Identification of Euphorbiae pekinensis Radix and its counterfeit and adulterated products based on DNA barcode, UPLC-Q-TOF-MS, UPLC fingerprint, and chemometrics.

Euphorbiae pekinensis Radix (EPR) is a traditional Chinese herb commonly used to treat edema, pleural effusion, and ascites. However, counterfeit and adulterated products often appear in the market because of the homonym phenomenon, similar appearance, and artificial forgery of Chinese herbs. This study comprehensively evaluated the quality of EPR using multiple methods. The DNA barcode technique was used to identify EPR, while the UPLC-Q-TOF-MS technique was utilized to analyze the chemical composition of EPR. A total of 15 tannin and phenolic acid components were identified. Furthermore, UPLC fingerprints of EPR and its common counterfeit products were established, and unsupervised and supervised pattern recognition models were developed using these fingerprints. The backpropagation artificial neural network and counter-propagation artificial neural network models accurately identified counterfeit and adulterated products, with a counterfeit ratio of more than 25%. Finally, the contents of the chemical markers 3,3'-di-O-methyl ellagic acid-4'-O-ß-D-glucopyranoside, ellagic acid, 3,3'-di-O-methyl ellagic acid-4'-O-ß-d-xylopyranoside, and 3,3'-di-O-methyl ellagic acid were determined to range from 0.05% to 0.11%, 1.95% to 8.52%, 0.27% to 0.86%, and 0.10% to 0.42%, respectively. This proposed strategy offers a general procedure for identifying Chinese herbs and distinguishing between counterfeit and adulterated products.

The first intermediate host of the invasive frog trematode Glypthelmins quieta in Japan.

Glypthelmins quieta is a frog trematode native to North and Central America. This trematode was recently detected in Japan in the American bullfrog Lithobates catesbeianus, which was introduced from North America to Japan. As the first intermediate host of G. quieta, typically a snail, has not yet been identified in Japan, we conducted a snail survey in eastern Japan to screen for an intermediate host using DNA barcoding based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1. We sampled 3 different snail species, Orientogalba ollula, Physella acuta, and Sinotaia quadrata histrica (157 individuals in total), and only the freshwater snail Physella acuta, which is also believed to have been introduced from North America to Japan, had sporocysts of G. quieta in its hepatopancreas. The introduction of the intermediate and definitive hosts from North America may have facilitated the invasion of G. quieta into Japan.

Delimiting species, revealing cryptic diversity, and population divergence in Qinghai-Tibet Plateau weevils through DNA barcoding.

The Leptomias group represents one of the most diverse taxonomic group of weevils in the Qinghai-Tibet Plateau and its adjacent areas. Despite the potential of hidden diversity, relatively few comprehensive studies have been conducted on species diversity in this taxonomic group. In this study, we performed DNA barcoding analysis for species of the Leptomias group using a comprehensive DNA barcode dataset that included 476 sequences representing 54 morphospecies. Within the dataset, our laboratory contributed 474 sequences, and 390 sequences were newly generated for this study. The average Kimura 2-parameter distances among morphospecies and genera were 0.76% and 19.15%, respectively. In 94.4% of the species, the minimum interspecific distances exceeded the maximum intraspecific distances, indicating the presence of barcode gaps in most species of Leptomias group. The application of Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Bayesian Poisson tree processes, jMOTU, and Neighbor-joining tree methods revealed 45, 45, 63, 54, and 55 distinct clusters representing single species, respectively. Additionally, a total of four morphospecies, Leptomias kangmarensis, L. midlineatus, L. siahus, and L. sp.9RL, were found to be assigned to multiple subclade each, indicating the geographical divergences and the presence of cryptic diversity. Our findings of this study demonstrate that Qinghai-Tibet Plateau exhibits a higher species diversity of the Leptomias group, and it is imperative to investigate cryptic species within certain morphospecies using integrative taxonomic approaches in future studies. Moreover, the construction of a DNA barcode reference library presented herein establishes a robust foundational dataset to support forthcoming research on weevil taxonomy, phylogenetics, ecology, and evolution.

