Freshwater Algae Chara L. Species Identification by Molecular Phylogenetics

Freshwater Algae Chara L. Species Identification by Molecular Phylogenetics | 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 Freshwater Algae Chara L. Species Identification by Molecular Phylogenetics ''Rekha Adimulam’’, And P Sujathamma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8002496/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The study focused on molecular phylogeny to identify Chara species using the rbcl genes. Chara species were found in both stream and brackish waters in Tirumala and Talakona, Andhra Pradesh, India. Chara DNA was isolated with DNeasy plant Mini Kit (Quiagen, Hilden, Germany). For quality assessment of isolated DNA 1.0% agarose gel were used. Chara species were identified using BLAST with the NCBI GenBank database's 'nr' database, Clustal W multiple alignment tools, and MEGA6 software. The Chara species identified as Chara zeylanica based on the highest identity score. This study is a pioneering attempt at molecular phylogeny for the Charophyceae of India. Chara zeylanica DNA sequencing Molecular phylogenetic Phylogenetic tree and rbcl gene Figures Figure 1 Figure 2 INTRODUCTION Class Charophyceae characteristics with macroscopic algae habitat in brook and briny water, the plant body shows great elaboration in their structure (Subramanian, 2018). It is erect, long, slender green main axis with nodes and internodes. The branching axis is central, each node arises a whorl of lateral branches. Sexual reproduction Oogamous and complex in Charophyceae (Vashishta, 2020). In India the family Characeae reported 5 genera i.e. Chara , Nitella , Lychnothamnus , Nitella and Tolypella. The Genus Chara reported about 27 species from India (Gupta, 2012 ). Chara is small, simple thalli compare land plants and ease manipulation and close relation to the land plant make them attractive research models in Plant physiology, Molecular biology, biochemistry and cell cycle (David et al , 2016; Rekha et al 2023). Charophyceae algae improve the water quality, control nutrient cycle, provide the food for fish and aquatic birds so, they have ecological value. So, this Charophyceae have the importance in ecological and economical context (Barbosa et al 2021, Rekha et al 2023). Availability of taxonomical details, diversity and occurrence is important for upcoming research work (Rekha et al 2023). Identification of algae and evolutionary relationships are studies under Systematics (Braun, 1834). Identification algae done by both morphological and molecular phylogenetic studies (Akash et al 2018). In the genome short gene in uniform locality used for the molecular phylogenetic identification of the algae. DNA barcode of each alga is unlike, so molecular phylogenetic studies have importance in taxonomical and evolution studies (Alberts et al, 2002 ). In some algae similar structure of organism also shows different molecular phylogenetic studies (Lorenz, Todd, 2012 ). In morphological method Chara species without sex organs most of the cases identification not possible (Subramanian, 2018). Molecular phylogenetic technique increased accurate identification organism, help in the effective nomenclature (Nair et al, 2024 ). MATERIAL AND METHODS Sample Collection The samples are collected from water resources of Talakona and Tirumala, Andhra Pradesh, India. Samples are collected three seasons in the study area about 10 different ponds, ditches and they named them as CH1 to CH20. The Chara samples are collected with soil and water where they grow. The samples are collected with soil and water where they grow and cultured for 75 days in vivo condition. The samples were separated from other algae, thoroughly washed with the water (Zulkarnain et al , 2016). Molecular Phylogenetic Chara DNA was isolated with DNeasy plant Mini Kit (Quiagen, Hilden, Germany). For quality assessment of isolated DNA 1.0% agarose gel were used. To Amplifying the fragments of rbcL gene primers rbcL-F and rbcL-R (Marit Mjelde et al 2021) were used. DNA sequencing forward and reverse reaction of PCR amplicon carried out with rbcL-F and rbcL-R primers. To carry out this reaction BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer used for removing impurities. PCR conditions at 1cycle 94 0 c for 3 min, 30 cycles for 94 0 for 1 min, 55 0 c for 40s, 72 0 c for 55 s and 1 cycle for 72 0 c for 7 min were maintained. Online pairwise alignment tool software used for generating consensus sequence of rbcL gene done by forward and reverse sequence. To find the Similarity sequence BLAST with the ‘nr’ database of NCBI GenBank database. Clustal W software used for alignment and phylogenetic tree constricted with help of MEGA6 Software. RESULTS AND DISCUSSION Morphological Characters This Chara species are robust, grow 750 to 780 µm in width, and 6- 10 branchlet in each whorl. Bracts and bracteole are short and stipulodes occur in 2 rows. Internodes grow 1 ½ time longer than branchlet. Collection number: Talkona surrounding areas and Number: 018 Distribution: First report from Southern India (Gupta, 2012) Molecular Phylogenetic The CH2 sample agarose gel analysis revealed a single band of high molecular weight of a high-molecular DNA. A Single distinct 690bp PCR amplicon was identified. rbcl genes were generated and used for BLAST through the ‘nr’ database of NCBI GenBank. Ten sequences were selected, aligned, and the phylogenetic tree was built according to the identity score. The CH2 sample shows 99.64% homology to the Chara zeylanica. The Ident of the Sample shows highly similar to the Chara sp. NIBRCR0000100094 but query cover shows only 98%. Whereas Chara zeylanica shows 99% query cover and 99.65% Ident, so Ch2 sample were Identified as Chara zeylanica . Table: 1. Sequences alignments for homology Sequences producing significant alignments Algae Identification S.No Description Max Score Total score Query cover E value Ident Accession 1. Chara zeylanica 1040 1040 99% 0 99.65% AY720934.1 2. Chara sp. NIBRCR0000100094 1038 1038 98% 0 100.00% KT343914.1 3. Chara zeylanica isolate Seosan 1 1038 1038 98% 0 100.00% KT343913.1 4. Chara zeylanica 1037 1037 99% 0 99.65% DQ076299.1 5. Chara zeylanica strain KGK0057b 1035 1035 98% 0 100.00% HQ380468.1 6. Chara haitensis isolate KGK0199 1031 1031 98% 0 99.82% KX431011.1 7. Chara zeylanica strain KGK0403 1029 1029 98% 0 99.82% HQ380476.1 8. Chara kenoyeri strain TP118 1029 1029 98% 0 99.82% HQ380465.1 9. Chara hydropitys strain KGK0213 1029 1029 98% 0 99.82% HQ380461.1 10. Chara haitensis strain KGK0483 1029 1029 98% 0 99.82% HQ380455.1 Phylogenetic Tree DISCUSSION Taxonomical investigations based on morphology may be result in misidentification (Karan et al, 2017). To avoid this, molecular approaches have been employed. The 18s RNA, matK, ITS2, and rbcl genes are frequently used for molecular phylogenetic analysis of plants and algae (Prakash and Sanjay, 2018 ). Charophyceae are subaquatic meadows that grow in relatively quiet, clean, fresh or brackish waters. The algal thallus has a very elaborate structure. Thallus are Chara erect, long, slender, green or grey main axis with nodes and internodes. The occurrence of morphological plasticity makes it challenging to recognize Chara members. The phenotypic plasticity is happened by the environmental changes and to overcome the phenotypic plasticity, DNA barcodes (ITS2, Mat K and rbcl) areas are useful to identify the Chara species (Schneider et al , 2015). Chara samples of about 20 are collected in the Tirumala and Talakona areas. These samples are then analyzed to assess their genetic diversity and ecological adaptations. The samples are morphologically dissimilar from different ponds and ditches. Some are stout, some are long, some are light green and thick green. Few samples show the reproductive parts, the remaining are without reproductive parts collected in the three seasons. Without sexual organs, the identification is difficult. Due to morphological plasticity and the absence of sexual organs in these samples, I have carried out molecular phylogenetics. MC Court et al . (1996) used eleven morphological characters and rbcl sequence data to reconstruct the phylogeny of the genus Chara. Monique Turmel et al. ( 2006 ) investigated the atpB, rbcl, 18S rRNA, and mitochondria of Chara vulgaris and concluded that Charales stay sister to the green plants of land. Hidetoshi (2009) identified Chara globularis using morphological and molecular phylogeny based on rbcl gene sequencing. Schneider et al. (2015) studied the rbcl, ITS2, SNP, MATK, and 18s rRNA markers used to identify species divergence in the Chara genus. Jacek and Michal ( 2017 ) investigated the atpB, matK, and rbcl sequences of Chara globularis var. tenuispina and determined that it is a distinct species from Chara globularis. The rbcl markers greatly rises the cost of sequencing the locus of barcode, it ideal for the Charophytes species level variability and sometime this marker alone not ideal for the identification (Hall,2010). The rbcl genes shares mutual codon for closely related species (Akash et al , 2018). So, rbcl based technologies can enhances efficacy of the algae identification and evolutionary relationship (Pheravut and Ywadee, 2014). These studies supported that rbcl genes and markers revealed the exact identification of Chara species through molecular phylogenetics. However, both molecular and morphological data together perfect for identification, when it separates this tool are not ideal (Sakayama 2008 ). From India total 10 sequence of Chara Zeyalnica are in the NCBI, which are identified with Maturase K genes 541 bp linear DNA (Balakrishnan and Rani, 2020). This study is a pioneering attempt at molecular phylogeny for the Charophyceae of India. CONCLUSION Chara species have been identified as Chara zeylanica based on molecular phylogenetic studies. To identify the rbcL gene sequence, BLAST was performed against the NCBI GenBank 'nr' database. The first 10 sequences were selected