Editor: Patricia Sobecky
Phylogenetic diversity of Synechococcus strains isolated from the East China Sea and the East Sea
Version of Record online: 22 JUN 2009
© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 69, Issue 3, pages 439–448, September 2009
How to Cite
Choi, D. H. and Noh, J. H. (2009), Phylogenetic diversity of Synechococcus strains isolated from the East China Sea and the East Sea. FEMS Microbiology Ecology, 69: 439–448. doi: 10.1111/j.1574-6941.2009.00729.x
- Issue online: 3 AUG 2009
- Version of Record online: 22 JUN 2009
- Received 12 February 2009; revised 11 May 2009; accepted 1 June 2009.Final version published online 14 July 2009.
- East Sea;
- East China Sea;
Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution.
Synechococcus are ubiquitously distributed throughout the world's oceans, ranging from equatorial to polar waters as well as from coastal to open waters. This wide distribution of Synechococcus, as opposed to Prochlorococcus, which live in oligotrophic open waters (Partensky et al., 1999), might be due to their ability to populate marine surface waters over a wide range of environmental conditions (Penno et al., 2006). Indeed, phylogenetic and physiological data have shown that Synechococcus comprise a very diverse group (Rocap et al., 2002; Fuller et al., 2003; Ahlgren & Rocap, 2006).
Phylogenetic analyses of Synechococcus have been performed using genetic markers including 16S rRNA gene, the 16S–23S rRNA gene internal transcribed spacer (ITS), the RNA polymerase gene (rpoC1), the phycoerythrin gene (cpeB), the nitrate reductase gene (narB), and the nitrogen regulator gene (ntcA) using cultured and natural samples from marine environments (Toledo & Palenik, 1997; Rocap et al., 2002; Fuller et al., 2003; Mühling et al., 2005; Ahlgren & Rocap, 2006; Jenkins et al., 2006; Penno et al., 2006; Haverkamp et al., 2008; Paerl et al., 2008). To date, 12 distinct Synechococcus lineages from culture isolates and at least four additional lineages from environmental clones have been described (Rocap et al., 2002; Fuller et al., 2003; Ahlgren & Rocap, 2006; Penno et al., 2006). Some of these phylogenetic clades can be differentiated from others by physiological traits such as motility (clade III), lack of accessory pigments, phycourobilin (clade VI) or both phycourobilin and phycoerythrobilin (clade VIII), ability of chromatic adaptation (clades I, III, IV, IX, XV, and XVI), and reduced efficiency in nitrate utilization (clades CRD1 and XV) (Toledo et al., 1999; Palenik, 2001; Moore et al., 2002; Fuller et al., 2003; Ahlgren & Rocap, 2006; Six et al., 2007).
It is well accepted that the distribution of Synechococcus lineages varies spatially and temporally (Fuller et al., 2003, 2005; Penno et al., 2006). Given that clades are hypothesized to represent physiologically and ecologically distinct populations or ecotypes (Ahlgren & Rocap, 2006; Dufresne et al., 2008), the distribution of Synechococcus likely varies in time and space to enable survival in each environmental condition. Recently, Dufresne et al. (2008) reported that the average nucleotide identity of genes shared between any pair of Synechococcus genomes belonging to distinct clades is significantly lower than the threshold value of c. 94%, which is equivalent to the currently accepted species threshold of 70% DNA–DNA hybridization. Thus, Synechococcus appears to be a genetically diverse group, and it would be useful to elucidate the extent of this diversity to understand their distribution in time and space, and thus their ecological roles in each niche.
Although many studies have examined the distribution of Synechococcus in various oceanic regions, few data exist for the western part of the Pacific Ocean, especially regarding Synechococcus diversity. We investigated the molecular phylogenic relationships and pigment characteristics of Synechococcus strains isolated from the marginal seas of the western Pacific Ocean, i.e., the East China Sea (ECS) and the East Sea (ES).
Materials and methods
The ECS is a marginal sea of the northwest Pacific Ocean (Fig. 1) that is influenced by diverse water masses, including the Kuroshio Current, Tsushima Warm Currents, cold Yellow Sea water, and fresh water from the Yangtze River (Beardsley et al., 1985). In addition, seasonal changes in solar radiation strongly affect the water temperatures of the ECS (Chen et al., 1994), and seasonal differences in the freshwater discharge from the Yangtze River influence salinity in the study area (Zhang et al., 1994). These environmental variations have a considerable impact on the biological conditions for phytoplankton growth (Guo, 1994). According to recent studies, Synechococcus is the overwhelmingly dominant group of autotrophic picoplankton in the ECS. In the warm period, Synechococcus concentrations can reach c. 106 cells mL−1, and cell abundances appear to reflect different environmental conditions (Jiao et al., 2002, 2005; Noh et al., 2005).
