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[dataset] Reynolds, N.K., Jusino, M.A., Stajich, J.E., Smith, M.E.; 2020; OSF (https://osf.io/cz3mh/); doi:
Figure 1. Violin plots of the average percentage of forward and reverse reads that were merged to form contigs from each primer set (ITS1F/ITS2, LROR/LR3 and LR22F/LR3). The number of total reads returned for each dataset is listed above the boxes and the number of total (i.e. fungal and non-fungal) OTUs found after filtering and quality control is given below.
Figure 2. Primer set variation (ITS1F/ITS2, LROR/LR3, LR22F/LR3) in OTU length and read number assigned to each OTU according to taxonomic assignment by Kingdom and fungal phylum using the hybrid (for ITS1F) or UTAX (for LSU) method (A, B) and the RDP classifier (C, D). Box plot height and whiskers represent OTU length range, whereas the box plot width represents the proportion of reads assigned to each group. The LROR primer set was almost entirely comprised of forward reads with a length of 283 bp, resulting in a flat line. SUPP FIG 4 contains additional graphs of the LROR data separately. Percentages indicate the proportion of non-fungal (A, C) and fungal (B, D) OTUs in each dataset. Note that Glomeromycotina is a subphylum within the Mucoromycota but is categorized separately for comparison with the ITS taxonomy.
Figure 3. Relative abundance of reads assigned to each phylum of early diverging fungi by sample type for each primer set.
Figure 4. Non-metric multi-dimensional scaling (NMDS) ordination plots for all fungi and early diverging fungal communities recovered by the primer sets (ITS1F/ITS2, LROR/LR3, LR22F/LR3) for all California (CA) and Florida (FL) environmental sampling sites. Point colors represent different sampling locations and point shapes indicate primer set. Stress values are listed for each dataset.
Figure 5. Comparison of alpha diversity measures for early diverging fungi for each primer set by site (colors) and sample type (shapes).
Figure 6. Maximum likelihood phylogenetic reconstruction of fungal LSU sequences including references from GenBank and newly sequenced isolates from this study. 50 OTUs identified only as “Fungi” from each of the LROR and LR22F datasets were included and the numbers are bolded. Analyses were performed in RAxML v 8 using the GTR + GAMMA model and 1,000 bootstrap replicates. Classes of fungi are colored if they include unknown LSU OTUs or shaded grey if they do not. The dark grey “BLAST match to protists” shading indicates that these clades are comprised of OTUs that had GenBank matches to protist sequences with the OTU IDs in red. Asterisks indicate early diverging clades. SUPP TABLE 5 has a list of all OTUs included in the phylogeny along with their BLAST matches.
Figure 7. Maximum likelihood phylogenetic reconstruction of Zoopagomycota LSU sequences including references from GenBank and newly sequenced isolates from this study. OTUs identified as Zoopagomycota species from each of the LROR and LR22F datasets were included and the numbers are bolded and include the order to which each OTU was classified. Analyses were performed in RAxML v 8 using the GTR + GAMMA model and 1,000 bootstrap replicates. Branch supports ≥70 are shown.
Table 1. Zoopagomycota mock community members and results of mock community OTU taxonomy comparisons across the ITS1F/ITS2, LROR/LR3, and LR22F/LR3 primer sets, including the target fragment length in base pairs (bp), GenBank accession numbers (ITS, LSU), and the primer columns list the number of OTUs of each mock community member detected by the RDP classifier taxonomy (outside parentheses) versus the RDP+SILVA LSU database (for LSU) or the AMPtk hybrid method (for ITS1F) (in parentheses).