Holistic genome assembly and analysis of the Tremella fuciformis interaction community uncovers intergenomic insights beyond dual genomes

Tremella fuciformis ( T. fuciformis ) is consistently found in association with Annulohypoxylon stygium ( A. stygium ) in natural environments. However, their interaction remains largely cryptic and requires a dedicated in situ sequencing approach for elucidation. Traditional genome sequencing and assembly yield genetic information for only one species at a time. In this study, the interacting community of T. fuciformis was sequenced as an integrated unit, obtaining three complete genomes in a single run, specifically two heterokaryotic genomes of T. fuciformis and one of A. stygium . Validated across four dimensions, these genomes showed excellent continuity, completeness, and accuracy. Interspecifically, the cell ratio of T. fuciformis to A. stygium was estimated at 1:1.09, and no genomic evidence supported DNA exchange through long-term symbiosis. Heterokaryotically, distinct chromosomal structural variations were observed between the core and accessory chromosomes of T. fuciformis , while internal transcribed spacer (ITS) fragment polymorphism indicated that single-locus ITS data may inadequately reflect genetic complexity. Using the community genome as molecular markers enabled strain identification and confirmed interactions. Overall, this study provides methods for studying interactive community genomes and their interspecific and internuclear connections.