Cytochrome C Oxidase I Barcode Research Articles

A DNA barcode reference library for the Tipulidae (Insecta, Diptera) of Germany.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination. In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries. Employing diverse species delimitation algorithms - including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script - we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes. This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.

DNA Barcoding of Economically Important Fruit Flies (Diptera: Tephritidae) from the Lower Gangetic Plains of Eastern India

The Lower Gangetic Plains are India's central horticultural crop production regions. Fruit flies (Diptera: Tephritidae) are a significant insect pest that significantly hinders the effective crop production of various commercially valuable fruit and vegetable crops. Therefore, rapid and precise species identification techniques are essential to controlling, managing, and quarantining these pests. Furthermore, reliably identifying fruit flies is exceedingly challenging due to their striking resemblances in morphological characters. In this work, we investigated the effectiveness of mitochondrial cytochrome c oxidase I (COI) gene sequences for identifying seven fruit fly species, involving only the genus Bactrocera from the lower Gangetic Plains in eastern India. The sequences of the studied species showed 99%–100% similarity with sequences from within and outside the country. COI barcodes were able to distinguish between species, with K2P intraspecific genetic divergences ranging from 0-1% and K2P interspecific genetic divergences ranging from 3.65-28.46%, thus maintaining a proper barcode gap. Phylogenetic analyses were performed, and the results showed moderate to high supported monophyly among species. Therefore, COI barcodes have proven a highly successful alternative for quickly identifying fruit flies. This is also the first account of molecular identification of the previously reported tephritid fruit flies infesting fruits and vegetables in eastern India's agriculturally important lower Gangetic Plains.

Balclutha jafara (Hemiptera: Cicadellidae): integrative identification of a species introduced in the Western Hemisphere, and notes on other Balclutha.

Leafhopper specimens of the genus Balclutha Kirkaldy, found in southern Florida (Palm Beach and Collier Counties), United States, beginning in 2020, and in shipments of plant products originating from Colombia and entering the United States beginning in 2019, are identified as B. jafara Webb. This species was previously known only from the Seychelles and Aldabra Islands, which are parts of the Seychelles archipelago in the Indian Ocean east of mainland Africa. Identifications were made by comparison with type specimens, both morphologically and through molecular analysis. Specimens in Palm Beach Co. were swept from commercial rice (Oryza sativa L.) paddies. Mitochondrial cytochrome c oxidase I (COI) barcodes of specimens from Florida and Colombia were closely matched to each other and to partial barcodes obtained from paratype specimens of B. jafara. The COI barcodes also closely matched sequences from previously unidentified Balclutha specimens in the Barcode of Life Data Systems (BOLD) from Kenya and South Africa, several of which were confirmed later morphologically as B. jafara. Previously unidentified museum specimens from South Sudan, Zambia, and Zimbabwe were determined as B. jafara. Together, these specimens show that B. jafara has a more widespread African distribution than was known previously, and that it arrived in the Western Hemisphere by 2019. Balclutha jafara is redescribed and illustrated. Further studies on the Balclutha fauna of Florida were performed. COI barcode data were generated for Floridian specimens of B. caldwelli Blocker, B. curvata Caldwell, B. flavescens (Baker), B. frontalis (Ferrari), B. incisa (Matsumura), and B. lucida (Butler). A phylogenetic analysis of COI data was conducted using publicly available sequences and those generated here. A key to the Balclutha species known from Florida is provided. The names that have been applied and mis-applied to Western Hemisphere species are discussed. To clarify the identity of some species, illustrations are given for: the female holotype and a male paratype of Eugnathodus virescens Osborn (=B. flavescens); the holotype of Nesosteles robustus Caldwell (=B. robusta); and the holotype of Balclutha curvata Caldwell. Additional barcoded specimens of Balclutha from Kenya and Pakistan were provided for examination by the BOLD research group and determined as B. sujawalensis Ahmed, previously known only from India and Pakistan, and this species is also illustrated here.

