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Article

Description of Cylindrospermum solincola sp. nov. from Jammu and Kashmir, India and Further Insights into the Ecological Distribution and Morphological Attributes of Cylindrospermum badium

by
Naresh Kumar
1,
Aniket Saraf
2,
Sagarika Pal
1 and
Prashant Singh
1,*
1
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India
2
Department of Biological Sciences, Ramniranjan Jhunjhunwala College, Mumbai 400086, India
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(5), 592; https://doi.org/10.3390/d15050592
Submission received: 14 February 2023 / Revised: 20 March 2023 / Accepted: 19 April 2023 / Published: 25 April 2023
(This article belongs to the Special Issue The Phylogenetic Diversity of Cyanobacteria and Algae)
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Abstract

:
Two cyanobacterial strains KUT1-PS and 18C-PS were collected from the soil surface and vernal pool, respectively, from the Basantgarh village, Udhampur district of the union territory of Jammu and Kashmir, India and characterized by a polyphasic approach. The morphological characterization indicated that both the strains showed typical Cylindrospermum-like morphology and probably belonged to the genus Cylindrospermum. Further, phylogenetic interpretations at the genus level were made using the 16S rRNA gene while the 16S-23S ITS region phylogenetic analysis and secondary structure analysis were conducted to enhance the resolution at the species level. The results from the comparative morphological analysis, the 16S rRNA gene percent similarity and phylogenetic analyses, the 16S-23S ITS percent dissimilarity and the ITS secondary structure analyses provided enough evidence that the strain 18C-PS is a representative of Cylindrospermum badium, providing further insights into its ecological distribution and morphological attributes. Additionally, the strain KUT1-PS was a novel species of the genus Cylindrospermum and is referred to herein as Cylindrospermum solincola sp. nov., in accordance with the International Code of Nomenclature for algae, fungi and plants. This study also discusses the importance of comparing the newly sequenced strains with previously established species before making final taxonomic interpretations.

1. Introduction

The genus Cylindrospermum was established by Kützing [1] with eight species (C. majus (=C. maius), C. conglobatum, C. polyspermum, C. spirale, C. riparium, C. limicola, C. humicola and C. arenicola), whereas Bornet and Flahult [2] included only five species, C. stagnale (including C. conglobatum, C. riparium and C. limicola), C. majus, C. licheniforme (=C. spirale), C. muscicola and C. catenatum, in their classification system. Furthermore, Bornet and Flahault [2] mentioned C. arenicola and C. humicola in the list of species to be investigated, and C. polyspermum in the list of species to be excluded. Interestingly, there has been a long-lasting debate around the type species of the genus Cylindrospermum and different studies have considered either C. maius or C. stagnale as the generitype [3,4,5,6,7,8,9]. Gardner [3] in his monograph chose C. maius to represent the type species of Cylindrospermum whereas; Geitler [4] chose C. stagnale as the type species. Since Gardner [3] has priority over Geitler [4], more recent treatments have considered C. maius CCALA 998 to be the generitype of Cylindrospermum [10,11].
The external morphology of the genus Cylindrospermum is a mostly amorphous, dull blue-green or brownish-coloured mucilaginous mat. The trichomes are short, unbranched, isopolar or uniformly broad, cylindrical, straight or slightly curved or coiled, and constricted at cross walls, only with very fine or thin, diffluent and colourless mucilage. The vegetative cells are cylindrical, barrel-shaped, or conical (terminal), longer to isodiametric or shorter than wide. Heterocytes are mostly solitary at both terminal ends or seldom at one end only, spherical or oval to ovoid and slightly larger than vegetative cells. Akinetes are always larger than vegetative cells and heterocytes, usually solitary or rarely in a row, adjacent to the heterocyte at one end or both ends of the trichome, oval or cylindrical to ellipsoidal, thick-walled and either smooth or ornamental with spines and warts [2,5,6]. The morphological characteristics of vegetative cells, heterocytes and akinetes, such as their shape and dimensions, the position of heterocytes and akinetes and the texture of akinete walls are considered to be crucial in differentiating morphologically related inter or intra-genic taxa [5,6,11,12,13].
Taxonomic studies using phylogenetic methods on members of the genus Cylindrospermum are limited [11]. Johansen et al. [11] in their phylogenetic analysis using the 16S rRNA gene found that all the five foundational species described by Bornet et Flahault [2] along with other strains isolated from temperate regions formed a monophyletic clade. This clade was designated to represent Cylindrospermum sensu stricto. Further, the authors also reported two different clades (Y and Z) consisting of Cylindrospermum strains isolated from non-temperate regions [11]. These clades were phylogenetically distant from Cylindrospermum sensu stricto, indicating the polyphyletic nature of the genus. In a recent study, Pal et al. [10] created a novel genus, Johanseniella, to accommodate the Cylindrospermum strains of the clade Y (as designated in Johansen et al. [11]). Furthermore, Tawong et al. [14] in their study found that their strains, which showed morphological resemblance to the genus Cylindrospermum, clustered distantly from Cylindrospermum sensu stricto, Johanseniella and clade Z of Johansen et al. [11]. Based on the phylogenetic evidence, Tawong et al. created a novel genus, Neocylindrospermum [14]. The recent studies of Pal et al. [10] and Tawong et al. [14] have contributed to resolving the phylogenetic inconsistencies of Cylindrospermum-like taxa to a certain extent, indicating the importance of the polyphasic approach in cyanobacterial taxonomic studies. Johansen et al. [11] emphasized the need for further studies using a polyphasic approach within the genus Cylindrospermum, but unfortunately, there has been no such study conducted to date.
Continuing our long-term pursuit of studying and unravelling the taxonomic complexities of Indian cyanobacteria dwelling in different habitats, we started to sample different regions of the Jammu division (Jammu and Kashmir, Union Territory) in 2019. Amongst the many strains that have been isolated and characterized, two Cylindrospermum-like strains were found to be interesting and hence were evaluated extensively using a polyphasic approach. Out of these two strains, KUT1-PS, emerged as a novel species in the genus Cylindrospermum and is being described as a novel species of Cylindrospermum, following the norms of the International Code of Nomenclature for algae, fungi and plants (ICN). The other strain, 18C-PS, turned out to be Cylindrospermum badium after a thorough polyphasic evaluation. The case study of 18C-PS reflects an interesting example in modern cyanobacterial taxonomy, in which strains isolated from different geographical and ecological realms turned out to represent the same species. The strain 18C-PS also exhibited interesting life-cycle events that will serve to bring more clarity into the morphological intricacies of the genus Cylindrospermum. The current study continues to bring more information on the cyanobacterial diversity from different habitats across India, which we believe is just the tip of the iceberg.

