Environmental DNA (eDNA) metabarcoding is increasingly used for marine biodiversity monitoring; yet, interpreting eDNA signals remains challenging due to transport processes in dynamic seas that weaken the link between eDNA detection and the location of source organisms. We evaluated whether sampling schemes designed for monitoring epibenthic fish using beam trawling are optimal for eDNA-based monitoring in hydrodynamically active systems. Across four sampling campaigns at 11 long-term monitoring stations in the Belgian Part of the North Sea (BPNS), eDNA data are coupled with Lagrangian particle tracking and hydrodynamic models. eDNA detected higher species richness, consistently reproduced spatial community structures established through long-term trawl monitoring, and revealed significant temporal patterns not evident in simultaneous beam trawl surveys. Model simulations indicated substantial variability in eDNA origin and dispersal linked to seasonal changes, with mean transport distances generally increasing from coastal (~10 km) to offshore sites (>15 km). Station-specific likely-origin areas largely corresponded with observed community similarity, suggesting that hydrodynamic transport likely shaped the observed eDNA community patterns. Our results show that the spatial resolution of eDNA signals is season dependent: despite longer transport distances, autumn dispersal remained more spatially confined within habitat zones, supporting clearer community structure, whereas winter dispersal extended more broadly across zones, reducing spatial resolution and interpretability of habitat-specific patterns. Both seasons nonetheless captured complementary, season-specific community signals. We provide operational guidance on sampling design and station spacing tailored to season and research objectives to support implementation of eDNA within existing monitoring frameworks, such as the Marine Strategy Framework Directive.

Coastal environments serve as essential nursing, feeding, and spawning grounds for commercially and ecologically important fish species, some of which use nearshore habitats as transitional steps in their ontogenetic migration. Understanding fish communities' spatial and temporal dynamics in coastal habitats is fundamental for sustainable ecosystems and fisheries management. Despite the importance of long-term monitoring to obtain information on fish movements and distribution, fine-scale temporal datasets on fish communities remain scarce due to the intense field work required. In the present study, we explored the use of eDNA 12S metabarcoding of seawater samples to monitor fine-scale temporal and spatial patterns in fish communities. In total, 168 samples were collected across 20 sampling campaigns conducted monthly between August 2021 and August 2023 within the 12 nautical miles of the Belgian part of the North Sea. eDNA patterns revealed no marked temporal or spatial patterns at the community levels due to the ubiquitous presence of the dominant Southern North Sea fish species linked to their non-migratory behavior and use of the coastal Belgian waters as nursing and spawning grounds. However, species-specific temporal patterns reflected their reproductive activity and seasonal migrations. Additionally, fish species spatial distribution was consistent with previous beam trawl and eDNA-based surveys conducted within the Belgian part of the North Sea and was mainly driven by the environmental gradient created by freshwater discharge from the Scheldt estuary. Our findings demonstrate that eDNA metabarcoding is a valuable biomonitoring tool and provides insight into fish distribution, migration, and reproductive activity.

Animals can use specific environmental cues to make informed decisions about whether and where to disperse. Patch conditions are known to affect the dispersal behavior of animals, but empirical studies investigating the impact of resource diversity on the dispersal of closely related species are largely lacking. In this study, we investigated how food diversity affects the dispersal behavior of three co-occurring cryptic species of the marine bacterivorous nematode complex Litoditis marina (Pm I, Pm III and Pm IV). Using microcosms composed of a local patch (inoculation plate), a connection tube, and a distant patch (dispersal plate), we examined nematode dispersal patterns with bacteria serving as the food source. Food treatments included low-, medium-, and high-diversity bacterial mixtures of 5, 10, and 15 bacterial strains, respectively. Additionally, a single-strain food resource Escherichia coli was used as a control treatment. Both local and distant patches had either identical food treatments (‘homogeneous patches’) or E. coli in the local patches and more diverse food (low-, medium-, or high-diversity food) in distant patches (‘heterogeneous patches’). Our results show that the dispersal behavior of the cryptic species varies depending on food diversity, indicating that L. marina acquire information about their environment when making dispersal decisions. All three cryptic species tend to disperse faster toward food patches that increase fitness. Pm I and Pm IV exhibited faster dispersal toward patches with a more diverse food source, while Pm III showed similar dispersal rates toward E. coli, medium-diversity, and high-diversity food. This indicates that resource diversity can alter the dispersal behavior of cryptic species and may be an important mechanism to achieve species coexistence in the field.

