Interspecific hybridization and bioactive alkaloid variation increases diversity in endophytic Epichloe species of Bromus laevipes

Studying geographic variation of microbial mutualists, especially variation in traits related to benefits they provide their host, is critical for understanding how these associations impact key ecological processes. In this study, we investigate the phylogenetic population structure of Epichloe species within Bromus laevipes, a native cool-season bunchgrass found predominantly in California. Phylogenetic classification supported inference of three distinct Epichloe taxa, of which one was nonhybrid and two were interspecific hybrids. Inheritance of mating-type idiomorphs revealed that at least one of the hybrid species arose from independent hybridization events. We further investigated the geographic variation of endophyte-encoded alkaloid genes, which is often associated with key benefits of natural enemy protection for the host. Marker diversity at the ergot alkaloid, loline, indole-diterpene, and peramine loci revealed four alkaloid genotypes across the three identified Epichloe species. Predicted chemotypes were tested using endophyte-infected plant material that represented each endophyte genotype, and 11 of the 13 predicted alkaloids were confirmed. This multifaceted approach combining phylogenetic, genotypic, and chemotypic analyses allowed us to reconstruct the diverse evolutionary histories of Epichloe species present within B.laevipes and highlight the complex and dynamic processes underlying these grass-endophyte symbioses.