J. Avian Biol. 41: 505514, 2010 doi: 10.1111/j.1600-048X.2009.04823.x # 2010 The Authors. J. Compilation # 2010 J. Avian Biol. Received 2 February 2009, accepted 20 July 2009 The evolution of black plumage from blue in Australian fairy-wrens (Maluridae): genetic and structural evidence Amy C. Driskell, Richard O. Prum and Stephen Pruett-Jones A. C. Driskell, Lab of Analytical Biology, National Museum of Natural History, Smithsonian Inst., 4210 Silver Hill Road, Suitland, MD 20746 USA.  R. O. Prum, Dept of Ecology and Evolutionary Biology, and Peabody Museum of Natural History, Yale Univ., Box 208105, New Haven, CT 06520 USA.  S. Pruett-Jones (pruett-jones@uchicago.edu), Dept of Ecology and Evolution, Univ. of Chicago, 1101 East 57th Street, Chicago, IL 60546 USA. 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. Identifying the genetic basis underlying adaptation is a central goal for evolutionary biologists. Related to this, coloration patterns in vertebrates have been studied exten- sively. In some instances, intraspecific variation in color has been shown to be controlled by a simple genetic mechan- ism. For example, variation at the melanocortin-1 receptor (MC1R) locus is known to affect plumage color in some species of birds (Takeuchi et al. 1996, Theron et al. 2001, Kerje et al. 2003, Ling et al. 2003, Mundy et al. 2003, 2004), coat coloration in some mammals (Majerus and Mundy 2003, Nachman et al. 2003), and hair coloration in humans (Schio ¨th et al. 1999). The receptor is expressed in the membrane of melanocytes, and is an important regulator of the synthesis of melanin pigments. Activation of the MC1R increases the production of brown and black eumelanin pigments (Mundy 2005). In a number of verte- brate species, non-synonymous substitutions in the MC1R gene are correlated with melanism (Takeuchi et al. 1996, Theron et al. 2001, Kerje et al. 2003, Ling et al. 2003, Majerus and Mundy 2003, Mundy et al. 2003, 2004, Nachman et al. 2003). In many of these cited cases, the mechanism by which melanism is triggered is essentially the same  mutations in the same region of the MC1R gene disrupt the hormone-recognition site and cause constitutive activation of the gene (Majerus and Mundy 2003). This results in the over-production of melanin that eclipses the normal coat or plumage color. One of the consistent features of avian species for which MC1R has been shown to be important is that the melanistic forms occur within the same population as non- melanistic forms (Theron et al. 2001, Mundy et al. 2004). Additionally, the expression of the melanistic phenotype is seldom sex limited  well-known examples exist in which both males and females are melanistic (Mundy 2005, Hoekstra 2006), although sex-specific effects of MC1R variation have been observed in the chicken (Ling et al. 2003). The extent to which MC1R affects coloration varies, but in general, the entire plumage of an individual is changed. For example, in the bananaquit Coereba flaveola all white and yellow colored areas are completely obscured by melanin (Theron et al. 2001); in the arctic skua Stercorarius parasiticus the entire plumage of the bird is 505