DNA Barcoding For Identification Research Articles

Genetic barcodes for species identification and phylogenetic estimation in ghost spiders (Araneae: Anyphaenidae: Amaurobioidinae).

The identification of spider species presents many challenges, since in most cases the characters used are from genital structures that are only fully developed in the adult stage, hence the identification of immatures is most often not possible. Additionally, these structures usually also present some intra-specific variability, which in some cases makes the identification of closely related species difficult. The genetic barcode technique (DNA barcodes), based on sequencing of the mitochondrial marker cytochrome c oxidase subunit I (COI ), has proven a useful, complementary tool to overcome these limitations. In this work, the contribution of DNA barcoding to the taxonomy of the subfamily Amaurobioidinae is explored using the refined single linkage analysis (RESL) algorithm for the delimitation of operational taxonomic units (OTUs), in comparison with the assemble species by automatic partitioning (ASAP) algorithm, and presented in conjunction with an updated molecular phylogenetic analysis of three other markers (28S rRNA, 16S rRNA, Histone H3 ), in addition to COI . Of a total of 97 included species identified by morphology, 82 species were concordant with the operational taxonomic units obtained from RESL, representing an 85% correspondence between the two methods. Similar results were obtained using the ASAP algorithm. Previous observations of morphological variation within the same species are supported, and this technique provides new information on genetic structure and potentially cryptic species. Most of the discrepancies between DNA barcoding and morphological identification are explained by low geographic sampling or by divergent or geographically structured lineages. After the addition of many specimens with only COI data, the multi-marker phylogenetic analysis is consistent with previous results and the support is improved. The markers COI , closely followed by 28S , are the most phylogenetically informative. We conclude that the barcode DNA technique is a valuable source of data for the delimitation of species of Amaurobioidinae, in conjunction with morphological and geographic data, and it is also useful for the detection of cases that require a more detailed and meticulous study.

DNA barcode identification of a tropical liver fluke (Fasciola gigantica) in cattle from Oyo, southwestern Nigeria

BackgroundFascioliasis is a major parasite illness that affects ruminants, both domesticated and free, and has an impact on public health and animals’ productivity. The genetic diversity of Fasciola species in cattle from Oyo, Oyo State, Nigeria is yet not well understood. In this study, the genetic diversity of Fasciola gigantica in slaughtered cattle in Oyo was examined using a molecular-based approach targeting the mitochondrial cytochrome C oxidase subunit I (COI) gene region.ResultsOne hundred flukes were gathered from slaughtered cattle, and their COI gene sequences were analyzed using maximum likelihood methods. The results of phylogenetic analysis showed genetic similarities between Nigerian F. gigantica and isolates from Algeria, Egypt, Iran, Sudan, China, Japan and Nigeria. The predominant DNA substitution was the A to T transversion, while the least common substitution was the G to A transition.ConclusionOur results show how useful the COI gene region is for examining intraspecific differences between F. gigantica isolates. The genetic similarity observed among the sampled F. gigantica populations suggests the value of mitochondrial DNA sequences as a marker for the accurate identification and characterization of Fasciola species across different ruminants.

Classification, identification, and DNA barcoding study for common cockroach species (Dictyoptera: Blattaria) from China

Cockroaches are well-known pests and quarantined organisms worldwide. Due to morphological diversity and a lack of molecular data, their classification and identification are facing challenges. This study performed classification, identification, and DNA barcoding for cockroaches collected from China. Seventy-six samples were morphologically identified as seven species of two superfamilies that included Blattella germanica, Eublaberus posticus and Blaptica dubia belonging to the superfamily Blaberoidea, and Periplaneta americana, Periplaneta lateralis, Periplaneta fuliginosa and Periplaneta australasiae belonging to the superfamily Blattoidea. Based on sequence alignments of nine ribosomal and mitochondrial genes across the order Blattaria retrieved from GenBank, rDNA ITS2-517 bp and mtDNA 16S-327 bp were screened as candidates for molecular identification. Universal primers were designed for PCR amplification, cloning, and sequencing of the 37 representative samples. Sequence alignments and phylogeny analysis showed that both ITS2 and 16S confirmed samples 1–9, 20–24, and 25–29 as B. germanica, P. americana, and P. lateralis, respectively; only 16S (not ITS2) confirmed samples 10–14, 15–19, 30–34, and 35–37 as E. posticus, Blap. dubia, P. fuliginosa, and P. australasiae, respectively, indicating that 16S was a better target than ITS2 for molecular identification of cockroaches. Conservative motif and divergence analysis further revealed that ITS2 sequences vary significantly among different taxa, whereas 16S sequences are relatively conserved. There is an obvious barcoding gap between maximum intraspecific divergence and minimum interspecific divergence (2.57 % vs. 5.62 %) for ITS2, but not for 16S (6.15 % vs. 2.63 %). Therefore, it was confirmed that ITS2 is an ideal DNA barcode for molecular identification of cockroaches at lower category.