Common sole (Solea solea) is a key commercial flatfish species in Europe, yet its stock identity in the southern Celtic Sea and southwest of Ireland (ICES area 7h and 7j) is uncertain, resulting in a precautionary approach to fisheries management and declining quota. Here, the structure of sole populations and their connectivity patterns were investigated from the southern North Sea to the Bay of Biscay spanning 10 ICES areas using 55 706 single nucleotide polymorphisms and five biological variables (sex, maturity, age, length, and weight). Our results confirmed the large-scale genetic differentiation between sole in the southern North Sea (ICES area 4c) and Bay of Biscay (8a, 8b). Sole from area 7h was genetically similar to sole from the Celtic Sea (7f and 7g) (both neutral and outlier loci), Western English Channel (7e, only neutral loci), and Irish Sea (7a, only neutral loci). Sole from area 7j showed significant neutral differentiation with sole from areas 7h and 7g, the Western English Channel (7e), and the Irish Sea (7a). These novel insights suggest a current mismatch between the biological populations and stock units of 7h and 7j, currently managed as a single stock, and provide a crucial basis for the re-evaluation of the current stock status, enabling more informed and effective fisheries management.

DNA-based methods and developments of sequencing technologies are integral to macrobenthos biodiversity studies, and their implementation as standardized monitoring methods is approaching. Evaluating the efficacy and reliability of these technological developments is crucial for macrobenthos biodiversity assessments. In this study, we compared three DNA-based techniques for assessing the diversity of bulk macrobenthos samples from the Belgian North Sea. Specifically, we compared amplicon sequencing using Illumina MiSeq and portable real-time sequencing of Oxford Nanopore versus shotgun sequencing using Illumina NovaSeq sequencing. The 313 bp mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding fragment served as the target region for the metabarcoding analysis. Our results indicate that Oxford Nanopore and MiSeq metabarcoding had similar performances in terms of alpha and beta diversity, revealing highly similar location-specific community compositions. The NovaSeq metagenomics method also resulted in similar alpha diversity, but slightly different community compositions compared to the metabarcoding approach. Despite these differences, location-specific community compositions were maintained across all platforms. Notably, read counts from the NovaSeq metagenomic analysis showed the weakest correlation to size corrected morphological abundance and there were mismatches between morphological identification and all DNA based findings which are likely caused by a combination of factors such as primer efficiency and an incomplete reference database. Our findings underscore the critical importance of database completeness prior to implementing DNA-based techniques as standardized monitoring method, especially for metagenomics. Nevertheless, our findings emphasize that Oxford Nanopore metabarcoding proves to be a viable alternative to the conventional Illumina MiSeq metabarcoding platform for macrobenthos biodiversity monitoring.

The hadal zone, the deepest part of the global ocean, encompasses several extreme ecosystems with elevated biological activity, contrasting the general trend of declining biomass and biodiversity with increasing water depth. These bathymetrically isolated habitats act as depocenters and are characterized by high hydrostatic pressure and unique environmental conditions that provide an opportunity to fundamentally explore biogeographic patterns and the genetic diversity of deep-sea meiofauna, which remain poorly understood. In this study, we compared nematode communities in two geographically distant trench systems, the Aleutian Trench (North Pacific) and the South Sandwich Trench (South Atlantic), separated by 17 000 km. Environmental DNA (eDNA) sequencing targeting the ribosomal 18S region was combined with a metaphylogeography approach. Comparative analyses were performed for intra- and inter-trench, biodiversity and genetic differentiation across environmentally characterised trench-specific habitats. Our findings reveal evidence for the meiofauna paradox in hadal settings, demonstrating distinct nematode communities that appear to be shaped by availability and quality of food. Furthermore, environmental filtering appeared to contribute to assembly of habitat specific communities according to localised environmental condition. Finally, we observed shared haplotypes among some dominating nematode genera, suggesting potential connectivity between habitats associated within or even across hadal trenches might exist. Overall, this study provides valuable insights on how environmental factors drive nematode biodiversity and genetic diversity in the hadal realm. Environmental heterogeneity plays a pivotal role in shaping nematode communities, influencing their population structure and connectivity by creating spatially variable habitats that drive diversification, local adaptation, and gene flow patterns.

