Laboratory protocols for mitochondrial markers
We extracted DNA from fresh tissue (pectoral muscle or blood) following the silica-based protocol described by Ivanova et al. (2006), using individual spin columns (Lijtmaer et al. 2012). We amplified two mitochondrial genes: cytochrome c oxidase I (COI) and cytochromeb (cyt b ). Primers used for the amplification of the 695 base pairs (bp) of the COI were BirdF1 (Hebert et al. 2004) and COIbirdR2 (Kerr et al. 2009) and to amplify 1007 bp of the cyt bwe used L14841 (Kocher et al. 1989) and H16065 (Lougheed et al. 2000).
PCR amplification reaction cocktails and the thermocycling profile for COI from fresh tissue followed Lijtmaer et al. (2012). To amplify the cyt b we followed the protocol in Arrieta et al. (2013) and the PCR cocktail was prepared in 20 μl volume with the following reagents: 3 μl of genomic DNA, 1x PCR buffer, 0.2 mM dNTPs, 3 mM Cl2Mg/SO4Mg, 0.5 μM primer forward, 0.5 μM primer reverse and 1U Taq DNA polymerase (Invitrogen). The PCR thermocycling profile was as follows: 3 min at 94 ºC; 40 cycles of 45 sec at 94 ºC, 30 sec at 53 ºC and 1 min at 72 ºC; and finally 10 min at 72 ºC.
Sequencing of both mitochondrial markers was conducted at Macrogen Korea (Seoul, Korea) and performed bidirectionally with the same primers used for amplification. GenBank accession numbers for all sequences generated in this study are included in Table S1.