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Sensory systems function as the interface between organisms and their environment, playing a pivotal role in animal biology. In vertebrates, habitat diversity and niche specialization contributed to the emergence of fascinating examples of sensory adaptation, allowing animals to better perceive, interact, and explore their surroundings. Recent advances have greatly expanded our understanding of vertebrate sensory biology, particularly regarding the molecular mechanisms of sensory transduction as well as the evolutionary processes underlying sensory adaptation. Here we review our current knowledge of the molecular evolution of vertebrate sensory systems.

Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution.

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin--the vertebrate visual pigment mediating the first step in phototransduction at low-light levels--via codon-based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well-studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.

Distantly related clades that occupy similar environments may differ due to the lasting imprint of their ancestors – historical contingency. The New World warblers (Parulidae) and Old World warblers (Phylloscopidae) are ecologically similar clades that differ strikingly in plumage coloration. We studied genetic and functional evolution of the short-wavelength sensitive visual pigments (SWS2 and SWS1) to ask if altered color perception could contribute to the plumage color differences between clades. We show SWS2 is short-wavelength shifted in birds that occupy open environments, such as finches, compared to those in closed environments, including warblers. Sequencing of opsin genes and phylogenetic reconstructions indicate New World warblers were derived from a finch-like form that colonized from the Old World 15-20Ma. During this process the SWS2 gene accumulated 6 substitutions in branches leading to New World warblers, inviting the hypothesis that passage through a finch-like ancestor resulted in SWS2 evolution. In fact, we show spectral tuning remained similar across warblers as well as the finch ancestor. Results reject the hypothesis of historical contingency based on opsin spectral tuning, but point to evolution of other aspects of visual pigment function. Using the approach outlined here, historical contingency becomes a generally testable theory in systems where genotype and phenotype can be connected.

Like many aquatic vertebrates, whales have blue-shifting spectral tuning substitutions in the dim-light visual pigment, rhodopsin, that are thought to increase photosensitivity in underwater environments. We have discovered that known spectral tuning substitutions also have surprising epistatic effects on another function of rhodopsin, the kinetic rates associated with light-activated intermediates. By using absorbance spectroscopy and fluorescence-based retinal release assays on heterologously expressed rhodopsin, we assessed both spectral and kinetic differences between cetaceans (killer whale) and terrestrial outgroups (hippo, bovine). Mutation experiments revealed that killer whale rhodopsin is unusually resilient to pleiotropic effects on retinal release from key blue-shifting substitutions (D83N and A292S), largely due to a surprisingly specific epistatic interaction between D83N and the background residue, S299. Ancestral sequence reconstruction indicated that S299 is an ancestral residue that predates the evolution of blue-shifting substitutions at the origins of Cetacea. Based on these results, we hypothesize that intramolecular epistasis helped to conserve rhodopsin's kinetic properties while enabling blue-shifting spectral tuning substitutions as cetaceans adapted to aquatic environments. Trade-offs between different aspects of molecular function are rarely considered in protein evolution, but in cetacean and other vertebrate rhodopsins, may underlie multiple evolutionary scenarios for the selection of specific amino acid substitutions.

Corticotrophin-releasing hormone (CRH) is the pivotal neuroendocrine peptide hormone associated with the regulation of the stress response in vertebrates. However, CRH-like peptides are also found in a number of invertebrate species. The origin of this peptide can be traced to a common ancestor of lineages leading to chordates and to arthropods, postulated to occur some 500 million years ago. Evidence indicates the presence of a single CRH-like receptor and a soluble binding protein system that acted to transduce and regulate the actions of the early CRH peptide. In vertebrates, genome duplications led to the divergence of CRH receptors into CRH1 and CRH2 forms in tandem with the development of four paralogous ligand lineages that included CRH; urotensin I/urocortin (Ucn), Ucn2 and Ucn3. In addition, taxon-specific genome duplications led to further local divergences in CRH ligands and receptors. Functionally, the CRH ligand–receptor system evolved initially as a molecular system to integrate early diuresis and nutrient acquisition. As multicellular organisms evolved into more complex forms, this ligand–receptor system became integrated with the organismal stress response to coordinate homoeostatic challenges with internal energy usage. In vertebrates, CRH and the CRH1 receptor became associated with the hypothalamo-pituitary–adrenal/interrenal axis and the initial stress response, whereas the CRH2 receptor was selected to play a greater role in diuresis, nutrient acquisition and the latter aspects of the stress response.

This study examines the pattern of opsin nucleotide and amino acid substitution among mimetic species «rings» of Heliconius butterflies that are characterized by divergent wing colour patterns. A long wavelength opsin gene, OPS1, was sequenced from each of seven species of Heliconius and one species of Dryas (Lepidoptera: Nymphalidae). A parsimony analysis of OPS1 nucleotide and amino acid sequences resulted in a phylogeny that was consistent with that presented by Brower & Egan in 1997, which was based on mitochondrial cytochrome oxidase I andII as well as nuclear wingless genes. Nodes in the OPS1 phylogeny were well supported by bootstrap analysis and decay indices. An analysis of specific sites within the gene indicates that the accumulation of amino acid substitutions has occurred independently of the morphological diversification of Heliconius wing colour patterns. Amino acid substitutions were examined with respect to their location within the opsin protein and their possible interactions with the chromophore and the G-protein. Of the 15 amino acid substitutions identified among the eight species, one nonconservative replacement (A226Q) was identified in a position that may be involved in binding with the G-protein.