EU directives (e.g. MSFD, Habitats Directive), along with OSPAR guidelines, mandate sustainable marine resource management across national borders. Benthic organisms are crucial for assessing marine ecosystem health, but their morphological identification is time-consuming and costly. High-throughput sequencing, particularly DNA metabarcoding, offers an alternative. However, DNA-based monitoring requires substantial investment in high-quality DNA reference libraries. The GEANS project (Genetic Tools for Ecosystem Health Assessment in the North Sea Region) aimed to develop efficient DNA-based tools for benthic biomonitoring. GEANS created a curated DNA reference library (COI) for species relevant to North Sea macrobenthos monitoring, using new sequences, non-public barcode sequences, and mined sequences from GenBank and BOLD. The library, stored in a dedicated BOLD project with photographs and metadata, includes DNA barcodes for 4005 specimens from 715 species, representing over 29% of North Sea macrobenthos species. Arthropoda is the most represented, while Bryozoa and Annelida have the lowest coverage. This DNA library is expected to facilitate fast, cost-effective environmental health assessments in the North Sea for public authorities and academics.

DNA-based techniques are a popular approach for assessing biodiversity in ecological research, especially for organisms which are difficult to detect or identify morphologically. Metabarcoding, the most established method for determining species composition and relative abundance in bulk samples, can be more sensitive and time- and cost-effective than traditional morphological approaches. However, one drawback of this method is PCR bias caused by between-species variation in the amplification efficiency of a marker gene. Metagenomics, bypassing PCR amplification, has been proposed as an alternative to overcome this bias. Several studies have already shown the promising potential of metagenomics, but they all indicate the unavailability of reference genomes for most species in any ecosystem as one of the primary bottlenecks preventing its wider implementation. In this study, we present a strategy that combines unassembled reads of low-coverage whole genome sequencing and publicly available reference genomes to construct a genomic reference database, thus circumventing high sequencing costs and intensive bioinformatic processing. We show that this approach is superior to metabarcoding for approximating relative biomass of macrobenthos species from bulk samples. Furthermore, these results can be obtained with a sequencing effort comparable to metabarcoding. The strategy presented here can thus accelerate the implementation of metagenomics in biodiversity assessments, as it should be relatively easy to adopt by laboratories familiar with metabarcoding and can be used as an accessible alternative.

Environmental impact assessments of marine aggregate extraction are traditionally conducted based on morphological characteristics of macrobenthos, which is time-consuming, labour-intensive and requires specific taxonomic expert knowledge. Bulk DNA metabarcoding is suggested as a promising alternative. This study compares the traditional morphological and the bulk DNA metabarcoding method to assess the impact of sand extraction activities on three sandbanks in the Belgian North Sea. Substantial differences in the detected species were observed between methods: Abundant and/or large macrobenthos species were detected by both methods, while small species or species with an exoskeleton were usually only detected by the morphological method. Taxa uniquely detected by bulk DNA metabarcoding could be explained by specimens identified at a higher taxonomic level by morphology, or by specimens with very low read numbers, probably representing species missed in the morphological sorting process, DNA traces on the specimens or false positives during PCR amplification efficiency. Despite the difference in detected species, comparable alpha and beta diversity patterns were observed by both methods, indicating that bulk DNA metabarcoding can effectively detect the overall ecological changes associated with sand extraction. We further demonstrate that bulk DNA metabarcoding reduces sample processing both in time (44 % faster) and cost (26 % cheaper) compared to the morphologybased identification. However, biomass quantification remains challenging for bulk DNA metabarcoding since of the ten most abundant genera, only two genera (Echinocardium and Ophelia) showed a significant positive correlation between biomass and read numbers. Additionally, bulk DNA metabarcoding does not provide information on life stages or size of the identified specimens. As such, our results underpin the complementary nature of both methods, wherein DNA-based analyses allow for rapid detection of community changes (as similar patterns in alpha and beta diversity and biotic index were observed), while morphology-based analyses provide additional information on e.g. secondary production (biomass) and size composition. We show how the strengths of both methods can be combined to assess the impact of sand extraction.

