Environmental DNA as a tool for ecological monitoring of fungal communities

AbstractInvestigation of the seasonal variation in the fungal community is essential for understanding biodiversity and its ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community compositions, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis, including both aquatic and terrestrial species, has been explored for its usefulness in biodiversity surveys. Examining eDNA may allow for the survey of the community over time with less disturbance to the ecosystem. In this study, we assessed whether seasonality of fungal communities can be detected with monitoring of eDNA in a flow-regulated stream in a restored forest. We conducted monthly water sampling in the stream over two years, and used DNA metabarcoding to estimate the taxonomic and functional groups of fungal eDNA in the water. The river water contained taxonomically and functionally diverse DNA from both aquatic and terrestrial fungi, such as plant decomposers, parasites, and mutualists. The DNA assemblages showed a distinct annual periodicity, meaning that the assemblages were similar to each other regardless of the year, in the same sampling season. These seasonal changes were partially explained by temperature alterations. Furthermore, the strength of the one-year periodicity may vary across functional groups. Our results suggest that forest streams act as a “natural trap” for fungal DNA and that studies of fungal DNA in stream water may provide information on the temporal variation of fungal communities inhabiting not only water but also the surrounding ecosystem.

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Communities of Fungi in Black Cherry Stumps and Effects of Herbicide

Plants ◽

10.3390/plants9091126 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1126

Author(s):

Robert Korzeniewicz ◽

Marlena Baranowska ◽

Hanna Kwaśna ◽

Gniewko Niedbała ◽

Jolanta Behnke-Borowczyk

Keyword(s):

Species Richness ◽

Environmental Dna ◽

Bioinformatic Analysis ◽

Diversity Indices ◽

Fungal Species ◽

Fungal Communities ◽

Reference Sequence ◽

Deciduous Tree ◽

Black Cherry ◽

5.8S Rdna

So far, there have been no studies on fungal communities in Prunus serotina (black cherry) wood. Our objectives were to characterize fungal communities from P. serotina wood and to evaluate effects of glyphosate (Glifocyd 360 SL) used on P. serotina stumps on abundance, species richness and diversity of those communities. In August 2016, in the Podanin Forest District, stumps of black cherry trees left after felling were treated with the herbicide. Control stumps were treated with water. Wood discs were cut from the surface of the stumps in May and July–August 2017. Eight treatment combinations (2 herbicide treatments × 2 disc sizes × 2 sample times) were tested. Sub-samples were pooled and ground in an acryogenic mill. Environmental DNA was extracted with a Plant Genomic DNA Purification Kit. The ITS1, 5.8S rDNA region was used to identify fungal species, using primers ITS1FI2 5′-GAACCWGCGGARGGATCA-3′ and 5.8S 5′-CGCTGCGTT CTTCATCG-3′. The amplicons were sequenced using the Illumina system. The results were subjected to bioinformatic analysis. Sequences were compared with reference sequences from the NCBI database using the BLASTn 2.8.0 algorithm. Abundance of fungi was defined as the number of Operational Taxonomic Units (OTUs), and diversity as the number of species in a sample. Differences between the number of OTUs and taxa were analyzed using the chi-squared test (χ2). Diversity in microbial communities was compared using diversity indices. A total of 54,644 OTUs were obtained. Culturable fungi produced 49,808 OTUs (91.15%), fungi not known from culture had 2571 OTUs (4.70%), non-fungal organisms had 1333 (2.44%) and organisms with no reference sequence in NCBI, 934 OTUs (1.71%). The total number of taxa ranged from 120 to 319. Fungi in stump wood were significantly more abundant in July–August than in May, in stumps >5 cm diameter than in stumps <5 cm diameter, in glyphosate-treated than in untreated stumps when sampled in May, and in untreated than in glyphosate-treated stumps when sampled in July–August. Species richness was significantly greater in July–August than in May, and in stumps >5 cm diameter than in stumps <5 cm diameter, either treated or untreated, depending on size. Herbicides can therefore affect the abundance and diversity of fungal communities in deciduous tree wood. The greater frequency of Ascomycota in herbicide-treated than in untreated stumps indicates their greater tolerance of glyphosate.

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Framing Cutting-Edge Integrative Deep-Sea Biodiversity Monitoring via Environmental DNA and Optoacoustic Augmented Infrastructures

Frontiers in Marine Science ◽

10.3389/fmars.2021.797140 ◽

2022 ◽

Vol 8 ◽

Author(s):

Sergio Stefanni ◽

Luca Mirimin ◽

David Stanković ◽

Damianos Chatzievangelou ◽

Lucia Bongiorni ◽

...

