Abstract
Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here we show that Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene and without any noteworthy changes to Bab protein coding sequences. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.