Genetic Profiling and Taxonomic Authentication of Indian Phyllanthus (Phyllanthaceae: Phyllantheae) Genus through Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions: A Comprehensive Analysis

Genetic Profiling and Taxonomic Authentication of Indian Phyllanthus (Phyllanthaceae: Phyllantheae) Genus through Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions: A Comprehensive Analysis | 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 Genetic Profiling and Taxonomic Authentication of Indian Phyllanthus (Phyllanthaceae: Phyllantheae) Genus through Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions: A Comprehensive Analysis P Raghavendra, Pushpalatha Ganesh, Gururaj Chalageri, R Kannan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3893650/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 Phyllanthus is a genus of plants that are both ecologically and medicinally valuable. This diversity highlights the need for accurate identification in order to support both conservation efforts and medical research. The escalating demand for Phyllanthus-derived herbal products raises concerns regarding market adulteration and misidentification. In response, our study employs DNA barcoding, specifically targeting the internal transcribed spacer 2 (ITS2) region, to authenticate Indian Phyllanthus species. The study underscores the ITS2 region's efficacy in identifying Indian Phyllanthus species, demonstrating substantial advancements in resolving genus relationships compared to prior analyses. To check if our plant DNA matches known ones, we used two tools: NCBI BLASTn and the ITS2 database. The results showed really high similarities, ranging from 98–100%. This helps us understand how closely related our plant is to others in the Phyllanthus family. We deposited the genetic data, particularly DNA sequences, of Phyllanthus plants into the NCBI GenBank repository. The construction of a phylogenetic tree through multiple sequence alignment of the ITS2 gene confirms clustering among Phyllanthus species, illuminating genetic relationships and diversity crucial for conservation. The ribosomal nuclear ITS2 region exhibits notable differences within and between species, validated by DNA barcodes and secondary structure analyses using minimum free energy calculations. This study underscores the effectiveness of ITS2-based DNA barcoding in accurately identifying Phyllanthus species, mitigating adulteration concerns, ensuring product quality, preserving biodiversity, and promoting sustainable utilization of these invaluable plant resources. Adulteration DNA barcode Genetic relationships ITS2 region ITS2 database NCBI BLASTn Phyllanthus species Phylogenetic tree Secondary structures Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The genus Phyllanthus, belonging to the Phyllanthaceae family, holds ecological and medicinal significance in India, known for its rich biodiversity. India is home to numerous Phyllanthus species, with around 800 recognized within the genus. Traditional medicine systems like Ayurveda have extensively utilized Phyllanthus species for their therapeutic properties, prompting scientific exploration (Mishra et al. 2011 ). Scientific investigations have unveiled diverse bioactive compounds within Phyllanthus species, including alkaloids, flavonoids, lignans, tannins, and phenolic compounds. These compounds exhibit various pharmacological activities, such as antimicrobial, antioxidant, anti-inflammatory, hepatoprotective, antidiabetic, immunomodulatory, and anticancer effects. In Asia, these plants have been traditionally employed in ayurvedic medicine for various ethnopharmacological purposes, including treating conditions such as astringency, abdominal pain, diuresis, fever, deobstruction, and as antiseptics. They are also utilized in the treatment of ailments related to digestion, genitourinary, respiratory, skin, hepatopathy, jaundice, and renal calculus (Kiran et al., 2021 ).Certain Phyllanthus species, in particular, have been studied for their hepatoprotective properties and their potential in treating liver disorders (Yang et al. 2020 ). Beyond medicinal uses, some Phyllanthus species have economic value as sources of timber, fiber, dye, ornamental plants, or edible fruits (Mishra et al. 2011 ). However, the popularity of Phyllanthus products in traditional medicine and other industries raises concerns about the adulteration of herbal preparations. Adulteration compromises the efficacy and safety of herbal products, hindering accurate taxonomic and pharmacological studies (Bhattarai et al. 2019 ). Adulteration can occur at various stages of the supply chain, involving the substitution of authentic plant materials with similar-looking but different species or unrelated plant materials. This introduces inconsistencies in therapeutic outcomes and challenges the accuracy of taxonomic and pharmacological studies, leading to misidentified or substituted samples (Bhattarai et al. 2019 ). To address this concern, reliable authentication methods, such as DNA barcoding, have been proposed. DNA barcoding, specifically targeting the ITS2 region, provides standardized and robust identification, less influenced by external factors (Hollingsworth et al. 2011). The ITS2 region, a non-coding DNA region between the 5.8S and 28S ribosomal RNA genes, serves as a unique molecular signature for different plant species. In the context of plant adulteration, ITS2 can distinguish between closely related plant species, aiding in the detection of unauthorized or fraudulent inclusion of plant material in food products. This approach ensures the authenticity and quality of botanical ingredients in various products like herbal supplements, teas, or food items. The ITS2 region's significance lies in its ability to contribute to accurate and reliable species identification, essential for addressing issues related to food fraud and adulteration. It is applicable to all plant life stages and processed materials, making it suitable for species authentication of herbal products (Newmaster et al. 2008 ). DNA barcoding has proven valuable in detecting potential adulterants and ensuring the quality and authenticity of herbal products (Stoeckle et al. 2004). The herbal commodity market is experiencing significant growth, driven by the perception that traditional medicine is inherently safer and conducive to promoting overall health and sustainable lifestyles. However, challenges such as the scarcity of authentic resources and a deficiency in taxonomic knowledge have led to an increase in cases of economically motivated or unintentional adulterations and substitutions (Raclariu et al. 2018a ). To safeguard consumers and ensure the integrity of herbal products, rigorous pharmacovigilance measures are imperative. However, existing regulatory guidelines on medicinal plants often lack clarity, varying significantly from one nation to another. Addressing this research gap requires regulatory bodies to adopt more dependable, universally applicable, and robust detection methods (Shetti et al. 2011 ). Such measures are essential to ensure the authenticity of herbal products and the safety of those who rely on them for their health and well-being. In this study, we aim to employ DNA barcoding techniques, specifically targeting the nrITS2 region, to molecularly identify Phyllanthus species. This approach offers a reliable solution to combat adulteration, ensuring the authenticity of herbal products and contributing significantly to taxonomic and pharmacological research. DNA barcoding provides a robust tool for accurate species identification, crucial for biodiversity conservation, evidence-based herbal medicine development, and sustainable Phyllanthus species utilization in India. Materials and Methods Collection of Plant Materials Mature plants of five Phyllanthus species, namely Phyllanthus maderaspatensis, Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus virgatus , and Phyllanthus tenellus , were systematically collected from diverse locations across India, with a keen focus on selecting healthy, fresh, and succulent plant parts (Fig. 1 ). To ensure the utmost accuracy in identification and authentication, the collected specimens underwent meticulous examination and verification at the Herbaria of the Himalaya Wellness, Bengaluru, a trusted repository of plant specimens housing authenticated reference materials for scientific purposes. This rigorous procedure guaranteed the researchers a source of reliable and authentic plant materials, underscoring the importance of adhering to stringent identification and authentication protocols to uphold the integrity and scientific validity of studies conducted on Phyllanthus species. Phyllanthus DNA Isolation and analysis DNA extraction was carried out utilizing the NucleoSpin® Plant II DNA Kit from MACHEREY-NAGEL, Germany, following the prescribed protocols. Initially, 2 grams of leaf samples were finely ground into a powder using liquid nitrogen. The finely ground material was then mixed meticulously with 500µl of lysis buffer (PL2), and 10µl of RnaseA solution was added. Subsequently, the mixture underwent a 45-minute incubation at 65°C in a water bath. After the incubation, 75µl of PL3 SDS precipitate buffer (potassium acetate) was introduced, thoroughly mixed, and left to incubate for 5 minutes at 4°C to induce SDS precipitation. A Nucleospin filter (violet ring) was placed within a fresh collection tube, and the lysate was transferred onto the column, followed by centrifugation at 11,000rpm for 2 minutes. Following this step, 450µl of precipitation buffer (PC) was mixed in, and the process was repeated using a Nucleospin Plant II column (green ring) in a new collection tube (2ml). In this step, 700µl of the sample was centrifuged, and the flow-through was discarded. Subsequently, washing buffer (PW1) and washing buffer (PW2) were introduced to the column. The column underwent a 2-minute centrifugation at 11,000rpm to ensure thorough drying of the silica membrane. The dried column (Green ring) was then placed in a fresh 1.5mL microcentrifuge tube, and 50µl of elution buffer (PE) was pipetted onto the membrane. The column was incubated for 5 minutes at 65°C and centrifuged for 1 minute at 11,000rpm for DNA elution. This elution step was repeated with another 50µl of PE buffer into the same tube. For qualitative analysis, 5 µL of the isolated DNA sample was combined with 5 µL of 6X loading dye (Himedia) and loaded onto a 0.8% agarose gel alongside a 1 kb DNA ladder. Electrophoresis was conducted at 100 V for 1 hour, and the gel was visualized using a UV Gel documentation unit (VilberLourmat, France) (Sambrook and Russel 2011). Quantification of the DNA sample was performed using a NanoDrop One (Thermo Scientific) spectrophotometer. For this, 2 µL of the DNA sample was placed on the instrument's pedestal, and OD readings were taken at 260/280nm. PCR Amplification of ITS2 Region The selected barcode candidates, nrITS2 loci, were used for PCR amplification, employing universal primers, including Forward primer ITS2-S2F-5’– ATGCGATACTTGGTGTGAAT-3’ (Chen et al. 2010 ) and Reverse primer ITS4R-5’-’ TCCTCCGCTTATTGATATGC-3’ (White et al. 1990). The PCR reaction was carried out using isolated DNA as the template in a Proflex™ thermal cycler (Applied Biosystems). The PCR mixture had a total volume of 25 µl, consisting of 10 µl of 2X PCR Taq mixture (MBT061- Himedia), 2.0 µl of each forward and reverse primer (10pM each, in equal proportions from Sigma), 5 µl of DNA template (20 ng/µl), and 6 µl of nuclease-free water (Himedia). The PCR program included initial denaturation at 94°C for 1 minute, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 20 seconds, extension at 72°C for 50 seconds, and a final extension step at 72°C for 5 minutes. Various purification methods were employed to obtain purified PCR products, including the use of commercial purification kits designed for DNA purification or ethanol precipitation, effectively removing any potential interfering substances. This PCR amplification, targeting the ITS2 region, facilitated the selective amplification of DNA fragments from the investigated Phyllanthus species, serving as valuable resources for subsequent molecular analyses, such as DNA sequencing or DNA barcoding. DNA Sequencing The amplified PCR products were assessed for successful amplification through 2% agarose gel electrophoresis, utilizing 2 microliters of the products. The remaining amplicons were subsequently forwarded to Eurofins Genomics India PVT.LTD, Bangalore, for Sanger sequencing (Sanger et al., 1977 ). Following sequencing, chromatograms were obtained from Eurofins Genomics and subsequently converted into FASTA format using Chromas software. Sequence Alignment and Data Analysis The ITS2 sequences obtained were aligned using Geneious Pro software, an established bioinformatics tool (Kearse et al. 2012 ). This alignment process involved comparing and organizing the sequences, enabling the identification of conserved regions and variations within the ITS2 region. To ensure high-quality sequences for subsequent analyses (Shendure and Ji, 2004), manual adjustments and trimming were performed to remove ambiguous ends that could result from sequencing errors or DNA sample variations. Consensus sequences were generated from the aligned ITS2 sequences, providing representative sequences for each investigated Phyllanthus species (Thompson et al. 1997 ), with a focus on maintaining at least 80% coverage of the original read length to ensure reliable representation. Geneious Pro software was also utilized for various statistical analyses, including composition, GC content, pairwise residue percentage, identical alignment percentage, minimum/maximum/mean lengths, and pairwise numbers (Drummond et al., 2017 ), offering insights into sequence characteristics and similarities. For species identity validation, the consensus sequences were compared to nucleotide databases using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (Altschul et al. 1990 ) ensuring the accuracy of species identification. Phylogenetic analysis In this study, we performed a comprehensive phylogenetic analysis of Phyllanthus species, specifically targeting Phyllanthus maderaspatensis, P. amarus, P. urinaria, P. virgatus , and P. tenellus . To construct the phylogenetic tree, we curated extensive datasets using MEGAX (Kumar et al., 2018 ) and Phylogeny Fr (Dereeper et al., 2008 ), amalgamating DNA sequences from two primary sources: sequences generated during our study and sequences retrieved from prior investigations available in the NCBI GenBank database. Our sequence acquisition process encompassed collecting DNA sequences from our own specimens, focusing on specific gene regions like the Internal Transcribed Spacer 2 (ITS2), as dictated by the study's objectives. Concurrently, we diligently gathered relevant sequences from the same gene regions in earlier research accessible in the NCBI GenBank, ensuring the inclusion of a diverse array of Phyllanthus species closely related to our study organisms. This meticulous curation aimed at providing a comprehensive representation of phylogenetic relationships within the Phyllanthus genus. Subsequent to sequence compilation, we conducted sequence alignments employing either MUSCLE or ClustalW within the MEGAX software suite (Kumar et al., 2018 ). Following alignment, the construction of the phylogenetic tree was executed utilizing the maximum likelihood (ML) method. The ML method facilitates the estimation of the optimal model for DNA evolution and calculates the likelihood of observing the sequences given this model. The resulting tree was inferred based on the highest likelihood score, offering insights into the most probable evolutionary connections among the Phyllanthus species. To gauge the robustness of the tree, bootstrapping was applied. This technique involved generating numerous replicate datasets by resampling the original sequence alignment and subsequently constructing individual ML trees for each of these resampled datasets. Support values for each branch within the final phylogenetic tree were derived from the frequency with which a specific branch appeared in the resampled trees, often expressed as a percentage. The resultant phylogenetic tree provided invaluable insights into the evolutionary relationships and genetic distances among the studied Phyllanthus species. It contributed significant information pertaining to their diversification, ancestral lineage, and interrelatedness within the broader context of the Phyllanthus genus. DNA Barcoding and ITS2 Secondary Structure Predictions In this study, we employed the Bio-Rad DNA barcode generator, which is accessible at http://biorad-ads.com/DNABarcodeWeb (accessed on 4 October 2023), to create DNA barcodes for the Phyllanthus genotypes under investigation. These barcodes were meticulously constructed based on the aligned DNA nucleotide sequences acquired through the utilization of ITS2 primers. Additionally, we conducted RNA secondary structure predictions using the nucleotide sequences derived from the same ITS2 primers. This prediction process was facilitated by tapping into the rRNA database hosted on the RNAfoldWebServer v2.4.18 platform, which can be found at http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi (accessed on 4 October 2023), following the methodology described by Lorenz et al. in 2011. These comprehensive molecular analyses were instrumental in characterizing the Phyllanthus genotypes, offering deep insights into their genetic compositions and structural attributes. Such investigations are pivotal for advancing our understanding of these Phyllanthus species at the molecular level. Results Phyllanthus Genomic DNA isolation The application of the state-of-the-art kit method proved highly effective in extracting genomic DNA from Phyllanthus species samples collected in India, resulting in robust DNA yields. The confirmation of these yields was conducted through nanodrop spectrophotometer analysis, as outlined in Table 1 and depicted in Fig. 2A. The method's efficiency was further underscored by its successful application in the amplification and sequencing of a well-established amplicon, thereby demonstrating its reliability and suitability for genomic DNA extraction in the study. Internal Transcribed Spacer 2 (ITS2) region gene amplification The ITS2 gene region, comprising approximately 545 base pairs, was successfully isolated using the ITS2-S2F and ITS4R primers for the genetic identification of Phyllanthus species. Gel electrophoresis clearly revealed that all PCR-treated samples exhibited amplification of the ITS2 region. In the agarose gel, the entire PCR product (25 μL) was mixed with 5 μL of 6X loading dye and loaded (Fig. 2B). The Figure illustrates the amplification of the ITS2 gene, approximately 545 base pairs in length, from a total of 75 insect Phyllanthus species samples collected from diverse locations. NCBI BLASTn and ITS2 database analysis Following the sequencing of the amplified ITS2 regions, the resultant nucleotide sequence data underwent comprehensive analysis using NCBI BLASTn. The results revealed a striking degree of sequence similarity between the samples and known Phyllanthus species. Specifically, Phyllanthus maderaspatensis exhibited an impressive 98.51% similarity, Phyllanthus amarus showcased a 98.54% resemblance, Phyllanthus urinaria displayed an impressive 99.70% congruence, Phyllanthus virgatus demonstrated a remarkable 99.64% similarity, and Phyllanthus tenellus manifested an exceptional 100% correspondence, respectively. Query cover, representing the percentage of the query sequence overlapping with the reference sequence, further validated the robustness of these alignments. It's worth noting that a high identity value within the range of 98-100% typically indicates a substantial sequence similarity. These BLASTn outcomes provide invaluable insights into the quality of the alignment, the potential existence of a biological relationship, and the likelihood that the observed similarity is not merely a product of chance. BLAST leverages statistical theory to furnish bit scores and expect values (E-values) for each alignment pair, further enhancing the depth of analysis and interpretation. The thorough BLAST analysis utilizing the ITS2 Database has unequivocally established the identity of our Phyllanthus species ITS2 sequence, unequivocally confirming its alignment with known Phyllanthus species. Several key parameters extracted from the analysis underscore the robustness of this match: notably, a Maximum Score of 561, indicative of a strong alignment with a well-documented reference sequence; a Coverage spanning an impressive range of 91% to 98%, signifying substantial overlap between the sequences in question; and an E-Value of 0.0, highlighting the high biological significance of this alignment. These findings were made possible through the extensive and invaluable ITS2 Database, which houses a wealth of data pertaining to the ITS2 region of ribosomal RNA genes. As such, the outcome of this analysis carries an exceptionally high degree of reliability, providing unequivocal evidence of the close genetic relationship that exists among Phyllanthus species. After successfully obtaining nucleotide sequences, we have diligently submitted and stored them in the NCBI GenBank Nucleotide Sequence Database. Each submitted sequence has been assigned a distinct accession number for reference purposes, ensuring their traceability and accessibility in subsequent research endeavors. The designated accession numbers for the deposited sequences are as follows: Phyllanthus maderaspatensis (OQ630483), Phyllanthus amarus (OQ630476), Phyllanthus urinaria (OQ630474), Phyllanthus virgatus (OQ630473), and Phyllanthus tenellus (OQ625750). These accession numbers serve as unequivocal and exclusive identifiers for each of the sequences in the repository, streamlining their retrieval and utilization in future scientific investigations.Top of Form Phylogenetic Analysis The ClustalW alignment of ITS gene sequences in Phyllanthus species genotypes, including Phyllanthus maderaspatensis, Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus virgatus, and Phyllanthus tenellus (Fig. 3). The alignment was conducted to compare and organize the sequences, enabling the identification of conserved regions and variations within the ITS2 region. The figure 3 highlights the high level of similarity observed between the analyzed Phyllanthus species and sequences from the NCBI database during BLASTn searches, indicating the consistency and reliability of the inferred phylogenetic relationships. This consistency validates the genetic affinities among the studied species and aligns with existing genetic data in the NCBI database. The ClustalW alignment is a crucial step in the DNA barcoding process, providing a standardized and robust identification method for Phyllanthus species. First, a maximum likelihood tree (MLT) was constructed using a Kimura 2-Parameter (K2P) model with 2000 bootstrap replicates. The high level of similarity observed between the analyzed Phyllanthus species and sequences from the NCBI database during BLASTn searches indicates the consistency and reliability of the inferred phylogenetic relationships. This consistency validates the genetic affinities among the studied species and aligns with existing genetic data in the NCBI database. Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution of Phyllanthus species depicted in fig.4. Additionally, a Neighbor-Joining (NJ) analysis was conducted, and the resulting tree topology closely mirrored the structure of the parsimony tree (Figure 4). This concordance between the NJ and parsimony trees is noteworthy as it signifies a high level of agreement between two distinct phylogenetic methods. The NJ method, known for its speed and simplicity in estimating evolutionary relationships, and the parsimony method, which minimizes the number of evolutionary changes needed to explain observed data, both produced similar tree topologies. This consistency between independent analytical approaches reinforces the accuracy and reliability of the phylogenetic analysis, enhancing the confidence in the inferred genetic relationships among the Phyllanthus species under investigation. The analysis focused on voucher specimens of Phyllanthus maderaspatensis (OQ630483), Phyllanthus amarus (OQ630476), Phyllanthus urinaria (OQ630474), Phyllanthus virgatus (OQ630473), and Phyllanthus tenellus (OQ625750). Here are the specific findings for each voucher specimen: Phyllanthus maderaspatensis (OQ630483): This specimen exhibited a high similarity of 98.09% with sequences from the Indian subcontinent, particularly OR073675 ( Phyllanthus maderaspatensis _ India: Dakshina Kannada, Karnataka ), KY079342 ( Nellica maderaspatensis _ India: Tamil Nadu, Madurai, Tirumangalam), and KF312391 ( Phyllanthus maderaspatensis_ Mysore:India) . These sequences clustered together in a single clade, indicating their close genetic relatedness. Interestingly, sequences from Austria and France also showed close relationships, all falling within the class Magnoliopsida, family Phyllanthaceae, and genus Phyllanthus. Phyllanthus amarus (OQ630476): The voucher specimen of Phyllanthus amarus (OQ630476) displayed the highest similarity of 98.54% with the ON026067 ( Phyllanthus amarus_ Odisha: India) sequence from Odisha, India, which was its closest phylogenetic relative. Other Indian sequences from various regions, including Karnataka, Kerala, Kolkata, Tamil Nadu, and Delhi, formed a single clade. Additionally, sequences from Austria, Netherlands, Brazil, Germany, Thailand, Vietnam, the UK, and Saudi Arabia were closely related. All sequences shared the classification of class Malpighiales, family Phyllanthaceae, and genus Phyllanthus. Phyllanthus urinaria (OQ630474): The voucher specimen of Phyllanthus urinaria (OQ630474) exhibited a remarkable 100% similarity with the KJ135018 ( Phyllanthus urinaria _ India: Manipal, Karnataka) sequence, indicating an extremely close genetic relationship. Other sequences from regions such as Karnataka (Mysore and Manipal), Kolkata, Odisha, China, Austria, Thailand, Korea, Netherlands, Brazil, and Germany also clustered together as a single clade. All these sequences shared the same classification within the class Malpighiales, family Phyllanthaceae, and genus Phyllanthus. Phyllanthus virgatus (OQ630473): The voucher specimen Phyllanthus virgatus (OQ630473) demonstrated a 99.59% similarity with the KJ135019 ( Phyllanthus virgatus _ India: Karkala, Karnataka) sequence from Mysore, Karnataka, making it the closest phylogenetic relative. Additionally, sequences from Taiwan, China, Austria, and Thailand clustered together as a single clade. These sequences shared the same classification within the