Keyword(s):

Deep Sea ◽

Environmental Dna ◽

Ecological Monitoring ◽

Environmental Data ◽

Marine Biodiversity ◽

Biodiversity Monitoring ◽

Imaging Data ◽

Optoacoustic Imaging ◽

Future Direction ◽

Critical Overview

Deep-sea ecosystems are reservoirs of biodiversity that are largely unexplored, but their exploration and biodiscovery are becoming a reality thanks to biotechnological advances (e.g., omics technologies) and their integration in an expanding network of marine infrastructures for the exploration of the seas, such as cabled observatories. While still in its infancy, the application of environmental DNA (eDNA) metabarcoding approaches is revolutionizing marine biodiversity monitoring capability. Indeed, the analysis of eDNA in conjunction with the collection of multidisciplinary optoacoustic and environmental data, can provide a more comprehensive monitoring of deep-sea biodiversity. Here, we describe the potential for acquiring eDNA as a core component for the expanding ecological monitoring capabilities through cabled observatories and their docked Internet Operated Vehicles (IOVs), such as crawlers. Furthermore, we provide a critical overview of four areas of development: (i) Integrating eDNA with optoacoustic imaging; (ii) Development of eDNA repositories and cross-linking with other biodiversity databases; (iii) Artificial Intelligence for eDNA analyses and integration with imaging data; and (iv) Benefits of eDNA augmented observatories for the conservation and sustainable management of deep-sea biodiversity. Finally, we discuss the technical limitations and recommendations for future eDNA monitoring of the deep-sea. It is hoped that this review will frame the future direction of an exciting journey of biodiscovery in remote and yet vulnerable areas of our planet, with the overall aim to understand deep-sea biodiversity and hence manage and protect vital marine resources.

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Spatial distribution of environmental DNA in a nearshore marine habitat

PeerJ ◽

10.7717/peerj.3044 ◽

2017 ◽

Vol 5 ◽

pp. e3044 ◽

Cited By ~ 61

Author(s):

James L. O’Donnell ◽

Ryan P. Kelly ◽

Andrew Olaf Shelton ◽

Jameal F. Samhouri ◽

Natalie C. Lowell ◽

...

Keyword(s):

Spatial Distribution ◽

Spatial Scales ◽

Environmental Dna ◽

Pcr Primers ◽

Ecological Monitoring ◽

Life History Stage ◽

Illumina Platform ◽

Marine Habitat ◽

Biological Communities ◽

Marine Surface

In the face of increasing threats to biodiversity, the advancement of methods for surveying biological communities is a major priority for ecologists. Recent advances in molecular biological technologies have made it possible to detect and sequence DNA from environmental samples (environmental DNA or eDNA); however, eDNA techniques have not yet seen widespread adoption as a routine method for biological surveillance primarily due to gaps in our understanding of the dynamics of eDNA in space and time. In order to identify the effective spatial scale of this approach in a dynamic marine environment, we collected marine surface water samples from transects ranging from the intertidal zone to four kilometers from shore. Using PCR primers that target a diverse assemblage of metazoans, we amplified a region of mitochondrial 16S rDNA from the samples and sequenced the products on an Illumina platform in order to detect communities and quantify their spatial patterns using a variety of statistical tools. We find evidence for multiple, discrete eDNA communities in this habitat, and show that these communities decrease in similarity as they become further apart. Offshore communities tend to be richer but less even than those inshore, though diversity was not spatially autocorrelated. Taxon-specific relative abundance coincided with our expectations of spatial distribution in taxa lacking a microscopic, pelagic life-history stage, though most of the taxa detected do not meet these criteria. Finally, we use carefully replicated laboratory procedures to show that laboratory treatments were remarkably similar in most cases, while allowing us to detect a faulty replicate, emphasizing the importance of replication to metabarcoding studies. While there is much work to be done before eDNA techniques can be confidently deployed as a standard method for ecological monitoring, this study serves as a first analysis of diversity at the fine spatial scales relevant to marine ecologists and confirms the promise of eDNA in dynamic environments.