2. Materials and Methods

2.1. Sampling, Isolation and Maintenance of the Isolates

Sampled specimens were collected in 50 mL falcon tubes containing 20 mL of sterile BG110 liquid medium [15]. Geographical coordinates, physicochemical parameters (pH, temperature, TDS and salinity) and the vegetation type of the habitat and surrounding areas were recorded at the time of sampling. The collected samples were observed under a microscope for the presence of cyanobacteria. Further, the samples were spread on solidified BG110 medium [15] with 1.2% agar. After an initial growth was observed, a small portion of the mat was taken from the Petri plate and transferred to a clean and sterile culture tube containing 5 mL sterile BG110 liquid medium. This step of subculturing from solid to liquid medium and vice versa was done until uni-cyanobacterial strains were obtained for both samples. The uni-cyanobacterial strains, 18C-PS and KUT1-PS were maintained in slants of solidified BG110 medium and also in 100 mL cotton-stopped flasks containing 50 mL liquid BG110 medium, at 22 °C ± 2 °C under the cool white light of approximately 50–55 μEm−2 s−1, with a photoperiod of a 14/10 h light/dark cycle. Both the strains, KUT1-PS and 18C-PS were deposited in the Global Collection of Cyanobacteria (https://ccinfo.wdcm.org/details?regnum=1165 (accessed on 30 January 2023)), Varanasi, India and are available under the accession numbers GCC20227 and GCC202228, respectively.

2.2. Morphological Analysis

Morphological analysis of both the strains characterized in this study (KUT1-PS and 18C-PS) was carried out with the help of an Olympus BX53 light microscope. Morphological characters such as the size and shape of vegetative cells and the position, size and shape of heterocytes and akinetes, etc. were carefully observed. The morphologies of both strains were matched and compared with the other species of Cylindrospermum, as described in the keys of Desikachary [5] and Komárek [6] and the other related research articles.

2.3. Molecular Analysis

Approximately 100–120 mg exponentially growing, uni-cyanobacterial culture was taken for each isolate (KUT1-PS and 18C-PS), washed 6–8 times with de-ionized water and subjected to genomic DNA isolation. DNA was isolated with the help of a FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) and visualized on 0.8% agarose gel. The 16S rRNA gene of both the strains was amplified with the primer pA (5′-AGAGTTTGATCCTGGCTCAG-3′) [16] and a cyanobacteria-specific B23S primer (5′-CTTCGCCTCTGTGTGCCTAGGT-3′) [17]. The PCR reaction was performed in a 25 µL volume consisting of dNTPs (250 µM) and Taq Buffer (1X), 1 µL of each primer (0.4 µM), 0.3 µL of Taq DNA polymerase (1U/µL) and 2 µL of genomic DNA (15–40 ng/µL). With the initial denaturation at 94 °C for 5 min and final extension at 72 °C for 3 min, the amplification of the 16S rRNA gene was performed for 34 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s with a Bio-Rad C1000TM Thermal Cycler. The 16S-23S ITS region was amplified using the primers and conditions mentioned by Iteman et al. [18]. The sequencing was performed by Sanger’s method with a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were assembled into contigs, and their quality was monitored using ChromasPro (ver. 2.1.10, Technelysium Pty. Ltd. Unit 406, 8 Cordelia St., South Brisbane, QLD, Australia). Finally, the sequences were submitted to NCBI GenBank and are available under the accession numbers OQ055346 and OQ055347.

2.4. Phylogenetic Analysis

Phylogenetic analysis of the strains KUT1-PS and 18C-PS was performed by using the 16S rRNA gene and 16S-23S ITS region. Phylogenetically related sequences of the 16S rRNA gene and the 16S-23S ITS region of both the strains under investigation were obtained by NCBI Blast search. The sequences for 16S rRNA gene phylogenetic analysis were aligned by MAFFT (version 7) [19] and manually curated to ensure that the secondary structure of the 16S rRNA gene was maintained. In addition, phylogenetic analysis was also inferred using the 16S-23S ITS region. The operons of the 16S-23S ITS region with both tRNAs were recovered for both KUT1-PS and 18C-PS and the phylogenetic analysis was inferred using the orthologous operons of the phylogenetically related strains. The sequences were aligned using MAFFT and manually curated to maintain the secondary structure of the conserved domains. Further, the phylogenetic trees for both analyses were reconstructed using Bayesian inference (BI), maximum likelihood (ML) and neighbour-joining (NJ) methods. The BI analysis was performed in Mr Bayes 3.2.2 [20]. For the 16S rRNA gene, the analysis was run for 4.7 million generations, whereas for the 16S-23S ITS region, the analysis was run for 0.5 million generations. A standard deviation of the split frequency of less than 0.01 was achieved for both analyses. The sampling frequency, print frequency and diagnostic frequency for both analyses were set to 1000. The estimated sample size (ESS) was above 100 and the potential scale reduction factor (PSRF) was near to 1 for all the parameters. The ML tree for each analysis was constructed in IQ-TREE [21], using the ultrafast bootstrap method with 1000 replications [22]. The BI and ML trees in both analyses were reconstructed using the GTR + G + I model, selected using jModelTest [23]. Furthermore, the NJ tree was inferred using MEGA X [24] for each analysis and the standard bootstrap method with 1000 replications was used to test the reliability of both the 16S rRNA gene and 16S-23S ITS region analyses [25]. Finally, the trees were visualised and edited with the help of iTOL (v6) [26] and Adobe Illustrator 2022 (version 23.0.2).

