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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Phylogenetic diversity of Synechococcus with different pigmentation in subtropical estuarine and coastal waters of Hong Kong was revealed by the phylogeny of cpcBA and cpeBA operons encoding for phycocyanin (PC) and phycoerythrin (PE). Synechococcus containing only PC (PC-rich Synechococcus) dominated at the estuarine station in summer, whereas PE-rich marine Synechococcus containing both PC and PE (PE-rich Synechococcus) dominated in the coastal waters. Our PC sequences are closely related to freshwater strains but differed from Baltic Sea strains, implying that they were from river discharge. Among PE-rich Synechococcus, clones grouping with strains containing only phycoerythrobilin (PEB-only) were abundant in July, while clones grouping with strains possessing a low content of phycourobilin (PUB) in addition to PEB (low PUB/PEB) were more abundant in January at both stations. Clones of high PUB/PEB types were only presented at the coastal station, but were not detected at the estuarine station. The much higher diversity of both PC-rich and PE-rich Synechococcus, as compared with the Baltic Sea, and the occurrence of the high PUB/PEB strains indicate the high dynamic nature of this subtropical estuarine-coastal environment with strong mixing of water masses ranging from Pearl River plume to oceanic South China Sea water. Our results of phylogenetic study agreed well with flow cytometric counts, which revealed the coexistence of PC-rich and PE-rich Synechococcus in the subtropical coastal waters and the dominance of the former type in the estuarine waters during summer high freshwater discharge. These results indicate that picocyanobacteria, particularly PC-rich Synechococcus, which has long been overlooked, are an important part of the primary production, and they could play an important role in the microbial food web in estuarine ecosystems.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

The importance of picocyanobacteria, including Prochlorococcus and Synechococcus, in global oceanic primary production and carbon cycle has been well recognized (Platt et al., 1983; Campbell et al., 1994; Liu et al., 1998; Richardson and Jackson, 2007; Lomas and Moran, 2011). Compared with Prochlorococcus, which is restricted to warm oceanic waters, Synechococcus are ubiquitously present in marine environments, ranging from the equator to the Arctic, and from coastal to oceanic waters (Partensky et al., 1999).

Synechococcus cells span a range of different colours, from blue-green to pink-orange depending on their pigment composition, specifically the phycobiliprotein in their photosynthetic antenna (Wood, 1985; Olson et al., 1990; Six et al., 2007; Stomp et al., 2007). All Synechococcus contain phycocyanin (PC), while some also contain phycoerythrin (PE). Synechococcus containing a high concentration of PE absorb green light effectively and have a red appearance, whereas strains that lack PE (PC-rich) absorb red light effectively and have a blue-green colour. Red Synechococcus are the dominant group in ocean waters (Olson et al., 1990), where green and particularly blue light penetrate deep into the water column. PE-containing marine Synechococcus spp. fall into two pigment groups: those containing both the red-coloured chromophore phycoerythrobilin (PEB) and the orange-coloured phycourobilin (PUB), and those composed of only PEB chromophore (Waterbury et al., 1986; Olson et al., 1988). PEB exhibits an absorption maximum at 550–570 nm, and PUB at about 500 nm, which appears advantageous for PUB-containing Synechococcus in deep oligotrophic waters where most of the light penetrating is blue. Olson and colleagues (1990) reported that high PUB:PEB Synechococcus was the major population in the North Atlantic and Pacific Oceans, whereas low PUB:PEB cells were found only in coastal waters. On the other hand, PE-lacking blue-green cyanobacteria dominate in turbid waters, where red light prevails (Stomp et al., 2007). Red and green picocyanobacteria can be found coexisting in waters of intermediate coloration, including estuarine and coastal seas and many freshwater lakes (Murrell and Lores, 2004; Stomp et al., 2007; Haverkamp et al., 2008).