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Towards Naples Ecological REsearch for Augmented Observatories (NEREA): The NEREA-Fix Module, a Stand-Alone Platform for Long-Term Deep-Sea Ecosystem Monitoring

Sensors ◽

10.3390/s20102911 ◽

2020 ◽

Vol 20 (10) ◽

pp. 2911

Author(s):

Emanuela Fanelli ◽

Jacopo Aguzzi ◽

Simone Marini ◽

Joaquin del Rio ◽

Marc Nogueras ◽

...

Keyword(s):

Deep Sea ◽

Human Impacts ◽

Environmental Dna ◽

Ecological Monitoring ◽

Mooring Line ◽

Tyrrhenian Sea ◽

Ecological Research ◽

Ecosystem Monitoring ◽

Wide Range

Deep-sea ecological monitoring is increasingly recognized as indispensable for the comprehension of the largest biome on Earth, but at the same time it is subjected to growing human impacts for the exploitation of biotic and abiotic resources. Here, we present the Naples Ecological REsearch (NEREA) stand-alone observatory concept (NEREA-fix), an integrated observatory with a modular, adaptive structure, characterized by a multiparametric video-platform to be deployed in the Dohrn canyon (Gulf of Naples, Tyrrhenian Sea) at ca. 650 m depth. The observatory integrates a seabed platform with optoacoustic and oceanographic/geochemical sensors connected to a surface transmission buoy, plus a mooring line (also equipped with depth-staged environmental sensors). This reinforced high-frequency and long-lasting ecological monitoring will integrate the historical data conducted over 40 years for the Long-Term Ecological Research (LTER) at the station “Mare Chiara”, and ongoing vessel-assisted plankton (and future environmental DNA-eDNA) sampling. NEREA aims at expanding the observational capacity in a key area of the Mediterranean Sea, representing a first step towards the establishment of a bentho-pelagic network to enforce an end-to-end transdisciplinary approach for the monitoring of marine ecosystems across a wide range of animal sizes (from bacteria to megafauna).

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Comparison of Water Sampling between Environmental DNA Metabarcoding and Conventional Microscopic Identification: A Case Study in Gwangyang Bay, South Korea

Applied Sciences ◽

10.3390/app9163272 ◽

2019 ◽

Vol 9 (16) ◽

pp. 3272

Author(s):

Kim ◽

Park ◽

Jo ◽

Kwak

Keyword(s):

South Korea ◽

Community Composition ◽

Late Summer ◽

Environmental Dna ◽

Ecological Monitoring ◽

Data Sampling ◽

Biological Communities ◽

Microscopic Identification ◽

Gwangyang Bay ◽

Study Sites

Our study focuses on methodological comparison of plankton community composition in relation to ecological monitoring and assessment with data sampling. Recently, along with the advancement of monitoring techniques, metabarcoding has been widely used in the context of environmental DNA (eDNA). We examine the applicability of eDNA metabarcoding for effective monitoring and assessment of community composition, compared with conventional observation using microscopic identification in a coastal ecosystem, Gwangynag Bay in South Korea. Our analysis is based primarily on two surveys at a total of 15 study sites in early and late summer (June and September) of the year 2018. The results of our study demonstrate the similarity and dissimilarity of biological communities in composition, richness and diversity between eDNA metabarcoding and conventional microscopic identification. It is found that, overall, eDNA metabarcoding appears to provide a wider variety of species composition, while conventional microscopic identification depicts more distinct plankton communities in sites. Finally, we suggest that eDNA metabarcoding is a practically useful method and can be potentially considered as a valuable alternative for biological monitoring and diversity assessments.

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Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities

Applied and Environmental Microbiology ◽

10.1128/aem.02637-17 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 11

Author(s):

Jaime Aguayo ◽

Céline Fourrier-Jeandel ◽

Claude Husson ◽

Renaud Ioos

Keyword(s):

Dna Extraction ◽

High Throughput ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

Linear Models ◽

Environmental Dna ◽

Illumina Miseq ◽

Fungal Communities ◽

Correct Identification ◽

Spore Trap

ABSTRACTTechniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCEThe advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

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Spatial distribution of environmental DNA in a nearshore marine habitat

10.7287/peerj.preprints.2608 ◽

2016 ◽

Author(s):

James L O'Donnell ◽

Ryan P Kelly ◽

Andrew Olaf Shelton ◽

Jameal F Samhouri ◽

Natalie C Lowell ◽

...

