Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

Abstract

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.

Under the canopy: Community-wide effects of invasive algae in Marine Protected Areas revealed by metabarcoding

“…Indeed, the use of several markers in metabarcoding is rapidly gaining momentum (e.g., Clarke et al, 2017;Harvey et al, 2017;Kelly et al, 2017). It is reassuring that the same general conclusions were reached with both markers used here.…”

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

“…Additionally, ribosomal markers often have limited taxonomic resolution, which is less of an issue for the COI barcoding marker (Meusnier et al, 2008;Clarke et al, 2014Clarke et al, , 2017.…”

Cryptic Zooplankton Diversity Revealed by a Metagenetic Approach to Monitoring Metazoan Communities in the Coastal Waters of the Okhotsk Sea, Northeastern Hokkaido

“…Although there are methodological limitations (e.g., PCR bias, gene copy numbers), sequence reads provide semi-quantitative information for zooplankton biomass (Lindeque et al, 2013;Hirai et al, 2015a;Albaina et al, 2016;Clarke et al, 2017), which is not directly comparable with the number of individuals in morphological analysis. We nevertheless observed similar seasonal patterns in diversity indexes, abundance-based community structures, and taxonomic compositions between the metagenetic and morphological analyses.…”

“…Metagenetic approaches provide a sensitive method for monitoring zooplankton diversity, as demonstrated in studies of zooplankton communities in the English Channel (Lindeque et al, 2013), tropical and subtropical Pacific (Hirai and Tsuda, 2015;Hirai et al, 2015a), station ALOHA (Sommer et al, 2017), Red Sea (Pearman et al, 2014;Pearman and Irigoien, 2015;Casas et al, 2017), Monterey Bay (Harvey et al, 2017), Canadian coastal areas (Chain et al, 2016), and Storm Bay (Clarke et al, 2017). Given the alignment of results generated from metagenetic and morphological methods, we conclude that metagenetic analysis was successfully used to monitor changes in zooplankton diversity and communities in the study area, and that this in turn corresponded with environmental changes, particularly water temperature.…”

“…Furthermore, we used the 18S V9 region because of its capacity to identify various metazoan zooplankton; however, marker choice affects the results of the metagenetic analysis. For example, although mitochondrial genes such as COI have high species-level resolution, primer mismatches remain common, particularly in highly variable regions of genes (Deagle et al, 2014;Clarke et al, 2017). Thus, molecular markers should be chosen according to the purpose of each study.…”