DNA Barcoding

Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes ...

Overfishing of wedgefish greatly affects its population and the balance of the marine ecosystem. This is exacerbated by their relatively low fecundity, slow growth, and late maturity results in one of the lowest population growth rate within elasmobranch species. However, lacking database information results in insufficient regulations and surveillance of wedgefish fishing. The current situation is feared to the risk of wedgefish’s survival, especially in Bangka Belitung Islands, Indonesia. Fundamental to a database is the accurate identification of wedgefish species based on mitochondrial DNA (mtDNA) analysis. This study aimed to use DNA barcodes to identify, determine the conservation status, and the status according to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). We collected samples including unidentified fin samples from confiscated illegal fishing catches, the traditional markets and fishing docks, South Bangka. In this research,...

The objective of the present study was to evaluate the species diversity of eels native to Aceh waters based on genetic data. Sampling was conducted in western coast waters of Aceh Province, Indonesia, from July to August 2016. Genomic DNA was extracted from the samples, a genomic region from the 5’ region of the cox1 gene was amplified and sequenced, and this was then used to analyse genetic variation. The genetic sequences were blasted into the NCBI database. Based on this analysis there were three valid species of eels that occurred in Aceh waters, namely Anguilla marmorata, A. bicolor bicolor, and A. bengalensis bengalensis.

Biodiversity of tropical Saturniidae, as measured through traditionally described and catalogued species, strongly risks pooling cryptic species under one name. We examined the DNA barcodes, morphology, habitus and ecology of 32 ‘well known’ species of dry forest saturniid moths from Area de Conservacion Guanacaste (ACG) in north-western Costa Rica and found that they contain as many as 49 biological entities that are probably separate species. The most prominent splitting of traditional species – Eacles imperialis, Automeris zugana, Automeris tridens, Othorene verana, Hylesia dalina, Dirphia avia, Syssphinx molina, Syssphinx colla, and Syssphinx quadrilineata – is where one species was believed to breed in dry forest and rain forest, but is found to be two biological entities variously distinguishable by DNA barcodes and morphology, habitus, and/or microecological distribution. This implies that ‘standard’ biological information about each traditional species may be an unconscious ...

Background: The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task. Methodology and Principal Findings: We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome-ITS, and three from plastid genome-trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation. Conclusions/Significance: We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case.

513 specimens of Leiopus nebulosus s.l. originating from different European countries, mainly Poland have been analyzed. Among all specimens 403 belong to L. linnei Wallin, Nylander et Kvamme and 110 to L. nebulosus (L.); precise data concerning localities, including presentation of Polish localities on UTM maps have been included. Host plants of both species have been listed including new

Talinum triangulare is a medicinal herb known to have originated from tropical Africa and is now widely cultivated in the humid tropical countries. The characterization of plant germplasm using molecular descriptors aids in describing the genotypic traits of the plant T. triangulare. Ten different accessions of T. triangulare were collected from nine districts of Tamil Nadu. High quality genomic DNA was isolated from the different samples, checked for purity and quantified. The DNA samples were subjected to PCR amplification using RAPD markers and by DNA barcoding technique. RAPD fingerprints were generated for the 10 different accessions of T. triangulare for the first time. Ten random decamer primers resulted in 73.08% of polymorphism by producing a total of 78 fragments, of which, 57 bands were determined as polymorphic loci. Phylogenetic relationship and intra-specific variation among the samples were established by constructing a dendrogram based on Jaccard's coefficient. DNA barcoding studies included the amplification of chloroplast large subunit of ribulosebisphosphate carboxylase (rbcL), trnH-psbA intergenic spacer (trnH-psbA), maturase K (matK) and nuclear internal transcribed spacer (ITS). The amplicons were gel eluted, sequenced and checked for homology by using the BLAST tool. However, no variation was detected among the samples after sequencing, proving that the accessions were identical at the genetic level.

DNA barcodes have been proposed for diverse applications as markers for species identification. One application that is not fully explored yet is their use for assessing the species biodiversity and presence of invasive alien species (IAS) in maritime biosecurity. The phylogeographical signals of the mitochondrial COI (mtCOI) gene have been sometimes used to infer the number of introductions and the origin of biological invasions. Here, we employed mtCOI barcodes of mollusks and acorn barnacles (N = 751) from ports of French Polynesia to infer the effect of port size, maritime traffic, and degree of openness in the risk of biological invasions. With 17.2% of non-indigenous species (NIS) recorded here, significant differences in diversity were found among docks and between long-time docked ships and their closest piers. A higher proportion of NIS was found from sheltered compared to open ports regardless of their size and traffic. Less frequent wave washing, a lower effect of currents, and partial isolation in sheltered ports could explain the difference. The results suggest that port biota surveys should focus first on ports sheltered from the open sea and emphasize the value of mtCOI barcodes for the early detection of potential invasive species and for prioritizing surveillance efforts.

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts.

The introduction of exotic species can have serious consequences for marine ecosystems. On the shores of the Cantabrian Sea (North of Spain) there are no routine examinations of seaweeds that combine molecular and morphological methods for early detection of exotic species making it difficult to assess in the early stages their establishment and expansion processes as a result of anthropogenic activities (e.g., shipping and/or aquaculture). In this work we used both morphological identification and molecular barcoding (COI-5P and rbcL genes) of red algae collected in Asturias, Bay of Biscay (Gijón and Candás harbours) and from the University of Oviedo's herbarium samples. The results confirmed the presence of exotic Asian seaweeds Pachymeniopsis gargiuli and Grateloupia turuturu Yamada on Cantabrian Sea shores. Several individuals of these species were fertile and developing cystocarps when collected, underlining the risk of possible expansion or continued establishment. This st...

Ports are gateways for aquatic invasions. New arrivals from maritime traffic and disturbed environmental conditions can promote the settlement of exotic species. Molluscs fall into the most prevalent group of invasive species and can have a tremendous impact on aquatic ecosystems. Here we have investigated exotic molluscs in three ports with different intensities of maritime traffic in the Cantabrian Sea. DNA barcodes were employed to identify the species using BLASTn and BOLD IDS assignment. Deep morphological analysis using diagnostic criteria confirmed BLAST species assignation based on COI and 16S rRNA genes. Results confirmed the usefulness of DNA barcoding for detecting exotic species that are visually similar to native species. Three exotic bivalves were identified: Ostrea stentina (dwarf oyster), the highly invasive Crassostrea gigas (Pacific oyster) and Xenostrobus securis (pygmy mussel). This is the first record of O. stentina in the Bay of Biscay and the second of X. securis in the Cantabrian Sea. Furthermore, we report on the presence of the cryptogenic mussel Mytilaster minimus in the central Cantabrian Sea. These exotic species might have been overlooked due to their phenotypic similarity with co-occurring oyster and mussel species. This study illustrates how combining morphological and DNA taxonomic analysis can help in port and marina biosecurity surveys.