class Equisetopsida, family Phyllanthaceae, and genus Phyllanthus. Phyllanthus tenellus (OQ625750): The voucher specimen Phyllanthus tenellus (OQ625750) exhibited a 100% similarity percentage identity with the KF312396 ( Phyllanthus tenellus _Mysore: India) sequence from Mysore, Karnataka, indicating an identical genetic match. Other sequences from Austria, Taiwan, Netherlands, the UK, and Saudi Arabia also grouped together. All these sequences shared the same classification within the class Malpighiales, family Phyllanthaceae, and genus Phyllanthus. DNA Barcoding and ITS2 Secondary Structure Predictions The DNA barcodes generated from the ITS2 sequences provided insights into the genetic variations among the tested Phyllanthus genotypes, including Phyllanthus maderaspatensis (OQ630483), Phyllanthus amarus (OQ630476), Phyllanthus urinaria (OQ630474), Phyllanthus virgatus (OQ630473), and Phyllanthus tenellus (OQ625750) (Fig. 5c,d). Notably, Phyllanthus virgatus (OQ630473) exhibited a relatively longer barcode with a length of 363 base pairs, followed by Phyllanthus maderaspatensis (OQ630483) with 356 base pairs, Phyllanthus urinaria (OQ630474) with 355 base pairs, Phyllanthus urinaria (OQ630474) with 354 base pairs, and Phyllanthus tenellus (OQ625750) with 326 base pairs. Furthermore, the predictions of the ITS2 secondary structures revealed distinct features among the tested Phyllanthus genotypes, represented by variations in the central ring's helical orientations (Fig. 5e,f). These differences included variations in loop number, position, size, and angle from the centroid in the secondary structures of the Phyllanthus genotypes. Phyllanthus virgatus, in particular, displayed a more complex secondary structure with diverse loop numbers and angles, distinguishing it from the other tested genotypes. The significance of these findings relates to the potential for developing species-specific primers to identify lesser-known Phyllanthus species more efficiently. The unique genetic structure observed in the conserved nuclear region of ITS2 offers an opportunity to design primers that can accurately differentiate between these species. This has significant implications for preventing adulteration in herbal products, as the ability to distinguish between closely related species is crucial for ensuring the authenticity and quality of herbal remedies. By using DNA barcoding and secondary structure predictions, the study contributes to the development of tools for verifying the identity of Phyllanthus species, thereby addressing concerns related to adulteration and supporting the herbal industry's integrity and consumer safety. Discussion The imperative for in-depth genetic profiling of Phyllanthus within the Indian subcontinent, emphasized by Sarin et al. ( 2014 ), stems from the species' constrained geographical distribution. This need is accentuated by the pivotal role Phyllanthus species play in India's herbal trade sector, with an annual trade volume ranging from 2000 to 5000 metric tons, signifying substantial economic importance (Sarin et al., 2014 ). Recognizing the restricted habitat and economic significance of Phyllanthus, this study seeks a nuanced understanding of its molecular characteristics and taxonomic relationships through rigorous genetic investigations. In the realms of ecology, medicine, and potential therapeutic applications, the diverse functions of Phyllanthus species, such as P. maderaspatensis, P. amarus, P. urinaria, P. virgatus , and P. tenellus , unfold. Safeguarding the quality and authenticity of herbal products derived from these species demands robust identification and authentication protocols due to the looming threat of adulteration. With certain species facing endangerment or extinction, conservation efforts become imperative for sustainable utilization. This study integrates into advancing our comprehension of the genetic landscape of Indian Phyllanthus species. Its overarching goal extends beyond taxonomy, reaching into conservation and medicinal research, enriching our understanding of these invaluable plants. In the initial phase of genetic profiling, successful isolation of genomic DNA from various Phyllanthus species has been accomplished using a kit-based extraction protocol. This crucial step lays the foundation for subsequent molecular analyses, enabling comprehensive exploration of the genetic makeup and diversity within the Phyllanthus genus. The utilization of a standardized kit method ensures efficiency, reliability, and reproducibility, minimizing variability across samples. Ensuring DNA integrity is critical for subsequent molecular investigations, including PCR amplification and sequencing. The introduction of liquid nitrogen into DNA extraction protocols showcases an innovative methodology that triumphs over conventional challenges. This method emerges as a cutting-edge technique, delivering high-quality genomic DNA efficiently and precisely, addressing practical hindrances associated with diverse Phyllanthus species. Liquid nitrogen's use highlights its flexibility and adaptability to the intricacies of Phyllanthus species. This method reliably captures the complex molecular subtleties of these plants, creating high-quality genomic material due to its unique properties, including precise destruction of plant cell structures, deactivation of damaging enzymes, and mitigation of DNA damage during extraction. The combination of liquid nitrogen-assisted DNA extraction and molecular research on Phyllanthus species opens new avenues for ecological and therapeutic research, increasing the dependability and effectiveness of molecular research. The method's superiority is demonstrated by the lack of smearing in electrophoresis bands and the single absorbance peak at 260 nm in NanoDrop measurements. The choice of focusing on the ITS2 gene region for DNA barcoding and systematic analysis is well-founded, given its well-documented exceptional attributes. Successfully amplifying the ITS2 region within Phyllanthus species, validated through a rigorous process, establishes a robust foundation. This achievement is pivotal for species identification, phylogenetic analyses, and aligns seamlessly with established molecular biology methodologies. The subsequent gene sequencing enhances the depth of analysis, providing insights into nucleotide composition and sequence variations, forming the basis for comprehensive phylogenetic studies. The integration of these molecular techniques with the ITS2 region advances our understanding of genetic diversity within the Phyllanthus genus, establishing a solid framework for DNA barcoding applications. The success of ITS2 region amplification and sequencing unequivocally validates its efficacy as a molecular marker for genetic profiling and taxonomic authentication of Indian Phyllanthus species. The incorporation of secondary structures within ITS2 sequences significantly elevates the accuracy of species delineation, providing crucial insights for cladistic inference of relationships among Phyllanthus species. This choice aligns with the broader consensus in the field, emphasizing the reliability and robustness of ITS2 in unraveling evolutionary connections within eukaryotes. Investigations by Xu et al. ( 2015 ) and Chen et al. ( 2018 ) emphasize the efficiency and cost-effectiveness of utilizing ITS2 minibarcodes in comparison to conventional sequencing methods. This scalability, facilitated by emulsion PCR, enables the simultaneous amplification of thousands of short DNA fragments, streamlining the sequencing process for diverse Phyllanthus species. The potential limitations of ITS2 in distinguishing closely related species are outweighed by its distinct advantages, as demonstrated in various studies. Notably, Hajibabaei et al. ( 2007 ) showcased the utility of ITS2 sequences in devising taxon-specific probes for rapid plant recognition, enhancing the practical applications of this gene region. Despite potential limitations, the ITS2 region stands out for its efficacy in identifying closely related species, especially in botanical and degraded DNA samples. Phylogenetic analysis contributes valuable insights into the minimal genetic variations among Phyllanthus genotypes, emphasizing their close genetic relatedness regardless of geographic origins. The NCBI BLAST analysis of Phyllanthus species yields compelling results, indicating remarkable similarity with Phyllanthus species closely associated with the Indian Phyllanthus gene deposits in the NCBI GenBank. The alignment without gaps and in a plus/plus orientation suggests a high degree of sequence conservation and accuracy in the generated data. The observed alignment underscores the robustness of the sequencing methodology employed and the meticulous nature of the subsequent analysis. To contribute to the broader scientific community, sequences generated in this investigation have been deposited in the NCBI GenBank, enhancing the reproducibility and traceability of the study. The deposition of sequences in the NCBI GenBank, along with specific accession numbers, enhances the reproducibility and traceability of the study, promoting transparency and collaboration in genomics research. The phylogenetic study of Phyllanthus species through DNA barcoding represents a significant stride in understanding the evolutionary relationships within this diverse genus. This methodological approach, rooted in molecular biology, holds the promise of unraveling the intricate genetic tapestry of Phyllanthus species, contributing valuable insights to taxonomy, conservation, and medicinal research. The choice of DNA barcoding for phylogenetic studies is well-founded, drawing inspiration from the pioneering work of Hebert et al. ( 2003 ) in proposing the use of a standardized DNA sequence for species identification. In the context of Phyllanthus, this methodology provides a robust and reliable means of discerning genetic variations and relationships among different species. Phylogenetic studies often require a marker with sufficient variability to capture distinctions between closely related species. In this regard, the Internal Transcribed Spacer 2 (ITS2) gene region emerges as a prime candidate, owing to its enhanced discriminatory power. The scalability of high-throughput sequencing methods plays a pivotal role in the success of large-scale DNA barcoding initiatives. Phylogenetic trees constructed based on DNA barcoding data yield valuable insights into the evolutionary context within the genus Phyllanthus. The clear definition of outgroups enhances the understanding of genetic relationships and aids in the identification of key evolutionary branches within the Phyllanthus genus. In the broader context of medicinal and pharmacological research, the phylogenetic study of Phyllanthus species assumes particular significance. Understanding the genetic diversity and relationships among species provides a foundation for exploring the potential therapeutic applications of these plants. This study augments the existing knowledge base on Phyllanthus phylogenetics by building upon prior research, notably that of Wurdack et al. ( 2004 ). Wurdack et al. conducted a molecular phylogenetic analysis of Phyllanthaceae, focusing on the Phyllanthoideae subfamily within the broader Euphorbiaceae family. Employing plastid rbcL DNA sequences, the researchers investigated evolutionary relationships and genetic diversity within this plant group. This research significantly contributes to our broader understanding of plant evolution and systematics, offering valuable insights into the molecular characteristics of Phyllanthaceae. Moreover, the collaborative nature of scientific inquiry is underscored by references to studies conducted by Manissorn et al. ( 2010 ), Gyana Ranjan Rout et al. ( 2010 ), Müller et al. ( 2007 ). This collaborative effort reinforces the cumulative progress achieved in unraveling the evolutionary complexities of Phyllanthus species through the application of barcoding techniques. The utilization of ITS2 secondary structures for primer design aligns with the broader consensus in molecular biology, as exemplified by studies conducted by Coleman ( 2003 ). These researchers emphasized the importance of incorporating secondary structure information for enhancing the accuracy of species delineation, a critical aspect illuminated by the present research within the context of Phyllanthus species. Furthermore, the concept of species-specific primers finds resonance in the work of Hajibabaei et al. ( 2007 ), where the utility of ITS2 sequences in designing taxon-specific probes for swift plant recognition was demonstrated. This innovative approach not only streamlines identification processes but also adds an extra layer of precision, particularly relevant in the complex taxonomic landscape of Phyllanthus species. The potential of DNA barcoding and predictive secondary structure analysis, as showcased in this study, is further validated by the findings of Engelmann et al. ( 2009 ). Their work elucidated the efficacy of ITS2 microarrays in discriminating species with high sequence identities, a testament to the robustness of such methodologies in addressing challenges related to degraded DNA and ensuring the authenticity of herbal products. In the broader landscape of genomics research, the pioneering spirit of this study aligns with the transformative potential of high-throughput sequencing methods, as