DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers

Abstract

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.

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

“…The hypothesis regarding number of putative S. spallanzanii sequences in metabarcoding data being a good predictor of a true detection was supported only for the COI assay applied to water samples, as evidenced by excellent response (AUC > 90%) of a binary classifier (targeted S. spallanzanii detection either by qPCR or ddPCR). This is probably a synergetic effect of higher detection efficiency from water samples and better taxonomic resolution, as well as more reliable taxonomic assignments attained by the COI marker (Cowart et al, ; Wangensteen et al, ) despite the cost of a higher proportion of unassigned taxa. There is a reasonable probability of type I error (false positive detections) when using metabarcoding, as evidenced by imperfect sensitivity (83.6%), therefore, additional investigation employing targeted assays and/or visual survey couple with morphological assessments to confirm species presence is recommended.…”

Non‐specific amplification compromises environmental DNA metabarcoding with COI

“…All PCR amplifications were done in duplicate reactions each with a unique 7/8‐mer oligo‐tag barcode, differing by at least three bases (Guardiola et al, ). In order to increase variability of the amplicon sequences, a variable number (two, three or four) of fully degenerate positions (Ns) were added at the 5′ end of the oligo tags (Wangensteen et al, ). For PCR amplification with the newly designed SeaDNA‐short and SeaDNA‐mid primers, a two‐step protocol was used, first using untagged primers, then tagged primers in a second PCR round.…”

“…After the second PCR, all tagged amplicons were pooled by marker, purified again using MinElute columns and eluted into a total volume of 45 µl, in order to concentrate the amplicons approximately 15 times. For 12S MiFish and Leray‐XT, we used a one‐step procedure with tagged PCR primers, with PCR cycling conditions following Miya et al () and Wangensteen et al (), respectively. Reagents and volumes were the same as for the two‐step protocol.…”

Cobble community DNA as a tool to monitor patterns of biodiversity within kelp forest ecosystems

“…Nevertheless, it is important to retain unclassified MOTUs in data analysis because public repositories are becoming increasingly updated with additional reference sequences (e.g. BioBlitz surveys; see MarineGEO: marinegeo.si.edu), enabling increased taxonomic resolution and monitoring (Wangensteen et al, ).…”

“…A two‐step PCR protocol (following methods described in O’Donnell, Kelly, Lowell, & Port, ) was used to amplify the mitochondrial cytochrome oxidase I (COI) marker using Leray et al’s () ‘mini‐barcode’ yielding a 313‐bp fragment (mICOIintF: GGWACWGGWTGAACWGTWTAYCCYCC, matched to jgHCO2198: TAIACYTCIGGRTGICCRAARAAYCA; Geller, Meyer, Parker, & Hawk, ). This versatile primer set has been used to amplify a wide range of phyla across eukaryotes (Leray et al, ) and algae (Wangensteen, Palacín, Guardiola, & Turon, ). The first PCR (PCR1) consisted of nonindexed primers, and the amplification was performed using 20 µl volumes containing 1× PCR buffer, 2 mM MgCl 2 , 4 mM dNTPs, 0.5 µM of each primer, 0.05 units of Taq DNA polymerase and 2 µl of DNA template (5–10 ng/µl).…”