Evolution Of Animal Color Patterns

Mammalian coat and bird plumage colors

Some of the most striking and best understood examples of phenotypic divergence are the color patterns of vertebrates. Mammalian coat, bird plumage, and fish scale coloration schemes are wonderfully diverse. Much progress has been made in understanding the genetic control of color formation and of differences within and between species.

The most widespread pigment in the animal kingdom is melanin. It occurs in various chemical forms that when polymerized produce black, brown, buff, tan or even reddish pigments. Melanism, the occurrence of dark morphs within a population or species, is one of the most common forms of phenotypic variation. In mammals, two types of melanin are produced in melanocytes (the pigment cells of the epidermis and hair follicles), eumelanin and phaeomelanin, which produce black/brown and red/yellow coloration respectively. The relative amounts of eumelanin and phaeomelanin are controlled by the products of several genes. Two key proteins are the melanocortin 1 receptor (MC1R) and the Agouti protein. During the hair growth cycle, a-melanocyte stimulating hormone (a-MSH) binds to the MC1R, which triggers elevated cAMP levels and activation of tyrosinase, the rate-limiting enzyme in melanin synthesis. The Agouti protein acts as an antagonist of this process by blocking a-MSH action, inhibiting eumelanin production, and allowing the default pathway

Kaiju Mammoth

Figure 7.1

Melanism in the jaguar

The two phases of Panthera anca are shown. Source: Copyright Nancy Vandermey, EFBC/FCC.

Figure 7.1

Melanism in the jaguar

The two phases of Panthera anca are shown. Source: Copyright Nancy Vandermey, EFBC/FCC.

of phaeomelanin synthesis. The true wild-type color of mouse fur arises from a banded coloration of each hair which is mostly black but with a subapical zone of yellow pigment. This zone is produced by a pulse of Agouti protein synthesis during a phase of the hair growth cycle. In the absence of Agouti function the hair is all black, while dominant gain of function agouti alleles produces a yellow coat. Mutations in the MC1R and agouti genes are associated with natural morphs of a variety of species and domestic breeds.

One striking example of melanism is found in the jaguar (Panthera anca) of Central and South America, which occurs in both the classic orange and a melanic phase (Fig. 7.1). Sequencing of melanic jaguar MC1R alleles revealed an in-frame five codon deletion with an adjacent amino acid replacement. All melanistic jaguars were found to be either homozygous or heterozygous for the deletion allele, while all orange animals were homozygous for the wild-type allele. Breeding studies have found that melanism in this cat is dominant, the melanistic allele appears to cause a constitutive activation of the MC1R protein. Similarly, melanism in the jaguarundi (Herpailurus yaguarondi) shows a dominant or semidominant

Figure 7.2

Melanism in the rock pocket mouse

Light and dark Chaetodipus intermediusfrom the Pinacate region of Arizona are shown on light and dark rock backgrounds. Source: Photograph courtesy of Michael Nachman. Nachman MW, Hoekstra HE, D'Agostino SL. Proc Natl Acad Sci USA 2003; 100: 5268-5273.

Figure 7.2

Melanism in the rock pocket mouse

Light and dark Chaetodipus intermediusfrom the Pinacate region of Arizona are shown on light and dark rock backgrounds. Source: Photograph courtesy of Michael Nachman. Nachman MW, Hoekstra HE, D'Agostino SL. Proc Natl Acad Sci USA 2003; 100: 5268-5273.

pattern of inheritance and is associated with an independent inframe deletion of MC1R that partially overlaps with the melanism-associated deletion in the jaguar. These findings demonstrate that dramatic changes in coat color can arise from mutations in a single gene, and be inherited and expressed in a dominant fashion in wild populations.

The factors that favor the spread of melanic forms of these cats are not clear. There is compelling field data to suggest that melanism involving the MC1R gene underlies adaptation in other species. For example, rock pocket mice are generally light-colored and live on light-colored rocks. However, in the American Southwest, populations of melanic mice are found that live on dark lava formations (Fig. 7.2) and field studies suggest that the dark coloration provides protection from predators such as owls. In an Arizona population, four amino acid replacements were found in the MC1R gene that showed a perfect association with dark coloration. Furthermore, a pattern of reduced nucleotide variation in the MC1R alleles of the dark population relative to the light animals suggested the recent action of positive selection at the MC1R locus.

Clear associations between MC1R mutations and melanism have also been found in other species. For example, the bananaquit is a widely distributed bird of the Neotropics. Most individuals have a bright yellow breast plumage and a white eye stripe but on the islands of Grenada and St Vincent and a few other locales, melanic forms occur that are almost completely black. A point mutation in MC1R that changes a single amino acid is always associated with melanism. This mutation causes the identical amino acid replacement associated with melanism in domestic chickens and mice. Thus, the same MC1R mutation has occurred independently in multiple lineages, and other mutations in MC1R have occurred at different sites, all of which cause constitutive activation of the melanocortin receptor (Fig. 7.3). The association of many cases of melanism in various mammals and birds with the same gene demonstrates that, at least in the case of coloration, evolution can and does repeat itself.

There are examples of melanism caused by mutations in genes other than MC1R. In the domestic cat, a small deletion in the agouti gene is responsible for a homozygous recessive form of melanism. Since Agouti acts as an inhibitor of melanism, homozygous loss of function mutations lead to melanism. Mutations in agouti are also associated with black coat color in nine different horse breeds.

There are other, as yet unknown, evolutionary pathways to melanism in vertebrates. In a number of cat species, including the leopard, no association exists between MC1R or agouti and melanism. Perhaps most surprisingly, in a second population of melanic rock pocket mice in New Mexico, no association was found between melanism and MC1R. These cases demonstrate that while similar phenotypic changes in different populations can be due to changes in the same gene, phenotypic convergence can also occur via independent genetic mechanisms. In the case of the rock pocket mice, the lava flows on which the two populations live are less that one million years old, indicating that convergence has occurred by different mechanisms in a relatively short timeframe.

Variation at the MC1R locus is also associated with other coat color phenotypes in addition to melanism. In the Pacific Northwest, two populations of the black bear occur. In addition to the more prevalent black morph, there is a striking white-phase "Kermode" bear (Fig. 7.4). Once thought to be separate species, it is now understood that the white-phased bear carries a single amino acid replacement in MC1R (Fig. 7.3). In this case, the black allele is dominant and the white allele is recessive, suggesting that white is due to a loss of MC1R function. Interestingly, the replacement in the Kermode bear MC1R protein occurs very near to a terminal deletion in MC1R that is associated in dogs with the red or yellow coat colors of yellow labradors, golden retrievers, and Irish setters, also thought to be due to a loss of MC1R function (Fig. 7.3). The different specific coat colors among these dog breeds are due to additional modifier genes that have not yet been identified.

It is important to note that all of these examples of coat color differences that are brought about by mutations in MC1R or agouti affect the entire coat. Of course, many coat colors are patterned in mammals, in stripes, spots, bands, etc. The molecular genetic basis of these patterns is generally not understood so it is not known whether coding changes in pigmentation pathway components, or differences in their spatial regulation, underlie the phenotypic divergence of more complex patterns. There is some suggestion from the mouse agouti gene as to how more complex coat patterns may be determined. One of the most common coat color schemes in rodents and other mammals is the lighter coloration of the underside. The

Kermode Mc1r

Figure 7.3

Location of mutations in the MC1R protein associated with melanism and other coat color forms

Figure 7.3

Location of mutations in the MC1R protein associated with melanism and other coat color forms

Partial diagram of the MC1R protein shows the location of amino acid replacements (orange colored circles) or deletions (dotted lines) associated with melanism in species indicated. The inset at left shows the general topology of the MC1R protein, with mutations nearer the amino terminus associated with melanism, and those nearer the carboxy terminus associated with white (Kermode Bear) or orange, yellow, or red coats (in various dogs).

Adapted from Eizirik E, Yuhki N, Johnson WE, et al. Curr Biol 2003; 13: 448-453. Copyright (2003), reprinted with permission from Elsevier.

Figure 7.4

The white coat of the Kermode bear

The two color morphs of the black bear found in the Pacific Northwest. Source: Photograph courtesy of Charlie Russell.

Figure 7.4

The white coat of the Kermode bear

The two color morphs of the black bear found in the Pacific Northwest. Source: Photograph courtesy of Charlie Russell.

agouti gene contains two different promoters, one which is active only in the dermal papilla of hair follicles on the ventral side of the animal, and one which is active in the midpoint of the hair cycle all over the body. The presence of this ventral-specific element suggests that it is likely that differences in the regulation of agouti in different body regions and other pigmentation pathway components could evolve through changes in cis-regulatory elements and contribute to coat color pattern diversity. The genetic architecture of the evolution of spatial coat color patterns is likely to be complex and, other than in the mouse, would be difficult to examine experimentally in most mammals. However, many groups of insects also display quite diverse color patterns, and the genetic basis of variation and divergence is beginning to be understood in model groups.

