BACKGROUND
an organism's ability to perceive, and respond to the challenges arising in its environment depends critically on the specific nature and design of its sensory system.
alterations in the molecular components of the transduction cascade can have profound consequences for sensory perception, offering the opportunity to better understand the evolution of these molecular pathways in the context of the environments in which an animal may reside.
sensory systems are therefore ideally suited for evolutionary investigations at the molecular level.
for example, it has been shown that fish living at increasing depths have adapted to the changing light environment by blue-shifting their visual sensitivities via specific amino acid substitutions in one of the visual pigments that form the first step in the sensory visual pathway  <cit> .
visual systems are particularly amenable to molecular evolutionary analysis not only because of the diversity of light environments and visually-mediated tasks in which vision may be required, but also because the details of the primary phototransduction cascade have been fairly well characterized.
in vertebrates, the first step in the visual cascade are the visual pigments, membrane-spanning protein complexes located in the outer segments of the rod and cone photoreceptor cells of the retina  <cit> .
they are composed of two parts, an opsin protein component, which is a member of the g-protein coupled receptor superfamily, and a vitamin a-derived chromophore that is covalently bound to the opsin via a schiff-base linkage.
absorbing a photon of light causes the chromophore to isomerize, triggering a conformational change in the opsin protein that allows it to activate the second messenger transducin, a heterotrimeric g-protein, which ultimately leads to a neural signal that light has been perceived  <cit> .
different visual pigments can be most sensitive to photons of differing wavelengths.
spectral tuning, the process by which a visual pigment's spectral sensitivity is modified, is usually achieved through amino acid substitutions that alter the environment of the chromophore within the opsin binding pocket  <cit> .
visual pigments in vertebrates are composed of five families of opsin genes  <cit> : the sws <dig>  sws <dig>  rh <dig>  and lws cone opsins that function under bright-light, or photopic conditions, and the rh <dig> rod opsins, or rhodopsins, that allow for vision under dim-light, or scotopic, conditions.
this dichotomy in the vertebrate visual system is reflected in the fact that cone and rod photoreceptors generally have distinct morphologies and physiological functions related to their divergent roles in day vs. night vision, with cones tending to be less photosensitive but having faster response kinetics than rods  <cit> .
at least some of these differences are thought to be mediated by differing components of the visual transduction pathways contained within each cell type.
in addition to cone visual pigments, cone-specific transducins  <cit> , cgmp phosphodiesterases  <cit> , cgmp-gated ion channels  <cit> , and arrestins  <cit>  have been found.
while many of the specifics of the molecular influences on photoreceptor response kinetics remain unknown, it seems likely that some of the differences in response are due to differences in g-protein activation, as cone visual pigments are known to be less efficient in activating transducin relative to rod pigments  <cit> .
the study of vision in fish has long attracted the attention of comparative and evolutionary biologists due to the varied and often extreme lighting conditions present in the underwater environment  <cit> .
in recent years, it has been discovered that many fishes have experienced several additional rounds of opsin duplication and divergence relative to other vertebrate groups.
for example, where the typical vertebrate might possess one member of each of the lws, sws <dig>  sws <dig>  and rh <dig> cone opsin families, the zebrafish  possesses two lws, one sws <dig>  one sws <dig>  and four rh <dig> opsins  <cit> , while the distantly related medaka  possesses two lws opsins, one sws <dig> opsin, two sws <dig> opsins, and three rh <dig> opsins  <cit> .
phylogenetic relationships suggest that these additional opsin duplication events occurred independently in the lineages leading to these two fishes  <cit> , though this pattern may also reflect concerted evolution, resulting in homogenized gene sequences within species  <cit> .
though it is tempting to suggest that these increased opsin numbers reflect one of the whole genome duplication events thought to have occurred in the fish lineage  <cit> , this does not appear to be the case; evidence from whole genome sequencing projects and the sequencing of opsins from genomic libraries suggests that the sws <dig> and lws opsins are consistently arranged in one tandem array, while the multiple rh <dig> opsins are arranged in another .
