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).     

Museomics Dissects the Genetic Basis for Adaptive Seasonal Coloration in the Least Weasel

Dissecting the link between genetic variation and adaptive phenotypes provides outstanding opportunities to understand fundamental evolutionary processes. Here, we use a museomics approach to investigate the genetic basis and evolution of winter coat coloration morphs in least weasels (Mustela nivalis), a repeated adaptation for camouflage in mammals with seasonal pelage color moults across regions with varying winter snow. Whole-genome sequence data were obtained from biological collections and mapped onto a newly assembled reference genome for the species. Sampling represented two replicate transition zones between nivalis and vulgaris coloration morphs in Europe, which typically develop white or brown winter coats, respectively. Population analyses showed that the morph distribution across transition zones is not a by-product of historical structure. Association scans linked a 200-kb genomic region to coloration morph, which was validated by genotyping museum specimens from intermorph experimental crosses. Genotyping the wild populations narrowed down the association to pigmentation gene MC1R and pinpointed a candidate amino acid change cosegregating with coloration morph. This polymorphism replaces an ancestral leucine residue by lysine at the start of the first extracellular loop of the protein in the vulgaris morph. A selective sweep signature overlapped the association region in vulgaris, suggesting that past adaptation favored winter-brown morphs and can anchor future adaptive responses to decreasing winter snow. Using biological collections as valuable resources to study natural adaptations, our study showed a new evolutionary route generating winter color variation in mammals and that seasonal camouflage can be modulated by changes at single key genes.     

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.     

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.     

Plumage coloration can be perceived as a multiple condition‐dependent signal by Great Tits Parus major

The evolution of multiple signals can be explained because they enhance the perception of a general message by recipients. Plumage coloration frequently acts as a condition‐dependent signal, so that species displaying different colour patches have the potential to transmit information on condition through a multiple signalling system. The Great Tit Parus major exhibits plumage colour patches generated by the main types of colour production, some of which, particularly those based on melanins and carotenoids, are known to be related to body condition. However, the colour expressions of all of the different colour types have never been investigated simultaneously to determine whether they function as multiple signals of condition. In addition, visual perception models have never been applied to a multiple signalling system in a wild population of birds. Here I present information that links body condition with the colour expression of almost all of the different plumage patches of male Great Tits captured during the winter. Birds in better condition had greater reflectance values at short wavelengths in all plumage patches, and this was especially so in the white (i.e. structural) colour of the cheeks. Plumage colour characteristics were calculated by means of avian visual models, suggesting that Great Tits have the capacity to perceive information contained in the plumage coloration of conspecifics. These results show that short‐wavelength reflectance has great potential to transmit biologically significant information on the body condition of birds, even in achromatic plumage patches.     

Beyond mean allelic effects: A locus at the major color gene MC1R associates also with differing levels of phenotypic and genetic (co)variance for coloration in barn owls

The mean phenotypic effects of a discovered variant help to predict major aspects of the evolution and inheritance of a phenotype. However, differences in the phenotypic variance associated to distinct genotypes are often overlooked despite being suggestive of processes that largely influence phenotypic evolution, such as interactions between the genotypes with the environment or the genetic background. We present empirical evidence for a mutation at the melanocortin‐1‐receptor gene, a major vertebrate coloration gene, affecting phenotypic variance in the barn owl, Tyto alba. The white MC1R allele, which associates with whiter plumage coloration, also associates with a pronounced phenotypic and additive genetic variance for distinct color traits. Contrarily, the rufous allele, associated with a rufous coloration, relates to a lower phenotypic and additive genetic variance, suggesting that this allele may be epistatic over other color loci. Variance differences between genotypes entailed differences in the strength of phenotypic and genetic associations between color traits, suggesting that differences in variance also alter the level of integration between traits. This study highlights that addressing variance differences of genotypes in wild populations provides interesting new insights into the evolutionary mechanisms and the genetic architecture underlying the phenotype.     

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.     