highlighted by Shendure and Ji ( 2008 ). The scalability and efficiency demonstrated in the current research resonate with the broader trends in genomics, emphasizing the need for advanced methodologies to handle large-scale projects effectively. Conclusions In summary, this investigation underscores the robustness of DNA barcoding, specifically utilizing the ITS2 region, for the precise identification and validation of Phyllanthus species in India. The ribosomal nuclear ITS2 region exhibits substantial levels of divergence, both within and between species, as evidenced by the comprehensive analysis of DNA barcodes and the determination of secondary structures through the minimum free energy principle. The meticulous implementation of DNA barcoding not only ensures the quality of herbal products but also serves as a critical tool in safeguarding Phyllanthus biodiversity and fostering the sustainable utilization of these invaluable plant resources. The genetic relationships and diversity unveiled among Indian Phyllanthus species, elucidated by the construction of a phylogenetic tree based on multiple sequence alignments of the ITS2 gene, provide vital insights that substantiate conservation efforts. This phylogenetic perspective underscores the impact of adulteration on genetic variation within the Phyllanthus genus. The deposited ITS2 gene sequences serve as indispensable genetic references, facilitating future research endeavors and contributing to stringent quality control measures. This, in turn, ensures the genuineness of herbal products derived from Phyllanthus species. Beyond its immediate applications, this study emphasizes the broader significance of accurate identification and conservation of the Phyllanthus genus. This genus encompasses a diverse array of ecologically and medicinally important plants. By addressing prevalent issues of adulteration and misidentification in the market, this research significantly contributes to the sustainable use and protection of Phyllanthus species, unlocking their full therapeutic potential. The findings underscore the paramount importance of integrating advanced molecular techniques in botanical research and conservation strategies, ultimately advancing our understanding of plant biodiversity and supporting the responsible utilization of natural resources. Declarations Acknowledgements We extend our gratitude to the Botanical Resource Center and Himalaya Wellness Company for their invaluable assistance and resources, pivotal in facilitating this research. Their dedication to biodiversity preservation and advocacy for sustainable living strongly resonates with our research objectives, playing a pivotal role in the success of this project. This contribution is a significant segment of the first author's PhD thesis. Author Contributions All authors participated in conceptualizing and designing the study. Ragavendra undertook material preparation, data collection, and analysis. The initial draft of the manuscript was authored by Raghavendra and Gururaj Chalageri, and subsequent versions were reviewed and commented upon by all authors: Raghavendra, Pushpalatha, Gururaj Chalageri, Kannan, and Babu. All authors critically reviewed, contributed to, and approved the final version of the manuscript. Competing interests The authors have no competing interests to declare that are relevant to the content of this article. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. doi: 10.1016/S0022-2836(05)80360-2. Bhattarai, S., Chandra Ghosh, D., Patra, A., Samanta, A., Mandal, S., Bandyopadhyay, A., & Sen, S. (2019). Adulteration of herbal products: challenges, solutions, and prospects. 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Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36(Web Server issue), W465-9. doi: 10.1093/nar/gkn180. Epub 2008 Apr 19. Drummond, A. J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., ... & Kearse, M. (2017). Geneious Pro. Version 10.0.4. Biomatters Ltd. Engelmann, J. C., Rahmann, S., & Wolf, M. (2009). Accurate and efficient genome-wide assembly of extremophilic ITS2 sequences. BMC Bioinformatics, 10 (Suppl 14), S7. Hajibabaei, M., Singer, G. A., Hebert, P. D., & Hickey, D. A. (2007). DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends in Genetics, 23 (4), 167-172. Hebert, P. D., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270 (1512), 313-321. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., ... & Drummond, A. (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28(12), 1647-1649. doi: 10.1093/bioinformatics/bts199. Kiran KR, Swathy PS, Paul B, Prasada KS, Rao MR, Joshi MB, Rai PS, Satyamoorthy K, Muthusamy A (2021) Untargeted metabolomics and DNA barcoding for discrimination of Phyllanthus species. Journal of Ethnopharmacology 273: 1–11. https://doi.org/10.1016/j.jep.2021.113928 Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547-1549. doi: 10.1093/molbev/msy096. Lorenz, R., Bernhart, S. H., Höner Zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for Molecular Biology, 6(1), 26. doi: 10.1186/1748-7188-6-26. Manisha, M., Prakash, A., Rajak, R. L., & Chakotiya, A. S. (2012). Phylogenetic analysis of Phyllanthus species collected from India based on nrDNA ITS and cpDNA psbA-trnH sequences. Biochemical Systematics and Ecology, 45 , 60-68. Manissorn, J., Sukkaew, P., & Sumranwanich, T. (2010). Molecular identification of medicinal Phyllanthus species (Euphorbiaceae) using internal transcribed spacer (ITS) sequences. Genetics and Molecular Research, 9 (2), 705-711. Mishra, A., Mishra, A. K., Bhargava, A., Pandey, A. K., & Kohli, D. V. (2011). Phyllanthus species: An overview. Journal of Pharmacy & Bioallied Sciences, 3(2), 361-379. doi: 10.4103/0975-7406.80770. Müller, K. F., Borsch, T., Legendre, L., Porembski, S., & Barthlott, W. (2007). Evolution of carnivory in Lentibulariaceae and the Lamiales. Plant Biology, 9 (03), 360-372. Newmaster, S. G., Fazekas, A. J., & Ragupathy, S. (2008). DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 86(8), 735-741. doi: 10.1139/B08-055. Orlandini, P., Pfeilstetter, E., Röser, M., & Hartmann, H. E. (2023). Phylogenetic relationships in the genus Phyllanthus (Phyllanthaceae): Evidence from chloroplast matK and nuclear ribosomal DNA ITS sequences. Plant Systematics and Evolution, 309 (1), 1-15. Raclariu, A. C., Heinrich, M., Ichim, M. C., de Boer, H., & Benefits, R. (2018a). Quality control of herbal medicines: From phytochemical screening to DNA metabarcoding. Frontiers in Pharmacology, 9, 1597. doi: 10.3389/fphar.2018.01597. Rout, G. R., Das, P., & Das, L. (2010). Molecular identification of Phyllanthus amarus using rDNA-ITS region. American-Eurasian Journal of Botany, 3 (4), 149-154. Rubinoff, D., Cameron, S., & Will, K. (2006). A genomic perspective on the shortcomings of mitochondrial DNA for "barcoding" identification. Journal of Heredity, 97 (6), 581-594. Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press. Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74(12), 5463-5467. doi: 10.1073/pnas.74.12.5463. Sarin, B., Verma, N., Martín, J. P., & Mohanty, A. (2014). An Overview of Important Ethnomedicinal Herbs of Phyllanthus Species: Present Status and Future Prospects. The Scientific World Journal, 2014, 839172. http://dx.doi.org/10.1155/2014/839172 Schuster, S. C. (2008). Next-generation sequencing transforms today's biology. Nature Methods, 5 (1), 16-18. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26 (10), 1135-1145. Shetti, A. A., Kumar, C. S., & Chandrashekar, A. (2011). Current regulatory scenario in marketing herbal products in India. International Journal of Pharmacy and Pharmaceutical Sciences, 3(4), 63-66. Song, C., Liu, Y., Song, A., Dong, G., Zhao, H., Sun, W., ... & Chen, S. (2012). The Chrysanthemum nankingense genome provides insights into the evolution and diversification of Chrysanthemum flowers and medicinal traits. Molecular Plant, 6 (3), 728-739. Stoeckle, M. Y., & Hebert, P. D. N. (2004). Barcoding life to conserve biological diversity: beyond the taxonomic imperative. PLoS Biol, 2(10), e372. doi: 10.1371/journal.pbio.0020372. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24), 4876-4882. doi: 10.1093/nar/25.24.4876. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. doi: 10.1093/nar/22.22.4673. Wurdack, K. J., Hoffmann, P., & Chase, M. W. (2004). Molecular phylogenetic analysis of Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) using plastid rbcL DNA sequences. American Journal of Botany, 91(12), 1882-1900. doi: 10.3732/ajb.91.12.1882. Xu, S., Li, D., Li, J., & Xiang, X. (2015). Rapid plant identification using species- and group-specific primers targeting chloroplast DNA. PLoS ONE, 10 (6), e0129585. Yang, S., Jin, H., Zhu, Y., Wan, J., & Chai, Y. (2020). Chemical constituents, pharmacological activities, and clinical applications of genus Phyllanthus. Evidence-Based Complementary and Alternative Medicine, 2020, 3176828. doi: 10.1155/2020/3176828. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3893650","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269707993,"identity":"87eca94a-5834-4344-b612-9c82845e160b","order_by":0,"name":"P Raghavendra","email":"data:image/png;base64,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","orcid":"","institution":"The Himalaya Wellness Company","correspondingAuthor":true,"prefix":"","firstName":"P","middleName":"","lastName":"Raghavendra","suffix":""},{"id":269707994,"identity":"c0388a00-7d14-4c59-86d0-f81c813b9658","order_by":1,"name":"Pushpalatha Ganesh","email":"","orcid":"","institution":"Centurion University of Technology and Management","correspondingAuthor":false,"prefix":"","firstName":"Pushpalatha","middleName":"","lastName":"Ganesh","suffix":""},{"id":269707995,"identity":"bffd4e33-e3df-4189-be41-360171d02aec","order_by":2,"name":"Gururaj Chalageri","email":"","orcid":"","institution":"The Himalaya Wellness Company","correspondingAuthor":false,"prefix":"","firstName":"Gururaj","middleName":"","lastName":"Chalageri","suffix":""},{"id":269707997,"identity":"bc5456e3-647f-448b-9af9-4e411c98169c","order_by":3,"name":"R Kannan","email":"","orcid":"","institution":"The Himalaya Wellness Company","correspondingAuthor":false,"prefix":"","firstName":"R","middleName":"","lastName":"Kannan","suffix":""},{"id":269707998,"identity":"5da309d5-c444-4f31-9dc2-b7ed2e93274b","order_by":4,"name":"Babu U V","email":"","orcid":"","institution":"The Himalaya Wellness Company","correspondingAuthor":false,"prefix":"","firstName":"Babu","middleName":"U","lastName":"V","suffix":""}],"badges":[],"createdAt":"2024-01-24 09:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3893650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3893650/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50298842,"identity":"fa175219-a19c-4e73-a5bb-65dc8ccd1d7f","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3377325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystematic Collection of Mature Phyllanthus Species and DNA Barcoding.\u003c/strong\u003e The systematic collection of mature plants of five Phyllanthus species, including Phyllanthus maderaspatensis, Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus virgatus, and Phyllanthus tenellus, from diverse locations across India is illustrated. The plants were carefully selected for their healthy, fresh, and succulent appearance. The subsequent steps involve DNA barcoding, targeting the ITS2 region, to molecularly identify the Phyllanthus species. The figure highlights the importance of the study in understanding the genetic diversity, evolutionary relationships, and medicinal potential of these species, which are essential for conservation efforts and sustainable utilization.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/844cd4c4684e6861029f96cc.png"},{"id":50298840,"identity":"33cff859-46a2-4c16-9dec-81cfb5a09977","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":333017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. Visualization of Genomic DNA Samples from Phyllanthus Species Using 0.8% Agarose Gel Stained with Ethidium Bromide.\u003c/strong\u003e \u003cstrong\u003eLegends:\u003c/strong\u003e \u003cstrong\u003eM-\u003c/strong\u003e Molecular Weight Marker (1.0kb Thermo Scientific, USA).\u003cstrong\u003eLane1-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus maderaspatensis;\u003c/em\u003e\u003cstrong\u003e Lane2-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus amarus\u003c/em\u003e; \u003cstrong\u003eLane3-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus urinaria\u003c/em\u003e; \u003cstrong\u003eLane4-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus virgatus\u003c/em\u003e; \u003cstrong\u003eLane5\u003c/strong\u003e-\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus tenellus\u003c/em\u003e\u003cstrong\u003e; Fig. 2B-\u003c/strong\u003e \u003cstrong\u003eGel Electrophoresis of PCR Products from Phyllanthus Species Using Internal Transcribed Spacer (ITS) Gene with 1.5% Agarose Gel. Legends:\u003c/strong\u003e \u003cstrong\u003eM-\u003c/strong\u003eMolecular Weight Marker (100 bp, Thermo Scientific, USA).