Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR

Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.Large numbers of viruses, an estimated abundance greater than 10³¹ viruses worldwide (11, 26), have been found in a variety of environments, including seawater (38), freshwater (19), sediments (25, 28), and soils (34). Viruses are not only abundant but also likely to significantly influence the population dynamics and genotypic composition of their bacterial host populations (29, 33). Process-level investigations of viral activity in sediments have shown that viruses are an active component of sediment microbial communities (23). Glud and Middelboe (23) found that bacterial growth rates and viral production increased in parallel with respiration, suggesting that viruses are active members of benthic microbial communities. Previous studies have shown that sediment viral abundance exceeds coexisting bacterial abundance by 10- to 1,000-fold (15, 17, 25), creating the potential for viral processes to influence the microbial ecology of aquatic sediments. However, with the exception of small-scale metagenomic investigations (4, 8), there exists little information on the genetic content of viriobenthos assemblages or how the composition of these assemblages changes over ecological gradients.Despite the high abundances of viruses in nature, the lack of a shared genetic marker creates a difficult problem when attempting to examine viral genetic diversity in environmental samples (31). Gene g20 encodes a multifunctional protein within the collar between the capsid and tail in T4-like bacteriophages and has been of significant importance in examining the genetic diversity of cyanomyoviruses (22, 24, 32). As well, others have been able to evaluate the diversity of unidentified aquatic picornavirus-like viruses using the RNA-dependent RNA polymerase gene (13). Other studies have attempted to examine phage genetic diversity based on the DNA polymerase gene (6, 21). Unfortunately, not all known phages contain these specific genes; hence, their use as universal markers is markedly inadequate. Thus, molecular methods that do not rely on polymorphism analysis of a single gene product must be used to circumvent these limitations.Recently, metagenomic approaches (i.e., sequencing of random genomic DNA fragments from whole microbial assemblages) have been used to examine genetic diversity within viral (18) and prokaryotic (10) assemblages. For sediment environments, metagenomic analysis has revealed that the viriobenthos is perhaps the most diverse of all viral assemblages, having been estimated to contain more than 10,000 genotypes per kg of sediment (4). Viral assemblages within a wide range of environments including marine (2, 8) and estuarine (3) waters, soils (20), stromatolites (16), and equine (9) and human feces (5, 40) have been examined. Overall, these studies have shown that a relatively low proportion (∼30%) of viral metagenome sequences are similar to sequences found in the nonredundant GenBank database (nr database), but the probability of detecting significant BLAST homologs increases twofold when queries against other viral metagenome sequence libraries are included (3). Thus, the function of most viral genes is currently unknown; however, these genes are broadly distributed among viruses.While large-scale metagenomics offers unprecedented resolution of the diversity and composition of a viral assemblage, the significant costs and computational requirements preclude routine application in a large collection of environmental samples. Recently, Winget and Wommack (36) introduced a new, low-cost, high-throughput means for genetic analysis of viral diversity utilizing random amplified polymorphic DNA PCR (RAPD-PCR). In this approach, a single 10-bp oligonucleotide serves as both the forward and reverse primers in a single thermocycler reaction. Target sequences in the template DNA are randomly selected; thus, development of a RAPD-PCR assay requires no prior information on the DNA coding content within the sample or organism—a significant advantage considering the largely unknown nature of most viral genes.In this study, we assess the potential of RAPD-PCR as a tool to examine genotype-scale compositional changes in the Chesapeake Bay viriobenthos and to explore the genetic diversity of viruses within Chesapeake Bay sediments. To our knowledge, this is the first study to use RAPD-PCR for evaluating sediment viral diversity and documenting compositional changes in viriobenthos assemblages over time and geographic location.     

