Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

The islands of the Caribbean are considered to be a “biodiversity hotspot.” Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology‐based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within “best match” and “best close match” criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor‐joining tree‐based comparisons. The performance of both markers seemed to be species‐specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.     

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。     

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.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

Nuclear simple sequence repeat markers are superior to DNA barcodes for identification of closely related Rhododendron species on the same mountain

Accurate species delimitation of sampled biological material is critical for a range of studies. Although the DNA barcodes developed in recent years are useful for identifying numerous well differentiated species that have not experienced frequent gene flow, they fail to delimit recently diverged species, especially those with extensive introgressions. Here we use five Rhododendron species growing together on the same mountain as a model system to compare the species delimitation effectiveness of the DNA barcodes (internal transcribed spacer, matK, psbA‐trnH, and rbcL) previously proposed versus 15 pairs of microsatellite markers. Using these markers, we genotyped 129 individuals, which were members of five species according to morphological identification. We identified five simple sequence repeat genetic clusters (independently evolving lineages) corresponding to the morphological identification. However, we found that numerous individuals contained cryptic hybrid introgressions from the other species. The four DNA barcodes could not delimit three out of four closely related species that showed clear morphological differentiation and cryptic introgressions. Even after excluding all cryptic hybrids, two closely related species could not be successfully identified. The low discrimination ability of the DNA barcodes for closely related Rhododendron species could result from two, not mutually exclusive factors: introgressive hybridization and incomplete lineage sorting. Our results highlight the importance of simple sequence repeat markers in delimiting closely related species and identifying cryptic introgressions in the absence of morphological changes.     

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。     

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.     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

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.     

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。     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

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.     

Botany without borders: barcoding in focus

This recent meeting, held on the campus of the University of British Columbia, attracted 1200 delegates and a vast array of talks, but was notable for a remarkable showing of talks and posters on DNA barcoding in plants, spread through many sessions. The Canadian Centre for DNA Barcoding defines barcoding as ‘species identification and discovery through the analysis of short, standardized gene regions known as DNA barcodes’. This approach is somewhat controversial in animals ( Rubinoff et al., 2006 ), although it has been shown to be useful and reliable in many metazoan taxa ( Meyer & Paulay 2005 ; Hajibabaei et al., 2007 ), in which the mitochondrial cytochrome oxidase I (COI) gene is used. However, in land plants, COI evolves far too slowly to be useful, and there is no obvious single universal alternative ( Fazekas et al., 2008 ). Genes that work well in one taxon may perform poorly in other taxa. Additionally, some perfectly good plant species, reproductively isolated and morphologically and ecologically distinct, are too young to show much sequence divergence at most loci. Nevertheless, as we saw at this conference, progress has been made towards identifying genes that serve many of the functions of DNA barcodes, at least in some plant taxa.     

Morphology and DNA barcoding reveal three species in one: description of Culicoides cryptipulicaris sp. nov. and Culicoides quasipulicaris sp. nov. in the subgenus Culicoides

Species of the genus Culicoides (Diptera: Ceratopogonidae) are well known for their importance in the field of medical and veterinary entomology. Culicoides spp. transmit a wide variety of pathogens, primarily viruses that affect animals and humans. In Europe, the most economically important disease transmitted by Culicoides is bluetongue (BT). Culicoides spp. have been recently involved as primary vectors for Schmallenberg disease. The taxonomy within the subgenus Culicoides has been historically difficult and reorganizations have been proposed regularly. The subgenus Culicoides includes species that are considered to be potential vectors for BT. High morphological intraspecific variability has been attributed to these species. This highlights the apparent presence of previously undetected cryptic species diversity in the subgenus. In the present study, a detailed morphological and molecular study of specimens belonging to Culicoides pulicaris s.l. and specimens resembling a cross between C. pulicaris and Culicoides punctatus revealed the presence of two new species: Culicoides cryptipulicaris and Culicoides quasipulicaris. Females of C. quasipulicaris and males of both species were morphologically distinguished from C. pulicaris (Linnaeus, 1758), whereas females of C. cryptipulicaris were identified using molecular techniques exclusively.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

Exploring the species diversity of Trichoderma in Norwegian drinking water systems by DNA barcoding

A total of 123 Trichoderma strains were isolated from Norwegian surface-sourced drinking water. The water samples included raw water, treated water, and water from private homes and hospital installations. Trichoderma species are difficult to differentiate morphologically, but recent molecular identification tools, including DNA barcoding, successfully distinguish between closely related species. The diversity of Trichoderma spp. was explored by DNA sequencing of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (TEF-1α). Sequence identification was performed in the TrichOKEY version 2.0 barcode program and in the multilocus similarity search database TrichoBLAST, combined with traditional blast searches in the EMBL/GenBank. A total of 11 known Trichoderma/Hypocrea species were identified. In addition, one group of unidentified Trichoderma strains was found to represent a separate, strongly supported subclade within the Pachybasium'A'/Hamatum clade, based on their TEF-1α haplotypes. Trichoderma viride comprised 49% of the identified strains, and was represented by four and eight slightly different ITS and TEF-1α haplotypes, respectively. Approximately 22% of the surface-derived water samples were positive for T. viride, and the species was frequently isolated throughout the surface-sourced drinking water distribution system. The results indicate that a broad range of Trichoderma species are present in Norwegian surface-sourced drinking. Water treatment has minor effect in removing Trichoderma from raw water, and active growth in the water distribution system is likely to occur.     

Diversity investigation by application of DNA barcoding: A case study of lepidopteran insects in Xinjiang wild fruit forests,China

To investigate the species diversity of lepidopteran insects in Xinjiang wild fruit forests, establish insect community monitoring systems, and determine the local species pool, we test the applicability of DNA barcoding based on cytochrome c oxidase subunit I (COI) gene for accurate and rapid identification of insect species. From 2017 to 2019, a total of 212 samples with ambiguous morphological identification were selected for DNA barcoding analysis. Five sequence‐based methods for species delimitation (ABGD, BINs, GMYC, jMOTU, and bPTP) were conducted for comparison to traditional morphology‐based identification. In total, 2,422 samples were recorded, representing 143 species of 110 genera in 17 families in Lepidoptera. The diversity analysis showed that the richness indices for Noctuidae was the highest (54 species), and for Pterophoridae, Cossidae, Limacodidae, Lasiocampidae, Pieridae, and Lycaenidae were the lowest (all with 1 species). The Shannon–Wiener species diversity index (H′) and Pielou''s evenness (J′) of lepidopteran insects first increased and then decreased across these 3 years, while the Simpson diversity index showed a trend of subtracted then added. For molecular‐based identification, 67 lepidopteran species within 61 genera in 14 families were identified through DNA barcoding. Neighbor‐joining (NJ) analysis showed that conspecific individuals were clustered together and formed monophyletic groups with a high support value, except for Lacanobia contigua (Denis & Schiffermüller, 1775) (Noctuidae: Hadeninae). Sixty‐seven morphospecies were classified into various numbers of MOTUs based on ABGD, BINs, GMYC, jMOTU, and bPTP (70, 96, 2, 71, and 71, respectively). In Xinjiang wild fruit forests, the family with the largest number of species is Noctuidae, followed by Geometridae, Crambidae, and the remaining families. The highest Shannon diversity index is observed for the family Noctuidae. Our results indicate that the distance‐based methods (ABGD and jMOTU) and character‐based method (bPTP) outperform GMYC. BINs is inclined to overestimate species diversity compared to other methods.