DNA Barcoding Methods Research Articles

Presence of <em>Notahypsibius pallidoides</em> (Tardigrada: Hypsibiidae) in the fauna of Russia confirmed with the methods of DNA barcoding

A brief report is given on the tardigrade species Notahypsibius pallidoides, discovered in a moss cushion in the vicinity of Saint Petersburg. Morphological identification of the species is supported with the analysis of obtained sequences of nuclear and mitochondrial DNA barcode genes (ITS-2 and COI). The current state of knowledge of Russian fauna of Tardigrada is characterised, and all available data on the distribution of Notahypsibius pallidoides and its genetic variability are summarised. The data obtained in this study by methods of DNA barcoding demonstrated that studied specimens of the tardigrada population isolated in Russia belong to the same species (Notahypsibius pallidoides) as the control specimens isolated in Austria though represent another haplotype thus confirming the presence of Notahypsibius pallidoides for the fauna of Russia.

Tapping the Potential of the Molecular Genetic Expert Research of Objects of Animal Origin in the Fight against Their Illegal Trafficking

Currently, the legal framework for protecting natural resources from human assault is being developed at the international level. However, the statistical data on the sentencing of persons convicted on the territory of the Russian Federation under Articles 258 and 258.1 of the Criminal Code of the Russian Federation in 2020 given in the article indicates: the crimes causing the reduction in the number of animal populations, and as a result, the loss of biodiversity, are challenging to prove; a small number of perpetrators are brought to criminal responsibility; punishment in the form of imprisonment is imposed only in isolated cases. However, using molecular genetic research methods for the biological material of wild animals, it is possible to fill in a significant part of the missing information or obtain new evidentiary information about the circumstances of crimes, including illegal hunting. The application of the DNA barcoding method, developed in the early 2000s, makes it possible to establish the taxonomic affiliation of living organisms when it is impossible to do this by classical methods, for example, if the organism is represented by a micro-number of biological tissues, and is also at the stages of the life cycle (egg, larva, embryo) with an insufficient number of morphological features. Thus, advanced DNA technologies allow us to obtain a considerable amount of previously inaccessible information.The issue of the illegal wild animal trafficking sold as “raised in captivity” is relevant for the pet industry and zoos and has implications both for animal welfare and the preservation of the natural environment. Methods based on the DNA analysis can also be applied for solving problems related to establishing the source of origin of the animal, in some cases – its habitat. The article provides cases from expert practice of ANO “Amur Tiger” when using molecular genetic methods of wild animal analysis allowed solving investigative and judicial tasks.

DNA Barcoding of Two Thymelaeaceae Species: Daphne mucronata Royle and Thymelaea hirsuta (L.) Endl.

Daphne mucronata Royle and Thymelaea hirsuta (L.) Endl both belong to the Thymelaeaceae family. Both species are used traditionally to treat several diseases along with various daily applications by Jordanian Bedouins. Traditionally, those species are identified through personal proficiency, which could be misleading due to human errors or lack of expertise. This study aims to investigate an effective DNA barcoding method to identify and characterize Daphne mucronata Royle and Thymelaea hirsuta plant species at the molecular level. Daphne mucronata Royle and Thymelaea hirsuta were collected from the ancient city of Petra in the Southern part of Jordan. Sequences of candidate DNA barcodes were amplified (rbcL, matK, and rpoC1), sequenced, and aligned to the blastn database. Moreover, the obtained sequences were compared with available sequences of related species at the GenBank database. Our results showed that DNA barcoding successfully identifies the two plant species using any of chloroplast genes (rbcL, matK, or rpoC1). The results emphasize the ability of DNA barcoding for identifying and characterizing different plant species through the recruitment of different barcode loci in molecular identification.

Developing an efficient DNA barcoding system to differentiate between Lilium species

BackgroundLilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties.ResultsTo develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG).ConclusionsA rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Characterizing Billbug (Sphenophorus spp.) Seasonal Biology Using DNA Barcodes and a Simple Morphometric Analysis.

