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Parsing ecological signal from noise in next generation amplicon sequencing

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... Such positive control is called a "mock community". Adding a mock community to a sequencing run can validate whether the expected abundances are recovered, and thus it can improve the quality of the sequencing results (Nguyen et al., 2015). A diverse mock community is preferred, meaning that it consists of a wide range of taxonomic groups (Flynn et al., 2015;Nguyen et al., 2015). ...
... Adding a mock community to a sequencing run can validate whether the expected abundances are recovered, and thus it can improve the quality of the sequencing results (Nguyen et al., 2015). A diverse mock community is preferred, meaning that it consists of a wide range of taxonomic groups (Flynn et al., 2015;Nguyen et al., 2015). Recently, a mock community comprised of representatives of 374 insect species has been assembled (Braukmann et al., 2019). ...
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This study aimed to establish a representative strain collection of dominant aerobic bacteria from black soldier fly larvae ( Hermetia illucens , BSFL). The larvae were fed either chicken feed or fiber-rich substrates to obtain a collection of BSFL-associated microorganisms. Via an approach based on only considering the highest serial dilutions of BSFL extract (to select for the most abundant strains), a total of 172 bacteria were isolated. Identification of these isolates revealed that all bacteria belonged to either the Proteobacteria (66.3%), the Firmicutes (30.2%), the Bacteroidetes (2.9%) or the Actinobacteria (0.6%). Twelve genera were collected, with the most abundantly present ones (i.e., minimally present in at least three rearing cycles) being Enterococcus (29.1%), Escherichia (22.1%), Klebsiella (19.8%), Providencia (11.6%), Enterobacter (7.6%), and Morganella (4.1%). Our collection of dominant bacteria reflects largely the bacterial profiles of BSFL already described in literature with respect to the most important phyla and genera in the gut, but some differences can be noticed depending on substrate, biotic and abiotic factors. Furthermore, this bacterial collection will be the starting point to improve in vitro digestion models for BSFL, to develop mock communities and to find symbionts that can be added during rearing cycles to enhance the larval performances, after functional characterization of the isolates, for instance with respect to enzymatic potential.
... Between each amplification step, the product was cleaned from reagents and primers using the NGS SPRI Bead clean-up kit (ABM). Negative PCR controls were sequenced along with the experimental samples [28]. We included a synthetic mock community for ITS1 as a positive control to recover tag-switching among samples [29]. ...
... We quality filtered the demultiplexed raw sequences (q > 30) setting the truncation of sequences 240 bp for forward and 200 bp for reverse sequences, and denoised the raw reads with DADA2 [32]. After quality filtering, we de novo clustered sequences into operational taxonomic units (OTU) with VSEARCH at a 97% sequence similarity followed by a re-clustering step with UCLUST at 97% to ensure the best recovery of our OTUs according to our input mock community [28]. We then excluded all sequences that were ≤ 85% coverage in the BLAST search since this primer set amplifies sequences that only partially match to a small part of the ITS gene. ...
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Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 °C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.
... High throughput sequencing has large biases due to PCR conditions and sequencing bioinformatics process. This is why we chose a strong singleton filter per sampling site to improve data reliability (Dickie, 2010;Nguyen et al., 2015a). The Molecular Operational Taxonomic Units (MOTU) table per sample was constructed using the pipeline reported in Bahram et al. (2016). ...
... Then, each MOTU was compared using Blast (Altschul et al., 1990) against the UNITE Database (version 7.2) (Abarenkov et al., 2010;Kõljalg et al., 2013;UNITE-Community, 2017). All fungal species with less than 10 sequences and the sequence counts found in negative controls and the Mock-community were removed by substracting them from each cell in the OTU table (Dickie, 2010;Hart et al., 2015;Nguyen et al., 2015a). ...
Article
Abies religiosa's forests are severely endangered as a result of climate change; to save this species and its biological interactions, population assisted migration is discussed in forest management, but not in the microbial ecology field. Our objectives were to analyze its mycorrhizal networks; and, with this data, to identify potential facilitator plants and it's most important mycorrhizal fungal links. This information could be used together in assisted migration programs to connect Abies religiosa saplings to their mycorrhizal network and improve their field establishment. We collected 47 rhizosphere samples from 19 plant species and sequenced their fungal ITS2 region by Illumina. In the whole fungal community, 464 species were mycorrhizal fungi with assigned guild (32%). In this subset, 85 fungi are arbuscular, 365 ectomycorrhizal and 14 from orchid-mycorriza. The Abies religiosa bipartite network is low nested and highly modular, and has a scale-free architecture. Besides Abies religiosa, the plants with the largest degree and the lowest average shortest path were Salix paradoxa, Muhlenbergia spp., and Baccharis conferta. The most important fungal nodes are species of Cortinarius, Genea, Rhodoscypha, Russula, and Tomentella. We suggest to evaluate the Abies' future establishment in the following scheme: in the first year reintroduce Muhlenbergia spp., and Baccharis conferta, in the second year Salix paradoxa, and in the third year–once the mycorrhizal network is reestablished– Abies religiosa' saplings in close proximity of these plants. This scheme is proposed using the data and network analyses of the present study. Authorized share link: https://authors.elsevier.com/a/1evxY,XbFFXEDn
... Our sequencing run included SynMock (Palmer et al., 2018), an equimolar spiked-in mock community control consisting of non-biological synthetic ITS sequences, and this control was used to parameterize our bioinformatics pipeline. The mock community allowed us to cluster and estimate our operational taxonomic units (OTUs) based on realistic parameters, an important and necessary component to any HTAS study of environmental samples (Nguyen et al., 2015;Palmer et al., 2018;Jusino et al., 2019). The synthetic mock community allows reliable detection of index bleed/barcode crossover, a documented problem across HTAS platforms (Schnell et al., 2015;Palmer et al., 2018). ...
... Additional issues such as chimera formation, index/barcode bleed, and lab contamination can also inflate estimates of diversity. Therefore, proper controls need to be taken in the field and laboratory, and positive mock community controls should be used to help parameterize downstream bioinformatics (Nguyen et al., 2015;Palmer et al., 2018;Jusino et al., 2019). ...
Article
Wood-decay fungi soften wood, putatively providing opportunities for woodpeckers to excavate an otherwise hard substrate, yet the fungal community composition in tree cavities and the specificity of these relationships is largely unknown. We used high-throughput amplicon sequencing of the fungal ITS2 region to examine the fungal communities associated with acorn woodpeckers (Melanerpes formicivorus) and their cavities in mature valley oak (Quercus lobata) and blue oak (Q. douglasii) trees in an oak savannah of central coastal California, USA. Acorn woodpeckers and their excavations harbored over 1500 fungal taxa, including more than 100 putative wood-decay fungi. The fungal communities found on the birds were more similar to those found in excavated cavities than those found in trees without excavated holes. These results suggest that symbiotic associations between acorn woodpeckers and fungi are highly diverse, with low specificity. Symbiotic associations between cavity-excavators and fungi are likely more common and widespread than previously thought.
... DNA was extracted from 0.25 g of frozen soil using the DNeasy PowerSoil kit (QIAGEN, Germany) following the manufacturer's standard protocol. Fungal and bacterial mock communities served as positive controls (45). Primer design and sample barcoding followed a two-step amplification, dual barcoding system using a combination of a P5/P7 Illumina Adapter, 8 bp barcodes attached to each adapter, and partial P5/P7 overhangs (Supplementary Figure 1). ...
... Furthermore, DADA2 ASVs overinflated fungal mock community richness (multiple OTUs per species) so it was necessary to further cluster these OTUs into 97% similarity OTUs using open reference clustering via the VSEARCH plugin (53). This clustering was essential to accurately recover the expected mock community diversity in the dataset (45). Hereafter both 16S DADA2 ASVs and ITS 97% clustered OTUs will be referred to simply as OTUs. ...
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Our current understanding suggests that nutrient management strategies applied to agricultural soils over multiple years are required to cause major and stable shifts in soil microbial communities. However, some studies suggest that agricultural soils can benefit even from sporadic, single additions of organic matter. Here we investigate how single additions of high-quality organic matter can cause significant shifts in microbial soil communities over multiple cropping cycles. We grew radishes in a tropical Oxisol soil for six crop cycles after a single application of a high-nitrogen compost or urea. At planting and before biomass harvest, we sampled soils influenced by the radish rhizosphere and sequenced bacterial and archaeal 16S and fungal ITS rDNA marker genes. We measured microbial richness and diversity, community composition and structure, and constructed correlation networks to predict cross-domain microbial interactions. We found that a single application of compost, compared to urea or control, resulted in a persistent improved plant biomass response and led to sustained changes in the soil microbial community throughout the duration of the 227-day study. Compost altered the structure of both the fungal and prokaryotic microbial communities, introduced new microorganisms that persisted in the resident soil system, and altered soil microbial correlation network structure and hub taxa. In contrast, fertilization with urea did not significantly alter the structure of soil microbial communities compared to the control but reduced network complexity and altered hub taxa. This study highlights the significant impacts that high-quality organic matter fertilization can exert on agricultural soil microbiomes and adds to the growing body of knowledge on using organic fertilizers as a way to steer the soil microbiome toward a healthier soil.
... Prediction of functional bacterial and fungal diversity within the 16S rRNA and ITS2 sequence libraries was performed using PICRUSt (Langille et al., 2013) and FUNGuild (Nguyen et al., 2015), respectively. PICRUSt predicts the potential metagenomic gene content of a 16S amplicon library based on genomic information of the bacteria represented within the Greengenes 16S database. ...
... FUNGuild assigns trophic modes to fungal taxa based on a comparison to a curated database of fungal lifestyles (sensu Tedersoo et al., 2014): pathotroph, symbiotroph, and saprotroph. Trophic mode refers to the mechanisms through which organisms obtain resources, providing putative information on the ecology of such organisms (Nguyen et al., 2015). Functional assignments through FUNGuild are based on taxonomy and are possible only if the taxa have been classified at the genus level or if the taxa belong to a fungal group with an exclusive lifestyle. ...
