DNA Barcoding Gap Research Articles

Exploring species level taxonomy and species delimitation methods in the facultatively self-fertilizing land snail genus Rumina (gastropoda: pulmonata).

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.

Identification of medical plants of 24 Ardisia species from China using the matK genetic marker

Background:Ardisia is a group of famous herbs in China, which has been used as medical plants for more than 900 years. However, the species from the genus are so analogous that it is difficult to discriminate them just by morphological characteristics. DNA barcoding is a new technique that uses a short and standard fragment of DNA sequences to identify species.Objective:Choose a suitable DNA marker to authenticate Ardisia species.Materials and Methods:Four markers (psbA-trnH, internal transcribed spacer 2 [ITS2], rbcL, matK) were tested on 54 samples of 24 species from genus Ardisia. The success rates of polymerase chain reaction amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and identification efficiency were used to evaluate the discrimination ability.Results:The results indicate that matK has the highest interspecific divergence and significant differences between inter- and intra-specific divergences, whereas psbA-trnH, ITS2 and rbcL have much lower divergence values. Matk possessed the highest species identification efficiency at 98.1% by basic local alignment search tool 1 [BLAST1], method and 91.7% by the nearest distance method.Conclusion:The matK region is a promising DNA barcode for the genus Ardisia.

DNA barcoding of populations of Fallopia multiflora, an indigenous herb in China

Fallopia multiflora, locally known as Heshouwu, is one of the most important and widely used Chinese medicinal herbs. However, there is still considerable confusion concerning its different provenances. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. We assessed the applicability of 4 candidate DNA barcodes (matK, rbcL, psbA-trnH, and ITS2) to identify populations of F. multiflora. To our knowledge, this is the first attempt involving the plant kingdom to apply DNA barcoding at a level lower than species. Four DNA loci (matK, rbcL, psbA-trnH, and ITS2) of 105 samples, including the wild F. multiflora distributed in 17 provinces of China and 4 cultivated F. multiflora lines, were amplified by PCR and sequenced. The 4 loci were evaluated by PCR amplification for sequence quality, extent of genetic divergence, DNA barcoding gap, and the ability to discriminate between populations by BLAST1 and Nearest Distance. We found that psbA-trnH was the best barcode, with significant inter-population variability and best potential for identifying F. multiflora. The combination of loci gave better performance for distinguishing populations than a single locus. We recommend using matK + rbcL + psbA-trnH + ITS2 or psbA-trnH alone for this species. This research demonstrates the utility of DNA barcoding for geoherbalism identifications.

DNA barcoding for identification of 'Candidatus Phytoplasmas' using a fragment of the elongation factor Tu gene.

BackgroundPhytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.Methodology/Principal FindingsWe designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.Conclusions/SignificanceThis study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.

Use of the potential DNA barcode ITS2 to identify herbal materials

A potential DNA barcode, ITS2, was studied to discriminate herbal materials to confirm their identities and ensure their safe application in pharmaceuticals. Here, a total of 4385 samples of 2431 species were collected, and these samples are from 61 commonly used herbs and their closely related species or adulterants. Based on assessments of the extent of genetic divergence, the DNA barcoding gap and the ability for species discrimination, our results suggest that ITS2 is a powerful tool for distinguishing herbs. For the first dataset including 61 herbs, ITS2 correctly identified 100% of them. For the second dataset containing 51 herbs and their 2382 closely related species, ITS2 could discriminate correctly 48 herbs from their closely related species. For the third dataset comprising 34 herbs and their 111 adulterants, ITS2 could distinguish successfully all the herbs from their adulterants. In conclusion, the ITS2 region is an efficient marker for the authentication of herbal materials, and our study will accelerate the process of the application of the DNA barcoding technique in differentiating herbs.

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA.

BackgroundInternal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.Methodology/Principal FindingsIn this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.ConclusionsIdentification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.

