Phylogenetics and association analyses illustrate substantial cryptic diversity of a newly isolated collection of Cenococcum geophilum

The ectomycorrhizal fungus Cenococcum geophilum is distributed worldwide across multiple climates and soil types and is known to positively associate with a multitude of plant genera, possibly contributing to plant ability to tolerate inorganic contaminants in a soil environment. New C. geophilum isolates are easily cultured from soils in a laboratory setting, making this an ideal candidate for a model species with which to study multiple plant-fungal effects across a collection of novel isolates. However, C. geophilum is also genetically complex and, at 178Mbp, features one of the largest fungal genomes, necessitating the use of the novel restriction-associated DNA sequencing (RADseq) technique which produces robust de novo single nucleotide polymorphism (SNP) detection. A preliminary investigation into the phylogenetic relationship of >200 new C. geophilum isolates from the United States Pacific Northwest (PNW) region using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) strongly resolved (>80%) 15 cryptic clades. An investigation of the worldwide C. geophilum collection using GAPDH resolved >30 cryptic clades. In both collections, at least two cryptic clades incorporated extreme spatial diversity in the form of cross-regional, cross-country, and international strains. Phylogenetic analyses of 171 PNW isolates conducted using RADseq strongly supported (>80%) the 15 PNW clades using the de novo dataset assembly at a per-site depth of at least 10%. Direct comparison of the PNW ITS and GAPDH gene regions indicated strong evidence of sexual recombination and additional analyses confirmed high levels of incongruency between the two genes. However, when these same analyses were conducted on the RADseq de novo dataset, no strong evidence of recombination was detected across the PNW collection, suggesting this collection represents a hybridized clonal population with rare localized sexual recombination. A association study linked heavy metal resistance of 56 C. geophilum isolates to significant SNP associations detected from the de novo and RADseq assembly, finding that 20 significant (p < 0.05) SNPs were detected in the presence of cadmium. These SNPs are linked to a series of metabolic, transcription and translation, and ion-binding protein coding regions as well as two proteins which may be directly involved in resistance to cadmium within isolates from two PNW sites.