this pattern is consistent with shared opsin duplication events occurring early in these fishes' evolution, but through local duplication events, not genome duplications.
in many cases, the duplicated opsins display numerous amino acid substitutions and substantial differences in spectral sensitivity , which is indicative of functional divergence following the duplication events.
in this paper we describe our investigation of the lws opsins of the guppy, poecilia reticulata.
the guppy has long served as a model system for researchers interested in natural and sexual selection, and is notable among fish in that it displays extremely high levels of variation for a number of traits, both behavioural and morphological  <cit> .
both male colour patterns  <cit>  and female mate preferences for these colour patterns  <cit> , for instance, vary dramatically among individuals within and among populations.
furthermore, microspectrophotometry  analysis of the guppy's photoreceptor cells revealed visual system variation; guppies appears to possess three photoreceptors maximally sensitive to different wavelengths of long-wavelength light, but the retina of individual fish may have different numbers and combinations of these three photoreceptors  <cit> .
it was suggested that these three different photoreceptors reflect the presence of two spectrally-distinct lws visual pigments, expressed either independently or in conjunction  <cit> .
we characterized the lws opsins of the guppy using degenerate primers to amplify lws opsin fragments from a cdna library constructed from a single individual.
surprisingly, our results indicate that guppy lws opsins have experienced several recent rounds of duplication and divergence, and reveal the presence of at least six expressed lws opsin sequences in a single individual.
RESULTS
we amplified and sequenced, from cdna obtained from a single guppy individual, six lws opsin sequences corresponding to a  <dig> bp fragment of the lws opsin .
this fragment of the opsin corresponds to lws exons  <dig> and  <dig>  and codes for the portion spanning from the beginning of the second extracellular loop to the middle of the seventh transmembrane domain .
variation within this portion of the gene is known to have a disproportionately large effect on visual pigment phenotype  <cit> .
most notably, this fragment contains transmembrane domains  <dig> and  <dig>  which include many of the known lws spectral tuning sites, including residues  <dig> and  <dig> that account for much of the difference between human red and green visual pigments  <cit> .
this fragment also contains the third cytoplasmic loop, which is thought to strongly influence the interaction between the activated visual pigment and the downstream g-protein  <cit> .
in order to eliminate errors due to enzyme misincorporations, template switching and other pcr artifacts, multiple clones from separate pcr reactions were sequenced, and an error rate of ~ <dig> % was allowed among the clones .
one of the opsins, lws <dig>  which may be expressed at low levels, was only isolated once from cdna, but several clones were isolated and sequenced from genomic dna.
the other five lws opsin sequences obtained from cdna were confirmed by designing primers specific to each lws sequence, and performing amplifications from genomic dna as well.
these genomic amplification products were then sequenced to confirm they were the same as those isolated from cdna.
among the six opsin sequences found, there were a total of  <dig> variable nucleotide sites and  <dig> variable amino acid sites .
percentage sequence differences among the guppy lws opsin sequences ranged from  <dig> % to  <dig>  %   <cit> ; additional file 1).
all guppy sequences had functionally critical residues known to be conserved among vertebrate opsins, such as the highly conserved lysine to which the chromophore is covalently bound via a schiff-base linkage , and a cysteine residue  involved in a disulphide linkage with a residue in the third transmembrane helix, cys <dig>  <cit> .
these observations, along with the fact that the sequences were obtained from mrna expressed in the head, suggest that the guppy lws opsins are functional.
we aligned the guppy lws opsins with a sample of teleost fish lws opsin sequences , including the medaka   <cit> , two cichids   <cit> , two pufferfish   <cit> , and a close relative of the guppy, the bluefin killifish   <cit> .
lws opsins from the more distantly related ayu smelt   <cit> , atlantic salmon   <cit> , and zebrafish   <cit>  were also included as outgroups.
figure  <dig> presents the results of phylogenetic analyses performed on this alignment.
though some nodes are weakly supported, the relationships among the ingroup acanthopterygian fish are consistent with current phylogenetic hypotheses for this group  <cit> .
