Molecular population genetics of the melanic plumage polymorphism in Arctic skuas (Stercorarius parasiticus): evidence for divergent selection on plumage colour

The Arctic skua (Stercorarius parasiticus) is a classic example of an avian plumage polymorphism, with variation in melanin‐based ventral plumage coloration defining pale, intermediate and dark morphs in adults of both sexes. However, despite several decades of field research, there is an incomplete understanding of how the polymorphism in ventral plumage colour is maintained and the selective forces involved. Here, we investigate selection on a locus (MC1R) that is strongly associated with plumage colour variation in Arctic skuas using patterns of nucleotide variation and comparison to neutral loci (nuclear introns and mtDNA). We find that three linked nonsynonymous mutations in MC1R, including the single mutation described previously, are associated with plumage colour in the Arctic skua. The position of nonsynonymous mutations on a MC1R haplotype network implies that divergent selection drove the initial evolution of the colour morphs. Comparisons of F_STs of MC1R vs. nuclear introns among five skua populations differing in proportion of dark morphs along an approximate north–south cline reveal a signature of divergent selection on MC1R. In contrast, we find limited evidence for balancing selection on MC1R within populations, although the power is low. Our results provide strong evidence for both past and ongoing selection on MC1R, and, by implication, plumage colour in Arctic skuas. The results suggest that a fruitful avenue for future ecological studies will be analysis of selection on morphs in colonies at the extremes along the morph ratio cline.     

Effect of the MC1R gene on sexual dimorphism in melanin‐based colorations

Variants of the melanocortin‐1 receptor (MC1R) gene result in abrupt, naturally selected colour morphs. These genetic variants may differentially affect sexual dimorphism if one morph is naturally selected in the two sexes but another morph is naturally or sexually selected only in one of the two sexes (e.g. to confer camouflage in reproductive females or confer mating advantage in males). Therefore, the balance between natural and sexual selections can differ between MC1R variants, as suggest studies showing interspecific correlations between sexual dimorphism and the rate of nonsynonymous vs. synonymous amino acid substitutions at the MC1R. Surprisingly, how MC1R is related to within‐species sexual dimorphism, and thereby to sex‐specific selection, has not yet been investigated. We tackled this issue in the barn owl (Tyto alba), a species showing pronounced variation in the degree of reddish pheomelanin‐based coloration and in the number and size of black feather spots. We found that a valine (V)‐to‐isoleucine (I) substitution at position 126 explains up to 30% of the variation in the three melanin‐based colour traits and in feather melanin content. Interestingly, MC1R genotypes also differed in the degree of sexual colour dimorphism, with individuals homozygous for the II MC1R variant being 2 times redder and 2.5 times less sexually dimorphic than homozygous individuals for the VV MC1R variant. These findings support that MC1R interacts with the expression of sexual dimorphism and suggest that a gene with major phenotypic effects and weakly influenced by variation in body condition can participate in sex‐specific selection processes.     

Testing whether macroevolution follows microevolution: Are colour differences among swans (<Emphasis Type="Italic">Cygnus</Emphasis>) attributable to variation at the <Emphasis Type="Italic">MC1R</Emphasis> locus?

Background  

The MC1R (melanocortin-1 receptor) locus underlies intraspecific variation in melanin-based dark plumage coloration in several unrelated birds with plumage polymorphisms. There is far less evidence for functional variants of MC1R being involved in interspecific variation, in which spurious genotype-phenotype associations arising through population history are a far greater problem than in intraspecific studies. We investigated the relationship between MC1R variation and plumage coloration in swans (Cygnus), which show extreme variation in melanic plumage phenotypes among species (white to black).     

Born blonde: a recessive loss‐of‐function mutation in the melanocortin 1 receptor is associated with cream coat coloration in Antarctic fur seals

Although the genetic basis of color variation has been extensively studied in humans and domestic animals, the genetic polymorphisms responsible for different color morphs remain to be elucidated in many wild vertebrate species. For example, hypopigmentation has been observed in numerous marine mammal species but the underlying mutations have not been identified. A particularly compelling candidate gene for explaining color polymorphism is the melanocortin 1 receptor (MC1R), which plays a key role in the regulation of pigment production. We therefore used Antarctic fur seals (Arctocephalus gazella) as a highly tractable marine mammal system with which to test for an association between nucleotide variation at the MC1R and melanin‐based coat color phenotypes. By sequencing 70 wild‐type individuals with dark‐colored coats and 26 hypopigmented individuals with cream‐colored coats, we identified a nonsynonymous mutation that results in the substitution of serine with phenylalanine at an evolutionarily highly conserved structural domain. All of the hypopigmented individuals were homozygous for the allele coding for phenylalanine, consistent with a recessive loss‐of‐function allele. In order to test for cryptic population structure, which can generate artefactual associations, and to evaluate whether homozygosity at the MC1R could be indicative of low genome‐wide heterozygosity, we also genotyped all of the individuals at 50 polymorphic microsatellite loci. We were unable to detect any population structure and also found that wild‐type and hypopigmented individuals did not differ significantly in their standardized multilocus heterozygosity. Such a lack of association implies that hypopigmented individuals are unlikely to suffer disproportionately from inbreeding depression, and hence, we have no reason to believe that they are at a selective disadvantage in the wider population.     