\u003cstrong\u003eLane1-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus maderaspatensis;\u003c/em\u003e\u003cstrong\u003e Lane2-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus amarus\u003c/em\u003e; \u003cstrong\u003eLane3-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus urinaria\u003c/em\u003e; \u003cstrong\u003eLane4-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus virgatus\u003c/em\u003e; \u003cstrong\u003eLane5\u003c/strong\u003e-\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePhyllanthus tenellus\u003c/em\u003e\u003cstrong\u003e;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/c8c9884d7040edb454bece8c.png"},{"id":50298843,"identity":"26984f95-8d46-4f76-8785-f48644958169","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ClustalW Alignment of ITS Gene Sequences from Phyllanthus Species Genotypes: A Comprehensive Comparison. \u003c/strong\u003eThis meaningful multiple sequence alignment (MSA) method, using ClustalW, aims to align sequences accurately, allowing for the visualization of identities, similarities, and differences among Phyllanthus species genotypes. Notably, the comparison of ITS gene sequences from various Phyllanthus species demonstrated a lack of mismatches, indicating the absence of sequencing errors and a strong alignment with the Phyllanthus genus.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/1d4763b6b6b57bdb67d9009e.png"},{"id":50298841,"identity":"2236e87a-bd4d-4a69-95c9-1ce58dd1d2a1","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":356387,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic Neighbor-Joining (NJ) Tree with Bootstrap Support. The Neighbor-Joining (NJ) tree, constructed with 2000 bootstrap replicates, illustrates the clustering of various Phyllanthus species collected from diverse geographical locations. The tree is based on the ribosomal sequences of the Internal Transcribed Spacer (ITS) gene. In this analysis, \u003cem\u003ePhyllanthus debilis\u003c/em\u003e was employed as an outgroup to provide context for the relationships among the studied Phyllanthus species. The bootstrap support values indicate the robustness of the tree's branching patterns, reflecting the reliability of the inferred phylogenetic relationships among these plant species.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/4286c3bdb256f7dbb70aee81.png"},{"id":50298839,"identity":"47534eff-0ed7-40bc-8533-8b33567e4216","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":719011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative Analysis of Phyllanthus Genotypes, (a) DNA Barcodes (b) Consensus Structure of ITS2 Region (c) Predicted Minimum Free Energy (MFE). \u003c/strong\u003eFigure 4 presents a comparative analysis of various aspects of Phyllanthus genotypes. Panel (a) displays the DNA barcodes, highlighting variations among the genotypes. Panel (b) represents the consensus structure of the ITS2 region, demonstrating the secondary structures of the genotypes. Panel (c) provides information on the predicted minimum free energy (MFE), indicating structural stability differences among the genotypes. This comprehensive analysis offers valuable insights into the genetic and structural attributes of Phyllanthus genotypes, aiding in their accurate identification and molecular characterization.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/dbe9392218726152cb9c5aaa.png"},{"id":55265560,"identity":"09df4582-1736-4bf7-8a5a-5a5de9c794ab","added_by":"auto","created_at":"2024-04-25 02:06:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4607917,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/549caea8-8b66-4cd3-ac6c-80125c86f67a.pdf"},{"id":50298844,"identity":"30b9b463-496c-45ab-bf8e-a30d93ea5f41","added_by":"auto","created_at":"2024-01-29 11:19:29","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11174912,"visible":true,"origin":"","legend":"","description":"","filename":"phyllanthustable.doc","url":"https://assets-eu.researchsquare.com/files/rs-3893650/v1/40c09254e7e376dc075d972c.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Profiling and Taxonomic Authentication of Indian Phyllanthus (Phyllanthaceae: Phyllantheae) Genus through Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions: A Comprehensive Analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus Phyllanthus, belonging to the Phyllanthaceae family, holds ecological and medicinal significance in India, known for its rich biodiversity. India is home to numerous Phyllanthus species, with around 800 recognized within the genus. Traditional medicine systems like Ayurveda have extensively utilized Phyllanthus species for their therapeutic properties, prompting scientific exploration (Mishra et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eScientific investigations have unveiled diverse bioactive compounds within Phyllanthus species, including alkaloids, flavonoids, lignans, tannins, and phenolic compounds. These compounds exhibit various pharmacological activities, such as antimicrobial, antioxidant, anti-inflammatory, hepatoprotective, antidiabetic, immunomodulatory, and anticancer effects. In Asia, these plants have been traditionally employed in ayurvedic medicine for various ethnopharmacological purposes, including treating conditions such as astringency, abdominal pain, diuresis, fever, deobstruction, and as antiseptics. They are also utilized in the treatment of ailments related to digestion, genitourinary, respiratory, skin, hepatopathy, jaundice, and renal calculus (Kiran et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).Certain Phyllanthus species, in particular, have been studied for their hepatoprotective properties and their potential in treating liver disorders (Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond medicinal uses, some Phyllanthus species have economic value as sources of timber, fiber, dye, ornamental plants, or edible fruits (Mishra et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, the popularity of Phyllanthus products in traditional medicine and other industries raises concerns about the adulteration of herbal preparations. Adulteration compromises the efficacy and safety of herbal products, hindering accurate taxonomic and pharmacological studies (Bhattarai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdulteration can occur at various stages of the supply chain, involving the substitution of authentic plant materials with similar-looking but different species or unrelated plant materials. This introduces inconsistencies in therapeutic outcomes and challenges the accuracy of taxonomic and pharmacological studies, leading to misidentified or substituted samples (Bhattarai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address this concern, reliable authentication methods, such as DNA barcoding, have been proposed. DNA barcoding, specifically targeting the ITS2 region, provides standardized and robust identification, less influenced by external factors (Hollingsworth et al. 2011). The ITS2 region, a non-coding DNA region between the 5.8S and 28S ribosomal RNA genes, serves as a unique molecular signature for different plant species. In the context of plant adulteration, ITS2 can distinguish between closely related plant species, aiding in the detection of unauthorized or fraudulent inclusion of plant material in food products. This approach ensures the authenticity and quality of botanical ingredients in various products like herbal supplements, teas, or food items. The ITS2 region's significance lies in its ability to contribute to accurate and reliable species identification, essential for addressing issues related to food fraud and adulteration. It is applicable to all plant life stages and processed materials, making it suitable for species authentication of herbal products (Newmaster et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). DNA barcoding has proven valuable in detecting potential adulterants and ensuring the quality and authenticity of herbal products (Stoeckle et al. 2004).\u003c/p\u003e \u003cp\u003eThe herbal commodity market is experiencing significant growth, driven by the perception that traditional medicine is inherently safer and conducive to promoting overall health and sustainable lifestyles. However, challenges such as the scarcity of authentic resources and a deficiency in taxonomic knowledge have led to an increase in cases of economically motivated or unintentional adulterations and substitutions (Raclariu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo safeguard consumers and ensure the integrity of herbal products, rigorous pharmacovigilance measures are imperative. However, existing regulatory guidelines on medicinal plants often lack clarity, varying significantly from one nation to another. Addressing this research gap requires regulatory bodies to adopt more dependable, universally applicable, and robust detection methods (Shetti et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Such measures are essential to ensure the authenticity of herbal products and the safety of those who rely on them for their health and well-being.\u003c/p\u003e \u003cp\u003eIn this study, we aim to employ DNA barcoding techniques, specifically targeting the nrITS2 region, to molecularly identify Phyllanthus species. This approach offers a reliable solution to combat adulteration, ensuring the authenticity of herbal products and contributing significantly to taxonomic and pharmacological research. DNA barcoding provides a robust tool for accurate species identification, crucial for biodiversity conservation, evidence-based herbal medicine development, and sustainable Phyllanthus species utilization in India.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Plant Materials\u003c/h2\u003e \u003cp\u003eMature plants of five Phyllanthus species, namely \u003cem\u003ePhyllanthus maderaspatensis, Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus virgatus\u003c/em\u003e, and \u003cem\u003ePhyllanthus tenellus\u003c/em\u003e, were systematically collected from diverse locations across India, with a keen focus on selecting healthy, fresh, and succulent plant parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To ensure the utmost accuracy in identification and authentication, the collected specimens underwent meticulous examination and verification at the Herbaria of the Himalaya Wellness, Bengaluru, a trusted repository of plant specimens housing authenticated reference materials for scientific purposes. This rigorous procedure guaranteed the researchers a source of reliable and authentic plant materials, underscoring the importance of adhering to stringent identification and authentication protocols to uphold the integrity and scientific validity of studies conducted on Phyllanthus species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhyllanthus DNA Isolation and analysis\u003c/h2\u003e \u003cp\u003eDNA extraction was carried out utilizing the NucleoSpin\u0026reg; Plant II DNA Kit from MACHEREY-NAGEL, Germany, following the prescribed protocols. Initially, 2 grams of leaf samples were finely ground into a powder using liquid nitrogen. The finely ground material was then mixed meticulously with 500\u0026micro;l of lysis buffer (PL2), and 10\u0026micro;l of RnaseA solution was added. Subsequently, the mixture underwent a 45-minute incubation at 65\u0026deg;C in a water bath. After the incubation, 75\u0026micro;l of PL3 SDS precipitate buffer (potassium acetate) was introduced, thoroughly mixed, and left to incubate for 5 minutes at 4\u0026deg;C to induce SDS precipitation. A Nucleospin filter (violet ring) was placed within a fresh collection tube, and the lysate was transferred onto the column, followed by centrifugation at 11,000rpm for 2 minutes. Following this step, 450\u0026micro;l of precipitation buffer (PC) was mixed in, and the process was repeated using a Nucleospin Plant II column (green ring) in a new collection tube (2ml). In this step, 700\u0026micro;l of the sample was centrifuged, and the flow-through was discarded. Subsequently, washing buffer (PW1) and washing buffer (PW2) were introduced to the column. The column underwent a 2-minute centrifugation at 11,000rpm to ensure thorough drying of the silica membrane. The dried column (Green ring) was then placed in a fresh 1.5mL microcentrifuge tube, and 50\u0026micro;l of elution buffer (PE) was pipetted onto the membrane. The column was incubated for 5 minutes at 65\u0026deg;C and centrifuged for 1 minute at 11,000rpm for DNA elution. This elution step was repeated with another 50\u0026micro;l of PE buffer into the same tube.