**3. Results**

#### *3.1. Bacterial Morphology and Localization*

The new HLB was found in macronucleus of *P. putrinum* YA111-52, isolated in the freshwater pond in Yakutia, Russia (Figure 1A). The infection was stable for at least three years under laboratory conditions. A small number of IFs could be found in the cytoplasm of the host cell (Figure 2), suggesting that there might be an intermediate state before the symbiont release into the environment. The endosymbionts were observed in two morphological forms of their life cycle: small (1–2 × 2–4 μm) bacteria undergoing binary fissions (RFs), and long (1–2 × 7–12 μm) IFs. Most observed IFs had straight rod-like shape with tapered ends, and some were slightly curved (Figure 1B). The symbionts were never observed in the micronucleus both in stably infected cultures and during the infection process. We also never observed the formation of the connecting piece during the host cell division (Figure 3), similar to what was previously described for species of the genus "*Ca.* Gortzia".

The endosymbionts are capable of infecting aposymbiotic cells of *P. putrinum*. IFs reach macronucleus and begin to divide in 20–30 h after infection forming chains of cells characteristic for HLB. Aposymbiotic cells of two *P. putrinum* clones belonging to the two different syngens were experimentally infected by IFs of the new symbiont. The native for the new HLB clone of *P. putrinum* YA111-52 belongs to the syngen 2. Infection of clones from both syngens remained stable for at least two months [20].

**Figure 1.** (**A**) *P. putrinum* with bacteria in the macronucleus; MA—macronucleus, MI—micronucleus; (**B**) Infectious forms of the new HLB released from the macronucleus.

**Figure 2.** *P. putrinum* with bacteria in the macronucleus, individual infectious forms in the cytoplasm shown with black arrowheads, white arrowhead shows IF presumably undergoing a binary fission. MA—macronucleus, MI—micronucleus.

**Figure 3.** *P. putrinum* during the division process. No connecting piece is observed.

## 3.1.1. Molecular Characterization

A total of 26 unique clones were sequenced, among which 21 were assigned to *Holospora*-like bacteria and the remaining 5 were affiliated with *Enterobacteriaceae*, according to the RDP classifier. A 1344–1346 bp long 16S rRNA sequences of the new HLB were deposited at GenBank under the accession numbers MT421875–MT421895. The similarity matrix calculated from multiple sequence alignment shows that the new HLB is closest to "*Ca*. G. shahrazadis" (98–98.2% similarity) and "*Ca*. G. infectiva" (97.4–97.7% similarity).

Using the FISH technique with the sequence-specific probe Gyak567 probe we detected many bacteria in the macronuclei of *P. putrinum* (Figure 4B). The Gyak567 probe bound to bacteria inside the macronucleus in our FISH experiments, thus demonstrating that the characterized 16S rRNA gene sequence had been derived from the new HLB. One of the IFs of the new HLB lies outside the macronucleus in the cytoplasm (marked with the arrowhead), thus confirming our observations, that IFs of the new endosymbiont can escape nucleus. The Eub338 probe was used as a positive control (Figure 4A), it hybridized with the new HLB, as well as with various bacteria in cytoplasm, which most likely are food bacteria.

**Figure 4.** Cells of *P. putrinum* with symbionts in macronuclei labeled with the probes Eub338 ( **A**) and Gyak567 (**B**). Single IF lying outside the macronucleus is shown with the white arrowhead.

## 3.1.2. Phylogenetic Analysis

The phylogenetic analysis confidently places the new HLB within the "*Ca.* Gortzia" branch as a sister taxon to two other "*Ca.* Gortzia" species, "*Ca.* G. infectiva" and "*Ca.* G. shahrazadis", macronuclear symbionts of *P. jenningsi* and *P. multimicronucleatum*, respectively. However, the level of sequence divergence of 1.8–2.0% and 2.3–2.5% of the new HLB with "*Ca.* G. shahrazadis" and "*Ca.* G. infectiva" respectively suggests that the new HLB is a separate species within the HLB clade and the genus "*Ca.* Gortzia". Two previously described "*Ca.* Gortzia" species show 0.7–0.9% divergence in their published 16S rRNA sequences. The difference of the new HLB with *Holospora* species ranges from 6.9% to 7.2% (Figure 5). Since HLBs are obligate endosymbionts and are not cultivable outside host cells, a complete culture-dependent characterization cannot be provided; hence, we propose the provisional name "*Ca.* Gortzia yakutica".

The phylogenetic tree shows a convincing monophyly of all *Holospora* and "*Ca.* Gortzia" species. (Figure 6).

**Figure 5.** Divergence of *Holospora*-like bacteria based on 16S rRNA gene. Pairwise sequence similarity was calculated from the trimmed multiple sequence alignment used for phylogeny inference.

**Figure 6.** Maximum likelihood phylogenetic tree of the order *Holosporales*. Bootstrap support values are shown on each branch. Clones, highly similar isolates, and 9 outgroup sequences from *Rickettsiales* are collapsed. Full list of Genbank sequence identifiers is available in Table S1.
