How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Quantifying the diversity of species in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, the reliability of biodiversity detection from eDNA samples can be low in marine environments because eDNA density is low and certainly patchy in this vast, heterogenous and dynamic environment. So, the number of sampling replicates and filtered volume necessary to obtain accurate estimates of biodiversity in rich tropical marine environments using eDNA metabarcoding is still unknown. Here, we used a paired sampling design of 30L per replicate on 68 reef transects from 8 sites in three tropical regions and identified fish Molecular Taxonomic Units (MOTUs) using a 12S marker. We quantified local biodiversity variation as MOTU richness, compositional turnover and compositional nestedness between replicated pairs of seawater samples. We report strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non-overlapping assemblages rather than subsets of one-another. As a result, localised diversity accumulation curves showed that even 6 replicates (180L) in the same location underestimated local diversity (for an area <1km). However, sampling of regional diversity using ~25 replicates in variable locations (often covering 10s of km) achieved saturation of biodiversity accumulation curves. Our results demonstrate high variability of diversity estimates perhaps arising from heterogeneous and local distribution of eDNA distribution in seawater or highly skewed frequencies of eDNA traces. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes of local assemblages. Future biomonitoring efforts could be strongly undermined by a high level of false-negative detections under low replication protocols. We reveal the need to increase replicates or increase sampled water volume to better inform management of marine biodiversity using eDNA.

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Environmental DNA diffusion of reef fishes along a distance gradient from four isolated islands of the Western Indian Ocean

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65060 ◽

2021 ◽

Vol 4 ◽

Author(s):

Mélissa Jaquier ◽

Camille Albouy ◽

Wilhelmine Bach ◽

Conor Waldock ◽

Viriginie Marques ◽

...

Keyword(s):

Distance Sampling ◽

Environmental Dna ◽

Western Indian Ocean ◽

Reef Fishes ◽

Species Distribution Modelling ◽

Model Systems ◽

Reference Database ◽

Developmental Phase ◽

Marine Environments ◽

Habitat Types

Islands have traditionally served as model systems to study ecological and evolutionary processes (Warren et al. 2015) and could also represent a relevant system to study environmental DNA (eDNA). Isolated island reefs that are affected by climatic threats would particularly benefit from cost- and time-efficient biodiversity surveys to set priorities for their conservation. Among time efficiency methods, eDNA has emerged as a novel molecular metabarcoding technique to detect biodiversity from simple environmental samples even in remote marine environments. However, eDNA monitoring techniques for marine environments are at a developmental phase, with a few remaining unknowns related to DNA residence time and movement. In particular, the redistribution of eDNA, via ocean currents, could blur the composition signal and its association with local environmental conditions (Goldberg et al. 2016). Here, we investigated the detection variation of eDNA along a distance gradient across four islands in the French Scattered Islands. We collected 30 L of surface water per filter at an increasing distance from the islands reefs (0m, 250m, 500m, 750m). Using a metabarcoding protocol, we used the teleo primers to target a fraction of 12S mitochondrial DNA to detect Actinopterygii and Elasmobranchii. We then applied a sequence clustering approach to generate Molecular Taxonomic Units (MOTUs), which were assigned to a taxonomic group using a reference database. By assigning eDNA sequences to species using a public reference database, we classified species according to their preferred habitat types between benthic/demersal and pelagic. Our results show no significant relationship between distance and MOTUs richness for both habitat types. By using a Joint Species Distribution Modelling approach (JSDM, Hierarchical Modelling of Species Communities), we retained the multidimensional information captured by eDNA and detect species- and family-specific responses to distance (Fig. 1). We showed that benthic MOTUs were found in closer proximity to the reef, while typical pelagic MOTUs were found at greater distances from the reef. Hence, MOTU-level analyses coupled with JSDM were more informative that when aggregating it into coarser richness. Altogether, our eDNA distance sampling gradient detected an ecological signal of habitat selection by fish species, which suggest that eDNA could help understand the behavior of species and their distribution in marine environments at a fine spatial scale.