Authentication of Illicium verum using a DNA barcode psbA-trnH

The objective of this research was to test an appropriate DNA barcode from four candidate barcoding loci (matK, rbcL, psbA-trnH and ITS2) for discrimination between Illicium verumand its congeneric toxic adulterants in trade. Twenty fresh samples from 8 species of the genera Illicium were collected from four areas of China, and two samples of medicinal material of I. verum were purchased respectively, from the market and pharmacy. In this study, PCR amplification success rate, sequencing efficiency, the intra- and inter-specific divergences, and DNA barcoding gap were employed to assess the ability of each barcode for the authentication of I. verum and its adultrants. The results showed that psbA-trnHexhibited the highest inter-specific divergence among the four DNA barcoding sequences and the highest species identification efficiency at 100% both by BLAST1 and the nearest distance method. In addition, the psbA-trnH query sequences of the two dried fruits were compared with the reference sequence by BLAST1, they were identified as I. verum. Our findings supported the notion that the psbA-trnH region could distinguish I. verum from other adulterating species. Key words: Illicium verum, psbA-trnH, identification, DNA barcoding, Illiciaceae, medicinal material.

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co-evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH-psbA, psbK-psbI, atpF-atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra- and inter-specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK-psbI and trnH-psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF-atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH-psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.

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.

DNA Barcoding the Dioscorea in China, a Vital Group in the Evolution of Monocotyledon: Use of matK Gene for Species Discrimination

Background Dioscorea is an important plant genus in terms of food supply and pharmaceutical applications. However, its classification and identification are controversial. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. In this study, the applicability of three candidate DNA barcodes (rbcL, matK, and psbA-trnH) to identify species within Dioscorea was tested.Methodology/Principal FindingsOne-hundred and forty-eight individual plant samples of Dioscorea, encompassing 38 species, seven varieties and one subspecies, representing majority species distributed in China of this genus, were collected from its main distributing areas. Samples were assessed by PCR amplification, sequence quality, extent of specific genetic divergence, DNA barcoding gap, and the ability to discriminate between species. matK successfully identified 23.26% of all species, compared with 9.30% for rbcL and 11.63% for psbA-trnH. Therefore, matK is recommended as the best DNA barcoding candidate. We found that the combination of two or three loci achieved a higher success rate of species discrimination than one locus alone. However, experimental cost would be much higher if two or three loci, rather than a single locus, were assessed.ConclusionsWe conclude that matK is a strong, although not perfect, candidate as a DNA barcode for Dioscorea identification. This assessment takes into account both its ability for species discrimination and the cost of experiments.

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA-trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non-flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA-trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non-flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra- and inter-specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non-flowering seed plants. In addition, we compared the abilities of the five most-recommended markers (psbA-trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non-flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non-flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.

Application of deoxyribonucleic acid barcoding in Lauraceae plants

Background:This study aims to determine the candidate markers that can be used as DNA barcode in the Lauraceae family.Material and Methods:Polymerase chain reaction amplification, sequencing efficiency, differential intra- and interspecific divergences, DNA barcoding gap, and identification efficiency were used to evaluate the four different DNA sequences of psbA-trnH, matK, rbcL, and ITS2. We tested the discrimination ability of psbA-trnH in 68 plant samples belonging to 42 species from 11 distinct genera and found that the rate of successful identification with the psbA-trnH was 82.4% at the species level. However, the correct identification of matK and rbcL were only 30.9% and 25.0%, respectively, using BLAST1. The PCR amplification efficiency of the ITS2 region was poor; thus, ITS2 was not included in subsequent experiments. To verify the capacity of the identification of psbA-trnH in more samples, 175 samples belonging to 117 species from the experimental data and from the GenBank database of the Lauraceae family were tested.Results:Using the BLAST1 method, the identification efficiency were 84.0% and 92.3% at the species and genus level, respectively.Conclusion:Therefore, psbA-trnH is confirmed as a useful marker for differentiating closely related species within Lauraceae.

DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India

We have checked the utility of DNA barcoding for species identification of nymphalid butterflies from Western Ghats of India by using 650 bp sequence of mitochondrial gene cytochrome c oxidase subunit I. Distinct DNA barcoding gap (i.e. difference between intraspecies and interspecies nucleotide divergence), exists between species studied here. When our sequences were compared with the sequences of the conspecifics submitted from different geographic regions, nine cases of deep intraspecies nucleotide divergences were observed. In spite of this, NJ (Neighbour Joining) clustering analysis successfully discriminated all species. Observed cases of deep intraspecies nucleotide divergences certainly warrant further study.

