SYNECHOCOCCUS SALSUS SP . NOV . ( CYANOBACTERIA ) : A NEW UNICELLULAR , COCCOID SPECIES FROM YUNCHENG SALT LAKE , NORTH CHINA

A new species of the genus Synechococcus C. Nägeli was described from extreme environment (high salinity) of the Yuncheng salt lake, North China. Morphological characteristics observed by light microscopy (LM) and transmission electron microscopy (TEM) were described. DNA barcodes (16S rRNA+ITS-1, cpcBA-IGS) were used to evaluate its taxonomic status. This species was identified as Synechococcus salsus H. Lv et S. Xie. It is characterized by unicellular, without common mucilage, cells with several dispersed or solitary polyhedral bodies, widely coccoid, sometimes curved or sigmoid, rounded at the ends, thylakoids localized along cells walls. Molecular analyses further support its systematic position as an independent branch. The new species Synechococcus salsus is closely allied to S. elongatus, C. Nägeli, but differs from it by having shorter cell with length 1.0–1.5 times of width. Introduction Synechococcus C. Nägeli (Synechococcaceae, Cyanobacteria) was first discovered in 1849 and is a botanical form-genus comprising rod-shaped to coccoid cyanobacteria with the diameter of 0.6–2.1 μm that divide in one plane. It is a group of ultra-structural photosynthetic prokaryote and has the close genetic relationship with Prochlorococcus (Johnson and Sieburth, 1979), and both of them are the most abundant phytoplankton in the world’s oceans (Huang et al., 2012). The genus is a taxon of unicellular cyanobacteria with an ongoing debate concerning its revision into several genera (Rippka et al., 1979). The genus Synechococcus is surely heterogeneous and its taxonomy needs to be solved by ultrastructural features and methods of molecular taxonomy (Komárek et al., 2014). The genetic diversity and phylogeny of Synechococcus in freshwater were determined by nucleotide sequence such as 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS-1) and cpcBA-IGS (phycocyanin operon) (Jasser et al., 2011). 16S rRNA gene is the most widely used gene sequence in the classification of cyanobacteria. Due to its highly conserved structure, it is only appropriate for evolutionary studies at the level of genera and above (Long and Chen, 2006). With the high variability and the rapid rate of change in molecular evolution, the internal transcribed spacer is often used to study the phylogenetic relationships at the taxa of genus, species, subspecies and so on (García-Martínez et al., 1996). In addition, cpcBA-IGS, the phycocyanin operon, can be used as the classification basis in identifying the level of species and the following (Bolch et al., 1996). Yuncheng salt lake is located in southern suburbs of Yuncheng city where the algal distribution has its particularity. On the north of beach of Yuncheng salt lake, there were nearly 2 × 10 m 1 Corresponding author: Email: xiesl@sxu.edu.cn 138 HONG-RUILV et al. waters extensively appeared rosy during June, 2015. In order to find the reasons why the water turns red, we specifically took water samples and a coccoid strain with flickering red light of Synechococcus species was isolated. In the present paper, we describe Synechococcus species isolated from Yuncheng salt lake as a new species Synechococcus salsus sp. nov. and discuss its taxonomic position based on morphological property observed by light microscopic and transmission electron microscopic, molecular data of 16S rRNA gene with the adjacent ITS, cpcBA-IGS and flanking regions were also used to support the entity as a new species. Materials and Methods Sample preparation and culture-dependent method The unicellular, coccoid strain in this study was collected from Yuncheng salt lake, Shanxi Province, North China, in June 2015. It was preliminarily identified belonging to the genus Synechococcus through light microscopic examination (Olympus BX-51, Tokyo, Japan). We prepared BG-11 medium and then added it to a 24-well cell culture plate, each hole with 2 ml. Pasteur Micropipette was used to pick individual cells (Rippka, 1988). Cyanobacteria were isolated into unialgal culture and they were inoculated on the medium in the 24-well cell culture plate after washing eight times by sterile water. Then, it was cultured in a light incubator (BSG300, Shanghai, China) at 25°C under a 12 h/12 h light/dark photoperiod. The light intensity was 25 μmol·m·s. Three weeks later, they reached exponential phase, then we examined them with microscope. In the case of growing well and axenic culture, the homogenates were transferred to conical flasks with 200 ml BG-11 liquid