CC BY
0
CHAMAESIPHON FONTINALIS SP. NOV., A NEW SPECIES OF UNICELLULAR ASYMMETRICALLY DIVIDING CYANOBACTERIA
Natalia V. Velichko1 * , Anastasia S. Makeeva1 , Svetlana G. Averina1 , Svetlana V. Smirnova2
1 Saint Petersburg State University, Russia
*e-mail: n.velichko@spbu.ru 2Komarov Botanical Institute RAS, Russia
Received: 22.02.2023. Revised: 26.06.2023. Accepted: 27.06.2023.
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.
Key words: Chamaesiphonaceae, morphotype, molecular-genetic features, polyphasic taxonomy, Protected Area
Introduction
Cyanobacteria form a morphologically heterogeneous group of photosynthetic bacteria, distributed in a variety of ecotopes (Komarek & An-agnostidis, 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; Komarek & Anag-nostidis, 1998; Hoffmann et al., 2005; Casamatta & Hasler, 2016). Chamaesiphon spp., together with the representatives of the genera Geitleribac-tron, Clastidium, and Cyanophanon (the last two have not yet been studied by molecular-genetic methods), constitute the family Chamaesiphona-ceae Borzi (phylum Cyanobacteria Stanier ex Cavalier-Smith, class Cyanophyceae Schaffner, order Gomontiellales Strunecky & Mares) (Hoffmann et al., 2005; Komarek et al., 2014; Guiry & Guiry, 2022; Strunecky 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 (Komarek & 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, 2015, 2017; Smirnova & Beljakova, 2016). This Protected Area (total area is about 1585 km2) 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/km2. 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, Lep-tolyngbya crassior (Skuja) Anagnostidis, Micro-coleus fonticola (Kirchner) Strunecky, Komarek & Johansen, Phormidium favosum Gomont, and Pseudanabaena minima Anagnostidis are abundant among filamentous cyanobacteria, along with some eukaryotic microalgae (Smirnova, 2021).
Chamaesiphon species lead a benthic or substrate-attached lifestyle and prefer siliceous or carbonate running waters of mountain streams (e.g. in the Alps and Carpathians) (Rott, 2008; Sant'Anna et al., 2011; Cantonati et al., 2015; Gutowski et al., 2015; Casamatta & Hasler, 2016; Loza et al., 2013a, 2018; Kurmayer et al., 2018). They are often settled on wet rocky surfaces along river banks (Rott & Wehr, 2016; Aigner et al., 2018). Analysis of Chamaesiphon spp. records on 429 sampling sites over a 6-year observation period has led to the conclusion that C. confervicola Braun, C. fuscus (Rostafinski) Hansgirg, C. incrustans Grunow, C. polymor-phus Geitler, and C. starmachii Kann can be considered as common species in periphyton of clear, fast-flowing waters in Germany (Gutowski et al., 2015). Therefore, Chamaesiphon species have been proposed for biomonitoring the ecological quality of streams and rivers in ac-
cordance with the European Water Framework Directive 2000/60/EC (Loza et al., 2013b; Gutowski et al., 2015). Representatives of the genus Chamaesiphon (about 40 species) are rare in Russia, and so far no new species have been discovered (Hollerbach et al., 1953; Melekhin et al., 2019). Moreover, only eight laboratory-cultivated strains (C. subglobosus PCC 7430, C. minutus PCC 6605C. polymorphus CCALA 037, C. polonicus SAG 32.87, C. geitleri №1023, C. sp. PCC 8308, C. investiens UAM 386, and C. cf. incrustans №1036) are known to date (Herd-man et al., 2015; Kurmayer et al., 2018).
The aim of this study is to describe a new species of Chamaesiphon found in the Valdaysky National Park by the polyphasic taxonomic approach (Komarek, 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.
Material and Methods
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.
Fig. 1. The location of Valdaysky National Park (a), spring Tekunok (b), and a view of brownish biofilms on stones in the sampling site (c). Scale bars: a - 100 km, b - 5 km, c - 25 cm.