\u003c/p\u003e \u003cp\u003eFor qualitative analysis, 5 \u0026micro;L of the isolated DNA sample was combined with 5 \u0026micro;L of 6X loading dye (Himedia) and loaded onto a 0.8% agarose gel alongside a 1 kb DNA ladder. Electrophoresis was conducted at 100 V for 1 hour, and the gel was visualized using a UV Gel documentation unit (VilberLourmat, France) (Sambrook and Russel 2011). Quantification of the DNA sample was performed using a NanoDrop One (Thermo Scientific) spectrophotometer. For this, 2 \u0026micro;L of the DNA sample was placed on the instrument's pedestal, and OD readings were taken at 260/280nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePCR Amplification of ITS2 Region\u003c/h2\u003e \u003cp\u003eThe selected barcode candidates, nrITS2 loci, were used for PCR amplification, employing universal primers, including Forward primer ITS2-S2F-5\u0026rsquo;\u0026ndash; ATGCGATACTTGGTGTGAAT-3\u0026rsquo; (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and Reverse primer ITS4R-5\u0026rsquo;-\u0026rsquo; TCCTCCGCTTATTGATATGC-3\u0026rsquo; (White et al. 1990). The PCR reaction was carried out using isolated DNA as the template in a Proflex\u0026trade; thermal cycler (Applied Biosystems). The PCR mixture had a total volume of 25 \u0026micro;l, consisting of 10 \u0026micro;l of 2X PCR Taq mixture (MBT061- Himedia), 2.0 \u0026micro;l of each forward and reverse primer (10pM each, in equal proportions from Sigma), 5 \u0026micro;l of DNA template (20 ng/\u0026micro;l), and 6 \u0026micro;l of nuclease-free water (Himedia). The PCR program included initial denaturation at 94\u0026deg;C for 1 minute, followed by 35 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 55\u0026deg;C for 20 seconds, extension at 72\u0026deg;C for 50 seconds, and a final extension step at 72\u0026deg;C for 5 minutes. Various purification methods were employed to obtain purified PCR products, including the use of commercial purification kits designed for DNA purification or ethanol precipitation, effectively removing any potential interfering substances. This PCR amplification, targeting the ITS2 region, facilitated the selective amplification of DNA fragments from the investigated Phyllanthus species, serving as valuable resources for subsequent molecular analyses, such as DNA sequencing or DNA barcoding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA Sequencing\u003c/h2\u003e \u003cp\u003eThe amplified PCR products were assessed for successful amplification through 2% agarose gel electrophoresis, utilizing 2 microliters of the products. The remaining amplicons were subsequently forwarded to Eurofins Genomics India PVT.LTD, Bangalore, for Sanger sequencing (Sanger et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Following sequencing, chromatograms were obtained from Eurofins Genomics and subsequently converted into FASTA format using Chromas software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSequence Alignment and Data Analysis\u003c/h2\u003e \u003cp\u003eThe ITS2 sequences obtained were aligned using Geneious Pro software, an established bioinformatics tool (Kearse et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This alignment process involved comparing and organizing the sequences, enabling the identification of conserved regions and variations within the ITS2 region. To ensure high-quality sequences for subsequent analyses (Shendure and Ji, 2004), manual adjustments and trimming were performed to remove ambiguous ends that could result from sequencing errors or DNA sample variations. Consensus sequences were generated from the aligned ITS2 sequences, providing representative sequences for each investigated Phyllanthus species (Thompson et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), with a focus on maintaining at least 80% coverage of the original read length to ensure reliable representation. Geneious Pro software was also utilized for various statistical analyses, including composition, GC content, pairwise residue percentage, identical alignment percentage, minimum/maximum/mean lengths, and pairwise numbers (Drummond et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), offering insights into sequence characteristics and similarities. For species identity validation, the consensus sequences were compared to nucleotide databases using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (Altschul et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) ensuring the accuracy of species identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eIn this study, we performed a comprehensive phylogenetic analysis of Phyllanthus species, specifically targeting \u003cem\u003ePhyllanthus maderaspatensis, P. amarus, P. urinaria, P. virgatus\u003c/em\u003e, and \u003cem\u003eP. tenellus\u003c/em\u003e. To construct the phylogenetic tree, we curated extensive datasets using MEGAX (Kumar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Phylogeny Fr (Dereeper et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), amalgamating DNA sequences from two primary sources: sequences generated during our study and sequences retrieved from prior investigations available in the NCBI GenBank database. Our sequence acquisition process encompassed collecting DNA sequences from our own specimens, focusing on specific gene regions like the Internal Transcribed Spacer 2 (ITS2), as dictated by the study's objectives. Concurrently, we diligently gathered relevant sequences from the same gene regions in earlier research accessible in the NCBI GenBank, ensuring the inclusion of a diverse array of Phyllanthus species closely related to our study organisms. This meticulous curation aimed at providing a comprehensive representation of phylogenetic relationships within the Phyllanthus genus.\u003c/p\u003e \u003cp\u003eSubsequent to sequence compilation, we conducted sequence alignments employing either MUSCLE or ClustalW within the MEGAX software suite (Kumar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Following alignment, the construction of the phylogenetic tree was executed utilizing the maximum likelihood (ML) method. The ML method facilitates the estimation of the optimal model for DNA evolution and calculates the likelihood of observing the sequences given this model. The resulting tree was inferred based on the highest likelihood score, offering insights into the most probable evolutionary connections among the Phyllanthus species. To gauge the robustness of the tree, bootstrapping was applied. This technique involved generating numerous replicate datasets by resampling the original sequence alignment and subsequently constructing individual ML trees for each of these resampled datasets. Support values for each branch within the final phylogenetic tree were derived from the frequency with which a specific branch appeared in the resampled trees, often expressed as a percentage. The resultant phylogenetic tree provided invaluable insights into the evolutionary relationships and genetic distances among the studied Phyllanthus species. It contributed significant information pertaining to their diversification, ancestral lineage, and interrelatedness within the broader context of the Phyllanthus genus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDNA Barcoding and ITS2 Secondary Structure Predictions\u003c/h2\u003e \u003cp\u003eIn this study, we employed the Bio-Rad DNA barcode generator, which is accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biorad-ads.com/DNABarcodeWeb\u003c/span\u003e\u003cspan address=\"http://biorad-ads.com/DNABarcodeWeb\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 4 October 2023), to create DNA barcodes for the Phyllanthus genotypes under investigation. These barcodes were meticulously constructed based on the aligned DNA nucleotide sequences acquired through the utilization of ITS2 primers. Additionally, we conducted RNA secondary structure predictions using the nucleotide sequences derived from the same ITS2 primers. This prediction process was facilitated by tapping into the rRNA database hosted on the RNAfoldWebServer v2.4.18 platform, which can be found at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi\u003c/span\u003e\u003cspan address=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 4 October 2023), following the methodology described by Lorenz et al. in 2011. These comprehensive molecular analyses were instrumental in characterizing the Phyllanthus genotypes, offering deep insights into their genetic compositions and structural attributes. Such investigations are pivotal for advancing our understanding of these Phyllanthus species at the molecular level.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhyllanthus Genomic DNA isolation\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe application of the state-of-the-art kit method proved highly effective in extracting genomic DNA from Phyllanthus species samples collected in India, resulting in robust DNA yields. The confirmation of these yields was conducted through nanodrop spectrophotometer analysis, as outlined in Table 1 and depicted in Fig. 2A. The method\u0026apos;s efficiency was further underscored by its successful application in the amplification and sequencing of a well-established amplicon, thereby demonstrating its reliability and suitability for genomic DNA extraction in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInternal Transcribed Spacer 2 (ITS2) region gene amplification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ITS2 gene region, comprising approximately 545 base pairs, was successfully isolated using the ITS2-S2F and ITS4R primers for the genetic identification of Phyllanthus species. Gel electrophoresis clearly revealed that all PCR-treated samples exhibited amplification of the ITS2 region. In the agarose gel, the entire PCR product (25 \u0026mu;L) was mixed with 5 \u0026mu;L of 6X loading dye and loaded (Fig. 2B). The Figure illustrates the amplification of the ITS2 gene, approximately 545 base pairs in length, from a total of 75 insect Phyllanthus species samples collected from diverse locations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNCBI BLASTn and ITS2 database analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the sequencing of the amplified ITS2 regions, the resultant nucleotide sequence data underwent comprehensive analysis using NCBI BLASTn. The results revealed a striking degree of sequence similarity between the samples and known Phyllanthus species. Specifically, \u003cem\u003ePhyllanthus maderaspatensis\u003c/em\u003e exhibited an impressive 98.51% similarity, \u003cem\u003ePhyllanthus amarus\u003c/em\u003e showcased a 98.54% resemblance, \u003cem\u003ePhyllanthus urinaria\u003c/em\u003e displayed an impressive 99.70% congruence, \u003cem\u003ePhyllanthus virgatus\u003c/em\u003e demonstrated a remarkable 99.64% similarity, and \u003cem\u003ePhyllanthus tenellus\u003c/em\u003e manifested an exceptional 100% correspondence, respectively. Query cover, representing the percentage of the query sequence overlapping with the reference sequence, further validated the robustness of these alignments. It\u0026apos;s worth noting that a high identity value within the range of 98-100% typically indicates a substantial sequence similarity. These BLASTn outcomes provide invaluable insights into the quality of the alignment, the potential existence of a biological relationship, and the likelihood that the observed similarity is not merely a product of chance. BLAST leverages statistical theory to furnish bit scores and expect values (E-values) for each alignment pair, further enhancing the depth of analysis and interpretation.\u003c/p\u003e\n\u003cp\u003eThe thorough BLAST analysis utilizing the ITS2 Database has unequivocally established the identity of our Phyllanthus species ITS2 sequence, unequivocally confirming its alignment with known Phyllanthus species. Several key parameters extracted from the analysis underscore the robustness of this match: notably, a Maximum Score of 561, indicative of a strong alignment with a well-documented reference sequence; a Coverage spanning an impressive range of 91% to 98%, signifying substantial overlap between the sequences in question; and an E-Value of 0.0, highlighting the high biological significance of this alignment. These findings were made possible through the extensive and invaluable ITS2 Database, which houses a wealth of data pertaining to the ITS2 region of ribosomal RNA genes. As such, the outcome of this analysis carries an exceptionally high degree of reliability, providing unequivocal evidence of the close genetic relationship that exists among Phyllanthus species.\u003c/p\u003e\n\u003cp\u003eAfter successfully obtaining nucleotide sequences, we have diligently submitted and stored them in the NCBI GenBank Nucleotide Sequence Database. Each submitted sequence has been assigned a distinct accession number for reference purposes, ensuring their traceability and accessibility in subsequent research endeavors. The designated accession numbers for the deposited sequences are as follows: \u003cem\u003ePhyllanthus maderaspatensis\u003c/em\u003e (OQ630483), \u003cem\u003ePhyllanthus amarus\u003c/em\u003e (OQ630476), \u003cem\u003ePhyllanthus urinaria\u003c/em\u003e (OQ630474), \u003cem\u003ePhyllanthus virgatus\u003c/em\u003e (OQ630473), and \u003cem\u003ePhyllanthus tenellus\u003c/em\u003e (OQ625750). These accession numbers serve as unequivocal and exclusive identifiers for each of the sequences in the repository, streamlining their retrieval and utilization in future scientific investigations.Top of Form\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ClustalW alignment of ITS gene sequences in Phyllanthus species genotypes, including Phyllanthus maderaspatensis, Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus virgatus, and Phyllanthus tenellus (Fig. 3). The alignment was conducted to compare and organize the sequences, enabling the identification of conserved regions and variations within the ITS2 region. The figure 3 highlights the high level of similarity observed between the analyzed Phyllanthus species and sequences from the NCBI database during BLASTn searches, indicating the consistency and reliability of the inferred phylogenetic relationships. This consistency validates the genetic affinities among the studied species and aligns with existing genetic data in the NCBI database. The ClustalW alignment is a crucial step in the DNA barcoding process, providing a standardized and robust identification method for Phyllanthus species.