Intraspecific competition and resource diversity are considered major drivers of niche differentiation, which are expected to promote population niche expansion by driving individuals to feed on alternative resources and/or by enhancing individual diet specialization. Nevertheless, experimental studies on the interaction effects of both factors on animal behavior and population dynamics remain scant. Here, we investigate how resource diversity alters the impact of intraspecific competition on resource preference and fitness of three co-occurring cryptic species of the marine nematode complex Litoditis marina (Pm I, Pm III and Pm IV). For each cryptic species, two competition regimes (‘low nematode density’ and ‘high nematode density’) were established in microcosms with varying resource diversity (E. coli, low-, medium- and high-diversity food). Our results show differences in resource preference and population fitness depending on intraspecific competition and resource diversity, but the response also varied considerably between cryptic species. Pm III did not exhibit resource preference under low intraspecific competition, but preferred the two most diverse food sources under high intraspecific competition. Pm IV also showed preference for medium-diversity food under high competition, whereas no resource preference was observed in Pm I regardless of competition regimes and resource diversity. Nevertheless, all cryptic species exhibited enhanced adult population growth on a more diverse food source under stronger intraspecific competition. These results indicate that resource diversity can alleviate intraspecific competition and affect niche diversification, which may impact diversity maintenance in ecological communities.

AbstractTo reach the renewable energy targets set by the European Commission, a tenfold expansion of the installed offshore wind farms is needed. Since the construction of offshore wind farms may affect local soft‐sediment fauna, an efficient monitoring technique is needed to monitor the potential effects on the marine ecosystem. Here, we assess whether eDNA metabarcoding is a suitable alternative to monitor fish and epibenthos biodiversity in these difficult to access marine habitats. Water sampling and trawl surveys were conducted in parallel in 12 coastal and 18 offshore sites, the latter located inside and outside two offshore wind farms in the Belgian part of the North Sea. 12S eDNA metabarcoding retrieved 85.7% of the fish species caught in the beam trawls, whereas the COI eDNA metabarcoding only identified 31.4% of the epibenthic invertebrate species. Furthermore, the 12S marker resulted in an additional detection of 26 unique fish species, whereas the COI marker detected an additional 90 invertebrate species. Spatial patterns in alpha diversity recovered with eDNA metabarcoding were not significantly different from those observed with morphological determination. Significant differences were found in fish and invertebrate community structures between the coastal, transition and offshore zones as well as on the smaller wind farm scales, which agreed with the morphological beam trawl data. Indicator species found with morphological beam trawl monitoring for each of the three zones (coastal, transition, offshore) were also detected with 12S eDNA metabarcoding, and the latter method detected an additional 31 indicator species. Our findings show the need for adequate quality control of the obtained species lists and reveal that 12S eDNA metabarcoding analyses offers a useful survey tool for the monitoring of fish communities in offshore wind farms, but the used COI assay did not adequately capture the epibenthic communities as observed with beam trawl data.

Marine sediments cover 70% of the Earth's surface, and harbour diverse bacterial communities critical for marine biogeochemical processes, which affect climate change, biodiversity and ecosystem functioning. Nematodes, the most abundant and species-rich metazoan organisms in marine sediments, in turn, affect benthic bacterial communities and bacterial-mediated ecological processes, but the underlying mechanisms by which they affect biogeochemical cycles remain poorly understood. Here, we demonstrate using a metatranscriptomic approach that nematodes alter the taxonomic and functional profiles of benthic bacterial communities. We found particularly strong stimulation of nitrogen-fixing and methane-oxidizing bacteria in the presence of nematodes, as well as increased functional activity associated with methane metabolism and degradation of various carbon compounds. This study provides empirical evidence that the presence of nematodes results in taxonomic and functional shifts in active bacterial communities, indicating that nematodes may play an important role in benthic ecosystem processes.

