Chemotypic diversity of the fungus <i>Epichloë brachyelytri</i> symbiotic with the wild forest grass <i>Brachyelytrum erectum</i>

ABSTRACT Many cool-season grasses (Poaceae subfam. Poöideae) host seed-transmissible symbionts (endophytes) in the fungal genus Epichloë , which can produce diverse alkaloids that protect against invertebrate and, in some cases, vertebrate herbivores. Rarely have population surveys been conducted to assess comprehensive alkaloid profiles and diversity of Epichloë in wild grasses. In this study, we surveyed Brachyelytrum erectum , which is a woodland grass in an early-diverging lineage of Poöideae, and commonly symbiotic with Epichloë brachyelytri . Analytical methods based on high-resolution UHPLC-MS/MS were refined to provide rapid, comprehensive detection, and quantitation of E. brachyelytri alkaloids, for six B. erectum populations in Kentucky. Chemotypes were identified with two or three of the alkaloids exo -1-acetamidopyrrolizidine ( 1 ), chanoclavine ( 2 ), and peramine ( 3 ). Both 1 and 2 are known as intermediates in biosynthetic pathways to more complex alkaloids, and chemotypes having both 1 and 2 as pathway end-products are novel. Such chemotypes were also identified in other species, and phylogenetic analysis indicated their multiple origins by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization. Alkaloid levels were comparable between most populations and at most plant developmental stages. Levels of 3 were compared between E. brachyelytri variants with and without 1 , providing evidence for competition between the pathways in young shoots, but not in older leaves or seeds. Furthermore, levels of 1 and 2 were moderate to high compared with their respective alkaloid classes in other grass- Epichloë symbiotic systems. We conclude that production of the alkaloids likely represents an important metabolic investment by E. brachyelytri . IMPORTANCE Defensive mutualisms, symbioses of hosts with organisms that defend them against parasites or predators, play important ecological roles. A widespread example is protection of cool-season grasses by symbiotic Epichloë species, which are fungi that transmit in seeds and produce several kinds of anti-insect alkaloids. Profiles of alkaloids evolve due to shifting balances of their benefits and the costs of producing them. In this study, Epichloë brachyelytri symbiotic with the wild forest grass Brachyelytrum erectum produced three alkaloids, of which two have been rarely reported. Furthermore, variations in its alkaloid profiles and quantities of each alkaloid at different plant growth stages and tissues suggested that occasional loss of its most abundant alkaloid can be adaptive due to the metabolic load of producing it. Although rare, similar alkaloid profiles were identified in several other species in which they arose by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization.