Intrathalline diversity of lichen-inhabiting fungi assessed by metabarcoding of ITS region

Lichen-inhabiting fungi are a large and diversified, but still relatively unknown, ecological group. They can live on host thalli symptomatically, showing characteristic phenotypes and different degrees of specificity (lichenicolous fungi), or asymptomatically (endolichenic fungi), detectable only through culture and molecular techniques. Here we aim at studying the whole diversity of lichen-associated fungi using metabarcoding analysis of the internaltranscribed-spacer regions, as parts of the fungal standard barcode, and comparing the results obtained by sequencing separately both the ITS1 and the ITS2 segments. In this way we avoid the analysis of group I intron occurring in some ITS1 fragments, hence achieving a higher resolution on species composition with the ITS2. We analyzed thalli with and without symptoms of fungal infection, collected from Alpine lichen communities on the Koralpe Massif (Austria). The first ITS1 amplicon sequencing highlighted a great taxonomic diversity. The majority of detected operational taxonomic units (OTUs) are fungi of the two main classes Dothideomycetes and Eurotiomycetes. Shared endolichenic fungal composition in the thalli varies in different lichen species, and does not correlate with the symptomatic fungal infections. Metabarcoding allows the exploration of lichens as ecological niches, revealing the organization of complex symbiotic communities, and, potentially, the existence of coherent patterns.