Deep cis-regulatory homology of the butterfly wing pattern groundplan

Butterfly wing patterns derive from a deeply conserved developmental groundplan, yet are highly diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the groundplan gene WntA in nymphalid butterflies. Comparative ATAC-seq and in vivo knockouts of 46 Cis-Regulatory Elements (CREs) across five species revealed extensive sequence homology of groundplan CREs, except in monarch butterflies. Most CRE perturbation assays showed effects spanning multiple elements and encoding both positive and negative regulatory functionality. Our results provide little support for models predicting rapid turnover of single-trait enhancers and suggest morphological change within a tissue is achieved by tuning deeply conserved and highly sensitive networks of interdependent CREs.