Cryptic Taxa Revealed through Combined Analysis of Chromosomes and DNA Barcodes: The Polyommatus ripartii Species Complex in Armenia and NW Iran.

The detection of cryptic species in complexes that have undergone recent speciation is often difficult, since many standard nuclear markers have not yet accumulated differences between closely related taxa, and differences in mitochondrial markers can be leveled out due to mitochondrial introgressions. In these cases, the use of derived chromosomal characters such as non-ancestral chromosomal numbers and/or unusual karyotype features may be a solution to the species delimitation problem. However, non-ancestral but similar karyotypes may arise secondarily as a result of homoplastic evolution, and their interpretation as homologies may lead to incorrect taxonomic conclusions. In our study, we show that the combined use of mitochondrial DNA barcodes and karyotypes helps to solve this problem and identifies cryptic species in situations where each of these markers does not work individually. Using this approach, we show that the fauna of Armenia and adjacent Iran includes the following cryptic taxa of the Polyommatus ripartii species complex (haploid chromosome number, n in parentheses): P. ripartii paralcestis (n = 90), P. ripartii kalashiani, subsp. nov (n close to 90), P. emmeli, sp. nov. (n = 77-79), P. keleybaricus, sp. nov. (n = 86), P. demavendi belovi (n = 73-75), P. demavendi antonius, subsp. nov. (n = 71-73), P. admetus anatoliensis (n = 79) and P. eriwanensis (n = 29-34). Polyommatus admetus yeranyani is synonymized with P. admetus anatoliensis.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths.

Background: The InBIO Barcoding Initiative (IBI) Dataset - DS-IBILP08 contains records of 2350 specimens of moths (Lepidoptera species that do not belong to the superfamily Papilionoidea). All specimens have been morphologically identified to species or subspecies level and represent 1158 species in total. The species of this dataset correspond to about 42% of mainland Portuguese Lepidoptera species. All specimens were collected in mainland Portugal between 2001 and 2022. All DNA extracts and over 96% of the specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. New information: The authors enabled "The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths" in order to release the majority of data of DNA barcodes of Portuguese moths within the InBIO Barcoding Initiative. This dataset increases the knowledge on the DNA barcodes of 1158 species from Portugal belonging to 51 families. There is an increase in DNA barcodes of 205% in Portuguese specimens publicly available. The dataset includes 61 new Barcode Index Numbers. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF).

Volatile Profiles and DNA Barcodes of Myrtaceae Species with Occurrence in the Brazilian Amazon.

Myrtaceae family includes many species with taxonomic challenges, making it one of the most complex families to identify. This study used DNA barcoding to find molecular markers for species authentication based on the Myrtaceae family's chemical composition and genetic diversity. Essential oils and genetic material were extracted from the leaves of six different species: Eugenia uniflora, E. patrisii, Myrcia splendens, Psidium guajava, P. guineense, and Psidium sp. The samples were analyzed based on compound classes and grouped into two categories. Group I included samples with high amounts of oxygenated sesquiterpenes (3.69-76.05 %) and fatty acid derivatives (0.04-43.59 %), such as E. uniflora, Myrcia splendens, and E. patrisii. Group II included samples P. guajava, P. guineense, and Psidium sp., which had a significant content of monoterpene hydrocarbons (0.69-72.35 %), oxygenated sesquiterpenes (8.06-68.1 %), phenylpropanoids (0.45-22.59 %), and sesquiterpene hydrocarbons (0.27-21.84 %). The PsbA-trnH gene sequences had a high genetic variability, allowing the species to be distinguished. A phylogenetic analysis showed two main clusters with high Bootstrap values corresponding to the subtribes Eugeniineae, Myrciinae, and Pimentinae. The results suggest a weak correlation between genetic and chemical data in these Myrtaceae species.

