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Article Terrestrial Species of Drouetiella (Cyanobacteria, Oculatellaceae) from the Russian Arctic and Subarctic Regions and Description of Drouetiella ramosa sp. nov. 1, 1 2 2 Denis Davydov *, Anna Vilnet , Irina Novakovskaya and Elena Patova Polar-Alpine Botanic Garden-Institute of Kola Science Center of the Russian Academy of Sciences, Botanical Garden, 184256 Kirovsk, Murmansk Region, Russia Institute of Biology of Komi Science Centre of the Ural Branch of the Russian Academy of Sciences, Kommunisticheskaya, 28, 167000 Syktyvkar, Komi Republic, Russia * Correspondence: email@example.com Abstract: The strains of Drouetiella species (Cyanobacteria, Oculatellaceae) from a terrestrial biotope were isolated and characterized using an integrative approach including molecular, morphological, and ecological information. The specimens were collected from the Arctic and Subarctic areas of European Russia. Drouetiella species possess morphological plasticity and can be confused with sim- ilar species of Oculatellaceae or Leptolyngbyaceae. The 16S rRNA gene phylogeny supported the strong monophyly of the genus Drouetiella with the separation of four linages corresponding to three known species and one to new taxon. The 16S-23S ITS rRNA sequences of the analyzed Drouetiella strains differ in length and nucleotide composition, which has had an effect on the hypothetical secondary structures of the D1–D1′, Box-B, V2, and V3 helices. As a result of complex study of the genus Drouetiella, a new species—Drouetiella ramosa sp. nov.—is described from the Subarctic of Eu- ropean Russia. Keywords: the Arctic; molecular phylogeny; 16S rRNA; 16S-23S ITS rRNA; filamentous cyanobac- teria Citation: Davydov, D.; Vilnet, A.; Novakovskaya, I.; Patova, E. Terrestrial Species of Drouetiella (Cyanobacteria, Oculatellaceae) from 1. Introduction the Russian Arctic and Subarctic Cyanobacteria are oxygenic micro-phototrophs present in all ecosystems. They are Regions and Description of often recognized as the most important primary producers , especially in polar ecosys- Drouetiella ramosa sp. nov. Diversity tems . Despite a long and rich history of cyanobacterial research in the Eurasian Arctic 2023, 15, 132. https://doi.org/ and Subarctic , the existence of diversity in these regions is still greatly underestimated. 10.3390/d15020132 An effort to widen the knowledge of cyanobacterial diversity in high latitudes is neces- Academic Editors: Alexander sary. Dvoretsky, Krivopalov Anton and Several studies of cyanobacteria in Svalbard [4–9], Murmansk Region [10–13], Kare- Miguel Ferrer lia Republic , Komi Republic, and Yamalo-Nenets Autonomous Okrug [15–19] sug- Received: 18 November 2022 gest that polar and boreal cyanobacterial floras probably contain many undiscovered spe- Revised: 12 January 2023 cies and genera. Accepted: 15 January 2023 In recent years, the cyanobacterial phylogeny has been intensely studied. Simple fil- Published: 18 January 2023 amentous cyanobacteria have undergone extensive taxonomic revision with an integra- tive approach that resulted in the transfer of the recently established genera Oculatella  and Timaviella  into a new family, Oculatellaceae, in which six new genera were sim- Copyright: © 2023 by the authors. Li- ultaneously described: Cartusia, Drouetiella, Kaiparowitsia, Komarkovaea, Pegethrix, Tildeni- censee MDPI, Basel, Switzerland. This article is an open access article ella . The subsequent taxonomical studies increased the number of known genera in distributed under the terms and con- Oculatellaceae to 15 with the description of Thermoleptolyngbya , Elainella , ditions of the Creative Commons At- Shackletoniella , Aerofilum , Trichotorquatus , Amphirytos , and Siamcapillus tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/). . Diversity 2023, 15, 132. https://doi.org/10.3390/d15020132 www.mdpi.com/journal/diversity Diversity 2023, 15, 132 2 of 19 Unfortunately, these new species and genera are quite difficult to identify by light microscopy due to a lack of clear morphological apomorphies. The genus Drouetiella, with three known species , could attend to such problem- atic taxa. This genus comprises a thin solitary filamentous false-branching form with col- orless sheaths and untapered trichomes, slightly constricted at the cross-walls. During the distinguishing of the new genus Drouetiella, Mai et al.  described within it two new species—Drouetiella fasciculata Mai, Johansen and Bohunická, and Drouetiella hepatica Mai, Johansen and Pietrasiak—from strains cultivated in the Algal Culture Collection at John Carroll University, Cleveland, USA. The authors also transferred Phormidium luridum Go- mont to Drouetiella lurida (Gomont) Mai, Johansen and Pietrasiak. That species was de- scribed as Leptothrix lurida Kützing , due to its origin in a Stuttgart waterbody. In our previous study, concerning the genus Phormidesmis’ systematics based on nu- cleotide sequence data of the 16S rRNA gene and 16S-23S ITS rRNA region , we addi- tionally tested five strains that were morphologically attributed to the genus Drouetiella; two of them, with major doubts concerning their morphological evidence, were attended to by D. hepatica, and three remained undetermined. The difficulties in morphological recognition of the Drouetiella strains cultivated in our laboratory forced us to sample pub- lished molecular data for Drouetiella spp., and