BACKGROUND
colour perception is one of the disciplines where birds excel.
in general, interspecific variation in avian colour vision falls into two discrete classes, which are characterised by the spectral sensitivity of the most shortwave sensitive of the four single cones, the sws <dig>  <cit>  .
the wavelength of maximum sensitivity  of the sws <dig> ranges either from 355- <dig> nm in the 'ultraviolet sensitive' class  or from 402- <dig> nm in the 'violet sensitive'  .
the uvs class is optimised for ultraviolet sensitivity, but the vs class also has some degree of sensitivity in the ultraviolet spectrum .
changes in spectral sensitivity of the sws <dig> are ecologically important, as spectral tuning affects the abilities of birds to accurately assess the quality of potential mates  <cit> , spot elusive prey or detect other food items  <cit>  and minimise visibility of social signals in plumage coloration to predators  <cit> .
non-conservative substitutions in the sws <dig> cone opsin protein that are located in the retinal binding pocket close enough to interact with the retinal chromophore may shift λmax from uvs to vs or vice versa  <cit> .
replacement of cysteine by serine in the 90th amino acid  position, the substitution cys90ser, or the reverse substitution, alone accounts for the whole shift .
despite the simple nature of the mechanism, very few shifts have been described so far , suggesting that the sws <dig> cone spectral sensitivity is under strong stabilising selection.
the vs class is both ancestral and the most common to birds; its members are distributed throughout the avian phylogeny  <cit> .
four or five independent shifts to uvs are currently known: in shorebirds, passerida passerines, parrots, the rhea and presumably trogons .
the most promising group of birds for further investigation into the spectral tuning of sws <dig> cone opsins is clearly the passerines .
this is the most species rich of all avian orders and the only one except the shorebirds  from which both classes of spectral sensitivity are known.
every investigated member of the passerida clade of passerines belongs to the uvs class  <cit>  and it has appeared that all other passerines can be placed in the vs category ).
recently, however, Ödeen et al.
<cit>  argued that the evolution of ultraviolet sensitive vision in passeriformes is more complex than a single vs to uvs opsin shift in an ancestor of passerida, presenting molecular evidence for additional uv shifts from vs colour vision outside passerida, in fairywrens, genus malurus.
since spectral tuning of the cone is under the genetic control of a few amino acid residues in the opsin protein, it is possible to quickly classify almost any bird as vs or uvs from a sample of genomic dna  <cit> .
the accuracy of this short-fragment genomic dna approach in distinguishing uvs from vs species has been validated against all published msp data  <cit> .
we have employed this method to search for gross differences in spectral tuning in a larger sample of passerine species than has been investigated before.
our aims with this survey were to assess how stable sws <dig> cone sensitivity has been in the course of passerine evolution and to trace the evolutionary sequence of shifts in spectral tuning.
as the basis for the study, we have inferred a molecular phylogeny with sequence data from genbank.
RESULTS
opsin sequencing
we amplified the target fragment of the sws <dig> opsin gene in  <dig> passerine species from  <dig> families and one falconiform species, northern crested caracara caracara cheriway .
cycle sequencing produced 50- <dig> bp long overlapping strands of identifiable nucleotides.
sequences have been deposited in the european nucleotide archive  .
amino acid translations spanning the spectral tuning sites  <dig>   <dig> and  <dig>  <cit>  are presented in additional file  <dig>  for unknown reasons, we failed to amplify the sws <dig> opsin in two species: blue jewel-babbler ptilorrhoa caerulescens  and white-bellied erpornis erpornis zantholeuca .
position  <dig> held either serine or cysteine residues, signifying vs and uvs opsins, respectively.
neither phenylalanine nor any other aa residues suggesting a potential major shift in spectral tuning through a non-conservative substitution was present at site  <dig> .
however, the rifleman acanthisitta chloris holds leucine in spectral tuning site  <dig> .
this state is previously undescribed in birds and probably the result of a non-conservative substitution .
phylogenetic reconstruction
the inferred tree  is mostly well resolved and, except for some short internal branches, well supported.