interestingly, the lws opsins of the guppy and the killifish, which is currently known to have at least two lws opsins, display trans-specific evolution; that is, guppy lws <dig> is most closely related to l. goodei lwsb while guppy lws1– <dig> are most closely related to l. goodei lwsa .
this pattern suggests that a lws opsin duplication event  pre-dated the guppy-killifish split, and that four more duplication events have occurred more recently within the guppy lineage.
whether or not all of the different guppy lws opsin sequences represent products of true gene duplication events, or instead divergence among alleles within a single gene locus, is not currently known, but the results suggest that the guppy has at least three and possibly as many as six lws opsin loci.
observing this many distinct lws opsin sequences in a single individual guppy is a highly surprising result.
not only is this number larger than the estimate derived from msp analysis of guppy lws photoreceptor cells  <cit> , but it is, to our knowledge, greater than has been found for any other fish studied to date.
whole genome sequencing or screening and mapping of genomic libraries has confirmed single lws opsins in two species of pufferfish  <cit> , and two lws opsins in both the zebrafish  <cit>  and medaka  <cit> , while screening and southern blotting of a genomic library suggests the presence of two  lws loci in the cavefish   <cit> .
in humans, the lws opsins are located tandemly along the x-chromosome; this tandem array expands and contracts as a result of non-homologous recombination events  <cit> , and individuals can have from one to six x-linked lws opsins per x chromosome  <cit> .
the genomic organization of guppy lws opsins is not currently known, but if guppy lws opsins are arranged in a tandem array then it is possible that similar mechanisms may have generated the larger number of guppy lws opsins.
evolutionary patterns of selective constraint were investigated in our data set using codon-based likelihood phylogenetic models, as implemented in paml  <cit> .
estimating the ratio of non-synonymous to synonymous rates  in a single parameter model  on the tree shown in fig.
<dig> revealed a low overall dn/ds ratio of  <dig>  .
this is indicative of fairly strong purifying selection across sites, a result typical of most studies of proteins with highly conserved functional roles  <cit> .
models that allow ω to vary across sites were implemented in order to test for sites that may be under positive selection  <cit> ; however, none of these tests were able to detect evidence of positive selection in our data set.
models incorporating site classes under positive selection were not found to be significantly better than neutral models in likelihood ratio tests, including m1a/m2a, and m7/m <dig> comparisons .
only the m0/m <dig> comparison was significant, though this is not a strict test of positive selection.
for the m <dig> model, the third site class was found to have a greatly elevated dn/ds ratio, though even in this case it remains below  <dig> .
branch  <cit>  and branch-sites  <cit>  models were also implemented in order to test for changes in selective constraint along lineages leading to the more divergent sequences, lws <dig> and lws <dig> .
both lineages were found to have a statistically significant, or nearly significant, increase in dn/ds ratio .
these ratios were found to be even more elevated in branch-sites models , although the addition of this site class was not found to be statistically significant in either lineage .
the significant changes in selective constraint in some of the lws opsin lineages led us to examine in further detail sites that may be important in shifting opsin function in our data set.
several lws opsin spectral tuning sites have been identified through comparative sequence analysis and site-directed mutagenesis studies  <cit> .
among the guppy lws opsins identified in this study, variation exists at several sites known to be functionally important .
most notable are the differences at sites  <dig> and 285; guppy lws <dig> possesses the same residues at those sites as the human green-lws opsin , while guppy lws1– <dig>  possess the same residues as the human red-lws opsin .
these two substitutions are known, through mutagenesis, to cause a relatively large spectral shift of approximately 15– <dig> nm, and are the primary substitutions responsible for the functional difference between human red and green lws opsins  <cit> .
this strongly suggests that the lws <dig> may shifted toward shorter wavelengths  relative to the other guppy lws opsins isolated in this study.
this shift in function may be the reason for the significantly elevated dn/ds ratio found in codon-based models allowing for an additional ω parameter along this branch .
moreover, branch-sites models identified both residues  <dig> and  <dig> as possible targets of positive selection, though inclusion of this class of positively selected sites was not found to be statistically significant .
