DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

DNA barcoding for identification of sand flies (Diptera: Psychodidae) in India

About 50 species of sand flies have been reported to be prevalent in India. We explored the utility of the DNA barcode approach towards species identification of these medically important insects. A total of 62 specimens belonging to seven morphologically identified species of two genera, Phlebotomus and Sergentomyia, collected from Puducherry Union Territory, Maharashtra and Rajasthan states of India were subjected to the analysis. Neighbor‐joining (NJ) analysis of DNA barcode sequences identified the individuals of seven morphological species into eight distinct species, as presented in the designed NJ tree. This methodology delineated morphologically identified species, S. bailyi, into two genetically isolated groups. Also, this study characterizes DNA barcodes of P. argentipes and P. papatasi, the vector species of leishmaniasis in India, for the first time.     

Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding

Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host - the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.     

Species identification of aphids (Insecta: Hemiptera: Aphididae) through DNA barcodes

A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.     

DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match

DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.     

Oligo(dT) is not a correct native PAGE marker for single-stranded DNA

Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility, and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length, and gel concentration. Depending on their length, oligo(dT) fragments migrate 2--4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g., determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.     

Unique expression of a sporophytic character on the gametophytes of notholaenid ferns (Pteridaceae)

? Premise of the study: Not all ferns grow in moist, shaded habitats; some lineages thrive in exposed, seasonally dry environments. Notholaenids are a clade of xeric-adapted ferns commonly characterized by the presence of a waxy exudate, called farina, on the undersides of their leaves. Although some other lineages of cheilanthoid ferns also have farinose sporophytes, previous studies suggested that notholaenids are unique in also producing farina on their gametophytes. For this reason, consistent farina expression across life cycle phases has been proposed as a potential synapomorphy for the genus Notholaena. Recent phylogenetic studies have shown two species with nonfarinose sporophytes to be nested within Notholaena, with a third nonfarinose species well supported as sister to all other notholaenids. This finding raises the question: are the gametophytes of these three species farinose like those of their close relatives, or are they glabrous, consistent with their sporophytes? ? Methods: We sowed spores of a diversity of cheilanthoid ferns onto culture media to observe and document whether their gametophytes produced farina. To place these species within a phylogenetic context, we extracted genomic DNA, then amplified and sequenced three plastid loci. The aligned data were analyzed using maximum likelihood to generate a phylogenetic tree. ? Key results: Here we show that notholaenids lacking sporophytic farina also lack farina in the gametophytic phase, and notholaenids with sporophytic farina always display gametophytic farina (with a single exception). Outgroup taxa never displayed gametophytic farina, regardless of whether they displayed farina on their sporophytes. ? Conclusions: Notholaenids are unique among ferns in consistently expressing farina across both phases of the life cycle.     

Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae

In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbK-psbI, trnH-psbA, and rpoB.The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter- and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belongingto 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.     

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

Assessment of loci for DNA barcoding in the genus Thrips (Thysanoptera:Thripidae)

Identification of the juveniles of economically important thrips species on imports by morphology alone can be challenging and culturing is usually required. In the case of EU quarantine species such as Thrips palmi, rapid and accurate identification is essential. DNA barcoding using the Cytochrome oxidase I (COI) gene has become a popular technique for species identification; however, in some invertebrate genera COI has been shown to provide insufficient variability for species discrimination. This study presents a comparison of five different loci to investigate their ability to discriminate a small number of Thrips species. All five loci discriminated the species by neighbour-joining tree and varying degrees of discrimination were determined upon further investigation of the intraspecific and interspecific distances. Two distinct COI clades were observed for T. Palmi and judged to be COI haplotypes when data from the other four additional loci and geographical collection data were taken into consideration. COI was shown to provide sufficient variation to be used in future DNA barcoding efforts within the genus Thrips.     

DNA条形码技术在青海海东地区小型兽类鉴定中的应用

为弥补传统形态分类方法的不足,探究应用DNA条形码技术进行分子生物学鉴定的可行性,本研究用DNA条形码技术检测了青海省海东地区3目6科14属18种110只小型兽类的COI基因部分序列。分析所测COI基因序列可知:种内遗传距离≤3%,种间遗传距离5-10%,属间遗传距离12-19%,种间遗传距离显著大于种内遗传距离。NJ树显示同种个体聚为有很高支持度的单一分支。有6个个体(4只黄胸鼠、2只小家鼠)在现场鉴定中被误定为其他种类。研究结果表明使用条形码技术能纠正形态学鉴定中的错误,也说明动物线粒体COI基因是一个有效的DNA条形码标准基因。     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.     

COI barcodes for identification of Merodon hoverflies (Diptera, Syrphidae) of Lesvos Island, Greece

DNA barcoding has become a useful system for linking different biological life stages, and for identification of species within a known taxonomic framework. In this study, we generated mitochondrial DNA COI barcodes using adult specimens of all 22 species of the hoverfly genus Merodon (Diptera, Syrphidae) occurring on Lesvos island (Greece). The generated COI barcodes could well discriminate between all Merodon taxa of Lesvos, except for M. loewi and M. papillus that shared the same haplotype, despite their clear morphological differences. In addition, the barcodes revealed two cases of hitherto unknown morphologically cryptic species close to M. avidus and M. nigritarsis, respectively. Because only few successful rearings of immature stages of Merodon hoverflies are available, the larval host plant remains unknown for these phytophagous taxa. The obtained COI barcode library for the Merodon spp. of Lesvos will constitute a tool to link any unknown immature stages with already known species, and thus provide important life-history information and promise for ecological studies.     

Comparative analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in ciliates (Alveolata,Ciliophora) and evaluation of its suitability as a biodiversity marker

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Phylogenetic Analysis of Brine Shrimp (Artemia) in China Using DNA Barcoding

DNA barcoding is a powerful approach for characterizing species of organisms,especially those with almost identical morphological features, thereby helping to to establish phylogenetic relationships and reveal evolutionary histories. In this study, we chose a 648-bp segment of the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), as a standard barcode region to establish phylogenetic relationships among brine shrimp (Artemia) species from major habitats around the world and further focused on the biodiversity of Artemia species in China, especially in the Tibetan Plateau. Samples from five major salt lakes of the Tibetan Plateau located at altitudes over 4,000 m showed clear differences from other Artemia populations in China. We also observed two consistent amino acid changes, 153A/V and 183L/F, in the COI gene between the high and low altitude species in China.Moreover, indels in the COI sequence were identified in cyst and adult samples unique to the Co Qen population from the Tibetan Plateau, demonstrating the need for additional investigations of the mitochondrial genome among Tibetan Artemia populations.     

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available.We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.     

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.     

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.