based on the highest identity score and then aligned using the multiple alignment software application Clustal W. MEGA6 was used to generate the phylogenetic tree. This study is a pioneering effort at molecular phylogeny for the Charophyceae of India. Declarations CONFLICT OF INTEREST The authors declare that there is no conflict of interest regarding the publication of this paper. Author Contribution ''Dr. Rekha Adimulam' corresponding author- wrote the conducted and wrote the manuscript.Prof P. Sujathamma guided and reviewed the manuscript Acknowledgments The authors gratefully acknowledge that the funding for this publication was provided by the Pradhan Mantri Uchchatar Shiksha Abhiyan (PM-USHA) , under the Multi-Disciplinary Education and Research Universities (MERU) Grant sanctioned to Sri Padmavati Mahila Visvavidyalayam, Tirupati . References Alberts B Johnson and A Lewis J, Molecular Biology of the Cell. Garland Science; Studying Gene Expression and Function, Garland Science, New York, (2002),https://www.ncbi.nlm.nih.gov/books/NBK26818/ . Balakrishnan Swetha and Rani Govindarajan, Environmental and Experimental Biology, 18: 169–174,(2020), http://doi.org/10.22364/eeb.18.17. Braun .A, Esquissemonographique du genre Chara . Ann. Sci. Nat., sér. II 1: 349-357, (1834). David. D, Iben.S and Zoe.P, Charophytes: Evolutionary Ancestors of plants and Emerging models for plant research, Frontiers, USA, P:1-5, (2017). David. D, Zoe.P and Iben.S, Charophytes: Evolutinory gaints and emerging model organisam, Frontiers, USA, P:6-10, (2016). Fawley MW and Fawley KP., Identification of Eukaryotic Microalgal Strains. Journal of Applied Phycology 32(5):2699-2709, (2020). doi: 10.1007/s10811-020-02190-5. Gupta, Algae of India Vol.2. Achecklist of Chlorophyceae, Xanthophyceae, Chryophyceae and Euglenophyceae, Botanical survey of India, Kolkata, 9-331, (2012). Hall, John & Fučíková, Karolina & Lo, Chien& Lewis, Louise & Karol, Kenneth, An assessment of proposed DNA barcodes in freshwater green algae. Cryptogamie Algologie, 31, 529-555, (2011). Sakayama H, Kasai F, Nozaki H, Watanabe MM, Kawachi M, Shigyo M, Nishihiro J, Washitani I, Krienitz L, Taxonomic re-examination of chara globularis (charales, charophyceae) from japan based on oospore morphology and rbcl gene sequences, and the description of c. leptospora sp, Journal of Phycology, 45: 917-927, (2009), DOI.org/10.1111/j.1529-8817.2009.00700.x. Jacek U and Hidetoshi S, Taxonomical analysis of closely related species of Chara L. Section Hartmania (Streptophyta: Charles ) based on morphological and molecular data, Fottea, 17:222-239, (2017), DOI: 10.5507/fot.2017.004. Jacek U and Michal C, Taxonomical status of Chratenupina A. Br. Based on LM morphology, matK, atpB, and rbcl of cpDNA sequences, Fottea, 17:20-33, (2017) DOI:10.5507/fot.2016.011. Karan, Erenler, Altunar, Isolation and Molecular identification of some blue green algae (cyanobacteria) from Fresh water sites in Tokat Province Turkey, Turkish Journal of Agriculture - Food Science and Technology, 5:1371-1378, (2017) doi: doi.org./10.24925/turjaf.v5i11.1371-1378.1470. Lorenz and Todd, Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies, Journal of visualized experiments , 63,(2012), doi:10.3791/3998. MaritMjelde, ThidaSwe, Anders Langangen& Andreas Ballot, A contribution to the knowledge of charophytes in Myanmar; morphological and genetic identification and ecology notes, Botany Letters, 168:1, 102-109, (2021), DOI: 10.1080/23818107.2020.1847189. McCourt, R. (1996). Phylogeny of extant genera in the family Characeae (Charales, Charophyceae) based on rbcL sequences and morphology. American Journal of Botany. Nair, Divya & Aseema, Pareeda & Saini, Khem. (2024). DNA barcoding: An effective molecular tool for species identification, molecular authentication and phylogeny studies in plant science research. Plant Science Today. 10.14719/pst.3183. Patel, Akash & Chaudhary, Spandan & Syed, Bakhtiyar & Gami, Bharat & Patel, Pankaj & Patel, Beena, Rbcl Marker Based Approach for Molecular Identification of Arthrospira and Dunaliella Isolates from Non-Axenic Cultures. 2. 24-34. Rbcl Marker based approach for molecular identification of Arthrospira and Dunaliella isolates from non- Axenic culture, Sryahwa publication, 2:24-34, (2018). Pheravut W and YwadeeP Molecular identification and phylogenetic relationship of green algae, Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcl markers, Saudi journals of Biological Sciences, 21:505-510, (2014), https://doi.org/ 10.1016/j.sjbs.2014.01.003. Prakash S and Sanjay K.V, Identification and characterization of green microalgae, Scenedesmus Sp. and Auctodesmus obliquus MCC33 isolated from Industrial polluted site using morphological and molecular markers, International Journal of Applied Sciences and Biotechnology, 5: 415-422,(2018), doi.10.3126/ijasbt.v5i4.18083. Rekha Adimulam and Sujathamma, P, 18S rRNA Approach for Identification of Chara L. Species. Biosciences Biotechnology Research Asia. 20. 