sites  <dig> and  <dig>  minor spectral tuning sites, are also variable in some of the sequences in our data set .
at site  <dig>  guppy lws <dig> possesses a threonine, while the rest possess an isoleucine; this mutation was found to alter spectral sensitivity by a few nm in human lws opsins  <cit> .
at site  <dig>  guppy lws3– <dig> possess a glycine, while the rest possess an alanine; mutagenesis studies have shown that substituting a serine for an alanine at this site does have a small effect on spectral sensitivity in human lws opsin  <cit> .
the effects of such substitutions on spectral sensitivity are small enough that they are often undetected by methods such as msp, which could explain why msp studies of guppy photoreceptor cells suggested a relatively low number of visual pigments  sensitive to long-wavelength light  <cit>  relative to the higher number of lws opsin sequences  found in this study.
there are also additional substitutions observed among guppy lws opsin sequences that have not yet been explored by mutagenesis methods which may influence spectral sensitivity as well .
such mutations will require experimental evaluation through site-directed mutagenesis and spectrophotometric analysis of expressed visual pigments  <cit> .
variation also exists among guppy lws opsin sequences at sites that likely influence other, non-spectral properties of the visual pigment .
the most notable differences occur within the third cytoplasmic loop, a portion of the visual pigment known to be critically important for binding and activating transducin following the absorption of a photon of light  <cit> .
guppy lws <dig> differs from the rest at seven aa sites within this loop.
the effect of several of these sites on transducin activation rates was experimentally tested in mutated bovine rhodopsin expressed in vitro  <cit>  .
the effect of most substitutions was to reduce transducin activation in the mutants relative to wildtype bovine rhodopsin.
the physiochemical properties of residues at sites  <dig>   <dig>  and  <dig>  guppy lws <dig> are more similar to the wildtype bovine rhodopsin, suggesting that guppy lws <dig> may have an enhanced transducin activating ability as compared with other guppy lws opsins.
however, the residues possessed by guppy lws <dig> at sites  <dig>   <dig>  and  <dig> appear to be unique among vertebrate opsins, with all other vertebrate opsins being either invariant or possessing residues with fairly different physiochemical properties.
the resemblance of the residues in guppy lws <dig> at key sites with rhodopsin, combined with the known importance of the third cytoplasmic loop in g-protein binding and activation  <cit> , raise the interesting possibility that the guppy lws <dig> opsin may have divergent function with regards to transducin activation relative to other lws opsins.
many of these same substitutions in the third cytoplasmic loop are present in l. goodei lwsb, the killifish opsin most closely related to guppy lws <dig>  including the unique substitutions at site  <dig>   <dig>  and  <dig> , suggesting that variation in transducin activating capacity is being selectively maintained.
this is consistent with our codon-based analyses using branch-specific models, which revealed a nearly statistically significant,  <dig> -fold increase in ω along the branch leading to guppy lws <dig> and l. goodei lwsb , even if the branch-site analysis of this lineage did not provide any evidence of positive selection .
interestingly, bayes empirical bayes  <cit>  analysis did identify five residues , all within the third cytoplasmic loop .
the evolutionary mechanisms that have promoted and maintained this diversity of lws opsins in the guppy remain to be investigated.
guppies are known to have highly variable colour patterns and mate preferences  <cit> .
the chroma of orange patches on male guppies varies among individuals, and it has been suggested that these differences may reflect differences in male foraging ability or parasite load  <cit> .
by having multiple lws opsins with slight variation in spectral sensitivity, female guppies may be able to better discriminate among differently coloured males  <cit>  or the brightly-coloured fruits that occasionally fall into the guppy's streams  <cit> .
also, it has been observed in the cichlid metriaclima zebra that a region of the cichlid genome that influences colour pattern is closely linked to the sws2-lws opsin tandem array  <cit> ; selection acting to maintain colour pattern variation could potentially lead to the maintenance of variation in physically linked opsin genes.
both directional  <cit>  and balancing  <cit>  selection have been shown to operate on variation in male colour patterns in the guppy, and could therefore function to influence visual variation if a similar linkage exists in the guppy.
in future work, we plan to investigate the relationship between behavioural ecology and opsin molecular biology in the guppy by studying the influence of opsin genes on individual behaviour and fitness.
