{"paper_id":"3e28d4ce-0732-47a7-9e92-fd0622016a74","body_text":"Uncovering cyanobacterial diversity in Northeast India: phylogenetic and structural insights from heterocystous Strains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Uncovering cyanobacterial diversity in Northeast India: phylogenetic and structural insights from heterocystous Strains Heisnam Sobhana Devi, Kamaljit Moirangthem, Nepuni Rinaldi, Ramachandran Muthaiyan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8830670/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract The North-East region of India, a recognised Indo-Myanmar biodiversity hotspot, remains underexplored for its cyanobacterial diversity. In this study, ten heterocystous cyanobacterial strains were isolated from freshwater-associated habitats using a polyphasic approach integrating morphological, molecular, and structural analyses. Morphological identification was complemented by 16S rRNA gene sequencing, phylogenetic reconstruction, and secondary structure prediction. The isolates were assigned to the genera Anabaena , Scytonema , Nostoc , Calothrix , Hapalosiphon , and Neowestiellopsis . Notably, one isolate corresponded to Neowestiellopsis sp. BTA1159 a taxon previously reported only from Iran marking the first record of this genus from Manipur and the second global report. Phylogenetic analysis using the neighbour-joining method (p-distance, Jukes Cantor correction) revealed that Neowestiellopsis sp. BTA1159 occupied a distinct polyphyletic position, while Calothrix sp. BTA1160 formed an outbranch. Minimum free energy (MFE) calculations for 16S rRNA secondary structures indicated that Neowestiellopsis sp. BTA1159 exhibited the highest structural stability among all isolates. These findings not only document novel cyanobacterial diversity from a globally significant hotspot but also demonstrate the utility of secondary structure analysis in complementing phylogenetic resolution. Cyanobacteria Heterocystous strains Indo-Myanmar biodiversity hotspot Neowestiellopsis Phylogenetic analysis 16S rRNA gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cyanobacteria commonly called blue-green algae are prokaryotic photoautotrophs capable of oxygenic photosynthesis. They thrive across diverse ecological niches from terrestrial to aquatic systems and are even found in extreme thermal and polar environments, making them globally abundant and ecologically significant. Many cyanobacteria also form symbiotic associations with plants, fungi (lichens), and certain eukaryotic algae (Whitton and Potts 2000). Traditionally, cyanobacterial taxonomy has relied on morphological traits such as trichome shape and size, cell differentiation (heterocysts, akinetes), and cell division patterns (Rippka et al. 1979). However, morphology-based classification is often unreliable traits can vary markedly under different growth conditions or developmental stages, even within axenic cultures (Anand 1988; Lyra et al. 2001; Nalewajko and Murphy 2001; Gugger et al. 2002; Gupta and Agrawal 2006; Gamage 2020; Overlingė et al. 2024). In response, the polyphasic approach integrating morphology, molecular markers, ultrastructure, biochemical and ecological traits have emerged as the gold standard in modern cyanobacterial taxonomy (Komárek 2013; Giovannoni 1991; Zampieri et al. 2025; Wang et al. 2025). Molecular data, especially 16S rRNA gene sequences, now form the backbone of phylogenetic classification. This marker is cultivation-independent, allows PCR from minimal DNA, and reveals evolutionary relationships often obscured by morphological plasticity (Gamage 2020; Wilmotte 1994; Strunecký et al. 2023). Notably, increasing 16S rRNA data availability has exposed polyphyletic groups and misclassifications highlighting the need to revise traditional taxonomic schemes (Strunecký et al. 2023; Pham et al. 2025). Recent studies have also emphasized the importance of ITS secondary structure analysis and additional markers like 16S-23S ITS to distinguish closely related cyanobacterial taxa with greater resolution (Zampieri et al. 2025; Wang et al. 2025). Further, amplicon-based sequencing reveals rich cyanobacterial diversity in under-studied habitats; e.g., a study of Bolivian Altiplano microbial mats identified numerous novel cyanobacterial lineages using 16S rRNA profiling (Hentschke et al. 2025). In India, applying this integrative approach is particularly crucial. A recent study characterized 15 Indian cyanobacterial strains exhibiting Nostoc-like morphology using a polyphasic framework, underscoring the uneven application of modern taxonomy within the region (Pal et al. 2024). The present study aimed to investigate ten heterocystous cyanobacterial strains isolated from North-Eastern India (Manipur and Meghalaya), focusing on morphological diversity, molecular identification via 16S rRNA sequencing, and their phylogenetic relationships. Additionally, our integration of structural (secondary structure) analysis aims to enhance taxonomic resolution and reveal novel diversity within this globally significant hotspot. Materials and methods Sampling, isolation and purification Ten cyanobacterial strains were isolated from environmental samples collected in the North-Eastern states of Manipur and Meghalaya, India. For each sampling site, metadata including latitude, longitude, and altitude were recorded. Samples were inoculated into 100 mL Erlenmeyer flasks containing sterile BG-11 broth (Stanier 1971) in two variants: nitrogen-supplemented (+ N) for non-heterocystous strains and nitrogen-free (-N) for heterocystous strains. Following initial growth, cultures were examined using a ZEISS Primo Star microscope (ZEISS, Germany) to confirm cyanobacterial presence, then streaked onto respective BG-11 agar plates (+/- N2). Pure cultures were obtained through repeated streaking and subsequently maintained in 100 mL BG-11 broth. All cultures were incubated at 28 ± 2°C under an irradiance of approximately 40 µmol photons m²/s from cool-white 40 W fluorescent lamps, with a 14 h light/10 h dark photoperiod. Identification of the strain Morphological Identification Taxonomic and morphological characterization was performed following standard monographs and descriptions (Desikachary 1959). Microscopic identification considered key phenotypic traits, including filament/trichome type, presence of sheath, cell shape, and occurrence of heterocysts and akinetes. Observations were conducted at 40× magnification using an Axio Scope A1 microscope (Carl Zeiss, Germany) fitted with an AxioCam MRc camera and AxioVision 4.7.2 software. Additional growth characteristics, including biomass color and growth pattern (wall-attached, bottom-settled, submerged, or floating), were also documented. Molecular identification DNA Extraction Genomic DNA was extracted from ~ 1 g of 15-20-day old cyanobacterial biomass using a modified glass bead method (Devi et al. 2015). The biomass was homogenized in an autoclaved mortar and pestle with 1 g of sterile glass powder, 10 mg of activated charcoal, and 2 mL of extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl; pH adjusted to 8.0).The homogenate was transferred to a 2 mL microtube containing 10 µL Proteinase K (20 mg/mL) and 50 µL RNase A (20 mg/mL), incubated at 65°C for 10 min, and centrifuged (12,000 g, 5 min, 4°C). To 500 µL supernatant, 100 µL sodium acetate and 400 µL PEG were added, incubated at -20°C for 20 min, and centrifuged. Pellets were resuspended in 500 µL TE buffer and extracted with 500 µL chloroform:isoamyl alcohol (24:1), followed by centrifugation. The aqueous fraction was mixed with 500 µL of chilled isopropanol, incubated at 4°C for 5 min, and centrifuged at 12,000 g for 10 min. The resulting pellets were rinsed with 70% ethanol, centrifuged again at 12,000 g for 2 min, air-dried, and finally resuspended in 50 µL of TE buffer (pH 8). 16S rRNA PCR amplification The 16S rRNA gene was amplified using forward primer 106F (5′-CGG ACG GGT GAG TAA CGC GTG A-3′) and an equimolar mixture of reverse primers 781Ra (5′-GAC TAC TGG GGT ATC TAA TCC CAT T-3′) and 781Rb (5′-GAC TAC AGG GGT ATC TAA TCC CTT T-3′) (IDT, USA). Each 50 µL PCR reaction contained 10 µL 5× Phusion HF buffer, 1 µL 10 mM dNTPs, 2.5 µL 10 µM of each primer, 0.5 µL Phusion DNA polymerase (2 U; ThermoFisher Scientific, USA), 2.5 µL template DNA (~ 50 ng), 1.5 µL DMSO, and 29.5 µL nuclease-free water. PCR amplification was conducted on a ProFlex thermocycler (Applied Biosystems, USA). The cycling conditions comprised an initial denaturation at 95°C for 3 min, followed by 34 cycles of 95°C for 30 s, 57°C for 40 s, and 72°C for 40 s, with a final extension at 72°C for 5 min. The resulting amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and visualized on 1.5% agarose gels. Visualization was performed using a Universal Hood II Gel Doc System (Bio-Rad, USA), and products were sequenced bidirectionally on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA). Phylogenetic Analysis Sequence data were analyzed using BioEdit v7.0.9 (Hall 1999). Homologous sequences (97–100% identity; E-value > 10⁻²⁰) were identified using the BLASTn tool against the NCBI nucleotide collection (nr/nt) database (Altschul et al. 1997). Sequences generated in this study were deposited in GenBank. Multiple sequence alignments of 16S rRNA gene sequences were performed using ClustalW in MEGA v4.0 (Tamura et al. 2007). Pairwise sequence divergence was estimated using the Jukes-Cantor substitution model (Jukes and Cantor 1969), and phylogenetic reconstruction was performed with the neighbour-joining algorithm (Saitou and Nei 1987) based on p-distance values. Bootstrap analysis was conducted with 1,000 replicates, and only values > 50% were displayed. Calothrix sp. BTA1160 served as the outgroup. Secondary RNA structures for all ten cyanobacterial strains were predicted using RNAfold software (Gruber et al. 2008). Results and Discussion Sample collection Algal samples were collected during 2019–2020 from four districts within the Indo-Myanmar biodiversity hotspot: East Khasi Hills and West Jaintia Hills (Meghalaya), and Tengnoupal and Senapati (Manipur). Sampling sites encompassed diverse habitats, including rivers, natural and artificial lakes, waterfalls, streams, and rock surfaces. Collected samples were cultured in BG-11 medium and sub-cultured on BG-11 agar plates to obtain pure isolates. A total of ten pure cultures were established and preserved in the Freshwater Cyanobacterial and Microalgal Repository at IBSD, Manipur, India. Metadata, including GPS coordinates, habitat type, and pH, are presented in Table 1 . Table 1 Metadata of 10 samples collected from different locations of NER used in this study Name of species with accession no Place Habitat Latitude Longitude Altitude (m) Hapalosiphon sp. BTA1153 Matilang, East Khasi Hills, Meghalaya Stream 25.54472 91.83195 1804.72 Scytonema sp. BTA1154 Jakrem, East Khasi Hills, Meghalaya Rock surface 25.38966 91.50979 435.86 Anabaena doliolum BTA1155 Barapani, East Khasi Hills, Meghalaya River 25.65956 91.89837 990.30 Nostochopsis sp. BTA1156 Wards, East Khasi Hills, Meghalaya Lake 25.57504 91.88744 1495 Anabaena variabilis BTA1157 Dawki, East Khasi Hills, Meghalaya River 25.18917 92.01827 48.77 Nostoc calcicola BTA1158 Kwatha, Tengnoupal, Manipur Rock surface 24.33725 94.27843 436.78 Neowestiellopsis sp. BTA1159 Kwatha, Tengnoupal, Manipur Stream 24.33725 94.27843 436.78 Calothrix sp. BTA1160 Jowai, West Jaintia Hills, Meghalaya Water fall 25.43385 92.18323 1291.74 Nostoc sp. BTA1161 Jowai, West Jaintia Hills, Meghalaya Rock surface 25.43385 92.18323 1291.74 Hapalosiphon sp. BTA1162 Mao, Senapati, Manipur Waterfall 25.52258 94.12332 1996.14 Morphological identification Initial cultures showed indistinct morphological features due to environmental debris. Through serial plating on agar, we isolated ten morphologically distinct strains. Microscopic examination (40×) evaluated cell shape and size, heterocyst and akinete morphology, and sheath presence complemented by growth patterns in flasks (Table 2 ) for preliminary taxonomic identification using the classification keys of Desikachary (1959). Representative micrographs are shown in Fig. 1 . This morphology-based approach remains common but increasingly recognized as insufficient on its own; recent studies advocate combining morphological, molecular, ultrastructural, and ecological traits for accurate classification (Komárek 2023; Jiang et al. 2023). Table 2 Morphological characteristics of the isolated cyanobacteria Name of the strains Thallus Filaments Cell Heterocyst Akinete Sheath Hapalosiphon sp.BTA1153 Dark green, bottom attach, clump and floccose biomass Filaments free, cells in one or two rows, sub-spherical, true branching. 5.8–8.41 µm long and 6.19–7.3 µm broad Intercalary and oblong, 6.37–10.35 µm long and 4.01–5.49 µm board Present Present Scytonema sp. BTA1154 Dark green, bottom attached, compact and clump biomass Filament false branching, sub-quadrate, 4.58–5.97 µm long and 3.78–4.8 µm broad Sub-quadrate, 6.12–7.18 µm long and 4.25–5.17 µm board Present Firm Anabaena sp. BTA1155 Brownish green, Submerged and attached at side of flask, reticulate biomass Barrel shaped, 1.89–4.92 µm long and 3.11–3.76 µm broad Spherical, 4.6-7 µm long and 4-5.59 µm board Present Present Nostochopsis sp.BTA1156 Dark green, bottom attached, initially caespitose and latter floccose biomass Barrel shape, 3.75–11.15 µm long and 4.87–10.21 µm broad Intercalary, 5.32-12-12 µm long and 3.89–7.37 µm board Absent Present Anabaena variabilis BTA1157 Dark green, side attached, submerged, filamentous and reticulate biomass Barrel-shaped, Cell 3.11–4.18 µm long and 2.77–4.05 µm broad Spherical, 6.22–7.94 µm long and 4.51–5.43 µm board Present Present Nostoccalcicola BTA1158 Dark green, submerged latter side attached, filamentous and reticulate biomass Barrel-shaped, 2.11–2.53 µm long and 2.14–2.44 µm broad Sub-spherical, 2.46–3.19 µm long and 2.14–3.23 µm board Present Present Neowestiellopis sp.BTA1159 Dark green, bottom attached, floccose and caespitose biomass Cell 5.06–5.8 µm long and 3.96–4.56µm broad. T-shaped branching on both sides, probable appearance of biseriate condition. 6.31–7.8µm long and 3.11–4.25µm board, irregularly shared –intercalary/elongate. Absent Present Calothrix sp. BTA1160 Dark green, bottom dweller, sheath like latter glomerate Sub-quadrate, 3.59–7.34 µm long and 4.18–7.13 µm broad Basal, 4.25–6.41 µm long and 5.04–6.3 µm board Present Present Nostoc sp. BTA1161 Dark green, submerged and reticulate biomass Cylindrical, 2.65–3.88 µm long and 2.32–2.59 µm broad Intercalary, 3.84–6.68 µm long and 3.18–4.13 µm board Present Present Hapolosiphon sp.BTA1162 Dark green, bottom attached, globose latter floccose Quadrate, 3.69–4.85 µm long and 5.14–6.22 µm broad. Intercalary, 3.41–8.17 µm long and 4.13–6.24 µm board Present Present Molecular identification Cyanobacterial identification under laboratory conditions is often hindered by morphological alterations and reduced interspecific variation caused by controlled growth environments (Dores and Parker 1988). To complement morphological taxonomy, molecular identification was performed through PCR amplification of cyanobacteria-specific 16S rRNA sequences. Genomic DNA from the ten isolates was obtained using a modified glass bead protocol, yielding high-purity DNA (A260/A280 = 1.28–2.09) at 12.2–65.2 ng/µL. The genomic DNA fragments measured approximately 9000-10,000 bp, while purified PCR amplicons of the 16S rRNA gene were 600–700 bp in size (Fig. 2 ). After successful PCR amplification, the 16S rRNA gene amplicons were sequenced using the Sanger method, and the obtained sequences were analyzed and submitted to the GenBank database at the National Center for Biotechnology Information (NCBI) (Table 3 ). Phylogenetic analysis of the partial 16S rRNA sequences placed the ten cyanobacterial isolates into two major clusters (Fig. 3 ). Cluster 1 comprised Anabaena doliolum , Anabaena variabilis , Scytonema sp., Nostoc sp., and Nostoc calcicola , while Cluster 2 included Neowestiellopsis sp., Hapalosiphon sp. (two isolates), and Nostochopsis sp. The outgroup was represented by Calothrix sp. Within Cluster 1, A. doliolum and A. variabilis shared 94% bootstrap support, and Scytonema sp. showed 94% similarity to these Anabaena species. The Nostoc sp. and N. calcicola grouping had 92% bootstrap support. In Cluster 2, Neowestiellopsis sp. showed 95% similarity with the Hapalosiphon clade, which in turn had 100% internal bootstrap support, while Nostochopsis sp. was supported by 91%. Both major clusters were strongly supported (92% bootstrap), and the phylogenetic tree confirmed identification of the isolates to the genus level. These high bootstrap values demonstrate sturdy phylogenetic resolution at the genus level consistent with findings that 16S rRNA-based trees are generally reliable for higher-order classification, though species-level resolution may vary (Vondrášková et al. 2025). Table 3 NCBI accession number of the isolated strains used in this study Sl.No. Strain Identity NCBI Accession no. Strain Identity NCBI Accession no. 1 Scytonema sp. BTA1154 MW354520 6 Calothrix sp. BTA1160 MW354522 2 Anabaena doliolum BTA1155 MW342607 7 Nostoc sp. BTA1161 MW354523 3 Hapalosiphon sp.BTA1153 MW354516 8 Nostoc calcicole BTA1158 MW354524 4 Anabaena variabilis BTA1157 MW354518 9 Neowestiellopis sp. BTA1159 MW354517 5 Nostochopsis sp.BTA1156 MW354521 10 Hapalosiphon sp. BTA1162 MW354519 Further phylogenetic interpretation revealed evolutionary distinctions among the isolates. Anabaena doliolum , A. variabilis , and Scytonema sp. formed a paraphyletic group, while Nostoc sp., N. calcicola , and Neowestiellopsis sp. appeared polyphyletic, consistent with recent reports that Nostocales taxa often require taxonomic revision (Wang et al. 2025; Komárek 2023). By contrast, Hapalosiphon isolates and Nostochopsis sp. formed a well-defined monophyletic clade. The close clustering of A . doliolum and A. variabilis despite clear morphological differences echoes previous findings that 16S rRNA similarity among Anabaena genotypes can reach ~ 94%, complicating species resolution (Valério et al. 2009). Increasingly, researchers recommend the integration of additional markers such as the 16S-23S ITS region or multi-locus sequence analysis to refine cyanobacterial systematics (Vondrášková et al. 2025; Jiang et al. 2023). Most significantly, Neowestiellopsis sp. was recorded here for the first time in Manipur, India, with strong bootstrap support (95%), representing only the second global report of this genus. This highlights the Northeast Indian Indo- Myanmar hotspot as an important but underexplored reservoir of cyanobacterial diversity. SEC-Structure The partial 16S rRNA gene sequences of all cyanobacterial genera were translated, and their RNA secondary structures were predicted. The folding patterns and minimum free energy (MFE) values indicated stable secondary structures, supporting the reliability of the predicted RNA conformations. Different structural folding shapes generated for all cyanobacterial isolates species of partial RNA sequences. The energy table showed (Table 4 ) that, the highest minimum free energy − 229.90 kcal/mol for Anabaena doliolum and Anabaena variabilis minimum free energy − 229.93 obtained. The Scytonema sp. structural potential free energy revealed − 213 kcal/mol was the lowest energy amongst others. Further the cyanobacterial isolate Nostoc sp. showed minimum free energy − 238.10 kcal/mol respectively. The Neowestiellopsis sp. structure potential energy was − 245.60 kcal/mol predicted. The Nostoc calcicola − 234.90 kcal/mol, Hapalosiphon sp. -237.60 kcal/mol and Hapalosiphon sp. -232.10 kcal/mol potential minimum free energies were predicted. For the Nostochopsis sp., structural free energy was − 221.50 kcal/mol was obtained from the RNA secondary structure. All the cyanobacterial isolates 16s rRNA secondary structures different level of energies with sequence positions wise represented clearly in energy plots shown in Fig. 4 . Table 4 Secondary Structure Stability of 16S rRNA Sequences in Cyanobacterial Strains Based on Minimum Free Energy (MFE) Sl. No Sample Name Sec-Struct (kcal/mol) 1 Anabaena doliolum BTA1155 -229.90 2 Anabaena variabilis