2.5. 16S-23S ITS Secondary Structure Analysis

The 16S-23S ITS regions of the strains under study along with their phylogenetically related strains were annotated by colour-coding the different ITS domains. The domains corresponding to the D1-D1′, V2, BoxB and V3 regions were folded using Mfold on UNAFold web server [27]. The folded secondary structures were downloaded and redrawn in Adobe Illustrator 2022 (version 23.0.2). Furthermore, the lengthwise comparison of different ITS domains (leader, D1-D1′, Spacer + D2, Spacer + D3 + Spacer, tRNAIle, Spacer + V2 + Spacer, tRNAAla, Spacer, BoxB + Spacer, BoxA, D4 + Spacer and V3 + end of ITS) was also done for our strains and their phylogenetically related taxa.

2.6. Calculations of 16S rRNA Gene Percent Similarity and 16S-23S ITS Percent Dissimilarity

p-distance values for calculating the 16S rRNA gene percent similarity and 16S-23S ITS percent dissimilarity was determined using MEGA X [24]. Further, the percent similarity of the 16S rRNA gene was calculated using the formula (1-p-distance) × 100, whereas the percent dissimilarity of the 16S-23S ITS region was calculated using the formula p-distance × 100.

2.7. Preparation of Holotype and Isotype

For holotype preparation, a portion of culture from each strain was preserved in a metabolically inactive form, provided with the accession number and stored in the Global Collection of Cyanobacteria (https://ccinfo.wdcm.org/details?regnum=1165 (accessed on 30 January 2023)), Varanasi, India. Isotype and herbaria preparation in duplicate for each strain were performed by taking a liquid culture, sieving onto a filter paper followed by air drying, keeping the filter paper in a small Petri plate and storing it in an acid-free envelope after proper labelling.

3. Results

3.1. Site Studied

The cyanobacterial samples were collected from the Basantgarh village of Udhampur district, in the Union territory of Jammu and Kashmir, India. This village falls in the subtropical region, has hilly terrain and is located at an altitude of ~2000 m above sea level in the Shivalik range of the Himalayas. Climatic conditions generally remain pleasant in the summers, with the average minimum and maximum temperature ranging between 24–37 °C, whereas the winters are extremely cold, with the average minimum and maximum temperature ranging between 5–15 °C. The area receives snowfall in the months of December to March and rainfall occurs in the months of July to September, with the annual rainfall in the region being about 40 mm. The vegetation of the area includes Deodar Forests with Berberis, Rubus, Rosa, Prinsepia, Sarcococca, Litsea, Wikstroemia, etc. and many other shrubs and plants in the wild. Apples, pears, walnuts, plums, peaches, kiwis, etc. are grown in the orchards and maize is grown as an annual crop.
Cylindrospermum badium 18C-PS was isolated from the cyanobacterial sample collected in November 2019, from a vernal pool (32°48′23′′ N, 75°32′36′′ E). The depth of the vernal pool was around one ft. and the sample collection was carried out at nearly 0.5 ft. depth from the surface of the pool. The temperature at the time of sample collection was 12.7 °C and the pH, electrical conductivity, total dissolved solids (TDS) and salinity of the pool were recorded at 5.5, 54.0 µS, 38.5 ppm and 27.3 ppm, respectively. Further, Cylindrospermum solincola KUT1-PS was isolated from the cyanobacterial sample collected in June 2021, from the surface of the soil found near an agricultural field. The temperature at the time of sample collection was 26 °C.

3.2. Morphological Characterization

The morphology of the natural sample of Cylindrospermum solincola KUT1-PS showed two different heterocytous cyanobacterial forms. One of the morphological forms exhibited a typical Cylindrospermum-like morphology, with isopolar trichomes, cylindrical-shaped vegetative cells, terminal heterocytes and paraheterocytic akinetes, whereas the other morphological form exhibited a Calothrix-like morphology, with heteropolar filaments and heterocytes at the basal end. In culture conditions, the trichomes of Cylindrospermum solincola KUT1-PS were isopolar and flexuous, with cylindrical (many times isodiametric too) vegetative cells. Heterocytes were solitary and terminal, sometimes developing asymmetrically at both ends of the trichome. Akinetes were solitary, large and always formed adjacent to the heterocyte. The exospore of mature akinetes was covered with spines (Figure 1, Table 1).
The morphology of the natural sample of Cylindrospermum badium 18C-PS also showed an assemblage of greenish and bluish-green isopolar and heteropolar filaments with heterocytes. The isopolar filaments were either long with terminal and intercalary heterocytes or short with only terminal heterocytes at both ends. The trichomes of Cylindrospermum badium 18C-PS were short, straight or slightly bent with cylindrical vegetative cells. Heterocytes were solitary, present either at one terminal end or both terminal ends of the trichome. Akinetes were large, solitary and always formed adjacent to heterocyte. Young akinetes appeared greenish in colour and were covered with a thin, transparent and diffluent layer of mucilage. The exospore of the mature akinete was thick, chestnut brown and did not have spines (Figure 2, Table 1).

3.3. Phylogenetic Analysis Based on 16S rRNA Gene and 16S-23S ITS Region

The 16S rRNA gene phylogenetic tree was reconstructed using 216 nucleotide sequences belonging to the heterocytous cyanobacteria and Chroococcidiopsis was used as an outgroup (Figure 3). Among the 216 nucleotide sequences corresponding to the order Nostocales, 43 sequences belonged to Cylindrospermum sensu stricto (Figure 3). The clustering of strains—KUT1-PS and 18C-PS within the Cylindrospermum sensu stricto indicated that both the strains under investigation were representatives of the genus Cylindrospermum (Figure 4).
C. solincola KUT1-PS clustered with C. alatosporum (CCALA 988/CCALA 994), C. maius CCALA 998 (reference strain), Cylindrospermum sp. CHAB2115 and Anabaena sp. 14-VSmolV10 (Figure 4). The strains of C. alatosporum were isolated from wildfire-impacted soil of the Riding Mountains National Park, Canada and from the gut of earthworms collected from soils above Amatérská Cave, Czech Republic. C. maius CCALA 998 was isolated from recultivated (top) soil after coal mining and with loblolly pine in the Pyramid State Recreation Area, Illinois, USA. The strain Cylindrospermum sp. CHAB2115 was isolated from benthic areas of freshwater Dali, China, while Anabaena 14-VSmolV10 was isolated from the eastern part of the Smolnice mine waste dump in the Czech Republic.
Cylindrospermum badium 18C-PS, along with Cylindrospermum badium CCALA 1000, Cylindrospermum sp. NIES-4074 and Cylindrospermum sp. YK2-01, clustered together at a single node (Figure 4). C. badium CCALA 1000 was isolated from the recultivated topsoil after coal mining with sweet gum in Pyramid State Recreational Area, Illinois, USA, whereas Cylindrospermum sp. YK2-01 was isolated from the Nostoc commune crust, Kanagawa, Japan and Cylindrospermum sp. NIES-4074 was isolated from soil in Yokohama, Kanagawa, Japan.
Furthermore, the 16S-23S ITS phylogenetic tree was reconstructed using 12 nucleotide sequences belonging to Cylindrospermum sensu stricto. C. badium 18C-PS, along with C. badium CCALA 1000, clustered together with strong probability/bootstrap support, whereas C. solincola KUT1-PS clustered separately at the bottom of the tree (Figure 5).

