Molecular identification of four Dunaliella strains from Ukraine via ITS2 marker

Keywords: Chlorophyta, Dunaliella, ITS2, DNA barcoding, phylogenetic analysis

Abstract

Species of the genus Dunaliella are model organisms in algal physiology and widely used in phycotechnologies for commercial production of bioactive compounds, particularly carotenoids (mainly β-carotene). Reliable species identification is challenging due to low morphological variability and the absence of distinct species-specific traits in Dunaliella. Common traits such as cell size, shape, and pigmentation are strongly influenced by environmental factors, often leading to misidentification in culture collections and complicating comparative and applied research. Molecular approaches have thus become essential for accurate species identification. However, molecular data on Dunaliella from Ukraine remain scarce. The internal transcribed spacer 2 (ITS2) region is particularly informative, combining conserved secondary structures with rapidly evolving primary sequences. This study performed molecular identification of four Dunaliella strains from the Cherniaev Herbarium MicroAlgae Culture Collection (CWU-MACC), V.N. Karazin Kharkiv National University, using the ITS2 marker. Species identification was performed based on sequence similarity and phylogenetic analysis. Among the four Ukrainian strains, only CWU-MACC-15 corresponded to D. salina, while the others, including CWU-MACC-16, previously identified as D. salina, were assigned to D. viridis. The ITS2 sequences of CWU-MACC-16 and CWU-MACC-20 were identical, whereas the Henichesk Lake isolate showed a minor ITS2 variation within D. viridis. These results provide a molecular basis for the taxonomy of Dunaliella strains from Ukraine and highlight the need for expanded sampling and additional molecular markers to further resolve species diversity and phylogenetic relationships.

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Author Biographies

K. Fursova, V. N. Karazin Kharkiv National University

4, Svobody square, 61022, Kharkiv, Ukraine, k.v.fursova@gmail.com

M. Horpynchenko, V. N. Karazin Kharkiv National University

4, Svobody square, 61022, Kharkiv, Ukraine, gorpynchenko@karazin.ua

O. Utevska, V. N. Karazin Kharkiv National University

4, Svobody square, 61022, Kharkiv, Ukraine, outevska@karazin.ua

V. Komarysta, V. N. Karazin Kharkiv National University

4, Svobody square, 61022, Kharkiv, Ukraine, v.p.komarysta@karazin.ua

References

Arora N., Tripathi S., Philippidis G. P., Kumar S. (2025). Thriving in extremes: Harnessing the potential of pH-resilient algal strains for enhanced productivity and stability. Environmental Science: Advances, 4(6), 884—900. https://doi.org/10.1039/D4VA00247D

Assunção P., Jaén-Molina R., Caujapé-Castells J., De La Jara A., Carmona L., Freijanes K., Mendoza H. (2012). Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling. Aquatic Biosystems, 8(1), 2. https://doi.org/10.1186/2046-9063-8-2

Assunção P., Jaén‐Molina R., Caujapé‐Castells J., Wolf M., Buchheim M. A., De La Jara A., Freijanes K., Carmona L., Mendoza H. (2013). Phylogenetic analysis of ITS2 sequences suggests the taxonomic re‐structuring of Dunaliella viridis (Chlorophyceae, Dunaliellales). Phycological Research, 61(2), 81—88. https://doi.org/10.1111/pre.12003

Barbosa M., Inácio L. G., Afonso C., Maranhão P. (2023). The microalga Dunaliella and its applications: A review. Applied Phycology, 4(1), 99—120. https://doi.org/10.1080/26388081.2023.2222318

Avron M., Ben-Amotz A. (Ed.). (1992). Dunaliella: Physiology, biochemistry, and biotechnology. CRC Press.