Population Dynamics of Chesapeake Bay Virioplankton: Total-Community Analysis by Pulsed-Field Gel Electrophoresis

Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i.e., transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales. To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle (1 year). Virioplankton in water samples collected at six stations along a transect running the length of the bay were concentrated 100-fold by ultrafiltration. Viruses were further concentrated by ultracentrifugation, and the concentrated samples were embedded in agarose. PFGE analysis of virus DNA in the agarose plugs yielded several distinct bands, ranging from 50 to 300 kb. Principal-component and cluster analyses of the virus PFGE fingerprints indicated that changes in virioplankton community structure were correlated with time, geographical location, and extent of water column stratification. From the results of this study, it is concluded that, based on the dynamic nature of the Chesapeake Bay virioplankton community structure, the clonal diversity of bacterio- and phytoplankton host communities is an important component of the virus community.     

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.     

Seasonal and annual reoccurrence in betaproteobacterial ammonia-oxidizing bacterial population structure

Microbes exhibit remarkably high genetic diversity compared with plant and animal species. Many phylogenetically diverse but apparently functionally redundant microbial taxa are detectable within a cubic centimetre of mud or a millilitre of water, and the significance of this diversity, in terms of ecosystem function, has been difficult to understand. Thus it is not known whether temporal and spatial differences in microbial community composition are linked to particular environmental factors or might modulate ecosystem response to environmental change. Fifty-three water and sediment samples from upper and lower Chesapeake Bay were analysed in triplicate arrays to determine temporal and spatial patterns and relationships between ammonia-oxidizing bacterial (AOB) communities and environmental variables. Thirty-three water samples (three depths) collected during April, August and October, 2001-2004, from the oligohaline upper region of the Bay were analysed to investigate temporal patterns in archetype distribution. Using a combination of a non-weighted discrimination analysis and principal components analysis of community composition data obtained from functional gene microarrays, it was found that co-varying AOB assemblages reoccurred seasonally in concert with specific environmental conditions, potentially revealing patterns of niche differentiation. Among the most notable patterns were correlations of AOB archetypes with temperature, DON and ammonium concentrations. Different AOB archetypes were more prevalent at certain times of the year, e.g. some were more abundant every autumn and others every spring. This data set documents the successional return to an indigenous community following massive perturbation (hurricane induced flooding) as well as the seasonal reoccurrence of specific lineages, identified by key functional genes, associated with the biogeochemically important process nitrification.     

Diverse and dynamic populations of cyanobacterial podoviruses in the Chesapeake Bay unveiled through DNA polymerase gene sequences

Many podoviruses have been isolated which infect marine picocyanobacteria, and they may play a potentially important role in regulating the biomass and population composition of picocyanobacteria. However, little is known about the diversity and population dynamics of autochthonous cyanopodoviruses in marine environments. Using a set of newly designed PCR primers which specifically amplify the DNA pol from cyanopodoviruses, a total of 221 DNA pol sequences were retrieved from eight Chesapeake Bay virioplankton communities collected at different times and locations. All DNA pol sequences clustered with the eight known podoviruses that infect different marine picocyanobacteria, and could be divided into at least 10 different subclusters (I-X). The presence of these cyanopodovirus genotypes based on PCR-amplification of DNA pol gene sequences was supported by the existence of similar DNA pol genotypes with metagenome libraries of Chesapeake Bay virioplankton assemblages. The composition of cyanopodoviruses in the Bay also exhibited distinct winter and summer patterns which were likely related to corresponding seasonal changes in the composition of cyanobacterial populations. Our study suggests that diverse and dynamic populations of cyanopodoviruses are present in the estuarine environment. The PCR method developed in this study provides a specific and sensitive tool to explore the abundance, distribution and phylogenetic diversity of cyanopodoviruses in aquatic environments. Linking the dynamics of host and viral populations in the natural environment is critical to broader characterization of the ecological role of virioplankton within microbial communities.     

Metagenomic Characterization of Chesapeake Bay Virioplankton

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.      

Novel N4 Bacteriophages Prevail in the Cold Biosphere

Coliphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a “genetic orphan” until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions.     

