A role for differential gene regulation in the rapid diversification of melanic plumage coloration in the dark-eyed junco (Junco hyemalis)

Color plays a prominent role in reproductive isolation, therefore understanding the proximal basis of pigmentation can provide insight into speciation. Subspecies of the dark-eyed junco (Junco hyemalis) have evolved marked differences in plumage coloration since the Last Glacial Maximum, yet whether color differences are caused by mutations in coding regions of expressed genes or are instead the result of regulatory differences remains unknown. To address this question, we studied the pigment composition and the genetic basis of coloration in two divergent subspecies, the slate-colored and Oregon juncos. We used HPLC and light microscopy to investigate pigment composition and deposition in feathers from four body areas. We then used RNAseq 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, MLANA), 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 color between the two subspecies showed differential expression of ASIP and three other genes (MFSD12, KCNJ13, HAND2) associated with pigmentation in other taxa. Sequence variation in the expressed genes was not related to color differences. Our findings support the hypothesis that differential regulation of a few genes can account for marked differences in coloration, a mechanism that may underlie the rapid diversification of juncos.