Coeloplanidae, the largest family of benthic ctenophores, comprises 33 species, all described based on traditional morphological characteristics, such as coloration, length, and number of aboral papillae, which are highly variable and can be affected by fixation methods and environmental conditions. Thus, there is a need for reliable genetic markers to complement the morphological identifications at the species level. Here, we analyzed 95 specimens from 11 morphologically distinct species of benthic ctenophores from the Red Sea and Sulu Sea, and tested selected regions of four genetic markers (ITS1, 18S rRNA, 28S rRNA and COI) for their ability to differentiate between species. We show that the barcoding region of the mitochondrial gene, cytochrome oxidase subunit I (COI), is highly variable among species of Coeloplanidae, and effectively discriminates between species in this family. The average Kimura-2-parameter (K2P) distance between species-level clades was 10%, while intraspecific variation was ~30 times lower (0.36%). COI-based phylogeny supported the delineation of four recently described new species from the Red Sea. The other nuclear markers tested were found to be too conserved in order to separate between species. We conclude that COI is a potential molecular barcode for the family Coeloplanidae and suggest to test it in pelagic ctenophores. Ctenophores represent a distinct phylum of invertebrates found in all marine environments. Most species within the phylum are planktonic gelatinous organisms, except the order Platyctenida, which is comprised of species that are benthic as adults and resemble flat worms (excluding the genus Ctenoplana Korotneff, 1886, which is also planktonic at the adult stage), and is composed of five different families (Coeloplanidae, Ctenoplanidae, Tjalfiellidae, Lyroctenidae and Savangiidae), with Coeloplanidae being the most species-rich 1. To date, there are 33 known species within Coeloplanidae, belonging to two genera, Coeloplana and Vallicula, all described based on classical taxonomic criteria, which mainly rely on morphological features 2 , including pigmentation pattern, number and arrangement of aboral papillae, maximal length along tentacular axis, location and shape of tentacular sheath, the presence of oral lappets, and an oral groove 2-6. Some of these morphological characteristics have been shown to be controversial or unreliable, as they tend to change depending on the individual's state (e.g., relaxed versus contracted), the fixation method, or the environmental conditions 2. As morphological characters used to designate species are altered post fixation, there is a need to develop molecular markers to aid in species descriptions and specimen identification for this group. The combination of such molecular markers and photographic records of live specimens will result in a more efficient and precise method for benthic ctenophore species delineation. To date, only 16 annotated sequences belonging to seven different species of benthic ctenophores from the order Platyctenida are available in the Nucleotide database of the National Center for Biotechnology Information (NCBI/GeneBank). These mainly consist of the 18S rDNA gene, while other genes, including the barcoding marker cytochrome oxidase subunit I (COI), have been sequenced for only two species (Table S1). Unfortunately, the 18S rDNA is a highly conserved gene in ctenophores 7 and is therefore not suitable for distinguishing between species or even genera in this phylum. COI is currently the most prevalent genetic barcode used for species identification in Metazoans 8 , with almost five million sequences stored in the Barcode of Life Database (BOLD, http://www.boldsystems.org/). Considering the extremely fast evolution rate of the mitochondrial genomes of the pelagic ctenophores' Mnemiopsis 9 and Pleurobrachia 10 , COI emerges as a promising tool for ctenophore barcoding. However, only one study has focused on COI variation in Ctenophora 11 , but the barcoding region of the COI gene was not examined. Thus, with only two COI sequences available for the Coeloplanidae, and only eight

Four species of Gasteromycetes collected in the Biosphere Reserve of Dja (Cameroon), are briefly described and commented. Two of them: Cyathus striatus and Sphaerobolus stellatus are well-known in Europe, being cosmopolitan, and the other two: Geastrum schweinitzii varo stipitatum and Phallus indusiatus show a typical pantropical distribution.

DNA barcoding can identify biological species and provides an important tool in diverse applications, such as conserving species and identifying pathogens, among many others. If combined with statistical tests, DNA barcoding can focus taxonomic scrutiny onto anomalous species identifications based on morphological features. Accordingly, we put nonparametric tests into a taxonomic context to answer questions about our sequence dataset of the formal fungal barcode, the nuclear ribosomal internal transcribed spacer (ITS). For example, does DNA barcoding concur with annotated species identifications significantly better if expert taxonomists produced the annotations? Does species assignment improve significantly if sequences are restricted to lengths greater than 500 bp? Both questions require a figure of merit to measure of the accuracy of species identification, typically provided by the probability of correct identification (PCI). Many articles on DNA barcoding use variants of PCI to measure the accuracy of species identification, but do not provide the variants with names, and the absence of explicit names hinders the recognition that the different variants are not comparable from study to study. We provide four variant PCIs with a name and show that for fixed data they follow systematic inequalities. Despite custom, therefore, their comparison is at a minimum problematic. Some popular PCI variants are particularly vulnerable to errors in species annotation, insensitive to improvements in a barcoding pipeline, and unable to predict identification accuracy as a database grows, making them unsuitable for many purposes. Generally, the Fractional PCI has the best properties as a figure of merit for species identification. The fungal genus Ramaria provides unusual taxonomic difficulties. As a case study, it shows that a good taxonomic background can be combined with the pertinent summary statistics of molecular results to improve the identification of doubtful samples, linking both disciplines synergistically.

