Phylogenetically distant clade of Nostoc-like taxa with the description of Aliinostoc gen. nov. and Aliinostoc morphoplasticum sp. nov.

Transcription

1 TAXONOMIC DESCRIPTION Bagchi et al., Int J Syst Evol Microbiol 2017;67: DOI /ijsem Phylogenetically distant clade of Nostoc-like taxa with the description of Aliinostoc gen. nov. and Aliinostoc morphoplasticum sp. nov. Suvendra Nath Bagchi, 1 Neelam Dubey 1 and Prashant Singh 2, * Abstract Nostoc is a complex and tough genus to differentiate, and its morphological plasticity makes it taxonomically complicated. Its cryptic diversity and almost no distinguishable morphological characteristics make this genus incredibly heterogeneous to evaluate on taxonomic scales. The strain NOS, isolated from a eutrophic water body, is being described as a new genus Aliinostoc with the strain showing motile hormogonia with gas as an atypical feature, which is currently considered as the diacritical feature of the genus but should be subjected to critical evaluation in the near future. The phylogenetic placement of Aliinostoc along with some other related sequences of Nostoc clearly separated this clade from Nostoc sensu stricto with high bootstrap support and robust topology in all the methods tested, thus providing strong proof of the taxa being representative of a new genus which morphologically appears to be Nostoc-like. Subsequent phylogenetic assessment using the rbcl, psba, rpoc1 and tufa genes was done with the aim of facilitating future multi-locus studies on the proposed genus for better taxonomic clarity and resolution. Folding of the 16S 23S internal transcribed spacer region and subsequent comparisons with members of the genera Nostoc, Anabaena, Aulosira, Cylindrospermum, Sphaerospermopsis, Raphidiopsis, Desmonostoc and Mojavia gave entirely new secondary structures for the D1-D1 and box-b helix. Clear and separate clustering from Nostoc sensu stricto supports the establishment of Aliinostoc gen. nov. with the type species being Aliinostoc morphoplasticum sp. nov. in accordance with the International Code of Nomenclature for algae, fungi and plants. INTRODUCTION Traditionally, the genus Nostoc has been placed under the order Nostocales, family Nostocaceae [1] and, by virtue of bacteriological classification, in subsection IV of heterocytous filamentous cyanobacteria [2]. Since the first official report of Nostoc published in the late nineteenth century (type species Nostoc commune) [3], until the latest updates available, more than 300 species of Nostoc have been documented [4]. The morphological description of Nostoc is based on the occurrence of unbranched uniseriate isopolar filaments with differentiated chains of akinetes and terminal and intercalary heterocytes embedded in mucilaginous material-forming colonies [1]. The diagnostic morphological characteristics distinguishing the taxa at a sub-generic level are twofold: rigidity of mucilaginous colonies and complexity in the life cycle [5]. The extracellular matrix may range from fragile and diffluent matter to distinguishable envelopes surrounding filaments and, in certain species, a thick peridermal layer capturing a mass of filaments. The two means of vegetative propagation are fragmentation of old filaments in hormogonia and germination of spores (akinetes). However, the morphological synapomorphies that distinguish members of Nostocales, e.g., branching pattern, special position of heterocytes, specialized terminal cell, trichome tapering, heteropolarity, gas etc., are either lacking or are highly plastic in Nostoc. Rajaniemi et al. [6] combined phylogenetic and molecular markers with morphological traits to re-assess the taxonomic position of a rather limited number of temperate-origin Nostoc species, revealing at a sub-generic level at least two separate clusters. Further, molecular and phenotypic analyses revealed Nostoc to be polyphyletic in origin, distinguishing between the well-established clade of Nostoc sensu stricto with laminar mucilaginous colonies [7, 8] and at least three different clades of cyanobacteria sharing Nostoc morphology [7, 9, 10]. These include the new genera Author affiliations: 1 Department of Biological Science, Rani Durgavati University, Jabalpur, Madhya Pradesh , India; 2 National Centre for Microbial Resource (NCMR) (formerly Microbial Culture Collection, MCC), National Centre for Cell Science (NCCS), Pune, India. *Correspondence: Prashant Singh, sps.bhu@gmail.com Keywords: Cyanobacteria; Nostoc; 16S rrna; ITS. Abbreviations: BIC, Bayesian information criterion; +G, gamma distribution; +I, invariable; ITS, internal transcribed spacer; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour joining. Accession numbers generated through this study: 16S rrna-ky403996, rbcl-ky401608, psba-ky401609, rpoc1-ky401610, tufa-ky Five supplementary figures and five supplementary tables are available with the online Supplementary Material ã 2017 IUMS 3329