medium. Similarly, they were placed in the same culture condition for large scale culture. To make them fully absorb nutrients and equally distribute, we manually shook the conical flasks every two days. The unialgal strain was deposited in Algae Culture Collection at Shanxi University. Morphological observations Morphological characteristics were examined under a light microscope when the cyanobacteria in large scale culture reached stationary phase. Photographs of main characters were taken with a digital camera (CAMEDIA C5060WZ, Olympus) and a CCD camera (DP72, Olympus) mounted on the microscope. Moreover, transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) was used to observe the ultrastructure through the process of centrifuging, fixation, dehydration, embedding, sectioning and dye (Lim et al., 2012). Molecular analyses Genomic DNA was extracted from 10 ml axenically exponential phase cultures according to the modified cetyltrimethyl ammonium bromide (CTAB) method (Doyle, 1987).PCR was carried out in 20 μl reaction volume containing 12.3 μl of double-distilled water, 2 μl of 10×Taq polymerase reaction buffer (Takara, Dalian, China), 0.2 μl of EasyTaq DNA polymerase (5 U/μl, Takara, China), 1.5 μl of each primer (10 μM), 2 μl dNTP mix (2.5 mM each; Takara) and 0.5 μl of undiluted genomic DNA. Part of the 16S ribosomal RNA gene (16S rRNA) and all of the 16S23S rRNA internal transcribed spacer region (16S-23S ITS) were amplified using the primers P1 (5’-CTC TGT GTG CCT AGG TAT CC-3’) and P2 (5’-GGG GAA TTT TCC GCA ATG GG-3’) as described by Boyer et al. (2001). The cpcBA-IGS region of the phycocyanin operon were amplified with the cyanobacterium-specific primer pairs (Crosbie et al., 2003a) cpcBF (5’-TAG TGT AAA ACG ACG GCC AGT TGY YTK CGC GAC ATG GA-3’) and cpcAR (5’-TAG CAG GAA ACA GCT ATG ACG TGG TGT ARG GGA AYT T-3’). Polymerase chain reaction (PCR) was performed in a My Cycler thermal cycler (Bio-Rad, Hercules, CA, USA). The program consisted of the following condition: 5 min at 95 °C, 35 cycles of 45 s at 94 °C, 45 s at 55 °C, 1 SYNECHOCOCCUS SALSUS SP. NOV. (CYANOBACTERIA): A NEW UNICELLULAR 139 min at 72 °C, and a final extensionstep of 10 min at 72 °C. Sequencing was carried out from purified PCR products by BGI (Beijing, China). Sequences generated from the research were deposited in GenBank (accessions KU925869 for 16S rRNA gene+ITS-1, KU925870 for phycocyanin operon). Sequences were aligned using Bioedit and then manually adjusted. Phylogenetic trees were constructed from the aligned gene sequences using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian (BI) methods. NJ, ML and BI analyses were performed through MEGA 5.0 (Tamura et al., 2011), PhyML 3.0 (Guindon and Gascuel, 2003) and MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003), respectively. Best fit models were selected under the Akaike information criterion (AIC) using Modeltest 3.7 (Posada and Crandall, 1998). For NJ analyses, evolutionary distances were computed using the Kimura 2-parameter method with 1000 bootstrap replicates. Treeview was used to view the phylogenetic trees. Final graphic refinement of all trees was done in Adobe Illustrator CS5 (Adobe Systems, San Jose, CA, USA). Results and Discussion The information and GenBank accession numbers for Synechococcus salsus sp. nov. and the other eighty-nine strains analyzed in this study were listed in Table 1. For the 16S rRNA+ITS-1 sequences, 44 strains of cyanobacteria were used and the aligned sequence dataset comprised 1321 nucleotides, of which 515 (38.99%) were variable sites and 455 (34.44%) were parsimonyinformative sites. The aligned phycocyanin operon (cpcBA-IGS) dataset was 449 bp long. Fortyeight aligned cpcBA-IGS sequences had 307 (68.37%) variable sites and 281 (62.58%) parsimony-informative sites. The best fit model used for each locus was presented in Table 2. For both the 16S rRNA+ITS-1 and the cpcBA-IGS data, tree topologies resulting from three methods had some similarities and differences. Based on 16S rRNA+ITS-1 sequences, the tree derived from the BI analysis is presented in Figure 1 to explain the relationships between Synechococcus salsus sp. nov. and the other accessions. Leptolyngbya sp. KIOST-1 was chosen as out group. S. salsus sp. nov. is grouped together with many Synechococcus strains. They are in a well-supported clade. Nevertheless, the bootstrap value by NJ method is less than 50%. The phylogenetic tree recovered by BI analysis of cpcBA-IGS is shown in Figure 2. Cyanidium caldarium was chosen as outgroup. In this topology, S. salsus sp. nov. and many other Synechococcus