The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal.

Background: The InBIO Barcoding Initiative (IBI) Orthoptera dataset contains records of 420 specimens covering all the eleven Orthoptera families occurring in Portugal. Specimens were collected in continental Portugal from 2005 to 2021 and were morphologically identified to species level by taxonomists. A total of 119 species were identified corresponding to about 77% of all the orthopteran species known from continental Portugal. New information: DNA barcodes of 54 taxa were made public for the first time at the Barcode of Life Data System (BOLD). Furthermore, the submitted sequences were found to cluster in 129 BINs (Barcode Index Numbers), 35 of which were new additions to the Barcode of Life Data System (BOLD). All specimens have their DNA barcodes publicly accessible through BOLD online database. Stenobothruslineatus is recorded for the first time for continental Portugal. This dataset greatly increases the knowledge on the DNA barcodes and distribution of Orthoptera from Portugal. All DNA extractions and most specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources.

Characterization and phylogenetic analysis of the complete chloroplast genome of Curcuma comosa and C. latifolia.

Members of the Curcuma genus, a crop in the Zingiberaceae, are widely utilized rhizomatous herbs globally. There are two distinct species, C. comosa Roxb. and C. latifolia Roscoe, referred to the same vernacular name "Wan Chak Motluk" in Thai. C. comosa holds economic importance and is extensively used as a Thai traditional medicine due to its phytoestrogenic properties. However, its morphology closely resembles that of C. latifolia, which contains zederone, a compound known for its hepatotoxic effects. They are often confused, which may affect the quality, efficacy and safety of the derived herbal materials. Thus, DNA markers were developed for discriminating C. comosa from C. latifolia. This study focused on analyzing core DNA barcode regions, including rbcL, matK, psbA-trnH spacer and ITS2, of the authentic C. comosa and C. latifolia species. As a result, no variable nucleotides in core DNA barcode regions were observed. The complete chloroplast (cp) genome was introduced to differentiate between the two species. The comparison revealed that the cp genomes of C. comosa and C. latifolia were 162,272 and 162,289 bp, respectively, with a total of 133 identified genes. The phylogenetic analysis revealed that C. comosa and C. latifolia exhibited a very close relationship with other Curcuma species. The cp genome of C. comosa and C. latifolia were identified for the first time, providing valuable insights for species identification and evolutionary research within the Zingiberaceae family.

Worldwide revision of synanthropic silverfish (Insecta: Zygentoma: Lepismatidae) combining morphological and molecular data.

Synanthropic silverfish are the best-known and most widely distributed insects of the order Zygentoma. However, there is a great gap in the knowledge and confusion about the geographic distribution and the diagnostic characteristics that allow their identification. In this work, we provide an exhaustive and deep analysis of the most common 9 synanthropic silverfish of the world, combining previously published and newly derived morphological and molecular data. Updated descriptions of Ctenolepisma calvum (Ritter, 1910) and Ctenolepisma (Sceletolepisma) villosum (Fabricius, 1775) are included, and morphological remarks, illustrations, and photographs of the remaining synanthropic species are provided to clarify their diagnosis and differentiation among them and from other free-living species. In addition, Ctenolepisma targionii (Grassi and Rovelli, 1889) is synonymized with C. villosum. A molecular phylogeny is presented based on the COI sequences of all the synanthropic species deposited in BOLD and GenBank, with 15 new sequences provided by this study. This has allowed us to detect and correct a series of identification errors based on the lack of morphological knowledge of several species. Moreover, 2 different lineages of Ctenolepisma longicaudatumEscherich, 1905 have also been detected. To help future studies, we also provide a taxonomic interpretation guide for the most important diagnostic characters of the order Zygentoma, as well as an identification key for all the Synanthropic studied species. Finally, an approximation of the global distribution of synanthropic silverfish is discussed. Several new records indicate that the expansion of these species, generally associated with the transport of goods and people, is still far from over.