Applications and challenges of DNA barcoding in rapid radiation groups: Rhodiola (Crassulaceae) as a case study

ObjectiveRhodiolae Crenulatae Radix et Rhizoma (Hongjingtian in Chinese, RCRR), the roots and rhizomes of Rhodiola crenulata and its application in the medicinal market is very chaotic. In this study, DNA barcoding database and identification engine of Rhodiola species were established, decoction pieces from the medicinal market were identified, and the application and challenges of DNA barcoding in the rapid radiation of Rhodiola species were analyzed. This study provides reference for the protection, rational development, and utilization of endangered resources within Rhodiola species. MethodsA total of 50 original plant samples from 20 species of the genus Rhodiola from Hebei, Xinjiang, Tibet, Jilin, and other major production areas were collected. Theses samples cover the typical distribution area (Qinghai-Tibetan Platea) of Rhodiola species and other scattered alpine regions (Changbai Mountain, Taibai Mountain, Lushan Mountain, etc.), it encompasses all Rhodiola species with thick rhizomes in China. ITS2 and psbA-trnH barcode of Rhodiola database (BORD) were established and an identification engine named Rhodiola-IDE was developed. The stability and accuracy of the standard DNA barcoding database were evaluated using two datasets. Rhodiola-IDE identified 31 decoction pieces of RCRR from the medicinal material market. ResultsThe BORD containing 1 532 sequences of 88 Rhodiola species has been established, and the identification efficiency results showed good accuracy and stability. According to the Chinese Pharmacopoeia (2020 edition), 23 samples (74.2%) were identified as authentic R. crenulata, while the rest of the marketed varieties were R. kirilowii, R. dumulosa, and R. fastigiata. The product label “Larger flower, Hongjingtian” was identified as R. crenulata. Samples labeled as “Smaller flower, Hongjingtian” were identified as R. crenulata, R. kirilowii, and R. fastigiata. ConclusionITS2 and psbA-trnH barcodes can identify monophyletic groups represented by R. crenulata. However, for non-monophyletic species, it is necessary to collect as many samples as possible and combine them with multiple markers for joint identification. This study discussed the application and challenges of DNA barcodes in Rhodiola under rapid radiation conditions, providing a scientific basis for the rational development and utilization of Rhodiola varieties.

Identification and characterization of a new species of Taxus — Taxus qinlingensis by multiple taxonomic methods

BackgroundThe taxonomy of Taxus Linn. remains controversial due to its continuous phenotypic variation and unstable topology, thus adversely affecting the formulation of scientific conservation strategies for this genus. Recently, a new ecotype, known as Qinling type, is mainly distributed in the Qinling Mountains and belongs to a monophyletic group. Here, we employed multiple methods including leaf phenotype comparison (leaf shapes and microstructure), DNA barcoding identification (ITS + trnL-trnF + rbcL), and niche analysis to ascertain the taxonomic status of the Qinling type.ResultsMultiple comparisons revealed significant differences in the morphological characters (length, width, and length/width ratio) among the Qinling type and other Taxus species. Leaf anatomical analysis indicated that only the Qinling type and T. cuspidata had no papilla under the midvein or tannins in the epicuticle. Phylogenetic analysis of Taxus indicated that the Qinling type belonged to a monophyletic group. Moreover, the Qinling type had formed a relatively independent niche, it was mainly distributed around the Qinling Mountains, Ta-pa Mountains, and Taihang Mountains, situated at an elevation below 1500 m.ConclusionsFour characters, namely leaf curvature, margin taper, papillation on midvein, and edges were put forward as primary indexes for distinguishing Taxus species. The ecotype Qingling type represented an independent evolutionary lineage and formed a unique ecological niche. Therefore, we suggested that the Qingling type should be treated as a novel species and named it Taxus qinlingensis Y. F. Wen & X. T. Wu, sp. nov.

