ABSTRACT Most plant species form symbioses with mycorrhizal fungi and this relationship is especially important for orchids. Fungi in the genera Tulasnella, Ceratobasidium, and Serendipita are critically important for orchid germination, growth and development. The goals of this study are to understand the phylogenetic relationships of mycorrhizal fungi and to improve the taxonomic resources for these groups. We identified 32 fungal isolates with the internal transcribed spacer region and used shallow genome sequencing to functionally annotate these isolates. We constructed phylogenetic trees from 408 orthologous nuclear genes for 50 taxa representing 14 genera, 11 families, and five orders in Agaricomycotina. While confirming relationships among the orders Cantharellales, Sebacinales, and Auriculariales, our results suggest novel relationships between families in the Cantharellales. Consistent with previous studies, we found the genera Ceratobasidium and Thanatephorus of Cerabotasidiaceae to not be monophyletic. Within the monophyletic genus Tulasnella , we found strong phylogenetic signals that suggest a potentially new species and a revision of current species boundaries (e.g. Tulasnella calospora ); however it is premature to make taxonomic revisions without further sampling and morphological descriptions. There is low resolution of Serendipita isolates collected. More sampling is needed from areas around the world before making evolutionary-informed changes in taxonomy. Our study adds value to an important living collection of fungi isolated from endangered orchid species, but also informs future investigations of the evolution of orchid mycorrhizal fungi
