*3.7. Intragenomic ITS Variation*

Our side-by-side comparison of the same samples sequenced with Illumina and Sanger sequencing yielded 89 specimens for this analysis. However, for 14 of these samples, the regions sequenced were non-overlapping, with less than 50 bp of overlap, or of low quality and, therefore, not included.

For the rest, or 75 samples, 55 (73%) of the Sanger sequence did not show any ambiguity, 11 (15%) showed one ambiguity (=fixed allele), while nine (12%) showed two or more ambiguities (Figure 12a). As for the ASVs, 290 ASVs were detected for these 75 samples, ranging from 1 to 11 per sample. About 30% of the ASVs matched the Sanger sequence exactly (=0 singletons), while most ASVs (61%) presented only one different base pair in comparison to the Sanger sequence (=1 singleton) (Figure 12b). Most samples produced multiple ASVs, but were usually dominated by one or two haplotypes.

**Figure 12.** ITS1 variation observed within specimens. The fixed alleles graph (**a**) shows the percentages in which Sanger sequences presented ambiguities (0–7) in the ITS1 (e.g., an "R" whereas the Illumina reads showed either an "A" or "G", the majority showing none). The singleton alleles graph (**b**) shows the percentage of ASVs in which Illumina reads differed from the Sanger sequences by 0–7 base pairs aside from the fixed alleles (e.g., 61% of all ASVs showed 1 bp different then the corresponding Sanger sequence they were being compared to).