CONCLUSIONS
our results demonstrated the presence of at least six expressed lws opsin sequences within a single guppy individual.
this number is substantially higher than the number found in any other fish to date, and higher than the minimum number previously estimated for the guppy by msp.
phylogenetic analysis reveals that these guppy lws opsins fall into two groups, one group of relatively closely related sequences, and another that appears to have descended from a duplication event pre-dating the guppy-killifish split.
many of the amino acid substitutions that distinguish these guppy lws opsins appear at sites thought to be functionally important, either for spectral tuning or transducin activation.
this is consistent with codon-based analyses, which indicate changes in selective constraint along two of the more divergent lineages of guppy lws opsins.

 an organism's ability to perceive, and respond to the challenges arising in its environment depends critically on the specific nature and design of its sensory system. alterations in the molecular components of the transduction cascade can have profound consequences for sensory perception, offering the opportunity to better understand the evolution of these molecular pathways in the context of the environments in which an animal may reside. sensory systems are therefore ideally suited for evolutionary investigations at the molecular level. for example, it has been shown that fish living at increasing depths have adapted to the changing light environment by blue-shifting their visual sensitivities via specific amino acid substitutions in one of the visual pigments that form the first step in the sensory visual pathway. visual systems are particularly amenable to molecular evolutionary analysis not only because of the diversity of light environments and visually-mediated tasks in which vision may be required, but also because the details of the primary phototransduction cascade have been fairly well characterized. in vertebrates, the first step in the visual cascade are the visual pigments, membrane-spanning protein complexes located in the outer segments of the rod and cone photoreceptor cells of the retina. they are composed of two parts, an opsin protein component, which is a member of the g-protein coupled receptor superfamily, and a vitamin a-derived chromophore that is covalently bound to the opsin via a schiff-base linkage. absorbing a photon of light causes the chromophore to isomerize, triggering a conformational change in the opsin protein that allows it to activate the second messenger transducin, a heterotrimeric g-protein, which ultimately leads to a neural signal that light has been perceived. different visual pigments can be most sensitive to photons of differing wavelengths. spectral tuning, the process by which a visual pigment's spectral sensitivity is modified, is usually achieved through amino acid substitutions that alter the environment of the chromophore within the opsin binding pocket. visual pigments in vertebrates are composed of five families of opsin genes: the sws sws rh and lws cone opsins that function under bright-light, or photopic conditions, and the rh rod opsins, or rhodopsins, that allow for vision under dim-light, or scotopic, conditions. this dichotomy in the vertebrate visual system is reflected in the fact that cone and rod photoreceptors generally have distinct morphologies and physiological functions related to their divergent roles in day vs. night vision, with cones tending to be less photosensitive but having faster response kinetics than rods. at least some of these differences are thought to be mediated by differing components of the visual transduction pathways contained within each cell type. in addition to cone visual pigments, cone-specific transducins, cgmp phosphodiesterases, cgmp-gated ion channels, and arrestins have been found. while many of the specifics of the molecular influences on photoreceptor response kinetics remain unknown, it seems likely that some of the differences in response are due to differences in g-protein activation, as cone visual pigments are known to be less efficient in activating transducin relative to rod pigments. the study of vision in fish has long attracted the attention of comparative and evolutionary biologists due to the varied and often extreme lighting conditions present in the underwater environment. in recent years, it has been discovered that many fishes have experienced several additional rounds of opsin duplication and divergence relative to other vertebrate groups. for example, where the typical vertebrate might possess one member of each of the lws, sws sws and rh cone opsin families, the zebrafish possesses two lws, one sws one sws and four rh opsins, while the distantly related medaka possesses two lws opsins, one sws opsin, two sws opsins, and three rh opsins. phylogenetic relationships suggest that these additional opsin duplication events occurred independently in the lineages leading to these two fishes, though this pattern may also reflect concerted evolution, resulting in homogenized gene sequences within species. though it is tempting to suggest that these increased opsin numbers reflect one of the whole genome duplication events thought to have occurred in the fish lineage, this does not appear to be the case; evidence