Fig. 11. The evolution of a novel pigmentation trait required cis-regulatory evolution at several realizator loci.

(A) In the embryo, Ddc expression (light blue) is upregulated around sites of epidermal wounding as part of a response that forms a melanized plug (black circle). Schematic depicts the anterior embryo. This gene function is conserved, and encoded in the activity of a CRE directly regulated by the Grh transcription factor. In D. willistoni, which serves as a proxy for the ancestrally monomorphic trait, Ddc expression is driven at a low level by the pleiotropic activity of the wound response CRE. This ancestral CRE possesses a direct binding site for Grh and is modestly-responsive to the Hox transcription factor Abd-B. In species with derived melanic abdomen pigmentation phenotypes, Ddc expression is robust throughout the abdomen (dark blue). This expression is in part due to cis-evolution at Ddc, which evolved a stronger, Hox-responsive CRE activity that is apparent for D. melanogaster, and this CRE seemingly evolved to be under the control of some trans-regulator(s) whose identity remains unknown and is represented here as factor “X”. Time scale is depicted in millions of years. Solid lines in cis-regulatory evolution indicate cases where the transcription factor directly binds to the Ddc CRE, and dashed lines indicate cases where the mechanism of transcription factor regulation remains unknown. (B) Model for the origin of the derived dimorphic pigmentation trait of D. melanogaster from an ancestral monomorphic state. Here, abdominal pale expression was ancestral, whereas Ddc, yellow, tan, and ebony each evolved novel expression through changes in CREs controlling their expression. The ancestral states upon which novel CREs evolved for yellow, tan, and ebony remain unknown (question marks). For Ddc, cis-evolution augmented an ancestral wound response CRE to have a robust pleiotropic activity.