Two new species of the genus Cheiracanthium C. L. Koch, 1839 (Araneae, Cheiracanthiidae) from China.

Two species of the long-legged sac spider genus Cheiracanthium C. L. Koch, 1839 collected from China are diagnosed and described as new to science: Cheiracanthiumbannaensissp. nov. (♂♀) from Yunnan Province and C.bifurcatumsp. nov. (♂♀) from Xinjiang Uyger Autonomous Region. Photos of the habitus and copulatory organs are given. In addition, DNA barcode information of the two new species is provided.

A new noctuid genus and species (Lepidoptera, Noctuidae, Amphipyrinae, Psaphidini, Triocnemidina) from New Mexico and Texas, United States of America.

Pooleagen. nov. is described for two noctuid species from southwestern United States: Pooleagrandimacula Barnes & McDunnough, comb. nov., previously in Oxycnemis Grote, and Pooleapsaphidoidessp. nov.Poolea is compared to Oxycnemis (Amphipyrinae, Psaphidini, Triocnemidina) and is retained in the same subtribe. Adult moths and male and female genitalia of Poolea species are illustrated along with those of Oxycnemisadvena Grote, the genus type species. Pertinent recent taxonomic changes to Amphipyrinae classification are reviewed.

Mfind: a tool for DNA barcode analysis in angiosperms and its relationship with microsatellites using a sliding window algorithm.

MAIN CONCLUSION: Mfind is a tool to analyze the impact of microsatellite presence on DNA barcode specificity. We found a significant correlation between barcode entropy and microsatellite count in angiosperm. Genetic barcodes and microsatellites are some of the identification methods in taxonomy and biodiversity research. It is important to establish a relationship between microsatellite quantification and genetic information in barcodes. In order to clarify the association between the genetic information in barcodes (expressed as Shannon's Measure of Information, SMI) and microsatellites count, a total of 330,809 DNA barcodes from the BOLD database (Barcode of Life Data System) were analyzed. A parallel sliding-window algorithm was developed to compute the Shannon entropy of the barcodes, and this was compared with the quantification of microsatellites like (AT)n, (AC)n, and (AG)n. The microsatellite search method utilized an algorithm developed in the Java programming language, which systematically examined the genetic barcodes from an angiosperm database. For this purpose, a computational tool named Mfind was developed, and its search methodology is detailed. This comprehensive study revealed a broad overview of microsatellites within barcodes, unveiling an inverse correlation between the sumz of microsatellites count and barcodes information. The utilization of the Mfind tool demonstrated that the presence of microsatellites impacts the barcode information when considering entropy as a metric. This effect might be attributed to the concise length of DNA barcodes and the repetitive nature of microsatellites, resulting in a direct influence on the entropy of the barcodes.

Generating 2D Barcode for DNA Barcode Sequences.

DNA barcode sequence is a short DNA sequence representing a sample from a particular species. The commonly used DNA barcodes are at least 200 bps long. This large number of characters cannot be encoded in two-dimensional codes for sample recognition and tracking. In the present study, we described a method that can be used to compress the DNA sequences and then generate the corresponding QR code. With the large numbers of software and hardware, the QR code can be used efficiently for printing, labeling, and scanning.

Principles for Constructing DNA Barcode Reference Libraries.

All DNA barcode methods rely on reference sequences linked to well-curated voucher specimens. Definitions for and locations of DNA barcode reference libraries are not standardized, and vary throughout the literature. Standardizing, and centralizing reference specimens would provide an unambiguous source, analogous to reference genomes, to reproduce identifications and improve a library. This chapter proposes a working definition of a DNA barcode reference library, consistent with DNA barcode data standards, along with principles and methods to consider when producing or using such a library. These methods allow explicit traceback to sequence-sources which elevate the value of voucher specimens, and create a potential for community curation.