3.4. Percent Similarity Analysis of 16S rRNA Gene

The 16S rRNA gene percent similarity of C. solincola KUT1-PS and C. badium 18C-PS with the other members of Cylindrospermum sensu stricto was in the range of 96.3–98.9% and 96.5–99.7% (Table S1), respectively, whereas the percent similarity between C. solincola KUT1-PS and C. badium 18C-PS was 96.3% (Table S1).

3.5. Secondary Structure Analysis of 16S-23S ITS Region

The D1-D1′ helix region of all the Cylindrospermum strains consisted of 64 nucleotides (Table S2). Furthermore, the folded secondary structures of all the strains were similar and consisted of a 5 bp basal stem followed by a 3′ unilateral bulge formed by 7 nucleotides. The unilateral bulge was followed by a bilateral bulge of 9 nucleotides and a terminal loop of 5 nucleotides (Figure 6). The lengths of the V2 helices and their folded secondary structures were variable among the Cylindrospermum species (Figure 7, Table S2). The folded secondary structure of the V2 helix of C. solincola KUT1-PS consisted of an 11 bp basal stem followed by two bilateral bulges and a terminal loop (Figure 7). On the other hand, the folded secondary structure of C. badium 18C-PS consisted of an 11 bp basal stem followed by a single bilateral bulge and a terminal loop (Figure 7).
Furthermore, the BoxB region consisted of 27 nucleotides in all the strains included in the analysis, except in C. stagnale PCC 7417, which consisted of 28 nucleotides (Figure 7). The folded secondary structure of the BoxB region of C. solincola KUT1-PS was found to be similar to that of C. alatosporum (CCALA 988, CCALA 994), consisting of a 4 bp basal stem followed by a bilateral bulge and a terminal loop (Figure 7). The folded secondary structure of C. badium 18C-PS was found to be similar to C. badium CCALA 1000, C. catenatum (CCALA 990, CCALA 991 and CCALA 996), C. muscicola SAG 44.79 and C. moravicum CCALA 993, comprising a 4 bp basal stem followed by a unilateral bulge opposed by adenine and a terminal loop (Figure 7). The length of the V3 helix within the Cylindrospermum varied from 33–41 nucleotides (Table S2). The folded secondary structures of the strains included in the analysis were similar, having a 3 bp basal stem followed by a bilateral bulge and a terminal loop, except for the folded secondary structure of C. moravicum, which consisted of two bilateral bulges (Figure 7).

3.6. Percent Dissimilarity Analysis of 16S-23S ITS Region

Furthermore, the percent dissimilarity of the 16S-23S ITS region of C. solincola KUT1-PS and C. badium was in the range of 12.2–15.5% and 4.1–16.6%, respectively, with their phylogenetically related strains (Table S3).

3.7. Taxonomic Description

Cylindrospermum solincola. N Kumar and P Singh sp. nov. (Figure 1A–G).
Description: Thallus dark blue-green mucilaginous mass in the natural environment and under the microscope, typical Cylindrospermum-like trichomes with a large paraheterocytic akinete were observed. In culture conditions, a greenish, thick, gelatinous mat observed on the surface of a Petri plate, olive at the centre and greenish spreading out. In liquid medium, a greenish, gelatinous mat formed, which turns brownish in older cultures. Trichomes cylindrical, uniseriate, isopolar, unbranched, short, flexuous, not straight, loosely tangled, greenish in colour and without any sheath. Vegetative cells slightly constricted at the ends, cylindrical, granulated and usually longer than wide, but some cells shorter than wide to isodiametric; 1.49–3.08 µm long and 1.72–2.11 µm wide. Heterocytes terminal, pale coloured, solitary, at both ends, usually oval, and very rarely spherical, 2.89–4.39 µm long and 2.61–3.33 µm wide. Akinetes large, yellowish, solitary, paraheterocytic, granulated, thick-walled, serrated with spines and with only one per filament; cylindrical when young and oblong when mature, 10.21–13.98 µm long and 5.04–6.41 µm wide.
Etymology: solincola (sol.in’co.la. L. neut. n. solum, soil; L. masc./fem. n. incola, an inhabitant; N.L. n. solincola, an inhabitant of soil).
Type locality: the sample was collected by Naresh Kumar in June 2021 from the village Basantgarh, Udhampur, Union Territory of Jammu and Kashmir, India (32°48′27′′ N 75°32′37′′ E).
Ecology of type locality: wet soil, found near a small agricultural field in which maize is cultivated annually. The sampling site was surrounded by a coniferous forest of Deodar, spiny bushes and grasses. Dark bluish-green mucilage found at the surface of the soil was collected as a sample.
Holotype (here designated): A portion of a culture of Cylindrospermum solincola has been preserved in a metabolically inactive form in the Global Collection of Cyanobacteria (https://ccinfo.wdcm.org/details?regnum=1165 (accessed on 30 January 2023)), Varanasi, India, and is available under the accession number GCC20227.
Isotype (here designated): Herbaria was deposited in the Global Collection of Cyanobacteria with the number GCC-botanybhu-20227.
Reference strain: KUT1-PS.
NCBI GenBank Accession number: OQ055347.

