Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

BACKGROUND: Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. MATERIALS AND METHODS: We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. RESULTS: The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. CONCLUSIONS: We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

The complete plastomes of seven Peucedanum plants: comparative and phylogenetic analyses for the Peucedanum genus.

BACKGROUND: The Peucedanum genus is the backbone member of Apiaceae, with many economically and medically important plants. Although the previous studies on Peucedanum provide us with a good research basis, there are still unclear phylogenetic relationships and many taxonomic problems in Peucedanum, and a robust phylogenetic framework of this genus still has not been obtained, which severely hampers the improvement and revision of taxonomic system for this genus. The plastid genomes possessing more variable characters have potential for reconstructing a robust phylogeny in plants. RESULTS: In the current study, we newly sequenced and assembled seven Peucedanum plastid genomes. Together with five previously published plastid genomes of Peucedanum, we performed a comprehensively comparative analyses for this genus. Twelve Peucedanum plastomes were similar in terms of genome structure, codon bias, RNA editing sites, and SSRs, but varied in genome size, gene content and arrangement, and border of SC/IR. Fifteen mutation hotspot regions were identified among plastid genomes that can serve as candidate DNA barcodes for species identification in Peucedanum. Our phylogenetic analyses based on plastid genomes generated a phylogeny with high supports and resolutions for Peucedanum that robustly supported the non-monophyly of genus Peucedanum. CONCLUSION: The plastid genomes of Peucedanum showed both conservation and diversity. The plastid genome data were efficient and powerful for improving the supports and resolutions of phylogeny for the complex Peucedanum genus. In summary, our study provides new sights into the plastid genome evolution, taxonomy, and phylogeny for Peucedanum species.

Dynamic Chloroplast Genome Rearrangement and DNA Barcoding for Three Apiaceae Species Known as the Medicinal Herb "Bang-Poong".

Three Apiaceae species Ledebouriella seseloides, Peucedanum japonicum, and Glehnia littoralis are used as Asian herbal medicines, with the confusingly similar common name "Bang-poong". We characterized the complete chloroplast (cp) genomes and 45S nuclear ribosomal DNA (45S nrDNA) sequences of two accessions for each species. The complete cp genomes of G. littoralis, L. seseloides, and P. japonicum were 147,467, 147,830, and 164,633 bp, respectively. Compared to the other species, the P. japonicum cp genome had a huge inverted repeat expansion and a segmental inversion. The 45S nrDNA cistron sequences of the three species were almost identical in size and structure. Despite the structural variation in the P. japonicum cp genome, phylogenetic analysis revealed that G. littoralis diverged 5-6 million years ago (Mya), while P. japonicum diverged from L. seseloides only 2-3 Mya. Abundant copy number variations including tandem repeats, insertion/deletions, and single nucleotide polymorphisms, were found at the interspecies level. Intraspecies-level polymorphism was also found for L. seseloides and G. littoralis. We developed nine PCR barcode markers to authenticate all three species. This study characterizes the genomic differences between L. seseloides, P. japonicum, and G. littoralis; provides a method of species identification; and sheds light on the evolutionary history of these three species.

A Single Standard to Determine Multi-Components Method Coupled with Chemometric Methods for the Quantification, Evaluation and Classification of Notopterygii Rhizoma et Radix from Different Regions.

An ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry is used to identify 33 compounds in Notopterygii rhizoma and radix, after which a single standard to determine multi-components method is established for the simultaneous determination of 19 compounds in Notopterygii rhizoma and radix using chlorogenic acid and notopterol as the internal standard. To screen the potential chemical markers among Notopterygii rhizoma and radix planted in its natural germination area and in others, the quantitative data of 19 compounds are analyzed via partial least-squares discriminant analysis (PLS-DA). Depending on the variable importance parameters (VIP) value of PLS-DA, six compounds are selected to be the potential chemical markers for the discrimination of Notopterygii rhizoma and radix planted in the different regions. Furthermore, the Fisher's discriminant analysis is used to build the models that are used to classify Notopterygii rhizoma and radix from the different regions based on the six chemical markers. Experimental results indicate that Notopterygii rhizoma and radix planted in the Sichuan province are distinguished successfully from those in other regions, reaching a 96.0% accuracy rating. Therefore, a single standard to determine multi-components method combined with a chemometrics method, which contains the advantages such as simple, rapid, economical and accurate identification, offers a new perspective for the quantification, evaluation and classification of Notopterygii rhizoma and radix from the different regions.

