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Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups-dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus-were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or 'barcoding', can be used to identify fish species.

The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon-indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, the morphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies.

With almost 40 000 species, the spiders provide important model systems for studies of sociality, mating systems, and sexual dimorphism. However, work on this group is regularly constrained by difficulties in species identification. DNA-based identification systems represent a promising approach to resolve this taxonomic impediment, but their efficacy has only been tested in a few groups. In this study, we demonstrate that sequence diversity in a standard segment of the mitochondrial gene coding for cytochrome c oxidase I (COI) is highly effective in discriminating spider species. A COI profile containing 168 spider species and 35 other arachnid species correctly assigned 100% of subsequently analyzed specimens to the appropriate species. In addition, we found no overlap between mean nucleotide divergences at the intra- and inter-specific levels. Our results establish the potential of COI as a rapid and accurate identification tool for biodiversity surveys of spiders.

The study of environmental DNA (eDNA) has the potential to revolutionize biodiversity science and conservation action by enabling the census of species on a global scale in near real time. To achieve this promise, technical challenges must be resolved. In this review, we explore the main uses of eDNA as well as the complexities introduced by its misuse. Current eDNA methods require refinement and improved calibration and validation along the entire workflow to lessen false positives negatives. Moreover, there is great need for a better understanding of the \"natural history\" of eDNA-its origins, state, lifetime, and transportation-and for more detailed insights concerning the physical and ecological limitations of eDNA use. Although eDNA analysis can provide powerful information, particularly in freshwater and marine environments, its impact is likely to be less significant in terrestrial settings. The broad adoption of eDNA tools in conservation will largely depend on addressing current uncertainties in data interpretation.

Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified.

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.

Although biomass values are critical for diverse ecological and evolutionary analyses, they are unavailable for most insect species. Museum specimens have the potential to address this gap, but the variation introduced by sampling and preservation methods is uncertain. This study quantifies species-level variation in the body mass of Canadian Coleoptera based on the analysis of 3,744 specimens representing 3,161 Barcode Index Number (BIN) clusters. Employing the BIN system as a proxy for species allows the inclusion of groups where the taxonomic impediment prevents the assignment of specimens to a Linnaean species. By validating the reproducibility of measurements and evaluating the error introduced by operational complexities such as curatorial practice and the loss of body parts, this study demonstrates that museum specimens can speed the assembly of a mass registry. The results further indicate that congeneric species of Coleoptera generally have limited variation in mass, so a genus-level identification allows prediction of the body mass of species that have not been weighed or measured. Building on the present results, the construction of a mass registry for all insects is feasible.

We evaluated sequence diversity in the mitochondrial cytochrome-c oxidase I (COI; EC 1.9.3.1) gene as a tool for resolving differences among species of Arctic springtails. The Collembola examined in this analysis were collected from Igloolik, Cornwallis, and Somerset islands and included representatives from all major families found in the Arctic. Members of 13 genera and 19 species were examined, including 4 species of the genus Folsomia and 3 species of the genus Hypogastrura. In all cases, species were successfully discriminated. Sequence divergences within species were generally less than 1%, whereas divergences between species were greater than 8% in all cases. Divergences among individuals of one species of Folsomia were much higher (up to 13%), but this likely represents the presence of an undescribed sibling species. We conclude that DNA barcoding is a powerful tool for identifying species of Collembola and should regularly be useful as a complement to traditional, morphological taxonomy.

While phylogeographic structure has been examined in many North American vertebrate species, insects have received much less attention despite their central ecological roles. The moth genus Malacosoma (Hübner, 1820), is an important group of forestry pests responsible for large-scale defoliation across much of the Nearctic and Palearctic. The present study uses sequence variation in the mitochondrial cytochrome c oxidase 1 (COI) gene to examine the population genetic structure of the three widespread Malacosoma species ( M. americana , M. californica , and M. disstria ). Populations of all three species showed highest diversity in the south, suggesting that modern populations derived from southern refugia with loss of variation as these lineages dispersed northwards. However, despite similar life histories and dispersal abilities, the extent of regional variation varied among the taxa. M. americana, a species restricted to eastern North America, showed much less genetic structure than the western M. californica or the widespread M. disstria . The regional differentiation in the latter reflects the likely derivation of modern lineages from several refugia, as well as taxonomic uncertainty in M. californica . In these respects, the three species of Malacosoma share phylogeographic patterns similar to those detected in vertebrates which are characterised by greater phylogeographic breaks in the western half of the continent and limited structure in the east.