Diversity of Synechococcus at the Martha’s Vineyard Coastal Observatory: Insights from Culture Isolations, Clone Libraries, and Flow Cytometry
The cyanobacterium Synechococcus is a ubiquitous, important phytoplankter across the world’s oceans. A high degree of genetic diversity exists within the marine group, which likely contributes to its global success. Over 20 clades with different distribution patterns have been identified. However, we do not fully understand the environmental factors that control clade distributions. These factors are likely to change seasonally, especially in dynamic coastal systems. To investigate how coastal Synechococcus assemblages change temporally, we assessed the diversity of Synechococcus at the Martha’s Vineyard Coastal Observatory (MVCO) over three annual cycles with culture-dependent and independent approaches. We further investigated the abundance of both phycoerythrin (PE)-containing and phycocyanin (PC)-only Synechococcus with a flow cytometric setup that distinguishes PC-only Synechococcus from picoeukaryotes. We found that the Synechococcus assemblage at MVCO is diverse (13 different clades identified), but dominated by clade I representatives. Many clades were only isolated during late summer and fall, suggesting more favorable conditions for isolation at this time. PC-only strains from four different clades were isolated, but these cells were only detected by flow cytometry in a few samples over the time series, suggesting they are rare at this site. Within clade I, we identified four distinct subclades. The relative abundances of each subclade varied over the seasonal cycle, and the high Synechococcus cell concentration at MVCO may be maintained by the diversity found within this clade. This study highlights the need to understand how temporal aspects of the environment affect Synechococcus community structure and cell abundance.
KeywordsCyanobacteria Microdiversity ntcA
The marine Synechococcus group of cyanobacteria is a globally important primary producer in the world’s oceans. This picophytoplankter (∼1 µm diameter) is responsible for up to 20 % of carbon fixation in coastal systems [19, 25]. Thus, it is important that we understand the factors that affect Synechococcus abundance and enable it to be ecologically important across a wide range of environmental conditions. One of these key factors appears to be the high level of diversity contained within the marine Synechococcus lineage. Studies of molecular phylogeny have resolved isolated strains and environmental sequences into a total of 20 well-defined clades distributed over three main subclusters (5.1, 5.2, and 5.3) [9, 42]. These clades have been supported by phylogenies constructed from a variety of loci, including rpoC1 [35, 51], ITS [1, 17], narB [30, 32, 33], ntcA [38, 40], and petB . Clade designation was recently shown to be congruent across these genetic markers  and multi-locus sequence analysis of core genes provides evidence that the clades are in fact distinct lineages .
This genetic diversity may be representative of physiological or ecological diversity, such that each clade (or closely related clades) corresponds to an ecotype that occupies a distinct niche . This relationship between genetic diversity and ecological physiology has been well documented in the sister genus, Prochlorococcus, where genetically distinct clades show differences in light acclimation and nutrient utilization [27, 28, 29, 41]. Differences in clade physiology explain vertical distributions of Prochlorococcus in the water column, as well as clade biogeography across ocean basins [3, 20, 55, 59].
Similar relationships between genetic designation and physiological characteristics have been shown for some clades of Synechococcus. For example, strains belonging to clade III exhibit a motility that is unique to this clade [52, 54]. Clades also exhibit differences in nitrogen (N) utilization; some clades are unable to grow on or demonstrate reduced growth rate with nitrate (clades CRD2 and XV, respectively; [1, 28]), while others are able to utilize different N sources, such as urea and amino acids . The response of growth rate to temperature can differ among clades as well as the response to temperature stress . Marine Synechococcus also exhibit differences in light harvesting pigments [1, 7, 15, 47, 53]. Many members of subcluster 5.1 contain phycoerythrin (PE) in their pigment complements, and some of these clades can chromatically adapt to different light environments (clades I, III, XV, and XVI; [1, 36]). Many members of subcluster 5.2 contain only phycocyanin (PC) as their light harvesting phycobilipigment (type-I pigment, ). These pigment distinctions are not absolute, however, as some strains from subclusters 5.2 and 5.3 contain PE, and some PC-only strains have been found in subcluster 5.1 (clade VIII). Six et al.  suggest that phycobilisome rod genes have evolved independently from the rest of the core genome and are likely to have undergone horizontal gene transfer.
