Combination of high throughput sequencing and morphological identification assess the diversity and distribution of key benthic taxa in anthropogenic environments

Abstract Port environments are both heavily impacted by human activities and of high societal importance, making their ecological monitoring essential. The assessment of marine macro invertebrate communities is an important part of such monitoring programs. In an era where molecular methods, such as metabarcoding of bulk DNA or environmental DNA (e-DNA), become increasingly integrated into benthic monitoring, it is crucial to evaluate their performance against morphology-based assessments and to develop robust protocols supported by comprehensive reference databases. We applied a combination of morphological and molecular methods to obtain an exhaustive checklist of the polychaete fauna present in four main Norwegian harbours. Relying on taxonomic expertise and a large sequencing effort, we considerably improved the reference databases for three molecular markers: COI, 16S and 28S with 22, 40 and 36 new sequences respectively, which is a key step in the implementation of molecular monitoring methods. In total, 141 species were morphologically identified. In contrast, integrating data from single-specimen barcoding and metabarcoding revealed 213 species. High-throughput sequencing (HTS) detected 84.4% of the species identified morphologically when all markers were combined. However, only 58.2% of the species detected using HTS could be assigned a species name. None of the sampled harbours showed the presence of non-indigenous species. Our results demonstrate that integrating morphology-based taxonomy with molecular approaches significantly improves biodiversity detection in harbour environments. The results also emphasize the critical need for expanded reference libraries to fully realize the potential of molecular monitoring tools. This in turn highlights the importance of alpha taxonomy and the need for increased taxonomic research.