Acetyl-coenzyme A synthases (ACS) are Ni–Fe–S containingenzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoAfrom CO2 + 2e-, CoA, and a methyl group, and contain5 types of subunits (α, β, γ, δ, and e). Class II enzymes catalyze essentially the reversereaction and have similar subunit composition. Class III ACS'scatalyze the same reaction as Class I enzymes, but use pyruvateas a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an α2β2 tetramerand a γδ heterodimer. Class IV enzymes catabolize CO to CO2 and are α-subunit monomers. Phylogeneticanalyses were performed on all five subunits. ACS α sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residuesthat may function as ligands to the B- and C-clusters wereidentified. Other residues exclusively conserved in Class I/IIsequences may be ligands to additional metal centers in Class I and II enzymes. ACS β sequences also separated into twogroups, but they were less divergent than the α's, and the separation was not as distinct. Class III-like β sequences contained ∼300 residues at their N-termini absent in Class I/II sequences. Conserved residues identifiedin β sequences may function as ligands to active siteresidues used for acetyl-CoA synthesis. ACS γ-sequencesseparated into 3 groups (Classes I, II, and III), while δ-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of α sequences. ACS e-sequence topology showed greaterdivergence and less consistency vis-a-vis the other subunits, possibly reflecting reduced evolutionary constraintsdue to the absence of metal centers. The α subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms mayhave evolved are discussed.

Tracing the history of molecular changes using phylogenetic methods can provide powerful insights into how and why molecules work the way they do. It is now possible to recreate inferred ancestral proteins in the laboratory and study the function of these molecules. This provides a unique opportunity to study the paths and the mechanisms of functional change during molecular evolution. What insights have already emerged from such phylogenetic studies of protein evolution and function, what are the impediments to progress and what are the prospects for the future?

The mating system of the Australian lycaenid butterfly, Jalmenus evagoras, is highly unusual compared to most other Lepidoptera. Characteristics of this system, which has been termed an ’explosive mating strategy,’ include the formation of an intensely competitive mating aggregation of males, a highly male biased operational sex ratio, a lack of discrimination and mate choice by both sexes, a high variance in male mating success, and female monogamy. In this study, we tested the hypothesis that multiple mating by males imposes physiological costs resulting in smaller spermatophores, and that this results in a fitness cost to females. We found that male J. evagoras transferred only 2.2% of their eclosion weight during their first mating, consistent with the hypothesis that males of monandrous species produce a relatively small investment. The wet weight of the ejaculate declined by an average of 27% at the second mating and the dry weight by 29%, and an intermating interval of 5–9 days was needed for the ejaculate to return to the size at the first mating, regardless of male size or age. Wet ejaculate mass increased proportionally with male size, though dry mass was proportionally larger in smaller males. Ejaculate mass tended to increase with male age at both first and second matings. Female characteristics, in general, did not affect ejaculate mass, although the wet weight of the ejaculate was positively associated with female weight at the second mating. Copulation duration increased from 2.4 h to approximately 3 h at the second mating, and to over 4 h at the third and fourth matings. Fecundity was positively correlated with female size but not with mating history, copulation duration, or any other characteristics measured for either males or females. Female longevity declined significantly as the number of times the male partner had previously mated increased. We conclude that despite the small male investment in ejaculate, the costs of multiple mating may nonetheless be significant, as indicated by the reduction in ejaculate mass, an increase in copulation duration, and reduction in female lifespan with increasing mating number.

The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding theApisblue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. TheApisblue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitiveDrosophilaRh3 and Rh4 opsins. To test whether these novelApisopsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1–6 photoreceptor cells of blindninaEmutantDrosophila, which lack the major opsin of the fly compound eye. We found that the expression of either theApisblue- or UV-sensitive opsin in transgenic flies rescued the visual defect ofninaEmutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings fromApisphotoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

The molecular mechanisms underlying the enormous diversity of visual pigment wavelength sensitivities found in nature have been the focus of many molecular evolutionary studies, with particular attention to the short wavelength-sensitive 1 (SWS1) visual pigments that mediate vision in the ultraviolet to violet range of the electromagnetic spectrum. Over a decade of study has revealed that the remarkable extension of SWS1 absorption maxima (λ max ) into the ultraviolet occurs through a deprotonation of the Schiff base linkage of the retinal chromophore, a mechanism unique to this visual pigment type. In studies of visual ecology, there has been mounting interest in inferring visual sensitivity at short wavelengths, given the importance of UV signaling in courtship displays and other behaviors. Since experimentally determining spectral sensitivities can be both challenging and time-consuming, alternative strategies such as estimating λ max based on amino acids at sites known to affect spectral tuning are becoming increasingly common. However, these estimates should be made with knowledge of the limitations inherent in these approaches. Here, we provide an overview of the current literature on SWS1 site-directed mutagenesis spectral tuning studies, and discuss methodological caveats specific to the SWS1-type pigments. We focus particular attention on contrasting avian and mammalian SWS1 spectral tuning mechanisms, which are the best studied among vertebrates. We find that avian SWS1 visual pigment spectral tuning mechanisms are fairly consistent, and therefore more predictable in terms of wavelength absorption maxima, whereas mammalian pigments are not well suited to predictions of λ max from sequence data alone.

