DROSOPHILA TRICHOME PATTERNS
Drosophila larvae are covered by a stereotypical, segmentally repeated pattern of single-cell cuticular projections (ventral denticles and dorsal trichomes). This pattern has undergone parallel evolutionary changes in different Drosophila lineages. A large fraction of the dorsal trichome field in every segment has been transformed into naked cuticle once in the melanogaster species group (in D. sechellia) and probably three times in the virilis species group (Dickinson et al. 1993; Sucena et al. 2003). Genetic mapping has shown that the loss of trichomes in D. sechellia is due to changes in a single gene, the transcription factor ovo/shavenbaby (svb) (Sucena and Stern 2000). svb is expressed in the embryonic epidermis and induces trichome formation in a cell-autonomous manner (Payre et al. 1999). In each abdominal segment, svb is expressed in three rows of 1° and 3° cells, which develop stout trichomes, and six to seven rows of 4° cells that form finer trichomes. In D. sechellia, svb expression in the 4° cells has been lost, and the lawn of fine trichomes is replaced by naked cuticle (Sucena and Stern 2000). A similar correlation between narrower svb expression and narrower trichome belts is observed in the virilis group species that show the derived cuticular pattern (Sucena et al. 2003). Genetic evidence, although not conclusive, suggests that cis-regulatory changes at the svb locus may be responsible for the partial loss of svb expression in the virilis group as well as in D. sechellia (Sucena et al. 2003).
Fine-scale genetic mapping and transgenic analysis reveal that the apparently simple monogenic basis of species differences is in fact due to the accumulation of multiple mutations at the svb gene (McGregor et al. 2007). svb expression in the dorsal epidermis is controlled by at least three enhancers spread over 50 kb. In D. sechellia, all three enhancers drive derived expression patterns, and each enhancer has undergone at least one change that contributes to the phenotypic differences between D. sechellia and its closest relatives (McGregor et al. 2007). This finding prompts an obvious question—if species differences are caused by the fixation of multiple mutations, why was their fixation confined to a single locus?
In the genetic pathway that controls trichome development, svb is positioned at the nexus point between upstream regulators and downstream effectors (Fig. 1 and Appendix). Stripes of svb expression are defined by a complex network of signaling pathways and transcription factors that control all aspects of segmental patterning and epithelial differentiation (Sanson 2001). In turn, svb cell-autonomously activates the expression of multiple structural genes that mediate different steps in trichome formation (Chanut-Delalande et al. 2006). A mutation that alters the expression of any upstream regulator of svb in the embryonic epidermis would disrupt not only trichome development, but also the formation of muscle attachments and other cellular processes (Fig. 1). Conversely, a mutation in any one of the downstream targets of svb would alter trichome morphology but not lead to the loss of trichomes. svb may be the only “optimally pleiotropic” locus where mutations have the capacity to abolish trichome development without changing other aspects of the segmental pattern (Delon and Payre 2004; McGregor et al. 2007). Indeed, loss of svb expression in D. sechellia is accompanied by predictable changes in all of its targets (Chanut-Delalande et al. 2006). Interestingly, although ectopic expression of svb in the naked cuticle region is sufficient to induce denticle formation, these ectopic denticles have abnormal shapes (Chanut-Delalande et al. 2006). This suggests that an evolutionary gain of a new trichome or denticle field, were it to occur, would require genetic changes at other loci in addition to svb.
Figure 1. A simplified schematic of the regulatory pathway that controls larval denticle and trichome development. Positive and negative regulatory interactions are shown by pointed and blunt arrows, respectively. In this and following figures, a star next to a gene indicates that mutations in this gene are responsible for phenotypic evolution.
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All genes involved in epithelial patterning and cell differentiation also play important roles in other, unrelated developmental pathways. For example, in addition to its function in the embryonic epidermis, svb is essential for female germline development (Garfinkel et al. 1994; Mevel-Ninio et al. 1995). However, given the modular organization of tissue-specific enhancers in developmentally controlled genes, pleiotropic gene functions can be easily uncoupled in the course of evolution through fixation of cis-regulatory mutations in different enhancers (Carroll 2008). This suggests that the role of each gene in the evolution of a particular developmental pathway depends on the extent of pleiotropy of this gene within that pathway, whereas its functions in other developmental contexts are likely irrelevant.