Randomly Amplified Polymorphic DNA Reveals Tight Links between Viruses and Microbes in the Bathypelagic Zone of the Northwestern Mediterranean Sea

The study site located in the Mediterranean Sea was visited eight times in 2005 and 2006 to collect samples from the epipelagic (5 m), mesopelagic (200 m, 600 m), and bathypelagic (1,000 m, 2,000 m) zones. Randomly amplified polymorphic DNA PCR (RAPD-PCR) analysis was used to obtain fingerprints from microbial and viral size fractions using two different primers each. Depending on the primer used, the number of bands in the water column varied between 12 to 24 and 6 to 19 for the microbial size fraction and between 16 to 26 and 8 to 22 for the viral size fraction. The majority of sequences from the microbial fraction was related to Alphaproteobacteria, Cyanobacteria, Gammaproteobacteria, Firmicutes, and Eukaryota. Only 9% of sequences obtained from the viral fraction were of identifiable viral origin; however, 76% of sequences had no close relatives in the nr database of GenBank. Only 20.1% of complete phage genomes tested in silico resulted in potential RAPD-PCR products, and only 12% of these were targeted by both primers. Also, in silico analysis indicated that RAPD-PCR profiles obtained by the two different primers are largely representative of two different subsets of the viral community. Also, correlation analyses and Mantel tests indicate that the links between changes in the microbial and viral community were strongest in the bathypelagic. Thus, these results suggest a strong codevelopment of virus and host communities in deep waters. The data also indicate that virus communities in the bathypelagic zone can exhibit substantial temporal dynamics.Viruses comprise the most abundant biological entities in the ocean (29) and play an integral part in global geochemical cycles (12, 35). Also, viruses have been proposed to influence the community composition of their hosts by selectively infecting the winners in the competition for nutrients (32, 33, 38). Indeed, the viral influence on prokaryotic community composition has been demonstrated in several studies (see, e.g., references 5, 26, and 41).The community composition of prokaryotes or eukaryotic single-celled organisms can be investigated by PCR-based fingerprinting techniques based on small subunit rRNA genes or their flanking regions. However, viruses lack such conserved genes, and consequently obtaining genetic data on the virus community in a water sample is challenging. Several authors have used primers specific for certain virus groups, e.g., Phycodnaviridae (8, 27) or picornaviruslike viruses (11). Undoubtedly, metagenomics currently offers the most comprehensive genetic data set on an entire viral community (see, for example, references 2, 4, and 6) and has become accessible to most research groups due to ever decreasing costs. Nevertheless, the challenge of adequately analyzing the large data sets yielded by metagenomics remains.Recently, Winget and Wommack (37) demonstrated the use of randomly amplified polymorphic DNA PCR (RAPD-PCR) to assess viral community composition. These authors showed that the banding patterns obtained by RAPD-PCR from viral communities are highly reproducible, making RAPD-PCR a valuable high-throughput and low-cost technique to assess viral community composition on a routine basis. Other studies have used RAPD-PCR for strain typing of closely related viruses (9) and to study the benthic viral community of the Chesapeake Bay (13), the pelagic viral community during an iron-induced phytoplankton bloom in the Southern Ocean (34), and virus-host interactions at hydrothermal vents (36). Since RAPD-PCR is a relatively novel fingerprinting approach to study complex viral communities, one may still ask the question of how to best interpret such results in an ecological context. To address this, we performed RAPD-PCR analysis on viral and microbial communities (prokaryotes and single-celled planktonic eukaryotes) at a station in the northwestern Mediterranean Sea to determine their temporal and depth variation. The results were coupled with in silico RAPD-PCR analysis of available whole viral genomes and sequence analysis of the DNA sequences obtained from selected RAPD-PCR bands. Finally, the RAPD-PCR banding patterns were used to relate changes in the viral and microbial communities with each other and with other microbial and environmental parameters to better understand the mechanism driving temporal and depth variation of viral and microbial communities at the study site.     

