Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the “Leray fragment” of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of the nuclear small subunit ribosomal RNA (18S). Eight different shallow marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. Despite applying stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignment of the detected sequences. In our dataset, 85% of 18S MOTUs and 64% of COI MOTUs could be identified to phylum or lower taxonomic level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples. Thus, eukaryotic metabarcoding emerges as a robust, fast, objective and affordable method to comprehensively characterize the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities.

Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp). We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column) that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100–2,250 m). We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla), Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm) of sediment was significantly different from deeper layers. We found that qualitative (presence-absence) and quantitative (relative number of reads) data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs) showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation efforts on these bottoms of ecological and economic importance.

We assessed spatio-temporal patterns of diversity in deep-sea sediment communities using metabarcoding. We chose a recently developed eukaryotic marker based on the v7 region of the 18S rRNA gene. Our study was performed in a submarine canyon and its adjacent slope in the Northwestern Mediterranean Sea, sampled along a depth gradient at two different seasons. We found a total of 5,569 molecular operational taxonomic units (MOTUs), dominated by Metazoa, Alveolata and Rhizaria. Among metazoans, Nematoda, Arthropoda and Annelida were the most diverse. We found a marked heterogeneity at all scales, with important differences between layers of sediment and significant changes in community composition with zone (canyon vs slope), depth, and season. We compared the information obtained from metabarcoding DNA and RNA and found more total MOTUs and more MOTUs per sample with DNA (ca. 20% and 40% increase, respectively). Both datasets showed overall similar spatial trends, but most groups had higher MOTU richness with the DNA template, while others, such as nematodes, were more diverse in the RNA dataset. We provide metabarcoding protocols and guidelines for biomonitoring of these key communities in order to generate information applicable to management efforts.

The allochthonous calcarean sponge Paraleucilla magna has proliferated in the western Mediterranean during the last decade, where it currently shows a highly patchy distribution with dense populations in the neighboring of sea farms and slightly eutrophised marinas, and more sparse populations in well-preserved habitats. To gain knowledge about the species invasive capacity, we studied spatial genetic differentiation and structure, clonality, and temporal differentiation, in three close populations of P. magna at the NE of the Iberian Peninsula, in three successive years. The study hypothesis was that the species is able to proliferate under favorable conditions in newly colonized habitats but populations can easily disappear where perturbations occur with some frequency. Samples were genotyped for nine polymorphic microsatellites. Spatial genetic structure was found in the three populations of 2006. One population disappeared in 2007, and the other two remained slightly differentiated, while the three populations were in place again in 2008, and showed very low (but significant) F ST values, and non-significant D values. Low but statistically significant differentiation also occurred for the three populations between years. Results showed high-allele diversity, but heterozygote deficit and changes in allele frequencies in the populations over the 3 years, which are consistent with some genetic drift. The whole population descriptors pointed to the species as a good opportunistic colonizer as it has been hypothesized, but highly sensitive to stochastic events affecting recruitment. This suggests a high impact of the species in favorable habitats (sea culture and sheltered zones) and a low-medium influence in native communities.

Among the complex ecosystems and habitats that form the deep sea, submarine canyons and open slope systems are regarded as potential hotspots of biodiversity. We assessed the spatial and temporal patterns of biodiversity in sediment communities of a NW Mediterranean Canyon and its adjacent open slope (Blanes Canyon) with DNA metabarcoding. We sampled three layers of sediment and four different depths (900–1750 m) at two seasons, and used a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI) as a metabarcoding marker. The final dataset contained a total of 15,318 molecular operational taxonomic units (MOTUs). Metazoa, Stramenopiles and Archaeplastida were the dominant taxa and, within metazoans, Arthropoda, Nematoda and Cnidaria were the most diverse. There was a trend towards decreasing MOTU richness and diversity in the first few cm (1 to 5) of the sediment, with only 26.3% of the MOTUs shared across sediment layers. Our results show the presence of heterogeneous communities in the studied area, which was significantly different between zones, depths and seasons. We compared our results with the ones presented in a previous study, obtained using the v7 region of the 18S rRNA gene in the same samples. There were remarkable differences in the total number of MOTUs and in the most diverse taxa. COI recovered a higher number of MOTUs, but more remained unassigned taxonomically. However, the broad spatio-temporal patterns elucidated from both datasets coincided, with both markers retrieving the same ecological information. Our results showed that COI can be used to accurately characterize the studied communities and constitute a high-resolution method to detect ecological shifts. We also highlight that COI reference databases for deep-sea organisms have important gaps, and their completeness is essential in order to successfully apply metabarcoding techniques.