ANTHOCYANIN PIGMENTATION IN PLANTS
Flower color often varies dramatically within and among species. This variation has profound adaptive consequences, because color largely determines the spectrum of pollinators attracted to flowers, and changes in color can lead to pollinator shifts (Bradshaw and Schemske 2003; Fenster et al. 2004; Whibley et al. 2006; Hoballah et al. 2007; Rausher 2008). Flower color is controlled in part by flavonoids, a group of plant secondary metabolites (Holton and Cornish 1995). Red, blue, and purple colors are conferred by a particular type of flavonoids called anthocyanins, although other factors such as vacuolar pH, ultrastructure of epidermal cells, and the presence of colorless co-pigments are also important (Mol et al. 1998; Koes et al. 2005). Anthocyanin synthesis is carried out in a stepwise fashion by several enzymes (Fig. 2 and Appendix 2). Importantly, this metabolic pathway includes a number of side branches that produce nonanthocyanin flavonoids that contribute to various physiological functions including pollen fertility, heat stress tolerance, UV resistance, pathogen and herbivore defense, etc. (Koes et al. 1994; Winkel-Shirley 2002; Strauss and Whittall 2006). Multiple steps in anthocyanin synthesis, ranging from several downstream reactions to the entire core pathway, are co-regulated as a single unit by a transcriptional complex including bHLH and MYB-domain transcription factors and a WD40-repeat scaffolding protein (Quattrocchio et al. 1998; Koes et al. 2005; Morita et al. 2006). Both the biosynthetic enzymes and the regulatory genes that control their expression are widely conserved among flowering plants, although many pathway components are encoded by paralogous gene families that expanded independently in different taxa (Holton and Cornish 1995; Winkel-Shirley 2001; Koes et al. 2005).
Figure 2. A generalized scheme of the anthocyanin synthesis pathway. Genes are shown in black and metabolites in gray. Transcriptional interactions (direct or indirect) are indicated by black arrows, and chemical reactions by gray arrows. The three upstream enzymes are co-regulated by the MYB/bHLH/WD-40 complex only in some plant lineages (dashed arrows).
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Genetic basis of evolutionary changes in flower color has been studied in several plant genera including Petunia, Antirrhinum (snapdragon), Ipomoea (morning glory), Aquilegia (columbine), and Mimulus (monkeyflower) (Quattrocchio et al. 1998; Bradshaw and Schemske 2003; Clegg and Durbin 2003; Schwinn et al. 2006; Whittall et al. 2006; Hoballah et al. 2007; Coberly and Rausher 2008; Streisfeld and Rausher 2009; D. L. Des Marais and M. D. Rausher, unpubl. ms). One of the most common changes is the loss of anthocyanin pigmentation, leading to the evolution of white or yellow flowers (Rausher 2008). For example, Petunia integrifolia has reddish-violet flowers and is pollinated primarily by solitary bees, whereas P. axillaris has white flowers and is pollinated by nocturnal hawkmoths. The loss of anthocyanins in P. axillaris is associated with mutations in the coding sequence of the an2 gene, which encodes a MYB-domain transcription factor (Hoballah et al. 2007). an2 is part of the protein complex that controls the expression of several enzymes in the downstream portion of the anthocyanin synthesis pathway (Fig. 2) (Quattrocchio et al. 1999). Remarkably, different isolates of P. axillaris carry at least six different nonsense or frameshift mutations in an2, indicating that loss-of-function alleles of this gene arose and were fixed repeatedly (Hoballah et al. 2007). In the absence of genome-wide QTL analysis, it is uncertain whether an2 was the only locus responsible for anthocyanin loss during species divergence. However, neither the enzyme genes regulated by an2 nor the other components of the regulatory complex (an1 and an11, which encode the bHLH and WD-40 proteins, respectively [de Vetten et al. 1997; Spelt et al. 2000]]) play major roles in the phenotypic differences between Petunia species. Similarly, in Antirrhinum, complete or partial loss of anthocyanin pigmentation in some species and subspecies is due to changes in the MYB-domain genes rosea and venosa, whereas delila and mut, the bHLH genes, do not make major contributions (Schwinn et al. 2006; Whibley et al. 2006).
