Identification Problems and Cryptic Diversity of Chlorella-Clade Microalgae (Chlorophyta)

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.