The genetic information describing the diversity of marine Synechococcus is rather rich, and more than a dozen of distinct clades have been identified based on 16S rRNA, rpoC1 and 16S–23S internal transcribed spacer (ITS) gene sequences (Rocap et al., 2002; Fuller et al., 2003; Ahlgren and Rocap, 2006), and their spatial and temporal variations were also revealed (Zwirglmaier et al., 2007; 2008; Tai and Palenik, 2009). In addition, genes encoding the major light-harvesting accessory pigment proteins, particularly the phycocyanin operon (cpcBA) and phycoerythrin operon (cpeBA), have been targeted for phylogenetic studies of marine and freshwater Synechococcus (Robertson et al., 2001; Haverkamp et al., 2008).

The phylogenies of Synechococcus based on sequence information of several loci, e.g. 16S rRNA, 16S–23S ITS and rpoC1 genes, are in general not consistent with their pigmentation type, indicating that there is no relationship between phylogeny and pigmentation (Fuller et al., 2003; Chen et al., 2006; Six et al., 2007; Haverkamp et al., 2008; Choi and Noh, 2009). A comparative genomic study suggests that the great diversity of phycobilisome pigmentation among marine Synechococcus is possibly the result of lateral transfer of phycobilisome rod genes or gene clusters between lineages during evolution (Six et al., 2007). While Synechococcus clades are hypothesized to represent ecotypes, physiological characterization of isolates from many clades is still rare, making it difficult to predict what distinct niches they may occupy (Ahlgren and Rocap, 2012). The clone libraries of partial gene sequences of the cpeBA and cpcBA, which encode for the pigments PE and PC, respectively, allow us to construct a phylogeny that differs from those based on the 16S rRNA-ITS operon, and to demonstrate the occurrence and dominance of PC-rich Synechococcus in the subtropical estuaries, which has long been overlooked.

Estuaries are usually turbid brackish waters that play an important role in transporting and transforming riverine nutrients and organic matter into the ocean. In the near-field of an estuary, the nutrient concentration is high, but chlorophyll and primary production is low due to the high water turbidity. In the mid- to far-field, phytoplankton bloom reaches its maximum as a result of the improved light environment as suspended particles settle (Liu and Dagg, 2003). With increasing turbidity, the underwater light spectrum is shifted towards the red part of the light spectrum. As a result the relative abundances of PC-rich species are expected to be higher in turbid estuaries, as PC is tuned to the orange-red light conditions in these water bodies, whereas PE pigments are optimally tuned to the blue-green light in clear waters (Stomp et al., 2007). However, the importance of PC-rich Synechococcus in the estuarine and coastal waters has not been studied intensively because common flow cytometry (with a 488 nm laser) cannot provide reliable information for the positive identification of PC-rich Synechococcus from other picocyanobacteria, e.g. Prochlorococcus, and the smallest picoeukaryotes (Collier, 2000). Early studies have observed the spatial gradient of PC- and PE-rich cyanobacteria in estuaries using epifluorescence microscopy (Ray et al., 1989; Dortch, 1998) and dual-laser flow cytometry (Collier, 2000; Murrell and Lores, 2004). However, there is a need to confirm the fluorescence-based identification, and genetic characterization using different genes is a reliable approach.

Pearl River, which is the second largest river in China in term of freshwater discharge, flows to the South China Sea on the west side of Hong Kong. Understanding the spatial and temporal variations in the distribution of various phylogenetic groups of Synechococcus in estuaries of large rivers is essential for the understanding of the biogeochemical cycle of carbon in estuaries, which play a key role in connecting the terrestrial and oceanic carbon cycles. Recently, by using the 16S–23S ITS gene as a phylogenetic tool, we have revealed high Synechococcus diversity, with clades clustered with possible PC-containing marine cluster B Synechococcus, in Hong Kong coastal waters (Jing et al., 2009b). In this study, we use the phycocyanin cpcBA operon and phycoerythrin cpeBA operon to study the diversity and phylogeny of PC- and PE-rich Synechococcus in subtropical estuarine and coastal waters of Hong Kong. Study in the Baltic Sea (BS) (Haverkamp et al., 2008) demonstrated that a phylogeny based on the operons encoding phycocyanin and phycoerythrin in picocyanobacteria differs from earlier phylogenies based on the 16S rRNA or ITS operons. Together with the results of a flow cytometer that is equipped with dual lasers of 488 nm and 635 nm (exciting phycocyanin), we demonstrate that PC-rich Synechococcus are abundant in subtropical estuarine waters.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Phylogeny of Synechococcus

Between 25 and 29 sequences were obtained for each of the four cpcBA and four cpeBA clone libraries (Tables 1 and 2). Based on 99% sequence similarity as cut-off value, the highest and the lowest numbers of operational taxonomic units (OTUs) identified from cpcBA clone libraries were found in January at the coastal site (Stn. PM7) and the estuarine site (Stn. NM3) respectively. The highest Shannon index was also obtained at Stn. PM7 in January, whereas the lowest appeared at Stn. NM3 in July (Table 1). For cpeBA phylogeny, the highest number of OTUs and diversity was also found at Stn. PM7 in January, while the lowest number of OTUs and diversity appeared at Stn. NM3 and Stn. PM7 in July respectively (Table 2). High species diversity at PM7 in winter reflected the strong interaction of different water masses as the intrusion of the China Coast Current intensified under the northeast monsoon, and the low diversity at the estuarine water (Stn. NM3) during summer indicated the dominance of river plume.

Table 1. Numbers of total sequences and OTUs (at 99% similarity) of two phylogenetic groups (PE-containing and PC-only) defined in the cpcBA phylogenetic tree in different clone libraries
 Stn. NM3Stn. PM7
January 2008July 2007January 2008July 2007
SequenceOTUSequenceOTUSequenceOTUSequenceOTU
PC-rich7124116353
PE-rich22102222132110
Total2911261328162613
H′ 2.15 2.03 2.59 2.27
Table 2. Numbers of total sequences and OTUs (at 99% similarity) of three groups of PE-rich Synechococcus defined by cpeBA gene operon in different clone libraries
 Stn. NM3Stn. PM7
January 2008July 2007January 2008July 2007
SequenceOTUSequenceOTUSequenceOTUSequenceOTU
PEB only8517464174
Low PUB/PEB1889416964
High PUB/PEB00007221
Total26132682915259
H′ 2.21 1.77 2.46 1.42

In general, two major sequence groups, each corresponding to a different pigmentation group, could be identified. One group comprised all clone sequences closely related to rpcBA genes encoding the subunits of R-PCII type phycocyanin (Ong and Glazer, 1987), which are found in all PE-containing Synechococcus. This is evident from the whole genome data analysis in Six and colleagues (2007) as the primers we used can target on both rpcBA and cpcBA genes. Another group includes sequences related to genes encoding the C-PC type phycocyanin, and these sequences belong to PC-rich type of picocyanobacteria. Here, clones grouping with PC-rich type sequences are mainly clustered with Group D, which are mostly composed of strains from Japanese lakes (Robertson et al., 2001) and were distinct from the reference BS clusters (Fig. 1).

figure

Figure 1. Phylogenetic relationship of the picocyanobacterial partial cpcBA genes sequences (439 positions) based on the neighbour-joining method using the Kimura two-parameter model of nucleotide substitution. Bootstrap values of > 50 (in the order of NJ, MP and ML) are shown at the nodes. Sequences obtained as part of this study are in bold, and their GenBank accession numbers were given in parentheses, followed by their abundance at Stn. NM3 and Stn. PM7 respectively. The group names are according to Robertson and colleagues (2001). Scale bars indicate Jukes–Cantor distances.

