For the list of primers see Table 1. We used part of mitochondrial Cytochrome C Oxidase I (COI) – the selected barcoding marker for animals (Hebert et al., 2003a), and amplified a part of 665 bp in length with the Lep primers (Hebert et al., 2004a). We also sequenced a section of 482 bp of the nuclear Elongation Factor 1-α (EF1-α) marker for most of the specimens. Initial attempts to amplify a 1240 bp fragment of this gene by using the primers (five sets) of Cho et al. (1995) largely failed. Only primer M44-1 with rcM52.6 (Cho et al., 1995) amplified a 701 bp fragment consistently for at least five different genera of Nepticulidae (Ectoedemia, Enteucha, Parafomoria, Trifurcula and Stigmella). Based on these results, Nepticulidae-specific primers, EF-NepF and EF-NepR (Table 1) that amplified a 482 bp fragment of this gene, were designed and used throughout this study.
For many specimens we used T7 promotor and T3 tailed primers for both COI and EF1-α, as this speeds up the work-flow and may improve results (Regier and Shi, 2005; Wahlberg and Wheat, 2008). For some older museum specimens, the DNA was too degraded for
amplifying sections over 400-bp long. For these we used internal primers for COI (Hajibabaei et al., 2006a, 2006b) and EF1-α (specially designed for Nepticulidae). As a consequence, for some specimens there is only a shorter sequence available, denoted with (p) for partial. These shorter sections are, respectively, for COI a 310-bp amplicon and for EF1-α a 251-bp amplicon. PCRThe PCR cycle consisted of 3 minutes initial denaturation at 94°C, 15 seconds cycle denaturation at 94°C, 30 seconds cycle at annealing temperature, 40 seconds cycle extension at 57°C for 40 cycles. A final extension at 57°C for 5 minutes occurred after all cycles had finished. The annealing temperature for COI was 50°C, for EF1-α 57°C. PCR was performed in volumes of 25 µl. For many samples the product was purified using the Promega Wizard Genomic Purification kit using the manufacturers ‘spin column protocol’, for others the purification was done by Macrogen with a Montage purification kit (Millipore). All samples were sequenced in both directions on an ABI 3730 by Macrogen.