Gene duplication plays a major role in evolution of novel gene functions as it provides a material basis for selection to act upon. How cis-regulatory landscapes are altered after duplication events to contribute to expression divergence is not well understood. Here we address this question by studying a recent duplicate pair originating at the base of drosophilids. The Drosophila Ly6 genes CG9336 and CG9338 arose through the tandem duplication of a single ortholog still present outside Drosophilidae. Despite their recent origin, the paralogs in D. melanogaster have diverged in embryonic tissue-specificities from each other as well as from the unduplicated ortholog. While retaining the ancestral expression in the glia, both genes acquired a novel expression in the embryonic heart and CG9338 alone in the hemocytes. Interestingly, these two novel expression domains are only present in subsets of Drosophila species examined, suggesting that their acquisitions may have been lineage-specific.
To elucidate the cis-regulatory basis of expression divergence, we first mapped the enhancer activities of the duplicated locus in D. melanogaster. We identified two enhancers driving the conserved glial expression, one located in the 3’ intergenic region of CG9336 and the other in the second intron of CG9338. Interestingly, the two fragments also showed activities in the heart and the hemocytes, respectively. Homologous regions from four additional Drosophila species all showed the conserved glial activity. However, they had a variable capacity to drive heart and hemocyte expression matching the species-specific mRNA expression. These results suggest that the pre-exiting glial enhancers inherited by each duplicate were differentially modified to acquire novel tissue-specific activities. Our study delineates a possible trajectory of enhancer evolution at a duplicated locus, which culminated in transcriptional divergence between the paralogs.