CHAMAESIPHON FONTINALIS SP. NOV., A NEW SPECIES OF UNICELLULAR ASYMMETRICALLY DIVIDING CYANOBACTERIA

Extensive brownish biofilms of algal-bacterial communities were found on stones in streams in the Valdaysky National Park (Russia). The dominant unicellular cyanobacteria in biofilms was determined as Chamaesiphon sp. Although species of this genus often occur in the periphyton of fast-flowing waters, many of them have a geographically limited distribution and occur mainly in mountain streams and rivers in Europe. The strain Chamaesiphon sp. CALU 1864 was isolated from epilithic biofilms in the spring Tekunok. We found that it differs from previously described species not only by its unusual purple pigmentation, but also by morphological features. Molecular-genetic analysis of the primary 16S rRNA sequence and secondary structures of the internal transcribed spacer of the ribosomal operon also confirmed its uniqueness. Phylogenetically, the strain represents an independent lineage on the evolutionary tree. Based on the differences found, which provide strong support for its delineation, we propose to consider strain CALU 1864 as a new species, Chamaesiphon fontinalis sp. nov., first discovered in Russia and described in accordance with the current rules of the International Code of Nomenclature for Algae, Fungi and Plants.


Introduction
Cyanobacteria form a morphologically heterogeneous group of photosynthetic bacteria, distributed in a variety of ecotopes (Komárek & Anagnostidis, 1998;Castenholz, 2015). Unicellular, asymmetrically dividing cyanobacteria, belonging to the genus Chamaesiphon Braun, are known as biofoulers, usually found epilithically (forming spots on stones) or epiphytically (as single cells or groups of cells attached to aquatic plants and filamentous algae) in freshwater springs, streams and ponds (Rabenhorst, 1865;Komárek & Anagnostidis, 1998;Hoffmann et al., 2005;Casamatta & Hašler, 2016). Chamaesiphon spp., together with the representatives of the genera Geitleribactron, Clastidium, and Cyanophanon (the last two have not yet been studied by molecular-genetic methods), constitute the family Chamaesiphonaceae Borzi (phylum Cyanobacteria Stanier ex Cavalier-Smith, class Cyanophyceae Schaffner, order Gomontiellales Strunecký & Mareš) (Hoff -) (Hoff - (Hoffmann et al., 2005;Komárek et al., 2014;Guiry & Guiry, 2022;Strunecký et al., 2023). Species of the family Chamaesiphonaceae differ by cell shape and size, as well as in the type of colonies. In addition, cells of the same species have various morphotypes, which alternate depending on the stage of development: mature cells are heteropolar (have a distinct apical and basal parts) and form small rounded daughter cells (exospores) by means of asymmetric binary fission, also called budding (Herdman et al., 2015). Thus, the cells of Chamaesiphon spp. can vary from round to ovoid or pear-shaped (cylindrical) during the life cycle (Komárek & Anagnostidis, 1998).
In order to expand our knowledge about the biodiversity of algae in water bodies of the Protected Areas of the Northwest Russia, several field expeditions have been carried out in the Valdaysky National Park since 2012 (Smirnova, 2014(Smirnova, , 2015(Smirnova, , 2017Smirnova & Beljakova, 2016). This Protected Area (total area is about 1585 km 2 ) is located on the conditional border of the subzones of southern taiga and coniferous-deciduous forests. In the Valdaysky National Park, there are 257 lakes (five are relatively large and the others are small) and many rivers belonging to the basins of the River Msta, River Volga, and River Western Dvina (Zapadnaya Dvina). The density of the river network is estimated at 0.80-0.98 km/km 2 . Most of these rivers are small, either fast-or slow-flowing (with varying degrees of the trophic state), with abundant aquatic vegetation. The majority of the water bodies are slightly saline, hydrocarbonate (salinity does not exceed 200 mg/l), with a pH range of 6.8-8.3 (Efimova & Frolova, 2013). Rivers in the Valdaysky National Park are mainly fed by springs, in which we often found stones, cov-ered by algal-bacterial communities in the form of brownish biofilms on their surface. In various samples collected in streams near the villages of Novotroitsy, Moiseevichi, and Russkie Novinki, we observed Chamaesiphon species that predominate among unicellular cyanobacteria, whereas Heteroleibleinia sp. (Geitler) Hoffmann, Leptolyngbya crassior (Skuja) Anagnostidis, Microcoleus fonticola (Kirchner) Strunecky, Komárek & Johansen, Phormidium favosum Gomont, and Pseudanabaena minima Anagnostidis are abundant among filamentous cyanobacteria, along with some eukaryotic microalgae (Smirnova, 2021).
The aim of this study is to describe a new species of Chamaesiphon found in the Valdaysky National Park by the polyphasic taxonomic approach (Komárek, 2016). For this purpose, a laboratory strain was obtained from a natural specimen; its morphology, physiology, pigment composition, and the molecular-genetic features (16S rRNA and secondary structures of the internal transcribed spacer sequences) were determined.