Why is repeated evolutionary loss of anthocyanin pigmentation associated with changes in the MYB genes rather than any other components of the regulatory complex or biosynthetic enzymes? The anthocyanin pathway is active in many vegetative tissues in addition to flowers, and the different flavonoids produced by this pathway perform many adaptive functions in addition to pollinator recruitment (Koes et al. 1994; Winkel-Shirley 2002). The expression of anthocyanin synthesis enzymes is controlled by tissue-specific regulatory complexes that share common bHLH and WD-40 components but include different MYB-domain proteins in different tissues (Koes et al. 2005). In Petunia, for example, an2 controls anthocyanin synthesis in the flower corolla, whereas its paralog an4 performs the same function in anthers (Quattrocchio et al. 1993; Spelt et al. 2000). Loss-of-function mutations in the bHLH or WD-40 genes (an1 and an11) result in completely white flowers, whereas an2 and an4 mutations have spatially restricted effects. an1 and an11 also have pleiotropic effects on seed coat development and other cellular processes (de Vetten et al. 1997; Spelt et al. 2002; Koes et al. 2005). At the same time, mutations in the MYB genes are sufficient to eliminate or severely reduce the expression of all enzymes regulated by the MYB/bHLH/WD-40 complexes in the flower corolla in both Petunia and Antirrhinum (Quattrocchio et al. 1999; Schwinn et al. 2006). Thus, tissue-specific MYB genes may be the “optimally pleiotropic” components of the anthocyanin pathway: mutations in these genes have the greatest potential to change pollinator recruitment while having the fewest deleterious side effects. For example, different white-flower alleles segregating in natural populations of Ipomoea purpurea are due to mutations in either an MYB transcription factor (the W locus), or in chalcone synthase (the A locus), the most upstream enzyme in the anthocyanin pathway. Consistent with the differential pleiotropy hypothesis, the white aa plants show reduced survival in the field, whereas the white ww individuals have apparently normal fitness (Chang et al. 2005; Coberly and Rausher 2008).
The optimally pleiotropic genes may be different in different plant taxa due to the changing organization and tissue-specific expression of paralogous gene families (Quattrocchio et al. 1993; Durbin et al. 2003; Des Marais and Rausher 2008), and to changes in the transcriptional co-regulation of the upstream and downstream parts of the anthocyanin pathway (Quattrocchio et al. 1998; Koes et al. 2005; Morita et al. 2006). However, changes in transcription factors that regulate anthocyanin synthesis appear to be a common evolutionary mechanism. In Mimulus aurantiacus, loss of anthocyanin pigmentation is associated with the loss of F3H, DFR, and ANS expression specifically in flowers, but not in vegetative tissues, and genetic analysis suggests that this change is controlled by a single trans-acting locus (Streisfeld and Rausher 2009). In Aquilegia, repeated losses of anthocyanin pigmentation are correlated with reduced expression of multiple enzymes that are known to be controlled by the MYB/bHLH/WD-40 complexes (F3H, DFR, ANS, and 3GT) (Whittall et al. 2006). Again, the genetic basis of color differences is monogenic in at least two different pairs of Aquilegia species (Prazmo 1965; Hodges et al. 2002), suggesting that reduced expression of the biosynthetic enzymes is due to mutations in a single trans-acting factor. Similarly, in Ipomoea, changes in flower color are also correlated with tissue-specific downregulation of multiple anthocyanin synthesis enzymes (Durbin et