中国果实蝇属种类的DNA条形码鉴定(双翅目,实蝇科)

将实验获得的25种果实蝇的155条COⅠ条形码序列,利用MEGA4.1的Kimura-2-Parameter模型进行了遗传距离分析和构建系统发育树,来检验线粒体COⅠ基因条形码序列对果实蝇属种类鉴定的有效性。研究表明COⅠ条形码序列能够对除桔小实蝇复合体外的中国果实蝇属种类进行准确鉴定。     

基于DNA条形码的中国普缘蝽属分类研究(半翅目:异翅亚目)

通过扩增中国普缘蝽属Plinachtus Stl(半翅目:异翅亚目:缘蝽科)已知的4个种:刺肩普缘蝽P.dissimilis Hsiao,1964,钝肩普缘蝽P.bicoloripes Scott,1874,黑普缘蝽P.acicularis(Fabricius,1803)和棕普缘蝽P.basalis(Westwood,1842)的COI(1338bp)序列,计算其种间/种内的遗传距离,并进行基于距离法、最大简约法和贝叶斯法的分析,结果均支持将刺肩普缘蝽和钝肩普缘蝽合并为1个种。     

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.     

High molecular and phenotypic diversity in the Merodon avidus complex (Diptera,Syrphidae): cryptic speciation in a diverse insect taxon

This paper examines molecular and phenotypic variability in the widely spread European hoverfly species complex Merodon avidus. Herein, based on the mitochondrial DNA (mtDNA) sequences of the cytochrome c oxidase subunit I (COI) and morphometric wing parameters, M. avidus is shown to comprise a complex of cryptic species, and one variety is redefined as a valid species: M. bicolor Gil Collado, 1930 (as var. of spinipes). The species M. bicolor, M. avidus A, and M. avidus B were clearly delimited based on their wing size. A total of 29 M. avidus and M. bicolor individuals presented 20 mtDNA haplotypes, four of which were shared by M. avidus A and M. avidus B, three were confined to M. bicolor, seven to M. avidus A, and six to M. avidus B. Sequence divergences between lineages occurring in the Balkan and in Spain ranged from 4.93 to 6.0 (uncorrected p in %) whereas divergences between M. avidus A and M. avidus B were 0.26 to 1.56. Divergence among morphologically identified individuals of M. avidus A and M. avidus B species ranged from 0.13 to 1.58, and from 0.13 to 0.52, respectively. The phenotypic substructuring and observed genetic uniqueness of populations in spatially and temporally fragmented M. avidus taxa were used to identify genetic units. The early split of two allopatric lineages, Spanish M. bicolor and Balkan M. avidus, was followed by diversification in each lineage. Present‐day morphological uniformity masks much of the genetic complexity of lineages within the M. avidus complex. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 819–833.     

云南常见药用石斛DNA条形码筛选

选择云南省内常见的12种药用石斛，采用分子生物学鉴定技术筛选其适用的DNA条形码序列。以12种药用石斛共36个样品为材料，提取样品总DNA，对核基因片段ITS和ITS2、叶绿体基因片段psbA-trnH和matK序列进行扩增、测序，结合GenBank下载部分石斛序列；利用生物信息学软件进行序列分析和系统发育分析。结果表明，4条序列扩增和测序成功率均为100%；4条序列没有明显的Barcoding Gap，但ITS序列与ITS2序列的种内和种间重叠部分较少，有偏向两端的趋势；系统发育树显示，ITS和psbA-trnH序列能成功区分12种云南常见的药用石斛，ITS2序列未能区分长距石斛(Dendrobium longicornu)和矮石斛(D. bellatulum)，matK序列仅区分6种石斛。建议以ITS和psbA-trnH序列作为云南药用石斛鉴定序列，为药用石斛的种源鉴定提供理论依据。     

Identifying Rattus species using mitochondrial DNA

In recent years, research has shown that geographical variation in mitochondrial DNA of commensal rats provides a strong signal of human dispersal and migration. However, interpretation of genetic variation is complicated by the presence of multiple species of Rattus especially in Island Southeast Asia, by the occurrence of some of these Rattus sp. as subfossils in archaeological and natural sites, and by the difficulty of osteological identification of these remains. Amplification of DNA from ancient sources usually yields only small fragments (～200 bp). We assessed whether we could identify Rattus sp. reliably with DNA barcoding using cytochrome oxidase I (COI) sequences, or tree‐based methods using D‐loop, cytochrome b and COI sequences. Species forming well‐differentiated clades in a molecular phylogeny were accurately identified by both methods, even when we used short DNA fragments. Identification was less accurate for paraphyletic and polyphyletic species. We suggest that taxonomic revisions that recognize cryptic or polytypic species will lead to even greater accuracy of DNA‐based identification methods.     