DNA barcodes of Saudi Arabian birds: Implications for species identification and diversity analysis

ObjectivesDNA barcoding using cytochrome c oxidase I (COI) gene is an effective tool for species identification with additional power of measuring molecular diversity and phylogenetic inference. In this study, we evaluated the performance of COI gene sequences for species identification and molecular diversity analysis of some Saudi Arabian birds. MethodsWe sequenced the 694 base pair segment of COI gene from 5 samples of white cheeked bulbul (Pycnonotus leucogenys), 4 samples of black scrub robin (Cercotrichas podobe), and 2 samples of crested lark (Galerida cristata), all belonged to the same Order. We also included all the COI sequences available in the GenBank for these birds with the aim of studying molecular diversity across geographies. For species identification, we enriched our dataset by including COI barcodes of other Saudi Arabian bird species, including Arabian partridge (Alectoris melanocephala), houbara bustard (Chlamydotis undulata macqueenii), green bee-eater (Merops orientalis), laughing dove (Streptopelia senegalensis), namaqua dove (Oena capensis) and collared dove (Streptopelia decaocto), representing different genera. ResultsWhite-cheeked bulbul from Saudi Arabia showed more diversity than the same birds from Iraq. The mean nucleotide difference and nucleotide diversity were 5.20 and 0.009 respectively with as Tajima’s D score of 1.6549 indicating the scarcity of rare alleles. The specimens of black scrub robin from Saudi Arabia and Djibouti showed mixed phylogeny with only two segregating sties and a near zero Tajima’s D score indicating no major selection. Out of 18 samples of crested lark, 13 birds from 4 different geographical regions showed identical sequences, while 3 birds from Russia and 1 from Cyprus grouped in a separate cluster and 1 crusted lark from Djibouti differed from all. The mean nucleotide difference and nucleotide diversity were 0.6993 and 0.0014, while a negative Tajima’s D of −1.1956 indicated the presence of rare alleles. Phylogenetic analysis of 9 different species of Saudi Arabian birds showed the discriminatory power of COI barcodes as all the different species grouped separately in specific clusters. ConclusionsCOI barcodes are not only indispensable tools for species identification but can also be used for analyzing molecular diversity and phylogenetic inference.

Chromosomes as Barcodes: Discovery of a New Species of Black Fly (Diptera: Simuliidae) from California, USA.

Simple SummaryThe discovery and elucidation of species, especially those that are structurally similar, often require special tools and approaches. The basic principle of a unique barcode, as used in retail stores and more recently as DNA sequences in taxonomy, is also inherent in the giant banded chromosomes of flies (Diptera). We used the sequence of chromosome bands—the barcode—in the discovery of a new species of black fly from California, USA. The chromosomal barcode indicates this species is unique among all others in the Simulium (Boreosimulium) annulus group to which it belongs. To provide an integrated taxonomic approach, we analyzed and described the larvae, pupae, males, and females of this new species and assigned the formal name Simulium ustulatum n. sp. We also provided a traditional DNA barcode using the cytochrome c oxidase I (COI) gene. The new species has the most rearranged chromosomes of all North American members of its species group and differs most conspicuously by having a large chromocenter from which all six chromosome arms radiate. The species is structurally unique in the larva and male. The COI barcode enables identification of the female and pupa, which are structurally similar to other species in the group. The new species is the only western North American member of the Simulium annulus group that inhabits low-elevation, disturbed landscapes.One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome c oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the Simulium (Boreosimulium) annulus species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species Simulium ustulatum n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the S. annulus group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California’s Central Valley could make it vulnerable, given climate change and increasing land development.

Integrative taxonomy clarifies species limits in the hitherto monotypic passion‐vine butterfly genera Agraulis and Dryas (Lepidoptera, Nymphalidae, Heliconiinae)