it agrees well with previously published passerine trees based on fewer loci, such as a large-scale study of passerine relationships based on rag- <dig> and - <dig>  <cit> , as well as with more densely sampled studies of the passerida based on odc, myo and β-fibrinogen intron  <dig>  <cit>  and of the 'core corvoidea' based on odc, myo, rag- <dig> and - <dig>  and nd <dig>  <cit> .
the character optimisation infers a minimum of six changes, two from vs to uvs, two from uvs to vs and two with uncertain direction due to three nodes with ambiguous ancestral states near the root .
the number of inferred transitions are reduced to five, if clades b and c in figure  <dig>  which are reconstructed with low support, are collapsed, and cnemophilus is placed as sister to the 'core corvoidea' clade .
on the contrary, if all 'basal' nodes with posterior probability <  <dig>  are collapsed, eight transitions are inferred .
the ancestor to all passerines is reconstructed as uvs when only parrots  form the outgroup, whereas it is ambiguous when falconiforms  are also included in the outgroup .
discussion
the evolution of colour vision in birds has apparently involved many more than the four to five independent shifts between sws <dig> cone opsin types that previously available data have indicated.
in passeriformes, the picture is indeed more complex than a single vs to uvs opsin shift in an ancestor of passerida; our phylogenetic trait mapping reveals more variation among the passerines than what is known from other birds combined.
the class of colour vision of the most recent common ancestor to the passerines is ambiguous as a result of different states in parrots  and falconiforms .
as parrots are suggested to be the sister group to the passerines  <cit> , the passerine ancestor is hypothesised to be uvs .
in the subsequent passerine radiation, colour vision has changed between uvs and vs at least eight times, including two shifts in the family maluridae   <cit> .
it is interesting to note that two major clades, the passerida/petroicidae and the suboscines, are homogeneous with respect to colour vision , whereas there is considerably more variation in the rest of the tree, particularly within maluridae.
it is not immediately apparent what the selective advantage of either vs or uvs might be, as both classes are present in taxa represented on all continents and in similar habitats.
for example, the ancestor to the mostly forest-dwelling new world vireonidae has changed from vs to uvs, whereas the mainly forest-inhabiting new world suboscines are vs. moreover, the uvs passerida/petroicidae clade is mainly distributed in afro-eurasia and the new world, with few representatives in australasia; the vs suboscines occur predominantly in the new world, with relatively few species in africa, madagascar, asia and australasia; the mostly vs, but also uvs, 'basalmost' oscines  plus philesturnus, cnemophilus and toxorhampus are chiefly australasian; and the 'core corvoidea' have representatives on all continents.
the ancestral area of the passerines has been inferred to be eastern gondwana, i.e.
proto-australasia  <cit> , whereas the philesturnus/cnemophilus/toxorhampus/'core corvoidea' clade has been inferred to have originated in the proto-papuan archipelago  <cit> .
unlike maluridae , none of the multi-sampled families from the present study are polytypic in gross tuning of the sws <dig> opsin, but either contain vs or uvs taxa.
in common with the australian-papuan maluridae, the african-eurasian genus motacilla  is rich in species and subspecies with strong differentiation in male plumage coloration.
yet, all the members of motacilla sampled have base-pair identical sws <dig> opsin gene fragments.
unknown differences in other traits than sws <dig> could explain why uvs-vs shifts are so unequally distributed between passerida/petroicidae and other passerines.
spectral tuning of the sws <dig> cone opsin seems to be under strong stabilising selection in birds  <cit> , likely partly due to gross spectral tuning shifts carrying costs, such as increased retinal photooxidation and ocular light scattering .
in addition, acute short wave colour vision is dependent on a complex of co-adapted physiological traits.
ultraviolet absorption by the ocular media is usually stronger in vs than in uvs species , and the sws <dig> cone class  is shifted toward shorter wavelengths in uvs than in vs birds .
a shortwave shift in λmax of a vs sws <dig> pigment will produce a virtually negligible increase in uv sensitivity, unless it is accompanied by an increased uv transmission in the ocular media  <cit> .