2 Mojavia, Trichormus and Nostoc group II. Nostoc muscorum and others falling in Nostoc group II made a highly inconsistent clade and were later brought appropriately under a separate genus Desmonostoc [11]. Komarek [4] presented an update of the phylogeny of Nostoc in which true Nostoc clusters displayed alignment in an order with Mojavia, Trichormus, Nostoc group II and Desmonostoc [4]. There are also reports of strains, e.g. those under the genus Halotia, with morphological resemblance to Nostoc, which upon phylogenetic analysis were positioned distantly from the Nostoc, Mojavia and Trichormus clades [12]. Recently, Genuario et al. [13], in their studies on Brazilian Nostoc-like morphotypes, described clade C-II, which seemed to represent a new polyphyletic lineage of Nostoc-like cyanobacteria. Nostoc therefore is an incredibly complicated and complex genus which is tough to differentiate morphologically; but at the same time, Nostoc can still be differentiated using proper sequencing and phylogenetic approaches. Hence, most of the above-mentioned studies have recommended a revision of the genus Nostoc, taking into account 16S rrna gene-based phylogenetic data. For phylogenetic analysis of Nostoc, apart from the 16S rrna gene, several diagnostic genes, namely rpoc1, hetr, rbclx and internal transcribed spacer (ITS) regions, were evaluated for sequence congruence, but the topologies thus generated were substantially different [14], indicating that these markers can be used as independent determinants with some coherence for taxonomic placement of new Nostoc species [15]. However, we believe that more conserved orthologs need to be assessed for better assessment of species identity. Owing to the highly conserved nature of the sequences [16], secondary structures based on D1-D1 sequences within the 16S 23S ITS region are instrumental in species-level identification of cyanobacteria, including that of Nostoc [9]. In the present work, strong phylogenetic support provided evidence that strain NOS is indeed a representative of a new genus having morphological similarities with Nostoc but being phylogenetically very distant from Nostoc sensu stricto. We began our study of strain NOS as being a putative species of the genus Nostoc, but analysis of the results obtained subsequently were found to be interesting. Taking into consideration the previous studies and our current data, we strongly recommend the creation of Aliinostoc gen. nov. with the type species being Aliinostoc morphoplasticum sp. nov. METHODS Sampling, culturing and phenotypic analysis Strain NOS was collected from a stagnant, eutrophic-polluted pond, among benthic rocks and other submerged substrates in Sihora, Jabalpur, India (23.48 N and E). Water analysis was performed by the M. P. Pollution Control Board, Jabalpur, by employing American Public Health Association methods [17]. The collected natural sample was immediately analysed microscopically to get an idea of the naturally occurring inherent microflora and to make an initial assessment of the morphology of the cyanobacteria present. This was followed by plating of the sample on 1.2 % solidified agar plates with BG-11 0 medium [18]. After 15 days of growth, one or two colonies were picked up, washed carefully with deionized water and transferred to 2 ml fresh liquid BG-11 0 medium. As the cultures started to disperse in the medium after days, intact new filaments, viewed under a 40 objective lens, were collected using sterilized 20 l micro-capillaries (Sigma Aldrich) and dispensed into new batches of 1 ml medium. This step was repeated until the purity of the culture was established by phase-contrast and bright-field microscopy, and by plating culture aliquots on Luria Bertani medium. This microscopic dilution method was adopted to establish axenicity of the culture. The cyanobacterial strain was then grown in 150 ml batches of the medium contained in cotton stoppered Erlenmeyer flasks (capacity 500 ml). The ph of the medium was adjusted to 7.2 and the culture was maintained in a culture room under illumination of approximately µem 2 s 1 with a photoperiod of 14/10 h light/dark cycle at 28±2 C. The culture was shaken twice a day. The initial identification of the cyanobacteria was done by using the keys of Desikachary [19]. Keys of Komarek [4] and Desikachary [19] were consulted to reach a final conclusion about the morphological description of strain NOS. Strain NOS was observed under a Nikon YS100 microscope. Microphotographs of the cyanobacteria were taken by using an Olympus BX53 microsope fitted with a ProgRes C5 camera (Jenoptik). The shape of the apical cells and the shape and size of the vegetative filaments, vegetative cells, heterocytes and akinetes were visualized and observed with 100 measurements being taken for each. The presence/absence of constrictions in the different cells and sheath, along with visibility of the sheath in different portions of the trichome were also carefully studied. Molecular analysis The DNA was isolated from 20-day-old log phase culture using the Himedia Ultrasensitive Spin Purification Kit (MB505) with some modifications in the lysis step in which incubation time was increased for lysis solutions AL and C1. Amplification of the 16S rrna gene was done using primers pa (5 -AGAGTTTGATCCTGGCTCAG-3 ) and B23S (5 -CTTCGCCTCTGTGTGCCTAGGT-3 ) [20, 21]. The amplification of the rbcl gene was done according to Singh et al. [22] while the psba gene was amplified as per Singh et al. [23]. The amplification of the rpoc1 gene was performed as per Glowacka et al. [24] while the tufa gene was amplified as per the recommendations of Fewer [25]. Sequence analysis Sequencing of the amplified products was carried out by using the Sanger method on a 3730xl automated sequencer (Applied BioSystems). For the 16S rrna gene and the ITS region, a 1760 bp sequence was generated and the similarity search was performed using the Identify option of the Eztaxon database ( [26] with only validated cyanobacterial strains. Appropriate sequences from NCBI were also selected for phylogenetic analysis using the 16S rrna gene. For the rbcl, psba, rpoc1 and tufa genes, 3330

