Integrating taxonomy and DNA reference barcoding substantially improves metabarcoding efficiency and detection of ecological patterns in beach meiofauna communities

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.

海滩是全球重要的生态系统，栖息着小型底栖动物（meiofauna）——一类生活于沙粒间隙中的微型动物，它们在滨海生物多样性与生态系统动态过程中发挥关键作用。然而，针对小型底栖动物的研究却面临诸多挑战：分类学专业人才匮乏，且采样覆盖度不足。本研究从荷兰西海岸采集的小型底栖动物样本中，新增生成了775条细胞色素c氧化酶亚基I（cytochrome c oxidase I, COI）DNA条形码，并将其与NCBI GenBank数据库进行整合。针对北海24处海滩的561份宏条形码（metabarcoding）测序样本，本研究分别使用扩充后的参考数据库，以及未添加新增条形码的原始NCBI数据库开展α多样性与β多样性分析——该区域是小型底栖动物研究的经典采样区。本研究结果显示，使用扩充后的数据库对宏条形码数据进行注释时，成功注释的序列数量提升2.5倍，物种级操作分类单元（Operational Taxonomic Units, OTUs）的识别数量翻倍。此外，分析结果揭示了潮间带区域OTU丰富度呈钟形分布的规律，这一结果与形态学分析的模式更为吻合；同时，我们还明确了潮上带与潮间带样点之间的群落相异性分布特征。本研究凸显了扩充分子参考数据库的重要性，以及将形态分类学与分子技术相结合用于生物多样性评估的价值，最终有助于深化我们对滨海生态系统的认知。