Effects of operational taxonomic unit inference methods on soil microeukaryote community analysis using long-read metabarcoding

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large-scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsangen Nature Reserve has drawn frequent visitors since Linnaeus's time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic-dry part that has a species rich grass-dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%-40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data

Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.publishedVersio

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

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

Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5

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

Connecting the multiple dimensions of global soil fungal diversity

15 páginas.- 5 figuras.- 99 referenciasHow 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.This work was supported by the Estonian Science Foundation: PRG632 (to L.T.), Estonian Research Council: PRG1615 (to R.D.), Estonian Research Council: PRG1170 (to U.K. and Ka.Po.), Estonian Science Foundation: MOBTP198 (to St.An.), Novo Nordisk Fonden: NNF20OC0059948 (to L.T.), Norway-Baltic financial mechanism: EMP442 (to L.T., K.-A.B., and M.T.), King Saud University: DFSP-2020-2 (to L.T.), King Saud University: Highly Cited Program (to L.T.), European Regional Development Fund: Centre of Excellence EcolChange TK131 (to M.O., M.Z., Ü.M., U.K., and M.E.), Estonian Research Council: PRG1789 (to M.O. and I.H.), British Ecological Society: LRB17\1019 (MUSGONET) (to M.D.-B.), Spanish Ministry of Science and Innovation: PID2020-115813RA-I00 (to M.D.-B.), Spanish Ministry of Science and Innovation: SOIL4GROWTH (to M.D.-B.), Marie Sklodowska-Curie: 702057 (CLIMIFUN) (to M.D.- B.), European Research Council (ERC): grant 647038 [BIODESERT] (to F.T.M.), Generalitat Valenciana: CIDEGENT/2018/041 (to F.T.M.), Spanish Ministry of Science and Innovation: EUR2022-134048 (to F.T.M.), Estonian Research Council: PRG1065 (to M.M. and M.Z.), Swedish Research Council Formas: 2020-00807 (to Mo.Ba.), Swedish Research Council: 2019-05191 (to Al. An.), Swedish Foundation for Strategic Environmental Research MISTRA: Project BioPath (to Al. An.), Kew Foundation (to Al.An.), EEA Financial Mechanism Baltic Research Programme in Estonia: EMP442 (to Ke.Ar. and Je.An.), Ghent University Special Research Fund (BOF): Metusalem (to N.S.), Estonian Research Council: PSG825 (to K.R.), European Research Council (ERC): 101096403 (MLTOM23415R) (to Ü.M.), European Regional Development Fund (ERDF): 1.1.1.2/VIAA/2/18/298 (to D.K.), Estonian Research Council: PUT1170 (to I.H.), German Federal Ministry of Education and Research (BMBF): 01DG20015FunTrAf (to K.T.I., M.P., and N.Y.), Proyecto SIA: SA77210019 (ANID—Chile) (to C.M.), Fondecyt: 1190642 (ANID—Chile) (to R.G.), European Research Council (ERC): Synergy Grant 856506—LIFEPLAN (to T.R.), Academy of Finland: grant 322266 (to T.R.), U.S. National Science Foundation: DEB-0918591 (to T.H.), U.S. National Science Foundation: DEB-1556338 (to T.H.), U.S. National Science Foundation: DEB 1737898 (to G.B.), UNAM-PAPIIT: IV200223 (to R.G.-O.), Czech Science Foundation: 21-26883S (to J.D.), Estonian Research Council: PRG352 (to M.E.), NERC core funding: the BAS Biodiversity, Evolution and Adaptation Team (to K.K.N.), NERC-CONICYT: NE/P003079/1 (to E.M.B.), Carlsberg Foundation: CF18-0267 (to E.M.B.), Qatar Petroleum: QUEX-CAS-QP-RD-18/19 (to Ju.Al.), Russian Ministry of Science and Higher Education: 075-15-2021-1396 (to V.F. and V.O.), Secretaria de Ciencia y Técnica (SECYT) of Universidad Nacional de Córdoba and CONICET (to E.N.), HighLevel Talent Recruitment Plan of Yunnan Province 2021:“High-End Foreign Experts” (to Pe.Mo.), AUA grant from research council of UAE University: G00003654 (to S.M.), Ghent University: Bijzonder Onderzoeksfonds (to A.V.), Ghent University: Bijzonder Onderzoeksfonds (BOF-PDO2017-001201) (to E.D.C.), Ghent University: The Faculty Committee Scientific Research, FCWO (to E.D.C. and A.V.), The King Leopold III Fund for Nature Exploration and Conservation (to A.V. and E.D.C.), The Research Foundation—Flanders (FWO) (to E.D.C. and A.V.), The High-Level Talent Recruitment Plan of Yunnan Provinces: “Young Talents” Program (to D.-Q.D.), The HighLevel Talent Recruitment Plan of Yunnan Provinces: “High-End Foreign Experts" Program (to N. N.W.), IRIS scholarship for progressive and ambitious women (to L.H.), Estonian University of Life Sciences: P190250PKKH (to Kr.Pa.), Hungarian Academy of Sciences: Lendület Programme (96049) (to J.G.), Eötvös Loránd Research Network (to J.G.), Botswana International University of Science and Technology (to C.N.), and Higher Education Commision (HEC, Islamabad, Pakistan): Indigenous and International research support initiative program (IRSIP) scholarship (to M.S.)Peer reviewe

