Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase

Abstract

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

Widespread convergence in toxin resistance by predictable molecular evolution

“…First, we reconstructed the evolutionary history of the multilocus Na (9,(15)(16)(17), with the exception of 111E proposed by Dobler et al (9) and 119D proposed and validated here (Fig. 4).…”

“…However, until now, comparisons of the molecular basis of resistance and mechanisms of action across the diverse range of resistant taxa reported (9,15,17,18) have been lacking. Here we present an analysis of convergent molecular changes to the Na + /K + -ATPase H1-H2 domain in cardiac glycoside-resistant invertebrates and vertebrates.…”

“…Epitomized by the writings of Gould (5) and Conway Morris (6), this debate centers on whether evolution is stochastic and unpredictable (5) or subject to constraints that limit the available options for evolution, resulting in frequent convergence and a degree of predictability (6). However, evidence of convergence at the genetic level, where similar molecular changes confer the same change in phenotype, is currently limited to only a few taxonomically restricted examples (7)(8)(9)(10)(11).…”

“…For example, milkweed, foxglove, and oleander plants produce "cardenolides" (e.g., ouabain) that protect against herbivorous insects (13), whereas bufonid toads secrete structurally and functionally similar defensive compounds, "bufotoxins" (e.g., bufalin), from their parotoid glands (14). Despite the toxicity of these molecules, natural resistance exists in many different herbivores and predators and is predominately mediated by molecular alterations to the H1-H2 extracellular domain of the Na + /K + -ATPase, resulting in targetsite insensitivity to cardiac glycosides (9,15,16). For example, we recently showed that two amino acid replacements in the Na…”

Recurrent specialization on a toxic fruit in an island Drosophila population

“…In herbivorous insects, host plant specialization also plays a major role in diversification (3), and spectacular examples of convergent evolution both in plant resistant toxins and insect toxin resistances spanning hundred million of years of divergence have been observed. For example, several unrelated flowering plants produce cardenolides that block activity of the ion gradient regulating enzyme (Na + +K + )ATPase in insects, but identical cardenolideresistant amino acid substitutions in this enzyme have independently arisen in beetles, butterflies, flies, and aphids specializing on such plants (4). Recent genomic studies have focused on parallel dietary shifts in early-diverging ecological races, pointing to a substantial degree of common molecular mechanisms (5)(6)(7)(8).…”