Despite the broad benefits that DNA barcoding can bring to a diverse range of biological disciplines, a number of shortcomings still exist in terms of the experimental design of studies incorporating this approach. One underlying reason for this lies in the confusion that often exists between species discovery and specimen identification, and this is reflected in the way that hypotheses are generated and tested. Although these aims can be associated, they are quite distinct and require different methodological approaches, but their conflation has led to the frequently inappropriate use of commonly used analytical methods such as neighbour-joining trees, bootstrap resampling and fixed distance thresholds. Furthermore, the misidentification of voucher specimens can also have serious implications for end users of reference libraries such as the Barcode of Life Data Systems, and in this regard we advocate increased diligence in the a priori identification of specimens to be used for this purpose. This commentary provides an assessment of seven deficiencies that we identify as common in the DNA barcoding literature, and outline some potential improvements for its adaptation and adoption towards more reliable and accurate outcomes.

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).

Metabarcoding extra‐organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci – COI, cytochrome b, 12S and 16S – in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs – three COI and one 12S – for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra‐organismal DNA at low template concentrations, both metazoan‐targeted and fish‐targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non‐specific amplification of prokaryotic and non‐target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome‐skimming existing tissue collections.

As environmental DNA (eDNA) becomes an increasingly valuable resource for marine ecosystem monitoring, understanding variation in its persistence across contrasting environments is critical. Here, we quantify the breakdown of macrobial eDNA over a spatio-temporal axis of locally extreme conditions, varying from ocean-influenced offshore to urban-inshore, and between winter and summer. We report that eDNA degrades 1.6 times faster in the inshore environment than the offshore environment, but contrary to expectation we find no difference over season. Analysis of environmental covariables show a spatial gradient of salinity and a temporal gradient of pH, with salinity—or the biotic correlates thereof—most important. Based on our estimated inshore eDNA half-life and naturally occurring eDNA concentrations, we estimate that eDNA may be detected for around 48 h, offering potential to collect ecological community data of high local fidelity. We conclude by placing these results in the context of previously published eDNA decay rates.

BackgroundPoorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species.Methodology/Principal FindingsWe sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90–99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii.Conclusions/SignificanceWe demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.