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.

Connecting the multiple dimensions of global soil fungal diversity.

How the multiple facets of soil fungal diversity vary worldwide remains virtually unknown, hindering the management of this essential species-rich group. By sequencing high-resolution DNA markers in over 4000 topsoil samples from natural and human-altered ecosystems across all continents, we illustrate the distributions and drivers of different levels of taxonomic and phylogenetic diversity of fungi and their ecological groups. We show the impact of precipitation and temperature interactions on local fungal species richness (alpha diversity) across different climates. Our findings reveal how temperature drives fungal compositional turnover (beta diversity) and phylogenetic diversity, linking them with regional species richness (gamma diversity). We integrate fungi into the principles of global biodiversity distribution and present detailed maps for biodiversity conservation and modeling of global ecological processes.

Structure of plant-associated microeukaryotes in roots and leaves of aquatic and terrestrial plants revealed by blocking peptide-nucleic acid (PNA) amplification.

Studies of plant-microbe interactions, including mutualistic, antagonistic, parasitic, or commensal microbes, have greatly benefited our understanding of ecosystem functioning. New molecular identification tools have increasingly revealed the association patterns between microorganisms and plants. Here, we integrated long-read PacBio single-molecule sequencing technology with a blocking protein-nucleic acid (PNA) approach to minimise plant amplicons in a survey of plant-eukaryotic microbe relationships in roots and leaves of different aquatic and terrestrial plants to determine patterns of organ, host, and habitat preferences. The PNA approach reduced the samples' relative amounts of plant reads and did not distort the fungal and other microeukaryotic composition. Our analyses revealed that the eukaryotic microbiomes associated with leaves and roots of aquatic plants exhibit a much larger proportion of non-fungal microorganisms than terrestrial plants, and leaf and root microbiomes are similar. Terrestrial plants had much stronger differentiation of leaf and root microbiomes and stronger partner specificity than aquatic plants.

A pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses.

Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.

Patterns in soil microbial diversity across Europe.

Factors driving microbial community composition and diversity are well established but the relationship with microbial functioning is poorly understood, especially at large scales. We analysed microbial biodiversity metrics and distribution of potential functional groups along a gradient of increasing land-use perturbation, detecting over 79,000 bacterial and 25,000 fungal OTUs in 715 sites across 24 European countries. We found the lowest bacterial and fungal diversity in less-disturbed environments (woodlands) compared to grasslands and highly-disturbed environments (croplands). Highly-disturbed environments contain significantly more bacterial chemoheterotrophs, harbour a higher proportion of fungal plant pathogens and saprotrophs, and have less beneficial fungal plant symbionts compared to woodlands and extensively-managed grasslands. Spatial patterns of microbial communities and predicted functions are best explained when interactions among the major determinants (vegetation cover, climate, soil properties) are considered. We propose guidelines for environmental policy actions and argue that taxonomical and functional diversity should be considered simultaneously for monitoring purposes.

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.

Effects of nitrogen deposition on carbon and nutrient cycling along a natural soil acidity gradient as revealed by metagenomics.

Nitrogen (N) deposition and soil acidification are environmental challenges affecting ecosystem functioning, health, and biodiversity, but their effects on functional genes are poorly understood. Here, we utilized metabarcoding and metagenomics to investigate the responses of soil functional genes to N deposition along a natural soil pH gradient. Soil N content was uncorrelated with pH, enabling us to investigate their effects separately. Soil acidity strongly and negatively affected the relative abundances of most cluster of orthologous gene categories of the metabolism supercategory. Similarly, soil acidity negatively affected the diversity of functional genes related to carbon and N but not phosphorus cycling. Multivariate analyses showed that soil pH was the most important factor affecting microbial and functional gene composition, while the effects of N deposition were less important. Relative abundance of KEGG functional modules related to different parts of the studied cycles showed variable responses to soil acidity and N deposition. Furthermore, our results suggested that the diversity-function relationship reported for other organisms also applies to soil microbiomes. Since N deposition and soil pH affected microbial taxonomic and functional composition to a different extent, we conclude that N deposition effects might be primarily mediated through soil acidification in forest ecosystems.

Global patterns in endemicity and vulnerability of soil fungi.

Fungi are highly diverse organisms, which provide multiple ecosystem services. However, compared with charismatic animals and plants, the distribution patterns and conservation needs of fungi have been little explored. Here, we examined endemicity patterns, global change vulnerability and conservation priority areas for functional groups of soil fungi based on six global surveys using a high-resolution, long-read metabarcoding approach. We found that the endemicity of all fungi and most functional groups peaks in tropical habitats, including Amazonia, Yucatan, West-Central Africa, Sri Lanka, and New Caledonia, with a negligible island effect compared with plants and animals. We also found that fungi are predominantly vulnerable to drought, heat and land-cover change, particularly in dry tropical regions with high human population density. Fungal conservation areas of highest priority include herbaceous wetlands, tropical forests, and woodlands. We stress that more attention should be focused on the conservation of fungi, especially root symbiotic arbuscular mycorrhizal and ectomycorrhizal fungi in tropical regions as well as unicellular early-diverging groups and macrofungi in general. Given the low overlap between the endemicity of fungi and macroorganisms, but high conservation needs in both groups, detailed analyses on distribution and conservation requirements are warranted for other microorganisms and soil organisms.

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.

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.

Sheltering Role of Well-Decayed Conifer Logs for Forest Floor Fungi in Long-Term Polluted Boreal Forests.

