The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding.

Methodology/Principal Findings

Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique.

Conclusions/Significance

A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.     

Reliability and Utility of Standard Gene Sequence Barcodes for the Identification and Differentiation of Cyst Nematodes of the Genus Heterodera

Difficulties inherent in the morphological identification of cyst nematodes of the genus Heterodera Schmidt, 1871, an important lineage of plant parasites, has led to broad adoption of molecular methods for diagnosing and differentiating species. The pool of publicly available sequence data has grown significantly over the past few decades, and over half of all known species of Heterodera have been characterized using one or more molecular markers commonly employed in DNA barcoding (18S, internal transcribed spacer [ITS], 28S, coxI). But how reliable are these data and how useful are these four markers for differentiating species? We downloaded all 18S, ITS, 28S, and coxI gene sequences available on the National Center for Biotechnology Information (NCBI) database, GenBank, for all species of Heterodera for which data were available. Using a combination of sequence comparison and tree-based phylogenetic methods, we evaluated this dataset for erroneous or otherwise problematic sequences and examined the utility of each molecular marker for the delineation of species. Although we find the rate of obviously erroneous sequences to be low, all four molecular markers failed to differentiate between at least one species pair. Our results suggest that while a combination of multiple markers is best for species identification, the coxI marker shows the most utility for species differentiation and should be favored over 18S, ITS, and 28S, where resources are limited. Presently, less than half the valid species of Heterodera have a sequence of coxI available, and only a third have more than one sequence of this marker.     

Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae)

Background

DNA barcoding, i.e. the use of a 648 bp section of the mitochondrial gene cytochrome c oxidase I, has recently been promoted as useful for the rapid identification and discovery of species. Its success is dependent either on the strength of the claim that interspecific variation exceeds intraspecific variation by one order of magnitude, thus establishing a "barcoding gap", or on the reciprocal monophyly of species.

Results

We present an analysis of intra- and interspecific variation in the butterfly family Lycaenidae which includes a well-sampled clade (genus Agrodiaetus) with a peculiar characteristic: most of its members are karyologically differentiated from each other which facilitates the recognition of species as reproductively isolated units even in allopatric populations. The analysis shows that there is an 18% overlap in the range of intra- and interspecific COI sequence divergence due to low interspecific divergence between many closely related species. In a Neighbour-Joining tree profile approach which does not depend on a barcoding gap, but on comprehensive sampling of taxa and the reciprocal monophyly of species, at least 16% of specimens with conspecific sequences in the profile were misidentified. This is due to paraphyly or polyphyly of conspecific DNA sequences probably caused by incomplete lineage sorting.

Conclusion

Our results indicate that the "barcoding gap" is an artifact of insufficient sampling across taxa. Although DNA barcodes can help to identify and distinguish species, we advocate using them in combination with other data, since otherwise there would be a high probability that sequences are misidentified. Although high differences in DNA sequences can help to identify cryptic species, a high percentage of well-differentiated species has similar or even identical COI sequences and would be overlooked in an isolated DNA barcoding approach.     

DNA barcoding of stylommatophoran land snails: a test of existing sequences

DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour‐joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny‐based methods for barcoding. There was, however, a large overlap between intra‐ and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2‐parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone.     

Identification of Channa species using the partial cytochrome c oxidase subunit I (COI) gene as a DNA barcoding marker

The snakehead fish of the genus Channa are an important food fish in China. However, the molecular identification and phylogeny of this genus is poorly understood. Here, we present the utility of partial sequences of the COI gene for use in DNA barcoding for the identification of Channa individuals, which includes four species: Channa argus, Channa maculata, Channa asiatica, and Channa striata. A total of 19 haplotypes were identified in this study. The interspecific K2P distances were higher than intraspecific distances. The lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. No intraspecific–interspecific distance overlaps were observed, and a distinct barcoding gap was found between intraspecific and interspecific distances in each species. Our results showed that the partial COI gene is an effective DNA barcoding marker for identifying Channa species.     

Applying DNA barcodes for identification of plant species in the family Araliaceae

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.     

Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences

An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL–F, and psbA–trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL + matK, psbA–trnH + ITS1, and trnL–F + ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序, 比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图, 使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力, 进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明, ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高, 其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势, 且纳入60个属316个种共1 228个样品的网上数据后, 其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高, 其鉴定成功率为63.2%, 并能鉴别一些ITS序列无法鉴别的种。实验结果表明, ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

DNA barcoding a regional fauna: Irish solitary bees

As the globally dominant group of pollinators, bees provide a key ecosystem service for natural and agricultural landscapes. Their corresponding global decline thus poses an important threat to plant populations and the ecosystems they support. Bee conservation requires rapid and effective tools to identify and delineate species. Here, we apply DNA barcoding to Irish solitary bees as the first step towards a DNA barcode library for European solitary bees. Using the standard barcoding sequence, we were able to identify 51 of 55 species. Potential problems included a suite of species in the genus Andrena, which were recalcitrant to sequencing, mitochondrial heteroplasmy and parasitic flies, which led to the production of erroneous sequences from DNA extracts. DNA barcoding enabled the assignment of morphologically unidentifiable females of the parasitic genus Sphecodes to their nominal taxa. It also enabled correction of the Irish bee list for morphologically inaccurately identified specimens. However, the standard COI barcode was unable to differentiate the recently diverged taxa Sphecodes ferruginatus and S. hyalinatus. Overall, our results show that DNA barcoding provides an excellent identification tool for Irish solitary bees and should be rolled out to provide a database for solitary bees globally.     

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (<Emphasis Type="Italic">Aphis gossypii</Emphasis>) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

Performance of distance-based DNA barcoding in the molecular identification of Primates

For comparative primatology proper recognition of basal taxa (i.e. species) is indispensable, and in this the choice of a suitable gene with high phylogenetic resolution is crucial. For the goals of species identification in animals, the cytochrome c oxidase subunit 1 (cox1) has been introduced as standard marker. Making use of the difference in intra- and interspecific genetic variation – the DNA barcoding gap – cox1 can be used as a fast and accurate marker for the identification of animal species. For the Order Primates we compare the performance of cox1 (166 sequences; 50 nominal species) in species-identification with that of two other mitochondrial markers, 16S ribosomal RNA (412 sequences, 92 species) and cytochrome b (cob: 547 sequences, 72 species). A wide gap exist between intra- and interspecific divergences for both cox1 and cob genes whereas this gap is less apparent for 16S, indicating that rRNA genes are less suitable for species delimitation in DNA barcoding. For those species where multiple sequences are available there are significant differences in the intraspecific genetic distances between different mitochondrial markers, without, however, showing a consistent pattern. We conclude that cox1 allows accurate differentiation of species and as such DNA barcoding may have an important role to play in comparative primatology.     

Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units

The lack of a robust taxonomy in the genus Saprolegnia is leading to the presence of incorrectly named isolates in culture collections and of an increasing number of misassigned sequences in DNA databases. Accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxonomic diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs). We have used 961 sequences of nrDNA ITS from culture collections (461 sequences) and GenBank (500 sequences), to perform phylogenetic and clustering optimization analyses. As result, we have identified 29 DNA-based MOTUs in agreement with phylogenetic studies. The resulting molecular clusters support the validity of 18 species of Saprolegnia and identify 11 potential new ones. We have also listed a number of incorrectly named isolates in culture collections, misassigned species names to GenBank sequences, and reference sequences for the species. We conclude that GenBank represents the main source of errors for identifying Saprolegnia species since it possesses sequences with misassigned names and also sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus.     

DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau

DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences — rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) — for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau.     

Deciphering amphibian diversity through DNA barcoding: chances and challenges

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species

Background

DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species.

Methodology/Principal Findings

In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob.

Conclusions/Significance

DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species.     

DNA barcoding as a complementary tool for conservation and valorisation of forest resources

Since the pre-historic era, humans have been using forests as a food, drugs and handcraft reservoir. Today, the use of botanical raw material to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on a selected group of forest tree species with the aim of contributing to the identification, conservation and trade control of these valuable plant resources.The “core barcode” for land plants (rbcL, matK, and trnH-psbA) was tested on 68 tree specimens (24 taxa). Universality of the method, ease of data retrieval and correct species assignment using sequence character states, presence of DNA barcoding gaps and GenBank discrimination assessment were evaluated. The markers showed different prospects of reliable applicability. RbcL and trnH-psbA displayed 100% amplification and sequencing success, while matK did not amplify in some plant groups. The majority of species had a single haplotype. The trnH-psbA region showed the highest genetic variability, but in most cases the high intraspecific sequence divergence revealed the absence of a clear DNA barcoding gap. We also faced an important limitation because the taxonomic coverage of the public reference database is incomplete. Overall, species identification success was 66.7%.This work illustrates current limitations in the applicability of DNA barcoding to taxonomic forest surveys. These difficulties urge for an improvement of technical protocols and an increase of the number of sequences and taxa in public databases.