Article
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Maize is one of the most important crops worldwide and is the number one arable crop in Portugal. A transition from the conventional farming system to organic agriculture requires optimization of cultivars and management, the interaction of plant–soil rhizosphere microbiota being pivotal. The objectives of this study were to unravel the effect of population genotype and farming system on microbial communities in the rhizosphere of maize. Rhizosphere soil samples of two open-pollinated maize populations (“SinPre” and “Pigarro”) cultivated under conventional and organic farming systems were taken during flowering and analyzed by next-generation sequencing (NGS). Phenological data were collected from the replicated field trial. A total of 266 fungi and 317 bacteria genera were identified in “SinPre” and “Pigarro” populations, of which 186 (69.9%) and 277 (87.4%) were shared among them. The microbiota of “Pigarro” showed a significant higher (P < 0.05) average abundance than the microbiota of “SinPre.” The farming system had a statistically significant impact (P < 0.05) on the soil rhizosphere microbiota, and several fungal and bacterial taxa were found to be farming system-specific. The rhizosphere microbiota diversity in the organic farming system was higher than that in the conventional system for both varieties. The presence of arbuscular mycorrhizae (Glomeromycota) was mainly detected in the microbiota of the “SinPre” population under the organic farming systems and very rare under conventional systems. A detailed metagenome function prediction was performed. At the fungal level, pathotroph–saprotroph and pathotroph–symbiotroph lifestyles were modified by the farming system. For bacterial microbiota, the main functions altered by the farming system were membrane transport, transcription, translation, cell motility, and signal transduction. This study allowed identifying groups of microorganisms known for their role as plant growth-promoting rhizobacteria (PGPR) and with the capacity to improve crop tolerance for stress conditions, allowing to minimize the use of synthetic fertilizers and pesticides. Arbuscular mycorrhizae (phyla Glomeromycota) were among the most important functional groups in the fungal microbiota and Achromobacter, Burkholderia, Erwinia, Lysinibacillus, Paenibacillus, Pseudomonas, and Stenotrophomonas in the bacterial microbiota. In this perspective, the potential role of these microorganisms will be explored in future research.
... There is a rise in the implementation of molecular-based approaches using DNA meta-barcoding of environmental samples (including faeces and gut contents), though they have not yet been widely employed in mycophagy studies (see: Nuske et al. 2019, Cloutier et al. 2019, Hopkins et al. 2021, Bradshaw et al. 2022. Detailed guidelines for fungal meta-barcoding are becoming abundant (see: Nguyen et al. 2015, Tedersoo & Lindahl 2016, Nilsson et al. 2019, and we strongly encourage researchers to standardise and publish detailed laboratory and bioinformatic protocols to make studies comparable between animal species and regions. Because of PCR biases toward certain fungal groups during the preparation of library amplicons, sequence abundance from next generation sequencing platforms is not directly equivalent to species, relative abundance and needs to be interpreted with caution (Pickles et al. 2020); this thus hinders detailed diet quantification. ...
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The consumption of fungi by animals is a significant trophic interaction in most terrestrial ecosystems, yet the role mammals play in these associations has been incompletely studied. In this review, we compile 1 154 references published over the last 146 years and provide the first comprehensive global review of mammal species known to eat fungi (508 species in 15 orders). We review experimental studies that found viable fungal inoculum in the scats of at least 40 mammal species, including spores from at least 58 mycorrhizal fungal species that remained viable after ingestion by mammals. We provide a summary of mammal behaviours relating to the consumption of fungi, the nutritional importance of fungi for mammals, and the role of mammals in fungal spore dispersal. We also provide evidence to suggest that the morphological evolution of sequestrate fungal sporocarps (fruiting bodies) has likely been driven in part by the dispersal advantages provided by mammals. Finally, we demonstrate how these interconnected associations are widespread globally and have far-reaching ecological implications for mammals, fungi and associated plants in most terrestrial ecosystems.
... For both bacteria and fungi, the number of sequences of each OUT present in the negative controls (PCR and DNA extraction kits) were manually subtracted from the total sequence abundance of that OUT in the experimental samples using R. This approach enables ecologically relevant OTUs to be retained while accounting for contamination and limiting noise associated with the sequence datasets (Nguyen et al. 2015). ...
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Manganese (Mn) is an essential plant micronutrient that influences photosynthesis, ecosystem productivity, and soil carbon storage. Our objective was to quantify how Mn uptake by forest vegetation relates to Mn release into soil solution through mineral dissolution. A greenhouse pot experiment was conducted to quantify Mn uptake by red maple saplings as a function of mineral solubility to test whether Mn uptake was limited by the supply of Mn to soil solution. Differences in soil microbial community composition between treatments, particularly amongst Mn cycling bacteria and fungi, were also evaluated to assess potential microbial impacts on observed Mn fluxes. Plant Mn uptake was highest in systems supplied with dissolved Mn(II) because it was not kinetically limited by mineral weathering. Mn uptake was also higher in systems supplied with a fast-weathering substrate (shale containing Mn(II)-bearing pyrite) than a slow-weathering substrate (Mn(IV)-oxide). However, vegetation accumulated a decreasing proportion of available Mn with increasing solubility, indicating that uptake was tempered relative to solubility. The presence of bacterial phyla containing known Mn-oxidizing bacteria indicates the potential for Mn-oxidizing bacteria to influence Mn solubility within Mn-oxide and dissolved Mn treatments. A relatively low abundance of these bacteria points to their limited capacity to oxidize and retain Mn in the shale treatments, consistent with substantial Mn leaching. We conclude that Mn uptake was primarily controlled by dissolution rates of Mn-bearing minerals but modified by biological processes. The quantitative framework presented here can guide understanding of how biogeochemical processes control element cycling between plants and soils.
... Sequences were demultiplexed and quality filtered (i.e. one mismatch per barcode was accepted, sequences with mean quality score < 20 with quality score window size of 50 bp and sequences shorter than 150 bp were removed along with primers and adapters). We employed a double de novo operational taxonomic unit (OTU) picking (Nguyen et al., 2015), first removing chimeras and clustering all sequences using USEARCH 6.1 (Edgar, 2010) followed by second clustering with UCLUST (Edgar, 2010) (at 97% sequence similarity level in QIIME). Taxonomy for each representative sequence was assigned using BLAST (Altschul et al., 1990) against dynamic release of UNITE v.7.0 (Kõljalg et al., 2013) with 90% similarity, and for the most common OTUs (over 1000 sequence reads) we also performed a second BLAST against National Center for Biotechnology Information GenBank with 95% similarity. ...
Article
Composition and functioning of arctic soil fungal communities may alter rapidly due to the ongoing trends of warmer temperatures, shifts in nutrient availability and shrub encroachment. In addition, the communities may also be intrinsically shaped by heavy grazing, which may locally induce an ecosystem change that couples with increased soil temperature and nutrients and where shrub encroachment is less likely to occur than in lightly grazed conditions. We tested how four years of experimental warming and fertilization affected organic soil fungal communities in sites with decadal history of either heavy or light reindeer grazing using high‐throughput sequencing of ITS2 rDNA region. Grazing history largely overrode the impacts of short‐term warming and fertilization in determining the composition of fungal communities. The less diverse fungal communities under light grazing showed more pronounced responses to experimental treatments when compared to the communities under heavy grazing. Yet, ordination approaches revealed distinct treatment responses under both grazing intensities. If grazing shifts the fungal communities in Arctic ecosystems to a different and more diverse state, this shift may dictate ecosystem responses to further abiotic changes. This inclines that the intensity of grazing cannot be left out when predicting future changes in fungi‐driven processes in the tundra.
... While most NGS platforms have some data analysis functionality, the automated bioinformatics workflow is seldom adequate or directly applicable to specific cases (Ahmed, 2016). The limitations can provide an over-or underestimated fungal community, depending on the region of the genome and the primers used for amplification (Tedersoo et al., 2015), the platform used for sequencing (with Illumina platforms dominating the market), and the method to assemble reads (Nguyen et al., 2015). Furthermore, PCR errors in HTS are common and can produce chimeras, which are caused by the incomplete extension of DNA strands during amplification that make up a recombination between two sequences; this can cause biases in diversity results (Mbareche, 2020). ...
Chapter
The concept of translational research refers to the process of harnessing knowledge from basic sciences to impacts beyond the academy. The process involves the transfer of knowledge over three major gaps: first, from knowledge generation to interpretation; second, from interpretation to implementation; and third, from implementation to impacts. In this chapter, we discuss how this process, usually described for medical and health care research, applies to the current research on the microbiomes of forest trees. The discussion is timely because while scientific knowledge about the potential of microbiome engineering to support tree resistance against pests and pathogens is rapidly increasing, practical solutions based on the utilization of the microbiome in forest protection have remained rare. Using the concept of translational research as a framework, we identify some major challenges in the translational process and suggest strategies to overcome them.
... Importantly, in this review, we do not discuss experimental design (including sample collection and storage [8,9]), laboratory inventory management systems (LIMS) (10), or bioinformatic approaches (e.g., see references [11][12][13][14]. We also do not compare sequencing instruments (including new long-read machines), though the multiplexing advances we describe require the use of the latest generation of short-read sequencers (e.g., the Illumina NovaSeq). ...
Article
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New approaches to characterizing microbiomes via high-throughput sequencing provide impressive gains in efficiency and cost reduction compared to approaches that were standard just a few years ago. However, the speed of method development has been such that staying abreast of the latest technological advances is challenging. Moreover, shifting laboratory protocols to include new methods can be expensive and time consuming. To facilitate adoption of new techniques, we provide a guide and review of recent advances that are relevant for single-locus sequence-based study of microbiomes-from extraction to library preparation-including a primer regarding the use of liquid-handling automation in small-scale academic settings. Additionally, we describe several amendments to published techniques to improve throughput, track contamination, and reduce cost. Notably, we suggest adding synthetic DNA molecules to each sample during nucleic acid extraction, thus providing a method of documenting incidences of cross-contamination. We also describe a dual-indexing scheme for Illumina sequencers that allows multiplexing of many thousands of samples with minimal PhiX input. Collectively, the techniques that we describe demonstrate that laboratory technology need not impose strict limitations on the scale of molecular microbial ecology studies. IMPORTANCE New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.
... Our bioinformatics pipeline, comprising quality filtering and taxonomic assignment, followed that described in the DADA2 ITS Pipeline Workflow V1.81 (https://benjjneb. github.io/dada2/ITS_workflow.html), with the following minor modifications: (1) due to lower quality, reverse reads were not used-discarding low-quality reverse reads is a common strategy that frequently gives better results than assembled reads (Nguyen et al. 2015;Pauvert et al. 2019) and (2) the R package decontam was used to identify and remove any contaminants identified in sequenced negative controls via the prevalence method (Davis et al. 2018). ...