BTA1157 -229.93 3 Scytonema sp. BTA1154 -219.40 4 Calothrix sp. BTA1160 -213.80 5 Nostoc sp. BTA1161 -238.10 6 Neowestiellopsis sp. BTA1159 -245.60 7 Nostoc calcicola BTA1158 -234.90 8 Hapalosiphon sp. BTA1161 -237.60 9 Hapalosiphon sp. BTA1153 -232.10 10 Nostochopsis sp. BTA1156 -221.50 16S rRNA partial genes sequences thermodynamically folding showed that substitutions of base pairing caused complex structural changes in the folding pattern. Pseudo nodes and nodes created mispairing sequence structural regions loss of stability of the structures. In Anabaena doliolum and Anabaena variabilis , 16S rRNA structures fold showed similarly with conserved. The Scytonema sp., Neowestiellopsis sp. and Calothrix sp. secondary structures created same folding conservations base pairs cause different potential energy and stability among the structures. Further Nostoc sp., Nostoc calcicola sp. and Nostochopsis sp. secondary structures showed same structural conserved bases interact with sequences formed helix structure and maintain the stability of structure. Node region of base pairs sites loss of interactions and potential structural stability. The two Hapalosiphon sp. 16S rRNA sequence secondary structure showed similar folding structures and the minimum free energy level by the conserved sequence base pairs sites. Secondary structure analyses like these provide valuable insights into cyanobacterial diversity. Not only do they reveal structural conservation and variability, but they also supplement phylogenetic resolution when used alongside molecular and morphological data (Klindworth et al. 2013). Indeed, the use of 16S-23S ITS secondary structure has previously proven effective for distinguishing closely related cyanobacterial taxa and resolving intrageneric relationships (e.g. Leptolyngbya corticola sp. nov.) (Johansen et al. 2011). In a broader molecular context, the 16S rRNA gene remains a cornerstone for phylogenetic analysis, though limitations at the species level are well acknowledged (Gamage 2020; Hassler et al. 2022). Conclusion Ten cyanobacterial strains were isolated from diverse habitats within the Indo-Myanmar biodiversity hotspot. These isolates exhibited marked variation in morphological and growth characteristics, which served as the primary basis for preliminary identification. Subsequent molecular characterisation and phylogenetic analysis, based on partial 16S rRNA gene sequences, corroborated the morphological findings. Comparative analysis of the predicted secondary structures of the 16S rRNA sequences revealed similarities in folding patterns and stability among certain strains. This study underscores the cyanobacterial diversity present in the examined region and reports, for only the second time globally, the occurrence of the recently classified genus Neowestiellopsis . Previously documented in Iran, this marks the first record of the genus from Manipur in North-East India. Declarations Competing interest: The authors declare no conflict of interest. Ethical approval and consent to participate This research work does not involve any animal or human participants. Author Contribution HSD: Writing–original draft; Methodology; performed the experiments; ValidationKM: Writing–original draft; Methodology; performed the experiments; ValidationNR: Data curationRM: Investigation; Data curationKKS: Writing–review & editing; Formal analysis; VisualizationCM: performed experiment; data curationAP: performed experiment; data curationNS: Writing–review & editing; Supervision; Funding acquisitionOSK: Writing–review & editing; conceived and designed the study; Formal analysis; Visualization Acknowledgement We are grateful to the local community and labmates for their assistance in sample collection. We also thank the Department of Biotechnology (DBT), Government of India, for financial assistance. We gratefully acknowledge the Department of Biotechnology, Government of India, for financial support under Grant No. BT/01/17/NE/TAX, provided through the Institute of Bioresources and Sustainable Development, Imphal, Manipur. 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Wang J, Zhang T, Guo S et al (2025) Novel species of Oculatellaceae (Oculatellales, Cyanobacteria) from Yunnan in China, based on the polyphasic approach. Diversity 17:170. Whitton B, Potts M (2000) The ecology of cyanobacteria: their diversity in time and space. In Kluwer Academic Publishers eBooks. http://ci.nii.ac.jp/ncid/BA47335651. Wilmotte A (1994) Molecular evolution and taxonomy of the cyanobacteria. In Springer eBooks, pp 1-25. Zampieri RM, Bizzotto E, Campanaro S, Caldara F, Bellucci M, La Rocca N (2025) Kovacikia euganea sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new chlorophyll f producing cyanobacterium from the Euganean Thermal District (Italy). Front Microbiol 16. https://doi.org/10.3389/fmicb.2025.1545008. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 02 Mar, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 19 Feb, 2026 Reviewers agreed at journal 16 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor assigned by journal 10 Feb, 2026 Submission checks completed at journal 09 Feb, 2026 First submitted to journal 09 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8830670\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":590742751,\"identity\":\"7b0cbbc3-b243-4735-b53b-d289d5204104\",\"order_by\":0,\"name\":\"Heisnam Sobhana Devi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Lovely Professional University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Heisnam\",\"middleName\":\"Sobhana\",\"lastName\":\"Devi\",\"suffix\":\"\"},{\"id\":590742753,\"identity\":\"69715d99-460c-45b3-bada-bba0225421be\",\"order_by\":1,\"name\":\"Kamaljit Moirangthem\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kamaljit\",\"middleName\":\"\",\"lastName\":\"Moirangthem\",\"suffix\":\"\"},{\"id\":590742756,\"identity\":\"db82d567-357b-4805-819e-7a78757635db\",\"order_by\":2,\"name\":\"Nepuni Rinaldi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nepuni\",\"middleName\":\"\",\"lastName\":\"Rinaldi\",\"suffix\":\"\"},{\"id\":590742758,\"identity\":\"2759d469-2873-4e81-87c3-5cd9bbda297f\",\"order_by\":3,\"name\":\"Ramachandran Muthaiyan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ramachandran\",\"middleName\":\"\",\"lastName\":\"Muthaiyan\",\"suffix\":\"\"},{\"id\":590742760,\"identity\":\"1424f945-3910-4b5c-bfd2-5c27b9ed99cb\",\"order_by\":4,\"name\":\"Khaidem Kennedy Singh\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Khaidem\",\"middleName\":\"Kennedy\",\"lastName\":\"Singh\",\"suffix\":\"\"},{\"id\":590742764,\"identity\":\"9c1bfdc1-6965-4c05-97c5-c61c54e33a44\",\"order_by\":5,\"name\":\"Chingoileima Maibam\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dhanamanjuri University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chingoileima\",\"middleName\":\"\",\"lastName\":\"Maibam\",\"suffix\":\"\"},{\"id\":590742765,\"identity\":\"b08dc125-7c6c-4677-8e89-6a72d8fba688\",\"order_by\":6,\"name\":\"Asha Pallujam\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Asha\",\"middleName\":\"\",\"lastName\":\"Pallujam\",\"suffix\":\"\"},{\"id\":590742766,\"identity\":\"e2df5177-d815-41d4-9b56-5b31ca17401b\",\"order_by\":7,\"name\":\"Nanaocha Sharma\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nanaocha\",\"middleName\":\"\",\"lastName\":\"Sharma\",\"suffix\":\"\"},{\"id\":590742767,\"identity\":\"854d9fdb-bcf2-4cc6-8a58-96da202d0612\",\"order_by\":8,\"name\":\"Ojit Singh Keithellakpam\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYFCCgw0MCRU2cvwgdkIBcVoaGx6cSTOWbABpMSDOGsbGh22HEzccALGJ0cLPeLj9QSJQy+bzqxM/PDBgkOcXO4Bfi2QD0GEJ59KNt914u1kC6DDDmbMT8GsxOADSUmYtu+3G2Q0gLQkGt4nSwsbMuHnG2c0/SNDS5qy4gb93G3G2gPwyIwEYyBI3eLdZJBhIEPYLv8TxBx9/gKKy/+zmm0CGPL80AS0MEgdgDLBKCQLKwdY0wBgH8KgaBaNgFIyCEQ0AytlQ+dOpKJMAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Institute of Bio-Resources and Sustainable Development\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Ojit\",\"middleName\":\"Singh\",\"lastName\":\"Keithellakpam\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-02-09 12:54:58\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8830670/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8830670/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":102962585,\"identity\":\"654cec1b-edb0-466f-885f-b33a38ff7461\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:09:56\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":594279,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhotomicrograph of the cyanobacterial strains used in this study.\\u003c/p\\u003e\\n\\u003cp\\u003eA,B -BTA1153 \\u003cem\\u003eHapalosiphon \\u003c/em\\u003esp.; C,D - BTA1154 \\u003cem\\u003eScytonema \\u003c/em\\u003esp.; E, F - BTA115 \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e;G,H -BTA1156 \\u003cem\\u003eNostochopsis \\u003c/em\\u003esp.; I,J - BTA1157 \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e; K,L BTA1158 - \\u003cem\\u003eNostoc calcicole\\u003c/em\\u003e; M,N -BTA1159 \\u003cem\\u003eNeowestiellopsis \\u003c/em\\u003esp.; O,P BTA1160 - \\u003cem\\u003eCalothrix \\u003c/em\\u003esp.; Q,R BTA1161 - \\u003cem\\u003eNostoc \\u003c/em\\u003esp.; S, T - BTA1162 \\u003cem\\u003eHapalosiphon \\u003c/em\\u003esp.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8830670/v1/d883e7c5a335d6ac957f8a05.png\"},{\"id\":102962701,\"identity\":\"5a4f73ae-7acb-4f36-b5bb-2f0e9eb43aa3\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:10:41\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":167008,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGel image of amplified products of 16S rRNA. \\u003cstrong\\u003e1\\u003c/strong\\u003e. \\u003cem\\u003eAnabaena variabilis \\u003c/em\\u003eBTA1157, \\u003cstrong\\u003e2\\u003c/strong\\u003e. \\u003cem\\u003eHapalosiphon\\u003c/em\\u003esp. BTA1153, \\u003cstrong\\u003e3\\u003c/strong\\u003e. \\u003cem\\u003eNostochopsis\\u003c/em\\u003esp. BTA1156, \\u003cstrong\\u003e4\\u003c/strong\\u003e. \\u003cem\\u003eScytonema\\u003c/em\\u003e sp. BTA1154, \\u003cstrong\\u003e5\\u003c/strong\\u003e. \\u003cem\\u003eAnabaena doliolum \\u003c/em\\u003eBTA1155, \\u003cstrong\\u003e6\\u003c/strong\\u003e. \\u003cem\\u003eNostoc\\u003c/em\\u003e sp. BTA1161, \\u003cstrong\\u003e7\\u003c/strong\\u003e. \\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160, \\u003cstrong\\u003e8\\u003c/strong\\u003e. \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp., BTA1159 \\u003cstrong\\u003e9\\u003c/strong\\u003e. \\u003cem\\u003eNostoc calcicola \\u003c/em\\u003eBTA1158, \\u003cstrong\\u003e10\\u003c/strong\\u003e. \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1162. Marker 1000 bp DNA marker.