Kessy Abarenkov

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

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.

Taxonomic Reliability of DNA Sequences in Public Sequence Databases: A Fungal Perspective

Background DNA sequences are increasingly seen as one of the primary information sources for species identification in many organism groups. Such approaches, popularly known as barcoding, are underpinned by the assumption that the reference databases used for comparison are sufficiently complete and feature correctly and informatively annotated entries. Methodology/Principal Findings The present study uses a large set of fungal DNA sequences from the inclusive International Nucleotide Sequence Database to show that the taxon sampling of fungi is far from complete, that about 20% of the entries may be incorrectly identified to species level, and that the majority of entries lack descriptive and up-to-date annotations. Conclusions The problems with taxonomic reliability and insufficient annotations in public DNA repositories form a tangible obstacle to sequence-based species identification, and it is manifest that the greatest challenges to biological barcoding will be of taxonomical, rather than technical, nature.

Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data

Summary 1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

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.

The ITS region as a target for characterization of fungal communities using emerging sequencing technologies

The advent of new high-throughput DNA-sequencing technologies promises to redefine the way in which fungi and fungal communities--as well as other groups of organisms--are studied in their natural environment. With read lengths of some few hundred base pairs, massively parallel sequencing (pyrosequencing) stands out among the new technologies as the most apt for large-scale species identification in environmental samples. Although parallel pyrosequencing can generate hundreds of thousands of sequences at an exceptional speed, the limited length of the reads may pose a problem to the species identification process. This study explores whether the discrepancy in read length between parallel pyrosequencing and traditional (Sanger) sequencing will have an impact on the perceived taxonomic affiliation of the underlying species. Based on all 39,200 publicly available fungal environmental DNA sequences representing the nuclear ribosomal internal transcribed spacer (ITS) region, the results show that the two approaches give rise to quite different views of the diversity of the underlying samples. Standardization of which subregion from the ITS region should be sequenced, as well as a recognition that the composition of fungal communities as depicted through different sequencing methods need not be directly comparable, appear crucial to the integration of the new sequencing technologies with current mycological praxis.

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.

ITS1 versus ITS2 as DNA metabarcodes for fungi

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.

Resistance and resilience of the forest soil microbiome to logging-associated compaction

Soil compaction is a major disturbance associated with logging, but we lack a fundamental understanding of how this affects the soil microbiome. We assessed the structural resistance and resilience of the microbiome using a high-throughput pyrosequencing approach in differently compacted soils at two forest sites and correlated these findings with changes in soil physical properties and functions. Alterations in soil porosity after compaction strongly limited the air and water conductivity. Compaction significantly reduced abundance, increased diversity, and persistently altered the structure of the microbiota. Fungi were less resistant and resilient than bacteria; clayey soils were less resistant and resilient than sandy soils. The strongest effects were observed in soils with unfavorable moisture conditions, where air and water conductivities dropped well below 10% of their initial value. Maximum impact was observed around 6–12 months after compaction, and microbial communities showed resilience in lightly but not in severely compacted soils 4 years post disturbance. Bacteria capable of anaerobic respiration, including sulfate, sulfur, and metal reducers of the Proteobacteria and Firmicutes, were significantly associated with compacted soils. Compaction detrimentally affected ectomycorrhizal species, whereas saprobic and parasitic fungi proportionally increased in compacted soils. Structural shifts in the microbiota were accompanied by significant changes in soil processes, resulting in reduced carbon dioxide, and increased methane and nitrous oxide emissions from compacted soils. This study demonstrates that physical soil disturbance during logging induces profound and long-lasting changes in the soil microbiome and associated soil functions, raising awareness regarding sustainable management of economically driven logging operations.