Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

Abstract

True fungi (Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

Sareomycetes: More Diversity than Meets the Eye

“…Finally, published nuITS sequences assignable to the Sareomycetes are variable at levels greater than the standard 3% threshold for species delimitation in fungi (Izzo et al 2005;Ciardo et al 2006;Blaalid et al 2013;Geml et al 2014;Gweon et al 2015), and greater than even the genus threshold (5.7% difference) suggested for lamentous fungi in a recent study (Vu et al 2019). While such thresholds are known to not be constant across kingdom Fungi (Nilsson et al 2008;Kõljalg et al 2013;Lücking et al 2020), this was particularly suggestive evidence of cryptic diversity.…”

Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations

“…Therefore, the taxonomic usefulness of this feature at genus level is unclear. Nevertheless, we consider the four-genus solution (in phylogenetic order: Trametes s.str., Artolenzites, Pycnoporus, Leiotrametes) a workable compromise at present, given the frequent lack of a clear, straightforward correlation between phenotype and phylogenetic relationships in many fungal lineages (Lücking & al. 2020).…”

“…2017;Lücking 2019). This renders their accurate identification based on phenotype often difficult or even impossible (Lücking & al. 2020).…”

“…In some cases, phenomena such as hybridization, introgression or gene duplication also complicate the use of the ITS barcoding marker (Lindner & Banki 2013;Li & al. 2013Lücking & al. 2020).…”

Accurate and sensitive detection of microbial eukaryotes from whole metagenome shotgun sequencing

“…As eukaryotic species do not have a 16S ribosomal RNA gene, 16S amplicon sequencing cannot detect them. In studies specifically targeting eukaryotes, amplicon sequencing of eukaryotic-specific (18S) or fungal-specific (ITS) genes are effective, but can have limited taxonomic resolution and often cannot disambiguate closely related species or strains (Schoch et al 2012;Lücking et al 2020). 18S or ITS sequencing is also performed less frequently than global shotgun methods of microbiome sequencing.…”