Figure Box2. Comparison of OTU-based (OTU-s , singletons removed) and ASV-based approaches for inferring taxonomic richness using the ITS2 subregion, full ITS region, and ITS + 28S based on the dataset of Furneaux et al. (2021), which was taxonomically re-analysed following Tedersoo et al. (in press). (a) average number of reads retained; (b) richness of OTUs/ASVs; (c) richness of fungal orders; and (d) kingdom and phylum-level distribution of OTUs/ASVs across the entire dataset. In (d), letters indicate statistically significant differences among groups based on Scheffe PostHoc tests following sample-wise testing of log-ratio transformed proportions using two-way ANOVAs including the two factors marker length and matrix type - OTU, OTUs without singletons (OTU-s) and ASV. We conclude that 1) the ASV approach and OTU-based approach with singletons removed recover lower proportions of non-Dikarya and non-fungal taxa compared with the OTU-based approach including singletons; 2) analyses based on the ITS2 region alone revealed a higher proportion of OTUs/ASVs that could not be identified to a fungal phylum or eukaryote kingdom compared with longer marker fragments due to less taxonomic information; 3) longer markers had fewer sequences passing the quality control and revealed relatively more singletons, suggesting accumulation of artefacts across the entire 1500-base amplicon; and 4) the ASV approach performs suboptimally for metabarcoding of fungi and particularly poorly for the full ITS region and longer markers.