Article
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Despite their recognized importance in terrestrial and aquatic ecosystems, marine fungi, especially those associated with host organisms, remain poorly studied. Corals contain diverse communities of microbes that play important roles in adapting to disturbance and promoting host health, but studies on coral-associated fungal communities are conspicuous by their absence. Here, we comprehensively characterized the fungal communities and diversity associated with 97 unrelated coral colonies of Pocillopora acuta from nine islands in Singapore. We depleted coral DNA prior to PCR amplification of the fungal barcoding region (ITS1) to minimize issues of preferential host DNA amplification. This approach produced over a hundred times greater proportion of reads that were of fungal origin (75%) than previous studies, allowing us to more precisely characterize the associated fungal communities. We found no spatial structuring of fungal communities based upon sampling location, and provide evidence that suggests coral-associated fungi are more than just pathogens within their hosts. Many have been putatively identified as saprotrophs that are likely involved in making nutrients available for their host, and others may be possible symbiotrophs. Characterization of host-associated fungal communities provides valuable understanding—including information on the coral holobiont and how it functions—on a frequently overlooked, yet important microbial group.
... Contaminating DNA can originate from various sources, including sampling procedures and environments [38,59,60], DNA extraction kits [34,35], and laboratory reagents like PCR mastermixes [61]. Cross-contamination occurs from other adjacent samples, sequencing runs, and barcode leakage [62]. Our data indicate that kits or reagents play an important role in the contamination of low biomass samples processed here. ...
Article
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Background The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities. Results We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 10 ⁵ CFU and that Benzonase digest is not sufficient to overcome this bias. Conclusions The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. Graphical abstract
... However, a quantitative meta-analysis found only a weak relationship between the two (Lamb et al., 2019). Read abundance can be profoundly affected by methodological biases at several steps during metabarcoding procedures, starting from the choice of primers through wet-lab methods, including sequencing, to bioinformatic pipelines (Lindahl et al., 2013;Nguyen et al., 2015;Song et al., 2015;Taylor et al., 2016). However, in our study, the main pathways affecting plants directly and not through fungal parameters remained present in both best-fitting models. ...
Article
Arctic plants are affected by many stressors. Root-associated fungi are thought to influence plant performance in stressful environmental conditions. However, the relationships are not well-known; do the number of fungal partners, their ecological functions and community composition mediate the impact of environmental conditions and/or influence host plant performance? To address these questions, we used a common arctic plant as a model system: Bistorta vivipara. Whole plants (including root system, n = 214) were collected from nine locations in Spitsbergen. Morphometric features were measured as a proxy for plant performance and combined with metabarcoding datasets of their root-associated fungi (amplicon sequence variants, ASVs), edaphic and meteorological variables. Seven biological hypotheses regarding fungal influence on plant measures were tested using structural equation modelling. The best-fitting model revealed that local temperature affected plants both directly (negatively aboveground and positively below-ground) and indirectly - mediated by fungal richness and the ratio of symbio- and saprotrophic ASVs. The influence of temperature on host plants is therefore complex and should be examined further. Fungal community composition did not impact plant measurements and plant reproductive investment was not influenced by any fungal parameters. The lack of impact of fungal community composition on plant performance suggests that the functional importance of fungi is more essential for the plant than their identity.
... To identify bacterial communities, we conducted 16S rRNA gene amplicon sequencing of the v4 region using F515 and R806 primers (~250 bp; Caporaso et al., 2010, according to Kozich et al., 2013Griffiths et al., 2018 We identified fungal communities by sequencing the ITS gene using ITS1F and ITS2 primers (~150-500 bp) using a modified protocol of Smith and Peay (2014) and Nguyen et al. (2015), as in Griffiths et al. (2019). We ran PCRs in duplicate using thermocycling conditions of 95°C for 10 min, followed by 28 cycles of 95°C ...
Article
Environmental impacts of the 1986 Chernobyl Nuclear Power Plant accident are much debated, but the effects of radiation on host microbiomes have received little attention to date. We present the first analysis of small mammal gut microbiome from the Chernobyl Exclusion Zone in relation to total absorbed dose rate and including caecum as well as faeces sample. We provide novel evidence that host species determines fungal community composition, and that associations between microbiome (both bacterial and fungal) communities and radiation exposure varies between host species. Using ambient versus total weighted absorbed dose rates in analyses produced different results, with the latter more robust for interpreting microbiome changes at the individual level. We found considerable variation between results for faecal and gut samples of bank voles, suggesting faecal samples are not an accurate indicator of gut composition. Associations between radiation exposure and microbiome composition of gut samples were not robust against geographical variation, although we identified families of bacteria (Lachnospiraceae and Muribaculaceae) and fungi (Steccherinaceae and Strophariaceae) in the guts of bank voles that may serve as biomarkers of radiation exposure. Further studies considering a range of small mammal species are needed to establish the robustness of these potential biomarkers.
... cgi). Finally, the maximum number of sequences of each OTU present in the negative controls (DNA extraction and PCRs) were subtracted from the sequence abundance of that OTU in the experimental samples (Nguyen et al., 2015). The final OTU table had 257 samples and 275 non-chimeric, non-singleton fungal OTUs, representing 4,811,621 quality sequences. ...
Thesis
Perennial plant dieback is an increasing and complex phenomenon. Perennial plants experience many interacting stressing events leading to final plant mortality. These interactions, and how they may change regarding climatic conditions and plant physiological status, are key in understanding the dieback process. Although dieback events are increasing worldwide, the knowledge on the dieback mechanisms are scarce, given the many technical challenges in studying complex interactions. In this thesis, we studied the interaction between two stresses frequently experienced by grapevines, one of the most important perennial crops: drought and esca (a vascular disease). Esca is a disease in which there are many competing hypotheses regarding its pathogenesis. One of the main hypothesis is that leaf symptoms and plant death are caused by hydraulic failure in xylem vessels. For this reason, drought is thought to contribute synergistically with esca to grapevine dieback. In this context, this thesis has primarily explored the hydraulic failure hypothesis during esca pathogenesis. We found that during leaf symptom expression both leaves and stems suffer from hydraulic failure causing (on average) 69% loss of hydraulic conductance in midribs, 55% in petioles, and 30% in stems. Differing from classical air embolism during drought, we observed that hydraulic failure during esca was caused by the presence of plant-derived vascular occlusions (i.e. tyloses and gels) produced at a distance from the pathogen niche in the trunk. After this discovering, we explored the interaction between esca and drought, subjecting naturally infected plants to drought. We found that drought totally inhibits esca leaf symptoms, as none of the plants under water deficit (at ΨPD ≈ -1MPa for three months) expressed leaf symptoms in two consecutive seasons. At the same time, in order to understand the interaction between esca and drought, we recorded the whole-plant water relations and carbon economy of grapevine under both stresses. We highlight the distinct physiology behind these two stresses, indicating that esca and drought present different underlying mechanisms, and induce different plant responses and physiological consequences. Esca (and subsequent stomatal conductance decline) does not result from decreases in water potential, and generates different gas exchange and non-structural carbohydrate seasonal dynamics compared to drought. Finally, we observed that esca affected the recorded plant physiology only seasonally, and not over the long-term. This thesis highlights the importance in finding the physiological thresholds triggering the different interactions during plant dieback. Together, the results open new scientific and agronomical perspectives on plant-pathogen-environment interactions and vineyard sustainability.
... As proposed by the authors of DADA2, we worked with ASVs rather than OTUs as (1) it is more beneficial if the merging of sequencing data of different sequencing runs is considered (Callahan et al., 2017) and (2) it is easier to distinguish unique contaminant sequences identified in the control samples. Discarding every single ASV detected in different negative controls would not be preferable, as it might result in the removal of ecologically valid sequences, making the biological interpretation harder (Nguyen et al., 2015). Therefore, only ASVs that were present in negative controls at a minimum of 0.5%, with at least 25 reads were discarded. ...
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The plant microbiome is an increasingly intensive research area, with significance in agriculture, general plant health, and production of bioactive natural products. Correlations between the fungal endophytic communities and plant chemistry can provide insight into these interactions, and suggest key contributors on both the chemical and fungal side. In this study, roots of various horseradish (Armoracia rusticana) accessions grown under the same conditions were sampled in two consecutive years and chemically characterized using a quality controlled, untargeted metabolomics approach by LC-ESI-MS/MS. Sinigrin, gluconasturtiin, glucoiberin, and glucobrassicin were also quantified. Thereafter, a subset of roots from eight accessions (n = 64) with considerable chemical variability was assessed for their endophytic fungal community, using an ITS2 amplicon-based metagenomic approach using a custom primer with high coverage on fungi, but no amplification of host internal transcribed spacer (ITS). A set of 335 chemical features, including putatively identified flavonoids, phospholipids, peptides, amino acid derivatives, indolic phytoalexins, a glucosinolate, and a glucosinolate downstream product was detected. Major taxa in horseradish roots belonged to Cantharellales, Glomerellales, Hypocreales, Pleosporales, Saccharomycetales, and Sordariales. Most abundant genera included typical endophytes such as Plectosphaerella, Thanatephorus, Podospora, Monosporascus, Exophiala, and Setophoma. A surprising dominance of single taxa was observed for many samples. In summary, 35.23% of reads of the plant endophytic fungal microbiome correlated with changes in the plant metabolome. While the concentration of flavonoid kaempferol glycosides positively correlated with the abundance of many fungal strains, many compounds showed negative correlations with fungi including indolic phytoalexins, a putative glucosinolate but not major glucosinolates and a glutathione isothiocyanate adduct. The latter is likely an in vivo glucosinolate decomposition product important in fungal arrest. Our results show the potency of the untargeted metabolomics approach in deciphering plant–microbe interactions and depicts a complex array of various metabolite classes in shaping the endophytic fungal community.
... After dereplication was conducted, the output, a feature table containing amplicon sequence variants (ASVs) and associated abundances, was generated based on forward reads. Chimeric and shifted sequences were removed through DADA2, and ASVs present in the environmental and negative controls were subtracted from all samples as previously described [17,18] using R (Additional file 1: Figure S1). For this purpose, ASVs have an advantage over traditional operating taxonomic units (OTUs) because while OTU-based approaches consolidate similar sequences into consensus units, the ASV approach treats exact sequences as unique units, thereby enabling the removal of contaminating sequences without significantly affecting relevant reads [16]. ...
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Background Currently, over half of breast cancer cases are unrelated to known risk factors, highlighting the importance of discovering other cancer-promoting factors. Since crosstalk between gut microbes and host immunity contributes to many diseases, we hypothesized that similar interactions could occur between the recently described breast microbiome and local immune responses to influence breast cancer pathogenesis. Methods Using 16S rRNA gene sequencing, we characterized the microbiome of human breast tissue in a total of 221 patients with breast cancer, 18 individuals predisposed to breast cancer, and 69 controls. We performed bioinformatic analyses using a DADA2-based pipeline and applied linear models with White’s t or Kruskal–Wallis H -tests with Benjamini–Hochberg multiple testing correction to identify taxonomic groups associated with prognostic clinicopathologic features. We then used network analysis based on Spearman coefficients to correlate specific bacterial taxa with immunological data from NanoString gene expression and 65-plex cytokine assays. Results Multiple bacterial genera exhibited significant differences in relative abundance when stratifying by breast tissue type (tumor, tumor adjacent normal, high-risk, healthy control), cancer stage, grade, histologic subtype, receptor status, lymphovascular invasion, or node-positive status, even after adjusting for confounding variables. Microbiome–immune networks within the breast tended to be bacteria-centric, with sparse structure in tumors and more interconnected structure in benign tissues. Notably, Anaerococcus , Caulobacter , and Streptococcus , which were major bacterial hubs in benign tissue networks, were absent from cancer-associated tissue networks. In addition, Propionibacterium and Staphylococcus , which were depleted in tumors, showed negative associations with oncogenic immune features; Streptococcus and Propionibacterium also correlated positively with T-cell activation-related genes. Conclusions This study, the largest to date comparing healthy versus cancer-associated breast microbiomes using fresh-frozen surgical specimens and immune correlates, provides insight into microbial profiles that correspond with prognostic clinicopathologic features in breast cancer. It additionally presents evidence for local microbial–immune interplay in breast cancer that merits further investigation and has preventative, diagnostic, and therapeutic potential.
... The normalization process should be selected to fit the size and organization of the datasets, as suggested by Weiss et al. (2017). Sufficient technical replicates should be performed in order to quantify sequencing error rates within an assay and between assays (Nguyen et al., 2015;Schloss et al., 2016). ...
Article
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Plants have always grown and evolved surrounded by numerous microorganisms that inhabit their environment, later termed microbiota. To enhance food production, humankind has relied on various farming practices such as irrigation, tilling, fertilization, and pest and disease management. Over the past few years, studies have highlighted the impacts of such practices, not only in terms of plant health or yields but also on the microbial communities associated with plants, which have been investigated through microbiome studies. Because some microorganisms exert beneficial traits that improve plant growth and health, understanding how to modulate microbial communities will help in developing smart farming and favor plant growth-promoting (PGP) microorganisms. With tremendous cost cuts in NGS technologies, metagenomic approaches are now affordable and have been widely used to investigate crop-associated microbiomes. Being able to engineer microbial communities in ways that benefit crop health and growth will help decrease the number of chemical inputs required. Against this background, this review explores the impacts of agricultural practices on soil- and plant-associated microbiomes, focusing on plant growth-promoting microorganisms from a metagenomic perspective.
... Recently, high-throughput sequencing technologies applied to environmental DNA have offered unprecedented insights into this amazing diversity (Roesch et al., 2007). The high volume and accuracy, as well as low-cost, in comparison to traditional culture-based methods provide these technologies tremendous advantages in covering the richness and abundance of various ecosystems (Bokulich et al., 2013;Nguyen et al., 2015). Across all ecosystems, soils harbor the most diverse microbiota from thousands to millions per gram of soil (Gans et al., 2005;Roesch et al., 2007;Schloss and Handelsman, 2006), and thus its diversity is most difficult to be fully characterized. ...
Article
Due to the massive quantity and broad phylogeny, an accurate measurement of microbial diversity is highly challenging in soil ecosystems. Initially, the deviation caused by sampling should be adequately considered. Here, we attempted to uncover the effect of different sampling strategies on α diversity measurement of soil prokaryotes. Four 1 m² sampling quadrats in a typical grassland were thoroughly surveyed through deep 16S rRNA gene sequencing (over 11 million reads per quadrat) with numerous replicates (33 soil sampling cores with total 141 replicates per quadrat). We found the difference in diversity was relatively small when pooling soil cores before and after DNA extraction and sequencing, but they were both superior to a non-pooling strategy. Pooling a small number of soil cores (i.e., 5 or 9) combined with several technical replicates is sufficient to estimate diversities for soil prokaryotes, and there is great flexibility in pooling original samples or data at different experimental steps. Additionally, the distribution of local α diversity varies with sampling core number, sequencing depth, and abundance distribution of the community, especially for high orders of Hill diversity index (i.e., Shannon entropy and inverse Simpson index). For each grassland soil quadrat (1 m²), retaining 100,000 reads after taxonomic clustering might be a realistic option, as these number of reads can efficiently cover the majority of common species in this area. Our findings provide important guidance for soil sampling strategy, and the general results can serve as a basis for further studies.
... Specifically, the restriction of hosts to a single plant family, combined with the relatively rapid speciation of the Asteraceae overall, could have led to an underestimation of host identity importance relative to spatial scale. Additionally, inclusion of negative and positive controls in our sequencing efforts (Nguyen et al., 2015) may have increased the confidence in community differences between host species by more accurately determining the presence or absence of OTUs between samples (Palmer et al., 2018). ...
Article
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Phylogenetic distance among host species represents a proxy for host traits that act as biotic filters to shape host‐associated microbiome community structure. However, teasing apart potential biotic assembly mechanisms, such as host specificity or local species interactions, from abiotic factors, such as environmental specificity or dispersal barriers, in hyperdiverse, horizontally transmitted microbiomes remains a challenge. In this study, we tested whether host phylogenetic relatedness among 18 native Asteraceae plant species and spatial distance between replicated plots in a common garden affects foliar fungal endophyte (FFE) community structure. We found that FFE community structure varied significantly among host species, as well as host tribes, but not among host subfamilies. However, FFE community dissimilarity between host individuals was not significantly correlated with phylogenetic distance between host species. There was a significant effect of spatial distance among host individuals on FFE community dissimilarity within the common garden. The significant differences in FFE community structure among host species, but lack of a significant host phylogenetic effect, suggest functional differences among host species not accounted for by host phylogenetic distance, such as metabolic traits or phenology, may drive FFE community dissimilarity. Overall, our results indicate that host species identity and the spatial distance between plants can determine the similarity of their microbiomes, even across a single experimental field, but that host phylogeny is not closely tied to FFE community divergence in native Asteraceae.
... The importance of these controls relies on the need to diminish the possible enrichment and presence of terrestrial species sequences in the collected samples, since a high abundance and diversity of facultative fungi has been detected in the deep-sea environment as discussed above. How to handle the contaminant sequences present in the controls will depend on the representation of these sequences in the samples, and since there is no consensus so far on how these sequences should be handled, different strategies could be applied: (1) delete all the sequences corresponding to the fungal OTUs present on the contaminant controls, or (2) eliminate a proportion of the sequences equal to the contaminant sequences present in the controls [109]. ...
Article
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The deep sea (>1000 m below sea level) represents one of the most extreme environments of the ocean. Despite exhibiting harsh abiotic conditions such as low temperatures, high hydrostatic pressure, high salinity concentrations, a low input of organic matter, and absence of light, the deep sea encompasses a great fungal diversity. For decades, most knowledge on the fungal diversity of the deep sea was obtained through culture-dependent techniques. More recently, with the latest advances of high-throughput next generation sequencing platforms, there has been a rapid increment in the number of studies using culture-independent techniques. This review brings into the spotlight the progress of the techniques used to assess the diversity and ecological role of the deep-sea mycobiota and provides an overview on how the omics technologies have contributed to gaining knowledge about fungi and their activity in poorly explored marine environments. Finally, current challenges and suggested coordinated efforts to overcome them are discussed.
... A few studies have addressed these fungal assemblages at the species level (Sun et al., 2016;Asemaninejad et al., 2017a;2017b;, which is crucial for nature conservation efforts, such as monitoring red-listed species and establishing indicator species. Apart from the well-known problems of overestimation of some fungal groups and underestimation or omission of others due to the choice of primers (Tedersoo et al., 2015), the reliability of singletons (Nguyen et al., 2015) and other technical issues (Zinger et al., 2019), an appropriate sampling strategy is also very important. ...
Article
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Fungi are a highly diverse group of organisms and play a significant role in decomposition and carbon cycling in boreal ecosystems. To determine how fungal communities are structured in peat bogs and how to obtain representative samples for monitoring fungal community changes, we separately sampled and sequenced (ITS2, Illumina MiSeq) peat, mixed litter and litter from individual dominant plant species in five permanent plots in raised bogs in the Jizerské Hory Mountains (Czech Republic). In total, we detected 68–103 OTUs per plot. The fungal assemblages were mostly influenced by substrate identity, whereas the effect of the site was minimal. Only a few identified OTUs behave like generalists. The most specific fungi were found in dead parts of Calluna vulgaris, Trichophorum cespitosum and Drosera rotundifolia. The high substrate heterogeneity means that sequencing soil core samples does not provide an exhaustive inventory of the fungal diversity in bogs. Poor correspondence was observed between the recorded OTUs and either visually inspected fruitbodies or literature reports on the fungal taxa associated with the analysed substrates.
... All of these procedures were performed in PipeCraft toolkit v1.0 (Anslan et al., 2017). In the final OTU × sample matrix, we subtracted the number of sequences found in negative controls and mock community from each sample, as in Nguyen et al. (2015). Fungal guilds were then assigned for OTUs using FUNGuild (Nguyen et al., 2016). ...
Article
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Climate change is increasing the frequency and intensity of catastrophic events such as hurricanes. Soil microbial communities regulate geochemical cycles and other important ecosystem processes. How the hurricanes affect soil microbial communities and their function, however, is largely unknown. Our aim was to describe the impact of a category 4 hurricane (Patricia) on the soil fungal community structure and diversity in a Mexican tropical dry forest. Soil fungal community was inferred sequencing the ITS2 rDNA of composite soil samples taken in a time series ranging one year before and two years after the hurricane. OTU richness before the hurricane and in the first sampling after were comparable, however a 20–40% decrease in richness was observed in later sampling times. There were also taxonomic shifts associated with the disturbance, changing from a higher richness and abundance of Ascomycota fungi to greater dominance of Basidiomycota and Glomeromycota fungi post-hurricane. Arbuscular mycorrhizal fungi and plant pathogens diversity increased immediately after the hurricane but decreased in subsequent years. Approximately 7% of the soil community, primarily composed of saprotrophic fungi, persisted across the hurricane and the harsh seasonality of this ecosystem. Soil fungal community was affected by the hurricane but had function stability and resilience through years.
