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.