3 the sequences obtained were analysed by using the NCBI web service with the BLASTN ( Blast.cgi) tool and submitted to the NCBI database using BankIt. The sequences were annotated for the coding regions using the NCBI ORF Finder ( gov/orffinder/). Phylogenetic analysis Multiple sequence alignment was performed using CLUSTAL X, version 1.81 [27]. With the aim of obtaining unambiguous data, the alignment was manually edited using DAMBE [28]. All the phylogenetic analyses were performed using the software package MEGA version 5 [29]. Analyses for the 16S rrna gene phylogeny were done using the validly published sequences from the Eztaxon database [26] along with selecting appropriate sequences from NCBI. The model of phylogeny was selected according to the lowest Bayesian information criterion (BIC) score, which led to the selection of the Kimura 2-parameter model (K2+G+I) [30] having the lowest BIC score of In case of the rbcl gene, the evolutionary history was inferred by using the maximum-likelihood (ML) method based on Kimura 2-parameter model (K2+G+I) with the BIC score being The psba gene was phylogenetically assessed by using the K2+G model with the BIC score being The rpoc1 gene was phylogenetically assessed by using the K2+G model with a BIC score of while the tufa gene was analysed using the K2+G+I model with a BIC score of Where there was non-uniformity of evolutionary rates among the sites, adjustments were modelled using a discrete Gamma distribution (+G) with five rate categories and by assuming that a certain fraction of sites were evolutionarily invariable (+I). Bootstrap values were deduced based on 1000 replications [31]. In order to evaluate the robustness of the tree topology, clustering was performed using three different methods: neighbour-joining (NJ), ML and maximum-parsimony (MP) [31 34]. 16S 23S secondary structure analysis Secondary structures of the 16S 23S ITS region were determined for all the closely related taxa present in the phylogenetic interpretations with comparisons made from other closely related members of the genera Nostoc, Anabaena, Aulosira, Cylindrospermum, Sphaerospermopsis, Raphidiopsis, Desmonostoc and Mojavia. The secondary structures were transcribed and folded for all the above-mentioned strains by using the Mfold web server [35]. RESULTS Habitat and morphological evaluation Various physicochemical characteristics of the water sample collected from the Matha Talab pond were measured so as to gain information about the general ecology of strain NOS. At the time of sampling, the physicochemical properties of the water (Table S1, available in the online Supplementary Material) signified high water conductivity owing to high ion concentrations, especially of ammonia and nitrite. The morphology of the naturally occurring sample was studied carefully and the shape and size of the vegetative cells and heterocytes were documented (data not shown). The morphology of the subsequent laboratory-grown culture of strain NOS was also studied at different intervals of growth and sub-culturing so as to keep a check on any life cycle pattern events or any culture contamination. Upon long-term growth in the same flask (150 ml BG-11 o medium for 6 months, 2 days) the culture exhibited akinete formation. However, we did not observe any akinetes in the natural sample. The morphology of strain NOS was assessed both individually and also compared with the available data of the rest of the strains of the Aliinostoc clade. Certain features, such as type of colonies, shape and size of vegetative cells, heterocytes, hormogonia and akinetes, were observed carefully with emphasis being placed on sheath presence, colour of sheath, presence/absence of sheath in apical portions, presence/absence of constrictions between the cross walls, and shape and size of the different cells. Motile hormogonia with gas were observed and this observation was found in many other (though not all) strains of the Aliinostoc clade also (Fig. 1 and Tables 1 and 2). Molecular evaluation Strain NOS showed % sequence similarity with strains belonging to the genus Aliinostoc and Nostoc elgonense TH3S05. With the exception of N. elgonense TH3S05 (AM711548), all the closely related strains were identified only to the genus level and thus had limited taxonomic clarity. The rbcl gene sequence similarity was found to be 96 % with Nostoc piscinale CENA21 (CP012036), while the psba gene sequence similarity was 95 % with Nostoc sp. PCC 7524 (CP003552). The rpoc1 gene sequence similarity was found to be 89 % with Anabaena variabilis ATCC (CP000117) while the pairwise similarity in case of the tufa gene was found at 91 % with Nostoc sp. PCC 7524 (CP003552). Phylogenetic assessment In the 16S rrna gene tree, strain NOS was placed in a close cluster with various strains of Aliinostoc (Fig. 2 and Table S2). The Aliinostoc clade comprised cyanobacteria from Thailand, India, USA, France and Brazil with none of the strains being taxonomically characterized [except perhaps Nostoc elgonense TH3S05 (AM711548)]. Overall, this clade, which contained strain NOS along with 14 other strains of Aliinostoc, was found to have robust phylogenetic support at all the pertinent nodes with the tree topology being similar in the NJ, ML and the MP methods. The Aliinostoc clade was also found to be sufficiently separated from Nostoc sensu stricto along with the other clades (Halotia, Mojavia, Desmonostoc) which had originated from the traditional Nostoc s. In all the 16S rrna gene trees, the branched heterocytous forms clustered together with good phylogenetic support while genera like Anabaenopsis, Cyanospira, Chrysosporum, Dolichospermum, Nodularia, 3331