Morphological Development and DNA Barcoding Identification of Pholis fangi Larvae and Juveniles in the Yellow Sea

Pholis fangi is a small, bottom-dwelling fish species endemic to the Yellow Sea and Bohai Sea in China. While previous research has focused on its population biology and molecular structure, little is known about the early developmental stages of this species. In this study, larval and juvenile P. fangi specimens were collected from the Sheyang Sea Area, Jiangsu Province, in 2017. Morphological features were examined using microscopy, and DNA barcoding was conducted to confirm species identification. The research documented detailed changes in yolk sac, fin development, and melanophore distribution patterns across larval and juvenile stages of P. fangi. Comparative analysis with other Pholis species revealed that melanophore distribution is a key distinguishing characteristic, allowing effective differentiation between larval and juvenile stages, as well as between Pholis species. This study provides valuable insights into the early life history of P. fangi, contributing to a better understanding of the genus Pholis. The findings demonstrate the utility of combining traditional morphological observation and molecular techniques for accurate species identification, particularly during the critical larval and juvenile developmental phases.

A study on the intertidal animal diversity of Xia-Sanheng Island by using morphological and DNA barcoding identification

Intertidal animals are an essential part of the ecosystem, and species diversity can reflect the state of the local ecological environment. However, traditional morphological identification is prone to corresponding classification errors or research limitations. With the development of molecular biology, many techniques and bioinformatics classification methods have been applied to identify species efficiently in recent years. This research aimed to examine the feasibility of DNA barcoding within the identification of intertidal animal species collected from the Xia-Sanheng Island. A total of 41 cox1 gene sequences were obtained by experimentalization or downloaded from the National Center for Biotechnology Information (NCBI) database. Then morphological classification, molecular identification, phylogenetic tree analysis, and Automatic Barcode Gap Discovery (ABGD) method were used to analyze the animal samples. We found that although the molecular classification of molluscs was not accurate enough, the collected specimens could be divided into 15 species, 12 families, and 4 phyla. Tetraclita japonica, Thais luteostoma, and Mytilus coruscus were the dominant species on the Xia-Sanheng Island. Additionally, there may be a new species, Platynereis sp., which needs further confirmation. We suggest that further identification of marine biodiversity should be carried out by combining morphological and molecular biological methods.

Axenic Culture and DNA Barcode Identification of Wood Decay Fungi from the Maltese Islands

Wood-decaying fungi are important study subjects for their ecological role as well as for their biotechnological applications. They break down lignin, cellulose, and hemicelluloses using enzymes that modify the chemical structure of these complex macromolecules. Due to their ability to degrade wood, these fungi can create structural damage to wooden structures and to trees, especially those with very low level of fitness. Previous studies on wood decay fungi in the Maltese Islands are limited to records and checklists described by a handful of authors. The aim of this study was to provide a comprehensive description of wood decay fungal diversity in the Maltese Islands including an updated checklist based on DNA barcoding, as well as to establish the first wood-decay fungal culture collection at the Biology Department Seed Bank of the University of Malta. Several surveys were carried out during the rainy season along wooded areas of the Maltese Islands as well as in historical gardens. Isolates were identified using macro- and micro-morphological features, dichotomous keys, as well as molecular data. Basidiomes were recorded growing on 14 different host plant species, 11 axenic cultures have been made and 9 species of wood decay fungi have been conclusively identified by DNA barcoding. The collection of the axenic isolates includes one of Aurificaria cf. euphoria, three of Ganoderma resinaceum sl., two of Laetiporus sulphureus, one of Inonotus sp., one of Inonotus rickii anamorph, one of Inocutis tamaricis, one of Stereum hirsutum, and one of Pleurotus eryngii. However, the mycelium of Coriolopsis gallica, though collected and identified, could not be isolated.

Molecular identification of DNA barcoding of Leguminous toxic species and quantitative analysis by ELISA kits

Molecular identification of DNA barcoding of Leguminous toxic species and quantitative analysis by ELISA kits

Evaluating the reliability of DNA Barcoding for Central American Pacific shallow water echinoderms identification: a molecular taxonomy and database accuracy analysis

