Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity

Abstract

Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2–5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).

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

“…This is in agreement with previous freshwater studies (Taib et al, 2013;Simon et al, 2014Simon et al, , 2015. The main reason of this large number might not be the abundance of the organisms themselves but the greater amount of rRNA gene copies in their genomes (Medinger et al, 2010;TorresMachorro et al, 2010;Pawlowski et al, 2011).…”

“…Cryptista appear to be moderately abundant in our samples, reaching up to 25% of the total eukaryotic abundance in the 2-8 µm size fraction in almost all samples (Figure 2, Supplementary Figures 1B, 2B). The abundance of this group is, however, not as large as in other surveys (Šlapeta et al, 2005;Taib et al, 2013;Simon et al, 2014). This pattern might be explained by the fluctuations of its abundance over time, variations in flow rate of the river or the presence of oxygenated waters (Šlapeta et al, 2005;Devercelli, 2006;Simon et al, 2015).…”

“…In fact, we overcame one of the main limitations of the V9 region, which is its short length (Amaral-Zettler et al, 2009;Pawlowski et al, 2011), by sequencing the V8 region as well. This strategy had already been proven to be reliable as demonstrated by Taib et al (2013). In addition, a combination of V4 and V8-9 regions compensate the existing primer bias (Amaral-Zettler et al, 2009;Pawlowski et al, 2011), leading to a more realistic picture of the eukaryotic diversity.…”

Geographic distance and mountain ranges structure freshwater protist communities on a European scalе

“…Considerable research effort has been focused on marine diversity (Countway et al 2005,Countway et al 2007, Campo et al 2016, Lovejoy et al 2006, López-García et al 2001, Massana and Pedrós-Alió 2008, Worden et al 2006. Despite the global importance of freshwater resources and the heterogeneity of freshwater systems, their biological diversity has been comparatively neglected (Filker et al 2016, Grossmann et al 2016a, Kammerlander et al 2015, Lepère et al 2007, Taib et al 2013, Triadó-Margarit and Casamayor 2012, Slapeta et al 2005. Our study revealed a high diversity of microbial freshwater eukaryotes and a high level of regional separation.…”

High habitat-specificity in fungal communities in oligo-mesotrophic, temperate Lake Stechlin (North-East Germany)

“…Studies in lakes have often concentrated on seasonal patterns in the water column (e.g., van Donk and Ringelberg 1983;Holfeld 1998;Lefèvre et al 2012;Rasconi et al 2012) or have compared different lakes (e.g. Zhao et al 2011;Lefèvre et al 2012;Taib et al 2013). Several studies have found evidence for vertical and horizontal structuring of fungal communities in the water column (Lefèvre et al 2007;Chen et al 2008; Lepère et al 2010), suggesting that there is an important spatial component of diversity.…”