Accumulation curves of environmental DNA sequences predict coastal fish diversity in the coral triangle

Environmental DNA (eDNA) has the potential to provide more comprehensive biodiversity assessments, particularly for vertebrates in species-rich regions. However, this method requires the completeness of a reference database (i.e. a list of DNA sequences attached to each species), which is not currently achieved for many taxa and ecosystems. As an alternative, a range of operational taxonomic units (OTUs) can be extracted from eDNA metabarcoding. However, the extent to which the diversity of OTUs provided by a limited eDNA sampling effort can predict regional species diversity is unknown. Here, by modelling OTU accumulation curves of eDNA seawater samples across the Coral Triangle, we obtained an asymptote reaching 1531 fish OTUs, while 1611 fish species are recorded in the region. We also accurately predict ( R ² = 0.92) the distribution of species richness among fish families from OTU-based asymptotes. Thus, the multi-model framework of OTU accumulation curves extends the use of eDNA metabarcoding in ecology, biogeography and conservation.

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Accumulation curves of environmental DNA sequences predict coastal fish diversity in the Coral Triangle

Authorea ◽

10.22541/au.158082947.75232617 ◽

2020 ◽

Author(s):

Jean Baptiste Juhel ◽

Rizkie Utama ◽

Virginie Marques ◽

Indra Vimono ◽

Hagi Sugeha ◽

...

Keyword(s):

Dna Sequences ◽

Environmental Dna ◽

Fish Diversity ◽

Coral Triangle ◽

Coastal Fish ◽

Accumulation Curves

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Environmental DNA in a Global Biodiversity Hotspot: Lessons from Coral Reef Fish Diversity Across the Indonesian Archipelago

10.1101/2021.02.19.432056 ◽

2021 ◽

Author(s):

Onny N. Marwayana ◽

Zachary Gold ◽

Paul H. Barber

Keyword(s):

Survey Methods ◽

Marine Ecosystem ◽

Sampling Site ◽

Environmental Dna ◽

Marine Biodiversity ◽

Fish Diversity ◽

Sampling Effort ◽

Coral Triangle ◽

Regional Patterns ◽

Biodiversity Survey

AbstractIndonesia is the heart of the Coral Triangle, the world’s most diverse marine ecosystem. Preserving the biological and economic value of this marine biodiversity requires efficient and economical ecosystem monitoring, yet our understanding of marine biodiversity in this region remains limited. This study uses environmental DNA (eDNA) to survey fish communities across a pronounced biodiversity gradient in Indonesia. A total of 12,939,690 sequence reads of MiFish12SrRNA from 39 sites spanning 7 regions of Indonesia revealed 4,146 Amplified Sequence Variants (ASVs). Regional patterns of fish diversity based on eDNA broadly conformed to expectations based on traditional biodiversity survey methods, with the highest fish biodiversity in Raja Ampat and generally lower diversity in Western Indonesia. However, eDNA performed relatively poorly compared to visual survey methods in site-by-site comparisons, both in terms of total number of taxa recovered and ability to assign species names to ASVs. This result stands in a stark contrast to eDNA studies of temperate and tropical ecosystems with lower diversity. Analyses show that while sequencing depth was sufficient to capture all fish diversity within individual seawater samples, variation among samples from individual localities was high, and sampling effort was insufficient to capture all fish diversity at a given sampling site. Interestingly, mean ASVs recovered per one-liter seawater was surprisingly similar across sites, despite substantial differences in total diversity, suggesting a limit to total ASVs (~200) per one-liter eDNA sample. Combined, results highlight two major challenges of eDNA in highly diverse ecosystems such as the Coral Triangle. First, reference databases are incomplete and insufficient for effective ASV taxonomic assignment. Second, eDNA sampling design developed from lower diversity temperate marine ecosystems are inadequate to fully capture diversity of biodiversity hotspots like the Coral Triangle.

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Overcoming limitations to environmental DNA studies: A coastal temperate reference sequence database for multiple chloroplast gene regions generated in a single assay.

10.22541/au.163252330.05592688/v1 ◽

2021 ◽

Author(s):

Nicole Foster ◽

Kor-jent Dijk ◽

Ed Biffin ◽

Jennifer Young ◽

Vicki Thomson ◽

...