Background One of the most striking features of avian vision is the variation in spectral sensitivity of the short wavelength sensitive (SWS1) opsins, which can be divided into two sub-types: violet- and UV- sensitive (VS & UVS). In birds, UVS has been found in both passerines and parrots, groups that were recently shown to be sister orders. While all parrots are thought to be UVS, recent evidence suggests some passerine lineages may also be VS. The great bowerbird (Chlamydera nuchalis) is a passerine notable for its courtship behaviours in which males build and decorate elaborate bower structures. Results The great bowerbird SWS1 sequence possesses an unusual residue combination at known spectral tuning sites that has not been previously investigated in mutagenesis experiments. In this study, the SWS1 opsin of C. nuchalis was expressed along with a series of spectral tuning mutants and ancestral passerine SWS1 pigments, allowing us to investigate spectral tuning mechanisms and explore the evolution of UV/violet sensitivity in early passerines and parrots. The expressed C. nuchalis SWS1 opsin was found to be a VS pigment, with a λmax of 403 nm. Bowerbird SWS1 mutants C86F, S90C, and C86S/S90C all shifted λmax into the UV, whereas C86S had no effect. Experimentally recreated ancestral passerine and parrot/passerine SWS1 pigments were both found to be VS, indicating that UV sensitivity evolved independently in passerines and parrots from a VS ancestor. Conclusions Our mutagenesis studies indicate that spectral tuning in C. nuchalis is mediated by mechanisms similar to those of other birds. Interestingly, our ancestral sequence reconstructions of SWS1 in landbird evolution suggest multiple transitions from VS to UVS, but no instances of the reverse. Our results not only provide a more precise prediction of where these spectral sensitivity shifts occurred, but also confirm the hypothesis that birds are an unusual exception among vertebrates where some descendants re-evolved UVS from a violet type ancestor. The re-evolution of UVS from a VS type pigment has not previously been predicted elsewhere in the vertebrate phylogeny.

Transducin is a heterotrimeric G protein that plays a critical role in phototransduction in the rod and cone photoreceptor cells of the vertebrate retina. Rods, highly sensitive cells that recover from photoactivation slowly, underlie dim-light vision, whereas cones are less sensitive, recover more quickly, and underlie bright-light vision. Transducin deactivation is a critical step in photoreceptor recovery and may underlie the functional distinction between rods and cones. Rods and cones possess distinct transducin α subunits, yet they share a common deactivation mechanism, the GTPase activating protein (GAP) complex. Here, we used codon models to examine patterns of sequence evolution in rod (GNAT1) and cone (GNAT2) α subunits. Our results indicate that purifying selection is the dominant force shaping GNAT1 and GNAT2 evolution, but that GNAT2 has additionally been subject to positive selection operating at multiple phylogenetic scales; phylogeny-wide analysis identified several sites in the GNAT2 helical domain as having substantially elevated dN/dS estimates, and branch-site analysis identified several nearby sites as targets of strong positive selection during early vertebrate history. Examination of aligned GNAT and GAP complex crystal structures revealed steric clashes between several positively selected sites and the deactivating GAP complex. This suggests that GNAT2 sequence variation could play an important role in adaptive evolution of the vertebrate visual system via effects on photoreceptor deactivation kinetics and provides an alternative perspective to previous work that focused instead on the effect of GAP complex concentration. Our findings thus further the understanding of the molecular biology, physiology, and evolution of vertebrate visual systems.

The vast diversity in spectral sensitivities in the vision of many organisms is mediated mostly (although not entirely) through variation in the photosensitive visual pigments (opsins) of the eye. Specifically, shifts in absorption maxima of visual pigments are thought to be a result of interactions within the binding pocket of the opsin, between amino acid side chains and the retinal chromophore, However, it has proven difficult to identify specific amino acid residues important in determining wavelength absorption maxima, especially for some of the short wavelength (blue) opsins. In this paper, a comparative phylogenetic approach was applied to opsin protein sequence data to identify residues important in opsin wavelength regulation. In essence, this approach consisted of interpreting evolutionary history as a series of experiments in which natural selection has repeatedly favored amino acid replacements of certain residues to shift the opsin absorption spectra to either shorter or longer wavelengths. Opsin protein sequences were obtained from GenBank, aligned, and used to reconstruct a phylogenetic tree. Amino acid replacements were traced along the branches of this opsin tree, focusing only on residues likely to reside within the chromophore-binding pocket. A number of functionally convergent, nonconservative amino acid replacements in independently evolved opsins with similar shifts in spectral properties were identified. In short, reconstruction of the phylogeny of the opsin molecule allowed us to track amino acid substitutions in specific sites within the opsin and to target those particular substitutions that are repeatedly associated with marked changes in peak absorbance, shifting the spectral sensitivity of the opsin toward shorter or longer wavelengths. Based on these results, we propose a model for blue shifts of opsin absorption spectra. Amino acid replacements of four polar and charged residues near the protonated Schiff base (SBH+) end of the chromophore are proposed to result in blue shifts of the opsin absorption spectra. This model may explain some of the diversity of blue opsins apparent in both vertebrates and invertebrates.