al. 2003). Interestingly, in Aquilegia, Petunia, and Antirrhinum, expression of enzymes that are regulated by the MYB/bHLH/WD-40 complex and act late in the anthocyanin pathway is more likely to be lost than the expression of early-acting enzymes that are not controlled by these genes (Quattrocchio et al. 1999; Durbin et al. 2003; Schwinn et al. 2006; Whittall et al. 2006). Interruption of this biosynthetic pathway at an upstream step could have negative pleiotropic effects because, in addition to flower color, it would affect the synthesis of nonanthocyanin flavonoids that are important for UV resistance, pathogen defense, and other physiological adaptations (Fig. 2) (Winkel-Shirley 2002; Strauss and Whittall 2006). Thus, the branched organization of the anthocyanin pathway makes later enzymatic steps more evolutionarily labile than earlier ones (Lu and Rausher 2003; Whittall et al. 2006). In Ipomoea, however, the entire pathway starting with CHS is co-regulated by the MYB/bHLH/WD-40 complex, so that even tissue-specific MYB mutations are perforce more widely pleiotropic than in other plants (Chang et al. 2005; Morita et al. 2006). It is intriguing that genetic changes in the downstream enzymes such as DFR, ANS, or 3GT do not appear to contribute to the evolutionary losses of anthocyanin pigmentation in Petunia, Ipomoea, Mimulus, or Antirrhinum. In principle, regulatory mutations in any of these genes could produce white or yellow flowers with relatively few pleiotropic consequences. One possible explanation is that these genes may lack tissue-specific cis-regulatory elements so that their expression cannot evolve independently in different tissues.
The loss of anthocyanins is not the only common mode of flower color evolution. Changes in the relative amounts of red, blue, and yellow pigments are associated with pollinator shifts in many plant genera (Fenster et al. 2004). In Ipomoea, the difference between blue, insect-pollinated and red, hummingbird-pollinated species is due to changes in the flavonoid 3′-hydroxylase (F3′H) gene (Zufall and Rausher 2004; D. L. Des Marais and M. D. Rausher, unpubl. ms). Expression of this enzyme, which converts a precursor of the red-colored pelargonidins into a precursor of the blue-colored cyanidins, is strongly reduced in the red-flowered compared to blue-flowered species. This change is observed in three independent lineages within Ipomoea that evolved red flowers from the blue ancestral state (M. A. Streisfeld and M. D. Rausher, unpubl. ms). In all cases, reduced expression of F3′H is observed in the floral, but not in vegetative tissues, and transgenic assays suggest that these changes are due to cis-regulatory mutations. Thus, evolutionary changes again appear to occur in the optimally pleiotropic component of the developmental pathway, but this component is different for different phenotypes. Continuing work in Ipomoea, Aquilegia, Mimulus, and other genera will tell which features of anthocyanin pathway evolution are truly universal, and what these features reveal about the role of pathway topology in shaping the fixation of natural genetic variation.
DROSOPHILA COLOR PATTERNS
Drosophila color patterns vary extensively both among and within species. Differences in pigmentation can be either global, where one species or morph is uniformly darker than the other, or local, where the taxa differ in the spatial distribution of darkly and lightly pigmented areas. Both types of differences have evolved repeatedly in many different lineages (Wittkopp et al. 2003a), making Drosophila color patterns an excellent metamodel for analyzing the genetic basis of phenotypic evolution.