Inter‐ and intraspecific variation in mycotoxin tolerance: A study of four Drosophila species

Many mycophagous Drosophila species have adapted to tolerate high concentrations of mycotoxins, an ability not reported in any other eukaryotes. Although an association between mycophagy and mycotoxin tolerance has been established in many Drosophila species, the genetic mechanisms of the tolerance are unknown. This study presents the inter‐ and intraspecific variation in the mycotoxin tolerance trait. We studied the mycotoxin tolerance in four Drosophila species from four separate clades within the immigrans‐tripunctata radiation from two distinct locations. The effect of mycotoxin treatment on 20 isofemale lines per species was studied using seven gross phenotypes: survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity. We observed interspecific variation among four species, with D. falleni being the most tolerant, followed by D. recens, D. neotestacea, and D. tripunctata, in that order. The results also revealed geographical variation and intraspecific genetic variation in mycotoxin tolerance. This report provides the foundation for further delineating the genetic mechanisms of the mycotoxin tolerance trait.     

江西新岗山森林昆虫种内遗传距离研究

DNA条形码目前广泛用于昆虫多样性研究。本研究采用DNA条形码（即线粒体细胞色素c氧化酶亚基I基因COI 5′端）,通过比较所获分子分类操作单元（Molecular operational taxonomic units,MOTU）的种内遗传距离,探究DNA条形码在亚热带森林（位于我国江西省新岗山）不同昆虫类群中的物种鉴定和界定效用。数据分析中结合数据库比对信息,采用jMOTU、ABGD、bPTP、GMYC 这4种物种界定方法获得MOTU,从而开展种内遗传距离分析。本研究共挑选出479个昆虫样本,获得475条COI序列,经NCBI、BOLD在线数据库比对属于6个目,与形态初步划分一致;物种界定分析获得288个MOTU,其中鳞翅目最多,达85个,膜翅目、双翅目、半翅目、鞘翅目次之,分别为80、74、21和20个,直翅目最少,仅8个。膜翅目和双翅目的种内遗传距离均值及标准偏差较大（膜翅目：0.89%±0.87%;双翅目：0.73%±0.58%）,鳞翅目的最小（0.28%±0.20%）。研究表明：不同昆虫类群的种内遗传距离虽然整体在一定范围,但仍然存在一定的差异,因此不能笼统地依靠遗传距离的距离阈值进行物种划分;现有数据库需要补充足够的昆虫物种信息,才能提升物种鉴定效率。本研究丰富了亚热带森林昆虫分子数据库,同时也为进一步探索基于分子分类学开展昆虫多样性研究提供了基础数据和参考。     

Identification of species within Tetrastigma (Miq.) Planch. (Vitaceae) based on DNA Barcoding Techniques

Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA, and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. hemsleyanum, could be identified precisely using DNA barcoding.     

COI-based DNA barcoding of Arcoida species (Bivalvia: Pteriomorphia) along the coast of China

DNA barcoding is a promising tool for the rapid and unambiguous identification of species. Some arcoid species are particularly difficult to distinguish with traditional morphological identification owing to phenotypic variation and the existence of closely related taxa. Here, we apply DNA barcoding based on mitochondrial cytochrome c oxidase I gene (COI) to arcoid species collected from the coast along China. Combining morphology with molecular data indicates the 133 specimens of Arcoida could be assigned to 24 species. Because of the deep genetic divergence within Tegillarca granosa, there was an overlap between genetic variation within species and variation between species. Nevertheless, NJ and Bayesian trees showed that all species fell into reciprocally monophyletic clades with high bootstrap values. Our results evidence that the COI marker can efficiently identify species, correct mistakes caused by morphological identification and reveal genetic differentiation among populations within species. This study provides a clear example of the usefulness of barcoding for arcoid identification. Furthermore, it also lays a foundation for other biological and ecological studies of Arcoida.     