AbstractThe Heliconiini genera Agraulis and Dryas are widely distributed throughout the Neotropics and into adjacent temperate regions, and although they are currently treated as monotypic, both show significant geographic phenotypic variation. In this work, we employ six genetic markers (4199 bp), two mitochondrial and four nuclear, to perform coalescent species delimitation analyses in Bayesian Phylogenetics and Phylogeography (BPP) and in integrated BPP (iBPP), the latter also includes linear measurements and wings landmarks. We also analyze cytochrome c oxidase I (COI) barcode sequences for each genus using genetic distances, haplotype networks and a character‐based approach. Based on the model testing results, complemented with data from previous studies, we performed morphometric analyses to compare fore and hindwing size, aspect ratio and shape among the new species. In addition, we compared the forewing spot pattern of hypothesizes species using the r package patternize and, for Dryas, compared the colour patterns of mature larvae. Model testing of the molecular species delimitation outputs favoured a seven species hypothesis for Agraulis and a four species hypothesis for Dryas. Average distances among COI barcode sequences of these groups were from 1.09 to 5.81% in Agraulis and from 1.09 to 3.44% in Dryas. Within‐group distances ranged from 0 to 1.11% and between 0 and 2.43%, respectively. NeighborNet haplotype networks showed that all but one of the species are monophyletic, and the character‐based approach found exclusive diagnostic positions for most species, while the rest can be recognized by unique combinations of the 44 informative nucleotide positions analysed. Morphometric analysis supported all species of Agraulis and Dryas based on wing shape, and also in several cases on wing sizes and aspect ratio (hindwing length–forewing length), including A. v. galapagensis, which was absent from the molecular study. The analysis of the forewing spot pattern also revealed differences among most species hypothesis. The colour pattern of the last instar larva is also diagnostic for each Dryas species hypothesis. Locality data for species in both genera show that most of them are allopatric albeit a few have contact zones being parapatric at some locations. Based on the observed genetic differences, which covary with morphology and geographical distribution, we recommend the recognition of eight species of Agraulis: A. incarnata, A. vanillae, A. forbesi new status, A. insularis reinstated status, A. maculosa new status, A. lucina reinstated status, A. galapagensis new status and one undescribed species, and four species of Dryas: D. iulia, D. dominicana revised status, D. lucia revised status and D. alcionea reinstated status. Further work is needed to investigate, which selective forces have led to the current configurations of venation and wing shape, the probable gene flow among species with a focus on Agraulis and gather more data on species ecology.LSIDurn: lsid: zoobank.org: pub: 9E8C9F6E‐91C3‐4FB2‐804D‐1A9C4BD2EABB.

DNA Barcoding and Diversity Analysis of 19 Economically Important Philippine Sea Cucumbers (Holothuroidea)

This study established the DNA barcodes of 19 economically important Philippine sea cucumbers belonging to Class Holothuroidea under Phylum Echinodermata using the cytochrome c oxidase I (COI) gene. These include sea cucumbers from the Family Holothuriidae under Order Aspidochirotida: Bohadschia marmorata, B. koellikeri, B. vitiensis, B. argus, Bohadschia sp. 1, Actinopyga echinites, A. lecanora, Holothuria scabra, H. fuscogilva, H. atra, H. impatiens, and H. albiventer; from the Family Stichopodiidae under Order Aspidochirotida: Stichopus horrens, S. monotuberculatus, S. vastus, S. hermanni, S. chloronotus, and Thelenota ananas; and from the Family Phyllophoriidae under Order Dendochirotida was Neocucumis proteus. Based on Kimura-2 pairwise (K2P) distances, low genetic variation within species of 0.005–0.018 was observed except for several species such as S. chloronotus, H. albiventer, A. echinites, and H. scabra – which had 0.057, 0.181, 0.207, and 0.215 within-species genetic variations, respectively. On the other hand, variation between species within a genus was 0.123 for Bohadschia, 0.18 for Actinopyga, 0.19 for Holothuria, and 0.071 for Stichopus. Phylogenetic tree using neighbor-joining analysis showed monophyletic clades for the genera Bohadschia, Actinopyga, Stichopus, Thelenota, and Neocucumis while paraphyletic clade was formed for the genus Holothuria. COI DNA sequences and barcodes were established for the first time for Neocucumis proteus and Holothuria albiventer.

Molecular evolution of cytochrome C oxidase-I protein of insects living in Saudi Arabia.

