Phylogenetic and Morphological Investigation of a Dunaliella Strain Isolated from Yuncheng Salt Lake, China

Plant 2014; 2(2): 20-26 Published online April 30, 2014 (http://www.sciencepublishinggroup.com/j/plant) doi: 10.11648/j.plant.20140202.12 Phylogenetic and morphological investigation of a Dunaliella strain isolated from Yuncheng Salt Lake, China Feipeng Wang, Jia Feng, Shulian Xie * School of Life Science, Shanxi University, Taiyuan 030006, China Email address: [email protected] (Shulian Xie) To cite this article: Feipeng Wang, Jia Feng, Shulian Xie. Phylogenetic and Morphological Investigation of a Dunaliella Strain Isolated from Yuncheng Salt Lake, China. Plant. Vol. 2, No. 2, 2014, pp. 20-26. doi: 10.11648/j.plant.20140202.12 Abstract: A Dunaliella strain was isolated from Yuncheng Salt Lake, Shanxi, China (111.05°E, 35.03°N). Morphological characteristics and molecular data were used to evaluate the relationship of this algal strain to other Dunaliella strains. Morphology of the isolated strain observed was close to Dunaliella salina . Phylogenetic trees were constructed from rbcL , psaB , ITS (ITS-1+5.8S rDNA+ITS-2) and 18S rDNA sequence data. Phylogenetic analysis of the four gene sequences revealed that the isolated Dunaliella strain is closely related to D. salina (Dunal) Teodoresco, D. peircei Nicolai et Baas-Becking, D. tertiolecta Butcher and more likely closer to D. salina . Combined morphological characteristics and phylogenetic analysis, the isolated Duanliella —designated here as D. sp YC01—should be a Dunalialla salina strain. Keywords: Dunaliella , Morphology, Phylogeny 1. Introduction The genus Dunaliella comprises a group of unicellular, characteristics, Massyuk [13-15] recognized 29 species, and biflagellated green algae that lack cell walls. Cells are ovoid, divided the genus into two subgenera, Pascheria and spherical, or ellipsoid [1]. Members of this genus are the Dunaliella . Members of the genus can adapt to extreme only eukaryotic, photosynthetic organisms known to grow environments. For example, D. acidophila is able to grow in over an extremely wide range of salt concentrations, varying highly acidic environments (pH 0–1), while a novel from 0.5 M NaCl up to saturation (5.5 M) [2, 3]. This salt subaerial species, D. atacamensis , has been described adaption ability is attributed to the production of growing on cave spider webs in the Atacama Desert [16]. intracellular glycerol, which acts as an osmotic compatible Because Dunaliella species lack cell walls, their solute [4]. Dunaliella may thus be a convenient model morphologies vary under different growing conditions, organism for studying salt adaptation in algae [5]. Under making characterization difficult. In spite of the excellent certain conditions, such as high light intensity exposure and work of Massyuk, several strains in culture collections are nutrient limitation, some Dunaliella species can accumulate misidentified, and some species described as separate high β-carotene levels equivalent to 10% of their dry entities may actually be polymorphic forms of the same biomass [6]. β-carotene is a major source of vitamin A, taxon [17]. Recent studies have confirmed that molecular necessary for proper retinal function, and affects many tissue methods are useful tools for distinguishing between types [7]. Humans cannot synthesize necessary carotenoids, Dunaliella taxa with similar morphologies at inter- and however, and must obtain them from their diets. Because intra-specific levels [18-19]. Combination of morphological Dunaliella is an ideal natural source of β-carotene, data with molecular methods should thus provide a better Dunaliella β-carotene production plants are in operation in understanding of Dunaliella taxonomy. Asia, North America, and Australia [8]. In an earlier study, Li and Xie [20] reported the existence Since the first formal description of Dunaliella by of Dunaliella in Yuncheng Salt Lake, Shanxi, China. In the Teodoresco [9], species of this genus have been described study reported here, we isolated and cultured the strain from from a wide variety of habitats [10-12]. Based on a Yuncheng Salt Lake, and performed a taxonomic and species-level analysis of morphological and structural phylogenetic analysis of this micro-alga using both features combined with physiological and biochemical morphological and molecular methods. Plant 2014; 2(2): 20-26 21 2. Material and Methods Dunaliella -specific primers MA1 (5'-CGGGATCCGTAGT- CATATGCTTGTCTC-3') and MA2 (5'-CGGAATTCCTT- 2.1. Sample Collection and Isolation CTGCAGGTTCACC-3') was used [23]. The chloroplast Samples were collected from Yuncheng Salt Lake, Shanxi, rbcL gene region was amplified using primers 475-497 China (111.05°E, 35.03°N; Fig. 1). Lake water pH was (5'-CGTGACAAACTAAACAAATATGG-3') and 