reference strains from the family Oculatel- laceae, to clarify the level of species variability and correlate this with morphological fea- tures. This study aimed to determine the diversity of the genus Drouetiella from the Arctic and Subarctic regions using a combined approach: morphological analysis, and phyloge- netic analysis, based on DNA sequence, genetic variability of the ribosomal gene, and sec- ondary structures of the 16S-23S ITS rRNA region, as well as by ecological characteriza- tion. 2. Materials and Methods 2.1. Sampling Five cyanobacterial samples were collected during the summers of 2009–2019 from different locations in the terrestrial habitats of the Russian Arctic and Subarctic area (Table 1, Figure 1). Table 1. The description of the sampling sites. Number of the Number Strain in Col- Longitud Latitude Elev. of Loca- lection (GB Ac- Locality Habitats e N (m) tion cession Num- E ber) Yamalo-Nenets Autonomous Okrug. The Polar Urals On the limestone KPABG 4163 1 Mountains. The Ochetyvis rocks and boulders 68.18995 65.67754 167 (ON897679) Valley. The left shore of the underwater. Ochetyvis River. Yamalo-Nenets Autonomous Okrug. The Polar Urals On a wet limestone KPABG 132178 2 Mountains. The Ochetyvis wall of rock, near the 68.19059 65.67815 154 (ON897678) Valley. The left shore of the water. Ochetyvis River. Murmansk Region. Ash dumps Anthropogenic KPABG 41662 3 of the Apatitsky Thermal Power habitat, crust on the 67.599972 33.48128 202 (ON897680) Plant. sludge. Next to a quartz SYKOA C-013- Komi Republic. Subpolar Urals 4 mine on damp 65.22639 60.24528 850 09 (ON897677) Mountains. quartz sand. Komi Republic. Subpolar Urals KPABG Grass–moss 5 Mountains. Near Maloe 65.155833 60.231833 690 610005/SYKOA community next to a Balbanty Lake. Diversity 2023, 15, 132 3 of 19 С-002-10 deer camp, in the (ON897681) soil. Figure 1. The position of the sample plots of Drouetiella strains; numbers of sample plots as outlined in Table 1. Two samples (Figure 1, plots 1, 2) were collected in the northern part of the Polar Urals Mountains in the tundra zone, which is primarily composed of tundra shrubs and moss–lichen communities, by D. Davydov. The samples were found in mats with other cyanobacteria. Two samples (Figure 1, plots 4, 5) were collected in the Subpolar Urals Mountains by Е. Patova and I. Novakovskaya, next to a quartz mine on damp quartz sand (plot 4), and in a grass–moss community next to a deer camp, in the soil (plot 5). The single specimen (Figure 1, plot 3) was obtained from the crust on the sludge of ash dumps at the Apatitsky Thermal Power Plant, by D. Davydov. The soil samples were collected within a 3–5 cm area and 2 cm deep. The cyanobacterial samples were taken and dried in sterile paper bags and then air-dried and stored in the laboratory before enrichment cultivation . 2.2. Isolation of Strains In the laboratory, the cyanobacterial strains were isolated from mixed cultures (the samples from plots 1–3 were cultivated on liquid Z8 medium [32,33], and the samples from plots 4–5 were cultivated on Agar medium with soil extract). Unialgal cultures were obtained by picking material from the edge of discrete colonies that had been growing for three weeks on solid BG11 media . Cultures were maintained under artificial −2 −1 illumination, 16 h light 35 μmol photons m s /8 h dark photoperiod, at 22 °C. The strains were deposited into the Collection of Cyanoprokaryotes at the Polar- Alpine Botanic Garden-Institute (KPABG), Apatity, Russia. Two of them were stored in the collection of microalgae at the Institute of Biology of Komi Scientific Centre (SYKOA), Syktyvkar, Russia. A portion of the growing material was dried and deposited in the herbarium at KPABG. The label information was included in the “L.” information system . Diversity 2023, 15, 132 4 of 19 2.3. Morphological Characterization The morphological characters of the strains were described from unialgal cultures using a Zeiss AxioScope A1 microscope (Jena, Germany) equipped with Nomarski DIC optics and an Olympus DP23 camera (Tokyo, Japan). Morphometric measurements were taken using an Olympus cellsSens Entry 3.2 (Tokyo, Japan). The diacritical morphological traits used in the species’ descriptions were considered, including the width and length of the cells, shape of cells, presence or absence of constrictions at the cross-wall, presence of necridic cells, color of the sheath, and the presence or absence of false-branching. 2.4. Specimens Sampling The sequences of the 16S rRNA gene and the single operon of the 16S-23S ITS rRNA region for the five strains of Drouetiella cultivated in KPABG were obtained during our previous study, using the protocols described there . These formed the base of the current estimation together with molecular data from five Drouetiella strains downloaded from GenBank ; this was also carried out for 46 of the predominantly referenced strains of allied taxa from Oculatellaceae and the outgroup. The GenBank accession num- bers for the Drouetiella strains from KPABG are given in Table 1. 