Maximum likelihood codon substitution models have proven useful for studying when and how protein function evolves, but they have recently been criticized on a number of fronts. The strengths and weaknesses of such methods must therefore be identified and improved upon. Here, using simulations, we show that the Clade model C versus M1a test for functional divergence among clades is prone to false positives under simple evolutionary conditions. We then propose a new null model (M2a_rel) that better accounts for among-site variation in selective constraint. We show that the revised test has an improved false-positive rate and good power. Applying this test to previously analyzed data sets of primate ribonucleases and mammalian rhodopsins reveals that some conclusions may have been misled by the original method. The improved test should prove useful for identifying patterns of divergence in selective constraint among paralogous gene families and among orthologs from ecologically divergent species.

The visual pigment rhodopsin (rh1) constitutes the first step in the sensory transduction cascade in the rod photoreceptors of the vertebrate eye, forming the basis of vision at low light levels. In most vertebrates, rhodopsin is a single-copy gene whose function in rod photoreceptors is highly conserved. We found evidence for a second rhodopsin-like gene (rh1-2) in the zebrafish genome. This novel gene was not the product of a zebrafish-specific gene duplication event and contains a number of unique amino acid substitutions. Despite these differences, expression ofrh1-2in vitroyielded a protein that not only bound chromophore, producing an absorption spectrum in the visible range (λmax≈ 500 nm), but also activated in response to light. Unlikerh1,rh1-2is not expressed during the first 4 days of embryonic development; it is expressed in the retina of adult fish but not the brain or muscle. Similarrh1-2sequences were found in two otherDaniospecies, as well as a more distantly related cyprinid,Epalzeorhynchos bicolor. While sequences were only identified in cyprinid fish, phylogenetic analyses suggest an older origin for this gene family. Our study suggests thatrh1-2is a functional opsin gene that is expressed in the retina later in development. The discovery of a new previously uncharacterized opsin gene in zebrafish retina is surprising given its status as a model system for studies of vertebrate vision and visual development.

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d(N)/d(S)), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d(S) is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand.

Previous investigations into the evolution of the Drosophila opsin gene family are extended by inter- and intraspecific DNA sequence comparisons of the Rh3 locus in the melanogaster subgroup and D. pseudoobscura. Two separate statistical tests of the neutral-mutation hypothesis suggest that random genetic drift is responsible for virtually all of the observed amino acid replacement substitutions within the melanogaster subgroup. Analyses incorporating the D. pseudoobscura sequences are enigmatic due to the accumulation of multiple substitutions, because the McDonald-Kreitman test is not applicable to species comparisons that approach mutational saturation. However, the data from D. pseudoobscura are not inconsistent with selective neutrality. The ratio of amino acid polymorphisms within species to fixed differences between species imply that there are approximately 31 possible neutral single-step amino-acid-replacement substitutions at this locus. Synonymous substitutions are unevenly distributed among the structural domains of the Rh3 gene. Patterns of synonymous polymorphism are analyzed with respect to GC content and codon bias, and are compared to other loci from the same species.

Background The Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome. Results Using gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype. Conclusion Our data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis.

The heterologous expression of membrane proteins such as G protein-coupled receptors can be a notoriously difficult task. We have engineered an expression vector, p1D4-hrGFP II, in order to efficiently express visual pigments in mammalian cell culture. This expression vector is based on pIRES-hrGFP II (Stratagene), with the addition of a C-terminal 1D4 epitope tag for immunoblotting and immunoaffinity purification. This vector employs the CMV promoter and hrGFP II, a co-translated reporter gene. We measured the effectiveness of pIRES-hrGFP II in expressing bovine rhodopsin, and showed a 3.9- to 5.7-fold increase in expression as measured by absorbance spectroscopy as compared with the pMT vector, a common choice for visual pigment expression. We then expressed zebrafish RH2-1 using p1D4-hrGFP II in order to assess its utility in expressing cone opsins, known to be less stable and more difficult to express than bovine rhodopsin. We show a λ 280 / λ MAX value of 3.3, one third of that reported in previous studies, suggesting increased expression levels and decreased levels of misfolded, non-functional visual pigment. Finally, we monitored HEK293T cell growth following transfection with pIRES-hrGFP II using fluorescence microscopy to illustrate the benefits of having a co-translated reporter during heterologous expression studies.

