Phylogenetic study of some strains of Dunaliella.

Dunaliella strains were isolated from a key site for salt production in Vietnam (Vinh Hao, Binh Thuan province). The strains were identified based on Internal Transcribed Spacer (ITS) markers. The phylogenetic tree revealed these strains belong to the clades of Dunaliella salina and Dunaliella viridis. Results of this study confirm the ubiquitous nature of Dunaliella and suggest that strains of Dunaliella salina might be acquired locally worldwide for the production of beta-carotene. The identification of these species infers the presence of other Dunaliella species (Dunaliella tertiolecta, Dunaliella primolecta, Dunaliella parva), but further investigation would be required to confirm their presence in Vietnam. We anticipate the physiological and biochemical characteristics of these local species will be compared with imported strains in a future effort. This will facilitate selection of strains with the best potential for exploitation in the food, aquaculture and biofuel industries. The Dunaliella strains isolated and identified in this study are maintained at the Laboratory of Algal Biotechnology, International University and will be made available for research and educational institutions.


INTRODUCTION
Unicellular green algae Dunaliella belong to the Chlorophytes (Oren, 2005). The algae was first described (Dunal, 1838), but it was not until 1905 that the name Dunaliella was given by Teodoresco (1905). There are currently 23 recognized species of Dunaliella (Pick, 1992;Oren, 2005). Dunaliella salina Teodoresco is the species whose vegetative cells turn yellow or orange with carotenoids in response to environmental stress such as high irradiance, high salinity, or low nutrient concentrations (Teodoresco, 1905;1906;Lamers et al., 2010;Fu et al., 2013;Tran et al., 2013). However, temperature, salinity and nutrients are limiting factors on the growth and development of Dunaliella (Polle et al., 2009).
Cells of Dunaliella are generally ovoid, 4-15 µm wide and 6-25 µm long, but depending on stages of growth or development and environmental conditions, the cell shape can vary from ovoid, ellipsoidal, cylindrical, pyriform, or fusiform to almost spherical (Butcher, 1959;Massjuk, 1973a;1973b). Dunaliella cells are motile with two equally long flagella. The main morphological characteristic of Dunaliella is the lack of a rigid polysaccharide wall (Gibbs and Duffus, 1976); instead, cells are covered by amorphous mucilaginous layer of variable thickness called a glycocalyx. Dunaliella cells contain a cup-shaped chloroplast with a pyrenoid in the center surrounded by starch which is the storage product. The nucleus is located in the colorless anterior portion of the cells (Baas-Becking, 1931).

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Currently Dunaliella is placed in the order of Chlamydomonadales and the family of Dunaliellaceae according to the NCBI database (Polle et al., 2009). Because the morphology of individual species of Dunaliella varies considerably under different stages of growth, development and environmental conditions, it is possible that some species have been previously misidentified (Borowitzka and Borowitzka, 1988;Gonzalez et al., 2005;Borowitzka and Siva, 2007;Tran et al., 2013). According to some more recent phylogenetic studies (Gonzalez et al., 2001;2005;Gomez and Gonzalez, 2004), it is believed that the total number of Dunaliella species may be less than 23. During the last decade, nuclear rDNA internal transcribed spacers ITS1 and ITS2 have been most commonly used to delineate Dunaliella species (Gonzalez et al., 2001;2005;Assuncao et al., 2013;Tran et al., 2013).
Dunaliella can be found on all continents in salterns and most hypersaline lakes and in oceans all over the world. Dunaliella has been found in the Great Salt Lake, the Dead Sea (Oren, 2005) and in Antarctic salt lakes, other salt lakes in America, Africa, Asia, Australia and Europe (Ginzburg, 1987;Borowitzka and Borowitzka, 1988;Tran et al., 2013). The Vinh Hao saltern (Binh Thuan province) is a key salt producing area in Vietnam. We were attracted there to investigate Dunaliella biodiversity, but more specifically to search for a strain of D. salina. Since the regional climate of this abundant seawater resource is also conducive to high marine algal productivity we were motivated to carry out this project. Our goal was to isolate a local strain of Dunaliella salina to compare with strains acquired elsewhere to identify the best candidates for the exploitation of beta-carotene, carbohydrate, lipid and protein production for the food, aquaculture and biofuels industries.
All cultures were maintained at room temperature (25°C), with a photon flux density of 50 µmoles photons/m 2 /s.

