Surprisingly high genetic divergence of the mitochondrial DNA barcode fragment (COI) within Central European woodlice species (Crustacea, Isopoda, Oniscidea).

DNA barcoding has become the most popular approach for species identification in recent years. As part of the German Barcode of Life project, the first DNA barcode library for terrestrial and freshwater isopods from Germany is presented. The analyzed barcode library included 38 terrestrial (78% of the documented species of Germany) and five freshwater (63%) species. A total of 513 new barcodes was generated and 518 DNA barcodes were analyzed. This analysis revealed surprisingly high intraspecific genetic distances for numerous species, with a maximum of 29.4% for Platyarthrushoffmannseggii Brandt, 1833. The number of BINs per species ranged from one (32 species, 68%) to a maximum of six for Trachelipusrathkii (Brandt, 1833). In spite of such high intraspecific variability, interspecific distances with values between 12.6% and 29.8% allowed a valid species assignment of all analyzed isopods. The observed high intraspecific distances presumably result from phylogeographic events, Wolbachia infections, atypical mitochondrial DNAs, heteroplasmy, or various combinations of these factors. Our study represents the first step in generating an extensive reference library of DNA barcodes for terrestrial and freshwater isopods for future molecular biodiversity assessment studies.

Barcoding a can of worms: testing cox1 performance as a DNA barcode of Nematoda.

Accurate taxonomic identifications and species delimitations are a fundamental problem in biology. The complex taxonomy of Nematoda is primarily based on morphology, which is often dubious. DNA barcoding emerged as a handy tool to identify specimens and assess diversity, but its applications in Nematoda are incipient. We evaluated cytochrome c oxidase subunit I (cox1) efficiency as a DNA barcode for nematodes scrutinising 5241 sequences retrieved from BOLD and GenBank. The samples included genera with medical, agricultural, or ecological relevance: Anguillicola, Caenorhabditis, Heterodera, Meloidogyne, Onchocerca, Strongyloides, and Trichinella. We assessed cox1 performance through barcode gap and Probability of Correct Identification (PCI) analyses, and estimated species richness through Automatic Barcode Gap Discovery (ABGD). Each genus presented distinct gap ranges, mirroring the evolutionary diversity within Nematoda. Thus, to survey the diversity of the phylum, a careful definition of thresholds for lower taxonomic levels should be considered. PCIs were around 70% for both databases, highlighting operational biases and challenges in nematode taxonomy. ABGD inferred higher richness than the taxonomic labels informed by databases. The prevalence of specimen misidentifications and dubious species delimitations emphasise the value of integrative approaches to nematode taxonomy and systematics. Overall, cox1 is a relevant tool for integrative taxonomy of nematodes.

A DNA barcode library for ground beetles of Germany: the genus Pterostichus Bonelli, 1810 and allied taxa (Insecta, Coleoptera, Carabidae).

Species of the ground beetle genus Pterostichus Bonelli, 1810 are some of the most common carabids in Europe. This publication provides a first comprehensive DNA barcode library for this genus and allied taxa including Abax Bonelli, 1810, Molops Bonelli, 1810, Poecilus Bonelli, 1810, and Stomis Clairville, 1806 for Germany and Central Europe in general. DNA barcodes were analyzed from 609 individuals that represent 51 species, including sequences from previous studies as well as more than 198 newly generated sequences. The results showed a 1:1 correspondence between BIN and traditionally recognized species for 44 species (86%), whereas two (4%) species were characterized by two BINs. Three BINs were found for one species (2%), while one BIN for two species was revealed for two species pairs (8%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for four species pairs. Haplotype sharing was found for two closely related species pairs: Pterostichus adstrictus Eschscholtz, 1823/Pterostichus oblongopunctatus (Fabricius, 1787) and Pterostichus nigrita Paykull, 1790/Pterostichus rhaeticus Heer, 1837. In contrast to this, high intraspecific sequence divergences with values above 2.2% were shown for three species (Molops piceus (Panzer, 1793), Pterostichus panzeri (Panzer, 1805), Pterostichus strenuus (Panzer, 1793)). Summarizing the results, the present DNA barcode library does not only allow the identification of most of the analyzed species, but also provides valuable information for alpha-taxonomy as well as for ecological and evolutionary research. This library represents another step in building a comprehensive DNA barcode library of ground beetles as part of modern biodiversity research.

DNA barcoding a unique avifauna: an important tool for evolution, systematics and conservation.

