Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila 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. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.

The repeated evolutionary specialization of distantly related insects to cardenolide-containing host plants provides a stunning example of parallel adaptation. Hundreds of herbivorous insect species have independently evolved insensitivity to cardenolides, which are potent inhibitors of the alpha-subunit of Na + ,K + -ATPase (ATPα). Previous studies investigating ATPα-mediated cardenolide insensitivity in five insect orders have revealed remarkably high levels of parallelism in the evolution of this trait, including the frequent occurrence of parallel amino acid substitutions at two sites and recurrent episodes of duplication followed by neo-functionalization. Here we add data for a sixth insect order, Orthoptera, which includes an ancient group of highly aposematic cardenolide-sequestering grasshoppers in the family Pyrgomorphidae. We find that Orthopterans exhibit largely predictable patterns of evolution of insensitivity established by sampling other insect orders. Taken together the data lend further support to the proposal that negative pleiotropic constraints are a key determinant in the evolution of cardenolide insensitivity in insects. Furthermore, analysis of our expanded taxonomic survey implicates positive selection acting on site 111 of cardenolide-sequestering species with a single-copy of ATPα, and sites 115, 118 and 122 in lineages with neo-functionalized duplicate copies, all of which are sites of frequent parallel amino acid substitution. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions’.

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Gene duplication is an important source of evolutionary innovation, but the adaptive division-of-labor between duplicates can be opposed by ongoing gene conversion between them. Here we document a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins while the other copy retained ancestral susceptibility. We show that the two Leptodactylus paralogs are distinguished by 12 amino acid substitutions that were maintained by strong selection that counteracted the homogenizing effect of gene conversion. Protein-engineering experiments show that two major-effect substitutions confer toxin resistance, whereas the 10 additional substitutions mitigate deleterious pleiotropic effects on enzyme function. Our results highlight how trans-specific, neofunctionalized gene duplicates can provide unique insights into interactions between adaptive substitutions and the genetic backgrounds on which they arise.One Sentence SummarySelection counteracts gene conversion to maintain an adaptive division-of-labor between tandemly duplicated genes.

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila 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. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.