Dunaliella Identification
Dunaliella were first identified based on morphological characteristics (Teodoresco, 1906;Butcher, 1959;Massjuk 1973a;1973b;Polle et al., 2009). The isolates were grown in batch culture until nutrient limitation, at which Dunaliella salina cells would change color to yellow or orange (Polle et al., 2009;Lamers et al., 2010;Fu et al., 2013). The genomic DNA of these cells was isolated using DNeasy plant mini kit following instructions from Qiagen (Cat.No 69104). Isolated DNA was checked by electrophoresis on 1% agarose gel in 1X TAE buffer (50X TAE: 242 g Tris-base, 57.1 mL acetic acid, 100 mL 0.5M EDTA) and was quantified by spectrophotometer at an OD of 260 nm and stored at -20°C. Internal Transcribed Spacer (ITS) covering ITS1, 5.8S, ITS2 were PCR amplified using GoTag PCR core system II (Cat. No M7665) from Promega with pairs of primers (Forward: 5'TCCGTAGGTGAACCTGCGG3', Reverse: 5'GCATCGATGAAGAACGCAGC3'). The products were checked by electrophoresis on 1% agarose gel in 1X TAE buffer and were purified using PCR clean-up kit from Promega (Cat. No A9281).

Isolation and Cell Description
Using a light microscope, various isolates grown under different salinities were scanned to match the morphological descriptions of Dunaliella (Teodoresco, 1906;Butcher, 1959;Massjuk, 1973a;1973b;Polle et al., 2009). Dunaliella could not be identified in salinities of 1M, 2M, 3M. However, five Dunaliella isolates (A, B, C, D, E) were obtained in samples of 4M, 5M NaCl (Fig. 1). Generally cells of the Dunaliella were green, ovoid, 10 µm wide and 25 µm long. However, cells in sample A subject to nutrient limitation (Fig. 1a) turned orange and grew larger, presumably due to beta-carotene accumulation (Fig. 2a). These cells were motile with two equally long flagella in each cell ( Fig. 2a and b) and contained a pyrenoid (d) surrounded by starch. Pyrenoid). Note that Dunaliella salina cell turned orange, with larger size due to beta-carotene accumulation under nutrient limit There was thick mucus (b) outside the cell membrane. To further confirm these isolates as Dunaliella and to distinguish them among the different Dunaliella species, it was necessary to use molecular marker ITS to delineate (Gonzalez et al., 2001;2005).

Molecular Identification
A phylogenetic tree was constructed using ITS sequences from five isolates (A, B, C, D and E) and other ITS sequences data obtained from NCBI ( Table 1). Three main clades were clearly separated (Fig. 3). Clade A showed isolates B, C, D and E belonging to Dunaliella viridis with high boottrap value (100%). Clade B contained isolate A with other strains of Dunaliella salina and Dunaliella bardawil from NCBI data which is believed to be a strain of Dunaliella salina (Polle et al., 2009) (Boottrap value 98%). Other Dunaliella species (D. tertiolecta, D. parva, D. primolecta) all together in clade C (boottrap value 100%), which were well separated from group of D. salina, D. bardawil and D. viridis, could possibly be the same species but have been wrongly named due to erroneous morphological identification (Borowitzka and Borowitzka, 1988;Gonzalez et al., 2005;Borowitzka and Siva, 2007). Sequences of Chlamydomonas obtained from NCBI were used as the out group which formed a separate clade D. This is our first attempt to identify Dunaliella salina and Dunaliella viridis from this productive Vietnamese saltern based on morphology and molecular markers. Together, morphology and ITS sequences indicated that all isolates were Dunaliella, of which the isolate A was Dunaliella salina and the other isolates B, C, D, E were Dunaliella viridis (Fig.  3). Locally isolated D. salina will be compared to D. salina strains acquired elsewhere in order to select the best candidate for beta-carotene, carbohydrate, lipid and protein production. This future work falls outside the funding constraints of this manuscript.

CONCLUSION
We identified Dunaliella salina and Dunaliella viridis for in Vietnam based on morphology, physiology and molecular markers. Results of this study confirm the ubiquitous nature of Dunaliella and suggest that strains of Dunaliella salina might be acquired locally worldwide for the production of beta-carotene. The identification of these species infers the presence of other Dunaliella species (Dunaliella tertiolecta, Dunaliella primolecta, Dunaliella parva), but further investigation would be required to confirm their presence in Vietnam. We anticipate the physiological and biochemical characteristics of these local species will be compared with imported strains in a future effort. This will facilitate selection of strains with the best potential for exploitation in the food, aquaculture and biofuel industries.

ACKNOWLEDGEMENT
The researchers are grateful for the funding of Nafosted (Funding number: 106. 16-2011.31) and the funding of

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HCM National University to carry out this research (Funding number: B2012-28-02/HD-DHQT-QLKH). The authors would like to thank anonymous reviewers of this manuscript for correction and improvement.