Pigmentation pattern diversity in Drosophila

Insect pigmentation offers some particular advantages as models for understanding the genetic, developmental, and molecular basis of character evolution. Foremost among these is the wealth of genetic and biochemical knowledge of pigmentation pattern development in Drosophila melanogaster. The fruit fly subfamily Drosophilinae to which D. melanogaster belongs displays a vast spectrum of pigmentation patterns, usually a mixture of light and dark pigments distributed in discrete spatial patterns on the thorax, abdomen, and sometimes, the wings. The cell biology of pigmentation is entirely different in insects and vertebrates, but some of the pigment chemistry is very similar. Black coloration is a polymer of dopamelanin and brown is a polymer of dopamine melanin. Other hues are created by chemical modifications of dopa and dopamine precursors. Pigmentation patterns are determined by a variety of transcription factors that control the expression of enzymatic components of the pigmentation pathway. A number of studies have identified individual genes associated with pigmentation variation and divergence among various groups of Drosophila species. Regulatory changes in both structural and regulatory genes contribute to pigmentation divergence, and in some cases, the regulation of the same gene has been modified many times independently in different lineages to produce divergent or similar patterns.

Evolution through a modular cis-regulatory element of a pigmentation gene

The yellow and ebony genes encode proteins that promote and inhibit black melanin formation, respectively, in D. melanogaster (the genes' names reflect the phenotypes of mutants). In many groups of related species, the degree of pigmentation of body parts differs considerably, and differences in Yellow and Ebony protein expression are associated with the pattern and/or degree of pigmentation among particular taxa. For example, in D. subobscura and D. virilis, dark pigmentation is more broadly distributed on the abdomen than in D. melanogaster. The level and pattern of expression of the Yellow protein correlates with dark pigmentation (Fig. 7.5).

The expression of yellow in various body parts (wing, body, bristles, larval mouthparts) is controlled by different cis-regulatory elements. Interspecific differences in Yellow expression are due, at least in part, to the evolution of a cis-regulatory element of the yellow gene. The function of the "body" enhancer of the yellow locus has diverged between D. melanogaster, D. subobscura, and D. virilis such that higher levels and a broader pattern of Yellow are produced in the latter two, more darkly pigmented species. This can be demonstrated using transgenic techniques by introducing the respective yellow genes or regulatory elements from different species into D. melanogaster. Because the cis-regulatory elements are independent modules, the pattern, level, and timing of yellow gene expression and pigmentation can evolve in selected body regions independently of other body parts.

Darker body pigmentation is not always associated with the Yellow protein. In two closely related species, the lightly colored D. novomexicana and its darkly colored sister species D. americana, the darker coloration is not associated with evolution at the yellow gene, but rather at the ebony gene. The darker D. americana expresses lower levels of the Ebony protein which allows for darker pigmentation. These two species can be crossed and hybrids backcrossed. Genetic association studies indicate that several other unidentified loci also contribute to the difference in pigmentation. Even though pigmentation is a relatively less complex character than the morphology of body parts, and single genes can have large effects, multiple loci are probably involved in the evolution of pigmentation differences between species.

Pigmentation Gene Evolution

Figure 7.5

Evolution of body color and yellow gene regulation in Drosophila species

Figure 7.5

Evolution of body color and yellow gene regulation in Drosophila species

(a,b) D. melanogasterhas a dark stripe of melanin near the posterior edge of each abdominal segment (arrow). (c) Yellow protein is present predominantly in the cells that will produce the stripe. (d,e,g,h) In D. subobscuraand D. virilis, the abdomen is more uniformly pigmented, with more melanin produced in D. subobscura. (f,i) Yellow protein is also present throughout the abdominal segments of both species, with higher levels of protein expressed in (f) D. subobscura. (b,e,h) Abdominal segments A3-A5, with anterior at the top and the dorsal midline in the center. (c,f,i) Yellow expression in the lateral A4 segment at approximately 72 hours after puparium formation.

Source: Wittkopp PJ, Vaccaro K, Carroll SB. Curr Biol 2002; 12: 1547-1556.