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 FeSO4 for ferrous citrate and 5-fold reduction in NaNO3. It also contained 250 mg x ml-1 cycloheximide (Oxoid, UK) to suppress eukary-otic microalgae growth. Samples were incubated under continuous illumination (10-15 pmol photons m-2 x 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 (Rip-pka 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 axeni-sation procedure, the obtained strain Chamaesi-phon 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-kollekciya-calu-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 BioSpec-trometer (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 Mares 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% OsO4 (at 10°C, 12 h). Fixed cells were washed with 0.15-M Na-phosphate 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 BioSpec-trometer (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 centrif-ugation (5000 g, 10 min). The phycobiliprotein (PBP) fraction was obtained from ~ 100 mg (wet weight) of pellet suspended in 500 pl 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 Na2-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 amplification of the nearly full-length 16S rRNA gene was performed using 27F/1492R primers (Lane, 1991) and the 16S-23S rRNA internal transcribed spacer (ITS) was amplified with 322F/340R primers (Iteman et al., 2000). PCR mixture (25 pl) contained 2.5 pl DreamTaq buffer (Thermo Fisher Scientific, Germany; 1 x commercial buffer with 20 mM MgCl2), 0.75 pl dNTPs (10 mM each), 0.75 pl of each primer (10 pM each), 0.15 pl of DreamTaq polymerase, 15.35 pl sterile water, and 2.5 pl of DNA template (30 ng).
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:
Similarity = 100% x (1 - p)
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 ultra-fast bootstrap and 1000 bootstrap replications (Tri-finopoulos 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.
The positions of the D1-D1', B-box, V2, V3, and putative D5 regions of 16S-23S ITS were determined as previously described (Iteman et al.,
2000; Johansen et al., 2011; Muhlsteinova et al., 2014). The Mfold web server was used to fold the helices with default settings (Zuker, 2003). tRNAs were found by the tRNAscan-SE 2.0 web server (Lowe & Chan, 2016).
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).
Taxonomic treatment
Phylum Cyanobacteria Stanier ex Cavalier-Smith, 2002
Class Cyanophyceae Schaffner, 1909
Order Gomontiellales Strunecky & Mares, 2023
Genus: Chamaesiphon Braun, 1864
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 dark-crimson 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 exo-spores. The cells can vary from round or ovoid to pear-shaped or cylindrical. Cells are 6.6-16.7(25.2) x 5.5-8.2(11.1) ^m in size with a length-to-width ratio close to 2 : 1. They have a thick (up to 2 ^m), colourless U-shaped pseudovagina (a multilayered extracellular sheath). After cell division, the pseudovagina delaminates at the apex, releasing exospores.
Fig. 2. Morphology of Chamaesiphon fontinalis sp. nov. (light micrographs). Scale bars: 500 |m (a), 20 |m (c), 10 |m (b, d, e, g, i), 5 |um (f, h, j, k).
Cell contents are heterogeneous, brownish-crimson in colour, with prominent cytoplasmic granules. Exospores are about 3 ^m in diameter; in the case of a large mother cell, they can reach 6 ^m. The cells showed parietal arrangement of thylakoids (3-5 layers) and the presence of carboxysomes, polyphosphates, and glycogen inclusions.
Type material. Holotype: herbarium material of Chamaesiphon strain CALU 1864 (dry biomass) is stored in the Herbarium of Koma-rov Botanical Institute RAS (LE), Saint Petersburg, Russia (LE A0002381).
Type locality. Biofilms occur on the surface of the upper part of stones in spring Teku-nok, 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: 0P415410 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.na'lis, 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 confer-vicola is an epiphyte of aquatic plants and green filamentous algae Cladophora Kutzing and Oedo-gonium Link ex Hirn. It usually grows as single cells or cells in groups, and has large elongated cells up to 200 |im in length (Komarek & Anag-nostidis, 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. star-machii is the most similar to C. fontinalis sp. nov. It also has a pear-shaped or ellipsoidal type of pseudo-sporangium, similar cell size (6-18 x 4.0-7.2 |im), a reddish-violet or purple colour of the cell content and prominent granules in the cytoplasm. The exo-spores are more or less rounded-polygonal, may remain attached 1-3 to the mother cell when the sheath is closed (Komarek & Anagnostidis, 1998). However, C. starmachii differs from C. fontinalis sp. nov. by a 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 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; Komarek & 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 p,m in length and 5.5-7.1 p,m 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 p,m in length, 6.0-8.2 p,m 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).
