Natural variation in pigmentation caused by cis -regulatory changes affecting expression of the ebony gene in Drosophila elegans species subgroup

Repetitive evolution has offered valuable insights into how similar traits can arise across different populations and species. The dimorphic body color morphs (brown and black) of geographically isolated Drosophila elegans , along with its closely related species D. gunungcola , which exhibits black morph, raise intriguing questions about whether these traits have emerged through independent evolution, introgression, or shared ancestral variants. To investigate the genetic and evolutionary origins of these differences, we perform quantitative trait locus (QTL) mapping on recombinant hybrids generated from reciprocal crosses between brown and black morphs D. elegans . QTL mapping reveals significant contributions from the X chromosome and Muller element E to intraspecific pigmentation polymorphism, with ebony identified as one candidate gene. Analysis of ebony expression, including allele-specific expression in F ₁ hybrids, and sequence variation suggests that the intra- and interspecific pigmentation differences within D. elegans species subgroup may have evolved independently from the lineage-specific region upstream of ebony . By identifying a shared candidate regulatory region associated with both intra- and interspecific variation, our findings suggest that recurrent use of the same genetic region may contribute to the emergence of similar traits across populations and species. Keywords: cis -regulatory divergence, Drosophila elegans , ebony , parallel evolution, pigmentation, QTL mapping.