Marine planktonic copepods are an ecologically important group with high species richness and abundance. Here, we propose a new metagenetic approach for revealing the community structure of marine planktonic copepods using 454 pyrosequencing of nuclear large subunit ribosomal DNA. We determined an appropriate similarity threshold for clustering pyrosequencing data into molecular operational taxonomic units (MOTUs) using an artificial community containing 33 morphologically identified species. The 99% similarity threshold had high species-level resolution for MOTU clustering but overestimated species richness. The artificial community was appropriately clustered into MOTUs at 97% similarity, with little inflation in MOTU numbers and with relatively high species-level resolution. The number of sequence reads of each MOTU was correlated with dry weight of that taxon, suggesting that sequence reads could be used as a proxy for biomass. Next, we applied the method to field-collected samples, and the results corresponded reasonably well with morphological analysis of these communities. Numbers of MOTUs were well correlated with species richness at 97% similarity, and large numbers of sequence reads were generally observed in MOTUs derived from species with large biomass. Further, MOTUs were successfully classified into taxonomic groups at the family level at 97% similarity; similar patterns of species richness and biomass were revealed within families with metagenetic and morphological analyses. At the 99% similarity threshold, MOTUs with high proportions of sequence reads were identified as biomass-dominant species in each field-collected sample. The metagenetic approach reported here can be an effective tool for rapid and comprehensive assessment of copepod community structure.

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Many oceanic zooplankton species have been described as cosmopolitan in distribution; however, recent molecular work has detected species complexity with highly divergent genetic lineages within several of these taxa. To further resolve the species complexity within these ecologically-important and widespread species, we performed both molecular and morphological analyses of the oceanic copepod Pleuromamma abdominalis using a comprehensive collection of material from 944 individuals collected at 46 sites across the global ocean. Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I (mtCOI) sequences detected eighteen divergent evolutionary lineages within P. abdominalis, with an additional four singleton specimens that were also genetically divergent. Two phylogenetically distinct groups, PLAB1 and PLAB2, were supported by concordant sequence variation in the nuclear large subunit ribosomal RNA gene (nLSU). Within PLAB1, two mtCOI clades, 1a-1 and 1b-1 were observed, and each clade contained geographically distinct sub-clades 1a-2 and 1b-2. PLAB2 was composed of sixteen well-supported mtCOI clades (2a-2p) as well as four singletons. High genetic divergence among the mtCOI lineages within both PLAB1 and PLAB2, ranging between 9.2-11.2% and 4.3-18.9% K2P distances respectively, suggests the presence of additional species within these groups. Significant differences were observed in the presence and shape of antennule spines of adult females between sympatric clades with genetic distances greater than 5.7-7.0% (K2P). The biogeographic distributions of mtCOI clades indicated greater specialization to particular oceanographic provinces than observed in the nominal species P. abdominalis, with mtCOI clades ranging from antitropical in subtropical waters of all three ocean basins (Atlantic, Pacific and Indian; clade 1b-1 and 2a) to taxa that are endemic to a particular ocean region, for example restricted to equatorial waters of the Atlantic Ocean (clade 1b-2 and 2b). We hypothesize that many of these mtCOI clades are likely distinct, and currently undescribed species. The well-known high dominance of P. abdominalis across a range of pelagic habitats may occur as a spatial composite of genetically-distinct species with more restricted distributions and greater 3 ecological specialization to particular marine habitats than was previously recognized.

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Monitoring zooplankton communities is important to understand dynamics in marine ecosystems. However, it is difficult to identify cryptic species and immature stages of zooplankton using morphological classification, which is time-consuming and requires high skill levels. Here, we conducted a metagenetic analysis of the 18S region in 101 zooplankton samples collected weekly throughout 2014 and 2015 at the Okhotsk Tower in Mombetsu, Hokkaido, Japan, and compared the results of this analysis with those provided by morphological analysis. The metagenetic analysis detected 561 molecular taxonomic units (MOTUs), whereas the morphological analysis detected 201 taxonomic groups. Zooplankton communities were dominated by copepods throughout the sampling period; however, non-copepod taxa, which comprised high proportions of both MOTUs (mean 51.1%) and sequence reads (mean 19.1%), were also important. Cryptic diversity detected by the metagenetic analysis was primarily driven by Copepoda and by the larvae of benthic taxa such as Bivalvia, Gastropoda, and Polychaeta. Community structure and diversity varied between periods of warm and cold water, indicating strong correlations with water temperature and thus seasonality. Furthermore, metagenetic analysis revealed detailed seasonal changes in dominant taxa, including larval stages of metazoans with high taxonomic resolutions; these included commercially important organisms such as Japanese scallops. The metagenetic analysis revealed that changes in both water mass and bentho-pelagic interactions sustain ecosystems rich in zooplankton diversity in this area. Metagenetic analysis provides novel insight into zooplankton diversity, and generates massive sequence data that may be used in future research; thus, it is considered an effective tool for monitoring zooplankton communities.

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A clear insight into the large-scale community structure of planktonic copepods is critical to understanding the mechanisms controlling diversity and biogeography of marine taxa in terms of their high abundance, ubiquity, and sensitivity to environmental changes. Here, we applied a 28S metabarcoding approach to large-scale communities of epipelagic and mesopelagic copepods at 70 stations across the Pacific Ocean and three stations in the Arctic Ocean. Major patterns of community structure and diversity, influenced by water mass structures, agreed with results from previous morphology-based studies. However, a largescale metabarcoding approach could detect community changes even under stable environmental conditions, including changes in the north/south subtropical gyres and east/west areas within each subtropical gyre. There were strong effects of the epipelagic environment on mesopelagic communities, and community subdivisions were observed in the environmentally stable mesopelagic layer. In each sampling station, higher operational taxonomic unit (OTU) numbers and lower phylogenetic diversity were observed in the mesopelagic layer than in the epipelagic layer, indicating a recent rapid increase in species numbers in the mesopelagic layer. The phylogenetic analysis utilizing representative sequences of OTUs revealed trends of recent emergence of cold-water OTUs, which are mainly distributed at high latitudes with low water temperatures. Conversely, the high diversity of copepods at low latitudes was suggested to have been formed through long evolution under high water temperature conditions. The metabarcoding results suggest that evolutionary processes have strong impacts on current patterns of copepod diversity, and support the "out of the tropics" theory explaining latitudinal diversity gradients of copepods. Diversity patterns in both epipelagic and mesopelagic copepods was highly correlated to sea surface temperature; thus, predicted global warming may have a significant impact on copepod diversity in both layers.