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Abstract Marine zooplankton are key players in pelagic food webs, central links in ecosystem function, useful indicators of water masses, and rapid responders to environmental variation and climate change. Characterization of biodiversity of the marine zooplankton assemblage is complicated by many factors, including systematic complexity of the assemblage, with numerous rare and cryptic species, and high local-to-global ratios of species diversity. The papers in this themed article set document important advances in molecular protocols and procedures, integration with morphological taxonomic identifications, and quantitative analyses (abundance and biomass). The studies highlight several overarching conclusions and recommendations. A primary issue is the continuing need for morphological taxonomic experts, who can identify species and provide voucher specimens for reference sequence databases, which are essential for biodiversity analyses based on molecular approaches. The power of metabarcoding using multi-gene markers, including both DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid)templates, is demonstrated. An essential goal is the accurate identification of species across all taxonomic groups of marine zooplankton, with particular concern for detection of rare, cryptic, and invasive species. Applications of molecular approaches include analysis of trophic relationships by metabarcoding of gut contents, as well as investigation of the underlying ecological and evolutionary forces driving zooplankton diversity and structure. 

Abstract Community-based diversity analyses, such as metabarcoding, are increasingly popular in the field of metazoan zooplankton community ecology. However, some of the methodological uncertainties remain, such as the potential inflation of diversity estimates resulting from contamination by pseudogene sequences. Furthermore, primer affinity to specific taxonomic groups might skew community composition and structure during PCR. In this study, we estimated OTU (operational taxonomic unit) richness, Shannon’s H’, and the phylum-level community composition of samples from a coastal zooplankton community using four approaches: complement DNA (cDNA) and genomic DNA (gDNA) mitochondrial COI (Cytochrome oxidase subunit I) gene amplicon, metatranscriptome sequencing, and morphological identification. Results of mismatch distribution demonstrated that 90% is good threshold percentage to differentiate intra- and inter-species. Moderate level of correlations appeared upon comparing the species/OTU richness estimated from the different methods. Results strongly indicated that diversity inflation occurred in the samples amplified from gDNA because of mitochondrial pseudogene contamination (overall, gDNA produced two times more richness compared with cDNA amplicons). The unique community compositions observed in the PCR-based methods indicated that taxonomic amplification bias had occurred during the PCR. Therefore, it is recommended that PCR-free approaches be used whenever resolving community structure represents an essential aspect of the analysis.