Sampling site location
The Chamaesiphon strain CALU 1864 was isolated from epilithic biofilm samples collected from stones in the forest spring Tekunok, flowing into Lake Golova in the Valdaysky National Park (58.114919° N, 33.290215° E) (Fig. 1). The water temperature in spring depends on the season and ranges from 4.0°C to 9.5°C; the pH value is around 7.0.

Strain isolation
Biofilm samples were scraped off the stones and placed in 25-ml glass tubes with BG11 medium (Stanier et al., 1971), modified by replacing FeSO 4 for ferrous citrate and 5-fold reduction in NaNO 3 . It also contained 250 mg × ml -1 cycloheximide (Oxoid, UK) to suppress eukaryotic microalgae growth. Samples were incubated under continuous illumination (10-15 µmol photons m -2 × s -1 ) with a cool-white light lamp, at 12°C. Strain isolation was performed by transferring cells with the Chamaesiphon morphotype to Petri dishes containing 1% agarised BG11, using standard microbiological methods (Rippka et al., 1979). Cultivation was carried out by periodic re-inoculations from agarised to liquid BG11 medium in 25-ml glass tubes or in 50-ml Erlenmeyer flasks. After undergoing the axenisation procedure, the obtained strain Chamaesiphon was deposited in the collection of microorganisms as CALU 1864 (Saint Petersburg State University, Russia, https://researchpark.spbu.ru/ en/collection-ccem-eng/1930-ccem-kollekciyacalu-eng), and was placed in the algal herbarium of Komarov Botanical Institute RAS (LE) as dry material.
To learn more about the strain physiology, cell growth curves were evaluated in batch liquid cultures incubated at various temperatures (7°C, 15°C, 25°C). Cells were mechanically suspended using a glass tube homogeniser and their density measured at 750 nm by BioSpectrometer (Eppendorf).

Morphological studies
Basic morphological features were analysed under a Leica DM 2500 microscope, equipped with a digital camera with the help of differential interference contrast. The average cell size was calculated by measuring about 100 cells of each morphotype. Transmission electron microscopy (TEM) was used to study the ultrastructure. Cell material was prepared as described by Mareš et al. (2019). Briefly, cells were fixed in 0.1-M Na-phosphate buffer (pH 7.0), with 2.5% glutaraldehyde for 2 h at 10°C and then post-fixed with 1.0% OsO 4 (at 10°C, 12 h). Fixed cells were washed with 0.15-M Naphosphate buffer, then dehydrated in a series of ethanol (50-100%) and embedded in LR White resin according to the manufacturer's protocol (Sigma-Aldrich). Ultrathin sections of cells were made by Leica EM UC7 microtome. Sec-tions were post-stained with 1% uranyl acetate and lead citrate according to Reynolds (1963). TEM microphotographs were taken with a JEM 1400 microscope at 80 kV. Cell structures and inclusions were determined as described by Stanier & Cohen-Bazire (1977).