BACKGROUND: DNA barcoding utilises a standardised region of the cytochrome c oxidase I (COI) gene to identify specimens to the species level. It has proven to be an effective tool for identification of avian samples. The unique island avifauna of New Zealand is taxonomically and evolutionarily distinct. We analysed COI sequence data in order to determine if DNA barcoding could accurately identify New Zealand birds. RESULTS: We sequenced 928 specimens from 180 species. Additional Genbank sequences expanded the dataset to 1416 sequences from 211 of the estimated 236 New Zealand species. Furthermore, to improve the assessment of genetic variation in non-endemic species, and to assess the overall accuracy of our approach, sequences from 404 specimens collected outside of New Zealand were also included in our analyses. Of the 191 species represented by multiple sequences, 88.5% could be successfully identified by their DNA barcodes. This is likely a conservative estimate of the power of DNA barcoding in New Zealand, given our extensive geographic sampling. The majority of the 13 groups that could not be distinguished contain recently diverged taxa, indicating incomplete lineage sorting and in some cases hybridisation. In contrast, 16 species showed evidence of distinct intra-species lineages, some of these corresponding to recognised subspecies. For species identification purposes a character-based method was more successful than distance and phylogenetic tree-based methods. CONCLUSIONS: DNA barcodes accurately identify most New Zealand bird species. However, low levels of COI sequence divergence in some recently diverged taxa limit the identification power of DNA barcoding. A small number of currently recognised species would benefit from further systematic investigations. The reference database and analysis presented will provide valuable insights into the evolution, systematics and conservation of New Zealand birds.

The Effect of Barcode Technology Use on Pathology Specimen Labeling Errors.

Pathology specimen labeling errors occur for a variety of reasons. We investigated the use of barcode technology as a method to improve the accuracy of pathology specimen labeling and patient safety. We also assessed nurses' perceptions of system quality, information quality, service quality, user satisfaction, and net benefits. Sixty-eight perioperative nurses who work in a teaching hospital in Taiwan completed the survey. Nurses scored net benefits as highly contributing to their satisfaction, whereas system quality contributed most to dissatisfaction. Further, we analyzed pathology specimen records before and after implementing the barcode system and found that specimen management errors significantly decreased. The use of a reliable barcode system could improve specimen labeling accuracy and enhance nurses' satisfaction with this technology.

A DNA barcode library for ground beetles of Germany: the genus Amara Bonelli, 1810 (Insecta, Coleoptera, Carabidae).

The genus Amara Bonelli, 1810 is a very speciose and taxonomically difficult genus of the Carabidae. The identification of many of the species is accomplished with considerable difficulty, in particular for females and immature stages. In this study the effectiveness of DNA barcoding, the most popular method for molecular species identification, was examined to discriminate various species of this genus from Central Europe. DNA barcodes from 690 individuals and 47 species were analysed, including sequences from previous studies and more than 350 newly generated DNA barcodes. Our analysis revealed unique BINs for 38 species (81%). Interspecific K2P distances below 2.2% were found for three species pairs and one species trio, including haplotype sharing between Amara alpina/Amara torrida and Amara communis/Amara convexior/Amara makolskii. This study represents another step in generating an extensive reference library of DNA barcodes for carabids, highly valuable bioindicators for characterizing disturbances in various habitats.

Re-evaluation of the discriminatory power of DNA barcoding on some specimens of African Cyprinidae (subfamilies Cyprininae and Danioninae).

Specimen identification in the absence of diagnostic morphological characters (e.g., larvae) can be problematic even for experts. The goal of the present study was to assess the performance of COI in discriminating specimens of the fish family Cyprinidae in Africa, and to explore whether COI-phylogeny can be reliably used for phylogenetic comparative analysis. The main objective was to analyse a matrix of COI sequences for 315 specimens from 15 genera of African Cyprinidae using various distance-based identification methods alongside multiple tests of DNA barcode efficacy (barcode gap, species monophyly on NJ tree). Some morphological and biological characters were also mapped on a COI-phylogeny reconstructed using Maximum Parsimony. First, the results indicated the existence of barcode gaps, a discriminatory power of COI ranging from 79 % to 92 %, and that most nodes form well-supported monophyletic clades on an NJ tree. Second, it was found that some morphological and biological characters are clustered on the COI-phylogeny, and this indicates the reliability of these characters for taxonomic discrimination within the family. Put together, our results provide not only an additional support for the COI as a good barcode marker for the African Cyprinidae but it also indicate the utility of COI-based phylogenies for a wide spectrum of ecological questions related to African Cyprinidae.

Estimating the barcoding gap in a global dataset of cox1 sequences for Odonata: close, but no cigar.

We evaluated the extent of intraspecific and interspecific genetic distances for two highly diverse infraorders of Odonata: Anisoptera and Zygoptera. All cytochrome c oxidase subunit I sequences (cox1), the region chosen for zoological DNA barcoding, present in GenBank for each infraorder were downloaded and curated. For Anisoptera, the final dataset consisted of 2,961 individual cox1 sequences for 536 species and the equivalent numbers for Zygoptera were 2,477 sequences for 497 species. More than 7 million individual genetic comparisons were made and the results indicated that there is a tendency towards a barcoding gap, but that the size of the gap may not be sufficient to robustly infer identities for some taxa. DNA barcoding may be of less use for some odonate taxa, perhaps pertaining to misidentifications in global databases. However, at local scales or with more confined taxonomical sampling, this tool may yet be beneficial in identifying these charismatic organisms.

Wrong Blood in Tube.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.

Testing the reliability of standard and complementary DNA barcodes for the monocot subfamily Alooideae from South Africa.