A key conclusion emerging from recent work is that genetic changes at different loci are responsible for color pattern differences in different Drosophila species (Table 1). Several genes involved in pigment synthesis (including yellow (y), tan (t), and ebony (e)) or in the spatial patterning of pigmentation (optomotor-blind (omb) and bric a brac (bab)) show an association with intra or interspecific pigmentation differences. For example, dark abdominal pigmentation is present in most species of the D. melanogaster species subgroup but has been lost in D. santomea (Llopart et al. 2002; Carbone et al. 2005). Genetic mapping and transgenic analysis have identified cis-regulatory changes at the t locus as one of the key causes of the difference in pigmentation between D. santomea and its closest relative, D. yakuba (Jeong et al. 2008). A similar difference in color pattern is found between D. m. malerkotliana and D. m. pallens in the ananassae subgroup. In this case, however, t makes no significant contribution to the phenotypic change (Ng et al. 2008). Similarly, omb and bab are associated with intraspecific variation in abdominal color patterns in D. polymorpha and D. melanogaster, respectively (Kopp et al. 2003; Brisson et al. 2004), but not in D. malerkotliana. More global differences in the intensity of pigmentation appear to be controlled by changes in ebony and tan in D. americana and D. novamexicana (Wittkopp et al. 2003b; P. Wittkopp, E. Stewart, L. Arnold, A. Neidert, B. Haerum, E. Thompson, S. Arkhas, G. Smith-Winberry, and L. Shefner, unpubl. ms), by ebony and yellow in D. elegans and D. gunungcola (S.-D. Yeh and J. True, pers. comm.), and by ebony in some populations of D. melanogaster (Pool and Aquadro 2007; Takahashi et al. 2007). None of these loci make a detectable contribution to the difference in pigmentation between D. m. malerkotliana and D. m. pallens (Ng et al. 2008). In general, the genetic architecture of color pattern differences ranges from a single Mendelian factor in D. kikkawai and D. jambulina (Ohnishi and Watanabe 1985) to polygenic systems involving complex gene interactions in D. arawakana and D. nigrodunni (Hollocher et al. 2000a,b). Most studied species fall somewhere between these extremes, and a moderately oligogenic basis of variation appears to be typical for this trait (Martinez and Cordeiro 1970; Spicer 1991; Wittkopp et al. 2003b; Carbone et al. 2005; Ng et al. 2008). In all cases that have been dissected at the molecular level, phenotypic changes are associated with regulatory mutations. This is probably not surprising, because many of the pigmentation enzymes and their products play additional roles in neurotransmission, so that coding sequence mutations are likely to have pleiotropic effects on nervous system function and behavior (True 2003).
Table 1. Genes associated with pigmentation differences in different Drosophila species.
|D. melanogaster (USA)||YES||NO||NO||NO||NO||1||(Kopp et al. 2003)|
|D. melanogaster (Africa)||nt2||nt||YES||nt||nt||2||(Pool and Aquadro 2007)|
|D. m. malerkotliana/D. m. pallens||NO||NO||NO||NO||NO||1||(Ng et al. 2008)|
|D. ananassae||NO||nt||YES||YES||NO||1||A. Kopp, unpubl. data|
|D. kikkawai||NO||NO||NO||NO||NO||1||A. Kopp, unpubl. data|
|D. serrata||NO||NO||NO||NO||NO||1||A. Kopp, unpubl. data|
|D. polymorpha||nt||YES||nt||nt||nt||2||(Brisson et al. 2004)|
|D. santomea/D. yakuba||NO||nt||nt||NO||YES||1||(Carbone et al. 2005; Jeong et al. 2008)|
|D. elegans/D. gunungcola||nt||nt||YES||YES||NO||1||S.-D. Yeh and J. True, pers. comm.|
|D. americana/D. novamexicana||NO||NO||YES||NO||YES||1||(Wittkopp et al. 2003b; P. Wittkopp, E. Stewart, L. Arnold, A. Neidert, B. Haerum, E. Thompson, S. Arkhas, G. Smith-Winberry, and L. Shefner, unpubl. ms)|
Different genetic basis of similar color patterns in different species may be explained by the branched organization of the Drosophila pigmentation pathway (Fig. 3 and Appendix 3). In this pathway, different metabolic reactions draw on a shared pool of soluble precursors to produce several distinct light and dark pigments (Wright 1987; True 2003; Wittkopp et al. 2003a). At least one of these reactions is reversible, with the opposing reactions catalyzed by the products of the ebony and tan loci (True et al. 2005). This nonlinear pathway structure means that changes in the output of different enzymatic reactions can produce similar phenotypes. For example, darker pigmentation can in principle be due either to increased expression or activity of enzymes required for the synthesis of dark pigments (e.g., Ddc, yellow, or tan), or to decreased expression or activity of enzymes involved in the synthesis of light pigments (such as ebony, black, or Dat). Increased or decreased expression of these enzymes can in turn be caused either by mutations in the regulatory regions of these loci, or to changes in the expression of their upstream regulators such as bab, omb, and Abd-B. Thus, the genetic target for producing any given phenotypic change is quite extensive, so that similar phenotypic adaptations can take distinct genetic paths in different evolutionary lineages.
