Genetic diversity and population structure based on mitochondrial markers
To perform the genetic analyses, we edited and aligned COI and cytb sequences using CodonCode Aligner 4.0.4 (CodonCode Corporation, Dedham, MA) and carefully checked the chromatograms for ambiguities and sequences to detect the presence of any stop codons, as well as alignment gaps.
Because mitochondrial sequences are linked within the same genome, COI and cyt b sequences were concatenated for the analyses. First, to analyze the genetic variation within the species and understand the relationship among haplotypes, we calculated the average p distances using MEGA 5.2 (Tamura et al. 2011) and generated haplotype networks using the median joining algorithm implemented in PopART 1.0 (< http://popart.otago.ac.nz >), respectively. We conducted an analysis of molecular variance (AMOVA) in Arlequin 3.5 (Excoffier and Lischer 2010) to explore the distribution of genetic variation within T. ruficapillus and specifically test whether there are differences in the frequency of mitochondrial haplotypes among subspecies. The ΦST values between pairs of subspecies were computed using uncorrected genetic distance matrices between haplotypes and significance was tested through 2,000 random permutations.