毛茛叶报春高分化的谱系地理结构

毛茛叶报春（Primula ranunculoides Chen）是我国特有的珍稀濒危花卉,其野生种质资源已十分稀少。为了揭示其谱系地理结构,本文对11个居群114个个体的叶绿体trnL-F和trnS-G基因片段进行了测序分析。结果共检测到10个单倍型,除YJPA和YJPB两居群共享一个单倍型（H9）外,其余各居群均仅有一个特有单倍型,居群内不存在单倍型多样性。毛茛叶报春具高分化的谱系地理结构,居群间的总平均遗传距离达0.015,单倍型间的平均突变次数高达31.2步,两者均显著高于同属的近缘种。其种子散布能力弱、生境片断化和地理隔离明显及两年生的生活史可能是导致其高分化谱系地理结构产生的主要原因。Samova、Structure以及TCS分析均支持将毛茛叶报春11个居群的遗传结构分为6组,它们与地形（山脉）关系密切对应,彼此间分化明显,因此建议不同山体的居群应视为不同的保护单元进行遗传管理。     

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.     

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.     

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.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Revision for the swordtail cricket genus Homoeoxipha (Grylloidea: Trigonidiidae: Trigonidiinae) from China

Species of Homoeoxipha are very small and always live on leaves of shrubs and grasses. This genus contains ten species worldwide and is distributed across five zoological regions, from Africa to New Guinea. Homoeoxipha lycoides is the type species of this genus and possesses the characteristic coloration for the genus. These coloration features resulted in some synonyms and disputes over species, because most Homoeoxipha species are ornamented with a similar coloration pattern. We compared and documented the differences between H. lycoides and its relatives in China. A new important character, the epiphallic transversal suture, is used to distinguish them. Based on this work, all of the Chinese species, H. lycoides, H. obliterata and H. nigripes, and one new species, H. eurylobus, with similar coloration to H. lycoides, are described and illustrated.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences ( KY468817 – KY468826 ) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae)

It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well-studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH-psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH-psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding.     

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.     

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.     

我国32种鸟类DNA条形码分析

DNA条形码是一种分子分类方法,近年来在物种鉴定方面得到迅速的发展和应用.本研究分析了我国27属32种鸟类(61只)的线粒体细胞色素c氧化酶亚基Ⅰ(COⅠ)基因的条形码片段,分别用阈值法、聚类法和诊断核苷酸进行了分析,探究DNA条形码鉴定我国鸟类的准确性.结果显示,种内CO Ⅰ序列变异很小,种间存在较多的变异位点,种间的遗传距离显著大于种内的遗传距离,DNA条形码序列能够鉴定所有鸟类.     

A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women''s health.

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.     

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。     

铁线莲属植物的引种栽培研究初报

对部分铁线莲属（Clematis L.)植物进行了引种和栽培,根据花枝的着生情况把铁线莲分为3组。通过对野生种Cl.ranunculoides去年采收的种子进行有性繁殖,结果表明进行春化处理后播种的种子萌发率最高,达71％。对最适合在昆明生长的野生种Cl.ranunculoides和栽培品种Cl.montana‘Vera’进行扦插试验。结果表明Cl.montann ‘Vera’的生根率较高;扦插基质山沙优于珍珠岩;经NAA或IBA处理过的插条一根数多于对照。对栽培土壤进行了分析,结果表明大部分铁线莲也可在偏酸性土壤中生长。     

Recognition and classification of histones using support vector machine.

Histones are DNA-binding proteins found in the chromatin of all eukaryotic cells. They are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3, and H4. Two copies of H2A, H2B, H3, and H4 bind to about 160 base pairs of DNA forming the core of the nucleosome (the repeating structure of chromatin) and H1/H5 bind to its DNA linker sequence. Overall, histones have a high arginine/lysine content that is optimal for interaction with DNA. This sequence bias can make the classification of histones difficult using standard sequence similarity approaches. Therefore, in this paper, we applied support vector machine (SVM) to recognize and classify histones on the basis of their amino acid and dipeptide composition. On evaluation through a five-fold cross-validation, the SVM-based method was able to distinguish histones from nonhistones (nuclear proteins) with an accuracy around 98%. Similarly, we obtained an overall >95% accuracy in discriminating the five classes of histones through the application of 1-versus-rest (1-v-r) SVM. Finally, we have applied this SVM-based method to the detection of histones from whole proteomes and found a comparable sensitivity to that accomplished by hidden Markov motifs (HMM) profiles.     

Italian odonates in the Pandora's box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale

The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA‐based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658‐bp 5′ end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi‐approach species delimitation analysis involving two distance (OT and ABGD) and four tree‐based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as ‘warnings’ (i.e. showing a mismatch between morphospecies assignment and DNA‐based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA‐based monitoring studies.     

Discrimination of three Typhlodromalus species (Acari: Phytoseiidae) using random amplified polymorphic DNA markers

We compared the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) banding patterns obtained from four laboratory cultures representing three phytoseiid mite species (Typhlodromalus limonicus (Garman and McGregor), two cultures of Typhlodromalus manihoti (de Moraes) and Typhlodromalus tenuiscutus (McMurtry and de Moraes). The RAPD-PCR was conducted on the pooled DNA from five adult female mites. For each culture, three samples of five females were analysed with each of eight RAPD-PCR primers. Five of the eight primers could be used individually to distinguish the species. To quantify the within- and between-species variation, genetic distances were calculated based on the proportion of shared scorable bands. The within-species genetic distances (0.072-0.186) were much lower than the between-species genetic distances (0.407-0.656). We believe that this technique could be used effectively to identify other cryptic mite species.