Introduction: Molecular divergence thresholds have been proposed to distinguish recently separated evolutive units, often displaying more accurate putative species assignments in taxonomic research compared to traditional morphological approaches. This makes DNA barcoding an attractive identification tool for a variety of marine invertebrates, especially for cryptic species complexes. Although GenBank and the Barcode of Life Data System (BOLD) are the major sequence repositories worldwide, very few have tested their performance in the identification of echinoderm sequences.
 Objective: We use COI echinoderm sequences from local samples and the molecular identification platforms from GenBank and BOLD, in order to test their accuracy and reliability in the DNA barcoding identification for Central American shallow water echinoderms, at genus and species level.
 Methods: We conducted sampling, tissue extraction, COI amplification, sequencing, and taxonomic identification for 475 specimens. The 348 obtained sequences were individually enquired with BLAST in GenBank as well as using the Identification System (IDS) in BOLD. Query sequences were classified depending on the best match result. McNemar’s chi-squared, Kruskal-Wallis’s and Mann-Whitney’s U tests were performed to prove differences between the results from both databases. Additionally, we recorded an updated list of species reported for the shallow waters of the Central American Pacific.
 Results: We found 324 echinoderm species reported for Central American Pacific shallow waters. Only 118 and 110 were present in GenBank and BOLD databases respectively. We proposed 325 solved morphology-based identities and 21 provisional identifications in 50 putative taxa. GenBank retrieved 348 molecular-based identifications in 58 species, including twelve provisional identifications in tree taxa. BOLD recovered 170 COI identifications in 23 species with one provisional identification. Nevertheless, 178 sequences retrieved unmatched terms (in 34 morphology-based taxa). Only 86 sequences (25 %) were retrieved as correct identifications and 128 (37 %) as identification errors in both platforms. We include 84 sequences for eleven species not represented in GenBank and 65 sequences for ten species in BOLD Echinoderm COI databases. The identification accuracy using BLAST (175 correct and 152 incorrect identifications) was greater than with IDS engine (110 correct and 218 identification errors), therefore GenBank outperforms BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001).
 Conclusions: Additional echinoderm sample references are needed to improve the utility of the evaluated DNA barcoding identification tools. Identification discordances in both databases may obey specific parameters used in each search algorithm engine and the available sequences. We recommend the use of barcoding as a complementary identification source for Central American Pacific shallow water echinoderm species.

DNA barcoding of herbal supplements on the US commercial market associated with the purported treatment of COVID-19.

The COVID-19 pandemic was associated with an increased global use of traditional medicines, including Ayurvedic herbal preparations. Due to their growing demand, their processed nature, and the complexity of the global supply chain, there is an increased risk of adulteration in these products. The objective of this study was to assess the use of DNA barcoding for species identification in herbal supplements on the US market associated with the Ayurvedic treatment of respiratory symptoms. A total of 54 commercial products containing Ayurvedic herbs were tested with four DNA barcoding regions (i.e., rbcL, matK, ITS2, and mini-ITS2) using two composite samples per product. Nine categories of herbs were targeted: amla, ashwagandha, cinnamon, ginger, guduchi, tribulus, tulsi, turmeric, and vacha. At least one species was identified in 64.8% of products and the expected species was detected in 38.9% of products. Undeclared plant species, including other Ayurvedic herbs, rice, and pepper, were detected in 19 products, and fungal species were identified in 12 products. The presence of undeclared plant species may be a result of intentional substitution or contamination during harvest or processing, while fungal DNA was likely associated with the plant material or the growing environment. The greatest sequencing success (42.6-46.3%) was obtained with the matK and rbcL primers. The results of this study indicate that a combination of genetic loci should be used for DNA barcoding of herbal supplements. Due to the limitations of DNA barcoding in identification of these products, future research should incorporate chemical characterization techniques.

A Measure of the DNA Barcode Gap for Applied and Basic Research.

DNA barcoding has largely established itself as a mainstay for rapid molecular taxonomic identification in both academic and applied research. The use of DNA barcoding as a molecular identification method depends on a "DNA barcode gap"-the separation between the maximum within-species difference and the minimum between-species difference. Previous work indicates the presence of a gap hinges on sampling effort for focal taxa and their close relatives. Furthermore, both theory and empirical work indicate a gap may not occur for related pairs of biological species. Here, we present a novel evaluation approach in the form of an easily calculated set of nonparametric metrics to quantify the extent of proportional overlap in inter- and intraspecific distributions of pairwise differences among target species and their conspecifics. The metrics are based on a simple count of the number of overlapping records for a species falling within the bounds of maximum intraspecific distance and minimum interspecific distance. Our approach takes advantage of the asymmetric directionality inherent in pairwise genetic distance distributions, which has not been previously done in the DNA barcoding literature. We apply the metrics to the predatory diving beetle genus Agabus as a case study because this group poses significant identification challenges due to its morphological uniformity despite both relative sampling ease and well-established taxonomy. Results herein show that target species and their nearest neighbor species were found to be tightly clustered and therefore difficult to distinguish. Such findings demonstrate that DNA barcoding can fail to fully resolve species in certain cases. Moving forward, we suggest the implementation of the proposed metrics be integrated into a common framework to be reported in any study that uses DNA barcoding for identification. In so doing, the importance of the DNA barcode gap and its components for the success of DNA-based identification using DNA barcodes can be better appreciated.