The ES is a semi-enclosed deep marginal sea in the northwestern Pacific (Fig. 1). It is surrounded by the Korean, Japanese, and Russian coasts and has an average depth of 1684 m. It is often referred to as a ‘miniature ocean’ because many of the physical processes and phenomena that take place in the open ocean also occur in the ES (Chang et al., 1998/1999). The upper layer of the southwestern ES is mainly influenced by the warm and saline Tsushima Warm Current transported through the Korea Strait and North Korean Cold Water. Similar to the ECS, the abundance of Synechococcus is the highest in summer, with c. 105 cells mL−1. We collected seawater samples for isolating Synechococcus strains 10 times over four seasons in the ECS and the ES between 2001 and 2006 (Table 1, Fig. 1).
|Clades||Strains||Isolation source||PUB : PEB ratio|
|V||KORDI-38||July 2004||32°07′N||125°10′E||0||No PUB|
|V||KORDI-48||October 2004||32°07′N||125°10′E||20||No PUB|
|V||KORDI-64||July 2006||32°00′N||124°31′E||0||No PUB|
|V||KORDI-65||July 2006||32°07′N||125°08′E||0||No PUB|
|V||KORDI-69||July 2006||32°59′N||124°14′E||0||No PUB|
|V||KORDI-11||September 2002||37°17′N||131°46′E||40||No PUB|
|V||KORDI-16||December 2002||36°30′N||129°42′E||30||No PUB|
|Subcluster 5.3||KORDI-15||December 2002||38°00′N||129°30′E||0||0.62|
|Sub-cluster 5.3||KORDI-30||October 2001||38°30′N||131°30′E||0||1.31|
|Subcluster 5.2 (CB5)||KORDI-78||October 2003||32°07′N||125°10′E||0||No PUB|
Seawater samples from surface and subsurface chlorophyll a maximum depth were filtered through 3.0-μm polycarbonate filters by gravity. Nutrients based on f/2-Si medium (f/2 medium without Na2SiO3·9H2O) supplemented with 100 μM (final concentration) of NH4Cl solution were added to the samples at an approximate dilution of 1 : 10, and the samples were incubated at 25 °C under illumination at c. 10 μE m−2 s−1. After c. 2 months, the culture samples were transferred to a fresh f/2-Si medium. These cultures were maintained by transferring small quantities to a fresh medium roughly every 1–2 months for several years. Cycloheximide was added to the cultures to restrict the growth of picoeukaryotes during the early culturing process. To obtain monoclonal or axenic cultures, a 1-mL aliquot of culture diluted to 0.2 cell mL−1 with fresh medium was dispensed into each well of a sterile 96-deep-well plate. The plate was incubated at 25 °C under c. 20 μE m−2 s−1. After 1–2 months, cell growth was observed in 5–20 wells, and 100 μL of culture was transferred from the wells with growing cells to a fresh medium and regrown. This dilution procedure was repeated using the isolate obtained from the first dilution.
DNA purification, PCR, and sequencing
Genomic DNA was extracted using a bead-beating protocol with zirconium beads, chloroform, and isopropanol (McBain et al., 2003). The 16S rRNA gene segments were amplified by PCR using several primers (27F, OXY359F, SYN1017R, OXY 1313R, and 1522R) as described by Fuller et al. (2003). ITS segments were amplified using the 16S-1247f and 23S-241r primer set (Rocap et al., 2002). PCR reactions (30 μL total volume) contained 3 μL Taq polymerase buffer, 0.2 mM of each dNTP, 0.3 μM of each primer, 1 U of Taq DNA polymerase (Bioneer, Daejeon, Korea), and 10–100 ng of template DNA. After purifying the PCR products using an AccuPrep PCR purification kit (Bioneer), the amplified DNA was sequenced bidirectionally using the above primers and an Applied Biosystems (Foster City, CA) automatic sequencher at Macrogen (Seoul, Korea) or Solgent (Daejeon, Korea).
