Redundant and Singular Regulatory Elements Underlie the Rapidly Evolving Pigmentation of Drosophila

A major hurdle in understanding the molecular changes responsible for metazoan diversity is the characterization of cis-regulatory elements (CREs) for gene regulatory networks (GRNs). CRE changes are suspected to be commonplace in trait evolution, since such changes circumvent deleterious effects of pleiotropy. A growing list of genes, though, are known to be regulated by redundant CREs. Such redundant CRE architectures complicates the characterization of GRN evolution, as it compounds the effort to characterize each locus, and raises the questions of how and whether genes with redundant architectures evolve expression. Here, we used the evolution of sexually dimorphic abdomen pigmentation of Drosophila (D.) melanogaster as a model to study the function and evolution of CREs. Numerous sequences were evaluated that were previously predicted as potential abdomen CREs. Most of these predictions were validated, including two, four, and ten that respectively reside in the homothorax, grainy head, and Eip74EF transcription factor loci. The homothorax CREs were found to be partially-redundant for this gene’s pigmentation function, and pupal-stage Homothorax expression and the CRE activities are conserved among Drosophila species with the derived dimorphic and ancestral monomorphic phenotypes. Similarly, the Eip74EF CREs are conserved in the monomorphic D. willistoni. Thus, this gene’s extensive CRE spatiotemporal redundancy has been conserved for over 30 million years, predating the dimorphic trait. Pigmentation evolution has been connected elsewhere to changes in non-redundant CREs. When these traits evolve, GRN changes may be biased towards the genes with singular non-redundant CREs, while the expression of redundantly regulated genes remain conserved.