Identification of hybrid individuals and direction of
backcrossing
Out of 37 individuals sampled in the contact zone, 13 were admixed at
the level between first generation backcrosses (with a hybrid index
between approx. 0.2 – 0.8, Fig. 3). Among the 13 admixed individuals we
identified one F1 hybrid (AR93112), while the remaining hybrids likely
had more complex ancestry (Table 3).
Backcrossing was thus asymmetric: autosomes of admixed individuals had a
higher percentage of extoni compared to pusillus genotype
than the Z chromosome based on their respective hybrid index (paired
t-test: df = 12, t = 2.55, P = 0.026, Table 3). This finding
suggests that hybrids result from females that have a higher proportion
of extoni ancestry mating with males with a relatively higher
proportion of pusillus ancestry, as found in 9 of 13 individuals
(compared to 2 with the reverse pattern and 2 with equal indexes). For
instance, female AR93167 had autosomal and Z hybrid indexes of 0.559 and
0.000 respectively, with an extoni mitochondrial haplotype (Table
3). With over 50% extoni autosomal hybrid index (andextoni mtDNA haplotype), her mother was likely a ‘pure’extoni and her father mostly pusillus with a hybrid index
= 0.559 and a Z chromosome hybrid index of 0.000 (Table 3).
The variant most strongly associated with crown score was an indel on
scaffold 50201 on chromosome 8, which aligns to the region 13-21 kbp
upstream of CYP2J19A in zebra finch, and 16-28 kbp upstream of
cytochrome P450 gene XM_009910004 in downy woodpecker (manually
annotated as CYP2J19 by Twyman et al. 2018a). All individuals
homozygous for the pusillus allele were scored with red
forecrowns, while heterozygotes were scored red, reddish or orange,
whatever their hybrid index (Fig. 3A). Forecrown score of homozygotes
with the extoni allele ranged from yellow and amber for
non-admixed individuals, through orange for admixed individuals to
reddish for one individual (AR93120) with 96% pusillus genotype.
Comparing hybrid index with interspecific heterozygosity in a triangle
plot revealed that backcrossing was asymmetric into the extonigenotype, with red to orange plumage coloration introgressing intoextoni carrying the pusillus allele at CYP2J19 (Fig. 3B).
There was also a very clear relationship between the genotype at the
CYP2J19 locus and crown hue. All heterozygotes at the locus had redder
hues than homozygotes at the locus with comparative hybrid index (Fig.
4). But we note that individual AR93120 homozygous for the extoniallele at this locus, with a red forecrown also based on her hue
measurement, was homozygous for the pusillus allele at two other
loci on chromosome 8 strongly associated with crown hue (on scaffolds
50344 and 50345; see supporting information).