4 (a) (b) (c) (d) (e) (f) (g) (h) Fig. 1. (a) Loosely arranged filaments with variable tendencies for coiling; spherical terminal heterocyte and ovate-shaped terminal heterocyte. (b) Well-constricted trichomes with differently shaped terminal heterocytes with one being oblong. (c) Well-constricted trichomes with terminal heterocytes having the same shape but vegetative cells being differently shaped. (d) Differently shaped vegetative cells in the same filament. (e) Oblong apoheterocytous akinetes. (f) Differently shaped vegetative cells with a few oblong cells. (g) Adjacent filaments with differently shaped terminal heterocytes with one being elliptical. (h) Constricted vegetative cells and motile four-celled hormogonia with gas. Scytonema, Brasilonema, Iphinoe, Cylindrospermum and Aulosira also exhibited tight clustering with sufficient support. A considerable degree of diversity was found to be occurring in the genera Nostoc, Anabaena and Trichormus in all the reconstructed trees. Interestingly, the rbcl gene tree also placed strain NOS in close proximity with strain Nostoc piscinale CENA 21 (CP012036) with the distance being indicative of its novel origin (Fig. S1 and Table S3). The psba tree clearly placed strain NOS in a close cluster with Nostoc sp. PCC 7524 (CP003552), and the phylogenetic distance indicated that it was different from Nostoc sp. PCC 7524 (CP003552) (Fig. S2). In case of the rpoc1 gene tree, strain NOS was placed at a different node from all the closely related strains of Nostoc (Fig. S3); while, in the tufa gene tree, its placement was again near Nostoc sp. PCC 7524 (CP003552) though the distance was again indicative of it being different from Nostoc sp. PCC 7524 (CP003552) (Fig. S4 and Table S4). 16S 23S secondary structure analysis Folding of the secondary structures of the D1-D1 and box- B helix was done for strain NOS and all the other closely related taxa (representatives from all the closely related taxa that appeared in the phylogenetic tree). There was a strong variation of both the sequences and the secondary structures when comparing strain NOS with all the other taxa (Fig. 3 and S5; Table S5). Unusually, the D1-D1 helix of strain NOS was found to be very variable with all the taxa being Table 1. Quantitative morphological traits of Aliinostoc morphoplasticum Type of cell Length (mm) Width (mm) Description Colonies 3 7 mm in Usually spherical, later scattered and bullose diameter Terminal vegetative Barrel-shaped to sometimes appearing almost spherical cells Intercalary vegetative Barrel-shaped to spherical to oblong cells Heterocytes Spherical to elliptical to ovate to oblong; observed more at the terminal positions and less at the intercalary positions Akinetes Oblong; apoheterocytous or intercalary 3332

5 Table 2. Comparative sequence similarities, habitat, origin and brief description of the various strains of the Aliinostoc clade Data of strains (except Aliinostoc morphoplasticum) taken from a previous report by Genuario et al. [13] and references therein. Strain Pairwise similarity (%) Habitat Origin Brief description Aliinostoc morphoplasticum (KY403996) Aliinostoc sp. PCC 8112 (AM711537) Aliinostoc sp. PCC 8976 (AM711525) Nostoc elgonense TH3S05 (AM711548) Aliinostoc sp. CENA175 (KC695867) Aliinostoc sp. CENA514 (KX458484) Aliinostoc sp. CENA513 (KX458483) Aliinostoc sp. CENA524 (KX458485) Aliinostoc sp. CENA543 (KX458489) Aliinostoc sp. CENA544 (KX458490) Aliinostoc sp. CENA88 (GQ259207) Aliinostoc sp. CENA511 (KX458482) Aliinostoc sp. CENA536 (KX458487) Aliinostoc sp. CENA548 (KX458492) Aliinostoc sp. CENA535 (KX458486) Eutrophic, polluted pond Jabalpur, India Motile hormogonia containing gas 98 Laundromat discharge Michigan, USA Motile hormogonia containing gas pond 97 Brackish marshland Mediterranean coast, France Hormogonia formation not seen 97 Rice field soil not flooded by water Thailand No data 97 Soil, mangrove Bertioga, S~ao Paulo, Brazil Motile hormogonia containing gas 97 Saline-alkaline lake Aquidauana, Mato Grosso do Sul Motile hormogonia containing gas 97 Saline-alkaline lake Aquidauana, Mato Grosso do Sul Motile hormogonia containing gas 97 Saline-alkaline lake Aquidauana, Mato Grosso do Sul Motile hormogonia containing gas 99 Freshwater reservoir Piracicaba, S~ao Paulo, Brazil Motile hormogonia containing gas compared. The most prominent visual difference was the presence of six loops (of different sizes) as compared to the usual presence of three to five loops in all the members of the family Nostocaceae. On closer inspection, more differences were observed which clearly differentiated strain NOS from all the previously known members of family Nostocaceae. The base of the stem in strain NOS was found to have 6 bp, which is also known to vary in the family Nostocaceae. There was no unilateral bulge in any of the six loops in strain NOS, though we observed that unilateral bulges were prominent in other members of the family Nostocaceae (although not always). Apart from the absence of a unilateral loop, we also found the absence of any large loop, which is also a common feature in many members of the family Nostocaceae. In the absence of a large loop, apart from the loops near the base of the stem and the terminal loop, four medium sized loops were found to be present, which was indeed a unique feature of strain NOS and which in comparison with any member(s) of the genera Nostoc, Anabaena, Aulosira, Cylindrospermum, Sphaerospermopsis, Raphidiopsis, Desmonostoc and Mojavia, was different. The box-b helix for strain NOS was also found to be distinct from all the closely related taxa (Fig. 4). For better coverage of comparisons, we analysed the box-b helix of strains belonging to the genus Nostoc and even Cylindrospermum, Aulosira and Desmonostoc. With the exception of the genus Desmonostoc, the basal structure was the same in all the taxa being compared. But