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.

Limited decadal growth of mountain birch saplings has minor impact on surrounding tundra vegetation.

Temperatures over the Arctic region are increasing at three times the rate of the global average. Consequently, Arctic vegetation is changing and trees are encroaching into the tundra. In this study, we examine the establishment and growth of mountain birch (Betula pubescens ssp. tortuosa), which forms the treeline in subarctic Europe, and its impact on community composition across the treeline ecotone nearby Abisko, Sweden. Birch advancement along elevational gradients was studied by comparing data collected in 2016 with data collected 10 and 15 years previously. Species identity, cover, and phylogenetic relatedness were used to assess the impact of birch encroachment on community composition. Our results show that birch occurrence above the treeline did not affect plant community composition, probably owing to the observed lack of significant growth due to herbivore browsing, nitrogen limitation, or a reduction in snow cover. Independent of birch performance, the tundra community structure shifted toward a novel community dissimilar from the forest plant community found below the treeline. Taken together, our findings are explained by species-specific responses to climate change, rather than by a linear forest advance. Future treeline advancements are likely more restricted than previously expected.

Dnabarcoder: An open-source software package for analysing and predicting DNA sequence similarity cutoffs for fungal sequence identification.

The accuracy and precision of fungal molecular identification and classification are challenging, particularly in environmental metabarcoding approaches as these often trade accuracy for efficiency given the large data volumes at hand. In most ecological studies, only a single similarity cutoff value is used for sequence identification. This is not sufficient since the most commonly used DNA markers are known to vary widely in terms of inter- and intraspecific variability. We address this problem by presenting a new tool, dnabarcoder, to predict local similarity cutoffs and measure the resolving powers of a biomarker for sequence identification for different clades of fungi. It was shown that the predicted similarity cutoffs varied significantly between the clades of a recently released ITS DNA barcode data set from the CBS culture collection of the Westerdijk Fungal Biodiversity Institute. When classifying a large public fungal ITS data set-the UNITE database-against the barcode data set, the local similarity cutoffs assigned fewer sequences than the traditional cutoffs used in metabarcoding studies. However, the obtained accuracy and precision were significantly improved. Our study showed that it might be better to extract the ITS region from the ITS barcodes to optimize taxonomic assignment accuracy. Furthermore, 15.3, 25.6, and 26.3% of the fungal species of the barcode data set were indistinguishable by full-length ITS, ITS1, and ITS2, respectively. Except for these indistinguishable species, the resolving powers of full-length ITS, ITS1, and ITS2 sequences were similar at the species level. Nevertheless, the complete ITS region had a better resolving power at higher taxonomic levels.

Best practices in metabarcoding of fungi: From experimental design to results.

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety of ecosystems. Here we provide an overview of current best practices in metabarcoding analysis of fungal communities, from experimental design through molecular and computational analyses. By reanalysing published data sets, we demonstrate that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) in recovering fungal diversity, a finding that is particularly evident for long markers. Additionally, analysis of the full-length ITS region allows more accurate taxonomic placement of fungi and other eukaryotes compared to the ITS2 subregion. Finally, we show that specific methods for compositional data analyses provide more reliable estimates of shifts in community structure. We conclude that metabarcoding analyses of fungi are especially promising for integrating fungi into the full microbiome and broader ecosystem functioning context, recovery of novel fungal lineages and ancient organisms as well as barcoding of old specimens including type material.

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.

Evidence for further non-coding RNA genes in the fungal rDNA region.

Non-coding RNA (ncRNA) genes play important, but incompletely understood, roles in various cellular processes, notably translation and gene regulation. A recent report on the detection of the ncRNA Signal Recognition Particle gene in the nuclear ribosomal internal transcribed spacer region of several species of three genera of ectomycorrhizal basidiomycetes prompted a more thorough bioinformatics search for additional ncRNA genes in the full fungal ribosomal operon. This study reports on the detection of three ncRNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of Auricularia and Inocybe. We verified their presence through resequencing of independent specimens. Two completed Auricularia genomes were found to lack these ncRNAs elsewhere than in the ribosomal operon, suggesting that these are functional genes. It seems clear that ncRNA genes play a larger role in fungal ribosomal genetics than hitherto thought.

Digging Up the Roots: Taxonomic and Phylogenetic Disentanglements in Corticiaceae s.s. (Corticiales, Basidiomycota) and Evolution of Nutritional Modes.