... Producing accurate diversity information from millions of reads is a primary requirement of all marker gene studies. To achieve this goal, many clustering methods based on different algorithms, have been implemented in recent years (Nguyen et al., 2015). However, inconsistency among algorithms is increased by the way they consider species with low abundances. ...
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Due to the tremendous diversity of microbial organisms in topsoil, the estimation of saturated richness in a belowground ecosystem is still challenging. Here, we intensively surveyed the 16S rRNA gene in four 1 m2 sampling quadrats in a typical grassland, with 141 biological or technical replicates generating over 11 million sequences per quadrat. Through these massive data sets and using both non-asymptotic extrapolation and non-parametric asymptotic approaches, results revealed that roughly 15 919±193, 27 193±1076 and 56 985±2347 prokaryotic species inhabited in 1 m2 topsoil, classifying by DADA2, UPARSE (97% cutoff) and Deblur, respectively, and suggested a huge difference among these clustering tools. Nearly 500 000 sequences were required to catch 50% species in 1 m2, while any estimator based on 500 000 sequences would still lose about a third of total richness. Insufficient sequencing depth will greatly underestimate both observed and estimated richness. At least ∼911 000, ∼3461 000, and ∼1 878 000 sequences were needed for DADA2, UPARSE, and Deblur, respectively, to catch 80% species in 1 m2 topsoil, and the numbers of sequences would be nearly twice to three times on this basis to cover 90% richness. In contrast, α-diversity indexes characterized by higher order of Hill numbers, including Shannon entropy and inverse Simpson index, reached saturation with fewer than 100 000 sequences, suggesting sequencing depth could be varied greatly when focusing on exploring different α-diversity characteristics of a microbial community. Our findings were fundamental for microbial studies that provided benchmarks for the extending surveys in large scales of terrestrial ecosystems.
... (b) ITS1F-2 and 16S rRNA amplicon sequencing Full details are provided in the electronic supplementary material. Briefly, we amplified the ITS1F-2 rRNA gene to identify fungal communities using single index reverse primers and a modified protocol of Smith & Peay [55] and Nguyen et al. [56], as detailed in Griffiths et al. [13]. To identify bacterial communities, we amplified DNA for the 16S rRNA V4 region using dual indexed forward and reverse primers according to Kozich et al. [57] and Griffiths et al. [53]. ...
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Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host–microbe interactions.
... In the past decade, amplicon metabarcoding with highthroughput sequencing approaches have allowed the identification of multiple groups of soil organisms [28][29][30]. However, PCR amplification has multiple layers of biases, including primer selection and bioinformatic processing, and the lack of universal primers means multiple primer sets are required to amplify taxonomically disparate groups [31][32][33]. An alternative approach is to use an amplification-independent method, such as shotgun RNA sequencing (RNA-Seq) for community analysis, which we call "community RNA-Seq". ...
Article
Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua . Plants were grown in ¹³ CO 2 -labeled microcosms amended with ¹⁵ N-root litter to identify the preferences of rhizosphere organisms for root exudates ( ¹³ C) versus decaying root biomass ( ¹⁵ N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.
... Although there are numerous indexing methods known for HTS (Gohl et al., 2016) but for diagnostic purposes, it is very indispensable to select an approach that can avoid unwanted recombination between molecules. The possible causes that can affect multiplexed sequencing include the formation of chimera during PCR (Schnell et al., 2015), excess quantities of primer/adapter oligonucleotide (Illumina, 2017), clusters formed on flow cell (Kircher et al., 2012), physical contamination during oligonucleotide synthesis or library preparation (Hanna and Doench, 2018;Nguyen et al., 2015). Index switching can cause taxa from one sample to extend to another leading to false-positive results for that particular taxon under investigation. ...
Book
Plant Ecogenomics offers a valuable introduction to plant ecology from a genomics point of view. The editors present a thorough foundation and summary of modern approaches, methodologies, research goals, and evidence of plant ecology in the modern genomic era. They also present important updated information on the most recent knowledge of the different aspects of plant populations. The volume begins with a general overview of the various approaches to unravelling the genetic basis underlying fundamental responses of plants to their natural environments. It also describes in detail the concepts, aims, and approaches of plant ecological genomics along with the genomic tools embraced by ecologists for mining various ecological problems. Recent advances and breakthroughs made in molecular markers along with their applications in plant ecogenomic studies are shared, and specific applications, techniques, and tools are described as well, such advanced molecular techniques, next-generation sequencing, eDNA metabarcoding, among others. The authors look at community and ecosystem responses in the form of phylogenetic systematics and the theory and methodology for inferring phylogenetic trees or cladograms, including parsimony, Bayesian, and maximum likelihood methods. The employability of QTL analysis and reverse genetics in plant ecogenomics is discussed. Other topics include the role of eco-genomics in conservation and management, models to study the population structure along with the genetic basis of speciation and adaptation in plants, and more. The volume looks at how genetic variability is assessed with the help of various available tools and how effective they are in the conservation of important plant species. Inbreeding and genetic load and the role of ecogenomics in landscape genetic restoration are covered as well. This volume offers a rich store of information on the interdisciplinary field of ecological genomics that will be valuable for molecular biologists, ecologists, as well as for researchers, scientists, and faculty and students in related areas.
... Nine fungal genera were identified in the negative control. These sequences were removed from the abundance of that OTU in the experimental samples [34]. According to the Good's coverage values, 96.9% of total species richness was accounted for in fungal communities (Supplementary Materials Table S2). ...
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Rootstocks are the link between the soil and scion in grapevines, can provide tolerance to abiotic and biotic stresses, and regulate yield and grape quality. The vascular system of grapevine rootstocks in nurseries is still an underexplored niche for research, despite its potential for hosting beneficial and pathogenic microorganisms. The purpose of this study was to investigate the changes in the composition of fungal communities in 110 Richter and 41 Berlandieri rootstocks at four stages of the grapevine propagation process. Taxonomic analysis revealed that the fungal community predominantly consisted of phylum Ascomycota in all stages of the propagation process. The alphadiversity of fungal communities differed among sampling times for both rootstocks, with richness and fungal diversity in the vascular system decreasing through the propagation process. The core microbiome was composed of the genera Cadophora, Cladosporium, Penicillium and Alternaria in both rootstocks, while the pathogenic genus Neofusicoccum was identified as a persistent taxon throughout the propagation process. FUNguild analysis showed that the relative abundance of plant pathogens associated with trunk diseases increased towards the last stage in nurseries. Fungal communities in the vascular system of grapevine rootstocks differed between the different stages of the propagation process in nurseries. Numerous genera associated with potential biocontrol activity and grapevine trunk diseases were identified. Understanding the large diversity of fungi in the rootstock vascular tissue and the interactions between fungal microbiota and grapevine will help to develop sustainable strategies for grapevine protection.
... Additionally, we identified which taxa might help us explain the differences between bat and fruit communities using the linear discriminant analysis (LDA) effect size (LEfSe) method [102]. We retained taxa with LDA scores > 4. In general, we placed more weight on the results from presence/absence data [100,103,104] over that from normalized abundance because we sought to determine whether the same fungal taxa in fruits were present in feces, and because of the intrinsic issues with misestimation of abundance in microbiome studies [99,[104][105][106][107][108]. ...
Article
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Background Bats are important long-distance dispersers of many tropical plants, yet, by consuming fruits, they may disperse not only the plant’s seeds, but also the mycobiota within those fruits. We characterized the culture-dependent and independent fungal communities in fruits of Ficus colubrinae and feces of Ectophylla alba to determine if passage through the digestive tract of bats affected the total mycobiota. Results Using presence/absence and normalized abundance data from fruits and feces, we demonstrate that the fungal communities were significantly different, even though there was an overlap of ca. 38% of Amplicon Sequence Variants (ASVs). We show that some of the fungi from fruits were also present and grew from fecal samples. Fecal fungal communities were dominated by Agaricomycetes, followed by Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Malasseziomycetes, while fruit samples were dominated by Dothideomycetes, followed by Sordariomycetes, Agaricomycetes, Eurotiomycetes, and Laboulbeniomycetes. Linear discriminant analyses (LDA) show that, for bat feces, the indicator taxa include Basidiomycota (i.e., Agaricomycetes: Polyporales and Agaricales), and the ascomycetous class Eurotiomycetes (i.e., Eurotiales, Aspergillaceae). For fruits, indicator taxa are in the Ascomycota (i.e., Dothideomycetes: Botryosphaeriales; Laboulbeniomycetes: Pyxidiophorales; and Sordariomycetes: Glomerellales). In our study, the differences in fungal species composition between the two communities (fruits vs. feces) reflected on the changes in the functional diversity. For example, the core community in bat feces is constituted by saprobes and animal commensals, while that of fruits is composed mostly of phytopathogens and arthropod-associated fungi. Conclusions Our study provides the groundwork to continue disentangling the direct and indirect symbiotic relationships in an ecological network that has not received enough attention: fungi-plants-bats. Findings also suggest that the role of frugivores in plant-animal mutualistic networks may extend beyond seed dispersal: they may also promote the dispersal of potentially beneficial microbial symbionts while, for example, hindering those that can cause plant disease.
... Nevertheless, ensuring the accuracy of detections must be a priority for applying metabarcoding to invasive species surveillance due to the stringent reporting requirements for regulated taxa and higher economic consequences of a false negative (Piper et al., 2019). During complex metabarcoding protocols, false positive detections can be introduced through laboratory contamination (Nguyen et al., 2015) and index switching (Schnell et al., 2015), while false negatives can arise through insufficient sequencing depth (Smith and Peay, 2014), stochastic sampling of molecules from low abundance specimens (Leray and Knowlton, 2017), and PCR biases (Deagle et al., 2014). Robust metabarcoding protocols thus require both technical replication and use of a detection threshold to resolve true positives from any low-abundance contaminant sequences (Zinger et al., 2019). ...
