Differential gene regulation underlies variation in melanic plumage coloration in the dark-eyed junco (Junco hyemalis).

Colour plays a prominent role in species recognition; therefore, understanding the proximate basis of pigmentation can provide insight into reproductive isolation and speciation. Colour differences between taxa may be the result of regulatory differences or be caused by mutations in coding regions of the expressed genes. To investigate these two alternatives, we studied the pigment composition and the genetic basis of coloration in two divergent dark-eyed junco (Junco hyemalis) subspecies, the slate-coloured and Oregon juncos, which have evolved marked differences in plumage coloration since the Last Glacial Maximum. We used HPLC and light microscopy to investigate pigment composition and deposition in feathers from four body areas. We then used RNA-seq to compare the relative roles of differential gene expression in developing feathers and sequence divergence in transcribed loci under common-garden conditions. Junco feathers differed in eumelanin and pheomelanin content and distribution. Within subspecies, in lighter feathers melanin synthesis genes were downregulated (including PMEL, TYR, TYRP1, OCA2 and MLANA), and ASIP was upregulated. Feathers from different body regions also showed differential expression of HOX and WNT genes. Feathers from the same body regions that differed in colour between the two subspecies showed differential expression of ASIP and three other genes (MFSD12, KCNJ13 and HAND2) associated with pigmentation in other taxa. Sequence variation in the expressed genes was not related to colour differences. Our findings support the hypothesis that differential regulation of a few genes can account for marked differences in coloration, a mechanism that may facilitate the rapid phenotypic diversification of juncos.