Amplification and Sequencing
The primers used to amplify the control region in North American specimens were initially designed to analyze the genetic diversity of the Australian population (Rollins et al, 2011). These two primers (svCRL1 and svPheH3) amplify the majority of the mitochondria control region (Table S1). These primers also were used to amplify DNA from the starling population in South Africa (Berthouly-Salazar et al., 2013). The thermocycling conditions used here were identical to those described in the original paper (Rollins et al, 2011). This includes a 5-min step at 94°C, 30 cycles of 94°C for 30s, 53°C for 15s and 72°C for 30s and a final extension step for 10 min at 72°C.
Rollins et al. (2011) also designed a series of nested primers to be utilized in the amplification of museum specimens or highly degraded samples (Table S1). We utilized these primers in order to sequence the control region in four overlapping segments to eliminate possibilities for error in the sequencing process and verify with confidence the base in each position. All sequences were sequenced in the forward direction. This was necessary due to a repeat region at the end of the sequence that would not amplify in the reverse direction. All sequences were sent to GENEWIZ, Inc (South Plainfield, NJ) for PCR cleanup via an enzymatic purification and to be sequenced. Primer extension sequencing was performed by GENEWIZ, Inc using Applied Biosystems BigDye version 3.1. The reactions were then run on Applied Biosystem’s 3730xl DNA Analyzer.