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

<i>Epichloë</i> 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 <i>Epichloë</i> species may reproduce sexually and transmit between host plants (horizontally), many <i>Epichloë</i> 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 (<i>LOL</i>) in many hybrids are from the haploid species <i>Epichloë bromicola</i>, but no <i>LOL</i>-containing <i>E. bromicola</i> strains were previously identified. We discovered <i>LOL</i>-containing <i>E. bromicola</i> from host grasses <i>Bromus tomentellus</i> and <i>Melica persica</i> in a Mediterranean-climate region (MCR) in Isfahan Province, Iran, and from <i>Thinopyrum intermedium</i> in Poland. The isolates from <i>B. tomentellus</i> and <i>M. persica</i> were closely related and had nearly identical alkaloid gene profiles, and their <i>LOL</i> clusters were most closely related to those of several <i>Epichloë</i> hybrids. In contrast, several <i>LOL</i> genes in the isolate from <i>T. intermedium</i> were phylogenetically more basal in genus <i>Epichloë</i>, indicating trans-species polymorphism. While identifying likely hybrid ancestors, this study also revealed novel host ranges in central Iran, with the first observation of <i>E. bromicola</i> in host tribe Meliceae and of <i>Epichloë festucae</i> in host tribe Bromeae. We discuss the possibility that MCRs may be hotspots for diversification of grass-<i>Epichloë</i> symbioses via extended host ranges and interspecific hybridization of the symbionts.