These physiological differences so far have not been sufficient to explain observed clade distributions in the ocean. Clades often co-occur [6, 11, 40], with reports of as many as six clades found at once . Nonetheless, biogeographical and time series studies have begun to identify environmental factors that may shape some clade distributions. For instance, clades I and IV are typically found in colder, nutrient-rich, coastal waters at latitudes greater than 30° N and 30° S , and members of clades CB4 and CB5 are also found in coastal waters and in estuaries [5, 6, 17]. Other clades seem to prefer warmer and more oligotrophic environments; clades II and III are typically found in tropical waters, with clade II having a much wider distribution into subtropical areas .
A complex set of interacting factors likely determine clade distributions. Abundance will be governed by bottom-up conditions, such as light, nutrient availability, and temperature; top-down factors, such as grazing and viral lysis [4, 30, 60]; as well as sideways interactions, such as with heterotrophic bacteria . These factors change over different time scales, such as across seasons and over water columns with variable mixing. The time scales of environmental changes may contribute to the ability of clades to simultaneously coexist. Consistent with this idea, time series studies of clade abundances have demonstrated shifting dominance. In California coastal waters, Tai and Palenik  found that clades I and IV were always dominant, but with changing relative abundance over the seasonal cycle, while clades II and III only appeared during autumn and even then at relatively low abundance. In the Gulf of Aqaba, Post et al.  observed a succession of clades across the transition from winter mixing to summer stratification, which led to insights of possible preferred nutrient environments for clades I, III, and V/XII. These studies highlight the need to understand how temporal aspects of the environment shape and maintain Synechococcus diversity. At present, there is little knowledge of how seasonal environmental changes affect clade abundances in North Atlantic coastal waters, including on the New England Shelf. To better understand how clade patterns may change over time, we used ntcA clone libraries and culture isolations to assess the diversity of Synechococcus at the Martha’s Vineyard Coastal Observatory (MVCO) over three annual cycles. We further investigated the abundance of both PE-containing and PC-only Synechococcus in these coastal waters by analyzing time series samples with a flow cytometric setup that separates PC-only Synechococcus from picoeukaryotes.
Materials and Methods
Flow Cytometry Analysis
A modified Epics V flow cytometer (FCM; Coulter Electronics Corp.) interfaced with a Cicero acquisition system (Cytomation, Inc.) was used to analyze preserved water samples. The instrument was equipped with a 5-W argon ion laser (Coherent Innova 90-5) and photomultipliers for three wavelengths of fluorescence detection and forward light scattering. Excitation was at 515 nm (300 mW) and a 540 long pass barrier filter was used to eliminate scattered laser light from the fluorescence detectors. Fluorescent emissions were split by successive dichroic mirrors and interference filters to measure wavelength bands of 562–588 nm (PE fluorescence), 610–660 nm (PC fluorescence), and 660–700 (chlorophyll fluorescence) (see Fig. S1 for schematic and filters used). Forward light scattering was measured at ∼3° − 19° above the axis of the laser beam. Samples were allowed to thaw in water before analysis and were injected into the sheath flow (MilliQ water, Millipore) by a peristaltic pump (Harvard Apparatus) at 0.1 mL min −1. Polystyrene microspheres (Polysciences, Inc.) of diameter 0.5 µm (polychromatic) and 1.0 µm (red-fluorescing) were measured as reference particles.
PE-containing Synechococcus were determined from characteristic PE fluorescence values and forward light scattering . PC-only Synechococcus were determined from values of PC fluorescence, PC-to-chlorophyll fluorescence ratio, and forward light scattering. These features allowed separation and enumeration of PC-only Synechococcus from picoeukaryotes. Values of these parameters fell within a well-confined range for cultures (Fig. S2), and these values were used to guide analysis of field samples. Specifically, an event was designated as a PC-only Synechococcus if it did not show any PE fluorescence, had minimum values of 1.5·104 arbitrary fluorescence units for both PC and chlorophyll fluorescence, and had a PC:chlorophyll fluorescence ratio of >0.5 but <1.0. Cell concentration was determined from sample flow rate (pump rate) and analysis time.
To characterize pigment types of selected cultures, in vivo fluorescence excitation and emission spectra were obtained for cell suspensions with a SpectraMax M3 (Molecular Devices) spectrofluorometer. For the emission spectra, excitation was at 515 nm and emission measured from 530 to 700 nm at 2 nm increments, with a 530 long pass cutoff filter. Emission at 680 nm (Chl maximum) was measured from 400–660 nm at 2 nm increments with a 665 long pass cutoff filter. Cultures were determined to contain only PC (designated as type 1 ) if they did not show a representative PE peak (emission maximum between 530 and 580 nm), but rather a peak between 600 and 650 nm .
To detect the presence of phycoerythrobilin (PEB) and phycobilins, PE emission at 585 nm was measured over the range 400-570 nm with a 570 nm long pass cutoff filter. Beginning at 550 nm, there was noticeable contribution due to stray light from the spectrofluorometer excitation source. This was between 2 and 16 % of the measured relative fluorescence value of isolate excitation spectra. To correct for this problem, excitation spectra of fresh media blanks (either SN or SNAV) were subtracted from these sample excitation spectra. Presence of PUB was determined if PE excitation spectra contained a peak or noticeable shoulder at ∼495 nm and if maximum Chl excitation fluorescence was between 540 and 550 nm. A PUB-lacking phenotype (PEB-only, designated as type 2) was characterized by no shoulder or peak at ∼495 nm and by shifted Chl excitation maximum towards 560–570 nm. PUB:PEB ratio was calculated as fluorescence excitation at 495 nm to that at 550 nm with corrected spectra. Correction to spectra did not influence pigment classification and ratio calculations varied less than 8 % before and after correction. Strains were designated as pigment type 3a if PUB:PEB ratio was low (∼0.4), and type 3b if PUB:PEB ratio was moderate (∼0.6–0.8) [10, 47].
Only a subset of the entire culture collection was characterized (∼ one strain representative per unique sequence). Strains that were not analyzed were assumed to have the same major phycobiliproteins as strains with the same culture color and clade type (i.e., if a strain color was peach and belonged to clade I, it was classified as containing PE, see Table S1).
Environmental Sample DNA Extraction and ntcA PCR Amplification
On selected days (dates marked on Figs. 2 and 7), 2–3 L of surface seawater was prefiltered through a 20- µm Nitex® mesh and then filtered onto 0.2- µm Sterivex® cartridge filters (Millipore) under vacuum pressure of no more than 40 kPa. Approximately 1.8 mL of DNA cell lysis buffer (Qiagen) was added to each cartridge before freezing and storage at -80 °C. For DNA extraction, samples were thawed on ice and, to break open cells, approximately 200 µL of 0.5-mm zirconia-silica beads (BioSpec Products) were added to the cartridges, which were then shaken vigorously at 2500 rpm for 10 min. Continued DNA extraction was carried out with Qiagen Purgene reagents but with the modified procedure described in Palacios et al. . DNA concentration was determined with a NanoDrop 2000 Spectrophotometer (ThermoScientific). Depending on the sample, 120 to 1200 ng of DNA template was added to PCR reactions with the degenerate 1F/4R primer pair . Final primer concentration was 2 µM in a total reaction volume of 50 µL with Qiagen Taq PCR Master Mix Kit reagents. BSA was added at 0.2 mg mL−1 final concentration. PCR reactions were performed on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems), with an initial denaturation period of 4 min at 94 °C; followed by 40 cycles of 1 min at 94 °C, 1 min at 45 °C, 1 min at 72 °C; and then a final extension step at 72 °C for 7 min.