2.5. Molecular Analyses Two datasets were produced for phylogenetic estimation in BioEdit 7.0.1. . The first dataset included the sequences of the 16S rRNA gene for 56 accessions with Gloeobac- ter kilaueensis JS1 (NR121745) as an outgroup. For the genus Drouetiella, the alignment of the 16S-23S ITS rRNA region for 10 strains was also constructed; Pegethrix olivacea GSE- PSE-MK46-15A was chosen as an outgroup. The length of the 16S rRNA gene alignment was 1181 sites; and for the 16S-23S ITS rRNA region: 619 sites. Molecular phylogenetic estimations were implemented through the maximum like- lihood (ML) with IQ-TREE  and the Bayesian approach with MrBayes v. 3.2.1 . The ML analysis of the 16S rRNA gene included a search for the best-fit evolutionary model of nucleotide substitutions using the incorporated option, ModelFinder , and ultrafast bootstrapping  with 1000 replicates. The K2P+I+G was selected as the best-fit evolu- tionary model with four rate categories of gamma distribution to evaluate the rate of het- erogeneity among the sites. For the ML analysis of the 16S-23S ITS rRNA region, the model TPM3u+F+G4 was selected, and 1000 replicates for ultrafast bootstrapping were imple- mented. The Bayesian analysis was only conducted for the 16S rRNA gene, using the GTR+I+G model and gamma distributions with four rate categories, as recommended by the program creators. Two independent runs of the Metropolis-coupled ΜCMC were used to sample the parameter values in proportion to their posterior probability. Each run included three heated chains and one unheated chain, and two starting trees were chosen randomly. Chains were run for one million generations and trees were sampled every 100th generation. The software tool Tracer  revealed an effective sample size of 1408.4793 and an auto-correlation time of 1278.116. The 10,000 were obtained in each run, and the first 2500 trees were discarded as burn-in. Thereafter, 15,000 trees were sampled from both runs. The average standard deviation of the split frequencies between the two runs was 0.007279. Bayesian posterior probabilities in both estimations were calculated from trees sampled after burn-in for each run as well. The majority rule (MJ) consensus tree for both datasets was calculated after combining the runs minus a burn-in of 25%. The infrageneric and infraspecific similarity of the 16S rRNA gene and 16S-23S ITS rRNA region of the tested Drouetiella strains were calculated with the following formula 100 × (1 − p), where p is as the average pairwise p-distances inferred from Mega 11 . The hypothetical secondary structures for the four conserved domains of the 16S-23S ITS rRNA region were determined using the program Mfold Ver. 3.1  and prepared for publication using Adobe CorelDRAW 22.214.171.1241. Diversity 2023, 15, 132 5 of 19 3. Results 3.1. Morphology All of the strains analyzed in this paper differ from the original species’ descriptions  concerning morphological features (Table 2). Table 2. Morphological comparison among the named Drouetiella strains. Information for the type strains were obtained from . D. ra- D. lurida D. hepatica D. fasciculata mosa Characteristic s/Species Lukesova KPABG KPABG KPABG Uher KPABG SYKOA GSE-PSE- 1986/6 4163 41662 610005 2000/2452 132178 C-013-09 MK29-07A False-branch- - + + - very rare - common - ing Cells elon- + + + + + + - + gated Constricted at not or not or not or not or distinctly slightly + not cross-walls slightly slightly slightly slightly Necridia - + + - frequent + + + Width of cells 1.7–2.1 1.8–2.5 1.6–2.4 1.6–3.4 1.5–3.0 1.6–3.1 2.3–4.3 1.5–2.4 (3.0) (2.1) 2.9–3.8 Length of cells 1.2–2.6 1.9–3 1.5–3.8(4) (2.2) 3.1–4.5 2.0–4.0 1.9–2.6 3.1–4.4 (5.4) (5.4) Meristematic - - - - + - - - zones Hormogonia - - - rare - - rare rare olive- blue- liver- green, Coloration olive-green olive-green green, ol- brownish olive-green blue-green brown blue- ive-green green The Rus- Slovakia, The Rus- Czech Re- sian Arc- The Rus- temperate The Rus- sian Sub- The Rus- public, tic/epilithic sian Sub- forest/on sian Sub- The USA, semi- Locality/ arctic/aero- sian Arc- temperate on a boul- arctic/aer- subaerial arctic/aer- arid/seep wall habitat phytic, tic/seep forest/aero- der in a ophytic, seep wall ophytic, and waterfall crust on the wall phytic, soil river, un- soil crusts and water- soil sludge derwater fall The strain KPABG 4163 fits well within the dimensions of Drouetiella lurida species. Macroscopically, mats of Drouetiella lurida KPABG 4163 are green-colored on an Agar plate. Trichomes with long or isodiametric cells (1.2–2.6 μm long) are distinctly constricted at the cross-walls (Figure 2a,b). The trichomes are characterized by rare false-branching, occasionally with necridic cells. Thylakoids: parietal. Cell: olive-green. The strain KPABG 41662 is similar to the strain KPABG 4163 (Figure 2c,d). It is char- acterized by long, straight trichomes with elongated or isodiametric cells (1.9–3 μm long, 1.6–2.4 μm wide) and rare false-branching. The investigated strain of Drouetiella lurida KPABG 610005 is characterized by long, curved or slightly waved filaments, containing one trichome, with or without sheath, 1.6– 3 (3.4) μm wide (Figure 2e,f). Mucilaginous sheaths are colorless, hyaline to firm, in older stages mostly firm. Trichomes indistinctly constricted at the cross-walls up to clearly con- stricted. Cells green or blue-green, from isodiametric to elongate, 1.5–3.8(4) μm long. Necridia cells absent. The principal morphological traits, such as the size and shape of cells, and presence of necridia cells, of the strain Drouetiella hepatica KPABG 132178 (Figure 2g) were very similar to the reference strain D. hepatica Uher 2000/2452 reported by Mai et al. . Minor differences in coloration and meristematic zone exist. Diversity 2023, 15, 132 6 of 19 Figure 2. Light micrographs of Drouetiella strains: (a,b) Drouetiella lurida KPABG 4163; (c,d) Drouetiella lurida KPABG 41662; (e,f) Drouetiella lurida KPABG 610005; (g) Drouetiella hepatica KPABG 132178; (b,d,f) unbranching filaments with thin, firm sheaths; (a,c) false-branching filaments. All photos are at the same magnification, scale = 10 μm. The strain SYKOA C-013-09 is distinguished by very common false-branching tri- chomes (Figure 3). Filaments: long and curved, containing one trichome. Sheath: firm, col- orless, thin, usually visible only during or following hormogonia formation and at the ends of filaments. Trichomes: isopolar, untapered, not or indistinctly constricted at the cross-walls, 2.3–4.3 μm wide. Cells: isodiametric or slightly longer or shorter than wide, olive-green or blue-green, 1.9–2.6 μm long. Apical cells: rounded, the same size as regular cells. Necridia: frequent. There was considerable overlap in the size ranges for all measured features (Table 2), and clear morphological separation of these taxa was not possible. Diversity 2023, 15, 132 7 of 19 Figure 3. Light micrographs of the Drouetiella SYKOA C-013-09 strain: (a,e) unbranching filaments with thin, firm sheaths; (b–d) false-branching filaments; (e) filaments with thin, firm sheaths. The arrow indicates a necridia. All photos are at the same magnification, scale = 10 μm. 3.2. Phylogeny The ML analysis of the 16S rRNA gene dataset resulted in a single tree with an arith- metic mean of Log likelihood of -7928.8266; in Bayesian analysis, the arithmetic means of Log likelihoods for each sampling run were −7957.81 and −7955.56. Trees from both phy- logenetic estimations revealed similar topologies; thus, Figure 4 demonstrates the ML to- pology with an indication of the bootstrap support values (BS) and Bayesian posterior probabilities (PP). The ML analysis of the 16S-23S ITS rRNA region resulted in a single tree with an arithmetic mean of Log likelihood of -2343.4528; the obtained topology is shown in Figure 5. The backbone phylogeny of the family Oculatellaceae, in the main course, agreed with that obtained previously and discussed in detail in the article . Here, we focus only on affinities within the genus Drouetiella. Diversity 2023, 15, 132 8 of 19 Figure 4. Phylogram obtained under the maximum likelihood approach for 56 accessions of Ocu- latellaceae based on the 16S rRNA gene. Bootstrap support values from the maximum likelihood and Bayesian posterior probabilities more than 50% (0.50) are indicated. The reference strain marked as r.s.; YNO: Yamalo-Nenets A.O. Diversity 2023, 15, 132 9 of 19 Figure 5. Phylogram obtained under the maximum likelihood approach for 10 accessions of Drouetiella based on the 16S-23S ITS region. Bootstrap support values of more than 50% are indi- cated. Ten strains of the genus Drouetiella formed a terminal clade with support BS = 94% and PP = 1.00 (or 94/1.00) in the reconstructed topology. The basal position in the clade belongs to the strain of Drouetiella fasciculata GSE-PSE-MK29-07A from the USA. Three unidentified Russian strains were found in the subclade (100/1.00) with the reference strain of Drouetiella lurida Lukesova 1986/6 from the Czech Republic. The closest relative to the reference strain was Drouetiella sp. KPABG 4163 from Yamalo-Nenets A.O. (99/0.99), the second divergence was presented by Drouetiella sp. KPABG 41662 from Murmansk Region (98/1.00), and the third by Drouetiella sp. KPABG 610005 from Komi Republic (100/1.00). The other Russian strain from Yamalo-Nenets A.O. revealed a sister affinity to the reference strain of Drouetiella hepatica Uher 2000/2452 from Slovakia (99/0.98). A sub- sequent relation to this subclade was composed of strains of Drouetiella sp. V16 from the USA (93/0.98) and Drouetiella sp. (Leptolyngbya frigida) ANT.L52.2 from Antarctica (95/0.78). The strain initially marked as Drouetiella sp. SYKOA C-013-09 from Komi Re- public was found to have a sister relation to D. hepatica-subclade (96/0.98), with a compar- atively long branch that supposed its separation. The topology arising from the ML analysis of the 16S-23S ITS rRNA region was highly similar to the topology obtained for the 16S rRNA gene in branching; however, the nodes received slighter bootstrap supports (Figure 5). The strains V16 and ANT.L52.2 re- vealed a sister relationship, supported by a BS = 72%. The position of strain SYKOA C- 013-09 was not supported. The variability of the sequence similarity values within the subclade containing the reference strain of Drouetiella lurida Lukesova 1986/6 was consistent in 99.17–99.90% cases in the 16S rRNA gene and 93.53–99.12% in the 16S-23S ITS rRNA region (Table 3). Table 3. The nucleotide sequence similarity for the genus Drouetiella strains, based on the nucleotide sequence data of the 16S rRNA gene and 16S-23S ITS rRNA region, %. Nucleotide Sequence Similarity, 16S/16S-23S ITS, % Taxon 1 2 3 4 5 6 7 8 9 1. D. lurida Lukesova 1986/6 2. D. lurida KPABG 99.90/ 4163 99.12 3. D. lurida KPABG 99.48/ 99.64/ 41662 94.83 94.13 4. D. lurida KPABG 99.17/ 99.37/ 99.28/ 610005 93.53 93.72 97.42 5. D. hepatica KPABG 97.82/ 98.21/ 98.40/ 98.66/ 132178 85.58 85.22 87.60 88.15 Diversity 2023, 15, 132 10 of 19 6. D. hepatica Uher 97.81/ 97.93/ 98.14/ 98.45/ 100.00/ 2000/2452 85.58 85.14 87.88 88.41 99.40 97.81/ 98.21/ 98.40/ 98.66/ 99.82/ 99.80/ 7. Drouetiella sp. V16 - 87.08 87.32 88.94 89.10 95.57 95.57 8. Drouetiella sp. 97.91/ 98.30/ 98.48/ 98.75/ 99.55/ 99.50/ 