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.

香柱菌属（<i>Epichloë</i>）物种是冷季型禾本科（Poaceae 早熟禾亚科 Pooideae）的系统性、常可经种子传播的共生内生真菌（endophytes），可合成至多四类具有生物保护活性的生物碱。单倍型香柱菌属物种可进行有性生殖并在宿主植物间水平传播，而多数该属物种为多倍体杂交体，仅能通过种子进行垂直传播。因此，单倍体与杂交体在化学型（生物碱）谱型及其多样性的产生与选择压力上理应存在差异。本研究对单倍体及多倍体杂交体开展了基因组水平分析，重点关注可合成洛林生物碱（loline）的杂交体——洛林生物碱是一类广谱强效的抗无脊椎动物生物碱，其积累量最高可达植物干重的2%。此前的系统发育分析表明，多数杂交体中的洛林生物碱基因簇（<i>LOL</i>）源自单倍体物种雀麦生香柱菌（<i>Epichloë bromicola</i>），但此前尚未发现携带<i>LOL</i>基因簇的<i>E. bromicola</i>菌株。我们在伊朗伊斯法罕省的地中海气候区（Mediterranean-climate region，简称MCR），从宿主植物绢雀麦（<i>Bromus tomentellus</i>）和波斯甜茅（<i>Melica persica</i>）中分离得到了携带<i>LOL</i>基因簇的雀麦生香柱菌，同时在波兰的中间偃麦草（<i>Thinopyrum intermedium</i>）中也分离到了该菌株。分离自绢雀麦和波斯甜茅的菌株亲缘关系紧密，生物碱基因谱几乎完全一致，其<i>LOL</i>基因簇与数种香柱菌属杂交体的基因簇亲缘关系最近。与之形成对比的是，分离自中间偃麦草的菌株中，数个<i>LOL</i>基因在系统发育上属于香柱菌属内更为基部的类群，这表明存在跨物种多态性（trans-species polymorphism）。本研究在确定潜在杂交祖先的同时，还揭示了伊朗中部地区新的宿主范围：首次在禾本科甜茅族（Meliceae）宿主中发现雀麦生香柱菌，并首次在雀麦族（Bromeae）宿主中发现羊茅草香柱菌（<i>Epichloë festucae</i>）。我们讨论了地中海气候区可能成为禾本科-香柱菌属共生体系多样化热点区域的可能性，这一过程依托于宿主范围的拓展以及共生体的种间杂交。