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Estimation accuracy of species abundance based on environmental DNA with relation to its production source, state, and assay methodology suggested by meta-analyses

10.22541/au.163490255.53353112/v1 ◽

2021 ◽

Author(s):

Toshiaki Jo ◽

Hiroki Yamanaka

Keyword(s):

Meta Analysis ◽

Empirical Studies ◽

Species Abundance ◽

Environmental Dna ◽

Efficient Estimation ◽

Estimation Accuracy ◽

Accurate Estimation ◽

Abundance Estimation ◽

Natural Environments ◽

Promising Tool

Environmental DNA (eDNA) analysis is a promising tool for non-disruptive and cost-efficient estimation of species abundance. However, its practical applicability in natural environments is limited because it is unclear whether eDNA concentrations actually represent species abundance in the field. Although the importance of accounting for eDNA dynamics, such as transport and degradation, has been discussed, the influences of eDNA characteristics, including production source and state, and methodology, including collection and quantification strategy and abundance metrics, on the accuracy of eDNA-based abundance estimation were entirely overlooked. We conducted a meta-analysis using 56 previous eDNA literature and investigated the relationships between the accuracy (R2) of eDNA-based abundance estimation and eDNA characteristics and methodology. Our meta-regression analysis found that R2 values were significantly lower for crustaceans than fish, suggesting that less frequent eDNA production owing to their external morphology and physiology may impede accurate estimation of their abundance via eDNA. Moreover, R2 values were positively associated with filter pore size, indicating that selective collection of larger-sized eDNA, which is typically fresher, could improve the estimation accuracy of species abundance. Furthermore, R2 values were significantly lower for natural than laboratory conditions, while there was no difference in the estimation accuracy among natural environments. Our findings shed a new light on the importance of what characteristics of eDNA should be targeted for more accurate estimation of species abundance. Further empirical studies are required to validate our findings and fully elucidate the relationship between eDNA characteristics and eDNA-based abundance estimation.

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Environmental DNA (eDNA) metabarcoding surveys extend the range of invasion for non-indigenous freshwater species in Eastern Europe.

10.1101/2021.05.16.444374 ◽

2021 ◽

Author(s):

Gert-Jan Jeunen ◽

Tatsiana Lipinskaya ◽

Helen Gajduchenko ◽

Viktoriya Golovenchik ◽

Michail Moroz ◽

...

Keyword(s):

Surface Water ◽

Water Samples ◽

False Negative ◽

Simultaneous Detection ◽

Environmental Dna ◽

Reference Database ◽

Indigenous Species ◽

Freshwater Species ◽

Traditional Approaches ◽

Surface Water Samples

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e., an ichthyological, hydrobiological, and eDNA survey, whereby DNA was extracted from 500 mL surface water samples and amplified with two 16S rRNA primer assays targeting the fish and macro-invertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous sediment-living macro-invertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two sediment-living macro-invertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey could be attributed to (i) preferential amplification of aquatic over sediment-living macro-invertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximize the probability of early detection of non-native organisms.

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False‐negative detections from environmental DNA collected in the presence of large numbers of killer whales ( Orcinus orca )

Environmental DNA ◽

10.1002/edn3.32 ◽

2019 ◽

Vol 1 (4) ◽

pp. 316-328 ◽

Cited By ~ 4

Author(s):

Róisín Pinfield ◽

Eileen Dillane ◽

Anne Kathrine W. Runge ◽

Alice Evans ◽

Luca Mirimin ◽

...

Keyword(s):

False Negative ◽

Environmental Dna ◽

Orcinus Orca ◽

Killer Whales ◽

Large Numbers

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Ancient DNA complements microfossil record in deep-sea subsurface sediments

Biology Letters ◽

10.1098/rsbl.2013.0283 ◽

2013 ◽

Vol 9 (4) ◽

pp. 20130283 ◽

Cited By ~ 67

Author(s):

Franck Lejzerowicz ◽

Philippe Esling ◽

Wojciech Majewski ◽

Witold Szczuciński ◽

Johan Decelle ◽

...