[Collection and sorting of medicinal plants in Chinese Apiaceae( Umbelliferae)].

The family Apiaceae( Umbelliferae) includes some of the world's most important medicinal plants,with more than 100 species recorded in the traditional Chinese medicine,of which more than ten species are commonly used medicinal materials. However,due to morphological similarities,high market demands and regional factors,substitutes and adulterants are often mixed with genuine in the medicinal market. Therefore,a comprehensive sorting for these poorly known plants has been done in this study by combining market survey with literature review,including its species,distribution,price and substitutes. According to the statistics,there are 65 genera and 262 species of medicinal plants of Apiaceae in China,with medicinal part mostly from radix and rhizoma. Sichuan province is the most abundant in distribution and planting resources,with about 137 species,followed by Yunnan,Hubei and Gansu provinces.Furthermore,we summarized the genuine and substitutes of 11 medicinal plants,e. g. Bupleurum,Angelica and Peucedanum etc.,which found that the medicinal plants of Apiaceae were substituted or mixed in different taxonomic ranks. This study would contribute to reduce the risk of medicine misuse,as well as explore other plants of Apiaceae with potential medicinal value,to achieve sustainable development of related industries.

Rampant polyphyly in the Arracacia clade (Apiaceae) and an assessment of the phylogenetic utility of 20 noncoding plastid loci.

The Arracacia clade (Apiaceae, Apioideae) is a heterogeneous assemblage of 12 genera, comprising 111 known species distributed in high montane temperate and sub-alpine habitats of meso- and South America. Previous studies have indicated that the genera Arracacia, Coulterophytum, and Prionosciadium are polyphyletic, but for the most part relationships among the members of the clade are largely unknown. Initially, cladistic analyses of nrDNA ITS sequences were carried out on 212 accessions (122 taxa), representing 92 species of the Arracacia clade and outgroups from the closely-related páramo genera Cotopaxia, Niphogeton, and Perissocoeleum and members of the Perennial Endemic North American clade and its allies. Using the ITS results to inform sampling of a small subset of taxa, a pilot study examining the phylogenetic utility of 20 noncoding chloroplast loci was subsequently performed to identify those regions most useful at resolving relationships. A cost-benefit analysis determined that five loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, psbD-trnT, ndhA intron) would maximize resolution and branch support in the clade. Cladistic analyses of four of these loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, ndhA intron) and the ITS region, separately and combined, revealed that Arracacia, Coaxana, Coulterophytum, Prionosciadium, and Rhodosciadium are each polyphyletic and that Donnellsmithia and Myrrhidendron are each monophyletic. Although most relationships in the Arracacia clade and among the closely-related genera Cotopaxia, Niphogeton, and Perissocoeleum are poorly resolved and supported, ten groups are recognized for future revisionary studies. Polyploidy and rapid species radiation have likely confounded generic circumscriptions and interpretation of relationships.

Genome-wide searches and molecular analyses highlight the unique evolutionary path of flavone synthase I (FNSI) in Apiaceae.

