*3.2. Culture Handling and Maintaining*

The "green cells" of the microalgal isolate BM1 were cultivated in 400 mL of BG11 medium in 600-mL glass columns (6.6 cm internal diameter, 1.5 L·volume). The cultures were grown under continuous photosynthetically active radiation (PAR) illumination of moderate (40 ΐE·mƺ2·sƺ1) intensity as measured with a LiCor 850 quantum sensor (LiCor, Lincoln, NE, USA) in the center of an empty column by white light emitting diode source. For the induction of carotenogenesis, the cells were incubated in distilled water at high (480 ΐE·mƺ2·sƺ1) PAR intensity. The cultures were continuously bubbled with air (1 v·vƺ1·minƺ1); the temperature was maintained at 27 ° ʈ. 

To obtain a preliminary estimation of salinity tolerance of BM1, the cells were cultured in 250-mL flasks containing 80 mL of BG-11 medium supplemented with 25 g·Lƺ1 NaCl (BG-11 lacking NaCl was used as the control) in a shaker incubator at 100 RPM, 27 °C and 40 ΐE·mƺ2·sƺ1 PAR. 

Cell dry weight (DW) was measured according to [20]. Cells were counted in a hemocytometer. 

## *3.3. Molecular Identification*

To destroy the tough cell wall, the 1.5-mg cell samples collected for DNA extraction were subjected to three cycles of freezing in liquid nitrogen and subsequent thawing at room temperature. The samples were incubated for one hour in 300 ΐL of citrate-phosphate buffer (pH 5.0) containing 10 mg·mLƺ1 cellulase (Fermentas, Vilnius, Lithuania), 10 mg·mLƺ1 pectinase (Fermentas, Vilnius, Lithuania) and 1mM EDTA at 37 °C for one hour. The samples were incubated with 2% sodium dodecyl sulfate for one hour at 40 °C. Next 400 ΐL of 1M NaCl were added and allowed to stand overnight for protein salting. Then standard phenolchloroform extraction was performed [25]. The DNA sample purity was evaluated by electrophoresis in 1.5% agarose gel with ethidium bromide. 

For amplification of the 18S rRNA gene fragment, the following primers were designed using Gene Runner 4.0.9.68: 5<sup>ȝ</sup>-tggctcattaaatcagttatag-3<sup>ȝ</sup>, 5<sup>ȝ</sup>ccaagaatttcacctctgaca-3<sup>ȝ</sup>. Polymerase chain reaction (PCR) was performed on a Bio-Rad DNA engine (PTC 200, Hercules, CA, USA) using the amplification profile of 94 °C for 3 min initial denaturation, 94 °C for 20 s, 60 °C for 25 s, 72 °C for 35 s, 30 cycles; final elongation—72 °C for 5 min. The PCR mixture contained 10 ng of the algae genomic DNA in 25 ΐL of 1× PCR Buffer for Tersus (Evrogen, Moscow, Russia) containing 200 ΐM of each dNTP, 0.2 ΐM of each primer and 0.5 ΐL of 50× Tersus Taq polymerase (Evrogen, Moscow, Russia). The PCR products were purified using a Cleanup Standard PCR purification kit (Evrogen, Moscow, Russia) and sequenced on ABI Prizm 3730 (Applied Biosystems, Life Technologies, Grand Island, NY, USA) in both directions. 

Sequences were searched against the NCBI GeneBank (nucleotide collection nr/nt database) using BLAST. For the data analysis, 24 sequences from GenBank were selected. Multiple alignment of the sequences were conducted using ClustalW2 online tool [26]. There were a total of 782 positions and 25 taxa in the final dataset. The following parameters of pair-wise alignment were used Alignment type: slow, DNA weight matrix: IUB, gap open: 100, gap extinction: 10.0. The parameters of multiple alignments are given below. DNA weight matrix: IUB, gap open: 100, gap extinction: 10.0, gap distances: 10, no end gaps, iteration type: "tree", number of iterations: 10. Phylogenetic trees for multiple alignments were designed with using maximum likelihood (ML) method [27] in PhyML 3.0 [28] and neighbor-joining method (NJ) [29] in BioNJ [30,31].The best of nearest neighbor interchange (NNI) and subtree pruning and regrafting (SPR) were used for tree improvement in PhyML 3.0. HKY85 model of DNA evolution [32] were used. For other parameters of the analysis, the default values were left. Trees were rendered using program TreeDyn 198.3 [33]. The accuracy of the tree topology was tested using bootstrap analysis [34] with 1000 replicates. 