Background: DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates. Methodology/Principal Findings: The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name. Conclusions/Significance: The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.

Significant morphological similarities within or in between species of insects has made reliable taxonomic identification difficult. DNA barcoding has appeared to be a useful tool in resolving the issues related to the identification of taxonomically difficult insect species. The North eastern region of India is one of the mega biodiversity hotspots in the world and the climatic conditions of the region are highly conducive for reproduction and multiplication of insects. The efforts were undertaken to study the biodiversity of insects and to develop molecular database by developing DNA barcodes of natural enemies of cole crops insect pests in mid hills of Meghalaya. Limited information is available on natural enemies in cole crops ecosystem in mid-hills of Meghalaya. A total of 17 natural enemies belonging to three insect orders viz., Coleoptera (4) Predator, Hymenoptera (5) (1 nymphal/pupal; 3 Larval parasitoid; 1 Pupal parasitoid), Diptera (8) (5 Predator, 3 pupal parasitoid) Hemiptera (2) were documented during the year 2014-2015. Natural enemies especially Predators of aphids viz., Coccinellid beetles, Syrphid flies were recorded, Parasitoids observed were; nymphal/adult Parasitoid of aphids, Diaeretiella rapae, larval parasitoid of cabbage butterfly, Hyposoter sp and Cotesia glomerata and Pupal parasitoid including Brachymeria femorata and Tachinidae sp. The collected species were identified by established taxonomic keys, by taxonomists and/or molecular basis. DNA was successfully extracted from multiple specimens of 17 insect species and molecular assays were also undertaken for presence of Wolbachia infection. The Wolbachia infected specimens were discarded and not used for further analysis. The DNA barcodes were successfully developed for 17 species by sequencing partial Cytochrome oxidase I (COI) gene of mitochondrial DNA. The molecular identity of the insect species was established through BLAST-n at NCBI. The total nucleotide length of barcodes varied from species to species (501bp to 682bp). All the analysed sequences were submitted to National Centre for Biotechnology Information (NCBI) and Accession numbers were obtained (KT175578 to KT 175606). The comprehensive taxonomical and molecular database developed in this study for a total of 17 species observed in cole crop ecosystem could be used as diagnostic guide at both morphological and molecular level.

The vegetable leaf miner, Liriomyza sativae (Diptera:Agromyzidae), is an invasive polyphagous species originally known to be found in America and now spread in many parts of Africa, Asia, and the Pacific region. During 2016, L. sativae was observed for the first time infesting tomato (Solanum lycopersicum L.) leaves in experimental farms of an institute at Umiam (Meghalaya state of northeastern India). Based on museum specimens, this species was reported from India on tomato during 1994. Nevertheless, no further information is hitherto available from India apart from just new record. Considering the pest status of L. sativae across the globe, it is crucial to understand its expansion range, severity, biological attributes, and seasonal incidence on tomato in India. Taxonomic Q3 identification of different species of Liriomyza leaf miners is very complex due to morphological resemblance, and consequently, species-level identification is often done incorrectly by mistaking one species for another. Therefore, we characterized L. sativae at the molecular level and developed species specific DNA barcodes by using mitochondrial cytochrome oxidase gene. Moreover, the information on the correct distribution, seasonal incidence, and basic biological attributes of different stages of L. sativae is reported and discussed.

Our current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species. This is due to inherent problems with obtaining high-quality arthropod data. Novel high throughput sequencing approaches are now emerging as powerful tools to overcome such limitations, and thus comprehensively address existing shortfalls in arthropod biodiversity data. Here we explore how, as a community, we might most effectively exploit these tools for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first review the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We consider how this can be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explore how these approaches can be impleme...

With the widespread adoption of barcoding and next-generation sequencing, metabarcoding 2 is emerging as a potential tool for detecting labelled and unlabelled plant species in herbal 2 products. In this study, newly designed rbcL and ITS2 metabarcode primers were validated 2 for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass 3 pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single 3 drugs and 15 polyherbal formulations procured from the Indian market. The rbcL 3 metabarcode demonstrated detection efficiencies of 86.7% and 71.7% in gDNA plant pools 3 and biomass mock controls, respectively, while the ITS2 metabarcode demonstrated 82.2% 3 and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative 3 detection efficiency increased by 100% and 90%, respectively. A total of 79% cumulative 3 detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was 3 observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted 3 plant species present within mock controls as well as in herbal formulations. Our results 3 demonstrated the applicability of multilocus strategies in metabarcoding as a potential tool 4 for detecting labelled and unlabelled plant species in herbal formulations.

In Vietnam, Anguilla eels have been documented before, but their species composition and biological characteristics are still relatively unknown. Research on the molecular phylogeny of Anguilla has been limited, and identification of the species of eels was usually based on morphological characteristics. This is the first time both cytochrome c oxydase subunit I (COI) and 16S ribosomal RNA (rRNA) genes were used to identify Anguilla eels in water bodies, where the eels can migrate downstream and upstream during different stages of the life cycle, in Thua Thien Hue province, Vietnam. Based on DNA sequence analysis of six specimens, two Anguilla species (including subspecies) were confirmed as A. marmorata and A. bicolor pacifica, which had not been identified based on morphological characteristics in previous analyses. ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 19 (4): gmr18722 K.T. Huyen et al. 2 Phylogenetic analysis showed that the Anguilla genus in Thua Thien Hue province belongs to the Indo-Pacific lineage and helped build phylogenetic trees generated from mtDNA data from GenBank for these eels.

The Teratosphaeriaceae represents a recently established family that includes numerous saprobic, extremophilic, human opportunistic, and plant pathogenic fungi. Partial DNA sequence data of the 28S rRNA and RPB2 genes strongly support a separation of the Mycosphaerellaceae from the Teratosphaeriaceae, and also provide support for the Extremaceae and Neodevriesiaceae, two novel families including many extremophilic fungi that occur on a diversity of substrates. In addition, a multi-locus DNA sequence dataset was generated (ITS, LSU, Btub, Act, RPB2, EF-1α and Cal) to distinguish taxa in Mycosphaerella and Teratosphaeria associated with leaf disease of Eucalyptus, leading to the introduction of 23 novel genera, five species and 48 new combinations. Species are distinguished based on a polyphasic approach, combining morphological, ecological and phylogenetic species concepts, named here as the Consolidated Species Concept (CSC). From the DNA sequence data generated, we show that each one of the five coding genes tested, reliably identify most of the species present in this dataset (except species of Pseudocercospora). The ITS gene serves as a primary barcode locus as it is easily generated and has the most extensive dataset available, while either Btub, EF-1α or RPB2 provide a useful secondary barcode locus.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome,...

In the last ten years, 14 species of cetaceans and five species of pinnipeds stranded along the Atlantic coast of Brittany in the North West of France. All species included, an average of 150 animals strand each year in this area. Based on reports from the stranding network operating along this coast, the most common stranding events comprise six cetacean species (Delphinus delphis, Tursiops truncatus, Stenella coeruleoalba, Globicephala melas, Grampus griseus, Phocoena phocoena) and one pinniped species (Halichoerus grypus). Rare stranding events include deep-diving or exotic species, such as arctic seals. In this study, our aim was to determine the potential contribution of DNA barcoding to the monitoring of marine mammal biodiversity as performed by the stranding network. We sequenced more than 500 bp of the 5’ end of the mitochondrial COI gene of 89 animals of 15 different species (12 cetaceans, and three pinnipeds). Except for members of the Delphininae, all species were unambi...

