Specimen collection, DNA extraction, PCR and sequencing
Specimens of the Eucalyptus snout beetle were collected by hand in various sites in the city of Quito. We removed three legs of the same side of nine individuals for molecular analyses. Voucher specimens were deposited in the invertebrate collection of the Zoology Museum (Quito Católica Zoología Invertebrados, QCAZ I) of Pontificia Universidad Católica del Ecuador (PUCE), Quito, Ecuador.
For DNA extraction, we left the legs in 500 µL of TE buffer for 24 hours at 4 °C. After rehydration, each softened leg was crushed with a pestle for 15 minutes, then 500 µL of CTAB-PVP and 16 µL of Proteinase K were added to each sample and left shaking at 56 °C for 24 hours. We then added 650 µL of chloroform to each sample and mixed them to form an emulsion; subsequently, samples were centrifuged for 10 minutes at 13000 rpm. We removed the supernatant from every sample and saved each in different 1.5 µL tubes. Around 600 µL of 100% Isopropanol were added to every sample for DNA precipitation, and then centrifuged for 10 minutes at 13000 rpm. The pellet was then washed with 70% ethanol and centrifuged again for 10 minutes at 13000 rpm. The pellet was left to dry, and then resuspended in 25 µL of 0.1X TE Buffer. DNA concentration was quantified using a Nanodrop 1000 from Thermo Scientific.
We amplified two fragments of the mitochondrial gene COI with the following pairs of primers: the forward primer Jerry (5′- CAA CAT TTA TTT TGA TTT TTT GG-3′) and the reverse primer Pat (5′- TCC AAT GCA CTA ATC TGC CAT ATT A-3′) (Simon et al., 1994) and the forward primer dgLCO1490 (5’- GGT CAA CAA ATC ATA AAG AYA TYG G-3’) and the reverse primer dgHCO2198 (5′- TAA ACT TCA GGG TGA CCA AAR AAY CA-3’) (Meyer et al., 2005). Each Polymerase Chain Reaction (PCR) contained 13 µL total volume: 2.5 µL of 10x PCR Rxn Buffer, 3 mM MgCl2, 0.2 µM of dNTPs, 0.2 M of forward primer, 0.2 M of reverse primer, 1 unit of Taq Platinum DNA Polymerase 5000U Invitrogen and 20 ng of DNA. Conditions for PCR amplification using the first pair of primers were the following: initial denaturation at 95 °C for 2 min; 40 cycles of 95 °C for 2 min, annealing at 46 °C for 1 min, and extension at 72 °C for 1 min; final extension at 72 °C for 5 min. Conditions for PCR amplification using the second pair of primers were the following: initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 45 sec, annealing at 47 °C for 30 sec, and extension at 72 °C for 1.5 min; final extension at 72 °C for 10 min. The results were confirmed through an agarose gel electrophoresis. The amplifications were then treated with ExoSAP before being sent to Macrogen Inc. for sanger sequencing.