Abstract—
The article considers the system of the Chlorella-clade based on morphological, ecological, and molecular genetic data. Diagnostic characteristics of some genera and species are discussed, as well as the systematic position of some taxa. Molecular phylogenetic analysis of members of the Chlorella-clade showed that the use of only the 18S rRNA gene, which is considered the main phylogenetic marker for green microalgae, does not reliably distinguish species and genera within the clade. Application of more variable spacers ITS1 and ITS2 resolved the tree topology and strengthened the phylogenetic signal. The polyphyly of the genus Chlorella was confirmed. Some of the species of the genus Chlorella formed a true clade of Chlorella species, including the type species C. vulgaris, C. heliozoae, C. pituita, C. chlorelloides, C. variabilis, and Lobosphaeropsis lobophora. Other species (C. pulchelloides, C. colonials, C. rotunda, C. singularis, C. elongata, C. sorokiniana, C. lewinii, C. volutis, and C. thermophila) were found to belong to other groups, and their taxonomic affiliation requires clarification. The CBC approach and the search for compensatory mutations in conservative ITS2 regions as a tool for distinguishing species was not effective for most Chlorella-clade representatives. Efficiency of using the intron as a criterion for separating closely related species was confirmed. Analysis of genetic distances made it possible to clearly identify the strains as species of the genera Didymogenes, Hindakia, and Heynigia. Members of the Chlorella-clade are characterized by a high cryptic diversity: none of the phenotypic characteristics considered separately was sufficient for unequivocal determination of the taxonomic position of members of the clade. Some morphological features were variable, often changed or became lost after instrumental manipulations during sample preparation for the water bodies monitoring. Using a combination of morphological, biochemical, ultrastructural, physiological, ecological, and molecular genetic features (a polyphasic approach), we were able to characterize eight groups within the Chlorella-clade and to make assumptions about the division of the genera and species within these groups.
Similar content being viewed by others
REFERENCES
Bock, C., Krienitz, L., and Pröschold, T., Taxonomic reassessment of the genus Chlorella (Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species, Fottea, 2011a, vol. 11, pp. 293–312.
Bock, C., Pažoutová, M., and Krienitz, L., Phylogenetic position of Coronastrum ellipsoideum and description of Parachlorella hussii sp. nov., Biologia, 2011b, vol. 66, pp. 585–594.
Bock, C., Pröschold, T., and Krienitz, L., Two new Dictyosphaerium-morphotype lineages of the Chlorellaceae (Trebouxiophyceae): Heynigia gen. nov. and Hindakia gen. nov., Eur. J. Phycol., 2010, vol. 45, pp. 267–277.
Caisová, L., Marin, B., and Melkonian, M., A consensus secondary structure of ITS2 in the Chlorophyta identified by phylogenetic reconstruction, Protist, 2013, vol. 164, pp. 482–496.
Coleman, A.W., The significance of a coincidence between evolutionary landmarks found in mating affinity and a DNA sequence, Protist, 2000, vol. 151, pp. 1–9.
Coleman, A.W., ITS2 is a double-edged tool for eukaryote evolutionary comparisons, Trends Genet., 2003, vol. 19, no. 7, pp. 370–375.
Coleman, A.W., Is there a molecular key to the level of “biological species” in eukaryotes?, A DNA guide, Mol. Phylogenet. Evol., 2009, vol. 50, pp. 197–203.
Chae, H., Lim, S., Kim, H., Choi, H.-G., and Kim, J.H., Morphology and phylogenetic relationships of Micractinium (Chlorellaceae, Trebouxiophyceae) taxa, including three new species from Antarctica, Algae, 2019, vol. 34, pp. 267–275.
Darienko, T., Rad-Menéndez, C., Campbel, C., and Pröschold, T., Are there any true marine Chlorella species? Molecular phylogenetic assessment and ecology of marine Chlorella-like organisms, including a description of Droopiella gen. nov., Syst. Biodivers., 2019, vol. 17, pp. 811–829.
Ettl, H. and Gärtner, G., Syllabus der Boden-, Luft- und Flechtenalgen, Stuttgart: Gustav Fischer, 1995.
Fawley, M.W., Dean, M.L., Dimmer, S.K., and Fawley, K.P., Evaluating the morphospecies concept in the Selenastraceae (Chlorophyceae, Chlorophyta), J. Phycol., 2006, vol. 42, pp. 142–154.
Guiry, M.D. and Guiry, G.M., AlgaeBase. World-Wide Electronic Publication, Galway: National University of Ireland, 2020. http://www.algaebase.org.
Hindák, F., The chlorococcal algal genus Didymogenes Schmidle 1905, Biologia, 1974, vol. 29, pp. 559–570.
Hoshina, R. and Fujiwara, Y., Molecular characterization of Chlorella cultures of the National Institute for Environment Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov. and Didymogenes soliella sp. nov. (Chlorellaceae, Trebouxiophyceae), Phycol.Res., 2013, vol. 61, pp. 124–132.
Hoshina, R., Iwataki, M., and Imamura, N., Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae, Trebouxiophyceae): redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year, Phycol.Res., 2010, vol. 58, pp. 188–210.
Hoshina, R., Kobayashi, M., Suzaki, T., and Kusuoka, Y., Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae, Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species, Phycol.Res., 2017, vol. 66, pp. 76–81.