Flavone synthase is a key enzyme for flavone biosynthesis and is encoded by two gene families: flavone synthase I (FNSI) and flavone synthase II (FNSII). FNSII is widely distributed in plants, while FNSI has been reported in rice (Oryza sativa) and seven species of Apiaceae. FNSI has likely evolved from the duplication of flavanone 3ß-hydroxylase (F3H). In this study, we used multiple bioinformatics tools to identify putative FNSI and F3H genes from 42 publicly available genome and transcriptome datasets. Results showed that rice FNSI does not share a common ancestral sequence with other known FNSI genes and that FNSI is absent from species outside of Apiaceae. Positive selection site identification analysis revealed that four sites within the FNSI tree branches of Apiaceae evolved under significant positive selection. The putative F3H genes identified in this study provide a valuable resource for further function analysis of flavone synthase.

Rapid discrimination of different Apiaceae species based on HPTLC fingerprints and targeted flavonoids determination using multivariate image analysis.

INTRODUCTION: Species of Apiaceae are used in folk medicine as spices and in officinal medicinal preparations of drugs. They are an excellent source of phenolics exhibiting antioxidant activity, which are of great benefit to human health. Discrimination among Apiaceae medicinal herbs remains an intricate challenge due to their morphological similarity. OBJECTIVE: In this study, a combined "untargeted" and "targeted" approach to investigate different Apiaceae plants species was proposed by using the merging of high-performance thin layer chromatography (HPTLC)-image analysis and pattern recognition methods which were used for fingerprinting and classification of 42 different Apiaceae samples collected from Egypt. METHODOLOGY: Software for image processing was applied for fingerprinting and data acquisition. HPTLC fingerprint assisted by principal component analysis (PCA) and hierarchical cluster analysis (HCA)-heat maps resulted in a reliable untargeted approach for discrimination and classification of different samples. The "targeted" approach was performed by developing and validating an HPTLC method allowing the quantification of eight flavonoids. RESULTS: The combination of quantitative data with PCA and HCA-heat-maps allowed the different samples to be discriminated from each other. CONCLUSION: The use of chemometrics tools for evaluation of fingerprints reduced expense and analysis time. The proposed method can be adopted for routine discrimination and evaluation of the phytochemical variability in different Apiaceae species extracts.

[Study on characteristics and microscopic identification of fruits of Notopterygium franchetii and N. forrestii].

Rhizoma et Radix Notopterygii is a rare and endangered Chinese medicine. In the collection of Notopterygium franchetii fruits, we collected a sample of N. forrestii , which is a spurious breed. Fruits of N. franchetii and N. forrestii are very similar in morphology and can be easily confused. Until now the morphological identification of the fruits of Notopterygium has not been reported. To provide a scientific basis for the identification of N. franchetii and N. forrestii fruits, the morphology and microscopic identification were studied in this paper. In this study, stereomicroscope and paraffin sections were used to compare the morphological characteristics and microscopic characteristics of these two fruits. Our results showed that these two fruits were different in size, surface texture and the number of vertical edges on the back. These traits can be used as diagnostic characteristic of these two fruits. The difference between the number of tubing and the endosperm cell contents can be used as microscopic identification features. The above discriminative characteristics can distinguish the two fruits and provide scientific basis for the identification and germplasm evaluation of Notopterygium fruits.

Chloroplast and ITS phylogenies to understand the evolutionary history of southern South American Azorella, Laretia and Mulinum (Azorelloideae, Apiaceae).

