BACKGROUND
understanding the predictability of the molecular changes associated with phenotypic transitions is a major goal in evolutionary biology.
evolutionary genetic studies across a wide range of organisms and traits have revealed that repeated phenotypic transitions are often caused by similar changes at the genetic level  <cit> .
in some cases, this molecular convergence extends not only to the locus but also to the type of mutation and even its nucleotide position .
these patterns suggest that the causes of phenotypic evolution may often be predictable at the molecular level, given knowledge of the genetic basis for the trait  <cit> .
indeed, for many traits, the mutations responsible for convergent phenotypes are concentrated in a small set of “hotspot” loci  <cit>  and are restricted to particular classes of mutations .
in addition to predictability in the mutations that cause phenotypic transitions, the genetic changes that follow phenotypic transitions can also be predictable.
for example, transitions to a parasitic or symbiotic habit are often followed by pseudogenization and molecular decay in particular functional gene categories  <cit> .
even genes which are conserved for other functions may experience an increase in evolutionary rate if the trait transition is associated with reduced breath or level of expression  <cit> .
predictable molecular patterns are also seen at the protein level, where substitutions that cause changes in function may be followed by compensatory mutations to offset fitness declines  <cit> , or by mutations to optimize the derived activity and/or increase stability of the new conformation .
despite having strong expectations about the genetic changes that are likely to follow many types of phenotypic transitions  <cit> , relatively few studies have tested these predictions at macroevolutionary scales  <cit> .
because trait losses occur relatively frequently across the tree of life , this type of transition provides a powerful tool for understanding the predictability of genetic changes following phenotypic evolution.
trait losses may lead to a range of outcomes for genes in pathways underlying those traits.
one possible outcome is the gradual erosion of pathway genes through the accumulation of inactivating mutations, leading to pseudogenization or even gene loss  <cit> .
this process is expected to occur over the span of  <dig>  to  <dig> million years  <cit>  and has been documented in association with many instances of trait loss, such as the loss of eyes in cavefish  <cit>  and teeth in edentulous mammals  <cit> .
however, molecular decay may be prevented if the underlying genes are involved in the development of multiple traits beyond the trait that was lost.
in this case, the genes may experience stabilizing selection to maintain their function or positive selection to improve function in those other contexts  <cit> .
another possible outcome for pathway genes following trait loss is recruitment for an entirely new function, although this pattern has never been documented.
to some extent, these possible evolutionary trajectories for genes after trait loss parallel those following gene duplication, in which new gene copies may undergo pseudogenization, subfunctionalization, neofunctionalization, or escape from adaptive conflict .
both evolutionary scenarios  share a reduction in constraint that alters the selective forces experienced by genes, and consequently, their evolutionary fates.
however, compared with the literature on gene duplication , much less attention has focused on the molecular fates of single-copy genes associated with cases of trait loss.
here we examine the molecular evolution of pigmentation pathway genes following repeated losses of floral pigmentation, focusing on the andean clade iochrominae in the tomato family, solanaceae.
this clade of roughly  <dig> species is well known for its diversity of floral colors  <cit> , and like many other angiosperms, these colors largely derive from red, purple, and blue anthocyanin pigments  <cit> .
these pigments are primarily produced in flowers, with little to no detectable presence in vegetative tissue in most taxa  <cit> .
over the estimated  <dig> million year history of iochrominae  <cit> , at least three lineages have lost floral anthocyanin pigmentation in association with transitions from the ancestral state of purple flowers to white or yellow flowers .
the oldest loss  is associated with a clade that has subsequently diversified to give rise to seven taxa , one of which has regained pigmentation.
the other two losses represent more recent events that occurred in single species .
the time span of these transitions  encapsulates the range over which we would expect to see molecular consequences of changes in selective constraint on pathway genes following trait loss  <cit> .
if selection maintained these genes primarily for their role in floral pigmentation, we expect losses of pigmentation to be followed by relaxed selective constraint and possibly pseudogenization.
alternatively, if the decay of these genes carries negative pleiotropic consequences, either for anthocyanins in other tissues or for the production of related compounds, the genes might remain under purifying selection, even in white-flowered lineages.
finally, if releasing genes from their role in floral pigmentation allows them to adopt new functions, then we might expect evidence of positive selection.fig.
<dig> distribution of floral anthocyanin pigmentation across iochrominae.
lineages with pigmentation are represented with black flowers and black lines; those without are represented with white flowers and gray lines.
generic names within iochrominae are abbreviated as follows: a.
= acnistus, d. = dunalia, e. = eriolarynx, i.
= iochroma, s. = saracha, and v. =vassobia.
phylogeny and estimated divergence times from sarkinen et al.
<cit>  and muchhala et al.
