The influence of tree genus, phylogeny, and richness on the specificity, rarity, and diversity of ectomycorrhizal fungi.

Partner specificity is a well-documented phenomenon in biotic interactions, yet the factors that determine specificity in plant-fungal associations remain largely unknown. By utilizing composite soil samples, we identified the predictors that drive partner specificity in both plants and fungi, with a particular focus on ectomycorrhizal associations. Fungal guilds exhibited significant differences in overall partner preference and avoidance, richness, and specificity to specific tree genera. The highest level of specificity was observed in root endophytic and ectomycorrhizal associations, while the lowest was found in arbuscular mycorrhizal associations. The majority of ectomycorrhizal fungal species showed a preference for one of their partner trees, primarily at the plant genus level. Specialist ectomycorrhizal fungi were dominant in belowground communities in terms of species richness and relative abundance. Moreover, all tree genera (and occasionally species) demonstrated a preference for certain fungal groups. Partner specificity was not related to the rarity of fungi or plants or environmental conditions, except for soil pH. Depending on the partner tree genus, specific fungi became more prevalent and relatively more abundant with increasing stand age, tree dominance, and soil pH conditions optimal for the partner tree genus. The richness of partner tree species and increased evenness of ectomycorrhizal fungi in multi-host communities enhanced the species richness of ectomycorrhizal fungi. However, it was primarily the partner-generalist fungi that contributed to the high diversity of ectomycorrhizal fungi in mixed forests.

The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

How, not if, is the question mycologists should be asking about DNA-based typification.

Fungal metabarcoding of substrates such as soil, wood, and water is uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside the scope of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings challenge the present stance of some in the mycological community - that the current situation is satisfactory and that no change is needed to "the code" - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. The present authors hope for a revitalized and deepened discussion on DNA-based typification, because to us it seems harmful and counter-productive to intentionally deny the overwhelming majority of extant fungi a formal standing under the International Code of Nomenclature for algae, fungi, and plants.

Host generalists dominate fungal communities associated with alpine knotweed roots: a study of Sebacinales.

Bistorta vivipara is a widespread herbaceous perennial plant with a discontinuous pattern of distribution in arctic, alpine, subalpine and boreal habitats across the northern Hemisphere. Studies of the fungi associated with the roots of B. vivipara have mainly been conducted in arctic and alpine ecosystems. This study examined the fungal diversity and specificity from root tips of B. vivipara in two local mountain ecosystems as well as on a global scale. Sequences were generated by Sanger sequencing of the internal transcribed spacer (ITS) region followed by an analysis of accurately annotated nuclear segments including ITS1-5.8S-ITS2 sequences available from public databases. In total, 181 different UNITE species hypotheses (SHs) were detected to be fungi associated with B. vivipara, 73 of which occurred in the Bavarian Alps and nine in the Swabian Alps-with one SH shared among both mountains. In both sites as well as in additional public data, individuals of B. vivipara were found to contain phylogenetically diverse fungi, with the Basidiomycota, represented by the Thelephorales and Sebacinales, being the most dominant. A comparative analysis of the diversity of the Sebacinales associated with B. vivipara and other co-occurring plant genera showed that the highest number of sebacinoid SHs were associated with Quercus and Pinus, followed by Bistorta. A comparison of B. vivipara with plant families such as Ericaceae, Fagaceae, Orchidaceae, and Pinaceae showed a clear trend: Only a few species were specific to B. vivipara and a large number of SHs were shared with other co-occurring non-B. vivipara plant species. In Sebacinales, the majority of SHs associated with B. vivipara belonged to the ectomycorrhiza (ECM)-forming Sebacinaceae, with fewer SHs belonging to the Serendipitaceae encompassing diverse ericoid-orchid-ECM-endophytic associations. The large proportion of non-host-specific fungi able to form a symbiosis with other non-B. vivipara plants could suggest that the high fungal diversity in B. vivipara comes from an active recruitment of their associates from the co-occurring vegetation. The non-host-specificity suggests that this strategy may offer ecological advantages; specifically, linkages with generalist rather than specialist fungi. Proximity to co-occurring non-B. vivipara plants can maximise the fitness of B. vivipara, allowing more rapid and easy colonisation of the available habitats.

DNA barcoding of fungal specimens using PacBio long-read high-throughput sequencing.