The matricellular glycoprotein SPARC is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals: a Ca(2+)-binding glutamic acid-rich acidic domain at the N-terminus (domain I), a follistatin-like module (domain II), and an extracellular Ca(2+)-binding (EC) module that contains two EF-hands and two collagen-binding epitopes (domain III). We report that four SPARC orthologs (designated nvSPARC1-4) are expressed by the genome of the starlet anemone Nematostella vectensis, a diploblastic basal cnidarian composed of an ectoderm and endoderm separated by collagen-based mesoglea. We also report that domain I is absent from all N. vectensis SPARC orthologs. In situ hybridization data indicate that N. vectensis SPARC mRNAs are restricted to the endoderm during post-gastrula development. The absence of the Ca(2+)-binding N-terminal domain in cnidarians and conservation of collagen-binding epitopes suggests that SPARC first evolved as a collagen-binding matricellular glycoprotein, an interaction likely to be dependent on the binding of Ca(2+)-ions to the two EF-hands in the EC domain. We propose that further Ca(2+)-dependent activities emerged with the acquisition of an acidic N-terminal module in triplobastic organisms.

Within the vertebrate eye, betagamma crystallins are extremely stable lens proteins that are uniquely adapted to increase refractory power while maintaining transparency. Unlike alpha crystallins, which are well-characterized, multifunctional proteins that have important functions both in and out of the lens, betagamma lens crystallins are a diverse group of proteins with no clear ancestral or contemporary nonlens role. We carried out phylogenetic and molecular evolutionary analyses of the betagamma-crystallin superfamily in order to study the evolutionary history of the gamma N crystallins, a recently discovered, biochemically atypical family suggested to possess a divergent or ancestral function. By including nonlens, betagamma-motif-containing sequences in our analysis as outgroups, we confirmed the phylogenetic position of the gamma N family as sister to other gamma crystallins. Using maximum likelihood codon models to estimate lineage-specific nonsynonymous-to-synonymous rate ratios revealed strong positive selection in all of the early lineages within the betagamma family, with the striking exception of the lineage leading to the gamma N crystallins which was characterized by strong purifying selection. Branch-site analysis, used to identify candidate sites involved in functional divergence between gamma N crystallins and its sister clade containing all other gamma crystallins, identified several positively selected changes at sites of known functional importance in the betagamma crystallin protein structure. Further analyses of a fish-specific gamma N crystallin gene duplication revealed a more recent episode of positive selection in only one of the two descendant lineages (gamma N2). Finally, from the guppy, Poecilia reticulata, we isolated complete gamma N1 and gamma N2 coding sequence data from cDNA and partial coding sequence data from genomic DNA in order to confirm the presence of a novel gamma N2 intron, discovered through data mining of two pufferfish genomes. We conclude that the function of the gamma N family likely resembles the ancestral vertebrate betagamma crystallin more than other betagamma families. Furthermore, owing to the presence of an additional intron in some fish gamma N2 crystallins, and the inferred action of positive selection following the fish-specific gamma N duplication, we suggest that further study of fish gamma N crystallins will be critical in further elucidating possible ancestral functions of gamma N crystallins and any nonstructural role they may have.

Allatostatins (ASTs) are a class of regulatory neuropeptides, with diverse functions, found in an array of invertebrate phyla. ASTs have complex gene structure, in which individual ASTs are cleaved from a precursor peptide. Little is known about the molecular evolution of AST structure and function, even in extensively studied groups such as cockroaches. This paper presents the application of a novel technique for the analysis of this system, that of ancestral reconstruction, whereby ancestral amino acid sequences are resurrected in the laboratory. We inferred the ancestral sequences of a well-characterized peptide, AST 7, for the insect ancestor, as well as several cockroach ancestors. Peptides were assayed for in vitro inhibition of JH production in Diploptera punctata and Periplaneta americana. Our results surprisingly, indicate a decrease in potency of the ancestral cockroach AST7 peptide in comparison with more ancient ones such as the ancestral insect peptide, as well as more recently evolved cockroach peptides. We propose that this unexpected decrease in peptide potency at the cockroach ancestor may be related to the concurrent increase in peptide copy number in the lineages leading to cockroaches. This model is consistent with current physiological data, and may be linked to the increased role of ASTs in the regulation of reproductive processes in the cockroaches.

Incursions of marine water into South America during the Miocene prompted colonization of freshwater habitats by ancestrally marine species and present a unique opportunity to study the molecular evolution of adaptations to varying environments. Freshwater and marine environments are distinct in both spectra and average intensities of available light. Here, we investigate the molecular evolution of rhodopsin, the photosensitive pigment in the eye that activates in response to light, in a clade of South American freshwater anchovies derived from a marine ancestral lineage. Using likelihood-based comparative sequence analyses, we found evidence for positive selection in the rhodopsin of freshwater anchovy lineages at sites known to be important for aspects of rhodopsin function such as spectral tuning. No evidence was found for positive selection in marine lineages, nor in three other genes not involved in vision. Our results suggest that an increased rate of rhodopsin evolution was driven by diversification into freshwater habitats, thereby constituting a rare example of molecular evolution mirroring large-scale palaeogeographic events.