DNA barcoding and morphological identification of Astragalus in Iran: Advantages to molecular approach

Astragalus is a crucial forage plant in the rangelands of northeastern Iran. However, identifying different species of Astragalus can be challenging due to their overlapping morphological features. In this study, we aimed to determine the relationships between 12 Astragalus species using both morphological and molecular traits. We also evaluated the effectiveness of DNA barcoding as a tool for identifying rangeland species of Astragalus in Northeast Iran. We examined five DNA barcodes, including three cpDNA regions (trnH-psbA, rpl32-trnL(UAG), and matK) and two nuclear sequences (ITS and ETS). We found that the plastid markers (rpl32-trnL(UAG, matK) were the most effective in differentiating between species. We also found that morphological data, as represented by the Neighbor-Net network and UPGMA dendrogram, had the potential to separate Astragalus species. We identified matK as the best and most accurate marker for barcoding, as trnH-psbA had some defects due to sequence size and alignment issues. Our study highlights the importance of using barcoding for quick and accurate recognition of plant species and shows the plastid markers were the most effective in differentiating between species.

DNA barcode identification of cultivated and wild tropical fruit species.

The online version contains supplementary material available at 10.1007/s13205-023-03848-w.

DNA Barcoding for Identification and Discovery of Fish Species in the Protected Mangroves of Hormozgan, Iran

DNA Barcoding for Identification and Discovery of Fish Species in the Protected Mangroves of Hormozgan, Iran

Application of Pb-Resistant Bacteria to Reduce Pb-Accumulation in Brassica sp. on Pb-Contaminated Soil

Brassica sp. is a horticultural crop with high demand for consumption. To meet the demand, farmers apply intensive farming to increase yields and prevent plant pests and diseases that cause yield loss. Agrochemical applications in the form of inorganic fertilizers and pesticides contribute to lead (Pb) contamination in agricultural soils and increase lead (Pb) content in the cultivated plants. Nowadays, using bacteria for remediation (bioremediation) is environmentally friendly and effective in cleaning pollutants by converting organic Pb into inorganic Pb which is less toxic. This study aims to explore Pb-resistant bacteria that can reduce Pb-accumulation on food crops such as Brassica sp. grown in Pb-contaminated soil. This study isolated 15 isolates that survived on nutrient agar containing 1,000 mg/L Pb(NO3)2. The study showed 2 potential Pb-resistant bacteria that reduced Pb accumulation in Brassica sp. up to 30.5%. The bacteria are gram-positive bacilli and non-human pathogens (PT-3 and PT-5). DNA barcode identification results showed the isolates identified as Bacillus altitudinis (PT-3) and Bacillus wiedmannii (PT-5). Aplication of the bacteria increases the shoot length and fresh weight of Brassica sp. Application of the bacteria improves food crops quality by reducing heavy metals accumulation, such as Pb. Thus, the bacteria are potential as biofertilizers for reducing agrochemicals use in intensive agriculture areas and preventing environmental destruction and food contamination. Keywords: Bioremediation, Brassica sp., Inorganic Fertilizers, Lead-Resistant Bacteria, Pesticides.

Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence

Plants of the genus Physalis are of economic interest because of their fleshy edible fruits with high nutritional value. Some species have high medicinal value with a long history of ethno-medicinal use to treat diverse diseases. There is therefore a need to correctly discriminate the different species of Physalis for proper utilization. Although most Physalis species have unique morphologies, their vegetative stages are identical, making it difficult to accurately identify them based on morphological characteristics. DNA barcoding has the potential to discriminate species accurately. In this study, ribulose bisphosphate carboxylase large (rbcL) and internal transcribed spacer 2 (ITS2) regions were used to discriminate Physalis species and to reveal their phylogenetic relationships and genetic diversity. Physalis plant samples were collected from seven counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. Genomic DNA was isolated from leaf samples of 64 Physalis accessions and used for PCR amplification and the sequencing of rbcL and ITS2 barcode regions. The discriminatory ability of the barcodes was based on BLASTn comparison, phylogenetic reconstruction and cluster analysis, and the determination of inter- and intra-specific distances. The nucleotide polymorphism, genetic diversity and distance of the identified Physalis species were determined using DnaSP and MEGA 11.0 software. Species discrimination was more robust using ITS2 sequences. The species identified and discriminated by ITS2 sequences were Physalis purpurea, Physalis peruviana and Physalis cordata. The rbcL sequences were only able to identify Physalis to the genus level. There was high interspecific and low intraspecific divergence within the identified Physalis species based on ITS2 sequences. The ITS2 barcode is an ideal DNA barcode for use in the discrimination of species, as well as in genetic diversity studies of Physalis accessions in Kenya.

