Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

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.     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

Incorporating trnH-psbA to the core DNA barcodes improves significantly species discrimination within?southern African Combretaceae

Recent studies indicate that the discriminatory power of the core DNA barcodes (rbcLa + matK) for land plants may have been overestimated since their performance have been tested only on few closely related species. In this study we focused mainly on how the addition of complementary barcodes (nrITS and trnH-psbA) to the core barcodes will affect the performance of the core barcodes in discriminating closely related species from family to section levels. In general, we found that the core barcodes performed poorly compared to the various combinations tested. Using multiple criteria, we finally advocated for the use of the core + trnH-psbA as potential DNA barcode for the family Combretaceae at least in southern Africa. Our results also indicate that the success of DNA barcoding in discriminating closely related species may be related to evolutionary and possibly the biogeographic histories of the taxonomic group tested.     

Critical factors for assembling a high volume of DNA barcodes

Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified.     

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).     

植物物种的快速鉴定与iFlora: DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比, DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（Pedicularis L.）植物具对生（轮生）叶的种类70%以上分布在中国,近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81.40%和89.57%,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera,Papilionidae)

Numerous studies have demonstrated that DNA barcoding is an effective tool for detecting DNA clusters, which can be viewed as operational taxonomic units (OTUs), useful for biodiversity research. Frequently, the OTUs in these studies remained unnamed, not connected with pre-existing taxonomic hypotheses, and thus did not really contribute to feasible estimation of species number and adjustment of species boundaries. For the majority of organisms, taxonomy is very complicated with numerous, often contradictory interpretations of the same characters, which may result in several competing checklists using different specific and subspecific names to describe the same sets of populations. The highly species-rich genus Parnassius (Lepidoptera: Papilionidae) is but one example, such as several mutually exclusive taxonomic systems have been suggested to describe the phenotypic diversity found among its populations. Here we provide an explicit flow chart describing how the DNA barcodes can be combined with the existing knowledge of morphology-based taxonomy and geography (sympatry versus allopatry) of the studied populations in order to support, reject or modify the pre-existing taxonomic hypotheses. We then apply this flow chart to reorganize the taxa within the Parnassius delphius species group, solving long-standing taxonomic problems.     

植物物种的快速鉴定与iFlora：DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比,DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（PedicularisL．）植物具对生（轮生）叶的种类70％以上分布在中国．近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH-psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81．40％和89．57％,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.     

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.     

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.     

Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding

By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user.     

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae)

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.     

DNA barcoding in closely related species: A case study of Primula L.sect.Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoCl, trnH-psbA, psbK-psbI, atpFatpH, and internal transcribed spacer (ITS)). The core combination rbcL + matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL +matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.     

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.     

DNA barcoding in closely related species: A case study of Primula L. sect. Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoC1, trnH-psbA, psbK-psbI, atpF-atpH, and internal transcribed spacer (ITS)). The core combination rbcL+matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL+matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.