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The cpeBA phylogeny yielded three clusters, representing Synechococcus with different composition of phycobilins. Micro-diversity is high within clusters, with average similarity over 80%. The majority of our sequences were aligned with the cluster containing low PUB/PEB PE type Synechococcus, whereas only a small number of sequences, obtained from the coastal station, were closely related to the high PUB/PEB species. Synechococcus sequences from the estuarine station dominated the PEB-only cluster. Noticeably, none of our sequences cluster with sequences from the BS (Fig. 2).

figure

Figure 2. Phylogenetic relationship of the picocyanobacterial partial cpeBA genes (534 positions) based on the neighbour-joining method using the Kimura two-parameter model of nucleotide substitution. Bootstrap values of > 50 (in the order of NJ, MP and ML) are shown at the nodes. Sequences obtained as part of this study are in bold, and their GenBank accession numbers were given in parentheses, followed by their abundance at Stn. NM3 and Stn. PM7 respectively. Scale bars indicate Jukes–Cantor distances.

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The current classification of cpcBA-based Synechococcus phylogeny is largely based on the work of Robertson and colleagues (Robertson et al., 2001), in which 38 freshwater Synechococcus species were sequenced. Their results showed that the phylogeny of cpc gene was largely consistent with those of 16S rRNA gene, and revealed a relationship between the primary PC DNA sequence and the phycobilin content of cells, with the PE-rich and PC-rich isolates distributed into different groups. In a recent study, Haverkamp and colleagues (Haverkamp et al., 2008) studied both the cpcBA and cpeBA genes of the picocyanobacteria in the BS, and showed widespread coexistence of red and green picocyanobacteria in the BS. Their results suggest that, based on their pigmentation, three different lineages, PC-rich, PE-rich and PUB containing PE-rich Synechococcus, occupy different ecological niches and strains from different lineages can coexist in light environments that overlap with their light absorption spectra. However, no PUB-containing Synechococcus were found in the BS.

The sequences of PC-rich Synechococcus in this study aligned with freshwater strains but differed with BS strains, implying they were from river discharge or strict brackish water habitant. However, both ITS (Jing et al., 2009b) and cpcBA (this study) gene sequences did not reveal any Cyanobium, a genus closely related to Synechococcus and mainly occurs in freshwater and brackish environments (Crosbie et al., 2003; Ernst et al., 2003), in Hong Kong coastal waters. Phylogenetic analysis of cpeBA clone libraries also indicates high abundance of PE sequences, and they differ with the BS assemblages. Moreover, the number of OTUs obtained from this study is about three times more than those reported from the BS (Haverkamp et al., 2008), along with the Shannon diversity index (Table 3). The sharp difference in cyanobacterial community composition between Hong Kong coastal water and the BS, and the much higher community diversity of the former, may reflect the high dynamic nature of our study sites, with strong mixing of various water masses ranging from oligotrophic South China Sea oceanic water to nutrient-rich coastal currents and freshwater plume of the Pearl River. It may also represent the differences between the river-impacted coastal community in subtropical and temperate environments.

Table 3. Comparison of cyanobacterial diversity between the Baltic Sea and Hong Kong estuarine and coastal waters based on phylogeny of phycocyanin cpcBA operon and phycoerythrin cpeBA operon
 Baltic SeaaHong Kongb
OTUs (99%)H′OTUs (99%)H′
  1. a

    Haverkamp et al. (2008).

  2. b

    This study.

cpcBA operon111.52422.47
cpeBA operon111.85312.66

Spatial and seasonal variation of Synechococcus phylotypes

Our cpcBA gene clone library data suggested that the clone sequences found at Stn. NM3 in July were composed mainly of PC-rich picocyanobacteria, while PE-rich sequences dominated in other three libraries (Table 1). Dominance of PC-rich Synechococcus in the estuary site in summer is coincident with the high riverine discharge. Furthermore, cpeBA phylogeny revealed that there were more OTUs of Synechococcus with low PUB/PEB ratio than those containing only PEB in January, while they were equally numbered in July (Table 2). High PUB/PEB OTUs only presented at Stn. PM7 in both seasons, but were not detected at Stn. NM3. Occurrence of high PUB/PEB strains at PM7 indicated the influence of oceanic water to this coastal site.

