A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

The Diversity of Parasitoids and Their Role in the Control of the Siberian Moth, Dendrolimus sibiricus (Lepidoptera: Lasiocampidae), a Major Coniferous Pest in Northern Asia.

The Siberian moth, Dendrolimus sibiricus Tschetv., 1908 (Lepidoptera: Lasiocampidae) is a conifer pest that causes unprecedented forest mortality in Northern Asia, leading to enormous ecological and economic losses. This is the first study summarizing data on the parasitoid diversity and parasitism of this pest over the last 118 years (1905-2022). Based on 860 specimens of freshly reared and archival parasitoids, 16 species from two orders (Hymenoptera and Diptera) were identified morphologically and/or with the use of DNA barcoding. For all of them, data on distribution and hosts and images of parasitoid adults are provided. Among them, the braconid species, Meteorus versicolor (Wesmael, 1835), was documented as a parasitoid of D. sibiricus for the first time. The eastern Palaearctic form, Aleiodes esenbeckii (Hartig, 1838) dendrolimi (Matsumura, 1926), status nov., was resurrected from synonymy as a valid subspecies, and a key for its differentiation from the western Palaearctic subspecies Aleiodes esenbeckii ssp. esenbecki is provided. DNA barcodes of 11 parasitoid species from Siberia, i.e., nine hymenopterans and two dipterans, represented novel records and can be used for accurate molecular genetic identification of species. An exhaustive checklist of parasitoids accounting for 93 species associated with D. sibirisus in northern Asia was compiled. Finally, the literature and original data on parasitism in D. sibiricus populations for the last 83 years (1940-2022) were analysed taking into account the pest population dynamics (i.e., growth, outbreak, decline, and depression phases). A gradual time-lagged increase in egg and pupal parasitism in D. sibiricus populations was detected, with a peak in the pest decline phase. According to long-term observations, the following species are able to cause significant mortality of D. sibiricus in Northern Asia: the hymenopteran egg parasitoids Telenomus tetratomus and Ooencyrtus pinicolus; the larval parasitoids Aleiodes esenbeckii sp. dendrolimi, Cotesia spp., and Glyptapanteles liparidis; and the dipteran pupal parasitoids Masicera sphingivora, Tachina sp., and Blepharipa sp. Their potential should be further explored in order to develop biocontrol programs for this important forest pest.

Taxonomic Structure and Wing Pattern Evolution in the Parnassius mnemosyne Species Complex (Lepidoptera, Papilionidae).

In our study, using the analysis of DNA barcodes and morphology (wing color, male genitalia, and female sphragis shape), we show that the group of species close to P. mnemosyne comprises the western and eastern phylogenetic lineages. The eastern lineage includes P. stubbendorfii, P. glacialis, and P. hoenei. The western lineage includes three morphologically similar species: P. mnemosyne (Western Eurasia), P. turatii (southwestern Europe), and P. nubilosusstat. nov. (Turkmenistan and NE Iran), as well as the morphologically differentiated P. ariadne (Altai). The latter species differs from the rest of the group in the presence of red spots on the wings. Parnassius mnemosyne s.s. is represented by four differentiated mitochondrial clusters that show clear association with specific geographic regions. We propose to interpret them as subspecies: P. mnemosyne mnemosyne (Central and Eastern Europe, N Caucasus, N Turkey), P. mnemosyne adolphi (the Middle East), P. mnemosyne falsa (Tian Shan), and P. mnemosyne gigantea (Gissar-Alai in Central Asia). We demonstrate that in P. ariadne, the red spots on the wing evolved as a reversion to the ancestral wing pattern. This reversion is observed in Altai, where the distribution areas of the western lineage, represented by P. ariadne, and the eastern lineage, represented by P. stubbendorfii, overlap. These two species hybridize in Altai, and we hypothesize that the color change in P. ariadne is the result of reinforcement of prezygotic isolation in the contact zone. The lectotype of Parnassius mnemosyne var. nubilosus Christoph, 1873, is designated.

Local eukaryotic and bacterial stream community assembly is shaped by regional land use effects.

With anticipated expansion of agricultural areas for food production and increasing intensity of pressures stemming from land-use, it is critical to better understand how species respond to land-use change. This is particularly true for microbial communities which provide key ecosystem functions and display fastest responses to environmental change. However, regional land-use effects on local environmental conditions are often neglected, and, hence, underestimated when investigating community responses. Here we show that the effects stemming from agricultural and forested land use are strongest reflected in water conductivity, pH and phosphorus concentration, shaping microbial communities and their assembly processes. Using a joint species distribution modelling framework with community data based on metabarcoding, we quantify the contribution of land-use types in determining local environmental variables and uncover the impact of both, land-use, and local environment, on microbial stream communities. We found that community assembly is closely linked to land-use type but that the local environment strongly mediates the effects of land-use, resulting in systematic variation of taxon responses to environmental conditions, depending on their domain (bacteria vs. eukaryote) and trophic mode (autotrophy vs. heterotrophy). Given that regional land-use type strongly shapes local environments, it is paramount to consider its key role in shaping local stream communities.