1039-1045, (2023), 10.13005/bbra/3154. Sakayama H Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material. Phycological Research 56(3): 202–215, (2008), https://doi.org/10.1111/j.1440-1835.2008.00502.x Susanne C. Schneider, Susanne C. Schneider, Anuar Rodrigues, Therese Fosholt Moe, Andreas Ballot, DNA barcoding the genus Chara : Molecular evidence recovers fewer taxa than the classical morphology approach, Journal of Phycology, 51:367-380, (2015), doi: 10.1111/jpy.12282 Subramanian, Indian Charophyta, MJP Publishers, Chennai, 45- 168, (2018). Tamura K. and Nei M., Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512-526, (1993). Tamura K.,Stecher G., and Kumar S., MEGA 11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution, (2021), https://doi.org/10.1093/molbev/msab120. Turmel M, Christian O, Claude L, The Chloroplast genome sequence of Chara vulgaris Sheds new light into the closet green algal relatives of land plants, Molecular biology and evolution, 23: 1324-1338, (2006), doi:10.1093/molbev/msk018. Vashishta (1960), Algae, S Chand and Company Limited, 324-325. Wongsawad P and Peerapornpisal Y., Molecular identification and phylogenetic relationship of green algae, Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers. Saudi Journal of Biological Sciences, 21(5):505-510, (2014), doi: 10.1016/j.sjbs.2014.01.003. Zulkarnain C,Neri Fadjria Armaini and Rahadian ZainulIsolation and molecular identification of Fresh water microalgae in Maninjau lake West Sumatra, Der Pharmacia Lettre, 8:177-187, (2016). Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":493112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChara zeylanica \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e160Pixel =4.23cm\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8002496/v1/0ee7ceb3891bb94c5b680a69.png"},{"id":95859502,"identity":"6b1ec2ff-7e10-48ff-aa4b-fb542e5a29a9","added_by":"auto","created_at":"2025-11-13 17:31:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular Phylogenetic analysis by Maximum Likelihood method.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8002496/v1/3b122454f3ac5e9973000198.png"},{"id":96362852,"identity":"1189b583-9483-46bd-80ce-44c04e03ffc1","added_by":"auto","created_at":"2025-11-20 10:01:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8002496/v1/f6b029ff-1220-4a2c-84a6-451cb030c607.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFreshwater Algae Chara L. Species Identification by Molecular Phylogenetics\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eClass Charophyceae characteristics with macroscopic algae habitat in brook and briny water, the plant body shows great elaboration in their structure (Subramanian, 2018). It is erect, long, slender green main axis with nodes and internodes. The branching axis is central, each node arises a whorl of lateral branches. Sexual reproduction Oogamous and complex in Charophyceae (Vashishta, 2020). In India the family Characeae reported 5 genera i.e. \u003cem\u003eChara\u003c/em\u003e, \u003cem\u003eNitella\u003c/em\u003e, \u003cem\u003eLychnothamnus\u003c/em\u003e, \u003cem\u003eNitella\u003c/em\u003e and \u003cem\u003eTolypella.\u003c/em\u003e The Genus \u003cem\u003eChara\u003c/em\u003e reported about 27 species from India (Gupta, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eChara\u003c/em\u003e is small, simple thalli compare land plants and ease manipulation and close relation to the land plant make them attractive research models in Plant physiology, Molecular biology, biochemistry and cell cycle (David \u003cem\u003eet al\u003c/em\u003e, 2016; Rekha \u003cem\u003eet al\u003c/em\u003e 2023). Charophyceae algae improve the water quality, control nutrient cycle, provide the food for fish and aquatic birds so, they have ecological value. So, this Charophyceae have the importance in ecological and economical context (Barbosa \u003cem\u003eet al\u003c/em\u003e 2021, Rekha \u003cem\u003eet al\u003c/em\u003e 2023). Availability of taxonomical details, diversity and occurrence is important for upcoming research work (Rekha \u003cem\u003eet al\u003c/em\u003e 2023).\u003c/p\u003e\u003cp\u003eIdentification of algae and evolutionary relationships are studies under Systematics (Braun, 1834). Identification algae done by both morphological and molecular phylogenetic studies (Akash et al 2018). In the genome short gene in uniform locality used for the molecular phylogenetic identification of the algae. DNA barcode of each alga is unlike, so molecular phylogenetic studies have importance in taxonomical and evolution studies (Alberts et al, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn some algae similar structure of organism also shows different molecular phylogenetic studies (Lorenz, Todd, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In morphological method Chara species without sex organs most of the cases identification not possible (Subramanian, 2018). Molecular phylogenetic technique increased accurate identification organism, help in the effective nomenclature (Nair et al, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample Collection\u003c/h2\u003e\u003cp\u003eThe samples are collected from water resources of Talakona and Tirumala, Andhra Pradesh, India. Samples are collected three seasons in the study area about 10 different ponds, ditches and they named them as CH1 to CH20. The Chara samples are collected with soil and water where they grow. The samples are collected with soil and water where they grow and cultured for 75 days in vivo condition. The samples were separated from other algae, thoroughly washed with the water (Zulkarnain \u003cem\u003eet al\u003c/em\u003e, 2016).