ITS2 metabarcoding analysis complements lichen mycobiome diversity data

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a wellcharacterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.