Keyword(s):

Ancient Dna ◽

Deep Sea ◽

Environmental Dna ◽

Small Subunit ◽

Marine Biodiversity ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Core Sediment ◽

Subsurface Sediments ◽

Sediment Layers

Deep-sea subsurface sediments are the most important archives of marine biodiversity. Until now, these archives were studied mainly using the microfossil record, disregarding large amounts of DNA accumulated on the deep-sea floor. Accessing ancient DNA (aDNA) molecules preserved down-core would offer unique insights into the history of marine biodiversity, including both fossilized and non-fossilized taxa. Here, we recover aDNA of eukaryotic origin across four cores collected at abyssal depths in the South Atlantic, in up to 32.5 thousand-year-old sediment layers. Our study focuses on Foraminifera and Radiolaria, two major groups of marine microfossils also comprising diverse non-fossilized taxa. We describe their assemblages in down-core sediment layers applying both micropalaeontological and environmental DNA sequencing approaches. Short fragments of the foraminiferal and radiolarian small subunit rRNA gene recovered from sedimentary DNA extracts provide evidence that eukaryotic aDNA is preserved in deep-sea sediments encompassing the last glacial maximum. Most aDNA were assigned to non-fossilized taxa that also dominate in molecular studies of modern environments. Our study reveals the potential of aDNA to better document the evolution of past marine ecosystems and opens new horizons for the development of deep-sea palaeogenomics.

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Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

Applied and Environmental Microbiology ◽

10.1128/aem.00796-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7253-7260 ◽

Cited By ~ 13

Author(s):

R. van Doorn ◽

M. M. Klerks ◽

M. P. E. van Gent-Pelzer ◽

A. G. C. L. Speksnijder ◽

G. A. Kowalchuk ◽

...

Keyword(s):

High Throughput ◽

Environmental Samples ◽

False Negative ◽

Environmental Dna ◽

Target Range ◽

Gene Target ◽

Soil Dna ◽

Control Approach ◽

Wide Range ◽

Accurate Quantification

ABSTRACT PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

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Effect of method of deduplication on estimation of differential gene expression using RNA-seq

PeerJ ◽

10.7717/peerj.3091 ◽

2017 ◽

Vol 5 ◽

pp. e3091 ◽

Cited By ~ 10

Author(s):

Anna V. Klepikova ◽

Artem S. Kasianov ◽

Mikhail S. Chesnokov ◽

Natalia L. Lazarevich ◽

Aleksey A. Penin ◽

...

Keyword(s):

Gene Expression ◽

Differential Gene Expression ◽

Cancer Genomics ◽

False Negative ◽

Read Length ◽

Rna Seq ◽

Negative Results ◽

Paired Samples ◽

Highly Expressed Genes ◽

Differential Gene

BackgroundRNA-seq is a useful tool for analysis of gene expression. However, its robustness is greatly affected by a number of artifacts. One of them is the presence of duplicated reads.ResultsTo infer the influence of different methods of removal of duplicated reads on estimation of gene expression in cancer genomics, we analyzed paired samples of hepatocellular carcinoma (HCC) and non-tumor liver tissue. Four protocols of data analysis were applied to each sample: processing without deduplication, deduplication using a method implemented in samtools, and deduplication based on one or two molecular indices (MI). We also analyzed the influence of sequencing layout (single read or paired end) and read length. We found that deduplication without MI greatly affects estimated expression values; this effect is the most pronounced for highly expressed genes.ConclusionThe use of unique molecular identifiers greatly improves accuracy of RNA-seq analysis, especially for highly expressed genes. We developed a set of scripts that enable handling of MI and their incorporation into RNA-seq analysis pipelines. Deduplication without MI affects results of differential gene expression analysis, producing a high proportion of false negative results. The absence of duplicate read removal is biased towards false positives. In those cases where using MI is not possible, we recommend using paired-end sequencing layout.

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Taxonomic identification of environmental DNA with informatic sequence classification trees.

10.7287/peerj.preprints.26812v1 ◽

2018 ◽

Cited By ~ 5

Author(s):

Shaun P Wilkinson ◽

Simon K Davy ◽

Michael Bunce ◽

Michael Stat

Keyword(s):

Classification Tree ◽

Probabilistic Method ◽

Query Sequence ◽

Dna Barcode ◽

Classification Trees ◽

Environmental Dna ◽

Training Data ◽

Marine Biodiversity ◽

Taxonomic Identification ◽

Sequence Classification

High-throughput sequencing of environmental DNA (eDNA) offers a simple and cost-effective solution for marine biodiversity assessments. Yet several analytical challenges remain, including the incorporation of statistical inference in the assignment of taxonomic identities. We developed a probabilistic method for DNA barcode classification that can be used for both eDNA and traditional single-source sampling. The pipeline involves: (1) compiling a primer-specific database of barcode sequences to be used as training data (obtained from GenBank and other sequence repositories), (2) generating a classification tree using an iterative learning algorithm that divisively sorts the training data into hierarchical clusters based on profile hidden Markov models, (3) assignment of each query sequence to a cluster using a recursive series of model-comparison tests, and (4) taxonomic identification of the query sequences based on the lowest common taxonomic rank of the training sequences within the cluster. This method compares favorably to other DNA classification methods when tested on benchmark datasets, and offers the added features of classifying at higher taxonomic ranks and returning interpretable confidence values in the form of the Akaike weight statistic. This bioinformatics pipeline is available as an open source R package called ‘insect’ (informatic sequence classification trees).

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eDNA Captures Microhabitat Partitioning in a Kelp Forest Ecosystem

10.1101/2021.06.01.446542 ◽

2021 ◽

Author(s):

Keira Monuki ◽

Paul Barber ◽

Zachary Gold

Keyword(s):

Southern California ◽

Surf Zone ◽

Habitat Preferences ◽

Kelp Forest ◽

Environmental Dna ◽

Marine Biodiversity ◽

Rocky Reef ◽

Temporal And Spatial Variability ◽

Community Assemblages ◽

Zone Sampling

Environmental DNA (eDNA) metabarcoding is an increasingly important tool for surveying biodiversity in marine ecosystems. However, the scale of temporal and spatial variability in eDNA signatures, and how this variation may impact eDNA-based marine biodiversity assessments, remains uncertain. To address this question, we systematically examined variation in vertebrate eDNA signatures across depth (0 m to 10 m) and horizontal space (nearshore and surf zone) over three successive days in a Southern California kelp forest. Across a broad range of marine vertebrates (teleosts, elasmobranchs, birds, and mammals), results showed significant variation in species richness and community assemblages across 4-5 m depth, reflecting microhabitat depth preferences of common Southern California nearshore rocky reef taxa. We also found significant differences in community assemblages between nearshore and surf zone sampling stations at the same depth, and across three sampling days. Patterns of microhabitat partitioning in eDNA signatures across space and time were largely consistent with known habitat preferences and species behavior. Results highlight the sensitivity of eDNA in capturing fine-scale vertical, horizontal, and temporal variation in marine vertebrate communities, demonstrating the ability of eDNA to capture a highly localized snapshot of marine biodiversity in dynamic coastal environments.

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Challenges to Implementing Environmental-DNA Monitoring in Namibia

Frontiers in Environmental Science ◽

10.3389/fenvs.2021.773991 ◽

2022 ◽

Vol 9 ◽

Author(s):

Iain Perry ◽

Ifan B. Jâms ◽

Roser Casas-Mulet ◽

Josefina Hamutoko ◽

Angela Marchbank ◽

...

Keyword(s):

Reference Data ◽

Sampling Technique ◽

Environmental Dna ◽

Cost Effective ◽

Trophic Levels ◽

Arid Ecosystems ◽

Test Field ◽

Local Conditions ◽

Promising Tool ◽

High Turbidity

By identifying fragments of DNA in the environment, eDNA approaches present a promising tool for monitoring biodiversity in a cost-effective way. This is particularly pertinent for countries where traditional morphological monitoring has been sparse. The first step to realising the potential of eDNA is to develop methodologies that are adapted to local conditions. Here, we test field and laboratory eDNA protocols (aqueous and sediment samples) in a range of semi-arid ecosystems in Namibia. We successfully gathered eDNA data on a broad suite of organisms at multiple trophic levels (including algae, invertebrates and bacteria) but identified two key challenges to the implementation of eDNA methods in the region: 1) high turbidity requires a tailored sampling technique and 2) identification of taxa by eDNA methods is currently constrained by a lack of reference data. We hope this work will guide the deployment of eDNA biomonitoring in the arid ecosystems of Namibia and neighbouring countries.

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