Chemotypic diversity of bioprotective grass endophytes based on genome analyses, with new insights from a Mediterranean-climate region in Isfahan Province, Iran

Epichloë species are systemic, often seed-transmissible symbionts (endophytes) of cool-season grasses (Poaceae subfam. Poöideae) that produce up to four classes of bioprotective alkaloids. Whereas haploid Epichloë species may reproduce sexually and transmit between host plants (horizontally), many Epichloë species are polyploid hybrids that are exclusively transmitted via seeds (vertically). Therefore, the generation of, and selection on, chemotypic (alkaloid) profiles and diversity should differ between haploids and hybrids. We undertook a genome-level analysis of haploids and polyploid hybrids, emphasizing hybrids that produce lolines, which are potent broad-spectrum anti-invertebrate alkaloids that can accumulate to levels up to 2% of plant dry mass. Prior phylogenetic analysis had indicated that loline alkaloid gene clusters (LOL) in many hybrids are from the haploid species Epichloë bromicola, but no LOL-containing E. bromicola strains were previously identified. We discovered LOL-containing E. bromicola from host grasses Bromus tomentellus and Melica persica in a Mediterranean-climate region (MCR) in Isfahan Province, Iran, and from Thinopyrum intermedium in Poland. The isolates from B. tomentellus and M. persica were closely related and had nearly identical alkaloid gene profiles, and their LOL clusters were most closely related to those of several Epichloë hybrids. In contrast, several LOL genes in the isolate from T. intermedium were phylogenetically more basal in genus Epichloë, indicating trans-species polymorphism. While identifying likely hybrid ancestors, this study also revealed novel host ranges in central Iran, with the first observation of E. bromicola in host tribe Meliceae and of Epichloë festucae in host tribe Bromeae. We discuss the possibility that MCRs may be hotspots for diversification of grass-Epichloë symbioses via extended host ranges and interspecific hybridization of the symbionts.