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。     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

基于mtDNA COI基因的离腹寡毛实蝇属常见种DNA条形码识别和系统发育分析

【目的】离腹寡毛实蝇属Bactrocera昆虫是最具经济重要性的实蝇类害虫,本研究依据mtDNA COI基因碱基序列对离腹寡毛实蝇属常见实蝇种类进行识别鉴定与系统发育分析。【方法】以口岸经常截获的离腹寡毛实蝇属8个亚属21种实蝇为对象,采用DNA条形码技术,通过对mtDNA COI基因片段 (约650 bp)的测序和比对,以MEGA软件的K2-P双参数模型计算种内及种间遗传距离,以邻接法(NJ) 构建系统发育树。【结果】聚类分析与形态学鉴定结果一致,除11种单一序列实蝇外,其他10种实蝇均各自形成一个单系,节点支持率为99%以上。种内（10种）遗传距离为0.0003~0.0068,平均为0.0043;种间（21种）遗传距离为0.0154~0.2395,平均为0.1540;种间遗传距离为种内遗传距离的35.8倍,而且种内、种间遗传距离没有重叠区域。【结论】基于mtDNA COI基因的DNA条形码技术可以用于离腹寡毛实蝇属昆虫的快速鉴定识别,该技术体系的建立对实蝇类害虫的检测监测具有重要意义。     

基于线粒体COI和16S片段序列的北部湾北部水螅水母DNA条形码分析

水螅水母类是浮游动物群落的重要组成部分,在近岸海洋生态系统物质循环和能量流动中扮演着重要角色。水螅水母类形态结构简单,但其物种的准确鉴定一直是分类工作中的难点。DNA条形码极大地促进了水螅水母物种的快速、准确鉴定。本研究扩增了北部湾北部28种水螅水母的线粒体COI和16S序列,分别为92条和116条;比较了2个基因片段的种内、种间K2P(Kimura 2-parameter)遗传距离;构建了基于这2个基因片段的系统发育邻接树(neighbor-joining phylogenetic tree);并结合矢量分析构建了Klee-diagram图。结果显示:COI序列的种内遗传距离为0.008±0.005(0–0.033),种间遗传距离为0.298±0.128(0.092–0.597);16S序列的种内遗传距离为0.006±0.010(0–0.047),种间遗传距离为0.394±0.195(0.068–0.898)。2个基因序列在所调查种类中,种内遗传差异均小于种间遗传差异,存在明显的条形码间隔(barcoding gap)。基于2个基因片段的NJ树均显示,单种所有个体都位于同一独立分枝。研究结果表明,以COI和16S作为DNA条形码均能对北部湾北部常见水螅水母类进行物种鉴定。     

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。     

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,p0.01,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。     

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。     

云南边境地区果实蝇属种类DNA条形码鉴定

【目的】果实蝇属Bactrocera中有国际上重要的检疫性害虫, 基于形态的物种鉴定有一定的局限性。另一方面, 云南边境地区为东南亚地区实蝇入侵我国的重要通道。因此, 对该地区实蝇分子鉴定方法的研究对于该属物种的快速准确鉴定具有重要意义。本研究旨在探讨DNA条形码技术在果实蝇属物种鉴定中的有效性。【方法】使用线粒体基因COI和COII序列的通用引物对果实蝇属20个物种60份样品进行PCR扩增、测序和序列分析; 采取距离方法和建树方法评价2种序列的鉴别能力。【结果】COI和COII序列平均长度分别为682 bp和339 bp, 种内和种间遗传差异较大, 有较明显的遗传距离间隔（barcoding gap）, 鉴定成功率分别为91.2%和90.7%。另外, 分子系统树表明华实蝇亚属Sinodacus不是单系群。【结论】COI和COII序列均能够将绝大多数果实蝇属物种进行准确鉴别, 应用COI或COII序列进行果实蝇属物种鉴定具有一定的可行性。     