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8830670/v1/45f403db3da8cf945a370b1b.png\"},{\"id\":102789031,\"identity\":\"2c8b3935-6a11-49a1-a8f6-f0d7623ba98b\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 16:42:59\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74512,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhylogenetics analysis of ten cyanobacterial strains genes of 16S rRNA along with the complete heterocytous cyanobacteria clade with the bootstrap values representing ML respectively. Bar, 0.02 changes per nucleotide position. All bootstrap values below 50 were deleted.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8830670/v1/4ec8039f40e32675d6da1d5f.png\"},{\"id\":102789028,\"identity\":\"0088d14c-fe00-4544-93e0-007266100709\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 16:42:58\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":199694,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe secondary structures folding conservations by complex rearrangement of base pairing and substitutions in 16S rRNA genes of all ten species shown in 1-10. [1] \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e BTA1155, [2] \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e BTA1157, [3] \\u003cem\\u003eScytonema \\u003c/em\\u003esp. BTA1154, [4] \\u003cem\\u003eCalothrix \\u003c/em\\u003esp BTA1160, [5] \\u003cem\\u003eNostoc \\u003c/em\\u003esp. BTA1161, [6] \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159, [7] \\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e BTA1158, [8] \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1161, [9] \\u003cem\\u003eHapalosiphon\\u003c/em\\u003esp. BTA1154, [10] \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. BTA1154. Each molecule was folded using a program based onthermodynamics and their respective showed in the minimum free energy of all cyanobacteria isolates shown in plots A-J. [A] \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e BTA1155, [B] \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e BTA1157,[C] \\u003cem\\u003eScytonema \\u003c/em\\u003esp. BTA1154, [D] \\u003cem\\u003eCalothrix \\u003c/em\\u003esp BTA1160, [E] \\u003cem\\u003eNostoc \\u003c/em\\u003esp. BTA1161, [F] \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159, [G] \\u003cem\\u003eNostoc calcicola \\u003c/em\\u003eBTA1158, [H] \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1161, [I] \\u003cem\\u003eHapalosiphon\\u003c/em\\u003esp. BTA1154, [J] \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. BTA1154.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8830670/v1/4d15944d432afedcebf7ecd5.png\"},{\"id\":102964939,\"identity\":\"17e81bcd-cf7e-4559-bbb4-c93538d9f546\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:29:28\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1993389,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8830670/v1/4029350c-c6a7-4189-a943-dabdceb86d94.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Uncovering cyanobacterial diversity in Northeast India: phylogenetic and structural insights from heterocystous Strains\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCyanobacteria commonly called blue-green algae are prokaryotic photoautotrophs capable of oxygenic photosynthesis. They thrive across diverse ecological niches from terrestrial to aquatic systems and are even found in extreme thermal and polar environments, making them globally abundant and ecologically significant. Many cyanobacteria also form symbiotic associations with plants, fungi (lichens), and certain eukaryotic algae (Whitton and Potts 2000). Traditionally, cyanobacterial taxonomy has relied on morphological traits such as trichome shape and size, cell differentiation (heterocysts, akinetes), and cell division patterns (Rippka et al. 1979). However, morphology-based classification is often unreliable traits can vary markedly under different growth conditions or developmental stages, even within axenic cultures (Anand 1988; Lyra et al. 2001; Nalewajko and Murphy 2001; Gugger et al. 2002; Gupta and Agrawal 2006; Gamage 2020; Overlingė et al. 2024). In response, the polyphasic approach integrating morphology, molecular markers, ultrastructure, biochemical and ecological traits have emerged as the gold standard in modern cyanobacterial taxonomy (Kom\\u0026aacute;rek 2013; Giovannoni 1991; Zampieri et al. 2025; Wang et al. 2025). Molecular data, especially 16S rRNA gene sequences, now form the backbone of phylogenetic classification. This marker is cultivation-independent, allows PCR from minimal DNA, and reveals evolutionary relationships often obscured by morphological plasticity (Gamage 2020; Wilmotte 1994; Struneck\\u0026yacute; et al. 2023). Notably, increasing 16S rRNA data availability has exposed polyphyletic groups and misclassifications highlighting the need to revise traditional taxonomic schemes (Struneck\\u0026yacute; et al. 2023; Pham et al. 2025).\\u003c/p\\u003e \\u003cp\\u003eRecent studies have also emphasized the importance of ITS secondary structure analysis and additional markers like 16S-23S ITS to distinguish closely related cyanobacterial taxa with greater resolution (Zampieri et al. 2025; Wang et al. 2025). Further, amplicon-based sequencing reveals rich cyanobacterial diversity in under-studied habitats; e.g., a study of Bolivian Altiplano microbial mats identified numerous novel cyanobacterial lineages using 16S rRNA profiling (Hentschke et al. 2025). In India, applying this integrative approach is particularly crucial. A recent study characterized 15 Indian cyanobacterial strains exhibiting Nostoc-like morphology using a polyphasic framework, underscoring the uneven application of modern taxonomy within the region (Pal et al. 2024). The present study aimed to investigate ten heterocystous cyanobacterial strains isolated from North-Eastern India (Manipur and Meghalaya), focusing on morphological diversity, molecular identification via 16S rRNA sequencing, and their phylogenetic relationships. Additionally, our integration of structural (secondary structure) analysis aims to enhance taxonomic resolution and reveal novel diversity within this globally significant hotspot.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSampling, isolation and purification\\u003c/h2\\u003e \\u003cp\\u003eTen cyanobacterial strains were isolated from environmental samples collected in the North-Eastern states of Manipur and Meghalaya, India. For each sampling site, metadata including latitude, longitude, and altitude were recorded. Samples were inoculated into 100 mL Erlenmeyer flasks containing sterile BG-11 broth (Stanier 1971) in two variants: nitrogen-supplemented (+\\u0026thinsp;N) for non-heterocystous strains and nitrogen-free (-N) for heterocystous strains. Following initial growth, cultures were examined using a ZEISS Primo Star microscope (ZEISS, Germany) to confirm cyanobacterial presence, then streaked onto respective BG-11 agar plates (+/- N2). Pure cultures were obtained through repeated streaking and subsequently maintained in 100 mL BG-11 broth. All cultures were incubated at 28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C under an irradiance of approximately 40 \\u0026micro;mol photons m\\u0026sup2;/s from cool-white 40 W fluorescent lamps, with a 14 h light/10 h dark photoperiod.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eIdentification of the strain\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMorphological Identification\\u003c/h2\\u003e \\u003cp\\u003eTaxonomic and morphological characterization was performed following standard monographs and descriptions (Desikachary 1959). Microscopic identification considered key phenotypic traits, including filament/trichome type, presence of sheath, cell shape, and occurrence of heterocysts and akinetes. Observations were conducted at 40\\u0026times; magnification using an Axio Scope A1 microscope (Carl Zeiss, Germany) fitted with an AxioCam MRc camera and AxioVision 4.7.2 software. Additional growth characteristics, including biomass color and growth pattern (wall-attached, bottom-settled, submerged, or floating), were also documented.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eMolecular identification\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDNA Extraction\\u003c/h2\\u003e \\u003cp\\u003eGenomic DNA was extracted from ~\\u0026thinsp;1 g of 15-20-day old cyanobacterial biomass using a modified glass bead method (Devi et al. 2015). The biomass was homogenized in an autoclaved mortar and pestle with 1 g of sterile glass powder, 10 mg of activated charcoal, and 2 mL of extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl; pH adjusted to 8.0).The homogenate was transferred to a 2 mL microtube containing 10 \\u0026micro;L Proteinase K (20 mg/mL) and 50 \\u0026micro;L RNase A (20 mg/mL), incubated at 65\\u0026deg;C for 10 min, and centrifuged (12,000 g, 5 min, 4\\u0026deg;C). To 500 \\u0026micro;L supernatant, 100 \\u0026micro;L sodium acetate and 400 \\u0026micro;L PEG were added, incubated at -20\\u0026deg;C for 20 min, and centrifuged. Pellets were resuspended in 500 \\u0026micro;L TE buffer and extracted with 500 \\u0026micro;L chloroform:isoamyl alcohol (24:1), followed by centrifugation. The aqueous fraction was mixed with 500 \\u0026micro;L of chilled isopropanol, incubated at 4\\u0026deg;C for 5 min, and centrifuged at 12,000 g for 10 min. The resulting pellets were rinsed with 70% ethanol, centrifuged again at 12,000 g for 2 min, air-dried, and finally resuspended in 50 \\u0026micro;L of TE buffer (pH 8).