Materials & Methods

Morphological Work

Character photos were taken using a Microptics Digital Lab XLT imaging system utilizing a Canon EOS 1 Ds camera and Microptics ML-1000 flash fibre optic illumination system. The computer freeware CombineZ (http://www.hadleyweb.pwp.blueyonder.co.uk/CZ5/combinez5.htm) was used to combine multiple photos taken at different focal points into high-resolution images. Pinned specimen habitus shots, plates and some character shots were taken using a Nikon D70s digital camera, a Nikkor 105mm Macro lens, and a Nikon SB800 flash unit. Live habitus photographs were taken using Nikon D70 or D2X cameras, with 105mm or 60mm lenses and various flash combinations. Photos were processed using Adobe Photoshop CS3 (AP CS3) for Windows; the online web pages were designed and built in Microsoft Powerpoint 2003. Further work on the web pages was done using Adobe Fireworks and Adobe Dreamweaver.
Most specimens used in pinned habitus shots are from the University of Guelph Insect Collection, although some specimen photos were of specimens borrowed from the CNCI and LEM. Specimens used for photos have been labeled accordingly and deposited.

Genetic Analysis

Total genomic DNA was extracted from a single leg according to the protocol developed by Ivanova et al. (2006). A 658-bp region near the 5 terminus of the CO1 gene was amplified using primers LepF1 5' -ATTCAACCAATCATAAAGATATTGG-3 and LepR1 5' -TAAACTTCTGGATGTCCAAAAAATCA-3.
PCRs were carried out in 96-well plates in 12.5- l reaction volumes containing 2.5 mM MgCl2, 5 pmol of each primer, 20 M dNTPs, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 10–20 ng (1–2 l) of genomic DNA, and 1 unit of TaqDNA polymerase using a thermocycling profile of one cycle of 2 min at 94°C; five cycles of 40 sec at 94°C, 40 sec at 45°C, and 1 min at 72°C; followed by 35 cycles of 40 sec at 94°C, 40 sec at 51°C, and 1 min at 72°C, with a final step of 5 min at 72°C. Products were visualized on a 2% agarose E-Gel 96-well system (Invitrogen), and samples containing clean single bands were bidirectionally sequenced by using BIGDYE 3.1 on an ABI 3730 DNA Analyzer (Applied Biosystems). Contigs were assembled by using SEQUENCHER 4.0.5 (Gene Codes). 

Sequences, trace files, and field data are available in the Teph (file name) file in the Completed Projects section of the Barcode of Life Database (BOLD; www.barcodinglife.org; Ratnasingham and Hebert, 2007). Additional collection information is deposited in the University of Guelph Insect Collection Database and all sequences have been deposited in the GenBank database (CO1: accession nos. EU484466–EU484568). Sequence divergences were calculated by using the K2P distance model (Kimura, 1980) and an NJ phenogram (Saitou & Nei, 1987) was created (as implemented in BOLD) to provide a graphic representation of the among-species divergences. A table of specimens sequenced for DNA barcoding has been included (Appendix I).

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