New faunistic data on Diptera (Hexapoda, Insecta) from the Ziarat Juniperus forest ecosystem (Pakistan).

Background: This study presents the first faunistic record and DNA barcoding for some Diptera species recorded from the Juniperus forest ecosystem of Balochistan, Pakistan. DNA barcoding was used to explore species diversity of Dipterans and collections carried out using a Malaise trap between December 2018 to December 2019. This process involved sequencing the 658 bp Cytochrome Oxidase I (COI) gene. New information: Amongst the collected Diptera specimens, nine families were identified, representing 13 genera. These species include Atherigonasoccata (Rondani, 1871), Atherigonavaria (Schiner, 1868), Chironomusdorsalis (Meigen, 1818), Eupeodescorollae (Linnaeus, 1758), Eristalistenax (Linnaeus,1758), Goniaornata (Meigen, 1826), Luciliasericata (Meigen, 1826), Paragusquadrifasciatus (Linnaeus, 1758), Polleniarudis (Fabricius, 1794), Raviniapernix (Thompson, 1869), Sarcophagadux (Thompson, 1869), Trupaneaamoena (Schiner, 1868) and Wohlfahrtiabella (Linnaeus, 1758). The families Syrphidae and Sarcophagidae exhibited the highest representation, each comprising three genera and three species. They were followed by the family Muscidae, which had a single genus and two species. Anthomyiidae, Chironomidae, Calliphoridae, Polleniidae, Tachinidae and Tephritidae were represented by only one genus and one species. A nique Barcode Index Number (BIN) was allotted to Tachinidae (specie i.e Goniaornata). The results indicated that barcoding through cytochrome oxidase I is an effective approach for the accurate identification and genetic studies of Diptera species. This discovery highlights the significant diversity of this insect order in study region. Furthermore, a comprehensive list of other Diptera species remains elusive because of difficulties in distinguishing them, based on morphology and a lack of professional entomological knowledge.

Initial study and phylogenetic analysis of hard ticks (Acari: Ixodidae) in Nantong, China along the route of avian migration.

The growing concern about migratory birds potentially spreading ticks due to global warming has become a significant issue. The city of Nantong in this study is situated along the East Asia-Australasian Flyway (EAAF), with numerous wetlands serving as roosting sites for migratory birds. We conducted an investigation of hard ticks and determined the phylogenetic characteristics of tick species in this city. We utilized three different genes for our study: the mitochondrial cytochrome oxidase subunit 1 (COX1) gene, the second internal transcribed spacer (ITS2), and the mitochondrial small subunit rRNA (12 S rRNA) gene. The predominant tick species were Haemaphysalis flava (H. flava) and Haemaphysalis longicornis (H. longicornis). Additionally, specimens of Haemaphysalis campanulata (H. campanulata) and Rhipicephalus sanguineus (R. sanguineus) were collected. The H. flava specimens in this study showed a close genetic relationship with those from inland provinces of China, as well as South Korea and Japan. Furthermore, samples of H. longicornis exhibited a close genetic relationship with those from South Korea, Japan, Australia, and the USA, as well as specific provinces in China. Furthermore, R. sanguineus specimens captured in Nantong showed genetic similarities with specimens from Egypt, Nigeria, and Argentina.

The InBIO Barcoding Initiative Database: DNA barcodes of Iberian Bees.