Flow cytometric analysis not only agreed well with the distribution patterns of the phylogenetic study, but also revealed that the abundance of both PE- and PC-rich Synechococcus was much higher in summer than in winter (Fig. 3). PE-rich Synechococcus occurred at the coastal site throughout the year, with maximum abundance of 3.8 × 105 and 5.7 × 105 cells ml−1 in the month of July 2007 and 2009, respectively, which were about three orders of magnitude higher than the winter abundance. Similar pattern was also observed for PE-rich Synechococcus at the estuarine site, but the maximum summer abundance there was more than one order of magnitude lower than that at the coastal site, and it was not detected during several winter months. On the other hand, PC-rich Synechococcus was only found at the estuarine site, with the exception of June 2009 when it was also detected at the coastal site. The maximum abundance of PC-rich Synechococcus in the estuarine water reached > 105 cells ml−1 in July of both years, which was much higher than the abundance of PE-rich Synechococcus (Fig. 3).

figure

Figure 3. Abundances of PC- and PE-rich Synechococcus at the estuarine station NM3 (upper panel) and coastal station PM7 (lower panel) during monthly surveys from February 2007 to February 2008, and from February 2009 to March 2010, measured by dual laser flow cytometry. Arrows indicate the months the clone libraries were constructed.

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Because of the comparable sizes and similar fluorescence signals under common flow cytometry (with 488 nm laser), it is impossible to separate PC-Synechococcus from Prochlorococcus (Collier, 2000). Without reliable genetic information, we believe that the reported so called Prochlorococcus-like cells in fresh and brackish waters (Corzo et al., 1999; Shang et al., 2007) are actually PE-lacking but PC-rich Synechococcus or Cyanobium (Stomp et al., 2004; Gin and Neo, 2005; Haverkamp et al., 2008). We have observed such populations in flow cytometric cytograms in many estuarine and coastal regions, including Mississippi River plumes, but most of these findings were not published due to uncertainty solely based on single-laser flow cytometric analysis (H. Liu, unpubl. data; Buskey et al., 2001).

Using a flow cytometer equipped with both 488 and 635 nm lasers allowed us to separate Synechococcus that contain only PC from those that contain both PC and PE, and from Prochlorococcus and eukaryotic picoplankton that lack both types of phycobilins (Collier, 2000; Murrell and Lores, 2004). Results from this study demonstrate that PC-rich Synechococcus dominate in turbid estuarine water in the Pearl River estuary during summer high freshwater discharge, whereas PE-rich Synechococcus dominate in the subtropical coastal waters throughout the year (Tables 1 and 2, Fig. 3; Chen et al., 2009). Our findings are in agreement with previous report on the shift of dominance by PC-rich and PE-rich piocyanobacteria along a gradient of salinity/turbidity in the subtropical estuary in the Gulf of Mexico (Murrell and Lores, 2004). Based on our results and the cytogram plots of Shang and colleagues (2007), there is no doubt that the so-called Prochlorococcus-like cells in the Changjiang Estuary reported by Shang and colleagues (2007) are PC-rich Synechococcus. Therefore, PC-rich picocyanobacteria are likely an important primary producer and a key player of microbial food web in estuaries.

The dominance of PC-rich Synechococcus in turbid estuarine water, especially during high discharge wet season (Fig. 3), is the result of niche differentiation as PC-rich cyanobacteria use the pigment phycocyanin to absorb red light, which is more prevailing in turbid waters (Stomp et al., 2004; 2007). High mineral suspended solids in river plume results a minimal diffuse attenuation coefficient in the 600 nm waveband and a maximum in the blue wavebands (Gallegos et al., 1990), providing an underwater environment suitable for PC-rich cyanobacteria. Given their high abundance, Synechococcus of both colours should play a significant role in the microbial food web and carbon and nitrogen cycles in estuary ecosystems from tropical–subtropical to temperate and arctic regions (Liu et al., 2004; Chen et al., 2006; Waleron et al., 2007; Jing et al., 2009a).