The ultrastructure of C. fontinalis sp. nov. is generally similar to other studied Chamaesi-phon species (Waterbury & Stanier, 1977; Gro-mov & Mamkaeva, 1980; Komarek & Ludvik, 1982; Wujek & Gretz, 1984; Kurmayer et al., 2018) (Fig. 3). The cell wall on TEM micrographs consists of three layers, typical for cya-nobacteria: the cytoplasmic membrane (CM), the outer membrane (OM) and the thick (up to 30-40 nm) peptidoglycan layer (PG) (Fig. 3i).
Fig. 3. Ultrastructure of Chamaesiphon fontinalis sp. nov. Designations: Cb - carboxysomes, CM - cytoplasmic membrane, GG - glycogen granules, OM - outer membrane, P - pores, PG - peptidoglycan layer, PP - polyphosphate granule, Sh - mul-tilayered sheath, T - thylakoid. Scale bars: 2 ^m (b, g), 1 ^m (c, e, f), 500 nm (a, d), 200 nm (h, i).
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 ^m in diameter) (Fig. 3a,b,c,d,f,g).
In strain CALU 1864 cell division occurs, as in other Chamaesiphon spp. (Waterbury & Stanier, 1977; Komarek & 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 C-phycocyanin (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 pmol m-2 x 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 pmol m-2 x 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 (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 subglo-bosus 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 lull-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 Anag-nostidis, Komarek and Crinalium epipsammum de Winder, Stal & Mur are the closest neighbours of this
Fig. 5. Absorption spectra of whole cells (black line) and methanol extract (blue line) of Chamaesiphon fontinalis sp. nov., grown under white light (WL) conditions (a). Absorption spectra of the water-soluble pigment fraction of the strain grown under white light (WL, black line), green light (GL, green line), and red light (RL, red line) (b).
Fig. 6. Maximum likelihood tree based on 16S rRNA gene sequences showing the phylogenetic position of strain CALU 1864. Numbers at nodes indicate posterior probability (> 0.50) and bootstrap values (> 50%) obtained from Bayesian Inference (BI) and maximum likelihood (ML) analyses, respectively. Support values are given in brackets after each clade in the following order: BI/ML at the same node. Asterisks (*) indicate maximum support values (posterior probability of 1.0 and bootstrap values of 100%).
The BLASTn search results that C. fontinalis sp. nov. has the highest similarity (98.0-98.2%) with the 16S rRNA sequences of uncultured cya-nobacteria 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 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 mono-phyletic 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. (Mares & 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; Per-ona 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-D1', 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 tRNAIle and tRNAA1A 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 B-box domain has common structure, but different nucleotide composition in the terminal and basal internal loops in all studied Chamaesi-phon 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, tRNAAla have a common structure, despite some substitutions in the T-loop and T-stem. At the same time, the tRNAIle 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).
