DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用

为了探讨DNA条形码技术在夜蛾物种鉴定中的可行性, 本研究利用条形码通用引物扩增了北京百花山地区43种夜蛾75个样本的线粒体细胞色素C氧化酶亚基I （mitochondrial cytochrome c oxidase subunit I, COI）基因序列, 以Kimura双参数模型进行种内种间遗传距离分析、 使用邻接法（neighbor-joining, NJ）和最大简约法（maximum parsimony, MP）分别构建系统发育树, 并利用分子序列差异阈值对样本进行分子可操作分类单元（molecular defined operational taxonomic units, MOTU）划分。结果表明： 所有夜蛾种类通过系统发育树可以成功区分; 种内平均遗传距离(0.03%)远远小于种间平均遗传距离(11.29%); 采用较为保守的1%的序列差异阈值将75个夜蛾样本分为42个MOTU, 正确率为95%, 除了MOTU04包含2个物种外, 剩余41个MOTU与形态种呈现一一对应的关系。结果显示, 基于COI基因的DNA条形码对于本研究中所涉及的夜蛾具有较好的区分, 可以作为一种有效的工具在夜蛾科昆虫物种鉴定中进行应用。     

戴云山国家级自然保护区访花昆虫DNA条形码数据集

访花和传粉昆虫对于维持生态系统功能具有重要作用,但我国相关昆虫类群的本底数据非常缺乏。作为基于特定基因序列的物种划分方法, DNA条形码在标本鉴定、新物种发现、生物多样性保护、种群遗传和进化等研究领域具有重要的应用价值。本文报道了福建戴云山国家级自然保护区双翅目、膜翅目和鞘翅目3个类群访花昆虫的815条线粒体COI条形码数据,并详细提供了所获样品的海拔分布信息。该数据集可为地区性昆虫多样性的DNA条形码数据库构建、隐存种发现、海拔梯度物种遗传多样性和生物多样性保护等方面研究提供帮助。     

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。     

基于三个分子标记的粉毛蚜亚科DNA条形码

【目的】粉毛蚜亚科昆虫是重要的林业害虫,但是由于蚜虫体型较小,形态特征趋于简化,可用于物种鉴定的有效特征非常有限,因此一般基于外部形态特征难以对蚜虫物种实现快速准确的鉴定。本研究获取该亚科2属10种的DNA条形码标准序列,解决部分物种的分类问题,同时比较了3种标记对粉毛蚜亚科（Pterocommatinae）物种快速鉴定的效率。【方法】基于蚜虫的线粒体细胞色素氧化酶C亚基I（cytochrome oxidase subunit I, COI）基因、细胞色素b（cytochrome b, Cytb）基因和蚜虫初级内共生菌Buchnera 6-磷酸葡萄糖酸脱氢酶（gluconate-6-phosphate dehydrogenase, gnd）基因,对2属10种共197号样品进行NJ分析、遗传距离的计算以及基于相似性的物种鉴定分析。【结果】与K-2P模型相比,基于p-distance模型计算得到的遗传距离更小,序列差异频次图上种内距离与种间距离的重叠区域也小于前者;COI序列的物种鉴定成功率最高。获取了粉毛蚜亚科近200条DNA条形码标准序列,并建立了基于3个标记的该亚科物种DNA条形码序列库。【结论】在粉毛蚜亚科DNA条形码研究中,p-distance模型要优于K-2P模型;COI序列具有最高的条形码分析效率;增毛卷粉毛蚜Plocamaphis assetacea可能为蜡卷粉毛蚜Plocamaphis flocculosa的同物异名。     

基于DNA条形码的四川老君山国家级自然保护区尺蛾科昆虫种类鉴定

为了解四川老君山国家级自然保护区鳞翅目Lepidoptera尺蛾科Geometridae资源,于2019年6—8月和2020年5—6月通过灯诱法采集2 402号标本,运用DNA条形码技术对标本进行分子鉴定。通过BOLD数据库,邻接法构建系统发育树和ABGD在线工具对获得的297条线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列进行物种界定,结合BOLD和GenBank中70条相关种序列共367条COⅠ基因序列分析遗传距离。发现297条序列隶属于191种,其中95种鉴定到种,35种鉴定到属;鉴定到属或种水平的序列隶属于92属。计算遗传距离发现,种内遗传距离为0～4.36%,平均种内遗传距离为0.63%,种间遗传距离为3.43%～19.12%,平均种间遗传距离为11.91%。结果表明,保护区尺蛾科昆虫种类丰富,基于COⅠ基因的DNA条形码技术能有效区分尺蛾科物种。     

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。     

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。     

DNA条形码在膜翅目昆虫中的应用分析

DNA条形码的提出,实现了分类学的一次质的飞跃,简便、快捷以及精确的优点使其被广泛应用在物种的分类工作中。膜翅目为昆虫纲的第3大目,其物种具有高度的多样性,种类鉴定工作复杂艰巨。DNA条形码在膜翅目中得到广泛应用。本文针对DNA条形码在膜翅目昆虫的物种分类鉴定、物种发现和隐存种、食物网与生物多样性等方面研究情况予以综述。     