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003e16S rRNA PCR amplification\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe 16S rRNA gene was amplified using forward primer 106F (5\\u0026prime;-CGG ACG GGT GAG TAA CGC GTG A-3\\u0026prime;) and an equimolar mixture of reverse primers 781Ra (5\\u0026prime;-GAC TAC TGG GGT ATC TAA TCC CAT T-3\\u0026prime;) and 781Rb (5\\u0026prime;-GAC TAC AGG GGT ATC TAA TCC CTT T-3\\u0026prime;) (IDT, USA). Each 50 \\u0026micro;L PCR reaction contained 10 \\u0026micro;L 5\\u0026times; Phusion HF buffer, 1 \\u0026micro;L 10 mM dNTPs, 2.5 \\u0026micro;L 10 \\u0026micro;M of each primer, 0.5 \\u0026micro;L Phusion DNA polymerase (2 U; ThermoFisher Scientific, USA), 2.5 \\u0026micro;L template DNA (~\\u0026thinsp;50 ng), 1.5 \\u0026micro;L DMSO, and 29.5 \\u0026micro;L nuclease-free water. PCR amplification was conducted on a ProFlex thermocycler (Applied Biosystems, USA). The cycling conditions comprised an initial denaturation at 95\\u0026deg;C for 3 min, followed by 34 cycles of 95\\u0026deg;C for 30 s, 57\\u0026deg;C for 40 s, and 72\\u0026deg;C for 40 s, with a final extension at 72\\u0026deg;C for 5 min. The resulting amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and visualized on 1.5% agarose gels. Visualization was performed using a Universal Hood II Gel Doc System (Bio-Rad, USA), and products were sequenced bidirectionally on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhylogenetic Analysis\\u003c/h2\\u003e \\u003cp\\u003eSequence data were analyzed using BioEdit v7.0.9 (Hall 1999). Homologous sequences (97\\u0026ndash;100% identity; E-value\\u0026thinsp;\\u0026gt;\\u0026thinsp;10⁻\\u0026sup2;⁰) were identified using the BLASTn tool against the NCBI nucleotide collection (nr/nt) database (Altschul et al. 1997). Sequences generated in this study were deposited in GenBank. Multiple sequence alignments of 16S rRNA gene sequences were performed using ClustalW in MEGA v4.0 (Tamura et al. 2007). Pairwise sequence divergence was estimated using the Jukes-Cantor substitution model (Jukes and Cantor 1969), and phylogenetic reconstruction was performed with the neighbour-joining algorithm (Saitou and Nei 1987) based on p-distance values. Bootstrap analysis was conducted with 1,000 replicates, and only values\\u0026thinsp;\\u0026gt;\\u0026thinsp;50% were displayed. \\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160 served as the outgroup. Secondary RNA structures for all ten cyanobacterial strains were predicted using RNAfold software (Gruber et al. 2008).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSample collection\\u003c/h2\\u003e \\u003cp\\u003eAlgal samples were collected during 2019\\u0026ndash;2020 from four districts within the Indo-Myanmar biodiversity hotspot: East Khasi Hills and West Jaintia Hills (Meghalaya), and Tengnoupal and Senapati (Manipur). Sampling sites encompassed diverse habitats, including rivers, natural and artificial lakes, waterfalls, streams, and rock surfaces. Collected samples were cultured in BG-11 medium and sub-cultured on BG-11 agar plates to obtain pure isolates. A total of ten pure cultures were established and preserved in the Freshwater Cyanobacterial and Microalgal Repository at IBSD, Manipur, India. Metadata, including GPS coordinates, habitat type, and pH, are presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eMetadata of 10 samples collected from different locations of NER used in this study\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eName of species with accession no\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePlace\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eHabitat\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eLatitude\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eLongitude\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eAltitude\\u003c/p\\u003e \\u003cp\\u003e(m)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1153\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMatilang, East Khasi Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eStream\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.54472\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e91.83195\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1804.72\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eScytonema\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1154\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eJakrem, East Khasi Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRock surface\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.38966\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e91.50979\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e435.86\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e BTA1155\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBarapani, East Khasi Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRiver\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.65956\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e91.89837\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e990.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1156\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWards, East Khasi Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eLake\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.57504\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e91.88744\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1495\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e BTA1157\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDawki, East Khasi Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRiver\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.18917\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e92.01827\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e48.77\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003eBTA1158\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eKwatha, Tengnoupal, Manipur\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRock surface\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e24.33725\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e94.27843\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e436.78\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003esp.\\u003c/p\\u003e \\u003cp\\u003eBTA1159\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eKwatha, Tengnoupal, Manipur\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eStream\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e24.33725\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e94.27843\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e436.78\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eJowai, West Jaintia Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWater fall\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.43385\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e92.18323\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1291.74\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1161\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eJowai, West Jaintia Hills, Meghalaya\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRock surface\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.43385\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e92.18323\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1291.74\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1162\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMao, Senapati, Manipur\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWaterfall\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e25.52258\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e94.12332\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1996.14\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMorphological identification\\u003c/h2\\u003e \\u003cp\\u003eInitial cultures showed indistinct morphological features due to environmental debris. Through serial plating on agar, we isolated ten morphologically distinct strains. Microscopic examination (40\\u0026times;) evaluated cell shape and size, heterocyst and akinete morphology, and sheath presence complemented by growth patterns in flasks (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) for preliminary taxonomic identification using the classification keys of Desikachary (1959). Representative micrographs are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. This morphology-based approach remains common but increasingly recognized as insufficient on its own; recent studies advocate combining morphological, molecular, ultrastructural, and ecological traits for accurate classification (Kom\\u0026aacute;rek 2023; Jiang et al. 2023).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eMorphological characteristics of the isolated cyanobacteria\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eName of the strains\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eThallus\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c6\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eFilaments\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCell\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eHeterocyst\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eAkinete\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eSheath\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003esp.BTA1153\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom attach, clump and floccose biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eFilaments free, cells in one or two rows, sub-spherical, true branching. 