the differences in the internal loop just after the basal stem were more significant in the base composition and the overall structure with no particular genus-specific trend being evident in this case. Differences were again observed in the stem between the internal loop and the terminal loop with the difference being either in overall length or even in the base pair composition (in cases where the length was the same). Finally, the terminal loop again varied in case of strain NOS as compared to the other closely related taxa. The box-b helix of Nostoc muscorum CENA61 was found to be very similar to that of strain NOS with the only difference being in the sequence of the terminal loop. With the aim of estimating the distance of the strains selected for the ITS analysis, we performed a p-distance calculation amongst all the strains and the results indicated that the strains were certainly different from each other. Overall, it was easy to distinguish the secondary structures of the D1-D1 and box-b helix for strain NOS as compared to all the closely related taxa whose secondary structures have been investigated. DISCUSSION Nostoc is a complex genus in which it is difficult to differentiate clearly between closely related taxa based solely on 3333

6 (a) /-/71 Anabaena planctonica NIES-816 (AY701548) 50/60/70 Aphanizomenon gracile 1tu26s2 (AJ630443) 80/76/97 Dolichospermum mucosum 1tu35s5 (AJ630425) 79/84/93 87/73/53 Dolichospermum affine NIES40 (AF247591) Anabaena cylindrica XP6B (AJ630414) 66/63/- 74/80/- Aphanizomenon gracile HEANEY/Camb (AJ630444) 100/-/95 99/97/96 Aphanizomenon gracile 1tu26s16 (AJ630445) Anabaena oscillarioides BECID22 (AJ630426) 76/-/- Cuspidothrix issatschenkoi 0tu37s7 (AJ630446) 100/99/98 Trichormus (4 OTUs) 100/99/99 Anabaena (5 OTUS) 99/100/99 Aulosira (2 OTUs) 96/73/85 Anabaenopsis (9 OTUs) 92/99/97 61/-/- Cyanospira (6 OTUs) 99/96/97 Cyanocohniella calida CCALA 1049 (KJ737427) 86/91/97 Aliinostoc (15 OTUs) 100/100/100 Chrysosporum (3 OTUs) 93/79/89 Nodularia (7 OTUs) 96/91/93 Halotia (9 OTUs) 100/100/100 Scytonema (3 OTUs) 100/100/100 68/-/- Iphinoe (3 OTUs) 97/96/87 Brasilonema (5 OTUs) 96/95/79 Synechococcus elongatus PCC 6301 (NR_074309) 87/91/94 Desmonostoc (25 OTUs) 98/99/98 97/99/99 Nostoc piscinale CENA21 (AY218832) Nostoc thermotolerans (KX252675) 56/-/67 Trichormus azollae Kom BAI/1983 (AJ630454) 99/100/99 Cylindrospermum (3 OTUs) 84/83/82 -/60/57 97/96/91 97/100/98 Nostoc sp. PCC 7524 (CP003552) Nostoc muscorum CENA61 (AY218828) Anabaena/Trichormus variabilis (4 OTUs) True branched heterocytous cyanobacteria (16 OTUs) (b) 66/79/93 Aliinostoc sp. PCC 8112 (AM711537) Aliinostoc sp. PCC 8976 (AM711525) Nostoc elgonense TH3S05 (AM711548) Aliinostoc sp. CENA175 (KC695867) 51/-/- 51/-/- Nostoc calcicola TH2S22 (AM711529) 81/-/62 Mojavia pulchra JT2-VF2 (AY577534) 92/98/97 Nostoc verrucosum (AB245144) 100/100/99 Nostoc verrucosum KU005 (AB494996) 94/90/93 Nostoc sensu stricto (15 OTUs) 64/-/- Aliinostoc sp. CENA514 (KX458484) 100/100/100 Aliinostoc sp. CENA513 (KX458483) 56/83/59 75/83/88 Aliinostoc sp. CENA524 (KX458485) Aliinostoc sp. CENA543 (KX458489) 100/99/99 Aliinostoc sp. CENA544 (KX458490) 61/64/- Aliinostoc sp. CENA88 (GQ259207) /99/73 Aliinostoc morphoplasticum (KY403996) Aliinostoc sp. CENA511 (KX458482) 84/81/73 100/99/99 Aliinostoc sp. CENA536 (KX458487) 70/-/68 Aliinostoc sp. CENA548 (KX458492) Aliinostoc sp. CENA535 (KX458486) Fig. 2. (a) Phylogenetic tree based on the 16S rrna gene of heterocytous cyanobacteria with the bootstrap values representing NJ, ML and MP, respectively. Bar, 0.01 changes per nucleotide position. All bootstrap values below 50 were deleted. (b) Complete Aliinostoc clade based on the 16S rrna gene with the bootstrap values representing NJ, ML and MP, respectively. Bar, changes per nucleotide position. All bootstrap values below 50 were deleted. morphology simply because of the huge amount of heterogeneity [15]. During the course of this study, we garnered strong evidence that strain NOS, isolated from a eutrophic freshwater body in Jabalpur, Madhya Pradesh, may be representative of a new lineage of Nostoc-like cyanobacteria which has not been described till now. 16S rrna gene sequencing showed a pairwise similarity ranging from 99 to 97 % with various Aliinostoc strains which had been 3334

7 Nostoc muscorum CENA61 (AY218828) Cylindrospermum badium CCALA 1000 (KF142524) Aulosira laxa NIES-50 (KJ920354) Anabaena bergii (JQ237772) Aliinostoc morphoplasticum (KY403996) Fig S-23S ITS folded structures of the D1-D1 helix of Aliinostoc morphoplasticum in comparison to closely related species. sequenced but not discussed together in phylogenetic perspectives. The phylogenetic positioning, distance and bootstrap support of strain NOS with reference to the closely related species of Nostoc, Desmonostoc and various other heterocytous cyanobacteria indicated that this strain might be a new member of a clade of Nostoc-like cyanobacteria which needs to be described as a new genus. Our 16S rrna gene phylogenetic results were completely in congruence with all past reports mentioning and recommending that the deviants of Nostoc morphotypes from Nostoc sensu stricto must be described as new genera [6 9, 11 13, 36, 37]. It is notable that such revisions based on the above-mentioned ideas have already been used to separate and describe new genera like Mojavia [9], Desmonostoc [11], Cyanocohniella [38] and Halotia [12]. Thus, our phylogenetic findings provide enough proof of concept, both from our current results and the previously described works of taxonomists, that the Aliinostoc clade in this study is indeed a new lineage that must be