Evolution of insensitivity to the toxic effects of cardiac glycosides has become a model in the study of convergent evolution, as five taxonomic orders of insects use the same few similar amino acid substitutions in the otherwise highly conserved Na,K-ATPase α. We show here that insensitivity in pyrgomorphid grasshoppers evolved along a slightly divergent path. As in other lineages, duplication of the Na,K-ATPase α gene paved the way for subfunctionalization: one copy maintains the ancestral, sensitive state, while the other copy is resistant. Nonetheless, in contrast with all other investigated insects, the grasshoppers' resistant copy shows length variation by two amino acids in the first extracellular loop, the main part of the cardiac glycoside-binding pocket. RT-qPCR analyses confirmed that this copy is predominantly expressed in tissues exposed to the toxins, while the ancestral copy predominates in the nervous tissue. Functional tests with genetically engineered Drosophila Na,K-ATPases bearing the first extracellular loop of the pyrgomorphid genes showed the derived form to be highly resistant, while the ancestral state is sensitive. Thus, we report convergence in gene duplication and in the gene targets for toxin insensitivity; however, the means to the phenotypic end have been novel in pyrgomorphid grasshoppers.

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