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Wangensteen OS, Palacín C, Guardiola M, Turon X.2017. Metabarcoding littoral hard-bottom communities: unexpected diversity and database gaps revealed by two molecular markers. PeerJ Preprints5:e3429v1https://doi.org/10.7287/peerj.preprints.3429v1
We developed a metabarcoding method for biodiversity characterization of structurally complex natural marine hard-bottom communities. Novel primer sets for two different molecular markers: the “Leray fragment” of mitochondrial cytochrome c oxidase, COI, and the V7 region of ribosomal RNA 18S were used to analyse eight different marine shallow benthic communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea). 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. We found unexpectedly high values for MOTU richness, 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 assignation of the detected sequences. Nevertheless, over 90% (in abundance) of the sequenced reads could be successfully assigned to phylum or lower taxonomical level. This identification rate might be significantly improved in the future, as reference databases are updated. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples, and emerges as a robust, fast, objective and affordable method for comprehensively characterizing the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans, allowing for standardized biomonitoring of these ecologically important communities. The 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.
Pie charts showing the reproducibility of metabarcoding results
Plots correspond to three ecological replicates (upper row), three DNA extraction replicates (middle row) and three PCR replicates (lower row) for COI (left) and 18S (right). Arrows indicate which replicate is used in the next level of study.
nMDS plots showing the ordination patterns of Bray-Curtis dissimilarities calculated from read abundances (fourth root transformed)
Data correspond to all MOTUs detected in eight different marine littoral communities and a set of sediment samples from a tidal lagoon. The size fractions (A, B and C) are indicated. Analyses from COI (left) and 18S (right) are shown.