Metabarcoding Reveals Rich and Diverse Aeromycobiota in Protected Oak Forests

Old-growth oak (Quercus robur) forests in Europe are biodiversity hotspots, yet their airborne fungal diversity remains poorly studied. We investigated aeromycobiota in three Lithuanian oak stands (Punia, Dūkštos and Šilinė) using passive spore traps combined with DNA metabarcoding. Weekly sampling between August and September 2022 yielded 75 spore samples, producing 262,755 high-quality fungal sequences clustered into 1,881 operational taxonomic units (OTUs) representing six phyla and 36 classes. Ascomycota (53.1% of OTUs) and Basidiomycota (44.3%) dominated in richness, while Basidiomycota prevailed in relative sequence abundance (62.8%). Major taxa included Exobasidiaceae sp., Cladosporium sp., Melampsora sp., and Thelephora terrestris. Airborne fungal communities exhibited a substantial core assemblage shared among stands, accompanied by stand-associated differences in species richness and relative sequence abundance. The Punia stand showed the highest richness and the greatest proportion of stand-specific OTUs. Temporal variation was detectable but moderate relative to spatial differences among stands. Functional guild analysis revealed dominance of saprotrophs (46.5% of assigned OTUs), followed by pathotrophs (14.5%) and symbiotrophs (13.2%). Overall, our results demonstrate that passive spore traps-based airborne metabarcoding captures high fungal diversity and resolves both widespread and stand-associated community patterns in old-growth oak forests. This study provides the first characterization of aeromycobiota in old-growth Q. robur forests of Northern Europe and highlights the value of airborne metabarcoding for fungal biodiversity assessment and long-term forest monitoring.