Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo.

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na,K-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction.