Aquatic Microbial Ecology 75:117

Vol. 75: 117–128, 2015 AQUATIC MICROBIAL ECOLOGY Published online June 4 doi: 10.3354/ame01752 Aquat Microb Ecol Metagenomic assessment of viral diversity in Lake Matoaka, a temperate, eutrophic freshwater lake in southeastern Virginia, USA Jasmin C. Green1, Faraz Rahman1, Matthew A. Saxton2,3, Kurt E. Williamson1,2,* 1Department of Biology, College of William & Mary, Williamsburg, Virginia 23185, USA 2Environmental Science and Policy Program, College of William & Mary, Williamsburg, Virginia 23185, USA 3Present address: Department of Marine Sciences, University of Georgia, Athens, Georgia 30602, USA ABSTRACT: Little is known about the composition and diversity of temperate freshwater viral communities. This study presents a metagenomic analysis of viral community composition, taxonomic and functional diversity of temperate, eutrophic Lake Matoaka in southeastern Virginia (USA). Three sampling sites were chosen to represent differences in anthropogenic impacts: the Crim Dell Creek mouth (impacted), the Pogonia Creek mouth (less impacted) and the main body of the lake (mixed). Sequences belonging to tailed bacteriophages were the most abundant at all 3 sites, with Podoviridae predominating. The main lake body harbored the highest virus genotype richness and included cyanophage and eukaryotic algal virus sequences not found at the other 2 sites, while the impacted Crim Dell Creek mouth showed the lowest richness. Cross-contig comparisons indicated that similar virus genotypes were found at all 3 sites, but at different rank- abundances. Hierarchical cluster analysis of multiple viral metagenomes indicated high genetic similarity between viral communities of related environments, with freshwater, marine, hypersaline, and eukaryote-associated environments forming into clear groups despite large geographic dis- tances between sampling locations within each environment type. These results support the con- clusion that freshwater viral communities are genetically distinct from virus assemblages in other environments. KEY WORDS: Virus · Freshwater · Lake · Temperate · Metagenome · Sequencing · Virome Resale or republication not permitted without written consent of the publisher INTRODUCTION allows the sequencing of all the DNA present in an environmental sample without relying on cultures, Viruses are important and active components of has revolutionized the study of viral ecology and the biosphere and can be found anywhere on the enabled unprecedented views of the taxonomic and planet where cellular life exists. Through infection of functional diversity of the global virome. microbial populations, viruses are significant drivers While viral metagenomics has been used to inves- of ecological change in terms of host community tigate a wide variety of sample types including fer- composition and biogeochemical cycling due to cell mented foods (Park et al. 2011), hotsprings (Schoen- lysis (Fuhrman 1999, Berdjeb et al. 2011), bacterial feld et al. 2008), and eukaryote-associated virus respiration rate due to lysis-induced changes in assemblies (Dinsdale et al. 2008, Cantalupo et al. organic matter quality (Bonilla-Findji et al. 2008, 2011), the majority of studies have focused on marine Ram et al. 2013), and host metabolic capabilities due samples (Breitbart et al. 2002, 2004, Angly et al. to horizontal gene transfer (Lindell et al. 2004, Kelly 2006). The current catalogue of viromes include rela- et al. 2013). The advent of metagenomics, which tively few freshwaters, and these tend to focus on *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 118 Aquat Microb Ecol 75: 117–128, 2015 exotic or extreme environments such as Antarctic lakes (López-Bueno et al. 2009), hypersaline lakes (Sime- Ngando et al. 2011), or ephemeral desert ponds (Fancello et al. 2013). Inland freshwaters provide a wide variety of ecosystem services, including (but not limited to) drinking water, irrigation, aquaculture, fish and shell- fish production, recreation, wildlife habitat, and aesthetic value (Postel & Carpenter 2012). In spite of their im- portance, the viral communities of only 2 temperate freshwater lakes from a single study have been described using high-throughput DNA sequencing (Roux et al. 2012). Fig. 1. Sampling sites at Lake Matoaka, Virginia, USA, and its watershed. MO, The goals of the present study were Matoaka open water; CDM, Crim Dell Mouth draining the William & Mary to investigate the viral community College campus (more impacted); PM, Pogonia Mouth draining the College Woods preserve (less impacted) composition, taxonomic and functional diversity of Lake Matoaka, a temperate, eutrophic lake in southeastern Virginia, USA. In Pogonia Creek and Strawberry Creek are 2 smaller particular, 3 sampling sites were chosen to represent streams that feed into the lake from the west (Fig. 1) a gradient of inputs and anthropogenic impacts: a and drain the minimally impacted ~150 ha College sub-watershed stream mouth draining unmanaged Woods preserve. The specific sample collection sites secondary growth forest, a sub-watershed stream were the Crim Dell Mouth (CDM; 37.267° N, mouth draining developed university campus, and 76.721° W), the Pogonia Mouth (PM; 37.268° N, the main body of the lake. Comparisons across these 76.727° W), and Matoaka open water (MO; 37.264° N, sites will provide insights as to how viral community 76.722° W). The Crim Dell Creek sub-watershed has composition may be influenced by human activities. substantial development present (the College of Furthermore, comparing our results to other viral William & Mary campus) while the Pogonia sub- meta genomes will improve our understanding of watershed is dominated by secondary-growth forest. how viral community structure varies by habitat type. Crim Dell Creek and Pogonia Creek contribute water to Lake Ma to aka at a base flow rate of 0.012 and 0.006 m3 s−1, respectively (http:// media.wm.edu/ MATERIALS AND METHODS content / as/kecklab / Weather/KeckWeather.htm). Sampling sites Viral metagenome construction Lake Matoaka is a freshwater impoundment in Williamsburg, Virginia, USA, covering roughly 16 ha Surface samples were collected from each site on with an average depth of 2.5 m (max. depth 4.75 m) 11 March 2013 in acid-washed polycarbonate bot- and water residence time of 75 d (Pensa & Chambers tles. A YSI-63 hand-held multimeter was used to 2004). The lake was established by English colonists measure temperature and conductivity and a YSI-55 in 1718 through damming of the original creek sys- hand-held probe was used to measure dissolved tem to form a mill-pond, making it the oldest man- oxygen in the field. Samples were placed on ice and made lake in Virginia and one of the oldest in the processed immediately upon return to the lab ac- New World. Lake Matoaka is currently fed by 5 cording to Thurber et al. (2009). Briefly, bacteria perennial streams and surface water runoff from its were removed by filtering 2 l samples through 600 ha watershed. College Creek to the north and 0.22 µm and the filtrate was precipitated using poly- Crim Dell Creek to the east are 2 major tributaries to ethylene glycol (PEG-8000) (10% w/v) and NaCl the lake and are more heavily impacted by the (1 M) and incubated at 4°C overnight. Viral particles nearby college campus and city of Williamsburg. from the PEG pellet were purified using CsCl den- Green et al.: Viral diversity of a temperate, eutrophic lake 119 sity gradient ultracentrifugation (1.7, 1.5, and 1.35 g Contig assembly ml−1 layers in SM buffer; virus particles collected from the 1.5−1.35 g ml−1 boundary) and DNAse Viral metagenomes were assembled using the GS treatment. Nucleic acids were extracted using the De Novo Assembler v. 2.8 (Roche Diagnostics). Open formamide procedure as previously de scribed reading frames (ORFs) were annotated in Metavir, (Thurber et al. 2009). Viral nucleic acids were which predicts ORFs for each contig through Meta- amplified using the Illustra Genomiphi V2 DNA GeneAnnotator and compares them to the RefSeq amplification kit (GE Healthcare Life Sciences). Virus protein database by BLASTp (E-value cutoff of Reactions were pooled and purified using the QIA- 10−3) and by HMMScan (Bit score cutoff of 30) to the gen DNeasy blood and tissue kit (QIAgen) before PFAM database (Roux et al. 2014). sequencing on a Roche Applied Sciences GS-FLX+ platform (454 Life Sciences). Viral community structure and diversity Environmental metadata Circonspect and GAAS (Genome relative Abun- dance and Average Size) (Angly et al. 2006, 2009) Viral and bacterial abundance was determined were installed on the SciClone computing cluster at using SYBR Gold-epifluorescence microscopy from the College of William & Mary (www. hpc. wm. edu/ triplicate subsamples (Noble & Fuhrman 1998, Hard- SciClone/Home) and run in the Unix environment. bower et al. 2012). Virus-to-bacterium ratios were Results were exported to PHACCS (Angly et al. 2005) calculated based on averages of both viral and bacte- and MaxiPhi (Angly et al. 2006), which were run rial abundances for each sample. Bacterial produc- using MATLAB (R2014.a). Contig spectra were gen- tion rate was determined based on 3H-Leu incorpora- erated by Circonspect, which was run using 10 000 tion rate from triplicate subsamples (Kirchman et al. randomly selected 100 bp reads and generating 1985, Chin-Leo & Kirchman 1988). Nutrient levels assemblies with Minimo using the default parame- (NO2 + NO3, NH4 and total phosphorus) were deter- ters of 35 bp overlap with 98% similarity. Community mined using colorimetric assays with water filtered structure and α-diversity were modelled in PHACCS through Whatman glass fiber filters (GF/F) (Parsons using the contig spectra from Circonspect and aver- et al. 1984). Chlorophyll a (chl a) was determined age genome size from GAAS. Power law, exponen- spectrophotometrically (Lorenzen 1967). Dissolved tial, logarithmic, and log-normal rank-abundance organic carbon was analyzed using GF/C (0.2 µm models were tested, and the best model was selected average pore size)-filtered water on a Shimadzu based on lowest error values.

Load more