Pigment analysis and chromatic adaptability
Cells were cultured in liquid BG11 under the previously described conditions for one month, followed by harvesting by centrifugation (5000 g, 10 min). Pigments were extracted from ~ 100 mg (wet weight) of cell pellets with 100% methanol at 10°C, 60 min. To remove large particles, the lysate was centrifuged (10 000 g, 20 min). The absorption spectra of the extracted pigments were recorded at 300-700 nm on a BioSpectrometer (Eppendorf).
Chromatic adaptation was studied by incubating liquid cultures under red or green Lee 108 and 124 light filters at maximum transmissions above 575 nm and at 475-560 nm, respectively. After two weeks, the cells were harvested by centrifugation (5000 g, 10 min). The phycobiliprotein (PBP) fraction was obtained from ~ 100 mg (wet weight) of pellet suspended in 500 μl of 0.1 M Na-phosphate buffer (pH = 7.0) by 3-4 repeated freeze/thaw cycles (from -18°C to 45°C). The resulting lysate was centrifuged (10 000 g, 20 min) to remove cell debris. The absorbance of the extracted PBPs was recorded at 300-700 nm on a BioSpectrometer (Eppendorf).

DNA isolation, PCR and sequencing conditions
Genomic DNA was obtained from harvested cell material (~ 200 mg of wet weight), washed twice with 10 mM TE buffer (Tris-HCl buffer, pH = 8.0 containing 1 mM Na 2 -EDTA). DNA was extracted by CTAB (cetyltrimethylammonium bromide) according to the protocol of Doyle & Doyle (1990), and then treated with RNAse A (Thermo Fisher Scientific, Germany) at 37°C, 15 min.
PCR-fragments were amplified on a T100 thermocycler (Bio-Rad, USA). The protocol included an initial denaturation at 95°C (3 min), followed by 35 cycles: 95°C, 30 s, annealing at 55°C, 30 s and elongation at 72°C, 1.3 min. PCR products and DNA sample were checked by gel electrophoresis on 1.0% (w/v) RA agarose (VWR Life Science, Amresco) in standard TBE (Tris-borate/ EDTA) electrophoresis buffer, and visualised by staining with 0.05% ethidium bromide. PCR products were sequenced with the Big Dye Terminator v3.1 Cycle Sequencing kit on an ABI Prism 310 Genetic Analyser (Applied BioSystems) according to the manufacturer's instructions, at the Research Centre «Molecular and Cell Technologies» (Saint Petersburg State University, Russia).

16S rRNA and 16S-23S ITS analysis
A pairwise sequence similarity search performed using the BLASTn tool (https://blast.ncbi. nlm.nih.gov). The p-distance of 16S rRNA was determined using Mega 5.0 (Tamura et al., 2011), and calculated as follows: Multiple sequence alignment (ClustalW) and phylogeny reconstruction were obtained using the CIPRES Science Gateway ver. 3.3 (Miller et al., 2010). For 16S rRNA phylogeny, the GTR + I + G model of nucleotide substitution with the lowest Bayesian Information Criterion (BIC) score was selected by jModeltest 2 (Darriba et al., 2012). For the Bayesian Inference (BI) tree, two runs of four Markov chains were used until the standard deviation of the splitting frequency was less than 0.01 (diagnosis was calculated every 1000 generations). The initial 25% of the sampled trees were discarded as burn-in and the rest was used to calculate the posterior probabilities (Ronquist et al., 2012). A maximum likelihood (ML) tree was calculated in IQ-TREE v. 6.1, generating 1000 samples for ultrafast bootstrap and 1000 bootstrap replications (Trifinopoulos et al., 2016). The resulting trees were visualised in FigTree v.1.4. Evolutionary distances in the ML/BI tree were calculated, and bootstrap values below 50 and probabilities below 95 were omitted. ML and BI phylogenetic trees with similar topology involved 52 sequences, with a total of 1067 positions in the final dataset. The robustness of our phylogenetic findings was achieved through different tree reconstruction approaches.