Azorella, Laretia and Mulinum are taxonomically complex, and good candidates to study evolutionary radiations in the Andes and the importance of hybridizations. Previous phylogenetic studies of subfamily Azorelloideae agree that Azorella and Mulinum as currently conceived are not monophyletic, and hence a revision of their circumscription is necessary. However, these phylogenies were based only on chloroplast DNA sequence data. Here, phylogenetic relationships within Azorelloideae were inferred using sequence data from five chloroplast DNA (rps16 intron, trnQ-rps16, rps16-trnKUUU 5' -exon, trnGGCC-trnSGCU and rpL32-trnLUAG), and from nuclear rDNA ITS regions to assess the monophyly of Azorella and Mulinum and discuss generic re-circumscriptions, determine hybridization and radiation events, identify and characterize important lineages, and propose hypotheses on evolution of key morphological characters. In total, 121 accessions of Azorelloideae were analyzed. Phylogenetic analyses of the different genomes were conducted separately and combined, with and without indels, using maximum parsimony, maximum likelihood, and Bayesian methods. To analyze the incongruence between plastid and nuclear-derived trees a consensus network from strongly supported nodes from cpDNA and ITS trees was constructed. Internode certainty values were calculated to evaluate the reliability of the relationships estimated from the individual cpDNA and ITS data sets and to examine the degree of conflict within the total evidence data set. Azorella and Mulinum were confirmed as not monophyletic. Except three Azorella species, the remaining azorellas, all species of Mulinum, and Laretia form a monophyletic group, designated here as Andean-Patagonian. The three species of Azorella that are not part of the Andean-Patagonian lineage are grouped together with Huanaca and Schizeilema in another lineage, designated here as Austral. Within the Andean-Patagonian clade, three major lineages can be recognized: Diversifolia, Trifurcata, and Spinosum. Each of these lineages have different leaf morpho-anatomies, Diversifolia species being more mesomorphic compared to species of Trifurcata, and species of Spinosum being the most xeromorphic. Hybridizations have been important in the evolution of the group, especially within Diversifolia, with at least six reticulation events resulting in putative homoploid and allopolyploid hybrid species. Evidence from branch lengths and low sequence divergences suggest a rapid radiation in the Spinosum group, probably associated with the acquisition of wings in the fruits.

[Identification of Notopterygium seeds using DNA barcoding method].

In order to guarantee the species correction of Notopterygium seeds, a molecular identification method with ITS2 as DNA barcode has been verified. In this study, 27 samples of Notopterygium seeds were collected from the main producing area of Notopterygium. The morphological characteristics of the Notopterygium seeds were firstly surveyed. Then the DNA extraction, PCR amplification, DNA sequencing and DNA assembly were carried out. The species identification for a Notopterygium seed was implemented through distance method, NJ-tree method and the DNA barcoding system for traditional Chinese medicine (www.tcmbarcode.cn). The results showed that the seeds of N. incisum and N. franchetii had similar morphological characteristics and were difficult to distinguish clearly based on morphological descriptions. With the results of molecular identification, 24 samples were genuine including 13 N. incisum seeds samples and 11 N. franchetii genuine seeds samples. In conclusion, DNA barcode technology can accurately and efficiently identify the species of Notopterygium seeds. Furthermore, this study will provide a new method for germplasm resources identification of medicinal materials and supplies some guidelines for establishing Chinese herbal seeds and seedlings quality standards.

[Correspondence analysis between traditional commercial specifications and quantitative quality indices of Notopterygii Rhizoma et Radix].

This paper aims to explore a comprehensive assessment method combined traditional Chinese medicinal material specifications with quantitative quality indicators. Seventy-six samples of Notopterygii Rhizoma et Radix were collected on market and at producing areas. Traditional commercial specifications were described and assigned, and 10 chemical components and volatile oils were determined for each sample. Cluster analysis, Fisher discriminant analysis and correspondence analysis were used to establish the relationship between the traditional qualitative commercial specifications and quantitative chemical indices for comprehensive evaluating quality of medicinal materials, and quantitative classification of commercial grade and quality grade. A herb quality index (HQI) including traditional commercial specifications and chemical components for quantitative grade classification were established, and corresponding discriminant function were figured out for precise determination of quality grade and sub-grade of Notopterygii Rhizoma et Radix. The result showed that notopterol, isoimperatorin and volatile oil were the major components for determination of chemical quality, and their dividing values were specified for every grade and sub-grade of the commercial materials of Notopterygii Rhizoma et Radix. According to the result, essential relationship between traditional medicinal indicators, qualitative commercial specifications, and quantitative chemical composition indicators can be examined by K-mean cluster, Fisher discriminant analysis and correspondence analysis, which provide a new method for comprehensive quantitative evaluation of traditional Chinese medicine quality integrated traditional commodity specifications and quantitative modern chemical index.