Analysis of intraspecific and interspecific variation in the genus Alternaria by the use of RAPD-PCR

The RAPD-PCR profiles of 13 phytopathogenic Alternaria species and two closely related outgroups were examined using six different primers. Each species produced a distinct pattern of DNA fragments which were used as a measure of the degree of relatedness between species. A. brassicae isolates of diverse origin showed high levels of similarity but little similarity was noted between other species. The closest interspecific genetic distances were recorded between A. citri, A. alternata and A. longipes. The outgroup genera Embellisia and Stemphylium, which are recognised as distinct, could not be clearly separated using RAPD banding criteria, suggesting a high level of genetic diversity amongst these groups of fungi.     

The campaign to DNA barcode all fishes, FISH-BOL

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System ( http://www.barcodinglife.org ). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.     

DNA barcodes for globally threatened marine turtles: a registry approach to documenting biodiversity

DNA barcoding is a global initiative that provides a standardized and efficient tool to catalogue and inventory biodiversity, with significant conservation applications. Despite progress across taxonomic realms, globally threatened marine turtles remain underrepresented in this effort. To obtain DNA barcodes of marine turtles, we sequenced a segment of the cytochrome c oxidase subunit I (COI) gene from all seven species in the Atlantic and Pacific Ocean basins (815 bp; n = 80). To further investigate intraspecific variation, we sequenced green turtles (Chelonia mydas) from nine additional Atlantic/Mediterranean nesting areas (n = 164) and from the Eastern Pacific (n = 5). We established character-based DNA barcodes for each species using unique combinations of character states at 76 nucleotide positions. We found that no haplotypes were shared among species and the mean of interspecific variation ranged from 1.68% to 13.0%, and the mean of intraspecific variability was relatively low (0–0.90%). The Eastern Pacific green turtle sequence was identical to an Australian haplotype, suggesting that this marker is not appropriate for identifying these phenotypically distinguishable populations. Analysis of COI revealed a north–south gradient in green turtles of Western Atlantic/Mediterranean nesting areas, supporting a hypothesis of recent dispersal from near equatorial glacial refugia. DNA barcoding of marine turtles is a powerful tool for species identification and wildlife forensics, which also provides complementary data for conservation genetic research.     

Phylogenetic relationships among species of the Neotropical genus Graomys (Rodentia: Cricetidae): contrasting patterns of skull morphometric variation and genetic divergence

Subtle differences of external traits characterize species of rodents in the Neotropical genus Graomys. On the other hand, the species differ markedly in chromosome number. In the present study, we evaluate the possible evolutionary forces involved in the evolution of the genus by assessing the degree of intra‐ and interspecific genetic and morphological variation. A phylogenetic analysis demonstrates the existence of at least three species with high levels of genetic distance (10%), which diverged between 1 and 1.5 Mya. Neither Graomys griseoflavus, nor Graomys chacoensis present marked phylogeographical structure. Regarding morphological characters, these species show shape differences in the skull that could be attributable to differences in the local conditions they inhabit, being more marked in G. griseoflavus than in G. chacoensis. The skull shape of G. chacoensis could have evolved under genetic drift, whereas evidence reported in the present study indicates that this character could be under selective pressures in G. griseoflavus. Reconstruction of the ancestral area suggests that G. griseoflavus originated in the central Monte desert, whereas G. chacoensis originated in the Chaco ecoregion surrounding the austral extreme of the Yungas rainforest. Subsequently, both species would have undergone demographic and geographical expansions almost simultaneously, starting approximately 150 000–175 000 years ago. The complex evolutionary history of the genus could be partly explained by the decoupling of morphological, karyological and molecular traits.     

Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae

Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell‐based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species‐rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin^® Tissue XS Kit, DNeasy^® Blood and Tissue kit, and QuickExtract^? DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments.     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

[目的]DNA条形码技术已成为生物分类鉴定的有力工具.DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性.我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类.[方法]针对大小蠹昆虫设计引物以提高PCR扩增效率.运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定.[结果]使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段.将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种.通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100％),与形态鉴定结果一致.[结论]结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类.该检测技术可以应用于其他经济重要性有害生物的检测鉴定.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.