The study underpins barcode characterization of insect species collected from Saudi Arabia and explored functional constraints during evolution at the DNA and protein levels to expect the possible mechanisms of protein evolution in insects. Codon structure designated AT-biased insect barcode of the cytochrome C oxidase I (COI). In addition, the predicted 3D structure of COI protein indicated tyrosine in close proximity with the heme ligand, depicted substitution to phenylalanine in two Hymenopteran species. This change resulted in the loss of chemical bonding with the heme ligand. The estimated nucleotide substitution matrices in insect COI barcode generally showed a higher probability of transversion compared with the transition. Computations of codon-by-codon nonsynonymous substitutions in Hymenopteran and Hemipteran species indicated that almost half of the codons are under positive evolution. Nevertheless, codons of COI barcode of Coleoptera, Lepidoptera and Diptera are mostly under purifying selection. The results reinforce that codons in helices 2, 5 and 6 and those in loops 2–3 and 5–6 are mostly conserved and approach strong purifying selection. The overall results argue the possible evolutionary position of Hymenopteran species among those of other insects.

Decision Theory-Based COI-SNP Tagging Approach for 126 Scombriformes Species Tagging.

The mitochondrial gene cytochrome c oxidase I (COI) is commonly used for DNA barcoding in animals. However, most of the COI barcode nucleotides are conserved and sequences longer than about 650 base pairs increase the computational burden for species identification. To solve this problem, we propose a decision theory-based COI SNP tagging (DCST) approach that focuses on the discrimination of species using single nucleotide polymorphisms (SNPs) as the variable nucleotides of the sequences of a group of species. Using the example of 126 teleost mackerel fish species (order: Scombriformes), we identified 281 SNPs by alignment and trimming of their COI sequences. After decision rule making, 49 SNPs in 126 fish species were determined using the scoring system of the DCST approach. These COI-SNP barcodes were finally transformed into one-dimensional barcode images. Our proposed DCST approach simplifies the computational complexity and identifies the most effective and fewest SNPs to resolve or discriminate species for species tagging.

A two-step DNA barcoding approach for delimiting moth species: moths of Dongling Mountain (Beijing, China) as a case study

DNA barcoding, based on a fragment of cytochrome c oxidase I (COI) mtDNA, is as an effective molecular tool for identification, discovery, and biodiversity assessment for most animals. However, multiple gene markers coupled with more sophisticated analytical approaches may be necessary to clarify species boundaries in cases of cryptic diversity or morphological plasticity. Using 339 moths collected from mountains surrounding Beijing, China, we tested a pipeline consisting of two steps: (1) rapid morphospecies sorting and screening of the investigated fauna with standard COI barcoding approaches; (2) additional analyses with multiple molecular markers for those specimens whose morphospecies and COI barcode grouping were incongruent. In step 1, 124 morphospecies were delimited into 116 barcode units, with 90% of the conflicts being associated with specimens identified to the genus Hypena. In step 2, 55 individuals representing all 12 Hypena morphospecies were analysed using COI, COII, 28S, EF-1a, Wgl sequences or their combinations with the BPP (Bayesian Phylogenetics and Phylogeography) multigene species delimitation method. The multigene analyses supported the delimitation of 5 species, consistent with the COI analysis. We conclude that a two-step barcoding analysis pipeline is able to rapidly characterize insect biodiversity and help to elucidate species boundaries for taxonomic complexes without jeopardizing overall project efficiency by substantially increasing analytical costs.

Life-stage association of black flies, using a fast-evolving nuclear gene sequence, and description of the larva of Simulium lampangense Takaoka & Choochote (Diptera: Simuliidae) from Thailand

Larval black flies, especially earlier instars, can be difficult to associate morphologically and chromosomally with other life stages. We used sequences of mitochondrial cytochrome c oxidase I (COI) and nuclear elongation complex protein 1 (ECP1) to associate unknown larvae with known species of the Simulium multistriatum species group in Thailand. COI barcode sequences failed to differentiate closely related species (S. chaliowae, S. lampangense, and S. fenestratum) and unknown larvae. In contrast, ECP1 sequences clearly separated these species, and revealed that the unknown larvae clustered with pupae of S. lampangense. The larva of S. lampangense is described morphologically for the first time. It is similar to that of S. chaliowae, but can be distinguished by a pair of dorsal protuberances on segments 3–7 rather than on segments 2–6.

Molecular identification of polydorid polychaetes (Annelida: Spionidae): is there a quick way to identify pest and alien species?

