The phylogenetic position and phenotypic changes of a Chlorella-like alga during 5-year microcosm culture

Abstract

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.

Effects of acidic conditions on the physiology of a green alga (Micractiniumsp.) before and after a 5-year interaction withTetrahymena thermophilain an experimental microcosm

“…In order to analyse the early evolutionary process of endosymbiosis between autotrophic and heterotrophic organisms, we developed an experimental ecosystem model, the CET microcosm, composed of a green alga (Micractinium sp., formerly described as Chlorella vulgaris), a bacterium (Escherichia coli) and a ciliate (Tetrahymena thermophila) Germond et al, 2013a). During the 3-year culture of this microcosm without transfer, we observed that ciliate cells harbouring living algal cells appeared at a frequency of up to 80-90% throughout the experiment .…”

“…and the ciliate (T. thermophila), and that at least some algal clones evolved a particular phenotype that benefits the ciliate. Genetic studies based on sequences of 18S and ITS regions (2700 bp length) showed no differences between the ancestral strains and 12 algal clones isolated from long-term microcosms (Germond et al, 2013a). Further studies using two algal clones (SC9-1 and SC10-2), isolated from within Tetrahymena cells, revealed that these clones released higher concentrations of glycerol and sucrose at pH 7.2 when compared with the ancestral strain at the same pH (Germond et al, 2013b).…”

“…Micractinium sp. Ehime, formerly described as Chlorella vulgaris, was used as the ancestral algal strain for the CET microcosm (Germond et al, 2013a). This alga was purified on agar before long-term microcosm culture and stored at −85°C in 15% glycerol.…”

“…The detailed procedure is described in Germond et al (2013a). The algal clones were purified and stored at −85°C in 15% glycerol.…”

“…To this end, we compared the phenotypes of the ancestral strain and the isolated algal clones under acidic conditions. So far, we have reported that the ancestral strain and some algal isolates among 12 clones had the ability to grow at pH 4.0 (Germond et al, 2013a).…”