Culture Isolate DNA Extraction and ntcA PCR Amplification
Approximately 2 mL of dense culture isolate was centrifuged at 9300 ×g for 6 min to pellet cells, and DNA was extracted from this pellet with a Qiagen DNeasy Plant Kit, following manufacturer’s instructions with the exception of final elution volume (75 µL). Approximately 10–30 ng of DNA was added to PCR reactions with 1AF and 4AR primers (targeted Synechococcus primers, ). Final primer concentration was 2.5 µM in a total reaction volume of 50 µL with Qiagen Taq PCR Master Mix Kit reagents. BSA was added at 0.2 mg mL−1 final concentration. Reactions were preformed on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems), with an initial denaturation period of 4 min at 94 °C; followed by 30 cycles of 1 min at 94 °C, 30 s at 55 °C, 30 s at 72 °C; and then a final extension step at 72 °C for 7 min.
ntcA Clone Libraries
All PCR products (an expected 449-bp fragment), from both the environmental and culture isolates, were gel purified with a Qiagen Qiaquick gel extraction kit. Cleaned products were cloned into TOPO vectors for sequencing (TOPO TA Kit, Invitrogen) and transformed into chemically competent E. coli TOP10 cells (Invitrogen) following manufacturer’s instructions. For isolates, at least five positive colonies (determined by blue/white selection on X-Gal, kanamycin LB plates) were picked. Plasmids were obtained via automated plasmid purification with a BiomekFX at the Josephine Bay Paul Center Keck Facility (Marine Biological Laboratory, Woods Hole, MA). Sequencing reactions used BigDye Terminator chemistry (Applied Biosystems) and contained at least 200 ng of purified plasmid and M13 reverse primer (15 µM, from TOPO kit). Sequencing was performed at the Josephine Bay Paul Center Keck Facility with a 3730 DNA Analyzer (Applied Biosystems). Primer and vector sequences were removed and resulting sequences were identified by BLAST search against ntcA accessions in NCBI Genbank. Sequences were manually evaluated and corrected if necessary from chromatograms. SynechococcusntcA sequences were deposited in NCBI GenBank with accession numbers KR360758–KR361175, KR364942 for environmental sequences, and KR361176–KR361318 for isolate sequences.
Sequences identified as ntcA were aligned with the ClustalW algorithm in BioEdit (version 7.2.0, ). Operational taxonomic unit (OTU) construction and rarefaction analysis was carried out in mothur v.1.23.1  with the furthest neighbor clustering algorithm. Distinct OTUs were designated at a 10 % dissimilarity cutoff. Fourteen sequences were found that could cluster with either OTU IC or OTU IB (see below) based on distance, and these sequences were randomly assigned to either OTU (see Table S2). Phylogenetic reconstructions were carried out in the ARB software package (version 5.3, Ludwig et al. 2004) with a maximum likelihood approach using RAxML  and a GTR GAMMA rate substitution model. Bootstrap analysis for support of tree branches was also carried out in ARB with rapid bootstrap analysis and 500 sample trees.
Temperature at MVCO exhibited large seasonal fluctuations from a minimum of around 0 °C up to a maximum of 22 °C during the 3-year period of this study (Fig. 7a). Salinity was typically within the range of 31–32.5. The concentration of nitrate + nitrite was usually below 1 µM, with the majority of the samples below 0.5 µM and often at the limit of detection for the autoanalyzer technique (0.05 µM) (Fig. 7b). Higher nitrate + nitrite concentrations (0.75–1 µM) occasionally occurred during fall. The concentration of phosphate was also usually low (typically < 0.25 µM).
Flow Cytometry Analysis
Consistent with our previous reports for this site , cell abundance of PE-containing Synechococcus followed a repeatable seasonal pattern of low wintertime concentrations of a few hundred cells mL−1 to greater than 105 cells mL−1 in summertime (Fig. 2). Large changes in abundance (an order of magnitude) were observed during the late summer and fall. By contrast, only a few samples over three seasonal cycles appeared to contain signatures that matched our criteria for identification as a PC-only Synechococcus (see Fig. S2 for an example). The maximum observed concentration (∼3000 cells mL−1) was roughly fourfold less than that of the concurrent PE-containing Synechococcus (∼13,800 cells mL−1). The majority of small (<∼2 µm), non-PE-containing cells were classified as picoeukaryotes (Fig. S2).