Although a standard DNA barcode has been identified for plants, it does not always provide species-level specimen identifications for investigating important ecological questions. In this study, we assessed the species-level discriminatory power of standard (rbcLa + matK) and complementary barcodes (ITS1 and trnH-psbA) within the subfamily Alooideae (Asphodelaceae), a large and recent plant radiation, whose species are important in horticulture yet are threatened. Alooideae has its centre of endemism in southern Africa, with some outlier species occurring elsewhere in Africa and Madagascar. We sampled 360 specimens representing 235 species within all 11 genera of the subfamily. With three distance-based methods, all markers performed poorly for our combined data set, with the highest proportion of correct species-level specimen identifications (30%) found for ITS1. However, when performance was assessed across genera, the discriminatory power varied from 0% for all single markers and combinations in Gasteria to 63% in Haworthiopsis, again for ITS1, suggesting that DNA barcoding success may be related to the evolutionary history of the lineage considered. Although ITS1 could be a good barcode for Haworthiopsis, the generally poor performance of all markers suggests that Alooideae remains a challenge. As species boundaries within Alooideae remain controversial, we call for continued search for suitable markers or the use of genomics approaches to further explore species discrimination in the group.

A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa.

As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well.

Does a global DNA barcoding gap exist in Annelida?

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications - false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for "Polychaeta", "Oligochaeta", and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 ("Polychaeta"), 3639 ("Oligochaeta"), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Looking back on a decade of barcoding crustaceans.

Species identification represents a pivotal component for large-scale biodiversity studies and conservation planning but represents a challenge for many taxa when using morphological traits only. Consequently, alternative identification methods based on molecular markers have been proposed. In this context, DNA barcoding has become a popular and accepted method for the identification of unknown animals across all life stages by comparison to a reference library. In this review we examine the progress of barcoding studies for the Crustacea using the Web of Science data base from 2003 to 2014. All references were classified in terms of taxonomy covered, subject area (identification/library, genetic variability, species descriptions, phylogenetics, methods, pseudogenes/numts), habitat, geographical area, authors, journals, citations, and the use of the Barcode of Life Data Systems (BOLD). Our analysis revealed a total number of 164 barcoding studies for crustaceans with a preference for malacostracan crustaceans, in particular Decapoda, and for building reference libraries in order to identify organisms. So far, BOLD did not establish itself as a popular informatics platform among carcinologists although it offers many advantages for standardized data storage, analyses and publication.

Metabarcoding vs. morphological identification to assess diatom diversity in environmental studies.

Diatoms are frequently used for water quality assessments; however, identification to species level is difficult, time-consuming and needs in-depth knowledge of the organisms under investigation, as nonhomoplastic species-specific morphological characters are scarce. We here investigate how identification methods based on DNA (metabarcoding using NGS platforms) perform in comparison to morphological diatom identification and propose a workflow to optimize diatom fresh water quality assessments. Diatom diversity at seven different sites along the course of the river system Odra and Lusatian Neisse from the source to the mouth is analysed with DNA and morphological methods, which are compared. The NGS technology almost always leads to a higher number of identified taxa (270 via NGS vs. 103 by light microscopy LM), whose presence could subsequently be verified by LM. The sequence-based approach allows for a much more graduated insight into the taxonomic diversity of the environmental samples. Taxa retrieval varies considerably throughout the river system, depending on species occurrences and the taxonomic depth of the reference databases. Mostly rare taxa from oligotrophic parts of the river systems are less well represented in the reference database used. A workflow for DNA-based NGS diatom identification is presented. 28 000 diatom sequences were evaluated. Our findings provide evidence that metabarcoding of diatoms via NGS sequencing of the V4 region (18S) has a great potential for water quality assessments and could complement and maybe even improve the identification via light microscopy.

Status and prospects of DNA barcoding in medically important parasites and vectors.

For over 10 years, DNA barcoding has been used to identify specimens and discern species. Its potential benefits in parasitology were recognized early, but its utility and uptake remain unclear. Here we review studies using DNA barcoding in parasites and vectors affecting humans and find that the technique is accurate (accords with author identifications based on morphology or other markers) in 94-95% of cases, although aspects of DNA barcoding (vouchering, marker implicated) have often been misunderstood. In a newly compiled checklist of parasites, vectors, and hazards, barcodes are available for 43% of all 1403 species and for more than half of 429 species of greater medical importance. This is encouraging coverage that would improve with an active campaign targeting parasites and vectors.

Feasibility of using microbeads with holographic barcodes to track DNA specimens in the clinical molecular laboratory.

We demonstrate the feasibility of using glass microbeads with a holographic barcode identifier to track DNA specimens in the molecular pathology laboratory. These beads can be added to peripheral blood specimens and are carried through automated DNA extraction protocols that use magnetic glass particles. We found that an adequate number of microbeads are consistently carried over during genomic DNA extraction to allow specimen identification, that the beads do not interfere with the performance of several different molecular assays, and that the beads and genomic DNA remain stable when stored together under regular storage conditions in the molecular pathology laboratory. The beads function as an internal, easily readable specimen barcode. This approach may be useful for identifying DNA specimens and reducing errors associated with molecular laboratory testing.