Barcoding Purposes Research Articles

Morphological and Molecular Analysis of Parasitoid Wasp, Cheiropachus quadrum (Hymenoptera: Chalcidoidea: Pteromalidae) Infesting Bark Beetles of Wide Host Trees

Cheiropachus quadrum Fabricius, 1787 (Hymenoptera: Chalcidoidea: Pteromalidae) is a Pteromalid parasitoid wasp infesting wood boring beetles predominantly family Scolytidae. Owing to their profound diversity and small size, their identification is bit tricky. The present study involves molecular analysis and morphological identification including new host record, distribution and key identification characters of species. Morphologically, Cheiropachus quadrum is easily characterised by its large propleura, maculate forewings and enlarged fore-femur. Cytochrome Oxidase gene 1 was used as marker gene for DNA barcoding purposes. Sequences edited and trimmed in BioEdit were submitted to GenBank for accession number generation. BLAST and MEGA tools were used for molecular analysis to confirm the corresponding taxonomy. Generated sequence was found to have no perfect identity matches and hence was morphologically confirmed and submitted as novel sequence to the database. Molecular characterisation involving phylogenetic analysis was performed with top hits from blast database using Neighbour Joining and Maximum Likelihood tree construction methods. It was found that the mean A+T content is 74.19% and estimated Transition/Transversion bias (R) is 0.71. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.02%) and minimum distance between species (3.6%). Phylogenetic analysis concluded DNA barcoding is helpful for identification of parasitoid species and complements morphological taxonomy for easy identification purposes.

BCdatabaser: on-the-fly reference database creation for (meta-)barcoding.

DNA barcoding and meta-barcoding have become irreplaceable in research and applications, where identification of taxa alone or within a mixture, respectively, becomes relevant. Pioneering studies were in the microbiological context, yet nowadays also plants and animals become targeted. Given the variety of markers used, formatting requirements for classifiers and constant growth of primary databases, there is a need for dedicated reference database creation. We developed a web and command-line interface to generate such on-the-fly for any applicable marker and taxonomic group with optional filtering, formatting and restriction specific for (meta-)barcoding purposes. Also, databases optionally receive a DOI, making them well-documented with meta-data, publicly sharable and citable. source code: https://www.github.com/molbiodiv/bcdatabaser, webservice: https://bcdatabaser.molecular.eco, documentation: https://molbiodiv.github.io/bcdatabaser.

New species and records of bioluminescent Mycena from Mexico

ABSTRACTSeven species of bioluminescent fungi are recorded from the cloud forests in Mexico. Six represent new species belonging to the genus Mycena, whereas Mycena globulispora is a new distribution record for the country. Descriptions, illustrations, photographs, and an identification key to bioluminescent fungi species from Mexico are provided. Sequences of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region were generated for barcoding purposes and comparisons with similar species.

Inclusion of Kickxia abhaica D.A. Sutton in the genus Nanorrhinum (Plantaginaceae): Evidence from its nuclear ribosomal DNA sequences

The nuclear ribosomal DNA (nrDNA) internal transcribed spacers (ITS) sequences is extensively used in the plant molecular phylogenetics for plant taxonomic identification and DNA barcoding purposes because the nrDNA ITS gene is easy to amplify by using the universal primers, its length is shorter and thus easy to sequence, and has strong discrimination power to distinguish the taxon at the species level. The present molecular phylogenetic analysis of ITS nrDNA sequences focuses to determine the taxonomic status of an unresolved endemic taxon Kickxia abhaica D.A. Sutton (Family Plantaginaceae, tribe Antirrhineae) reported from Saudi Arabia. The analysis supports the transfer of K. abhaica under the genus Nanorrhinum.

New luminescent mycenoid fungi (Basidiomycota, Agaricales) from São Paulo State, Brazil

Four species of mycenoid fungi are reported as luminescent (or putatively luminescent) on the basis of specimens collected from São Paulo State, Brazil. Two of them represent new species (Mycena oculisnymphae, Resinomycena petarensis), and two represent new reports of luminescence in previously described species (M. deformis, M. globulispora). Comprehensive descriptions, illustrations, photographs, and comparisons with phenetically similar species are provided. Sequences of nuc rDNA internal transcribed spacer regions were generated for barcoding purposes and for comparisons with similar species.

Comparison of manual and semi-automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae)

The barcoding of life initiative provides a universal molecular tool to distinguish animal species based on the amplification and sequencing of a fragment of the subunit 1 of the cytochrome oxidase (COI) gene. Obtaining good quality DNA for barcoding purposes is a limiting factor, especially in studies conducted on small-sized samples or those requiring the maintenance of the organism as a voucher. In this study, we compared the number of positive amplifications and the quality of the sequences obtained using DNA extraction methods that also differ in their economic costs and time requirements and we applied them for the genetic characterization of louse flies. Four DNA extraction methods were studied: chloroform/isoamyl alcohol, HotShot procedure, Qiagen DNeasy(®) Tissue and Blood Kit and DNA Kit Maxwell(®) 16LEV. All the louse flies were morphologically identified as Ornithophila gestroi and a single COI-based haplotype was identified. The number of positive amplifications did not differ significantly among DNA extraction procedures. However, the quality of the sequences was significantly lower for the case of the chloroform/isoamyl alcohol procedure with respect to the rest of methods tested here. These results may be useful for the genetic characterization of louse flies, leaving most of the remaining insect as a voucher.

Testing the performance of a fragment of the COI gene to identify western Palaearctic stag beetle species(Coleoptera, Lucanidae).

The taxonomy of stag beetles (Coleoptera: Lucanidae) remains challenging, mainly due to the sexual dimorphism and the strong allometry in males. Such conjecture confounds taxonomic based conservation efforts that are urgently needed due to numerous threats to stag beetle biodiversity. Molecular tools could help solve the problem of identification of the different recognized taxa in the “Lucanus cervus complex” and in some related Palaearctic species. We investigated the potential use of a 670 bp region at the 3’ end of the mitochondrial cytochrome c oxidase subunit I gene (COI) for barcoding purposes (different from the standard COI barcoding region). Well resolved species and subspecies were L. tetraodon, L. cervusakbesianus, L. c. laticornis, as well as the two eastern Asian outgroup taxa L. formosanus and L. hermani. Conversely, certain taxa could not be distinguished from each other based on K2P-distances and tree topologies: L. c. fabiani / L. (P.) barbarossa, L. c. judaicus / an unknown Lucanus species, L. c. cervus / L. c. turcicus / L. c. pentaphyllus / L. (P.) macrophyllus / L. ibericus. The relative roles of phenotypic plasticity, recurrent hybridisation and incomplete lineage sorting underlying taxonomic and phylogenetic discordances are discussed.

Colletotrichum - current status and future directions.

A review is provided of the current state of understanding of Colletotrichum systematics, focusing on species-level data and the major clades. The taxonomic placement of the genus is discussed, and the evolution of our approach to species concepts and anamorph-teleomorph relationships is described. The application of multilocus technologies to phylogenetic analysis of Colletotrichum is reviewed, and selection of potential genes/loci for barcoding purposes is discussed. Host specificity and its relation to speciation and taxonomy is briefly addressed. A short review is presented of the current status of classification of the species clusters that are currently without comprehensive multilocus analyses, emphasising the orbiculare and destructivum aggregates. The future for Colletotrichum biology will be reliant on consensus classification and robust identification tools. In support of these goals, a Subcommission on Colletotrichum has been formed under the auspices of the International Commission on Taxonomy of Fungi, which will administer a carefully curated barcode database for sequence-based identification of species within the BioloMICS web environment.

Rapid DNA barcoding analysis of large datasets using the composition vector method

BackgroundSequence alignment is the rate-limiting step in constructing profile trees for DNA barcoding purposes. We recently demonstrated the feasibility of using unaligned rRNA sequences as barcodes based on a composition vector (CV) approach without sequence alignment (Bioinformatics 22:1690). Here, we further explored the grouping effectiveness of the CV method in large DNA barcode datasets (COI, 18S and 16S rRNA) from a variety of organisms, including birds, fishes, nematodes and crustaceans.ResultsOur results indicate that the grouping of taxa at the genus/species levels based on the CV/NJ approach is invariably consistent with the trees generated by traditional approaches, although in some cases the clustering among higher groups might differ. Furthermore, the CV method is always much faster than the K2P method routinely used in constructing profile trees for DNA barcoding. For instance, the alignment of 754 COI sequences (average length 649 bp) from fishes took more than ten hours to complete, while the whole tree construction process using the CV/NJ method required no more than five minutes on the same computer.ConclusionThe CV method performs well in grouping effectiveness of DNA barcode sequences, as compared to K2P analysis of aligned sequences. It was also able to reduce the time required for analysis by over 15-fold, making it a far superior method for analyzing large datasets. We conclude that the CV method is a fast and reliable method for analyzing large datasets for DNA barcoding purposes.