7.2–7.6, and its salinity was above 0.1 g ml −1 . Isolation and 1181-1160 (5'-AAGATTTCAACTAAAGCTGGCA-3'). purification of Dunaliella cells was performed according to The nuclear rDNA ITS region (ITS-1+5.8S rDNA+ITS-2) previously described methods [21]. The isolated Dunaliella was amplified using universal primers AB28 strain was designated as D. sp. YC01 in this study. (5'-GGGATCCATATGCTTAAGTTCAGCGGGT-3') and TW81 (5'-GGGATCCGTTTCCGTAGGTGAACCTGC-3'). The chloroplast psaB gene was amplified using the primers DunpsaBFw22 (5'-ATTTGGGATCCACATTTTGGT-3') and DunapsaBRv753 (5'-TACTGAAGCTAAAGCTAAA-3') [16]. Polymerase chain reactions (PCRs) were performed in a MyCycler thermal cycler (Bio-Rad, California, USA). PCR amplifications of 18S rDNA, psaB , and rbcL regions were carried out as follows: initial denaturation at 94°C for 5 min, followed either by 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min for 18S rDNA, 30 cycles of 93°C for 1 min, 52°C for 1 min, and 72°C for 1 min for psaB , or 30 cycles of 94°C for 1 min, 62°C for 50 s, and 72°C for 1 min for rbcL , with a final extension of 8 min at 72°C. For the ITS (ITS-1+5.8S rDNA+ITS-2) region, PCRs were performed as described previously [19]. PCR products were analyzed by 1% agarose gel electrophoresis and purified using a Sangon agarose gel DNA extraction kit. The purified products were sent to Sangon Biotech (Shanghai, China) for sequencing. 2.4. Phylogenetic Analyses Phylogenetic analyses were performed using 18S rDNA, rbcL , psaB , and ITS sequence data. Multiple alignments incorporating sequences of other Dunaliella strains obtained a. The map of China. The shaded part is Shanxi; b. The map of Shanxi. The shaded part is the location of Yuncheng Salt Lake; c. The map of Yuncheng from the National Center for Biotechnology Information Salt Lake. The marked part is the sample collection location. (NCBI) nucleotide database (Table 1) were performed using Figure 1. Sample collection location CLUSTALW [24], with manual adjustments as needed. Phylogenetic trees were constructed from the aligned gene 2.2. Culture Conditions sequences using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian (BI) methods. NJ, ML, and The isolated and purified alga was cultured on medium BI analyses were performed using MEGA5 [25], PhyML 3.0 containing 2.05 M NaCl 17.64 mM, NaNO 3, 0.22 mM [26], and MrBayes version 3.1.2 [27], respectively. For NJ K2HPO 4, 0.3 mM MgSO 4·7H 2O, 0.32 mM CaCl 2·2HO 2, analyses, evolutionary distances were computed using the 0.18 mM Na 2CO 3, 0.02 mM C 6H8O7·H 2O, 10 µM Kimura 2-parameter method with 1000 bootstrap replicates. Fe(NH 4)3(C 6H2O7), 46.1 µM H 3BO 3, 9.39 µM MnCl 2·4H 2O, The program jModeltest [28] was used to determine the 0.77µM ZnSO 4·7H 2O, 1.61 µM Na 2MoO 4·2H 2O, 0.32µM best-fitting models of sequence evolution for ML and BI CuSO 4·5H 2O, and 0.172 µM Co(NO 3)·6H 2O. Dunaliella sp. methods. The model selected for the 18S rDNA data was YC01 was grown in a 250-ml Erlenmeyer flask containing TrN+I+Γ, with the following parameters used: invariant sites 100 ml medium at 25±1°C, which was manually shaken = 0.8532, gamma distribution = 0.6780; base frequencies A twice daily and subjected to a 12h:12h light/dark = 0.2494, C = 0.2100, G = 0.2755, T = 0.2651; rate matrix photoperiod provided by cool white fluorescent lamps. A-C = A-T = C-G = G-T = 1, A-G = 4.5699, C-T = 11.4595. For the rbcL gene, we used a TPM3uf +I+Γ model, with the 2.3. DNA Methods following parameters: invariant sites = 0.5750, gamma After approximately 3 weeks, cultured cells were distribution = 0.5830; base frequencies A = 0.2879, C = harvested by centrifugation for 10 min at 5000 x g. Genomic 0.2084, G = 0.1878, T = 0.3159; rate matrix A-C = C-G = DNA was extracted using the CTAB protocol [22]. For 0.0970, A-G = C-T = 1.1090, A-T = C-T = 1. Analysis of amplification of the nuclear 18S rDNA gene region, the psaB gene data was carried out under a GTR+Γ model, with gamma distribution = 0.2230 and base frequencies A = 22 Feipeng Wang et al. : Phylogenetic and Morphological Investigation of a Dunaliella Strain Isolated from Yuncheng Salt Lake, China 0.2417, C = 0.1841, G = 0.1850, and T = 03892. The model selected for the ITS region was TrNef+Γ, with gamma distribution = 0.4337, rate matrix A-C = A-T = C-G = G-T = 1, A-G = 2.5767, and C-T = 7.0137. D. sp. YC01 18S rDNA, rbcL , ITS, and psaB sequences were deposited in GenBank with accession numbers KF054056, KF054057, KF054058, and KF054059, respectively. 3 Results 3.1. Morphological Observations Dunaliella sp. YC01 cells were examined and photographed under an optical microscope (Olympus BX-51, Japan). The cells were varied in shape from pyriform to round or ovoid. The cells contained one cup-shaped Figure 2.

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