DNA metabarcoding is a promising method to increase cost and time efficiency of marine monitoring. While substantial evidence exists that bulk DNA samples adequately reflect diversity patterns of marine macrobenthos, the potential of eDNA in the ethanol preservative of benthic samples for biodiversity monitoring remains largely unexplored. We investigated species detection in bulk DNA and eDNA from the ethanol preservative in samples from four distinct macrobenthic communities in the North Sea. Bulk DNA and eDNA were extracted with different extraction kits and five COI primer sets were tested. Despite the availability of a nearly complete reference database, at most 22% of the amplicon sequence variants (ASVs) were assigned taxonomy at the phylum level. However, the unassigned ASVs represented only a small fraction of the total reads (13%). The Leray primer set outperformed the four other primer sets in the number of non-chimeric reads and species detected, and in the recovery of beta diversity patterns. Community composition differed significantly between bulk DNA and eDNA samples, but both sample types were able to differentiate the four communities. The probability of detecting a species in the eDNA from the ethanol preservative was significantly lower than for bulk DNA for macrobenthos species having small to medium body size and for species having chitine or CaCO3 in their cuticula. Detection in the bulk DNA samples was not affected by the investigated morphological traits, indicating that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

DNA metabarcoding is a promising method to increase cost and time efficiency of marine monitoring, providing that the impact of methodological choices on the reliability and reproducibility of results are well understood. Here, we investigated the impact of primer choice, DNA source (bulk DNA or eDNA from the ethanol preservative) and morphological traits (body size and body skeleton) on species detection in four distinct macrobenthos communities from the North Sea. We generated a reference database with COI sequences for macrobenthos from the North sea and applied DNA metabarcoding using five COI primer sets. At most 22% of the ASVs were assigned taxonomy at the phylum level, despite the availability of a nearly complete reference database. However, the unassigned ASVs represented only a small fraction of the total reads (13%). The Leray primer set outperformed the four other primer sets in the number of non-chimeric reads and species detected, and in the recovery of beta diversity patterns. Community composition differed significantly between bulk DNA and eDNA samples, but both sample types were able to differentiate the four communities. Importantly, the probability of detecting a species in the eDNA from the ethanol preservative was significantly lower than for bulk DNA for macrobenthos species with small to medium body size and for species with chitine or CaCO3 in their skeleton. Detection in the bulk DNA samples was not affected by the traits investigated, indicating that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos.

Benthic macroinvertebrates are key components in environmental impact assessments. Nevertheless, their use as bioindicators can be constrained by the time- and cost-consuming processes needed for their morphological identification. The recent advances in high-throughput sequencing, particularly DNA metabarcoding can provide an alternative to morphology-based approaches. The main limitation for DNA-based tools to be implemented in biomonitoring projects is the considerable investment needed to build high-quality and curated taxonomic reference DNA sequence libraries for species identification. To begin addressing this shortage, Interreg NSR project GEANS aim in developing a curated DNA reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for the North Sea macrobenthos that will serve as the backbone of all the molecular protocols. For the collection of the macroinvertebrates, a targeted sampling campaign is underway across the North Sea Region, while existing collections are being scanned. Macrobenthic specimens are being processed following a series of standardised work flows covering sampling, morphological identifications, molecular lab processing and data handling. GEANS' reference library, currently holds DNA barcodes for 3443 specimens collected from various areas of the North Sea. In total 11 phyla, 28 classes, 80 orders, 280 families, 428 genera and 586 species (37 non-indigenous species) are represented within the library. Species-level identification is taxonomically verified for over 90 % of the species, while genus-level identifications are verified for almost 100 % of the genera. GEANS reference library covers so far over 30 % of North Sea species and its constantly enriched. All specimens are photographed and together with the accompanying sequences, collection and taxonomic data are archived in a dedicate BOLD project. Voucher specimens and DNA extracts are archived at three different institutes in Germany (Senckenberg am Meer), the Netherlands (Naturalis) and Belgium (ILVO). Once available, this DNA-library will be based on taxonomically well-curated specimens and it will support the implementation of fast, cost-efficient and reliable DNA-based identifications and subsequently environmental health assessments in the North Sea.

Macrobenthos is a good indicator to evaluate the potential effects of human activities on the marine benthic ecosystem. In environmental impact assessments (EIAs), macrobenthic species identification is typically based on morphological characteristics, a time-consuming and labor-intensive process for which specific taxonomic knowledge and experts are needed. DNA metabarcoding can circumvent most of these shortcomings. However, to be applicable in EIAs and to be adapted by policy, a standardized protocol that allows for reproducible and reliable DNA metabarcoding results is a prerequisite. Here, three research questions were investigated as part of the international Interreg NSR project GEANS: 1) “How many replicates of DNA extractions and PCR products are needed to capture most of the macrobenthic species in a sample?”, 2) “Is a ‘fixed’ DNA metabarcoding protocol repeatable across different institutes?” and 3) “What is the impact of small changes in this DNA metabarcoding protocol on alpha diversity?”. These are important steps to convince stakeholders that this efficient and quick method generates reliable and comparable results. First, variation in macrobenthic species across technical replicates was investigated in three biological replicates from three macrobenthic communities in the Belgian Part of the North Sea (BPNS) with high, medium and low diversity. For each biological replicate, six DNA replicates were taken and one of these DNA replicates was used to assess variation between three replicates for PCR amplification. Three DNA replicates were needed in locations with a high and medium diversity to pick up at least 80% of the species diversity present in the six replicates, while four DNA replicates were needed in the location with low diversity. Variation in the detected species between PCR replicates was high, illustrating the importance of including at least three PCR replicates in the lab protocol. Second, we conducted a ring test where subsamples of 12 bulk macrobenthos samples, originating from four different macrobenthic communities in the BPNS (differing in species density and diversity), were distributed to four institutes located in Belgium, the Netherlands, Germany and Denmark. Samples were processed using the same standardized lab protocol and the resulting datasets were processed bioinformatically by one institute. The number of ASVs and the number of species reflected the morphological diversity patterns, i.e. highest values for the replicates from the highly diverse macrobenthic community, lowest numbers in the low diversity replicates and intermediate values in the samples from the medium diversity community. These patterns were identical between the four institutes, showing high repeatability for alpha diversity when using the same protocol. In total, 100 macrobenthic species were detected through DNA metabarcoding, of which 60 species were picked up by all four institutes, while 0-14 species were recorded by only one of the four institutes. Beta diversity patterns were also comparable between the four institutes, as the nMDS plot clearly showed clustering based on the macrobenthic communities, independent of the institute that conducted the work. Finally, small changes to the lab protocol (different DNA extraction kit, different high fidelity polymerases for PCR amplification, different reagents for clean-up) resulted in only minor changes in alpha diversity: similar number of species were detected as with the fixed protocol in all samples and 70% - 75% of the species were shared between the ‘fixed’ and adjusted protocols. This study shows for the first time that DNA metabarcoding offers a highly repeatable assessment of alpha and beta diversity patterns, which supports the suitability of DNA metabarcoding for monitoring of marine macrobenthos. These results are highly valuable to establish a harmonized and uniform DNA metabarcoding protocol, to be used by all institutions in Europe when implemented as a new standard method in EIAs of the benthic ecosystem.