The 16S rRNA gene sequences were aligned with those of other Synechococcus strains obtained from the GenBank database based on known 16S rRNA gene secondary structure information using the program jphydit (Jeon et al., 2005). The ITS sequences were aligned using the arb software package (Ludwig et al., 2004) and a database (from Dr A. Martiny, UCI) containing aligned Synechococcus ITS sequences. For phylogenetic analysis of ITS sequences, 16S and 23S rRNA gene sequences and two tRNA sequences were excluded. In addition, a base frequency filter of >50% identity was applied to select the most homologous positions for phylogenetic analysis. The selected sequences (537 bp) were exported in FASTA format, and phylogenetic trees were constructed using the neighbor-joining and the maximum parsimony methods. The Jukes–Cantor model was used to create an evolutionary distance with the neighbor-joining method (Jukes & Cantor, 1969). The robustness of tree topologies was assessed by bootstrap analyses based on 1000 replications. Phylogenetic analyses and inter- and intraclade sequence distance were carried out using mega 4 (Tamura et al., 2007).
The absorption and excitation spectra of pigments were analyzed using both spectrophotometry and spectrofluorometry, respectively. A 5-mL aliquot of culture was filtered on GF/F filters (25-mm diameter, Whatman). An in vivo absorption spectrum was measured from 400 to 800 nm using a dual-beam spectrophotometer (Lambda 19, Perkin-Elmer). A wetted filter without cells was used as a reference. An in vivo excitation spectrum was obtained at a wavelength between 400 and 570 nm (emission: 585 nm, excitation and emission slits: 5 nm) using a spectrofluorometer (LS-50B, Perkin-Elmer). Ratios of phycourobilin to phycoerythrobilin were calculated from the excitation spectra.
Isolation of strains
In total, 33 strains were isolated from the study areas (Table 1): 14 from the ES and 19 from the ECS. Among these, 16 and 17 strains were isolated from surface and subsurface chlorophyll maximum depths, respectively, and most of the strains (24 strains) were isolated during the summer and autumn.
16S rRNA gene and ITS phylogeny
Based on their 16S rRNA gene sequences, the Synechococcus isolates from the ECS and the ES clustered into 10 clades, which comprised eight previously designated clades and two new clades that did not cluster with any of the 10 known clades (Fig. 2). Among the 33 culture isolates examined, 16 belonged to clades V and VI and the others to clades II (four strains), III (one), VIII (three), IX (two), and subcluster 5.3 (two), CB5 in Synechococcus subcluster 5.2 (one), and two novel clades (three strains in clade WPC1 and one in clade WPC2). The KORDI-52 strain of the novel WPC2 clade did not form a robust clade with any other strains in phylogenetic trees based on 16S rRNA gene sequences constructed by using neighbor-joining and maximum-parsimony methods (Fig. 2). The three strains in the novel clade WPC1 formed a robust clade with the clone sequence of SAR7. However, the strains closest to those in clade WPC1, based on 16S rRNA gene similarity, belonged to clade IV rather than with SAR7. In addition to strains belonging to marine subcluster 5.1, the KORDI-78 strain affiliated with the CB5 clade was also isolated in the ECS. The strain formed a robust clade with a clone sequence collected from the middle of Chesapeake Bay, with a salinity of 19 p.s.u. (Chen et al., 2006).
The phylogenetic relationships of the ITS sequences were almost congruent with those inferred by the 16S rRNA gene sequences (Fig. 3). Similar to the 16S rRNA gene tree, two novel clades were also revealed in the ITS tree. The intraclade distance (0.002) of the novel clade WPC1 calculated using the selected ITS sequences (see Materials and methods) was lower than the distances to its closest neighboring clades II and III (0.073 and 0.067, respectively). The intraclade distance (0.002) of clade WPC2 including a strain KORDI-52 and two environmental clones obtained from the ECS was also lower than the interclade distance (0.041) between clade WPC2 and its closest neighboring clade II. The strain KORDI-78 clustered into clade CB5 with a high bootstrap value in both the ITS tree and the 16S rRNA gene tree (Figs 2 and 3).
The pigment patterns of our strains were divided into four types based on absorption spectra (Fig. 4). Strains belonging to clade VIII did not exhibit absorption maxima characteristics of phycoerythrin and thus conformed to the type 1 pigment type, which carries phycocyanin only. Specifically, they did not exhibit any peaks between 490 and 570 nm. All strains belonging to clade V showed the type 2 pattern, which has phycoerythrobilin (peak between 554 and 561 nm), but not phycourobilin. Strains in clade VI exhibited low phycourobilin : phycoerythrobilin ratios (type 3), showing two peaks between 495 and 498 nm and between 541 and 547 nm, corresponding to phycourobilin and phycoerythrobilin, respectively. Strain KORDI-28 belonging to clade III and KORDI-52 in clade WPC2 exhibited both phycourobilin (494 nm) and phycoerythrobilin (544 nm) with a relatively higher phycourobilin : phycoerythrobilin ratio (type 4) than the type 3 strains. The other strains in clades II, IX, and WPC1 also showed two peaks classified as type 3. Two strains belonging to subcluster 5.3 showed different pigment types. Strain KORDI-15 showed a type 3 pattern, whereas strain KORDI-30 showed a type 4 pattern (Fig. 4). Strain KORDI-78 affiliated with the CB5 clade showed a type 2 pattern (Fig. 4). The excitation spectra confirmed that the strains isolated from the ES and the ECS could be classified into four types of pigment patterns (Fig. 4). Pigment type 1 strains showed only the C-phycocyanin peak at 623 nm measured at an emission wavelength of 650 nm (Fig. 4). Pigment type 2 strains showed only one phycoerythrobilin peak between 553 and 554 nm (567 nm for KORDI-78). Strains belonging to type 3 pigment patterns showed both phycourobilin and phycoerythrobilin peaks between 492 and 500 nm and between 542 and 544 nm, respectively, with low phycourobilin : phycoerythrobilin ratios (Fig. 4). However, strains exhibiting pigment type 4 showed two peaks between 491 and 492 nm, and at 544 nm with high phycourobilin : phycoerythrobilin ratios (Fig. 4).
Synechococcus strains affiliated with diverse lineages including two novel clades were isolated from the ECS and the ES. In addition, the pigment patterns of strains belonging to several clades were not consistent with previous reports. These results suggest that there could be a greater phenotypic and genetic diversity in Synechococcus assemblages than previously recognized.
Two novel clades designated as clades WPC1 and WPC2 were identified in this study. The strain KORDI-52 could be considered as a member of novel clade WPC2 on the basis of phylogenetic analyses of 16S rRNA gene sequences (Fig. 2). Further, strain KORDI-52 formed a robust clade with two environmental clones from the ECS in the ITS tree (Fig. 3). The novelty of clade WPC1, which contained three strains from the ECS and ES, could be confirmed by the sequence similarities and phylogenetic analyses (Figs 2 and 3). In both 16S rRNA gene and ITS trees, the strains formed a robust clade with high bootstrap values and were not clustered with any other previously designated clades. Further, the similarity of ITS sequences among the strains and the closest relative in GenBank was very low (c. 87% to Synechococcus sp. UW95). The existence of additional clades, with the exception of the 16 previously known Synechococcus lineages, was reported from environmental clone sequences from samples collected in the Costa Rica upwelling dome (Saito et al., 2005). However, the sequences of the novel clades identified in this study did not form a clade with those environmental clones (Fig. 3). These novel sequences, which are phylogenetically distant from sequences recovered from other oceanic areas, indicate that these strains could have endemic distributions in the ECS and the ES. The presence of an unidentified lineage was also suggested by the observation that the sum of abundances for known clades does not fully account for the total community abundance (Fuller et al., 2003, 2005). Indeed, the 16S rRNA gene sequences that formed novel clades did not match any of the clade-specific probe sequences (Fuller et al., 2003; data not shown).
The pigment patterns of the strains belonging to clades V, VI, and CB5 are noteworthy. Pigment types of Synechococcus strains could be largely classified into three types: no phycoerythrin, phycoerythrin with only phycoerythrobilin, and phycoerythrin with both phycourobilin and phycoerythrobilin (Six et al., 2007). Strains affiliated with clade VI exhibited phycourobilin-lacking phycoerythrin with only phycoerythrobilin, whereas strains in clade V showed both phycourobilin and phycoerythrobilin (Fuller et al., 2005; Six et al., 2007). In this study, however, the pigment patterns of clades V and VI were different from those reported in previous studies. Specifically, clade V showed phycourobilin-lacking phycoerythrin, whereas clade VI exhibited phycoerythrin with both phycourobilin and phycoerythrobilin (Fig. 4). Given that in this study, the strains affiliated with clades V and VI were isolated by time and space and the same pigment patterns were found in all tested strains, these results are very intriguing. Such discrepancies in pigment patterns might have resulted from chromatic adaptation and/or variation of phycourobilin : phycoerythrobilin ratios induced by variations in incubation conditions. However, it is well accepted that these strains are not chromatic adapters (Six et al., 2007). Additionally, to test the latter hypothesis, the two reference strains Synechococcus sp. WH7803 and WH8018 belonging to clades V and VI, respectively, were incubated under the same conditions as our strains and their excitation spectra were examined. The pigment patterns of the reference strains did not change in our incubation conditions (Fig. 5). Thus, the different pigment patterns between our strains and the reference strains could not be explained by different incubation conditions.