<cit> .
note that the timescale is split to accommodate the shallow divergences within iochrominae as well as the deeper splits among the outgroups
in order to test these possible effects of trait loss on molecular evolution, we focus on a set of three anthocyanin pathway genes, chi , f3h , and dfr .
the dfr gene represents the most downstream step among these three, and it appears to be exclusively involved in anthocyanin biosynthesis in solanaceae .
thus, it might be predicted to experience the strongest effects of relaxed constraint following loss of floral anthocyanins.
by contrast, the other two upstream enzymes are required for the production of multiple flavonoids, including anthocyanins, flavones, and flavonols .
the uncolored flavones and flavonols are primarily involved in responses to uv stress  <cit> , but also play a role in male fertility and signaling in some species  <cit> .
thus, chi and f3h might remain under purifying selection despite the loss of floral pigmentation because of their pathway position and functional significance.
the dynamics of chi evolution may also be influenced by the fact that, unlike the single copy dfr and f3h, this enzyme is encoded by two loci   <cit> .
in this analysis, we focus on the chi-a copy  that is required for floral pigmentation in solanaceae  <cit> .
the extent to which these genes experience relaxed selective pressures following the loss of floral pigmentation has important consequences for the evolutionary trajectory of these plant lineages, as the molecular decay of any of these loci would significantly reduce the potential for future regain of this trait.fig.
<dig> core anthocyanin biosynthetic pathway adapted from rausher  <cit> .
enzymes include: chs, chalcone synthase; chi, chalcone isomerase; f3h, flavanone-3-hydroxylase; dfr, dihydroflavonol reductase; ans, anthocyanidin synthase; and uf3gt, udp-glucose flavonoid 3-o-glucosyl transferase.
several of the steps required for anthocyanin pigment production are shared with other uncolored flavonoid compounds .
the enzymes examined in this study are shaded in gray
RESULTS
sequence variation in chi, f3h, and dfr across iochrominae
we recovered complete coding regions for the three anthocyanin pathway genes chi , f3h , and dfr  from all of the sampled pigmented and unpigmented iochrominae .
within iochrominae, we observed no length variation .
across all loci, pairwise divergence was low, with less than 3 % difference at the nucleotide level and less than  <dig>  % at the amino acid level across iochrominae.
divergence was higher at the family level, ranging from 8 % at the amino acid level in dfr across solanaceae to 20 % in chi, the most rapidly evolving of the three genes .
however, we did not observe any clear inactivating mutations, such as premature stop codons, or frameshifts, in any of the sequences .
variation in selective constraint across genes and across lineages
we applied codon-based maximum likelihood methods to characterize patterns of molecular evolution across these loci and test for the effect of pigment loss on selective constraint.
models with a single ratio of nonsynonymous to synonymous substitution rates  for each locus resulted in values between  <dig>  and  <dig> , suggesting that these loci have predominantly experienced purifying selection .
among the three loci, chi has the highest dn/ds ratio, followed by dfr and f3h.
the values for chi and dfr are statistically indistinguishable, while that for f3h is significantly lower .
the range of dn/ds ratios for these genes in our study is similar to that found for anthocyanin transcription factors and other core anthocyanin pathway genes in previous studies  <cit> .fig.
<dig> patterns of variation in the dn/ds ratio  across genes and between pigmented and unpigmented lineages.
the upper panel shows ω values for the single ratio model for each of the three loci .
* represents a locus with a significantly lower ω value than the other two loci.
the lower panel shows ω values for pigmented  and unpigmented  lineages under the two-ratio model.
* represents cases in which the unpigmented lineages have significantly higher ω values
we next estimated branch models with separate dn/ds ratios for pigmented and unpigmented lineages in order to test the hypothesis that these flower color transitions have altered the selective pressures acting on pigmentation genes.
the two-ratio models revealed higher dn/ds ratios in unpigmented lineages for all of the loci , consistent with relaxed selective constraint.
the ratio for unpigmented lineages was nearly twice as high as that for pigmented lineages in both chi  and f3h , and the two ratio models for these genes resulted in significantly higher likelihoods than the single ratio models .
although dn/ds is slightly elevated in unpigmented lineages for dfr, a single ratio model could not be rejected .
considering that the timing of losses of floral pigmentation varies across the sampled taxa , we examined two additional models allowing for different ratios among the unpigmented lineages.
in the first of these, we divided the unpigmented lineages between those in the outgroups  versus those in the ingroup .
we found the same pattern as in the two ratio models; the unpigmented lineages in both the ingroup and outgroup have elevated dn/ds ratios relative to the background ratio in the pigmented lineages for both chi and f3h.
however, this division did not represent a significant improvement over the two ratio models .
we also tested models separating the clade which has diversified since a recent loss  from the remaining unpigmented lineages and recovered the same result .
these analyses suggest that the pattern of elevated dn/ds ratios following losses of floral anthocyanins is not taxon-specific  but rather shared across all of the sampled lineages without floral anthocyanins.
testing for positive selection in unpigmented lineages
the finding of elevated dn/ds ratios following losses of floral pigmentation could be attributed either to reduced selective constraint or positive selection acting on multiple sites across the genes.
we implemented an additional series of maximum likelihood models to distinguish among these explanations.
first, we fit alternative branch-site models to test the hypothesis that a proportion of sites within the unpigmented lineages has experienced positive selection .
in these comparisons, we found no evidence of positively selected sites in the unpigmented lineages .
the majority of the sites  are inferred to have experienced purifying selection in both pigmented and unpigmented lineages while the remainder are evolving neutrally in one or both .
as additional confirmation of this pattern, we fit simpler sites models , and these analyses were also consistent with a lack of positive selection.
models including a proportion of sites under positive selection did not result in a significantly higher likelihood for chi or f3h .
although the addition of a proportion of positively selected sites did significantly improve the likelihood for dfr, no positively selected sites were identified in the using the bayes empirical bayes analysis  .
given previous work on dfr, we suspect the better fit of models with a category of positively selected sites reflects selection associated with shifts among different pigment types  as opposed to gains and losses of pigments.
