Dna extraction

DNA extraction , amplification and sequencing

Animals were placed in a drop of propylene glycol on a concave slide to allow the dissection of the animal without compromising the DNA ( Moreau et al., 2013) . Whole specimen or body segments of the abdomen were dissected under a stereo microscope using tungsten needles and used for the extraction. Specimens were labelled individually with a unique number and measured using an eyepiece micrometer or the measurement tool of the Leica montage software LAS V4.13.

DNA was extracted using Qiagen DNeasy Blood and Tissue kit, following the manufacturer’s instructions for tissue samples ( Alda et al., 2007) except that the digestion step was increased to 16 hours and the final DNA elution volume was reduced to 80 µL. PCR was used to amplify three gene regions using the primers listed in Table 1. Reaction mixtures consisted of 12.5 µL MyTaq RedMix (Bioline), 0.5–1 µL of each primer (10 µM), 2–5 µL DNA and adjusted to 25 µL with PCR grade water. The PCR amplification conditions for the COI region were the same as those listed in ( Folmer et al., 1994) except that for the C1J1718/HCO2198 and C1J1718/C1J2329 primer combinations the initial denaturation and the extension phases were increased to 5 min and 1 min, respectively. The reaction conditions for the 12S region were: (i) an initial 5 min denaturation phase at 95 °C, followed by (ii) 35–40 cycles of 30s denaturation at 95 °C, 30s annealing at 47 °C and 45 s extension at 72 °C with (iii) a final 10 min extension at 72 °C. These same conditions were used for the 18S region except that the annealing temperature was 49 °C. The outcomes of PCR assays were assessed using either 2 % Invitrogen E-gels or 1.5 % agarose gels stained with SYBR Safe (Invitrogen). Successfully amplified PCR products were sent to the Australian Genome Research Facility (AGRF) for Exo-SAP purification and bidirectional sequencing.

The raw chromatograms were imported into Geneious Prime software ( Kearse et al., 2012). Forward and reverse reads were assembled, checked by eye and edited. The consensus sequences were extracted, blasted against GenBank and aligned using the MAFFT algorithm (Multiple Alignment using Fast Fourier Transform) ( Katoh et al., 2002) with default parameters.