**Algal Diversity in Identification, Detection of** *Choricystis parasitica***, and Assessment of the Interaction Specificity**

#### **Felicitas E. Flemming 1, Alexey Potekhin 2,3, Thomas Pröschold 4 and Martina Schrallhammer 1,\***


Received: 6 May 2020; Accepted: 19 July 2020; Published: 23 July 2020

**Abstract:** The 'green' ciliate *Paramecium bursaria* lives in mutualistic symbiosis with green algae belonging to the species *Chlorella variabilis* or *Micractinium conductrix*. We analysed the diversity of algal endosymbionts and their *P. bursaria* hosts in nine strains from geographically diverse origins. Therefore, their phylogenies using different molecular markers were inferred. The green paramecia belong to different syngens of *P. bursaria*. The intracellular algae were assigned to *Chl. variabilis*, *M. conductrix* or, surprisingly, *Choricystis parasitica*. This usually free-living alga co-occurs with *M. conductrix* in the host's cytoplasm. Addressing the potential status of *Chor. parasitica* as second additional endosymbiont, we determined if it is capable of symbiosis establishment and replication within a host cell. Symbiont-free *P. bursaria* were generated by cycloheximid treatment. Those aposymbiotic *P. bursaria* were used for experimental infections to investigate the symbiosis specificity not only between *P. bursaria* and *Chor. parasitica* but including also *Chl. variabilis* and *M. conductrix*. For each algae we observed the uptake and incorporation in individual perialgal vacuoles. These host-symbiont associations are stable since more than five months. Thus, *Chor. parasitica* and *P. bursaria* can form an intimate and long-term interaction. This study provides new insights into the diversity of *P. bursaria* algal symbionts.

**Keywords:** *Chlorella*; endosymbiosis; intracellular algae; *Micractinium*; photobiont; infection; syngen