Borowitzka M. A., Siva C. J. (2007). The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. Journal of Applied Phycology, 19(5), 567—590. https://doi.org/10.1007/s10811-007-9171-x

Buchheim M. A., Keller A., Koetschan C., Förster F., Merget B., Wolf M. (2011). Internal Transcribed Spacer 2 (nu ITS2 rRNA) Sequence-structure phylogenetics: Towards an automated reconstruction of the green algal tree of life. PLoS ONE, 6(2), e16931. https://doi.org/10.1371/journal.pone.0016931

Coleman A. W. (2009). Is there a molecular key to the level of “biological species” in eukaryotes? A DNA guide. Molecular Phylogenetics and Evolution, 50(1), 197—203. https://doi.org/10.1016/j.ympev.2008.10.008

Dunaliella Salina. (n.d.). CCAP. Retrieved January 2026 from https://www.ccap.ac.uk/catalogue/strain-19-18?mfp=61-archived%5BAvailable%5D&search=Dunaliella

Gordillo F. J. L., Jiménez C., Chavarría, J., Xavier Niell F. (2001). Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis. Photosynthesis Research, 68(3), 225—235. https://doi.org/10.1023/A:1012969324756

Highfield A., Ward A., Pipe R., Schroeder D. C. (2021). Molecular and phylogenetic analysis reveals new diversity of Dunaliella salina from hypersaline environments. Journal of the Marine Biological Association of the United Kingdom, 101(1), 27—37. https://doi.org/10.1017/S0025315420001319

Hoang D. T., Chernomor O., Von Haeseler A., Minh B. Q., Vinh L. S. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35(2), 518—522. https://doi.org/10.1093/molbev/msx281

Jalili V., Afgan E., Gu Q., Clements D., Blankenberg D., Goecks J., Taylor J., Nekrutenko A. (2020). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2020 update. Nucleic Acids Research, 48(14), 8205—8207. https://doi.org/10.1093/nar/gkaa554

Keller A., Schleicher T., Schultz J., Müller T., Dandekar T., Wolf M. (2009). 5.8S-28S rRNA interaction and HMM-based ITS2 annotation. Gene, 430(1—2), 50—57. https://doi.org/10.1016/j.gene.2008.10.012

Komarysta V.P. (2021) Collection of live cultures of microalgae in the herbarium of V.N. Karazin Kharkiv National University (CWU). Herbaria of the 21st century: achievements and challenges. Proceedings of International Scientific Conference dedicated to the 100th anniversary of the National Herbarium of Ukraine (KW) – the Herbarium of M.G. Kholodny Institute of Botany, NAS of Ukraine (1 October 2021, Kyiv). – Kyiv: M.G. Kholodny Institute of Botany, NAS of Ukraine, 2021. р. 64-68. (In Ukrainian)

Leung C., Grulois D., Chevin L. (2022). Plasticity across levels: Relating epigenomic, transcriptomic, and phenotypic responses to osmotic stress in a halotolerant microalga. Molecular Ecology, 31(18), 4672—4687. https://doi.org/10.1111/mec.16542

Li W., Godzik A. (2006). Cd-hit: A fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 22(13), 1658—1659. https://doi.org/10.1093/bioinformatics/btl158

Massjuk N.P. (1973). Morphology, taxonomy, ecology and geographic distribution of the genus Dunaliella Teod. and prospects for its potential utilization. Kiev, Naukova Dumka.

Massjuk N.P., Lilitska G.G. (2011). Dunaliellales. In: Algae of Ukraine: diversity, nomenclature, taxonomy, ecology and geography. Volume 3: Chlorophyta. (Tsarenko P.M., Wasser S.P., Nevo E. Eds), pp. 152-157. Ruggell: A.R.A. Gantner Verlag K.-G.