strains also constitute one cluster. The strain Synechococcus PCC9005 AF223465 was confirmed as closely related to S. salsus sp. nov., but the support value was low. Synechococcus salsus H. R. Lv et S. L. Xie, sp. nov. (Fig 3). Diagnosis: LM observations-Unicellular, cells solitary or agglomerated in groups (Figs A, B), but without common mucilage. Cells widely coccoid, sometimes curved or sigmoid, rounded at the ends, with a mean diameter of 2.0–3.5 μm and a length of 3–5 μm, usually pale blue-green, rarely olive-green, bright blue-green or pinkish, division always by binary fission, perpendicular to the longer axis of the cell, reproduction by solitary cells (Figs C, D). TEM observations-Cells with several dispersed or solitary polyhedral bodies, thylakoids localized along cells walls, photosynthetic pigments attached t


Introduction
Synechococcus C. Nägeli (Synechococcaceae, Cyanobacteria) was first discovered in 1849 and is a botanical form-genus comprising rod-shaped to coccoid cyanobacteria with the diameter of 0.6-2.1 µm that divide in one plane.It is a group of ultra-structural photosynthetic prokaryote and has the close genetic relationship with Prochlorococcus (Johnson and Sieburth, 1979), and both of them are the most abundant phytoplankton in the world's oceans (Huang et al., 2012).The genus is a taxon of unicellular cyanobacteria with an ongoing debate concerning its revision into several genera (Rippka et al., 1979).
The genus Synechococcus is surely heterogeneous and its taxonomy needs to be solved by ultrastructural features and methods of molecular taxonomy (Komárek et al., 2014).The genetic diversity and phylogeny of Synechococcus in freshwater were determined by nucleotide sequence such as 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS-1) and cpcBA-IGS (phycocyanin operon) (Jasser et al., 2011).16S rRNA gene is the most widely used gene sequence in the classification of cyanobacteria.Due to its highly conserved structure, it is only appropriate for evolutionary studies at the level of genera and above (Long and Chen, 2006).With the high variability and the rapid rate of change in molecular evolution, the internal transcribed spacer is often used to study the phylogenetic relationships at the taxa of genus, species, subspecies and so on (García-Martínez et al., 1996).In addition, cpcBA-IGS, the phycocyanin operon, can be used as the classification basis in identifying the level of species and the following (Bolch et al., 1996).Yuncheng salt lake is located in southern suburbs of Yuncheng city where the algal distribution has its particularity.On the north of beach of Yuncheng salt lake, there were nearly 2 × 10 5 m 2 waters extensively appeared rosy during June, 2015.In order to find the reasons why the water turns red, we specifically took water samples and a coccoid strain with flickering red light of Synechococcus species was isolated.
In the present paper, we describe Synechococcus species isolated from Yuncheng salt lake as a new species Synechococcus salsus sp.nov.and discuss its taxonomic position based on morphological property observed by light microscopic and transmission electron microscopic, molecular data of 16S rRNA gene with the adjacent ITS, cpcBA-IGS and flanking regions were also used to support the entity as a new species.

Sample preparation and culture-dependent method
The unicellular, coccoid strain in this study was collected from Yuncheng salt lake, Shanxi Province, North China, in June 2015.It was preliminarily identified belonging to the genus Synechococcus through light microscopic examination (Olympus BX-51, Tokyo, Japan).We prepared BG-11 medium and then added it to a 24-well cell culture plate, each hole with 2 ml.Pasteur Micropipette was used to pick individual cells (Rippka, 1988).Cyanobacteria were isolated into unialgal culture and they were inoculated on the medium in the 24-well cell culture plate after washing eight times by sterile water.Then, it was cultured in a light incubator (BSG-300, Shanghai, China) at 25°C under a 12 h/12 h light/dark photoperiod.The light intensity was 25 µmol•m -2 •s -1 .Three weeks later, they reached exponential phase, then we examined them with microscope.In the case of growing well and axenic culture, the homogenates were transferred to conical flasks with 200 ml BG-11 liquid medium.Similarly, they were placed in the same culture condition for large scale culture.To make them fully absorb nutrients and equally distribute, we manually shook the conical flasks every two days.The unialgal strain was deposited in Algae Culture Collection at Shanxi University.