described as a new genus of cyanobacteria. Our work also gains support from the recent work of Genuario et al. [13] who mentioned that this clade may be a new lineage of Nostoc-like cyanobacteria (clade C- II). We also propose to reclassify all the Nostoc-like cyanobacteria listed in the clade as strains of Aliinostoc. We would like to recommend that studies using DNA DNA hybridization (DDH) should be employed in some cases for resolving taxonomic issues of cyanobacteria subject to the relative similarities of the 16S rrna, phylogenetic clustering and also considering the availability of proper type materials. Whole genome sequencing could be another effective alternative in resolving ambiguities of cyanobacterial taxonomy but they must be used prudently in cases where the clustering and similarity values become ineffective. In this case, the clustering of strain NOS was so distant that DDH or whole genome sequencing was not needed. It is well known that the database for functional genes of cyanobacteria is limited and, hence, there is limited scope for multi locus studies being attempted to solve the taxonomic issues of closely related cyanobacteria. This drawback has been indicated in some of our previous studies [39]. In the present work, when we aimed to describe a new genus, we extended our molecular analyses by using the rbcl, psba, rpoc1 and tufa genes with the aim of creating a robust database for at least a newly described genus so that, in future, taxonomic studies focusing on this genus could gain support from a well standardized multi locus plan of work. As a result of database inconsistencies, our rbcl, psba, rpoc1 and tufa gene phylogenies proved inadequate in giving any clear picture in this case (Figs S1 S4). We performed the folding of the 16S 23S ITS region and the secondary structures obtained were used for comparison of strain NOS with the closely related cyanobacterial taxa in the phylogenetic tree. The folded secondary structures have been found to be useful in resolving closely related species in cyanobacterial taxonomy [9, 40 42]. The D1-D1 helix and box-b helix were folded and it was very easy to demarcate the entirely different secondary structure of strain NOS as compared to many other closely related taxa belonging to the genera Nostoc, Anabaena, Aulosira, Cylindrospermum, Sphaerospermopsis, Raphidiopsis, Desmonostoc and Mojavia. 3335

8 Aulosira sp. ISB-2 (EU532189) Nostoc commune NC1 (EU784149) Nostoc muscorum CENA61 (AY218828) Cylindrospermum badium CCALA 1000 (KF142524) Nostoc commune HHCP (EU586724) Nostoc commune NC3-K1 (EU586722) Aulosira laxa NIES-50 (KJ920354) Bagchi et al., Int J Syst Evol Microbiol 2017;67: Desmonostoc vinosum HA7617 LM4 (KF417429) Aliinostoc morphoplasticum (KY403996) Fig S-23S ITS folded structures of the box-b helix of Aliinostoc morphoplasticum in comparison to closely related species. The strain Nostoc muscorum CENA61 was found to have an almost identical box-b structure with the only differences being in the terminal loop of the sequence. The p-distance calculations also clearly showed that the strains were distant and, hence, there were no two species that were identical. In fact, the distance between strain NOS and Nostoc muscorum CENA61 was found to be while the overall mean distance between all the strains was noted at with the standard error (SE) being In this study, we compared the morphology of a natural sample and a laboratory grown culture of strain NOS and found almost no differences between them. However, on continued growth in a suspension culture for around 6 months, we did observe formation of akinetes, which we did not find in the natural sample. The water body from which the sample was isolated had a huge amount of ammonia and nitrite and was in fact higher than the recommended level even for polluted waters, and this attribute seems to have contributed to high water conductivity. Some of the atypical morphological features of strain NOS that deserve particular attention include bullose colonies which appeared yellowish to brownish. It was further noticed that there were significant variations in the size of the vegetative cells, heterocytes and akinetes. One of the most prominent differences that deserves special consideration was the presence of heterocytes of different shapes, which clearly demarcated strain NOS from other closely related sub-generic entities of Aliinostoc. Our attempt to compare certain morphological features, namely presence/absence of motile hormogonia with gas amongst the related Aliinostoc strains, gave some interesting results where the presence of hormogonia with gas was observed in all the strains except Aliinostoc sp. PCC 8976 (a brackish marshland isolate) and Nostoc elgonense TH3S05 (no data). The rest of the strains of the Aliinostoc clade were reported to have motile hormogonia with gas vesicle formation being evident and we believe that, as of now, this may be considered as the morphological autapomorphic diacritical character of this clade. The consistency and usefulness of motile hormogonia with gas as a diacritical feature has also been indicated in some previous works [4, 13]. It is always advisable to further evaluate this character when describing new strains of the Aliinostoc clade. Even though evidence of atypical morphological features specific to the taxon is at the moment not very strong, we believe that, on the basis of robust phylogenetic evidence and interesting 16S 23S ITS folded structures complemented by satisfactory morphological and ecological observations, there is enough evidence to support our conclusion that Aliinostoc should be recognized as a new genus. DESCRIPTION OF ALIINOSTOC BAGCHI ET AL. GEN. NOV. Aliinostoc (A.li.i.nos toc. L. adj. and pronoun alius, other, another, different; N.L. neut. n. Nostoc, a cyanobacterial genus name; N.L. neut. n. Aliinostoc the other Nostoc). Widely spread genus with members comprising strains from rice fields of Thailand, water bodies of India and saline-alkaline lakes of Brazil. All members show close morphological resemblance to the genus Nostoc with almost no distinguishable differences being evident. A promising diacritical feature is the presence of motile hormogonia with gas. The filaments are loosely arranged with variable tendencies for coiling. The ecology of this genus is diverse, and it is possible to assign a specific habitat for most of the 3336