\u003c/p\u003e\n\u003cp\u003eFirst, a maximum likelihood tree (MLT) was constructed using a Kimura 2-Parameter (K2P) model with 2000 bootstrap replicates. The high level of similarity observed between the analyzed Phyllanthus species and sequences from the NCBI database during BLASTn searches indicates the consistency and reliability of the inferred phylogenetic relationships. This consistency validates the genetic affinities among the studied species and aligns with existing genetic data in the NCBI database. Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution of Phyllanthus species depicted in fig.4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, a Neighbor-Joining (NJ) analysis was conducted, and the resulting tree topology closely mirrored the structure of the parsimony tree (Figure 4). This concordance between the NJ and parsimony trees is noteworthy as it signifies a high level of agreement between two distinct phylogenetic methods. The NJ method, known for its speed and simplicity in estimating evolutionary relationships, and the parsimony method, which minimizes the number of evolutionary changes needed to explain observed data, both produced similar tree topologies. This consistency between independent analytical approaches reinforces the accuracy and reliability of the phylogenetic analysis, enhancing the confidence in the inferred genetic relationships among the Phyllanthus species under investigation.\u003c/p\u003e\n\u003cp\u003eThe analysis focused on voucher specimens of \u003cem\u003ePhyllanthus maderaspatensis\u003c/em\u003e (OQ630483), \u003cem\u003ePhyllanthus amarus\u003c/em\u003e (OQ630476), \u003cem\u003ePhyllanthus urinaria\u003c/em\u003e (OQ630474), \u003cem\u003ePhyllanthus virgatus\u003c/em\u003e (OQ630473), and \u003cem\u003ePhyllanthus tenellus\u003c/em\u003e (OQ625750). Here are the specific findings for each voucher specimen:\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cem\u003ePhyllanthus maderaspatensis\u003c/em\u003e (OQ630483):\u0026nbsp;This specimen exhibited a high similarity of 98.09% with sequences from the Indian subcontinent, particularly OR073675 (\u003cem\u003ePhyllanthus maderaspatensis\u003c/em\u003e\u003cstrong\u003e_\u003c/strong\u003e India: Dakshina Kannada, Karnataka\u003cstrong\u003e),\u0026nbsp;\u003c/strong\u003eKY079342 (\u003cem\u003eNellica maderaspatensis\u003c/em\u003e\u003cstrong\u003e_\u0026nbsp;\u003c/strong\u003eIndia: Tamil Nadu, Madurai, Tirumangalam), and KF312391 (\u003cem\u003ePhyllanthus maderaspatensis_\u003c/em\u003eMysore:India)\u0026nbsp;. These sequences clustered together in a single clade, indicating their close genetic relatedness. Interestingly, sequences from Austria and France also showed close relationships, all falling within the class Magnoliopsida, family Phyllanthaceae, and genus Phyllanthus.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003ePhyllanthus amarus\u003c/em\u003e (OQ630476):\u0026nbsp;The voucher specimen of \u003cem\u003ePhyllanthus amarus\u003c/em\u003e (OQ630476) displayed the highest similarity of 98.54% with the\u0026nbsp;ON026067 (\u003cem\u003ePhyllanthus amarus_\u003c/em\u003eOdisha: India)\u0026nbsp;sequence from Odisha, India, which was its closest phylogenetic relative. Other Indian sequences from various regions, including Karnataka, Kerala, Kolkata, Tamil Nadu, and Delhi, formed a single clade. Additionally, sequences from Austria, Netherlands, Brazil, Germany, Thailand, Vietnam, the UK, and Saudi Arabia were closely related. All sequences shared the classification of class Malpighiales, family Phyllanthaceae, and genus Phyllanthus.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003ePhyllanthus urinaria\u003c/em\u003e (OQ630474):\u0026nbsp;The voucher specimen of Phyllanthus urinaria (OQ630474) exhibited a remarkable 100% similarity with the KJ135018 (\u003cem\u003ePhyllanthus urinaria\u003c/em\u003e_\u0026nbsp;India: Manipal, Karnataka) sequence, indicating an extremely close genetic relationship. Other sequences from regions such as Karnataka (Mysore and Manipal), Kolkata, Odisha, China, Austria, Thailand, Korea, Netherlands, Brazil, and Germany also clustered together as a single clade. All these sequences shared the same classification within the class Malpighiales, family Phyllanthaceae, and genus Phyllanthus.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003ePhyllanthus virgatus\u003c/em\u003e (OQ630473):\u0026nbsp;The voucher specimen Phyllanthus virgatus (OQ630473) demonstrated a 99.59% similarity with the KJ135019 (\u003cem\u003ePhyllanthus virgatus\u003c/em\u003e_\u0026nbsp;India: Karkala, Karnataka)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esequence from Mysore, Karnataka, making it the closest phylogenetic relative. Additionally, sequences from Taiwan, China, Austria, and Thailand clustered together as a single clade. These sequences shared the same classification within the class Equisetopsida, family Phyllanthaceae, and genus Phyllanthus.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003ePhyllanthus tenellus\u003c/em\u003e (OQ625750):\u0026nbsp;The voucher specimen \u003cem\u003ePhyllanthus tenellus\u003c/em\u003e (OQ625750) exhibited a 100% similarity percentage identity with the KF312396 (\u003cem\u003ePhyllanthus tenellus\u003c/em\u003e_Mysore: India) sequence from Mysore, Karnataka, indicating an identical genetic match. Other sequences from Austria, Taiwan, Netherlands, the UK, and Saudi Arabia also grouped together. All these sequences shared the same classification within the class Malpighiales, family Phyllanthaceae, and genus Phyllanthus.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Barcoding and ITS2 Secondary Structure Predictions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA barcodes generated from the ITS2 sequences provided insights into the genetic variations among the tested Phyllanthus genotypes, including Phyllanthus maderaspatensis (OQ630483), Phyllanthus amarus (OQ630476), Phyllanthus urinaria (OQ630474), Phyllanthus virgatus (OQ630473), and Phyllanthus tenellus (OQ625750) (Fig. 5c,d). Notably, Phyllanthus virgatus (OQ630473) exhibited a relatively longer barcode with a length of 363 base pairs, followed by Phyllanthus maderaspatensis (OQ630483) with 356 base pairs, Phyllanthus urinaria (OQ630474) with 355 base pairs, Phyllanthus urinaria (OQ630474) with 354 base pairs, and Phyllanthus tenellus (OQ625750) with 326 base pairs.\u003c/p\u003e\n\u003cp\u003eFurthermore, the predictions of the ITS2 secondary structures revealed distinct features among the tested Phyllanthus genotypes, represented by variations in the central ring\u0026apos;s helical orientations (Fig. 5e,f). These differences included variations in loop number, position, size, and angle from the centroid in the secondary structures of the Phyllanthus genotypes. Phyllanthus virgatus, in particular, displayed a more complex secondary structure with diverse loop numbers and angles, distinguishing it from the other tested genotypes.\u003c/p\u003e\n\u003cp\u003eThe significance of these findings relates to the potential for developing species-specific primers to identify lesser-known Phyllanthus species more efficiently. The unique genetic structure observed in the conserved nuclear region of ITS2 offers an opportunity to design primers that can accurately differentiate between these species. This has significant implications for preventing adulteration in herbal products, as the ability to distinguish between closely related species is crucial for ensuring the authenticity and quality of herbal remedies. By using DNA barcoding and secondary structure predictions, the study contributes to the development of tools for verifying the identity of Phyllanthus species, thereby addressing concerns related to adulteration and supporting the herbal industry\u0026apos;s integrity and consumer safety.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe imperative for in-depth genetic profiling of Phyllanthus within the Indian subcontinent, emphasized by Sarin et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), stems from the species' constrained geographical distribution. This need is accentuated by the pivotal role Phyllanthus species play in India's herbal trade sector, with an annual trade volume ranging from 2000 to 5000 metric tons, signifying substantial economic importance (Sarin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecognizing the restricted habitat and economic significance of Phyllanthus, this study seeks a nuanced understanding of its molecular characteristics and taxonomic relationships through rigorous genetic investigations. In the realms of ecology, medicine, and potential therapeutic applications, the diverse functions of Phyllanthus species, such as \u003cem\u003eP. maderaspatensis, P. amarus, P. urinaria, P. virgatus\u003c/em\u003e, and \u003cem\u003eP. tenellus\u003c/em\u003e, unfold.\u003c/p\u003e \u003cp\u003eSafeguarding the quality and authenticity of herbal products derived from these species demands robust identification and authentication protocols due to the looming threat of adulteration. With certain species facing endangerment or extinction, conservation efforts become imperative for sustainable utilization.\u003c/p\u003e \u003cp\u003eThis study integrates into advancing our comprehension of the genetic landscape of Indian Phyllanthus species. Its overarching goal extends beyond taxonomy, reaching into conservation and medicinal research, enriching our understanding of these invaluable plants.\u003c/p\u003e \u003cp\u003eIn the initial phase of genetic profiling, successful isolation of genomic DNA from various Phyllanthus species has been accomplished using a kit-based extraction protocol. This crucial step lays the foundation for subsequent molecular analyses, enabling comprehensive exploration of the genetic makeup and diversity within the Phyllanthus genus. The utilization of a standardized kit method ensures efficiency, reliability, and reproducibility, minimizing variability across samples.\u003c/p\u003e \u003cp\u003eEnsuring DNA integrity is critical for subsequent molecular investigations, including PCR amplification and sequencing. The introduction of liquid nitrogen into DNA extraction protocols showcases an innovative methodology that triumphs over conventional challenges. This method emerges as a cutting-edge technique, delivering high-quality genomic DNA efficiently and precisely, addressing practical hindrances associated with diverse Phyllanthus species.\u003c/p\u003e \u003cp\u003eLiquid nitrogen's use highlights its flexibility and adaptability to the intricacies of Phyllanthus species. This method reliably captures the complex molecular subtleties of these plants, creating high-quality genomic material due to its unique properties, including precise destruction of plant cell structures, deactivation of damaging enzymes, and mitigation of DNA damage during extraction.\u003c/p\u003e \u003cp\u003eThe combination of liquid nitrogen-assisted DNA extraction and molecular research on Phyllanthus species opens new avenues for ecological and therapeutic research, increasing the dependability and effectiveness of molecular research. The method's superiority is demonstrated by the lack of smearing in electrophoresis bands and the single absorbance peak at 260 nm in NanoDrop measurements.\u003c/p\u003e \u003cp\u003eThe choice of focusing on the ITS2 gene region for DNA barcoding and systematic analysis is well-founded, given its well-documented exceptional attributes. Successfully amplifying the ITS2 region within Phyllanthus species, validated through a rigorous process, establishes a robust foundation. This achievement is pivotal for species identification, phylogenetic analyses, and aligns seamlessly with established molecular biology methodologies.\u003c/p\u003e \u003cp\u003eThe subsequent gene sequencing enhances the depth of analysis, providing insights into nucleotide composition and sequence variations, forming the basis for comprehensive phylogenetic studies. The integration of these molecular techniques with the ITS2 region advances our understanding of genetic diversity within the Phyllanthus genus, establishing a solid framework for DNA barcoding applications. The success of ITS2 region amplification and sequencing unequivocally validates its efficacy as a molecular marker for genetic profiling and taxonomic authentication of Indian Phyllanthus species.\u003c/p\u003e \u003cp\u003eThe incorporation of secondary structures within ITS2 sequences significantly elevates the accuracy of species delineation, providing crucial insights for cladistic inference of relationships among Phyllanthus species. This choice aligns with the broader consensus in the field, emphasizing the reliability and robustness of ITS2 in unraveling evolutionary connections within eukaryotes.\u003c/p\u003e \u003cp\u003eInvestigations by Xu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and Chen et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) emphasize the efficiency and cost-effectiveness of utilizing ITS2 minibarcodes in comparison to conventional sequencing methods. This scalability, facilitated by emulsion PCR, enables the simultaneous amplification of thousands of short DNA fragments, streamlining the sequencing process for diverse Phyllanthus species.\u003c/p\u003e \u003cp\u003eThe potential limitations of ITS2 in distinguishing closely related species are outweighed by its distinct advantages, as demonstrated in various studies. Notably, Hajibabaei et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) showcased the utility of ITS2 sequences in devising taxon-specific probes for rapid plant recognition, enhancing the practical applications of this gene region.