a shortwave shifted sws <dig> will cause an uneven distribution of sensitivities across the spectrum, possibly deteriorating colour discriminability, if not followed by a shift of the sws <dig> pigment towards the range vacated by sws <dig>  a plausible evolutionary scenario is that increased uv transmittance in an ancestor of passerines relaxed stabilising selection on sws <dig> spectral tuning, allowing a vs to uvs shift of that opsin to reach fixation  .
later, a shortwave shift in sws <dig> spectral tuning in an ancestor of passerida/petroicidae may have improved spectral discriminability of uvs retinae and thereby reinstated stabilising selection on sws <dig> spectral tuning.
in agreement with this scenario, passerida species where this cone has been examined by msp mostly show shortwave shifted sws <dig> λmax compared to vs species from other avian orders , but unfortunately the character state is not known from any vs or non-passerida passerine.
the realised spectral sensitivities of the two shortwave sensitive cones in non-passerida passerines may be different from that of characteristic vs and uvs types .
due to a general paucity of data on sws <dig> λmax and ocular transmission, one can merely speculate at this point.
shifts from uvs to vs in passerines seem to be controlled exclusively by the cys90ser substitution.
carvalho et al.
<cit>  has reported a significant short wavelength tuning effect of substitution ser86phe, but we did not find phenylalanine in position  <dig>  in any of the species we sequenced.
the effects of all tuning site substitutions found in this study are known  <cit> , with one exception: a leucine residue in the spectral tuning site  <dig> of the rifleman acanthisitta chloris.
the most likely evolutionary event given the phylogeny in figure  <dig> is the substitution thr93leu.
this is a non-conservative substitution, changing from polar and mildly hydrophilic threonine to nonpolar, hydrophobic leucine.
site  <dig> is located in the retinal binding pocket, on the inner side of the opsin's alpha-helices close enough to interact with the retinal chromophore.
thr93leu is therefore potentially important to spectral tuning , but its effect in the avian sws <dig> opsin is presently unknown.
the reconstruction of the evolution of colour vision in passerines is conditional on the true phylogeny having been inferred.
the tree inferred here is based on a larger number of loci than any previous study of passerine relationships.
as it is overall well supported by the data, and in good agreement with previous analyses of different datasets .
with respect to the poorly supported node a, switching position between pomatostomus and orthonyx, as is possible if this node is collapsed, would require one more step in the vs/uvs optimisation and result in eight further internal nodes having ambiguous states.
further collapsing of poorly supported 'basal' nodes leads to more steps in the uv optimisation.
accordingly, the opsin data lend further support to the present topology, and the favoured topology results in a more parsimonious optimisation than the alternative topologies.
however, if the insignificantly supported nodes b and c are collapsed, and cnemophilus is placed as sister to the 'core corvoidea', one fewer change is required to explain the evolution of colour vision.
more sequence data might resolve this issue in the future.
CONCLUSIONS
spectral tuning of the sws <dig> cone opsin in birds is a trait of great ecological importance.
it appears to be under strong stabilising selection and varies categorically between different clades in the avian phylogenetic tree.
in the radiation of the very species rich order passeriformes, sequencing of a small gene fragment allows us to map and trace the change from one stable state to the other.
clades with variation in the colour vision system are nested among clades with a seemingly stable vs or uvs state, providing a rare opportunity to understand how an ecologically important trait under simple genetic control may co-evolve with, and be stabilised by, associated traits in a character complex.

