Basidiomycete yeasts have recently been reported as stably associated secondary fungal symbionts (SFS) of many lichens, but their role in the symbiosis remains unknown. Attempts to sequence their genomes have been hampered both by the inability to culture them and their low abundance in the lichen thallus alongside two dominant eukaryotes (an ascomycete fungus and chlorophyte alga). Using the lichen Alectoria sarmentosa, we selectively dissolved the cortex layer in which SFS are embedded to enrich yeast cell abundance and sequenced DNA from the resulting slurries as well as bulk, pulverized lichen thallus. In addition to yielding a near-complete genome of the filamentous ascomycete using both methods, metagenomes from cortex slurries yielded a 36- to 84-fold increase in coverage and near-complete genomes for two basidiomycete species, members of Cystobasidiomycetes and Tremellomycetes. The ascomycete possesses the largest gene inventory of the three. It is enriched in proteases often associated with pathogenicity and harbours the majority of predicted secondary metabolite clusters. The basidiomycete genomes possess ~35% fewer predicted genes than the ascomycete and have reduced secretomes even compared to close relatives, while exhibiting signs of nutrient limitation and scavenging. Furthermore, both basidiomycetes are enriched in genes coding for secreted acidic polysaccharides, representing a potential contribution to the shared extracellular matrix. All three fungi appear to retain genes involved in dimorphic switching, despite the ascomycete not being known to possess an anamorphic state. The basidiomycete genomes are an important new resource for exploration of lifestyle and function in fungal-fungal interactions in lichen symbioses.
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