Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems.

Cyprus Marine and Maritime Institute CMMI House Larnaca Cyprus

Department of Biodiversity Ecology and Evolution Universidad Complutense de Madrid Madrid Spain

Department of Biology Geology and Environmental Science University of Tennessee at Chattanooga Chattanooga Tennessee USA

Department of Chemical Engineering Cyprus University of Technology Limassol Cyprus

Department of Ecology Faculty of Science Charles University Prague 2 Czech Republic

Department of Environmental Biology Institute of Environmental Sciences Leiden University Leiden The Netherlands

Department of Life Sciences University of Modena and Reggio Emilia Modena Italy

Department of Zoology Swedish Museum of Natural History Stockholm Sweden

Dipartimento di Medicina Veterinaria Università di Sassari Sassari Italy

Institute for Biodiversity and Ecosystem Dynamics University of Amsterdam Amsterdam The Netherlands

Laboratory of Environmental Microbiology Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

National Biodiversity Future Center Palermo Italy

National Research Council of Italy Verbania Pallanza Italy

Natural History Museum of Denmark University of Copenhagen Copenhagen Denmark

Naturalis Biodiversity Center Marine Biodiversity Leiden The Netherlands

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