成都天府国际机场地表昆虫群落及其与边界生境的关系

2022年3月—2023年2月,对成都天府国际机场围界内草坪地表昆虫群落特征进行了调查,探究了与机场4种边界生境相邻的机场内草坪间、不同距离梯度处地表昆虫分布差异。（1）共鉴定出10目49科101属119种昆虫,优势类群为直翅目Orthoptera、膜翅目Hymenoptera、鞘翅目Coleoptera和双翅目Diptera。（2）不同边界生境相邻草坪间,地表昆虫优势度和多样性指数无显著差异;地表昆虫物种丰富度：林地相邻草坪（Ⅱ）>灌丛相邻草坪（Ⅰ）>农田相邻草坪（Ⅳ）>裸地相邻草坪（Ⅲ）,Ⅰ、Ⅱ显著高于Ⅲ;地表昆虫物种多度：Ⅱ>Ⅳ>Ⅰ>Ⅲ,Ⅱ显著高于Ⅲ。（3）不同距离梯度下,地表昆虫丰富度、多度和多样性从机场围界向草坪内部呈递减趋势,而优势度则相反。（4）地表昆虫丰富度和多度夏季最高,冬季以直翅目和双翅目为主,夏、秋季主要为直翅目、膜翅目和鞘翅目。研究结果为机场昆虫的综合防治提供了科学依据。     

南京地区外来植物一年蓬上访花昆虫的多样性及其访花选择性的影响因素分析

为揭示外来植物一年蓬Erigeron annuus上的本土访花昆虫多样性和影响访花行为的因素, 本研究在南京郊区进行了连续2年的野外调查, 采用跨栏模型分析了环境因素如何影响昆虫的访花选择性, 即接受概率（测度是否接受一年蓬花）和访问频数（测度接受一年蓬花的程度）。调查发现, 访问一年蓬花的昆虫共计9目54科145种, 其中, 科丰富度占优势的是膜翅目、 鳞翅目和鞘翅目（均占总科数的20.75%）, 其次是双翅目（18.87%）和半翅目（13.21%）; 物种丰富度占优势的是双翅目（26.39%）, 其次是膜翅目（18.75%）、 半翅目（18.75%）、 鞘翅目（17.36%）和鳞翅目（15.38%）。多数目的物种丰富度在6-7月最高, 9月最低, 仅双翅目（食蚜蝇为主）在5月最高。运用跨栏模型对物种优势度最大的半翅目、 膜翅目和双翅目等的访花个体数量及其影响因素的分析结果表明： 影响半翅目和膜翅目对一年蓬花访问倾向（接受概率）的因素多于影响其访问频数的因素, 由此预测这些访花昆虫可能参考较多因素做出是否接受一年蓬花, 而依据较少线索做出访问程度的访花行为决策; 一年蓬植株密度影响半翅目和膜翅目昆虫的接受概率, 而花密度影响半翅目和双翅目昆虫的接受概率和访问频数, 说明靶标植物花的特性可能对访花昆虫的访花行为决策起主要作用。     

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.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae)

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.     

Species diversity of freshwater shrimp in Henan Province,China, based on morphological characters and COI mitochondrial gene

Freshwater shrimp are a rich species group, with a long and problematic taxonomic history attributed to their wide distribution and similar morphological characteristics. Shrimp diversity and species identification are important cornerstones for fisheries management. However, identification based on morphological characteristics is a difficult task for a nonspecialist. Abundant freshwater shrimp species are distributed in the waters of Henan Province, but investigations of freshwater shrimp are limited in this region, especially concerning molecular features. Here, we combined morphology and DNA barcodes to reveal the species diversity of freshwater shrimp in Henan province. A total of 1,200 freshwater shrimp samples were collected from 46 sampling sites, and 222 samples were chosen for further microscopic examination and molecular delimitation. We used tree‐based methods (NJ, ML, and bPTP) and distance‐based methods (estimation of the paired genetic distances and ABGD) to delimit species. The results showed that there were nine morphospecies based on morphological characteristics; all could effectively be defined by molecular methods, among which bPTP and ABGD defined 13 and 8 MOTUs, respectively. The estimation of the paired genetic distances of K2P and the p‐distances had similar results. Mean K2P distances and p‐distances within species were both equal to 1.2%. The maximum intraspecific genetic distances of all species were less than 2%, with the exception of Palaemon modestus and M. maculatum. Various analyses have shown that P. modestus and M. maculatum have a large genetic differentiation, which may indicate the existence of cryptic species. By contrast, DNA barcoding could unambiguously discriminate 13 species and detect cryptic diversity. Our results demonstrate the high efficiency of DNA barcoding to delimit freshwater shrimp diversity and detect the presence of cryptic species.     

DNA barcode library and its efficacy for identifying food‐associated insect pests in Korea

Food‐associated insect pests are of great economic and hygienic importance. However, their identification requires expert knowledge and excessive time. Such pests are discovered in food as body parts or immature stages, which further complicates the identification process. In this study, we constructed a DNA barcode dataset of insect pests that can be detected in food. We also tested the efficacy of these DNA barcode sequences for identifying food‐associated insect pests. A 658 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was analyzed from 55 species of food‐associated insect pests in Korea. The results indicated that this portion of the COI gene effectively discriminated >90% of the food‐associated insect pests. Mean genetic divergences among individuals belonging to one species/between species belonging to one genus of the five orders, Blattaria, Coleoptera, Hymenoptera, Lepidoptera and Diptera, were 0.59%/13.18%, 0.84%/20.10%, 0.02%/22.61%, 0.24%/3.48% and 0.17%/15.90%, respectively. In conclusion, we established the first DNA barcode dataset and confirmed its efficiency for identifying food‐associated insect pests in Korea.     

Comparative performances of DNA barcoding across insect orders

Background  

Previous studies on insect DNA barcoding provide contradictory results and suggest not consistent performances across orders. This work aims at providing a general evaluation of insect DNA barcoding and "mini-barcoding" by performing simulations on a large database of 15,948 DNA barcodes. We compared the proportions of correctly identified queries across a) six insect orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera and Orthoptera), b) four identification criteria (Best Match: BM; Best Close Match: BCM; All Species Barcodes: ASB; tree-based identification: NJT), and c) reference databases with different taxon coverage (100, 500, 1,000, 1,500 and 1,995 insect species).