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.     

The utility of DNA barcoding as a tool to assess the success of ecological restoration using Hemiptera as a biological indicator

DNA barcoding uses a short, standardized DNA fragment to sort individuals into species. This molecular technique has applications in fields including ecology, evolution, conservation, and biogeography. In ecological applications such as species monitoring and habitat restoration, its potential has not been fully realized and implemented. Invertebrates are excellent biological indicators, as changes in species diversity or community assemblage provide important insights into the condition of, or changes in, the environment. This information is particularly useful within the context of restoration ecology. In this study, DNA barcoding is used to assess the potential of Hemiptera as a biological indicator of restoration success for the Buffelsdraai Landfill Site Community Reforestation Project (Durban, South Africa). A total of 393 Hemiptera specimens were collected from sites reforested at distinct phases (plots reforested in 2010, 2012, and 2015) and two reference sites (natural forest and grassland). The Hemiptera species composition and assemblage were assessed by analyzing diversity indices, ordination, unweighted pair‐group average cluster analysis, and phylogenetic analysis. Hemiptera species composition varied significantly across the chronologically different reforested sites, with a higher species richness observed in the older reforested plots. This suggests that Hemiptera diversity can be used to track restoration success, even over the small temporal scale used in this study. This study highlights the utility of DNA barcoding as a taxonomic sorting tool both to monitor ecological restoration and to discover specific taxa within Hemiptera that may be useful biological indicators.     

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.     

Bioinformatic challenges for DNA metabarcoding of plants and animals

Almost all empirical studies in ecology have to identify the species involved in the ecological process under examination. DNA metabarcoding, which couples the principles of DNA barcoding with next generation sequencing technology, provides an opportunity to easily produce large amounts of data on biodiversity. Microbiologists have long used metabarcoding approaches, but use of this technique in the assessment of biodiversity in plant and animal communities is under-explored. Despite its relationship with DNA barcoding, several unique features of DNA metabarcoding justify the development of specific data analysis methodologies. In this review, we describe the bioinformatics tools available for DNA metabarcoding of plants and animals, and we revisit others developed for DNA barcoding or microbial metabarcoding. We also discuss the principles and associated tools for evaluating and comparing DNA barcodes in the context of DNA metabarcoding, for designing new custom-made barcodes adapted to specific ecological question, for dealing with PCR and sequencing errors, and for inferring taxonomical data from sequences.     

An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding

DNA barcoding has become a well-funded, global enterprise since its proposition as a technique for species identification, delimitation and discovery in 2003. However, the rapid development of next generation sequencing (NGS) has the potential to render DNA barcoding irrelevant because of the speed with which it generates large volumes of genomic data. To avoid obsolescence, the DNA barcoding movement must adapt to use this new technology. This review examines the DNA barcoding enterprise, its continued resistance to improvement and the implications of this on the future of the discipline. We present the consistent failure of DNA barcoding to recognize its limitations and evolve its methodologies, reducing the usefulness of the data produced by the movement and throwing into doubt its ability to embrace NGS.     

DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs.     

DNA宏条形码技术在食草动物食性研究中的应用

随着测序技术的快速发展,整合DNA条形码和高通量测序的DNA宏条形码技术已经成为当前研究热点之一,在食草动物的食性鉴定中有很大潜力。放牧动物食性研究是动物营养学和草地生态学领域的重要研究内容。而与传统食性研究方法相比,宏条形码技术可通过对植物DNA条形码的高通量测序,获得样本中的物种组成进而分析动物食性。介绍了传统食性分析手段的局限,重点综述了DNA宏条形码技术的产生、操作原理以及在食草类动物食性鉴定领域中的应用,同时还简述了可能存在的挑战,并对该技术今后的发展方向进行了展望。     

Prospects of barcoding the Italian wild dendroflora: oaks reveal severe limitations to tracking species identity