Coarse woody debris (CWD) provides food and shelter to a large proportion of forest biota and is considered vital for biodiversity during periods of harsh weather. However, its importance in long-term stressed ecosystems remains largely unknown. In this work, we explored the contribution of CWD to fungal diversity along the gradient of boreal forest degradation caused by 77 years of heavy industrial emissions. We analyzed the diversity and composition of fungi in 270 samples of well-decayed Picea abies and Abies sibirica logs, as well as forest litter both adjacent to and distant from the logs. Compared with forest litter, the wood had higher water content and possessed substantially lower concentrations of heavy metals, which suggests its potential favorability for biota in polluted areas. The pollution-induced loss of fungal diversity in forest litter reached 34% and was stronger in the microhabitats not influenced by CWD. Meanwhile, wood fungal communities lost less than 10% of their total richness and even increased in alpha diversity. These processes led to the diversity and compositional convergence of fungal communities from different microhabitats and substrates in polluted areas. Despite this, the importance of wood and CWD-influenced microhabitats for fungal diversity maintenance was low. Apart from wood-associated fungi, the taxa whose diversity increased in the wood of polluted areas were ectomycorrhizal fungi and eurytopic soil saprotrophs (Mucoromycota, Mortierellomycota, Eurotiomycetes, and Helotiales) that easily tolerate highly toxic litter. Within the majority of pollution-sensitive soil saprotrophic groups, only terricolous Tricholomataceae benefit from CWD as microrefugia. Upon considering the ecological variability within low-rank taxa, the importance of decayed logs as safe sites can be high for certain soil-inhabiting fungal groups in polluted areas.

Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms.

With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the 'noisy' reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost.

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.

Bird diversity and dissimilarity show contrasting patterns along heavy metal pollution gradients in the Urals, Russia.

The effects of industrial pollution on bird diversity have been widely studied using traditional diversity measures, which assume all species to be equivalent. We compared species richness and Shannon index with distance-based measures of taxonomic, functional, and phylogenetic diversity (the abundance-weighted mean nearest taxon distances), which describe within-community dissimilarity at terminal branches. Analysis of dissimilarity can shed light on the processes underlying community assembly, i.e., environmental filtering decreases dissimilarity whereas competitive exclusion increases it. In the 2-year study near Karabash and Revda copper smelters in Russia, point counts of nesting birds and habitat descriptions were taken at 10 sites (40 plots) along each pollution gradient. The abundance and diversity of birds showed good repeatability in both regions. The total density of birds, number of species per plot, and Shannon diversity decreased at high toxic load in both regions. The taxonomic, functional, and phylogenetic nearest taxon distances showed the same pattern within regions. Species dissimilarity within communities increased with pollution in Karabash (due to loss of functionally similar species), but did not change in Revda (due to mass replacement of forest species by species of open habitats). Pollution-induced changes in bird communities near Karabash were greater due to the stronger deterioration of the forest ecosystems and less favorable natural conditions (more arid climate, lower diversity and vitality of the tree stand and understorey) compared to Revda. This study emphasizes the need for a multi-level approach to the analysis of bird communities using traditional indices of diversity, functional, taxonomic, or phylogenetic distances between species and environmental variables.

Genetic variation and selfing rate in Lychnis flos-cuculi along an industrial pollution gradient.

We studied nine populations of a meadow mixed-mating plant Lychnis flos-cuculi growing in a gradient of copper smelter emissions. We hypothesize that metal tolerant populations in the polluted areas have experienced a loss of genetic variation and are more selfing than the populations from the unpolluted areas. One hundred and thirty-five parental plants and 1059 offspring were genotyped with six microsatellite markers. Selfing rates were assessed manually, with Rmes, Mltr and Colony2. Soil toxicity, population density and pollinators' activity were estimated in the studied areas. Populations from the heavily polluted area have experienced a strong founder effect. However, at present, they are characterized by high densities. A recent genetic explosion was registered for the population from the most polluted site, probably due to forest thinning under pollution effects. Selfing rates estimated with different approaches agreed well only for populations with high genetic variation; they comprised 0-0.23 and were similar between polluted and clean areas. Self-fertilization in L. flos-cuculi hardly represents a mechanism for the fixation of advantageous alleles and a barrier for gene flow from non-tolerant populations. The employment of different methods of selfing rate estimation in populations with low genetic variation appears to be necessary, though not a guarantee of reliable conclusions.

European phylogeography of the epiphytic lichen fungus Lobaria pulmonaria and its green algal symbiont.

In lichen symbiosis, fungal and algal partners form close associations, often codispersed by vegetative propagules. Due to the particular interdependence, processes such as colonization, dispersal or genetic drift are expected to result in congruent patterns of genetic structure in the symbionts. To study the population structure of an obligate symbiotic system in Europe, we genotyped the fungal and algal symbionts of the epiphytic lichen Lobaria pulmonaria at eight and seven microsatellite loci, respectively, and analysed about 4300 L. pulmonaria thalli from 142 populations from the species' European distribution range. Based on a centroid approach, which localizes centres of genetic differentiation with a high frequency of geographically restricted alleles, we identified the South Italy-Balkan region as the primary glacial refugial area of the lichen symbiosis. Procrustean rotation analysis and a distance congruence test between the fungal and algal population graphs indicated general concordance between the phylogeographies of the symbionts. The incongruent patterns found in areas of postglacial recolonization may show the presence of an additional refugial area for the fungal symbiont, and the impact that horizontal photobiont transmission and different mutation rates of the symbionts have on their genotypic associations at a continental scale.