Clustering of fungal community internal transcribed spacer sequence data obscures taxonomic diversity

Summary Next‐generation DNA sequencing has enabled a rapid expansion in the size of molecular fungal ecology studies employing the nuclear internal transcribed spacer ( ITS ) region. Many sequence‐processing pipelines and protocols require sequence clustering to generate operational taxonomic units ( OTUs ) based on sequence similarity as a step to reduce total data quantity and complexity prior to taxonomic assignment. However, the consequences of ITS sequence clustering in regard to sample taxonomic coverage have not been carefully examined. Here we demonstrate that typically used clustering thresholds for fungal ITS sequences result in statistically significant losses in taxonomic coverage. Analyses using environmentally derived fungal sequences indicated an average of 3.1% of species went undetected ( P  <   0.05) if the sequences were denoised and clustered at