Table 1. The percentage of similarity (p-distance) matrix of Chamaesiphon and Geitleribactron strains based on partial 16S rRNA sequences (1207 nt)
Strain 1 2 3 4 5 6 7 8 9 10
1 Chamaesiphon fontinalis sp. nov. strain CALU 1864 (OP415410) 100
2 Chamaesiphon cf. incrustans No 1036 (KY704115.1) 92.5 100
3 Chamaesiphon geitleri No 1023 (KY704109.1) 98.2 93.7 100
4 Chamaesiphon investiens UAM 386 (JQ070061.1) 96.8 93.2 97.2 100
5 Chamaesiphon minutus PCC 6605 (KY704112.1) 96.9 92.8 97.4 98.3 100
6 Chamaesiphon polonicus SAG 32.87 (KY704111.1) 96.9 92.8 97.5 97.8 98.9 100
7 Chamaesiphon polymorphus CCALA 037 (KY704110.1) 97.1 93.1 97.5 97.8 93.0 98.9 100
8 Chamaesiphon sp. PCC 8308 (KY704110.1) 93.0 98.9 93.4 94.1 95.9 94.7 95.2 100
9 Chamaesiphon subglobosus PCC 7430 (KY704113.1) 97.9 93.1 98.3 97.7 98.0 97.8 98.3 93.9 100
10 Geitleribactron purpureum Tovel-4 (KT819293.1) 89.4 88.8 90.6 90.6 90.6 90.8 90.6 87.9 90.2 100
Strain ITS region C. fontinalis sp. nov. PCR clone st_23_NB C. geitleri №1023 C. minutus PCC 6605 C.subglobosus PCC 7430 C. sp. PCC 8308 C. polonicus SAG 32.87 C. polymorphus CCALA 037
Complete 567 600 591 587 592 574 594 632
Leader 8 8 8 8 8 8 8 8
D1-D1' 62 62 62 62 63 62 62 62
D2 + pre-D2 spacer 33 33 33 33 33 32 33 33
Spacer + D3 + spacer 23 23 23 22 24 20 24 22
tRNABe gene 72 72 72 74 74 74 74 74
V2 71 71 68 62 61 57 61 61
tRNAAla gene 73 73 73 73 73 73 73 73
Pre-B-box spacer 62 56 52 50 54 51 59 49
B-box 54 53 53 54 53 54 54 51
Post-B-box spacer 5 6 5 5 5 5 5 5
A-box 12 12 12 12 12 12 12 12
D4 7 7 7 7 7 7 7 7
Spacer 14 14 14 14 10 10 10 14
V3 30 30 30 31 38 38 40 29
Spacer + D5 + spacer 41 80 79 80 77 71 72 132
Table 2. Comparison of the 16S-23S ITS conserved domains lengths (nt) of Chamaesiphon strains
Fig. 7. Secondary structures of the 16S-23S ITS regions. Chamaesiphon fontinalis sp. nov. (a), PCR clone 23_st_NB (b), C. geitleri №1023 (c), C. minutus PCC 6605 (d), C. subglobosus PCC 7430 (e), Chamaesiphon sp. PCC 8308 (f), C. polonicus SAG 32.87 (g), C. polymorphus CCALA 037 (h).
Conclusions
Chamaesiphon species are predominantly epiphytes of aquatic plants (in particular, Cladopho-ra and Oedogonium) and biofoulers of stones in periphyton. They are widely distributed in freshwater bodies and streams in Europe and Russia (Hollerbach et al., 1953; Komarek & Anagnos-tidis, 1998; Rott, 2008; Sant'Anna et al., 2011; Loza et al., 2013a, 2018; Cantonati et al., 2015; Gutowski et al., 2015; Casamatta & Hasler, 2016; Kurmayer et al., 2018). It is worth to mention that the type species of this genus, C. confervicola, like the vast majority of about 40 species, have only a morphological description (Guiry & Guiry, 2022). They still have to be verified in accordance with the current taxonomic requirements used for new cyanobacterial taxa, including morphological, physiological and genetic methods (Van-damme et al., 1996; Iteman et al., 2000; Johan-sen & Casamatta, 2005; Ramasamy et al., 2014; Dvorak et al., 2015; Komarek, 2016; Raina et al., 2019; Perona et al., 2022).
In this paper, a new species of Chamae-siphon found in the Valdaysky National Park (Russia) is investigated, using a polyphasic taxonomic approach. Chamaesiphon fontinalis sp. nov. was identified based on the 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.
Acknowledgements
We are very grateful to the Saint Petersburg University (Russia) Research Centres «Molecular and Cell Technologies», «Cultivation of microorganisms», «Chromas», and «Biobank» for technical assistance. The research was supported by the Russian Science Foundation, project №22-24-00590.