Simple SummaryBillbugs (Sphenophorus spp.) are a group of grass-feeding weevils considered to be one of the most important and widespread insect pests of turfgrass. However, our limited understanding of regional variation in billbug species composition and inability to identify the damaging larval stage to species level, has hindered our ability to resolve the seasonal biology of many billbug species and constrained development of effective management approaches. In this study, we developed a robust DNA barcoding approach for identification of morphologically cryptic billbug larvae. Using this molecular tool combined with larval head capsule measurements we characterized regional variation in billbug species and developed larval phenology charts. Our approach provides researchers with the molecular tools necessary to fill critical gaps in our understanding of billbug seasonal biology and will facilitate the development of improved turfgrass pest management programs.Billbugs (Sphenophorus spp.) are a complex of grass-feeding weevil species that reduce the aesthetic and functional qualities of turfgrass. Effective billbug monitoring and management programs rely on a clear understanding of their seasonal biology. However, our limited understanding of regional variation in the species compositions and seasonal biology of billbugs, stemming primarily from our inability to identify the damaging larval stage to species level, has hindered efforts to articulate efficient IPM strategies to growers. We used a combination of DNA barcoding methods and morphometric measures to begin filling critical gaps in our understanding of the seasonal biology of the billbug species complex across a broad geographic range. First, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Utah and Arizona. Next, we used our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. Finally, we combined our DNA barcoding approach with larval head capsule diameter, a proxy for developmental instar, to develop larval phenology charts. Adult COI sequences varied among billbug species, but variation was not influenced by geography, indicating that this locus alone was useful for resolving larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better visualization of billbug species composition and seasonal biology emerged. This approach will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology and facilitate the development of turfgrass pest management programs.

DNA barcoding of Iranian leeches (Annelida: Clitellata: Hirudinida)

The diversity of freshwater leeches (Clitellata; Hirudinida) of Iran was estimated by employing both DNA barcoding and species delimitation methods. Phylogenetic relationships of arhynchobdellid (including Hirudinidae, Praobdellidae, Haemopidae, and Erpobdellidae) and rhynchobdellid (Glossiphoniidae) leeches were reconstructed, based on the cytochrome c oxidase subunit I (COI) locus, using both new sequence data and those available from GenBank. Our results suggest that each of Helobdella stagnalis (Linnaeus, 1758), Glossiphonia concolor (Aphathy, 1888), Erpobdella borisi Cichocka & Bielecki, 2015, Dina lineata (O.F. Müller, 1774), Hirudo orientalis Utevsky and Trontelj, 2005, Haemopis sanguisuga (Linnaeus, 1758), Limnatis paluda (Tennent, 1859), and two unidentified species of Dina and Trocheta (these did not find species-level matches in GenBank) are present in Iran. A potential case of phenotypic change in response to ecological adaptation was observed in E. borisi insofar as two genetically identical sub- and super-terranean morphotypes were distinguished. The glossiphoniids of Iran and Europe are admixed in the phylogenetic tree, revealing low COI variation and no divergence within species between the continents for these taxa.

Assessment of classification ability for rbcL and trnL barcode in some Stemonaceae samplescollected from northern Vietnam

Stemona tuberosa Lour. a precious medicinal plant that belongs to the Stemonaceae family, has high applicability to human life. Extracts from leaves or roots of this herb contain many bioactive compounds with great value for both medical application and economy. However, there is not enough data related to evaluation and classification for Stemona species in Vietnam. Therefore, in this study, the authors used the DNA barcoding method to evaluate at the molecular level for four samples of Stemona tuberosa Lour. in northern Vietnam compared with other reference sequences from other species in the Stemonaceae family from GenBank based on two chloroplast barcodes rbcL and trnL. The primary results of genetics distance analysis and phylogenetic trees showed the close relationship between the four studied samples with Stemona tuberosa Lour. (sequenced from GenBank). The trnL with 1100 bp in length also proves the better classification ability of Stemona than the rbcLregion with 600 bp in length. These outcomes would be the first promising step for further studies about Stemona and other herbs, thus contributing to both scientific research and practical applicability demands as classification and conservation.

