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 old fi eld mouse ( Peromyscus polionotus ), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida ’ s Gulf and Atlantic coasts as camou fl age from predators. To facilitate genomic analyses, we fi rst generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus . Next, in a uniquely variable mainland population ( Peromyscus polionotus albifrons ), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We fi nd that pigment variation is strongly associated with an ∼ 2-kb region ∼ 5 kb upstream of the Agouti sig- naling protein coding region. Using a reporter-gene assay, we demonstrate that this reg- ulatory 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 Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fi xed in both Gulf and Atlantic coast beach mice, despite these populations being separated by > 1,000 km. Together, our results suggest that this identi fi ed Agouti enhancer allele has been main- tained 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.