References
Aigner S., Herburger K., Holzinger A., Karsten U. 2018. Epi-lithic Chamaesiphon (Synechococcales, Cyanobacteria) species in mountain streams of the Alps - interspecific differences in photo-physiological traits. Journal of AppliedPhycology 30(2): 1125-1134. DOI: 10.1007/ s10811-017-1328-7 Bryant D.A. 1982. Phycoerythrocyanin and phycoerythrin: properties and occurrence in cyanobacteria. Journal of General Microbiology 128(4): 835-844. DOI: 10.1099/00221287-128-4-835 Cantonati M., Komarek J., Montejano G. 2015. Cyanobacteria in ambient springs. Biodiversity and Conservation 24(4): 865-888. DOI: 10.1007/s10531-015-0884-x Casamatta D.A., Hasler P. 2016. Blue-green algae (Cyanobacteria) in rivers. In: O. Necchi (Ed.): River Algae. Cham: Springer. P. 5-34. DOI: 10.1007/978-3-319-31984-1_2 Castenholz R.W. 2015. General characteristics of the Cyanobacteria. In: Bergey's Manual of Systematic of Archaea and Bacteria. Hobolken, NJ: Wiley. P. 1-23. DOI: 10.1002/9781118960608.cbm00019 Darriba D., Taboada G.L., Doallo R., Posada D. 2012. jModelT-est 2: more models, new heuristics and parallel computing. Nature Methods 9(8): 772. DOI: 10.1038/nmeth.2109
Doyle J.J., Doyle J.L. 1990. Isolation of plant DNA from
fresh tissue. Focus 12: 13-15. Dvorak P., Poulickova A., Hasler P., Belli M., Casamatta D.A., Papini A. 2015. Species concepts and speciation factors in cyanobacteria, with connection to the problems of diversity and classification. Biodiversity and Conservation 24: 739-757. DOI: 10.1007/s10531-015-0888-6 Efimova L.E., Frolova N.L. 2013. Hydrological monitoring within specially protected natural areas. Water: Chemistry and Ecology 5: 20-28. [In Russian] Gromov B.V, Mamkaeva K.A. 1980. Cell cycle characteristics of Chamaesiphon confervicola, a cyanobacterium forming exospores. Mikrobiologiia 49(4): 551-554. Guiry M.D., Guiry G.M. 2022. AlgaeBase. Worldwide electronic publication. Galway: National University of Ireland. Available from https://www.algaebase.org Gutowski A., Foerster J., Doege A., Paul M. 2015. Cha-maesiphon species in soft-water streams in Germany: occurrence, ecology and use for bioindication. Algological Studies 148: 33-56. DOI: 10.1127/ algol_stud/2015/0220 Herdman M., Castenholz R.W., Waterbury J.B., Rippka R. 2015. Form-Chamaesiphon. In: Bergey's Manual of Systematic of Archaea and Bacteria. Hobolken, NJ: Wiley. P. 1-3. DOI: 10.1002/9781118960608. gbm00410
Hirose Y., Katayama M., Ohtsubo Y., Misawa N., Iioka E., Suda W., Oshima K., Hanaoka M., Tanaka K., Eki T., Ikeuchi M., Kikuchi Y., Ishida M., Hattori M. 2015. Complete genome sequence of cyanobacterium Geminocystis sp. strain NIES-3708, which performs type II complementary chromatic acclimation. Genome Announcements 3(3): e00357-15. DOI: 10.1128/genomeA.00357-15 Hoffmann L., Komarek J., Kastovsky J. 2005. System of cyanoprokaryotes (cyanobacteria) state in 2004. Al-gological Studies 117: 95-115. DOI: 10.1127/18641318/2005/0117-0095 Hollerbach M.M., Kosinskaya E.K., Polyansky V.I. 1953. Key to freshwater algae of the USSR. Vol. 2: Blue-Green Algae. Moscow: Sovetskaya Nauka. 652 p. [In Russian]
Iteman I., Rippka R., Tandeau de Marsac N., Herdman M. 2000. Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria. Microbiology 146: 1275-1286. DOI: 10.1099/00221287-146-6-1275 Johansen J.R., Casamatta D.A. 2005. Recognizing cya-nobacterial diversity through adoption of a new species paradigm. Algological Studies 117: 71-93. DOI: 10.1127/1864-1318/2005/0117-0071 Johansen J.R., Kovacik L., Casamatta D.A., Ikova K.F., Kastovsky J. 2011. Utility of 16S-23S ITS sequence and secondary structure for recognition of intrageneric and intergeneric limits within cyanobacterial taxa: Lep-tolyngbya corticola sp. nov. (Pseudanabaenaceae, Cyanobacteria). Nova Hedwigia 92(3-4): 283-302. DOI: 10.1127/0029-5035/2011/0092-0283 Kim M., Oh H.S., Park S., Chun J. 2014. Towards a taxo-nomic coherence between average nucleotide identity
