Toward a global DNA barcode reference library of the intolerant nonbiting midge genus Rheocricotopus Brundin, 1956

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time‐ and cost‐efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the “Barcode Gap Analysis” and neighbor‐joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well‐separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota.     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

Building a DNA Barcode Reference Library for the True Butterflies (Lepidoptera) of Peninsula Malaysia: What about the Subspecies?

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.     

FuzzyID2: A software package for large data set species identification via barcoding and metabarcoding using hidden Markov models and fuzzy set methods

Species identification through DNA barcoding or metabarcoding has become a key approach for biodiversity evaluation and ecological studies. However, the rapid accumulation of barcoding data has created some difficulties: for instance, global enquiries to a large reference library can take a very long time. We here devise a two‐step searching strategy to speed identification procedures of such queries. This firstly uses a Hidden Markov Model (HMM) algorithm to narrow the searching scope to genus level and then determines the corresponding species using minimum genetic distance. Moreover, using a fuzzy membership function, our approach also estimates the credibility of assignment results for each query. To perform this task, we developed a new software pipeline, FuzzyID2, using Python and C++. Performance of the new method was assessed using eight empirical data sets ranging from 70 to 234,535 barcodes. Five data sets (four animal, one plant) deployed the conventional barcode approach, one used metabarcodes, and two were eDNA‐based. The results showed mean accuracies of generic and species identification of 98.60% (with a minimum of 95.00% and a maximum of 100.00%) and 94.17% (with a range of 84.40%–100.00%), respectively. Tests with simulated NGS sequences based on realistic eDNA and metabarcode data demonstrated that FuzzyID2 achieved a significantly higher identification success rate than the commonly used Blast method, and the TIPP method tends to find many fewer species than either FuzztID2 or Blast. Furthermore, data sets with tens of thousands of barcodes need only a few seconds for each query assignment using FuzzyID2. Our approach provides an efficient and accurate species identification protocol for biodiversity‐related projects with large DNA sequence data sets.     

DNA条形码参考数据集构建和序列分析相关的新兴技术

近年来DNA条形码技术迅速发展, 产生的条形码的数量及其应用范围都呈指数性增长, 现已广泛用于物种鉴定、食性分析、生物多样性评估等方面。本文重点总结并讨论了构建条形码参考数据库和序列聚类相关的信息分析的技术和方法, 包括: 基于高通量测序(high throughput sequencing, HTS)平台以高效并较低的成本获取条形码序列的方法; 同时还介绍了从原始测序序列到分类操作单元(operational taxonomic units, OTUs)过程中的一些计算逻辑以及被广泛采用的软件和技术。这是一个较新并快速发展的领域, 我们希望本文能为读者提供一个梗概, 了解DNA条形码技术在生物多样性研究应用中的方法和手段。     

BOLD’s role in barcode data management and analysis: a response

DNA barcoding is a very effective tool for the identification of specimens when a carefully validated and taxonomically comprehensive library of reference DNA barcodes is available. Libraries meeting this criterion are now available for some taxonomic groups in some geographic regions, provoking their use as a tool for the identification of samples that would otherwise remain as unknowns. In this article, we emphasize the need for caution in the interpretation of identifications based on a reference library with entries that have seen limited validation. We also emphasize the need for the deposition of sequence records for ‘unknowns’ so that presumptive identifications can be tested by other researchers and updated as the barcode reference library gains increased coverage and validation.     

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.     

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available.We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.     

DNA barcoding in autotrophic euglenids: evaluation of COI and 18s rDNA

Autotrophic euglenids (Euglenophyceae) are a common and abundant group of microbial eukaryotes in freshwater habitats. They have a limited number of features, which can be observed using light microscopy, thus species identification is often problematic. Establishing a barcode for this group is therefore an important step toward the molecular identification of autotrophic euglenids. Based on the literature, we selected verified species and used a plethora of available methods to validate two molecular markers: COI and 18S rDNA (the whole sequence and three fragments separately) as potential DNA barcodes. Analyses of the COI gene were performed based on the data set of 43 sequences (42 obtained in this study) representing 24 species and the COI gene was discarded as a DNA barcode mainly due to a lack of universal primer sites. For 18S rDNA analyses we used a data set containing 263 sequences belonging to 86 taxonomically verified species. We demonstrated that the whole 18S rDNA is too long to be a useful marker, but from the three shorter analyzed variable regions we recommend variable regions V2V3 and V4 of 18S rDNA as autotrophic euglenid barcodes due to their high efficiency (above 95% and 90%, respectively).     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

From barcodes to genomes: extending the concept of DNA barcoding

DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach.     

Employing DNA barcoding as taxonomy and conservation tools for fish species censuses at the southeastern Mediterranean,a hot-spot area for biological invasion

This study evaluates the utility of DNA barcoding (mitochondrial cytochrome oxidase subunit I; COI) as a biodiversity and conservation applied tool for identifying fish fauna from the southeastern Mediterranean (the continental coast of Israel), a hot-spot area for biological invasion, also with an eye to establish the foundation for follow-up studies that will use environmental DNA (eDNA) tracks of native and invasive species, and for the application of eDNA concepts and methodologies in nature conservation. We established a dataset of 280 DNA barcodes, representing 110 marine fish species (all identified by a taxonomist), belonging to 75 native and 35 Lessepsian migratory species that were tested within and against the BOLD system database. Most of the query sequences showed 98% matches with reference DNA barcodes in the BOLD system excluding two Trachurus species, three Dasyatis pastinaca and two Rhinobatos rhinobatos individuals. Relatively high intraspecific genetic distances were observed in two Elasmobranchii species (8.83%–18%), suggesting possible cryptic species. In contrast, relatively low interspecific genetic distances were found between two Actinopterygii species (1.54%). Gobiidae family members were observed as being the most scattered on the Bayesian tree. Out of the 110 fish species, 48 were taxonomically discordant with the BOLD BINs (4 at the family level, 15 at the genus level and 29 at the species level), 53 were concordant and 9 were singletons. Discordancy was noted for some Diplodus and Fistularia species during the BOLD BIN analysis. Apogon queketti, Champsodon nudivittis and Cheilodipterus novemstriatus were uploaded to the BOLD system for the first time. We elucidated 177 haplotypes, 122 for the native fish species and 55 for the Lessepsian species. The results will allow the evaluation of invasive species success and will help developing improved policies for the conservation of Mediterranean biodiversity.     

