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.