The structure of population genetic diversity in <Emphasis Type="Italic">Vallisneria</Emphasis><Emphasis Type="Italic">americana</Emphasis> in the Chesapeake Bay: implications for restoration

Submersed aquatic macrophyte beds provide important ecosystem services, yet their distribution and extent has declined worldwide in aquatic ecosystems. Effective restoration of these habitats will require, among other factors, reintroduction of genetically diverse source material that can withstand short- and long-term environmental fluctuations in environmental conditions. We examined patterns of genetic diversity in Vallisneria americana because it is a cosmopolitan freshwater submersed aquatic macrophyte and is commonly used for restoring freshwater habitats. We sampled 26 naturally occurring populations of V. americana in the Chesapeake Bay estuary and its tributaries and found that the majority of populations have high genotypic diversity and are not highly inbred. Fourteen of the populations had high allelic and genotypic diversity and could serve as source sites for restoration material. However, substantial geographic structuring of genetic diversity suggests that caution should be used in moving propagules to locations distant from their source. In particular, we suggest that propagules at least be limited within four primary geographic areas that correspond to freshwater tidal and non-tidal, oligohaline, and seasonally mesohaline areas of the Chesapeake Bay.     

Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 10(6) and 4.0 × 10(7) viruses m(-3), which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions.     

Evaluation of Two Approaches for Assessing the Genetic Similarity of Virioplankton Populations as Defined by Genome Size

Viral production estimates show that virioplankton communities turn over rapidly in aquatic ecosystems. Thus, it is likely that the genetic identity of viral populations comprising the virioplankton also change over temporal and spatial scales, reflecting shifts in viral-host interactions. However, there are few approaches that can provide data on the genotypic identity of viral populations at low cost and with the sample throughput necessary to assess dynamic changes in the virioplankton. This study examined two of these approaches—T4-like major capsid protein (g23) gene polymorphism and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting—to ask how well each technique could track differences in virioplankton populations over time and geographic location. Seasonal changes in overall virioplankton composition were apparent from pulsed-field gel electrophoresis (PFGE) analysis. T4-like phages containing similar g23 proteins were found within both small- and large-genome populations, including populations from different geographic locations and times. The surprising occurrence of T4-like g23 within small genomic groups (23 to 64 kb) indicated that the genome size range of T4-like phages may be broader than previously believed. In contrast, RAPD-PCR fingerprinting detected high genotypic similarity within PFGE bands from the same location, time, and genome size class without the requirement for DNA sequencing. Unlike g23 polymorphism, RAPD-PCR fingerprints showed a greater temporal than geographic variation. Thus, while polymorphism in a viral signature gene, such as g23, can be a powerful tool for inferring evolutionary relationships, the degree to which this approach can capture fine-scale variability within virioplankton populations is less clear.     

Interannual dynamics of viriobenthos abundance and morphological diversity in Chesapeake Bay sediments

Despite significant implications of viral activity in sediment ecosystems, there are limited data describing how sediment viral assemblages respond to broader ecosystem changes. To document this, the spatial and temporal dynamics of viral and bacterial abundance (BA) and changes in the morphological distribution of viruses were examined within three salinity regions over 2 years. Viral abundances (VA) ranged from 0.2 to 17 × 10(10) viruses mL(-1) sediment while direct bacterial counts ranged from 3.8 to 37 × 10(8) cells mL(-1) sediment. Peaks and valleys in the abundance of extracted viruses and bacteria from surface sediments occurred simultaneously, with lows in February 2004 and highs in April 2003. Across all samples, viral and BA were positively correlated (P < 0.001). Vertical profiles showed a decrease in viral and BA with depth in sediments. Based on transmission electron microscopy results, viruses with diminutive capsids (20-50 nm) and from the Myoviridae and Podoviridae viral family types were dominant within surface sediments. The most morphologically diverse viral assemblages occurred in autumn samples from the sandy, polyhaline station and spring samples from the mesohaline station. Seasonal changes showed an average 72% decrease in VA from spring to winter. These observations support the view that viriobenthos assemblages are responsive to seasonal environmental changes and that viral processes have significant implications for the biogeochemical processes mediated by bacterial communities within Bay sediments.     