Abstract Sustainable fisheries management requires regular scientific monitoring of fish stocks. When information on certain fish stocks is limited, environmental DNA (eDNA) holds promise to complement traditional monitoring surveys. However, a better understanding of how eDNA concentrations relate to fish abundance and biomass is needed. Here, eDNA quantification of two commercially important flatfish species in the North‐East Atlantic, common sole (Solea solea) and European plaice (Pleuronectes platessa), was assessed. First, species‐specific, probe‐based assays for plaice and sole targeting the mitochondrial cytochrome b and cytochrome c oxidase subunit I gene, respectively, were developed (for sole) and validated (for both species). Subsequently, two mesocosm experiments revealed a significant and positive relationship between both abundance and biomass and eDNA concentrations for both species at three eDNA emission time periods (5 min, 1 h, and 24 h). Larger plaice shed significantly more eDNA (copies L−1) than smaller conspecifics. Finally, eDNA was obtained from seawater collected during research surveys in the Belgian part of the North Sea in spring 2020 (i.e., local scale) and the southwestern North Sea in autumn 2020 and 2021 (i.e., regional scale). eDNA concentrations were compared to the observed abundance (individuals per km2) and fish density in terms of biomass (kg per km2) as observed in the trawl at the same station. Local eDNA concentrations of both sole and plaice were positively correlated with observed abundance and fish density. The correlation between regional eDNA concentrations and fish density was positive and significant for sole in 2020 and 2021 and for plaice in 2020, but not in 2021. The correlation between regional eDNA concentrations and observed abundance was positive and significant for sole and plaice in 2020, but not in 2021. These results illustrate the potential of eDNA to estimate abundance and biomass parameters for stock assessments of flatfishes in the North Sea.

The integration of eDNA metabarcoding into monitoring programs provides valuable information about fish community structures. Despite the growing body of evidence supporting the method's effectiveness in distinguishing fine-scale eDNA signals, there is a limited understanding of eDNA distribution in shallow, well-mixed environments, especially related to sampling depth. We analyzed 167 samples collected from the surface and bottom water at 17 locations of the Belgian Part of the North Sea (BPNS), where the deepest sampling point was 31 m, and compared this to beam trawl catch data collected simultaneously at the same locations. eDNA metabarcoding identified an additional 22 species compared to beam trawl catch data. Diversity measures and patterns were very similar between surface and bottom samples and revealed community patterns that were previously described by long-term beam trawl catch data. Surface and bottom samples had 39 fish species in common, while six and eight rare species were uniquely detected, respectively. Our results demonstrate that eDNA metabarcoding effectively identifies spatial community patterns of fishes in the highly dynamic environment of the BPNS regardless of sampling depth. Out of the six most common species tested, eDNA metabarcoding read abundances correlated strongly with catch-based abundance data for one species, but moderately for two others, indicating that inferring fish abundance and biomass via eDNA metabarcoding remains challenging.

Seafood is a major food source worldwide, but it is prone to fraudulent activities such as species substitution. DNA barcoding is currently the most used tool to identify processed seafood, but remains expensive, time-consuming, and requires heavy and expensive lab equipment and expert knowledge. Here we compared the Nucleospin (R) Food kit with a dipstick-based, a paramagnetic bead-based, and an alkaline-based DNA extraction method on tissue of common sole (Solea solea). The alkaline-based method was the most reliable and cheapest, with the lowest hands-on time. A S. solea-specific loop-mediated isothermal amplification (LAMP) assay was designed using the cytochrome b (cytb) gene with a limit of detection (LOD) of 0.1 ng. The alkaline-based DNA extraction and LAMP was validated using ten sole fillets under different preservation conditions (fresh, frozen, and ethanol stored), and ten previously identified sole dishes. A combination of the alkaline-based DNA extraction and the LAMP assay detects common sole in seafood products within an hour in the field, and at a cost of less than half a euro. The method developed in this study is applicable in large-scale audits of sole products. Similar methods may emerge for other seafood species allowing seafood fraud studies in labs short on resources.

Seafood is the world's most highly traded food commodity. Due to the non-stop demand of the ever-increasing human population, the supply of certain species is not guaranteed, and mislabelling practices arise. Fillets and heavily processed products that cannot be easily authenticated are particularly vulnerable to fraudulent practices. In order to face this challenge, as a cost-effective, rapid, and easy handling tool, the MinION sequencer from Oxford Nanopore Technologies (ONT) coupled with DNA (meta)barcoding represents a suitable option to assess seafood fraud. In this study, we evaluated the feasibility of MinION sequencing to authenticate mixed seafood products by (1) evaluating MinION generated barcodes from single species that have already been Sanger sequenced and (2) performing MinION sequencing of mixed samples containing commercial seafood species in different amounts to evaluate whether all species can be detected, even when they are low abundant. Using the mitochondrial DNA gene markers Cytb and COI, 86% and 76% of the single species were correctly identified, respectively, of which 26% and 29% also showed non-target species. For the mixed samples, not all species were detected regardless of the composition of the mixtures or the marker gene used. Nevertheless, the MinION sequencing platform can be considered an additional tool to complement currently available approaches to authenticate mixed seafood samples providing that adequate measures are taken to reduce false positives and false negatives.

