Tian, S.; Zhou, J. Y.; Mazo-Vargas, A.

Phenotypes evolve through changes in gene regulatory networks (GRNs), but how changes in network topology contribute to phenotypic evolution is still poorly understood. Butterfly wing color patterns provide a tractable system to answer this question. Here, we compared the expression of four color pattern genes, WntA, pdm3, ivory, and Optix, in five nymphalid butterflies, and tested their regulatory connections through genetic perturbations in three species spanning 80 million years of divergence. We found broadly conserved 1) regulatory hierarchy linking early pattern instruction via WntA signaling to subsequent color readout via ivory and Optix, 2) positive autoregulation in WntA signaling, and 3) positive regulation from WntA signaling to pdm3. Notably, we discovered lineage-specific negative feedback from pdm3 to WntA signaling in Vanessa cardui, which closes a reciprocal WntA-pdm3 feedback loop that refines complex pattern elements. We show that developmental programs can evolve by rewiring internal interactions among pre-existing GRN nodes, emphasizing the importance of network topology in phenotypic evolution.