Species-Level Para- and Polyphyly in DNA Barcode Gene Trees: Strong Operational Bias in European Lepidoptera.

The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer" to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric-conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.

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.

Measuring mass: variation among 3,161 species of Canadian Coleoptera and the prospects of a mass registry for all insects.

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.

A molecular-based identification resource for the arthropods of Finland.

To associate specimens identified by molecular characters to other biological knowledge, we need reference sequences annotated by Linnaean taxonomy. In this study, we (1) report the creation of a comprehensive reference library of DNA barcodes for the arthropods of an entire country (Finland), (2) publish this library, and (3) deliver a new identification tool for insects and spiders, as based on this resource. The reference library contains mtDNA COI barcodes for 11,275 (43%) of 26,437 arthropod species known from Finland, including 10,811 (45%) of 23,956 insect species. To quantify the improvement in identification accuracy enabled by the current reference library, we ran 1000 Finnish insect and spider species through the Barcode of Life Data system (BOLD) identification engine. Of these, 91% were correctly assigned to a unique species when compared to the new reference library alone, 85% were correctly identified when compared to BOLD with the new material included, and 75% with the new material excluded. To capitalize on this resource, we used the new reference material to train a probabilistic taxonomic assignment tool, FinPROTAX, scoring high success. For the full-length barcode region, the accuracy of taxonomic assignments at the level of classes, orders, families, subfamilies, tribes, genera, and species reached 99.9%, 99.9%, 99.8%, 99.7%, 99.4%, 96.8%, and 88.5%, respectively. The FinBOL arthropod reference library and FinPROTAX are available through the Finnish Biodiversity Information Facility (www.laji.fi) at https://laji.fi/en/theme/protax. Overall, the FinBOL investment represents a massive capacity-transfer from the taxonomic community of Finland to all sectors of society.

Integrative taxonomy of Nearctic and Palaearctic Aleocharinae: new species, synonymies, and records (Coleoptera, Staphylinidae).

A long tradition of separate Nearctic and Palaearctic taxonomic studies of the diverse aleocharine rove beetles (Coleoptera: Staphylinidae) has obscured the recognition of Holarctic species and detection of adventive species in both regions. Recently, integrated study of the two regions through detailed morphological comparisons and development of an authoritatively identified DNA barcode reference library has revealed the degree to which these two aleocharine faunas are interconnected, both naturally and through human activity. Here this approach is adopted to recognize new species, reveal Holarctic species, and recognize adventive species in both North America and Europe. The following new species are described: Isoglossa triangularis Klimaszewski, Brunke & Pentinsaari, sp. nov. from British Columbia; Gnypeta impressicollis Klimaszewski, Brunke & Pentinsaari, sp. nov., from Ontario, Maryland and North Carolina; Aloconota pseudogregaria Klimaszewski, Brunke & Pentinsaari, sp. nov., from Ontario and Virginia; and Philhygra pseudolaevicollis Klimaszewski, Brunke & Pentinsaari, sp. nov. from eastern Canada. Dasygnypeta velata and Philhygra angusticauda are revealed to be Holarctic species, resulting in the following synonymies: Dasygnypeta velata (Erichson, 1839) = Gnypeta minuta Klimaszewski & Webster, 2008, syn. nov. and Philhygra angusticauda (Bernhauer, 1909) = Atheta (Philhygra) pinegensis Muona, 1983, syn. nov. The Nearctic species Hylota ochracea (and genus Hylota), Thecturota tenuissima, and Trichiusa robustula are newly reported from the Palaearctic region as adventive, resulting in the following synonymies: Hylota ochracea Casey, 1906 = Stichoglossa (Dexiogyia) forticornis Strand, 1939, syn. nov.; Thecturota tenuissima Casey, 1893 = Atheta marchii Dodero, 1922, syn. nov.; and Trichiusa robustula Casey, 1893 = T. immigrata Lohse, 1984, syn. nov. The Palaearctic species Amarochara forticornis, Anomognathus cuspidatus, Oligota pumilio, and Parocyusa rubicunda are newly confirmed from the Nearctic region as adventive, resulting in the following synonymies: Parocyusa rubicunda (Erichson, 1837) = Chilopora americana Casey, 1906, syn. nov. and Anomognathus cuspidatus (Erichson, 1839) = Thectura americana Casey, 1893, syn. nov. The genus Dasygnypeta, sensu nov. is newly reported from North America, Paradilacra is newly reported from eastern North America, and Haploglossa is newly reported from Canada, resulting in the following synonymy: Paradilacra densissima (Bernhauer, 1909) = Gnypeta saccharina Klimaszewski & Webster, 2008, syn. nov. Native Cyphea wallisi is newly reported from across Canada and C. curtula is removed from the Nearctic fauna. The status of both Gyrophaena affinis and Homalota plana is uncertain but these species are no longer considered to be adventive in North America. Three new combinations are proposed: Dasygnypeta baranowskii (Klimaszewski, 2020) and D. nigrella (LeConte, 1863) (both from Gnypeta) and Mocyta scopula (Casey, 1893) (from Acrotona). Dolosota Casey, 1910, syn. nov. (type species Eurypronota scopula Casey), currently a subgenus of Acrotona, is therefore synonymized with Mocyta Mulsant & Rey, 1874. Additionally, four new Canadian records and 18 new provincial and state records are reported.

