AbstractEcological studies of species pairs demonstrated that biotic interactions promote phenotypic change and eco-evolutionary feedbacks. However, we have a limited understanding of how phenotypes respond to interactions with multiple taxa. We investigate how interactions with a network of prey species contribute to spatially structured variation in the skin toxins of the Neotropical poison frog Oophaga pumilio. Specifically, we assess how beta-diversity of alkaloid-bearing arthropod prey assemblages (68 ant species) and evolutionary divergence among populations (from a neutral genetic marker) contribute to frog poison dissimilarity (toxin profiles composed of 230 different lipophilic alkaloids sampled from 934 frogs at 46 sites). We show that ant assemblage turnover predicts alkaloid turnover and unique toxin combinations across the range of O. pumilio. By contrast, evolutionary relatedness is barely correlated with toxin variation. We discuss how the analytical framework proposed here can be extended to other multi-trophic systems, coevolutionary mosaics, microbial assemblages, and ecosystem services.