Identification of hybrid individuals and direction of backcrossing
In order to determine whether individuals with intermediate ancestry were progeny of recent interbreeding or historical events, we used Faststructure (Raj et al. 2014) with k = 2 to estimate pusillusand extoni ancestry (i.e., hybrid index) based on all loci, and separately for loci on scaffolds that aligned to the zebra finch Z chromosome. We estimated interspecific heterozygosity of each individual based on a genome-wide subset of 1,723 SNPs with allele frequency difference > 0.95 between allopatric pusillus andextoni populations, and compared this to the hybrid index across the whole genome and the Z chromosome to identify F1 hybrids, first generation backcrosses and the direction of backcrossing for early and later-generation backcrosses.
To illustrate the relationship between hybrid index and interspecific heterozygosity, we produced triangle plots, which would reveal F1 hybrids as individuals with intermediate hybrid index and the highest levels of interspecific heterozygosity at loci diagnostic for each species. F2 hybrids and backcrosses would be expected to have intermediate levels of interspecific heterozygosity and fall in the middle and sides of the triangle respectively, in accordance with simulations (Toews et al., 2018), with non-admixed individuals falling in the lower corners of the triangle.