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular Phylogenetic\u003c/h3\u003e\n\u003cp\u003eChara DNA was isolated with DNeasy plant Mini Kit (Quiagen, Hilden, Germany). For quality assessment of isolated DNA 1.0% agarose gel were used. To Amplifying the fragments of rbcL gene primers rbcL-F and rbcL-R (Marit Mjelde \u003cem\u003eet al\u003c/em\u003e 2021) were used. DNA sequencing forward and reverse reaction of PCR amplicon carried out with rbcL-F and rbcL-R primers. To carry out this reaction BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer used for removing impurities. PCR conditions at 1cycle 94\u003csup\u003e0\u003c/sup\u003ec for 3 min, 30 cycles for 94\u003csup\u003e0\u003c/sup\u003e for 1 min, 55\u003csup\u003e0\u003c/sup\u003e c for 40s, 72 \u003csup\u003e0\u003c/sup\u003e c for 55 s and 1 cycle for 72\u003csup\u003e0\u003c/sup\u003e c for 7 min were maintained.\u003c/p\u003e\u003cp\u003eOnline pairwise alignment tool software used for generating consensus sequence of rbcL gene done by forward and reverse sequence. To find the Similarity sequence BLAST with the \u0026lsquo;nr\u0026rsquo; database of NCBI GenBank database. Clustal W software used for alignment and phylogenetic tree constricted with help of MEGA6 Software.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eMorphological Characters\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis \u003cem\u003eChara\u0026nbsp;\u003c/em\u003especies are robust, grow 750 to 780 \u0026micro;m in width, and 6- 10 branchlet in each whorl. \u0026nbsp;Bracts and bracteole are short and stipulodes occur in 2 rows. Internodes grow 1 \u0026frac12; time longer than branchlet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollection number: Talkona surrounding areas and Number: 018\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDistribution: First report from Southern India \u0026nbsp; (Gupta, 2012)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Phylogenetic\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CH2 sample agarose gel analysis revealed a single band of high molecular weight of a high-molecular DNA. A Single distinct 690bp PCR amplicon was identified. rbcl genes were generated and used for BLAST through the \u0026lsquo;nr\u0026rsquo; database of NCBI GenBank. Ten sequences were selected, aligned, and the phylogenetic tree was built according to the identity score.\u0026nbsp;The CH2 sample shows 99.64% homology to the \u003cem\u003eChara zeylanica. \u0026nbsp;\u003c/em\u003eThe Ident of the Sample shows highly similar to the\u0026nbsp;\u003cem\u003eChara\u003c/em\u003e sp. NIBRCR0000100094 but query cover shows only 98%. Whereas \u003cem\u003eChara zeylanica\u003c/em\u003e shows 99% query cover and 99.65% Ident, so Ch2 sample were Identified as \u003cem\u003eChara zeylanica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable: 1. Sequences alignments for homology\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"616\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"bottom\" style=\"width: 414px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequences producing significant alignments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlgae Identification\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMax Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eQuery cover\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eE value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara zeylanica\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1040\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1040\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.65%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAY720934.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003esp. NIBRCR0000100094\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1038\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1038\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKT343914.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara zeylanica\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;isolate Seosan 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1038\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1038\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKT343913.