Background Vertebrate SWS1 visual pigments mediate visual transduction in response to light at short wavelengths. Due to their importance in vision, SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals. The SWS1 genes exhibit many of the characteristics of genes typically targeted for phylogenetic analyses. This study investigates both the utility of SWS1 as a marker for inferring vertebrate phylogenetic relationships, and the characteristics of the gene that contribute to its phylogenetic utility. Results Phylogenetic analyses of vertebrate SWS1 genes produced topologies that were remarkably congruent with generally accepted hypotheses of vertebrate evolution at both higher and lower taxonomic levels. The few exceptions were generally associated with areas of poor taxonomic sampling, or relationships that have been difficult to resolve using other molecular markers. The SWS1 data set was characterized by a substantial amount of among-site rate variation, and a relatively unskewed substitution rate matrix, even when the data were partitioned into different codon sites and individual taxonomic groups. Although there were nucleotide biases in some groups at third positions, these biases were not convergent across different taxonomic groups. Conclusion Our results suggest that SWS1 may be a good marker for vertebrate phylogenetics due to the variable yet consistent patterns of sequence evolution exhibited across fairly wide taxonomic groups. This may result from constraints imposed by the functional role of SWS1 pigments in visual transduction.

Why are humans susceptible to certain illnesses but not others? Diseases such as gout and hypertension have plagued us for centuries. The prevalence of gout in the United States has risen to 3.9% in recent years (1), and a staggering one in three adults have hypertension (2). It has often been assumed that some of the less than desirable aspects of our physiology are relics of our evolutionary past, but rarely have these ideas been subjected to experimental investigation, and many of these theories have been surprisingly difficult to prove (3). Although the idea of resurrecting ancient proteins using sequence information from extant organisms was first proposed half a century ago by Pauling and Zuckerkandl (4), it is only in the last decade or so that advances in phylogenetic likelihood and Bayesian models (5), combined with fast, inexpensive gene synthesis technology, have made this approach increasingly feasible. These methods have since been used with great success to investigate a variety of topics, including the evolution of protein regulatory complexes in developmental pathways (6), vertebrate steroid hormone receptors (7), paleoenvironments and the thermophilicity of early life (8), archosaur vision (9), vaccine development (10), yeast transcriptional circuits following gene duplication (11), and the evolution of complexity in coral fluorescent proteins (12). Evolutionary questions can be notoriously intractable to experimental investigation, but the recreation of ancestral proteins provides us a window to the past through which we can directly test hypotheses regarding our evolutionary history.

Recreating ancestral proteins in the laboratory increasingly is being used to study the evolutionary history of protein function. More efficient gene synthesis techniques and the decreasing costs of commercial oligosynthesis are making this approach both simpler and less expensive to perform. Developments in ancestral reconstruction methods, particularly more realistic likelihood models of molecular evolution, allow for the accurate reconstruction of more ancient proteins than previously possible. This chapter reviews phylogenetic methods of ancestral inference, strategies for investigating alternative reconstructions, gene synthesis, and design, and an application of these methods to the reconstruction of an ancestor in the green fluorescent protein family.

Recreating ancestral proteins in the laboratory increasingly is being used to study the evolutionary history of protein function. More efficient gene synthesis techniques and the decreasing costs of commercial oligosynthesis are making this approach both simpler and less expensive to perform. Developments in ancestral reconstruction methods, particularly more realistic likelihood models of molecular evolution, allow for the accurate reconstruction of more ancient proteins than previously possible. This chapter reviews phylogenetic methods of ancestral inference, strategies for investigating alternative reconstructions, gene synthesis, and design, and an application of these methods to the reconstruction of an ancestor in the green fluorescent protein family.

In proteins homologous to the green fluorescent protein (GFP), formation of red fluorescence requires three autocatalytic steps, whereas only two are needed for green fluorescence. Multiple red/green color diversification events in the GFP superfamily may reflect convergent evolution of the more complex three-step pathway. In the great star coral Montastraea cavernosa , a recreated common ancestor of green and red proteins turned out to be green, indicating that in this case red proteins evolved their color independently from most other homologous red proteins. Furthermore, red color appears to have evolved gradually by small incremental transitions.

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pK a compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110–Cys187); this process may be a general element of G protein-coupled receptor activation.

The ancestors of the archosaurs, a major branch of the diapsid reptiles, originated more than 240 MYA near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods and explored different models of evolution for inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Three different types of maximum likelihood models were used: nucleotide-based, amino acid-based, and codon-based models. Where possible, within each type of model, likelihood ratio tests were used to determine which model best fit the data. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement, except for three amino acid residues at which one reconstruction differed from the other two. To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind to 11-cis-retinal to yield stable photoactive pigments with lambda(max) values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.