Keyword(s):

Dna Sequences ◽

Dna Barcode ◽

Environmental Dna ◽

Reference Sequence ◽

Reference Database ◽

Chloroplast Gene ◽

Coastal Plants ◽

Reference Databases ◽

Targeted Capture ◽

Comprehensive Reference

A proliferation in environmental DNA (eDNA) research has increased the reliance on reference sequence databases to assign unknown DNA sequences to known taxa. Without comprehensive reference databases, DNA extracted from environmental samples cannot be correctly assigned to taxa, limiting the use of this genetic information to identify organisms in unknown sample mixtures. For animals, standard metabarcoding practices involve amplification of the mitochondrial Cytochrome-c oxidase subunit 1 (CO1) region, which is a universally amplifyable region across majority of animal taxa. This region, however, does not work well as a DNA barcode for plants and fungi, and there is no similar universal single barcode locus that has the same species resolution. Therefore, generating reference sequences has been more difficult and several loci have been suggested to be used in parallel to get to species identification. For this reason, we developed a multi-gene targeted capture approach to generate reference DNA sequences for plant taxa across 20 target chloroplast gene regions in a single assay. We successfully compiled a reference database for 93 temperate coastal plants including seagrasses, mangroves, and saltmarshes/samphire’s. We demonstrate the importance of a comprehensive reference database to prevent species going undetected in eDNA studies. We also investigate how using multiple chloroplast gene regions impacts the ability to discriminate between taxa.

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Seeing through sedimented waters: environmental DNA reduces the phantom diversity of sharks and rays in turbid marine habitats

BMC Ecology and Evolution ◽

10.1186/s12862-021-01895-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yin Cheong Aden Ip ◽

Jia Jin Marc Chang ◽

Kelvin K. P. Lim ◽

Zeehan Jaafar ◽

Benjamin J. Wainwright ◽

...

Keyword(s):

High Throughput Sequencing ◽

Environmental Dna ◽

Historical Collections ◽

Operational Taxonomic Units ◽

Marine Habitats ◽

The Pacific ◽

Elasmobranch Species ◽

Reef Sharks ◽

Seawater Samples ◽

Diversity Estimates

Abstract Background Sharks and rays are some of the most threatened marine taxa due to the high levels of bycatch and significant demand for meat and fin-related products in many Asian communities. At least 25% of shark and ray species are considered to be threatened with extinction. In particular, the density of reef sharks in the Pacific has declined to 3–10% of pre-human levels. Elasmobranchs are thought to be sparse in highly urbanised and turbid environments. Low visibility coupled with the highly elusive behaviour of sharks and rays pose a challenge to diversity estimation and biomonitoring efforts as sightings are limited to chance encounters or from carcasses ensnared in nets. Here we utilised an eDNA metabarcoding approach to enhance the precision of elasmobranch diversity estimates in urbanised marine environments. Results We applied eDNA metabarcoding on seawater samples to detect elasmobranch species in the hyper-urbanised waters off Singapore. Two genes—vertebrate 12S and elasmobranch COI—were targeted and amplicons subjected to Illumina high-throughput sequencing. With a total of 84 water samples collected from nine localities, we found 47 shark and ray molecular operational taxonomic units, of which 16 had species-level identities. When data were compared against historical collections and contemporary sightings, eDNA detected 14 locally known species as well as two potential new records. Conclusions Local elasmobranch richness uncovered by eDNA is greater than the seven species sighted over the last two decades, thereby reducing phantom diversity. Our findings demonstrate that eDNA metabarcoding is effective in detecting shark and ray species despite the challenges posed by the physical environment, granting a more consistent approach to monitor these highly elusive and threatened species.

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Documenting decapod biodiversity in the Caribbean from DNA barcodes generated during field training in taxonomy

Biodiversity Data Journal ◽

10.3897/bdj.8.e47333 ◽

2020 ◽

Vol 8 ◽

Cited By ~ 2

Author(s):

Dagoberto Venera-Pontón ◽

Amy Driskell ◽

Sammy De Grave ◽

Darryl Felder ◽

Justin Scioli ◽

...

Keyword(s):

Cryptic Species ◽

Dna Sequences ◽

Marine Invertebrates ◽

Graduate Training ◽

Dna Barcode ◽

Reference Database ◽

Diagnostic Features ◽

Operational Taxonomic Units ◽

As Species ◽

Coi Sequences

DNA barcoding is a useful tool to identify the components of mixed or bulk samples, as well as to determine individuals that lack morphologically diagnostic features. However, the reference database of DNA barcode sequences is particularly sparsely populated for marine invertebrates and for tropical taxa. We used samples collected as part of two field courses, focused on graduate training in taxonomy and systematics, to generate DNA sequences of the barcode fragments of cytochrome c oxidase subunit I (COI) and mitochondrial ribosomal 16S genes for 447 individuals, representing at least 129 morphospecies of decapod crustaceans. COI sequences for 36% (51/140) of the species and 16S sequences for 26% (37/140) of the species were new to GenBank. Automatic Barcode Gap Discovery identified 140 operational taxonomic units (OTUs) which largely coincided with the morphospecies delimitations. Barcode identifications (i.e. matches to identified sequences) were especially useful for OTUs within Synalpheus, a group that is notoriously difficult to identify and rife with cryptic species, a number of which we could not identify to species, based on morphology. Non-concordance between morphospecies and barcode OTUs also occurred in a few cases of suspected cryptic species. As mitochondrial pseudogenes are particularly common in decapods, we investigate the potential for this dataset to include pseudogenes and discuss the utility of these sequences as species identifiers (i.e. barcodes). These results demonstrate that material collected and identified during training activities can provide useful incidental barcode reference samples for under-studied taxa.