Molecular methods are increasingly used to identify species that lack conspicuous macro- or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency of long-read high throughput sequencing (HTS) as opposed to the traditionally used Sanger method for taxonomic identification of multiple vouchered fungal specimens. We also evaluated whether this method can provide reference information about intraindividual gene polymorphism. We developed a workflow based on a test set of 423 basidiomycete specimens (representing 195 species), the PacBio HTS method, and ribosomal rRNA operon internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers. The PacBio HTS had a higher success rate than Sanger sequencing at a comparable cost. Species identification based on PacBio reads was usually straightforward, because the dominant operational taxonomic unit (OTU) typically represented the targeted organism. The PacBio HTS also enabled us to detect widespread polymorphism within the ITS marker. We conclude that multiplex DNA barcoding of the fungal ITS and LSU markers using PacBio HTS is a useful tool for taxonomic identification of large amounts of collected voucher specimens at a competitive price. Furthermore, PacBio HTS accurately recovers various alleles and paralogues, which can provide crucial information for species delimitation and population-level studies.

The curse of the uncultured fungus.

The international DNA sequence databases abound in fungal sequences not annotated beyond the kingdom level, typically bearing names such as "uncultured fungus". These sequences beget low-resolution mycological results and invite further deposition of similarly poorly annotated entries. What do these sequences represent? This study uses a 767,918-sequence corpus of public full-length fungal ITS sequences to estimate what proportion of the 95,055 "uncultured fungus" sequences that represent truly unidentifiable fungal taxa - and what proportion of them that would have been straightforward to annotate to some more meaningful taxonomic level at the time of sequence deposition. Our results suggest that more than 70% of these sequences would have been trivial to identify to at least the order/family level at the time of sequence deposition, hinting that factors other than poor availability of relevant reference sequences explain the low-resolution names. We speculate that researchers' perceived lack of time and lack of insight into the ramifications of this problem are the main explanations for the low-resolution names. We were surprised to find that more than a fifth of these sequences seem to have been deposited by mycologists rather than researchers unfamiliar with the consequences of poorly annotated fungal sequences in molecular repositories. The proportion of these needlessly poorly annotated sequences does not decline over time, suggesting that this problem must not be left unchecked.

The Taxon Hypothesis Paradigm-On the Unambiguous Detection and Communication of Taxa.

Here, we describe the taxon hypothesis (TH) paradigm, which covers the construction, identification, and communication of taxa as datasets. Defining taxa as datasets of individuals and their traits will make taxon identification and most importantly communication of taxa precise and reproducible. This will allow datasets with standardized and atomized traits to be used digitally in identification pipelines and communicated through persistent identifiers. Such datasets are particularly useful in the context of formally undescribed or even physically undiscovered species if data such as sequences from samples of environmental DNA (eDNA) are available. Implementing the TH paradigm will to some extent remove the impediment to hastily discover and formally describe all extant species in that the TH paradigm allows discovery and communication of new species and other taxa also in the absence of formal descriptions. The TH datasets can be connected to a taxonomic backbone providing access to the vast information associated with the tree of life. In parallel to the description of the TH paradigm, we demonstrate how it is implemented in the UNITE digital taxon communication system. UNITE TH datasets include rich data on individuals and their rDNA ITS sequences. These datasets are equipped with digital object identifiers (DOI) that serve to fix their identity in our communication. All datasets are also connected to a GBIF taxonomic backbone. Researchers processing their eDNA samples using UNITE datasets will, thus, be able to publish their findings as taxon occurrences in the GBIF data portal. UNITE species hypothesis (species level THs) datasets are increasingly utilized in taxon identification pipelines and even formally undescribed species can be identified and communicated by using UNITE. The TH paradigm seeks to achieve unambiguous, unique, and traceable communication of taxa and their properties at any level of the tree of life. It offers a rapid way to discover and communicate undescribed species in identification pipelines and data portals before they are lost to the sixth mass extinction.

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe.

Soil microbiome has a pivotal role in ecosystem functioning, yet little is known about its build-up from local to regional scales. In a multi-year regional-scale survey involving 1251 plots and long-read third-generation sequencing, we found that soil pH has the strongest effect on the diversity of fungi and its multiple taxonomic and functional groups. The pH effects were typically unimodal, usually both direct and indirect through tree species, soil nutrients or mold abundance. Individual tree species, particularly Pinus sylvestris, Picea abies, and Populus x wettsteinii, and overall ectomycorrhizal plant proportion had relatively stronger effects on the diversity of biotrophic fungi than saprotrophic fungi. We found strong temporal sampling and investigator biases for the abundance of molds, but generally all spatial, temporal and microclimatic effects were weak. Richness of fungi and several functional groups was highest in woodlands and around ruins of buildings but lowest in bogs, with marked group-specific trends. In contrast to our expectations, diversity of soil fungi tended to be higher in forest island habitats potentially due to the edge effect, but fungal richness declined with island distance and in response to forest fragmentation. Virgin forests supported somewhat higher fungal diversity than old non-pristine forests, but there were no differences in richness between natural and anthropogenic habitats such as parks and coppiced gardens. Diversity of most fungal groups suffered from management of seminatural woodlands and parks and thinning of forests, but especially for forests the results depended on fungal group and time since partial harvesting. We conclude that the positive effects of tree diversity on overall fungal richness represent a combined niche effect of soil properties and intimate associations.

