**4. Discussion**

#### *4.1. Symbiotic Relationships and Specificity of Paramecium bursaria and Its Green Algal Endosymbionts*

Previous studies showed that aposymbiotic *P. bursaria* are able to re-establish symbiosis with their intracellular microalgae [19,72]. After uptake via phagocytosis, most of the algae are incorporated in digestive vacuoles. Some manage to escape the host's lysosomal fusion by 'budding off' into the cytoplasm being enclosed in perialgal vacuoles. Subsequently, those horizontally transmitted algae are distributed throughout the host cell as well as beneath the cell cortex [73]. Based on the characterization of natural *P. bursaria*-algae symbioses, it was speculated that the occurrence of certain intracellular algae species might depend on the geographic origin of *P. bursaria*. The observed endosymbionts separated into two groups [10,12,13,74–76], i.e., the "American/Southern" (later on described as *Chl. variabilis*) and the "European/Northern" (*M. conductrix*) group. Thus, a correlation between endosymbiont and host population was assumed. Another recent study presents contradicting results reporting the first *Chl. variabilis* from European *P. bursaria* [50].

Actual testing the specificity of symbiotic partners accepted by *P. bursaria* ([77], this study) revealed that all tested intracellular algae derived from this ciliate were accepted as endosymbionts. On the other hand, free-living algae and those obtained from *Hydra* were digested [77]. A competition experiment revealed a clear preference of the aposymbiotic *P. bursaria* strain for its native *Chlorella* strain [77]. In the here conducted re- and cross-infection experiments without of a competitive setting, algae-free *P. bursaria* did not show a preference for their original symbiont or symbiont species. Aposymbiotic strains formerly harbouring *Chl. variabilis* maintained *M. conductrix* as endosymbiont, and vice versa. The picoalga *Chor. parasitica*, naturally sharing the cytoplasm of its host with *M. conductrix*, was able to form a stable symbiosis independently of co-occurring microalgae and with cells naturally harbouring *Chl. variabilis*. Even though *P. bursaria* establishes an intimate and long-term stable symbiotic association with *Chor. parasitica*, the role of the picoalga for *P. bursaria* remains unclear. An evaluation of the symbiotic relationship between *P. bursaria* and the intracellular picoalga *Chor. parasitica* awaits further detailed analyses as current findings do not allow a placement of this interaction on the mutualism-parasitism continuum.

#### *4.2. Molecular Characterization of Paramecium bursaria and Its Green Algal Endosymbionts*

Unsurprisingly, the novel isolates of 'green' ciliates were unambiguously confirmed as *P. bursaria*. The phylogenetic tree inferred from SSU sequences is in overall good agreemen<sup>t</sup> with those previously published [5,42,44]. It already indicates intraspecific diversity, which becomes obvious in the analysis of the ITS region whose results reflect previous findings [47,50]. The analysis of the currently available ITS data might be interpreted that certain *P. bursaria* syngens harbour preferentially certain algal symbiont species. On the other hand, the number of symbiotic systems with both partners fully characterized is small, therefore this result should be considered very cautiously. It is likely that the picture will change with an increased amount of characterized strains. A revision of the *P. bursaria* species complex and conclusions about the suitability of certain syngens as hosts for specific algae await future studies.

The here characterized algal symbionts *Chl. variabilis* and *M. conductrix* are morphologically nearly identical [50]. However, they clearly differ in size from the additional algal symbiont detected in strain Frieds. The observed sizes in case of the microalgae were at the minimal range described for *M. conductrix* (5.0–12.0 μm, [10]) and in the typical size range for intracellular *Chor. parasitica* (1.5–3.0 μm) in case of the picoalgae. In the phylogenetic inference based on SSU-ITS sequences, the genera *Chlorella* and *Micractinium* were recovered as monophyletic and maximum support was obtained for the associations of the here studied microalgae with either *Chl. variabilis* or *M. conductrix*. The tree topology is nearly identical to that of previous studies [62,68] except for small differences lacking support in each analysis. The recovery of both algal species as endosymbiont of *P. bursaria* was not surprising, the detection of picoalgae in strain Frieds and their identification as *Chor. parasitica* more so. This is the first report of these picoalgae co-occurring with *M. conductrix* in the cytoplasm of *P. bursaria* and only the third report of *Chor. parasitica* as potential endosymbiont of *P. bursaria*. In previous observations the co-occuring primary symbiont was identified as *Chlorella* [31,40].
