Phylogenetic diversity of 200+ isolates of the ectomycorrhizal fungusCenococcum geophilumassociated withPopulus trichocarpasoils in the Pacific Northwest, USA and comparison to globally distributed representatives

The ectomycorrhizal fungal symbiont Cenococcum geophilum is of great interest as it is globally distributed, associates with many plant species, and has resistance to multiple environmental stressors. C. geophilum is only known from asexual states but is often considered a cryptic species complex, since extreme phylogenetic divergence is often observed within morphologically identical strains. Alternatively, C. geophilum may represent a highly diverse single species, which would suggest cryptic but frequent recombination. Here we describe a new isolate collection of 229 C. geophilum isolates from soils under Populus trichocarpa at 123 collection sites spanning a ~283 mile north-south transect in Washington and Oregon, USA (PNW). To further understanding of the phylogenetic relationships within C. geophilum , we performed maximum likelihood phylogenetic analyses to assess divergence within the PNW isolate collection, as well as a global GAPDH phylogenetic analysis of 789 isolates with publicly available data from the United States, Europe, Japan, and other countries. Phylogenetic analyses of the PNW isolates revealed three distinct phylogenetic groups, with 15 clades that strongly resolved at >80% bootstrap support based on a GAPDH phylogeny and one clade segregating strongly in two principle component analyses. The abundance and representation of PNW isolate clades varied greatly across the North-South range, including a monophyletic group of isolates that spanned nearly the entire gradient at ~250 miles. A direct comparison between the GAPDH and ITS phylogenies combined with additional analyses revealed stark incongruence between the ITS and GAPDH gene regions, implicating probable intra-species recombination between PNW isolates. In the global isolate collection phylogeny, 34 clades were strongly resolved at >80% bootstrap support, with some clades having intra- and intercontinental distributions. These data are highly suggestive of divergence within multiple cryptic species, however additional analyses such as higher resolution genotype-by-sequencing approaches are needed to distinguish potential species boundaries and recombination patterns.