Gene loss associated with plasticity-first evolution in Heliconius butterflies

Phenotypic plasticity occurs when a genotype can produce more than one phenotype under different environmental conditions. Genetic accommodation allows plastic phenotypes to be tuned to new environments and eventually to even be lost. The colourful and toxic Heliconiini butterflies have biochemical plasticity - they either sequester their cyanogenic glucosides (CG) from their larval hostplant or biosynthesize them when compounds for sequestration are not available. Here, we trace the evolution of CG biosynthesis in Heliconiini butterflies, a fundamental component of this biochemical plasticity. We first reconstructed the evolutionary history of biochemical plasticity in Heliconiini butterflies using chemical data from over 700 individuals, demonstrating that plasticity was ancestral in the tribe and further lost in a few clades, such as the Sapho clade that only sequester CGs. In lepidopterans, CG biosynthesis has been fully characterized in the moth Zygaena filipendulae, and is composed of CYP405A2, CYP332A3, and UGT33A1. Thus, we CRISPR-edited the gene CYP405 in Heliconius erato, and confirmed that CYP405-knockout caterpillars do not biosynthesize CGs. We identified the CYP405s and CYP332s in other lepidopterans and found that both genes were independently co-opted into CG biosynthesis in the Heliconiinae butterflies and Zygaena moths. Most lepidopterans have a CYP332, but CYP405 is mostly restricted to butterflies and has been duplicated in all Heliconius species. Although several CYP405 copies were found in the Sapho clade, they lack structurally important P450 domains, which explain they loss of biochemical plasticity via specialization in CG sequestration. Our findings represent one of the few examples of plasticity-first evolution in which the genetic mechanisms associated with its accommodation/assimilation are known.