and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology 64: 346351. DOI: 10.1099/ijs.0.059774-0 Komárek J. 2016. A polyphasic approach for the taxonomy of cyanobacteria: principles and applications. European Journal of Phycology 51(3): 346-353. DOI: 10.1080/09670262.2016.1163738 Komárek J., Anagnostidis K. 1998. Cyanoprokaiyota, 1 Teil: Chroococcales. In: Süsswasserflora von Mitteleuropa. B. 19/1. Jena: Gustav Fischer. 548 p. Komárek J., Ludvik J. 1982. Cell structure and reproduction process in the Blue-Green alga Chamaesiphon. Plant Systematics and Evolution 139: 267-277. DOI: 10.1007/BF00989329 Komárek J., Kastovsky J., Mares J., Johansen J.R. 2014. Taxonomic classification of cyanoprokaryotes (cya-nobacterial genera) 2014, using a polyphasic approach. Preslia 86(4): 295-335. Kurmayer R., Christiansen G., Holzinger A., Rott E. 2018. Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp. Hydrobiologia 811(1): 61-75. DOI: 10.1007/s10750-017-3295-z Lane D.J. 1991. 16S/23S rRNA sequencing. In: Nucleic acid techniques in bacterial systematics. Chichester: John Wiley and Sons. P. 115-175. Loza V, Berrendero E., Perona E., Mateo P. 2013 a. Polypha-sic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama River (Spain): morphological, molecular, and ecological approaches. Journal of Phycology 49(2): 282-297. DOI: 10.1111/jpy.12036 Loza V., Perona E., Mateo P. 2013b. Molecular fingerprinting of cyanobacteria from river biofilms as a water quality monitoring tool. Applied and Environmental Microbiology 79(5): 1459-1472. DOI: 10.1128/AEM.03351-12 Loza V., Morales A., Perona E., Muñoz-Martín M.A., Mateo P. 2018. Fingerprinting Chamaesiphon populations as an approach to assess the quality of running waters. River Research and Applications 34(6): 595-605. DOI: 10.1002/rra.3277 Lowe T.M., Chan P.P. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44(W1): 54-57. DOI: 10.1093/nar/gkw413 Mares J., Cantonati M. 2016. Phylogenetic position of Geitleribactron purpureum (Synechococcales, Cyano-bacteria / Cyanophyceae) and its implications for the taxonomy of Chamaesiphonaceae and Leptolyngbyace-ae. Fottea 16(1): 104-111. DOI: 10.5507/fot.2016.002 Mares J., Strunecky O., Bucinská L., Wiedermannová J. 2019. Evolutionary patterns of thylakoid architecture in Cyanobacteria. Frontiers in Microbiology 10: 277. DOI: 10.3389/fmicb.2019.00277 Melekhin A.V., Davydov D.A., Borovichev E.A., Shalygin S.S., Konstantinova N.A. 2019. CRIS - service for input, storage and analysis of the biodiversity data of the cryptogams. Folia Cryptogamica Estonica 56: 99-108. DOI: 10.12697/fce.2019.56.10
Miller M., Pfeiffer W.T., Schwartz T. 2010. Creating the CIPRES science gateway for inference of large phy-logenetic trees. Proceedings of the Gateway Computing Environments Workshop 14: 1-8. DOI: 10.1109/ GCE.2010.5676129 Morita R.Y. 1975. Psychrophilic bacteria. Bacteriological