Alien ectomycorrhizal plants differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in novel sites.

Alien plants represent a potential threat to environment and society. Understanding the process of alien plants naturalization is therefore of primary importance. In alien plants, successful establishment can be constrained by the absence of suitable fungal partners. Here, we used 42 independent datasets of ectomycorrhizal fungal (EcMF) communities associated with alien Pinaceae and Eucalyptus spp., as the most commonly introduced tree species worldwide, to explore the strategies these plant groups utilize to establish symbioses with EcMF in the areas of introduction. We have also determined the differences in composition of EcMF communities associated with alien ectomycorrhizal plants in different regions. While alien Pinaceae introduced to new regions rely upon association with co-introduced EcMF, alien Eucalyptus often form novel interactions with EcMF species native to the region where the plant was introduced. The region of origin primarily determines species composition of EcMF communities associated with alien Pinaceae in new areas, which may largely affect invasion potential of the alien plants. Our study shows that alien ectomycorrhizal plants largely differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in sites of introduction, which may potentially affect their invasive potential.

FungalRoot: global online database of plant mycorrhizal associations.

Testing of ecological, biogeographical and phylogenetic hypotheses of mycorrhizal traits requires a comprehensive reference dataset about plant mycorrhizal associations. Here we present a database, FungalRoot, which summarizes publicly available data about vascular plant mycorrhizal type and intensity of root colonization by mycorrhizal fungi, accompanied with rich metadata. We compiled and digitized data about plant mycorrhizal colonization in nine widespread languages. The present version of the FungalRoot database contains 36 303 species-by-site observations for 14 870 plant species, tripling the previously available compiled information about plant mycorrhizal associations. Based on these data, we provide a recommended list of genus-level plant mycorrhizal associations, based on the majority of data for species and careful analysis of conflicting data. The majority of ectomycorrhizal and ericoid mycorrhizal plants are trees (92%) and shrubs (85%), respectively. The majority of arbuscular and nonmycorrhizal plant species are herbaceous (50% and 70%, respectively). Our publicly available database is a powerful resource for mycorrhizal scientists and ecologists. It features possibilities for dynamic updating and addition of data about plant mycorrhizal associations. The new database will promote research on plant and fungal biogeography and evolution, and on links between above- and belowground biodiversity and ecosystem functioning.

Fungal functional ecology: bringing a trait-based approach to plant-associated fungi.

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro-organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function. Trait-based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and -omics-based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (FunFun ). FunFun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait-based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.

The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications.

UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets the formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ∼1 000 000 public fungal ITS sequences for reference. These are clustered into ∼459 000 species hypotheses and assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house and web-based third-party sequence curation and annotation have resulted in more than 275 000 improvements to the data over the past 15 years. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include redesigned handling of unclassifiable species hypotheses, integration with the taxonomic backbone of the Global Biodiversity Information Facility, and support for an unlimited number of parallel taxonomic classification systems.

Protax-fungi: a web-based tool for probabilistic taxonomic placement of fungal internal transcribed spacer sequences.

Incompleteness of reference sequence databases and unresolved taxonomic relationships complicates taxonomic placement of fungal sequences. We developed Protax-fungi, a general tool for taxonomic placement of fungal internal transcribed spacer (ITS) sequences, and implemented it into the PlutoF platform of the UNITE database for molecular identification of fungi. With empirical data on root- and wood-associated fungi, Protax-fungi reliably identified (with at least 90% identification probability) the majority of sequences to the order level but only around one-fifth of them to the species level, reflecting the current limited coverage of the databases. Protax-fungi outperformed the Sintax and Rdb classifiers in terms of increased accuracy and decreased calibration error when applied to data on mock communities representing species groups with poor sequence database coverage. We applied Protax-fungi to examine the internal consistencies of the Index Fungorum and UNITE databases. This revealed inconsistencies in the taxonomy database as well as mislabelling and sequence quality problems in the reference database. The according improvements were implemented in both databases. Protax-fungi provides a robust tool for performing statistically reliable identifications of fungi in spite of the incompleteness of extant reference sequence databases and unresolved taxonomic relationships.

