Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity. Melanic (darkly pigmented) phenotypes in mammals provide a potent system in which to study the genetic basis of naturally occurring mutant phenotypes because
melanism occurs in many mammals, and the mammalian pigmentation pathway is well understood. Spontaneous alleles of a few key pigmentation loci are known to cause
melanism in domestic or laboratory populations of mammals, but in natural populations, mutations at one gene, the
melanocortin-1 receptor (Mc1r), have been implicated in the vast majority of cases, possibly due to its minimal pleiotropic effects. To investigate whether mutations in this or other genes cause
melanism in the wild, we investigated the genetic basis of
melanism in the rodent genus Peromyscus, in which melanic mice have been reported in several populations. We focused on two genes known to cause
melanism in other taxa, Mc1r and its antagonist, the
agouti signaling protein (Agouti). While variation in the Mc1r coding region does not correlate with
melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color. In a second population from Alaska, we find that a
premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype. These results show that
melanism has evolved independently in these populations through mutations in the same gene, and suggest that
melanism produced by mutations in genes other than Mc1r may be more common than previously thought.