Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: a case study on beach meiofauna

Abstract

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.

Metacommunity theory and metabarcoding reveal the environmental, spatial, and biotic drivers of meiofaunal communities in sandy beaches

“…Following bioinformatic processing with APSCALE and quality filtering, we retained 14,822,456 mitochondrial cytochrome c oxidase I (COI) sequences and 566 Operative Taxonomic Units (OTUs). Following taxonomic annotation using NCBI GenBank and meiofauna reference barcodes reported in 26 , we retained 11,029,442 sequences of 127 OTUs. Of these, 42 were Nematoda, 21 Copepoda, 16 Clitellata, 12 Polychaeta, 9 Gastrotricha, 8 Platyhelminthes, 7 Acoela, 4 Collembola, 3 Rotifera, 2 Branchiopoda, 1 Nemertea, 1 Tardigrada, and 1 Arachnida.…”

“…We removed 11 samples with less than 3000 reads per sample during bioinformatic processing, retaining samples for which 1 read corresponds to >0.03% of the total reads number. We performed taxonomic assignment using NCBI GenBank expanded with meiofauna COI barcodes generated as part of associated taxonomic workshops in Leiden 26 . Taxonomic ranks were assigned to OTUs using established identity thresholds: >97%: species, >95%: genus, >90%: family, >85% order 105 .…”

“…Despite their ecological importance, a thorough understanding and study of beach meiofauna communities remains challenging due to difficulties in morphological identification and a lack of ecological knowledge 22 . The advent of metabarcoding, a molecular technique that enables amplification and identification of many species from environmental samples, has changed meiofauna research by offering a cost-effective and efficient method of understanding meiofaunal biodiversity [23][24][25] , especially when combined with morpho-taxonomic approaches 26 .…”