Balancing selection is frequently invoked as a mechanism to maintain variation within and across populations. However, rigorous tests demonstrating balancing selection operating in nature are scarce, particularly on complex traits, which frequently display high levels of variation. Leveraging a focal polymorphism, leaf chemical profile in a perennial wildflower (Boechera stricta, Brassicaceae), we investigated the ecological and genetic mechanisms that may influence the maintenance of variation in this trait. A suite of common garden and greenhouse experiments showed that the alleles underlying variation in chemical profile have contrasting fitness effects across environments, implicating two ecological drivers of selection on chemical profile: herbivory and drought. Phenotype-environment associations and molecular genetic analyses revealed additional evidence of past selection by these drivers. Together, these data are consistent with balancing selection on chemical profile, likely caused by pleiotropic effects of genes that influence secondary chemical biosynthesis on herbivore defense and drought response.

The studies reported here include: analytical chemistry, field transplant experiments, field array experiments, a lab herbivory assay, two drought tolerance dry-down experiments, Sanger sequencing analysis, and analyses of genomic data to characterize linkage disequilibrium, flanking genes, and F_ST. Full details regarding the study design for each of these components are provided in Methods, Results, and Supplementary Information of the manuscript.