DNA barcoding may be particularly important in influencing ecology, economic issues, and the fundamental crisis facing biodiversity as a standardized, species-level identification tool for taxonomy assessment. Trees play important roles in the conservation of many land ecosystems, the wood trade, and the definition of biogeographical processes; nevertheless, peculiar biological, evolutionary and taxonomical features will probably constitute an intriguing challenge to barcoders. We examined whether four marker regions (trnh-psba, rbcL, rpoc1, matK) proposed by the Consortium for the Barcode of Life (CBOL) matched species taxonomy in a preliminary tree biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co-occurrence of the species in the main forest ecosystems. Fifty-two species were included in a floristic study. We obtained 73% total discrimination success, with trnH-psbA as the best performing marker and oaks as the least responsive plants to the markers used. A further taxon-based study of Quercus (thirty specimens, 12 species) revealed that this genus is refractory to barcoding (0% discrimination success), a probable consequence of low variation rate at the plastid genome level, hybridization, and the incidence of biogeography. We conclude that some species-rich tree genera in small geographical regions may prove exceptionally difficult to barcode. Until more efficient markers are developed, we recommend that improved and diversified sampling (multiple locations of sympatric and co-occurring congenerics) be embraced as a timely and important goal for the precise assessment of haplotype specificity to facilitate the productive application of barcoding in practice.     

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。     

After 7 years and 1000 citations: comparative assessment of the DNA barcoding and the DNA taxonomy proposals for taxonomists and non-taxonomists

In 2003, two different approaches-DNA taxonomy and DNA barcoding-were simultaneously proposed to overcome some of the perceived intrinsic weaknesses of the traditional morphology-based taxonomical system, and to help non-taxonomists to resolve their crucial need for accurate and rapid species identification tools. After 7 years, it seems unlikely that a completely new taxonomical system based on molecular characters only (DNA taxonomy) will develop in the future. It is more likely that both morphological and molecular data will be simultaneously analyzed, developing what has been coined as "integrative taxonomy". Concerning DNA barcoding, it is now clear that it does not focus on building a tree-of-life nor to perform DNA taxonomy, but rather to produce a universal molecular identification key based on strong taxonomic knowledge that is collated in the barcode reference library. The indisputable success of the DNA barcoding project is chiefly due to the fact that DNA barcoding standards considerably enhance current practices in the molecular identification field, and standardization offers virtually endless applications for various users.     

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule‐coalescent (GMYC) model‐based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA‐barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large‐scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.     

Reading the complex skipper butterfly fauna of one tropical place

Background

An intense, 30-year, ongoing biodiversity inventory of Lepidoptera, together with their food plants and parasitoids, is centered on the rearing of wild-caught caterpillars in the 120,000 terrestrial hectares of dry, rain, and cloud forest of Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Since 2003, DNA barcoding of all species has aided their identification and discovery. We summarize the process and results for a large set of the species of two speciose subfamilies of ACG skipper butterflies (Hesperiidae) and emphasize the effectiveness of barcoding these species (which are often difficult and time-consuming to identify).

Methodology/Principal Findings

Adults are DNA barcoded by the Biodiversity Institute of Ontario, Guelph, Canada; and they are identified by correlating the resulting COI barcode information with more traditional information such as food plant, facies, genitalia, microlocation within ACG, caterpillar traits, etc. This process has found about 303 morphologically defined species of eudamine and pyrgine Hesperiidae breeding in ACG (about 25% of the ACG butterfly fauna) and another 44 units indicated by distinct barcodes (n = 9,094), which may be additional species and therefore may represent as much as a 13% increase. All but the members of one complex can be identified by their DNA barcodes.

Conclusions/Significance

Addition of DNA barcoding to the methodology greatly improved the inventory, both through faster (hence cheaper) accurate identification of the species that are distinguishable without barcoding, as well as those that require it, and through the revelation of species “hidden” within what have long been viewed as single species. Barcoding increased the recognition of species-level specialization. It would be no more appropriate to ignore barcode data in a species inventory than it would be to ignore adult genitalia variation or caterpillar ecology.     

Current state and perspectives of fungal DNA barcoding and rapid identification procedures

Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.     

Assessing the performance of DNA barcoding using posterior predictive simulations

Accurate estimates of biodiversity are required for research in a broad array of biological subdisciplines including ecology, evolution, systematics, conservation and biodiversity science. The use of statistical models and genetic data, particularly DNA barcoding, has been suggested as an important tool for remedying the large gaps in our current understanding of biodiversity. However, the reliability of biodiversity estimates obtained using these approaches depends on how well the statistical models that are used describe the evolutionary process underlying the genetic data. In this study, we utilize data from the Barcode of Life Database and posterior predictive simulations to assess the performance of DNA barcoding under commonly used substitution models. We demonstrate that the success of DNA barcoding varies widely across DNA substitution models and that model choice has a substantial impact on the number of operational taxonomic units identified (changing results by ~4–31%). Additionally, we demonstrate that the widely followed practice of a priori assuming the Kimura 2‐parameter model for DNA barcoding is statistically unjustified and should be avoided. Using both data‐based and inference‐based test statistics, we detect variation in model performance across taxonomic groups, clustering algorithms, genetic divergence thresholds and substitution models. Taken together, these results illustrate the importance of considering both model selection and model adequacy in studies quantifying biodiversity.     

