Leho Tedersoo

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.

Global diversity and geography of soil fungi

Introduction The kingdom Fungi is one of the most diverse groups of organisms on Earth, and they are integral ecosystem agents that govern soil carbon cycling, plant nutrition, and pathology. Fungi are widely distributed in all terrestrial ecosystems, but the distribution of species, phyla, and functional groups has been poorly documented. On the basis of 365 global soil samples from natural ecosystems, we determined the main drivers and biogeographic patterns of fungal diversity and community composition. Direct and indirect effects of climatic and edaphic variables on plant and fungal richness. Line thickness corresponds to the relative strength of the relationships between the variables that affect species richness. Dashed lines indicate negative relationships. MAP, mean annual precipitation; Fire, time since last fire; Dist. equator, distance from the equator; Ca, soil calcium concentration; P, soil phosphorus concentration; pH, soil pH. Rationale We identified soil-inhabiting fungi using 454 Life Sciences (Branford, CN) pyrosequencing and through comparison against taxonomically and functionally annotated sequence databases. Multiple regression models were used to disentangle the roles of climatic, spatial, edaphic, and floristic parameters on fungal diversity and community composition. Structural equation models were used to determine the direct and indirect effects of climate on fungal diversity, soil chemistry, and vegetation. We also examined whether fungal biogeographic patterns matched paradigms derived from plants and animals—namely, that species’ latitudinal ranges increase toward the poles (Rapoport’s rule) and diversity increases toward the equator. Last, we sought group-specific global biogeographic links among major biogeographic regions and biomes using a network approach and area-based clustering. Results Metabarcoding analysis of global soils revealed fungal richness estimates approaching the number of species recorded to date. Distance from equator and mean annual precipitation had the strongest effects on richness of fungi, including most fungal taxonomic and functional groups. Diversity of most fungal groups peaked in tropical ecosystems, but ectomycorrhizal fungi and several fungal classes were most diverse in temperate or boreal ecosystems, and many fungal groups exhibited distinct preferences for specific edaphic conditions (such as pH, calcium, or phosphorus). Consistent with Rapoport’s rule, the geographic range of fungal taxa increased toward the poles. Fungal endemicity was particularly strong in tropical regions, but multiple fungal taxa had cosmopolitan distribution. Conclusions Climatic factors, followed by edaphic and spatial patterning, are the best predictors of soil fungal richness and community composition at the global scale. Richness of all fungi and functional groups is causally unrelated to plant diversity, with the exception of ectomycorrhizal root symbionts, suggesting that plant-soil feedbacks do not influence the diversity of soil fungi at the global scale. The plant-to-fungi richness ratio declined exponentially toward the poles, indicating that current predictions—assuming globally constant ratios—overestimate fungal richness by 1.5- to 2.5-fold. Fungi follow similar biogeographic patterns as plants and animals, with the exception of several major taxonomic and functional groups that run counter to overall patterns. Strong biogeographic links among distant continents reflect relatively efficient long-distance dispersal compared with macro-organisms. Fungi play major roles in ecosystem processes, but the determinants of fungal diversity and biogeographic patterns remain poorly understood. Using DNA metabarcoding data from hundreds of globally distributed soil samples, we demonstrate that fungal richness is decoupled from plant diversity. The plant-to-fungus richness ratio declines exponentially toward the poles. Climatic factors, followed by edaphic and spatial variables, constitute the best predictors of fungal richness and community composition at the global scale. Fungi show similar latitudinal diversity gradients to other organisms, with several notable exceptions. These findings advance our understanding of global fungal diversity patterns and permit integration of fungi into a general macroecological framework. Global metagenomics detects hotspots of fungal diversity and macroecological patterns and indicates that plant and fungal diversity are uncoupled. [Also see Perspective by Wardle and Lindahl] Assessing fungal diversity worldwide Fungi are hyperdiverse but poorly known, despite their ecological and economic impacts. Tedersoo et al. collected nearly 15,000 topsoil samples from 365 sites worldwide and sequenced their genomes (see the Perspective by Wardle and Lindahl). Overall, they found a striking decline in fungal species richness with distance from the equator. For some specialist groups though, diversity depended more on the abundance of host plants than host diversity or geography. The findings reveal a huge gap between known and described species and the actual numbers of distinct fungi in the worlds soils. Science, this issue 10.1126/science.1256688; see also p. 1052

Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages

The ectomycorrhizal (EcM) symbiosis involves a large number of plant and fungal taxa worldwide. During studies on EcM diversity, numerous misidentifications, and contradictory reports on EcM status have been published. This review aims to: (1) critically assess the current knowledge of the fungi involved in the EcM by integrating data from axenic synthesis trials, anatomical, molecular, and isotope studies; (2) group these taxa into monophyletic lineages based on molecular sequence data and published phylogenies; (3) investigate the trophic status of sister taxa to EcM lineages; (4) highlight other potentially EcM taxa that lack both information on EcM status and DNA sequence data; (5) recover the main distribution patterns of the EcM fungal lineages in the world. Based on critically examining original reports, EcM lifestyle is proven in 162 fungal genera that are supplemented by two genera based on isotopic evidence and 52 genera based on phylogenetic data. Additionally, 33 genera are highlighted as potentially EcM based on habitat, although their EcM records and DNA sequence data are lacking. Molecular phylogenetic and identification studies suggest that EcM symbiosis has arisen independently and persisted at least 66 times in fungi, in the Basidiomycota, Ascomycota, and Zygomycota. The orders Pezizales, Agaricales, Helotiales, Boletales, and Cantharellales include the largest number of EcM fungal lineages. Regular updates of the EcM lineages and genera therein can be found at the UNITE homepage http://unite.ut.ee/EcM_lineages. The vast majority of EcM fungi evolved from humus and wood saprotrophic ancestors without any obvious reversals. Herbarium records from 11 major biogeographic regions revealed three main patterns in distribution of EcM lineages: (1) Austral; (2) Panglobal; (3) Holarctic (with or without some reports from the Austral or tropical realms). The holarctic regions host the largest number of EcM lineages; none are restricted to a tropical distribution with Dipterocarpaceae and Caesalpiniaceae hosts. We caution that EcM-dominated habitats and hosts in South America, Southeast Asia, Africa, and Australia remain undersampled relative to the north temperate regions. In conclusion, EcM fungi are phylogenetically highly diverse, and molecular surveys particularly in tropical and south temperate habitats are likely to supplement to the present figures. Due to great risk of contamination, future reports on EcM status of previously unstudied taxa should integrate molecular identification tools with axenic synthesis experiments, detailed morphological descriptions, and/or stable isotope investigations. We believe that the introduced lineage concept facilitates design of biogeographical studies and improves our understanding about phylogenetic structure of EcM fungal communities.

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.

Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers.

Ectomycorrhizal (ECM) symbiosis is a widespread plant nutrition strategy in Australia, especially in semiarid regions. This study aims to determine the diversity, community structure and host preference of ECM fungi in a Tasmanian wet sclerophyll forest. Ectomycorrhizal fungi were identified based on anatomotyping and rDNA internal transcribed spacer (ITS)-large subunit (LSU) sequence analysis using taxon-specific primers. Host tree roots were identified based on root morphology and length differences of the chloroplast trnL region. A total of 123 species of ECM fungi were recovered from root tips of Eucalyptus regnans (Myrtaceae), Pomaderris apetala (Rhamnaceae) and Nothofagus cunninghamii (Nothofagaceae). The frequency of two thirds of the most common ECM fungi from several lineages was significantly influenced by host species. The lineages of Cortinarius, Tomentella-Thelephora, Russula-Lactarius, Clavulina, Descolea and Laccaria prevailed in the total community and their species richness and relative abundance did not differ by host species. This study demonstrates that strongly host-preferring, though not directly specific, ECM fungi may dominate the below-ground community. Apart from the richness of Descolea, Tulasnella and Helotiales and the lack of Suillus-Rhizopogon and Amphinema-Tylospora, the ECM fungal diversity and phylogenetic community structure is similar to that in the Holarctic realm.