DNA barcoding of marine fish species in the waters surrounding Hainan Island, northern South China Sea

IntroductionHainan Island is encompassed within the shallow waters of the tropical continental shelf in China, which is recognized as a significant hotspot for fish biodiversity. Despite extensive research conducted on marine fish taxonomy surrounding Hainan Island, there remains a substantial gap between our current understanding and the actual fish diversity within this oceanic area.MethodsIn this study, we employed DNA barcoding and molecular identification approaches to explore the species diversity and distribution pattern of marine fish in both the northern and southern sea areas of Hainan Island in the northern South China Sea.ResultsA total of 186 sequences were obtained from the collected marine fish samples in the two sea areas surrounding Hainan Island. Through DNA identification, it was confirmed that all 186 sequences corresponded to typical fish species found in the northern South China Sea, all sequences represented a total of 56 species, 47 genera, 34 families, and 17 orders. The average Kimura 2-parameter (K2P) distances within species, genus, family, order and class were 0.15%, 6.53%, 13.17%, 16.95% and 24.81%, respectively.DiscussionOur investigation in the northern sea areas of Hainan Islands identified a total of 33 distinct species, while the southern sea areas exhibited 29 distinct species, with only 5 species found to be shared between both regions. These findings clearly indicate a significant disparity in the species composition of fish communities between the northern and southern sea regions.

First record of Leptopilina japonica Novković & Kimura, 2011 (Hymenoptera: Figitidae) in Germany, a parasitoid of the Spotted Wing Drosophila Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae)

AbstractTwo years after the first European record in Italy, we report the first occurrence of the parasitoid wasp Leptopilina japonica Novković & Kimura, 2011 (Hymenoptera: Figitidae) in Germany. The species is a larval‐pupal parasitoid of Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae), which is a widespread invasive and economically important pest of soft‐skinned fruit. In total, we found 29 specimens of L. japonica in five different locations in southern and western Germany in the years 2021, 2022 and 2023. We examined the specimens morphologically and generated their DNA barcodes for identification. In three of the locations, L. japonica was sampled from raspberries. In two locations, L. japonica was caught in two and three consecutive years, respectively, which indicates adventive establishment. As D. suzukii and L. japonica originate from the same region in Asia, the possible establishment of L. japonica could be a case of unintentional biological control in Germany. In addition to this first record in Germany, we present a diagnosis of L. japonica to distinguish the species from the rest of the European Leptopilina fauna.

Morpho-anatomical, Molecular, and Chemical Standardization of Trillium govanianum Wall. ex D. Don: An Endangered Medicinal Herb Native to the Himalayas

Background Trillium govanianum Wall. ex D. Don occurring at high altitudes in the Himalayas and known by the common name ‘Nag Chhatri,’ has high medicinal value. Its rhizome samples have substantial therapeutic potential and possess anti-cancer and anti-aging properties. Due to similar common names and morphology, there is an issue with adulteration and misidentification of actual rhizome samples with rhizomes of other species. So, there is a need to develop valid scientific methods to identify raw herbal samples of authentic ‘Nagchatri’ ( T. govanianum). Objectives The present study focused on developing botanical, molecular, and chemical standards for identifying raw herbal samples of T. govanianum. Materials and Methods Samples were collected from four locations in the Northwestern Himalayas. Botanical characters were studied by stereomicroscope and compound microscope. Molecular identification was done by DNA barcoding and chemical identification by chemical fingerprinting (TLC and HPTLC), LC-MS profiling, and NMR studies. Results The botanical characters involved detailed morpho-anatomical characterization of herbal samples. The DNA barcoding exhibited a 100% identification match of generated barcode sequences with NCBI database sequences for all the selected markers. Chemical profiling revealed diosgenin, makisterone A, and 20-hydroxy ecdysone as major constituents in the rhizome sample, confirming the authenticity of the species. Conclusion The integrated authentication approach employing botanical standardization, DNA barcoding, and chemical profiling will be a promising tool for accurately identifying the genuine raw herbal material of T. govanianum.