Future perspectives and conclusions
Metabarcoding analyses have recently opened new frontiers in many fields of mycology including assessment of functionality, integration into the entire microbiome analyses, detection of novel fungal taxa and ancient organisms and providing sequence data for taxonomically valuable specimens. Metabarcoding analyses using SIP of labelled substrates (Hannula et al., 2017) or coupled with metatranscriptomics (Zifcakova et al., 2016) and proteomics have revealed functionally active fungal organisms and their activity in situ. Parallel studies of fungi, bacteria and protists have revealed antagonistic interactions (Bork et al., 2015; Bahram et al., 2018), the structure of the micro- and mycobiome web (Tipton et al., 2018) and mechanisms of community assembly (Zinger et al., 2019b). HTS-derived data have revealed several groups of previously undescribed (or unsequenced) order- and class-level fungal lineages (Tedersoo et al., 2020b; Zhang et al., 2021). These reads offer material for constructing taxon-specific primers and probes for visualizing the cells (Chambouvet et al., 2019) and help discovery and characterisation of fungal dark matter. Increasing read length and precision of HTS methods enhance taxonomic precision and highlight a venue for population-level studies based on eDNA (Byrne et al., 2017; Turon et al., 2020). This may be of particular relevance to fungal taxa that are difficult to culture and form no fruit-bodies (Lücking et al., 2021). Metabarcoding of short fragments furthermore sheds light into ancient DNA including potentially ancient fungi (Balint et al., 2018; Talas et al., 2021), but methods distinguishing organisms representing ancient, recently dead and active organisms should be refined (e.g., metabarcoding along a marker length series, RNA vs. DNA approaches and analyses of chemically modified DNA fraction). Similarly, metabarcoding approaches are useful in generating DNA barcodes from century-old fruit-body specimens including valuable type material where extra care is needed to identify and dismiss air-borne contaminants (Forin et al., 2018).
Metabarcoding of fungi is a rapidly advancing research topic, which has benefited from initial methods and development in clinical microbiology and environmental microbiology - disciplines that are an order of magnitude larger. Thus, fungal ecologists have been able to learn from technological breakthroughs and pitfalls and develop unique solutions for analysis of the ITS marker, the present mainstay of fungal taxonomy and ecology. Through short-read metabarcoding, fungal diversity has been analysed in nearly all habitats on Earth, including extreme environments (Nilsson et al., 2018). We hope that with the assistance of growing reference databases, studies involving fungal taxonomic, phylogenetic and functional composition will flourish, because many principal aspects in evolutionary and functional (e.g., trait-based) ecology remain poorly known so far. These studies should be supplemented by rigorous experiments to validate the findings and infer causality. It somehow seems unacceptable that studies on one of the most important groups of nutrient cyclers - the fungi - be based on anything but the best and most up-to-date methodological recommendations, and we sincerely hope that this review has contributed to that effect.