Abstract
<jats:p>Insect herbivores have driven plants to evolve complex chemical defenses, including mimics of the arthropod molting hormone 20-hydroxyecdysone (20E). Many distantly related plants produce 20E, suggesting multiple, independent evolutionary origins. However, despite the prevalence of 20E in arthropods and plants, complete biosynthetic pathways have remained elusive. Here, we combine genomics, gene co-expression, and enzyme characterization to discover convergent 20E biosynthetic pathways from spinach and quinoa (amaranth family) and Ajuga species (mint family). We reveal that these families use distinct biosynthetic gene clusters to produce 20E and related phytoecdysteroids, highlighting both biochemical and genetic convergence of 20E biosynthesis in plants. Our work establishes the first complete 20E pathways in any organism and provides foundational insight into the evolution of this animal hormone across kingdoms of life.</jats:p>