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara zeylanica\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1037\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1037\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.65%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDQ076299.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara zeylanica\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain KGK0057b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1035\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1035\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHQ380468.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara haitensis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eisolate KGK0199\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1031\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1031\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKX431011.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara zeylanica\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain KGK0403\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHQ380476.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara kenoyeri\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain TP118\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHQ380465.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara hydropitys\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain KGK0213\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHQ380461.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eChara haitensis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003estrain KGK0483\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1029\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHQ380455.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Tree\u003c/strong\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTaxonomical investigations based on morphology may be result in misidentification (Karan et al, 2017). To avoid this, molecular approaches have been employed. The 18s RNA, matK, ITS2, and rbcl genes are frequently used for molecular phylogenetic analysis of plants and algae (Prakash and Sanjay, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCharophyceae are subaquatic meadows that grow in relatively quiet, clean, fresh or brackish waters. The algal thallus has a very elaborate structure. Thallus are Chara erect, long, slender, green or grey main axis with nodes and internodes.\u003c/p\u003e\u003cp\u003eThe occurrence of morphological plasticity makes it challenging to recognize Chara members. The phenotypic plasticity is happened by the environmental changes and to overcome the phenotypic plasticity, DNA barcodes (ITS2, Mat K and rbcl) areas are useful to identify the \u003cem\u003eChara\u003c/em\u003e species (Schneider \u003cem\u003eet al\u003c/em\u003e, 2015).\u003c/p\u003e\u003cp\u003eChara samples of about 20 are collected in the Tirumala and Talakona areas. These samples are then analyzed to assess their genetic diversity and ecological adaptations. The samples are morphologically dissimilar from different ponds and ditches. Some are stout, some are long, some are light green and thick green. Few samples show the reproductive parts, the remaining are without reproductive parts collected in the three seasons. Without sexual organs, the identification is difficult. Due to morphological plasticity and the absence of sexual organs in these samples, I have carried out molecular phylogenetics.\u003c/p\u003e\u003cp\u003eMC Court \u003cem\u003eet al\u003c/em\u003e. (1996) used eleven morphological characters and rbcl sequence data to reconstruct the phylogeny of the genus Chara. Monique Turmel et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) investigated the atpB, rbcl, 18S rRNA, and mitochondria of Chara vulgaris and concluded that Charales stay sister to the green plants of land. Hidetoshi (2009) identified Chara globularis using morphological and molecular phylogeny based on rbcl gene sequencing. Schneider et al. (2015) studied the rbcl, ITS2, SNP, MATK, and 18s rRNA markers used to identify species divergence in the Chara genus. Jacek and Michal (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) investigated the atpB, matK, and rbcl sequences of Chara globularis var. tenuispina and determined that it is a distinct species from Chara globularis.\u003c/p\u003e\u003cp\u003eThe rbcl markers greatly rises the cost of sequencing the locus of barcode, it ideal for the Charophytes species level variability and sometime this marker alone not ideal for the identification (Hall,2010). The rbcl genes shares mutual codon for closely related species (Akash \u003cem\u003eet al\u003c/em\u003e, 2018). So, rbcl based technologies can enhances efficacy of the algae identification and evolutionary relationship (Pheravut and Ywadee, 2014). These studies supported that rbcl genes and markers revealed the exact identification of \u003cem\u003eChara\u003c/em\u003e species through molecular phylogenetics. However, both molecular and morphological data together perfect for identification, when it separates this tool are not ideal (Sakayama \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom India total 10 sequence of \u003cem\u003eChara Zeyalnica\u003c/em\u003e are in the NCBI, which are identified with Maturase K genes 541 bp linear DNA (Balakrishnan and Rani, 2020). This study is a pioneering attempt at molecular phylogeny for the Charophyceae of India.