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

BACKGROUND: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects. RESULTS: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. CONCLUSION: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

Chloroplast capture and intra- and inter-continental biogeographic diversification in the Asian - New World disjunct plant genus Osmorhiza (Apiaceae).

Osmorhiza Raf. (Apiaceae) contains about 12 species disjunctly distributed in temperate Asia, and North, Central to South America. Phylogenetic and biogeographic analyses were carried out applying sequences of two nuclear and nine plastid loci from eleven recognized Osmorhiza species. The nuclear ITS and ETS and the plastid data fully resolved the infrageneric relationships, yet the two phylogenies were largely incongruent. Comparisons of nuclear and plastid phylogenies revealed several interspecific chloroplast transfer events in Osmorhiza, one of which involved an extinct or an unsampled lineage. This genus was inferred to have originated in the Old World during the late Miocene (11.02mya, 95% HPD: 9.13-12.93mya), and the crown of the genus was dated to be in the late Miocene (5.51mya, 95% HPD: 2.81-8.37mya). Species of Osmorhiza were inferred to have migrated from the Old World into North America across the Bering land bridge during the late Miocene, and they then diversified in the New World through multiple dispersal and divergence events. The intraspecific amphitropical disjunctions between North and South America, and the eastern and western North American disjunctions within O. berteroi and O. depauperata were hypothesized to be via recent long-distance dispersals most likely facilitated by birds.

DNA-based identification of Peucedanum ostruthium specimens and detection of common adulterants by high-resolution melting curve analysis.

Masterwort (Peucedanum ostruthium, syn. Imperatoria ostruthium, Apiaceae) is an old economic plant in Alpine countries cultivated as ornamental plant and used for spirits and in folk medicine. P. ostruthium is a species that has often been confused with related Apiaceae species or morphologically similar roots or tubers resulting in products of minor quality. Masterwort can be distinguished from other Apiaceae species by nrDNA (ITS1 and ITS2). The analysed chloroplast markers (trnK 5' intron, trnT-trnL, and psbA-trnH), however, showed no species-specific mutations. With the application of two primer pairs amplifying parts of ITS and developed for high-resolution melting curve analysis (HRM) the target species was distinguishable from the other Peucedanum and Apiaceae species of our reference set. A multiplex PCR/HRM was developed to detect adulterations with Gentiana spp., Aconitum napellus and Veratrum album.

Comparison of two species of Notopterygium by GC-MS and HPLC.

Notopterygii Rhizoma et Radix (Qianghuo), including Notopterygium incisum Ting ex H. T. Chang (NI) and Notopterygium franchetii H. de Boiss (NF), is an important traditional Chinese medicine. Of these two plants, NI, is more commonly used and has a much higher price in the marketplace. To compare these two plants, a combination of gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) was carried out, thus obtaining an overall characterization for both volatile and none-volatile compounds. Combined with hierarchical cluster analysis (HCA) and principal component analysis, GC-MS was successfully applied to distinguish NF and NI. The chemical constitutes of volatile oil in NI and NF were firstly compared in detail, and 1R-alpha-pinene, beta-pinene and 4-isopropyl-1-methyl-1,4-cyclohexadiene had great contribution to the discrimination. Fingerprints of 14 batches of Qinghuo samples were also established based on HPLC, and an obvious difference was found between the two species. The chromatographic fingerprints were further analyzed by similarity analysis and HCA. The present study is the first reported evaluation of two origins of Notopterygii Rhizoma et Radix by GC-MS and HPLC, which will facilitate quality control and its clinical application.

[ITS sequences variation and phylogenetic analysis on 31 geographical populations of Notopterygium incisum].