 colour perception is one of the disciplines where birds excel. in general, interspecific variation in avian colour vision falls into two discrete classes, which are characterised by the spectral sensitivity of the most shortwave sensitive of the four single cones, the sws. the wavelength of maximum sensitivity of the sws ranges either from 355- nm in the'ultraviolet sensitive' class or from 402- nm in the'violet sensitive'. the uvs class is optimised for ultraviolet sensitivity, but the vs class also has some degree of sensitivity in the ultraviolet spectrum. changes in spectral sensitivity of the sws are ecologically important, as spectral tuning affects the abilities of birds to accurately assess the quality of potential mates, spot elusive prey or detect other food items and minimise visibility of social signals in plumage coloration to predators. non-conservative substitutions in the sws cone opsin protein that are located in the retinal binding pocket close enough to interact with the retinal chromophore may shift λmax from uvs to vs or vice versa. replacement of cysteine by serine in the 90th amino acid position, the substitution cys90ser, or the reverse substitution, alone accounts for the whole shift. despite the simple nature of the mechanism, very few shifts have been described so far, suggesting that the sws cone spectral sensitivity is under strong stabilising selection. the vs class is both ancestral and the most common to birds; its members are distributed throughout the avian phylogeny. four or five independent shifts to uvs are currently known: in shorebirds, passerida passerines, parrots, the rhea and presumably trogons. the most promising group of birds for further investigation into the spectral tuning of sws cone opsins is clearly the passerines. this is the most species rich of all avian orders and the only one except the shorebirds from which both classes of spectral sensitivity are known. every investigated member of the passerida clade of passerines belongs to the uvs class and it has appeared that all other passerines can be placed in the vs category ). recently, however, Ödeen et al. argued that the evolution of ultraviolet sensitive vision in passeriformes is more complex than a single vs to uvs opsin shift in an ancestor of passerida, presenting molecular evidence for additional uv shifts from vs colour vision outside passerida, in fairywrens, genus malurus. since spectral tuning of the cone is under the genetic control of a few amino acid residues in the opsin protein, it is possible to quickly classify almost any bird as vs or uvs from a sample of genomic dna. the accuracy of this short-fragment genomic dna approach in distinguishing uvs from vs species has been validated against all published msp data. we have employed this method to search for gross differences in spectral tuning in a larger sample of passerine species than has been investigated before. our aims with this survey were to assess how stable sws cone sensitivity has been in the course of passerine evolution and to trace the evolutionary sequence of shifts in spectral tuning. as the basis for the study, we have inferred a molecular phylogeny with sequence data from genbank.  opsin sequencing we amplified the target fragment of the sws opsin gene in passerine species from families and one falconiform species, northern crested caracara caracara cheriway. cycle sequencing produced 50- bp long overlapping strands of identifiable nucleotides. sequences have been deposited in the european nucleotide archive. amino acid translations spanning the spectral tuning sites and are presented in additional file for unknown reasons, we failed to amplify the sws opsin in two species: blue jewel-babbler ptilorrhoa caerulescens and white-bellied erpornis erpornis zantholeuca. position held either serine or cysteine residues, signifying vs and uvs opsins, respectively. neither phenylalanine nor any other aa residues suggesting a potential major shift in spectral tuning through a non-conservative substitution was present at site. however, the rifleman acanthisitta chloris holds leucine in spectral tuning site. this state is previously undescribed in birds and probably the result of a non-conservative substitution. phylogenetic reconstruction the inferred tree is mostly well resolved and, except for some short internal branches, well supported. it agrees well with previously published passerine trees based on fewer loci, such as a large-scale study of passerine relationships based on rag- and -, as well as with more densely sampled studies of the passerida based on odc, myo and β-fibrinogen intron and of the'core corvoidea' based on odc, myo, rag- and - and nd. the character optimisation infers a minimum of six changes, two from vs to uvs, two from uvs to vs and two with uncertain direction due to three nodes with ambiguous ancestral states near the root. the number of inferred transitions are reduced to five, if clades b and c in figure which are reconstructed with low support, are collapsed, and cnemophilus is placed as sister to the'core corvoidea' clade. on the contrary, if all'basal' nodes with posterior probability < are collapsed, eight transitions are inferred. the ancestor to all passerines is reconstructed as uvs when only parrots form the outgroup, whereas it is ambiguous when falconiforms