\u003c/p\u003e \u003cp\u003eDespite potential limitations, the ITS2 region stands out for its efficacy in identifying closely related species, especially in botanical and degraded DNA samples. Phylogenetic analysis contributes valuable insights into the minimal genetic variations among Phyllanthus genotypes, emphasizing their close genetic relatedness regardless of geographic origins.\u003c/p\u003e \u003cp\u003eThe NCBI BLAST analysis of Phyllanthus species yields compelling results, indicating remarkable similarity with Phyllanthus species closely associated with the Indian Phyllanthus gene deposits in the NCBI GenBank. The alignment without gaps and in a plus/plus orientation suggests a high degree of sequence conservation and accuracy in the generated data. The observed alignment underscores the robustness of the sequencing methodology employed and the meticulous nature of the subsequent analysis. To contribute to the broader scientific community, sequences generated in this investigation have been deposited in the NCBI GenBank, enhancing the reproducibility and traceability of the study. The deposition of sequences in the NCBI GenBank, along with specific accession numbers, enhances the reproducibility and traceability of the study, promoting transparency and collaboration in genomics research.\u003c/p\u003e \u003cp\u003eThe phylogenetic study of Phyllanthus species through DNA barcoding represents a significant stride in understanding the evolutionary relationships within this diverse genus. This methodological approach, rooted in molecular biology, holds the promise of unraveling the intricate genetic tapestry of Phyllanthus species, contributing valuable insights to taxonomy, conservation, and medicinal research.\u003c/p\u003e \u003cp\u003eThe choice of DNA barcoding for phylogenetic studies is well-founded, drawing inspiration from the pioneering work of Hebert et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) in proposing the use of a standardized DNA sequence for species identification. In the context of Phyllanthus, this methodology provides a robust and reliable means of discerning genetic variations and relationships among different species. Phylogenetic studies often require a marker with sufficient variability to capture distinctions between closely related species. In this regard, the Internal Transcribed Spacer 2 (ITS2) gene region emerges as a prime candidate, owing to its enhanced discriminatory power. The scalability of high-throughput sequencing methods plays a pivotal role in the success of large-scale DNA barcoding initiatives.\u003c/p\u003e \u003cp\u003ePhylogenetic trees constructed based on DNA barcoding data yield valuable insights into the evolutionary context within the genus Phyllanthus. The clear definition of outgroups enhances the understanding of genetic relationships and aids in the identification of key evolutionary branches within the Phyllanthus genus. In the broader context of medicinal and pharmacological research, the phylogenetic study of Phyllanthus species assumes particular significance. Understanding the genetic diversity and relationships among species provides a foundation for exploring the potential therapeutic applications of these plants.\u003c/p\u003e \u003cp\u003eThis study augments the existing knowledge base on Phyllanthus phylogenetics by building upon prior research, notably that of Wurdack et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Wurdack et al. conducted a molecular phylogenetic analysis of Phyllanthaceae, focusing on the Phyllanthoideae subfamily within the broader Euphorbiaceae family. Employing plastid rbcL DNA sequences, the researchers investigated evolutionary relationships and genetic diversity within this plant group.\u003c/p\u003e \u003cp\u003eThis research significantly contributes to our broader understanding of plant evolution and systematics, offering valuable insights into the molecular characteristics of Phyllanthaceae. Moreover, the collaborative nature of scientific inquiry is underscored by references to studies conducted by Manissorn et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), Gyana Ranjan Rout et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), M\u0026uuml;ller et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This collaborative effort reinforces the cumulative progress achieved in unraveling the evolutionary complexities of Phyllanthus species through the application of barcoding techniques.\u003c/p\u003e \u003cp\u003eThe utilization of ITS2 secondary structures for primer design aligns with the broader consensus in molecular biology, as exemplified by studies conducted by Coleman (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). These researchers emphasized the importance of incorporating secondary structure information for enhancing the accuracy of species delineation, a critical aspect illuminated by the present research within the context of Phyllanthus species.\u003c/p\u003e \u003cp\u003eFurthermore, the concept of species-specific primers finds resonance in the work of Hajibabaei et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), where the utility of ITS2 sequences in designing taxon-specific probes for swift plant recognition was demonstrated. This innovative approach not only streamlines identification processes but also adds an extra layer of precision, particularly relevant in the complex taxonomic landscape of Phyllanthus species.\u003c/p\u003e \u003cp\u003eThe potential of DNA barcoding and predictive secondary structure analysis, as showcased in this study, is further validated by the findings of Engelmann et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Their work elucidated the efficacy of ITS2 microarrays in discriminating species with high sequence identities, a testament to the robustness of such methodologies in addressing challenges related to degraded DNA and ensuring the authenticity of herbal products.\u003c/p\u003e \u003cp\u003eIn the broader landscape of genomics research, the pioneering spirit of this study aligns with the transformative potential of high-throughput sequencing methods, as highlighted by Shendure and Ji (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The scalability and efficiency demonstrated in the current research resonate with the broader trends in genomics, emphasizing the need for advanced methodologies to handle large-scale projects effectively.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this investigation underscores the robustness of DNA barcoding, specifically utilizing the ITS2 region, for the precise identification and validation of Phyllanthus species in India. The ribosomal nuclear ITS2 region exhibits substantial levels of divergence, both within and between species, as evidenced by the comprehensive analysis of DNA barcodes and the determination of secondary structures through the minimum free energy principle. The meticulous implementation of DNA barcoding not only ensures the quality of herbal products but also serves as a critical tool in safeguarding Phyllanthus biodiversity and fostering the sustainable utilization of these invaluable plant resources. The genetic relationships and diversity unveiled among Indian Phyllanthus species, elucidated by the construction of a phylogenetic tree based on multiple sequence alignments of the ITS2 gene, provide vital insights that substantiate conservation efforts. This phylogenetic perspective underscores the impact of adulteration on genetic variation within the Phyllanthus genus. The deposited ITS2 gene sequences serve as indispensable genetic references, facilitating future research endeavors and contributing to stringent quality control measures. This, in turn, ensures the genuineness of herbal products derived from Phyllanthus species. Beyond its immediate applications, this study emphasizes the broader significance of accurate identification and conservation of the Phyllanthus genus. This genus encompasses a diverse array of ecologically and medicinally important plants. By addressing prevalent issues of adulteration and misidentification in the market, this research significantly contributes to the sustainable use and protection of Phyllanthus species, unlocking their full therapeutic potential. The findings underscore the paramount importance of integrating advanced molecular techniques in botanical research and conservation strategies, ultimately advancing our understanding of plant biodiversity and supporting the responsible utilization of natural resources.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the Botanical Resource Center and Himalaya Wellness Company for their invaluable assistance and resources, pivotal in facilitating this research. Their dedication to biodiversity preservation and advocacy for sustainable living strongly resonates with our research objectives, playing a pivotal role in the success of this project. This contribution is a significant segment of the first author\u0026apos;s PhD thesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors participated in conceptualizing and designing the study. Ragavendra undertook material preparation, data collection, and analysis. The initial draft of the manuscript was authored by Raghavendra and Gururaj Chalageri, and subsequent versions were reviewed and commented upon by all authors: Raghavendra, Pushpalatha, Gururaj Chalageri, Kannan, and Babu. All authors critically reviewed, contributed to, and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltschul, S. F., Gish, W., Miller, W., Myers, E. W., \u0026amp; Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. doi: 10.1016/S0022-2836(05)80360-2.\u003c/li\u003e\n\u003cli\u003eBhattarai, S., Chandra Ghosh, D., Patra, A., Samanta, A., Mandal, S., Bandyopadhyay, A., \u0026amp; Sen, S. (2019). Adulteration of herbal products: challenges, solutions, and prospects. Journal of Pharmaceutical Analysis, 9(4), 205-211. doi: 10.1016/j.jpha.2018.12.003.\u003c/li\u003e\n\u003cli\u003eChen, S., Pang, X., Song, J., et al. (2014). 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Chemical constituents, pharmacological activities, and clinical applications of genus Phyllanthus. Evidence-Based Complementary and Alternative Medicine, 2020, 3176828. doi: 10.1155/2020/3176828.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\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":"Adulteration, DNA barcode, Genetic relationships, ITS2 region, ITS2 database, NCBI BLASTn, Phyllanthus species, Phylogenetic tree, Secondary structures","lastPublishedDoi":"10.21203/rs.3.rs-3893650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3893650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhyllanthus is a genus of plants that are both ecologically and medicinally valuable. This diversity highlights the need for accurate identification in order to support both conservation efforts and medical research. The escalating demand for Phyllanthus-derived herbal products raises concerns regarding market adulteration and misidentification. In response, our study employs DNA barcoding, specifically targeting the internal transcribed spacer 2 (ITS2) region, to authenticate Indian Phyllanthus species. The study underscores the ITS2 region's efficacy in identifying Indian Phyllanthus species, demonstrating substantial advancements in resolving genus relationships compared to prior analyses. To check if our plant DNA matches known ones, we used two tools: NCBI BLASTn and the ITS2 database. The results showed really high similarities, ranging from 98\u0026ndash;100%. This helps us understand how closely related our plant is to others in the Phyllanthus family. We deposited the genetic data, particularly DNA sequences, of Phyllanthus plants into the NCBI GenBank repository. The construction of a phylogenetic tree through multiple sequence alignment of the ITS2 gene confirms clustering among Phyllanthus species, illuminating genetic relationships and diversity crucial for conservation. The ribosomal nuclear ITS2 region exhibits notable differences within and between species, validated by DNA barcodes and secondary structure analyses using minimum free energy calculations. This study underscores the effectiveness of ITS2-based DNA barcoding in accurately identifying Phyllanthus species, mitigating adulteration concerns, ensuring product quality, preserving biodiversity, and promoting sustainable utilization of these invaluable plant resources.\u003c/p\u003e","manuscriptTitle":"Genetic Profiling and Taxonomic Authentication of Indian Phyllanthus (Phyllanthaceae: Phyllantheae) Genus through Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions: A Comprehensive Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 11:19:24","doi":"10.21203/rs.3.rs-3893650/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","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}}],"origin":"","ownerIdentity":"31dd11c6-650f-4086-9571-1890c935dd9f","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-16T09:18:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-29 11:19:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3893650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3893650","identity":"rs-3893650","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}