Corticiaceae is one of the traditional families of the Agaricomycetes and served for a long time as a convenient placement for basidiomycetes with a resupinate, corticioid form of fruiting body. Molecular studies have helped to assign many corticioid fungi to diverse families and orders; however, Corticiaceae still lacks a phylogenetic characterization and modern circumscription. Here, we provide the first comprehensive phylogenetic and taxonomic revision of the family Corticiaceae based on extensive type studies and sequences of nLSU, ITS, IGS, nSSU, and mtSSU regions. Our analyses support the recognition of ten monophyletic genera in the Corticiaceae, and show that nutritional mode is not a robust basis for generic delimitations in the family. The genus Mycobernardia and the species Corticium thailandicum, Erythricium vernum, and Marchandiomyces allantosporus are described as new to science, and five new combinations are proposed. Moreover, ancestral character state reconstruction revealed that saprotrophy is the plesiomorphic nutritional mode in the Corticiaceae, while several transitions have occurred to diverse nutritional modes in this family. Identification keys are provided to the genera in Corticiaceae s.s. as well as to the species in Corticium, Erythricium, Laetisaria, and Marchandiomyces.

Assessing Biotic and Abiotic Interactions of Microorganisms in Amazonia through Co-Occurrence Networks and DNA Metabarcoding.

Species may co-occur due to responses to similar environmental conditions, biological associations, or simply because of coincident geographical distributions. Disentangling patterns of co-occurrence and potential biotic and abiotic interactions is crucial to understand ecosystem function. Here, we used DNA metabarcoding data from litter and mineral soils collected from a longitudinal transect in Amazonia to explore patterns of co-occurrence. We compared data from different Amazonian habitat types, each with a characteristic biota and environmental conditions. These included non-flooded rainforests (terra-firme), forests seasonally flooded by fertile white waters (várzeas) or by unfertile black waters (igapós), and open areas associated with white sand soil (campinas). We ran co-occurrence network analyses based on null models and Spearman correlation for all samples and for each habitat separately. We found that one third of all operational taxonomic units (OTUs) were bacteria and two thirds were eukaryotes. The resulting networks were nevertheless mostly composed of bacteria, with fewer fungi, protists, and metazoans. Considering the functional traits of the OTUs, there is a combination of metabolism modes including respiration and fermentation for bacteria, and a high frequency of saprotrophic fungi (those that feed on dead organic matter), indicating a high turnover of organic material. The organic carbon and base saturation indices were important in the co-occurrences in Amazonian networks, whereas several other soil properties were important for the co-exclusion. Different habitats had similar network properties with some variation in terms of modularity, probably associated with flooding pulse. We show that Amazonian microorganism communities form highly interconnected co-occurrence and co-exclusion networks, which highlights the importance of complex biotic and abiotic interactions in explaining the outstanding biodiversity of the region.

When mycologists describe new species, not all relevant information is provided (clearly enough).

Taxonomic mycology struggles with what seems to be a perpetual shortage of resources. Logically, fungal taxonomists should therefore leverage every opportunity to highlight and visualize the importance of taxonomic work, the usefulness of taxonomic data far beyond taxonomy, and the integrative and collaborative nature of modern taxonomy at large. Is mycology really doing that, though? In this study, we went through ten years' worth (2009-2018) of species descriptions of extant fungal taxa - 1,097 studies describing at most ten new species - in five major mycological journals plus one plant journal. We estimated the frequency at which a range of key words, illustrations, and concepts related to ecology, geography, taxonomy, molecular data, and data availability were provided with the descriptions. We also considered a range of science-demographical aspects such as gender bias and the rejuvenation of taxonomy and taxonomists as well as public availability of the results. Our results show that the target audience of fungal species descriptions appears to be other fungal taxonomists, because many aspects of the new species were presented only implicitly, if at all. Although many of the parameters we estimated show a gradual, and in some cases marked, change for the better over time, they still paint a somewhat bleak picture of mycological taxonomy as a male-dominated field where the wants and needs of an extended target audience are often not understood or even considered. This study hopes to leave a mark on the way fungal species are described by putting the focus on ways in which fungal taxonomy can better anticipate the end users of species descriptions - be they mycologists, other researchers, the public at large, or even algorithms. In the end, fungal taxonomy, too, is likely to benefit from such measures.

The pitfalls of biodiversity proxies: Differences in richness patterns of birds, trees and understudied diversity across Amazonia.

