The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut-off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut-off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under- or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.

We investigated changes in the root-associated fungal communities associated with the ectomycorrhizal herb Bistorta vivipara along a primary succession gradient using 454 amplicon sequencing. Our main objective was to assess the degree of variation in fungal richness and community composition as vegetation cover increases along the chronosequence. Sixty root systems of B. vivipara were sampled in vegetation zones delimited by dated moraines in front of a retreating glacier in Norway. We extracted DNA from rinsed root systems, amplified the ITS1 region using fungal-specific primers and analysed the amplicons using 454 sequencing. Between 437 and 5063 sequences were obtained from each root system. Clustering analyses using a 98.5% sequence similarity cut-off yielded a total of 470 operational taxonomic units (OTUs), excluding singletons. Between eight and 41 fungal OTUs were detected within each root system. Already in the first stage of succession, a high fungal diversity was present in the B. vivipara root systems. Total number of OTUs increased significantly along the gradient towards climax vegetation, but the average number of OTUs per root system stayed unchanged. There was a high patchiness in distribution of fungal OTUs across root systems, indicating that stochastic processes to a large extent structure the fungal communities. However, time since deglaciation had impact on the fungal community structure, as a systematic shift in the community composition was observed along the chronosequence. Ectomycorrhizal basidiomycetes were the dominant fungi in the roots of B. vivipara, when it comes to both number of OTUs and number of sequences.

There is growing evidence that root-associated fungi have important roles in Arctic ecosystems. Here, we assess the diversity of fungal communities associated with roots of the ectomycorrhizal perennial herb Bistorta vivipara on the Arctic archipelago of Svalbard and investigate whether spatial separation and bioclimatic variation are important structuring factors of fungal community composition. We sampled 160 plants of B. vivipara from 32 localities across Svalbard. DNA was extracted from entire root systems, and 454 pyrosequencing of ITS1 amplicons was used to profile the fungal communities. The fungal communities were predominantly composed of Basidiomycota (55% of reads) and Ascomycota (35%), with the orders Thelephorales (24%), Agaricales (13.8%), Pezizales (12.6%) and Sebacinales (11.3%) accounting for most of the reads. Plants from the same site or region had more similar fungal communities to one another than plants from other sites or regions, and sites clustered together along a weak latitudinal gradient. Furthermore, a decrease in per-plant OTU richness with increasing latitude was observed. However, no statistically significant spatial autocorrelation between sites was detected, suggesting that environmental filtering, not dispersal limitation, causes the observed patterns. Our analyses suggest that while latitudinal patterns in community composition and richness might reflect bioclimatic influences at global spatial scales, at the smaller spatial scale of the Svalbard archipelago, these changes more likely reflect varied bedrock composition and associated edaphic factors. The need for further studies focusing on identifying those specific bioclimatic and edaphic factors structuring root-associated fungal community composition at both global and local scales is emphasized.

Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis.

Glacier chronosequences are important sites for primary succession studies and have yielded well-defined primary succession models for plants that identify environmental resistance as an important determinant of the successional trajectory. Whether plant-associated fungal communities follow those same successional trajectories and also respond to environmental resistance is an open question. In this study, 454 amplicon pyrosequencing was used to compare the root-associated fungal communities of the ectomycorrhizal (ECM) herb Bistorta vivipara along two primary succession gradients with different environmental resistance (alpine versus arctic) and different successional trajectories in the vascular plant communities (directional replacement versus directional non-replacement). At both sites, the root-associated fungal communities were dominated by ECM basidiomycetes and community composition shifted with increasing time since deglaciation. However, the fungal community's successional trajectory mirrored the pattern observed in the surrounding plant community at both sites: the alpine site displayed a directional-replacement successional trajectory, and the arctic site displayed a directional-non-replacement successional trajectory. This suggests that, like in plant communities, environmental resistance is key in determining succession patterns in root-associated fungi. The need for further replicated study, including in other host species, is emphasized.