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。     

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.     

DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

DNA条形码技术在植物中的研究现状

DNA条形码技术（DNA barcoding）是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I（COI）进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

华北地区四种梢斑螟的遗传分化

根据线粒体COⅠ基因序列,对华北地区梢斑螟属Dioryctria Zeller的微红梢斑螟D.rubella Hampson、芽梢斑螟D.yiai Mutuura&Munroe、大梢斑螟D.magnifica Munroe和冷杉梢斑螟D.abietella Denis & Schiffermüller共4种,9个不同地理种群进行遗传多样性研究,并以同族的渣云翅斑螟Oncocera faecella(Zeller)为外群探讨它们之间的系统发育关系。结果表明:(1)微红梢斑螟和大梢斑螟的亲缘关系较近,与芽梢斑螟和冷杉梢斑螟构成的姐妹群分别形成华北地区梢斑螟属的两大支系;(2)种内不同地理种群间的遗传距离(0.000～0.016)明显小于种间遗传距离(0.029～0.089),说明利用线粒体COⅠ基因序列研究梢斑螟属昆虫的系统发育关系是可行的。     

Half of the European fruit fly species barcoded (Diptera,Tephritidae); a feasibility test for molecular?identification

A feasibility test of molecular identification of European fruit flies (Diptera: Tephritidae) based on COI barcode sequences has been executed. A dataset containing 555 sequences of 135 ingroup species from three subfamilies and 42 genera and one single outgroup species has been analysed. 73.3% of all included species could be identified based on their COI barcode gene, based on similarity and distances. The low success rate is caused by singletons as well as some problematic groups: several species groups within the genus Terellia and especially the genus Urophora. With slightly more than 100 sequences – almost 20% of the total – this genus alone constitutes the larger part of the failure for molecular identification for this dataset. Deleting the singletons and Urophora results in a success-rate of 87.1% of all queries and 93.23% of the not discarded queries as correctly identified. Urophora is of special interest due to its economic importance as beneficial species for weed control, therefore it is desirable to have alternative markers for molecular identification.We demonstrate that the success of DNA barcoding for identification purposes strongly depends on the contents of the database used to BLAST against. Especially the necessity of including multiple specimens per species of geographically distinct populations and different ecologies for the understanding of the intra- versus interspecific variation is demonstrated. Furthermore thresholds and the distinction between true and false positives and negatives should not only be used to increase the reliability of the success of molecular identification but also to point out problematic groups, which should then be flagged in the reference database suggesting alternative methods for identification.     

Molecular-based estimate of species number,phylogenetic relationships and divergence times for the genus Stenotaenia (Chilopoda,Geophilomorpha) in the Italian region

Stenotaenia is one of the largest and most widespread genera of geophilid centipedes in the Western Palearctic, with a very uniform morphology and about fifteen species provisionally recognized. For a better understanding of Stenotaenia species-level taxonomy, we have explored the possibility of using molecular data. As a preliminary assay, we sampled twelve populations, mainly from the Italian region, and analyzed partial sequences of the two genes COI and 28S. We employed a DNA-barcoding approach, complemented by a phylogenetic analysis coupled with divergence time estimation. Assuming a barcoding gap of 10–16% K2P pairwise distances, we found evidence for the presence of at least six Stenotaenia species in the Italian region, which started diverging about 50 million years ago, only partially matching with previously recognized species. We found that small-sized oligopodous species belong to a single clade that originated about 33 million years ago, and obtained some preliminary evidence of the related genus Tuoba being nested within Stenotaenia.     

Phylogeny of the green lacewing Chrysoperla nipponensis species‐complex (Neuroptera: Chrysopidae) in China,based on mitochondrial sequences and AFLP data

Abstract Several studies have indicated that the green lacewing, Chrysoperla nipponensis (Neuroptera: Chrysopidae) may include more than one valid species. We investigated the phylogenetic status of Chrysoperla nipponensis s.l. in China and Japan using mitochondrial sequences and AFLP data. The molecular phylogenetic analyses based on mitochondrial genes showed that the C. nipponensis species‐complex comprises four clades, each having high support values. In addition, the phylogenetic tree based on AFLP data indicates that the species‐complex comprises three groups. These results confirm that C. nipponensis s.l. comprises at least three genetically distinct clades and suggests that two of these clades may be closely related to populations of C. nipponensis in Japan. However, these clades cannot be recognized as species until analysis of their courtship songs has been completed.     

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。     

Species diversity of Onchidiidae (Eupulmonata: Heterobranchia) on the mainland of China based on molecular data

The identification of members of the Onchidiidae is based on morphological characters; this is often time-consuming and can be inconclusive. In order to explore the species diversity of onchidiids in China, we provide a phylogeny constructed using partial sequences of two mitochondrial genes (16S rRNA and COI) and one nuclear ribosomal RNA gene (28S rRNA) from 32 samples comprising five genera. The topology, using both Bayesian and Maximum Likelihood inference methods, showed that the taxa clustered in two main groups of six species, one of which included Platevindex mortoni, Platevindex sp. and Onchidium ‘struma’; the other included Paraoncidium reevesii, Onchidella sp. and Peronia verruculata. It is clear that COI will be useful in discriminating onchidiid species-group taxa.     

Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding

Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host - the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.     

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。     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

基于COI基因5′端与3′端序列田间常见粉虱的分子鉴定

【目的】粉虱种类繁多,个体微小,其种类识别与鉴定常需借助分子生物学技术。本研究旨在明确线粒体COI基因(mitochondrial cytochrome c oxidase subunit I gene) 5′端和3′端序列对常见种类粉虱识别鉴定的可行性。【方法】以我国田间常见的16种粉虱为对象,以COI基因5′端(641 bp)和3′端(738 bp)序列为靶标进行比对分析,以MEGA 5.10软件的K2-P模型计算种内与种间遗传距离,以邻接法(NJ法)构建进化树并进行系统发育分析。【结果】当以5′端为靶标时,16种粉虱的种内平均遗传距离为0.0015,种间平均遗传距离为0.2897,种间遗传距离为种内遗传距离的193.1倍;而且种内、种间遗传距离没有重叠区域。当以3′端为靶标时种内平均遗传距离为0.0007,种间平均遗传距离为0.2817,种间遗传距离为种内遗传距离的402.4倍;但桑粉虱Pealius mori与烟粉虱Bemisia tabaci Asia II 1的种内和种间遗传距离重叠。系统发育分析结果显示,以5′端为靶标时,16种粉虱可以形成独立的进化分支;以3′端为靶标时,除桑粉虱与传统分类学不一致外,其余种类均可形成独立的分支。【结论】结果表明,5′端序列更适用于基于DNA条形码技术的物种识别鉴定研究。     

中国与北美大陆的高卢蜜环菌系统发育分析

在中国和北美大陆分别收集53和15株高卢蜜环菌Armillaria gallica菌株,并用ISSR（inter-simple sequence repeat）分子标记对这些菌株进行了亲缘关系及系统发育分析。结果表明：中国和北美大陆的A. gallica因地理隔离产生明显的遗传分异,在系统发育树上分别形成了各自的进化分支;北美大陆的分支内部遗传分化尚不明显,而中国的进化分支遗传分化程度相对较大且更为古老,中国菌株可能是两个大陆A. gallica的祖先株系。