A novel enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable coat-color variation. Dorsal coats range from dark brown in mice inhabiting mainland habitat to near white on the white-sand beaches of the southeastern US, where light pelage has evolved independently on Florida’s Gulf and Atlantic coasts as an adaptation to visually hunting predators. To facilitate genomic analyses in this species, we first generated a high-quality, chromosome-level genome assembly of P. polionotus subgriseus. Next, in a uniquely variable mainland population that occurs near beach habitat (P. p. albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that variation in pigmentation is strongly associated with a ~2 kb region approximately 5 kb upstream of the Agouti-signaling protein (ASIP) coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this region of Agouti indicate that the light allele has experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000km. Given the evolutionary history of this species, our results suggest that this newly identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there has spread to, and been selected in, two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.