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.

Metabarcoding of soil environmental DNA to estimate plant diversity globally.

Introduction: Traditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods. Methods: We sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data. Results: Large-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region. Discussion: eDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region.

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.

Global diversity and distribution of nitrogen-fixing bacteria in the soil.

Our knowledge of microbial biogeography has advanced in recent years, yet we lack knowledge of the global diversity of some important functional groups. Here, we used environmental DNA from 327 globally collected soil samples to investigate the biodiversity patterns of nitrogen-fixing bacteria by focusing on the nifH gene but also amplifying the general prokaryotic 16S SSU region. Globally, N-fixing prokaryotic communities are driven mainly by climatic conditions, with most groups being positively correlated with stable hot or seasonally humid climates. Among soil parameters, pH, but also soil N content were most often shown to correlate with the diversity of N-fixer groups. However, specific groups of N-fixing prokaryotes show contrasting responses to the same variables, notably in Cyanobacteria that were negatively correlated with stable hot climates, and showed a U-shaped correlation with soil pH, contrary to other N-fixers. Also, the non-N-fixing prokaryotic community composition was differentially correlated with the diversity and abundance of N-fixer groups, showing the often-neglected impact of biotic interactions among bacteria.

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.

Analysis of culturable airborne fungi in outdoor environments in Tianjin, China.

BACKGROUND: Fungal spores dispersed in the atmosphere may become cause of different pathological conditions and allergies for human beings. A number of studies have been performed to analyze the diversity of airborne fungi in different environments worldwide, and in particular in many urban areas in China. We investigated, for the first time, the diversity, concentration and distribution of airborne fungi in Tianjin city. We sampled 8 outdoor environments, using open plate method, during a whole winter season. Isolated fungi were identified by morphological and molecular analysis. Environmental factors which could influence the airborne fungi concentration (temperature, humidity, wind speed, and air pressure) were monitored and analyzed. The effect of different urban site functions (busy areas with high traffic flow and commercial activities vs. green areas) on airborne fungal diversity was also analyzed. RESULTS: A total of 560 fungal strains, belonging to 110 species and 49 genera of Ascomycota (80 %), Basidiomycota (18 %), and Mucoromycota (2 %) were isolated in this study. The dominant fungal genus was Alternaria (22 %), followed by Cladosporium (18.4 %), Naganishia (14.1 %), Fusarium (5.9 %), Phoma (4.11 %), and Didymella (4.8 %). A fungal concentration ranging from 0 to 3224.13 CFU m- 3 was recorded during the whole study. Permutational multivariate analysis showed that the month was the most influential factor for airborne fungal community structure, probably because it can be regarded as a proxy of environmental variables, followed by wind speed. The two analyzed environments (busy vs. green) had no detectable effect on the air fungal community, which could be related to the relatively small size of parks in Tianjin and/or to the study season. CONCLUSIONS: Our study shed light on the highly diverse community of airborne fungi characterizing the outdoor environments of Tianjin, and clarified the role that different environmental factors played in shaping the analyzed fungal community. The dominant presence of fungi with potential hazardous effect on human health, such as Alternaria, Cladosporium and Naganishia, deserves further attention. Our results may represent a valuable source of information for air quality monitoring, microbial pollution control, and airborne diseases prevention.

Temperature and pH define the realised niche space of arbuscular mycorrhizal fungi.

The arbuscular mycorrhizal (AM) fungi are a globally distributed group of soil organisms that play critical roles in ecosystem function. However, the ecological niches of individual AM fungal taxa are poorly understood. We collected > 300 soil samples from natural ecosystems worldwide and modelled the realised niches of AM fungal virtual taxa (VT; approximately species-level phylogroups). We found that environmental and spatial variables jointly explained VT distribution worldwide, with temperature and pH being the most important abiotic drivers, and spatial effects generally occurring at local to regional scales. While dispersal limitation could explain some variation in VT distribution, VT relative abundance was almost exclusively driven by environmental variables. Several environmental and spatial effects on VT distribution and relative abundance were correlated with phylogeny, indicating that closely related VT exhibit similar niche optima and widths. Major clades within the Glomeraceae exhibited distinct niche optima, Acaulosporaceae generally had niche optima in low pH and low temperature conditions, and Gigasporaceae generally had niche optima in high precipitation conditions. Identification of the realised niche space occupied by individual and phylogenetic groups of soil microbial taxa provides a basis for building detailed hypotheses about how soil communities respond to gradients and manipulation in ecosystems worldwide.

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.

Structure and function of the global topsoil microbiome.

