**3. Results**

#### *3.1. Microscopic Investigation of the Intracellular Algae*

Microscopic observations of the nine *P. bursaria* strains (Table 1) confirmed the presence of intracellular green algae (data not shown). In case of strain Frieds, algal cells belonging to two size categories were detected (Figure 2). Algae belonging to the larger category showed cell diameters of 4.08 ± 0.02 μm (referred to as microalgae) while the smaller algae had a diameter of 1.40 ± 0.20 μm (picoalgae). The picoalgae were localized individually throughout the cytoplasm, especially near the host's cell cortex. In addition, several of these picoalgae were observed aggregated in digestive vacuoles (Figure 2B). In a single occasion we even followed the fate of a digestive vacuole comprising more than 20 algal cells being emptied into the surrounding medium and thus *Chor. parasitica* were expelled.

**Figure 2.** *Paramecium bursaria* and its intracellular algae. (**A**) *P. bursaria* cell of strain Frieds with micro- and picoalgae distributed throughout the cytoplasm. (**B**) Digestive vacuole of the same cell incorporating multiple picoalgae. (**C**) Micro- and picoalgae simultaneously situated within the host's cytoplasm. Arrows indicate intracellular picoalgae. (**D**) Autofluorescence signal of the chloroplast from intracellular picoalgae of *P. bursaria* strain Frieds. Scale bars: 20 μm.

#### *3.2. Paramecium bursaria and the Five Syngens*

The phylogenetic inference of *Paramecium* species based on SSU rRNA gene sequences (Figure 3) recovers the expected topology and composition of the five subgenera of this genus. All here obtained sequences cluster within the monophyletic and maximal supported subgenus *Chloroparamecium*. Subgroups within the *P. bursaria* clade can be observed (Figure 3), but the achieved resolution does not allow unambiguous identification of syngen affiliations. In order to discriminate between the five syngens of *P. bursaria*, we analysed the ITS1-5.8S-ITS2-5'LSU region alone (Figure 4). While the here characterized sequences span from SSU till the beginning of the LSU (except for strain Ard10), the majority of publicly available sequences of *P. bursaria* strains with known syngen affiliation are limited to the ITS region. Sequences obtained here cluster either with (Figure 4) *P. bursaria* syngen R1 (Old-Pf and Scot), R2 (Bob2, Ek, Frieds, and RanNy), R3 (JPN and Tue2015), or R4 (Ard10). A potential correlation between the host's syngen affiliation and the present algal symbiont can be observed. Strains affiliated to syngens R3, R4, and R5 harbour *Chl. variabilis* while those assigned to syngen R2 contain *M. conductrix*. Only in case of syngen R1 both algae have been described as symbionts.

**Figure 3.** Molecular phylogeny of the genus *Paramecium* based on SSU rRNA gene sequences. Maximum likelihood tree based on 1493 aligned positions. GTR +I+G was used as evolutionary model. The respective subgenera are highlighted. Other members of Peniculida were used as outgroup. Bootstrap values above 70% and Bayesian Interference values above 0.95 are indicated. Asterisks indicate maximum support in both analyses. Numbers in brackets represent the number of sequences included in collapsed groups. Sequences marked in bold were obtained in this study.


**Figure 4.** Syngen affiliation of *Paramecium bursaria* strains based on the internal transcribed spacer (ITS) region spanning ITS1-5.8S-ITS2. Unrooted Maximum likelihood tree based on 506 aligned positions is shown. GTR+I was used as evolutionary model. Bootstrap values above 70 % and Bayesian Inference values above 0.95 are indicated. The five syngens are highlighted as R1 to R5, a plus marks a strain whose mating behaviour was previously experimentally determined. The identity of the algal symbiont if known is provided. Sequences marked in bold were obtained in this study.

#### *3.3. Chlorella variabilis, Micractinium conductrix, and Choricystis parasitica as Paramecium's Endosymbionts*

Phylogenetic inference of the intracellular microalgae of *P. bursaria* based on sequences spanning the SSU-ITS1-5.8S-ITS2 region (Figure 5) reveals the genera *Chlorella* and *Micractinium* as two distinct monophyletic clades. The intracellular microalgal strains JPN and Tue2015 cluster with other *Chl. variabilis* strains with maximum support in the phylogenetic analyses. Frieds, Old-Pf, and Scot affiliate with sequences belonging to *M. conductrix* with maximum support.