The molecular basis of the plumage colour polymorphism in the Tahiti reed‐warbler Acrocephalus caffer

The endemic Tahiti reed‐warbler Acrocephalus caffer occurs in two distinct morphs, a typical or ‘yellow’ morph and a melanic or ‘dark’ morph, which are found together in the valleys of the eastern and central parts of the island of Tahiti (Society Islands, French Polynesia). We investigated the molecular basis of the plumage colour polymorphism in this species using sequences of the melanocortin‐1 receptor (MC1R), a gene often found associated to melanism in birds. We found that the MC1R genotype was perfectly associated with plumage colour in the Tahiti reed‐warbler, with the same nonsynonymous substitution that showed a correlation with phenotype in the Caribbean bananaquit Coereba flaveola. An heterozygous reed‐warbler at this site presented a melanic phenotype, suggesting that the melanic allele is dominant. All other Polynesian reed‐warbler species, which do not have a melanic morph, shared the ‘yellow’ nucleotide at this position. These results suggested that the same mutation point was linked to a melanic polymorphism in two unrelated passerine birds.     

Melanocortin‐1 receptor (MC1R) genotypes do not correlate with size in two cohorts of medium‐to‐giant congenital melanocytic nevi

Congenital melanocytic nevi (CMN) are cutaneous malformations whose prevalence is inversely correlated with projected adult size. CMN are caused by somatic mutations, but epidemiological studies suggest that germline genetic factors may influence CMN development. In CMN patients from the U.K., genetic variants in MC1R, such as p.V92M and loss‐of‐function variants, have been previously associated with larger CMN. We analyzed the association of MC1R variants with CMN characteristics in two distinct cohorts of medium‐to‐giant CMN patients from Spain (N = 113) and from France, Norway, Canada, and the United States (N = 53), similar at the clinical and phenotypical level except for the number of nevi per patient. We found that the p.V92M or loss‐of‐function MC1R variants either alone or in combination did not correlate with CMN size, in contrast to the U.K. CMN patients. An additional case–control analysis with 259 unaffected Spanish individuals showed a higher frequency of MC1R compound heterozygous or homozygous variant genotypes in Spanish CMN patients compared to the control population (15.9% vs. 9.3%; p = .075). Altogether, this study suggests that MC1R variants are not associated with CMN size in these non‐UK cohorts. Additional studies are required to define the potential role of MC1R as a risk factor in CMN development.     

Non‐synonymous SNPs in MC1R gene are associated with the extended black variant in domestic ducks (Anas platyrhynchos)

In this study, we performed a sequence characterization of the duck melanocortin 1 receptor (alpha‐melanocyte stimulating hormone receptor) (MC1R) gene to analyze the relationship between MC1R polymorphism and the extended black variant in domestic ducks based on the extended black (E) and non‐extended black (e⁺) allele hypothesis of the duck MC1R gene. Both c.52G>A and c.376G>A substitutions are highly associated with the duck extended black variant (P < 0.01), but the novel c.52G>A substitution is more of a fit for the allele hypothesis of the duck MC1R gene.     

A stable,genetically determined colour dimorphism in the dung beetle Aphodius depressus: patterns and mechanisms

1. Melanism – the occurrence of dark morphs – in insects has been attributed to differences in, among other things, thermoregulation and immune defence. Dark individuals are hypothesised to perform better in colder areas, and to exhibit stronger melanin‐based immune defence. 2. In the present study, the geographical distribution of two colour morphs in Aphodius depressus (Kugelann), its climatic correlates, and temporal stability was described. Underlying mechanisms were then targeted through experiments: the inheritance of colour through controlled crosses, heating rates by thermal imaging, physiological tolerance by critical thermal limits, and immune efficiency by melanisation of implants. 3. In A. depressus, colour appears inherited by simple Mendelian principles, with red dominating over black. The frequency of two colour morphs forms a large‐scale cline. In the South West of Finland, all individuals are black, whereas, in the North East, most are red. This pattern has remained constant over 13 years (1996–2008). 4. The geographical pattern was not attributable to thermoregulation: black morphs were more abundant in warmer rather than colder parts of the country. In experiments, we found no differences in the heating rate of the two morphs, or in their upper temperature maxima. Neither did the morphs differ in their response to artificial objects inserted in their haemolymph. 5. Overall, colour variation in A. depressus occurs as a stable, genetically determined dimorphism, governed by Mendelian inheritance. Yet, no support for prevailing theory of factors sustaining melanism was found. The reasons for colour polymorphism in insects may thus be complex, and should be sought on a case‐by‐case basis.     

