Genetic changes contributing to phenotypic differences within or between species have been identified for a handful of traits, but the relationship between alleles underlying intraspecific polymorphism and interspecific divergence is largely unknown. We found that noncoding changes in the tan gene, as well as changes linked to the ebony gene, contribute to pigmentation divergence between closely related Drosophila species. Moreover, we found that alleles linked to tan and ebony fixed in one Drosophila species also contribute to variation within another species, and that multiple genotypes underlie similar phenotypes even within the same population. These alleles appear to predate speciation, which suggests that standing genetic variation present in the common ancestor gave rise to both intraspecific polymorphism and interspecific divergence.

Pigmentation is one of the most variable traits within and between Drosophila species. Much of this diversity appears to be adaptive, with environmental factors often invoked as selective forces. Here, we describe the geographic structure of pigmentation in Drosophila americana and evaluate the hypothesis that it is a locally adapted trait. Body pigmentation was quantified using digital images and spectrometry in up to 10 flies from each of 93 isofemale lines collected from 17 locations across the United States and found to correlate most strongly with longitude. Sequence variation at putatively neutral loci showed no evidence of population structure and was inconsistent with an isolation-by-distance model, suggesting that the pigmentation cline exists despite extensive gene flow throughout the species range, and is most likely the product of natural selection. In all other Drosophila species examined to date, dark pigmentation is associated with arid habitats; however, in D. americana, the darkest flies were collected from the most humid regions. To investigate this relationship further, we examined desiccation resistance attributable to an allele that darkens pigmentation in D. americana. We found no significant effect of pigmentation on desiccation resistance in this experiment, suggesting that pigmentation and desiccation resistance are not unequivocally linked in all Drosophila species.