Evolution and spectral tuning of visual pigments in birds and mammals

Variation in the types and spectral characteristics of visual pigments is a common mechanism for the adaptation of the vertebrate visual system to prevailing light conditions. The extent of this diversity in mammals and birds is discussed in detail in this review, alongside an in-depth consideration of the molecular changes involved. In mammals, a nocturnal stage in early evolution is thought to underlie the reduction in the number of classes of cone visual pigment genes from four to only two, with the secondary loss of one of these genes in many monochromatic nocturnal and marine species. The trichromacy seen in many primates arises from either a polymorphism or duplication of one of these genes. In contrast, birds have retained the four ancestral cone visual pigment genes, with a generally conserved expression in either single or double cone classes. The loss of sensitivity to ultraviolet (UV) irradiation is a feature of both mammalian and avian visual evolution, with UV sensitivity retained among mammals by only a subset of rodents and marsupials. Where it is found in birds, it is not ancestral but newly acquired.     

Evolution of visual pigments in geckos

Most geckos are nocturnal forms and possess rod retinas, but some diurnal genera have pure-cone retinas. We isolated cDNAs encoding the diurnal gecko opsins, dg1 and dg2, similar to nocturnal gecko P521 and P467, respectively. Despite the large morphological differences between the diurnal and nocturnal gecko photoreceptor types, they express phylogenetically closely related opsins. These results provide molecular evidence for the reverse transmutation, that is, rods of an ancestral nocturnal gecko have backed into cones of diurnal geckos. The amino acid substitution rates of dgl and dg2 are higher than those of P521 and P467, respectively. Changes of behavior regarding photic environment may have contributed to acceleration of amino acid substitutions in the diurnal gecko opsins.     

Spectral tuning of avian violet- and ultraviolet-sensitive visual pigments

The violet- and ultraviolet-sensitive visual pigments of birds belong to the same class of pigments as the violet-sensitive (so-called blue) pigments of mammals. However, unlike the pigments from mammals and other vertebrate taxa which, depending on species, have lambda(max) values of either around 430 nm or around 370 nm, avian pigments are found with lambda(max) values spread across this range. In this paper, we present the sequences of two pigments isolated from Humbolt penguin and pigeon with intermediate lambda(max) values of 403 and 409 nm, respectively. By comparing the amino acid sequences of these pigments with the true UV pigments of budgerigar and canary and with chicken violet with a lambda(max) value of 420 nm, we have been able to identify five amino acid sites that show a pattern of substitution between species that is consistent with differences in lambda(max). Each of these substitutions has been introduced into budgerigar cDNA and expressed in vitro in COS-7 cells. Only three resulted in spectral shifts in the regenerated pigment; two had relatively small effects and may account for the spectral shifts between penguin, pigeon, and chicken whereas one, the replacement of Ser by Cys at site 90 in the UV pigments, produced a 35 nm shortwave shift that could account for the spectral shift from 403 nm in penguin to around 370 nm in budgerigar and canary.     

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.     

Reconstitution of ancestral green visual pigments of zebrafish and molecular mechanism of their spectral differentiation

We previously reported that zebrafish have four tandemly duplicated green (RH2) opsin genes (RH2-1, RH2-2, RH2-3, and RH2-4). Absorption spectra vary widely among the four photopigments reconstituted with 11-cis retinal, with their peak absorption spectra (lambda(max)) being 467, 476, 488, and 505 nm, respectively. In this study, we inferred the ancestral amino acid (aa) sequences of the zebrafish RH2 opsins by likelihood-based Bayesian statistics and reconstituted the ancestral opsins by site-directed mutagenesis. The ancestral pigment (A1) to the four zebrafish RH2 pigments and that (A3) to RH2-3 and RH2-4 showed lambda(max) at 506 nm, while that (A2) to RH2-1 and RH2-2 showed a lambda(max) at 474 nm, indicating that a spectral shift had occurred toward the shorter wavelength on the evolutionary lineages A1 to A2 by 32 nm, A2 to RH2-1 by 7 nm, and A3 to RH2-3 by 18 nm. Pigment chimeras and site-directed mutagenesis revealed a large contribution (approximately 15 nm) of glutamic acid to glutamine substitution at residue 122 (E122Q) to the A1 to A2 and A3 to RH2-3 spectral shifts. However, the remaining spectral differences appeared to result from complex interactive effects of a number of aa replacements, each of which has only a minor spectral contribution (1-3 nm). The four zebrafish RH2 pigments cover nearly an entire range of lambda(max) distribution among vertebrate RH2 pigments and provide an excellent model to study spectral tuning mechanisms of RH2 in vertebrates.     

