Phylogenetic resolution of different OTU generation methods within kingdom Fungi
For a more detailed analysis of kingdom Fungi, phylogenetic reconstruction using the LSU and 5.8S regions of all fungal OUT representative sequences from the three OTU generation methods (Table 1) was used to analyze the phylogenetic signal of estimated species richness for the three different OTU generation methods (Supplementary datafile 2). Within kingdom Fungi, we identified 1,590 genus-level clusters (GH_90), the vast majority of which were monophyletic, indicating good concordance between phylogenetic inference based on conserved LSU and 5.8 regions and sequence similarity in the ITS2 region of individual sequences. The nine GH_90 clusters that were polyphyletic in the fungal tree were found in lineages with short branch lengths separating terminal nodes (Fig. S12a). In these lineages, the existing variation within conserved regions, which may be in part due to sequencing errors, provides low phylogenetic resolution, resulting in collapse to random order in the tree. The same pattern applies to cases of polyphyletic SH_97 and SH_99 clusters since different sequence regions were used for phylogenetic inference and similarity clustering (Supplementary datafile 2).
As expected, different OTU generation methods detect different levels of genetic variation within and between taxa in the sequenced fungal community. For rare taxa, OTU_A completely fails to even detect phylum level diversity, as in the case of Glomeromycota, that was recovered in six OTUs across OTU_C and OTU_S, all of which represent rare taxa in the dataset (Fig. S12b). In abundant taxa on the other hand, intra-species variation is captured with several OTU_A sequences per SH_99 or SH_97 (Fig. S10), while the other methods identify one or two variants as exemplified by a single Morteriella SH_99, containing 12 OTU_As (Fig. S12c).