Identifying gastropod spawn from DNA barcodes: possible but not yet practicable

Identifying life stages of species with complex life histories is problematic as species are often only known and/or described from a single stage. DNA barcoding has been touted as an important tool for linking life-history stages of the same species. To test the current efficacy of DNA barcodes for identifying unknown mollusk life stages, 24 marine gastropod egg capsules were collected off the Philippines in deep water and sequenced for partial fragments of the COI, 16S and 12S mitochondrial genes. Two egg capsules of known shallow-water Mediterranean species were used to calibrate the method. These sequences were compared to those available in GenBank and the Barcode of Life Database (BOLD). Using COI sequences alone, only a single Mediterranean egg capsule was identified to species, and a single Philippine egg capsule was identified tentatively to genus; all other COI sequences recovered matches between 76% and 90% with sequences from BOLD and GenBank. Similarity-based identification using all three markers confirmed the Mediterranean specimens' identifications. A phylogenetic approach was also implemented to confirm similarity-based identifications and provide a higher-taxonomic identification when species-level identifications were not possible. Comparison of available GenBank sequences to the diversity curve of a well-sampled coral reef habitat in New Caledonia highlights the poor taxonomic coverage achieved at present in existing genetic databases, emphasizing the need to develop DNA barcoding projects for megadiverse and often taxonomically challenging groups such as mollusks, to fully realize its potential as an identification and discovery tool.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Application of DNA barcoding to the identification of Hymenoptera parasitoids from the soybean aphid (Aphis glycines) in China

Aphis glycines Matsumura is an important pest of soybean in Asia and North America. Hymenoptera parasitoids play a key role in the control of the soybean aphid. The correct identification of parasitoids is a critical step that precedes the assessment of their potential biological control agents. Accurate identification of the majority of the species attacking the soybean aphid often requires elaborate specimen preparation and expert taxonomic knowledge. In this study, we facilitated the identification of soybean aphid parasitoids by applying a DNA barcoding approach following a preliminary morphological identification. We generated DNA sequence data from the mitochondrial COI gene and the D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species emerging from the soybean aphid in China. Fifteen Hymenoptera parasitoid species belonging to 10 genera of five families were identified with little intra‐specific variation (0.09% ± 0.06% for 28S and 0.36% ± 0.18% for COI) and large inter‐specific divergence (30.46% ± 3.42% for 28S and 20.4% ± 1.20% for COI).     

Identification of gobies (Teleostei: Perciformes: Gobiidae) from the North and Baltic Seas combining morphological analysis and DNA barcoding

Gobies are difficult to identify, as they are very similar in appearance. Here, we identified (sub)adult specimens of 12 goby species from the North Sea and the Baltic Sea by carefully analysing meristic characters, coloration patterns, papillae row patterns and morphometric measurements. The results of the morphological identifications were congruent with those obtained with the analysis of COI DNA barcodes; sequences from morphological conspecific specimens were clustered together in clades with bootstrap values ≥ 99%. Mean intra‐ and interspecific distance (uncorrected p) was 0.37 and 18.97%, respectively. A gap between the maximum intraspecific distance and the distance to the nearest neighbour was apparent in every species and ranged from 2.35 to 16.11%. The Barcode Index Number (BIN) analysis performed on the Barcode of Life Data Systems (BOLD) web platform, assigned the DNA barcodes to 12 separate clusters corresponding to sequence‐ and morphology‐based identification. In 25% of the investigated species, the BIN clusters showed taxonomic discordances, as they contained sequences assigned to more than one species. This result demonstrates the importance of accurate morphological species identification at the beginning of the barcoding pipeline. © 2014 The Linnean Society of London     

Unexpected problem in aphid DNA barcoding by universal primers

DNA barcode (mitochondrial COI) sequences have allowed for species identification of aphids. In this study, we newly found a DNA barcoding problem in a part of the DNA sequences for Sitobion avenae. Five S. avenae individuals showed differences of, on average, 32.60% in the DNA sequences from other conspecific individuals, and a BLAST search revealed that the five sequences are similar to those of aphid parasitoids such as Aphidius, Ephedrus and Praon spp. (Hymenoptera: Braconidae). Based on these results, we concluded that the universal primers used in aphid DNA barcodes can amplify barcode sequences from parasitoid species within host aphids.     

