A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.

The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658-bp barcode fragment from the 5′-end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life-cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well-differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life-cycle characteristics, indicating a need for further taxonomic analysis.

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided.

Pentalonia nigronervosa (sensu Hardy 1931) samples from banana and from Zingiberaceae and Araceae species exhibit fixed differences in DNA sequence in mitochondrial cytochrome oxidase subunit 1 (“DNA barcode”) and in the nuclear gene elongation factor 1α, and have morphometric differences, including non-overlapping ranges in the length of the distal rostral segment. It is thus proposed that the name P. nigronervosa Coquerel be restricted to banana-feeding ‘nigronervosa’ specimens, and that the name Pentalonia caladii van der Goot be restored to full species status for specimens typically feeding on Zingiberaceae and Araceae.

Morphometric techniques, DNA mitochondrial cytochrome c oxidase subunit 1 gene (COI) barcoding, and microsatellite flanking region sequences were used to assess the reliability of suggested morphological characters in distinguishing the green apple aphid (Aphis pomi De Geer) from the spirea aphid (Aphis spiraecola Patch), and to assess variation within these species. Both molecular approaches clearly distinguished two groups corresponding to the morphologically defined species. Differences in the length of the distal rostral segment and the number of lateral tubercles were found to be robust indicators of species membership, performing as well as multivariate approaches. Among A. pomi samples, microsatellite flanking region sequences were relatively uniform, whereas A. spiraecola exhibited much variability, which suggests that North American populations of the latter species are genetically much more complex.Résumé-Des techniques morphométriques et l'utilisation de codes à barres d'ADN de COI et de séquences de la région flanquante des microsatellites nous ont servi à évaluer les caractères morphologiques qu'on a proposés pour distinguer le puceron vert du pommier (Aphis pomi De Geer) du puceron de la spirée (Aphis spiraecola Patch), et à déterminer la variation au sein de ces espèces. Les deux approches moléculaires permettent de distinguer clairement les deux groupes qui correspondent aux espèces définies morphologiquement. Les différences dans la longueur du segment distal du rostre et le nombre de tubercules latéraux sont des indicateurs robustes de l'identité spécifique; l'utilisation de ces caractères fonctionne aussi bien que les méthodes multidimensionnelles. Les séquences de la région flanquante des microsatellites sont relativement uniformes chez A. pomi, mais elles sont très variables chez A. spiraecola, ce qui laisse croire que les populations nord-américaines de cette dernière espèce sont génétiquement beaucoup plus complexes.[Traduit par la Rédaction]