ITS alchemy: On the use of ITS as a DNA marker in fungal ecology

High throughput sequencing of PCR amplicons derived from environmental DNA (aka DNA metabarcoding) has become an integral part of fungal ecology, enabling in-depth characterization of fungal communities. In most cases, the rDNA Internal Transcribed Spacer (ITS) region, which has a long history as a target in fungal systematics, is used as a DNA barcode marker. Despite improvements in sequencing techniques and bioinformatics approaches, there are inherent limitations associated with the use of a single-locus DNA marker that are often ignored. In this text, I discuss both inherent biological and methodological limitations associated with the use of the ITS marker. For example, proper species delimitation is often not possible with a single marker, and a significant DNA barcoding gap (i.e. interspecific divergence) is often missing between sister taxa in ITS. Further, we can rarely be fully confident about the assigned species-level taxonomy based on available reference sequences. In addition to the inherent limitations, an extra layer of complexity and variation is blended into DNA metabarcoding data due to PCR and sequencing errors that may look similar to natural molecular variation. The bioinformatics processing of ITS amplicons must take into account both the basic properties of the ITS region, as well as the generated errors and biases. In this regard, we cannot adopt approaches and settings from other markers, such as 16S and 18S, blindly. For example, due to intraspecific variability in the ITS region, and sometimes intragenomic variability, ITS sequences must be clustered to approach species level resolution in community studies. Therefore, I argue that the concept of amplicon sequence variants (ASVs) is not applicable. Although the ITS region is by far the best option as a general DNA (meta)barcoding marker for fungi, this contribution is meant to remind against a naive or simplistic use of the ITS region, and for stimulating further discussions.