9 Aliinostoc strains. The water resources from where the organisms are isolated are rich in total dissolved ions, salts etc., as revealed from high water conductivity. Another striking feature is that more new species belonging to this genus could be anticipated from both southern and northern latitudes on both sides of the Equator indicating a possible pattern of biogeographical distribution centering around the tropical regions. The type species is Aliinostoc morphoplasticum. DESCRIPTION OF ALIINOSTOC MORPHOPLASTICUM BAGCHI ET AL. SP. NOV. Aliinostoc mophoplasticum (mor.pho.pla sti.cum. Gr. n. morph^e, form, shape; Gr. adj. plastikos, ^e, on, fit for moulding, plastic; N.L. neut. adj. morphoplasticum, having a shape that can vary or can adapt, because the strain shows a variety of heterocyte shapes). Macroscopic mats in its natural habitat; colonies are spherical, 3 7 mm in diameter, later scattered and bullose, leathery and irregular, yellowish to brownish, no distinct periderm, filaments not entangled and not coalescent together; loosely arranged filaments with variable tendencies for coiling; slight amount of sheath present which is usually colourless and also present in the apical portions of the filament; cells almost barrel-shaped to sometimes appearing even isodiametric; cells prominently constricted at the cross walls; terminal vegetative cells µm in length to µm in width while the intercalary vegetative cells are µm in length to µm in width; heterocytes varying in shape from being circular to slightly cylindrical with tapering at one end; observed more at the terminal positions and lesser at the intercalary positions; good frequency of filaments having heterocytes at both the ends; polar nodules clearly visible in many of the filaments; µm in length to µm in width; akinetes oblong, µm in length to µm in width, apoheterocytous or intercalary, brownish inner and greenish outer cell wall. This species separates itself from other closely related subgeneric taxonomic entities of Nostoc and Aliinostoc in having differently sized vegetative cells and differently shaped heterocytes and akinetes. The heterocytes particularly show varying shapes even in the same filament. The presence of motile hormogonia with gas is an important character that is a common feature in the Aliinostoc clade. In addition, it has low identity of the 16S rrna gene in comparison to other closely related strains along with showing typical separate clustering in the complete Aliinostoc clade with robust bootstrap support and strong tree topologies using various methods. Folding of the secondary structures of the ITS region clearly demonstrate a new structure of the D1-D1 helix and box-b helix on being compared to closely related strains in the phylogenetic tree having sequences of the above-mentioned regions. Type locality: Sihora town, district Jabalpur, Madhya Pradesh, India (23.48 N and E). Habitat: In stagnant, eutrophic-polluted water, attached to the benthic rocks and other submerged substrates. Isolated from pond. Holotype: Culture of Aliinostoc morphoplasticum was deposited to Microbial Culture Collection (MCC), National Centre for Cell Science (NCCS), Pune, India as Nostoc sp. The culture was again authenticated at MCC using 16S rrna sequencing. After authentication, cryopreserved culture is being maintained in MCC with the Accession Number MCC Funding information This work was supported by the Department of Biotechnology (DBT; Grant no. BT/PR/0054/NDB/52/94/2007), the Government of India, under the project Establishment of Microbial Culture Collection. Acknowledgements We thank the Director NCCS for facilities and encouragement. We thank Professor Aharon Oren and Dr Stefano Ventura for help in the scientific names and etymology. The authors thank the anonymous reviewers for the thorough critical review of the work. P. S. is thankful to the Department of Science and Technology (DST), India for the project YSS/2014/ S. N. B. and N. D. thank the Head, Department of Biological Science, Rani Durgavati University for extending the laboratory facilities and M.P. Pollution Control Board for providing the data on water analysis. Conflicts of interest The authors declare that there are no conflicts of interest References 1. Komarek J, Anagnostidis K. Modern approach to the classification system of the cyanophytes 4 - Nostocales. Algol Stud 1989;56: Rippka R, Castenholz RW, Herdman M. Subsection IV (Formerly Nostocales Castenholz 1989b sensu Rippka, Deruelles, Waterbury, Herdman and Stanier 1979). In: Boone DR and Castenholz RW (editors). Bergey s Manual of Systematic Bacteriology. New York, NY: Springer-Verlag; pp Bornet É, Flahault C. Revision des Nostocacees heterocystees continues dans les principaux herbiers de France (quatrie me et dernier fragment). Ann Sci Nat Bot 1888;7: Komarek J. Cyanoprokaryota 3. Heterocytous genera. In: G artner G, Krienitz L and Schagerl M (editors). Sübwasserflora Von Mitteleuropa/Freshwater Flora of Central Europe. Heidelberg: Springer; pp Mateo P, Perona E, Berrendero E, Leganes F, Martín M et al. Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers. FEMS Microbiol Ecol 2011;76: Rajaniemi P, Hrouzek P, Kastovska K, Willame R, Rantala A et al. Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (Nostocales, Cyanobacteria). Int J Syst Evol Microbiol 2005;55: Hrouzek P, Ventura S, Lukešova A, Mugnai MA, Turicchia S et al. Diversity of soil Nostoc strains: phylogenetic and phenotypic variability. Arch Hydrobiol Suppl Algol Stud 2005;117: Papaefthimiou D, Hrouzek P, Mugnai MA, Lukesova A, Turicchia S et al. Differential patterns of evolution and distribution of the symbiotic behaviour in nostocacean cyanobacteria. Int J Syst Evol Microbiol 2008;58: Řehakova K, Johansen JR, Casamatta DA, Xuesong L, Vincent J. Morphological and molecular characterization of selected desert 3337

10 soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. Nov. Phycologia 2007;46: Fernandez-Martínez MA, de Los Ríos A, Sancho LG, Perez-Ortega S. Diversity of endosymbiotic Nostoc in Gunnera magellanica from Tierra del Fuego, Chile [corrected]. Microb Ecol 2013;66: Hrouzek P, Lukesova A, Mares J, Ventura S. Description of the cyanobacterial genus Desmonostoc gen. nov. including D. muscorum comb. nov. as a distinct, phylogenetically coherent taxon related to the genus Nostoc. Fottea 2013;13: Genuario DB, Vaz MG, Hentschke GS, Sant anna CL, Fiore MF. Halotia gen. nov., a phylogenetically and physiologically coherent cyanobacterial genus isolated from marine coastal environments. Int J Syst Evol Microbiol 2015;65: Genuario DB, Andreote AP, Vaz MG, Fiore MF. Heterocyte-forming cyanobacteria from Brazilian saline-alkaline lakes. Mol Phylogenet Evol 2017;109: Han D, Fan Y, Hu Z. An evaluation of four phylogenetic markers in Nostoc: implications for cyanobacterial phylogenetic studies at the intrageneric level. Curr Microbiol 2009;58: Singh P, Shaikh ZM, Gaysina LA, Suradkar A, Samanta U. New species of Nostoc (cyanobacteria) isolated from Pune, India, using morphological, ecological and molecular attributes. Plant Syst Evol 2016;302: Johansen JR, Kovacik L, Casamatta DA, Ikova KF, Kaštovský J. Utility of 16S-23S ITS sequence and secondary structure for recognition of intrageneric and intergeneric limits within cyanobacterial taxa: Leptolyngbya corticola sp. nov. (Pseudanabaenaceae, Cyanobacteria). Nova Hedwigia 2011;92: American Public Health Association (APHA). Standard Methods for the Examination of Water and Waste Water. Washington, DC; Rippka R, Stanier RY, Deruelles J, Herdman M, Waterbury JB. Generic assignments, strain histories and properties of pure cultures of Cyanobacteria. Microbiology 1979;111: Desikachary TV. Cyanophyta. New Delhi: ICAR monographs on algae; Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 1989;17: Gkelis S, Rajaniemi P, Vardaka E, Moustaka-Gouni M, Lanaras T et al. Limnothrix redekei (van goor) Meffert (Cyanobacteria) strains from lake Kastoria, Greece form a separate phylogenetic group. Microb Ecol 2005;49: Singh P, Fatma A, Mishra AK. Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data. Ann Microbiol 2015;65: Singh P, Singh SS, Aboal M, Mishra AK. Decoding cyanobacterial phylogeny and molecular evolution using an evonumeric approach. Protoplasma 2015;252: Głowacka J, Szefel-Markowska M, Waleron M, Łojkowska E, Waleron K. Detection and identification of potentially toxic cyanobacteria in polish water bodies. Acta Biochim Pol 2011;58: Fewer D. Molecular evidence for the antiquity of group I introns interrupting transfer RNA genes in cyanobacteria. M.Sc. Thesis, Göttingen: Universit at zu Göttingen; Kim OS, Cho YJ, Lee K, Yoon SH, Kim M et al. Introducing EzTaxon-e: a prokaryotic 16S rrna gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 2012;62: Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25: Xia X. DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution. Mol Biol Evol 2013;30: Tamura K, Peterson D, Peterson N, Stecher G, Nei M et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28: Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16: Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39: Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 1981;17: Fitch WM. Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 1971;20: Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4: Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31: Genuario DB, Silva-Stenico ME, Welker M, Beraldo Moraes LA, Fiore MF. Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc. Toxicon 2010;55: Lukesova A, Johansen JR, Martin MP, Casamatta DA, Lukesova A et al. Aulosira bohemensis sp. nov.: further phylogenetic uncertainty at the base of the Nostocales (Cyanobacteria). Phycologia 2009;48: Kaštovský J, Berrendero-Gomez E, Hladil J, Johansen JR. Cyanocohniella calida gen. nov. et spec. nov. (Cyanobacteria: Aphanizomenonaceae) a new cyanobacterium from the thermal springs from Karlovy Vary, Czech Republic. Phytotaxa 2014;181: Singh P, Minj RA, Kunui K, Shaikh ZM, Suradkar A et al. A new species of Scytonema isolated from Bilaspur, Chhattisgarh, India. J Syst Evol 2016;54: Boyer SL, Flechtner VR, Johansen JR. Is the 16S-23S rrna internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol Biol Evol 2001;18: Boyer SL, Johansen JR, Flechtner VR. Characterization of the 16S rrna gene and associated 16S-23S ITS region in Microcoleus: evidence for the presence of cryptic species in desert soils. J Phycol 2002;38: Johansen JR, Casamatta DA. Recognizing cyanobacterial diversity through adoption of a new species paradigm. Arch Hydrobiol Suppl Algol Stud 2005;117: