Karlijn Doorenspleet

The North Sea is one of the most productive seas globally and contains several biogenic reefs that host a range of fish and invertebrate species. Yet the North Sea is heavily used, putting substantial pressure on its habitats and marine communities. In response, several conservation and restoration measures, as well as nature-inclusive designs in industry, are in place and need to be guided by robust, transparent monitoring methods. eDNA metabarcoding holds great promise for marine biodiversity assessments, and novel sequencing platforms, such as Oxford Nanopore sequencing, can facilitate long-read metabarcoding and potentially improve the resolution of species detection for monitoring the North Sea. In five chapters, new molecular platforms and methods were verified and applied to explore the biodiversity of natural and artificial habitats of the North Sea.

Chapter 2 compares short-read metabarcoding using Oxford Nanopore sequencing with metabarcoding using a conventional sequencing platform Illumina MiSeq and metagenomics, and assesses their performance for macrobenthos biodiversity assessments in offshore windfarms in the Belgian part of the North Sea. Using marine invertebrate specific markers, Nanopore and MiSeq yielded very similar alpha and beta diversity compositions and were consistent with observed location-specific community patterns. Novaseq metagenomics yielded slightly different community compositions compared to metabarcoding and correlated poorly with size-corrected morphological abundance data. All methods had mismatches with classical monitoring methods, based on morphological identification of species, and likely reflect primer biases and an incomplete reference database. Overall, this chapter emphasises that nanopore-based metabarcoding is a viable alternative to Illumina MiSeq for macrobenthos biodiversity monitoring.

Chapter 3 presents a long-read eDNA metabarcoding method using a 2kb mitochondrial primer targeting the 12S and 16S mitochondrial rRNA and the DECONA bioinformatics processing pipeline to handle error-prone nanopore reads. The results from the 2kb primer were compared with a widely used universal fish (MiFish) primer. Mock communities and aquarium eDNA showed improved species-level resolution with the 2kb fragment. Still, North Sea samples from shipwrecks, the Borkum reef grounds, and a wind park yielded more species detections with the MiFish primer. This is likely due to a lower presence of longer reads in the environment, as they degrade to shorter fragments over time. This demonstrates the potential of long-read metabarcoding to provide a more accurate picture of species that were recently present.

Chapter 4 presents an eRNA-based metabarcoding method to assess the vertebrate biodiversity on a natural Horse mussel (Modiolus modiolus) reef in a marine protected area in the Scottish part of the North Sea. By comparing short-read eDNA, long-read eDNA and short-read eRNA fragments, this chapter evaluates how nucleic acid type and fragment length influence community resulting species community detected. Short-read metabarcoding detected the highest diversity and was the only method that distinguished communities between high and low reef density. eRNA was similar to short-read eDNA but missed many large vertebrates, such as sharks and rays, suggesting that eRNA reflects the active local benthic community. Long fragments from the 2kb primer showed the lowest detectability, contradicting earlier statements that long-read metabarcoding may provide a more temporally accurate signal.

Chapter 5 reviews current and future monitoring tools for assessing North Sea biodiversity and effective monitoring. The chapter outlines the strengths and limitations of existing molecular methods, including eDNA metabarcoding, and metagenomics, and describes improvements to their application. This chapter also explores emerging DNA and RNA-based methods, such as epigenetics and metatranscriptomics, that can potentially improve understanding of ecosystem functioning. The chapter also discusses the challenges of reference database curation and standardisation, and concludes with recommendations for further integrating novel molecular methods into harmonised, long-term biodiversity monitoring of the North Sea.

Chapter 6 applies metabarcoding methods to study marine vertebrate diversity in two North Sea shellfish reefs: a mixed shellfish reef in the Dutch Voordelta and a Horse mussel reef at Noss Head, Scotland, to evaluate their role as essential fish habitats. By comparing stations inside and outside the reef using eDNA and baited remote underwater video (BRUVs), this chapter shows that vertebrate diversity was consistently higher inside the reef and that richness declined with increasing distance from it. The reef community included reef-associated species such as gobies, gunnels, and clingfish, as well as commercially important species like cod and pollock. eDNA detected more species overall, but BRUVs captured juvenile ling individuals, as verified by eDNA, demonstrating the value of complementary methods for monitoring.

Chapter 7 discusses the thesis outcomes by examining the effectiveness of novel molecular methods for biodiversity assessment and monitoring in the North Sea. The findings indicate that i) Nanopore metabarcoding is comparable to Illumina metabarcoding for assessing macrobenthos, making it a viable option; ii) Long-read and eRNA metabarcoding enhance species identification and active community detection; and iii) eDNA metabarcoding reveals distinct biodiversity patterns linked to reef density and natural reefs. In summary, this thesis highlights the value of these methods for improved, standardised, non-invasive monitoring of the North Sea ecosystem.