Background: The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition. Methodology: Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra-and interspecific distances: 99.3% of all interspecific comparisons were .0.05, while 99.5% of all intraspecific comparisons were ,0.05 K2P distance. Conclusion: The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

Genus Spodoptera of family Noctuidae, order Lepidoptera comes under Superfamily Noctuidea. This superfamily comprises a large clade of economically important agricultural species known as “pest clade” causing a serious infestation in crops. The species of the Spodoptera genus found in Indo-Australian tropics and New Guinea mostly feeds on grasses and sometimes on Shorea curtisii (Dipterocarpaceae) seeds. The current study presents the molecular-based identification and first DNA barcode sequence of Spodoptera pecten. The first DNA barcode sequence of Spodoptera pecten from India has shown matching similarity with the COI sequences previously deposited from Pakistan, Papua New Guinea, and Japan.

We describe and illustrate Anthophila variabilis Falck, Karsholt & Rota, sp. n. (Choreutidae) from Tenerife (Canary Islands, Spain). The new species is outstanding due to the variability of its male genitalia. It is closely related to A. fabriciana (Linnaeus, 1767), and more distantly related to Anthophila threnodes (Walsingham, 1910), which is endemic to Madeira. Based on the DNA barcode, the new species is molecularly very distinct from its closest relative, A. fabriciana, with a pairwise K2P distance of more than 6.5%. The previous record of A. fabriciana from the Canary Islands is based on misidentification, and the species should be removed from the list of Lepidoptera found in the Canary Islands.

Here, we present a new approach for increasing the rate and lowering the cost of identifying, cataloging, and monitoring global biodiversity. These advances, which we call Closed-Tube Barcoding, are one application of a suite of proven PCR-based technologies invented in our laboratory. Closed-Tube Barcoding builds on and aims to enhance the profoundly important efforts of the International Barcode of Life initiative. Closed-Tube Barcoding promises to be particularly useful when large numbers of small or rare specimens need to be screened and characterized at an affordable price. This approach is also well suited for automation and for use in portable devices.

DNA barcoding is a technology used for the identification of any biological species by sequencing obtained through amplification of DNA. Here, we seek a standardized protocol along with the choice of markers used for different microbial species: bacteria, fungi, algae, plants, and animals. Various projects, consorts, tools, and databases have been discussed. With the advancement of technology, the future scope and applications have also been identified with an argument on the limitations along with the concept of universality on the DNA barcoding.

Dissomphalus is a cosmopolitan genus of Bethylidae and has 269 Neotropical species divided into 32 species-groups, mostly defined by the genital and the tergal process structures. Dissomphalus rectilineus and D. concavatus are sympatric species in the ulceratus species-group. Members of the species-group share many similarities in the morphology of the head, hypopygium, tergal process and genitalia, but may be distinguished by the structure of the hypopygium. Previous studies have found intermediate structures of the hypopygium in the sympatric areas and raised questions about the distinctiveness of these two species. We sequenced 340 bp of the mitochondrial gene cytochrome oxidase I of 29 specimens from Brazil and Paraguay, calculated the genetic divergence among specimens, and recovered the phylogenetic relationships between taxa. In addition, we compared the morphology of the hypopygium to evaluate its use as a species-specific diagnostic character using the genetic divergence values. We recovered three well-supported monophyletic groups (intraclade divergence from 1.3 to 13.4%) and three hypopygium morphologies associated with each clade, two of them associated with D. rectilineus and D. concavatus (as described in the literature); the third one is new, not associated with any known species. The divergence between the D. rectilineus and D. concavatus clades was 19%, while the third clade is divergent from each species by 19-20%. If fully described, the hypopygium shape associated with the COI sequence will represent an extremely promising approach to the diagnosis of Dissomphalus species.

Dissomphalus is a cosmopolitan genus of Bethylidae and has 269 Neotropical species divided into 32 species-groups, mostly defined by the genital and the tergal process structures. Dissomphalus rectilineus and D. concavatus are sympatric species in the ulceratus species-group. Members of the species-group share many similarities in the morphology of the head, hypopygium, tergal process and genitalia, but may be distinguished by the structure of the hypopygium. Previous studies have found intermediate structures of the hypopygium in the sympatric areas and raised questions about the distinctiveness of these two species. We sequenced 340 bp of the mitochondrial gene cytochrome oxidase I of 29 specimens from Brazil and Paraguay, calculated the genetic divergence among specimens, and recovered the phylogenetic relationships between taxa. In addition, we compared the morphology of the hypopygium to evaluate its use as a species-specific diagnostic character using the genetic divergence va...