4. Discussion

The aim of this study was to characterize two Cylindrospermum-like strains isolated from the Jammu division of the Jammu and Kashmir Union territory of India. This is the first study after the work of Yajnavalkya Bharadwaja, in which a novel species of Cylindrospermum from Jammu and Kashmir is described. Bharadwaja in 1931 described C. muscicola var. kashmiriensis growing on Myriophyllum in a shallow pond situated in Srinagar, Kashmir [28]. Both the strains investigated in this study differ from C. kashmiriensis in more than one feature, such as the shape and size of the vegetative cells, heterocytes and akinetes, the position of the heterocytes and the ecology of the habitat (Table 1).
C. solincola KUT1-PS was related to C. gorakhpurense, C. dobrudjense and C. ecballiisporum in terms of the ecology and morphology of the akinetes. The aforementioned species, including C. solincola KUT1-PS, were isolated from wet/moist soils (except C. ecballiisporum, which was isolated from the edge of wet peat) and had spines on their akinetes. However, the smaller size of vegetative cells, heterocytes and akinetes along with minor differences in their shapes distinguished C. solincola KUT1-PS from C. gorakhpurence, C. dobrudjense and C. ecballiisporum (Table 1).
Further, C. badium 18C-PS was morphologically similar to C. badium CCALA 1000 in terms of trichome features, the shape of vegetative cells, the basal position and shape of heterocytes, the paraheterocytic position and the very typical chestnut brown smooth exospore of large akinetes in later stages of growth. Both the strains differed from each other only in the dimensions of vegetative cells, heterocytes and akinetes, this is understandable given the slight differences in ecology. Thus, this particular instance of the two strains representing C. badium casts intriguing reflections on the biogeographical importance of these findings. The evolution of cyanobacterial species and their subsequent spread to different geographical regions is something that has still not been clearly understood and hence must be studied in the near future. Another interesting observation related to C. badium 18C-PS was the extremely clear exhibition of different life cycle events (Figure 2). Various events were observed, including the rupturing of the exospore, the release of the spore and the germination of the spore into hormogonia (Figure 2I–K).
In the 16S rRNA gene phylogenetic analysis, C. solincola KUT1-PS and C. badium 18C-PS clustered within the Cylindrospermum sensu stricto clade with strong and well-supported posterior probabilities/bootstrap support. Unlike all the previous phylogenetic studies based on the 16S rRNA gene on the genus Cylindrospermum or its related taxa [7,10,11,14], Cylindrospermum sensu stricto clustered with good posterior probabilities/bootstrap support in the current phylogenetic analysis (Figure 3). However, similar to the above studies, C. alatosporum, C. licheniforme, C. stagnale C. catenatum and C. muscicola were found to be polyphyletic (Figure 4). The clustering of these strains again clearly emphasizes the need for taxonomic revisions inside the Cylindrospermum sensu stricto.
All the phylogenetically related strains of C. solincola KUT1-PS and C. badium 18C-PS were isolated from different habitats located in different localities. Amongst the phylogenetically related strains of C. solincola KUT1-PS, two strains, Anabaena sp. 14-VSmolV10 and Cylindrospermum sp. CHAB2115, were not identified up to the rank of species level. Further, the absence of oval or rhomboidal-shaped large-sized akinetes with smooth walls and smaller sizes of vegetative cells and heterocytes differentiated C. solincola KUT1-PS from C. alatosporum (Table 1). Similarly, the smaller size of vegetative cells, heterocytes and akinetes and the presence of spines on the wall of akinetes differentiated C. solincola KUT1-PS from C. maius (Table 1).
The 16S rRNA gene percent similarity of C. solincola KUT1-PS was found to be the highest, at 98.8%, with C. alatosporum CCALA 994. This value is higher than the threshold value recommended by Yarza et al. [29] for the delimitation of prokaryotic species (Table S1). However, Johansen et al. [11] also reported exceptionally high 16S rRNA gene percent similarity within the Cylindrospermum sensu stricto. The authors concluded that the morphological diversity within the genus Cylindrospermum exceeds variations observed in the 16S rRNA gene, suggesting recent rapid divergence within the genus [11]. The present study also supports the observations made in the study of Johansen et al. [11].
Furthermore, the phylogenetic positioning of 18C-PS in the 16S rRNA gene tree and the percent similarity of 18C-PS with C. badium CCALA 1000 was 99.7%. The results from the 16S rRNA gene phylogenetic analysis and 16S rRNA gene percent similarity indicate that the strain 18C-PS is a representative of C. badium, supporting the results obtained from morphological characterization. Moreover, the results from the 16S-23S ITS phylogenetic analysis also indicated that 18C-PS is a representative of C. badium and C. solincola. KUT1-PS is a novel species of the genus Cylindrospermum (Figure 5), providing further support to the results from the 16S rRNA gene phylogenetic analysis.
Furthermore, the 16S-23S ITS region’s folded secondary structure analysis was conducted to enhance the resolution at the species level. The D1-D1′ helix of C. solincola KUT1-PS was highly similar to other Cylindrospermum species and differed in only one base at the top of the bilateral bulge (Figure 6). However, the folded secondary structures of the V2, BoxB and V3 helices of C. solincola KUT1-PS were completely different from the other phylogenetically related strains included in the analysis (Figure 7).
The folded secondary structure of the D1-D1′ helix of C. badium 18C-PS was identical to C. moravicum CCALA 993 and was similar to C. badium CCALA 1000, with minor differences in the middle region, the bilateral bulge and the terminal loop (Figure 6). Similarly, the folded secondary structure of the BoxB region of C. badium 18C-PS was identical to C. catenatum (CCALA 990, CCALA 991, CCALA 996, CCALA 999) and showed one nucleotide difference in the terminal loop of C. badium CCALA 1000 (Figure 7). Further, the folded secondary structure of the V3 region of C. badium 18C-PS was completely identical to C. badium CCALA 1000 (Figure 7). Amongst all the compared motifs of the 16S-23S ITS, only the V2 helix was found to be unique in C. badium 18C-PS (Figure 6 and Figure 7).
Additionally, the percent dissimilarity of the 16S-23S ITS of C. solincola KUT1-PS was >7% from the other phylogenetically related members of the Cylindrospermum sensu stricto (Table S3). Notably, percent dissimilarity values ≥7% strongly indicate that the strains belong to separate species [30,31,32]. However, the percent dissimilarity of C. badium 18C-PS was <7% with C. badium CCALA 1000 (4.1%) and C. moravicum CCALA 993 (5.5%) and >7% with the other members of the Cylindrospermum sensu stricto (Table S3).
Thus, based on the results obtained from the comparative morphological analysis, phylogenetic analysis based on the 16S rRNA gene and 16S-23S ITS region, the 16S-23S ITS secondary structure analysis and the 16S-23S ITS percent dissimilarity, we gathered enough evidence indicating that C. solincola KUT1-PS represents a novel species of the genus Cylindrospermum. On the other hand, based on the morphological similarities, the phylogenetic positioning in the 16S rRNA gene and 16S-23S ITS region, the percent similarity of the 16S rRNA gene and the percent dissimilarity of the 16S-23S ITS region, it was established that strain 18C-PS was a representative of C. badium.
This study serves as an important reminder, especially to modern cyanobacterial taxonomists, to always compare newly sequenced strains with previously established species before going ahead with the creation of a novel species. Molecular studies should always be supplemented with morphological assessments in order to avoid ambiguous situations that could arise in the future. This fact has been mentioned very clearly in Komárek [33] and we fully agree that the polyphasic approach must always have molecular criterion as the primary and morphological/ecological evidence as a secondary but essential criterion in order to properly establish any taxonomic entity. In a similar vein, we also feel that phylogenomic interpretations could be employed to bring an enhanced resolution and separate the various strains that are present inside the Cylindrospermum cluster. However, unless and until all the taxa inside this cluster are sequenced, taxa-limited phylogenomic studies will bring in additional confusing interpretations. Issues related to limited taxon sampling and ambiguities emerging even after phylogenomic interpretations have already started to emerge in heterocytous cyanobacteria (the Amazonocrinis story, Alvarenga et al. [34], and Kumar et al. [35]), and we believe that with time, as more data emerges from the tropical and subtropical regions, taxonomic efforts will fuel interesting stories and debates. Hence, caution must be adopted while deriving taxonomic conclusions from whole genome efforts. Over-reliance on any method, modern or classical, must always be avoided and in fact, striking a careful balance between these methods constitutes the basic spirit of the polyphasic approach.
Further, recent studies on Cylindrospermum and Cylindrospermum-like taxa [7,10,11,14] clearly indicate that the diversity of these taxa is underrepresented and more taxonomic studies, based on a polyphasic approach, on the cyanobacterial strains that are morphologically similar to the genus Cylindrospermum need to be conducted in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15050592/s1, Table S1. Comparative percent similarity matrix of the 16S rRNA gene of C. solincola KUT1-PS and C. badium 18C-PS with their phylogenetically related taxa based on 16S rRNA gene; Table S2. Comparison of nucleotide length of the 16S-23S ITS regions of C. solincola KUT1-PS and C. badium 18C-PS with their phylogenetically related taxa based on 16S rRNA gene; Table S3. Comparative percent dissimilarity matrix of the 16S-23S ITS regions of C. solincola KUT1-PS and C. badium 18C-PS with their phylogenetically related taxa based on 16S rRNA gene.

