鸡爪槭种下分类群的DNA条形码筛选

槭属鸡爪槭(Acer palmatum Thunb.)是北温带广泛分布的一类园艺观赏树种,由于频繁的种内杂交渐渗导致其种下分类群的形态性状特征趋同,致使传统的形态学分类难以准确鉴定,新兴的DNA条形码技术为快速、准确的鉴定鸡爪槭种下分类群提供了新的思路。本研究采用5个叶绿体DNA片段(rpl16、psbA-trnH、trnL-trnF、rbcL、matK)和核基因组ITS片段,运用PWG-distance和Tree-Building两种方法对鸡爪槭的8个分类群共32个个体进行DNA条形码分析。结果显示,单个叶绿体基因组片段(分辨率为0%~25%)或核rDNA ITS片段(12.5%)的分辨率较低,不同组合的叶绿体DNA片段(0%~62.5%)、叶绿体片段与核rDNA ITS片段(12.5%~50%)的分辨率则相对较高。其中,rpl16+psbA-trnH+trnL-trnF片段组合的分辨率最高(62.5%),较为符合DNA条形码快速、准确鉴定的要求,因此建议将其作为鸡爪槭种下分类群鉴定的DNA条形码。     

蒟蒻薯属 (薯蓣科) 植物DNA条形码研究

蒟蒻薯属（Tacca）植物种间在形态上差别不大,导致分类上存在一定的困难。DNA条形码是一种利用短的DNA标准片段来鉴别和发现物种的方法。本研究利用核基因ITS片段和叶绿体基因trnH psbA, rbcL, matK片段对蒟蒻薯属6个种的DNA条形码进行研究,对4个DNA片段可用性,种内种间变异,barcode gap进行了分析,采用Tree based和BBA两种方法比较不同序列的鉴定能力。结果显示：单片段ITS正确鉴定率最高,片段组合rbcL+matK正确鉴定率最高。支持CBOL植物工作组推荐的条码组合rbcL+matK可作为蒟蒻薯属物种鉴定的标准条码,建议ITS片段作为候选条码。丝须蒟蒻薯Tacca integrifolia采自西藏的居群与马来西亚居群形成了2个不同的遗传分支,且两者在形态上也存在一定的差异,很可能是一个新种。     

蒟蒻薯属(薯蓣科)植物DNA条形码研究

蒟蒻薯属(Tacca)植物种间在形态上差别不大,导致分类上存在一定的困难.DNA条形码是一种利用短的DNA标准片段来鉴别和发现物种的方法.本研究利用核基因ITS片段和叶绿体基因trn H-psbA,rbcL,matK片段对蒟蒻薯属6个种的DNA条形码进行研究,对4个DNA片段可用性,种内种间变异,barcode gap进行了分析,采用Tree-based和BBA两种方法比较不同序列的鉴定能力.结果显示:单片段ITS正确鉴定率最高,片段组合rbcL+matK正确鉴定率最高.支持CBOL植物工作组推荐的条码组合rbcL+matK可作为蒟蒻薯属物种鉴定的标准条码,建议ITS片段作为候选条码.丝须蒟蒻薯Tacca integrifolia采自西藏的居群与马来西亚居群形成了2个不同的遗传分支,且两者在形态上也存在一定的差异,很可能是一个新种.     

地黄属植物的DNA条形码研究

地黄属(Rehmannia)为玄参科(Scrophulariaceae)药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段(trn L-trn F、trn M-trn V和trn S-trn G)及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明:单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低(0%~20%),组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G+ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合(trn L-trn F+trn M-trn V+trn S-trn G+ITS)的辨别率相同,因此推荐trn S-trn G+ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。     

DNA Barcoding of Celyphidae (Diptera) from Vietnam

The COI gene fragment was first examined in representatives of the family Celyphidae. The presence of a considerable hiatus between interspecific and intraspecific distances was demonstrated, which enabled using the barcoding method to distinguish species of the family Celyphidae. The results of the analysis showed that different species of Celyphidae clustered together. Within the Celyphus (Hemiglobus) porosus cluster, some haplotype heterogeneity, which was, however, within the limits of intraspecific variability, was observed.     

ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of an Endangered Slipper Orchid,Paphiopedilum micranthum (Orchidaceae)

Paphiopedilum micranthum is an endangered pink slipper orchid mainly distributed in the limestone areas of southwestern China. Wild populations of this species have been seriously threatened by excessive collections, rampant smuggling for export, and habitat destruction. We used 15 ISSR markers and 11 SRAP markers to investigate the genetic diversity and structure of 15 natural populations. A high degree of diversity was observed at the species level (ISSR: PPB=9166%, He=03839; SRAP: PPB=9929%, He=02806). Certain degree of genetic differentiation among populations (ISSR: Gst=02577; SRAP: Gst=02383) was detected maybe caused by low gene flow (ISSR: Nm=07201; SRAP: Nm=07991). Consistent with the results of Principal Coordinate Analysis, the UPGMA dendrogram analysis divided the 15 populations into two main clades. In addition to geographic distance, the difference in elevation was another natural factor contributing to this differentiation. Knowledge about genetic diversity and structure gained from our study will be beneficial for the development of reasonable and efficient strategies to conserve this endangered species.     

DNA Barcoding Works in Practice but Not in (Neutral) Theory

Background

DNA barcode differences within animal species are usually much less than differences among species, making it generally straightforward to match unknowns to a reference library. Here we aim to better understand the evolutionary mechanisms underlying this usual “barcode gap” pattern. We employ avian barcode libraries to test a central prediction of neutral theory, namely, intraspecific variation equals 2 Nµ, where N is population size and µ is mutations per site per generation. Birds are uniquely suited for this task: they have the best-known species limits, are well represented in barcode libraries, and, most critically, are the only large group with documented census population sizes. In addition, we ask if mitochondrial molecular clock measurements conform to neutral theory prediction of clock rate equals µ.

Results

Intraspecific COI barcode variation was uniformly low regardless of census population size (n = 142 species in 15 families). Apparent outliers reflected lumping of reproductively isolated populations or hybrid lineages. Re-analysis of a published survey of cytochrome b variation in diverse birds (n = 93 species in 39 families) further confirmed uniformly low intraspecific variation. Hybridization/gene flow among species/populations was the main limitation to DNA barcode identification.

Conclusions/Significance

To our knowledge, this is the first large study of animal mitochondrial diversity using actual census population sizes and the first to test outliers for population structure. Our finding of universally low intraspecific variation contradicts a central prediction of neutral theory and is not readily accounted for by commonly proposed ad hoc modifications. We argue that the weight of evidence–low intraspecific variation and the molecular clock–indicates neutral evolution plays a minor role in mitochondrial sequence evolution. As an alternate paradigm consistent with empirical data, we propose extreme purifying selection, including at synonymous sites, limits variation within species and continuous adaptive selection drives the molecular clock.     

DNA Barcoding of Japanese Click Beetles (Coleoptera,Elateridae)

Click beetles (Coleoptera: Elateridae) represent one of the largest groups of beetle insects. Some click beetles in larval form, known as wireworms, are destructive agricultural pests. Morphological identification of click beetles is generally difficult and requires taxonomic expertise. This study reports on the DNA barcoding of Japanese click beetles to enable their rapid and accurate identification. We collected and assembled 762 cytochrome oxidase subunit I barcode sequences from 275 species, which cover approximately 75% of the common species found on the Japanese main island, Honshu. This barcode library also contains 20 out of the 21 potential pest species recorded in Japan. Our analysis shows that most morphologically identified species form distinct phylogenetic clusters separated from each other by large molecular distances. This supports the general usefulness of the DNA barcoding approach for quick and reliable identification of Japanese elaterid species for environmental impact assessment, agricultural pest control, and biodiversity analysis. On the other hand, the taxonomic boundary in dozens of species did not agree with the boundary of barcode index numbers (a criterion for sequence-based species delimitation). These findings urge taxonomic reinvestigation of these mismatched taxa.     

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.     

Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae)

Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25–26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color gradient between the setae submenti and genal setae. DNA barcodes performances were high: average accuracy was ~89% and precision of ~99%. On the basis of the present data, we can thus conclude that molecular identification represents a promising tool that could be effectively adopted in evaluating biodiversity of high-altitude streams.     

Analyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Barcoding

Background

Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications.

Methodology/Principal Findings

Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010–2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0–2.4%, while congeneric species showed from 2.3–17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments.

