PCR and Sequencing
Three gene segments were amplified using previously described primers for the nuclear genes Na-K-ATPase (NaKA) and elongation factor 1α (EF1) (Wares, Pansky, Pitombo, Daglio, & Achituv, 2009). PCR reactions were carried out in 25-µl reaction volumes containing 1X PCR buffer (including 1.5 mM MgCl2), 0.2 mM of each dNTP, 1 µM of each primer, 1 unit of Super-Term Taq polymerase (Hoffmann-La Roche), and about 100 ŋg template DNA. PCR reactions were processed in an MJ Research thermal cycler with the following thermal regime: an initial step of 2 min at 95°C followed by 35 cycles of 0.5 min at 94°C, 0.5 min at 57°C, and 1 min at 72°C, followed by 3 min at 72°C and then held at 15°C. PCR products were visualized on 1.5% agarose gels and sequenced bidirectionally using the PCR primers on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.