The role of common and rare MC4R variants and FTO polymorphisms in extreme form of obesity

Melanocortin 4 receptor (MC4R) is an important regulator of food intake and number of studies report genetic variations influencing the risk of obesity. Here we explored the role of common genetic variation from MC4R locus comparing with SNPs from gene FTO locus, as well as the frequency and functionality of rare MC4R mutations in cohort of 380 severely obese individuals (BMI > 39 kg/m²) and 380 lean subjects from the Genome Database of Latvian Population (LGDB). We found correlation for two SNPs—rs11642015 and rs62048402 in the fat mass and obesity-associated protein (FTO) with obesity but no association was detected for rs17782313 located in the MC4R locus in these severely obese individuals. We sequenced the whole gene MC4R coding region in all study subjects and found five previously known heterozygous non-synonymous substitutions V103I, I121T, S127L, V166I and I251L. Expression in mammalian cells showed that the S127L, V166I and double V103I/S127L mutant receptors had significantly decreased quantity at the cell surface compared to the wild type MC4R. We carried out detailed functional analysis of V166I that demonstrated that, despite low abundance in plasma membrane, the V166I variant has lower EC₅₀ value upon αMSH activation than the wild type receptor, while the level of AGRP inhibition was decreased, implying that V166I cause hyperactive satiety signalling. Overall, this study suggest that S127L may be the most frequent functional MC4R mutation leading to the severe obesity in general population and provides new insight into the functionality of population based variants of the MC4R.     

A genome-wide epistatic network underlies the molecular architecture of continuous color variation of body extremities

Deciphering the molecular architecture of coat coloration for a better understanding of the biological mechanisms underlying pigmentation still remains a challenge. We took advantage of a rabbit French experimental population in which both a pattern and a gradient of coloration from white to brown segregated within the himalayan phenotype. The whole experimental design was genotyped using the high density Affymetrix® AxiomOrcun? SNP Array and phenotyped into 6 different groups ordered from the lighter to the darker. Genome-wide association analyses pinpointed an oligogenic determinism, under recessive and additive inheritance, involving genes already known in melanogenesis (ASIP, KIT, MC1R, TYR), and likely processed pseudogenes linked to ribosomal function, RPS20 and RPS14. We also identified (i) gene-gene interactions through ASIP:MC1R affecting light cream/beige phenotypes while KIT:RPS responsible of dark chocolate/brown colors and (ii) a genome-wide epistatic network involving several others coloration genes such as POT1 or HPS5. Finally, we determined the recessive inheritance of the English spotting phenotype likely involving a copy number variation affecting at least the end of the coding sequence of the KIT gene. Our analyses of coloration as a continuous trait allowed us to go beyond much of the established knowledge through the detection of additional genes and gene-gene interactions that may contribute to the molecular architecture of the coloration phenotype.     

Unmelanized plumage patterns in Old World leaf warblers do not correspond to sequence variation at the melanocortin-1 receptor locus (MC1R)

Evolutionary changes in patterns and coloration of plumage are likely to represent a major mechanism for speciation among birds, yet the molecular basis for such changes remains poorly understood. Recently much attention has focused on the melanocortin-1 receptor (MC1R) as a candidate locus for determining the level and extent of epidermal melanin deposition. We tested the hypothesis that MC1R sequence variation is associated with interspecific variation in unmelanized plumage pattern elements in Old World leaf warblers (genus Phylloscopus). This genus is characterized by a variety of plumage patterns that nonetheless vary along similar lines. Species vary in the presence or absence of pale (unmelanized) pattern elements against a dark background, and these patterns are used in species recognition and courtship. We sequenced most of the MC1R coding region for eight Phylloscopus species, representing the full range of plumage patterns found in this genus. Although MC1R sequence varied among species, this variation was not related to melanin-based plumage variation. Rather, evolution of this locus in these birds appears to be conservative. Ratios of nonsynonymous to synonymous substitutions (dN/dS) were consistently low, suggesting that strong purifying selection has operated at this locus, and likelihood ratio testing revealed no evidence of variable selective pressures among lineages or across codons. Adaptive evolution at MC1R may be constrained by the adaptive importance of plumage pattern elements in this genus.     