Soils harbour some of the most diverse microbiomes on Earth and are essential for both nutrient cycling and carbon storage. To understand soil functioning, it is necessary to model the global distribution patterns and functional gene repertoires of soil microorganisms, as well as the biotic and environmental associations between the diversity and structure of both bacterial and fungal soil communities1-4. Here we show, by leveraging metagenomics and metabarcoding of global topsoil samples (189 sites, 7,560 subsamples), that bacterial, but not fungal, genetic diversity is highest in temperate habitats and that microbial gene composition varies more strongly with environmental variables than with geographic distance. We demonstrate that fungi and bacteria show global niche differentiation that is associated with contrasting diversity responses to precipitation and soil pH. Furthermore, we provide evidence for strong bacterial-fungal antagonism, inferred from antibiotic-resistance genes, in topsoil and ocean habitats, indicating the substantial role of biotic interactions in shaping microbial communities. Our results suggest that both competition and environmental filtering affect the abundance, composition and encoded gene functions of bacterial and fungal communities, indicating that the relative contributions of these microorganisms to global nutrient cycling varies spatially.

Host preference and network properties in biotrophic plant-fungal associations.

Analytical methods can offer insights into the structure of biological networks, but mechanisms that determine the structure of these networks remain unclear. We conducted a synthesis based on 111 previously published datasets to assess a range of ecological and evolutionary mechanisms that may influence the plant-associated fungal interaction networks. We calculated the relative host effect on fungal community composition and compared nestedness and modularity among different mycorrhizal types and endophytic fungal guilds. We also assessed how plant-fungal network structure was related to host phylogeny, environmental and sampling properties. Orchid mycorrhizal fungal communities responded most strongly to host identity, but the effect of host was similar among all other fungal guilds. Community nestedness, which did not differ among fungal guilds, declined significantly with increasing mean annual precipitation on a global scale. Orchid and ericoid mycorrhizal fungal communities were more modular than ectomycorrhizal and root endophytic communities, with arbuscular mycorrhizal fungi in an intermediate position. Network properties among a broad suite of plant-associated fungi were largely comparable and generally unrelated to phylogenetic distance among hosts. Instead, network metrics were predominantly affected by sampling and matrix properties, indicating the importance of study design in properly inferring ecological patterns.

Tree diversity and species identity effects on soil fungi, protists and animals are context dependent.

Plant species richness and the presence of certain influential species (sampling effect) drive the stability and functionality of ecosystems as well as primary production and biomass of consumers. However, little is known about these floristic effects on richness and community composition of soil biota in forest habitats owing to methodological constraints. We developed a DNA metabarcoding approach to identify the major eukaryote groups directly from soil with roughly species-level resolution. Using this method, we examined the effects of tree diversity and individual tree species on soil microbial biomass and taxonomic richness of soil biota in two experimental study systems in Finland and Estonia and accounted for edaphic variables and spatial autocorrelation. Our analyses revealed that the effects of tree diversity and individual species on soil biota are largely context dependent. Multiple regression and structural equation modelling suggested that biomass, soil pH, nutrients and tree species directly affect richness of different taxonomic groups. The community composition of most soil organisms was strongly correlated due to similar response to environmental predictors rather than causal relationships. On a local scale, soil resources and tree species have stronger effect on diversity of soil biota than tree species richness per se.

FUNGAL BIOGEOGRAPHY. Response to Comment on "Global diversity and geography of soil fungi".

Schadt and Rosling (Technical Comment, 26 June 2015, p. 1438) argue that primer-template mismatches neglected the fungal class Archaeorhizomycetes in a global soil survey. Amplicon-based metabarcoding of nine barcode-primer pair combinations and polymerase chain reaction (PCR)-free shotgun metagenomics revealed that barcode and primer choice and PCR bias drive the diversity and composition of microorganisms in general, but the Archaeorhizomycetes were little affected in the global study. We urge that careful choice of DNA markers and primers is essential for ecological studies using high-throughput sequencing for identification.

Fungal biogeography. Global diversity and geography of soil fungi.

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 biogeography of Alnus-associated Frankia actinobacteria.

Macroecological patterns of microbes have received relatively little attention until recently. This study aimed to disentangle the determinants of the global biogeographic community of Alnus-associated actinobacteria belonging to the Frankia alni complex. By determining a global sequence similarity threshold for the nitrogenase reductase (nifH) gene, we separated Frankia into operational taxonomic units (OTUs) and tested the relative effects of Alnus phylogeny, geographic relatedness, and climatic and edaphic variables on community composition at the global scale. Based on the optimal nifH gene sequence similarity threshold of 99.3%, we distinguished 43 Frankia OTUs from root systems of 22 Alnus species on four continents. Host phylogeny was the main determinant of Frankia OTU-based community composition, but there was no effect on the phylogenetic structure of Frankia. Biogeographic analyses revealed the strongest cross-continental links over the Beringian land bridge. Despite the facultative symbiotic nature of Frankia, phylogenetic relations among Alnus species play a prominent role in structuring root-associated Frankia communities and their biogeographic patterns. Our results suggest that Alnus species exert strong phylogenetically determined selection pressure on compatible Actinobacteria.

