**3. Results**

The isolation of the green algal endosymbionts using the method described above (Figure 2) was successful for some of the *Paramecium bursaria* strains (SAG 27.96, CIL-16, CIL-19, and CIL-20). However, it was not always successful and was too time-consuming. Therefore, we used the diagnostic PCR approach for the identification of the endosymbionts. The primer combinations CvarF/ITS055R and MconF/ITS055R were highly species-specific as shown in Figure 4 for both the isolated algae CCAP 211/84 (=NC64A) *Chlorella variabilis* and SAG 241.80 *Micractinium conductrix* and their host organisms CIL-16 and SAG 27.96.

**Figure 4.** Diagnostic PCR using the primer combinations CvarF/ITS055R ( **A**) and MconF/ITS055R (**B**). Mcon—*Micractinium conductrix* (SAG 241.80); Cvar—*Chlorella variabilis* (CCAP 211/84); reference hosts: *Paramecium bursaria* strains SAG 27.96 and strain CIL-16.

We tested this approach using these primer combinations on 18 *Paramecium bursaria* strains from di fferent geographical origin and various syngens (see Figure 5 for the origin of the strains). Additionally, we sequenced the SSU and ITS rDNA sequences of the ciliate strains (the accession numbers are given in Figure 5). Despite their low genetic variability among the isolates (only 1.7%), the phylogenetic analyses (Figure 5) of the SSU and ITS rDNA sequences confirmed their subdivision of *P. bursaria* strains into five lineages representing the known syngens R1–R5 [9], which was well or moderately supported in all bootstrap analyses.

**Figure 5.** Molecular phylogeny of the *Paramecium bursaria* based on SSU and ITS rDNA sequence comparisons. The phylogenetic tree shown was inferred using the maximum likelihood method based on the datasets (2197 aligned positions of 19 taxa) using computer program PAUP 4.0a167. For the analyses, the best model was calculated by PAUP 4.0a167. The setting of the best model was given as follows: GTR + I (base frequencies: A 0.2994, C 0.1832, G 0.2278, T 0.2896; rate matrix A-C 2.4955,

A-G 4.2183, A-U 4.9756, C-G 0.7513, C-U 9.6155, G-U 1.0000) with the proportion of invariable sites (I = 0.9303). The branches in bold are highly supported in all bootstrap analyses (bootstrap values > 50% calculated with PAUP using the maximum likelihood, neighbor-joining, and maximum parsimony). The clades are named after the syngens (color-coded) proposed by Greczek-Stachura et al. [9]. The accession numbers are given after the strain numbers. The endosymbiotic green algae identified using the diagnostic PCR are highlighted (Mcon—*Micractinium conductrix* and Cvar—*Chlorella variabilis*) after the origin of the *Paramecium bursaria* strains. The reference strain of each syngen is marked with an asterisk. The green algal endosymbionts isolated and available for further investigations are highlighted in blue.

Among the di fferent syngens, both endosymbionts were discovered using the diagnostic PCR approach. All ciliate syngens but not all strains harbored *C. variabilis*, whereas *M. conductrix* was only present in syngens R1 and R2, which originated from Europe (Figure 5). The ITS-2 sequences of these *Chlorella variabilis* (12 obtained with the primers CvarF/ITS055R) and *Micractinium conductrix* (6 obtained with the primers MconF/ITS055R) were identical to those of the reference strains NC64A = CCAP 211/84 (FN298923; [1]) and SAG 241.80 (FM205851; [1]), respectively.