Morphological observations
Morphological characteristics were examined under a light microscope when the cyanobacteria in large scale culture reached stationary phase.Photographs of main characters were taken with a digital camera (CAMEDIA C5060WZ, Olympus) and a CCD camera (DP72, Olympus) mounted on the microscope.Moreover, transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) was used to observe the ultrastructure through the process of centrifuging, fixation, dehydration, embedding, sectioning and dye (Lim et al., 2012).
Sequences were aligned using Bioedit and then manually adjusted.Phylogenetic trees were constructed from the aligned gene sequences using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian (BI) methods.NJ, ML and BI analyses were performed through MEGA 5.0 (Tamura et al., 2011), PhyML 3.0 (Guindon and Gascuel, 2003) and MrBayes version 3.1.2(Ronquist and Huelsenbeck, 2003), respectively.Best fit models were selected under the Akaike information criterion (AIC) using Modeltest 3.7 (Posada and Crandall, 1998).For NJ analyses, evolutionary distances were computed using the Kimura 2-parameter method with 1000 bootstrap replicates.Treeview was used to view the phylogenetic trees.Final graphic refinement of all trees was done in Adobe Illustrator CS5 (Adobe Systems, San Jose, CA, USA).

Results and Discussion
The information and GenBank accession numbers for Synechococcus salsus sp.nov.and the other eighty-nine strains analyzed in this study were listed in Table 1.For the 16S rRNA+ITS-1 sequences, 44 strains of cyanobacteria were used and the aligned sequence dataset comprised 1321 nucleotides, of which 515 (38.99%) were variable sites and 455 (34.44%) were parsimonyinformative sites.The aligned phycocyanin operon (cpcBA-IGS) dataset was 449 bp long.Fortyeight aligned cpcBA-IGS sequences had 307 (68.37%) variable sites and 281 (62.58%) parsimony-informative sites.The best fit model used for each locus was presented in Table 2.
For both the 16S rRNA+ITS-1 and the cpcBA-IGS data, tree topologies resulting from three methods had some similarities and differences.Based on 16S rRNA+ITS-1 sequences, the tree derived from the BI analysis is presented in Figure 1 to explain the relationships between Synechococcus salsus sp.nov.and the other accessions.Leptolyngbya sp.KIOST-1 was chosen as out group.S. salsus sp.nov. is grouped together with many Synechococcus strains.They are in a well-supported clade.Nevertheless, the bootstrap value by NJ method is less than 50%.The phylogenetic tree recovered by BI analysis of cpcBA-IGS is shown in Figure 2. Cyanidium caldarium was chosen as outgroup.In this topology, S. salsus sp.nov.and many other Synechococcus strains also constitute one cluster.The strain Synechococcus PCC9005 AF223465 was confirmed as closely related to S. salsus sp.nov., but the support value was low.A, B), but without common mucilage.Cells widely coccoid, sometimes curved or sigmoid, rounded at the ends, with a mean diameter of 2.0-3.5 µm and a length of 3-5 µm, usually pale blue-green, rarely olive-green, bright blue-green or pinkish, division always by binary fission, perpendicular to the longer axis of the cell, reproduction by solitary cells ( Figs C, D).TEM observations-Cells with several dispersed or solitary polyhedral bodies, thylakoids localized along cells walls, photosynthetic pigments attached to it ( Figs E, F).The new species is closely allied to S. elongatus C. Nägeli, but differs from it by having shorter cell with length 1.0-1.5 times of width, and cell length of S. elongatusis 1.5-3.0times of width.

Gene
Etymology: The specific epithet refers to its living environment with the high salinity.
Habitat and distribution: Synechococcus salsus was discovered in Yuncheng salt lake in Shanxi Province, North China, which is located at 34°48′ to 35°30′ N and 110°12′ to 111°41′ E. The lake is about 1.3×10 8 m 2 in area, with a salinity of 6.8%and pH 7.54 in June.
Note: The genus Synechococcus belongs to Cyanobacteria, Cyanophyceae, Synechococcophycideae, Synechococcales, Synechococcaceae.Synechococcus is polyphyletic and currently recognized as a distinct genus.The Synechococcus species are unicellular and rodshaped to coccoid.Such tiny cells can be identified through the fluorescence of the main pigments phycoerythrin (PE) they contain, which is the evidence with respect to the phenomenon that Yuncheng salt lake appears rosy during June, 2015.According to Algae Base (http://www.algaebase.org/),38 species have been flagged as currently accepted taxonomically.Zhao et al. (2010) studied spatial and temporal patterns of plankton assemblage structure of the saline lake Namuka Co in Northern Tibet and found Synechococcus strain.In the study of taxonomic and functional diversity in the saline Qinghai Lake, Huang et al. (2014) pointed out that Synechococcus was the dominant genus.Among the reports about cyanobacteria in Yuncheng Salt Lake region, only one Synechococcus species (S. aeruginosus Nägeli) was reported (Li and Xie, 2006).However, it is generally accepted belonging to the genus Cyanothece Komárek, namely C. aeruginosa (Nägeli) Komárek.Synechococcus salsus sp.nov.has no homology with it.