BOLD and GenBank revisited - Do identification errors arise in the lab or in the sequence libraries?

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.

A DNA Barcoding Survey of an Arctic Arthropod Community: Implications for Future Monitoring.

Accurate and cost-effective methods for tracking changes in arthropod communities are needed to develop integrative environmental monitoring programs in the Arctic. To date, even baseline data on their species composition at established ecological monitoring sites are severely lacking. We present the results of a pilot assessment of non-marine arthropod diversity in a middle arctic tundra area near Ikaluktutiak (Cambridge Bay), Victoria Island, Nunavut, undertaken in 2018 using DNA barcodes. A total of 1264 Barcode Index Number (BIN) clusters, used as a proxy for species, were recorded. The efficacy of widely used sampling methods was assessed. Yellow pan traps captured 62% of the entire BIN diversity at the study sites. When complemented with soil and leaf litter sifting, the coverage rose up to 74.6%. Combining community-based data collection with high-throughput DNA barcoding has the potential to overcome many of the logistic, financial, and taxonomic obstacles for large-scale monitoring of the Arctic arthropod fauna.

Coleoptera of Canada.

The beetle fauna of Canada was assessed, including estimates of yet unreported diversity using information from taxonomists and COI sequence clusters in a BOLD (Barcode of Life Datasystems) COI dataset comprising over 77,000 Canadian records. To date, 8302 species of Coleoptera have been recorded in Canada, a 23% increase from the first assessment in 1979. A total of 639 non-native beetle species have become established in Canada, with most species in the Staphylinidae (153 spp.), Curculionidae (107 spp.), Chrysomelidae (56 spp.) and Carabidae (55 spp.). Based on estimates from the taxonomic community and our BOLD dataset, we estimate that slightly more than 1000 beetle species remain to be reported from Canada, either as new records or undescribed species. Renewed enthusiasm toward and financial support for surveys, especially in the central and western provinces of Canada will be critical for detecting, documenting and describing these species. The Barcode of Life database is still far from comprehensive for Canadian Coleoptera but substantial progress has been made and the number of Barcode Index Numbers (BINs) (as candidate species) has reached nearly 70% of the number of species reported from Canada. Comparison of BINs to observed species in a group of Canadian Staphylinidae suggests that BINs may provide a good estimate of species diversity within the beetles. Histeridae is a diverse family in Canada that is notably underrepresented in BOLD. Families such as Mordellidae, Scraptiidae, Latridiidae, Ptiliidae and Scirtidae are poorly known taxonomically in Canada and are represented in our BOLD dataset by many more BINs than recorded species.

DNA barcodes reveal 63 overlooked species of Canadian beetles (Insecta, Coleoptera).

This study demonstrates the power of DNA barcoding to detect overlooked and newly arrived taxa. Sixty-three species of Coleoptera representing 25 families are studied based on DNA barcode data and morphological analysis of the barcoded specimens. Three of the species involve synonymies or previous taxonomic confusion in North America, while the first Canadian records are published for 60 species. Forty-two species are adventive in North America, and 40 of these adventive species originate from the Palaearctic region. Three genera are recorded from the Nearctic region for the first time: Coelostoma Brullé, 1835 (Hydrophilidae), Scydmoraphes Reitter, 1891 (Staphylinidae), and Lythraria Bedel, 1897 (Chrysomelidae). Two new synonymies are established: Mycetoporus triangulatus Campbell, 1991 (Staphylinidae) is a junior synonym of Mycetoporus reichei Pandellé, 1869, syn. nov. while Bledius philadelphicus Fall, 1919 (Staphylinidae) is a junior synonym of Bledius gallicus (Gravenhorst, 1806), syn. nov. The previously suggested move of Ctenicera tigrina (Fall, 1901) to the genus Pseudanostirus Dolin, 1964 (Elateridae) is formalized, resulting in Pseudanostirus tigrinus (Fall, 1901), comb. nov.

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.

Algorithmic single-locus species delimitation: effects of sampling effort, variation and nonmonophyly in four methods and 1870 species of beetles.

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode-based OTU-picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.

Molecular evolution of a widely-adopted taxonomic marker (COI) across the animal tree of life.

DNA barcodes are widely used for identification and discovery of species. While such use draws on information at the DNA level, the current amassment of ca. 4.7 million COI barcodes also offers a unique resource for exploring functional constraints on DNA evolution. Here, we explore amino acid variation in a crosscut of the entire animal kingdom. Patterns of DNA variation were linked to functional constraints at the level of the amino acid sequence in functionally important parts of the enzyme. Six amino acid sites show variation with possible effects on enzyme function. Overall, patterns of amino acid variation suggest convergent or parallel evolution at the protein level connected to the transition into a parasitic life style. Denser sampling of two diverse insect taxa revealed that the beetles (Coleoptera) show more amino acid variation than the butterflies and moths (Lepidoptera), indicating fundamental difference in patterns of molecular evolution in COI. Several amino acid sites were found to be under notably strong purifying selection in Lepidoptera as compared to Coleoptera. Overall, these findings demonstrate the utility of the global DNA barcode library to extend far beyond identification and taxonomy, and will hopefully be followed by a multitude of work.

Barcoding beetles: a regional survey of 1872 species reveals high identification success and unusually deep interspecific divergences.

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.