Interplay between developmental flexibility and determinism in the evolution of mimetic heliconius wing patterns

To what extent can we predict how evolution occurs? Do genetic architectures and developmental processes canalize the evolution of similar outcomes in a predictable manner? Or do historical contingencies impose alternative pathways to answer the same challenge? Examples of Mullerian mimicry between distantly related butterfly species provide natural replicates of evolution, allowing us to test whether identical wing patterns followed parallel or novel trajectories. Here, we explore the role that the signaling ligand WntA plays in generating mimetic wing patterns in Heliconius butterflies, a group with extraordinary mimicry-related wing pattern diversity. The radiation is relatively young, and numerous cases of wing pattern mimicry have evolved within the last 2.5-4.5 Ma. WntA is an important target of natural selection and is one of four major effect loci that underlie much of the pattern variation in the group. We used CRISPR/Cas9 targeted mutagenesis to generate WntA-deficient wings in 12 species and a further 10 intraspecific variants, including three comimetic pairs. In all tested butterflies, WntA knockouts affect pattern broadly and cause a shift among every possible scale cell type. Interestingly, the comimics lacking WntA were very different, suggesting that the gene networks that pattern a wing have diverged considerably among different lineages. Thus, although natural selection channeled phenotypic convergence, divergent developmental contexts between the two major Heliconius lineages opened different developmental routes to evolve resemblance. Consequently, even under very deterministic evolutionary scenarios, our results underscore a surprising unpredictability in the developmental paths underlying convergence in a recent radiation292339964009We thank Oscar Paneso, Cruz Batista, Lissette Rivera, Lissette Jimenez, and Clement Aubert for technical support with rearing of butterfly stocks and larvae resulting from experimental procedures. We are also grateful to Krzysztof “Chris” Kozak, Michael Logan, and Eric Haag for thoughtful discussions and feedback on the manuscript. We thank Robert Reed for sharing anti-optix antibodies to produce pupal wing immunostainings and to Sebastian Mena for taking professional photo images of mutant and WT butterflies. This research was funded by a Smithsonian Institute Pell Grant to W.O.M.; Smithsonian Institute Scholarly Studies Award to W.O.M. and C.C.; Smithsonian Institute Biodiversity Genomics Fellowship Award to C.C.; the National Science Foundation awards IOS-1656553and IOS-1755329 to A.M.; National Science Foundation awards IOS-1656389 to R.P. and OIA 1736026 to B.C. and R.P.; funds from Universidad del Rosario for M.L., C.P.-D., and C.S.; and a Leverhulme Trust project grant RPG-2014-167 to C.D.J. We would like to dedicate this article to the memory of Sabrina Ferrara, who was an intern in our team and died before she could see the publication of this work. She was a kind person, and we remember her with affectio