Limitations of common molecular markers in fungal biodiversity analysis and the benefits of their synergistic use

High-throughput sequencing of the Internal Transcribed Spacer (ITS) regions is the primary method for estimating fungal diversity from environmental DNA. However, reliance solely on ITS markers is complicated by its high variability in sequence length and the presence of multiple variants within a single genome, which can bias diversity estimates. The core objective of the dataset is to provide a comparative analysis of the ITS regions (ITS1 and ITS2) against five alternative markers: Mitochondrial Large Subunit ribosomal DNA (mtLSU), the second largest subunit of RNA polymerase II (rpb2a and rpb2b regions), the translation elongation factor 1-alpha (ef1-α), and the minichromosome maintenance complex component 7 (mcm7). The data comprises raw Illumina MiSeq amplicon sequences (FASTQ format) derived from a mock community of 413 fungal species belonging to Ascomycota, Basidiomycota, Mucoromycota, and Zoopagomycota. It also includes manually curated reference sequences (FASTA format) ..., , This dataset supports the study titled \"Limitations of common molecular markers in fungal biodiversity analysis and the benefits of their synergistic use\", which evaluates the performance of seven molecular markers for fungal metabarcoding ([https://doi.org/10.1111/1755-0998.70123](https://doi.org/10.1111/1755-0998.70123)).

# Mock community

The mock community consisted of an equimolar mixture of genomic DNA extracted from 676 fungal specimens obtained either from pure cultures (613) or field collections (63). All these fungi belonged to the group Eumycota: 103 Ascomycota strains, 554 Basidiomycota strains, 18 Mucoromycota strains and 1 Zoopagomycota strain, corresponding to 413 different species, 202 genera, 96 families, 32 orders and 11 classes. Altogether, 71 genera were represented by several species, and 125 species were represented by more than one specimen.

All fungal specimens were identified based on morphology and full-length ITS region sequences. We received biological mate..., ,