The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32-trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA ''universal'' barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed.

DNA barcode sequences are accumulating in large data sets. A barcode is generally a sequence larger than 1000 base pairs and generates a computational burden. Although the DNA barcode was originally envisioned as straightforward species tags, the identification usage of barcode sequences is rarely emphasized currently. Single-nucleotide polymorphism (SNP) association studies provide us an idea that the SNPs may be the ideal target of feature selection to discriminate between different species. We hypothesize that SNP-based barcodes may be more effective than the full length of DNA barcode sequences for species discrimination. To address this issue, we tested a ribulose diphosphate carboxylase (rbcL) SNP barcoding (RSB) strategy using a decision tree algorithm. After alignment and trimming, 31 SNPs were discovered in the rbcL sequences from 38 Brassicaceae plant species. In the decision tree construction, these SNPs were computed to set up the decision rule to assign the sequences into 2 groups level by level. After algorithm processing, 37 nodes and 31 loci were required for discriminating 38 species. Finally, the sequence tags consisting of 31 rbcL SNP barcodes were identified for discriminating 38 Brassicaceae species based on the decision tree-selected SNP pattern using RSB method. Taken together, this study provides the rational that the SNP aspect of DNA barcode for rbcL gene is a useful and effective sequence for tagging 38 Brassicaceae species.

Identification of jellyfish species is challenging, even for taxonomic experts, due to their fragile forms and complex, morphologically-distinct life-history stages. DNA barcoding along with the morphological characters would complement the species identification. In the present study, a jellyfish collected from the Juhu beach, Mumbai coast, Maharashtra, India was identified based on the morphology and molecular markers. Visual morphological characters indicate that the specimen belongs to the genus Rhopilema. The partial gene of nuclear Internal Transcribed Spacer 1 was amplified, sequenced and subjected to similarity analysis with the NCBI GenBank database. The analysis showed a similarity of 99 % with the reported Rhopilema hispidum and confirmed the species identity. In the Neighbour-joining tree, the present study specimen is clustered with R. hispidum reported from Malaysia.

Aphids were surveyed in Diyarbakir province of Turkey between 1998-2000. As a result of the survey studies performed, 61 species of aphids were determined on different host plants. Their distribution in Turkey, host plants, collecting dates and occurrence were given for each species. Of them, 30 species were the first record for Diyarbakir province.

Chemical and biological principles of medicinal plants have lead to the discovery of newer and costeffective drugs. Lantana camara has been used in various folk medicinal preparations and its chemical composition and pharmacological activities have been elaborated recently. However there remains still a huge scope for use of modern scientific methods genomics, proteomics, metabolomics and bioinformatics in this plant. Bioinformatics shall facilitate analysis and integration of information from these related fields to enable the identification of genes and gene products and elucidate the functional relationships between genotype and observed phenotype. This review provides a state-of-the-art overview of bioinformatics study of Lantana camara with emphasis on the current progress and future directions, which shall provide tools and resources necessary to understand and promote advances in this important field.

Sundaland constitutes one of the largest and most threatened biodiversity hotspots; however, our understanding of its biodiversity is afflicted by knowledge gaps in taxonomy and distribution patterns. The subfamily Rasborinae is the most diversified group of freshwater fishes in Sundaland. Uncertainties in their taxonomy and systematics have constrained its use as a model in evolutionary studies. Here, we established a DNA barcode reference library of the Rasborinae in Sundaland to examine species boundaries and range distributions through DNA-based species delimitation methods. A checklist of the Rasborinae of Sundaland was compiled based on online catalogs and used to estimate the taxonomic coverage of the present study. We generated a total of 991 DNA barcodes from 189 sampling sites in Sundaland. Together with 106 previously published sequences, we subsequently assembled a reference library of 1097 sequences that covers 65 taxa, including 61 of the 79 known Rasborinae species of...

