The ontogeny of color: developmental origins of divergent pigmentation in D rosophila americana and D . novamexicana

SUMMARY Pigmentation is a model trait for evolutionary and developmental analysis that is particularly amenable to molecular investigation in the genus D rosophila . To better understand how this phenotype evolves, we examined divergent pigmentation and gene expression over developmental time in the dark‐bodied D . americana and its light‐bodied sister species D . novamexicana . Prior genetic analysis implicated two enzyme‐encoding genes, tan and ebony , in pigmentation divergence between these species, but questions remain about the underlying molecular mechanisms. Here, we describe stages of pupal development in both species and use this staging to determine when pigmentation develops and diverges between D . americana and D . novamexicana . For the developmental stages encompassing pigment divergence, we compare m RNA expression of tan and ebony over time and between species. Finally, we use allele‐specific expression assays to determine whether interspecific differences in m RNA abundance have a cis ‐regulatory basis and find evidence of cis ‐regulatory divergence for both tan and ebony . cis ‐regulatory divergence affecting tan had a small effect on m RNA abundance and was limited to a few developmental stages, yet previous data suggests that this divergence is likely to be biologically meaningful. Our study suggests that small and developmentally transient expression changes may contribute to phenotypic diversification more often than commonly appreciated. Recognizing the potential phenotypic impact of such changes is important for a scientific community increasingly focused on dissecting quantitative variation, but detecting these types of changes will be a major challenge to elucidating the molecular basis of complex traits.