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.     

Searching for evidence of selection in avian DNA barcodes

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

DNA barcodes provide new evidence of a recent radiation in the genus Sporophila (Aves: Passeriformes)

The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.     

Low resolution of mitochondrial COI barcodes for identifying species of the genus Larus (Charadriiformes: Laridae)

We evaluated whether cytochrome c oxidase subunit I (COI) barcodes that have been previously suggested for birds are useful for identifying species of the genus Larus, which are resident or migratory birds in Korea. We found 31 intra- or interspecific COI haplotypes from 12 of 13 Larus species in Korea. Haplotype analyses showed that the COI barcodes could not distinguish some interspecific haplotypes from 6 of 12 Larus species because there were no nucleotide substitutions among their COI haplotypes. The neighbor-joining tree formed shallow branches in the clades expected for L. saundersi, L. crassirostris, L. canus, L relictus, and L. ridbundus. In the nine Larus species, COI haplotypes were not grouped as distinct entities that were correctly assigned to their corresponding species, resulting in polytypic clades. These results indicate that the COI sequences need to be cautiously selected as a DNA barcode for identifying species of Korean Larus birds.     

Nuclear gene sequences resolve species phylogeny and mitochondrial introgression in Leptocarabus beetles showing trans-species polymorphisms

We studied the phylogenetic relationships among Japanese Leptocarabus ground beetles, which show extensive trans-species polymorphisms in mitochondrial gene genealogies. Simultaneous analysis of combined nuclear data with partial sequences from the long-wavelength rhodopsin, wingless, phosphoenolpyruvate carboxykinase, and 28S rRNA genes resolved the relationships among the five species, although separate analyses of these genes provided topologies with low resolution. For both the nuclear gene tree resulting from the combined data from four genes and a mitochondrial cytochrome oxidase subunit I (COI) gene tree, we applied a Bayesian divergence time estimation using a common calibration method to identify mitochondrial introgression events that occurred after speciation. Three mitochondrial lineages shared by two or three species were likely subject to introgression due to interspecific hybridization because the coalescent times for these lineages were much shorter than the corresponding speciation times estimated from nuclear gene sequences. We demonstrated that when species phylogeny is fully resolved with nuclear gene sequence data, comparative analysis of nuclear and mitochondrial gene trees can be used to infer introgressive hybridization events that might cause trans-species polymorphisms in mitochondrial gene trees.     

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。     

DNA barcoding of Dutch birds

The mitochondrial cytochrome c oxidase subunit I (COI) can serve as a fast and accurate marker for the identification of animal species, and has been applied in a number of studies on birds. We here sequenced the COI gene for 387 individuals of 147 species of birds from the Netherlands, with 83 species being represented by > 2 sequences. The Netherlands occupies a small geographic area and 95% of all samples were collected within a 50 km radius from one another. The intraspecific divergences averaged 0.29% among this assemblage, but most values were lower; the interspecific divergences averaged 9.54%. In all, 95% of species were represented by a unique barcode, with 6 species of gulls and skua (Larus and Stercorarius) having at least one shared barcode. This is best explained by these species representing recent radiations with ongoing hybridization. In contrast, one species, the Lesser Whitethroat Sylvia curruca showed deep divergences, averaging 5.76% and up to 8.68% between individuals. These possibly represent two distinct taxa, S. curruca and S. blythi, both clearly separated in a haplotype network analysis. Our study adds to a growing body of DNA barcodes that have become available for birds, and shows that a DNA barcoding approach enables to identify known Dutch bird species with a very high resolution. In addition some species were flagged up for further detailed taxonomic investigation, illustrating that even in ornithologically well-known areas such as the Netherlands, more is to be learned about the birds that are present.     

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.     

