet sensitive (UVS) pigments. In this paper, we show by site directed mutagenesis of avian VS pigments that Ser86 is required in an avian VS pigment to maintain violet-sensitivity and therefore underlies the evolution of avian VS pigments. The major mechanism for the evolution of avian UVS pigments from an ancestral avian VS pigment is undoubtedly a Ser90Cys substitution. However, Phe86, as found in the Blue-crowned trogon, will also short-wave shift the pigeon VS pigment into the UV whereas Ala86 and Cys86 which are also found in natural avian pigments do not generate short-wave shifts when substituted into the pigeon pigment. From available data on avian SWS1 pigments, it would appear that UVS pigments have evolved on at least 5 separate occasions and utilize 2 different mechanisms for the short-wave shift.     

Depth‐dependent plasticity in opsin gene expression varies between damselfish (Pomacentridae) species

Phenotypic plasticity plays an important role in adapting the visual capability of many animal species to changing sensory requirements. Such variability may be driven by developmental change or may result from environmental changes in light habitat, thereby improving performance in different photic environments. In this study, we examined inter‐ and intraspecific plasticity of visual sensitivities in seven damselfish species, part of the species‐rich and colourful fish fauna of the Great Barrier Reef in Australia. Our goal was to test whether the visual systems of damselfish were tuned to the prevailing light environment in different habitats and/or other aspects of their lifestyle. More specifically, we compared the opsin gene expression levels from individuals living in different photic habitats. We found that all species expressed rod opsin (RH1) used for dim‐light vision, and primarily three cone opsins (SWS1, RH2B and RH2A) used for colour vision. While RH1 levels changed exclusively following a diurnal cycle, cone opsin expression varied with depth in four of the seven species. Estimates of visual pigment performance imply that changes in opsin expression adjust visual sensitivities to the dominant photic regime. However, we also discovered that some species show a more stable opsin expression profile. Further, we found indication that seasonal changes, possibly linked to changes in the photic environment, might also trigger opsin expression. These findings suggest that plasticity in opsin gene expression of damselfish is highly species‐specific, possibly due to ecological differences in visual tasks or, alternatively, under phylogenetic constraints.     

Cone visual pigments in two marsupial species: the fat-tailed dunnart (Sminthopsis crassicaudata) and the honey possum (Tarsipes rostratus)

Uniquely for non-primate mammals, three classes of cone photoreceptors have been previously identified by microspectrophotometry in two marsupial species: the polyprotodont fat-tailed dunnart (Sminthopsis crassicaudata) and the diprotodont honey possum (Tarsipes rostratus). This report focuses on the genetic basis for these three pigments. Two cone pigments were amplified from retinal cDNA of both species and identified by phylogenetics as members of the short wavelength-sensitive 1 (SWS1) and long wavelength-sensitive (LWS) opsin classes. In vitro expression of the two sequences from the fat-tailed dunnart confirmed the peak absorbances at 363 nm in the UV for the SWS1 pigment and 533 nm for the LWS pigment. No additional expressed cone opsin sequences that could account for the middle wavelength cones could be amplified. However, amplification from the fat-tailed dunnart genomic DNA with RH1 (rod) opsin primer pairs identified two genes with identical coding regions but sequence differences in introns 2 and 3. Uniquely therefore for a mammal, the fat-tailed dunnart has two copies of an RH1 opsin gene. This raises the possibility that the middle wavelength cones express a rod rather than a cone pigment.     

Ultraviolet photopigment sensitivity and ocular media transmittance in gulls,with an evolutionary perspective

Gulls (Laridae excluding Sternidae) appear to be the only shorebirds (Charadriiformes) that have a short wavelength sensitive type 1 (SWS1) cone pigment opsin tuned to ultraviolet (UV) instead of violet. However, the apparent UV-sensitivity has only been inferred indirectly, via the interpretation that the presence of cysteine at the key amino acid position 90 in the SWS1 opsin confers UV sensitivity. Unless the cornea and the lens efficiently transmit UV to the retina, gulls might in effect be similar to violet-sensitive birds in spectral sensitivity even if they have an ultraviolet sensitive (UVS) SWS1 visual pigment. We report that the spectral transmission of the cornea and lens of great black-backed Larus marinus and herring gulls L. argentatus allow UV-sensitivity, having a λ_T0.5 value, 344 nm, similar to the ocular media of UV sensitive birds. By molecular sequencing of the second α-helical transmembrane region of the SWS1 opsin gene we could also infer that 15 herring gulls and 16 yellow-legged gulls L. michahellis, all base-pair identical, are genetically UV-sensitive.     

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.     

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.     