Finally, phylogenetic analysis in this study revealed that PC-rich Synechococcus occurred in both estuarine and coastal water during both winter and summer, but flow cytometry could only detect this type of cells in estuarine water during summer when they were abundant (Fig. 3). It is suggestive that molecular-based analysis, such as quantitative polymerase chain reaction (qPCR), may provide a more sensitive method to quantify PC-rich picocyanobacteria.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Sample collection

Seawater samples were collected from two Hong Kong marine stations with contrasting hydrographical and trophic conditions. Stn. NM3 located in the Pearl River estuary represents eutrophic estuarine water, while Stn. PM7 at Port Shelter on the east waters represents the more oceanic-influenced coastal water (Fig. 4, Table S1). For molecular study, 500 ml of surface water were collected in July 2007 (wet season) and January 2008 (dry season) by filtering first through a 3 μm and then a 0.22 μm pore-size polycarbonate filter. The filters were preserved at −80°C until DNA extraction. Samples for flow cytometric analysis were collected monthly at the same station during the period from February 2007 to February 2008, and from February 2009 to March 2010. One milliliter seawater was fixed with 0.2% (final concentration) paraformaldehyde and frozen in −80°C until analysis.

figure

Figure 4. Location of the sampling stations of NM3 and PM7. The former represents eutrophic estuarine water, whereas the latter represents oceanic influenced mesotrophic coastal water.

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DNA isolation and amplification

Total genomic DNAs were recovered from biomass collected with the 0.22 μm filters by phenol : chloroform : isoamyl alcohol (25:24:1) extraction at 60°C after lysis with cetyltrimethyl ammonium bromide (CTAB) buffer containing RNase A (10 mg ml−1) and lysozyme (50 mg ml−1). Extracted DNAs were stored at −80°C after they were precipitated with isopropanol. Two primer sets were applied in this study: SyncpcB-Fw (5′-ATGGCTGCTTGCCTGCG-3′) and SyncpcA-Rev (5′-ATCTGGGTGGTGTAGGG-3′) for the cpcBA gene (Haverkamp et al., 2008); B3FW (5′-TCAAGGAGACCTACATCG-3′) and SynA1R (5′-CAGTAGTTGATCAGRCGCAGGT-3′) for the cpeBA gene (Everroad and Wood, 2006). PCR reaction was carried out with 50 μl master mix including 5 μl of 10× buffer, 2 μl of MgCl2 (25 mM), 4 μl of dNTPs (2.5 mM), 0.2 μl of Taq polymerase (5 U) and 1 μl of each primer (10 μM) with the same PCR programmes for cpcBA and cpeBA genes as described by Haverkamp and colleagues (2008). PCR products were stained with ethidium bromide and visualized on 1% agarose gel.

Clone library construction and sequencing

Independent PCR products from triplicate reactions were pooled to reduce the chances of PCR artefacts and then purified by PureLinkTM Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA). Purified amplicons were cloned into the PCR4.0 vector by using the TOPO TA cloning kit (Invitrogen). In total, eight clone libraries were constructed; from each, 30 white clones were randomly picked. The correct insertion was identified by direct amplification of the inserted DNA fragment with the original PCR primer set. Positive clones were purified with the PurelineTM Quick gel extraction kit (Invitrogen) and sequenced by an Applied Biosystems 3730 genetic analyser using the BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems, Carlsbad, CA, USA) with the QM13 forward primer.