The second extracellular loop of rhodopsin folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore. A carboxylic acid side chain from this loop, Glu181, points toward the center of the retinal polyene chain. We studied the role of Glu181 in bovine rhodopsin by characterizing a set of site-directed mutants. Sixteen of the 19 single-site mutants expressed and bound 11-cis-retinal to form pigments. The lambda(max) value of mutant pigment E181Q showed a significant spectral red shift to 508 nm only in the absence of NaCl. Other substitutions did not significantly affect the spectral features of the mutant pigments in the dark. Thus, Glu181 does not contribute significantly to spectral tuning of the ground state of rhodopsin. The most likely interpretation of these data is that Glu181 is protonated and uncharged in the dark state of rhodopsin. The Glu181 mutants displayed significantly increased reactivity toward hydroxylamine in the dark. The mutants formed metarhodopsin II-like photoproducts upon illumination but many of the photoproducts displayed shifted lambda(max) values. In addition, the metarhodopsin II-like photoproducts of the mutant pigments had significant alterations in their decay rates. The increased reactivity of the mutants to hydroxylamine supports the notion that the second extracellular loop prevents solvent access to the chromophore-binding pocket. In addition, Glu181 strongly affects the environment of the retinylidene Schiff base in the active metarhodopsin II photoproduct.

Publisher Summary A novel and important application of synthetic gene technology in studies of G protein-coupled receptors (GPCRs) is the recreation of ancestral receptors. The superfamily of GPCRs consists of a large number of related seven transmembrane proteins of highly varied function. Recreating fossil receptors for functional studies in the laboratory can provide important insights into the constraints that shaped molecular structure and function in this diverse superfamily that are difficult to attain by using more traditional mutagenesis methods. In addition, in studying receptor function via heterologous expression of mutant receptors, it is often useful to create chimeric receptors in which putative functional domains are exchanged. This chapter presents a summary of methods and applications of synthetic gene technology, with an emphasis on synthesizing ancestral genes. Algorithms for inferring ancestral sequences and general considerations for designing synthetic genes are discussed in detail in the chapter. Detailed laboratory procedures for the various steps in gene synthesis, from oligonucleotide preparation to cassette mutagenesis, are also given.

Two spurious nodes were found in phylogenetic analyses of vertebrate rhodopsin sequences in comparison with well-established vertebrate relationships. These spurious reconstructions were well supported in bootstrap analyses and occurred independently of the method of phylogenetic analysis used (parsimony, distance, or likelihood). Use of this data set of vertebrate rhodopsin sequences allowed us to exploit established vertebrate relationships, as well as the considerable amount known about the molecular evolution of this gene, in order to identify important factors contributing to the spurious reconstructions. Simulation studies using parametric bootstrapping indicate that it is unlikely that the spurious nodes in the parsimony analyses are due to long branches or other topological effects. Rather, they appear to be due to base compositional bias at third positions, codon bias, and convergent evolution at nucleotide positions encoding the hydrophobic residues isoleucine, leucine, and valine. LogDet distance methods, as well as maximum-likelihood methods which allow for nonstationary changes in base composition, reduce but do not entirely eliminate support for the spurious resolutions. Inclusion of five additional rhodopsin sequences in the phylogenetic analyses largely corrected one of the spurious reconstructions while leaving the other unaffected. The additional sequences not only were more proximal to the corrected node, but were also found to have intermediate levels of base composition and codon bias as compared with neighboring sequences on the tree. This study shows that the spurious reconstructions can be corrected either by excluding third positions, as well as those encoding the amino acids Ile, Val, and Leu (which may not be ideal, as these sites can contain useful phylogenetic signal for other parts of the tree), or by the addition of sequences that reduce problems associated with convergent evolution.

Rhodopsins (Rh), G-protein-coupled receptors with seven transmembrane (TM) helices, form the first step in visual transduction in most organisms. Although many long-wavelength (LW) vertebrate opsin sequences are known, less information is available for invertebrate LW sequences. By a combination of RT-PCR and cDNA library screening, we have cloned and sequenced the honeybee LW Rh gene. The deduced protein is composed of 378 amino acids (aa), appears to have seven TM regions, and contains many of the structures and key aa thought to be important for Rh function. Phylogenetic analysis of this sequence in relation to other invertebrate Rh reveals it to be a member of a new group of insect LW Rh.

The aim of this study is to measure human mitochondrial sequence variability in the relatively slowly evolving mitochondrial gene cytochrome oxidase subunit II (COII) and to estimate when the human common ancestral mitochondrial type existed. New COII gene sequences were determined for five humans (Homo sapiens), including some of the most mitochondrially divergent humans known; for two pygmy chimpanzees (Pan paniscus); and for a common chimpanzee (P. troglodytes). COII sequences were analyzed with those from another relatively slowly evolving mitochondrial region (ND4-5). From class 1 (third codon position) sequence data, a relative divergence date for the human mitochondrial ancestor is estimated as 1/27 th of the human-chimpanzee divergence time. If it is assumed that humans and chimpanzees diverged 6 Mya, this places a human mitochondrial ancestor at 222,000 years, significantly different from 1 Myr (the presumed time of an H. erectus emergence from Africa). The mean coalescent time estimated from all 1,580 sites of combined mitochondrial data, when a 6-Mya human-chimpanzee divergence is assumed, is 298,000 years, with 95% confidence interval of 129,000-536,000 years. Neither estimate is compatible with a 1-Myr-old human mitochondrial ancestor. The mitochondrial DNA sequence data from COII and ND4-5 regions therefore do not support this multiregional hypothesis for the emergence of modern humans.