99.74/ ANTL522 86.67 86.35 87.53 87.45 94.15 94.22 96.98 9. D. ramosa SYKOA 98.02/ 98.39/ 98.03/ 98.30/ 98.84/ 98.65/ 98.84/ 98.92/ C-013-09 88.99 87.73 88.10 88.89 89.41 89.24 91.57 90.18 10. D. fasciculata GSE- 96.42/ 96.51/ 96.17/ 98.43/ 96.52/ 96.54/ 96.82/ 96.90/ 96.60/ PSE-MK29-07A 79.57 78.57 80.14 79.91 80.59 80.59 83.33 80.41 80.14 The strain KPABG 4163 was highly similar to the reference strain of the type species; strains KPABG 41662 and KPABG 610005 were similar to each other. The level of similar- ity for both strain pairs was 99.17–99.64% in the 16S rRNA gene and 93.53–94.83% in the 16S-23S ITS rRNA region. The similarity within the subclade containing the reference strain of Drouetiella hepatica Uher 2000/2452 was higher and achieved 99.50–100% in the 16S rRNA and 94.15–99.40% in the 16S-23S ITS rRNA region. Yarza et al. , pointed out that a similarity of more than 98.7% in the 16S rRNA gene allows us to consider allied strains as members of a single cyanobacterial species. Gonzalez-Resendiz et al.  sug- gested that less than 3% difference in ITS agrees with a level of infraspecific variability, and a difference of more than 7% determines a different species. In terms of Droutiella study, the level of 16S rRNA gene similarity fits well in a species concept, whereas in the 16S-23S ITS rRNA region, similarity takes an intermediate position within 3–7%. Accord- ing to the trees’ topology, the tested strains from Russia could be attributed to the corre- sponding species Drouetiella lurida (KPABG 4163, 41662, 610005) and D. hepatica (KPABG 132178). The sequence similarity of both species was 97.81–98.75% in the 16S rRNA and 85.14–89.10% in the 16S-23S ITS rRNA locus. The strain Drouetiella sp. V16 was highly similar in its 16S rRNA gene sequence with Drouetiella hepatica Uher 2000/2452 (99.80%) and Drouetiella hepatica KPABG 132178 (99.82%), and in its 16S-23S ITS rRNA, with a 95.57% similarity for both strains. The strain Drouetiella sp. ANT.L52.2 had ≥ 99.55% iden- tity in the 16S rRNA gene and ≥ 94.15% in the 16S-23S ITS rRNA, compared with other strains of the Drouetiella hepatica clade. Evidently, the level of similarity of both previously sequenced strains suggests them as belonging to Drouetiella hepatica. The strain Drouetiella sp. SYKOA C-013-09 was similar to both of the mentioned Drouetiella species with 98.02–98.92% in the 16S rRNA gene and 87.73–91.57% in the 16S- 23S ITS rRNA region. Drouetiella fasciculata appears to be the most isolated species in the genus that revealed a similarity with the rest of the taxa, with 96.17–98.43% in the 16S rRNA gene and 78.57–83.33% in the 16S-23S ITS rRNA region. The comparatively low level of sequence similarity in strain SYKOA C-013-09 compared to Drouetiella lurida and D. hepatica, additionally suggests its separation from known species. 3.3. The Secondary Structure of Conserved Domains of the 16S-23S ITS rRNA The sequence length variation in the conserved domains of 16S-23S ITS rRNA is quite different within the genus Drouetiella. The length of the V3 helix was stable for all strains of Drouetiella and consisted of 50 base pairs (b.p.). The length of the D1-D1′ helix was only 65 b.p. for D. fasciculata, whereas for the other tested strains, it was 64 b.p. The sequence length variability was slightly varied in the Box-B region: with 33 b.p. counted for the strains D. lurida Lukesova 1986/6, and KPABG 4163, and 32 b.p. for the strains KPABG 41662 and KPABG 610005; in addition, there were 34 b.p. for all strains allied to D. hepatica Uher 2000/2452 and SYKOA C-013-09; and 39 b.p. for D. fasciculata GSE-PSE-MK29-07A. The greatest sequence length variability was found in the V2 region. Among the strains attended to by Drouetiella lurida, the length of the helices changed twice: from 16 b.p. in D. lurida Lukesova 1986/6 and KPABG 4163, to 25 b.p. in strain KPABG 610005, and 30 b.p. in strain KPABG 41662. All strains of the D. hepatica affinity had 21 b.p. in the V2 Diversity 2023, 15, 132 11 of 19 helix, except for the strain SYKOA C-013-09, which had 23 b.p. Evidently, this region is absent in the D. fasciculata spacer. The D1-D1’ helix possesses three loops; only strain KPABG 610005 has four loops (Figure 6). The D1-D1’ helix secondary structures in the Drouetiella lurida clade strains (Lukesova 1986/6, KPABG 4163, KPABG 41662) are characterized by a long stem between the first and second loops, whereas strains of D. hepatica, D. fasciculata, and SYKOA C-013- 09 have only two steps in the corresponding region. There are three types of motives in the terminal loop in D. lurida Lukesova 1986/6, and two in D. hepatica. The secondary structures of D1-D1′ Drouetiella lurida Lukesova 1986/6 and D. lurida KPABG 4163 are highly similar: they have a substitution in the 10th position U/G and in the 42nd position С/A. The subterminal loop of D. lurida KPABG 610005 is similar to that of SYKOA C-013-09. The strain Drouetiella hepatica KPABG 132178 has only one substitution in the 12th position С/U compared with D. hepatica UHER 2000/2452. The strain SYKOA C-013-09 differs by the substitution of two nucleotides in the ter- minal loop, a substitution of one nucleotide in the middle loop (position 34 A/G), and substitutions of four nucleotides in the stem between the middle and basal loops (9th-12th positions) compared with the reference strain Drouetiella hepatica UHER 2000/2452. Drouetiella fasciculata has a unique motive of a terminal