Results and Discussion
A new microorganism belonging to the genus Chamaesiphon was discovered as a result of field research in the Valdaysky National Park. The strain differs from the currently described species by morphological features, such as the size and shape of pseudosporangia and an unusual crimson-brown colour of the cells. The evolutionary divergence of the strains was verified by phylogenetic analysis. The relatively low similarity of the 16S rRNA gene with other Chamaesiphon species and the specific organisation of secondary structures 16S-23S ITS regions confirm its uniqueness. On this basis, we propose that strain CALU 1864 to be considered as a new species, described in accordance with the rules of the International Code of Nomenclature for algae, fungi, and plants (Turland et al., 2018).

Description of the new species Chamaesiphon fontinalis Velichko, Makeeva, Averina & Smirnova
Diagnosis. In nature it occurs in extensive brownish biofilms on the upper surface of stones in a cold stream (spring). The laboratory strain in liquid medium forms a dense darkcrimson or reddish-brown biofilm on the walls and bottom of glassware, or grows as convex, round-lobed, brown colonies on solidified medium (Fig. 2).
Cell size and shape depend on the stage of the life cycle: unequal binary fission usually occurs near the apical pole of the cell and results in 1-2 (rarely 3) small rounded exospores. The cells can vary from round or ovoid to pear-shaped or cylindrical. Cells are 6.6-16.7(25.2) × 5.5-8.2(11.1) µm in size with a length-to-width ratio close to 2 : 1. They have a thick (up to 2 µm), colourless U-shaped pseudovagina (a multilayered extracellular sheath). After cell division, the pseudovagina delaminates at the apex, releasing exospores.
Cell contents are heterogeneous, brownishcrimson in colour, with prominent cytoplasmic granules. Exospores are about 3 µm in diameter; in the case of a large mother cell, they can reach 6 µm. The cells showed parietal arrangement of thylakoids (3-5 layers) and the presence of carboxysomes, polyphosphates, and glycogen inclusions.
Type locality. Biofilms occur on the surface of the upper part of stones in spring Tekunok, located in the Valdaysky National Park (58.114919° N, 33.290215° E), Novgorod Region, Russia.
Reference strain. Chamaesiphon fontinalis strain CALU 1864 was deposited in the collection of cyanobacteria and algae of the Saint Petersburg State University, Saint Petersburg, Russia. GenBank accession numbers: OP415410 for 16S rRNA and OP429223 for 16S-23S ITS. Isotypes. These are LE A0003534, LE A0003535 and LE A0003536 herbarium material.
Etymology. The choice of the specific epithet for C. fontinalis sp. nov. is based on the fact that the species was found in a cold stream (spring) (L. masc. adj. fon.ti.naʹlis, L. masc. nom. fons, originating from spring).
Ecology. It was found only in the type habitat in epilithic periphyton. The water temperature in the spring on the sampling day was 9.0°C, and the pH was about 7.0.

Differential diagnosis
The new species can be clearly distinguished from the type species, C. confervicola, by cell morphology and habitat type. Chamaesiphon confervicola is an epiphyte of aquatic plants and green filamentous algae Cladophora Kützing and Oedogonium Link ex Hirn. It usually grows as single cells or cells in groups, and has large elongated cells up to 200 µm in length (Komárek & Anagnostidis, 1998). Chamaesiphon confervicola has been described only morphologically so far, and we have no data on its successful cultivation.
Chamaesiphon fontinalis sp. nov. has similar morphology with C. geitleri Luther and C. fuscus (Rostafinski) Hansgirg: oval, oval-elongated or cylindrical pseudosporangium, with a length to width ratio of 2 : 1. But the new species differs from them in the type of colonies (not bushy-like), the size of the pseudosporangium (it is on average smaller), and the shape (rarely club-shaped cell), the cell colour (brownish-red, rather than pale) and the colour of the pseudovagina (colourless, in contrast to brownish or blackish). The morphospecies C. starmachii is the most similar to С. fontinalis sp. nov. It also has a pear-shaped or ellipsoidal type of pseudosporangium, similar cell size (6-18 × 4.0-7.2 µm), a reddish-violet or purple colour of the cell content and prominent granules in the cytoplasm. The exospores are more or less rounded-polygonal, may remain attached 1-3 to the mother cell when the sheath is closed (Komárek & Anagnostidis, 1998). However, C. starmachii differs from C. fontinalis sp. nov. by a thicker black pseudovagina (Kurmay-thicker black pseudovagina (Kurmayer et al., 2018). Notably, the dark pigmented coatings of C. geitleri and C. starmachii are thought to be due to the presence of UV-protectors such as scytonemin (Aigner et al., 2018).

Morphology and culture properties
Chamaesiphon fontinalis sp. nov. forms con-forms convex, round-lobed, slightly slimy, brown colonies of 1-2 mm in diameter that exhibit the ability to grow towards a light source under laboratory conditions on solidified medium (Fig. 2a). In a liquid unstirred culture, strain CALU 1864 grows on the walls of glassware and forms a brownish or dark crimson biofilm, in which cells are arranged irregularly in 1-3 layers (Fig. 2b,c,d) or can form rosette-like structures (Fig. 2e). They are tightly pressed together in the biofilm and have a polygonal shape when viewed from above (Fig. 2b). It is noteworthy that strains of C. subglobosus PCC 7430 and C. polonicus SAG 32.87 grow in liquid cultures in irregular groups, and are unable to form colonies, as in nature (Waterbury & Stanier, 1977;Komárek & Anagnostidis, 1998;Kurmayer et al., 2018).
Morphological features such as cell size and shape are very important for distinguishing species of the genus Chamaesiphon, but they change during the life cycle. In this regard, the laboratory cultivation is therefore of great importance. Our long-term observations have shown that the shape and size of pseudosporangia of C. fontinalis sp. nov. depend on the age of the studied culture. In young cultures (up to 4-6 months), cells are round or almost ovoid (6.6-8.6 μm in length and 5.5-7.1 μm in width; the average length to width ratio is 1.4 : 1.0) and covered with a colourless sheath (Fig. 2f,g,h). It is noteworthy that, unlike novel strain, C. subglobosus PCC 7430, C. minutus PCC 6605, and C. cf. incrustans №1036 lost their sheaths under laboratory conditions (Herdman et al., 2015;Kurmayer et al., 2018). In old cultures (over six months), most cells of C. fontinalis sp. nov. are pear-shaped, club-shaped or elongated (8.0-16.7 μm in length, 6.0-8.2 μm in width; average length to width ratio is 2 : 1) and have an extracellular coating (Fig. 2i,j,k). In mature cells, the sheath laminates at the apical pole after budding to release exospores. Unequal cell division leads to the formation of 1-2 (rarely 3) small, rounded exospores, which can be arranged in short rows (Fig. 2i) or lie in pairs inside a closed pseudovagina (Fig. 2g,h).
Numerous pores penetrating the cell wall through the peptidoglycan layer were found in the basal part of the cell and in the area of cell division (Fig 3i). Mucilaginous sheath (pseudovagina) has a clearly defined layered structure (Fig.  3). Cells have 3-5 layers of thylakoids arranged at the periphery parallel to the cytoplasmic membrane, but 1-2 layers may invaginate into the central cytoplasmic zone. Cells usually contain 3-6 polyhedral bodies (carboxysomes) about 200 nm in size (Fig. 3a,d,g), small glycogen granules (10-20 nm, repeatedly occurring among thylakoids) (Fig. 3h,i), and 5-10 rounded polyphosphate inclusions (large black spots, up to 500 μm in diameter) (Fig. 3a,b,c,d,f,g).
In strain CALU 1864 cell division occurs, as in other Chamaesiphon spp. (Waterbury & Stanier, 1977;Komárek & Ludvik, 1982;Wujek & Gretz, 1984). Septate and constrictive binary fission proceeds asymmetrically: it initiates at the apical pole of the mother cell and begins with ingrowth from the cell wall and build-up of the peptidoglycan septa (Fig. 3f,h). This leads to an unequal division and the formation of exospores (Fig. 3g).
Chamaesiphon fontinalis sp. nov. grows very slowly under laboratory conditions and has a delayed initial growth phase (Fig. 4). Intensive growth was observed after 30-35 days of cultivation in liquid medium at 7°C and 15°C, while at 25°C cells do not remain viable for a long time. This indicates that the strain has a temperature optimum of growth below 15°C, as observed in psychrophilic forms of bacteria (Morita, 1975;Nadeau & Castenholz, 2000). Psychrophilic cyanobacteria are known to be much less common than psychrotolerant ones, possibly due to the poor nutritional adaptability or specificity of regulatory processes (Morita, 1975;Wynn-Williams, 1990;Nadeau & Castenholz, 2000).

