Abstract
Cis-regulatory evolution is an important engine of organismal diversification. Although recent studies have looked at genomic patterns of regulatory evolution between species, we still have a poor understanding of the magnitude and nature of regulatory variation within species. Here, we examine the evolution of regulatory element activity over wing development in three Heliconius erato butterfly populations to determine how regulatory variation is associated with population structure. We show that intraspecific divergence in chromatin accessibility and regulatory activity is abundant, and that regulatory variants are spatially clustered in the genome. Regions with strong population structure are highly enriched for regulatory variants, and enrichment patterns are associated with developmental stage and gene expression. We also found that variable regulatory elements are particularly enriched in species-specific genomic regions and long interspersed nuclear elements. Our findings suggest that genome-wide selection on chromatin accessibility and regulatory activity is an important force driving patterns of genomic divergence within Heliconius species. This work also provides a resource for the study of gene regulatory evolution in H. erato and other heliconiine butterflies.