Single-Thallus Genomics of Ejectosporus trisporus, an Unculturable Stonefly Gut Fungal Symbiont.

Microorganisms play essential roles in global ecosystems, yet much of their diversity, particularly among fungi, remains unexplored due to challenges in culturing and genomic characterisation. Trichomycetes, an early-diverging lineage of obligate gut symbionts of aquatic insects, exemplify this 'microbial dark matter', as most taxa cannot be maintained in axenic culture. Here, we present the first culture-independent genome assembly of Ejectosporus trisporus, an unculturable Harpellales fungus isolated from the hindgut of a winter stonefly (Allocapnia sp.) in Rouge National Urban Park, Canada. Using a single-thallus genomic approach based on multiple displacement amplification and Illumina short-read sequencing, we generated a 29.3 Mb genome assembly with 76.6% BUSCO completeness, comparable to existing culture-based Harpellales genomes. Phylogenomic analyses using 1241 conserved orthologs placed E. trisporus in a well-supported clade with Zancudomyces culisetae and Capniomyces stellatus, confirming its taxonomic position. Scanning electron microscopy further revealed detailed ultrastructural features of thalli, trichospores, and zygospores. This study demonstrates the feasibility of single-thallus genomics for unculturable fungi and provides the first genomic resource for an unculturable trichomycete species. Our study establishes a valuable basis for future large-scale genomic investigations of early-diverging fungi, enabling further exploration of the symbiosis and ecological roles of these cryptic gut-dwelling fungi.