Taxonomic annotation of public fungal ITS sequences from the built environment - a report from an April 10-11, 2017 workshop (Aberdeen, UK).

Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS barcode sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.

ToxGen: an improved reference database for the identification of type B-trichothecene genotypes in Fusarium.

Type B trichothecenes, which pose a serious hazard to consumer health, occur worldwide in grains. These mycotoxins are produced mainly by three different trichothecene genotypes/chemotypes: 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) and NIV (nivalenol), named after these three major mycotoxin compounds. Correct identification of these genotypes is elementary for all studies relating to population surveys, fungal ecology and mycotoxicology. Trichothecene producers exhibit enormous strain-dependent chemical diversity, which may result in variation in levels of the genotype's determining toxin and in the production of low to high amounts of atypical compounds. New high-throughput DNA-sequencing technologies promise to boost the diagnostics of mycotoxin genotypes. However, this requires a reference database containing a satisfactory taxonomic sampling of sequences showing high correlation to actually produced chemotypes. We believe that one of the most pressing current challenges of such a database is the linking of molecular identification with chemical diversity of the strains, as well as other metadata. In this study, we use the Tri12 gene involved in mycotoxin biosynthesis for identification of Tri genotypes through sequence comparison. Tri12 sequences from a range of geographically diverse fungal strains comprising 22 Fusarium species were stored in the ToxGen database, which covers descriptive and up-to-date annotations such as indication on Tri genotype and chemotype of the strains, chemical diversity, information on trichothecene-inducing host, substrate or media, geographical locality, and most recent taxonomic affiliations. The present initiative bridges the gap between the demands of comprehensive studies on trichothecene producers and the existing nucleotide sequence databases, which lack toxicological and other auxiliary data. We invite researchers working in the fields of fungal taxonomy, epidemiology and mycotoxicology to join the freely available annotation effort.

Sequence-based classification and identification of Fungi.

Fungal taxonomy and ecology have been revolutionized by the application of molecular methods and both have increasing connections to genomics and functional biology. However, data streams from traditional specimen- and culture-based systematics are not yet fully integrated with those from metagenomic and metatranscriptomic studies, which limits understanding of the taxonomic diversity and metabolic properties of fungal communities. This article reviews current resources, needs, and opportunities for sequence-based classification and identification (SBCI) in fungi as well as related efforts in prokaryotes. To realize the full potential of fungal SBCI it will be necessary to make advances in multiple areas. Improvements in sequencing methods, including long-read and single-cell technologies, will empower fungal molecular ecologists to look beyond ITS and current shotgun metagenomics approaches. Data quality and accessibility will be enhanced by attention to data and metadata standards and rigorous enforcement of policies for deposition of data and workflows. Taxonomic communities will need to develop best practices for molecular characterization in their focal clades, while also contributing to globally useful datasets including ITS. Changes to nomenclatural rules are needed to enable validPUBLICation of sequence-based taxon descriptions. Finally, cultural shifts are necessary to promote adoption of SBCI and to accord professional credit to individuals who contribute to community resources.

Determining threshold values for barcoding fungi: lessons from Cortinarius (Basidiomycota), a highly diverse and widespread ectomycorrhizal genus.

Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%-2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches.

Stochastic distribution of small soil eukaryotes resulting from high dispersal and drift in a local environment.

A central challenge in ecology is to understand the relative importance of processes that shape diversity patterns. Compared with aboveground biota, little is known about spatial patterns and processes in soil organisms. Here we examine the spatial structure of communities of small soil eukaryotes to elucidate the underlying stochastic and deterministic processes in the absence of environmental gradients at a local scale. Specifically, we focus on the fine-scale spatial autocorrelation of prominent taxonomic and functional groups of eukaryotic microbes. We collected 123 soil samples in a nested design at distances ranging from 0.01 to 64 m from three boreal forest sites and used 454 pyrosequencing analysis of Internal Transcribed Spacer for detecting Operational Taxonomic Units of major eukaryotic groups simultaneously. Among the main taxonomic groups, we found significant but weak spatial variability only in the communities of Fungi and Rhizaria. Within Fungi, ectomycorrhizas and pathogens exhibited stronger spatial structure compared with saprotrophs and corresponded to vegetation. For the groups with significant spatial structure, autocorrelation occurred at a very fine scale (<2 m). Both dispersal limitation and environmental selection had a weak effect on communities as reflected in negative or null deviation of communities, which was also supported by multivariate analysis, that is, environment, spatial processes and their shared effects explained on average <10% of variance. Taken together, these results indicate a random distribution of soil eukaryotes with respect to space and environment in the absence of environmental gradients at the local scale, reflecting the dominant role of drift and homogenizing dispersal.