are also included in the outgroup. discussion the evolution of colour vision in birds has apparently involved many more than the four to five independent shifts between sws cone opsin types that previously available data have indicated. in passeriformes, the picture is indeed more complex than a single vs to uvs opsin shift in an ancestor of passerida; our phylogenetic trait mapping reveals more variation among the passerines than what is known from other birds combined. the class of colour vision of the most recent common ancestor to the passerines is ambiguous as a result of different states in parrots and falconiforms. as parrots are suggested to be the sister group to the passerines, the passerine ancestor is hypothesised to be uvs. in the subsequent passerine radiation, colour vision has changed between uvs and vs at least eight times, including two shifts in the family maluridae. it is interesting to note that two major clades, the passerida/petroicidae and the suboscines, are homogeneous with respect to colour vision, whereas there is considerably more variation in the rest of the tree, particularly within maluridae. it is not immediately apparent what the selective advantage of either vs or uvs might be, as both classes are present in taxa represented on all continents and in similar habitats. for example, the ancestor to the mostly forest-dwelling new world vireonidae has changed from vs to uvs, whereas the mainly forest-inhabiting new world suboscines are vs. moreover, the uvs passerida/petroicidae clade is mainly distributed in afro-eurasia and the new world, with few representatives in australasia; the vs suboscines occur predominantly in the new world, with relatively few species in africa, madagascar, asia and australasia; the mostly vs, but also uvs,'basalmost' oscines plus philesturnus, cnemophilus and toxorhampus are chiefly australasian; and the'core corvoidea' have representatives on all continents. the ancestral area of the passerines has been inferred to be eastern gondwana, i.e. proto-australasia, whereas the philesturnus/cnemophilus/toxorhampus/'core corvoidea' clade has been inferred to have originated in the proto-papuan archipelago. unlike maluridae, none of the multi-sampled families from the present study are polytypic in gross tuning of the sws opsin, but either contain vs or uvs taxa. in common with the australian-papuan maluridae, the african-eurasian genus motacilla is rich in species and subspecies with strong differentiation in male plumage coloration. yet, all the members of motacilla sampled have base-pair identical sws opsin gene fragments. unknown differences in other traits than sws could explain why uvs-vs shifts are so unequally distributed between passerida/petroicidae and other passerines. spectral tuning of the sws cone opsin seems to be under strong stabilising selection in birds, likely partly due to gross spectral tuning shifts carrying costs, such as increased retinal photooxidation and ocular light scattering. in addition, acute short wave colour vision is dependent on a complex of co-adapted physiological traits. ultraviolet absorption by the ocular media is usually stronger in vs than in uvs species, and the sws cone class is shifted toward shorter wavelengths in uvs than in vs birds. a shortwave shift in λmax of a vs sws pigment will produce a virtually negligible increase in uv sensitivity, unless it is accompanied by an increased uv transmission in the ocular media. a shortwave shifted sws will cause an uneven distribution of sensitivities across the spectrum, possibly deteriorating colour discriminability, if not followed by a shift of the sws pigment towards the range vacated by sws a plausible evolutionary scenario is that increased uv transmittance in an ancestor of passerines relaxed stabilising selection on sws spectral tuning, allowing a vs to uvs shift of that opsin to reach fixation. later, a shortwave shift in sws spectral tuning in an ancestor of passerida/petroicidae may have improved spectral discriminability of uvs retinae and thereby reinstated stabilising selection on sws spectral tuning. in agreement with this scenario, passerida species where this cone has been examined by msp mostly show shortwave shifted sws λmax compared to vs species from other avian orders, but unfortunately the character state is not known from any vs or non-passerida passerine. the realised spectral sensitivities of the two shortwave sensitive cones in non-passerida passerines may be different from that of characteristic vs and uvs types. due to a general paucity of data on sws λmax and ocular transmission, one can merely speculate at this point. shifts from uvs to vs in passerines seem to be controlled exclusively by the cys90ser substitution. carvalho et al. has reported a significant short wavelength tuning effect of substitution ser86phe, but we did not find phenylalanine in position in any of the species we sequenced. the effects of all tuning site substitutions found in this study are known, with one exception: a leucine residue in the spectral tuning site of the rifleman acanthisitta chloris. the most likely evolutionary event given the phylogeny in figure is the substitution thr93leu. this is a non-conservative substitution, changing from polar and mildly hydrophilic threonine to nonpolar, hydrophobic leucine. site