Diversity of Environmental Sequences
Diversity of Isolates
From the 17 enrichments, 143 isolates were identified from their ntcA gene sequences. Of these, 50 unique strains were identified (i.e., different ntcA sequences). Isolates with identical ntcA sequences did occur not only within the same enrichment but also across different enrichments, with original sample collection dates sometimes separated by months (see Table S1). Analysis of ntcA phylogeny showed that the strains belonged to 12 different clades that spanned the three subclusters (Fig. 5). Isolates mapped to known clades I, II, III, VI, VII, and VIII of subcluster 5.1; three clades of subcluster 5.2, including CB4 and CB5; and one clade of subcluster 5.3 (5.3I/X). Strains also clustered into two other clades, belonging to subcluster 5.2 that did not match to other known strains. These clades have been labeled as 5.2MV1 and 5.2MV2 until confirmation of their status as either novel or known can be determined (strain representatives may exist for which phylogenetic markers other than ntcA have been sequenced). Strains of clade 5.2MV1 contained PC as their primary pigment, whereas the one strain of clade 5.2MV2, MV1218, contained PE (see Table S1). We have designated isolates that clustered closely with reference strain WH5701 as clade 5.2MV3.
Surprisingly, we also isolated strains considered more common to subtropical and tropical waters (i.e., clades II, III, and VII [40, 59]). Isolates of such clade members only occurred in late summer and early fall. Clade II types were isolated during September (2011 and 2012) and October (2012) and clade III was isolated during August and September (2012). Clade VI isolates were also only obtained in late summer and early fall and clade VII was only found during the fall. In general, isolations of clades II, III, VI, VII, CB5, and 5.3I tended to occur when water temperature was relatively warm (17–20 °C) and nitrate + nitrite concentration was relatively low (<0.5 µM, Fig. 7). The frequency of isolation of these clades was much lower than for clade I representatives (Fig. S4).
We were able to culture PC-only pigment type strains from all but a few of the enrichments from MVCO (Fig. S4). Although not all of these isolates were sequenced (Fig. S4), those that were fell into clades VIII, CB4, 5.2MV1, and 5.2MV3 (Fig. 5). Isolation of these PC-only strains over the entire enrichment time series suggests that they were persistent members of the Synechococcus community with a year round presence at MVCO.
Synechococcus Diversity at MVCO
From culture-dependent and culture-independent approaches, we find that the Synechococcus assemblage observed throughout the year at MVCO is diverse. Members of 13 different clades spanning all 3 known subclusters of marine Synechococcus were identified, but members of clade I dominate the Synechococcus population over the entire year. Below we discuss and highlight possible reasons for the prevalence and occurrence of these clades at MVCO.
Clade I and Subclade Diversity
This dominance of clade I is consistent with the known biogeography of clade I, which is primarily found in cooler, higher nutrient, coastal waters [7, 17, 51, 59], and has been shown to make up a majority of the Synechococcus assemblage in surrounding shelf areas . While a preference for cooler water is likely to be a key factor explaining the presence of clade I at MVCO, the ability to survive colder winter water temperatures (∼0–4 °C) may also contribute to clade I dominance at this site. Recently, Pittera et al.  demonstrated that clade I strains were able to grow at temperatures lower (∼10–15 °C) than tolerated by strains from clades II and V. These authors also demonstrated that clade I strains were more tolerant of cold stress. At MVCO, strains of clade I were isolated from water at a range of temperatures (Fig. 7), but notably from water at ∼4 °C, indicating that cells were still viable during this time. The PE-containing Synechococcus population reaches a minimum cell concentration of a few hundred cells per mL−1 during winter (Fig. 2), and the ability to survive these colder temperatures may be an important factor that allows this population to “overwinter” until more favorable spring conditions.
Within clade I, we also found significant diversity at the subclade level, such that four different subclades could be resolved in the clone library sequences (Figs. 6 and S3). While not strictly quantitative, relative abundances of sequences in our clone libraries suggest that not all of these subclades are equally represented when they co-occur. While subclade IC appears to be dominant throughout the year, subclades IB and IE appeared to increase in relative abundance at different times of year (spring-summer, summer-early winter, respectively). This may indicate possible differences in environmental preference, but the small number of clone libraries in our study (n = 11), combined with the possible biases in this data type, preclude definitive investigation of relationships between environmental factors and subclade relative abundances. Given that clade I appears to be dominant, it will be important to understand how each subclade affects and contributes to this dominance as well as seasonal cell abundance patterns.