Reviews 39: 144-167. Mühlsteinová R., Johansen J.R., Pietrasiak N., Martin M.P., Osorio-Santos K., Warren S.D. 2014. Poly-phasic characterization of Trichocoleus desertorum sp. nov. (Pseudanabaenales, Cyanobacteria) from desert soils and phylogenetic placement of the genus Trichocoleus. Phytotaxa 163(5): 241-261. DOI: 10.11646/phylotaxa.163.5.1 Nadeau T.L., Castenholz R.W. 2000. Characterization of psy-chrophilic oscillatorians (cyanobacteria) from Antarctic meltwater ponds. Journal of Phycology 36(5): 914-923. DOI: 10.1046/j.1529-8817.2000.99201.x Perona E., Muñoz-Martín M.Á., Berrendero Gómez E. 2022. Chapter 1 - Recent trends of polyphasic approach in taxonomy and cyanobacterial diversity. In: Expanding horizon of cyanobacterial Biology. A volume of developments in microbiology. Elsevier. P. 1-49. DOI: 10.1016/B978-0-323-91202-0.00008-7 Rabenhorst L. 1865. Sectio II. Algas phycochromaceas comple-ctens. In: Flora europaea algarum aquae dulcis et subma-rinae. Leipzig: Apud Eduardum Kummerum. P. 148-149. Raina V., Nayak T., Ray L., Kumari K., Suar M. 2019. Chapter 9 - A polyphasic taxonomic approach for designation and description of novel microbial species. In: Microbial Diversity in the Genomic Era. Academic Press. P. 137-152. DOI: 10.1016/B978-0-12-814849-5.00009-5 Ramasamy D., Mishra A.K., Lagier J., Padhmanabhan R., Rossi M., Sentausa E., Raoult D., Fournier P.E. 2014. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. International Journal of Systematic and Evolutionary Microbiology 64: 384-391. DOI: 10.1099/ijs.0.057091-0 Reynolds E.S. 1963. Use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17(1): 208-212. DOI: 10.1083/jcb.17.1.208 Rippka R., Deruelles J., Waterbury J.B., Herdman M., Sta-nier R.Y. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111(1): 1-61. DOI: 10.1099/00221287-111-1-1 Ronquist F., Teslenko M., van der Mark P., Ayres D.L., Darling A.E., Höhna S., Larget B.R., Liu L., Suchard M.A., Huelsenbeck J.P. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. DOI: 10.1093/sysbio/sys029 Rott E. 2OO8. Chamaesiphon komarekii species nova, a new benthic freshwater chroococcalean species (Cyanophyta/ Cyanobacteria) from western coniferous forest streams in British Columbia, Canada. Algological Studies 12б: 37-46. DOI: 10.1127/1864-1318/2008/0126-0037
Rott E., Wehr J.D. 2016. The spatio-temporal development of macroalgae in rivers. In: O. Necchi (Ed.): River Algae. Cham: Springer. P. 159-195. DOI: 10.1007/978-3-319-31984-1_8 Sant'Anna C.L., Gama W.A., Azevedo M.T., Komarek J. 2011. New morphospecies of Chamaesiphon (Cyanobacteria) from Atlantic rainforest, Brazil. Fottea 11: 25-30. DOI: 10.5507/FOT.2011.004 Smirnova S.V 2014. Planktonic Cyanoprokaryota from waterbodies of the National Park «Valdaiskiy». No-vosti Systematiki Nizshikh Rastenii 48: 89-103. DOI: 10.31111/nsnr/2014.48.89 [In Russian] Smirnova S.V 2015. Benthic Cyanoprokaryota from water-bodies of the National Park «Valdaisky» (Novgorod Region). Novosti Systematiki Nizshikh Rastenii 49: 52-74. DOI: 10.31111/nsnr/2015.49.52 [In Russian] Smirnova S.V. 2017. Analysis of cyanoprokaryote flora in waterbodies of «Valdaiskiy» National Park (Novgorod Region). Botanicheskii Zhurnal 102(5): 598-617. DOI: 10.1134/S0006813617050027. [In Russian] Smirnova S.V. 2021. Cyanoprokaryotes from waterbodies of the National Park «Valdaysky» (Novgorod Region). PhD Thesis. Saint Petersburg. 202 p. [In Russian] Smirnova S.V., Beljakova R.N. 2016. New species of genus Stichosiphon (Cyanoprokaryota) from waterbodies of Valdaysky National Park (Novgorod Region). Botanicheskii Zhurnal 101(12): 1466-1481. DOI: 10.1134/S0006813616120073 [In Russian] Stanier R.Y., Cohen-Bazire G. 1977. Phototrophic prokaiyotes: the Cyanobacteria. Annual Reviews in Microbiology 31: 225-274. DOI: 10.1146/annurev.mi.31.100177.001301 Stanier R.Y., Kunisawa R., Mandel M., Cohen-Bazire G. 