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.

PlutoF—a Web Based Workbench for Ecological and Taxonomic Research, with an Online Implementation for Fungal ITS Sequences

DNA sequences accumulating in the International Nucleotide Sequence Databases (INSD) form a rich source of information for taxonomic and ecological meta-analyses. However, these databases include many erroneous entries, and the data itself is poorly annotated with metadata, making it difficult to target and extract entries of interest with any degree of precision. Here we describe the web-based workbench PlutoF, which is designed to bridge the gap between the needs of contemporary research in biology and the existing software resources and databases. Built on a relational database, PlutoF allows remote-access rapid submission, retrieval, and analysis of study, specimen, and sequence data in INSD as well as for private datasets though web-based thin clients. In contrast to INSD, PlutoF supports internationally standardized terminology to allow very specific annotation and linking of interacting specimens and species. The sequence analysis module is optimized for identification and analysis of environmental ITS sequences of fungi, but it can be modified to operate on any genetic marker and group of organisms. The workbench is available at http://plutof.ut.ee.

Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae.

Several forest understorey achlorophyllous plants, termed mycoheterotrophs (MHs), obtain C from their mycorrhizal fungi. The latter in turn form ectomycorrhizas with trees, the ultimate C source of the entire system. A similar nutritional strategy occurs in some green forest orchids, phylogenetically close to MH species, that gain their C via a combination of MH and photosynthesis (mixotrophy). In orchid evolution, mixotrophy evolved in shaded habitats and preceded MH nutrition. By generalizing and applying this to Ericaceae, we hypothesized that green forest species phylogenetically close to MHs are mixotrophic. Using stable C isotope analysis with fungi, autotrophic, mixotrophic and MH plants as comparisons, we found the first quantitative evidence for substantial fungi-mediated mixotrophy in the Pyroleae, common ericaceous shrubs from boreal forests close to the MH Monotropoideae. Orthilia secunda, Pyrola chlorantha, Pyrola rotundifolia and Chimaphila umbellata acquired between 10.3 and 67.5% of their C from fungi. High N and 15N contents also suggest that Pyroleae nutrition partly rely on fungi. Examination of root fungal internal transcribed spacer sequences at one site revealed that 39 species of mostly endophytic or ectomycorrhizal fungi, including abundant Tricholoma spp., were associated with O. secunda, P. chlorantha and C. umbellata. These fungi, particularly ectomycorrhizal associates, could thus link mixotrophic Pyroleae spp. to surrounding trees, allowing the C flows deduced from isotopic evidence. These data suggest that we need to reconsider ecological roles of understorey plants, which could influence the dynamics and composition of forest communities.

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.

Species abundance distributions and richness estimations in fungal metagenomics – lessons learned from community ecology

Results of diversity and community ecology studies strongly depend on sampling depth. Completely surveyed communities follow log‐normal distribution, whereas power law functions best describe incompletely censused communities. It is arguable whether the statistics behind those theories can be applied to voluminous next generation sequencing data in microbiology by treating individual DNA sequences as counts of molecular taxonomic units (MOTUs). This study addresses the suitability of species abundance models in three groups of plant‐associated fungal communities – phyllosphere, ectomycorrhizal and arbuscular mycorrhizal fungi. We tested the impact of differential treatment of molecular singletons on observed and estimated species richness and species abundance distribution models. The arbuscular mycorrhizal community of 48 MOTUs was exhaustively sampled and followed log‐normal distribution. The ectomycorrhizal (153 MOTUs) and phyllosphere (327 MOTUs) communities significantly differed from log‐normal distribution. The fungal phyllosphere community in particular was clearly undersampled. This undersampling bias resulted in strong sensitivity to the exclusion of molecular singletons and other rare MOTUs that may represent technical artefacts. The analysis of abundant (core) and rare (satellite) MOTUs clearly identified two species abundance distributions in the phyllosphere data – a log‐normal model for the core group and a log‐series model for the satellite group. The prominent log‐series distribution of satellite phyllosphere fungi highlighted the ecological significance of an infrequent fungal component in the phyllosphere community.