DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller’s classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.     

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.     

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.     

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.     

Testing candidate plant barcode regions in the Myristicaceae

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon - UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.     

Slow Mitochondrial COI Sequence Evolution at the Base of the Metazoan Tree and Its Implications for DNA Barcoding

The evolution rates of mtDNA in early metazoans hold important implications for DNA barcoding. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and Scyphozoa) using a data set of 619 sequences from 224 species. We found variation within and between species to be much lower in Porifera and Anthozoa compared to Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans. Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution could have originated independently in Medusozoa and Bilateria or been acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding. Caution is also advised for Porifera and Hydrozoa because of relatively low identification success rates as even threshold divergence that maximizes the “barcoding gap” does not improve identification success. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Wolbachia and DNA barcoding insects: patterns, potential, and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.     

The complete mitochondrial genome of the verongid sponge Aplysina cauliformis: implications for DNA barcoding in demosponges

DNA “barcoding,” the determination of taxon-specific genetic variation typically within a fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene, has emerged as a useful complement to morphological studies, and is routinely used by expert taxonomists to identify cryptic species and by non-experts to better identify samples collected during field surveys. The rate of molecular evolution in the mitochondrial genomes (mtDNA) of nonbilaterian animals (sponges, cnidarians, and placozoans) is much slower than in bilaterian animals for which DNA barcoding strategies were developed. If sequence divergence among nonbilaterian mtDNA and specifically cox1 is too slow to generate diagnostic variation, alternative genes for DNA barcoding and species-level phylogenies should be considered. Previous study across the Aplysinidae (Demospongiae, Verongida) family of sponges demonstrated no nucleotide substitutions in the traditional cox1 barcoding fragment among the Caribbean species of Aplysina. As the mitochondrial genome of Aplysina fulva has previously been sequenced, we are now able to make the first comparisons between complete mtDNA of congeneric demosponges to assess whether potentially informative variation exists in genes other than cox1. In this article, we present the complete mitochondrial genome of Aplysina cauliformis, a circular molecule 19620 bp in size. The mitochondrial genome of A. cauliformis is the same length as is A. fulva and shows six confirmed nucleotide differences and an additional 11 potential SNPs. Of the six confirmed SNPs, NADH dehydrogenase subunit 5 (nad5) and nad2 each contain two, and in nad2 both yield amino acid substitutions, suggesting balancing selection may act on this gene. Thus, while the low nucleotide diversity in Caribbean aplysinid cox1 extends to the entire mitochondrial genome, some genes do display variation. If these represent interspecific differences, then they may be useful alternative markers for studies in recently diverged sponge clades.     

Limitations of mitochondrial gene barcoding in Octocorallia

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.     

COI-based DNA barcoding of Arcoida species (Bivalvia: Pteriomorphia) along the coast of China

DNA barcoding is a promising tool for the rapid and unambiguous identification of species. Some arcoid species are particularly difficult to distinguish with traditional morphological identification owing to phenotypic variation and the existence of closely related taxa. Here, we apply DNA barcoding based on mitochondrial cytochrome c oxidase I gene (COI) to arcoid species collected from the coast along China. Combining morphology with molecular data indicates the 133 specimens of Arcoida could be assigned to 24 species. Because of the deep genetic divergence within Tegillarca granosa, there was an overlap between genetic variation within species and variation between species. Nevertheless, NJ and Bayesian trees showed that all species fell into reciprocally monophyletic clades with high bootstrap values. Our results evidence that the COI marker can efficiently identify species, correct mistakes caused by morphological identification and reveal genetic differentiation among populations within species. This study provides a clear example of the usefulness of barcoding for arcoid identification. Furthermore, it also lays a foundation for other biological and ecological studies of Arcoida.     

DNA barcoding of the main cultivated yams and selected wild species in the genus Dioscorea

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.     

The Rosy Region of Drosophila Melanogaster and Drosophila Simulans. I. Contrasting Levels of Naturally Occurring DNA Restriction Map Variation and Divergence

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.     