Synechococcus is an important type genus and species of this genus grow well in many different mediums.Most are freshwater species, and some grow within mats and colonies of other algae, or form fine colonies on wet substrates including mud, wood, stones, etc.A group of species is known from the metaphyton and periphyton of thermal and mineral springs (Dor, 1967), and few are described from aerophytic sites (Skuja, 1964).There are also some marine species (Jao, 1948;Komárek, 1956).Interesting picoplanktonic or planktonic species were found in oceans as well as in freshwater reservoirs (lakes) (Komárek and Anagnostidis, 1995).
In the past, the classification of cyanobacteria only relied on traditional morphological criteria.However, it is difficult to distinguish the members of Synechococcus because of their small differences, and its taxonomy should be resolved by help of ultrastructural procedures and molecular approaches.With the development of electron microscope technique and molecular phylogeny, the accuracy of describing the phylogenetic relationship about cyanobacteria has been greatly improved (Komárek, 2010).16S rRNA gene encodes the small subunit ribosomal in prokaryote.Because of its high conservation and the largest database available for comparison, it has been widely chosen as one of the molecular markers for phylogeny (Gupta, 2009).However, even for species with distinct physiological differences, 16S rRNA does not have enough distinguishing ability in solving the relationship problems (Jaspers and Overmann, 2004).ITS (internal transcribed sequences) with relatively high mutation rate, when it comes to some closely related species, is quite effective.Despite three pure cultures of Synechococcus strains and a Prochlorococcus strain was close to each other in the phylogenetic tree based on the 16S rRNA, but the length of their ITS sequences differ greatly (Laloui et al., 2002).Rocap et al. (2002) also indicated that the length of the ITS sequences and G+C content vary greatly among different strains.It is always used to distinguish Synechococcus strains with highly similar sequences but different in physiological characteristics (e.g.different pigment composition, of light and nutrients of different growth response, etc.).The phycocyanin operon is composed of the two genes (cpcB and cpcA) that code for phycocyanin, a short and quite variable intergenic region (IGS) and three linker polypeptides (Belknap and Haselkorn, 1987).It can avoid many non-specific amplified products when used to study the diversity of Synechococcus.This study relatively accurately evaluated the taxonomic status of S. salsus sp.nov.based on the 16S rRNA+ITS-1 gene sequence and phycocyanin operon (cpcBA-IGS).Based on 16S rRNA+ITS-1 sequences, the tree showed the relationships between Synechococcus salsus sp.nov.and the other accessions.The members of Synechococcus are gathered together into one cluster, which is in a well-supported rate.But in the Synechococcus-clade, it is quite obvious that S. salsus sp.nov. is separated from the other Synechococcus strains and alone in a small clade.On the phylogenetic tree recovered by BI analysis of cpcBA-IGS, the genus Synechococcus is not a monophyletic group.The strain Synechococcus PCC9005 AF223465 was confirmed as closely related to S. salsus sp.nov., but the support values were weak.From the above, it is reasonable to make it as a new Synechococcus species.Finally, salt lake (salinity > 0.5 g/L) is a special habitat.Although the salinity cannot be a criterion to justify a new species (Anagnostidis and Komarek, 2005), it may indeed contain many special organisms.

Fig. 1 .
Fig. 1.Phylogenetic tree reconstructed using Bayesian inference from the aligned 16S rRNA+ITS-1 sequence.Numbers at nodes represent maximum likelihood bootstrap/neighbor-joining bootstrap/Bayesian posterior probabilities values.Support values less than 50% are not shown.Scale bar represents 0.1 substitutions per site.

Fig. 2 .
Fig. 2. Phylogenetic tree reconstructed using Bayesian inference from the aligned cpcBA-IGS sequence.Numbers at nodes represent maximum likelihood bootstrap/neighbor-joining bootstrap/Bayesian posterior probabilities values.Support values less than 50% are not shown.Scale bar represents 0.1 substitutions per site.