Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

DNA条形码技术在毒品原植物大麻鉴定中的应用

准确鉴定毒品原植物大麻的种属及品种具有重要的理论和实践意义。为了探讨DNA条形码技术用于毒品原植物大麻种属鉴定及品种鉴定的可行性,该研究以60份大麻原植物(分别采自内蒙、黑龙江、陕西延安、陕西榆林4个地区的栽培大麻雌雄各6株及新疆玛纳斯地区的野生大麻雌雄各6株)为材料,通过从其叶片中提取的DNA为模版,利用核糖体DNA基因间隔区的通用引物ITS2和叶绿体DNA的通用引物psbAtrnH进行PCR扩增,对扩增片段进行双向测序,将测序结果进行人工矫正和比对。结果显示:所有大麻样本的ITS2扩增片段序列没有变异完全一致,但psbA-trnH扩增片段变异较大共检测出8种cpDNA单倍型,用MEGE5.1软件计算种间遗传距离,并构建NJ系统聚类树可以有效把这五个地区的大麻样本区别开来,因此证明DNA条形码技术在毒品原植物大麻的种属鉴定方面具有可行性,但其用于大麻的种属鉴定的准确性、可靠性及在其来源地鉴定及品种鉴定中的可能性还有待进一步深入地研究。     

DNA条形码在鳞翅目昆虫中的应用

2003年,Hebert等提出DNA条形码后,快速而精确的特点使它在物种鉴定中得到了广泛的应用。鳞翅目是昆虫纲中第二大目,其物种鉴定任务复杂而艰巨,因此DNA条形码具有广阔的应用前景。该文主要针对DNA条形码概况以及近年来它在鳞翅目昆虫中的研究情况予以综述。     

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.     

Cryptic diversity revealed by DNA barcoding in Colombian illegally traded bird species

Colombia is the country with the largest number of bird species worldwide, yet its avifauna is seriously threatened by habitat degradation and poaching. We built a DNA barcode library of nearly half of the bird species listed in the CITES appendices for Colombia, thereby constructing a species identification reference that will help in global efforts for controlling illegal species trade. We obtained the COI barcode sequence of 151 species based on 281 samples, representing 46% of CITES bird species registered for Colombia. The species analysed belong to nine families, where Trochilidae and Psittacidae are the most abundant ones. We sequenced for the first time the DNA barcode of 47 species, mainly hummingbirds endemic of the Northern Andes region. We found a correct match between morphological and genetic identification for 86–92% of the species analysed, depending on the cluster analysis performed (BIN, ABGD and TaxonDNA). Additionally, we identified eleven cases of high intraspecific divergence based on K2P genetic distances (up to 14.61%) that could reflect cryptic diversity. In these cases, the specimens were collected in geographically distant sites such as different mountain systems, opposite flanks of the mountain or different elevations. Likewise, we found two cases of possible hybridization and incomplete lineage sorting. This survey constitutes the first attempt to build the DNA barcode library of endangered bird species in Colombia establishing as a reference for management programs of illegal species trade, and providing major insights of phylogeographic structure that can guide future taxonomic research.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

DNA barcoding for the identification of smoked fish products

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.     

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.     

DNA barcoding and haplotyping in different species of Andrographis

The genus Andrographis, belonging to the family Acanthaceae, contains several species of medicinal importance. Species, such as Andrographis alata, Andrographis echioides, Andrographis glandulosa, Andrographis lineata, Andrographis nallamalayana and Andrographis paniculata, with several bio-active compounds are being extensively used in folk medicine. However, difference of opinion exists with regard to inclusion of the species echioides into the genus Andrographis. The present study, using rbcL and matK sequences, for the first time established DNA barcodes for these six species. The nucleotide sequence of rbcL provided species-specific haplotypes for A. alata, A. lineata, and A. paniculata. Despite the differences with regard to nucleotide sequence, all the six species showed conserved amino acid sequence. However, all the six species showed distinct haplotypes in nucleotide sequence of matK and facilitated the identification and discrimination of these species. The phylogenetic tree generated with combined sequence of rbcL and matK revealed grouping of all the six species into a single clade confirming the positioning of the species echioides into the genus Andrographis.     

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Identification of species within Tetrastigma (Miq.) Planch.(Vitaceae) based on DNA barcoding techniques

Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. Hemsleyanum, could be identified precisely using DNA barcoding.     

Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between‐species divergence from within‐species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single‐locus or multiloci sequence data. Here, we use simulations to demonstrate three features of an MSC method implemented in the program bpp . First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be misidentified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analysing rare taxa (singletons) are unfounded. Currently, barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large data sets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Identifying Chinese species of Gammarus （Crustacea： Amphipoda） using DNA barcoding

Using a standard cytochrome c oxidase I sequence, DNA barcoding has been shown to be effective to distinguish known species and to discover cryptic species. Here we assessed the efficiency of DNA barcoding for the amphipod genus Gammarus from China. The maximum intraspecific divergence for widespread species, Gammarus lacustris, was 3.5%, and mean interspecific divergence reached 21.9%. We presented a conservative benchmark for determining provisional species using maximum intraspecific divergence of Gammarus lacustris. Thirty-one species possessed distinct barcode clusters. Two species were comprised of highly divergent clades with strong neighbor-joining bootstrap values, and likely indicated the presence of cryptic species. Although DNA barcoding is effective, future identification of species of Gammarus should incorporate DNA barcoding and morphological detection[Current Zoology 55(2):158-164,2009].     

The impact of genetic diversity on the accuracy of DNA barcoding to identify species: A study on the genus Phellodendron

DNA barcoding is widely used in species identification, but there is considerable controversy regarding the extent of sampling in research methods. Some scholars have proposed that this small sample size underestimates the intraspecific genetic diversity, which would impact on the accuracy of DNA barcoding to identify species. In study, we selected all Phellodendron species (including P. amurense Rupr., P. chinense Schneid., and P. chinense var. glabriusculum Schneid.) as the materials, collected 59 P. amurense samples from 35 populations greatly to represent the genetic diversity, and analyzed the haplotype, genetic distance, barcoding gap, and Neighbor‐Joining (NJ) trees based on psbA‐trnH and internal transcribed spacer gene sequences. Additionally, a sampling simulation was conducted to assess the correlation between genetic diversity and the number of populations. Finally, analysis of critical geographical populations was performed. Based on analysis of haplotype, genetic distance, barcoding gap, and NJ trees, we found that eight P. amurense samples impacted on the effectiveness of DNA barcoding, which genetic information were very important to identify Phellodendron species. Moreover, the result of the NJ tree analysis performed the small‐scale P. amurense sample size did not completely match the objective phylogenetic relationship in Phellodendron. In simulation sampling analysis, the data showed the genetic diversity indexes at the same population level gradually decreased and stabilized as the number of simulation sampling populations increased. We found that 1–2 samples from over 24 populations based on uniform geographical distribution could represent 80% of the genetic diversity of P. amurense and ensure authenticity and reliability of DNA barcoding. Thus, we proposed it is particularly important adequately samples to cover infraspecific genetic diversity in order to ensure identification accuracy of DNA barcoding.