**3. Results**

*Micractinium tetrahymenae* Pröschold, Pitsch, & Darienko sp. nov. (Figure 1A)

**Figure 1. A.** Morphology and phenotypic plasticity of *Micractinium tetrahymenae*, strain SAG 2587, **B.-C.** *Tetrahymena utriculariae* under anoxic (B) and oxygenic (C) conditions.

**Description**: Young cells are solitary, ellipsoidal up to broadly ellipsoidal; 3.1–4.2 μm in size. Mature vegetative cells are broadly ellipsoidal up to spherical, 4.8 × 4.9 μm up to 7.1 × 7.6 μm in size; rarely pyriform under suboptimal condition, 8.5 × 5.3 μm. Old cells are spherical up to 9.3 μm in diameter. Chloroplast is parietal cup-shaped possessing a single pyrenoid surrounded by starch grains. Cytoplasm is vacuolized. Asexual reproduction by autosporulation. The autospores are produced by 2–4 per cell. Autosporangia are 4.6 × 6.2 μm up to 6.3 × 7.4 μm. Release of autospores occurs after rupture of the mother cell wall. Bristle formation was not observed.

**Diagnosis**: Differs from morphologically similar *M. conductrix* and other free-living species of *Micractinium* through genetic signatures in SSU and ITS-2 rDNA sequences as well as in ITS-2 Barcode (see Section 4.2).

**Holotype** (designated here): The authentic strain SAG 2587 is cryopreserved in a metabolically inactive state at SAG under the number Z000694542.

**Type locality**: Facultative endosymbiont of *Tetrahymena utriculariae* (Oligohymenophorea, Ciliophora). **Etymology**: The name reflected the appearance in the host organism.

**Phylogenetic position and genetic signatures of the endosymbiont of** *Tetrahymena utriculariae***:** The SSU and ITS rDNA sequences of strain SAG 2587 (MT359915) were completely identical with those deposited in GenBank by Pitsch et al. [14] under the number LT605003. This endosymbiont clearly is the sister of *Micractinium pusillum*, based on the phylogenetic analyses of SSU and ITS rDNA sequences (Figure 2). The genus *Micractinium* is only highly supported in Bayesian analyses using the complex evolutionary models, which included the doublet and RNA7D functions (secondary structure

models implemented in MrBayes and PHASE, respectively; see details in Material and Methods). The maximum likelihood analyses using bootstrapping resulted in a high to moderate support for the genus *Micractinium*. In contrast, the common branch of the genus *Chlorella* was not supported in Bayesian analyses and only go<sup>t</sup> moderate values in bootstrap calculations. All analyses showed that the separation of *Micractinium* and *Chlorella* is not supported using simple evolutionary models and distance or parsimony methods (data not shown). However, both genera together were highly supported in all analyses questioning the separation into two genera. The other genera belonging to the *Chlorella* clade were highly supported in all of our analyses.

Within *Micractinium*, *M. tetrahymenae* sp. nov. is closely related to *M. pusillum*. The genetic variability of SSU rDNA among the species of *Micractinium* was very low (only 28 variable positions of 1783 bp = 1.6%). Even variable regions such as V4 showed only little changes (5 bases). Only the V9 region was partly diagnostic (Figure 3), being unique for both, *M. conductrix* and *M. pusillum*. In contrast, The V9 of *M. tetrahymenae*/*M. belenophorum* and *M. inermum*/*M. simplicissimum*/*M. singulare*/*M.variabile* were identical, respectively. The variability among the species was higher in the ITS-1 and ITS-2. The general structures of *M. tetrahymenae* are presented in Figure 4 and were similar to those of the members of *Micractinium* and other genera of the *Chlorella* clade. The ITS-1 and ITS-2 showed the typical four helices called helices 1–4 of ITS-1 and helices I-IV for ITS-2 according to Coleman and Mai [28]. The differences among the species in ITS-1 and ITS-2 showed that all species could be distinguished by characteristic compensatory base changes (CBCs and HCBCs) and loops (highlighted in white boxes in Figures 5 and 6). The base pair differences of V9 (SSU) and the conserved region of ITS-2 among the *Micractinium* species are summarized in Figure 7. In total, ten CBCs, seven HCBCs, and six insertion/deletions could be discovered (highlighted with an asterisk in Figure 7). By replacing base pairs with a number code, representatives of *Micractinium* received a unique barcode based on which species could be clearly recognized.

**Figure 2.** Comparison of the V9 of SSU and the conserved region of ITS-2 among the eight *Micractinium* species. Compensatory base changes (CBCs and HCBCs) and insertion/deletion are marked with an asterisk.

**Figure 3.** Molecular phylogeny of the Chlorellaceae based on SSU and ITS sequence comparisons. The phylogenetic trees shown were inferred using the maximum likelihood method based on the data sets (2602 aligned positions of 40 taxa), using PAUP 4.0a167. For the analyses, the best model was calculated by PAUP. The setting of the best model was given as follows: GTR + I + G (base frequencies: A 0.2112, C 0.2784, G 0.2743, T 0.2361; rate matrix A-C 0.7316, A-G 0.9716, A-U 0.9475, C-G 0.6216, C-U 3.2173, G-U 1.0000) with the proportion of invariable sites (I = 0.7266) and gamma shape parameter (G = 0.6963). The branches in bold are highly supported in all analyses (Bayesian values > 0.95 calculated with MrBayes and PHASE, 10 million generations; bootstrap values > 50%, calculated with PAUP, 1000 replicates using maximum likelihood, neighbor-joining, and maximum parsimony). The endosymbiotic species are marked with a green circle. The accession and strain numbers are given.

**Figure 4.** Secondary structure of the V9 region (Helix 49) of the SSU rDNA among the *Micractinium* species. The variable region within the V9 are highlighted in white boxes.


**Figure 5.** Secondary structure of the ITS-1 (**A**) and ITS-2 (**B**) rDNA of *Micractinium tetrahymenae*. The regions used for barcoding are highlighted in white boxes.


**Figure 6.** Variability of ITS-1 among the eight *Micractinium* species.

**Figure 7.** Variability of ITS-2 among the eight *Micractinium* species. The characteristic features within the conserved regions are highlighted in white boxes.