Background: Bees are important actors in terrestrial ecosystems and are recognised for their prominent role as pollinators. In the Iberian Peninsula, approximately 1,100 bee species are known, with nearly 100 of these species being endemic to the Peninsula. A reference collection of DNA barcodes, based on morphologically identified bee specimens, representing 514 Iberian species, was constructed. The "InBIO Barcoding Initiative Database: DNA Barcodes of Iberian bees" dataset contains records of 1,059 sequenced specimens. The species of this dataset correspond to about 47% of Iberian bee species diversity and 21% of endemic species diversity. For peninsular Portugal only, the corresponding coverage is 71% and 50%. Specimens were collected between 2014 and 2022 and are deposited in the research collection of Thomas Wood (Naturalis Biodiversity Center, The Netherlands), in the FLOWer Lab collection at the University of Coimbra (Portugal), in the Andreia Penado collection at the Natural History and Science Museum of the University of Porto (MHNC-UP) (Portugal) and in the InBIO Barcoding Initiative (IBI) reference collection (Vairão, Portugal). New information: Of the 514 species sequenced, 75 species from five different families are new additions to the Barcode of Life Data System (BOLD) and 112 new BINs were added. Whilst the majority of species were assigned to a single BIN (94.9%), 27 nominal species were assigned to multiple BINs. Although the placement into multiple BINs may simply reflect genetic diversity and variation, it likely also represents currently unrecognised species-level diversity across diverse taxa, such as Amegillaalbigena Lepeletier, 1841, Andrenarussula Lepeletier, 1841, Lasioglossumleucozonium (Schrank, 1781), Nomadafemoralis Morawitz, 1869 and Sphecodesalternatus Smith, 1853. Further species pairs of Colletes, Hylaeus and Nomada were placed into the same BINs, emphasising the need for integrative taxonomy within Iberia and across the Mediterranean Basin more broadly. These data substantially contribute to our understanding of bee genetic diversity and DNA barcodes in Iberia and provide an important baseline for ongoing taxonomic revisions in the West Palaearctic biogeographical region.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market.

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

Molecular characterization and demographic insights into soybean bud borer (Lepidoptera: Tortricidae) in Brazil.

The soybean bud borer, a soybean pest in Brazil, was initially identified as Crocidosema aporema (Walsingham 1914) (Lepidoptera: Tortricidae). Outbreaks of this species have recently increased, but identification of this pest remains uncertain, and the historical factors associated with its geographic distribution in Brazil are little known. Here, we conducted a species characterization and phylogeographic analysis based on molecular and morphological evidence. Ninety individuals of bud-borers Lepidoptera were collected in different regions of Brazil. We sequenced COI and COII mitochondrial genes and examined wing patterns and male genital morphology. DNA barcoding approach revealed that 10 individuals were Argyrotaenia sphaleropa (Meyrick 1909) (Lepidoptera: Tortricidae) and 80 were a species of the genus Crocidosema Zeller. The morphology of the adult genitalia and wings proved to be insufficient to confirm the identification of Brazilian individuals as C. aporema, a species originally described from a high-elevation site in Costa Rica. Furthermore, the genetic distance between putative C. aporema specimens from Brazil and Costa Rica (ranging from 5.2% to 6.4%) supports the hypothesis that the Brazilian specimens are not referable to C. aporema. Our analysis revealed a single genetic strain (i.e., species) with low genetic diversity on soybean crops. We found no indication that the genetic structure was related to geographic distance among populations or edaphoclimatic regions. The population expansion of the soybean bud borer coincides with the increase in the area of soybean production in Brazil, suggesting that expanded soybean farming has allowed a significant increase in the effective population size of this pest.

Optimizing Nucleic Acid Delivery Systems through Barcode Technology.

Conventional biological experiments often focus on in vitro assays because of the inherent limitations when handling multiple variables in vivo, including labor-intensive and time-consuming procedures. Often only a subset of samples demonstrating significant efficacy in the in vitro assays can be evaluated in vivo. Nonetheless, because of the low correlation between the in vitro and in vivo tests, evaluation of the variables under examination in vivo and not solely in vitro is critical. An emerging approach to achieve high-throughput in vivo tests involves using a barcode system consisting of various nucleotide combinations. Unique barcodes for each variant enable the simultaneous testing of multiple entities, eliminating the need for separate individual tests. Subsequently, to identify crucial parameters, samples were collected and analyzed using barcode sequencing. This review explores the development of barcode design and its applications, including the evaluation of nucleic acid delivery systems and the optimization of gene expression in vivo.