One alga to rule them all: unrelated mixotrophic testate amoebae (amoebozoa, rhizaria and stramenopiles) share the same symbiont (trebouxiophyceae).

Endosymbiosis is a central and much studied process in the evolution of eukaryotes. While plastid evolution in eukaryotic algae has been extensively studied, much less is known about the evolution of mixotrophy in amoeboid protists, which has been found in three of the five super groups of Eukaryotes. We identified the green endosymbionts in four obligate mixotrophic testate amoeba species belonging to three major eukaryotic clades, Hyalosphenia papilio and Heleopera sphagni (Amoebozoa: Arcellinida), Placocista spinosa (Rhizaria: Euglyphida), and Archerella flavum (Stramenopiles: Labyrinthulomycetes) based on rbcL (ribulose-1,5-diphosphate carboxylase/oxygenase large subunit) gene sequences. We further investigated whether there were different phylotypes of algal endosymbionts within single H. papilio cells and the degree of host-symbiont specificity by amplifying two genes: COI (mitochondrial cytochrome oxydase subunit 1) from the testate amoeba host, and rbcL from the endosymbiont. Results show that all studied endosymbionts belong to genus Chlorella sensu stricto, closely related to Paramecium bursaria Chlorella symbionts, some lichen symbionts and also several free-living algae. Most rbcL gene sequences derived from symbionts from all testate amoeba species were almost identical (at most 3 silent nucleotides difference out of 780 bp) and were assigned to a new Trebouxiophyceae taxon we named TACS (Testate Amoeba Chlorella Symbionts). This "one alga fits all mixotrophic testate amoeba" pattern suggests that photosynthetic symbionts have pre-adaptations to endosymbiosis and colonise diverse hosts from a free-living stage.