loop in the genus, a substitution in the middle loop compared with D. hepatica and SYKOA C-013-09, and a longer stem between the middle and basal loops. Figure 6. The secondary structure of D1-D1′ helices of the 16S-23S ITS rRNA region of Drouetiella strains. Circles indicate differences. Diversity 2023, 15, 132 12 of 19 The secondary structures of the V2 helices, in spite of their great length variability, possess a common view within the genus—a stem with a terminal loop (Figure 7). Three types of V2 helices were found in the Drouetiella lurida clade that reflected sufficient length diversity of the nucleotide sequences. The type strain of D. lurida and D. lurida KPABG 4163 has an identical structure with the shortest stem, by only six steps. The strains D. lurida KPABG 610005 and D. lurida KPABG 41662 have 9 and 12 steps in the stem, corre- spondingly, and different motives in the terminal loop. Four strains from the Drouetiella hepatica clade, with an identical length of V2 helix, have stems with nine steps, but each strain is characterized by a unique motive in the loop region presented, in each case, by only three nucleotides. The strain SYKOA C-013-09 has eight steps in a stem, a single un- paired base, and a loop composed of six nucleotides, which sufficiently differentiated this strain from multiplied sampled Drouetiella hepatica, with its quite stable structure. The strain Drouetiella fasciculata GSE-PSE-MK29-07A has the shortest region between the two tRNA genes that do not contain a V2 helix. Figure 7. The secondary structure of the V2 helices of the 16S-23S ITS rRNA region of the Drouetiella strains. Circles indicate differences. There are two types of Box-B helix structures in the genus: all strains of D. lurida clade are characterized by two loops; strains of the rest of the taxa only have a terminal loop (Figure 8). Three types of motives in the terminal loop are registered in the D. lurida clade (one type in a pair, Drouetiella lurida Lukesova 1986/6 and D. lurida KPABG 4163; the sec- ond in a pair, D. lurida KPABG 610005 and D. lurida KPABG 41662; and the third in a strain, Drouetiella sp. V16). The strains of D. hepatica possess a single motive; the stem structures for all strains are identical. The strain SYKOA C-013-09 differs from all D. he- patica strains only by the single substitution G/A in the 18th position in the terminal loop. Diversity 2023, 15, 132 13 of 19 The strain Drouetiella fasciculata GSE-PSE-MK29-07A differs from other species by the mo- tive of a terminal loop and a longer stem. The secondary structure of the V3 helices reveals three loops for all tested strains of the genus (Figure 9). For the type strain Drouetiella lurida Lukesova 1986/6, the secondary structure of the V3 region could not be reconstructed due to the absence of appropriate nucleotide sequence data. Two types of motives in the terminal loop are found for strains of Drouetiella lurida (the first type: D. lurida KPABG 4163 and D. lurida KPABG 610005; the second type: D. lurida KPABG 41662). The insertion U-A in the 15th/36th position of the stem between the terminal and middle loops is presented in strain KPABG 610005. Two substitutions—G/U in the 28th and U/C in the 31st position—are marked in strain KPABG 41662. The length of the stem between the terminal and middle loops counts 10 and 11 steps. Figure 8. The secondary structure of the Box-B helices of the 16S-23S ITS rRNA region of the Drouetiella strains. Circles indicate differences. Two types of motives in the terminal loop have been obtained for strains of Drouetiella hepatica. The strain KPABG 132178 differs from the type strain by a single substitution U/C in the base of the terminal loop. The middle loop of the Drouetiella hepatica strains and SYKOA C-013-09 is common and differs from this region in Drouetiella lurida strains by three substitutions. The strain SYKOA C-013-09 has a unique motive of a terminal loop, and the stem between the ter- minal and middle loop is shorter by one step compared with strains of Drouetiella hepatica. The secondary structure of the V3 helix of Drouetiella fasciculata differs from other taxa in the motives of loops and stems. Diversity 2023, 15, 132 14 of 19 The hypothetical secondary structures of conserved helices allow us to note signifi- cant differences between known species of the genus Drouetiella lurida, D. hepatica, and D. fasciculata, especially in terms of the D1-D1’, V2, and Box-B stem loops, whereas V3 seems to be more conservative. The variability of the secondary structures in all four regions is marked for both multiplied sampled Drouetiella lurida and D. hepatica. The features of the secondary structures for strain SYKOA C-013-09 are unique and not registered fully in the sister allied species Drouetiella hepatica, which also allows us to segregate it taxonomically. Taking into account the morphological features, position on the phylogenetic tree, nucleotide sequence dissimilarity, and the secondary structures of the conserved helices, we suggest that the strain SYKOA C-013-09 is a species new to science, and which will be described here. Figure 9. The secondary structure of the V3 helices of the 16S-23S ITS region in Drouetiella strains. Circles indicate differences. 