Base frequencies for heterozygous sites were inferred from filtered Samtools pile-up results. For each predicted heterozygous site, the number of each of the four possible alternative sites over the read coverage of this base was determined and rounded to the second decimal place by a custom Perl script. Base frequencies were binned into 101 categories of 0.01 each (0–1.00), and an approximate distribution of base frequencies for each chromosome of L. braziliensis and L. mexicana was plotted in Excel (Supplemental Fig. S3). Frequencies of zero were not included in the plot.