Sequence Polymorphism in Candidate Genes for Differences in Winter Plumage between Scottish and Scandinavian Willow Grouse (Lagopus lagopus)

Background

Population variation in the degree of seasonal polymorphism is rare in birds, and the genetic basis of this phenomenon remains largely undescribed. Both sexes of Scandinavian and Scottish Willow grouse (Lagopus lagopus) display marked differences in their winter phenotypes, with Scottish grouse retaining a pigmented plumage year-round and Scandinavian Willow grouse molting to a white morph during winter. A widely studied pathway implicated in vertebrate pigmentation is the melanin system, for which functional variation has been characterised in many taxa.

Methodology/Principal Findings

We sequenced coding regions from four genes involved in melanin pigmentation (DCT, MC1R, TYR and TYRP1), and an additional control involved in the melanocortin pathway (AGRP), to investigate the genetic basis of winter plumage in Lagopus. Despite the well documented role of the melanin system in animal coloration, we found no plumage-associated polymorphism or evidence for selection in a total of ∼2.6 kb analysed sequence.

Conclusions/Significance

Our results indicate that the genetic basis of alternating between pigmented and unpigmented seasonal phenotypes is more likely explained by regulatory changes controlling the expression of these or other loci in the physiological pathway leading to pigmentation.     

Seasonal Changes in Colour: A Comparison of Structural,Melanin- and Carotenoid-Based Plumage Colours

Background

Plumage coloration is important for bird communication, most notably in sexual signalling. Colour is often considered a good quality indicator, and the expression of exaggerated colours may depend on individual condition during moult. After moult, plumage coloration has been deemed fixed due to the fact that feathers are dead structures. Still, many plumage colours change after moult, although whether this affects signalling has not been sufficiently assessed.

Methodology/Principal Findings

We studied changes in coloration after moult in four passerine birds (robin, Erithacus rubecula; blackbird, Turdus merula; blue tit, Cyanistes caeruleus; and great tit, Parus major) displaying various coloration types (melanin-, carotenoid-based and structural). Birds were caught regularly during three years to measure plumage reflectance. We used models of avian colour vision to derive two variables, one describing chromatic and the other achromatic variation over the year that can be compared in magnitude among different colour types. All studied plumage patches but one (yellow breast of the blue tit) showed significant chromatic changes over the year, although these were smaller than for a typical dynamic trait (bill colour). Overall, structural colours showed a reduction in relative reflectance at shorter wavelengths, carotenoid-based colours the opposite pattern, while no general pattern was found for melanin-based colours. Achromatic changes were also common, but there were no consistent patterns of change for the different types of colours.

Conclusions/Significance

Changes of plumage coloration independent of moult are probably widespread; they should be perceivable by birds and have the potential to affect colour signalling.     

Changes in carotenoid‐based plumage colour in relation to age in European Serins Serinus serinus

Yearling birds generally display duller colours than adults. This may be due to selection favouring birds with more intensely coloured plumage or to an increase in colour after the first complete moult. Most research to date on the topic has been carried out on species with structural plumage coloration or with carotenoid‐based coloration that is produced by the unmodified deposition of pigments. However, no study has been carried out on species whose carotenoids are metabolically modified before deposition. In this study, we assess age‐related changes in the carotenoid‐based coloration of European Serins, a species that metabolically processes carotenoids before they can be deposited into feathers. Birds were captured over consecutive years and we carried out both cross‐sectional and longitudinal analysis. Adults had significantly greater values of lightness and chroma than yearling birds. However, there were no changes in plumage colour when analysing the same individuals captured in subsequent seasons. Plumage lightness and chroma of adult males after moult were related to body mass, suggesting a role of body condition on plumage coloration. Our results suggest that changes in plumage coloration with age in European Serins are due to a selection process that favours more intensely coloured individuals.     