Biogeography of ectomycorrhizal fungi associated with alders (Alnus spp.) in relation to biotic and abiotic variables at the global scale.

· Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa. · This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods. · Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness. · Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.

Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi.

We aimed to enhance understanding of the molecular diversity of arbuscular mycorrhizal fungi (AMF) by building a new global dataset targeting previously unstudied geographical areas. In total, we sampled 96 plant species from 25 sites that encompassed all continents except Antarctica. AMF in plant roots were detected by sequencing the nuclear SSU rRNA gene fragment using either cloning followed by Sanger sequencing or 454-sequencing. A total of 204 AMF phylogroups (virtual taxa, VT) were recorded, increasing the described number of Glomeromycota VT from 308 to 341 globally. Novel VT were detected from 21 sites; three novel but nevertheless widespread VT (Glomus spp. MO-G52, MO-G53, MO-G57) were recorded from six continents. The largest increases in regional VT number were recorded in previously little-studied Oceania and in the boreal and polar climatic zones - this study providing the first molecular data from the latter. Ordination revealed differences in AM fungal communities between different continents and climatic zones, suggesting that both biogeographic history and environmental conditions underlie the global variation of those communities. Our results show that a considerable proportion of Glomeromycota diversity has been recorded in many regions, though further large increases in richness can be expected in remaining unstudied areas.

Infrageneric variation in partner specificity: multiple ectomycorrhizal symbionts associate with Gnetum gnemon (Gnetophyta) in Papua New Guinea.

Majority of autotrophic plants and fungi associate with multiple mycorrhizal partners, with notable exceptions being Gnetum africanum, Pisonia grandis, and Alnus spp from the phytobiont perspective. We hypothesized that an understorey tree species Gnetum gnemon hosts a narrow range of mycobionts as shown in G. africanum and suggested for South American species. Sampling and molecular analysis of G. gnemon root tips revealed that besides Scleroderma spp. this gymnosperm tree associates with several fungal species from unrelated lineages. However, all Scleroderma isolates that associate with Gnetum spp. belong to a narrow clade close to Scleroderma sinnamariense. Our results demonstrate for the first time that specificity for mycobionts may substantially differ within an ectomycorrhizal plant genus.

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi.

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.

Regional and local patterns of ectomycorrhizal fungal diversity and community structure along an altitudinal gradient in the Hyrcanian forests of northern Iran.

• Altitudinal gradients strongly affect the diversity of plants and animals, yet little is known about the altitudinal effects on the distribution of microorganisms, including ectomycorrhizal fungi. • By combining morphological and molecular identification methods, we addressed the relative effects of altitude, temperature, precipitation, host community and soil nutrient concentrations on species richness and community composition of ectomycorrhizal fungi in one of the last remaining temperate old-growth forests in Eurasia. • Molecular analyses revealed 367 species of ectomycorrhizal fungi along three altitudinal transects. Species richness declined monotonically with increasing altitude. Host species and altitude were the main drivers of the ectomycorrhizal fungal community composition at both the local and regional scales. The mean annual temperature and precipitation were strongly correlated with altitude and accounted for the observed patterns of richness and community. • The decline of ectomycorrhizal fungal richness with increasing altitude is consistent with the general altitudinal richness patterns of macroorganisms. Low environmental energy reduces the competitive ability of rare species and thus has a negative effect on the richness of ectomycorrhizal fungi. Because of multicollinearity with altitude, the direct effects of climatic variables and their seasonality warrant further investigation at the regional and continental scales.

A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi.

Ectomycorrhizal fungi (EcMF) form diverse communities and link different host plants into mycorrhizal networks, yet little is known about the magnitude of mycobiont diversity of a single tree individual. This study addresses species richness and spatial structure of EcMF in the root system of a single European aspen (Populus tremula) individual in an old-growth boreal mixed forest ecosystem in Estonia. Combining morphological and molecular identification methods for both plant and fungi, 122 species of EcMF were recovered from 103 root samples of the single tree. Richness estimators predicted the total EcMF richness to range from 182 to 207 species, reflecting the observation of 62.3% singletons and doubletons within the community. Fine-scale genetic diversity in Cenococcum geophilum indicates the presence of 23 internal transcribed spacer genotypes. EcMF community was significantly spatially autocorrelated only at the lineage level up to 3 m distance, but not at the species level. Proximity of other hosts had a significant effect on the spatial distribution of EcMF lineages. This study demonstrates that a single tree may host as many EcMF species and individuals as recovered on multiple hosts in diverse communities over larger areas.