3.4. Taxonomic Description Drouetiella ramosa Davydov, Vilnet, Novakovskaya and Patova sp. nov. (Figure 3) Diagnosis: D. ramosa is phenotypically distinct from other Drouetiella species due to its very common false-branching and short cells. It is different from any other represent- atives of the genus Drouetiella by its phylogenetic position based on 16S rRNA and 16S- 23S ITS rRNA gene phylogenies, with differences in the secondary structures of D1-D1’, Box-B, V2, and V3 helices. The V2 and V3 helices have the unique motive of a terminal loop (Figures 7 and 9). The percent dissimilarity between the 16S-23S ITS rRNA of this species and the other taxa is >8% (Table 3). Diversity 2023, 15, 132 15 of 19 Description: Colony green or olive-green, forming mucilaginous mats in liquid cul- ture. Filaments long to short, curved, with or without sheaths, containing one trichome. Sheath firm, colorless, thin, usually visible only during or following hormogonia for- mation and at the ends of filaments. Trichomes: isopolar, untapered, not or indistinctly constricted near the transverse cell walls with very common false-branching, 2.3–4.3 μm wide. Cells: isodiametric or slightly longer or shorter than wide, olive-green or blue-green, 1.9–2.6 μm long. Apical cells: rounded, the same size as regular cells. Necridia: frequent. Etymology: D. ramosa N.L. fem. Adj. = branched, bearing branches. Holotype designated here: dried specimen deposited into the herbarium of the Polar- Alpine Botanic Garden-Institute (KPABG), Apatity, Russia, under the following accession number: KPABG 4511. Type locality: Russia. Komi Republic. Subpolar Urals Mountains, 65.22639 N, 60.24528 E, elevation: 850 m. Next to a quartz mine on damp quartz sand, collected by E. Patova on August 5, 2009. Reference strain: SYKOA C-013-09 (isolated into culture by I. Novakovskaya), depos- ited in the Culture Collection of Algae at the Institute of Biology of Komi Scientific Centre under the number SYKOA C-013-09. NCBI GenBank Accession number: ON897677. 4. Discussion Similarly to many other Oculatellaceae and Leptolyngbyaceae species, morphologi- cal convergence and a low level of plasticity complicate the morphological identification of species in the genus Drouetiella. A comparison of the morphological features suggested that the Drouetiella species showed an indistinguishable shape and structure of the tri- chomes and a variability in cell sizes between them; as such, we cannot distinguish them with confidence based on morphological data only. In the original description, the type species Drouetiella lurida is characterized by purple and blackish-violet mats . After- wards, the authors noted brown and reddish-brown colonies [22,46]. Filaments of Drouetiella lurida are characterized as lacking false-branching, with elongate cells of a brownish color . The investigated strains KPABG 4163, KPABG 41662, and KPABG 610005 are similar to Drouetiella lurida, with their long and wide cells; however, our strains have trichomes characterized by false-branching (KPABG 4163, KPABG 41662), have distinctly con- stricted cross-walls (KPABG 4163, KPABG 610005), and have olive-green cell coloration. Such variability in color and branching could be associated with the origin of the native samples and could only be postulated by enlarged number of studied samples. The 16S rRNA and 16S-23S ITS rRNA genes phylogenies suggest a relationship between the tested strains to the type strain Drouetiella lurida Lukesova 1986/6. The strain KPABG 4163 is closer and more similar, regarding the secondary structure of its conservative helices in the 16S-23S ITS rRNA, to the type strain, whereas the strains KPABG 41662 and KPABG 610005 possess common features and differ from the first strains’ pair. The 16S rRNA gene similarity for all tested strains was high and corresponded more closely with the level of infraspecific variation (< 1.3%), whereas the similarity of the 16S-23S ITS rRNA had an interval of 3-7%, whereby it is hard to solve the problem of whether the strains belong to the same species, or to different taxa. The greatest diversity in the secondary structures of the ITS regions in the Drouetiella lurida clade was registered for the D1-D1’ and V2 helices, whereas Box-B and V3 appear to be more conservative. Previously, we illustrated the widely varied secondary structures in the helices among a large number of geographically remote strains of other filamentous species, Phormidesmis nigrescens, P. priestley, and P. communis, wherein the 16S rRNA gene similarity within each species was less than 1.3%, and the 16S-23S ITS rRNA was approximately 7% . Evidently, additional strain sam- pling of Drouetiella lurida would allow us to estimate the species’ genetic diversity and to make a robust taxonomical conclusion; however, the data in hand suggests that the at- tending strains, KPABG 4163, KPABG 41662, and KPABG 610005, belong to a single Diversity 2023, 15, 132 16 of 19 species, Drouetiella lurida, with a wide distribution stretching from East Europe to the Mur- mansk Region, and the Polar and Subpolar Ural Mountains. Drouetiella hepatica is similar to Drouetiella lurida by the color of its colony and tri- chomes; however, this species is distinguished from the other ones by its meristematic zones and false-branching. However, the strain KPABG 132178 does not have meriste- matic zones and is characterized by olive-green cell coloration in contrast with the type material, though it reveals its identity in the 16S rRNA and has the highest similarity of 16S-23S ITS rRNA genes with the type strain Drouetiella hepatica UHER 2000/2452. Based on the analyses of an