Figure 3. A simplified schematic of the regulatory pathway that controls Drosophila pigmentation. Genes are shown in black and metabolites in gray. Transcriptional interactions (direct or indirect) are indicated by black arrows, and chemical reactions by gray arrows. Positive and negative regulatory interactions are shown by pointed and blunt arrows, respectively. Hypothesized regulatory interactions are indicated by dashed lines.
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COAT AND PLUMAGE COLOR IN VERTEBRATES
Pigmentation is as variable in vertebrates as it is in insects, with some intraspecific color variation found in most species. In particular, melanic phenotypes segregate in many mammals, birds, and reptiles (Majerus 1998). Decades of research in mice (Hoekstra 2006), chickens (Smyth 1990), and other domestic animals have identified over a hundred genes affecting coat and plumage color, and the pathway responsible for vertebrate pigmentation is understood in some detail (Fig. 4 and Appendix 4). Unlike insects, vertebrate melanin pigments are produced not by all epithelial cells but by migratory cells (melanocytes) derived from the neural crest, so that genetic changes that affect melanocyte migration lead to altered color patterns (Jackson 1994; Parichy 2006). Subsequent steps in the development of pigmentation are controlled by an endocrine signaling mechanism involving peptide hormones generated as cleavage products of the POMC protein (Fig. 4) (Pritchard and White 2007). Melanocortin hormones are widely pleiotropic, affecting behavior, metabolism, and immunity in addition to pigmentation (Ducrest et al. 2008). Melanin synthesis is regulated by the binding of melanocortins and their paracrine antagonist Agouti to the MC1R receptor expressed in melanocytes (Garcia-Borron et al. 2005). A signal transduction cascade initiated by MC1R activates the expression of MITF, a transcription factor that regulates multiple aspects of melanocyte differentiation (Lin and Fisher 2007). MITF regulates multiple genes involved in pigment synthesis, including the Tyrosinase enzyme that catalyzes the rate-limiting step in this process (Slominski et al. 2004).
Figure 4. A simplified schematic of the regulatory pathway that controls vertebrate pigmentation. Genes are shown in black and metabolites in gray. Transcriptional interactions (direct or indirect) are indicated by black arrows, and chemical reactions by gray arrows. Positive and negative regulatory interactions are shown by pointed and blunt arrows, respectively.
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Genetic basis of color variation has been studied in dozens of mammal, bird, and reptile species. A key result to emerge from these studies is the frequent involvement of MC1R. Changes in the coding sequence of MC1R, leading to altered receptor activity, are associated with melanism in pocket mice, snow geese, jaguars, lizards, and other vertebrates (Eizirik et al. 2003; Nachman et al. 2003; Mundy et al. 2004; Rosenblum et al. 2004). There are at least 16 independent cases of genetic association between MC1R and melanism in natural populations, and more examples exist in domesticated species (Hoekstra 2006; Ducrest et al. 2008). Although MC1R mutations have mainly been characterized in relation to melanism, this gene is also involved in the evolution of light coat color in beach mice (Steiner et al. 2007). In this case, MC1R is only one of two major QTLs responsible for phenotypic variation; an even greater contribution is made by the Agouti locus. In contrast to MC1R, genetic changes at Agouti are regulatory rather than coding (Steiner et al. 2007). Interestingly, skin color variation in humans is associated with both MC1R and Agouti loci (Valverde et al. 1995; Bonilla et al. 2005), and Agouti may also be responsible for coat color differences in deer mice (Hoekstra 2006).