Nguyen L.-T., Schmidt, H. A., Von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution, 32(1), 268—274. https://doi.org/10.1093/molbev/msu300

O'Leary N. A., Wright M. W., Brister J. R., Ciufo S., Haddad D., McVeigh R., Rajput B., Robbertse B., Smith-White B., Ako-Adjei D., Astashyn A., Badretdin A., Bao Y., Blinkova O., Brover V., Chetvernin V., Choi J., Cox E., Ermolaeva O., Farrell C. M., Goldfarb T., Gupta T., Haft D., Hatcher E., Hlavina W., Joardar V. S., Kodali V. K., Li W., Maglott D., Masterson P., McGarvey K. M., Murphy M. R., O'Neill K., Pujar S., Rangwala S. H., Rausch D., Riddick L. D., Schoch C., Shkeda A., Storz S. S., Sun H., Thibaud-Nissen F., Tolstoy I., Tully R. E., Vatsan A. R., Wallin C., Webb D., Wu W., Landrum M. J., Kimchi A., Tatusova T., DiCuccio M., Kitts P., Murphy T. D., Pruitt K. D. (2016). Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Research, 44(D1), D733—D745. https://doi.org/10.1093/nar/gkv1189

Oren A. (2005). A hundred years of Dunaliella research: 1905–2005. Saline Systems, 1(1), 2. https://doi.org/10.1186/1746-1448-1-2

Oren A. (2014). The ecology of Dunaliella in high-salt environments. Journal of Biological Research-Thessaloniki, 21(1), 23. https://doi.org/10.1186/s40709-014-0023-y

Pasiuga O.S., Antonenko S.P., Komaristaya V.P., Rudas A.N. (2013). Variability of cultural and morphological traits of Dunaliella salina Teod. from different habitats. The Journal of V. N. Karazin Kharkiv National University. Series Biology, 18 (1079), 54–63.

Phytozome. (n.d.). Phytozome. Retrieved January 2026 from https://phytozome-next.jgi.doe.gov/info/Dsalina_v1_0

Preetha K., John L., Subin C. S., Vijayan K. K. (2012). Phenotypic and genetic characterization of Dunaliella (Chlorophyta) from Indian salinas and their diversity. Aquatic Biosystems, 8(1), 27. https://doi.org/10.1186/2046-9063-8-27

Ramachandran P., Pandey N. K., Yadav R. M., Suresh P., Kumar, A., Subramanyam R. (2023). Photosynthetic efficiency and transcriptome analysis of Dunaliella salina under hypersaline: A retrograde signaling mechanism in the chloroplast. Frontiers in Plant Science, 14, 1192258. https://doi.org/10.3389/fpls.2023.1192258

SAG 44.89 Dunaliella viridis. (n.d.). SAG catalogue of strains. Retrieved January 2026 from https://sagdb.uni-goettingen.de/detailedList.php?str_number=44.89

Schultz J., Maisel S., Gerlach D., Müller T., Wolf M. (2005). A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA, 11(4), 361—364. https://doi.org/10.1261/rna.7204505

Sun Z., Chen S. (2013). Identification of cortex herbs using the DNA barcode nrITS2. Journal of Natural Medicines, 67(2), 296—302. https://doi.org/10.1007/s11418-012-0681-8

Will S. (2024). LocARNA 2.0: Versatile simultaneous alignment and folding of RNAs. In R. Lorenz (Ed.), RNA Folding (V. 2726, P. 235—254). Springer US. https://doi.org/10.1007/978-1-0716-3519-3_10

Yu N., Wei Y., Zhang X., Zhu N., Wang Y., Zhu Y., Zhang H., Li F., Yang L., Sun J., Sun A. (2017). Barcode ITS2: A useful tool for identifying Trachelospermum jasminoides and a good monitor for medicine market. Scientific Reports, 7(1), 5037. https://doi.org/10.1038/s41598-017-04674-w

Zhang W., Yuan Y., Yang S., Huang J., Huang L. (2015). ITS2 secondary structure improves discrimination between medicinal “Mu Tong” species when using DNA barcoding. PLOS ONE, 10(7), e0131185. https://doi.org/10.1371/journal.pone.0131185
Published
2026-05-31
Cited
How to Cite
Fursova, K., Horpynchenko, M., Utevska, O., & Komarysta, V. (2026). Molecular identification of four Dunaliella strains from Ukraine via ITS2 marker. The Journal of V.N.Karazin Kharkiv National University. Series «Biology», 46, 6-15. https://doi.org/10.26565/2075-5457-2026-46-1
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BOTANY