基于通用引物的多重PCR对小圆胸小蠹的分子鉴定

本研究利用通用引物的多重PCR方法开展小圆胸小蠹Euwallacea fornicatus的分子鉴定,以期探究多重PCR在昆虫分子鉴定中的可行性,并为开展小圆胸小蠹的有效、准确鉴定及综合防治等提供重要依据。使用多重PCR方法扩增了小圆胸小蠹的COI、16S和28S的3个分子片段,并将获得的目的序列在GenBank中进行BLAST比对;利用MEGA 7计算方胸小蠹属不同种间的遗传距离,并基于邻接法和最大似然法分别构建单基因系统发育树。结果表明：多重PCR可以用于小圆胸小蠹分子序列的获取;基于COI和16S的遗传距离分析表明了小圆胸小蠹的种内遗传距离均小于2%;基于单个基因构建的系统发育树均显示本研究扩增的小圆胸小蠹COI和16S序列与GenBank中获取的小圆胸小蠹COI和16S序列聚为一支。多重PCR可以应用于小圆胸小蠹的分子鉴定,该方法不仅可以提高物种鉴定的准确率,还可以减少PCR过程中的时间和DNA消耗。     

苹果园鳞翅目夜蛾科DNA条形码鉴定

为了检验DNA条形码在鳞翅目夜蛾科蛾类鉴定中的可行性,本文对采自北京昌平苹果园内的夜蛾科14种71头蛾类标本分别提取了DNA,并扩增了线粒体cox1及核基因28S,利用系统发育树、遗传距离、阈值等方法进行了鉴定和比较分析。同时,检验了目前BOLD系统的鉴定成功率。实验表明,基于cox1基因和BOLD系统的鉴定成功率达到了100%,而基于28S则很低,为64.8%。用不同方法构建的系统发育树,鉴定结果均相同。93%的种内遗传距离小于1%,94%的种间遗传距离为大于3%,种内种间的遗传距离形成明显的3%阈值现象。     

Applying DNA barcoding for the study of geographical variation in host-parasitoid interactions

Studies on the biogeography of host-parasitoid interactions are scarce, mainly because of technical difficulties associated with rearing and species identification. DNA barcoding is increasingly recognized as a valuable tool for taxon identification, allowing to link different life history stages of a species. We evaluate the usefulness of a protocol based on cytochrome oxidase I (COI) sequencing for the study of geographical variation of host-parasitoid interactions. Larvae of Acroclita subsequana (Lepidoptera: Tortricidae) were collected in Macaronesia and dissected to search for parasitoid larvae. Both hosts and parasitoids were sequenced and assigned to molecular operational taxonomic units (MOTUs) based on pairwise genetic distances, tree-based and similarity-based methods. Hosts were grouped into six MOTUs, usually with an allopatric distribution, while parasitoids clustered into 12 MOTUs, each of which was mostly found attacking a single host MOTU. Available COI sequence databases failed to provide identification to species level for these MOTUs. Three challenges related to the applicability of DNA barcoding in this type of studies are identified and discussed: (i) more suitable primers need to be developed for both parasitoids and hosts; (ii) the most commonly used approaches for inferring MOTUs have different limitations (e.g. arbitrary nature of defining a threshold to separate MOTUs) and need to be improved or replaced by other techniques; and (iii) for the identification of MOTUs, it is imperative to increase the range of sequenced taxa in the currently available reference databases. Finally, in spite of these difficulties, we discuss how DNA barcoding will help ecological and biogeographical studies of host-parasitoid interactions.     