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eChara species have been identified as Chara zeylanica based on molecular phylogenetic studies. To identify the rbcL gene sequence, BLAST was performed against the NCBI GenBank 'nr' database. The first 10 sequences were selected based on the highest identity score and then aligned using the multiple alignment software application Clustal W. MEGA6 was used to generate the phylogenetic tree. This study is a pioneering effort at molecular phylogeny for the Charophyceae of India.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e''Dr. Rekha Adimulam' corresponding author- wrote the conducted and wrote the manuscript.Prof P. Sujathamma guided and reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge that the funding for this publication was provided by the \u003cb\u003ePradhan Mantri Uchchatar Shiksha Abhiyan (PM-USHA)\u003c/b\u003e, under the \u003cb\u003eMulti-Disciplinary Education and Research Universities (MERU) Grant\u003c/b\u003e sanctioned to Sri \u003cb\u003ePadmavati Mahila Visvavidyalayam, Tirupati\u003c/b\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlberts B Johnson and A Lewis J, Molecular Biology of the Cell. Garland Science; Studying Gene Expression and Function, Garland Science, New York, (2002),https://www.ncbi.nlm.nih.gov/books/NBK26818/\u003cu\u003e.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eBalakrishnan Swetha and Rani Govindarajan, Environmental and Experimental Biology, 18: 169–174,(2020), http://doi.org/10.22364/eeb.18.17.\u003c/li\u003e\n \u003cli\u003eBraun .A, Esquissemonographique du genre \u003cem\u003eChara\u003c/em\u003e. \u003cem\u003eAnn. Sci. Nat., sér. \u003c/em\u003e\u003cem\u003eII\u003c/em\u003e 1: 349-357, (1834).\u003c/li\u003e\n \u003cli\u003eDavid. D, Iben.S and Zoe.P, Charophytes: Evolutionary Ancestors of plants and Emerging models for plant research, Frontiers, USA, P:1-5, (2017).\u003c/li\u003e\n \u003cli\u003eDavid. D, Zoe.P and Iben.S, Charophytes: Evolutinory gaints and emerging model organisam, Frontiers, USA, P:6-10, (2016).\u003c/li\u003e\n \u003cli\u003eFawley MW and Fawley KP., Identification of Eukaryotic Microalgal Strains. Journal of Applied Phycology 32(5):2699-2709, (2020). \u003cu\u003edoi: 10.1007/s10811-020-02190-5. \u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eGupta, Algae of India Vol.2. Achecklist of Chlorophyceae, Xanthophyceae, Chryophyceae and Euglenophyceae, Botanical survey of India, Kolkata, 9-331, (2012).\u003c/li\u003e\n \u003cli\u003eHall, John \u0026amp; Fučíková, Karolina \u0026amp; Lo, Chien\u0026amp; Lewis, Louise \u0026amp; Karol, Kenneth, An assessment of proposed DNA barcodes in freshwater green algae. Cryptogamie Algologie, 31, 529-555, (2011).\u003c/li\u003e\n \u003cli\u003eSakayama H, Kasai F, Nozaki H, Watanabe MM, Kawachi M, Shigyo M, Nishihiro J, Washitani I, Krienitz L, Taxonomic re-examination of chara globularis (charales, charophyceae) from japan based on oospore morphology and rbcl gene sequences, and the description of c. leptospora sp, Journal of Phycology, 45: 917-927, (2009), \u003cu\u003eDOI.org/10.1111/j.1529-8817.2009.00700.x.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eJacek U and Hidetoshi S, Taxonomical analysis of closely related species of \u003cem\u003eChara\u003c/em\u003eL. Section Hartmania (Streptophyta: Charles ) based on morphological and molecular data, Fottea, 17:222-239, (2017), \u003cu\u003eDOI: 10.5507/fot.2017.004.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eJacek U and Michal C, Taxonomical status of \u003cem\u003eChratenupina\u003c/em\u003e A. Br. Based on LM morphology, matK, atpB, and rbcl of cpDNA sequences, Fottea, 17:20-33, (2017) \u003cu\u003eDOI:10.5507/fot.2016.011.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eKaran, Erenler, Altunar, Isolation and Molecular identification of some blue green algae (cyanobacteria) from Fresh water sites in Tokat Province Turkey, Turkish Journal of Agriculture - Food Science and Technology, 5:1371-1378, (2017) \u003cu\u003edoi: doi.org./10.24925/turjaf.v5i11.1371-1378.1470.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eLorenz and Todd, Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies, Journal of visualized experiments\u003cem\u003e, \u003c/em\u003e63,(2012), \u003cu\u003edoi:10.3791/3998.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eMaritMjelde, ThidaSwe, Anders Langangen\u0026amp; Andreas Ballot, A contribution to the knowledge of charophytes in Myanmar; morphological and genetic identification and ecology notes, Botany Letters, 168:1, 102-109, (2021), DOI: \u003cu\u003e10.1080/23818107.2020.1847189.