Metabarcoding Is Powerful yet Still Blind: A Comparative Analysis of Morphological and Molecular Surveys of Seagrass Communities

Abstract

In the context of the sixth wave of extinction, reliable surveys of biodiversity are increasingly needed to infer the cause and consequences of species and community declines, identify early warning indicators of tipping points, and provide reliable impact assessments before engaging in activities with potential environmental hazards. DNA metabarcoding has emerged as having potential to provide speedy assessment of community structure from environmental samples. Here we tested the reliability of metabarcoding by comparing morphological and molecular inventories of invertebrate communities associated with seagrasses through estimates of alpha and beta diversity, as well as the identification of the most abundant taxa. Sediment samples were collected from six Zostera marina seagrass meadows across Brittany, France. Metabarcoding surveys were performed using both mitochondrial (Cytochrome Oxidase I) and nuclear (small subunit 18S ribosomal RNA) markers, and compared to morphological inventories compiled by a long-term benthic monitoring network. A sampling strategy was defined to enhance performance and accuracy of results by preventing the dominance of larger animals, boosting statistical support through replicates, and using two genes to compensate for taxonomic biases. Molecular barcodes proved powerful by revealing a remarkable level of diversity that vastly exceeded the morphological survey, while both surveys identified congruent differentiation of the meadows. However, despite the addition of individual barcodes of common species into taxonomic reference databases, the retrieval of only 36% of these species suggest that the remaining were either not present in the molecular samples or not detected by the molecular screening. This finding exemplifies the necessity of comprehensive and well-curated taxonomic reference libraries and multi-gene surveys. Overall, results offer methodological guidelines and support for metabarcoding as a powerful and repeatable method of characterizing communities, while also presenting suggestions for improvement, including implementation of pilot studies prior to performing full “blind” metabarcoding assessments to optimize sampling and amplification protocols.

Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment

“…The accuracy of metabarcoding-based taxonomic inferences relies on the retrieval of a wide range of taxonomic groups from a given environmental sample using the appropriate barcode, primers, and amplification conditions (Deagle et al, 2014;Kress et al, 2015), and on the completeness of the reference database (Zepeda Mendoza et al, 2015). Some attempts have been performed to compare morphological vs. metabarcodingbased taxonomic inferences; yet, results are inconclusive as some studies do not apply both approaches to the same sample and/or have focused on a particular taxonomic group Carew et al, 2013;Zhou et al, 2013;Gibson et al, 2014;Cowart et al, 2015;Zimmermann et al, 2015). A recent study (Gibson et al, 2015) has performed morphological and metabarcoding-based taxonomic identification on the same freshwater aquatic invertebrate samples, but limited their visual identifications to family level.…”

Genetic and Manual Survey Methods Yield Different and Complementary Views of an Ecosystem

“…One means of doing so is to survey multiple genetic markers ("loci") simultaneously. Just as the results from any single genetic marker may differ from those of manual or visual sampling techniques, so too may the results differ among genetic markers (Cowart et al, 2015). Using multiple markers allows the researcher to assess the degree to which the detected ecological communities vary as a result of the primer set used, and where the taxa amplified by multiple markers do overlap, offers a chance to cross-validate detections of particular taxa of interest.…”

DNA Sequencing as a Tool to Monitor Marine Ecological Status

“…Amplification of 18S rRNA regions is used for a variety of communities, including phytoplankton and other microeukaryotes (Dunthorn et al, 2012;Hugerth et al, 2014;Johnson and Martiny, 2015;del Campo et al, 2016;Giner et al, 2016), and amplification of the 23S rRNA gene is used to classify organisms such as zooplankton (Hirai et al, 2015a;Bucklin et al, 2016). Amplification of mitochondrial DNA is used to identify an assortment of organisms, with cytochrome oxidase I (COI), cytochrome b, and mitochondrial 16S as examples of popularly employed target regions (Dauble et al, 2012;Pawlowski et al, 2014;Cowart et al, 2015;Guo et al, 2015;Harada et al, 2015;Johnson and Martiny, 2015;Aylagas et al, 2016;Bucklin et al, 2016;Creer et al, 2016;Leray and Knowlton, 2016;Thompson et al, 2016;Trivedi et al, 2016). Newer approaches are being used to identify fish (Miya et al, 2015) and marine mammals (Foote et al, 2012;Ma et al, 2016) from seawater samples.…”

“…Studies demonstrate DNA metabarcoding to be a reliable method for biodiversity assessment with potential for inferring biotic indices for marine ecosystem quality assessment (Aylagas et al, 2014;Pawlowski et al, 2014;Cowart et al, 2015;Elbrecht and Leese, 2015;Visco et al, 2015;Ferrera et al, 2016). Comparable results between molecular and traditional approaches are reported in a number of studies (Hirai et al, 2015a;Lejzerowicz et al, 2015;Aylagas et al, 2016Aylagas et al, , 2017Valentini et al, 2016), suggesting that assessment programs that require manually intensive sorting and visual inspection will be among the first to formally integrate molecular techniques.…”

“…Lack of intercalibration between morphological and molecular methods presents an additional issue, and transferability across taxa is a concern, particularly if the initial assessment used to establish the database is limited to one or two taxonomic groups (Kelly, 2016). Although comparable molecular and traditional approaches are reported (Lejzerowicz et al, 2015;Aylagas et al, 2016Aylagas et al, , 2017Hänfling et al, 2016;Valentini et al, 2016), other studies show discrepancies (Cowart et al, 2015;Hirai et al, 2015b;Mohrbeck et al, 2015;Giner et al, 2016;Kelly et al, 2017); therefore, it has been advised to use both approaches in a complementary fashion rather than outright substitution of morphotaxonomic approaches (Borja et al, 2008;Pedersen et al, 2015;Thomsen and Willerslev, 2015). Although such an approach may deliver a holistic perspective of marine ecological status, it would not produce the desired effect of streamlining monitoring efforts, which is needed to transition research into practice (de Jonge et al, 2006).…”