Keyword(s):

Spatial Distribution ◽

Massively Parallel Sequencing ◽

Spatial Scales ◽

Environmental Dna ◽

Ecological Monitoring ◽

Life History Stage ◽

Marine Habitat ◽

Biological Communities ◽

The Face ◽

Marine Surface

In the face of increasing threats to biodiversity, the advancement of methods for surveying biological communities is a major priority for ecologists. Recent advances in molecular biological technologies have made it possible to detect and sequence DNA from environmental samples (environmental DNA or eDNA); however, eDNA techniques have not yet seen widespread adoption as a routine method for biological surveillance primarily due to gaps in our understanding of the dynamics of eDNA in space and time. In order to identify the effective spatial scale of this approach in a dynamic marine environment, we collected marine surface water samples from transects ranging from the intertidal zone to 4 kilometers from shore. Using massively parallel sequencing of 16S amplicons, we identified a diverse community of metazoans and quantified their spatial patterns using a variety of statistical tools. We find evidence for multiple, discrete eDNA communities in this habitat, and show that these communities decrease in similarity as they become further apart. Offshore communities tend to be richer but less even than those inshore, though diversity was not spatially autocorrelated. Taxon-specific relative abundance coincided with our expectations of spatial distribution in taxa lacking a microscopic, pelagic life-history stage, though most of the taxa detected do not meet these criteria. Finally, we use carefully replicated laboratory procedures to show that laboratory treatments were remarkably similar in most cases, while allowing us to detect a faulty replicate, emphasizing the importance of replication to metabarcoding studies. While there is much work to be done before eDNA techniques can be confidently deployed as a standard method for ecological monitoring, this study serves as a first analysis of diversity at the fine spatial scales relevant to marine ecologists and confirms the promise of eDNA in dynamic environments.

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Spatial distribution of environmental DNA in a nearshore marine habitat

10.7287/peerj.preprints.2608v1 ◽

2016 ◽

Author(s):

James L O'Donnell ◽

Ryan P Kelly ◽

Andrew Olaf Shelton ◽

Jameal F Samhouri ◽

Natalie C Lowell ◽

...

Keyword(s):

Spatial Distribution ◽

Massively Parallel Sequencing ◽

Spatial Scales ◽

Environmental Dna ◽

Ecological Monitoring ◽

Life History Stage ◽

Marine Habitat ◽

Biological Communities ◽

The Face ◽

Marine Surface

In the face of increasing threats to biodiversity, the advancement of methods for surveying biological communities is a major priority for ecologists. Recent advances in molecular biological technologies have made it possible to detect and sequence DNA from environmental samples (environmental DNA or eDNA); however, eDNA techniques have not yet seen widespread adoption as a routine method for biological surveillance primarily due to gaps in our understanding of the dynamics of eDNA in space and time. In order to identify the effective spatial scale of this approach in a dynamic marine environment, we collected marine surface water samples from transects ranging from the intertidal zone to 4 kilometers from shore. Using massively parallel sequencing of 16S amplicons, we identified a diverse community of metazoans and quantified their spatial patterns using a variety of statistical tools. We find evidence for multiple, discrete eDNA communities in this habitat, and show that these communities decrease in similarity as they become further apart. Offshore communities tend to be richer but less even than those inshore, though diversity was not spatially autocorrelated. Taxon-specific relative abundance coincided with our expectations of spatial distribution in taxa lacking a microscopic, pelagic life-history stage, though most of the taxa detected do not meet these criteria. Finally, we use carefully replicated laboratory procedures to show that laboratory treatments were remarkably similar in most cases, while allowing us to detect a faulty replicate, emphasizing the importance of replication to metabarcoding studies. While there is much work to be done before eDNA techniques can be confidently deployed as a standard method for ecological monitoring, this study serves as a first analysis of diversity at the fine spatial scales relevant to marine ecologists and confirms the promise of eDNA in dynamic environments.

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Metabarcoding on both environmental DNA and RNA highlights differences between fungal communities sampled in different habitats

PLoS ONE ◽

10.1371/journal.pone.0244682 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244682

Author(s):

Martino Adamo ◽

Samuele Voyron ◽

Matteo Chialva ◽

Roland Marmeisse ◽

Mariangela Girlanda

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Environmental Dna ◽

Taxonomic Composition ◽

Fungal Communities ◽

Rna Sequences ◽

Operational Taxonomic Units ◽

Dna And Rna ◽

Filamentous Microorganisms ◽

Active Communities

In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based (total) and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either “active” or “total” microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone.

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