Invertebrate COI Barcoding Protocol

The primers LCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ [72 (link)] were used to amplify the COI gene barcode region, according to Bourke et al. [35 (link)]. Each reaction was performed in a total volume of 25 μL containing 2 μL of DNA, 1× PCR buffer (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 0.2 mM of each dNTP (Amresco), 0.1 μM of each primer, and 0.625 U of Taq Platinum polymerase (Invitrogen), and the remaining volume consisted of ultrapure water. The PCR thermal conditions consisted of 94 °C for 3 min, five cycles of 94 °C for 30 s, 45 °C for 90 s, 68 °C for 60 s, followed by 35 cycles of 94 °C for 30 s, 51 °C for 30 s, 68 °C for 60 s, and a final extension at 68 °C for 10 min. PCR products were purified by PEG precipitation (20% polyethylene glycol 8000/2.5 M NaCl).