Spatial-temporal variability in diazotroph assemblages in Chesapeake Bay using an oligonucleotide nifH microarray

The distribution of nitrogen-fixing microorganisms in the Chesapeake Bay was investigated using fingerprints from a nifH microarray comprised of 706 60-mer oligonucleotide nifH probes representing cultivated organisms and environmental clones from different nifH clusters. Diverse nifH targets, amplified from samples using degenerate nifH primers, were detected in water column and sediment samples collected in April and October, 2001-2002. Total nifH richness and diversity (Simpson's and Shannon indices) were highest at the most riverine, oligohaline North Bay station. In most samples, the highest diversity was in nifH Cluster 3, which includes many anaerobes, while Cluster 1 (alpha-, beta- gamma- Proteobacteria, Cyanobacteria) targets had the greatest microarray signal intensities. In a multidimensional scaling analysis, deep water communities from April and October were similar within each of the sampling sites, while the surface communities had more variability. Diazotroph communities in the water column in the North Bay were distinct from the Mid- and South Bay communities, and there was a gradual change in sediment diazotroph assemblages from the North to the South Bay. Diazotrophic assemblages from the majority of the water column samples from the Mid- and South Bay clustered with the sediment assemblage in Mid-Bay. Dissolved inorganic nitrogen, salinity, dissolved organic carbon and dissolved organic phosphorus had a significant relationship with the diazotrophic bacterioplankton community. Higher diversity in the freshwater end of the system may reflect variability in disturbance rates and environmental conditions such as forms and concentrations of organic matter, nutrients and oxygen.     

Diel and daily fluctuations in virioplankton production in coastal ecosystems

Viruses saturate the world around us, yet a basic understanding of how viral impacts on microbial host organisms vary over days to hours, which typify the replication cycles of aquatic viruses, remains elusive. Thus, diel patterns of viral production (VP) in Chesapeake Bay surface waters were examined on five sampling dates. Day-to-day variations in VP in the Chesapeake and coastal California surface waters were also investigated. Significant variations in VP were detected over 24 h cycles during four of five studies, but rates did not vary significantly over the course of a few days in either location. Diel patterns of VP displayed seasonality with shorter viral assemblage turnover times and shorter times to maximum viral abundance in summer, implying shorter replication cycles for virus–host systems in warmer months. No correlation was found between VP and time of day, likely due to seasonal changes in the diel patterns of VP. This analysis significantly increases our knowledge of the short-term patterning of in situ VP, and thus viral impacts, and suggests that variations in viral biology in response to changes in host communities or physio-chemical properties affect both diel and seasonal cycles and magnitudes of VP.     

Attached bacterial populations shared by four species of aquatic angiosperms

Symbiotic relationships between microbes and plants are common and well studied in terrestrial ecosystems, but little is known about such relationships in aquatic environments. We compared the phylogenetic diversities of leaf- and root-attached bacteria from four species of aquatic angiosperms using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of PCR-amplified 16S rRNA genes. Plants were collected from three beds in Chesapeake Bay at sites characterized as freshwater (Vallisneria americana), brackish (Potomogeton perfoliatus and Stuckenia pectinata), and marine (Zostera marina). DGGE analyses showed that bacterial communities were very similar for replicate samples of leaves from canopy-forming plants S. pectinata and P. perfoliatus and less similar for replicate samples of leaves from meadow-forming plants Z. marina and V. americana and of roots of all species. In contrast, bacterial communities differed greatly among plant species and between leaves and roots. DNA sequencing identified 154 bacterial phylotypes, most of which were restricted to single plant species. However, 12 phylotypes were found on more than one plant species, and several of these phylotypes were abundant in clone libraries and represented the darkest bands in DGGE banding patterns. Root-attached phylotypes included relatives of sulfur-oxidizing Gammaproteobacteria and sulfate-reducing Deltaproteobacteria. Leaf-attached phylotypes included relatives of polymer-degrading Bacteroidetes and phototrophic Alphaproteobacteria. Also, leaves and roots of three plant species hosted relatives of methylotrophic Betaproteobacteria belonging to the family Methylophilaceae. These results suggest that aquatic angiosperms host specialized communities of bacteria on their surfaces, including several broadly distributed and potentially mutualistic bacterial populations.     

