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.

Phylosymbiosis, the association between the phylogenetic relatedness of hosts and the composition of their microbial communities, is a widespread phenomenon in diverse animal taxa. However, the generality of the existence of such a pattern has been questioned, and there seems evidence against its occurrence in small-sized aquatic animals, for which the microbiota composition is mostly shaped by local environmental factors. This study aims to investigate the microbial communities associated with poorly known marine interstitial nemerteans to uncover their microbiota diversity and assess the occurrence of phylosymbiosis. Through a comprehensive approach, specimens from various Central American sites were analyzed using morphology-based taxonomy and molecular techniques targeting the host 18S rRNA gene whereas their microbial association was analyzed by targeting the prokaryotic 16S rRNA gene. Phylogenetic and statistical analyses were conducted to examine the potential effects of host nemertean taxa and sampling locations on the host-associated microbial communities. The results provide compelling evidence of phylosymbiosis in meiofaunal nemerteans, emphasizing the significant impact of host genetic relatedness on microbiome diversity also in small-sized animals. Additional research is needed to fully unravel potential symbiotic relationships as well as the complex mechanisms that govern the relationships between hosts and their microbiota across various organisms and ecological settings. The remarkable diversity of meiofaunal animals, spanning various animal phyla with different lifestyles and inhabiting diverse ecosystems, combined with the advancements in multi-omics approaches, offers a promising avenue for comprehensive understanding of the evolutionary and ecological interactions between hosts and their microbiota throughout the animal tree of life.