because the dn/ds based tests may have low power when selection has not acted repeatedly on the same sites  <cit> , we employed alternative methods that focus on physiochemical properties  <cit> .
unlike dn/ds based tests, which treat all non-synonymous substitutions equally, these methods consider the physiochemical effects of amino acid changes and the magnitude of their predicted impacts on protein function.
positively selected sites are identified by comparison with a expected distribution of effects based on codon frequencies  <cit> .
focusing on the unpigmented lineages, we found zero substitutions with significant physiochemical effects in f3h .
only one such substitution was identified in dfr, and this change occurred in one of the two unpigmented outgroup taxa .
we found six sites with non-conservative substitutions in unpigmented lineages in chi  although a far greater number  were found among the pigmented lineages .
thus, the proportion of radical changes on unpigmented lineages  is less than the proportion of unpigmented branches in the phylogeny .
these patterns suggest that positive selection is not elevated in the unpigmented lineages compared to pigmented ones, but that overall, chi has experienced a more dynamic evolutionary history than the other two loci, as evidenced by its longer branch lengths .
discussion
following evolutionary losses of floral anthocyanin production, we predicted a range of possible outcomes for genes in the anthocyanin pathway.
on one extreme, if the role of these genes was limited to flower color, we would predict that losses of floral pigmentation would lead to relaxed constraint and possibly pseudogenization.
however, to the extent that these genes are important for traits beyond flower color, we might expect them to experience purifying selection, even in lineages without floral anthocyanins.
in addition, the genes could undergo positive selection to optimize their non-floral functions or to acquire new functions in lineages which no longer produce floral anthocyanins.
our results indicated that all three of the pathway genes remain under purifying selection and have not experienced positive selection following multiple losses of pigmentation across the phylogeny.
while the ratio of non-synonymous to synonymous substitutions was elevated in lineages without floral anthocyanins, we did not find evidence for positive selection, as would have been anticipated if the genes had shifted to new functions.
these results suggest that while losses of flower color relax the level of purifying selection acting on pigment genes, they do not lead to irreversible molecular decay.
conservation of anthocyanin pathway genes
our study supports previous findings that the core genes of the anthocyanin pathway are highly conserved, likely due to their wide range of functions in plant physiology.
anthocyanins and related flavonoids are found across all land plants and thus, the structural elements of the pathway trace back to their common ancestor over  <dig> million years ago  <cit> .
depending on the taxon, these compounds may play a role in pigmentation, uv stress response, signaling, and defense  <cit> .
accordingly, a loss of function of any of the core genes of the pathway  would likely affect many aspects of plant fitness.
consistent with this hypothesis, we found that chi, f3h, and dfr have remained conserved through multiple rounds of flower color loss at varying timescales .
all of the white and yellow flowered lineages presented complete coding regions, without premature stop codons or frameshift mutations, suggesting that these copies have retained their function.
although additional experiments would be required to confirm their function , the presence of uncolored flavonoids  in the flowers of several of the unpigmented taxa  <cit>  supports the conclusion that chi and f3h have been conserved.
the factors responsible for the conservation of dfr are less clear as it is only known to function in anthocyanin biosynthesis in solanaceae  <cit>  and anthocyanins have thus far not been detected in non-floral tissues in the white and yellow-flowered lineages of iochrominae  <cit> .
one possibility is that dfr contributes to pigment production in these lineages but only under particular conditions .
alternately, this enzyme may participate in additional biochemical reactions beyond those presently identified in the literature .
although this represents the one of the few systematic studies of anthocyanin genes across a clade , the strong conservation of chi, f3h, and dfr is consistent with findings from population level studies.
decades of research on flower color variation in natural populations have failed to uncover any segregating loss-of-function mutations at chi or f3h and only two for dfr  <cit> .
moreover, the individuals carrying the known dfr mutations in both cases are exceptionally rare relative to their pigmented counterparts.
given that such loss-of-function mutations are known to be quite common among horticultural varieties, these patterns at micro and macroevolutionary scales suggest that selection strongly disfavors mutations which disrupt the function of any of the core genes, preventing their fixation  <cit> .
the conservation of these core genes stands in contrast to other elements of the larger flavonoid pathway, which have the potential to rapidly pseudogenize.
in particular, the f3’5’h gene, which hydroxylates dihydroflavonols to produce the precursors of blue anthocyanins, has been found to repeatedly experience pseudogenization or deletion following transitions to red flower colors  <cit> .
in both well-studied cases, these color transitions have been associated with floral radiations occurring on the order of  <dig> to  <dig> million years ago  <cit> .
compared to the core genes examined in this study, which are required for production of anthocyanins and many other flavonoids, the role of f3’5’h appears to be limited to the blue anthocyanins, lessening the potential pleiotropic effect of its loss  <cit> .
this contrast between the evolutionary history of f3’5’h and the core genes examined here suggests that the timespan of the iochrominae radiation  would be sufficient to observe pseudogenization of chi, f3h, or dfr if these genes were not maintained by selection.
relaxed constraint following losses of floral pigmentation
although the core genes have been conserved in lineages without floral anthocyanins, our analyses indicate that chi and f3h have experienced altered selection pressures.
models allowing different dn/ds ratios for pigmented and unpigmented branches of the phylogeny resulted in a significantly better fit to the data , and in both cases, the ratio for the unpigmented branches was roughly twice the ratio for the pigmented branches .