In this study, 31 Notopterygium incisum populations were analyzed using ITS sequences to investigate the genetic structure. The results showed that: the ITS region ranged in size from 634 to 635 bp and base composition was with high G + C content of 57.8%. Thirty-one polymorphic sites were detected from 402 sequences of 31 populations of N. incisum, and the proportion of polymorphic sites was 4.88%, in which parsimony informative sites were up to 12. And 31 haplotypes were identified based on these polymorphic sites. Molecular variance analysis (AMOVA) indicated that high genetic differentiation (57%) existed among population, and gene flow was low (N(m) = 0.38) among populations. Phylogenetic relationships of 31 haplotypes were analyzed using NJ method with N. forbesiias an out-group. Phylogenetic analysis showed that 31 haplotypes from different populations mixed together and did not form distinct geographically separated clades.

--Essential-oil composition of the fruits of six Heracleum L. species from Iran: chemotaxonomic significance.

The fruit essential oils of Heracleum persicum, H. rechingeri, H. gorganicum, H. rawianum, H. pastinacifolium, and H. anisactis from Iran were obtained by hydrodistillation and characterized by GC-FID and GC/MS analyses. The oils of the six species were compared to determine the similarities and differences among their compositions. Overall, 36 compounds were identified in the fruit oils, accounting for 92.40-96.74% of the total oil compositions. Aliphatic esters constituted the main fraction of the oils (86.61-94.31%), with octyl acetate and hexyl butyrate as the major components. The oil compositions of species belonging to section Pubescentia (H. persicum, H. gorganicum, and H. rechingeri) were discriminated by equally high contents of both octyl acetate (13.84-20.48%) and hexyl butyrate (17.73-38.36%). On the other hand, the oils of H. rawianum, H. pastinacifolium and H. anisactis, belonging to section Wendia, showed lower hexyl butyrate contents (3.62-6.6%) and higher octyl acetate contents (48.71-75.36%) than the former. Moreover, isoelemicin was identified at low amounts (0.10-2.51%) only in the oils of the latter species. The differences in the oil composition among the six species were investigated by hierarchical cluster and principal component analyses, which indicated that the oil composition confirmed well the taxonomical classification based on the morphological and botanical data, and, thus, may provide a reliable marker to discriminate Heracleum species at the intersectional level.

[Identification of peucedani radix, peucedani decursivi radix and its adulterants using ITS2 sequence].

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.

Morphology and ITS sequences provide insights into the phylogeny of Tongoloa (Apiaceae) from China.

BACKGROUND: Tongoloa is a genus comprising approximately 20 species, primarily distributed in the mountainous regions of southwest China. The insufficiency of specimen materials and morphological similarities among species render it a taxonomically challenging genus within the Apiaceae family. To elucidate the phylogenetic relationships and taxonomy of Chinese Tongoloa, this study utilized a total of 115 nrITS sequences, including 47 recently obtained sequences, for phylogenetic reconstruction. RESULTS: Phylogenetic relationships reconstructed from ITS sequences indicate that the East Asia Clade and the Komarovia Clade are sister groups, and Tongoloa belongs to the East Asia Clade. Species of Tongoloa are subdivided into 3 distinct groups, all sharing similar fruit morphologies and are clearly differentiated from related taxa. Several Tongoloa-like members classified under other genera are interpreted to be closely related to Tongoloa. Morphological and molecular data indicate that Tongoloa, Sinolimprichtia subclade and Chinese Trachydium subclade are separate yet genetically contiguous taxa. It is confirmed that Tongoloa zhongdianensis belongs to the Hymenidium Clade, while Sinocarum is classified within the Acronema Clade. Two new taxa are found in the Hengduan Mountains. CONCLUSION: Tongoloa is a genus within the East Asia Clade of Apiaceae, and the phylogeny reconstructed based on ITS sequences divides it into 3 main groups. By integrating fruit morphology and molecular phylogenetic analyses, we preliminary clarified the intricate taxonomic relationships among Tongoloa and related taxa. These results provide valuable opportunities for a deeper understanding of the phylogeny of Tongoloa.