5.8\\u0026ndash;8.41 \\u0026micro;m long and 6.19\\u0026ndash;7.3 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eIntercalary and oblong, 6.37\\u0026ndash;10.35 \\u0026micro;m long and 4.01\\u0026ndash;5.49 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eScytonema\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1154\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom attached, compact and clump biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eFilament false branching,\\u003c/p\\u003e \\u003cp\\u003esub-quadrate, 4.58\\u0026ndash;5.97 \\u0026micro;m long and 3.78\\u0026ndash;4.8 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSub-quadrate, 6.12\\u0026ndash;7.18 \\u0026micro;m long and 4.25\\u0026ndash;5.17 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eFirm\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1155\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBrownish green, Submerged and attached at side of flask, reticulate biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBarrel shaped, 1.89\\u0026ndash;4.92 \\u0026micro;m long and 3.11\\u0026ndash;3.76 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSpherical, 4.6-7 \\u0026micro;m long and 4-5.59 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp.BTA1156\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom attached, initially caespitose and latter floccose biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBarrel shape, 3.75\\u0026ndash;11.15 \\u0026micro;m long and 4.87\\u0026ndash;10.21 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eIntercalary, 5.32-12-12 \\u0026micro;m long and 3.89\\u0026ndash;7.37 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eAbsent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e BTA1157\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, side attached, submerged,\\u003c/p\\u003e \\u003cp\\u003efilamentous and reticulate biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBarrel-shaped, Cell 3.11\\u0026ndash;4.18 \\u0026micro;m long and 2.77\\u0026ndash;4.05 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSpherical, 6.22\\u0026ndash;7.94 \\u0026micro;m long and 4.51\\u0026ndash;5.43 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoccalcicola\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003eBTA1158\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, submerged latter side attached, filamentous and reticulate biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBarrel-shaped, 2.11\\u0026ndash;2.53 \\u0026micro;m long and 2.14\\u0026ndash;2.44 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSub-spherical, 2.46\\u0026ndash;3.19 \\u0026micro;m long and 2.14\\u0026ndash;3.23 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNeowestiellopis\\u003c/em\\u003e sp.BTA1159\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom attached, floccose and caespitose biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCell 5.06\\u0026ndash;5.8 \\u0026micro;m long and 3.96\\u0026ndash;4.56\\u0026micro;m broad. T-shaped branching on both sides, probable appearance of biseriate condition.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e6.31\\u0026ndash;7.8\\u0026micro;m long and 3.11\\u0026ndash;4.25\\u0026micro;m board, irregularly shared \\u0026ndash;intercalary/elongate.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eAbsent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCalothrix\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1160\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom dweller, sheath like latter glomerate\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSub-quadrate, 3.59\\u0026ndash;7.34 \\u0026micro;m long and 4.18\\u0026ndash;7.13 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eBasal, 4.25\\u0026ndash;6.41 \\u0026micro;m long and 5.04\\u0026ndash;6.3 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc\\u003c/em\\u003e sp.\\u003c/p\\u003e \\u003cp\\u003eBTA1161\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, submerged and reticulate biomass\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCylindrical, 2.65\\u0026ndash;3.88 \\u0026micro;m long and 2.32\\u0026ndash;2.59 \\u0026micro;m broad\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eIntercalary, 3.84\\u0026ndash;6.68 \\u0026micro;m long and 3.18\\u0026ndash;4.13 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapolosiphon\\u003c/em\\u003e sp.BTA1162\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDark green, bottom attached, globose latter floccose\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eQuadrate, 3.69\\u0026ndash;4.85 \\u0026micro;m long and 5.14\\u0026ndash;6.22 \\u0026micro;m broad.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eIntercalary, 3.41\\u0026ndash;8.17 \\u0026micro;m long and 4.13\\u0026ndash;6.24 \\u0026micro;m board\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ePresent\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular identification\\u003c/h2\\u003e \\u003cp\\u003eCyanobacterial identification under laboratory conditions is often hindered by morphological alterations and reduced interspecific variation caused by controlled growth environments (Dores and Parker 1988). To complement morphological taxonomy, molecular identification was performed through PCR amplification of cyanobacteria-specific 16S rRNA sequences. Genomic DNA from the ten isolates was obtained using a modified glass bead protocol, yielding high-purity DNA (A260/A280\\u0026thinsp;=\\u0026thinsp;1.28\\u0026ndash;2.09) at 12.2\\u0026ndash;65.2 ng/\\u0026micro;L. The genomic DNA fragments measured approximately 9000-10,000 bp, while purified PCR amplicons of the 16S rRNA gene were 600\\u0026ndash;700 bp in size (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAfter successful PCR amplification, the 16S rRNA gene amplicons were sequenced using the Sanger method, and the obtained sequences were analyzed and submitted to the GenBank database at the National Center for Biotechnology Information (NCBI) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Phylogenetic analysis of the partial 16S rRNA sequences placed the ten cyanobacterial isolates into two major clusters (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Cluster 1 comprised \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e, \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e, \\u003cem\\u003eScytonema\\u003c/em\\u003e sp., \\u003cem\\u003eNostoc\\u003c/em\\u003e sp., and \\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e, while Cluster 2 included \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp., \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. (two isolates), and \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. The outgroup was represented by \\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. Within Cluster 1, \\u003cem\\u003eA. doliolum\\u003c/em\\u003e and \\u003cem\\u003eA. variabilis\\u003c/em\\u003e shared 94% bootstrap support, and \\u003cem\\u003eScytonema\\u003c/em\\u003e sp. showed 94% similarity to these \\u003cem\\u003eAnabaena\\u003c/em\\u003e species. The \\u003cem\\u003eNostoc\\u003c/em\\u003e sp. and \\u003cem\\u003eN. calcicola\\u003c/em\\u003e grouping had 92% bootstrap support. In Cluster 2, \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. showed 95% similarity with the \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e clade, which in turn had 100% internal bootstrap support, while \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. was supported by 91%. Both major clusters were strongly supported (92% bootstrap), and the phylogenetic tree confirmed identification of the isolates to the genus level. These high bootstrap values demonstrate sturdy phylogenetic resolution at the genus level consistent with findings that 16S rRNA-based trees are generally reliable for higher-order classification, though species-level resolution may vary (Vondr\\u0026aacute;škov\\u0026aacute; et al. 2025).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eNCBI accession number of the isolated strains used in this study\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSl.No.\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eStrain Identity\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eNCBI Accession no.\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eStrain Identity\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eNCBI Accession no.\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eScytonema\\u003c/em\\u003esp. BTA1154\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMW354520\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eMW354522\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e BTA1155\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMW342607\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc\\u003c/em\\u003e sp. BTA1161\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eMW354523\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003esp.BTA1153\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMW354516\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc calcicole\\u003c/em\\u003e BTA1158\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eMW354524\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003eBTA1157\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMW354518\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e9\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNeowestiellopis\\u003c/em\\u003e sp. BTA1159\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eMW354517\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostochopsis\\u003c/em\\u003esp.BTA1156\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMW354521\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1162\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eMW354519\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eFurther phylogenetic interpretation revealed evolutionary distinctions among the isolates. \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e, \\u003cem\\u003eA. variabilis\\u003c/em\\u003e, and \\u003cem\\u003eScytonema\\u003c/em\\u003e sp. formed a paraphyletic group, while \\u003cem\\u003eNostoc\\u003c/em\\u003e sp., \\u003cem\\u003eN. calcicola\\u003c/em\\u003e, and \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. appeared polyphyletic, consistent with recent reports that Nostocales taxa often require taxonomic revision (Wang et al. 2025; Kom\\u0026aacute;rek 2023). By contrast, \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e isolates and \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. formed a well-defined monophyletic clade. The close clustering of \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003edoliolum\\u003c/em\\u003e and \\u003cem\\u003eA. variabilis\\u003c/em\\u003e despite clear morphological differences echoes previous findings that 16S rRNA similarity among \\u003cem\\u003eAnabaena\\u003c/em\\u003e genotypes can reach\\u0026thinsp;~\\u0026thinsp;94%, complicating species resolution (Val\\u0026eacute;rio et al. 2009). Increasingly, researchers recommend the integration of additional markers such as the 16S-23S ITS region or multi-locus sequence analysis to refine cyanobacterial systematics (Vondr\\u0026aacute;škov\\u0026aacute; et al. 2025; Jiang et al. 2023). Most significantly, \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. was recorded here for the first time in Manipur, India, with strong bootstrap support (95%), representing only the second global report of this genus. This highlights the Northeast Indian Indo- Myanmar hotspot as an important but underexplored reservoir of cyanobacterial diversity.