even with this increase, all of the dn/ds values fall into the range expected for genes that are under purifying selection .
for dfr, we also found an increase in dn/ds in unpigmented lineages, but the addition of this extra parameter did not improve the likelihood .
while such an elevated dn/ds could be due to the action of positive selection, our subsequent analyses suggest that instead the pattern is due to relaxed selective constraint.
branch-site models that allow a proportion of sites to experience positive selection  did not provide a better fit to the data and identified no positively selected sites in unpigmented lineages .
given that these analyses do not account for the potential functional consequences of mutations, we compared these results to an alternative physiochemical approach to identifying positive selection  <cit> .
these analyses found little evidence of radical amino acid substitutions in dfr and f3h, and none within the white or yellow flowered iochrominae.
while chi experienced a larger number of radical changes, most of these occurred in pigmented lineages, and in particular, in the long branches of the outgroups .
thus, although some functional evolution has likely occurred across the phylogeny, these analyses do not implicate losses of flower pigmentation in driving these changes.
as with our study, many instances of trait loss have been followed by relaxed selection on the underlying genes , while positive selection on these genes has never been reported.
theoretically, once a gene is no longer required for a particular task, it has the potential to be co-opted for a new function, in a similar fashion to what has been observed for duplicate copies of genes .
one explanation for the lack of empirical examples may simply be the relative paucity of traits for which the genetic basis is sufficiently well known to test the effects of trait loss on molecular evolution of the underlying pathway.
we suspect that cases may be uncovered as our knowledge of the genotype-phenotype map expands to include a wider array of traits and taxa.
pathway position and rate variation
studies of biochemical pathways have shown that properties of the interacting genes often predict patterns of molecular evolution.
for example, more highly connected enzymes  tend to evolve more slowly than those which are less connected .
in the case of the anthocyanin pathway, the upstream genes have been predicted to experience stronger evolutionary constraint than downstream genes because their control on flux through the pathway and because of the number of products that require their activity  <cit> .
indeed, previous studies of anthocyanin genes have found evidence for this positional effect, where upstream genes generally evolve more slowly due to differences in the selective constraints  <cit> .
while our study found significant variation in rates of evolution across the genes, we did not find that most upstream genes experienced the highest constraint.
chi was the most rapidly evolving gene with the highest dn/ds ratio among the three  despite the fact that it occupies the most upstream position.
partitioned analyses of the three gene dataset showed that the dn/ds ratio for chi is significantly higher than for f3h, and statistically indistinguishable from that for dfr .
this high rate of evolution for chi may be related to several unique aspects of its function and evolutionary history.
first, unlike the other two loci, chi has at least two closely related duplicates  <cit> , which may allow for reduced constraint on the individual copies.
second, the reaction catalyzed by chi, namely the cyclization of chalcone into naringenin, can occur spontaneously  and thus is likely to occur in cells where chs is active even if chi is not present  <cit> .
these factors may help to explain why chi evolves as quickly as the downstream genes like dfr, a result also found in rausher et al.
<cit> .
future studies examining the full pathway  would clarify the extent to which the pathway position predicts rate variation and whether indeed chi is a strong outlier among upstream genes.
CONCLUSIONS
on a phylogenetic scale, trait losses have commonly been associated with molecular decay of the underlying pathway  <cit> .
however, such decay is expected to be mitigated when the genes of the pathway are required for multiple functions  <cit> .
in the case of floral anthocyanin pigmentation, our study shows that the core genes of the anthocyanin pigmentation pathway have been conserved through repeated losses of floral pigmentation, a result that likely reflects the importance of these genes for functions beyond flower color.
the conservation of these genes preserves the potential for regain of flower color following loss as observed in iochroma  and other flowering plant clades  <cit> .
despite this overall structural conservation, we identify patterns consistent with relaxed selective constraint in two of the three pathway genes  following losses of floral pigmentation.
given that the upstream products of these two genes, flavones and flavonols, are often produced in flowers and leaves  <cit> , the elevated dn/ds ratios we observe are not likely to indicate a decrease in functionality of the enzymes.
instead, this molecular evolutionary pattern may be a consequence of changes in the expression of these genes.
specifically, reduced gene expression is well known to result in higher rates of protein evolution due to relaxed selection for translational efficiency and robustness  <cit> .
although it is not known whether losses of floral pigmentation in iochrominae are associated with reduced expression of chi and f3h, such downregulation has been documented in other taxa  <cit> .
ongoing studies in the iochrominae system aimed at integrating analyses of molecular evolution, gene expression, and flavonoid production along the phylogeny will contribute to a better understanding of both the causes and consequences of phenotypic transitions.