An update on DNA barcoding: low species coverage and numerous unidentified sequences

DNA barcoding was proposed in 2003, the Consortium for the Barcode of Life was established in 2004, and the movement has since attracted more than $80 million funding. Here we investigate how many species of multicellular animals have been barcoded. We compare the numbers in a public database (GenBank as of January 2012) with those in the Barcode of Life Database (BOLD) and find that GenBank contains COI (cytochrome c oxidase subunit 1) sequences for ca. 60 000 species while BOLD reports barcodes for ca. 150 000 species. The discrepancy is likely due to a large amount of unpublished data in BOLD. Overall, the species coverage remains sparse, growth rates are low, and the barcode accumulation curve for Metazoa is linear with only 4788 species having been added in 2011. In addition, the vast majority of species in the public database (73%) were barcoded by projects that are unlikely to be related to the DNA barcoding movement. Particularly surprising was the large number of DNA barcodes in GenBank that were not identified to species (Jan 2012: 74%), with insect barcodes often being identified only to order. Of these several hundred thousand have since been suppressed by NCBI because they did not satisfy the iBOL/GenBank early release agreement. Species coverage is considerably better for target taxa of DNA barcoding campaigns (e.g. birds, fishes, Lepidoptera), although it also falls short of published campaign targets. © The Willi Hennig Society 2012     

山西翅果油树上三种重要鳞翅目害虫的形态和分子鉴定及生活史观察

[目的]明确山西翅果油树Elaeagnus mollis上发生危害的3种鳞翅目害虫形态鉴定特征及生活史特性,并基于mtDNA COI基因DNA条形码对这3个种进行快速物种识别鉴定.[方法]通过观察山西翅果油树上3种鳞翅目害虫成虫外部形态和解剖拍照雌、雄性外生殖器特征,利用PCR扩增对待测样本COI基因DNA条形码序列进...     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes

Background

DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity.

Methodology/Principal Findings

A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%–18.74%), most of them of high commercial relevance, suggesting possible cryptic species.

Conclusion/Significance

We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.     

Introduced ants reduce interaction diversity in a multi‐species,ant–aphid mutualism

Mutualisms contribute in fundamental ways to the origin, maintenance and organization of biological diversity. Introduced species commonly participate in mutualisms, but how this phenomenon affects patterns of interactions among native mutualists remains incompletely understood. Here we examine how networks of interactions among aphid‐tending ants, ant‐tended aphids, and aphid‐attacking parasitoid wasps differ between 12 spatially paired riparian study sites with and without the introduced Argentine ant Linepithema humile in southern California. To resolve challenges in species identification, we used DNA barcoding to identify aphids and screen for parasitoid wasps (developing inside their aphid hosts) from 170 aphid aggregations sampled on arroyo willow Salix lasiolepis. Compared to uninvaded sites, invaded sites supported significantly fewer species of aphid‐tending ants and ant‐tended aphids. At invaded sites, for example, we found only two species of ant‐tended aphids, which were exclusively tended by L. humile, whereas at uninvaded sites we found 20 unique ant–aphid interactions involving eight species of ant‐tended aphids and nine species of aphid‐tending ants. Ant–aphid linkage density was thus significantly lower at invaded sites compared to uninvaded sites. We detected aphid parasitoids in 14% (28/198) of all aphid aggregations. Although the level of parasitism did not differ between invaded and uninvaded sites, more species of wasps were detected within uninvaded sites compared to invaded sites. These results provide a striking example of how the assimilation of introduced species into multi‐species mutualisms can reduce interaction diversity with potential consequences for species persistence.     

