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.

Metabarcoding is by now a well‐established method for biodiversity assessment in terrestrial, freshwater, and marine environments. Metabarcoding data sets are usually used for α‐ and β‐diversity estimates, that is, interspecies (or inter‐MOTU [molecular operational taxonomic unit]) patterns. However, the use of hypervariable metabarcoding markers may provide an enormous amount of intraspecies (intra‐MOTU) information—mostly untapped so far. The use of cytochrome oxidase (COI) amplicons is gaining momentum in metabarcoding studies targeting eukaryote richness. COI has been for a long time the marker of choice in population genetics and phylogeographic studies. Therefore, COI metabarcoding data sets may be used to study intraspecies patterns and phylogeographic features for hundreds of species simultaneously, opening a new field that we suggest to name metaphylogeography. The main challenge for the implementation of this approach is the separation of erroneous sequences from true intra‐MOTU variation. Here, we develop a cleaning protocol based on changes in entropy of the different codon positions of the COI sequence, together with co‐occurrence patterns of sequences. Using a data set of community DNA from several benthic littoral communities in the Mediterranean and Atlantic seas, we first tested by simulation on a subset of sequences a two‐step cleaning approach consisting of a denoising step followed by a minimal abundance filtering. The procedure was then applied to the whole data set. We obtained a total of 563 MOTUs that were usable for phylogeographic inference. We used semiquantitative rank data instead of read abundances to perform AMOVAs and haplotype networks. Genetic variability was mainly concentrated within samples, but with an important between seas component as well. There were intergroup differences in the amount of variability between and within communities in each sea. For two species, the results could be compared with traditional Sanger sequence data available for the same zones, giving similar patterns. Our study shows that metabarcoding data can be used to infer intra‐ and interpopulation genetic variability of many species at a time, providing a new method with great potential for basic biogeography, connectivity and dispersal studies, and for the more applied fields of conservation genetics, invasion genetics, and design of protected areas.

The population structure of the edible Atlanto-Mediterranean sea urchin Paracentrotus lividus is described by analysing sequence variation in a fragment of the mitochondrial gene cytochrome c oxidase subunit I in 127 individuals from 12 localities across south-west Europe. The study revealed high levels of genetic diversity but low levels of genetic structure, suggesting a large degree of gene flow between populations and panmixis within each, the Mediterranean and Atlantic basins. However, we found significant genetic differentiation between the two basins probably due to restricted gene flow across the geographical boundary imposed by the area of the Strait of Gibraltar. Populations of P. liuidus appeared to have experienced a recent demographic expansion in the late Pleistocene. We provide new evidence on the population structure of this commercial species, predicting a healthy stock of this sea urchin on the Mediterranean and Atlantic coasts.