Timing and body condition of dichromatic Black Redstarts during autumn migration

Individual variation in postjuvenile molt in male Black Redstart is pronounced with about 90% of young males retaining female‐like coloration (cairei plumage type) and about 10% acquiring adult male‐like feathers (paradoxus plumage type). We examined whether autumn migration timing and body condition differed between individuals of the two plumage types. We used the data of 10,977 Black Redstarts captured during autumn at a ringing site in northern Switzerland where a protocol to record plumage types of captures has been applied since 1980. As cairei individuals cannot be distinguished from young females while sexing is comparatively easy for paradoxus individuals, the proportion of missing data on sex was likely to be higher for cairei individuals than for paradoxus individuals. We formally accounted for captures with unidentified sex using a Bayesian approach and conducted a simulation study to show that our approach was able to provide unbiased results even if the proportion of unsexed captures was high. Applying the method to the Black Redstart data, we found that the proportion of individuals with paradoxus plumage type increased from 7.6% in 1980 to 18.1% in 2013. Individuals with the paradoxus plumage type were on average 0.25 g heavier and had 0.62 mm longer third primaries than individuals with the cairei plumage type. However, we found no support for our expectation of later migration of paradoxus males compared to cairei individuals based on the assumption that paradoxus individuals should occupy autumn territories like adult males. Our results shed new light on the understudied timing of autumn migration in birds and are in line with available studies on Black Redstarts, suggesting a molt‐constraint that allows only young males in good body condition to molt into adult‐like 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.     

Evolution of dark colour in toucans (Ramphastidae): a case of molecular adaptation?

In the last decades, researchers have been able to determine the molecular basis of some phenotypes, to test for evidence of natural selection upon them, and to demonstrate that the same genes or genetic pathways can be associated with convergent traits. Colour traits are often subject to natural selection because even small changes in these traits can have a large effect on fitness via camouflage, sexual selection or other mechanisms. The melanocortin‐1 receptor locus (MC1R) is frequently associated with intraspecific coat colour variation in vertebrates, but it has been far harder to demonstrate that this locus is involved in adaptive interspecific colour differences. Here, we investigate the contribution of the MC1R gene to the colour diversity found in toucans (Ramphastidae). We found divergent selection on MC1R in the clade represented by the genus Ramphastos and that this coincided with the evolution of darker plumage in members of this genus. Using phylogenetically corrected correlations, we show significant and specific relationships between the rate of nonsynonymous change in MC1R (dN) and plumage darkness across Ramphastidae, and also between the rate of functionally significant amino acid changes in MC1R and plumage darkness. Furthermore, three of the seven amino acid changes in MC1R that occurred in the ancestral Ramphastos branch are associated with melanism in other birds. Taken together, our results suggest that the dark colour of Ramphastos toucans was related to nonsynonymous substitutions in MC1R that may have been subject to positive selection or to a relaxation of selective pressure. These results also demonstrate a quantitative relationship between gene and phenotype evolution, representing an example of how MC1R molecular evolution may affect macroevolution of plumage phenotypes.     

The effects of elevated testosterone on plumage hue in male House Finches

The majority of studies examining the role of hormones in the proximate mechanisms of plumage coloration in birds have focused on intersexual differences (plumage dichromatism) and on structural- or melanin-based plumage coloration. The relationship between hormones and carotenoid-based plumage color, and in particular intrasexual plumage color variation, has received little attention. We manipulated testosterone levels of both captive and wild male House Finches to determine whether testosterone influences the expression of male plumage color in this species. We found that in captive male House Finches elevated testosterone delayed molt and resulted in drabber, less red plumage, even when birds were supplemented with dietary carotenoids. Elevated testosterone also resulted in drab plumage color in wild males, and appeared to delay molt in wild birds as well. Wild males implanted with testosterone showed wide variation in expression of plumage coloration. Those implanted early in the year molted plumage similar in color to their pre-treatment plumage, but those implanted later molted substantially duller plumage, possibly because delayed molt resulting from elevated testosterone caused these males to molt when carotenoid pigments were not available in sufficient amounts. These observations have the potential to explain previously reported relationships between plumage color and behavior in male House Finches, and highlight the importance of considering the proximate mechanisms of plumage coloration in avian sexual selection.     