from whole genome sequencing projects and the sequencing of opsins from genomic libraries suggests that the sws and lws opsins are consistently arranged in one tandem array, while the multiple rh opsins are arranged in another. this pattern is consistent with shared opsin duplication events occurring early in these fishes' evolution, but through local duplication events, not genome duplications. in many cases, the duplicated opsins display numerous amino acid substitutions and substantial differences in spectral sensitivity, which is indicative of functional divergence following the duplication events. in this paper we describe our investigation of the lws opsins of the guppy, poecilia reticulata. the guppy has long served as a model system for researchers interested in natural and sexual selection, and is notable among fish in that it displays extremely high levels of variation for a number of traits, both behavioural and morphological. both male colour patterns and female mate preferences for these colour patterns, for instance, vary dramatically among individuals within and among populations. furthermore, microspectrophotometry analysis of the guppy's photoreceptor cells revealed visual system variation; guppies appears to possess three photoreceptors maximally sensitive to different wavelengths of long-wavelength light, but the retina of individual fish may have different numbers and combinations of these three photoreceptors. it was suggested that these three different photoreceptors reflect the presence of two spectrally-distinct lws visual pigments, expressed either independently or in conjunction. we characterized the lws opsins of the guppy using degenerate primers to amplify lws opsin fragments from a cdna library constructed from a single individual. surprisingly, our results indicate that guppy lws opsins have experienced several recent rounds of duplication and divergence, and reveal the presence of at least six expressed lws opsin sequences in a single individual.  we amplified and sequenced, from cdna obtained from a single guppy individual, six lws opsin sequences corresponding to a bp fragment of the lws opsin. this fragment of the opsin corresponds to lws exons and and codes for the portion spanning from the beginning of the second extracellular loop to the middle of the seventh transmembrane domain. variation within this portion of the gene is known to have a disproportionately large effect on visual pigment phenotype. most notably, this fragment contains transmembrane domains and which include many of the known lws spectral tuning sites, including residues and that account for much of the difference between human red and green visual pigments. this fragment also contains the third cytoplasmic loop, which is thought to strongly influence the interaction between the activated visual pigment and the downstream g-protein. in order to eliminate errors due to enzyme misincorporations, template switching and other pcr artifacts, multiple clones from separate pcr reactions were sequenced, and an error rate of ~ % was allowed among the clones. one of the opsins, lws which may be expressed at low levels, was only isolated once from cdna, but several clones were isolated and sequenced from genomic dna. the other five lws opsin sequences obtained from cdna were confirmed by designing primers specific to each lws sequence, and performing amplifications from genomic dna as well. these genomic amplification products were then sequenced to confirm they were the same as those isolated from cdna. among the six opsin sequences found, there were a total of variable nucleotide sites and variable amino acid sites. percentage sequence differences among the guppy lws opsin sequences ranged from % to %; additional file 1). all guppy sequences had functionally critical residues known to be conserved among vertebrate opsins, such as the highly conserved lysine to which the chromophore is covalently bound via a schiff-base linkage, and a cysteine residue involved in a disulphide linkage with a residue in the third transmembrane helix, cys. these observations, along with the fact that the sequences were obtained from mrna expressed in the head, suggest that the guppy lws opsins are functional. we aligned the guppy lws opsins with a sample of teleost fish lws opsin sequences, including the medaka, two cichids, two pufferfish, and a close relative of the guppy, the bluefin killifish. lws opsins from the more distantly related ayu smelt, atlantic salmon, and zebrafish were also included as outgroups. figure presents the results of phylogenetic analyses performed on this alignment. though some nodes are weakly supported, the relationships among the ingroup acanthopterygian fish are consistent with current phylogenetic hypotheses for this group. interestingly, the lws opsins of the guppy and the killifish, which is currently known to have at least two lws opsins, display trans-specific evolution; that is, guppy lws is most closely related to l. goodei lwsb while guppy lws1– are most closely related to l. goodei lwsa. this pattern suggests that a lws opsin duplication event pre-dated the guppy-killifish split, and that four more duplication events have occurred more recently within the guppy lineage. whether or not all of the different guppy lws opsin sequences represent products