Partner specificity is a well-known phenomenon in biotic interactions, but little is known about biotic and abiotic factors that determine specificity in plant-fungal associations. Using PacBio sequencing of soils from monospecific and mixed forest stands, we determined the predictors driving partner specificity in both ectomycorrhizal plants and fungi. Fungal guilds differed strongly in the patterns of partner preference and avoidance, and specificity to particular tree genera. Specialist ectomycorrhizal fungi dominated in belowground communities, and most species preferred one of their partner trees - mostly at the plant genus level. Furthermore, all tree genera (sometimes species) displayed preference towards certain fungal groups. Partner specificity was unrelated to rarity of fungi or plants or environmental conditions except soil pH. Depending on partner taxon, specificity in fungi tended to increase with dominance and optimal pH of the partner tree genus and stand age. Partner tree richness and increased evenness of ectomycorrhizal fungi in multi-host communities promotes species richness. However, mainly partner-generalist fungi contribute to the high diversity in mixed forests. Our results further suggest that reforestation with mixed tree species promotes soil biodiversity, and that besides conserving mixed forests, protection of old pure stands may be particularly important for conserving partner-specific ectomycorrhizal fungi.

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety of ecosystems. Here we provide an overview about current best practices in metabarcoding analysis of fungal communities, from experimental design through molecular and computational analyses. By re-analysing published datasets, we find that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) in recovering fungal diversity, which is particularly evident for long markers. Additionally, analysis of the full-length ITS region allows more accurate taxonomic placement of fungi and other eukaryotes compared with the ITS2 subregion. We conclude that metabarcoding analyses of fungi are especially promising for co-analyses with the functional metagenomic or transcriptomic data, integrating fungi in the entire microbiome, recovery of novel fungal lineages and ancient organisms as well as barcoding of old specimens including type material.

Fungi form diverse communities and play essential roles in many terrestrial ecosystems, yet there are methodological challenges in taxonomic and phylogenetic placement of fungi from environmental sequences. To address such challenges we investigated spatio-temporal structure of a fungal community using soil metabarcoding with four different sequencing strategies: short amplicon sequencing of the ITS2 region (300–400\ bp) with Illumina MiSeq, Ion Torrent Ion S5, and PacBio RS II, all from the same PCR library, as well as long amplicon sequencing of the full ITS and partial LSU regions (1200–1600\ bp) with PacBio RS II. Resulting community structure and diversity depended more on statistical method than sequencing technology. The use of long-amplicon sequencing enables construction of a phylogenetic tree from metabarcoding reads, which facilitates taxonomic identification of sequences. However, long reads present issues for denoising algorithms in diverse communities. We present a solution that splits the reads into shorter homologous regions prior to denoising, and then reconstructs the full denoised reads. In the choice between short and long amplicons, we suggest a hybrid approach using short amplicons for sampling breadth and depth, and long amplicons to characterize the local species pool for improved identification and phylogenetic analyses.

Fungi form diverse communities and play essential roles in many terrestrial ecosystems, yet there are methodological challenges in taxonomic and phylogenetic placement of fungi from environmental sequences. To address such challenges we investigated spatio-temporal structure of a fungal community using soil metabarcoding with four different sequencing strategies: short amplicon sequencing of the ITS2 region (300-400 bp) with Illumina MiSeq, Ion Torrent Ion S5, and PacBio RS II, as well as long amplicon sequencing of the full ITS and partial LSU regions (1200-1600 bp) with PacBio RS II. Resulting community structure and diversity depended more on statistical method than sequencing technology. The use of long-amplicon sequencing enables construction of a phylogenetic tree from metabarcoding reads, which facilitates taxonomic identification of sequences. However, long reads present issues for denoising algorithms in diverse communities. We present a solution that splits the reads into shorter homologous regions prior to denoising, and then reconstructs the full denoised reads. In the choice between short and long amplicons, we suggest a hybrid approach using short amplicons for sampling breadth and depth, and long amplicons to characterize the local species pool for improved identification and phylogenetic analyses.