Gene duplication is an important source of evolutionary innovation, but the functional distinction between duplicates can be opposed by ongoing gene conversion between them. Here we document a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) sharedby 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 paralog retained ancestral susceptibility. Frequent non-allelic gene conversion homogenized most of the paralog sequences,yet the two Leptodactylusparalogs are distinguished by 12 amino acid substitutions that were maintained by strong selection that counteracted gene conversion. Protein-engineering experiments show that two substitutions substantially increase toxin resistance, whereas the 10 additional substitutions mitigate deleterious pleiotropic effects on enzyme function. Our results highlight how neofunctionalized gene duplicates can help pinpoint functional substitutions and their interactions with the genetic backgrounds on which they arise.

"NKA Enzyme Assays Raw Data" - raw plate reader data and downstream calculations for enzyme assays used in this study

"Mohammadi_ATP1A1_alignment" - alignment of ATP1A1 gene sequences used to generate phylogenetic trees in this study

"NKA_Enzyme_Assays_Raw_Data_Readme" - ReadMe file for the enzyme assay raw data