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSEC-Structure\\u003c/h2\\u003e \\u003cp\\u003eThe partial 16S rRNA gene sequences of all cyanobacterial genera were translated, and their RNA secondary structures were predicted. The folding patterns and minimum free energy (MFE) values indicated stable secondary structures, supporting the reliability of the predicted RNA conformations.\\u003c/p\\u003e \\u003cp\\u003eDifferent structural folding shapes generated for all cyanobacterial isolates species of partial RNA sequences. The energy table showed (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) that, the highest minimum free energy\\u0026thinsp;\\u0026minus;\\u0026thinsp;229.90 kcal/mol for \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e and \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e minimum free energy\\u0026thinsp;\\u0026minus;\\u0026thinsp;229.93 obtained. The \\u003cem\\u003eScytonema\\u003c/em\\u003e sp. structural potential free energy revealed\\u0026thinsp;\\u0026minus;\\u0026thinsp;213 kcal/mol was the lowest energy amongst others. Further the cyanobacterial isolate \\u003cem\\u003eNostoc\\u003c/em\\u003e sp. showed minimum free energy\\u0026thinsp;\\u0026minus;\\u0026thinsp;238.10 kcal/mol respectively. The \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. structure potential energy was \\u0026minus;\\u0026thinsp;245.60 kcal/mol predicted. The \\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e\\u0026thinsp;\\u0026minus;\\u0026thinsp;234.90 kcal/mol, \\u003cem\\u003eHapalosiphon sp.\\u003c/em\\u003e -237.60 kcal/mol and \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. -232.10 kcal/mol potential minimum free energies were predicted. For the \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp., structural free energy was \\u0026minus;\\u0026thinsp;221.50 kcal/mol was obtained from the RNA secondary structure. All the cyanobacterial isolates 16s rRNA secondary structures different level of energies with sequence positions wise represented clearly in energy plots shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSecondary Structure Stability of 16S rRNA Sequences in Cyanobacterial Strains Based on Minimum Free Energy (MFE)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSl. No\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSample Name\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSec-Struct (kcal/mol)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e BTA1155\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-229.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e BTA1157\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-229.93\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eScytonema\\u003c/em\\u003esp. BTA1154\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-219.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-213.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc\\u003c/em\\u003e sp. BTA1161\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-238.10\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-245.60\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e BTA1158\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-234.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1161\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-237.60\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e9\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. BTA1153\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-232.10\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. BTA1156\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-221.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e16S rRNA partial genes sequences thermodynamically folding showed that substitutions of base pairing caused complex structural changes in the folding pattern. Pseudo nodes and nodes created mispairing sequence structural regions loss of stability of the structures. In \\u003cem\\u003eAnabaena doliolum\\u003c/em\\u003e and \\u003cem\\u003eAnabaena variabilis\\u003c/em\\u003e, 16S rRNA structures fold showed similarly with conserved. The \\u003cem\\u003eScytonema\\u003c/em\\u003e sp., \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. and \\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. secondary structures created same folding conservations base pairs cause different potential energy and stability among the structures. Further \\u003cem\\u003eNostoc\\u003c/em\\u003e sp., \\u003cem\\u003eNostoc calcicola\\u003c/em\\u003e sp. and \\u003cem\\u003eNostochopsis\\u003c/em\\u003e sp. secondary structures showed same structural conserved bases interact with sequences formed helix structure and maintain the stability of structure. Node region of base pairs sites loss of interactions and potential structural stability. The two \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e sp. 16S rRNA sequence secondary structure showed similar folding structures and the minimum free energy level by the conserved sequence base pairs sites.\\u003c/p\\u003e \\u003cp\\u003eSecondary structure analyses like these provide valuable insights into cyanobacterial diversity. Not only do they reveal structural conservation and variability, but they also supplement phylogenetic resolution when used alongside molecular and morphological data (Klindworth et al. 2013). Indeed, the use of 16S-23S ITS secondary structure has previously proven effective for distinguishing closely related cyanobacterial taxa and resolving intrageneric relationships (e.g. \\u003cem\\u003eLeptolyngbya corticola\\u003c/em\\u003e sp. nov.) (Johansen et al. 2011). In a broader molecular context, the 16S rRNA gene remains a cornerstone for phylogenetic analysis, though limitations at the species level are well acknowledged (Gamage 2020; Hassler et al. 2022).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eTen cyanobacterial strains were isolated from diverse habitats within the Indo-Myanmar biodiversity hotspot. These isolates exhibited marked variation in morphological and growth characteristics, which served as the primary basis for preliminary identification. Subsequent molecular characterisation and phylogenetic analysis, based on partial 16S rRNA gene sequences, corroborated the morphological findings. Comparative analysis of the predicted secondary structures of the 16S rRNA sequences revealed similarities in folding patterns and stability among certain strains. This study underscores the cyanobacterial diversity present in the examined region and reports, for only the second time globally, the occurrence of the recently classified genus \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e. Previously documented in Iran, this marks the first record of the genus from Manipur in North-East India.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003cstrong\\u003eCompeting interest:\\u003c/strong\\u003e \\u003cp\\u003eThe authors declare no conflict of interest.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eEthical approval and consent to participate\\u003c/strong\\u003e \\u003cp\\u003eThis research work does not involve any animal or human participants.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eHSD: Writing\\u0026ndash;original draft; Methodology; performed the experiments; ValidationKM: Writing\\u0026ndash;original draft; Methodology; performed the experiments; ValidationNR: Data curationRM: Investigation; Data curationKKS: Writing\\u0026ndash;review \\u0026amp; editing; Formal analysis; VisualizationCM: performed experiment; data curationAP: performed experiment; data curationNS: Writing\\u0026ndash;review \\u0026amp; editing; Supervision; Funding acquisitionOSK: Writing\\u0026ndash;review \\u0026amp; editing; conceived and designed the study; Formal analysis; Visualization\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eWe are grateful to the local community and labmates for their assistance in sample collection. We also thank the Department of Biotechnology (DBT), Government of India, for financial assistance. We gratefully acknowledge the Department of Biotechnology, Government of India, for financial support under Grant No. BT/01/17/NE/TAX, provided through the Institute of Bioresources and Sustainable Development, Imphal, Manipur.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAltschul SF, Madden TL, Sch\\u0026auml;ffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-3402.\\u003c/li\\u003e\\n\\u003cli\\u003eAnand N (1988) Culture studies and taxonomy of blue-green algae: Certain identification problems. Arch Hydrobiol Suppl Algol Stud, Supplement Volumes, pp 141-147.\\u003c/li\\u003e\\n\\u003cli\\u003eDesikachary TV (1959) Cyanophyta, Monograph on blue green algae, Indian Council of Agricultural Research, New Delhi, pp 1-686.\\u003c/li\\u003e\\n\\u003cli\\u003eDevi SG, Fathima AA, Radha S, Arunraj R, Curtis WR, Ramya M (2015) A rapid and economical method for efficient DNA extraction from diverse soils suitable for metagenomic applications. PloS One 10: e0132441.\\u003c/li\\u003e\\n\\u003cli\\u003eDores MP, Parker DL (1988) Properties of \\u003cem\\u003eMicrocystis aeruginosa\\u003c/em\\u003e and \\u003cem\\u003eM\\u003c/em\\u003e. \\u003cem\\u003eflosaquaein\\u003c/em\\u003e culture taxonomic implication. J Phycol 24:502-508.