Assemblage Structure of the Ichthyoplankton and Its Relationship With Environmental Factors in Spring and Autumn off the Pearl River Estuary

Ichthyoplankton assemblages and their relationship with environmental variables are investigated in waters off the Pearl River Estuary in spring and autumn of 2019. Of 80 ichthyoplankton taxa identified using DNA barcode and morphological methods, 61 are identified to species. The most abundance families (Carangidae, Trichiuridae, Mullidae, and Scombridae) account for 61.34% of the horizontal total catch in spring, while Menidae and Carangidae are the most abundant families identified in autumn, accounting for 89.72% of the horizontal total catch. Cluster analysis identifies three species assemblages in spring, and four in autumn based on horizontal trawls. Relationships between assemblage structure and environmental variables (in situ and remote sensed) are determined by canonical correspondence analysis. Ichthyoplankton assemblage structure appears to be strongly influenced by sea level anomalies, salinity, water depth, temperature at 10 m depth, and distance from shore. We demonstrate the efficacy of using DNA barcode to identify ichthyoplankton, and suggest how these data can be used to protect fish spawning grounds in waters off the Pearl River Estuary.

Species Delimitation of Asteropyrum (Ranunculaceae) Based on Morphological, Molecular, and Ecological Variation.

Objectively evaluating different lines of evidence within a formalized framework is the most efficient and theoretically grounded approach for defining robust species hypotheses. Asteropyrum Drumm. et Hutch. is a small genus of perennial herb containing two species, A. cavaleriei and A. peltatum. The distinction of these two species mainly lies in the shape and size of leaf blades. However, these characters have been considered labile and could not differentiate the two species reliably. In this study, we investigated the variation of the leaf blades of 28 populations across the whole range of Asteropyrum using the landmark-based geometric morphometrics (GMM), sought genetic gaps within this genus using DNA barcoding, phylogenetic reconstruction and population genetic methods, and compared the predicted ecological niches of the two species. The results showed that the leaf form (shape and size) was overlapped between the two species; barcode gap was not detected within the genus Asteropyrum; and little ecological and geographical differentiation was found between the two taxa. Two genetic clusters detected by population genetic analysis did not match the two morphospecies. The results suggest that there are no distinct boundaries between the two species of Asteropyrum in terms of morphology, genetics and ecology and this present classification should be abandoned. We anticipate that range-wide population genomic studies would properly delineate the species boundaries and help to understand the evolution and speciation within Asteropyrum.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Molecular Identification, Nutritional Profile and Heavy Metals Content of Edible Caulerpa from Binuangeun Coast, Banten

Two species of Caulerpa, locally known as “Pedesan” and “Latuh” have been traditionally consumed by coastal communities at Binuangeun, Banten. This study aimed to identify “Pedesan” and “Latuh” using the DNA barcoding method and to evaluate their nutrient and heavy metal contents. Fatty acids were determined by Gas Chromatography Flame Ionization Detector (GC FID), amino acids using Ultra Performance Liquid Chromatography (UPLC), and minerals using Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Based on the tufA gene sequences, the “Pedesan” was identified as Caulerpa racemosa var. macrophysa and “Latuh” as Caulerpa chemnitzia. Thirteen fatty acids were detected in C. racemosa var. macrophysa and twelve fatty acids in C. chemnitzia. Of the total fatty acid content, C. racemosa var. macrophysa contained 41.0% unsaturated fatty acids, dominated by linolenic acid and eicosapentaenoic acid. Meanwhile, C. chemnitzia contained 47.5%, dominated by oleic acid. Both seaweeds contained fatty acids with the w6/w3 ratio lower than 10, which could prevent heart disease risk based on World Health Organization (WHO) recommendation. The primary amino acids content in C. racemosa var. macrophysa were glutamic acid, alanine, serine and aspartic acid, while those in C. chemnitzia were glutamic acid, serine, aspartic acid, and glycine. The high content of glutamic acid in both samples indicated their potential use as food flavor enhancer. The Na/K ratio of C. racemosa var. macrophysa (40.31) and C. chemnitzia (27.48) were higher than those recommended by WHO. Heavy metals were not detected in either “Pedesan” nor “Latuh”, indicating that they are safe for consumption.

Isoëtes sabatina (Isoëtaceae, Lycopodiopsida): Taxonomic distinctness and preliminary ecological insights

Abstract Isoëtes sabatina is the rarest aquatic quillwort in Europe. Although recently found (2013) in Lake Bracciano (central Italy), the species is just one step away from extinction with an estimated population not exceeding 400 individuals and a spatial range of a few hundred square metres. Lake Bracciano is a deep, oligo‐mesotrophic Mediterranean volcanic lake that has been subjected to human activities. From January to October 2017, the lake experienced a dramatic water level decrease (up to −1.50 m), which significantly affected the littoral zone and the habitat of I. sabatina. To improve the chances of survival of I. sabatina, the first eco‐taxonomic investigation on this species was carried out to describe its genetic distinctness, physical and chemical requirements and companion species. The phylogenetic position of I. sabatina was investigated by applying standard DNA barcoding methods. Simultaneously, during summer 2019, the physical and chemical features of water and sediments of the I. sabatina population and five small Alpine lakes colonized by Isoëtes echinospora – a supposed close relative – were characterized. These data were then compared with the available data on the trophic requirements of the target obligate aquatic Isoëtes, together with Isoëtes lacustris and Isoëtes malinverniana. The present survey confirmed the taxonomic and ecological distinctness of I. sabatina – providing the first evidence of genetic differentiation from I. echinospora. Isoëtes sabatina grows in waters with temperature, conductivity and total alkalinity up to 30°C, 561 μS cm−1 and 3.45 meq L−1, respectively. The edaphic requirements of I. sabatina confirm its outstanding conservation value, and this study offers a basic understanding of how to prevent its extinction. Now, all possible actions must be taken immediately to save this species.

Phylogenetic Analysis of Mosquito (Diptera: Culicidae) Species with Mitochondrial Cytochrome Oxidase Subunit 1 Gene Distributed in Kocaeli

Mosquitoes, which are in the Culicidae family and have blood-sucking properties, infect humans and animals with many diseases. In the present study, it was aimed that mosquitoes spreading in the Kocaeli province were detected species identification based on DNA barcoding method. DNA isolation was performed with the samples of mosquitoes collected in the Kocaeli province between June 2017 and September 2018. Then, Polymerase Chain Reaction (PCR) and DNA sequence analysis were applied by using universal primers of the mitochondrial COI gene. The sequences in FASTA format obtained with the Chromas program were compared with those of other mosquito species in the world through the NCBI-BLAST database. For phylogenetic analysis, the sequences were uploaded into the MEGA X program, and phylogenetic trees were created in the Maximum Likelihood method, Tamura-Nei Model (Tamura & Nei, 1993), Bootstrap 1000. Among mosquitoes collected in this study, 7 species belonging to Aedes, Anopheles, Culiseta and Culex genera were identified and characterized as Aedes geniculatus (n = 10), Aedes albopictus (n = 7), Anopheles funestus (n = 1), Anopheles plumbeus (n = 1), Culiseta longiareolata (n = 1), Cx. torrentium (n = 1) and Cx. pipiens complex sp. (n = 33). Within them, Cx. pipiens complex sp. was found as the dominant species in Kocaeli. In conclusion, this study revealed the first molecular research of mosquito species spreading in Kocaeli and provided records to GenBank.

Characterizing Industrial and Artisanal Fishing Vessel Catch Composition Using Environmental DNA and Satellite-Based Tracking Data.

The decline in wild-caught fisheries paired with increasing global seafood demand is pushing the need for seafood sustainability to the forefront of national and regional priorities. Validation of species identity is a crucial early step, yet conventional monitoring and surveillance tools are limited in their effectiveness because they are extremely time-consuming and require expertise in fish identification. DNA barcoding methods are a versatile tool for the genetic monitoring of wildlife products; however, they are also limited by requiring individual tissue samples from target specimens which may not always be possible given the speed and scale of seafood operations. To circumvent the need to individually sample organisms, we pilot an approach that uses forensic environmental DNA (eDNA) metabarcoding to profile fish species composition from the meltwater in fish holds on industrial and artisanal fishing vessels in Ecuador. Fish identified genetically as present were compared to target species reported by each vessel’s crew. Additionally, we contrasted the geographic range of identified species against the satellite-based fishing route data of industrial vessels to determine if identified species could be reasonably expected in the catch.

Exploring DNA in biochemistry lab courses: DNA barcoding and phylogenetic analysis.

DNA structure has been leveraged in a variety of facets that allow scientists to perform a range of assays, including ones for identification of species, establishing evolutionary relationships between taxa, or even identifying individuals. Here, we present a DNA barcoding method as practical, hands-on approach that connects several experimental techniques in one sequence to teach the principles behind DNA isolation, purification, PCR, sequencing, and phylogeny analysis. Our set of exercises is designed for a teaching university laboratory setting. The three laboratory class assignments utilize DNA from a mushroom (can be purchased at a supermarket) and provide a pipeline to guide students through the process of identifying an unknown sample, like in many research laboratories. The third assignment can be used as a stand-alone exercise on phylogeny analysis and can be taught remotely. Students explore the theory behind the standard molecular techniques and apply it in a hands-on setting that involves experimental design, sample preparation, and use of hallmark molecular instruments.

Biological identification of Eudistoma species (Polycitoridae: Ascidiacea) from Gulf of Mannar, India through COI gene sequences

Members of the family Polycitoridae (subphylum Tunicata, class Ascidiacea) including the most diverse species of Eudistoma are one among the potential sources of bioactive compounds. Due to the production of several alkaloids including eudistomins, exhibiting significant biological activity, they occupy a key position among ascidians. However, identification of this genus via morphology usually is difficult and complex for a non-specialist to perform due to apparent lack of internal morphological variations in the zooids. DNA barcodes or the partial sequences of the cytochrome c oxidase I (CO1) gene were used to confirm the identity of Eudistoma species and also to infer phylogeny among them. In this study, the efficiency of mt gene, CO1, in delineating closely resembling species belong to the genus Eudistoma has been examined. A total of thirty six new CO1 sequences from 39 samples of Eudistoma species found in Mandapam coast, Gulf of Mannar were generated from this study. They represent eight species including one new species. Only 25% of our sequences had matches in GenBank. Thus, this study adds the number of CO1 gene sequences of these Eudistoma species and genetic divergence in both GenBank and BOLD database. The mean interspecific divergence (13.4%) is larger than the mean intraspecific divergence of 0.56% indicating the efficiency of DNA barcoding method to identify the Eudistoma species and could be used to manage this resource for pharmacological study in India in the future.

Observing frugivores or collecting scats: a method comparison to construct quantitative seed dispersal networks

Mutualistic interactions form the basis for many ecological processes and are often analyzed within the framework of ecological networks. These interactions can be sampled with a range of methods and first analyses of pollination networks sampled with different methods showed differences in common network metrics. However, it is yet unknown if metrics of seed dispersal networks are similarly affected by the sampling method and if different methods detect a complementary set of frugivores. This is necessary to better understand the (dis‐)advantages of each method and to identify the role of each frugivore for the seed dispersal process.Here, we compare seed removal networks based on the observation of 2189 frugivore visits on ten focal plant species with seed deposition networks constructed by DNA barcoding of plant seeds in 3094 frugivore scats. We were interested in whether both methods identify the same disperser species and if species‐level network metrics of plant species were correlated between network types.Both methods identified the same avian super‐generalist frugivores, which accounted for the highest number of dispersed seeds. However, only with DNA barcoding, we detected elusive but frequent mammalian seed dispersers. The overall networks created by both methods were congruent but the plant species' degree, their interaction frequency and their specialization index (d′) differed.Our study suggests that DNA barcoding of defecated and regurgitated seeds can be used to construct quantitative seed deposition networks similar to those constructed by focal observations. To improve the overall completeness of seed dispersal networks it might be useful to combine both methods to detect interactions by both birds and mammals. Most importantly, the DNA barcoding method provides information on the post‐dispersal stage and thus on the qualitative contribution of each frugivore for the plant community thereby linking species interactions to regeneration dynamics of fleshy‐fruited plant species.

DNA Barcoding: Applications

The method of DNA barcoding has become a reliable tool that allows fast and accurate identification and verifies biodiversity in the hands of an experienced taxonomist. This method is very popular in various fields of human activity because of its ease of use and economic benefit. Examples of use are presented and the potential of the DNA barcoding method of living organisms in areas such as environmental monitoring (identification of invasive species, parasites and their carriers, pests), food and pharmaceutical industries (detection of counterfeit products, determination of product quality), and forensics are shown.

Application of DNA barcoding and metabarcoding for species identification in salmon products

ABSTRACT Mislabelling is a significant manifestation of food fraud. Traditional Sanger sequencing technology is the gold standard for seafood species identification. However, this method is not suitable for analysing processed samples that may contain more than one species. This study tested the feasibility of next-generation sequencing in identifying mixed salmon products. Salmon samples containing up to eight species were amplified using 16S rRNA mini-barcode primers, and sequenced on an Illumina HiSeq2500 platform. All species were accurately identified, and mixtures as low as 1% (w/w) could be detected. Furthermore, this study conducted a market survey of 32 products labelled as salmon. For pure and mixed fish products, Sanger and next-generation sequencing techniques were respectively used for species identification, and for NGS results, we also used real-time PCR method to cross-validate the mixed products to further verify the accuracy of the DNA metabarcoding technology established in this study. DNA barcoding and metabarcoding of commercial salmon food products revealed the presence of mislabelling in 16 of 32 (50%) samples. The developed DNA barcoding and metabarcoding methods are useful for the identification of salmon species in food and can be used for quality control of various types of salmon products.

Is Your Açaí Really from Amazon? Using DNA Barcoding to Authenticate Commercial Products

Acai is an Amazon superfruit with antioxidant and anti-inflammatory properties used in the preparation of energy drinks consumed mainly in Brazil and lately exported to the whole world. It is obtained from two palm species, Euterpe oleracea (mainly) and Euterpe precatoria (secondarily). However, it may be mistaken with the fruit of a non-Amazon sister species (Euterpe edulis), called jucara, due to its morphological similarities. As species identification is fundamental in the confirmation of commercial product authenticity, and most of the time the morphological identification is impossible, the molecular biology analysis represents a powerful method in food science. DNA barcoding, for example, represents an efficient method for detecting plant-based adulterants. In this study, we established a DNA barcoding method for discriminating the three Euterpe species, and it was successfully applied to authenticate acai commercial products sold in the Brazilian market. After testing nine regions as DNA barcoding candidates in reference samples, psbK-I region was elected for the authentication method. As result, 88.6% of the samples were classified as authenticated and 11.4% were classified as adulterated products. Authenticated acai products showed clustered with E. oleracea reference plants. Four adulterated acai products showed clustered with E. edulis, a different species from the label. No products showed clustered with E. precatoria. These results bring us concern about the correct identification of species in food and about the occurrence of misleading advertising on labeled acai products.