is located in the retinal binding pocket, on the inner side of the opsin's alpha-helices close enough to interact with the retinal chromophore. thr93leu is therefore potentially important to spectral tuning, but its effect in the avian sws opsin is presently unknown. the reconstruction of the evolution of colour vision in passerines is conditional on the true phylogeny having been inferred. the tree inferred here is based on a larger number of loci than any previous study of passerine relationships. as it is overall well supported by the data, and in good agreement with previous analyses of different datasets. with respect to the poorly supported node a, switching position between pomatostomus and orthonyx, as is possible if this node is collapsed, would require one more step in the vs/uvs optimisation and result in eight further internal nodes having ambiguous states. further collapsing of poorly supported'basal' nodes leads to more steps in the uv optimisation. accordingly, the opsin data lend further support to the present topology, and the favoured topology results in a more parsimonious optimisation than the alternative topologies. however, if the insignificantly supported nodes b and c are collapsed, and cnemophilus is placed as sister to the'core corvoidea', one fewer change is required to explain the evolution of colour vision. more sequence data might resolve this issue in the future.  spectral tuning of the sws cone opsin in birds is a trait of great ecological importance. it appears to be under strong stabilising selection and varies categorically between different clades in the avian phylogenetic tree. in the radiation of the very species rich order passeriformes, sequencing of a small gene fragment allows us to map and trace the change from one stable state to the other. clades with variation in the colour vision system are nested among clades with a seemingly stable vs or uvs state, providing a rare opportunity to understand how an ecologically important trait under simple genetic control may co-evolve with, and be stabilised by, associated traits in a character complex. 
interspecific variation in avian colour vision falls into two discrete classes: violet sensitive and ultraviolet sensitive. the phylogenetic distribution of sws cone opsin types in passeriformes reveals a much higher degree of complexity in avian colour vision evolution than what was previously indicated from the limited data available. clades with variation in the colour vision system are nested among clades with a seemingly stable vs or uvs state, providing a rare opportunity to understand how an ecologically important trait under simple genetic control may co-evolve with, and be stabilised by, associated traits in a character complex. changes in spectral sensitivity of the sws are ecologically important, as they affect the abilities of birds to accurately assess potential mates, find food and minimise visibility of social signals to predators. they are characterised by the spectral sensitivity of the most shortwave sensitive of the four single cones, the sws which is seemingly under direct control of as little as one amino acid substitution in the cone opsin protein. sequencing a small gene fragment has allowed us to trace the trait changing from one stable state to another through the radiation of the passeriform birds. still, available data have indicated that shifts between classes are rare, with only four to five independent acquisitions of uv sensitivity in avian evolution. in the subsequent radiation, colour vision changed between uvs and vs at least eight times.
 interspecific variation in avian colour vision falls into two discrete classes: violet sensitive and ultraviolet sensitive. they are characterised by the spectral sensitivity of the most shortwave sensitive of the four single cones, the sws which is seemingly under direct control of as little as one amino acid substitution in the cone opsin protein. changes in spectral sensitivity of the sws are ecologically important, as they affect the abilities of birds to accurately assess potential mates, find food and minimise visibility of social signals to predators. still, available data have indicated that shifts between classes are rare, with only four to five independent acquisitions of uv sensitivity in avian evolution.  we have classified a large sample of passeriform species as vs or uvs from genomic dna and mapped the evolution of this character on a passerine phylogeny inferred from published molecular sequence data. sequencing a small gene fragment has allowed us to trace the trait changing from one stable state to another through the radiation of the passeriform birds. their ancestor is hypothesised to be uvs. in the subsequent radiation, colour vision changed between uvs and vs at least eight times.  the phylogenetic distribution of sws cone opsin types in passeriformes reveals a much higher degree of complexity in avian colour vision evolution than what was previously indicated from the limited data available. clades with variation in the colour vision system are nested among clades with a seemingly stable vs or uvs state, providing a rare opportunity to understand how an ecologically important trait under simple genetic control may co-evolve with, and be stabilised by, associated traits in a character complex. 