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.

No abstract

For the first time, a nearly complete barcode library for European Gelechiidae is provided. DNA barcode sequences (COI gene – cytochrome c oxidase 1) from 751 out of 865 nominal species, belonging to 105 genera, were successfully recovered. A total of 741 species represented by specimens with sequences ≥ 500bp and an additional ten species represented by specimens with shorter sequences were used to produce 53 NJ trees. Intraspecific barcode divergence averaged only 0.54% whereas distance to the Nearest-Neighbour species averaged 5.58%. Of these, 710 species possessed unique DNA barcodes, but 31 species could not be reliably discriminated because of barcode sharing or partial barcode overlap. Species discrimination based on the Barcode Index System (BIN) was successful for 668 out of 723 species which clustered from minimum one to maximum 22 unique BINs. Fifty-five species shared a BIN with up to four species and identification from DNA barcode data is uncertain. Finally, 65 clusters with a unique BIN remained unidentified to species level. These putative taxa, as well as 114 nominal species with more than one BIN, suggest the presence of considerable cryptic diversity, cases which should be examined in future revisionary studies.

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species-level taxonomy. We found five COI sub-clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub-clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.

DNA barcodes of European tortricid moths identified as Epinotia nisella (Clerck, 1759) were found to comprise two ge-netically distinct clusters. These coincided with E. nisella and E. cinereana (Haworth, 1811) (sp. rev.), the latter havingbeen considered a synonym of the former for several decades. Comparing these DNA barcodes with those of North Amer-ican Epinotia showed that both species are Holarctic. The North American Proteopteryx criddleana Kearfott, 1907 is anew junior synonym of E. cinereana (syn. nov.). The two species also show distinct differences in male and female gen-italia. North American populations of both species show moderate differences in barcodes from their respective Europeanpopulations but there are no morphological differences correlated with the intraspecific barcode clusters. Tortrix petranaHübner, 1813 is considered as a junior synonym of E. cinereana (syn. rev.). Epinotia nisella has several synonyms reflect-ing its highly variable forewing colour pattern. We discuss its variation and the status of the names associated with it. The biology of both species in Europe and North America is summarized. Adults and genitalia are illustrated.