Metabarcoding of Insect-Associated Fungal Communities: A Comparison of Internal Transcribed Spacer (ITS) and Large-Subunit (LSU) rRNA Markers

Abstract Complex communities of fungi are regularly characterised by deep sequencing with standard barcode markers, especially the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus. Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations or for early detection of pathogens and invasive species, but reliance on a single short sequence fragment may be problematic due to various technological and analytical obstacles. Here we conducted a side-by-side comparison of fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens, using ITS and LSU. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type). Identification against the respective reference databases using various classifiers revealed similar higher-level taxonomic composition, but decreasing resolution towards lower levels, especially at the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, a phylogenetic analysis (focused on the epidemiologically important Sordariomycetes) placed OTUs relative to reference sequences spanning both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. The analysis of congruence in both markers also suggested the biologically most defensible threshold values for OTU delimitation (98% for ITS, 99% for LSU). Studies of complex fungal communities using the canonical ITS metabarcode marker require corroboration across additional loci, and benefit from phylogenetic analyses and their greater precision compared to conventional taxonomic classifiers, even in the face of incomplete and partially identified reference databases.