Conserved signaling pathways antagonize and synergize with co-opted doublesex to control development of novel mimetic butterfly wing patterns

Abstract

Novel phenotypes are increasingly recognized to have evolved by co-option of conserved genes into new developmental contexts, yet the impact of co-option on existing developmental programs remains obscure. Here we provide insight into this process by characterizing the consequences of doublesex co-option on wing color pattern development in Papilio swallowtail butterflies. doublesex is the master regulator of insect sex differentiation but has been co-opted to control the switch between discrete mimetic and non-mimetic, male-like color patterns in Papilio polytes and its close relatives. Here we show that development of the mimetic color pattern in P. polytes is caused by a pulse of dsx expression early in female wing development that results in a corresponding pulse of differential expression that both alters color pattern development and quickly becomes decoupled from dsx expression itself. Differentially expressed genes were enriched in canonical Wnt and Hedgehog signaling pathway genes, but case studies of key genes using RNAi and antibody stains suggested opposing, novel roles for the two pathways in mimetic color pattern development. The pulse of Dsx expression caused Engrailed, the key transcription factor effector of Hh signaling, to gain anterior expression in early pupal wing development. However, Dsx and En became decoupled by mid-pupal development when En pre-figured melanic and red patterns and Dsx pre-figured white patterns. In contrast, Wnt signaling antagonizes Dsx in restricted regions of the wing to refine the mimetic color pattern. Our results therefore provide strong experimental evidence that dsx co-option significantly altered spatiotemporal activities of conserved wing patterning pathways to promote and refine the development of a novel adaptive color pattern. Altogether, our findings provide strong evidence for how co-opted genes can both cause and elicit changes to established gene regulatory networks during the evolution and development of novel phenotypes.