Extended Data Fig. 1: Substitutions at ATPα amino acid residues 111, 119 and 122 are directly or indirectly associated with insect specialization on plants that produce cardiac glycosides.

a, The number of occurrences of each substitution across the 21 lineages in which specialization evolved independently. b, TraitRateProp analysis of the H1–H2 loop of ATPα across insects shows amino acid residues that are strongly associated with feeding on cardiac glycoside-producing plants and toxin sequestration. Bayes factor values in the top histogram indicate per-site associations between feeding and sequence rate evolution, values in the bottom histogram indicate per-site associations between sequestration and sequence rate evolution. Values over 10 were considered different (asterisks). For information on the species included in the analysis please see Supplementary Text. Colours in the multi-sequence alignment represent individual amino acids. c, BGM shows the correlated evolution of amino acid sites within the H1–H2 loop of ATPα. The table shows the marginal posterior probabilities (PP) between amino acid interactions, where the PP exceeds a default cut-off of 0.5. The residue interactions are depicted graphically, with amino acid sites represented by the nodes and the PP associated with a given epistatic or co-evolutionary interaction indicated by the values at the arrows. Nodes circled in orange indicate amino acid sites that are the focus of experiments in this study. Sites 111 and 122 are very strongly associated with feeding and sequestering, and site 119 co-evolves with site 111.