Article
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The spotted wing drosophila ( Drosophila suzukii , Matsumara) is a rapidly spreading global pest of soft and stone fruit production. Due to the similarity of many of its life stages to other cosmopolitan drosophilids, surveillance for this pest is currently bottlenecked by the laborious sorting and morphological identification of large mixed trap catches. DNA metabarcoding presents an alternative high-throughput sequencing (HTS) approach for multi-species identification, which may lend itself ideally to rapid and scalable diagnostics of D. suzukii within unsorted trap samples. In this study, we compared the qualitative (identification accuracy) and quantitative (bias toward each species) performance of four metabarcoding primer pairs on D. suzukii and its close relatives. We then determined the sensitivity of a non-destructive metabarcoding assay (i.e., which retains intact specimens) by spiking whole specimens of target species into mock communities of increasing specimen number, as well as 29 field-sampled communities from a cherry and a stone fruit orchard. Metabarcoding successfully detected D. suzukii and its close relatives Drosophila subpulchrella and Drosophila biarmipes in the spiked communities with an accuracy of 96, 100, and 100% respectively, and identified a further 57 non-target arthropods collected as bycatch by D. suzukii surveillance methods in a field scenario. While the non-destructive DNA extraction retained intact voucher specimens, dropouts of single species and entire technical replicates suggests that these protocols behave more similarly to environmental DNA than homogenized tissue metabarcoding and may require increased technical replication to reliably detect low-abundance taxa. Adoption of high-throughput metabarcoding assays for screening bulk trap samples could enable a substantial increase in the geographic scale and intensity of D. suzukii surveillance, and thus likelihood of detecting a new introduction. Trap designs and surveillance protocols will, however, need to be optimized to adequately preserve specimen DNA for molecular identification.
... Sequences with ≥97% of similarity were assigned to the same operational taxonomic unit (OTUs). OTUs represented by <10 reads were discarded as sequencing errors (Nguyen et al., 2015). ...
Article
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A long-standing hypothesis in biogeography predicts that a species’ abundance is highest at the center of its geographical range and decreases toward its edges. In this study, we test the abundant-center hypothesis of ectomycorrhizal (ECM) fungal communities associated with Picea crassifolia , an endemic species widely distributed in northwest China. We analyzed the taxonomic richness and the relative abundance of ECM fungi in four main distribution areas, from center to edges. In total, 234 species of ECM fungi were detected, and of these, 137 species were shared among all four sites. Inocybe , Sebacina , Tomentella , and Cortinarius were the dominant genera. ECM fungal richness and biodiversity were highest at the central and lower at peripheral sites. Our results indicated that ECM fungal species richness was consistent with the abundant-center hypothesis , while the relative abundances of individual fungal genera shifted inconsistently across the plant’s range.
... When FUNGuild failed to assign a guild as a result of taxonomic uncertainty, we assigned them manually when BLAST hits matched a UNITE "Species Hypothesis" with > 97% similarity and > 90% coverage. The OTU table was filtered based on positive and negative controls (Nguyen et al., 2015) and dubious OTUs with abundance < 0.05% of total reads per sample were filtered out. ...
Article
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Competition for resources between arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) plants can alter belowground mycorrhizal communities, but few studies have investigated host effects on both AM and ECM communities. In Central Mexico, the AM plant Juniperus deppeana is frequently used for reforesting areas affected by soil erosion, while the surrounding native forests are dominated by ECM oak trees. Oaks are capable of associating with both AM and ECM fungi during part of their life cycle (a feature known as dual mycorrhization) but it is unclear whether junipers possess such ability. To assess how juniper planting may affect belowground fungal interactions with oaks, we investigated mycorrhizal associations in J. deppeana and Quercus rugosa seedlings along a disturbance gradient: a native oak forest, a mixed Juniperus-Quercus population in secondary vegetation and a juniper site severely degraded by mining extraction. We measured root colonization and identified fungal communities using soil and root meta-barcoding of the ITS2 rDNA region. ECM fungal community composition was strongly affected by disturbance (regardless of host), while the community composition of AM fungi was mostly host-dependent, with a higher AM fungal richness in J. deppeana . Importantly, the fungal communities associated with Q. rugosa seedlings significantly changed in the vicinity of juniper trees, while those of J. deppeana seedlings were not affected by the presence of oak trees. Even though ECM fungal richness was higher in Q. rugosa and in the native forest, we detected a variety of ECM fungi associated exclusively with J. deppeana seedlings, suggesting that this plant species may be colonized by ECM fungi. Our results indicate that J. deppeana can alter ECM native fungal communities, with implications for its use in reforestation of mixed oak forests.
... The main problem with ITS is size polymorphism, with a size range of 361 to 1475 bases in UNITE 7.1 [3] (unlike 16S where 95% of the sequences have a length between 1205 and 1556 bases). Most studies describing ITS data analyses process either (i) paired-end reads but filter out non-overlapping, non-mergeable reads, thus systematically discarding taxa with longer ITS, or (ii) single-end reads, thus limiting taxonomic resolution and losing the benefit of information contained in longer sequences [4,5]. In both cases, the tools used, which only support mergeable paired-end reads or single-end reads, are unable to process all the sequences produced. ...
Article
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Fungi are present in all environments. They fulfil important ecological functions and play a crucial role in the food industry. Their accurate characterization is thus indispensable, particularly through metabarcoding. The most frequently used markers to monitor fungi are ITSs. These markers are the best documented in public databases but have one main weakness: polymerase chain reaction amplification may produce non-overlapping reads in a significant fraction of the fungi. When these reads are filtered out, traditional metabarcoding pipelines lose part of the information and consequently produce biased pictures of the composition and structure of the environment under study. We developed a solution that enables processing of the entire set of reads including both overlapping and non-overlapping, thus providing a more accurate picture of fungal communities. Our comparative tests using simulated and real data demonstrated the effectiveness of our solution, which can be used by both experts and non-specialists on a command line or through the Galaxy-based web interface.
... The ITS1 rDNA subunit of each soil and hyphal sample, plus a mock community (Nguyen et al., 2015) and negative controls, were PCR amplified following Smith and Peay (2014). Specifically, KAPA HIFI Taq (Roche) with 10 uM (initial concentration) barcoded fungal-specific ITS1F-ITS2 primer set were combined with 12 ng (on average) of DNA per sample in a 25 μL total reaction volume. ...
Article
Atmospheric nitrogen (N) pollution visibly and rapidly changes forest systems, for example impacting the diversity of forest ectomycorrhizal (EcM) fungi. EcM fungi are measurable by components that vary in longevity, hence, reflecting different temporal scales of presence: EcM root-tips are viable for, at most, a couple growing seasons, compared to fungal structures that may persist in soil as DNA for decades or longer. In-growth mesh bags, in contrast, typically capture a single growing season of hyphal growth. The different components might portray different diversity responses by EcM fungi to large-scale N pollution, as well as any subsequent reductions from improved environmental standards. Within an established oak forest study system, we examined the impact of sustained and recently reduced experimental N addition on EcM fungal diversity and composition, measured in three main fungal components (root-free soil, root-tips, and mesh bag hyphae). We hypothesized that elevated soil N would reduce EcM fungal diversity, and that composition would change, but with differences among fungal components related to the temporal longevity of the components. Our expectations were largely met, in that richness primarily declined with increased soil N, and all trends were most pronounced with the soil EcM fungi (the only component potentially reflective of long-term fungal presence). We discovered that abatement of the experimental N treatment did not revert fungal trends to those of the same-site plots with ambient N treatment. Instead, the stochastic nature of local-scale disturbances, related to invasive earthworms and forest stand dynamics, likely impacted N levels and, thus, EcM fungal trends. Due to the context-dependency of localized disturbance(s), assessing the effects of reduced large-scale N deposition on EcM fungi can prove to be challenging.
... This could be a result of biological (e.g., intraspecific rDNA copy number variation) and/or methodological (e.g., inappropriate OTU clustering identity threshold) factors. Artificial inflation of the number of ITS OTUs has been shown for various taxa in mock communities in metabarcoding studies (Castaño et al., 2020;De Filippis et al., 2017;Jusino et al., 2019;Nguyen et al., 2015;Větrovský et al., 2016). These results underscore the importance of mock communities for detecting methodological errors and refining bioinformatic parameters such as clustering thresholds (Caporaso et al., 2011;Palmer et al., 2018;Taylor et al., 2016). ...
Article
Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with “standard” metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.
... To reduce potential sequencing errors, we omitted rare OTUs with sequence counts ≤10 Nguyen et al., 2015;Oliver et al., 2015). In mothur, we iteratively calculated Good's coverage We tested for latitudinal gradients in fungal composition using perMANOVA in Primer v. 6.1.10 ...
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Aim Roots and rhizospheres host diverse microbial communities that can influence the fitness, phenotypes, and environmental tolerances of plants. Documenting the biogeography of these microbiomes can detect the potential for a changing environment to disrupt host-microbe interactions, particularly in cases where microbes buffer hosts against abiotic stressors. We evaluated whether root-associated fungi had poleward declines in diversity, tested whether fungal communities in roots shifted near host plant range edges, and determined the relative importance of environmental and host predictors of root fungal community structure. Location North American plains grasslands. Taxon Foundation grasses – Andropogon gerardii, Bouteloua dactyloides, B. eriopoda, B. gracilis, and Schizachyrium scoparium and root fungi. Methods At each of 24 sites representing three replicate 17°–latitudinal gradients, we collected roots from 12 individuals per species along five transects spaced 10 m apart (40 m × 40 m grid). We used next-generation sequencing of ITS2, direct fungal culturing from roots, and microscopy to survey fungi associated with grass roots. Results Root-associated fungi did not follow the poleward declines in diversity documented for many animals and plants. Instead, host plant identity had the largest influence on fungal community structure. Edaphic factors outranked climate or host plant traits as correlates of fungal community structure; however, the relative importance of environmental predictors differed among plant species. As sampling approached host species range edges, fungal composition converged in similarity among individual plants of each grass species. Main conclusions Environmental predictors of root-associated fungi depended strongly on host plant species identity. Biogeographic patterns in fungal composition suggested a homogenizing influence of stressors at host plant range limits. Results predict that communities of non-mycorrhizal, root-associated fungi in the North American plains will be more sensitive to future changes in host plant ranges and edaphic factors than to the direct effects of climate.
... Bonferroni corrections were applied to p values to account for multiple comparisons. Since read counts are nor representative of species abundances [37,38], we constructed a Raup-Crick dissimilarity matrix based on OTU presence/ absence (after eliminating OTUs only present in one sample) and visualized bacterial and fungal community composition by non-metric multidimensional scaling (NMDS). The effect of organ and condition on bacterial and fungal community composition was tested with PERMANOVA and 999 random permutations (Adonis tests). ...