One factor that probably contributes to the prominent roles of MC1R and Agouti in color variation is the structure of the vertebrate pigmentation pathway (Fig. 4). Although multiple melanocortin receptors are expressed in most tissues and regulate many physiological processes, MC1R is the main if not the only receptor expressed in melanocytes, and its functions in other cell types appear to be relatively minor (Pritchard and White 2007; Ducrest et al. 2008). Agouti is more widely pleiotropic, but its expression is controlled by multiple cis-regulatory elements (Dinulescu and Cone 2000) and evolutionary changes are associated with regulatory rather than coding Agouti mutations (Steiner et al. 2007). In contrast, the upstream parts of the melanocortin pathway are based on endocrine signaling, and any changes in the expression or processing of POMC are bound to have pleiotropic effects on physiology and behavior (Pritchard and White 2007; Ducrest et al. 2008). Signal transduction downstream of MC1R is mediated by a ubiquitous secondary messenger system, and its main downstream target MITF regulates many aspects of melanocyte function including survival (Steingrimsson et al. 2004; Levy et al. 2006). Thus, MC1R and Agouti may well be the “optimally pleiotropic” components of this pathway: genetic changes at these loci alone are sufficient to produce major changes in pigmentation while having the fewest pleiotropic effects (Mundy 2005; Hoekstra 2006).
However, a serious ascertainment bias undoubtedly contributes to the central position of MC1R coding changes in pigmentation literature. MC1R is a small, conserved, single-exon gene, making its coding sequence by far the easiest target for genetic association studies (Mundy 2005; Hoekstra 2006). Other loci, or, indeed, regulatory changes at MC1R are more difficult to study. Thus, the vast majority of papers on the genetic basis of color variation consist of two types: those that report an association between MC1R and pigmentation, and those that report a lack of such association. The frequency of these outcomes is roughly equal; there are at least 24 cases in which color variation is not linked to MC1R (Ducrest et al. 2008). In fact, the two alternative scenarios may be found in the same species: MC1R is associated with melanism in one natural population of rock pocket mice, but not in three others (Hoekstra and Nachman 2003; Nachman et al. 2003); a similar situation is found in different populations of beach mice (Steiner et al. 2007, 2009). Despite clear evidence that MC1R is only part of the story, no concerted effort has been made to identify the other genes, leaving us with a highly biased picture of the genetic basis of evolutionary change (but see [Steiner et al. 2007] for a welcome exception).
What could these other genes be? Pigment synthesis enzymes could well play a role in the evolution of color patterns in vertebrates, as they do in flies. Although these enzymes are essential in many tissues, their expression in different cell types is controlled by independent cis-regulatory elements. In particular, Tyrosinase and Tyrp1 expression in melanocytes is conferred by melanocyte-specific enhancers (Murisier and Beermann 2006). Null Tyrosinase alleles cause oculocutaneous albinism that would be strongly deleterious in nature, but hypomorph mutations lead to subtle changes in coat color (Beermann et al. 2004). Thus, regulatory changes that cause increased or decreased expression of Tyrosinase or other enzymes specifically in melanocytes could become fixed in natural populations. In humans, for example, noncoding variation at the Aim-1 enzyme locus is associated with natural skin color variation (Graf et al. 2007).
More upstream pathway components may also play a role in evolutionary change. Color patterns play an important adaptive role as a defense against visual predators (Caro 2005; Hoekstra 2006). However, due to the pleiotropy of the melanocortin system, pigmentation is often associated with a wide range of physiological and behavioral phenotypes, especially if it involves changes in the upstream (endocrine) part of the pathway (Ducrest et al. 2008). In lions, dark mane color correlates with high testosterone levels, fighting ability, and sexual activity (West and Packer 2002). In alpine swifts, plumage color correlates with stress resistance and body size (Roulin et al. 2008), whereas in great tits it correlates with aggression, body size, and resting metabolic rate (Roskaft et al. 1986; Kölliker et al. 1999). Melanic pigmentation itself may also be antagonistically pleiotropic due, for example, to increasing heat stress (West and Packer 2002; Caro 2005). Thus, evolutionary changes in color patterns may tend to have a different genetic basis depending not only on the selective regime, but also on what trait is under selection—pigmentation itself, or the behavioral syndromes associated with pigmentation. In cases in which physiological or behavioral changes are favorable or at least neutral, the evolution of pigmentation may be due to mutations in the upstream, highly pleiotropic components of the melanocortin pathway. In contrast, selection on color patterns is more likely to fix mutations in the “nexus” components such as MC1R and Agouti, especially when physiological changes that could result from increased melanocortin production are deleterious.