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.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

Flavonoid pigments in swallowtail butterflies

Flavonoid pigments have been identified in the swallowtail butterfly Eurytides marcellus and its larval foodplant Asimina triloba (Annonaceae). Although quercetin 3-glycoside, quercetin 3-rutinoside and quercetin 3-rutinoside-7-glucoside are present in the plant, only quercetin 3-glucoside is sequestered by the insect. Flavonoids have also been found in 10 out of 27 other papilionid species examined. These were mainly 3- and 7-glycosides of the flavonols quercetin and kaempferol. The sequestration of flavonoids by papilionid butterflies appears to be related both to the phylogeny of the Papilionidae and to the choice of larval foodplants by the various phylogenetic groups.     

Gene duplication and spectral diversification of cone visual pigments of zebrafish

Zebrafish is becoming a powerful animal model for the study of vision but the genomic organization and variation of its visual opsins have not been fully characterized. We show here that zebrafish has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes in the genome, among which LWS-2, RH2-2, and RH2-3 are novel. SWS2, LWS-1, and LWS-2 are located in tandem and RH2-1, RH2-2, RH2-3, and RH2-4 form another tandem gene cluster. The peak absorption spectra (lambdamax) of the reconstituted photopigments from the opsin cDNAs differed markedly among them: 558 nm (LWS-1), 548 nm (LWS-2), 467 nm (RH2-1), 476 nm (RH2-2), 488 nm (RH2-3), 505 nm (RH2-4), 355 nm (SWS1), 416 nm (SWS2), and 501 nm (RH1, rod opsin). The quantitative RT-PCR revealed a considerable difference among the opsin genes in the expression level in the retina. The expression of the two red opsin genes and of three green opsin genes, RH2-1, RH2-3, and RH2-4, is significantly lower than that of RH2-2, SWS1, and SWS2. These findings must contribute to our comprehensive understanding of visual capabilities of zebrafish and the evolution of the fish visual system and should become a basis of further studies on expression and developmental regulation of the opsin genes.     

Mechanisms of spectral tuning in the RH2 pigments of Tokay gecko and American chameleon

At present, molecular bases of spectral tuning in rhodopsin-like (RH2) pigments are not well understood. Here, we have constructed the RH2 pigments of nocturnal Tokay gecko (Gekko gekko) and diurnal American chameleon (Anolis carolinensis) as well as chimeras between them. The RH2 pigments of the gecko and chameleon reconstituted with 11-cis-retinal had the wavelengths of maximal absorption (lambda(max)'s) of 467 and 496 nm, respectively. Chimeric pigment analyses indicated that 76-86%, 14-24%, and 10% of the spectral difference between them could be explained by amino acid differences in transmembrane (TM) helices I-IV, V-VII, and amino acid interactions between the two segments, respectively. Evolutionary and mutagenesis analyses revealed that the lambda(max)'s of the gecko and chameleon pigments diverged from each other not only by S49A (serine to alanine replacement at residue 49), S49F (serine to phenylalanine), L52M (leucine to methionine), D83N (aspartic acid to asparagine), M86T (methionine to threonine), and T97A (threonine to alanine) but also by other amino acid replacements that cause minor lambda(max)-shifts individually.     

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.     

Molecular mechanism of spectral tuning in sensory rhodopsin II.

Sensory rhodopsin II (SRII) is unique among the archaeal rhodopsins in having an absorption maximum near 500 nm, blue shifted roughly 70 nm from the other pigments. In addition, SRII displays vibronic structure in the lambda(max) absorption band, whereas the other pigments display fully broadened band maxima. The molecular origins responsible for both photophysical properties are examined here with reference to the 2.4 A crystal structure of sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis. We use semiempirical molecular orbital theory (MOZYME) to optimize the chromophore within the chromophore binding site, and MNDO-PSDCI molecular orbital theory to calculate the spectroscopic properties. The entire first shell of the chromophore binding site is included in the MNDO-PSDCI SCF calculation, and full single and double configuration interaction is included for the chromophore pi-system. Through a comparison of corresponding calculations on the 1.55 A crystal structure of bacteriorhodopsin (bR), we identify the principal molecular mechanisms, and residues, responsible for the spectral blue shift in NpSRII. We conclude that the major source of the blue shift is associated with the significantly different positions of Arg-72 (Arg-82 in bR) in the two proteins. In NpSRII, this side chain has moved away from the chromophore Schiff base nitrogen and closer to the beta-ionylidene ring. This shift in position transfers this positively charged residue from a region of chromophore destabilization in bR to a region of chromophore stabilization in NpSRII, and is responsible for roughly half of the blue shift. Other important contributors include Asp-201, Thr-204, Tyr-174, Trp-76, and W402, the water molecule hydrogen bonded to the Schiff base proton. The W402 contribution, however, is a secondary effect that can be traced to the transposition of Arg-72. Indeed, secondary interactions among the residues contribute significantly to the properties of the binding site. We attribute the increased vibronic structure in NpSRII to the loss of Arg-72 dynamic inhomogeneity, and an increase in the intensity of the second excited (1)A(g)(-) -like state, which now appears as a separate feature within the lambda(max) band profile. The strongly allowed (1)B(u)(+)-like state and the higher-energy (1)A(g)(-) -like state are highly mixed in NpSRII, and the latter state borrows intensity from the former to achieve an observable oscillator strength.     