MOTU analysis of ichthyoplankton biodiversity in the upper Yangtze River,China

Identification of ichthyoplankton is difficult because fish during early life stages often lack stable morphological characteristics; such difficulty in species identification can be a major hindrance in conducting ichthyoplankton surveys for fish biodiversity investigations. Here, we evaluated the feasibility of a molecular operational taxonomic unit (MOTU) approach for ichthyoplankton investigations, and describe fish biodiversity in the Jinshajiang section of the upper Yangtze River, China. The MOTUs were established by grouping specimens diverging less than 1.00% Kimura two‐parameter (K2P) distance units from their nearest neighbor within the same MOTU, based on previous work on between‐species divergences of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Taxonomic assignment of the MOTUs was performed by comparing the MOTU sequences with the COI sequences of taxonomic species. Sixty‐eight MOTUs were inferred from 818 COI sequences of ichthyoplankton in the Jinshajiang river section. Among those, one MOTU was composed of two identified taxonomic species, and each of the other MOTUs was linked to a single, identified taxonomic species. Only 26 MOTUs were successfully identified to taxonomic species due to the limited reference database. Our results demonstrate that the MOTU approach can be applied successfully for analyzing biodiversity and identifying species of freshwater ichthyoplankton. Compared with previous ichthyoplankton investigations the richness of ichthyoplankton was very high. High diversity of ichthyoplankton noted in our study suggests that the Jinshajiang section should be an important target for fish biodiversity conservation in the Yangtze River.     

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。     

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis.     

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.     

MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

A metagenetic approach for revealing community structure of marine planktonic copepods

Marine planktonic copepods are an ecologically important group with high species richness and abundance. Here, we propose a new metagenetic approach for revealing the community structure of marine planktonic copepods using 454 pyrosequencing of nuclear large subunit ribosomal DNA. We determined an appropriate similarity threshold for clustering pyrosequencing data into molecular operational taxonomic units (MOTUs) using an artificial community containing 33 morphologically identified species. The 99% similarity threshold had high species‐level resolution for MOTU clustering but overestimated species richness. The artificial community was appropriately clustered into MOTUs at 97% similarity, with little inflation in MOTU numbers and with relatively high species‐level resolution. The number of sequence reads of each MOTU was correlated with dry weight of that taxon, suggesting that sequence reads could be used as a proxy for biomass. Next, we applied the method to field‐collected samples, and the results corresponded reasonably well with morphological analysis of these communities. Numbers of MOTUs were well correlated with species richness at 97% similarity, and large numbers of sequence reads were generally observed in MOTUs derived from species with large biomass. Further, MOTUs were successfully classified into taxonomic groups at the family level at 97% similarity; similar patterns of species richness and biomass were revealed within families with metagenetic and morphological analyses. At the 99% similarity threshold, MOTUs with high proportions of sequence reads were identified as biomass‐dominant species in each field‐collected sample. The metagenetic approach reported here can be an effective tool for rapid and comprehensive assessment of copepod community structure.     

Mind the gap! Integrating taxonomic approaches to assess ant diversity at the southern extreme of the Atlantic Forest

Understanding patterns of species diversity relies on accurate taxonomy which can only be achieved by long‐term natural history research and the use of complementary information to establish species boundaries among cryptic taxa. We used DNA barcoding to characterize the ant diversity of Iguazú National Park (INP), a protected area of the Upper Paraná Atlantic Forest ecoregion, located at the southernmost extent of this forest. We assessed ant diversity using both cytochrome c oxidase subunit 1 (COI) sequences and traditional morphological approaches, and compared the results of these two methods. We successfully obtained COI sequences for 312 specimens belonging to 124 species, providing a DNA barcode reference library for nearly 50% of the currently known ant fauna of INP. Our results support a clear barcode gap for all but two species, with a mean intraspecific divergence of 0.72%, and an average congeneric distance of 17.25%. Congruently, the library assembled here was useful for the discrimination of the ants of INP and allowed us to link unidentified males and queens to their worker castes. To detect overlooked diversity, we classified the DNA barcodes into Molecular Operational Taxonomic Units (MOTUs) using three different clustering algorithms, and compared their number and composition to that of reference species identified based on morphology. The MOTU count was always higher than that of reference species regardless of the method, suggesting that the diversity of ants at INP could be between 6% and 10% higher than currently recognized. Lastly, our survey contributed with 78 new barcode clusters to the global DNA barcode reference library, and added 36 new records of ant species for the INP, being 23 of them new citations for Argentina.