Development of 11 polymorphic microsatellite markers in a macrophyte of conservation concern, Vallisneria americana Michaux (Hydrocharitaceae)

Vallisneria americana Michaux (wild celery) is currently a target of submersed aquatic vegetation restoration efforts in the Chesapeake Bay watershed. To aid these efforts, we have developed 11 polymorphic microsatellite markers to assess the distribution and degree of genetic diversity in both restored and naturally occurring populations in the Chesapeake Bay. In 59 individuals from two populations, we detected two to 10 total alleles per locus. Observed heterozygosity ranged from 0.125 to 0.929, and two loci exhibited significant deviations from Hardy-Weinberg equilibrium in at least one of the populations assayed.     

Seasonal variations in PCR-DGGE fingerprinted viruses infecting phytoplankton in large and deep peri-alpine lakes

Double-stranded DNA viruses infecting eukaryotic algae (e.g., phycodnaviruses) and cyanobacteria (e.g., cyanophages) are now recognized as widespread and ubiquitous in aquatic environments. However, both the diversity and functional roles of these viruses in fresh waters are still poorly understood. We conducted a year-long study in 2011 of the community structure of planktonic virus groups in the upper lit layer of two important freshwater natural ecosystems in France, Lake Annecy (oligotrophic) and Lake Bourget (oligo-mesotrophic). Using PCR-DGGE to target a number of different structural and functional signature genes, i.e.,g20, g23, psbA, polB, and mcp, the phytoplankton viruses were shown to display temporal and spatial variability. There were marked seasonal changes in community structure for all viral groups in Lake Bourget, but only for T4-like myoviruses and psbA-containing cyanophages in Lake Annecy. The multivariate statistical analyses revealed that (1) various environmental factors can directly or indirectly explain the community structure observed for each phytoplankton viral group, and (2) temporal patterns of T4-like myovirus community structure were similar between the two lakes. In general, our results (1) suggest that the observed algal virus patterns were associated with significant shifts in phytoplankton biomass and/or structure, which in turn were shaped by the abiotic environment, and (2) support the Bank model proposed by Breitbart and Rohwer (Trends Microbiol 13:278–284, 2005). This study provides new evidence that freshwater lakes contain a significant diversity of algal viruses, and that the distribution of these viruses strongly mirrors that of their hosts.     

Predator Assemblage Structure and Temporal Variability of Species Richness and Abundance in Forests of High Tree Diversity

Predators significantly affect ecosystem functions, but our understanding of to what extent findings can be transferred from experiments and low‐diversity systems to highly diverse, natural ecosystems is limited. With a particular threat of biodiversity loss at higher trophic levels, however, knowledge of spatial and temporal patterns in predator assemblages and their interrelations with lower trophic levels is essential for assessing effects of trophic interactions and advancing biodiversity conservation in these ecosystems. We analyzed spatial and temporal variability of spider assemblages in tree species‐rich subtropical forests in China, across 27 study plots varying in woody plant diversity and stand age. Despite effects of woody plant richness on spider assemblage structure, neither habitat specificity nor temporal variability of spider richness and abundance were influenced. Rather, variability increased with forest age, probably related to successional changes in spider assemblages. Our results indicate that woody plant richness and theory predicting increasing predator diversity with increasing plant diversity do not necessarily play a major role for spatial and temporal dynamics of predator assemblages in such plant species‐rich forests. Diversity effects on biotic or abiotic habitat conditions might be less pronounced across our gradient from medium to high plant diversity than in previously studied less diverse systems, and bottom‐up effects might level out at high plant diversity. Instead, our study highlights the importance of overall (diversity‐independent) environmental heterogeneity in shaping spider assemblages and, as indicated by a high species turnover between plots, as a crucial factor for biodiversity conservation at a regional scale in these subtropical forests.     

Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91-103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.