Lichen-associated Cystobasidiomycetes have been reported from a wide range of lichens worldwide. Previous studies proposed that lichen-associated Cystobasidiomycetes may be involved in the development of the thallus cortex, suggesting also a contribution to the construction of thallus architecture within the Parmeliaceae. However, Parmeliaceae do not include species with ecorticate thalli. In contrast, Arthoniales encompass a wide diversity of thallus growth forms, including both corticate and ecorticate types, making this order a suitable model to test this hypothesis across a broader evolutionary context. Furthermore, Cystobasidiomycetes have not yet been studied in Arthoniales. This study aims therefore to (1) characterize the diversity of Arthoniales-associated Cystobasidiomycetes; (2) investigate their selectivity with respect to mycobiont partners and the ecological factors shaping their distribution, and (3) test whether their distribution is consistent with a proposed role in cortex formation and thallus architecture in Arthoniales. The ITS region of Cystobasidiomycetes was sequenced from 169 lichen samples (in majority Arthoniales) and an exhaustive maximum-likelihood phylogeny, including our sequences and all sequences found in GenBank, was inferred using IQ-tree. The effects of substratum, climate, host taxonomy and geographic location on the distribution of Cystobasidiomycetes were assessed using variation partitioning. Cystobasidiomycetes were found in 60 of our samples. Our sequences belonged to 39 Cystobasidiomycetes OTUs, 21 of which were detected in Arthoniales thalli, and eight of them represent unknown lineages. Globally, OTU selectivity was variable. However, most OTU's exhibited a preference for particular groups of mycobionts. Mycobiont taxonomy emerged as the primary factor shaping lichen-derived Cystobasidiomycetes community composition. Climate and substrate likely also influence their distribution, although their effects may be underestimated, due to an unbalanced dataset, dominated by sequences from extra-tropical and corticolous specimens. Finally, no evidence was found to support a correlation between Cystobasidiomycetes detection, thallus growth form or cortex presence in Arthoniales.

Overall, these results suggest that lichen-derived Cystobasidiomycetes OTUs largely form lineages distinct from those represented by Cystobasidiomycetes from other sources. However, the lifestyle of many sequences remains uncertain because they were obtained through environmental sequencing. Despite their generally low selectivity, a consistent pattern of mycobiont preference was observed across Cystobasidiomycetes orders. This pattern was not associated with thallus growth form or the presence of a cortex.