 understanding the predictability of the molecular changes associated with phenotypic transitions is a major goal in evolutionary biology. evolutionary genetic studies across a wide range of organisms and traits have revealed that repeated phenotypic transitions are often caused by similar changes at the genetic level. in some cases, this molecular convergence extends not only to the locus but also to the type of mutation and even its nucleotide position. these patterns suggest that the causes of phenotypic evolution may often be predictable at the molecular level, given knowledge of the genetic basis for the trait. indeed, for many traits, the mutations responsible for convergent phenotypes are concentrated in a small set of “hotspot” loci and are restricted to particular classes of mutations. in addition to predictability in the mutations that cause phenotypic transitions, the genetic changes that follow phenotypic transitions can also be predictable. for example, transitions to a parasitic or symbiotic habit are often followed by pseudogenization and molecular decay in particular functional gene categories. even genes which are conserved for other functions may experience an increase in evolutionary rate if the trait transition is associated with reduced breath or level of expression. predictable molecular patterns are also seen at the protein level, where substitutions that cause changes in function may be followed by compensatory mutations to offset fitness declines, or by mutations to optimize the derived activity and/or increase stability of the new conformation. despite having strong expectations about the genetic changes that are likely to follow many types of phenotypic transitions, relatively few studies have tested these predictions at macroevolutionary scales. because trait losses occur relatively frequently across the tree of life, this type of transition provides a powerful tool for understanding the predictability of genetic changes following phenotypic evolution. trait losses may lead to a range of outcomes for genes in pathways underlying those traits. one possible outcome is the gradual erosion of pathway genes through the accumulation of inactivating mutations, leading to pseudogenization or even gene loss. this process is expected to occur over the span of to million years and has been documented in association with many instances of trait loss, such as the loss of eyes in cavefish and teeth in edentulous mammals. however, molecular decay may be prevented if the underlying genes are involved in the development of multiple traits beyond the trait that was lost. in this case, the genes may experience stabilizing selection to maintain their function or positive selection to improve function in those other contexts. another possible outcome for pathway genes following trait loss is recruitment for an entirely new function, although this pattern has never been documented. to some extent, these possible evolutionary trajectories for genes after trait loss parallel those following gene duplication, in which new gene copies may undergo pseudogenization, subfunctionalization, neofunctionalization, or escape from adaptive conflict. both evolutionary scenarios share a reduction in constraint that alters the selective forces experienced by genes, and consequently, their evolutionary fates. however, compared with the literature on gene duplication, much less attention has focused on the molecular fates of single-copy genes associated with cases of trait loss. here we examine the molecular evolution of pigmentation pathway genes following repeated losses of floral pigmentation, focusing on the andean clade iochrominae in the tomato family, solanaceae. this clade of roughly species is well known for its diversity of floral colors, and like many other angiosperms, these colors largely derive from red, purple, and blue anthocyanin pigments. these pigments are primarily produced in flowers, with little to no detectable presence in vegetative tissue in most taxa. over the estimated million year history of iochrominae, at least three lineages have lost floral anthocyanin pigmentation in association with transitions from the ancestral state of purple flowers to white or yellow flowers. the oldest loss is associated with a clade that has subsequently diversified to give rise to seven taxa, one of which has regained pigmentation. the other two losses represent more recent events that occurred in single species. the time span of these transitions encapsulates the range over which we would expect to see molecular consequences of changes in selective constraint on pathway genes following trait loss. if selection maintained these genes primarily for their role in floral pigmentation, we expect losses of pigmentation to be followed by relaxed selective constraint and possibly pseudogenization. alternatively, if the decay of these genes carries negative pleiotropic consequences, either for anthocyanins in other tissues or for the production of related compounds, the genes might remain under purifying selection, even in white-flowered lineages. finally, if releasing genes from their role in floral pigmentation allows them to adopt new functions, then we might expect evidence of positive selection.fig. distribution of floral anthocyanin pigmentation across iochrominae. lineages with pigmentation are represented with black flowers and black lines; those without are represented with white flowers and gray lines. generic names within iochrominae are abbreviated as follows: a. = acnistus, d. = dunalia, e. = eriolarynx, i. = iochroma, s. = saracha, and v. =vassobia. phylogeny and estimated divergence times from sarkinen et al. and muchhala et al.. note that the timescale is split to accommodate the shallow divergences within iochrominae as well as the deeper splits among the outgroups in order to test these possible effects of trait loss on molecular evolution, we focus on a set of three anthocyanin pathway genes, chi, f3h, and dfr. the dfr gene represents the most downstream step among these three, and it appears to be exclusively involved in anthocyanin biosynthesis in solanaceae. thus, it might be predicted to experience the strongest effects of relaxed constraint following loss of floral anthocyanins. by contrast, the other two upstream enzymes are required for the production of multiple flavonoids, including anthocyanins, flavones, and flavonols. the uncolored flavones and flavonols are primarily involved in responses to uv stress, but also play a role in male fertility and signaling in some species. thus, chi and f3h might remain under purifying selection despite the loss of floral pigmentation because of their pathway position and functional significance. the dynamics of chi evolution may also be influenced by the fact that, unlike the single copy dfr and f3h, this enzyme is encoded by two loci. in this analysis, we focus on the chi-a copy that is required for floral pigmentation in solanaceae. the extent to which these genes experience relaxed selective pressures following the loss of floral pigmentation has important consequences for the evolutionary trajectory of these plant lineages, as the molecular decay of any