Comparing the conserved regions of ITS2 helices I-III of *Chl. variabilis* (barcode CVAR, [50]) to that of strains JPN and Tue2015, respectively *M. conductrix* (barcode MCON, [50]) to strains Frieds, Old-Pf, and Scot, no differences were observed (data not shown).

The sequence of the second intracellular alga detected in strain Frieds clusters with other sequences of the genus *Choricystis* as monophyletic sister group to the *Elliptochloris* clade with high support in both analyses (Figure 6). The obtained sequence is identical to that of several other *Chor. parasitica* strains.

**Figure 5.** Molecular identification of microalgal endosymbionts. Phylogeny of members of the Chlorellaceae based on sequences covering the SSU rRNA gene and the internal transcribed spacer region spanning ITS1-5.8S-ITS2. The shown tree was inferred by the Neighbour Joining method based on 2224 aligned positions. GTR+I+G was used as evolutionary model. Bootstrap values above 70 % and Bayesian Interference values above 0.95 are indicated (ML/BI/NJ). Asterisks indicate maximum support in all analyses. Members of the *Parachlorella* clade were selected as outgroup. Black circles indicate species capable of living in symbiosis. The sequences marked in bold were obtained in this study.

#### *3.4. Establishment of Symbioses*

We followed the fate of isolated algae after their uptake and differentiated between digestion, expulsion, and endosymbiotic maintenance by aposymbiotic *P. bursaria* cells via fluorescence microscopy. Within five days after exposure of aposymbiotic *P. bursaria* cells (Figure 7A,D), digestive vacuoles were observed in all paramecia predominantly occurring in the central part of the *Paramecium* cell with more than one enclosed alga regardless of the supplied species. Multiple perialgal vacuoles enclosing single algal cells were observed throughout the entire host cytoplasm. After 12 to 15 days p.i., numerous perialgal vacuoles were localized in the cytoplasm near the host's cell cortex in each examined *Paramecium* cell (Figure 7B,E). Successful re-establishment of symbiosis was achieved for all tested combinations (Supplementary Table S4) regardless of host background (former *Chlorella* or *Micractinium* host), syngen affiliation, and algal identity (*Chl. variabilis*, *M. conductrix*, or *Chor. parasitica*). Thus, we observed no differences between re- versus cross-infection and no preference of certain *P. bursaria* syngens for specific algae. After four months of cultivation, infected *P. bursaria* cells harbour numerous (200–500) intracellular algal symbionts. The observed symbiotic conditions are comparable to the natural state (Figure 7C,F). These cultures are stable since over five months and are maintained to date in the laboratory.

**Figure 6.** Molecular phylogeny of *Choricystis parasitica* and closely related members of the Chlorellaceae based on partial SSU gene sequences. Maximum likelihood tree based on 948 aligned positions. GTR+I+G was used as evolutionary model. Bootstrap values above 70 % and Bayesian Inference values above 0.95 are indicated. Asterisks indicate maximum support in both analyses. Dark circles indicate species capable of living in symbiosis, in case of *Choricystis* the symbiotic strains are highlighted. Species belonging to the *Prasiola* and *Neocystis* clades were chosen as outgroup. The obtained sequence is marked in bold.

**Figure 7.** Establishment of symbiosis between aposymbiotic *Paramecium bursaria* and intracellular algae. Re-establishment of symbiosis between aposymbiotic *P. bursaria* RanNy with *Micractinium conductrix* obtained from strain Scot; (**A**–**C**) and aposymbiotic *P. bursaria* JPN with *Choricystis parasitica* (**D**–**F**). Aposymbiotic *Paramecium* cells (**A**,**D**) obtained after treatment with cycloheximide. The white outline corresponds to the ciliate's cell shape based on bright field microscopy (not shown). Symbiotic status of *P. bursaria* after four to seven days (**B**,**E**). Successfully established symbiosis after two (**C**) to four (**F**) weeks. Representative examples are shown. Arrows indicate digestive vacuoles. Scale bars: 20 μm.