Author Contributions

Conceptualization, N.K., A.S., S.P. and P.S.; methodology, N.K. and P.S.; software, N.K. and S.P.; validation, A.S. and P.S.; formal analysis, A.S., S.P. and P.S.; investigation, A.S., S.P. and P.S.; resources, A.S. and P.S.; data curation, A.S., S.P. and P.S.; writing—original draft preparation, N.K.; writing—review and editing, A.S. and P.S.; visualization, N.K., A.S., S.P. and P.S.; supervision, P.S.; project administration, P.S.; funding acquisition, P.S.; All authors have read and agreed to the published version of the manuscript.

Funding

The work was funded by the Department of Science and Technology (DST-SERB), India, through the Core Research Grant Project CRG/2018/004111. The Banaras Hindu University is also acknowledged for providing Seed Grant under the Institution of Eminence (IoE) Scheme.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank the Head of the Department of Botany at Banaras Hindu University (BHU) for providing the necessary facilities and encouragement. NK thanks the University Grants Commission (UGC) for the Senior Research Fellowship (SRF). SP thanks the Department of Science and Technology for the DST-INSPIRE Senior Research Fellowship (SRF). We thank Aharon Oren for his help with the Latin etymology of the scientific name.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Figure 1. Microscopic plate showing the morphological features of Cylindrospermum solincola KUT1-PS. (A) trichomes short, isopolar, slightly bent or irregularly coiled; (B,C) vegetative cells cylindrical, longer or shorter than wide or isodiametric, and heterocytes terminal, solitary at both ends of the trichome; (D,E) heterocytes oval or spherical and a large, young, solitary akinete adjoining to the heterocyte at one end of the trichome; (F,G) mature, oblong, granulated, exospore thick akinete, serrated with short spines. Scale bars: (A) 20 µm; (B–E) 10 µm and (F,G) 5 µm.
Figure 1. Microscopic plate showing the morphological features of Cylindrospermum solincola KUT1-PS. (A) trichomes short, isopolar, slightly bent or irregularly coiled; (B,C) vegetative cells cylindrical, longer or shorter than wide or isodiametric, and heterocytes terminal, solitary at both ends of the trichome; (D,E) heterocytes oval or spherical and a large, young, solitary akinete adjoining to the heterocyte at one end of the trichome; (F,G) mature, oblong, granulated, exospore thick akinete, serrated with short spines. Scale bars: (A) 20 µm; (B–E) 10 µm and (F,G) 5 µm.
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Figure 2. Microscopic plate showing the morphological features of Cylindrospermum badium 18C-PS. (A) trichomes short, isopolar, straight or slightly bent, cells cylindrical, isodiametric to longer than wide, heterocytes solitary, terminal, one or both ends, oval to ovoid with a broad base; (B) heterocyte with thread-like projections; (C) development of proakinetes; (D,E) proakinetes, cylindrical, free or attached, adjoining to heterocyte at one end of trichome, constricted or unconstricted at the centre; (F,G) young, oval to oblong akinetes, solitary with a thin, transparent, diffluent layer of mucilage; (H) mature, oblong akinetes, exospore chestnut brown, thick and smooth; (I,J) rupturing of akinetes wall, release of akinetes; (K) germination of akinete. Scale bars: (A) 10 µm and (B–K) 5 µm.
Figure 2. Microscopic plate showing the morphological features of Cylindrospermum badium 18C-PS. (A) trichomes short, isopolar, straight or slightly bent, cells cylindrical, isodiametric to longer than wide, heterocytes solitary, terminal, one or both ends, oval to ovoid with a broad base; (B) heterocyte with thread-like projections; (C) development of proakinetes; (D,E) proakinetes, cylindrical, free or attached, adjoining to heterocyte at one end of trichome, constricted or unconstricted at the centre; (F,G) young, oval to oblong akinetes, solitary with a thin, transparent, diffluent layer of mucilage; (H) mature, oblong akinetes, exospore chestnut brown, thick and smooth; (I,J) rupturing of akinetes wall, release of akinetes; (K) germination of akinete. Scale bars: (A) 10 µm and (B–K) 5 µm.
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Figure 3. Phylogenetic position of Cylindrospermum sensu stricto with morphologically related and unrelated taxa of heterocytous cyanobacteria based on 16S rRNA gene sequences of 216 OTUs of heterocytous cyanobacteria, rooted with 4 OTUs of Chroococcidiopsis and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped on to the nodes in the BI analysis. Scale bar represents 0.2 changes per nucleotide position.
Figure 3. Phylogenetic position of Cylindrospermum sensu stricto with morphologically related and unrelated taxa of heterocytous cyanobacteria based on 16S rRNA gene sequences of 216 OTUs of heterocytous cyanobacteria, rooted with 4 OTUs of Chroococcidiopsis and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped on to the nodes in the BI analysis. Scale bar represents 0.2 changes per nucleotide position.
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Figure 4. Phylogenetic position of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS within the Cylindrospermum sensu stricto clade, based on 16S rRNA gene and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped onto the nodes in the BI analysis. Scale bar represents 0.1 changes per nucleotide position.
Figure 4. Phylogenetic position of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS within the Cylindrospermum sensu stricto clade, based on 16S rRNA gene and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped onto the nodes in the BI analysis. Scale bar represents 0.1 changes per nucleotide position.
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Figure 5. Phylogenetic analysis based on 16S-23S ITS region with both tRNA operons, showing the phylogenetic positioning of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped onto the nodes in the BI analysis. Scale bar representing 0.20 changes per nucleotide position.
Figure 5. Phylogenetic analysis based on 16S-23S ITS region with both tRNA operons, showing the phylogenetic positioning of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS and posterior probability values/bootstrap values (0.50/≥50%), representing BI/ML/NJ mapped onto the nodes in the BI analysis. Scale bar representing 0.20 changes per nucleotide position.
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Figure 6. Comparative folded secondary structure analysis of D1-D1′ helix of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS, with their phylogenetically related strains based on the 16S rRNA gene.