Interestingly, both pigment types 2 and 3a (no PUB and low PUB, respectively) were found among clade I isolates for both subclade IC and IE. To our knowledge, this is the first instance of PEB-only clade I strains. Other clade I representatives examined to date have contained PUB [1, 11, 53]. It is unknown whether this spectral phenotype results from a lack of PE-II subunit or associated genes  or can be achieved by chromatic adaption (CA), as clade I strains have been shown to exhibit type IV CA [36, 47]. The prevalence of low or no PUB phenotype among these isolates may reflect a strong pressure for efficient absorption of green light, which often predominates at coastal locations. It has long been appreciated that there are different geographical distributions of PUB-containing PEs, with higher PUB:PEB types dominating in the open ocean and low or no PUB types more prevalent nearshore [16, 31, 45, 57, 58]. This is thought to be an adaptation (or acclimation) to the in situ light environment, where PUB allows better absorption of blue, open water (PUB maximum absorption ∼495 nm), while PEB better absorbs greener, coastal water (maximum absorption ∼550 nm).
Isolation and Detection of Other Clades
We were able to culture representatives of clades that have typically been found in either warmer or more oligotrophic environments (e.g., clades II, III, VII, 5.3I), but only in late summer and early fall. Ahlgren and Rocap  found clades II and 5.3I at an outer shelf location (304 km south of MVCO) and detected clade III in samples from the Gulf Stream. One possibility for these clade occurrences at MVCO is that they are advected onto the inner New England shelf from more offshore locations and can only survive at this location during late summer and fall. The general circulation on the New England Shelf is part of the larger shelf circulation of the Middle Atlantic Bight, which is characterized by a southwestward along-shelf flow of relatively fresh water, with across-shelf offshore currents at the surface and bottom and onshore currents in the middle of the water column . Shelf water is separated from saltier slope water by a shelf-slope front, but exchanges between these water types can occur due to frontal instabilities , eddies , warm-core ring shelf interactions [8, 21], and saline intrusions at the seasonal pycnocline . Locally, there is a counterclockwise recirculation just south of MVCO, which is strongest in the summer months . Slope water intrusions plus this recirculation feature could make it possible for clades growing in warmer, saltier water to be advected and then retained near MVCO. During summer, water temperature is warm for this location (∼20 °C) and nitrate levels are typically low or undetectable (Fig. 7b), such that conditions may allow persistence of clades that would not typically thrive in coastal waters at other times of the year.
Clades VI, VIII, and CB5 were also only isolated during late summer and fall. Noticeably, this is when water temperature was relatively warm. This is most apparent for clade VI, which was cultured from seven different enrichments, but only when water temperature at time of sampling was greater than 16 °C. The global distribution of clade VI is ambiguous (previous studies utilized probes that could not separate V, VI, and VII from one another ). In general, clade VI representatives have been isolated from coastal environments (Woods Hole Harbor, ; East Sea and East China Sea, ), suggesting tolerance or preference of coastal conditions. Clade CB5 has also been isolated from estuarine or coastal locations [6, 7]. Representatives of both VI and CB5 were of pigment type 2, which as stated above, can allow better absorption of green light that is prevalent in coastal waters, and may contribute to their presence at MVCO.
Interestingly, clade IV, which has been reported to co-occur with clade I in other coastal waters , may have lower relative abundance at MVCO. Strain representatives were not able to be isolated and only ∼2 % of the clone library sequences belonged to this clade. While we cannot be sure that biases in clone libraries and culture isolations are not a factor, it may be that this clade is very rare at MVCO. This is consistent with observations of Ahlgren and Rocap  who found clade IV to be much less abundant than clade I at outer shelf locations (80–304 km south of MVCO). This is different from other coastal locations for which the abundance of clade IV usually matches or exceeds that of clade I . The potentially low abundance of clade IV at MVCO raises interesting questions as to the physiological differences between these two clades and why, for this coastal system, clade I is much more abundant. As highlighted by Ahlgren and Rocap , these questions are relevant for the wider shelf region of the northern Middle Atlantic Bight.
