Department of Biology, Hamburg University, 20146 Hamburg, Germany.
Proc Natl Acad Sci U S A. 2012 Aug 7;109(32):13040-5. doi: 10.1073/pnas.1202111109. Epub 2012 Jul 23.
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,K-ATPase 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.
趋同分子进化的程度在很大程度上是未知的，但对于理解适应的遗传学至关重要。靶标对卡地洛尔的不敏感性是研究分子趋同的主要候选者，因为六个昆虫目级的食草动物已经专门针对这些植物毒素进行了特化，这些毒素通过阻断一种必需的跨膜载体，即钠泵（Na，K-ATPase）来获得毒性。我们研究了 18 种以含有卡地洛尔的植物为食的昆虫的 Na，K-ATPaseα亚基的基因序列，以筛选可能降低对卡地洛尔敏感性的氨基酸取代。先前在黑脉金斑蝶（Danaus plexippus）和 Chrysochus 叶甲虫中显示出抗性的 N122H 取代在另外四个物种中被发现，即 Oncopeltus fasciatus 和 Lygaeus kalmii（半翅目，叶甲科），Labidomera clivicollis（鞘翅目，金龟科）和 Liriomyza asclepiadis（双翅目，叶蝇科）。因此，在 3 亿年的昆虫分化中，对含有卡地洛尔的植物的特化导致了可能涉及共同进化的适应的分子趋同。我们的筛选揭示了与哺乳动物中卡地洛尔结合有关的许多其他取代。我们通过在 Ouabain 选择条件下将果蝇基因的五个不同构建体引入敏感的真核细胞，证实了一些特定的取代可以提供对卡地洛尔的抗性。这些功能测定表明，Q（111）和 N（122）的联合取代是协同的，其抗性比单独任何一个取代都高出两倍以上，比野生型高出 12 倍以上。因此，即使在深的系统发育分支中，进化的自由度似乎也受到生理限制的限制，使得相同的分子取代赋予适应。