Applying plant DNA barcodes for Rosaceae species identification.

© The Willi Hennig Society 2010. ABSTRACT: The Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG) established the use of matK+rbcL as core barcodes and ITS2 as one of the supplementary loci for differentiating plants at the Third International Barcoding Conference. Here, we tested the applicability of four DNA regions (rbcL, matK, rpoC1 and ITS2) as the barcodes for identifying species within Rosaceae. Based on assessments of the success rates of PCR amplifications, the sequence quality, extent of specific genetic divergence, DNA barcoding gap and ability for species discrimination, our results suggest that ITS2 is the best of the four loci tested for barcoding Rosaceae. We further evaluated the effectiveness of ITS2 for identifying a wide range of species within Rosaceae. Of the 1410 plant samples collected from 893 species in 96 diverse genera, ITS2 successfully identified 78 and 100% of them at the species and genus levels, respectively. Therefore, our research indicated that the ITS2 region is a powerful, though not perfect, barcode for Rosaceae identification that also contributes valuable information for identifying closely related species in other plant taxonomic groups.

Exploring the Use of Cytochrome Oxidase c Subunit 1 (COI) for DNA Barcoding of Free-Living Marine Nematodes

BackgroundThe identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.MethodologyBoth partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.ConclusionThe I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

DNA barcoding in medicinal plants Caprifoliaceae

To determine the candidate sequences which can be used as DNA barcode to identify species in Caprifoliaceae family by screening out from four different DNA fragments sequences. PCR amplification, sequencing efficiency, differential intra- and interspecific divergences, the DNA barcoding gap and identification efficiency were used to evaluate these loci. The ITS2 was used as a candidate sequence of DNA barcode to identify the species in Caprifoliaceae family, whose rate of success in identification in genera level was 100% and in species 96.6%, and psbA-trnH as a complementary barcode to ITS2 for Caprifoliaceae.

Identification of Fabaceae Plants Using the DNA BarcodematK

In this study, we tested the applicability of the core DNA barcode MATK for identifying species within the Fabaceae family. Based on an evaluation of genetic variation, DNA barcoding gaps, and species discrimination power, MATK is a useful barcode for Fabaceae species. Of 1355 plant samples collected from 1079 species belonging to 409 diverse genera, MATK precisely identified approximately 80 % and 96 % of them at the species and genus levels, respectively. Therefore, our research indicates that the MATK region is a valuable marker for plant species within Fabaceae.

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention. Here, seven regions (psbA-trnH, matK, ycf5, rpoC1, rbcL, ITS2, and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and barcoding gaps were assessed. We found that the ITS2 region exhibited the highest inter-specific divergence, and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests. The ITS2 locus had the highest identification efficiency among all tested regions. In a previous study, we found that ITS2 was able to discriminate a wide range of plant taxa, and here we confirmed that ITS2 was also able to discriminate a number of closely related species. Therefore, we propose that ITS2 is a promising candidate barcode for plant species identification.

Using DNA Barcoding to Identify Species within Euphorbiaceae

In this study, we tested the applicability of four DNA regions (rbcL, matK, ITS, and ITS2) as barcodes for identifying species within Euphorbiaceae. Based on assessments of the specific genetic divergence, the DNA barcoding gap, and the ability for species discrimination, the present results affirmed that ITS/ITS2 is a potential barcode for the Euphorbiaceae species. This study also provided a large-scale test to evaluate the effectiveness of ITS/ITS2 for differentiating species within Euphorbiaceae. Of the 1183 plant samples collected from 871 species in 66 diverse genera, ITS/ITS2 successfully identified > 90% and 100% of them at the species and genus levels, respectively. Therefore, our research indicates that use of the ITS/ITS2 region is a powerful technique for Euphorbiaceae identification.

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

BackgroundThe plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.Methodology/Principal FindingsHere, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.ConclusionsThe ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.