of these loci would significantly reduce the potential for future regain of this trait.fig. core anthocyanin biosynthetic pathway adapted from rausher. enzymes include: chs, chalcone synthase; chi, chalcone isomerase; f3h, flavanone-3-hydroxylase; dfr, dihydroflavonol reductase; ans, anthocyanidin synthase; and uf3gt, udp-glucose flavonoid 3-o-glucosyl transferase. several of the steps required for anthocyanin pigment production are shared with other uncolored flavonoid compounds. the enzymes examined in this study are shaded in gray  sequence variation in chi, f3h, and dfr across iochrominae we recovered complete coding regions for the three anthocyanin pathway genes chi, f3h, and dfr from all of the sampled pigmented and unpigmented iochrominae. within iochrominae, we observed no length variation. across all loci, pairwise divergence was low, with less than 3 % difference at the nucleotide level and less than  % at the amino acid level across iochrominae. divergence was higher at the family level, ranging from 8 % at the amino acid level in dfr across solanaceae to 20 % in chi, the most rapidly evolving of the three genes. however, we did not observe any clear inactivating mutations, such as premature stop codons, or frameshifts, in any of the sequences. variation in selective constraint across genes and across lineages we applied codon-based maximum likelihood methods to characterize patterns of molecular evolution across these loci and test for the effect of pigment loss on selective constraint. models with a single ratio of nonsynonymous to synonymous substitution rates for each locus resulted in values between and, suggesting that these loci have predominantly experienced purifying selection. among the three loci, chi has the highest dn/ds ratio, followed by dfr and f3h. the values for chi and dfr are statistically indistinguishable, while that for f3h is significantly lower. the range of dn/ds ratios for these genes in our study is similar to that found for anthocyanin transcription factors and other core anthocyanin pathway genes in previous studies.fig. patterns of variation in the dn/ds ratio across genes and between pigmented and unpigmented lineages. the upper panel shows ω values for the single ratio model for each of the three loci. * represents a locus with a significantly lower ω value than the other two loci. the lower panel shows ω values for pigmented and unpigmented lineages under the two-ratio model. * represents cases in which the unpigmented lineages have significantly higher ω values we next estimated branch models with separate dn/ds ratios for pigmented and unpigmented lineages in order to test the hypothesis that these flower color transitions have altered the selective pressures acting on pigmentation genes. the two-ratio models revealed higher dn/ds ratios in unpigmented lineages for all of the loci, consistent with relaxed selective constraint. the ratio for unpigmented lineages was nearly twice as high as that for pigmented lineages in both chi and f3h, and the two ratio models for these genes resulted in significantly higher likelihoods than the single ratio models. although dn/ds is slightly elevated in unpigmented lineages for dfr, a single ratio model could not be rejected. considering that the timing of losses of floral pigmentation varies across the sampled taxa, we examined two additional models allowing for different ratios among the unpigmented lineages. in the first of these, we divided the unpigmented lineages between those in the outgroups versus those in the ingroup. we found the same pattern as in the two ratio models; the unpigmented lineages in both the ingroup and outgroup have elevated dn/ds ratios relative to the background ratio in the pigmented lineages for both chi and f3h. however, this division did not represent a significant improvement over the two ratio models. we also tested models separating the clade which has diversified since a recent loss from the remaining unpigmented lineages and recovered the same result. these analyses suggest that the pattern of elevated dn/ds ratios following losses of floral anthocyanins is not taxon-specific but rather shared across all of the sampled lineages without floral anthocyanins. testing for positive selection in unpigmented lineages the finding of elevated dn/ds ratios following losses of floral pigmentation could be attributed either to reduced selective constraint or positive selection acting on multiple sites across the genes. we implemented an additional series of maximum likelihood models to distinguish among these explanations. first, we fit alternative branch-site models to test the hypothesis that a proportion of sites within the unpigmented lineages has experienced positive selection. in these comparisons, we found no evidence of positively selected sites in the unpigmented lineages. the majority of the sites are inferred to have experienced purifying selection in both pigmented and unpigmented lineages while the remainder are evolving neutrally in one or both. as additional confirmation of this pattern, we fit simpler sites models, and these analyses were also consistent with a lack of positive selection. models including a proportion of sites under positive selection did not result in a significantly higher likelihood for chi or f3h. although the addition of a proportion of positively selected sites did significantly improve the likelihood for dfr, no positively selected sites were identified in the using the bayes empirical bayes analysis. given previous work on dfr, we suspect the better fit of models with a category of positively selected sites reflects selection associated with shifts among different pigment types as opposed to gains and losses of pigments. because the dn/ds based tests may have low power when selection has not acted repeatedly on the same sites, we employed alternative methods that focus on physiochemical properties. unlike dn/ds based tests, which treat all non-synonymous substitutions equally, these methods consider the physiochemical effects of amino acid changes and the magnitude of their predicted impacts on protein function. positively selected sites are identified by comparison with a expected distribution of effects based on codon frequencies. focusing on the unpigmented lineages, we found zero substitutions with significant physiochemical effects in f3h. only one such substitution was identified in dfr, and this change occurred in one of the two unpigmented outgroup taxa. we found six sites with non-conservative substitutions in unpigmented lineages in chi although a far greater number were found among the pigmented lineages. thus, the proportion of radical changes on unpigmented lineages is less than the proportion of unpigmented branches in the phylogeny. these patterns suggest that positive selection is not elevated in the unpigmented lineages compared to pigmented ones, but that overall, chi has experienced a more dynamic evolutionary history than the other two loci, as evidenced by its longer branch lengths. discussion following evolutionary losses of floral anthocyanin production, we predicted a range of possible outcomes for genes in the anthocyanin pathway. on one