Gene expression divergence is coupled to evolution of DNA structure in coding regions

Sequence changes in coding region and regulatory region of the gene itself (cis) determine most of gene expression divergence between closely related species. But gene expression divergence between yeast species is not correlated with evolution of primary nucleotide sequence. This indicates that other factors in cis direct gene expression divergence. Here, we studied the contribution of DNA three-dimensional structural evolution as cis to gene expression divergence. We found that the evolution of DNA structure in coding regions and gene expression divergence are correlated in yeast. Similar result was also observed between Drosophila species. DNA structure is associated with the binding of chromatin remodelers and histone modifiers to DNA sequences in coding regions, which influence RNA polymerase II occupancy that controls gene expression level. We also found that genes with similar DNA structures are involved in the same biological process and function. These results reveal the previously unappreciated roles of DNA structure as cis-effects in gene expression.     

Things fall apart: biological species form unconnected parsimony networks

The generality of operational species definitions is limited by problematic definitions of between-species divergence. A recent phylogenetic species concept based on a simple objective measure of statistically significant genetic differentiation uses between-species application of statistical parsimony networks that are typically used for population genetic analysis within species. Here we review recent phylogeographic studies and reanalyse several mtDNA barcoding studies using this method. We found that (i) alignments of DNA sequences typically fall apart into a separate subnetwork for each Linnean species (but with a higher rate of true positives for mtDNA data) and (ii) DNA sequences from single species typically stick together in a single haplotype network. Departures from these patterns are usually consistent with hybridization or cryptic species diversity.     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

DNA barcoding Bromeliaceae: achievements and pitfalls

Background

DNA barcoding has been successfully established in animals as a tool for organismal identification and taxonomic clarification. Slower nucleotide substitution rates in plant genomes have made the selection of a DNA barcode for land plants a much more difficult task. The Plant Working Group of the Consortium for the Barcode of Life (CBOL) recommended the two-marker combination rbcL/matK as a pragmatic solution to a complex trade-off between universality, sequence quality, discrimination, and cost.

Methodology/Principal Findings

It is expected that a system based on any one, or a small number of plastid genes will fail within certain taxonomic groups with low amounts of plastid variation, while performing well in others. We tested the effectiveness of the proposed CBOL Plant Working Group barcoding markers for land plants in identifying 46 bromeliad species, a group rich in endemic species from the endangered Brazilian Atlantic Rainforest. Although we obtained high quality sequences with the suggested primers, species discrimination in our data set was only 43.48%. Addition of a third marker, trnH–psbA, did not show significant improvement. This species identification failure in Bromeliaceaecould also be seen in the analysis of the GenBank''s matK data set. Bromeliaceae''s sequence divergence was almost three times lower than the observed for Asteraceae and Orchidaceae. This low variation rate also resulted in poorly resolved tree topologies. Among the three Bromeliaceae subfamilies sampled, Tillandsioideae was the only one recovered as a monophyletic group with high bootstrap value (98.6%). Species paraphyly was a common feature in our sampling.

Conclusions/Significance

Our results show that although DNA barcoding is an important tool for biodiversity assessment, it tends to fail in taxonomy complicated and recently diverged plant groups, such as Bromeliaceae. Additional research might be needed to develop markers capable to discriminate species in these complex botanical groups.     

Searching for evidence of selection in avian DNA barcodes

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species.     

DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.     

DNA条形码在中国金合欢属中的应用

根据形态特征难以准确地辨别金合欢属植物,DNA条形码技术提供了一种准确地鉴定物种的方法。本文利用条形码技术对中国金合欢属物种的序列(psbA trnH、matK、rbcL和ITS)及其不同组合进行比较,通过计算种内和种间变异进行barcoding gap分析,运用Wilcoxon秩和检验比较不同序列的变异性,构建系统树。结果表明：4个片段均存在barcoding gap,ITS序列种间变异率较psbA trnH、rbcL和matK序列有明显优势,单片段ITS正确鉴定率最高,ITS+rbcL片段联合条码的正确鉴定率最高,因此我们认为ITS片段或条形码组合ITS+rbcL是金合欢属的快速鉴别最理想的条码。     

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.