For T. rathkei, the putative mitochondrial reads from each individual were mapped to the reference mitochondrial genome generated above using bwa. For C. convexus, all sequencing reads were mapped to the reference mitochondrial genome generated above using Bowtie2 (Langmead and Salzberg 2012). Full pileup files were generated for each species using the samtools pileup command. All positions with at least 100× coverage in which a nonreference allele was present at a frequency greater than 0.2 were identified as putative polymorphisms.