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In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na + /K + –ATPase), each playing a central role in milkweed–insect coevolution. The four-eyed milkweed beetle ( Tetraopes tetrophthalmus ) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle’s Na + /K + –ATPase to cardenolide extracts from roots versus leaves of its main host ( Asclepias syriaca ), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes’ enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle’s enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na + /K + –ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes ’ Na + /K + –ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes’ enhanced enzymatic tolerance of cardenolides. Thus, milkweed’s tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore. 

Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host’s range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed ( Asclepias syriaca ) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na + /K + -ATPase); there was little variation among compounds in inhibition of an unadapted Na + /K + -ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltu s. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration. 

Dormancy has repeatedly evolved in plants, animals, and microbes and is hypothesized to facilitate persistence in the face of environmental change. Yet previous experiments have not tracked demography and trait evolution spanning a full successional cycle to ask whether early bouts of natural selection are later reinforced or erased during periods of population dormancy. In addition, it is unclear how well short-term measures of fitness predict long-term genotypic success for species with dormancy. Here, we address these issues using experimental field populations of the plantOenothera biennis, which evolved over five generations in plots exposed to or protected from insect herbivory. While populations existed above ground, there was rapid evolution of defensive and life-history traits, but populations lost genetic diversity and crashed as succession proceeded. After >5 y of seed dormancy, we triggered germination from the seedbank and genotyped >3,000 colonizers. Resurrected populations showed restored genetic diversity that reduced earlier responses to selection and pushed population phenotypes toward the starting conditions of a decade earlier. Nonetheless, four defense and life-history traits remained differentiated in populations with insect suppression compared with controls. These findings capture key missing elements of evolution during ecological cycles and demonstrate the impact of dormancy on future evolutionary responses to environmental change.