Conclusions

As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations.     

DNA条形码在中国金合欢属中的应用

根据形态特征难以准确地辨别金合欢属植物,DNA条形码技术提供了一种准确地鉴定物种的方法。本文利用条形码技术对中国金合欢属物种的序列(psbA trnH、matK、rbcL和ITS)及其不同组合进行比较,通过计算种内和种间变异进行barcoding gap分析,运用Wilcoxon秩和检验比较不同序列的变异性,构建系统树。结果表明：4个片段均存在barcoding gap,ITS序列种间变异率较psbA trnH、rbcL和matK序列有明显优势,单片段ITS正确鉴定率最高,ITS+rbcL片段联合条码的正确鉴定率最高,因此我们认为ITS片段或条形码组合ITS+rbcL是金合欢属的快速鉴别最理想的条码。     

DNA条形码及其在生物多样性研究中的应用

DNA条形码是利用生物体内标准的、有足够变异的、易扩增且相对较短的DNA片段对物种进行快速准确鉴定的技术。自2003年DNA条形码相关概念提出以来广受关注,国内外相继开展了DNA条形码及信息系统建设研究,为DNA条形码技术的发展提供了坚实的研究基础和生物信息学分析平台。DNA条形码技术弥补了传统分类学的不足,为生物多样性研究提供了新的思路和方法。本文介绍了DNA条形码的产生与发展过程,国内外DNA条形码技术与信息系统建设研究进展,重点阐述了DNA条形码技术在物种鉴定、濒危物种保护、隐存种发现、生物多样性评估等研究领域中的应用。最后结合DNA条形码技术目前存在的问题,对其在相关研究领域的应用前景进行了展望。     

藻类DNA条形码研究进展

DNA barcode,又称为DNA条形码,是指利用短的标准DNA序列的核苷酸多样性进行物种的鉴定和快速识别.目前该方法在动物分类研究中应用广泛,其中线粒体的细胞色素c氧化酶亚基1(cytochrome c oxidase subunit 1,COI或cox 1)基因中的约700bp长度的一段被用来作为标准DNA片段.在陆地植物条形码研究中,生命-植物条形码联盟会(Consortium for the Barcode of Life-Plant Working Group,CBOL-Plant Working Group)近期推荐将植物叶绿体中的两个基因片段rbcL+ matK作为初步的陆生植物条形码,此组合能在70％的程度上进行植物物种的鉴别.在海藻的分类研究中,DNA条形码的应用较少,已有的研究主要集中在硅藻、红藻和褐藻,尚没有学者明确提出适合藻类的DNA条形码.总结了能够作为藻类DNA条形码的序列特点、应用流程及分析方法,综述了DNA条形码在藻类中的研究现状和存在的问题,展望了藻类DNA条形码的应用前景.     

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。     

DNA条形码研究进展

DNA条形码是应用有足够变异的标准化短基因片段对物种进行快速、准确鉴定的新的生物身份识别系统.2003年,加拿大Guelph大学Hebert等首次正式提出了DNA条形码概念,2004年成立了生物条形码联盟,目前有来自50个国家的两百多个组织成为其成员,2007年5月加拿大Guelph大学组建了世界上第一个DNA barcoding鉴定中心,2009年1月正式启动"国际生命条形码计划",中国科学院代表中国与加拿大、美国和欧盟共同为iBOL 4个中心节点.线粒体细胞色素C氧化酶基因COⅠ具有引物通用性高和进化速率快等优点,是理想的动物DNA条形码,不过,COⅠ在植物中应用效果较差,因此,核糖体ITS序列和质体rbcL、matK和trnH-psbA等序列也相继被引入植物的DNA条形码研究.虽然DNA条形码研究还处于起步阶段,面临巨大挑战,但是,越来越多的研究表明DNA条形码可以广泛应用于生物的分类和鉴定,是一种简便、高效、准确的物种鉴定技术,已经在动物、植物和微生物等研究中取得了显著成果,是生命科学领域发展最快的学科前沿之一.本文从DNA条形码的开发、应用、国内相关文献研究现状、DNA条形码面临的挑战以及发展前景等进行了综合分析,以期推动我国DNA条形码和分类学研究的发展.     

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.