Pigment composition and complementary chromatic adaptation
The pigment composition of C. fontinalis sp. nov. is similar to cyanobacteria in general (Herdman et al., 2015;Aigner et al., 2018). Spectrometric characterisations of whole cells and methanol extracts are shown in Fig. 5a. In the absorption spectrum of whole cells, the peaks at 450 nm and 675 nm correspond to chlorophyll a, and there are also peaks of some carotenoids (475-525 nm). Among phycobiliproteins (PBPs), C-phycoerythrin (C-PE, peak 525-560 nm) and Cphycocyanin (PC, peak 625-630 nm) were identified. An additional shoulder in the region of 570-575 nm may probably belong to a special spectral form of PE previously identified in Gloeothece sp. PCC 6909 and Oscillatoria sp. PCC 7823 or it can be considered as some phycoerythrocyanine (PEC, peak 570-595) (Bryant, 1982). Under low light conditions (10-15 μmol m -2 × s -1 , cool-white lamp), strain CALU 1864 has a purple to dark crimson colour and is relatively rich in PEC (data are not shown). At high levels of irradiation (30-40 μmol m -2 × s -1 , cool-white lamp), the strain exhibited photosensitivity, which led to a colour change from intense red-violet to yellowish-green.
Cyanobacteria are known to be able to change the composition of PBPs in light-harvesting antennae (phycobilisomes) in response to changes of light spectral composition (green or red light). This phenomenon is called complementary chromatic adaptation (CA) (Tandeau de Marsac, 1977). Water-soluble cells extracts of C. fontinalis sp. nov., grown under green or red light, were analysed to determine capa-bility to CA. We have established that Chamaesiphon fontinalis sp. nov. can only regulate PE content (de-can only regulate PE content (decreases PE, but does not increase PC, when exposed to red light) (Fig. 5b). This fact indicates the CA type 2 (Tandeau de Marsac, 1977;Hirose at al., 2015). In contrast, the reference strains Chamaesiphon subglobosus PCC 7430 and Chamaesiphon minutus PCC 6605 exhibit type 1 and type 3 of chromatic adaptation, respectively (Herdman et al., 2015).