Adult plasticity in African cichlids: Rapid changes in opsin expression in response to environmental light differences

Phenotypic plasticity allows organisms to adapt quickly to local environmental conditions and could facilitate adaptive radiations. Cichlids have recently undergone an adaptive radiation in Lake Malawi where they inhabit diverse light environments and tune their visual sensitivity through differences in cone opsin expression. While cichlid opsin expression is known to be plastic over development, whether adults remain plastic is unknown. Adult plasticity in visual tuning could play a role in cichlid radiations by enabling survival in changing environments and facilitating invasion into novel environments. Here we examine the existence of and temporal changes in adult visual plasticity of two closely related species. In complementary experiments, wild adult Metriaclima mbenji from Lake Malawi were moved to the lab under UV‐deficient fluorescent lighting; while lab raised M. benetos were placed under UV‐rich lighting designed to mimic light conditions in the wild. Surprisingly, adult cichlids in both experiments showed significant changes in the expression of the UV‐sensitive single cone opsin, SWS1, in only 3 days. Modeling quantum catches in the light environments revealed a possible link between the light available to the SWS1 visual pigment and SWS1 expression. We conclude that adult cichlids can undergo rapid and significant changes in opsin expression in response to environmental light shifts that are relevant to their habitat and evolutionary history in Lake Malawi. This could have contributed to the rapid divergence characteristic of these fantastic fishes.     

Plasticity of opsin gene expression in cichlids from Lake Malawi

Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild‐caught fish to lab‐reared F₁ that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild‐caught fish were also expressed in lab‐reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the SWS2B (violet) opsin, which was always expressed at higher levels in lab‐reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi.     

Evolution of opsin expression in birds driven by sexual selection and habitat

Theories of sexual and natural selection predict coevolution of visual perception with conspecific colour and/or the light environment animals occupy. One way to test these theories is to focus on the visual system, which can be achieved by studying the opsin-based visual pigments that mediate vision. Birds vary greatly in colour, but opsin gene coding sequences and associated visual pigment spectral sensitivities are known to be rather invariant across birds. Here, I studied expression of the four cone opsin genes (Lws, Rh2, Sws2 and Sws1) in 16 species of New World warblers (Parulidae). I found levels of opsin expression vary both across species and between the sexes. Across species, female, but not male Sws2 expression is associated with an index of sexual selection, plumage dichromatism. This fits predictions of classic sexual selection models, in which the sensory system changes in females, presumably impacting female preference, and co-evolves with male plumage. Expression of the opsins at the extremes of the light spectrum, Lws and Uvs, correlates with the inferred light environment occupied by the different species. Unlike opsin spectral tuning, regulation of opsin gene expression allows for fast adaptive evolution of the visual system in response to natural and sexual selection, and in particular, sex-specific selection pressures.     

Multiple shifts between violet and ultraviolet vision in a family of passerine birds with associated changes in plumage coloration

Colour vision in diurnal birds falls into two discrete classes, signified by the spectral sensitivity of the violet- (VS) or ultraviolet-sensitive (UVS) short wavelength-sensitive type 1 (SWS1) single cone. Shifts between sensitivity classes are rare; three or four are believed to have happened in the course of avian evolution, one forming UVS higher passerines. Such shifts probably affect the expression of shortwave-dominated plumage signals. We have used genomic DNA sequencing to determine VS or UVS affinity in fairy-wrens and allies, Maluridae, a large passerine family basal to the known UVS taxa. We have also spectrophotometrically analysed male plumage coloration as perceived by the VS and UVS vision systems. Contrary to any other investigated avian genus, Malurus (fairy-wrens) contains species with amino acid residues typical of either VS or UVS cone opsins. Three bowerbird species (Ptilonorhynchidae) sequenced for outgroup comparison carry VS opsin genes. Phylogenetic reconstructions render one UVS gain followed by one or more losses as the most plausible evolutionary scenario. The evolution of avian ultraviolet sensitivity is hence more complex, as a single shift no longer explains its distribution in Passeriformes. Character correlation analysis proposes that UVS vision is associated with shortwave-reflecting plumage, which is widespread in Maluridae.     

Evolution of ultraviolet vision in shorebirds (Charadriiformes)

Diurnal birds belong to one of two classes of colour vision. These are distinguished by the maximum absorbance wavelengths of the SWS1 visual pigment sensitive to violet (VS) and ultraviolet (UVS). Shifts between the classes have been rare events during avian evolution. Gulls (Laridae) are the only shorebirds (Charadriiformes) previously reported to have the UVS type of opsin, but too few species have been sampled to infer that gulls are unique among shorebirds or that Laridae is monomorphic for this trait. We have sequenced the SWS1 opsin gene in a broader sample of species. We confirm that cysteine in the key amino acid position 90, characteristic of the UVS class, has been conserved throughout gull evolution but also that the terns Anous minutus, A. tenuirostris and Gygis alba, and the skimmer Rynchops niger carry this trait. Terns, excluding Anous and Gygis, share the VS conferring serine in position 90 with other shorebirds but it is translated from a codon more similar to that found in UVS shorebirds. The most parsimonious interpretation of these findings, based on a molecular gene tree, is a single VS to UVS shift and a subsequent reversal in one lineage.     

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.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.