Urmas Kõljalg

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third‐party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web‐based sequence management system in UNITE.

Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide

Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.

Regional and local patterns of ectomycorrhizal fungal diversity and community structure along an altitudinal gradient in the Hyrcanian forests of northern Iran

• Altitudinal gradients strongly affect the diversity of plants and animals, yet little is known about the altitudinal effects on the distribution of microorganisms, including ectomycorrhizal fungi. • By combining morphological and molecular identification methods, we addressed the relative effects of altitude, temperature, precipitation, host community and soil nutrient concentrations on species richness and community composition of ectomycorrhizal fungi in one of the last remaining temperate old-growth forests in Eurasia. • Molecular analyses revealed 367 species of ectomycorrhizal fungi along three altitudinal transects. Species richness declined monotonically with increasing altitude. Host species and altitude were the main drivers of the ectomycorrhizal fungal community composition at both the local and regional scales. The mean annual temperature and precipitation were strongly correlated with altitude and accounted for the observed patterns of richness and community. • The decline of ectomycorrhizal fungal richness with increasing altitude is consistent with the general altitudinal richness patterns of macroorganisms. Low environmental energy reduces the competitive ability of rare species and thus has a negative effect on the richness of ectomycorrhizal fungi. Because of multicollinearity with altitude, the direct effects of climatic variables and their seasonality warrant further investigation at the regional and continental scales.

Biogeography of ectomycorrhizal fungi associated with alders (Alnus spp.) in relation to biotic and abiotic variables at the global scale

· Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa. · This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods. · Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness. · Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.

Enzymatic activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and exploration types in an afrotropical rain forest

Ectomycorrhizal (ECM) fungi obtain both mineral and simple organic nutrients from soil and transport these to plant roots. Natural abundance of stable isotopes (¹⁵N and ¹³C) in fruit bodies and potential enzymatic activities of ECM root tips provide insights into mineral nutrition of these mutualistic partners. By combining rDNA sequence analysis with enzymatic and stable isotope assays of root tips, we hypothesized that phylogenetic affinities of ECM fungi are more important than ECM exploration type, soil horizon and host plant in explaining the differences in mineral nutrition of trees in an African lowland rainforest. Ectomycorrhizal fungal species belonging to extraradical mycelium-rich morphotypes generally displayed the strongest potential activities of degradation enzymes, except for laccase. The signature of ¹⁵N was determined by the ECM fungal lineage, but not by the exploration type. Potential enzymatic activities of root tips were unrelated to ¹⁵N signature of ECM root tip. The lack of correlation suggests that these methods address different aspects in plant nutrient uptake. Stable isotope analysis of root tips could provide an additional indirect assessment of fungal and plant nutrition that enables enhancement of taxonomic coverage and control for soil depth and internal nitrogen cycling in fungal tissues.

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

Spatial structure and the effects of host and soil environments on communities of ectomycorrhizal fungi in wooded savannas and rain forests of Continental Africa and Madagascar

Mycorrhizal fungi play a key role in mineral nutrition of terrestrial plants, but the factors affecting natural distribution, diversity and community composition of particularly tropical fungi remain poorly understood. This study addresses shifts in community structure and species frequency of ectomycorrhizal (EcM) fungi in relation to host taxa, soil depth and spatial structure in four contrasting African ecosystems. We used the rDNA and plastid trnL intron sequence analysis for identification of fungi and host plants, respectively. By partitioning out spatial autocorrelation in plant and fungal distribution, we suggest that African EcM fungal communities are little structured by soil horizon and host at the plant species and family levels. These findings contrast with patterns of vegetation in these forests and EcM fungal communities in other tropical and temperate ecosystems. The low level of host preference indirectly supports an earlier hypothesis that pioneer Phyllanthaceae may facilitate the establishment of late successional Fabaceae and potentially other EcM host trees by providing compatible fungal inoculum in deforested and naturally disturbed ecosystems of tropical Africa.

Towards standardization of the description and publication of next‐generation sequencing datasets of fungal communities

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The distance decay of similarity in communities of ectomycorrhizal fungi in different ecosystems and scales

Summary 1. Despite recent advances in understanding community ecology of ectomycorrhizal fungi, little is known about their spatial patterning and the underlying mechanisms driving these patterns across different ecosystems. 2. This meta-study aimed to elucidate the scale, rate and causes of spatial structure of ectomycorrhizal fungal communities in different ecosystems by analysing 16 and 55 sites at the local and global scales, respectively. We examined the distance decay of similarity relationship in species- and phylogenetic lineage-based communities in relation to sampling and environmental variables. 3. Tropical ectomycorrhizal fungal communities exhibited stronger distance-decay patterns compared to non-tropical communities. Distance from the equator and sampling area were the main determinants of the extent of distance decay in fungal communities. The rate of distance decay was negatively related to host density at the local scale. At the global scale, lineage-level community similarity decayed faster with latitude than with longitude. 4. Synthesis. Spatial processes play a stronger role and over a greater scale in structuring local communities of ectomycorrhizal fungi than previously anticipated, particularly in ecosystems with greater vegetation age and closer to the equator. Greater rate of distance decay occurs in ecosystems with lower host density that may stem from increasing dispersal and establishment limitation. The relatively strong latitude effect on distance decay of lineage-level community similarity suggests that climate affects large-scale spatial processes and may cause phylogenetic clustering of ectomycorrhizal fungi at the global scale.