2.3 | Amplification and Sequencing
Fragments of 18S rDNA were amplified using primers MitchA and
MitchB (Medlin et al. 1988). Both 18S and COIfragments were amplified using Phusion High-Fidelity PCR Master Mix with
HF buffer (New England BioLabs, Ipswich, MA) in a Veriti PCR thermal
cycler (Life Technologies, Carlsbad, CA). Annealing temperatures were
most successful at 50ºC for most species. Gene fragments were sequenced
bi-directionally with PCR primers and the BigDyeTerminator v3.1
sequencing kit according to the manufacturer’s protocol and analyzed on
a 3500xL Genetic Analyzer (Life Technologies, Carlsbad, CA).