DOI: 10.5061/dryad.g79cnp5t7
title: Extensive intragenomic variation in the internal transcribed spacer (ITS) region of fungi

description: Fungi are among the most biodiverse organisms in the world. Accurate
 species identification is imperative for studies on fungal ecology and
 evolution. The internal transcribed spacer (ITS) rDNA region has been
 widely accepted as the universal barcode for fungi. However, several
 recent studies have uncovered intragenomic sequence variation within the
 ITS in multiple fungal species. Here, we mined the genome of 2414 fungal
 species to determine the prevalence of intragenomic variation and found
 that the genomes of 641 species, about one-quarter of the 2414 species
 examined, contained multiple ITS copies. Of those 641 species, 419 (~65%)
 contained variation among copies revealing that intragenomic variation is
 common in fungi. We proceeded to show how these copies could result in the
 erroneous description of hundreds of fungal species and skew studies
 evaluating eDNA especially when making diversity estimates. Additionally,
 many genomes were found to be contaminated, especially those of
 unculturable fungi.
description: Data were mined from at least one genome assembly of every fungal
 species from September-December of 2021 on GenBank. Data mining was
 accomplished by extracting the multiple ITS copies from a given assembly
 and then aligning and analyzing the extracted copies for variation.
 Detailed methods are as follows: (1) A list of all taxa
 with publicly available assemblies was compiled. (2)
 For each taxon, GenBank’s nucleotide database was searched for a fully
 annotated ITS region. (3) The GenBank accession number
 determined from (2) was GenBank blasted (blastn) to ensure the taxon was
 identified correctly. (4) The ITS region from (2) was
 trimmed to include only nucleotides present in the ITS1+5.8S+ITS2
 region. (5) A genome assembly was chosen for each
 fungal species on GenBank. If multiple assemblies for a given taxon were
 available, the assembly with the smallest number of scaffolds/contigs was
 evaluated first. (6) A genome assembly was GenBank
 blasted (blastn) with the trimmed ITS region. For example, in
 Supplementary Dataset 1, column A (‘Assembly Reference’) was GenBank
 blasted with column E (‘GenBank Accession Number of ITS Region used to
 blast assembly’); if no ITS region was located or if it was very
 fragmented other assemblies were checked. (7) The
 results of the assembly blast were downloaded into Geneious version
 2021.2.2 and aligned. (8) ITS copies from the genome
 assembly that were ~ &gt;50 bases shorter than the length of the ITS
 region determined in step (4) were discarded to eliminate short contigs
 and to keep the data consistent. (9) Alignments for
 these taxa are available here in both a .geneious and .fasta file
 format. (10) The number of ITS copies in the assembly,
 identical site % and pairwise identity % among the different copies were
 calculated in Geneious and recorded. (11) The ITS
 accessions used to blast the assemblies were downloaded into Geneious and
 their GC content was recorded. (12) The remaining data
 from the assemblies were recorded from GenBank (Taxa ID, assembly method,
 sequencing technology used, genome coverage, contigs, scaffolds, assembly
 GC content (%), assembly release date, and genome size).
description: Geneious or any .fasta file reader