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Environmental DNA (eDNA) metabarcoding: Diversity study around the Pondok Dadap fish landing station, Malang, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d201241 ◽

2019 ◽

Vol 20 (12) ◽

Cited By ~ 1

Author(s):

Sapto Andriyono ◽

MD. JOBAIDUL ALAM ◽

HYUN-WOO KIM

Keyword(s):

Marine Fish ◽

Molecular Identification ◽

Environmental Dna ◽

Fish Diversity ◽

Morphological Identification ◽

Fish Samples ◽

Identification Of Species ◽

Seawater Samples ◽

Next Generation Sequencing Ngs ◽

Diversity Study

Abstract. Andriyono S, Jobaidul Alam Md, Kim HW. 2019. Environmental DNA (eDNA) metabarcoding: Diversity study around the Pondok Dadap fish landing station, Malang, Indonesia. Biodiversitas 20: 3772-3781. Molecular identification of species is now fast growing and currently widely applied method in the diversity estimation of aquatic biota; even though morphological identification is still carried out. The molecular approach is beneficial complementing on regular surveys, e.g. use of nets, traps, fishing rods, and even with poisons. In this study, the eDNA metabarcoding was applied to water samples around the Pondok Dadap fish landing station, Indonesia to determine the diversity of fish around the waters and also to identify marine fish landed in this area. Molecular identification was carried out on fish samples obtained from the fish market improved GenBank database on COI and ITS. While, seawater samples were carried out by using the next-generation sequencing (NGS) platform to obtain the eDNA metabarcoding data for the first time. Molecular identification obtained 34 species (68 sequences of COI and ITS regions) belonging to 28 genera, 18 families, 4 orders, while the eDNA metabarcoding approach identified 53 marine fish species by using the MiFish pipeline representing 38 genera, 27 families, and 7 orders. From the present study, we can able to estimated fish diversity by eDNA metabarcoding, and this finding will be helpful for baseline data preparation for future effective management of resources in this area.

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An Optimized Metabarcoding Method for Mimiviridae

Microorganisms ◽

10.3390/microorganisms8040506 ◽

2020 ◽

Vol 8 (4) ◽

pp. 506

Author(s):

Florian Prodinger ◽

Hisashi Endo ◽

Yasuhiro Gotoh ◽

Yanze Li ◽

Daichi Morimoto ◽

...

Keyword(s):

Dna Polymerase ◽

Environmental Samples ◽

Environmental Dna ◽

Degenerate Primer ◽

Polymerase Gene ◽

Operational Taxonomic Units ◽

Ecological Relevance ◽

Seawater Samples ◽

Multiple Samples ◽

Pcr Conditions

Mimiviridae is a group of viruses with large genomes and virions. Ecological relevance of Mimiviridae in marine environments has been increasingly recognized through the discoveries of novel isolates and metagenomic studies. To facilitate ecological profiling of Mimiviridae, we previously proposed a meta-barcoding approach based on 82 degenerate primer pairs (i.e., MEGAPRIMER) targeting the DNA polymerase gene of Mimiviridae. The method detected a larger number of operational taxonomic units (OTUs) in environmental samples than previous methods. However, it required large quantities of DNA and was laborious due to the use of individual primer pairs. Here, we examined coastal seawater samples using varying PCR conditions and purification protocols to streamline the MEGAPRIMER method. Mixing primer pairs in “cocktails” reduced the required amount of environmental DNA by 90%, while reproducing the results obtained by the original protocol. We compared the results obtained by the meta-barcoding approach with quantifications using qPCR for selected OTUs. This revealed possible amplification biases among different OTUs, but the frequency profiles for individual OTUs across multiple samples were similar to those obtained by qPCR. We anticipate that the newly developed MEGAPRIMER protocols will be useful for ecological investigation of Mimiviridae in a larger set of environmental samples.

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Assessing accuracy and completeness of GenBank for eDNA metabarcoding: towards a reliable marine fish reference database

ARPHA Conference Abstracts ◽

10.3897/aca.4.e64671 ◽

2021 ◽

Vol 4 ◽

Author(s):

Cristina Claver ◽

Oriol Canals ◽

Naiara Rodriguez-Ezpeleta

Keyword(s):

Ribosomal Rna ◽

Gap Analysis ◽

Environmental Dna ◽

Reference Database ◽

Fish Diversity ◽

Cyt B ◽

Taxonomic Assignment ◽

Reference Databases ◽

Taxonomic Groups ◽

High Level

Environmental DNA (eDNA) metabarcoding, the process of sequencing DNA collected from the environment for producing biodiversity inventories, is increasingly being applied to assess fish diversity and distribution in marine environments. Yet, the successful application of this technique deeply relies on accurate and complete reference databases used for taxonomic assignment. The most used markers for fish eDNA metabarcoding studies are the cytochrome C oxidase subunit 1 (COI), 16S ribosomal RNA (16S), the 12S ribosomal RNA (12S) and cytochrome b (cyt b) genes, whose sequences are usually retrieved from GenBank, the largest DNA sequence database that represents a worldwide public resource for genetic studies. Thus, the completeness and accuracy of GenBank is critical to derive reliable estimations from fish eDNA metabarcoding data. Here, we have i) compiled the checklist of European marine fishes, ii) performed a gap analysis of the four genes and, within COI and 12S, also of the most used barcodes for fish, and iii) developed a workflow to detect potentially incorrect records in GenBank. We found that from the 1965 species in the checklist (1761 Actinopterygii, 189 Elasmobranchii, 9 Holocephali, 4 Petromyzonti and 2 Myxini), about 70% have sequences for COI, whereas less have sequences for 12S, 16S and cyt b (45-55%). Among the species for which COI ad 12S sequences are available, about 60% and 40% have sequences covering the most used barcodes respectively. The analysis of pairwise distances between sequences revealed pairs belonging to the same species with significantly low similarity and pairs belonging to different high level taxonomic groups (class, order) with significantly large similarity. In light of this further confirmation of presence of a substantial number of incorrect records in GenBank, we propose a method for identifying and removing spurious sequences to create reliable and accurate reference databases for eDNA metabarcoding.

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Toward a global reference database of COI barcodes for marine zooplankton

Marine Biology ◽

10.1007/s00227-021-03887-y ◽

2021 ◽

Vol 168 (6) ◽

Author(s):

Ann Bucklin ◽

Katja T. C. A. Peijnenburg ◽

Ksenia N. Kosobokova ◽

Todd D. O’Brien ◽

Leocadio Blanco-Bercial ◽

...

Keyword(s):

Species Diversity ◽

Dna Sequences ◽

Reference Sequence ◽

Global Ocean ◽

Reference Database ◽

Data Repositories ◽

Marine Zooplankton ◽

The North ◽

Coi Sequences ◽

Taxonomic Groups

AbstractCharacterization of species diversity of zooplankton is key to understanding, assessing, and predicting the function and future of pelagic ecosystems throughout the global ocean. The marine zooplankton assemblage, including only metazoans, is highly diverse and taxonomically complex, with an estimated ~28,000 species of 41 major taxonomic groups. This review provides a comprehensive summary of DNA sequences for the barcode region of mitochondrial cytochrome oxidase I (COI) for identified specimens. The foundation of this summary is the MetaZooGene Barcode Atlas and Database (MZGdb), a new open-access data and metadata portal that is linked to NCBI GenBank and BOLD data repositories. The MZGdb provides enhanced quality control and tools for assembling COI reference sequence databases that are specific to selected taxonomic groups and/or ocean regions, with associated metadata (e.g., collection georeferencing, verification of species identification, molecular protocols), and tools for statistical analysis, mapping, and visualization. To date, over 150,000 COI sequences for ~ 5600 described species of marine metazoan plankton (including holo- and meroplankton) are available via the MZGdb portal. This review uses the MZGdb as a resource for summaries of COI barcode data and metadata for important taxonomic groups of marine zooplankton and selected regions, including the North Atlantic, Arctic, North Pacific, and Southern Oceans. The MZGdb is designed to provide a foundation for analysis of species diversity of marine zooplankton based on DNA barcoding and metabarcoding for assessment of marine ecosystems and rapid detection of the impacts of climate change.

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Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

Antarctic Science ◽

10.1017/s0954102021000274 ◽

2021 ◽

pp. 1-10

Author(s):

Micheline Carvalho-Silva ◽

Luiz Henrique Rosa ◽

Otávio H.B. Pinto ◽

Thamar Holanda Da Silva ◽

Diego Knop Henriques ◽

...

Keyword(s):

High Throughput Sequencing ◽

Crater Lake ◽

Environmental Dna ◽

Food Sources ◽

South Shetland Islands ◽

Deception Island ◽

Shetland Islands ◽

Operational Taxonomic Units ◽

First Time ◽

Impacted Site

Abstract The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

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