Coalescing molecular evolution and DNA barcoding

The DNA barcoding concept (Woese et al. 1990 ; Hebert et al. 2003 ) has considerably boosted taxonomy research by facilitating the identification of specimens and discovery of new species. Used alone or in combination with DNA metabarcoding on environmental samples (Taberlet et al. 2012 ), the approach is becoming a standard for basic and applied research in ecology, evolution and conservation across taxa, communities and ecosystems (Scheffers et al. 2012 ; Kress et al. 2015 ). However, DNA barcoding suffers from several shortcomings that still remain overlooked, especially when it comes to species delineation (Collins & Cruickshank 2012 ). In this issue of Molecular Ecology, Barley & Thomson ( 2016 ) demonstrate that the choice of models of sequence evolution has substantial impacts on inferred genetic distances, with a propensity of the widely used Kimura 2‐parameter model to lead to underestimated species richness. While DNA barcoding has been and will continue to be a powerful tool for specimen identification and preliminary taxonomic sorting, this work calls for a systematic assessment of substitution models fit on barcoding data used for species delineation and reopens the debate on the limitation of this approach.     

Molecular identification of species in Juglandaceae: A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces

The development of DNA barcoding from faeces represents a promising method for animal diet analysis. However, current studies mainly rely on prior knowledge of prey diversity for a specific predator rather than on a range of its potential prey species. Considering that the feeding behaviour of teleosts may evolve with their environment, it could prove difficult to establish an exhaustive listing of their prey. In this article, we extend the DNA barcoding approach to diet analysis to allow the inclusion of a wide taxonomic range of potential prey items. Thirty-four ecological clade-specific primer sets were designed to cover a large proportion of prey species found in European river ecosystems. Selected primers sets were tested on isolated animal, algal or plant tissues and thereafter on fish faeces using nested PCR to increase DNA detection sensitivity. The PCR products were sequenced and analysed to confirm the identity of the taxa and to validate the method. The methodology developed here was applied to a diet analysis of three freshwater cyprinid species that are assumed to have similar feeding behaviour [Chondrostoma toxostoma toxostoma (Vallot 1837), Chondrostoma nasus nasus (Linnaeus, 1758) and Barbus barbus, (Linneaus 1758)]. These three species were sampled in four different hydrographic basins. Principal Component Analysis based on prey proportions identified distinct perilithon grazer and benthophagous behaviours. Furthermore, our results were consistent with the available literature on feeding behaviour in these fish. The simplicity of the PCR-based method and its potential generalization to other freshwater organisms may open new perspectives in food web ecology.     

Molecular approach to the identification of fish in the South China Sea

Background

DNA barcoding is one means of establishing a rapid, accurate, and cost-effective system for the identification of species. It involves the use of short, standard gene targets to create sequence profiles of known species against sequences of unknowns that can be matched and subsequently identified. The Fish Barcode of Life (FISH-BOL) campaign has the primary goal of gathering DNA barcode records for all the world''s fish species. As a contribution to FISH-BOL, we examined the degree to which DNA barcoding can discriminate marine fishes from the South China Sea.

Methodology/Principal Findings

DNA barcodes of cytochrome oxidase subunit I (COI) were characterized using 1336 specimens that belong to 242 species fishes from the South China Sea. All specimen provenance data (including digital specimen images and geospatial coordinates of collection localities) and collateral sequence information were assembled using Barcode of Life Data System (BOLD; www.barcodinglife.org). Small intraspecific and large interspecific differences create distinct genetic boundaries among most species. In addition, the efficiency of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cytb), and one nuclear ribosomal gene, 18S rRNA (18S), was also evaluated for a few select groups of species.

Conclusions/Significance

The present study provides evidence for the effectiveness of DNA barcoding as a tool for monitoring marine biodiversity. Open access data of fishes from the South China Sea can benefit relative applications in ecology and taxonomy.