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Because many animal species are undescribed, and because the identification of known species is often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By assigning individuals to presumptive species, called operational taxonomic units (OTUs), these systems speed investigations into the patterning of biodiversity and enable studies that would otherwise be impossible. Although OTUs have conventionally been separated through their morphological divergence, DNA-based delineations are not only feasible, but have important advantages. OTU designation can be automated, data can be readily archived, and results can be easily compared among investigations. This study exploits these attributes to develop a persistent, species-level taxonomic registry for the animal kingdom based on the analysis of patterns of nucleotide variation in the barcode region of the cytochrome c oxidase I (COI) gene. It begins by examining the correspondence between groups of specimens identified to a species through prior taxonomic work and those inferred from the analysis of COI sequence variation using one new (RESL) and four established (ABGD, CROP, GMYC, jMOTU) algorithms. It subsequently describes the implementation, and structural attributes of the Barcode Index Number (BIN) system. Aside from a pragmatic role in biodiversity assessments, BINs will aid revisionary taxonomy by flagging possible cases of synonymy, and by collating geographical information, descriptive metadata, and images for specimens that are likely to belong to the same species, even if it is undescribed. More than 274,000 BIN web pages are now available, creating a biodiversity resource that is positioned for rapid growth.

Applications of biological knowledge, such as forensics, often require the determination of biological materials to a species level. As such, DNA-based approaches to identification, particularly DNA barcoding, are attracting increased interest. The capacity of DNA barcodes to assign newly encountered specimens to a species relies upon access to informatics platforms, such as BOLD and GenBank, which host libraries of reference sequences and support the comparison of new sequences to them. As parameterization of these libraries expands, DNA barcoding has the potential to make valuable contributions in diverse applied contexts. However, a recent publication called for caution after finding that both platforms performed poorly in identifying specimens of 17 common insect species. This study follows up on this concern by asking if the misidentifications reflected problems in the reference libraries or in the query sequences used to test them. Because this reanalysis revealed that missteps in acquiring and analyzing the query sequences were responsible for most misidentifications, a workflow is described to minimize such errors in future investigations. The present study also revealed the limitations imposed by the lack of a polished species-level taxonomy for many groups. In such cases, applications can be strengthened by mapping the geographic distributions of sequence-based species proxies rather than waiting for the maturation of formal taxonomic systems based on morphology.

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.

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area. This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0-26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%-46.5%), and showed a significant positive correlation with nearest neighbour distances. DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.

With 400 K described species, beetles (Insecta: Coleoptera) represent the most diverse order in the animal kingdom. Although the study of their diversity currently represents a major challenge, DNA barcodes may provide a functional, standardized tool for their identification. To evaluate this possibility, we performed the first comprehensive test of the effectiveness of DNA barcodes as a tool for beetle identification by sequencing the COI barcode region from 1872 North European species. We examined intraspecific divergences, identification success and the effects of sample size on variation observed within and between species. A high proportion (98.3%) of these species possessed distinctive barcode sequence arrays. Moreover, the sequence divergences between nearest neighbor species were considerably higher than those reported for the only other insect order, Lepidoptera, which has seen intensive analysis (11.99% vs up to 5.80% mean NN divergence). Although maximum intraspecific divergence increased and average divergence between nearest neighbors decreased with increasing sampling effort, these trends rarely hampered identification by DNA barcodes due to deep sequence divergences between most species. The Barcode Index Number system in BOLD coincided strongly with known species boundaries with perfect matches between species and BINs in 92.1% of all cases. In addition, DNA barcode analysis revealed the likely occurrence of about 20 overlooked species. The current results indicate that DNA barcodes distinguish species of beetles remarkably well, establishing their potential to provide an effective identification tool for this order and to accelerate the discovery of new beetle species.

This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections.

High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.

The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families. This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species. The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity.

DNA barcoding has proven an effective tool for species identification in varied groups of marine invertebrates including crustaceans, molluscs, polychaetes and echinoderms. In this study, we further validate its utility by analyzing almost half of the 300 species of Echinodermata known from Canadian waters. COI sequences from 999 specimens were assigned to 145 BINs. In most cases, species discrimination was straightforward due to the large difference (25-fold) between mean intra- (0.48%) and inter- (12.0%) specific divergence. Six species were flagged for further taxonomic investigation because specimens assigned to them fell into two or three discrete sequence clusters. The potential influence of larval dispersal capacity and glacial events on patterns of genetic diversity is discussed for 19 trans-oceanic species. Although additional research is needed to clarify biogeographic patterns and resolve taxonomic questions, this study represents an important step in the assembly of a DNA barcode library for all Canadian echinoderms, a valuable resource for future biosurveillance programs.

The nuclear genomes of most animal species include NUMTs, segments of the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study of their frequency/attributes in the most diverse group of terrestrial organisms, insects. This study examines NUMTs derived from a 658 bp 5’ segment of the cytochrome c oxidase I (COI) gene, the barcode region for the animal kingdom. This assessment is important because unrecognized NUMTs can elevate estimates of species richness obtained through DNA barcoding and derived approaches (eDNA, metabarcoding). This investigation detected nearly 10,000 COI NUMTs ≥ 100 bp in the genomes of 1,002 insect species (range = 0–443). Variation in nuclear genome size explained 56% of the mitogenome-wide variation in NUMT counts. Although insect orders with the largest genome sizes possessed the highest NUMT counts, there was considerable variation among their component lineages. Two thirds of COI NUMTs possessed an IPSC (indel and/or premature stop codon) allowing their recognition and exclusion from downstream analyses. The remainder can elevate species richness as they showed 10.1% mean divergence from their mitochondrial homologue. The extent of exposure to “ghost species” is strongly impacted by the target amplicon’s length. NUMTs can raise apparent species richness by up to 22% when a 658 bp COI amplicon is examined versus a doubling of apparent richness when 150 bp amplicons are targeted. Given these impacts, metabarcoding and eDNA studies should target the longest possible amplicons while also avoiding use of 12S/16S rDNA as they triple NUMT exposure because IPSC screens cannot be employed.