Mutations in ASIP and MC1R: dominant black and recessive black alleles segregate in native Swedish sheep populations

By studying genes associated with coat colour, we can understand the role of these genes in pigmentation but also gain insight into selection history. North European short‐tailed sheep, including Swedish breeds, have variation in their coat colour, making them good models to expand current knowledge of mutations associated with coat colour in sheep. We studied ASIP and MC1R, two genes with known roles in pigmentation, and their association with black coat colour. We did this by sequencing the coding regions of ASIP in 149 animals and MC1R in 129 animals from seven native Swedish sheep breeds in individuals with black, white or grey fleece. Previously known mutations in ASIP [recessive black allele: g.100_105del (D₅) and/or g.5172T>A] were associated with black coat colour in Klövsjö and Roslag sheep breeds and mutations in both ASIP and MC1R (dominant black allele: c.218T>A and/or c.361G>A) were associated with black coat colour in Swedish Finewool. In Gotland, Gute, Värmland and Helsinge sheep breeds, coat colour inheritance was more complex: only 11 of 16 individuals with black fleece had genotypes that could explain their black colour. These breeds have grey individuals in their populations, and grey is believed to be a result of mutations and allelic copy number variation within the ASIP duplication, which could be a possible explanation for the lack of a clear inheritance pattern in these breeds. Finally, we found a novel missense mutation in MC1R (c.452G>A) in Gotland, Gute and Värmland sheep and evidence of a duplication of MC1R in Gotland sheep.     

Melanocortin 1 receptor (MC1R) polymorphisms’ influence on size and dermoscopic features of nevi

The melanocortin 1 receptor (MC1R) is a highly polymorphic gene. The loss‐of‐function MC1R variants (“R”) have been strongly associated with red hair color phenotype and an increased melanoma risk. We sequenced the MC1R gene in 175 healthy individuals to assess the influence of MC1R on nevus phenotype. We identified that MC1R variant carriers had larger nevi both on the back [p‐value = .016, adjusted for multiple parameters (adj. p‐value)] and on the upper limbs (adj. p‐value = .007). Specifically, we identified a positive association between the “R” MC1R variants and visible vessels in nevi [p‐value = .033, corrected using the FDR method for multiple comparisons (corrected p‐value)], dots and globules in nevi (corrected p‐value = .033), nevi with eccentric hyperpigmentation (corrected p‐value = .033), a high degree of freckling (adj. p‐value = .019), and an associative trend with presence of blue nevi (corrected p‐value = .120). In conclusion, the MC1R gene appears to influence the nevus phenotype.     

Geographic variation of color polymorphism in two sibling ladybird species,Harmonia yedoensis and H. axyridis (Coleoptera: Coccinellidae)

Geographical variation of elytra color pattern in two sibling ladybird species, Harmonia yedoensis and H. axyridis (Coleoptera: Coccinellidae), was examined. The two species are distributed sympatrically in central Japan; however, only H. yedoensis and H. axyridis occur in the Ryukyu Islands (southern Japan) and Hokkaido island (northern Japan), respectively. The frequency of elytra color patterns was significantly different between the two species in all sympatric locations and our results were inconsistent with the classical theory on Müllerian mimicry. The most dominant pattern of H. axyridis was the least dominant of H. yedoensis in all sympatric populations. Furthermore, the frequency of the non‐melanic form (red ground color with or without black spots) increased towards the south in H. yedoensis. This tendency was in contrast to the known geographical cline in H. axyridis in which the melanic form (black ground color with red spots) was gradually displaced with the non‐melanic form northwards in the Japanese archipelago. We discuss possible selective factors including predator avoidance, thermal adaptation and reproductive character displacement, all of which might contribute to the maintenance of the color polymorphism in the two Harmonia species.     