DNA metabarcoding can be used in marine environmental monitoring if results are reproducible between labs and robust against modifications to the lab protocol. In this interlaboratory study, we conducted a ring test where subsamples of blended macrobenthos samples were distributed to four laboratories located in Belgium, the Netherlands, Germany and Denmark. Samples were processed by a standardized lab protocol and by an adapted protocol, and the resulting datasets were analyzed with the same bioinformatics pipeline. Different biodiversity indicators were calculated. Our results show that bulkDNA metabarcoding of marine macrobenthos offers a highly reproducible assessment of alpha diversity patterns when using a standardized protocol, since comparable species numbers, Shannon indices and Inverse Simpson indices were found between laboratories. Especially high abundant species and species with large body sizes were shared between the laboratories. The need for using a standardized protocol to enhance comparability in alpha diversity between different studies was shown. Beta diversity patterns are less subjected to changes in the metabarcoding protocol and were almost identical between different laboratories, as the main clustering was always based on the macrobenthic community, independent of the used protocol or the laboratory that conducted the work. We conclude that DNA metabarcoding for marine environmental monitoring is an appropriate method when the aim is to study changes in community patterns and advocate its implementation in routine monitoring programs of national and European authorities, providing that a standardized protocol is implemented and/or a detailed description of the protocol is available.

Identifying and understanding patterns of biological diversity is crucial at a time when even the most remote and pristine marine ecosystems are threatened by resource exploitation such as deep-seabed mining. Metabarcoding provides the means through which one can perform comprehensive investigations of diversity by examining entire assemblages simultaneously. Nematodes commonly represent the most abundant infaunal metazoan group in marine soft sediments. In this meta-analysis, we compiled all publicly available metabarcoding datasets targeting the 18S rRNA v1-v2 region from sediment samples to conduct a global-scale examination of nematode amplicon sequence variant (ASV) alpha diversity patterns and phylogenetic community structure at different depths and habitats. We found that nematode ASV richness followed a parabolic trend, increasing from the intertidal to the shelf, reaching a maximum in the bathyal and decreasing in the abyssal zone. No depth- or habitat-specific assemblages were identified as a large fraction of genera were shared. Contrastingly, the vast majority of ASVs were unique to each habitat and/or depth zone; genetic diversity was thus highly localized. Overwhelmingly, nematode ASVs in all habitats exhibited phylogenetic clustering, pointing to environmental filtering as the primary force defining community assembly rather than competitive interactions. This finding stresses the importance of habitat preservation for the maintenance of marine nematode diversity.

Cichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how they can coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, females of O. nasuta behaved differently towards conspecific or heterospecific males, whereas females of O. ventralis did not. When presented with a heterospecific male, females of O. nasuta performed a lower number of ‘ram’ behaviours. Additionally, they never displayed the ‘flee’ behaviour, a component of the species’ mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour.

Identifying and understanding patterns of biological diversity is crucial at a time when even the most remote and pristine marine ecosystems are threatened by resource exploitation such as deep-seabed mining. Metabarcoding provides the means through which to perform comprehensive investigations of diversity by examining entire assemblages simultaneously. Nematodes commonly represent the most abundant infaunal metazoan group in marine soft sediments. In this meta-analysis, we compiled all publicly available metabarcoding datasets targeting the 18S rRNA v1-v2 region from sediment samples to conduct a global-scale examination of nematode Amplicon Sequence Variant (ASV) alpha diversity patterns, evolutionary distinctiveness (ED) and phylogenetic community structure at different depths and environments. We found that nematode ASV richness followed a parabolic trend, increasing from the intertidal to the shelf, reaching a maximum in the bathyal and decreasing in the abyssal zone. No depth- or environment-specific assemblages were identified as a large fraction of genera were shared. Contrastingly, the vast majority of ASVs were unique to each environment and/or depth zone; genetic diversity was thus highly localised. The intertidal and abyssal samples had the highest ED values, indicating that both a dynamic, fluctuating ecosystem, as well as a relatively stable yet very old one, can produce highly diversified assemblages. Overwhelmingly, nematode ASVs in all environments exhibited phylogenetic clustering, pointing to environmental filtering as the primary force defining community assembly rather than competitive interactions. This finding stresses the importance of habitat preservation for the maintenance of marine nematode diversity.