Trait-mediated responses of caterpillar communities to spongy moth outbreaks and subsequent tebufenozide treatments.

Outbreaks of the spongy moth Lymantria dispar can have devastating impacts on forest resources and ecosystems. Lepidoptera-specific insecticides, such as Bacillus thuringiensis var. kurstaki (BTK) and tebufenozide, are often deployed to prevent heavy defoliation of the forest canopy. While it has been suggested that using BTK poses less risk to non-target Lepidoptera than leaving an outbreak untreated, in situ testing of this assumption has been impeded by methodological challenges. The trade-offs between insecticide use and outbreaks have yet to be addressed for tebufenozide, which is believed to have stronger side effects than BTK. We investigated the short-term trade-offs between tebufenozide treatments and no-action strategies for the non-target herbivore community in forest canopies. Over 3 years, Lepidoptera and Symphyta larvae were sampled by canopy fogging in 48 oak stands in southeast Germany during and after a spongy moth outbreak. Half of the sites were treated with tebufenozide and changes in canopy cover were monitored. We contrasted the impacts of tebufenozide and defoliator outbreaks on the abundance, diversity, and functional structure of chewing herbivore communities. Tebufenozide treatments strongly reduced Lepidoptera up to 6 weeks after spraying. Populations gradually converged back to control levels after 2 years. Shelter-building species dominated caterpillar assemblages in treated plots in the post-spray weeks, while flight-dimorphic species were slow to recover and remained underrepresented in treated stands 2 years post-treatment. Spongy moth outbreaks had minor effects on leaf chewer communities. Summer Lepidoptera decreased only when severe defoliation occurred, whereas Symphyta declined 1 year after defoliation. Polyphagous species with only partial host plant overlap with the spongy moth were absent from heavily defoliated sites, suggesting greater sensitivity of generalists to defoliation-induced plant responses. These results demonstrate that both tebufenozide treatments and spongy moth outbreaks alter canopy herbivore communities. Tebufenozide had a stronger and longer lasting impact, but it was restricted to Lepidoptera, whereas the outbreak affected both Lepidoptera and Symphyta. These results are tied to the fact that only half of the outbreak sites experienced severe defoliation. This highlights the limited accuracy of current defoliation forecast methods, which are used as the basis for the decision to spray insecticides.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

A workflow for expanding DNA barcode reference libraries through 'museum harvesting' of natural history collections.

Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society - through, for example, digitisation or the construction of a validated, global DNA barcode library - is of crucial importance. To this end, we developed and streamlined a workflow for 'museum harvesting' of authoritatively identified Diptera specimens from the Smithsonian Institution's National Museum of Natural History. Our detailed workflow includes both on-site and off-site processing through specimen selection, labelling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly-generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data Systems.

Tracing the invasion of a leaf-mining moth in the Palearctic through DNA barcoding of historical herbaria.

The lime leaf-miner, Phyllonorycter issikii is an invasive micromoth with an unusually higher number of haplotypes in the invaded area (Europe, Western Siberia) compared to its putative native region (East Asia). The origin of the genetic diversity in the neocolonized region remains unclear. We surveyed over 15 thousand herbarium specimens of lime trees (Tilia spp.) collected across the Palearctic over a period of 252 years (1764-2016) looking for preserved larvae within the archival leaf mines. We found 203 herbarium specimens with leaf mines of Ph. issikii collected in East Asia, one of them dating back to 1830, i.e. 133 years before the description of the species. In contrast, only 22 herbarium specimens collected in the West Palearctic in the last three decades (1987-2015) carried leaf mines. DNA barcoding of archival specimens revealed 32 haplotypes out of which 23 were novel (not known from modern populations) and found exclusively in East Asia. Six haplotypes are shared between both native and invaded areas and only two were responsible for the recent invasion of the Western Palearctic. The remarkable number of newly discovered haplotypes in archival populations supports East Asia as the native region and the source area of invasion.

A DNA barcode library for the butterflies of North America.

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.

Torix Rickettsia are widespread in arthropods and reflect a neglected symbiosis.

BACKGROUND: Rickettsia are intracellular bacteria best known as the causative agents of human and animal diseases. Although these medically important Rickettsia are often transmitted via haematophagous arthropods, other Rickettsia, such as those in the Torix group, appear to reside exclusively in invertebrates and protists with no secondary vertebrate host. Importantly, little is known about the diversity or host range of Torix group Rickettsia. RESULTS: This study describes the serendipitous discovery of Rickettsia amplicons in the Barcode of Life Data System (BOLD), a sequence database specifically designed for the curation of mitochondrial DNA barcodes. Of 184,585 barcode sequences analysed, Rickettsia is observed in ∼0.41% of barcode submissions and is more likely to be found than Wolbachia (0.17%). The Torix group of Rickettsia are shown to account for 95% of all unintended amplifications from the genus. A further targeted PCR screen of 1,612 individuals from 169 terrestrial and aquatic invertebrate species identified mostly Torix strains and supports the "aquatic hot spot" hypothesis for Torix infection. Furthermore, the analysis of 1,341 SRA deposits indicates that Torix infections represent a significant proportion of all Rickettsia symbioses found in arthropod genome projects. CONCLUSIONS: This study supports a previous hypothesis that suggests that Torix Rickettsia are overrepresented in aquatic insects. In addition, multiple methods reveal further putative hot spots of Torix Rickettsia infection, including in phloem-feeding bugs, parasitoid wasps, spiders, and vectors of disease. The unknown host effects and transmission strategies of these endosymbionts make these newly discovered associations important to inform future directions of investigation involving the understudied Torix Rickettsia.