It is also possible that the geographical differentiation between the strains led to variable pigment patterns. Despite the limited data, the two geographically isolated groups, i.e., from the western Pacific Ocean and the Atlantic Ocean, might show different pigment compositions. Recently, using comparative genome analyses of phycobilisome, Six et al. (2007) suggested that acquisition of phycoerythrin I components of phycoerythrin was attained before the separation of the marine Synechococcus branch from other cyanobacteria, whereas acquisition of the phycoerythrin II component related to phycourobilin occurred after the differentiation of the marine Synechococcus lineage, probably by lateral gene transfer. Indeed, our strains belonging to clade VI have the mpeBA operon (unpublished data), but not for strains from the previous studies. Thus, the different pigment patterns among clades V and VI found in this study might support this suggestion. To confirm this hypothesis, however, further genetic analyses of phycobiliprotein operons for more Synechococcus strains (Six et al., 2007; Haverkamp et al., 2008) would be essential. In addition to the diverse pigment patterns in both clades V and VI, strain KORDI-78, which clustered into the CB5 clade belonging to Synechococcus subcluster 5.2 (Chen et al., 2006), could be distinguished from other strains in the clade because it showed phycourobilin-lacking phycoerythrin (type 2). Chen et al. (2006) defined two clades (CB4 and CB5) in subcluster 5.2 and found that five phycoerythrin-rich strains and four PC-rich strains clustered into the CB4 clade. Likewise, strain KORDI-78 is the first member of clade CB5 that contains phycoerythrin. Thus, phycoerythrin seems to be widely distributed even in Synechococcus subcluster 5.2. Thus, pigmentation patterns cannot be used as a key characteristic to differentiate clades, at least in clades V, VI, and subcluster 5.2. These results are consistent with the contention that some phycobilisome genes evolved independent of core genome phylogeny, possibly by lateral gene transfer of phycobilisome rod genes between Synechococcus strains (Six et al., 2007).
Recently, a phycoerythrin-containing novel cyanobacterium, Rubidibacter lacunae, which is phylogenetically distant from the Synechococcus group, was found in tropical waters (Choi et al., 2008). Thus, the phycoerythrin seems to distribute more widely in the cyanobacterial group than previously known. In this respect, a phylogenetic study of phycoerythrin pigments is necessary to understand the evolutionary history (Apt et al., 1995), such as the acquisition of phycoerythrin, of marine Synechococcus.
In addition to two novel clades in subcluster 5.1 and clade CB5 in subcluster 5.2, strains belonging to seven different clades were recovered in this study. Given that the study sites are influenced by various coastal and open water masses and that Synechococcus are cosmopolitan species, the isolation of diverse Synechococcus groups in the ECS and ES is not surprising. As we isolated the strains after growing them in an enriched medium, the isolates would represent dominant strains and/or at least winners among competing strains. Thus, the spatial and seasonal distribution of Synechococcus appears to be diverse in the ECS and the ES, and these areas would be ideal for future studies on the diversity and ecology of Synechococcus.
In conclusion, strains belonging to diverse Synechococcus lineages including two novel clades were isolated in the ECS and the ES. Pigment analyses revealed that the pigment patterns of clades V and VI as well as CB5 clades of Synechococcus subcluster 5.2 are different from those reported previously. The different pigment patterns raise a question in terms of the evolution of phycoerythrin in marine Synechococcus and suggest the existence of a geographical barrier between the studied areas (Ramette & Tiedje, 2007). Despite the ubiquitous and cosmopolitan distribution of Synechococcus species, these results indicate that there might be unseen barriers among the world's oceans, and thus several genotypes of Synechococcus might restrict their habitat to narrow areas of marine environments, resulting in endemic species. To further examine this hypothesis, additional studies that examine the distribution of Synechococcus species and their evolution are required.
We are grateful to Dr A. Martiny (UCI) for providing the arb file with aligned ITS sequences. This study was supported by research programs (PM54850, PE98312, PM53901, PM54880) of the Korea Ocean Research and Development Institute (KORDI) and the Korean Ministry of Land, Transportation and Maritime Affairs (MLTMA).
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