Molecular phylogeny assessment
Phylogenetic relationships were reconstructed using the nearly full-length (1403 nt) 16S rRNA gene sequence of C. fontinalis sp. nov., all cultured strains of Chamaesiphon spp. and reference strains of cyanobacteria (Fig. 6). Chamaesiphon fontinalis sp. nov. represents the separate phylogenetic lineage within the cluster «Chamaesiphon» (see Fig. 6). Members of the family Gomontiellaceae Elenkin ex Geitler, 1942, in particular, Hormoscilla pringsheimii Anagnostidis, Komárek and Crinalium epipsammum de Winder, Stal & Mur are the closest neighbours of this cluster (Kurmayer et al., 2018).  The BLASTn search results that C. fontinalis sp. nov. has the highest similarity (98.0-98.2%) with the 16S rRNA sequences of uncultured cyanobacteria found in various geographic regions, including the Antarctica. The greatest resemblance (98.2%) has the uncultured PCR clone st_23 NB (KY704135.1), derived from a single colony isolated from epilithic biofilm found in the stream Nederbach, Austrian Alps, and attributed to C. starmachii, based on morphological features (Kurmayer et al., 2018). The 16S rRNA similarity of C. fontinalis sp. nov. with other Chamaesiphon spp. ranges from ~ 93.0% to 98.2% (Table 1). In accordance with the established level of 16S rRNA similarity of 98.65% as a criterion for separating a new bacterial species (Kim et al., 2014), and taking into account the distinct phylogenetic position of the strain CALU 1864, we define it as a new Chamaesiphon species.
Intraspecific analysis of p-distances of 16S rRNAs within the Chamaesiphon spp. showed that C. fontinalis sp. nov. has the highest simi-the highest similarity with C. geitleri №1023 (98.2%), and the least with Chamaesiphon cf. incrustans №1036 (92.5%). The last one groups separately from all sequences of Chamaesiphon spp. and probably belongs to a different genus. In this case, maximum level of 16S rRNA species dissimilarity for the remaining strains of the monophyletic cluster «Chamaesiphon» is 7.2%. The Geitleribactron purpureum Tovel-4 strain was also taken for 16S rRNAs pairwise comparison to show the genetic distance (mean 10.65%) between genera of Chamaesiphonaceae. Geitleribactron purpureum was previously found to cluster distantly from all Chamaesiphon spp. (Mareš & Cantonati, 2016).

16S-23S ITS secondary structure evaluation
The secondary structures of 16S-23S ITS regions are very useful for comparative taxonomy at the intrageneric level and can provide insight into whether a taxon belongs to a new species (Iteman et al., 2000;Johansen et al., 2011;Perona et al., 2022). We compared the secondary structures of conserved and variable regions of 16S-23S ITS in Chamaesiphon strains. The total length of the ITS region was found to be the shortest for C. fontinalis sp. nov. (567 nt) and the longest for C. polymorphus CCALA 037 (632 nt) ( Table 2).
Nucleotide sequences and secondary structures of 16S-23S ITS (domains D1-D1ʹ, B-box, and V2) of C. fontinalis sp. nov. differ considerably from other species (Fig. 7). It should be noted that all 16S-23S ITS of Chamaesiphon spp., including the strain CALU 1864, had alternative conserved sequences of D2 (CTTTCAAACTT instead of CTTTCAAAC-TA) and A-box (GCACCTTGACAA instead of GAACCTTGAAAA), previously described in Iteman et al. (2000). The V2 domain and pre-B-box spacer differ in length in Chamaesiphon strains. The V2 region located between tRNA Ile and tRNA A1A varied from 57 nt to 71 nt in length, while the pre-B box spacer varied from 49 nt to 62 nt in length ( Table 2). The Bbox domain has common structure, but different nucleotide composition in the terminal and basal internal loops in all studied Chamaesiphon species (Fig. 7). Chamaesiphon fontinalis sp. nov. and PCR clone 23_st_NB have similar V2 structures (except for CUU/UUC inversion, marked by circles in Fig. 7), but quite different in helices from other species (Fig. 7). In all Chamaesiphon spp. we analysed, tRNA Ala have a common structure, despite some substitutions in the T-loop and T-stem. At the same time, the tRNA Ile in C. fontinalis sp. nov., PCR clone 23_st_NB and C. geitleri №1023 is two nucleotides shorter (72 nt) than in other strains (74 nt) ( Table 2).
In this paper, a new species of Chamaesiphon found in the Valdaysky National Park (Russia) is investigated, using a polyphasic taxonomic approach. Chamaesiphon fontinalis sp. nov. was identified based on the morpho-morphology, type of chromaic adaptation, physiology and molecular-genetic features of strain CALU 1864. It was found to be unique in the specific organisation of secondary structures of 16S-23S ITS regions. Strain divergence is supported phylogenetically, by a relatively low 16S rRNA gene similarity with other Chamaesiphon spp.
Our findings extend the present understanding on the diversity of the genus Chamaesiphon and represent a novel species, discovered for the first time in Russia.