An isotope-shift, Zeeman-effect atomic absorption spectrometer has been demonstrated to have sufficient sensitivity to continuously monitor the total mercury content of ambient air. At present, the minimum total mercury concentration detectable with this device is 0.2 microgram per cubic meter of air-one fifth of the proposed federal guideline. This is the first technique which responds to both mercury vapor and mercury in particulates available for continuous monitoring at this concentration.

Background Synonymous codon usage can affect many cellular processes, particularly those associated with translation such as polypeptide elongation and folding, mRNA degradation/stability, and splicing. Highly expressed genes are thought to experience stronger selection pressures on synonymous codons. This should result in codon usage bias even in species with relatively low effective population sizes, like mammals, where synonymous site selection is thought to be weak. Here we use phylogenetic codon-based likelihood models to explore patterns of codon usage bias in a dataset of 18 mammalian rhodopsin sequences, the protein mediating the first step in vision in the eye, and one of the most highly expressed genes in vertebrates. We use these patterns to infer selection pressures on key translational mechanisms including polypeptide elongation, protein folding, mRNA stability, and splicing. Results Overall, patterns of selection in mammalian rhodopsin appear to be correlated with post-transcriptional and translational processes. We found significant evidence for selection at synonymous sites using phylogenetic mutation-selection likelihood models, with C-ending codons found to have the highest relative fitness, and to be significantly more abundant at conserved sites. In general, these codons corresponded with the most abundant tRNAs in mammals. We found significant differences in codon usage bias between rhodopsin loops versus helices, though there was no significant difference in mean synonymous substitution rate between these motifs. We also found a significantly higher proportion of GC-ending codons at paired sites in rhodopsin mRNA secondary structure, and significantly lower synonymous mutation rates in putative exonic splicing enhancer (ESE) regions than in non-ESE regions. Conclusions By focusing on a single highly expressed gene we both distinguish synonymous codon selection from mutational effects and analytically explore underlying functional mechanisms. Our results suggest that codon bias in mammalian rhodopsin arises from selection to optimally balance high overall translational speed, accuracy, and proper protein folding, especially in structurally complicated regions. Selection at synonymous sites may also be contributing to mRNA stability and splicing efficiency at exonic-splicing-enhancer (ESE) regions. Our results highlight the importance of investigating highly expressed genes in a broader phylogenetic context in order to better understand the evolution of synonymous substitutions.

Background Gene duplications play an important role in the evolution of functional protein diversity. Some models of duplicate gene evolution predict complex forms of paralog divergence; orthologous proteins may diverge as well, further complicating patterns of divergence among and within gene families. Consequently, studying the link between protein sequence evolution and duplication requires the use of flexible substitution models that can accommodate multiple shifts in selection across a phylogeny. Here, we employed a variety of codon substitution models, primarily Clade models, to explore how selective constraint evolved following the duplication of a green-sensitive (RH2a) visual pigment protein (opsin) in African cichlids. Past studies have linked opsin divergence to ecological and sexual divergence within the African cichlid adaptive radiation. Furthermore, biochemical and regulatory differences between the RH2aα and RH2aβ paralogs have been documented. It thus seems likely that selection varies in complex ways throughout this gene family. Results Clade model analysis of African cichlid RH2a opsins revealed a large increase in the nonsynonymous-to-synonymous substitution rate ratio (ω) following the duplication, as well as an even larger increase, one consistent with positive selection, for Lake Tanganyikan cichlid RH2aβ opsins. Analysis using the popular Branch-site models, by contrast, revealed no such alteration of constraint. Several amino acid sites known to influence spectral and non-spectral aspects of opsin biochemistry were found to be evolving divergently, suggesting that orthologous RH2a opsins may vary in terms of spectral sensitivity and response kinetics. Divergence appears to be occurring despite intronic gene conversion among the tandemly-arranged duplicates. Conclusions Our findings indicate that variation in selective constraint is associated with both gene duplication and divergence among orthologs in African cichlid RH2a opsins. At least some of this variation may reflect an adaptive response to differences in light environment. Interestingly, these patterns only became apparent through the use of Clade models, not through the use of the more widely employed Branch-site models; we suggest that this difference stems from the increased flexibility associated with Clade models. Our results thus bear both on studies of cichlid visual system evolution and on studies of gene family evolution in general.

Background The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns. Results We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages. Conclusion The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppy's behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.