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      Integration of DNA-Based Approaches in Aquatic Ecological Assessment Using Benthic Macroinvertebrates

      , , , ,
      Water
      MDPI AG

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          Abstract

          Benthic macroinvertebrates are among the most used biological quality elements for assessing the condition of all types of aquatic ecosystems worldwide (i.e., fresh water, transitional, and marine). Current morphology-based assessments have several limitations that may be circumvented by using DNA-based approaches. Here, we present a comprehensive review of 90 publications on the use of DNA metabarcoding of benthic macroinvertebrates in aquatic ecosystems bioassessments. Metabarcoding of bulk macrozoobenthos has been preferentially used in fresh waters, whereas in marine waters, environmental DNA (eDNA) from sediment and bulk communities from deployed artificial structures has been favored. DNA extraction has been done predominantly through commercial kits, and cytochrome c oxidase subunit I (COI) has been, by far, the most used marker, occasionally combined with others, namely, the 18S rRNA gene. Current limitations include the lack of standardized protocols and broad-coverage primers, the incompleteness of reference libraries, and the inability to reliably extrapolate abundance data. In addition, morphology versus DNA benchmarking of ecological status and biotic indexes are required to allow general worldwide implementation and higher end-user confidence. The increased sensitivity, high throughput, and faster execution of DNA metabarcoding can provide much higher spatial and temporal data resolution on aquatic ecological status, thereby being more responsive to immediate management needs.

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          Biological identifications through DNA barcodes.

          Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
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            Exact sequence variants should replace operational taxonomic units in marker-gene data analysis

            Recent advances have made it possible to analyze high-throughput marker-gene sequencing data without resorting to the customary construction of molecular operational taxonomic units (OTUs): clusters of sequencing reads that differ by less than a fixed dissimilarity threshold. New methods control errors sufficiently such that amplicon sequence variants (ASVs) can be resolved exactly, down to the level of single-nucleotide differences over the sequenced gene region. The benefits of finer resolution are immediately apparent, and arguments for ASV methods have focused on their improved resolution. Less obvious, but we believe more important, are the broad benefits that derive from the status of ASVs as consistent labels with intrinsic biological meaning identified independently from a reference database. Here we discuss how these features grant ASVs the combined advantages of closed-reference OTUs—including computational costs that scale linearly with study size, simple merging between independently processed data sets, and forward prediction—and of de novo OTUs—including accurate measurement of diversity and applicability to communities lacking deep coverage in reference databases. We argue that the improvements in reusability, reproducibility and comprehensiveness are sufficiently great that ASVs should replace OTUs as the standard unit of marker-gene analysis and reporting.
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              A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

              Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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                Author and article information

                Contributors
                Journal
                WATEGH
                Water
                Water
                MDPI AG
                2073-4441
                February 2021
                January 29 2021
                : 13
                : 3
                : 331
                Article
                10.3390/w13030331
                ff99c114-0e91-4000-bf7d-8372d8baf6b0
                © 2021

                https://creativecommons.org/licenses/by/4.0/

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