Strains of Synechococcus that only have PC as their light-harvesting pigment have been isolated previously from either estuarine or nearshore coastal waters [6, 7, 11, 15, 53], and sequences that match these strain representatives have been found in similar regions [5, 6, 17]. PC-only Synechococcus are well suited to absorb the quality of light found in these more turbid waters [49, 50]. Given the nearshore location of MVCO, it is not surprising then that PC-only Synechococcus strains were isolated. However, consistently low to undetectable cell concentrations from flow cytometry analysis suggest that these strains may not be ecologically relevant at MVCO. These strains may have been transported from more estuarine sites and then grow poorly in the environmental conditions at this location. Although the site is exposed to the open shelf, it is located on the inner shelf only 3 km from the south shore of the island of Martha’s Vineyard. Another hypothesis is that these organisms are found in the benthos at MVCO (water column 15 m deep), such that storms or other mixing events could transport them to the surface. The sudden appearance and then rapid decline of PC-only Synechococcus in samples from October 2010 (Fig. 2, separated only by a week) is consistent with a hypothesis of a population advected to the site (either from shore or the bottom) but not able to thrive.
The PC-only Synechococcus strains appeared to flourish, however, in the culture conditions, and in fact dominated many enrichments (Fig. S4). The media recipe used to culture and maintain the strains contained only 75 % seawater. Many members of subcluster 5.2 are halotolerant, such that they do not require elevated salt requirements for growth . If these strains grow better in lower salinity, then the enrichment conditions may have selected for these representatives. Anecdotal observations from our enrichments also suggest that these strains may persist at background levels for months in a low-nutrient enrichment that is dominated by another pigment type (as judged by color of the culture). Once supplied with higher nutrient concentrations (i.e., when we switched to SN media), these PC-only strains were able to quickly out compete PE-containing strains that appeared to dominate originally. This highlights important questions about the factors that either allow clades to coexist or certain groups to dominate. In particular, little is known about the ability of certain strains to survive unfavorable conditions and how variation in this ability can impact clade distributions in nature.
Comparison of Diversity from Clone Libraries and Culture Isolations
It is noteworthy that the occurrence of certain clades differed among enrichments that were separated only by a few weeks in time. For example, the samples for enrichments 15–17 came from similar nutrient and temperature conditions, but each of these yielded a very different array of clade representatives (Fig. S4). While there are many biases in culturing, these differences between enrichments may hint at fast changing dynamics either in the field or in the enrichment culture (probably during the first few weeks). This illustrates some of the challenges faced when attempting to isolate and culture novel strains of Synechococcus or other organisms. We do not yet understand all the factors that determine how an organism will grow in isolation under laboratory conditions, and caution is needed in extending findings to natural dynamics.
Many questions remain not only concerning how environmental (temperature, light, nutrients) and ecological (grazers, viruses, heterotrophic bacteria) factors govern clade distributions but also how changes in these factors over a seasonal cycle affects the abundance of different clades. We emphasize the importance of investigating the temporal aspects of diversity patterns, especially in dynamic coastal systems. On the New England Shelf, Synechococcus cell concentration undergoes a dramatic (3 orders of magnitude) seasonal cycle, and it is likely that some of the abundance patterns are determined by which clades are favored under different conditions. With isolates in culture from this location, we are poised to begin exploration into some of the differences between clade and subclade types. Ultimately, high frequency monitoring of clade diversity, coupled with physiological and ecological knowledge of representative strains, will allow a greater insight into how diversity of this genus is maintained and how that diversity is linked to overall population dynamics.