Fungal communities of West African ectomycorrhizal woodlands

Ectomycorrhizal symbiosis is a widespread mutualist relationship between fungi and plants, often trees. It is most well-known in temperate and boreal forests, but also occurs in a variety of tropical ecosystems, including Sudanian savanna woodlands and gallery forests in West Africa. In addition to their important role in nutrient cycling, many ectomycorrhizal fungi also produce edible mushrooms as their fruit-bodies. In this thesis, I explore spatial patterns of above-ground and below-ground diversity in West African ectomycorrhizal communities, as well as the use of wild edible fungi by the local human population. In Papers I and II, I used soil DNA metabarcoding to investigate fungal communities in their vegetative state. Paper I used measurements of the scale of spatial autocorrelation within the fungal community in Sudanian woodlands in Benin as a test to compare different high-throughput DNA sequencing strategies, including short (≈350 bp) amplicon barcoding using the Illumina, Ion Torrent, and PacBio sequencing platforms, as well as long (≈1550 bp) amplicon barcoding using PacBio. There were some differences in species richness and community composition recovered by the two amplicon lengths, attributable to known primer biases for the short amplicons, but these did not lead to different ecological results. Additionally, Paper I introduced new software packages for analysis of long-amplicon metabarcoding data and integrating phylogenetic information into sequence-based taxonomic identification. In Paper II we sampled both Sudanian woodland and gallery forest sites in five countries across West Africa using long-amplicon metabarcoding with PacBio. We found significant differences in fungal community composition between Sudanian woodland and gallery forest sites, but not between different tree species within each vegetation type. Papers III and IV focused above-ground to study the natural production and human use of the mushrooms themselves. In Paper III we exhaustively collected mushrooms from nine Sudanian woodland plots in Benin during three consecutive rainy seasons. We measured the total biomass produced of each morphospecies, as well as environmental variables related to microclimate, host tree availability, and soil chemistry. Mushroom production and diversity were negatively correlated with soil nitrogen levels, and positively correlated with soil phosphorus levels. Although there were no clear differences in the fungal communities associated with the four host trees present in our plots, greater host tree diversity was associated with greater fungal diversity and productivity. Finally, Paper IV combined interviews with local people from four ethnic groups in five villages near the study sites from Paper III about their knowledge and preferences for edible mushrooms with DNA barcoding of specimens. Knowledge and preferences for different mushrooms varied between ethnic groups, but people living in a village where their ethnic group is a minority tended to absorb knowledge about mushrooms from the majority group in the village. Women over age 35 were the most knowledgeable about mushrooms, but the degree of gender difference varied between groups. We compiled a list of the most choice edible mushrooms in the area, which can inform the possibility for commercial trade in wild mushrooms to supplement the income of rural people and encourage sustainable forest management

Long- and short-read metabarcoding technologies reveal similar spatiotemporal structures in fungal communities

Fungi form diverse communities and play essential roles in many terrestrial ecosystems, yet there are methodological challenges in taxonomic and phylogenetic placement of fungi from environmental sequences. To address such challenges, we investigated spatiotemporal structure of a fungal community using soil metabarcoding with four different sequencing strategies: short-amplicon sequencing of the ITS2 region (300-400 bp) with Illumina MiSeq, Ion Torrent Ion S5 and PacBio RS II, all from the same PCR library, as well as long-amplicon sequencing of the full ITS and partial LSU regions (1200-1600 bp) with PacBio RS II. Resulting community structure and diversity depended more on statistical method than sequencing technology. The use of long-amplicon sequencing enables construction of a phylogenetic tree from metabarcoding reads, which facilitates taxonomic identification of sequences. However, long reads present issues for denoising algorithms in diverse communities. We present a solution that splits the reads into shorter homologous regions prior to denoising, and then reconstructs the full denoised reads. In the choice between short and long amplicons, we suggest a hybrid approach using short amplicons for sampling breadth and depth, and long amplicons to characterize the local species pool for improved identification and phylogenetic analyses