The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.

Assessment of snake DNA barcodes based on mitochondrial COI and Cytb genes revealed multiple putative cryptic species in Thailand

DNA barcodes of mitochondrial cytochrome c oxidase I (COI), cytochrome b (Cytb) genes, and their combined data sets were constructed from 35 snake species in Thailand. No barcoding gap was detected in either of the two genes from the observed intra- and interspecific sequence divergences. Intra- and interspecific sequence divergences of the COI gene differed 14 times, with barcode cut-off scores ranging over 2%–4% for threshold values differentiated among most of the different species; the Cytb gene differed 6 times with cut-off scores ranging over 2%–6%. Thirty-five specific nucleotide mutations were also found at interspecific level in the COI gene, identifying 18 snake species, but no specific nucleotide mutation was observed for Cytb in any single species. This suggests that COI barcoding was a better marker than Cytb. Phylogenetic clustering analysis indicated that most species were represented by monophyletic clusters, suggesting that these snake species could be clearly differentiated using COI barcodes. However, the two-marker combination of both COI and Cytb was more effective, differentiating snake species by over 2%–4%, and reducing species numbers in the overlap value between intra- and interspecific divergences. Three species delimitation algorithms (general mixed Yule-coalescent, automatic barcoding gap detection, and statistical parsimony network analysis) were extensively applied to a wide range of snakes based on both barcodes. This revealed cryptic diversity for eleven snake species in Thailand. In addition, eleven accessions from the database previously grouped under the same species were represented at different species level, suggesting either high genetic diversity, or the misidentification of these sequences in the database as a consequence of cryptic species.

Hybridization relics complicate barcode-based identification of species in earthworms.

Introgressive hybridization results in mito-nuclear discordance which could obscure the delimitation of closely related taxa. Although such events are increasingly reported, they have been poorly studied in earthworms. Here, we propose a method for investigating the degree of introgressive hybridization between three taxa of the Allolobophora chlorotica aggregate within two field populations (N=67 and N=105) using a reference data set including published DNA barcoding and microsatellite data of all known A.chlorotica lineages (N=85). For this, we used both molecular phylogenetic and population genetic approaches. The test of correspondence between mitochondrial cytochrome c oxidase I (COI) lineages and clusters of nuclear microsatellite genotypes allowed individuals to be sorted in three categories (matching, admixed and nonmatching) and additional markers (mitochondrial NADH dehydrogenase subunit 1, nuclear Histone 3 and Internal transcribed Spacer Region 2) were used for phylogenetic reconstructions in order to check assignments. Although 15 admixed individuals were observed, no early-generation hybrids were detected within the two populations. Interestingly, 14 nonmatching individuals (i.e. with a mtDNA haplotype that did not correspond to their nuclear cluster) were detected, a pattern that would result after multiple generations of unidirectional hybridization of female from one taxon to male of the other taxon. Because earthworms are simultaneous hermaphrodites, these events of unidirectional hybridization suggest sterility of the male function in several crosses and highlight that some individuals can be misidentified if reliance is placed on COI barcodes alone. These findings could improve the use of these barcodes in earthworms for species delineation.

Morphological identification and COI barcodes of adult flies help determine species identities of chironomid larvae (Diptera, Chironomidae).

Establishing reliable methods for the identification of benthic chironomid communities is important due to their significant contribution to biomass, ecology and the aquatic food web. Immature larval specimens are more difficult to identify to species level by traditional morphological methods than their fully developed adult counterparts, and few keys are available to identify the larval species. In order to develop molecular criteria to identify species of chironomid larvae, larval and adult chironomids from Western Lake Erie were subjected to both molecular and morphological taxonomic analysis. Mitochondrial cytochrome c oxidase I (COI) barcode sequences of 33 adults that were identified to species level by morphological methods were grouped with COI sequences of 189 larvae in a neighbor-joining taxon-ID tree. Most of these larvae could be identified only to genus level by morphological taxonomy (only 22 of the 189 sequenced larvae could be identified to species level). The taxon-ID tree of larval sequences had 45 operational taxonomic units (OTUs, defined as clusters with >97% identity or individual sequences differing from nearest neighbors by >3%; supported by analysis of all larval pairwise differences), of which seven could be identified to species or 'species group' level by larval morphology. Reference sequences from the GenBank and BOLD databases assigned six larval OTUs with presumptive species level identifications and confirmed one previously assigned species level identification. Sequences from morphologically identified adults in the present study grouped with and further classified the identity of 13 larval OTUs. The use of morphological identification and subsequent DNA barcoding of adult chironomids proved to be beneficial in revealing possible species level identifications of larval specimens. Sequence data from this study also contribute to currently inadequate public databases relevant to the Great Lakes region, while the neighbor-joining analysis reported here describes the application and confirmation of a useful tool that can accelerate identification and bioassessment of chironomid communities.

