We used pheromone-baited traps to survey the distribution of winter moth, Operophtera brumata (L.) (Lepidoptera: Geometridae), a new invasive defoliator from Europe in eastern New England. The traps also attracted Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera: Geometridae), native to North America. We distinguished between the two species by examining male genitalia and sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene, the DNA barcoding region. In 2005, we recovered winter moths at sites stretching from eastern Long Island, southeastern Connecticut, all of Rhode Island, eastern Massachusetts, coastal New Hampshire, and southern coastal Maine. At sites further west and north we captured only Bruce spanworm. In 2006, we confirmed that both winter moth and Bruce spanworm are present in Nova Scotia and in coastal Maine, but only Bruce spanworm was recovered in coastal New Brunswick, Canada; Pennsylvania; Vermont; or Quebec City, Canada. In 2007, we collected Bruce spanworm, but no winter moths, in New Brunswick and the interior areas of Maine, New Hampshire, and New York. Winter moth and Brace spanworm differed in the COI sequence by 7.45% of their nucleotides. The prevalence of intermediate genitalia in the zone of overlap suggested that hybridization between the two species may be occurring. To confirm the presence of hybrids, we sequenced the nuclear gene, glucose-6phosphate dehydrogenase (G6PD). We identified six nucleotides that routinely distinguished winter moth and Bruce spanworm, of which three were always diagnostic. We showed that eggs produced by hybridizing the two species in the laboratory contained copies of both species at these six sites. We found that most of the moths collected in the field with intermediate genitalia had winter moth CO1 and G6PD sequences and thus were not hybrids (or at least F1 hybrids). We found three hybrids out of 158 moths with intermediate genitalia in the region where both species were caught. We conclude that hybrids occur in nature, but are not as common as previously reported. Introgression of genes between the two species may still be significant.

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Estimating appropriate sample sizes to measure species abundance and richness is a fundamental problem for most biodiversity research. In this study, we explore a method to measure sampling sufficiency based on haplotype diversity in the ray-finned fishes (Animalia: Chordata: Actinopterygii). To do this, we use linear regression and hypothesis testing methods on haplotype accumulation curves from DNA barcodes for 18 species of fishes, in the statistics platform R. We use a simple mathematical model to estimate sampling sufficiency from a sample-number based prediction of intraspecific haplotype diversity, given an assumption of equal haplotype frequencies. Our model finds that haplotype diversity for most of the 18 fish species remains largely unsampled, and this appears to be a result of small sample sizes. Lastly, we discuss how our overly simple model may be a useful starting point to develop future estimators for intraspecific sampling sufficiency in studies using DNA barcodes.

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.