The authors are indebted to Rob Olson and Alexi Shalapyonok for assistance with the flow cytometry setup. We are also grateful to John Waterbury for training and assistance in culture isolations and to Kasia Hammar, Erin Banning, and Heather Landry for assistance with clone library construction and troubleshooting. We thank Taylor Crockford, Janet Fredericks and the MVCO Operations Team, captain and mate of the R/V Tioga, and Paul Henderson of the WHOI Nutrient Analytical Facility. We would also like to thank Jennie C. Hunter-Cevera and Athena Aicher for culture collection assistance and Michael G. Neubert and Penny Chisholm for helpful discussions. We further thank the anonymous reviewers, whose comments and suggestions improved this manuscript. This work was supported in part by NSF Chemical Oceanography award 1155566 to AFP; Gordon and Betty Moore Foundation grant 934, NASA Ocean Biology and Biogeochemistry Program Grant NNX11AF07G, NSF OCE grant 0530830 to HMS; and a WHOI Ocean Ventures Fund award, a WHOI Coastal Ocean Institute award, private donation, and a National Defense Science and Engineering Graduate Fellowship to KRHC.
- 2.Ahlgren NA, Rocap G (2012) Diversity and distribution of marine Synechococcus: multiple gene phylogenies for consensus classification and development of qPCR assays for sensitive measurement of clades in the ocean. Front Microbiol 3(213)Google Scholar
- 11.Fuller NJ, Marie D, Partensky F, Vaulot D, Post AF, Scanlan DJ (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl Environ Microbiol 69(5):2430–2443PubMedCentralCrossRefPubMedGoogle Scholar
- 13.Gawarkiewicz G, Brink KH, Bahr F, Beardsley RC, Caruso M, Lynch JF, Chiu CS (2004) A large-amplitude meander of the shelfbreak front during summer south of New England: observations from the shelfbreak PRIMER experiment. J Geophys Res Oceans (1978–2012) 109(C3)Google Scholar
- 14.Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
- 21.Joyce TM, Bishop JK, Brown OB (1992) Observations of offshore shelf-water transport induced by a warm-core ring. Deep Sea Res Part A 39:S97—S113Google Scholar
- 22.Kirincich AR, Lentz SJ, Farrar JT, Ganju NK (2013) The spatial structure of tidal and mean circulation over the inner shelf south of Martha’s Vineyard, Massachusetts. J Phys Oceanogr 43(9)Google Scholar
- 23.Lentz S (2003) A climatology of salty intrusions over the continental shelf from Georges Bank to Cape Hatteras. J Geophys Res Oceans (1978–2012) 108(C10)Google Scholar
- 24.Lentz SJ (2008) Observations and a model of the mean circulation over the Middle Atlantic Bight continental shelf. J Phys Oceanogr 38(6)Google Scholar
- 33.Paerl RW, Johnson KS, Welsh RM, Worden AZ, Chavez FP, Zehr JP (2011) Differential distributions of Synechococcus subgroups across the California current system. Front Microbiol Microbiol 2(59)Google Scholar
- 40.Post AF, Penno S, Zandbank K, Paytan A, Huse SM, Welch DM (2011) Long term seasonal dynamics of Synechococcus population structure in the Gulf of Aqaba, Northern Red Sea. Front Microbiol 2(131)Google Scholar
- 41.Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P, Ahlgren NA, Arellano A, Coleman M, Hauser L, Hess WR, Johnson ZI, Land M, Lindell D, Post AF, Regala W, Shah M, Shaw SL, Steglich C, Sullivan MB, Ting CS, Tolonen A, Webb EA, Zinser ER, Chisholm SW (2003) Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424(6952):1042–1047CrossRefPubMedGoogle Scholar
- 44.Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75 (23):7537–7541PubMedCentralCrossRefPubMedGoogle Scholar
- 45.Shalapenok L, Shalapenok A (1997) Heterogeneous pigment composition of phycoerythrin-containing picocyanobacteria Synechococcus spp. in the black sea. Microbiology 66(1):80–84Google Scholar
- 53.Waterbury JB, Watson SW, Valois FW, Franks DG (1986) Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can Bull Fish Aquat Sci 214:71–120Google Scholar
- 59.Zwirglmaier K, Jardillier L, Ostrowski M, Mazard S, Garczarek L, Vaulot D, Not F, Massana R, Ulloa O, Scanlan DJ (2008) Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes. Environ Microbiol 10(1):147–161PubMedGoogle Scholar