Previously unknown and highly divergent ssDNA viruses populate the oceans

The ISME Journal (2013) 7, 2169–2177 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE Previously unknown and highly divergent ssDNA viruses populate the oceans Jessica M Labonté1,5 and Curtis A Suttle1,2,3,4 1 Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada; Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, BC, Canada; 3Department of Botany, University of British Columbia, Vancouver, BC, Canada and 4Canadian Institute of Advanced Research, University of British Columbia, Vancouver, BC, Canada 2 Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton. The ISME Journal (2013) 7, 2169–2177; doi:10.1038/ismej.2013.110; published online 11 July 2013 Subject Category: Evolutionary genetics Keywords: ssDNA viruses; microbial diversity; viral diversity Introduction Single-stranded DNA (ssDNA) viruses are major pathogens of plants and animals. There are seven families of ssDNA viruses that are recognized by the International Committee on Virus Taxonomy (King et al., 2012) based on the host range and the type of ssDNA (segmented or not-segmented, positive-sense or negative-sense, circular or linear) composing the genome. Thus, there are two families of bacteriophages (Inoviridae and Microviridae) and five families of viruses infecting eukaryotes (Nanoviridae and Geminiviridae infecting plants; Circoviridae, Parvoviridae and Anelloviridae infecting animals). Correspondence: CA Suttle, Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia, 2207 Main Mall, Vancouver, BC, Canada V6T 1Z4. E-mail: 5 Current address: Single Cell Genomic Center, Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, East Boothbay, ME 04544, USA. Received 30 October 2012; revised 30 May 2013; accepted 4 June 2013; published online 11 July 2013 The genomes are small (between 1.4 and 8.5 kb), and can encode as few as two genes, a capsid and a replication initiator. Viruses are the most abundant (Suttle, 2005) and genetically diverse (Breitbart et al., 2002; Angly et al., 2006) life forms in the biosphere; yet, little is known about the diversity of ssDNA viruses in natural systems, the evolutionary relationships among them and with characterized viruses, and the role they have in ecosystems. Sequences with similarity to ssDNA viruses have been found in metagenomic data from multiple environments (reviewed in Rosario and Breitbart, 2011; Rosario et al., 2012). For example, sequences from the Microviridae and Circoviridae have been observed in marine environments (Angly et al., 2006; Rosario et al., 2009), freshwater (López-Bueno et al., 2009) and modern stromatolites (Desnues et al., 2008), and similar to those from the Circoviridae, Geminiviridae, Nanoviridae and Parvoviridae were observed in corals (Thurber et al., 2008). However, the identification of ssDNA viruses relies on comparative analysis with sequences in databases that do not Previously unknown diversity of ssDNA viruses JM Labonté and CA Suttle 2170 adequately reflect the diversity of ssDNA in nature; hence, the diversity of ssDNA viruses remains poorly characterized. Here we present a comprehensive metagenomic study of marine ssDNA viruses and reveal their genetic diversity in samples from temperate and subtropical waters. Our results greatly extend the existing view of diversity in ssDNA viruses by uncovering new groups of ssDNA viruses that are divergent enough at the sequence level that they could represent new families. Our study demonstrates that the oceans harbor hundreds of previously unknown genetically distinct groups of ssDNA viruses that are likely significant pathogens of the phytoplankton and microzooplankton underlying marine food webs. Materials and methods Collection and preparation of samples Samples were collected from five distinct geographic regions (Supplementary Table 1) as follows: the coastal waters of British Columbia Strait of Georgia (SOG, 82 samples), the Gulf of Mexico (GOM, 41 samples) and Saanich Inlet (SI, 11 samples). Water samples (B20 l for SI; B200 l for the others) were collected using GO-FLO or Niskin bottles mounted either on a rosette (SOG and GOM) or directly on a hydrographic wire (SI). For each sample, the viruses were concentrated B10 to 100-fold (B200 ml final volume) using ultrafiltration (Suttle et al., 1991). Briefly, particulate matter was removed by pressure filtering (o17 kPa) the samples through 142-mm-diameter glass fiber (MFS GC50, nominal pore size 1.2 mm) and polyvinylidene difluoride (Millipore (Billerica, MA, USA) GVWP, pore size 0.22 mm) filters connected in series. The viral size fraction in the filtrate was then concentrated by ultrafiltration through a 30-kDa-molecularweight cutoff cartridge (Amicon S1Y30, Millipore), and stored at 4˚ C in the dark until processed. In order to integrate variation within a region, virus concentrates (VCs) collected from different locations and at different times within a geographic region were combined into a single mix (Supplementary Table 1). Two of these mixes (GOM, SOG) correspond to GOM and BBC, respectively, used in the study by Angly et al. (2006), in which marine viral ssDNA sequences were first observed. Two ml from each VC collected from SOG and neighboring inlets and bays were pooled into three mixes based on the year of collection (BC1—1999, 23 samples; BC3—2000, 26 samples; BC4—2004, 16 samples) and one mix based on salinity (BC2—l (...truncated)