enlarged dataset, and with the possibility of estimating the infraspe- cific variability with strains from Europe, the Russian Arctic, Alaska and Antarctica, we assume it is appropriate to treat the unnamed strains of Drouetiella (V16 and ANT.L52.2) as belonging to Drouetiella hepatica, taking into account a <1.3% variation in 16S rRNA genes, <7% variation in the 16S-23S ITS rRNA, and minor changes in the helices’ struc- tures; this is in opposition to the study by Mai et al. ; wherein, strain ANT.L52.2 is proposed as a candidate for a new species. Possibly, Drouetiella hepatica is a more molecu- larly conservative species, opposite to that of Drouetiella lurida. The single tested strain of Drouetiella fasciculata from the USA is clearly distinct from other Drouetiella species due to its bright blue-green color and fasciculation of trichomes . The type strain of this species shares only 96.42% identity with the Drouetiella lurida type strain and 96.54% identity with the D. hepatica type strain in the 16S rRNA, 79.57% and 80.59% in 16S-23S ITS rRNA, correspondingly; Drouetiella fasciculata is also distin- guished by its secondary structures of D1-D1’ and V3 helices. Taking into account the 16S rRNA sequence divergence between the recently described sister-related genera Pegethrix and Cartusia (98.0% ± 0.57), or Pegethrix and Elainella (97.0% ± 0) , Drouetiella fasciculata could be supposed to transfer to a distinct genus. The new species described here, Drouetiella ramosa, is morphologically the most sim- ilar to D. lurida, though it differs mainly by its very common false-branching and shorter cells. Molecularly, Drouetiella ramosa is allied to the D. hepatica clade, from both tested nu- cleotide loci. The differences in the D. ramosa and D. hepatica count, 1.35–1.38% in the 16S rRNA gene and 8.43–10.76% in the 16S-23S ITS rRNA, that demonstrated more high level of differentiation than level of infraspecific variability in both D. lurida and D. hepatica. The D1-D1′, Box-B, and V2 and V3 helices’ structures are similar to D. hepatica, but are not identical in the number of nucleotide substitutions, reflected in the unique motives in the terminal loops (Figures 6–9). We suggest that D. ramosa fits well in concept, of delimitation of a new species according to the published and mostly accepted criteria of similarity be- tween cyanobacterial species [44,45]. The puzzling combination of morphological and mo- lecular features of Drouetiella ramosa makes its identification difficult, and can only be ro- bustly resolved by testing the DNA sequence data. Drouetiella species occur in several types of habitats. Evidently, Drouetiella lurida is widely distributed in different ecological conditions: moist soils (KPABG 41662, 610005) , stagnant water and lake littoral zones , epilithic on a boulder in a river (KPABG 4163), and in thermal springs [47,48]. Both of the Drouetiella hepatica strains occurred on a subaerial seep wall near the water. Drouetiella fasciculata was found in subaerophytic hab- itats in wet walls. Drouetiella ramosa grows in aerophytic habitats in biological soil crusts. Drouetiella lurida was found in different regions and could be characterized as a worldwide distributed species . The occurrence of Drouetiella lurida at high latitudes has not been recorded previously. In Russia the species was only noted in the Caucasus . The distribution of Drouetiella hepatica, considering the inclusion of the species’ strains V16 and ANT.L52.2, comprises both polar regions. The occurrence of Drouetiella hepatica in the Polar Urals has been recorded for the first time for this Russian flora. Obvi- ously, reliable Arctic and Subarctic cyanobacterial species identification remains ex- tremely important to understand their geographic distribution. Diversity 2023, 15, 132 17 of 19 Author Contributions: Conceptualization, D.D.; methodology, D.D. and A.V.; formal analysis, D.D. and A.V.; investigation, D.D., I.N., A.V. and E.P.; resources, D.D.; data curation, D.D. and A.V.; writing—original draft preparation, D.D. and A.V.; writing—review and editing, D.D., A.V., I.N. and E.P.; visualization, D.D. and A.V.; supervision, D.D.; project administration, D.D.; funding ac- quisition, D.D. and A.V. All authors have read and agreed to the published version of the manu- script. Funding: This research was funded by the Russian Science Foundation, grant number 21-14-00029 (https://rscf.ru/project/21-14-00029/ accessed on 18 November 2022). The research was partially car- ried out within the framework of the state tasks Polar-Alpine Botanic Garden-Institute No. 1021071612832-8-1.6.11 (FMER-2021-0001) and IB FRC Komi SC UB RAS No. 122040600026-9. The research was performed using the large-scale research facilities of the herbarium at the Polar-Alpine Botanic Garden-Institute (KPABG; Kirovsk, Russia) reg. No. 499397. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data supporting the reported results can be found in the publicly archived dataset of the “L” Information system https://isling.org/ (accessed on 10 November 2022). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results. References 1. Whitton, B.A.; Potts, M. 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Diversity – Multidisciplinary Digital Publishing Institute
Published: Jan 18, 2023
Keywords: the Arctic; molecular phylogeny; 16S rRNA; 16S-23S ITS rRNA; filamentous cyanobacteria
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