Evolution at a regulatory locus associated with diverse sex-specific and segment-specific pigmentation patterns

Most members of the melanogaster species group exhibit sexually dimorphic pigmentation where the last two abdominal segments are heavily pigmented in males but not in females (Fig. 7.6, top). This sex-specific and segment-specific pattern is a fairly recent innovation, as the abdomens of both sexes are identically pigmented in most other members of the subgenus Sophophora (e.g. D. willistoni, Fig. 7.6). In D. melanogaster, the sexually dimorphic pattern is controlled by the bric- a -brac (bab) locus, which encodes two related transcription

Figure 7.6

Evolution of sex- and segment-specific pigmentation patterns through regulatory evolution at the bric-à-brac locus of Drosophila

Abdominal pigmentation differs between Drosophila species. In D. melanogaster, the intense pigmentation in segments A5 and A6 in males is due to repression of bab expression in these segments. This repression evolved in the melanogaster species group because in other members of the subgenus Sophophora, such as D. willistoni, both sexes exhibit the same patterns of pigmentation and bab expression. In more distant species, the lack of pigmentation on the midline (D. funebris) or the presence of pigmentation in spots on the midline (D. tripunctata), are associated with differences in bab2regulation. Source: Adapted from Gompel N, Carroll SB. Nature 2003; 424: 931-935.

Figure 7.6

Evolution of sex- and segment-specific pigmentation patterns through regulatory evolution at the bric-à-brac locus of Drosophila

Abdominal pigmentation differs between Drosophila species. In D. melanogaster, the intense pigmentation in segments A5 and A6 in males is due to repression of bab expression in these segments. This repression evolved in the melanogaster species group because in other members of the subgenus Sophophora, such as D. willistoni, both sexes exhibit the same patterns of pigmentation and bab expression. In more distant species, the lack of pigmentation on the midline (D. funebris) or the presence of pigmentation in spots on the midline (D. tripunctata), are associated with differences in bab2regulation. Source: Adapted from Gompel N, Carroll SB. Nature 2003; 424: 931-935.

factors that act as repressors of pigmentation. In D. melanogaster, female Bab proteins are expressed throughout the developing abdominal epidermis, but in males the Bab proteins are repressed in the fifth and sixth abdominal segments (Fig. 7.6), allowing for pigmentation of these segments. The sex- and segment-specific regulation of bab is controlled by the Doublesex and Abdominal-B proteins of the sex determination and homeotic segment identity systems, respectively. In males, Abd-B represses bab in the fifth (A5) and sixth (A6)

abdominal segments while in females the female-specific isoform of the Doublesex protein activates bab in A5 and A6 to sufficient levels to suppress pigmentation.

Comparison of Bab expression in a large number of species reveals that, in most species with male-specific pigmentation, Bab is expressed in a similar sex- and segment-specific pattern. In species in which pigmentation is identical between the sexes, Bab is expressed throughout the abdomens of both sexes. This suggests that evolutionary changes in the regulation of bab expression in the melanogaster species group played a key role in the origin of sexually dimorphic pigmentation. Because DSX and Abd-B expression are conserved across species, the favored model for this innovation would be the evolution of binding sites for the DSX and Abd-B transcription factors in the cz's-regulatory control element(s) of the bab gene that govern expression in the developing abdomen.

Further support for a central role of the bab locus in the evolution of abdominal pigment patterns comes from studies of both intraspecific variation in D. melanogaster and interspecific divergence throughout the entire subfamily Drosophilanae. In D. melanogaster, females exhibit considerable variation in the degree of pigmentation in the posterior abdomen. Quantitative genetic analysis has indicated that variation at the bab locus accounts for approximately 70% of the phenotypic variation in females. This finding has two important implications. First, it suggests that variation at bab has a major effect on pigmentation and that selection on the bab locus is a likely scenario for the origin of sexually dimorphic pigmentation in the melanogasterspecies group. Second, it also indicates that other loci contribute to variation and divergence.

Bab's role in the diversification of pigmentation patterns is broader than just within the melanogasterspecies group. A survey of a large number of species from across the subfamily with conspicuous and diverse patterns of abdominal pigmentation revealed a correlation between bab expression and melanism patterns in a majority of species. This indicates the evolutionary changes in bab have occurred multiple times in independent lineages to create different patterns. For example, in D. tripunctata, bab is repressed in circular patches of cells along the midline of the A4, A5, and A6 segments in both sexes, which correlates very well with the development of intense pigmentation in these patches (Fig. 7.6). In D. funebris and other species, Bab is expressed at high levels along the midline of the abdomen, which correlates with repression of melanin formation (Fig. 7.6). Dimorphic regulation of bab expression in the posterior abdomen has occurred several times in the course of Drosophila evolution.

Importantly, there are exceptions where bab is not associated with abdominal pigmentation, even for sexually dimorphic patterns. These findings illustrate that while evolution at bab is the most frequently exploited path to abdominal pigmentation pattern diversification, it is not the only path. The genetic architecture of pigmentation regulation in fruit flies is such that variation at other loci can be selected upon if evolution at bab is constrained, just as we saw for evolution of the MC1R and agouti genes in the evolution of mammalian fur colors.

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