Local scale DNA barcoding of bivalves (Mollusca): a case study

Divergence in cytochrome c oxidase 1 (COI), the genetic marker proposed for DNA barcoding, was investigated in marine bivalves from the genera Ennucula , Nucula , Yoldiella and Thyasira . No overlap in levels of intra- and interspecific variation was found. The levels of divergence found suggest that barcodes from COI will be useful in distinguishing between the species investigated in this study. The insufficiency of blast searches in GenBank to assign many of the obtained sequences to correct phylum was noted and clearly demonstrates the need for better search strategies specifically targeted at identification using DNA barcodes.     

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

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.     

Cytochrome c oxidase subunit I barcoding of the green bee-eater (Merops orientalis)

DNA barcoding using mitochondrial cytochrome c oxidase subunit I (COI) is regarded as a standard method for species identification. Recent reports have also shown extended applications of COI gene analysis in phylogeny and molecular diversity studies. The bee-eaters are a group of near passerine birds in the family Meropidae. There are 26 species worldwide; five of them are found in Saudi Arabia. Until now, GenBank included a COI barcode for only one species of bee-eater, the European bee-eater (Merops apiaster). We sequenced the 694-bp segment of the COI gene of the green bee-eater M. orientalis and compared the sequences with those of M. apiaster. Pairwise sequence comparison showed 66 variable sites across all the eight sequences from both species, with an interspecific genetic distance of 0.0362. Two and one within-species variable sites were found, with genetic distances of 0.0005 and 0.0003 for M. apiaster and M. orientalis, respectively. This is the first study reporting barcodes for M. orientalis.     

DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)

In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications.     

DNA barcoding of endoparasitoid wasps in the genus Anicetus reveals high levels of host specificity (Hymenoptera: Encyrtidae)

The genus Anicetus includes economically important biocontrol agents that are introduced for control of soft and wax scale insect agricultural pests (Ceroplastes spp.). Understanding of host–parasitoid associations is critical to the successful outcome of their utilization in biological control projects. However, identification of these parasitoids is often difficult because of their small size and generally similar morphological features, and hence, studies on the host–parasitoid associations. Here, nucleotide sequence data were generated from the mitochondrial COI gene and the D2 region of 28S rRNA to assess genetic variation within and between species of Anicetus occurring in China. The results of this study support the use of the COI and the D2 region of 28S rRNA gene as useful markers in separating species of Anicetus, even in cases where morphological differences are subtle. On the other hand, the COI gene is also useful in recognizing species with much variation in morphology. DNA barcoding reveals high levels of host specificity of endoparasitoids wasps in the genus Anicetus. Our results indicate that each Anicetus species is adapted to a limited set of host species, or even are monospecific in their host choice.     

DNA barcoding of billfishes

DNA barcoding is a method promising fast and accurate identification of animal species based on the sequencing of the mitochondrial c oxidase subunit (COI) gene. In this study, we explore the prospects for DNA barcoding in one particular fish group, the billfishes (suborder Xiphioidei--swordfish, marlins, spearfishes, and sailfish). We sequenced the mitochondrial COI gene from 296 individuals from the 10 currently recognized species of billfishes, and combined these data with a further 57 sequences from previously published projects. We also sequenced the rhodopsin gene from a subset of 72 individuals to allow comparison of mitochondrial results against a nuclear marker. Five of the 10 species are readily distinguishable by COI barcodes. Of the rest, the striped marlin (Kajikia audax) and white marlin (K. albida) show highly similar sequences and are not unambiguously distinguishable by barcodes alone, likewise are the three spearfishes Tetrapturus angustirostris, T. belone, and T. pfluegeri. We discuss the taxonomic status of these species groups in light of our and other data, molecular and morphological.     

Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.     

DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller’s classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.