Phylogenetic and diversity analysis

blast searches of the GenBank database (http://www.ncbi.nlm.nih.gov) were performed to identify closely related sequences. For the purpose of this study, 99% sequence similarity was used as a cut-off value. Sequences were also checked for chimeric properties by using Chimera_CHECK from the RDP- II (Ribosomal Database Project II). The coding regions of the cpcBA and cpeBA operons were both used in phylogenetic analyses. Neighbour joining trees were generated separately for cpcBA gene sequences (about 439 bp) and cpeBA gene sequences (about 534 bp) with programme Phylip 3.63 (http://evolution.genetics.washington.edu/Phylip%203.63) after they were aligned by Clustal X 1.80. Sequence segments of the intergenic spacer were excluded for the phylogenetic tree of cpcBA gene sequences. Additional phylogenetic trees were reconstructed using maximum parsimony and maximum likelihood methods, and bootstrap values for all trees were obtained with 1000 resamplings, and clades with greater than 50% bootstrap value were shown on the nodes of branches. Genetic diversity was assessed by the number of different OTUs and the Shannon–Weaver index (H′).

Nucleotide sequence accession numbers

Sequences of all the colonies obtained from this study were deposited in GenBank under accession numbers JN128193 to JN128234 for cpcBA gene sequences, and JN128162 to JN128192 for cpeBA gene sequences.

Flow cytometric analysis

Upon return to the lab, cell abundances of PC-, PE-Synechococcus were enumerated using a Becton–Dickinson FACSCalibur cytometer equipped with dual lasers of 488 nm and 635 nm. Forward and right-angle light scattering and four fluorescences signals (FL1: 530/30 BP, FL2: 585/42 BP, FL3: 670 LP, FL4: 661/16BP, BP: bandpass, LP: longpass) were collected, saved and analysed using WinMDI 2.9 developed by Joseph Trotter. PC-, PE-rich Synechococcus were distinguished from other eukaryotic picoplankton by orange fluorescence (FL2) and red fluorescence (FL4; induced by the 635 nm laser) respectively (Fig. 5). In order to confirm that the PC-rich Synechococcus was not Prochlorococcus, we mixed PC-rich Synechococcus cultures isolated from Hong Kong coastal waters (culture kept in f/2 medium) with a field sample collected from the South China Sea, where Prochlocococcus is abundant. Flow cytometric analysis of the mixed samples showed convincing separation between Prochlocococcus and PC-rich Synechococcus by the strong FL4 signal of the latter (Fig. S1). Yellow-green fluorescent beads (1 μm, Polysciences, Warrington, PA, USA) were added to each sample as an internal standard. The exact flow rate was calibrated by weighing a tube filled with distilled water before and after running for certain time intervals, and the flow rate was estimated as the slope of a linear regression curve between elapsed time and weight differences (Li and Dickie, 2001).

figure

Figure 5. Flow cytometric signatures of PC- and PE-rich Synechococcus using a flow cytometer with 488 nm and 635 nm dual lasers. Sample was collected from NM3 at 0 m on 25 May 2009. Red fluorescence excited by 488 nm laser indicates chlorophyll a, whereas red fluorescence excited by 635 nm is from PC. Orange fluorescence represents PE, and side scattering is an indicator of cell size.

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Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Xihan Chen for collecting the DNA samples. Comments from three anonymous reviewers helped improve the quality of this paper. This research is supported by GRF grants from Hong Kong RGC to H.L. (661610, 661912 and 661813). H.M. J. thanks the grant support of SIDSSE-BR-201301.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
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emi412111-sup-0001-si.doc485K

Table S1. Hydrographic parameters and chlorophyll a concentrations at two sampling stations representing subtropical estuarine (NM3) and coastal (PM7) waters of Hong Kong.

Fig. S1. A cytogram of a field sample collected from the South China Sea with abundant Prochlocococcus, mixed with a PC-rich Synechococcus cultures isolated from Hong Kong coastal waters (culture kept in f/2 medium), to demonstrate the cytometric difference between Prochlorococcus and PC-rich Synechococcus when analysed by a flow cytometer equipped with 488 nm and 635 nm dual lasers. The sample was taken from station E503 (112.28665 E; 19.1978 N) at 25 m on 11 August 2012.

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