extreme, if the role of these genes was limited to flower color, we would predict that losses of floral pigmentation would lead to relaxed constraint and possibly pseudogenization. however, to the extent that these genes are important for traits beyond flower color, we might expect them to experience purifying selection, even in lineages without floral anthocyanins. in addition, the genes could undergo positive selection to optimize their non-floral functions or to acquire new functions in lineages which no longer produce floral anthocyanins. our results indicated that all three of the pathway genes remain under purifying selection and have not experienced positive selection following multiple losses of pigmentation across the phylogeny. while the ratio of non-synonymous to synonymous substitutions was elevated in lineages without floral anthocyanins, we did not find evidence for positive selection, as would have been anticipated if the genes had shifted to new functions. these results suggest that while losses of flower color relax the level of purifying selection acting on pigment genes, they do not lead to irreversible molecular decay. conservation of anthocyanin pathway genes our study supports previous findings that the core genes of the anthocyanin pathway are highly conserved, likely due to their wide range of functions in plant physiology. anthocyanins and related flavonoids are found across all land plants and thus, the structural elements of the pathway trace back to their common ancestor over million years ago. depending on the taxon, these compounds may play a role in pigmentation, uv stress response, signaling, and defense. accordingly, a loss of function of any of the core genes of the pathway would likely affect many aspects of plant fitness. consistent with this hypothesis, we found that chi, f3h, and dfr have remained conserved through multiple rounds of flower color loss at varying timescales. all of the white and yellow flowered lineages presented complete coding regions, without premature stop codons or frameshift mutations, suggesting that these copies have retained their function. although additional experiments would be required to confirm their function, the presence of uncolored flavonoids in the flowers of several of the unpigmented taxa supports the conclusion that chi and f3h have been conserved. the factors responsible for the conservation of dfr are less clear as it is only known to function in anthocyanin biosynthesis in solanaceae and anthocyanins have thus far not been detected in non-floral tissues in the white and yellow-flowered lineages of iochrominae. one possibility is that dfr contributes to pigment production in these lineages but only under particular conditions. alternately, this enzyme may participate in additional biochemical reactions beyond those presently identified in the literature. although this represents the one of the few systematic studies of anthocyanin genes across a clade, the strong conservation of chi, f3h, and dfr is consistent with findings from population level studies. decades of research on flower color variation in natural populations have failed to uncover any segregating loss-of-function mutations at chi or f3h and only two for dfr. moreover, the individuals carrying the known dfr mutations in both cases are exceptionally rare relative to their pigmented counterparts. given that such loss-of-function mutations are known to be quite common among horticultural varieties, these patterns at micro and macroevolutionary scales suggest that selection strongly disfavors mutations which disrupt the function of any of the core genes, preventing their fixation. the conservation of these core genes stands in contrast to other elements of the larger flavonoid pathway, which have the potential to rapidly pseudogenize. in particular, the f3’5’h gene, which hydroxylates dihydroflavonols to produce the precursors of blue anthocyanins, has been found to repeatedly experience pseudogenization or deletion following transitions to red flower colors. in both well-studied cases, these color transitions have been associated with floral radiations occurring on the order of to million years ago. compared to the core genes examined in this study, which are required for production of anthocyanins and many other flavonoids, the role of f3’5’h appears to be limited to the blue anthocyanins, lessening the potential pleiotropic effect of its loss. this contrast between the evolutionary history of f3’5’h and the core genes examined here suggests that the timespan of the iochrominae radiation would be sufficient to observe pseudogenization of chi, f3h, or dfr if these genes were not maintained by selection. relaxed constraint following losses of floral pigmentation although the core genes have been conserved in lineages without floral anthocyanins, our analyses indicate that chi and f3h have experienced altered selection pressures. models allowing different dn/ds ratios for pigmented and unpigmented branches of the phylogeny resulted in a significantly better fit to the data, and in both cases, the ratio for the unpigmented branches was roughly twice the ratio for the pigmented branches. even with this increase, all of the dn/ds values fall into the range expected for genes that are under purifying selection. for dfr, we also found an increase in dn/ds in unpigmented lineages, but the addition of this extra parameter did not improve the likelihood. while such an elevated dn/ds could be due to the action of positive selection, our subsequent analyses suggest that instead the pattern is due to relaxed selective constraint. branch-site models that allow a proportion of sites to experience positive selection did not provide a better fit to the data and identified no positively selected sites in unpigmented lineages. given that these analyses do not account for the potential functional consequences of mutations, we compared these results to an alternative physiochemical approach to identifying positive selection. these analyses found little evidence of radical amino acid substitutions in dfr and f3h, and none within the white or yellow flowered iochrominae. while chi experienced a larger number of radical changes, most of these occurred in pigmented lineages, and in particular, in the long branches of the outgroups. thus, although some functional evolution has likely occurred across the phylogeny, these analyses do not implicate losses of flower pigmentation in driving these changes. as with our study, many instances of trait loss have been followed by relaxed selection on the underlying genes, while positive selection on these genes has never been reported. theoretically, once a gene is no longer required for a particular task, it has the potential to be co-opted for a new function, in a similar fashion to what has been observed for duplicate copies of genes. one explanation for the lack of empirical examples may simply be the relative paucity of traits for which the genetic basis is sufficiently well known to test the effects of trait loss on molecular evolution of the underlying pathway. we suspect that cases may be uncovered as our knowledge of the genotype-phenotype map expands