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.

In this article, the system of the green microalgal genus Micractinium, based on morphological, physiological, ecological and molecular data, is considered. The main diagnostic species characteristics and the taxonomic placement of some taxa are also discussed. Phylogenetic analysis showed that the genus Micractinium is characterized by high cryptic diversity. The algorithms used for species delimitation had different results on the number of potentially species-level clusters allocated. The ABGD method was less “sensitive”. The tree-based approaches GMYC and PTP showed a more feasible taxonomy of the genus Micractinium, being an effective additional tool for distinguishing species. The clustering obtained by the latter two methods is in good congruence with morphological (cell size and shape, ability to form colonies, production of bristles, chloroplast type), physiological (vitamin requirements, reaction to high and low temperatures), molecular (presence of introns, level of genetic differences, presence of CBCs or special features of the secondary structure in ITS1 and ITS2) and ecological characteristics (habitat). The polyphyly of the holotype of the genus M. pusillum as well as M. belenophorum is shown. The intron was effective as an additional tool for distinguishing species, and the results of the intron analysis should be taken into account together with other characteristics. The CBC approach, based on the search for compensatory base changes in conservative ITS2 regions, was successful only for distinguishing cryptic species from “true” members of M. pusillum. Therefore, to distinguish species, it is more effective to take into account all the CBC in ITS1 and ITS2 and analyze characteristic structural differences (molecular signatures) in the secondary structure of internal transcribed spacers. The genetic distances analysis of 18S–ITS1–5.8S–ITS2 nucleotide sequences showed that intraspecific differences in the genus ranged from 0 to 0.5 % and interspecific differences, from 0.6 to 4.7 %. Due to the polyphasic approach, it was possible to characterize 29 clusters and phylogenetic lines at the species level within the genus Micractinium and to make assumptions about the species.