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The worldwide production of vanilla, a native orchid from Mexico, is greatly affected by stem and root rot disease (SRD), typically associated with Fusarium oxysporum fungi. We hypothesized that the presence of Fusarium species in vanilla is not sufficient for the plant to express symptoms of the disease. We described the taxonomic composition of endophytic microbiomes in symptomatic and asymptomatic vanilla plants using 16S and ITS rDNA metabarcoding, and ITS Sanger sequences generated from fungal isolates. We compared the bacterial and fungal diversity in vanilla plants from a long-term plantation, and from feral plants found near abandoned plantations that did not present SRD symptoms. No significant differences were found in the species richness of the bacterial and fungal microbiome among feral, or asymptomatic and symptomatic cultivated vanilla. However, significant differences were detected in both fungal and bacterial diversity from different organs in the same plant, with roots being more diverse than stems. We found that Proteobacteria and Actinobacteria, as well as the fungal families Nectriaceae and Xylariaceae, constitute the core of the vanilla microbiome that inhabits the root and stem of both cultivated and feral plants. Our work provides information on the microbial diversity associated to root and stem rot in vanilla and lays the groundwork for a better understanding of the role of the microbiome in vanilla fungal diseases.
... New technologies and tactics are in development to remedy many of these issues. Spike-in internal DNA standards for fungal community analysis ameliorate some of the issues associated with abundance estimates from high-throughput sequencing 112,113 . Advances in metatranscriptomics coupled with improved databases of fungal functional activities have the potential to link genetic diversity to functional diversity 75,114 , and metagenomic and amplicon studies (such as those now compiled in the GlobalFungi database) will aid in assessing biogeographic frequency 115 . ...
Article
Fungi have successfully established themselves across seemingly every possible niche, substrate, and biome. They are fundamental to biogeochemical cycling, interspecies interactions, food production, and drug bioprocessing, as well as playing less heroic roles as difficult to treat human infections and devastating plant pathogens. Despite community efforts to estimate and catalog fungal diversity, we have only named and described a minute fraction of the fungal world. The identification, characterization, and conservation of fungal diversity is paramount to preserving fungal bioresources, and to understanding and predicting ecosystem cycling and the evolution and epidemiology of fungal disease. Although species and ecosystem conservation are necessarily the foundation of preserving this diversity, there is value in expanding our definition of conservation to include the protection of biological collections, ecological metadata, genetic and genomic data, and the methods and code used for our analyses. These definitions of conservation are interdependent. For example, we need metadata on host specificity and biogeography to understand rarity and set priorities for conservation. To aid in these efforts, we need to draw expertise from diverse fields to tie traditional taxonomic knowledge to data obtained from modern -omics-based approaches, and support the advancement of diverse research perspectives. We also need new tools, including an updated framework for describing and tracking species known only from DNA, and the continued integration of functional predictions to link genetic diversity to functional and ecological diversity. Here, we review the state of fungal diversity research as shaped by recent technological advancements, and how changing viewpoints in taxonomy, -omics, and systematics can be integrated to advance mycological research and preserve fungal biodiversity.
... Here, we analyzed only forward sequence reads because lower quality and quantity of reverse reads resulted in a nearly 50% reduction in total sequence reads after quality filtering of the assembled paired-end reads (Appendix S1a). Discarding low-quality reverse reads is a common strategy that often provides better results than assembled paired-end reads (Nguyen et al., 2015;Pauvert et al., 2019). One chimera was removed. ...
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Premise: Host-plant genetic variation can shape associated communities of organisms. These community-genetic effects include (1) genetically similar hosts harboring similar associated communities (i.e., the genetic similarity rule) and (2) host-plant heterozygosity increasing associated community diversity. Community-genetic effects are predicted to be less prominent in plant systems with limited genetic variation, such as those at distributional range limits. Yet, empirical evidence from such systems is limited. Methods: We sampled a natural population of a mangrove foundation species (Avicennia germinans) at an expanding range limit in Florida, USA. We measured genetic variation within and among 40 host trees with 24 nuclear microsatellite loci and characterized their foliar endophytic fungal communities with internal transcribed spacer (ITS1) gene amplicon sequencing. We evaluated relationships among host-tree genetic variation, host-tree spatial location, and the associated fungal communities. Results: Genetic diversity was low across all host trees (mean: 2.6 alleles per locus) and associated fungal communities were relatively homogeneous (five sequence variants represented 78% of all reads). We found (1) genetically similar host trees harbored similar fungal communities, with no detectable effect of interhost geographic distance. (2) Host-tree heterozygosity had no detectable effect, while host-tree absolute spatial location affected community alpha diversity. Conclusions: This research supports the genetic similarity rule within a range limit population and helps broaden the current scope of community genetics theory by demonstrating that community-genetic effects can occur even at expanding distributional limits where host-plant genetic variation may be limited. Our findings also provide the first documentation of community-genetic effects in a natural mangrove system.
... The most common, and cost-effective, method is the amplification and sequencing of targeted genetic elements. Amplicon sequencing of taxonomic marker genes such as 16S rRNA [1], the ITS region [2] or 18S rRNA [3] provides a census of a community. Functional diversity can be probed by targeting functional genes [4]. ...
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Microbial communities are commonly characterized by amplifying and sequencing target genes, but errors limit the precision of amplicon sequencing. We present DADA2, a software package that models and corrects amplicon errors. DADA2 identified more real variants than other methods in Illumina-sequenced mock communities, some differing by a single nucleotide, while outputting fewer spurious sequences. DADA2 analysis of vaginal samples revealed a diversity of Lactobacillus crispatus strains undetected by OTU methods.
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Microorganisms play essential roles in agricultural systems, and their abundance, diversity and activity can be influenced by management practices. Crop rotational diversity is known to influence both soil microorganisms and crop productivity, yet the specific contributions of microorganisms to rotational benefits are unknown. To facilitate monitoring soil biological processes that support vigorous and high yielding crops, we studied maize (Zea mays L.) and soybean (Glycine max L.), and their associated microorganisms within a two-year maize-soybean rotation and within four-year crop rotations with varying crop sequences. We hypothesized that rhizosphere microbial communities are strong predictors of crop productivity contingent on rotational diversity and previous crop legacy. Sampling at seedling and flowering stages, we assessed rhizosphere bacterial and fungal communities, soil and plant tissue nutrients, aboveground biomass, and yield. Rhizosphere communities varied with rotational diversity and previous crop legacy. Concurrently, maize and soybean yield and biomass were approximately 15-25% larger in more diverse rotations and with different crop legacies, but there were no crop rotational effects on tissue or soil nutrients. Yield differences across rotational diversity or previous crop legacy were better predicted when microbial communities were considered. Fungal communities predicted lower maize seedlings biomass when following soybean, and lower soybean seedling biomass when following maize, independent of rotational diversity. Further, for maize, fungal communities predicted lower maize yield in the maize-soybean rotation, while in the four-year diverse rotations, bacterial communities predicted maize recovery from a soybean legacy by flowering. These results suggest that benefits of four-year rotations in maize and soybean production are driven by changes in plant pathogenic communities.
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Rapid changes of the biosphere observed in recent years are caused by both small and large scale drivers, like shifts in temperature, transformations in land-use, or changes in the energy budget of systems. While the latter processes are easily quantifiable, documentation of the loss of biodiversity and community structure is more difficult. Changes in organismal abundance and diversity are barely documented. Censuses of species are usually fragmentary and inferred by often spatially, temporally and ecologically unsatisfactory simple species lists for individual study sites. Thus, detrimental global processes and their drivers often remain unrevealed. A major impediment to monitoring species diversity is the lack of human taxonomic expertise that is implicitly required for large-scale and fine-grained assessments. Another is the large amount of personnel and associated costs needed to cover large scales, or the inaccessibility of remote but nonetheless affected areas. To overcome these limitations we propose a network of Automated Multisensor stations for Monitoring of species Diversity (AMMODs) to pave the way for a new generation of biodiversity assessment centers. This network combines cutting-edge technologies with biodiversity informatics and expert systems that conserve expert knowledge. Each AMMOD station combines autonomous samplers for insects, pollen and spores, audio recorders for vocalizing animals, sensors for volatile organic compounds emitted by plants (pVOCs) and camera traps for mammals and small invertebrates. AMMODs are largely self-containing and have the ability to pre-process data (e.g. for noise filtering) prior to transmission to receiver stations for storage, integration and analyses. Installation on sites that are difficult to access require a sophisticated and challenging system design with optimum balance between power requirements, bandwidth for data transmission, required service, and operation under all environmental conditions for years. An important prerequisite for automated species identification are databases of DNA barcodes, animal sounds, for pVOCs, and images used as training data for automated species identification. AMMOD stations thus become a key component to advance the field of biodiversity monitoring for research and policy by delivering biodiversity data at an unprecedented spatial and temporal resolution.
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Marine Protected Areas (MPAs) have been adopted globally as a tool to combat biodiversity loss and restore marine ecosystems. Successful application of MPAs will be predicated on the ability to monitor biodiversity in a synoptic and non-invasive manner. Environmental DNA (eDNA) methods have important advantages over traditional biodiversity survey methods for monitoring conservation areas. To evaluate the efficacy of eDNA metabarcoding for fish biodiversity monitoring, we sampled 19 coastal eelgrass beds in Canada, as eelgrass beds are known for high biodiversity and significant conservation value. At each site, beach seines were used to survey fish and water samples were collected contemporaneously for eDNA metabarcoding. In total, beach seining caught 32,672 individuals across 59 fish taxa and eDNA detected 129 fish taxa. eDNA captured site-level variation and detected higher species richness at both site and regional levels compared to seining. eDNA abundance had a positive association with capture abundance. Collectively these results highlight how eDNA metabarcoding offers an efficient approach for monitoring fish biodiversity in coastal eelgrass beds, thus providing a valuable and non-invasive tool for MPA planning and coastal monitoring.
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Fungal communities associated with plants often decrease in similarity as the distance between sampling sites increases (i.e., they demonstrate distance decay). In the southwestern USA, forests occur in highlands separated from one another by warmer, drier biomes with plant and fungal communities that differ from those at higher elevations. These disjunct forests are broadly similar in climate to one another, offering an opportunity to examine drivers of distance decay in plant-associated fungi across multiple ecologically similar yet geographically disparate landscapes. We examined ectomycorrhizal and foliar endophytic fungi associated with a dominant forest tree (Pinus ponderosa) in forests across ca. 550 km of geographic distance from northwestern to southeastern Arizona (USA). Both guilds of fungi showed distance decay, but drivers differed for each: ectomycorrhizal fungi are constrained primarily by dispersal limitation, whereas foliar endophytes are constrained by specific environmental conditions. Most ectomycorrhizal fungi were found in only a single forested area, as were many endophytic fungi. Such regional-scale perspectives are needed for baseline estimates of fungal diversity associated with forest trees at a landscape scale, with attention to the sensitivity of different guilds of fungal symbionts to decreasing areas of suitable habitat, increasing disturbance, and related impacts of climate change.
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This article is a Commentary on Hagenbo et al. (2021), 230: 1609–1622.
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Biodiversity surveys are crucial to monitor the health of threatened aquatic ecosystems, such as tropical estuaries and mangroves. Conventional monitoring methods are intrusive, time-consuming, substantially expensive and frequently only provide rough assessments in complex habitats. Recent advanced molecular methods such as environmental DNA (eDNA) using high-throughput sequencing technology are promising although only few applications in tropical estuarine ecosystems have been reported. In this study, we explore the advantages and limitations of an eDNA metabarcoding survey on the fish community of the Merbok Estuary (Peninsular Malaysia). COI and 12S eDNA metabarcoding assays collectively detected 178 species belonging to 127 genera, 68 families, and 25 orders. This approach captured significantly more species than in any previous traditional surveys, including a few species of conservation importance. However, we highlight three limitations: (1) in the absence of a comprehensive reference database the identities of several species are unresolved; (2) a fraction of previously documented specimen-based diversity was not captured by the current method, may be as a consequence of PCR primer specificity, and (3) the detection of non-resident species; stenohaline freshwater taxa (e.g. cyprinids, channids, osphronemids) and marine coral reef taxa (e.g. some sharks, holocentrids and syngnathids), not known to frequent estuaries, leading to the supposition that their DNA have drifted into the estuary through water movement. The community analysis revealed that fish diversity along the estuary is not homogenous, with the upstream more diverse than further downstream. This may be the consequence of the salinity or pollution gradients. In summary, we demonstrated the practicality of eDNA metabarcoding in assessing fish community and structure within a complex and rich tropical environment within a short sampling period. However, some limitations need to be considered and addressed to fully exploit the efficacy of this approach, in particular the development of a comprehensive reference genetic database.
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Arbuscular mycorrhizal fungi (AMF) are keystone symbionts of agricultural soils but agricultural intensification has negatively impacted AMF communities. Increasing crop diversity could ameliorate some of these impacts by positively affecting arbuscular mycorrhizal fungi. However, the underlying relationship between plant diversity and AMF community composition has not been fully resolved. ●We examined how greater crop diversity affected AMF across farms in an intensive agricultural landscape, defined by high nutrient input, low crop diversity, and high tillage frequency. We assessed AMF communities across 31 field sites that were either monocultures or polycultures (growing >20 different crop types) in three ways: (1) richness, (2) diversity, and (3) composition. We also determined root colonization across these sites. ●We found polycultures drive the available AMF community into richer and more diverse communities while soil properties structure AMF community composition. AMF root colonization did not vary by farm management (monocultures versus polycultures), but did vary by crop host. ●We demonstrate that crop diversity enriches AMF communities, counteracting the negative effects of agricultural intensification on AMF, providing the potential to increase agroecosystem functioning and sustainability.
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The genus Malassezia is part of the normal skin mycobiota of a wide range of warm-blooded animals. In this genus, M. cuniculi is the only species described from rabbits. However, Malassezia species are rarely studied in lagomorphs. In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples. Although no growth was observed in the cultured plates, cytological examination revealed the presence of round cells similar to those of Malassezia yeasts. For metagenomics analysis, the D1/D2 domain of the large subunit of the ribosomal DNA (LSU rDNA) was PCR amplified and the resulting reads were mapped against a custom-made cured database of 26S fungal sequences. NGS analysis revealed that Basidiomycota was the most abundant phylum in all the samples followed by Ascomycota. Malassezia was the most common genus presenting the highest abundance in the external ear canal. Malassezia phylotype 131 and M. cuniculi were the main sequences detected in the external auditory canal of rabbits. The study included both lop-eared and erect-eared rabbits and no differences were observed in the results when comparing both groups. This is the first attempt to study the external ear canal mycobiome of rabbits of different breeds using NGS. Lay Summary In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples.
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The interest in studying wood-inhabiting fungal communities has grown in recent years. This interest has mainly been motivated by the important roles of wood-inhabiting fungi in ecosystem functioning (e.g. nutrient cycling) and conservation biology (e.g. their sensitivity to forest management). In this paper, I argue that another important, but yet largely unexplored motivation for studying wood-inhabiting fungal communities, is their potential to advance fundamental community ecology. One major advantage of wood-inhabiting fungi as model systems is that they are organized as spatially well-defined metacommunities, thus conforming to the assumptions of many theoretical frameworks. Another major advantage is that they allow observations and manipulations over large numbers of local communities (habitat patches). After reviewing recent approaches in theoretical community ecology, I discuss how past empirical studies on wood-inhabiting fungal communities relate to community assembly processes, and provide future research directions on how the still unstudied assembly processes could be tackled using wood-inhabiting fungi as a model system.
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Recent advances in molecular approaches and DNA sequencing have greatly progressed the field of ecology and allowed for the study of complex communities in unprecedented detail. Next generation sequencing (NGS) can reveal powerful insights into the diversity, composition, and dynamics of cryptic organisms, but results may be sensitive to a number of technical factors, including molecular practices used to generate amplicons, sequencing technology, and data processing. Despite the popularity of some techniques over others, explicit tests of the relative benefits they convey in molecular ecology studies remain scarce. Here we tested the effects of PCR replication, sequencing depth, and sequencing platform on ecological inference drawn from environmental samples of soil fungi. We sequenced replicates of three soil samples taken from pine biomes in North America represented by pools of either one, two, four, eight, or sixteen PCR replicates with both 454 pyrosequencing and Illumina MiSeq. Increasing the number of pooled PCR replicates had no detectable effect on measures of α- and β-diversity. Pseudo-β-diversity - which we define as dissimilarity between re-sequenced replicates of the same sample - decreased markedly with increasing sampling depth. The total richness recovered with Illumina was significantly higher than with 454, but measures of α- and β-diversity between a larger set of fungal samples sequenced on both platforms were highly correlated. Our results suggest that molecular ecology studies will benefit more from investing in robust sequencing technologies than from replicating PCRs. This study also demonstrates the potential for continuous integration of older datasets with newer technology.
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Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis.
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Biological communities are often structured by environmental factors even at small spatial scales. Fungi are no exception, though the patterns and mechanisms underlying their community structure are usually unknown. Previous work documented zonation in fungi under tree canopies primarily through their fruiting patterns. Here we investigate the existence of zonation patterns in fungal communities around isolated Pinus muricata trees of different ages in northern coastal California. Using a combination of ingrowth bags and pyrosequencing to target underground mycelium we found highly diverse soil fungal communities associated with single trees. Both ectomycorrhizal and non-ectomycorrhizal fungi were present in all samples, but the latter were more species rich, dominated the samples by sequence read abundance, and showed partitioning by canopy-defined zones and tree age. Soil chemistry was correlated with fungal zonation, but host root density was not. Our results indicate different guilds of fungi partition space differently and are driven by distinct environmental parameters.
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High throughput sequencing has become a powerful tool for fungal ecologists to explore the diversity and composition of fungal communities. However, various biases and errors are associated with the new sequencing techniques that must be handled properly. We here provide evidence for a source of error that has not yet been taken into account. During amplicon pyrosequencing we incorporate tags in both ends of the amplicons, which allows us to check for tag coherence after sequencing. In several studies we have observed that a small proportion of the resulting sequences possess novel tag combinations. Our observations cannot be explained by primer contamination or PCR chimaeras. This indicates that some DNA fragments switch tags during laboratory setup. If not controlled for, this will cause numerous false positives in downstream analyses. In most amplicon pyrosequencing studies of fungal communities, amplicons are typically tagged in one end only. We suggest that amplicons should be tagged in both ends before pyrosequencing to control for tag switching.
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The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference dataset, and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g., Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term “species hypothesis” (SH) for the taxa discovered in clustering on different similarity tresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such species hypothesis. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, e.g., local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new web-based sequence management system in UNITE.This article is protected by copyright. All rights reserved.
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Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.
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Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.
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Protists are ubiquitous members of soil microbial communities, but the structure of these communities, and the factors that influence their diversity, are poorly understood. We used barcoded pyrosequencing to survey comprehensively the diversity of soil protists from 40 sites across a broad geographic range that represent a variety of biome types, from tropical forests to deserts. In addition to taxa known to be dominant in soil, including Cercozoa and Ciliophora, we found high relative abundances of groups such as Apicomplexa and Dinophyceae that have not previously been recognized as being important components of soil microbial communities. Soil protistan communities were highly diverse, approaching the extreme diversity of their bacterial counterparts across the same sites. Like bacterial taxa, protistan taxa were not globally distributed, and the composition of these communities diverged considerably across large geographic distances. However, soil protistan and bacterial communities exhibit very different global-scale biogeographical patterns, with protistan communities strongly structured by climatic conditions that regulate annual soil moisture availability.The ISME Journal advance online publication, 13 December 2012; doi:10.1038/ismej.2012.147.
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With recent methodological advances, molecular markers are increasingly used for semi-quantitative analyses of fungal communities. The aim to preserve quantitative relationships between genotypes through PCR places new demands on primers to accurately match target sites and provide short amplicons. The internal transcribed spacer (ITS) region of the ribosome encoding genes is a commonly used marker for many fungal groups. Here, we describe three new primers - fITS7, gITS7 and fITS9, which may be used to amplify the fungal ITS2 region by targeting sites in the 5.8S encoding gene. We evaluated the primers and compared their performance with the commonly used ITS1f primer by 454-sequencing of both artificially assembled templates and field samples. When the entire ITS region was amplified using the ITS1f/ITS4 primer combination, we found strong bias against species with longer amplicons. This problem could be overcome by using the new primers, which produce shorter amplicons and better preserve the quantitative composition of the template. In addition, the new primers yielded more diverse amplicon communities than the ITS1f primer.
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Illumina paired-end reads are used to analyse microbial communities by targeting amplicons of the 16S rRNA gene. Publicly available tools are needed to assemble overlapping paired-end reads while correcting mismatches and uncalled bases; many errors could be corrected to obtain higher sequence yields using quality information. PANDAseq assembles paired-end reads rapidly and with the correction of most errors. Uncertain error corrections come from reads with many low-quality bases identified by upstream processing. Benchmarks were done using real error masks on simulated data, a pure source template, and a pooled template of genomic DNA from known organisms. PANDAseq assembled reads more rapidly and with reduced error incorporation compared to alternative methods. PANDAseq rapidly assembles sequences and scales to billions of paired-end reads. Assembly of control libraries showed a 4-50% increase in the number of assembled sequences over naïve assembly with negligible loss of "good" sequence.
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