2.1 DNA extraction, PCR amplification, sequencing and alignment
The targeted taxa were sorted and dissected under a stereo microscope. Thorax and one pair of legs were used for genomic DNA extraction. All extraction procedures followed the Qiagen DNeasy Blood and Tissue kit protocol provided by the manufacturer. DNA was extracted from the thorax and head in a buffered solution with the enzyme proteinase-K at 56 °C overnight, and otherwise followed the manufacturer’s protocol, except using a final elution volume of 100 μl. After digestion, the exoskeletons were removed carefully using a fine-tipped forceps and washed with 96% ethanol before mounting in Euparal on the same microscope slide as its corresponding head, antennae, wings, legs and abdomen following the procedure outlined by Sæther (1969).
A 658 bp fragment of the COI region was PCR-amplified in 25 µL reactions and containing 2 µL DNA template (concentration not measured), 2.5 µL 5X buffer, 2 µL MgCl2 in 25 µM concentration, 0.2 µL of dNTPs in 10 mM concentration, 1 µL of each of the universal standard barcode primers (Folmer et al., 1994) LCO1490 (50-GGTCAACAAATCATAAAGATATTGG-30) and HCO2198 (50-TAAACTTCAGGGTGACCAAAAAATCA-30), in 10 µM concentration, 0.2 µL of HotStarTaq (Qiagen, Germany) and 16.1 µL of ddH2O. PCR amplification was performed in a thermocycler with an initial denaturation step of 95 ºC for 15 minutes, then followed by five cycles of 94 ºC for 30 seconds, 45 ºC for 30 seconds, 72 ºC for 1 minute, followed by 35 cycles of 94 ºC for 30 seconds, 51 ºC for 30 seconds, 72 ºC for 1 minute, and one cycle at 72 ºC for 5 minutes, then held at 4 ºC.
PCR products were checked visually by electrophoresis on a 1.5% agarose gel and purified using shrimp alkaline phosphatase and exonuclease I (USB Corp., USA). For bidirectional sequencing, we used the ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Kit (Life Technologies, USA), and cycle sequencing reactions were performed on ABI PRISM 3130xl or 3730xl automated sequencers (Life Technologies, USA) at Harvard University, or shipped to EurofinsGenomics (Ebersberg, Germany). Raw sequences were assembled and edited using Geneious 2021.2.2 (Kearse et al. , 2012), checked for stop codons and aligned as translated amino acids using the MUSCLE algorithm (Edgar, 2004) on amino acids as implemented in MEGA11 (Tamura et al. , 2021). The nucleotide compositions were calculated in MEGA11, while the pairwise genetic distances for each individual sequence were determined in BOLD, both using the K2P model (Kimura, 1980).