Molecular determinants of spectral properties and signal transduction in the visual pigments.

Site-directed mutagenesis of the visual pigment rhodopsin has provided a wealth of information regarding amino acid residues responsible for the determination of the spectral properties of the chromophore, the amino acids involved in activation and inactivation of the protein, and the effect of amino acid substitutions found in patients with retinitis pigmentosa. In addition, cell culture systems have now been established for expression of the three human color vision pigments, opening the way for a similar attack on the structure and function of these important proteins.     

Carotenoids and bile pigments in Danaid and swallowtail butterflies

The carotenoids found in adult kite swallowtails ( Graphium spp.) and the Danaid glassy tigers ( Ideopsis, Tirumala and Parantica ), butterflies with exposed blue/green pigmented wing membranes (which contain pterobilins) and a modified scale vestiture, were compared with those of related species of Papilios and Danaids with concealed wing membranes an an entire scale vestiture. It was found that both the kite swallowtails contained only lutein, and even lacked β-carotene, the first known instance among butterflies. This was presumably due to selective storage by the larva since the food-plant was found to be rich in varied carotenoids. A possible link between the high concentration of pterobilins in the wing membrane and the lack of carotenoids is discussed.     

The cone visual pigments of an Australian marsupial,the tammar wallaby (Macropus eugenii): sequence,spectral tuning,and evolution

Studies on marsupial color vision have been limited to very few species. There is evidence from behavioral, electroretinographic (ERG), and microspectrophotometric (MSP) measurements for the existence of both dichromatic and trichromatic color vision. No studies have yet investigated the molecular mechanisms of spectral tuning in the visual pigments of marsupials. Our study is the first to determine the mRNA sequence, infer the amino acid sequence, and determine, by in vitro expression, the spectra of the cone opsins of a marsupial, the tammar wallaby (Macropus eugenii). This yielded some information on mechanisms and evolution of spectral tuning of these pigments. The tammar wallaby retina contains only short-wavelength sensitive (SWS) and middle-wavelength sensitive (MWS) pigment mRNAs. This predicts dichromatic color vision, which is consistent with conclusions from previous behavioral studies ( Hemmi 1999). We found that the wallaby has a SWS1 class pigment of 346 amino acids. Sequence comparison with eutherian SWS pigments predicts that this SWS1 pigment absorbs maximally (lambdamax) at 424 nm and, therefore, is a blue rather than a UV pigment. This (lambdamax) is close to that of the in vitro-expressed wallaby SWS pigment (lambdamax of 420 +/- 2 nm) and to that determined behaviorally (420 nm). The difference from the mouse UV pigment (lambdamax of 359 nm) is largely accounted for by the F86Y substitution, in agreement with in vitro results comparing a variety of other SWS pigments. This suggests that spectral tuning employing F86Y substitution most likely arose independently in the marsupials and ungulates as a result of convergent evolution. An apparently different mechanism of spectral tuning of the SWS1 pigments, involving five amino acid positions, evolved in primates. The wallaby MWS pigment has 363 amino acids. Species comparisons at positions critical to spectral tuning predict a lambdamax near 530 nm, which is close to that of the in vitro-expressed pigment (529 +/- 1 nm), but quite different from the value of 539 nm determined by microspectrophotometry. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species.     

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.     

Molecular basis of spectral tuning in the red- and green-sensitive (M/LWS) pigments in vertebrates

Vertebrate vision is mediated by five groups of visual pigments, each absorbing a specific wavelength of light between ultraviolet and red. Despite extensive mutagenesis analyses, the mechanisms by which contemporary pigments absorb variable wavelengths of light are poorly understood. We show that the molecular basis of the spectral tuning of contemporary visual pigments can be illuminated only by mutagenesis analyses using ancestral pigments. Following this new principle, we derive the "five-sites" rule that explains the absorption spectra of red and green (M/LWS) pigments that range from 510 to 560 nm. Our findings demonstrate that the evolutionary method should be used in elucidating the mechanisms of spectral tuning of four other pigment groups and, for that matter, functional differentiations of any other proteins.     

Fly visual pigments difference in visual pigments of blowfly and dronefly peripheral retinula cells

Journal of Comparative Physiology A - The visual pigments of peripheral retinula cells in fly eyes have been investigated by microspectrophotometry in vivo. Since flies have a pupil mechanism...     

Evolutionary analysis of rhodopsin and cone pigments: connecting the three-dimensional structure with spectral tuning and signal transfer

Extensive sequence data and structural sampling of expressed proteins from different species lead to the idea that entire molecules or specific domain folds belong to large superfamilies of proteins. A subset of G protein-coupled receptors, one of the largest families involved in cellular signaling, rod and cone opsins are involved in phototransduction in photoreceptor cells. Here, the evolutionary analysis of opsin sequences and structures predicts key residues involved in the transmission of the signal from the binding site of the chromophore to the cytoplasmic surface and residues that are involved in the spectral tuning of opsins to short wavelengths of light.