\\u003c/li\\u003e\\n\\u003cli\\u003eGamage SMKW (2020) The 16S rRNA gene and cyanobacterial taxonomy; current problems and future prospects. J Univ Ruhuna 8:60-66.\\u003c/li\\u003e\\n\\u003cli\\u003eGiovannoni SJ (1991) The polymerase chain reaction. 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In Nucleic Acids Symp Ser 41:95-98. \\u003c/li\\u003e\\n\\u003cli\\u003eHassler HB, Probert B, Moore C et al (2022) Phylogenies of the 16S rRNA gene and its hypervariable regions lack concordance with core genome phylogenies. Microbiome 10:104.\\u003c/li\\u003e\\n\\u003cli\\u003eHentschke GS, Semedo M, Ciancas J et al (2025) Cyanobacterial mats and their associated microbiomes in saline and freshwater lakes from the Bolivian Altiplano. Front Microbiol 16. https://doi.org/10.3389/fmicb.2025.1650455.\\u003c/li\\u003e\\n\\u003cli\\u003eJiang Y, Tang J, Liu X, Daroch M (2023) Polyphasic characterization of a novel hot-spring cyanobacterium \\u003cem\\u003eThermocoleostomius\\u003c/em\\u003e \\u003cem\\u003esinensis\\u003c/em\\u003e gen et sp. nov. and genomic insights into its carbon concentration mechanism. Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1176500.\\u003c/li\\u003e\\n\\u003cli\\u003eJohansen JR, Kovacik L, Casamatta DA, Ikov\\u0026aacute; KF, Ka\\u0026scaron;tovsk\\u0026yacute; J (2011) Utility of 16S-23S ITS sequence and secondary structure for recognition of intrageneric and intergeneric limits within cyanobacterial taxa: \\u003cem\\u003eLeptolyngbya corticola\\u003c/em\\u003e sp. nov. (Pseudanabaenaceae, Cyanobacteria). Nova Hedwig 92:283-302. \\u003c/li\\u003e\\n\\u003cli\\u003eJukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism, Academic Press, New York, 21-132.\\u003c/li\\u003e\\n\\u003cli\\u003eKlindworth A, Pruesse E, Schweer T et al (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1.\\u003c/li\\u003e\\n\\u003cli\\u003eKom\\u0026aacute;rek J (2013) Cyanoprokaryota: 3rd Part: Heterocystous Genera. 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Int J Syst Evol Microbiol 51:513-526. \\u003c/li\\u003e\\n\\u003cli\\u003eNalewajko C, Murphy TP (2001) Effects of temperature, and availability of nitrogen and phosphorus on the abundance of \\u003cem\\u003eAnabaena\\u003c/em\\u003e and \\u003cem\\u003eMicrocystis\\u003c/em\\u003e in Lake Biwa, Japan: an experimental approach. Limnology 2:45-48.\\u003c/li\\u003e\\n\\u003cli\\u003eOverlingė D, Toruńska-Sitarz A, Cegłowska M, Szubert K, Mazur-Marzec H (2024) Phylogenetic and molecular characteristics of two \\u003cem\\u003eAphanizomenon\\u003c/em\\u003e strains from the Curonian Lagoon, Southeastern Baltic Sea and their biological activities. Sci Rep 14:24686.\\u003c/li\\u003e\\n\\u003cli\\u003ePal S, Saraf A, Kumar N et al (2024) Polyphasic characterization of 15 heterocytous cyanobacterial isolates from different habitats of India and description of 9 novel species belonging to the genera \\u003cem\\u003eDesikacharya\\u003c/em\\u003e, \\u003cem\\u003eAliinostoc\\u003c/em\\u003e, and \\u003cem\\u003eDesmonostoc\\u003c/em\\u003e. Algal Res 86:103873.\\u003c/li\\u003e\\n\\u003cli\\u003ePham HTL, Ngo TT, Tran, TV et al (2025) Classification of \\u003cem\\u003eNostoc\\u003c/em\\u003e-like cyanobacteria isolated from paddy soil into \\u003cem\\u003eAliinostoc\\u003c/em\\u003e, \\u003cem\\u003eAulosira\\u003c/em\\u003e, and \\u003cem\\u003eDesmonostoc\\u003c/em\\u003e. Front Microbiol 16:1581725. \\u003c/li\\u003e\\n\\u003cli\\u003eRippka R, Stanier RY, Deruelles J, Herdman M, Waterbury JB (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111:1-61.\\u003c/li\\u003e\\n\\u003cli\\u003eSaitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425.\\u003c/li\\u003e\\n\\u003cli\\u003eStanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171-205. \\u003c/li\\u003e\\n\\u003cli\\u003eStruneck\\u0026yacute; O, Ivanova AP, Mare\\u0026scaron; J (2023). An updated classification of cyanobacterial orders and families based on phylogenomic and polyphasic analysis. J Phycol 59(1):12-51.\\u003c/li\\u003e\\n\\u003cli\\u003eTamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software Version 4.0. Mol Biol Evol 24:1596-1599.\\u003c/li\\u003e\\n\\u003cli\\u003eVal\\u0026eacute;rio E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R (2009) Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs. Microbiology 155:642-656.\\u003c/li\\u003e\\n\\u003cli\\u003eVondr\\u0026aacute;\\u0026scaron;kov\\u0026aacute; A, Hauer T, Bengtsson R, Ka\\u0026scaron;tovsk\\u0026yacute; J, Johansen JR (2025) Molecular characterization of two cyanobacterial generitypes from their type localities in Scandinavia. J Phycol 61:119-131.\\u003c/li\\u003e\\n\\u003cli\\u003eWang J, Zhang T, Guo S et al (2025) Novel species of Oculatellaceae (Oculatellales, Cyanobacteria) from Yunnan in China, based on the polyphasic approach. Diversity 17:170. \\u003c/li\\u003e\\n\\u003cli\\u003eWhitton B, Potts M (2000) The ecology of cyanobacteria: their diversity in time and space. In Kluwer Academic Publishers eBooks. http://ci.nii.ac.jp/ncid/BA47335651.\\u003c/li\\u003e\\n\\u003cli\\u003eWilmotte A (1994) Molecular evolution and taxonomy of the cyanobacteria. In Springer eBooks, pp 1-25.\\u003c/li\\u003e\\n\\u003cli\\u003eZampieri RM, Bizzotto E, Campanaro S, Caldara F, Bellucci M, La Rocca N (2025) \\u003cem\\u003eKovacikia\\u003c/em\\u003e \\u003cem\\u003eeuganea\\u003c/em\\u003e sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new chlorophyll f producing cyanobacterium from the Euganean Thermal District (Italy). Front Microbiol 16. https://doi.org/10.3389/fmicb.2025.1545008.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-microbiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"aomi\",\"sideBox\":\"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)\",\"snPcode\":\"203\",\"submissionUrl\":\"https://submission.nature.com/new-submission/203/3\",\"title\":\"Archives of Microbiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Cyanobacteria, Heterocystous strains, Indo-Myanmar biodiversity hotspot, Neowestiellopsis, Phylogenetic analysis, 16S rRNA gene\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8830670/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8830670/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe North-East region of India, a recognised Indo-Myanmar biodiversity hotspot, remains underexplored for its cyanobacterial diversity. In this study, ten heterocystous cyanobacterial strains were isolated from freshwater-associated habitats using a polyphasic approach integrating morphological, molecular, and structural analyses. Morphological identification was complemented by 16S rRNA gene sequencing, phylogenetic reconstruction, and secondary structure prediction. The isolates were assigned to the genera \\u003cem\\u003eAnabaena\\u003c/em\\u003e, \\u003cem\\u003eScytonema\\u003c/em\\u003e, \\u003cem\\u003eNostoc\\u003c/em\\u003e, \\u003cem\\u003eCalothrix\\u003c/em\\u003e, \\u003cem\\u003eHapalosiphon\\u003c/em\\u003e, and \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e. Notably, one isolate corresponded to \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159 a taxon previously reported only from Iran marking the first record of this genus from Manipur and the second global report. Phylogenetic analysis using the neighbour-joining method (p-distance, Jukes Cantor correction) revealed that \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159 occupied a distinct polyphyletic position, while \\u003cem\\u003eCalothrix\\u003c/em\\u003e sp. BTA1160 formed an outbranch. Minimum free energy (MFE) calculations for 16S rRNA secondary structures indicated that \\u003cem\\u003eNeowestiellopsis\\u003c/em\\u003e sp. BTA1159 exhibited the highest structural stability among all isolates. These findings not only document novel cyanobacterial diversity from a globally significant hotspot but also demonstrate the utility of secondary structure analysis in complementing phylogenetic resolution.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Uncovering cyanobacterial diversity in Northeast India: phylogenetic and structural insights from heterocystous Strains\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-16 16:42:39\",\"doi\":\"10.21203/rs.3.rs-8830670/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-03-02T08:13:22+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-23T17:35:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-19T08:00:20+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"77189291804538287923993124830265014180\",\"date\":\"2026-02-16T13:17:01+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"61147435314892801000307373494397488025\",\"date\":\"2026-02-13T08:26:10+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"73724401654817292684466588284286580498\",\"date\":\"2026-02-13T05:00:10+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"31771855562022743321399067070699770452\",\"date\":\"2026-02-11T16:32:09+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"3949512844831763225477281020006106111\",\"date\":\"2026-02-11T06:34:45+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-11T03:42:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-02-10T06:27:16+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-02-10T02:59:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Archives of Microbiology\",\"date\":\"2026-02-09T12:30:38+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-microbiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"aomi\",\"sideBox\":\"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)\",\"snPcode\":\"203\",\"submissionUrl\":\"https://submission.nature.com/new-submission/203/3\",\"title\":\"Archives of Microbiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"05d8f3c2-fc83-4e88-8a1c-7f2f58c7b2c1\",\"owner\":[],\"postedDate\":\"February 16th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"in-revision\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-02T08:24:10+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-16 16:42:39\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8830670\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8830670\",\"identity\":\"rs-8830670\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}