Differentiation of a free-living alga into forms with ecto- and endosymbiotic associations with heterotrophic organisms in a 5-year microcosm culture

The ecological mechanisms underlying the diversification of autotrophic species into endosymbiotic lifestyles and the ways in which the evolution of endosymbiotic species is ecologically and evolutionarily affected by sister lineages/lines that are adapted to extra-host environments remain unclear. In this paper, we investigated a differentiation process of algal species in which an endosymbiotic type was differentiated phenotypically from a free-living ancestral clone, by using an experimental model called the CET microcosm, which contains a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila) cultured together without an external resource supply for over 5 years. We then analyzed the algal diversification process by comparing algal phenotypic properties regarding cell-aggregate formation and their effects on the survival of Tetrahymena (using a clone isolated on day 2668) in the absence of bacteria. We examined 13 Micractinium clones, including both ancestral and derived clones isolated from long-term (day 1819-1847) CET microcosm cultures. The results revealed that the free-living ancestral algal strain diversified in sympatry into an aggregate-forming type that associates with E. coli, and a non-aggregate-forming type that associates with Tetrahymena. Furthermore, a competition experiment revealed that the endosymbiotic (non-aggregate-forming) type was less adapted to the extracellular environment than the aggregate-forming type. This result suggests that severe competition with a nonendosymbiotic sister line in the extra-host environment favors the host-benefiting phenotype in an endosymbiotic line, because such symbionts can enhance the longevity of the host, thereby enabling the survival and reproduction of the symbiont within the host.

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