Temporal Detection Limits of Remnant Larval Bloodmeals in Nymphal Ixodes scapularis (Say, Ixodida: Ixodidae) Using Two Next-Generation Sequencing DNA Barcoding Assays.

Using next-generation sequencing DNA barcoding, we aimed to determine: 1) if the larval bloodmeal can be detected in Ixodes scapularis nymphs and 2) the post-moult temporal window for detection of the larval bloodmeal. Subsets of 30 nymphs fed on a domestic rabbit (Oryctolagus cuniculus Linnaeus, Lagomorphia: Leporidae) as larvae were reared and frozen at 11 time points post-moult, up to 150 d. Vertebrate DNA was amplified using novel universal (UP) and species-specific primers (SSP) and sequenced for comparison against cytochrome c oxidase subunit I barcodes to infer host identification. Detectable bloodmeals decreased as time since moult increased for both assays. For the SSP assay, detection of bloodmeals decreased from 96.7% (n = 29/30) in day 0 nymphs to 3.3% (n = 1/30) and 6.7% (n = 2/30) at 4- and 5-mo post-moult, respectively. A shorter temporal detection period was achieved with the UP assay, declining from 16.7% (n = 5/30) in day 0 nymphs to 0/30 in 3-d-old nymphs. Bloodmeal detection was nonexistent for the remaining cohorts, with the exception of 1/30 nymphs at 2-mo post-moult. Host detection was significantly more likely using the SSP assay compared to the UP assay in the first three time cohorts (day 0: χ 2 = 39.1, P < 0.005; day 2: χ 2 = 19.2, P < 0.005; day 3: χ 2 = 23.3, P < 0.005). Regardless of the primer set used, the next-generation sequencing DNA barcoding assay was able to detect host DNA from a larval bloodmeal in the nymphal life stage; however, a short window with a high proportion of detection post-moult was achieved.

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.

Validation of COI metabarcoding primers for terrestrial arthropods.

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).

Metabarcoding a diverse arthropod mock community.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.

Characterization and comparison of poorly known moth communities through DNA barcoding in two Afrotropical environments in Gabon 1.

Biodiversity research in tropical ecosystems-popularized as the most biodiverse habitats on Earth-often neglects invertebrates, yet invertebrates represent the bulk of local species richness. Insect communities in particular remain strongly impeded by both Linnaean and Wallacean shortfalls, and identifying species often remains a formidable challenge inhibiting the use of these organisms as indicators for ecological and conservation studies. Here we use DNA barcoding as an alternative to the traditional taxonomic approach for characterizing and comparing the diversity of moth communities in two different ecosystems in Gabon. Though sampling remains very incomplete, as evidenced by the high proportion (59%) of species represented by singletons, our results reveal an outstanding diversity. With about 3500 specimens sequenced and representing 1385 BINs (Barcode Index Numbers, used as a proxy to species) in 23 families, the diversity of moths in the two sites sampled is higher than the current number of species listed for the entire country, highlighting the huge gap in biodiversity knowledge for this country. Both seasonal and spatial turnovers are strikingly high (18.3% of BINs shared between seasons, and 13.3% between sites) and draw attention to the need to account for these when running regional surveys. Our results also highlight the richness and singularity of savannah environments and emphasize the status of Central African ecosystems as hotspots of biodiversity.

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding 1.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21 194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds.

PREMISE OF THE STUDY: The detection of environmental DNA (eDNA) using high-throughput sequencing has rapidly emerged as a method to detect organisms from environmental samples. However, eDNA studies of aquatic biomes have focused on surveillance of animal species with less emphasis on plants. Pondweeds are important bioindicators of freshwater ecosystems, although their diversity is underestimated due to difficulties in morphological identification and monitoring. METHODS: A protocol was developed to detect pondweeds in water samples using atpB-rbcL and ITS2 markers. The water samples were collected from the Grand River within the rare Charitable Research Reserve, Ontario (RARE). Short fragments were amplified using primers targeting pondweeds, sequenced on an Ion Torrent Personal Genome Machine, and assigned to the taxonomy using a local DNA reference library and GenBank. RESULTS: We detected two species earlier documented at the experimental site during ecological surveys (Potamogeton crispus and Stuckenia pectinata) and three species new to the RARE checklist (P. foliosus, S. filiformis, and Zannichellia palustris). DISCUSSION: Our targeted approach to track the species composition of pondweeds in freshwater ecosystems revealed underestimation of their diversity. This result suggests that eDNA is an effective tool for monitoring plant diversity in aquatic habitats.

A Sequel to Sanger: amplicon sequencing that scales.

BACKGROUND: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. RESULTS: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). CONCLUSIONS: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America.

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences.

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.