Speciation history of the North American funnel web spiders, Agelenopsis (Araneae: Agelenidae): phylogenetic inferences at the population-species interface

Intra- and interspecific relationships of 12 out of 13 described species as well as a potential new species in the spider genus Agelenopsis (Araneae: Agelenidae) were analyzed using sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and 16S ribosomal RNA. Approximately half of the species examined formed well-supported monophyletic groups, whereas the rest of the species were part of well-supported monophyletic species groups. Rather than viewing cases where species were not identified as being monophyletic as poor taxonomy, these cases more likely represent recent speciation and offer insights into the process of speciation. The clade with the lowest levels of interspecific sequence divergence was found in eastern North America, whereas western species displayed much higher levels of interspecific divergence. These patterns appear to extend below the species level as well, with southwestern species exhibiting the highest levels of intraspecific sequence divergence and geographic structuring. The relationship between Agelenopsis and Barronopsis, a genus once considered a sub-genus of Agelenopsis, was also examined. The two genera are reciprocally monophyletic but more generic level sampling is needed to confirm an apparent sister relationship between the two.     

Cytochrome c oxidase subunit 1 barcode data of fish of the Nayband National Park in the Persian Gulf and analysis using meta-data flag several cryptic species

We provide cytochrome c oxidase subunit 1 (COI) barcode sequences of fishes of the Nayband National Park, Persian Gulf, Iran. Industrial activities, ecological considerations and goals of The Fish Barcode of Life campaign make it crucial that fish species residing in the park be identified. To the best of our knowledge, this is the first report of barcoding data on fishes of the Persian Gulf. We examined 187 individuals representing 76 species, 56 genera and 32 families. The data flagged potentially cryptic species of Gerres filamentosus and Plectorhinchus schotaf. 16S rDNA data on these species are provided. Exclusion of these two potential cryptic species resulted in a mean COI intraspecific distance of 0.18%, and a mean inter- to intraspecific divergence ratio of 66.7. There was no overlap between maximum Kimura 2-parameter distances among conspecifics (1.66%) and minimum distance among congeneric species (6.19%). Barcodes shared among species were not observed. Neighbour-joining analysis showed that most species formed cohesive sequence units with little variation. Finally, the comparison of 16 selected species from this study with meta-data of conspecifics from Australia, India, China and South Africa revealed high interregion divergences and potential existence of six cryptic species. Pairwise interregional comparisons were more informative than global divergence assessments with regard to detection of cryptic variation. Our analysis exemplifies optimal use of the expanding barcode data now becoming available.     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。     

Using COI barcodes to identify forensically and medically important blowflies

Abstract.  The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658-bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0–0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458–9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.     

Identification of Birds through DNA Barcodes

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.     

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.     

The dark side of pseudoscorpion diversity: The German Barcode of Life campaign reveals high levels of undocumented diversity in European false scorpions

DNA barcoding is particularly useful for identification and species delimitation in taxa with conserved morphology. Pseudoscorpions are arachnids with high prevalence of morphological crypsis. Here, we present the first comprehensive DNA barcode library for Central European Pseudoscorpiones, covering 70% of the German pseudoscorpion fauna (35 out of 50 species). For 21 species, we provide the first publicly available COI barcodes, including the rare Anthrenochernes stellae Lohmander, a species protected by the FFH Habitats Directive. The pattern of intraspecific COI variation and interspecific COI variation (i.e., presence of a barcode gap) generally allows application of the DNA barcoding approach, but revision of current taxonomic designations is indicated in several taxa. Sequences of 36 morphospecies were assigned to 74 BINs (barcode index numbers). This unusually high number of intraspecific BINs can be explained by the presence of overlooked cryptic species and by the accelerated substitution rate in the mitochondrial genome of pseudoscorpions, as known from previous studies. Therefore, BINs may not be an appropriate proxy for species numbers in pseudoscorpions, while partitions built with the ASAP algorithm (Assemble Species by Automatic Partitioning) correspond well with putative species. ASAP delineated 51 taxonomic units from our data, an increase of 42% compared with the present taxonomy. The Neobisium carcionoides complex, currently considered a polymorphic species, represents an outstanding example of cryptic diversity: 154 sequences from our dataset were allocated to 23 BINs and 12 ASAP units.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

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...     

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two‐parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour‐joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (～10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli.