Halotetraselmis gen. nov. and Halotetraselmis indica comb. nov. (Chlorodendrophyceae, Chlorophyta): New Halophilic Green Algae from Geographically Distant Hypersaline Habitats

To date, the taxonomic revision of the class Chlorodendrophyceae remains incomplete; the diversity of its halophilic representatives is poorly studied, and the precise phylogenetic position of Tetraselmis indica, its only known halophilic species, remains unresolved. Two novel halophilic green microalgal strains related to Tetraselmis indica, isolated from geographically distant hypersaline habitats (Crimean lakes and rivers of the Lake Elton basin), were examined using an integrative approach combining morpho-physiological characterization with multilocus molecular phylogenetic analysis (18S rRNA gene, ITS1-5.8S-ITS2 region, 28S rRNA gene, and rbcL gene); ITS2 metabarcoding was additionally used to clarify their distribution in the temperate zone of Eurasia. As a result, Halotetraselmis gen. nov. and H. indica comb. nov. were described. The early-diverging (basal) position of Halotetraselmis within Chlorodendrophyceae, the substantial genetic distances separating it from Tetraselmis, and its obligate halophily, together with the previously described morphological and chemotaxonomic differences, consistently support recognition of this lineage at the genus level. Genetic markers determined for H. indica for the first time (the 28S rRNA gene, the rbcL gene, and the ITS1-5.8S-ITS2 region) showed a differing diagnostic value for genus/subgenus-level versus species-level differentiation. Analysis of the ITS1-5.8S-ITS2 region and of ITS2 secondary structure, used for delimitation within Halotetraselmis, revealed potentially novel species among unidentified cultured strains. The combination of culture-based methods and metabarcoding yielded new data on the geographic distribution and ecology of the genus. These findings broaden current understanding of the diversity, phylogeny, and ecology of the class Chlorodendrophyceae and represent an important step toward a comprehensive taxonomic revision of the class.