A stuttering start to plant DNA barcoding: microsatellites present a previously overlooked problem in non‐coding plastid regions

AbstractPlastid microsatellites are simple mono/dinucleotide repeats that can cause in‐vitro taq polymerase errors leading to 'stutter' peaks that reduce the clarity and reliability of electropherograms. As an efficient and automated barcoding protocol requires high‐quality raw data, this paper investigates the suitability of the psbA‐trnH and atpF‐H regions of the plastid genome for automated barcoding purposes. Amplification of 92 individuals from species pairs and triplets was undertaken. Of these, 59% of atpF‐H species samples and 36% of psbA‐trnH species samples showed evidence of microsatellite‐induced errors in electropherogram base calling. The downstream necessity of human intervention renders these non‐coding regions sub‐optimal for barcoding purposes.

A test of psbK-psbI and atpF-atpH as potential plant DNA barcodes using the flora of the Kruger National Park (South Africa) as a model system

AbstractA DNA barcode consists of a standardized short sequence of DNA (400-800bp) used to identify the taxonomic species a small organic fragment belongs to. Even though it has been easy to discriminate animal species by using the mitochondrial gene cox1, this is still difficult for plants seeing that the mitochondrial genome is not variable enough on the species level. During the Second International Barcode of Life Conference in Tapei (September 2007), different plastid regions were proposed as potential plant DNA barcodes, such as atpF-atpH and psbK-psbI, but no consensus on which region to use was reached during the meeting. The largest plant DNA barcoding study to date proposed matK as the best candidate and suggested that in combination with trnH-psbA a slight increase in performance could be achieved. However, no study has tested the suitability of the newly proposed psbK-psbI and atpF-atpH for plant barcoding purposes. Four potential DNA barcodes, matK, trnH-psbA, atpF-atpH, and psbK-psbI, were amplified and sequenced for a selective sampling including mainly trees and shrubs of the flora of the Kruger National Park Africa (South Africa). The performance of each region and also each possible combination of these were tested by applying a battery of metrics and statistical tests. Our results confirm that the second half (5’ end) of matK is the best candidate in a single locus barcoding approach reaching 87.5% of species correctly identified. Combining matK with trnH-psbA and psbK-psbI increased only slightly the performance in discriminating species. The results from this study show that the use of a ‘three-region barcode’ does not significantly outperform matK in a single-locus barcoding approach. We therefore argue against the ‘multiple barcode approach’ proposed by the plant working group, and instead propose to keep barcoding plants in line with the approach taken for animals, i.e. using one barcode: cox1 for animals and matK for plants.

Spatiotemporal analysis of population genetic structure in Geomonhystera disjuncta (Nematoda, Monhysteridae) reveals high levels of molecular diversity

Species identification in the phylum Nematoda is complicated due to the paucity of easily obtainable diagnostic morphological features. Furthermore, the cosmopolitan distribution of several species despite low dispersal abilities makes cryptic diversity potentially substantial within this phylum. We conducted a population genetic survey in the marine nematode Geomonhystera disjuncta in Belgium and The Netherlands in two seasons. The mitochondrial cytochrome oxidase c subunit 1 (COI) gene was screened with the single-strand conformation polymorphism method in 759 individuals. The 43 haplotypes were grouped into five lineages, with low divergences within (<3%) and high divergences between lineages (>14%). Analysis of the nuclear ITS region yielded concordant tree topologies, indicating the presence of five cryptic taxa within G. disjuncta. Analysis of Molecular Variance (AMOVA) illustrated a significant structuring in all lineages and temporal fluctuations in haplotype frequencies within and between locations. Metapopulation dynamics and/or priority effects best explained this structuring. Finally, our data indicate that the COI gene may be useful for DNA barcoding purposes.

Ribosomal RNA as molecular barcodes: a simple correlation analysis without sequence alignment

We explored the feasibility of using unaligned rRNA gene sequences as DNA barcodes, based on correlation analysis of composition vectors (CVs) derived from nucleotide strings. We tested this method with seven rRNA (including 12, 16, 18, 26 and 28S) datasets from a wide variety of organisms (from archaea to tetrapods) at taxonomic levels ranging from class to species. Our results indicate that grouping of taxa based on CV analysis is always in good agreement with the phylogenetic trees generated by traditional approaches, although in some cases the relationships among the higher systemic groups may differ. The effectiveness of our analysis might be related to the length and divergence among sequences in a dataset. Nevertheless, the correct grouping of sequences and accurate assignment of unknown taxa make our analysis a reliable and convenient approach in analyzing unaligned sequence datasets of various rRNAs for barcoding purposes. The newly designed software (CVTree 1.0) is publicly available at the Composition Vector Tree (CVTree) web server http://cvtree.cbi.pku.edu.cn.

Use of DNA barcodes to identify flowering plants.

Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.