Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics

et al. (2012) Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics. PLoS ONE 7(3): e33641. doi:10.1371/journal.pone.0033641 Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics Simon Roux 0 Francois Enault 0 Agne` s Robin 0 Viviane Ravet 0 Se bastien Personnic 0 Se bastien 0 Theil 0 Jonathan Colombet 0 Te lesphore Sime-Ngando 0 Didier Debroas 0 Darren P. Martin, Institute of Infectious Disease and Molecular Medicine, South Africa 0 1 Laboratoire ''Microorganismes: Ge nome et Environnement'', Clermont Universite , Universite Blaise Pascal, Clermont-Ferrand, France , 2 CNRS, UMR 6023, LMGE, Aubie`re , France Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale. To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time. - Funding: This study was funded by an EC2CO CNRS program grant (Metavir). SR was supported by a PhD grant from the French defense procurement agency (DGA, Direction Generale de lArmement). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Despite the large population sizes of microbes, their high reproductive rates and the potential for long-distance passive dispersal, an increasing amount of studies are showing that the transitions between marine and fresh waters are infrequent [1]. Indeed, marine and freshwater microbes are usually not closely related, often grouping into distinct marine and freshwater clades among bacteria [2] or eukaryotes [3]. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Despite the paucity of molecular data from freshwater viruses, recent studies comparing freshwater and marine viruses have concluded on the existence of distinct clades [4,5]. Nevertheless, these PCRmediated analyses are restricted to chosen viral groups as no gene is universally conserved among viruses. In addition, part of the existing diversity of the viral groups studied is missed as PCR primers are based on previously known sequences described in public databases. Thus, both the diversity of freshwater viral communities and its distinction with marine viruses still need to be demonstrated on a community scale by studying not one but all the major viral families. Viral metagenomics is a methodology capable of providing an exhaustive view of viral diversity [6], and it has so far revealed an important unknown diversity and an unexpected richness of viral communities [7]. Virome studies on freshwater environments were conducted on aquaculture facilities [8], and a polar lake in Antarctica [9], but never on temperate freshwater lakes. The viral diversity retrieved in these analyses was contrasted : aquaculture facilities were mainly composed of bacteriophages (Myoviridae and Podoviridae), whereas eukaryotic viruses, including phycodnaviruses and single-stranded DNA viruses accounted for a large proportion of the Antarctic viral communities. Thus, a fine-grained and exhaustive description of viral diversity in temperate freshwater lakes is needed to improve our knowledge about these communities and to offer the opportunity of identifying potential viral populations specific of those environments. To assist in these goals, we performed a characterization of freshwater viromes from two french lakes: the lake Pavin and the lake Bourget which exhibit different trophic status, morphological and hydrological features (Table S1). Because species compositions and abundances of potential hosts have been shown to vary with lake trophic status, depth, watershed or size [3,10], suggesting possible similar variations for viral communities, these two lakes are expected to be complementary systems for studies on freshwater viromes. The two viromes were analyzed according to the following procedure : (i) the characteristics and richness indices of the two viral communities were determined, (ii) freshwater viromes were crosscompared to a set of previously published viromes in terms of sequence similarity and richness, (iii) the composition of the two communities were determined and phylogenetic analyses of the major families were computed in order to accurately describe the genetic diversity in these families. Overview of the two freshwater viromes After 454 pyrosequencing and data filtering, viromes of 593,084 and 649,290 reads with an average length of 420 bp were available for Lake Bourget and Lake Pavin, respectively. The proportion of reads similar to protein sequences of the nonredundant NCBI database (NR) were 26.4% and 14.3% for Lakes Bourget and Pavin, respectively (Figure 1A). These proportions of known reads (reads with a BLAST hit against NR) are among the highest compa (...truncated)