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eMcCourt, R. (1996). Phylogeny of extant genera in the family Characeae (Charales, Charophyceae) based on rbcL sequences and morphology. American Journal of Botany.\u003c/li\u003e\n \u003cli\u003eNair, Divya \u0026amp; Aseema, Pareeda \u0026amp; Saini, Khem. (2024). DNA barcoding: An effective molecular tool for species identification, molecular authentication and phylogeny studies in plant science research. Plant Science Today. 10.14719/pst.3183.\u003c/li\u003e\n \u003cli\u003ePatel, Akash \u0026amp; Chaudhary, Spandan \u0026amp; Syed, Bakhtiyar \u0026amp; Gami, Bharat \u0026amp; Patel, Pankaj \u0026amp; Patel, Beena, Rbcl Marker Based Approach for Molecular Identification of Arthrospira and Dunaliella Isolates from Non-Axenic Cultures. 2. 24-34. Rbcl Marker based approach for molecular identification of \u003cem\u003eArthrospira \u003c/em\u003eand \u003cem\u003eDunaliella \u003c/em\u003eisolates from non- Axenic culture, Sryahwa publication, 2:24-34, (2018).\u003c/li\u003e\n \u003cli\u003ePheravut W and YwadeeP Molecular identification and phylogenetic relationship of green algae, \u003cem\u003eSpirogyra ellipsospora\u003c/em\u003e (Chlorophyta) using ISSR and rbcl markers, Saudi journals of Biological Sciences, 21:505-510, (2014), https://doi.org/ 10.1016/j.sjbs.2014.01.003.\u003c/li\u003e\n \u003cli\u003ePrakash S and Sanjay K.V, Identification and characterization of green microalgae, \u003cem\u003eScenedesmus\u003c/em\u003e Sp. and \u003cem\u003eAuctodesmus obliquus\u003c/em\u003e MCC33 isolated from Industrial polluted site using morphological and molecular markers, International Journal of Applied Sciences and Biotechnology, 5: 415-422,(2018), \u003cu\u003edoi.10.3126/ijasbt.v5i4.18083.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eRekha Adimulam and Sujathamma, P, 18S rRNA Approach for Identification of Chara L. Species. Biosciences Biotechnology Research Asia. 20. 1039-1045, (2023), 10.13005/bbra/3154.\u003c/li\u003e\n \u003cli\u003eSakayama H Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material. Phycological Research 56(3): 202–215, (2008), \u003cu\u003ehttps://doi.org/10.1111/j.1440-1835.2008.00502.x\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eSusanne C. Schneider, Susanne C. Schneider, Anuar Rodrigues, Therese Fosholt Moe, Andreas Ballot, DNA barcoding the genus \u003cem\u003eChara\u003c/em\u003e: Molecular evidence recovers fewer taxa than the classical morphology approach, Journal of Phycology, 51:367-380, (2015), \u003cu\u003edoi: 10.1111/jpy.12282\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eSubramanian, Indian Charophyta, MJP Publishers, Chennai, 45- 168, (2018).\u003c/li\u003e\n \u003cli\u003eTamura K. and Nei M., Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512-526, (1993).\u003c/li\u003e\n \u003cli\u003eTamura K.,Stecher G., and Kumar S., MEGA 11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution, (2021), https://doi.org/10.1093/molbev/msab120. \u003c/li\u003e\n \u003cli\u003eTurmel M, Christian O, Claude L, The Chloroplast genome sequence of \u003cem\u003eChara vulgaris\u003c/em\u003e Sheds new light into the closet green algal relatives of land plants, Molecular biology and evolution, 23: 1324-1338, (2006), \u003cu\u003edoi:10.1093/molbev/msk018.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eVashishta (1960), Algae, S Chand and Company Limited, 324-325.\u003c/li\u003e\n \u003cli\u003eWongsawad P and Peerapornpisal Y., Molecular identification and phylogenetic relationship of green algae, Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers. Saudi Journal of Biological Sciences, 21(5):505-510, (2014), \u003cu\u003edoi: 10.1016/j.sjbs.2014.01.003.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eZulkarnain C,Neri Fadjria Armaini and Rahadian ZainulIsolation and molecular identification of Fresh water microalgae in Maninjau lake West Sumatra, Der Pharmacia Lettre, 8:177-187, (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chara zeylanica, DNA sequencing, Molecular phylogenetic, Phylogenetic tree and rbcl gene","lastPublishedDoi":"10.21203/rs.3.rs-8002496/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8002496/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study focused on molecular phylogeny to identify Chara species using the rbcl genes. Chara species were found in both stream and brackish waters in Tirumala and Talakona, Andhra Pradesh, India. Chara DNA was isolated with DNeasy plant Mini Kit (Quiagen, Hilden, Germany). For quality assessment of isolated DNA 1.0% agarose gel were used. Chara species were identified using BLAST with the NCBI GenBank database's 'nr' database, Clustal W multiple alignment tools, and MEGA6 software. The Chara species identified as \u003cem\u003eChara zeylanica\u003c/em\u003e based on the highest identity score. This study is a pioneering attempt at molecular phylogeny for the Charophyceae of India.\u003c/p\u003e","manuscriptTitle":"Freshwater Algae Chara L. 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