to include a wider array of traits and taxa. pathway position and rate variation studies of biochemical pathways have shown that properties of the interacting genes often predict patterns of molecular evolution. for example, more highly connected enzymes tend to evolve more slowly than those which are less connected. in the case of the anthocyanin pathway, the upstream genes have been predicted to experience stronger evolutionary constraint than downstream genes because their control on flux through the pathway and because of the number of products that require their activity. indeed, previous studies of anthocyanin genes have found evidence for this positional effect, where upstream genes generally evolve more slowly due to differences in the selective constraints. while our study found significant variation in rates of evolution across the genes, we did not find that most upstream genes experienced the highest constraint. chi was the most rapidly evolving gene with the highest dn/ds ratio among the three despite the fact that it occupies the most upstream position. partitioned analyses of the three gene dataset showed that the dn/ds ratio for chi is significantly higher than for f3h, and statistically indistinguishable from that for dfr. this high rate of evolution for chi may be related to several unique aspects of its function and evolutionary history. first, unlike the other two loci, chi has at least two closely related duplicates, which may allow for reduced constraint on the individual copies. second, the reaction catalyzed by chi, namely the cyclization of chalcone into naringenin, can occur spontaneously and thus is likely to occur in cells where chs is active even if chi is not present. these factors may help to explain why chi evolves as quickly as the downstream genes like dfr, a result also found in rausher et al.. future studies examining the full pathway would clarify the extent to which the pathway position predicts rate variation and whether indeed chi is a strong outlier among upstream genes.  on a phylogenetic scale, trait losses have commonly been associated with molecular decay of the underlying pathway. however, such decay is expected to be mitigated when the genes of the pathway are required for multiple functions. in the case of floral anthocyanin pigmentation, our study shows that the core genes of the anthocyanin pigmentation pathway have been conserved through repeated losses of floral pigmentation, a result that likely reflects the importance of these genes for functions beyond flower color. the conservation of these genes preserves the potential for regain of flower color following loss as observed in iochroma and other flowering plant clades. despite this overall structural conservation, we identify patterns consistent with relaxed selective constraint in two of the three pathway genes following losses of floral pigmentation. given that the upstream products of these two genes, flavones and flavonols, are often produced in flowers and leaves, the elevated dn/ds ratios we observe are not likely to indicate a decrease in functionality of the enzymes. instead, this molecular evolutionary pattern may be a consequence of changes in the expression of these genes. specifically, reduced gene expression is well known to result in higher rates of protein evolution due to relaxed selection for translational efficiency and robustness. although it is not known whether losses of floral pigmentation in iochrominae are associated with reduced expression of chi and f3h, such downregulation has been documented in other taxa. ongoing studies in the iochrominae system aimed at integrating analyses of molecular evolution, gene expression, and flavonoid production along the phylogeny will contribute to a better understanding of both the causes and consequences of phenotypic transitions. 
phenotypic transitions, such as trait gain or loss, are predicted to carry evolutionary consequences for the genes that control their development. for example, trait losses can result in molecular decay of the pathways underlying the trait. focusing on the iochrominae clade, we examine how repeated losses of floral anthocyanin pigmentation associated with flower color transitions have affected the molecular evolution of three anthocyanin pathway genes. we recovered intact coding regions for the three genes in all of the lineages that have lost floral pigmentation, suggesting that molecular decay is not associated with these flower color transitions. maximum likelihood analyses suggest that this increase is due to relaxed constraint on anthocyanin genes in the unpigmented lineages as opposed to positive selection. however, two of the three genes show significantly elevated dn/ds ratios in lineages without floral pigmentation. moreover, this conservation maintains the potential for regain of flower color, and indicates that evolutionary losses of floral pigmentation may be readily reversible. molecular decayreversibilitytrait evolutionpredictabilitygene fateevolutionary trajectoryhttp://dx.doi.org/ /100000001national science foundation1355518smith stacey d. issue-copyright-statement© the author 2016 the broad conservation of anthocyanin pathway genes across lineages with and without floral anthocyanins is consistent with the growing consensus that losses of pigmentation are largely achieved by changes in gene expression as opposed to structural mutations.
 phenotypic transitions, such as trait gain or loss, are predicted to carry evolutionary consequences for the genes that control their development. for example, trait losses can result in molecular decay of the pathways underlying the trait. focusing on the iochrominae clade, we examine how repeated losses of floral anthocyanin pigmentation associated with flower color transitions have affected the molecular evolution of three anthocyanin pathway genes.  we recovered intact coding regions for the three genes in all of the lineages that have lost floral pigmentation, suggesting that molecular decay is not associated with these flower color transitions. however, two of the three genes show significantly elevated dn/ds ratios in lineages without floral pigmentation. maximum likelihood analyses suggest that this increase is due to relaxed constraint on anthocyanin genes in the unpigmented lineages as opposed to positive selection. despite the increase, the values for dn/ds in both pigmented and unpigmented lineages were consistent overall with purifying selection acting on these loci.  the broad conservation of anthocyanin pathway genes across lineages with and without floral anthocyanins is consistent with the growing consensus that losses of pigmentation are largely achieved by changes in gene expression as opposed to structural mutations. moreover, this conservation maintains the potential for regain of flower color, and indicates that evolutionary losses of floral pigmentation may be readily reversible. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords molecular decayreversibilitytrait evolutionpredictabilitygene fateevolutionary trajectoryhttp://dx.doi.org/ /100000001national science foundation1355518smith stacey d. issue-copyright-statement© the author 2016 