Hoshina, R. and Nakada, T., Carolibrandtia nom. nov. as a replacement name for Brandtia Hoshina (Chlorellaceae, Trebouxiophyceae), Phycol.Res., 2018, vol. 66, pp. 82–83.
Katana, A., Kwiatowski, J., Spalik, K., Zakryś, B., Szalacha, E., and Szymańska, H., Phylogenetic position of Koliella (Chlorophyta) as inferred from nuclear and chloroplast small subunit rDNA, J. Phycol., 2001, vol. 37, pp. 443–451.
Komárek, J. and Fott B., Chlorophyceae (Grünalgen). Ordnung Chlorococcales, in Die Binnengewässer, Stuttgart: Huber-Pestalozzi, 1983, vol. 16, pt. 7 (1st half).
Korneva, L.G., Fitoplankton vodokhranilishch basseina Volgi (Phytoplankton of Reservoirs of the Volga Basin), Kopylov, A.I., Ed., Kostroma: Kostromskoi Pechatnyi Dom, 2015.
Krienitz, L., Hegewald, E.H., Hepperle, D., Huss, V.A.R., Rohr, T., and Wolf, M., Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae), Phycologia, 2004, vol. 43, pp. 529–542.
Krivina, E.S., Transformation of phytoplankton of small water bodies of urban territories under conditions of changing anthropogenic pressure, Extended Abstract of Cand. Sci. (Biol.) Dissertation, Togliatti, 2018.
Luo, W., Pflugmacher, S., Pröschold, T., Walz, N., and Krienitz, L., Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae), Protist, 2006, vol. 157, pp. 315–333.
Luo, W., Pröschold, T., Bock, C., and Krienitz, L., Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae), Plant Biol., 2010, vol. 12, pp. 545–553.
Ma, S., Han, B., Huss, V.A.R., Hu, X., Sun, X., and Zhang, J., Chlorella termophyla (Trebouxiophyceae, Chlorophyta), a novel thermo-tolerant Chlorella species isolated from an occupied rooftop incubator), Hydrobiologia, 2015, vol. 760, pp. 81–89.
Metodika izucheniya biogeotsenozov vnutrennikh vodoemov (Methods of Studying Biogeocenoses of Inland Water Bodies), Mordukhai-Boltovskii, F.D., Ed., Moscow: Nauka, 1975.
Müller, T., Philippi, N., Dandekar, T., Schultz, J., and Wolf, M., Distinguishing species, RNA, 2007, vol. 13, pp. 1469–1472.
Pröschold, T., Bock, C., Luo, W., and Krienitz, L., Polyphyletic distribution of bristle formation in Chlorellaceae: Micractinium, Diacanthos, Didymogenes and Hegewaldia gen. nov. (Trebouxiophyceae, Chlorophyta), Phycol.Res., 2010, vol. 58, pp. 1–8.
Pröschold, T., Darienko, T., Silva, P. C., Reisser, W., and Krienitz, L., The systematics of “Zoochlorella” revisited employing an integrative approach, Environ. Microbiol., 2011, vol. 13, pp. 350–364.
Shihira, I. and Krauss, R.W., Chlorella. Physiology and Taxonomy of Forty-One Isolates, Maryland: University of Maryland, College Park, 1965.
White, T.J., Bruns, T., Lee, S., and Taylor, J.W., Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in PCR Protocols: A Guide to Methods and Applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., Eds., San Diego: Acad. Press, 1990, vol. 3, pp. 315–322.
Wolf, M., Krienitz, L., and Hepperle, D., Phylogenetic position of Actinastrum hantzschii Lagerheim (Chlorophyta, Trebouxiophyceae), Algol. Stud., 2002, vol. 104, pp. 59–67.
Yamamoto, M., Kurihara, I., and Kawano, S., Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae), Planta, 2005, vol. 221, pp. 766–775.
Zou, S., Fei, C., Song, J., Bao, Y., He, M., and Wang, C., Combining and comparing coalescent, distance and character-based approaches for barcoding microalgaes: a test with Chlorella-like species (Chlorophyta), PLoS One, 2016, vol. 11, pp. 1–24, e0153833.
ACKNOWLEDGMENTS
The authors are especially grateful to Dr. M.A. Sinetova, the Head of the IPPAS Collection of Microalgae and Cyanobacteria (Institute of Plant Physiology, Russian Academy of Sciences) for providing Chlorella sp. IPPAS C1210.
Funding
This study was funded by the Russian Foundation for Basic Research (project no. 19-34-60002) and was partially supported by the Ministry of Science and Higher Education of the Russian Federation (project no. AAAA-A18-118013190177-9).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interests. The authors declare that they have no conflict of interest.
Statement on the welfare of animals. This article does not contain any studies involving animals or human participants performed by any of the authors.
Additional information
Translated by A. Panyushkina
Supplementary material
Rights and permissions
About this article
Cite this article
Krivina, E.S., Temraleeva, A.D. Identification Problems and Cryptic Diversity of Chlorella-Clade Microalgae (Chlorophyta). Microbiology 89, 720–732 (2020). https://doi.org/10.1134/S0026261720060107
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026261720060107