A metagenetic approach for revealing community structure of marine planktonic copepods

Abstract

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.

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

“…In this study, we examined the relationship for calanoid copepods as all markers were capable of PCR‐amplifying this group, and we could estimate biomass from morphology‐based counts using the approach of Hirai et al. (2015). We found all three markers displayed a positive relationship between biomass and HTS reads for calanoid families.…”

“…Counts for each sex of each species were converted to dry weights (μg) based on sex‐specific prosome lengths from the literature using the approach of Hirai et al. (2015). HTS read counts and dry weights were summarized at family‐level and converted to the percentage of total calanoid HTS reads or dry weight.…”

“…Recent research has begun to validate metabarcoding as a time and cost‐efficient method for biodiversity surveys in terrestrial, freshwater, and marine ecosystems (Hirai, Kuriyama, Ichikawa, Hidaka, & Tsuda, 2015; Ji et al., 2013; Thomsen et al., 2012; Valentini et al., 2016). The results of metabarcoding studies depend on the markers used providing sufficient taxonomic coverage and resolution for the taxa of interest.…”

DNA Sequencing as a Tool to Monitor Marine Ecological Status

“…For example, amplification of a nuclear 16S rRNA gene segment is typical for taxonomic classification of marine prokaryotic assemblages (Klindworth et al, 2013). Amplification of 18S rRNA regions is used for a variety of communities, including phytoplankton and other microeukaryotes (Dunthorn et al, 2012;Hugerth et al, 2014;Johnson and Martiny, 2015;del Campo et al, 2016;Giner et al, 2016), and amplification of the 23S rRNA gene is used to classify organisms such as zooplankton (Hirai et al, 2015a;Bucklin et al, 2016). Amplification of mitochondrial DNA is used to identify an assortment of organisms, with cytochrome oxidase I (COI), cytochrome b, and mitochondrial 16S as examples of popularly employed target regions (Dauble et al, 2012;Pawlowski et al, 2014;Cowart et al, 2015;Guo et al, 2015;Harada et al, 2015;Johnson and Martiny, 2015;Aylagas et al, 2016;Bucklin et al, 2016;Creer et al, 2016;Leray and Knowlton, 2016;Thompson et al, 2016;Trivedi et al, 2016).…”

“…Studies demonstrate DNA metabarcoding to be a reliable method for biodiversity assessment with potential for inferring biotic indices for marine ecosystem quality assessment (Aylagas et al, 2014;Pawlowski et al, 2014;Cowart et al, 2015;Elbrecht and Leese, 2015;Visco et al, 2015;Ferrera et al, 2016). Comparable results between molecular and traditional approaches are reported in a number of studies (Hirai et al, 2015a;Lejzerowicz et al, 2015;Aylagas et al, 2016Aylagas et al, , 2017Valentini et al, 2016), suggesting that assessment programs that require manually intensive sorting and visual inspection will be among the first to formally integrate molecular techniques. For example, larval rockfish time-series are currently used to quantify spawning stock biomass off California, USA and are integral components of rockfish stock assessment .…”