Immune defence,a possible nonvisual selective factor behind the industrial melanism of moths (Lepidoptera)

It is generally believed that industrial melanism in Lepidoptera is mainly caused by differential predation by birds. In polluted areas, melanic individuals are favoured by natural selection because they are better camouflaged than pale moths on lichen‐free and sooty tree trunks. In this article, we show that, in the black arches moth (Lymantria monacha), melanic morphs have a stronger encapsulation response than pale morphs against nylon monofilament implants. This indicates that the melanic and pale morphs differ in the strength of their immune defence. The same chemical precursors and their end product, melanin pigment, are involved in the encapsulation response and in the external coloration. Thus, it seems that there may be two possible, not mutually exclusive, explanations for the frequency changes observed in the industrial melanism of moths. The dominant gene causes an increase in the amount of melanin pigment and its precursors. This increase causes two changes: an intensified immune defence as a form of improved encapsulation ability of foreign objects, and the well‐known protective dark coloration (a case of relational pleiotropy). It seems possible that industrial melanism is a by‐product of selection on the strength of immunity. In the field, these pleiotropic aspects are exceedingly difficult to distinguish from each other, and the factors may even be compensatory. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 831–838.     

Melanism in guinea fowl (Numida meleagris) is associated with a deletion of Phenylalanine‐256 in the MC1R gene

We have characterized a deletion in the MC1R gene causing the loss of one amino acid (p.Phe256del), which is perfectly associated with melanism in guinea fowl (Numida meleagris). Co‐segregation of the p.Phe256del with melanism was confirmed in 25 offspring born from a cross of two heterozygote birds; therefore we suggest that this mutation is responsible for the black phenotype. Interestingly, this is the first case of recessive melanism linked to MC1R.     

Investigating the Role of the Melanocortin-1 Receptor Gene in an Extreme Case of Microgeographical Variation in the Pattern of Melanin-Based Plumage Pigmentation

The Réunion grey white-eye (Zosterops borbonicus) is a single-island endemic passerine bird that exhibits striking geographically structured melanic polymorphism at a very small spatial scale. We investigated the genetic basis of this color polymorphism by testing whether the melanocortin-1 receptor (MC1R), a gene often involved in natural melanic polymorphism in birds, was associated with the observed plumage variation. Although we found three non-synonymous mutations, we detected no association between MC1R variants and color morphs, and the main amino-acid variant found in the Réunion grey white-eye was also present at high frequency in the Mauritius grey white-eye (Zosterops mauritianus), its sister species which shows no melanic polymorphism. In addition, neutrality tests and analysis of population structure did not reveal any obvious pattern of positive or balancing selection acting on MC1R. Altogether these results indicate that MC1R does not play a role in explaining the melanic variation observed in the Réunion grey white-eye. We propose that other genes such as POMC, Agouti or any other genes involved in pigment synthesis will need to be investigated in future studies if we are to understand how selection shapes complex patterns of melanin-based plumage pigmentation.

Trial Registration

All sequences submitted to Genbank. Accession number: {"type":"entrez-nucleotide-range","attrs":{"text":"JX914505 to JX914564","start_term":"JX914505","end_term":"JX914564","start_term_id":"429326239","end_term_id":"429326357"}}JX914505 to JX914564.     

Sex‐specific allelic transmission bias suggests sexual conflict at MC1R

Sexual conflict arises when selection in one sex causes the displacement of the other sex from its phenotypic optimum, leading to an inevitable tension within the genome – called intralocus sexual conflict. Although the autosomal melanocortin‐1‐receptor gene (MC1R) can generate colour variation in sexually dichromatic species, most previous studies have not considered the possibility that MC1R may be subject to sexual conflict. In the barn owl (Tyto alba), the allele MC1R_WHITE is associated with whitish plumage coloration, typical of males, and the allele MC1R_RUFOUS is associated with dark rufous coloration, typical of females, although each sex can express any phenotype. Because each colour variant is adapted to specific environmental conditions, the allele MC1R_WHITE may be more strongly selected in males and the allele MC1R_RUFOUS in females. We therefore investigated whether MC1R genotypes are in excess or deficit in male and female fledglings compared with the expected Hardy–Weinberg proportions. Our results show an overall deficit of 7.5% in the proportion of heterozygotes in males and of 12.9% in females. In males, interannual variation in assortative pairing with respect to MC1R explained the year‐specific deviations from Hardy–Weinberg proportions, whereas in females, the deficit was better explained by the interannual variation in the probability of inheriting the MC1R_WHITE or MC1R_RUFOUS allele. Additionally, we observed that sons inherit the MC1R_RUFOUS allele from their fathers on average slightly less often than expected under the first Mendelian law. Transmission ratio distortion may be adaptive in this sexually dichromatic species if males and females are, respectively, selected to display white and rufous plumages.     