Most knowledge on biodiversity derives from the study of charismatic macro-organisms, such as birds and trees. However, the diversity of micro-organisms constitutes the majority of all life forms on Earth. Here, we ask if the patterns of richness inferred for macro-organisms are similar for micro-organisms. For this, we barcoded samples of soil, litter and insects from four localities on a west-to-east transect across Amazonia. We quantified richness as Operational Taxonomic Units (OTUs) in those samples using three molecular markers. We then compared OTU richness with species richness of two relatively well-studied organism groups in Amazonia: trees and birds. We find that OTU richness shows a declining west-to-east diversity gradient that is in agreement with the species richness patterns documented here and previously for birds and trees. These results suggest that most taxonomic groups respond to the same overall diversity gradients at large spatial scales. However, our results show a different pattern of richness in relation to habitat types, suggesting that the idiosyncrasies of each taxonomic group and peculiarities of the local environment frequently override large-scale diversity gradients. Our findings caution against using the diversity distribution of one taxonomic group as an indication of patterns of richness across all groups.

Exploring the taxonomic composition of two fungal communities on the Swedish west coast through metabarcoding.

BACKGROUND: Fungi are heterotrophic, unicellular or filamentous organisms that exhibit a wide range of different lifestyles as, e.g., symbionts, parasites, and saprotrophs. Mycologists have traditionally considered fungi to be a nearly exclusively terrestrial group of organisms, but it is now known that fungi have a significant presence in aquatic environments as well. We know little about most fungi in limnic and marine systems, including aspects of their taxonomy, ecology, and geographic distribution. The present study seeks to improve our knowledge of fungi in the marine environment. The fungal communities of two coastal marine environments of the Kattegat sea, Sweden, were explored with metabarcoding techniques using the nuclear ribosomal internal transcribed spacer 2 (ITS2) metabarcode. Our data add new information to the current picture of fungal community composition in benthic and coastal habitats in Northern Europe. NEW INFORMATION: The dataset describes the number of operational taxonomic units (OTUs) and their taxonomic affiliations in two littoral gradients sampled on the Swedish west coast, Gothenburg municipality. Our data include basic diversity indices as well as chemical and edaphic sediment/soil parameters of the sampling sites. From the sites, 3470 and 4315 fungal OTUs, respectively, were recovered. The number of reads were 673,711 and 779,899, respectively, after quality filtering. Within the benthic sites, more than 80% of the sequences could not be classified taxonomically. The phylum composition of the classifiable sequences was dominated in both localities by Dikarya, which made up around 33% of the OTUs. Within Dikarya, Ascomycota was the dominant phylum. Guild assignment failed for more than half of the classifiable OTUs, with undefined saprotrophs being the most common resolved guild. This guild classification was slightly more common in the ocean sediment samples than in the terrestrial ones. Our metadata indicated that ocean sites contain organisms at a lower trophic level and that there are predominantly endophytic, parasitic, and pathogenic fungi in the marine environments. This hints at the presence of interesting and currently poorly understood fungus-driven ecological processes. It is also clear from our results that a very large number of marine fungi are in urgent need of taxonomic study and formal description.

Biodiversity assessments in the 21st century: the potential of insect traps to complement environmental samples for estimating eukaryotic and prokaryotic diversity using high-throughput DNA metabarcoding 1.

The rapid loss of biodiversity, coupled with difficulties in species identification, call for innovative approaches to assess biodiversity. Insects make up a substantial proportion of extant diversity and play fundamental roles in any given ecosystem. To complement morphological species identification, new techniques such as metabarcoding make it possible to quantify insect diversity and insect-ecosystem interactions through DNA sequencing. Here we examine the potential of bulk insect samples (i.e., containing many non-sorted specimens) to assess prokaryote and eukaryote biodiversity and to complement the taxonomic coverage of soil samples. We sampled 25 sites on three continents and in various ecosystems, collecting insects with SLAM traps (Brazil) and Malaise traps (South Africa and Sweden). We then compared our diversity estimates with the results obtained with biodiversity data from soil samples from the same localities. We found a largely different taxonomic composition between the soil and insect samples, testifying to the potential of bulk insect samples to complement soil samples. Finally, we found that non-destructive DNA extraction protocols, which preserve insect specimens for morphological studies, constitute a promising choice for cost-effective biodiversity assessments. We propose that the sampling and sequencing of insect samples should become a standard complement for biodiversity studies based on environmental DNA.