Molecular identification of Reesa vespulae (Milliron) (Coleoptera: Dermestidae), a newly recorded species from Korea

A museum pest belonging to the family Dermestidae was found in the Museum of Sunchon National University, Sunchon, Korea and identified using its morphological characteristics and a DNA-based analysis of the mitochondrial cytochrome c oxidase I (COI) gene. This marks the first recorded appearance of this pest, which is identified as Reesa vespulae (Milliron), in the Korean Peninsula. We provide the details of the morphological diagnosis with the habitus of its adult and larva and its COI barcode data, and introduce historical data about its invasion around the world.

DNA barcoding detected improper labelling and supersession of crab food served by restaurants in India.

Detection of improper labelling of raw and processed seafood is of global importance for reducing commercial fraud and enhancing food safety. Crabs are crustaceans with intricate morphological as well as genetic divergence among species and are popular as seafood in restaurants. Owing to the high number of crab species available, it can be difficult to identify those included in particular food dishes, thus increasing the chance of supersession. DNA barcoding is an advanced technology for detecting improper food labelling and has been used successfully to authenticate seafood. This study identified 11 edible crab species from India by classical taxonomy and developed molecular barcodes with the cytochrome c oxidase I (COI) gene. These barcodes were used as reference barcodes for detecting any improper labelling of 50 restaurant crab samples. Neighbour-joining tree analysis with COI barcodes showed distinct clusters of restaurant samples with respective reference species. The study demonstrated 100% improper labelling of restaurant samples to cover up acts of inferior crab supersession. DNA barcoding successfully identified 11 edible crabs in accordance with classical taxonomy and discerned improper crab food labelling in restaurants of India.

TreeParser-aided Klee diagrams display taxonomic clusters in DNA barcode and nuclear gene datasets.

Indicator vector analysis of a nucleotide sequence alignment generates a compact heat map, called a Klee diagram, with potential insight into clustering patterns in evolution. However, so far this approach has examined only mitochondrial cytochrome c oxidase I (COI) DNA barcode sequences. To further explore, we developed TreeParser, a freely-available web-based program that sorts a sequence alignment according to a phylogenetic tree generated from the dataset. We applied TreeParser to nuclear gene and COI barcode alignments from birds and butterflies. Distinct blocks in the resulting Klee diagrams corresponded to species and higher-level taxonomic divisions in both groups, and this enabled graphic comparison of phylogenetic information in nuclear and mitochondrial genes. Our results demonstrate TreeParser-aided Klee diagrams objectively display taxonomic clusters in nucleotide sequence alignments. This approach may help establish taxonomy in poorly studied groups and investigate higher-level clustering which appears widespread but not well understood.

DNA barcodes of eight species in genus Sebastes

DNA barcode is effective for biological taxonomy and is able to identify species from any life-history stage. In the present study, eight species which belong to four different subgenera of genus Sebastes found in China sea waters were identified by cytochrome c oxidase I (COI) barcode. The results indicated that the intra-species variation in DNA barcode was less than inter-species variation. When the phylogenetic trees were reconstructed by neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods, all the species clustered in their groups distinguishable by high bootstrap values, which proved that COI barcode is a powerful means to differentiate species of Sebastes and supports their identification. When the molecular tree was compared to the morphological tree, only Sebastes trivittatus in subgenus Sebastocles settled in the different positions. It is suggested that S. trivittatus is one of the shallowest occurring species in the Northwest Pacific due to its life characters.

Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.