Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

Abstract

High-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescence in situ hybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances. IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescence in situ hybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed. Protists are key components of marine ecosystems, being major players in the global respiration and production budgets (1, 2) and playing central roles in marine food webs (3). Despite their importance and ubiquity, it was only during the past decade that environmental studies, based on molecular (i.e., culture-independent) techniques, revealed an unsuspected protist diversity in a large variety of marine ecosystems (4-13). These studies we...

Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina

“…The differences in the proportion of richness and abundance between both regions in some eukaryotic groups are likely to be due to primer bias effect. In addition to that, indels or variation among sequences can also change the properties of primers favoring the amplification of some groups above others, explaining the unequal recovery of diversity and novelty in eukaryotes (Pawlowski et al, 2011;Giner et al, 2016).…”

“…These clean reads were then clustered into Operational Taxonomic Units (OTUs) using UPARSE with a 97% similarity threshold, resulting in a total of 4,598 OTUs for V4 and 4,176 OTUs for V8-9 region. Even though there is no clear consensus on the optimal threshold to use in clustering algorithms Brown et al, 2015), we decided to use 97% because it has been widely used (Stoeck et al, 2010;Kammerlander et al, 2015;Massana et al, 2015;Giner et al, 2016;Kim et al, 2016). We are aware that this threshold might group species or even genus into the same OTU.…”

DNA Sequencing as a Tool to Monitor Marine Ecological Status

“…The ocean microbiome, an assemblage of bacteria, archaea, microeukaryotes, and viruses (Stulberg et al, 2016), is the heart of marine food webs. Although the need for food web assessments in marine ecosystem status is recognized (Zampoukas et al, 2014;Caruso et al, 2016), microbes are not readily amenable to visual identification (Bowers et al, 2016;Giner et al, 2016), hindering inclusion as a BQE in monitoring frameworks. Instead, marine monitoring programs are often restricted to gross measurements such as chlorophyll to capture this critical segment of the ecosystem.…”

“…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).…”

“…Lack of intercalibration between morphological and molecular methods presents an additional issue, and transferability across taxa is a concern, particularly if the initial assessment used to establish the database is limited to one or two taxonomic groups (Kelly, 2016). Although comparable molecular and traditional approaches are reported (Lejzerowicz et al, 2015;Aylagas et al, 2016Aylagas et al, , 2017Hänfling et al, 2016;Valentini et al, 2016), other studies show discrepancies (Cowart et al, 2015;Hirai et al, 2015b;Mohrbeck et al, 2015;Giner et al, 2016;Kelly et al, 2017); therefore, it has been advised to use both approaches in a complementary fashion rather than outright substitution of morphotaxonomic approaches (Borja et al, 2008;Pedersen et al, 2015;Thomsen and Willerslev, 2015). Although such an approach may deliver a holistic perspective of marine ecological status, it would not produce the desired effect of streamlining monitoring efforts, which is needed to transition research into practice (de Jonge et al, 2006).…”