Genome diversification of symbiotic fungi in beetle-fungus mutualistic symbioses.

Ambrosia beetles and their fungal symbionts represent a widespread and diverse insect-fungus mutualism. This study investigates the genomic adaptations associated with the evolution of the ambrosia lifestyle across multiple fungal lineages. We performed comparative genomic analyses on 70 fungal genomes from four families (Irpicaceae, Ceratocystidaceae, Nectriaceae, and Ophiostomataceae), including 24 ambrosia and 34 nonambrosia lineages. Our phylogenomic analyses reveal multiple independent colonization of insect vectors by the fungi, spanning from the mid-Cretaceous (114.6 Ma) to the early Quaternary (1.9 Ma). Contrary to expectations for obligate symbionts, ambrosia fungi showed no significant genome-wide modification in size, gene count, or secreted protein repertoire compared to their nonsymbiotic relatives. Instead, we observed conservation of most assessed genomic features; where genome traits differ between free-living relatives and ambrosia fungi, the changes are lineage-specific, not convergent. Key findings include lineage-specific expansions in carbohydrate-active enzyme families (AA4 in Nectriaceae, CE4 in Ophiostomataceae, and GH3 in Ophiostomataceae and Ceratocystidaceae), suggesting potential enhancement or loss of lignin modification, hemicellulose deacetylation, and cellulose degradation in different ambrosia lineages. Repeat-induced point mutation analysis revealed family-specific patterns rather than lifestyle-associated differences. These results highlight the diverse genomic strategies employed by ambrosia fungi, demonstrating that symbiont evolution can proceed through refined, lineage-specific changes rather than genome-wide or convergent alterations. Our genomic analyses do not reveal patterns typically associated with domestication in these ambrosia fungi, suggesting they may represent free-living fungi that co-opted wood-boring beetles as vectors through subtle, lineage-specific adaptations.