show Abstracthide AbstractAposematic color pattern mimicry in Heliconius butterflies provides a well-known example of adaptation via selection on a few genes of large effect. To understand how selection at individual genes can drive the evolution of complex traits, we functionally characterized five novel enhancers of the color pattern gene, optix. In Heliconius erato we found that wing pattern enhancers are largely ancestral, pleiotropic, functionally interdependent, and introgressed between populations. Remarkably, many of these enhancers are also associated with regional pattern variation in the distantly related co-mimics Heliconius melpomene and Heliconius timareta. Our findings provide a case study of how parallel co-evolution of ancient, multifunctional regulatory elements can facilitate the rapid diversification of complex phenotypes, and provide a counterpoint to many widespread assumptions of cis-regulatory evolution. Overall design: Examination of chromatin accessibility in wing tissue between populations of H. melpomene.