The extent of convergent molecular evolution is largely unknown, yet is critical to understanding the genetics of adaptation. Target site insensitivity to cardenolides is a prime candidate for studying molecular convergence because herbivores in six orders of insects have specialized on these plant poisons, which gain their toxicity by blocking an essential transmembrane carrier, the sodium pump (Na,K-ATPase). We investigated gene sequences of the Na,KATPase α-subunit in 18 insects feeding on cardenolide-containing plants (spanning 15 genera and four orders) to screen for amino acid substitutions that might lower sensitivity to cardenolides. The replacement N122H that was previously shown to confer resistance in the monarch butterfly (Danaus plexippus) and Chrysochus leaf beetles was found in four additional species, Oncopeltus fasciatus and Lygaeus kalmii (Heteroptera, Lygaeidae), Labidomera clivicollis (Coleoptera, Chrysomelidae), and Liriomyza asclepiadis (Diptera, Agromyzidae). Thus, across 300 Myr of insect divergence, specialization on cardenolide-containing plants resulted in molecular convergence for an adaptation likely involved in coevolution. Our screen revealed a number of other substitutions connected to cardenolide binding in mammals. We confirmed that some of the particular substitutions provide resistance to cardenolides by introducing five distinct constructs of the Drosophila melanogaster gene into susceptible eucaryotic cells under an ouabain selection regime. These functional assays demonstrate that combined substitutions of Q 111 and N 122 are synergistic, with greater than twofold higher resistance than either substitution alone and >12-fold resistance over the wild type. Thus, even across deep phylogenetic branches, evolutionary degrees of freedom seem to be limited by physiological constraints, such that the same molecular substitutions confer adaptation.insect-plant interactions | specialist herbivores | target site insensitivity | toxin resistance | cardiac glycosides

Author contributions M.K. co-designed and implemented the overall strategy for the creation of the knock-in fly lines, designed and implemented the bioassays, the RT-qPCR experiments and the RMO analysis, performed statistical analyses and co-wrote the manuscript. S.C.G. designed and implemented the overall strategy for the creation of the knock-in fly lines, prepared the sequence data and metadata for the phylogenetic analyses, co-designed all other experiments, and co-wrote the manuscript. F.S. performed the structural modelling and docking site analyses. J.N.P. performed the phylogenetic, ancestral state and co-evolutionary analyses. K.I.V. conducted crosses, genotyping, and feeding experiments, and co-designed the qPCR experiments. J.M.A. and S.L.B. conducted crosses and genotyping, and feeding and sequestration experiments. A.P.H. performed the in vitro physiological experiments and sequestration analyses. T.M. conducted feeding experiments M.A. performed the RMO analysis with M.K., and conducted genotyping and feeding experiments. G.G. completed the RMO and ouabain dietary survival analyses. F.R. supervised the structural modelling and docking site analyses. S.D. oversaw and interpreted in vitro cell line analyses, helped to design the overall project and co-wrote the manuscript. A.A.A. helped to design the overall project, oversaw the in vitro physiological and sequestration experiments, and co-wrote the manuscript. N.K.W. led the overall collaboration, the project design and its integration, creation of fly lines and statistical analyses, and co-wrote the manuscript. Peer review information Nature thanks Joseph W. Thornton and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Online content Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at

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Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed

Insect‐plant interactions have played a prominent role in investigating phylogenetic constraints in the evolution of ecological traits. The patterns of host association among specialized insects have often been described as highly conservative, yet not all specialized herbivorous insect lineages display the same degree of fidelity to their host plants. In this paper, we present an estimate of the evolutionary history of the leaf beetle genus Oreina. This genus displays an amazing flexibility in several aspects of its ecology and life history: (1) host plant switches in Oreina occurred between plant families or distantly related tribes within families and thereby to more distantly related plants than in several model systems that have contributed to the idea of parallel cladogenesis; (2) all species of the genus are chemically defended, but within the genus a transition between autogenous production of defensive toxins and sequestration of secondary plant compounds has occurred; and (3) reproductive strategies in the genus range from oviparity to viviparity including all intermediates that could allow the gradual evolution of viviparity. Cladistic analysis of 18 allozyme loci found two most parsimonious trees that differ only in the branching of one species. According to this phylogeny estimate, Oreina species were originally associated with Asteraceae, with an inclusion of Apiaceae in the diet of one oligophagous species and an independent switch to Apiaceae in a derived clade. The original mode of defense appears to be the autogenous production of cardenolides as previously postulated; the additional sequestration of pyrrolizidine alkaloids could have either originated at the base of the genus or have arisen three times independently in all species that switched to plants containing these compounds. Viviparity apparently evolved twice in the genus, once without matrotrophy, through a retention of the eggs inside the female's oviducts, and once in combination with matrotrophy. We hypothesize that the combination of autogenous defense and a life history that involves mobile externally feeding larvae allowed these beetles to switch host plants more readily than has been reported for highly conservative systems.