Figure 6. Comparative folded secondary structure analysis of D1-D1′ helix of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS, with their phylogenetically related strains based on the 16S rRNA gene.
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Figure 7. Comparative folded secondary structure analysis of V2, BoxB and V3 helices of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS, with their phylogenetically related strains based on the 16S rRNA gene.
Figure 7. Comparative folded secondary structure analysis of V2, BoxB and V3 helices of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS, with their phylogenetically related strains based on the 16S rRNA gene.
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Table
Table 1. Comparison of morphological features of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS with their morphologically, geographically and phylogenetically related taxa (based on 16S rRNA gene) of the genus Cylindrospermum. Note: Species studied in the present investigation are marked in bold.
Table 1. Comparison of morphological features of Cylindrospermum solincola KUT1-PS and Cylindrospermum badium 18C-PS with their morphologically, geographically and phylogenetically related taxa (based on 16S rRNA gene) of the genus Cylindrospermum. Note: Species studied in the present investigation are marked in bold.
SpeciesHabitatVegetative Cell (Size; LxW)Heterocyte (Size; LxW)Akinete (Size; LxW)
Cylindrospermum solincola KUT1-PSWet soilCylindrical, often longer than wide, some isodiametric and shorter than wide, 1.49–3.08 × 1.72–2.11 µmTerminal, solitary, both terminal ends, oval, rarely spherical, 2.89–4.39 × 2.61–3.33 µmLarge, solitary, at one terminal end, adjoining to heterocyte, young cylindrical, mature oblong, wall serrated with spines, yellowish in colour, 10.21–13.98 × 5.04–6.41 µm
Cylindrospermum gorakhpurenceMoist soils in paddy fieldsCylindrical, 7–16.5 × 3.5–5 µmTerminal, solitary, both terminal ends, ellipsoidal, 13.2–16.5 × 6–7.8 µmSubterminal, ellipsoidal with rounded ends, wall thick, with delicate, needle-like projections, 26.4–33 × 13.2–20 µm
Cylindrospermum dobrudjenseAgricultural soils (Medicago sativa)Cylindrical, longer than wide, 6.6–9.2 × 4 µmTerminal, both terminal ends, elongated ellipsoidal, conical or subspherical, 9.4–13.4 × 6.7 µmSolitary, elongated, ellipsoidal, with rounded ends, wall with very fine and short spines, 22.8–26.6 (28.8) × 9.2–10.5 µm
Cylindrospermum ecballiisporumEdge of wetted, peaty, partly periodically submerged pools and ditchesCylindrical, ± isodiametric or longer than wide, end cells (without heterocytes) cylindrical and rounded 2.5–7.5 (8) × 2–3.4 µmTerminal, almost spherical, ellipsoidal, short cylindrical or ovoid, slightly wider than vegetative cells, 5–10 × 3.5–6 µmSolitary, rarely in pairs, adjoining to heterocytes, cylindrical-oval, rarely ellipsoidal, wall thick, with spines, (14) 20–33 (40) × (9) 11–14.5 (16) µm
Cylindrospermum maius CCALA 998Recultivated soil after coal miningCylindrical, longer than wide, isodiametric to shorter than wide, apical cell rounded to cylindrical, 3.7–6.5 × 3.9–5 µmTerminal, spherical to elongated, 4–10 × 4.5–6 µmSolitary, adjoining to heterocyte, cylindrical, oval to ellipsoidal, wall smooth, initially colourless, later brown and layered 1–1.5 µm wide, (18) 21–36 × 10–15 (16) µm
Cylindrospermum badium 18C-PSVernal poolCylindrical, isodiametric to longer than wide, 1.44–2.92 1.47–1.88 µmTerminal, solitary, one or both terminal ends, oval to ovoid with broad base, 2.59–3.74 × 2.04–2.51 µmLarge, solitary, at one terminal end, adjoining to heterocyte, young oval to oblong, covered with a thin, transparent and diffluent layer of mucilage, mature oblong, wall thick, smooth and chestnut brown, 6.57–10.63 × 4.31–7.19 µm
Cylindrospermum badium CCALA 1000Recultivated soil after coal mining with sweet gumCylindrical, isodiametric to longer than wide, 3.5–7.5 × 3.0–4.8 µmTerminal, solitary, almost spherical, elongated or slightly conical, 5–10 (13) ×, 3–5 µmSolitary, paraheterocytic, oval broadly, flattened at ends, wall should be granulated or coarse instead of smooth, wide and chestnut-coloured, 17–30 × 10.0–14.4 µm
Cylindrospermum musicola Var. kashmiriensisEpiphytic on Myriophyllum in shallow pondBarrel-shaped, longer than broad, 2.6–8.4 × 2.6–3.9 µmTerminal and intercalary, terminal, solitary, one or both terminal ends, oval or ellipsoidal, intercalary, solitary or in pairs, shorter or longer than vegetative cells, 5.2–10.5 × 3.9–5.9 µmAdjoining to terminal heterocyte, barrel-ellipsoidal, wall thick and smooth, 9.5–13.6 × 5.2–7.8 µm
Cylindrospermum stagnaleMarshes, pools, ponds, lakesCylindrical, isodiametric or double longer than wide, (3) 3.8–4.5 (6?) µm wide, apical cylindrical-roundedUsually oval, ovoid, or cylindrical, with rounded ends, rarely almost spherical, 7–16 × (4.5) 6–7 µmSolitary, cylindrical or cylindrical-oval, rounded at the ends, smooth-walled, 32–40 × 10–16 µm
Cylindrospermum skujaeWet sandy soils or sand, sometimes among mosses, in drying pools with a sandy bottomCylindrical, rarely isodiametric mainly 1.5–2 (4) × longer as wide, 1.6–5 (7) × 2.2–3 µm, terminal conical or cylindrical and roundedTerminal, at one end only, oval, ovoid to cylindrical with rounded ends, (4.8) 6–8 × 2.8–4.5 µmSolitary or pairs or up to 3 in a row oval–cylindrical, wall smooth, (12) 14–36 × 7–11.5 µm
Cylindrospermum muscicolaWet soils, among mosses, shallow ditches and pools with water vegetationMore or less cylindrical, ± isodiametric or slightly longer or shorter than wide, terminal conical rounded or cylindrical and rounded, 3.6–5.4 (6) × 3–5.3 µmElongated, 5–7 (10.5) × (3.6) 4–5 (7) µmSolitary, oval or ovoid, rounded at ends, wall smooth, 10–20 (21) × (8.2) 9–12 µm
Cylindrospermum alatosporumSoil or guts of earthwormsCells isodiametric or longer than wide, rounded at ends, 3–7 (8) × 3.5–5.0 µmRounded cylindrical, elongated or almost spherical, 4–9 (11) × 3.5–7.0 µmSolitary or in pair, oval–rhomboid, wall smooth, 20–32 × (6.5) 10.0–13.0 (17.5) µm
Cylindrospermum licheniformePrairie remnant
soil		Cylindrical, shorter than wide to isodiametric, 3.1–5.1 × 3.6–4.8 µmTerminal, solitary, oval to elongated to bluntly conical, 5.4–9 × 4.0–5.2 µmSolitary, paraheterocytic, oval broadly to elongated, wall smooth, 13–23 × 7.0–12.4 µm
Cylindrospermum moravicumCave sedimentCylindrical, rarely concave or irregular, isodiametric, longer than wide, 2.7–5 × 3.5–7 µmTerminal, solitary, spherical to cylindrical elongated, 5.0–9.0 (11) × 2.7–6.0 µmSolitary, paraheterocytic, granulated, cylindrical, with widened end toward the heterocyte, wall wide with colourless to golden-brown, smooth, internally structured or lamellate exospores, (18) 22–32 × 9–13 µm
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MDPI and ACS Style

