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1. Recommendations for interpreting zooplankton metabarcoding and integrating molecular methods with morphological analyses

   https://doi.org/10.1093/icesjms/fsab107  

   Matthews, Stephanie A ; Goetze, Erica ; Ohman, Mark D ( June 2021 , ICES Journal of Marine Science)

   Hauser, Lorenz (Ed.)

   Abstract Metabarcoding of zooplankton communities is becoming more common, but molecular results must be interpreted carefully and validated with morphology-based analyses, where possible. To evaluate our metabarcoding approach within the California Current Ecosystem, we tested whether physical subsampling and PCR replication affects observed community composition; whether community composition resolved by metabarcoding is comparable to morphological analyses by digital imaging; and whether pH neutralization of ethanol with ammonium hydroxide affects molecular diversity. We found that (1) PCR replication was important to accurately resolve alpha diversity and that physical subsampling can decrease sensitivity to rare taxa; (2) there were significant correlations between relative read abundance and proportions of carbon biomass for most taxonomic groups analyzed, but such relationships showed better agreement for the more dominant taxonomic groups; and (3) ammonium hydroxide in ethanol had no effect on molecular diversity. Together, these results indicate that with appropriate replication, paired metabarcoding and morphological analyses can characterize zooplankton community structure and biomass, and that metabarcoding methods are to some extent indicative of relative community composition when absolute measures of abundance or biomass are not available. 

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2. COI Metabarcoding of Zooplankton Species Diversity for Time-Series Monitoring of the NW Atlantic Continental Shelf

   https://doi.org/10.3389/fmars.2022.867893  

   Bucklin, Ann ; Batta-Lona, Paola G. ; Questel, Jennifer M. ; Wiebe, Peter H. ; Richardson, David E. ; Copley, Nancy J. ; O’Brien, Todd D. ( April 2022 , Frontiers in Marine Science)

   Marine zooplankton are rapid-responders and useful indicators of environmental variability and climate change impacts on pelagic ecosystems on time scales ranging from seasons to years to decades. The systematic complexity and taxonomic diversity of the zooplankton assemblage has presented significant challenges for routine morphological (microscopic) identification of species in samples collected during ecosystem monitoring and fisheries management surveys. Metabarcoding using the mitochondrial Cytochrome Oxidase I (COI) gene region has shown promise for detecting and identifying species of some – but not all – taxonomic groups in samples of marine zooplankton. This study examined species diversity of zooplankton on the Northwest Atlantic Continental Shelf using 27 samples collected in 2002-2012 from the Gulf of Maine, Georges Bank, and Mid-Atlantic Bight during Ecosystem Monitoring (EcoMon) Surveys by the NOAA NMFS Northeast Fisheries Science Center. COI metabarcodes were identified using the MetaZooGene Barcode Atlas and Database ( https://metazoogene.org/MZGdb ) specific to the North Atlantic Ocean. A total of 181 species across 23 taxonomic groups were detected, including a number of sibling and cryptic species that were not discriminated by morphological taxonomic analysis of EcoMon samples. In all, 67 species of 15 taxonomic groups had ≥ 50 COI sequences; 23 species had >1,000 COI sequences. Comparative analysis of molecular and morphological data showed significant correlations between COI sequence numbers and microscopic counts for 5 of 6 taxonomic groups and for 5 of 7 species with >1,000 COI sequences for which both types of data were available. Multivariate statistical analysis showed clustering of samples within each region based on both COI sequence numbers and EcoMon counts, although differences among the three regions were not statistically significant. The results demonstrate the power and potential of COI metabarcoding for identification of species of metazoan zooplankton in the context of ecosystem monitoring. 

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3. Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding

   https://doi.org/10.1002/ece3.8239  

   Shirazi, Sabrina ; Meyer, Rachel S. ; Shapiro, Beth ( November 2021 , Ecology and Evolution)
4. Richness and resilience in the Pacific: DNA metabarcoding enables parallelized evaluation of biogeographic patterns

   https://doi.org/10.1111/mec.16575  

   Kennedy, Susan ; Calaor, Jerilyn ; Zurápiti, Yazmín ; Hans, Julian ; Yoshimura, Masashi ; Choo, Juanita ; Andersen, Jeremy C. ; Callaghan, Jackson ; Roderick, George K. ; Krehenwinkel, Henrik ; et al ( December 2023 , Molecular Ecology)

   Islands make up a large proportion of Earth's biodiversity, yet are also some of the most sensitive systems to environmental perturbation. Biogeographic theory predicts that geologic age, area, and isolation typically drive islands' diversity patterns, and thus potentially impact non‐native spread and community homogenization across island systems. One limitation in testing such predictions has been the difficulty of performing comprehensive inventories of island biotas and distinguishing native from introduced taxa. Here, we use DNA metabarcoding and statistical modelling as a high throughput method to survey community‐wide arthropod richness, the proportion of native and non‐native species, and the incursion of non‐natives into primary habitats on three archipelagos in the Pacific – the Ryukyus, the Marianas and Hawaii – which vary in age, isolation and area. Diversity patterns largely match expectations based on island biogeography theory, with the oldest and most geographically connected archipelago, the Ryukyus, showing the highest taxonomic richness and lowest proportion of introduced species. Moreover, we find evidence that forest habitats are more resilient to incursions of non‐natives in the Ryukyus than in the less taxonomically rich archipelagos. Surprisingly, we do not find evidence for biotic homogenization across these three archipelagos: the assemblage of non‐native species on each island is highly distinct. Our study demonstrates the potential of DNA metabarcoding to facilitate rapid estimation of biogeographic patterns, the spread of non‐native species, and the resilience of ecosystems.

    

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5. Perspectives from Ten Years of Protist Studies by High‐Throughput Metabarcoding

   https://doi.org/10.1111/jeu.12813  

   Santoferrara, Luciana ; Burki, Fabien ; Filker, Sabine ; Logares, Ramiro ; Dunthorn, Micah ; McManus, George B. ( July 2020 , Journal of Eukaryotic Microbiology)

   During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding. 

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