Structural innovation in the evolution of plant chemical defense

Abstract

Chemical defenses are fundamental in organismal biology and widely used in medicine and agriculture. Plant defense chemistry evolves in response to various selective pressures, particularly herbivory, and theory has emphasized predicting toxin abundance and diversity. Here we test hypotheses about the evolution of structural innovation in chemical defense by combining molecular complexity metrics, metabolomics, molecular docking, and phylogenetic analyses, using milkweed cardenolides, steroidal glycosides that inhibit animal Na + /K + -ATPases. We identify the addition of a nitrogen–sulfur (N,S) heterocycle in highly substituted cardenolides as a major structural innovation that restores toxicity against coevolved natural enemies, such as the monarch butterfly. This toxicity is likely achieved by rigidifying the cardenolide scaffold and creating additional nonelectrostatic interactions within the Na + /K + -ATPase binding pocket, thereby enhancing binding affinity despite target-site resistance. Two biosynthetically distinct N,S-cardenolides, uscharin and labriformin, rank among the most complex structures in this chemical class and show divergent macroevolutionary histories: uscharin represents an ancestral character state with repeated losses, whereas labriformin has independently evolved multiple times in later-diverging lineages. This pattern across Asclepiadoideae indicates that the structural innovation evolved repeatedly, apparently limited by lineage-specific biosynthetic constraints among precursor pathways. N,S-cardenolides occur in over 75% of the 59 Asclepias species examined here, and species producing N,S-cardenolides exhibit greater cardenolide abundance, richness, metabolomic space, and toxicity against adapted organisms. More generally, structural innovation defines a distinct evolutionary axis in plant chemistry, enabling defense diversification and adaptive recovery of toxicity. Such innovations are predicted to build on existing molecular scaffolds in response to ecological challenges, here driven by coevolving specialist herbivores.