The campaign to DNA barcode all fishes, FISH-BOL

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System ( http://www.barcodinglife.org ). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.     

大型经济真菌的DNA条形码研究——以我国剧毒鹅膏为例

鹅膏属（Amanita）部分物种为重要食用真菌,而另外部分物种则是剧毒的,在我国及其他许多国家,每年都有因误食剧毒鹅膏而导致中毒甚至死亡的事件发生。DNA条形码是用一段或几段短的DNA序列来对物种进行快速、准确鉴定的方法。本研究选取三个候选片段,即核糖体大亚基（nLSU）、内转录间隔区（ITS）和翻译延长因子1-α（tef1-α）,使用真核生物通用引物,测试我国已知的7种剧毒鹅膏及2种易混的可食鹅膏,并将欧美分布的但与黄盖鹅膏（A.subjunquillea）亲缘关系密切的绿盖鹅膏（A.phalloides）纳入分析中。nLSU的PCR扩增和测序成功率均为100%,但种内和种间遗传变异偶有重叠。ITS的PCR扩增和测序成功率分别达到100%和85.7%,且具有高的种间变异和低的种内变异。tef1-α的PCR扩增和测序成功率分别达到85.7%和100%,种间和种内遗传分化均高于ITS和nLSU。三个片段的物种分辨率均较高,但与nLSU相比,ITS和tef1-α具有更为明显的barcode gap。鉴于ITS可能会成为真菌界的通用条码,故建议将ITS作为鹅膏属的核心条形码,tef1 α和nLSU作为该属的辅助条形码。     

Quantitative bias in Illumina TruSeq and a novel post amplification barcoding strategy for multiplexed DNA and small RNA deep sequencing

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias.     

AeDNA: 水生生物eDNA数据库

环境 DNA (eDNA) 技术是一种生态和生物多样性监测和评价的新手段, 完整和准确的参考序列库是eDNA技术应用于水生生物多样性调查的基础。当前, 不同水生生物eDNA参考序列还存在诸多问题, 如不同类群使用的标记基因不同且资源较为分散, 部分参考序列分类不准确, 以及针对我国各类水体中水生生物eDNA参考序列不多等。针对上述问题, 研究构建了水生生物eDNA数据库(AeDNA, http://aedna.ihb.ac.cn/)。 AeDNA整合了DNA条形码和基因组两种类型参考序列。其中18S、28S、ITS、COΙ、12S、rbcL 等各类DNA条形码60余万条, 涉及2万余种鱼类、1万余种水生植物、1万余种底栖动物、1万余种浮游动物和1万余种浮游植物; 基因组包含线粒体、叶绿体等细胞器基因组6199个及万种鱼类基因组计划和万种原生生物基因组计划所产生的物种基因组。涉及的生境有江、河、湖、海、冰川和温泉等各类水环境, 尤其数据库构建团队贡献的6万余条参考序列, 具有我国丰富的各类水体生境信息。总体来说, AeDNA是一个数据量大、类群覆盖全、准确性高且具有我国水生生物特色的综合性eDNA参考序列库, 是水生态和水生生物多样性监测的重要基础资源。     

DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes

Background

DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity.

Methodology/Principal Findings

A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%–18.74%), most of them of high commercial relevance, suggesting possible cryptic species.

Conclusion/Significance

We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.     

Plant DNA barcode library for native flowering plants in the arid region of northwestern China

DNA barcoding is a well-established tool for rapid species identification and biodiversity monitoring. A reliable and traceable DNA barcode reference library with extensive coverage is necessary but unavailable for many geographical regions. The arid region in northwestern China, a vast area of about 2.5 million km², is ecologically fragile and often overlooked in biodiversity studies. In particular, DNA barcode data from the arid region in China are lacking. We develop and evaluate the efficacy of an extensive DNA barcode library for native flowering plants in the arid region of northwestern China. Plant specimens were collected, identified and vouchered for this purpose. The database utilized four DNA barcode markers, namely rbcL, matK, ITS and ITS2, for 1816 accessions (representing 890 species from 385 genera and 72 families), and consisted of 5196 barcode sequences. Individual barcodes varied in resolution rates: species- and genus-level rates for rbcL, matK, ITS and ITS2 were 79.9%–51.1%/76.1%, 79.9%–67.2%/88.9%, 85.0%–72.0%/88.2% and 81.0%–67.4%/84.9%, respectively. The three-barcode combination of rbcL + matK + ITS (RMI) revealed a higher species- and genus-level resolution (75.5%/92.1%, respectively). A total of 110 plastomes were newly generated as super-barcodes to increase species resolution for seven species-rich genera, namely Astragalus, Caragana, Lactuca, Lappula, Lepidium, Silene and Zygophyllum. Plastomes revealed higher species resolution compared to standard DNA barcodes and their combination. We suggest future databases include super-barcodes, especially for species-rich and complex genera. The plant DNA barcode library in the current study provides a valuable resource for future biological investigations in the arid regions of China.