of true gene duplication events, or instead divergence among alleles within a single gene locus, is not currently known, but the results suggest that the guppy has at least three and possibly as many as six lws opsin loci. observing this many distinct lws opsin sequences in a single individual guppy is a highly surprising result. not only is this number larger than the estimate derived from msp analysis of guppy lws photoreceptor cells, but it is, to our knowledge, greater than has been found for any other fish studied to date. whole genome sequencing or screening and mapping of genomic libraries has confirmed single lws opsins in two species of pufferfish, and two lws opsins in both the zebrafish and medaka, while screening and southern blotting of a genomic library suggests the presence of two lws loci in the cavefish. in humans, the lws opsins are located tandemly along the x-chromosome; this tandem array expands and contracts as a result of non-homologous recombination events, and individuals can have from one to six x-linked lws opsins per x chromosome. the genomic organization of guppy lws opsins is not currently known, but if guppy lws opsins are arranged in a tandem array then it is possible that similar mechanisms may have generated the larger number of guppy lws opsins. evolutionary patterns of selective constraint were investigated in our data set using codon-based likelihood phylogenetic models, as implemented in paml. estimating the ratio of non-synonymous to synonymous rates in a single parameter model on the tree shown in fig. revealed a low overall dn/ds ratio of. this is indicative of fairly strong purifying selection across sites, a result typical of most studies of proteins with highly conserved functional roles. models that allow ω to vary across sites were implemented in order to test for sites that may be under positive selection; however, none of these tests were able to detect evidence of positive selection in our data set. models incorporating site classes under positive selection were not found to be significantly better than neutral models in likelihood ratio tests, including m1a/m2a, and m7/m comparisons. only the m0/m comparison was significant, though this is not a strict test of positive selection. for the m model, the third site class was found to have a greatly elevated dn/ds ratio, though even in this case it remains below. branch and branch-sites models were also implemented in order to test for changes in selective constraint along lineages leading to the more divergent sequences, lws and lws. both lineages were found to have a statistically significant, or nearly significant, increase in dn/ds ratio. these ratios were found to be even more elevated in branch-sites models, although the addition of this site class was not found to be statistically significant in either lineage. the significant changes in selective constraint in some of the lws opsin lineages led us to examine in further detail sites that may be important in shifting opsin function in our data set. several lws opsin spectral tuning sites have been identified through comparative sequence analysis and site-directed mutagenesis studies. among the guppy lws opsins identified in this study, variation exists at several sites known to be functionally important. most notable are the differences at sites and 285; guppy lws possesses the same residues at those sites as the human green-lws opsin, while guppy lws1– possess the same residues as the human red-lws opsin. these two substitutions are known, through mutagenesis, to cause a relatively large spectral shift of approximately 15– nm, and are the primary substitutions responsible for the functional difference between human red and green lws opsins. this strongly suggests that the lws may shifted toward shorter wavelengths relative to the other guppy lws opsins isolated in this study. this shift in function may be the reason for the significantly elevated dn/ds ratio found in codon-based models allowing for an additional ω parameter along this branch. moreover, branch-sites models identified both residues and as possible targets of positive selection, though inclusion of this class of positively selected sites was not found to be statistically significant. sites and minor spectral tuning sites, are also variable in some of the sequences in our data set. at site guppy lws possesses a threonine, while the rest possess an isoleucine; this mutation was found to alter spectral sensitivity by a few nm in human lws opsins. at site guppy lws3– possess a glycine, while the rest possess an alanine; mutagenesis studies have shown that substituting a serine for an alanine at this site does have a small effect on spectral sensitivity in human lws opsin. the effects of such substitutions on spectral sensitivity are small enough that they are often undetected by methods such as msp, which could explain why msp studies of guppy photoreceptor cells suggested a relatively low number of visual pigments sensitive to long-wavelength light relative to the higher number of lws opsin sequences found in this study. there are also additional substitutions observed among guppy lws opsin sequences that have not yet been explored by mutagenesis methods which may influence spectral sensitivity as well. such mutations will require experimental evaluation through site-directed mutagenesis and spectrophotometric analysis of expressed visual pigments. variation also exists among guppy lws opsin sequences at sites that likely influence other, non-spectral properties of the visual pigment. the most notable differences occur within the third cytoplasmic loop, a portion of the visual pigment known to be critically important for binding and activating transducin following the absorption of a photon of light. guppy lws differs from the rest at seven aa sites within this loop. the effect of several of these sites on transducin activation rates was experimentally tested in mutated bovine rhodopsin expressed in vitro. the effect of most substitutions was to reduce transducin activation in the mutants relative to wildtype bovine rhodopsin. the physiochemical properties of residues at sites and guppy lws are more similar to the wildtype bovine rhodopsin, suggesting that guppy lws may have an enhanced transducin activating ability as compared with other guppy lws opsins. however, the residues possessed by guppy lws at sites and appear to be unique among vertebrate opsins, with all other vertebrate opsins being either invariant or possessing residues with fairly different physiochemical properties. the resemblance of the residues in guppy lws at key sites with rhodopsin, combined with the known importance of the third cytoplasmic loop in g-protein binding and activation, raise the interesting possibility that the guppy lws opsin may have divergent function with regards to transducin activation relative to other lws opsins. many of these same substitutions in the third cytoplasmic loop are present in l. goodei lwsb, the killifish opsin most closely related to guppy lws including the unique substitutions at site and, suggesting that variation in transducin activating capacity is being selectively maintained. this is consistent with our codon-based analyses using branch-specific models, which revealed a nearly statistically significant, -fold increase in ω along the branch leading to guppy lws and l. goodei lwsb, even if the branch-site analysis of this lineage did not provide any evidence of positive selection. interestingly, bayes empirical bayes analysis did identify five residues, all within the third cytoplasmic loop. the evolutionary mechanisms that have promoted and maintained this diversity of lws opsins in the guppy remain to be investigated. guppies are known to have highly variable colour patterns and mate preferences. the chroma of orange patches on male guppies varies among individuals, and it has been suggested that these differences may reflect differences in male foraging ability or parasite load. by having multiple lws opsins with slight variation in spectral sensitivity, female guppies may be able to better discriminate among differently coloured males or the brightly-coloured fruits that occasionally fall into the guppy's streams. also, it has been observed in the cichlid metriaclima zebra that a region of the cichlid genome that influences colour pattern is closely linked to the sws2-lws opsin tandem array; selection acting to maintain colour pattern variation could potentially lead to the maintenance of variation in physically linked opsin genes. both directional and balancing selection have been shown to operate on variation in male colour patterns in the guppy, and could therefore function to influence visual variation if a similar linkage exists in the guppy. in future work, we plan to investigate the relationship between behavioural ecology and opsin molecular biology in the guppy by studying the influence of opsin genes on individual behaviour and fitness.  our results demonstrated the presence of at least six expressed lws opsin sequences within a single guppy individual. this number is substantially higher than the number found in any other fish to date, and higher than the minimum number previously estimated for the guppy by msp. phylogenetic analysis reveals that these guppy lws opsins fall into two groups, one group of relatively closely related sequences, and another that appears to have descended from a duplication event pre-dating the guppy-killifish split. many of the amino acid substitutions that distinguish these guppy lws opsins appear at sites thought to be functionally important, either for spectral tuning or transducin activation. this is consistent with codon-based analyses, which indicate changes in selective constraint along two of the more divergent lineages of guppy lws opsins. 
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. 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. 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. 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. likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy lws opsin lineages. 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. 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. 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. we identified six lws opsins expressed within a single individual. 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 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.  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.  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. 15– march first international conference on phylogenomics sainte-adèle, québec, canada 