Recent phylogenetic studies of Chlorella-like algae revealed that this group has diversified into free-living and endosymbiotic niches, the latter within protists and invertebrate organisms. Our previous studies using a long-term culture composed of an alga (Chlorella vulgaris, rDNA sequence unknown), a bacterium (Escherichia coli) and a ciliate (Tetrahymena thermophila), suggested that some clones in the algal population developed an endosymbiotic ability with T. thermophila while others developed an ectosymbiotic ability with E. coli from a non-associated stage. In this paper, an rDNA (18S, ITS1, 5.8S, ITS2)-based phylogeny of the ancestral strain and derived clones isolated from 5-year microcosm cultures was constructed and revealed that the alga belongs to the genus Micractinium. This result was supported by analysis of the secondary structure of the rRNA gene. No difference was observed in the sequence between the ancestral and derived clones. On the other hand, some morphological and physiological traits of the ancestral strain and the derived clones were analysed and revealed that several phenotypic changes had occurred among the algal clones. In particular, the derived clones obtained from long-term cultures had increased in cell size, changed in their ability to grow at pH 4.0, and developed a tendency for cell-aggregation or sedimentation.

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