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriological Reviews 35(2): 171-205. DOI: 10.1128/ BR.35.2.171-205.1971 Strunecky O., Ivanova A.P., Mares J. 2023. An updated classification of cyanobacterial orders and families based on phylogenomic and polyphasic analysis. Journal of Phycology 59(1): 12-51. DOI: 10.1111/jpy.13304 Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739. DOI: 10.1093/molbev/msr121 Tandeau de Marsac N. 1977. Occurrence and nature of chromatic adaptation in cyanobacteria. Journal of Bacteriology 130(1): 82-91. DOI: 10.1128/jb.130.1.82-91.1977 Trifinopoulos J., Nguyen L.T., von Haeseler A., Minh B.Q. 2016. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research 44(W1): 232-235. DOI: 10.1093/nar/gkw256 Turland N.J., Wiersma J.H., Barrie F.R., Greuter W., Hawk-sworth D.L., Herendeen P.S., Knapp S., Kusber W.-H., Li D.-Z., Marhold K., May T.W., McNeill J., Monro A.M., Prado J., Price M.J., Smith G.F. (Eds.). 2018. International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth Internation-
al Botanical Congress Shenzhen, China, July 2017. In: Regnum Vegetabile. Vol. 159. Glashütten: Koeltz Botanical Books. 254 p. DOI: 10.12705/Code.2018 Vandamme P., Pot B., Gillis M., de Vos P., Kersters K., Swings J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiology Reviews 60(2): 407-438. DOI: 10.1128/mr.60.2.407-438.1996 Waterbury J.B., Stanier R.Y. 1977. Two unicellular cyano-bacteria which reproduce by budding. Archives of Microbiology 115: 249-257. DOI: 10.1007/BF00446449
Wujek D.E., Gretz M.R. 1984. Ultrastructure of the blue-green alga Chamaesiphon incrustans. Cytobios 40(158): 87-93. Wynn-Williams D.W. 1990. Ecological Aspects of Antarctic Microbiology. In: K.C. Marshall (Ed.): Advances in Microbial Ecology. Vol. 11. Boston: Springer. P. 71-146. DOI: 10.1007/978-1-4684-7612-5_3 Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31(13): 3406-3415. DOI: 10.1093/nar/gkg595
CHAMAESIPHON FONTINALIS SP. NOV., НОВЫЙ ВИД ОДНОКЛЕТОЧНЫХ АССИММЕТРИЧНО ДЕЛЯЩИХСЯ ЦИАНОБАКТЕРИЙ
Н. В. Величко1'* , А. С. Макеева1 , С. Г. Аверина1 , С. В. Смирнова2
1 Санкт-Петербургский государственный университет, Россия *e-mail: n.velichko@spbu.ru 2Ботанический институт имени В.Л. Комарова РАН, Россия
На камнях ручьев и родников национального парка «Валдайский» (Россия) были обнаружены обширные коричневатые биопленки альго-бактериальных сообществ. Доминирующими одноклеточными ци-анобактериями в исследованных биопленках являлись морфотипы Chamaesiphon sp. Хотя виды этого рода часто встречаются в перифитоне проточных вод, многие из них имеют географически ограниченное распространение и часто встречаются в горных ручьях и реках Европы. Штамм Chamaesiphon CALU 1864 был выделен из эпилитных биопленок родника Текунок. Мы обнаружили, что он отличается от ранее описанных видов не только необычной малиновой окраской, но и некоторыми морфологическими особенностями. Молекулярно-генетический анализ первичной последовательности 16S рРНК и вторичных структур внутреннего транскрибируемого спейсера рибосомального оперона подтвердил его уникальность. Филогенетически штамм представляет собой независимую линию на эволюционном древе. На основании выявленных отличий мы предлагаем рассматривать штамм CALU 1864 как новый вид, Chamaesiphon fontinalis sp. nov., впервые обнаруженный в России и описанный в соответствии с действующими правилами Международного кодекса номенклатуры водорослей, грибов и растений.
Ключевые слова: Chamaesiphonaceae, молекулярно-генетические признаки, морфотип, особо охраняемая природная территория, полифазная таксономия
