**5. Conclusions**

*Paramecium bursaria* is well adapted to harbour green algae as symbionts. Almost exclusively, either *Chl. variabilis* or *M. conductrix* are found in high abundances in the cytoplasm of this ciliate. One route to address the specificity of these associations is the molecular characterizations of host and symbionts. This approach provides information which symbioses are ecologically and evolutionary successful in the studied habitat. It is important to stress that therefore host and symbiont should be characterized in parallel, which is not always the case. Molecular approaches need to be combined with microscopical observations to ensure that indeed all partners are accounted for and none is overlooked or dismissed as environmental contamination. This might easily happen with minor symbionts in multiple infections or with much smaller and intracellularly less abundant picoalgae. Experiments to test the ability for symbiosis formation provide an additional perspective to the observed occurrence of associations in the environment. Infection assays can examine which interactions can be formed at e.g., species or genotype level. Thus, they provide insights into the genetic broadness of potentially realizable symbioses under constant laboratory conditions and allow to entangle the impact of additional biotic or abiotic factors shaping the formation and occurrence of symbioses. The present work can serve as a roadmap how such analyses can be conducted in regard to molecular as well as physiological characterization.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/8/287/s1, Table S1: Primer combinations and their PCR program specifications. Table S2: General PCR program to amplify the SSU rRNA gene. Table S3: Model parameters as defined by PAUP. Table S4: Combinations of aposymbiotic

*P. bursaria* strains and algae used in the re- and cross-infection experiments in this study. Figure S1: Diagnostic PCR for identification of *Micractinium conductrix*.

**Author Contributions:** Conceptualization and supervision, M.S.; methodology, F.E.F., T.P., and M.S.; formal analysis, F.E.F.; resources, A.P., T.P., and M.S.; data curation, F.E.F. and M.S.; visualization, F.E.F.; funding acquisition, F.E.F., T.P., and M.S.; project administration, T.P. and M.S.; writing—original draft preparation, F.E.F. and M.S.; writing—review and editing, F.E.F. and M.S. All authors read and approved the final version of the manuscript.

**Funding:** This research received external funding from the Albert Ludwig University of Freiburg (Innovationsfond, project number 2100297401; MS), the Austrian Science Fund (FWF, project P28333-B25; TP), the Wissenschaftliche Gesellschaft Freiburg i. Breisgau (MS), the Grünewald-Zuberbier-Stiftung (FEF), and from the European Community's H2020 Programme H2020-MSCA-RISE 2019 under gran<sup>t</sup> agreemen<sup>t</sup> number 872767 (MS).

**Acknowledgments:** We thank Bettina Sonntag for the opportunity to work at the Limnological Research Station Mondsee, Austria. We also thank Tatyana Darienko for her kind assistance with the morphology of green algae and in addition Birgit Süssenbach for preliminary molecular work and Chantal Bleile for her contributions to graphical design.

**Conflicts of Interest:** The authors declare no conflict of interest.