The relationship between plumage coloration and aggression in female tree swallows

Intrasexual competition is an important selective force that can favor the evolution of honest signals of fighting ability. Research has focused predominantly on male birds, but many female birds also possess plumage ornaments that could mediate the outcome of competitive interactions. We examined the relationship between blue and white structural coloration and aggression in female tree swallows Tachycineta bicolor. Tree swallows are secondary cavity nesters and females show delayed plumage maturation which may be related to intense competition for nest sites. We compared plumage reflectance of second‐year (SY) and after‐second year (ASY) females, and within‐individual changes in plumage reflectance of ASY females in two successive years. We assessed aggression by placing a caged SY female 2 m from the nest box of an ASY female and quantified the ASY female's response in relation to her own plumage coloration. We found substantial differences in plumage reflectance of SY and ASY females, but found that ASY females became greener with duller white coloration in the second year. This may be due to poor weather that made reproduction particularly costly in the first year of the study and suggests coloration is influenced by condition. We found no relationship between dorsal coloration and aggressiveness. Rather, brighter white females spent more time on their nest box and within 2 m of the intruder than females with dull breasts. Our findings suggest that brighter white ASY females may perceive a SY female as less of a threat or that there may be a trade‐off associated with aggression and parental care that is related to white brightness.     

Genetic Association Study of Adiposity and Melanocortin-4 Receptor (MC4R) Common Variants: Replication and Functional Characterization of Non-Coding Regions

Common genetic variants 3′ of MC4R within two large linkage disequilibrium (LD) blocks spanning 288 kb have been associated with common and rare forms of obesity. This large association region has not been refined and the relevant DNA segments within the association region have not been identified. In this study, we investigated whether common variants in the MC4R gene region were associated with adiposity-related traits in a biracial population-based study. Single nucleotide polymorphisms (SNPs) in the MC4R region were genotyped with a custom array and a genome-wide array and associations between SNPs and five adiposity-related traits were determined using race-stratified linear regression. Previously reported associations between lower BMI and the minor alleles of rs2229616/Val103Ile and rs52820871/Ile251Leu were replicated in white female participants. Among white participants, rs11152221 in a proximal 3′ LD block (closer to MC4R) was significantly associated with multiple adiposity traits, but SNPs in a distal 3′ LD block (farther from MC4R) were not. In a case-control study of severe obesity, rs11152221 was significantly associated. The association results directed our follow-up studies to the proximal LD block downstream of MC4R. By considering nucleotide conservation, the significance of association, and proximity to the MC4R gene, we identified a candidate MC4R regulatory region. This candidate region was sequenced in 20 individuals from a study of severe obesity in an attempt to identify additional variants, and the candidate region was tested for enhancer activity using in vivo enhancer assays in zebrafish and mice. Novel variants were not identified by sequencing and the candidate region did not drive reporter gene expression in zebrafish or mice. The identification of a putative insulator in this region could help to explain the challenges faced in this study and others to link SNPs associated with adiposity to altered MC4R expression.     

Pigeons in the sun: Thermal constraints of eumelanic plumage in the rock pigeon (Columba livia)

In wild vertebrates, several species exhibit eumelanic color polymorphism with the coexistence of dark and light morphs. The maintenance of such polymorphism suggests the existence of a selective balance between the morphs and a large body of literature has reported the costs and benefits of darker plumage coloration in birds. Among them, it has been suggested that melanin and dark plumage could entail high energetic costs especially under hot and sunny climates. However, to my knowledge, the thermal constraints of sun exposure have rarely been studied in polymorphic species. Here, we tested the impact of eumelanic plumage coloration on plumage and body temperatures, and evaporative cooling behavior in the polymorphic rock pigeon (Columbia livia). We experimentally exposed light and dark pigeons to direct sun radiation for 1 h while a few birds were maintained in the shade as controls. We found that sun exposure was associated with increased plumage temperature, and this effect was greater for darker pigeons. In addition, we found that sun exposure was also associated with higher cloacal temperature but for dark pigeons only. Finally, light and dark pigeons were more likely to show cooling evaporative behavior when exposed to sun and as their cloacal temperature increases. Altogether, these results suggest that darker pigeons may have a lower ability to cope with heat and solar radiations and that dark plumage can be associated with thermal costs in this polymorphic species.