The evolution of black plumage from blue in Australian fairy‐wrens (Maluridae): genetic and structural evidence

Genetic variation in the melanocortin‐1 receptor (MC1R) locus is responsible for color variation, particularly melanism, in many groups of vertebrates. Fairy‐wrens, Maluridae, are a family of Australian and New Guinean passerines with several instances of dramatic shifts in plumage coloration, both intra‐ and inter‐specifically. A number of these color changes are from bright blue to black plumage. In this study, we examined sequence variation at the MC1R locus in most genera and species of fairy‐wrens. Our primary focus was subspecies of the white‐winged fairy‐wren Malurus leucopterus in which two subspecies, each endemic to islands off the western Australian coast, are black while the mainland subspecies is blue. We found fourteen variable amino acid residues within M. leucopterus, but at only one position were alleles perfectly correlated with plumage color. Comparison with other fairy‐wren species showed that the blue mainland subspecies, not the black island subspecies, had a unique genotype. Examination of MC1R protein sequence variation across our sample of fairy‐wrens revealed no correlation between plumage color and sequence in this group. We thus conclude that amino acid changes in the MC1R locus are not directly responsible for the black plumage of the island subspecies of M. leucopterus. Our examination of the nanostructure of feathers from both black and blue subspecies of M. leucopterus and other black and blue fairy‐wren species clarifies the evolution of black plumage in this family. Our data indicate that the black white‐winged fairy‐wrens evolved from blue ancestors because vestiges of the nanostructure required for the production of blue coloration exist within their black feathers. Based on our phylogeographic analysis of M. leucopterus, in which the two black subspecies do not appear to be each other's closest relatives, we infer that there have been two independent evolutionary transitions from blue to black plumage. A third potential transition from blue to black appears to have occurred in a sister clade.     

Nymphal colour/pattern polymorphism in the leafhoppers Eupteryx urticae (F.) and E. cyclops Matsumura (Hemiptera: Auchenorrhyncha): spatial and temporal variation in morph frequencies

Variation in colour/pattern morph frequencies in Eupteryx urticae and E. cyclops is described for various field populations. Eupteryx urticae populations in S Wales exhibit a steep morph-ratio cline, such that black morph frequencies are positively correlated with altitude. High melanic frequencies at high-altitude sites, and the absence of the two darker morphs in lowland populations, suggest a similar trend in E. cyclops , but the data are insufficient to confirm this statistically. No differences in morph frequencies were detected on different parts of the primary host plant or on alternative host species. Similarly, there were no consistent trends within or between the two annual generations of either species, although melanic morph frequencies in one E. urticae population were heterogeneous over 10 generations. It is suggested that the polymorphism in E. urticae is strongly influenced by climate selection, darker morphs being at an advantage in cooler environments where their coloration enhances absorption of solar radiation. The advantage gained through thermal melanism is probably balanced by visual selection against black morphs by entomophagous parasitoids.     

Genetic polymorphisms in the 5'-flanking region of the melanocortin 1 receptor (<Emphasis Type="Italic">MC1R</Emphasis>) gene in foxes

The one of the key pigment genes, the melanocortin 1 receptor (MC1R) gene, plays a fundamental role in the determination of coat color in a variety of mammals. However, so far there has been no report regarding the genetic variants of the MC1R promoter region and the potential association of its mutations with coat color in foxes. This work aimed to characterize 5'-flanking region of the MC1R gene and its mutations associated with coat color variations in foxes. A total of 76 individuals including 64 red foxes (Vulpes vulpes), representing 11 color morphs, and 12 arctic foxes (Vulpes lagopus), representing 2 color morphs were studied. To explore the potential cause of coat color variation in foxes, an 1105 bp region located upstream of the MC1R gene coding region was sequenced in 76 foxes. In the present study, a 1267 bp 5'-flanking region of fox MC1R gene was obtained using a PCR-mediated chromosome-walking technique and a 1105 bp segment was sequenced. A total of 8 novel SNPs and an insertion/deletion of 4 nucleotides were detected. The results of mutations analysis indicated that SNPs g.-52G>A, g.-266A>G, g.-297T>C, g.-300G>A and the insertion/deletion spaning positions g.-382~-379 were important in distinguishing V. vulpes and V. lagopus. This work, for the first time, described and confirmed the different variants existed in the 5'-flanking region of MC1R gene between red foxes and arctic foxes. These findings may be extremely helpful for further exploring the alternative splicings or promoter activity of MC1R gene for different coat-colored foxes.