From 2019 to 2021, scientific field campaigns have been organised in Corsica by the Muséum national d'Histoire naturelle, the Office français de la biodiversité and the Collectivité de Corse as part of the "Our Planet Reviewed" naturalist exploration programme. This paper presents the context, the state of biogeographical and taxonomic knowledge prior to our expeditions, and the objectives, the methods and the first results obtained. The aim was to establish a modern survey of the species present in a selection of sites representative of different Corsican ecosystems, and to further develop the natural history collections through depositing new specimens and species with associated standard DNA barcodes, useful for their identification. Over a period of three years, nineteen sites were surveyed with a semi-standardised protocol and a large-scale trapping scheme was organised in three of these. Sampling efforts focused on forest habitats at higher altitudes and on coastal dune and lowland marshland habitats. A vast array of methods was used to collect invertebrates, with a specific effort on flightinterception traps and pan traps. A total of 34 experts participated to the field surveys and more than 80 further contributed to the study of the specimens. Occurrence data are available in the Inventaire national du patrimoine naturel (http://www.inpn.fr) and, for specimens processed through DNA barcoding, specimen and DNA sequencing data will be accessible in the Barcode of Life datasystems (BOLD : http://www.boldsystems.org). In early 2023, the assembled datasets included 31,100 occurrence data for 3,900 taxa of terrestrial arthropods, representing a 53% increase in publicly available data for the island. More than 6,800 DNA barcode sequences have been produced for arthropods, representing a 14-fold increase in available sequences of Corsican insects compared to those available before the start of the programme. So far, these efforts resulted in producing the first Corsican records for 148 species and in the description of 12 species new to science. Résumé.-La Planète Revisitée en Corse 2019-2021 : un grand inventaire de la biodiversité négligée dans une île méditerranéenne. De 2019 à 2021, des missions scientifiques de terrain ont été organisées en Corse par le Muséum national d'Histoire naturelle, l'Office français de la biodiversité et la Collectivité de Corse dans le cadre du programme d'exploration naturaliste "La Planète Revisitée". Cet article présente le contexte, l'état des connaissances biogéographiques et taxinomiques avant le début du programme, les objectifs, les méthodes ainsi que les premiers résultats obtenus. L'objectif était d'établir un inventaire moderne des espèces présentes

Fungi are among the most abundant and diverse organisms on Earth. However, a substantial amount of the species diversity, relationships, habitats, and life strategies of these microorganisms remain to be discovered and characterized. One important factor hindering progress is the difficulty in correctly identifying fungi. Morphological and molecular characteristics have been applied in such tasks. Later, DNA barcoding has emerged as a new method for the rapid and reliable identification of species. The nrITS region is considered the universal barcode of Fungi, and the ITS1 and ITS2 sub-regions have been applied as metabarcoding markers. In this study, we performed a large-scale analysis of all the available Basidiomycota sequences from GenBank. We carried out a rigorous trimming of the initial dataset based in methodological principals of DNA Barcoding. Two different approaches (PCI and barcode gap) were used to determine the performance of the complete ITS region and sub-regions. F...

Water mites (Acari, Hydrachnidia) are a significant component of freshwater ecosystems inhabiting a wide range of aquatic habitats. This study provides a first comprehensive DNA barcode library for the water mites of Montenegro. DNA barcodes were analysed from 233 specimens of water mites morphologically assigned to 86 species from 28 genera and 15 families. In the course of the study, four species, i.e. Lebertia reticulata (Koenike, 1919),

Water mites (Acari, Hydrachnidia) are a significant component of freshwater ecosystems inhabiting a wide range of aquatic habitats. This study provides a first comprehensive DNA barcode library for the water mites of Montenegro. DNA barcodes were analysed from 233 specimens of water mites morphologically assigned to 86 species from 28 genera and 15 families. In the course of the study, four species, i.e. Lebertia reticulata (Koenike, 1919),