High ascomycete diversity and novel fungal associations found in expanding Epipactis helleborine populations outside their native range.

Epipactis helleborine is an orchid species that has been introduced to North America and continues to expand its range. Numerous studies have investigated the mycorrhizal specificity of these orchids in their native range and found broad associations with Ascomycota, particularly Wilcoxina. Similar studies in North America are rare. We assessed the fungal diversity associated with populations of E. helleborine, and the surrounding soil, in mixed habitats on Vancouver Island (Canada) to make comparisons with findings from the species native range. After collecting 45 rhizomes and 36 soil samples, we amplified the nuclear ribosomal internal transcribed spacer (ITS2) region to assess alpha and beta diversity. BLAST searches were conducted, and phylogenetic reconstructions of high-matching fungal sequences from other E. helleborine studies were created. We demonstrate that E. helleborine populations continue to associate with a diverse group of Ascomycota fungi, particularly Wilcoxina spp. as they continue to expand their range northward in western North America. However, we also detected a novel association among E. helleborine rhizomes and Rhexocercosporidium, a genus containing known saprophytes and plant pathogens. Our results indicate that this orchid can pair with an extensive range of fungal partners, likely by taking advantage of varying degrees of phylogenetic niche conservatism among different fungal groups, which could have wide-reaching implications in terms of further geographic expansion and introductions to new continents.