Mycobiome diversity: high-throughput sequencing and identification of fungi.

Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous - and largely uncharted - taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.

Introducing ribosomal tandem repeat barcoding for fungi.

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third-generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets.

Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding.

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

High-throughput metabarcoding reveals the effect of physicochemical soil properties on soil and litter biodiversity and community turnover across Amazonia.

BACKGROUND: Knowledge on the globally outstanding Amazonian biodiversity and its environmental determinants stems almost exclusively from aboveground organisms, notably plants. In contrast, the environmental factors and habitat preferences that drive diversity patterns for micro-organisms in the ground remain elusive, despite the fact that micro-organisms constitute the overwhelming majority of life forms in any given location, in terms of both diversity and abundance. Here we address how the diversity and community turnover of operational taxonomic units (OTU) of organisms in soil and litter respond to soil physicochemical properties; whether OTU diversities and community composition in soil and litter are correlated with each other; and whether they respond in a similar way to soil properties. METHODS: We used recently inferred OTUs from high-throughput metabarcoding of the 16S (prokaryotes) and 18S (eukaryotes) genes to estimate OTU diversity (OTU richness and effective number of OTUs) and community composition for prokaryotes and eukaryotes in soil and litter across four localities in Brazilian Amazonia. All analyses were run separately for prokaryote and eukaryote OTUs, and for each group using both presence-absence and abundance data. Combining these with novel data on soil chemical and physical properties, we identify abiotic correlates of soil and litter organism diversity and community structure using regression, ordination, and variance partitioning analysis. RESULTS: Soil organic carbon content was the strongest factor explaining OTU diversity (negative correlation) and pH was the strongest factor explaining community turnover for prokaryotes and eukaryotes in both soil and litter. We found significant effects also for other soil variables, including both chemical and physical properties. The correlation between OTU diversity in litter and in soil was non-significant for eukaryotes and weak for prokaryotes. The community compositions of both prokaryotes and eukaryotes were more separated among habitat types (terra-firme, várzea, igapó and campina) than between substrates (soil and litter). DISCUSSION: In spite of the limited sampling (four localities, 39 plots), our results provide a broad-scale view of the physical and chemical correlations of soil and litter biodiversity in a longitudinal transect across the world's largest rainforest. Our methods help to understand links between soil properties, OTU diversity patterns, and community composition and turnover. The lack of strong correlation between OTU diversity in litter and in soil suggests independence of diversity drives of these substrates and highlights the importance of including both measures in biodiversity assessments. Massive sequencing of soil and litter samples holds the potential to complement traditional biological inventories in advancing our understanding of the factors affecting tropical diversity.

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.

Metaxa2 Database Builder: enabling taxonomic identification from metagenomic or metabarcoding data using any genetic marker.

Motivation: Correct taxonomic identification of DNA sequences is central to studies of biodiversity using both shotgun metagenomic and metabarcoding approaches. However, no genetic marker gives sufficient performance across all the biological kingdoms, hampering studies of taxonomic diversity in many groups of organisms. This has led to the adoption of a range of genetic markers for DNA metabarcoding. While many taxonomic classification software tools can be re-trained on these genetic markers, they are often designed with assumptions that impair their utility on genes other than the SSU and LSU rRNA. Here, we present an update to Metaxa2 that enables the use of any genetic marker for taxonomic classification of metagenome and amplicon sequence data. Results: We evaluated the Metaxa2 Database Builder on 11 commonly used barcoding regions and found that while there are wide differences in performance between different genetic markers, our software performs satisfactorily provided that the input taxonomy and sequence data are of high quality. Availability and implementation: Freely available on the web as part of the Metaxa2 package at http://microbiology.se/software/metaxa2/. Supplementary information: Supplementary data are available at Bioinformatics online.

New light on names and naming of dark taxa.

A growing proportion of fungal species and lineages are known only from sequence data and cannot be linked to any physical specimen or resolved taxonomic name. Such fungi are often referred to as "dark taxa" or "dark matter fungi". As they lack a taxonomic identity in the form of a name, they are regularly ignored in many important contexts, for example in legalisation and species counts. It is therefore very urgent to find a system to also deal with these fungi. Here, issues relating to the taxonomy and nomenclature of dark taxa are discussed and a number of questions that the mycological community needs to consider before deciding on what system/s to implement are highlighted.