Kumar, N.; Saraf, A.; Pal, S.; Singh, P. Description of Cylindrospermum solincola sp. nov. from Jammu and Kashmir, India and Further Insights into the Ecological Distribution and Morphological Attributes of Cylindrospermum badium. Diversity 2023, 15, 592. https://doi.org/10.3390/d15050592

AMA Style

Kumar N, Saraf A, Pal S, Singh P. Description of Cylindrospermum solincola sp. nov. from Jammu and Kashmir, India and Further Insights into the Ecological Distribution and Morphological Attributes of Cylindrospermum badium. Diversity. 2023; 15(5):592. https://doi.org/10.3390/d15050592

Chicago/Turabian Style

Kumar, Naresh, Aniket Saraf, Sagarika Pal, and Prashant Singh. 2023. "Description of Cylindrospermum solincola sp. nov. from Jammu and Kashmir, India and Further Insights into the Ecological Distribution and Morphological Attributes of Cylindrospermum badium" Diversity 15, no. 5: 592. https://doi.org/10.3390/d15050592

APA Style

Kumar, N., Saraf, A., Pal, S., & Singh, P. (2023). Description of Cylindrospermum solincola sp. nov. from Jammu and Kashmir, India and Further Insights into the Ecological Distribution and Morphological Attributes of Cylindrospermum badium. Diversity, 15(5), 592. https://doi.org/10.3390/d15050592

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