DISCUSSION
We found that CYP2J19 on chromosome 8 was within one of three
regions associated with crown colour across a natural hybrid zone in
tinkerbirds. Indeed, it was the only region of the genome that was
significantly associated with both hue, determined from spectral
reflectance, and plumage colour score. Only extoni individuals
homozygous for the CYP2J19 -linked SNP exhibited a yellow
forecrown, while all individuals homozygous for the pusillusallele had red forecrowns. All heterozygotes at theCYP2J19 -linked locus had either red, reddish or orange crowns.CYP2J19 is a cytochrome P450 enzyme, which has been implicated as
encoding the ketolase that mediates red coloration in birds (Lopes et
al., 2016; Mundy et al., 2016). Ketolases convert dietary yellow
carotenoids into red ketocarotenoids in birds with red feathers (Toews
et al., 2017; Norden & Price, 2018). CYP2J19 has also been
associated with red retinal oil droplets influencing colour vision in
birds (Mundy et al., 2016), proposed to be its ancestral function, with
its role in red coloration having evolved in some but not other species
(Twyman et al., 2018a). It has also been shown to function in red
integumental coloration in turtles (Twyman et al., 2016).
CYP2J19 is not the only gene shown to function in red coloration,
however, with recent work finding that a mutation in the beta-carotenoid
oxygenase 2 gene (BCO2) functions in red bare part coloration in birds
(Gazda et al., 2020). The CYP2J19 linked SNP on chromosome 8 was
also not the only region associated with red forecrown colour in
tinkerbirds, with chromosome 20 associated with crown score and
chromosome 24 with crown hue. BC02 is on chromosome 24, and in
addition to its association with crown hue, a single SNP on scaffold
70216 on chromosome 24 was also significantly associated with crown
score. However, the scaffolds significantly associated with forecrown
colour in tinkerbirds on chromosome 24 aligned to the interval from 5.8
to 6.0 Mbp on zebra finch chromosome 24, while BCO2 spans from
1.53 to 1.54, suggesting that the SNPs associated with forecrown colour
are not linked to BCO2 . We found that forecrown colour could be
intermediate between red and yellow, with some heterozygotes at theCYP2J19 locus displaying orange forecrowns. This suggests
possible additive effects of the gene rather than dominance of one
allele over the other, and/or interactions with genes at other regions
we found associated with forecrown colour. There was a cluster of SNPs
on adjacent scaffolds on chromosome 8 that overlapped with several genes
in zebra finch, but to our knowledge there are presently no known
functional associations of those genes with carotenoid-based coloration
determination.
We found no significant association with crown chroma. Unlike in hue,
the two species were shown to overlap in chroma between sympatric forms
by Nwankwo et al. (2019), with both species showing much more variation
in this measure of colour intensity than in the measures of hue and
brightness. We did, however, find SNPs significantly associated with
supercilium colour, but not the extent of yellow on the wing bar. The
SNPs associated with supercilium colour were on a different chromosome
to those associated with forecrown colour, indicating that a different
pathway is involved in mediating carotenoid-based coloration in those
traits. This is not unexpected. Those plumage patches varied between
white and shades of yellow, while CYP2J19 has been shown to
function in red feather colour determination (Lopes et al., 2016; Mundy
et al., 2016).
In another pair of sister taxa of Pogoniulus tinkerbird,
yellow-rumped (P. bilineatus ) and yellow-throated tinkerbird
(P. subsulphureus ), which meet in the forests of west and central
Africa, reproductive isolation is maintained through character
differences such as throat colour and song, which have diverged in
sympatry (Kirschel et al., 2009; Kirschel et al., 2020). By contrast,
yellow-fronted and red-fronted tinkerbird hybridise extensively, and
hybridisation is asymmetric (Nwankwo et al., 2019). The pattern of
asymmetry in putative hybrids with extoni ancestry and
predominantly pusillus Z chromosome suggests red-fronted males
are much more likely to mate with yellow-fronted females than the other
way around. Even when the reverse scenario does happen, based on the
proportion of genotypically extoni individuals in the contact
zone with red forecrowns (Nwankwo et al., 2019), a high proportion of
genotypically extoni males mating with pusillus females
might indeed sport reddish forecrowns.
Such asymmetry between mitochondrial haplotype and hybrid index of the Z
chromosome could result from a genetic incompatibility in the reverse
cross, i.e. pusillus haplotype females interbeeding withextoni males. Yet, such crosses do occur – two individuals in
our sample with pusillus haplotype had extoni fathers
(AR93115 and AR93163), and both individuals are females – the
heterogametic sex - theoretically more likely to suffer from inviability
and infertility (Haldane, 1922). Instead, we suggest the asymmetry is a
consequence of female preference for males with red forecrowns inpusillus and potentially in extoni females (c.f.
Baldassarre et al., 2014). The pusillus mitochondrial haplotype
is less than one quarter as common as the extoni haplotype in the
contact zone (8/37 individuals, see Nwankwo et al. (2019)). Yet femalepusillus appear to put the extra effort into findingpusillus males, outnumbered four-to-one by their extonicounterparts. By contrast, female extoni appear to breed with
males of either genotype at rates equivalent to their frequency in the
population. Mate choice experiments would be needed to test whether
female extoni would choose male pusillus (or otherwise
males with extoni ancestry but with introgressed red forecrowns)
over yellow-fronted male extoni , when given a choice. But it does
appear to be the failure of female extoni to mate assortatively
with males of their own species and phenotype likely drives
introgressive hybridisation between these species. Premating isolation
between the two species remains weak in spite of 4 million years of
divergence, according to their mitochondrial DNA phylogeny (Nwankwo et
al., 2019). The choosiness of female pusillus to mate
assortatively on the other hand might be the main factor maintaining a
narrow tension zone that does not progress into panmixia and species
collapse (e.g., Kleindorfer et al., 2014; Kearns et al., 2018).
Red feathers result from the conversion of dietary carotenoids to red
ketocarotenoids and such converted carotenoids have been shown to be an
honest indicator of fitness that could function in mate choice (Weaver
et al., 2018). Here, female red-fronted tinkerbirds might only mate with
males that display red forecrowns because they consider
yellow-forecrowned males of lower quality. Greater extents of difference
in plumage between related species have been proposed to play a role in
premating isolation (Scordato et al., 2017). Here, though yellow-fronted
females instigate asymmetric introgression by not mating assortatively
with yellow-fronted males. If there was a sensory bias for red plumage
coloration the two species would surely collapse into one if there is no
loss of fitness in hybrids, and with heterozygotes at the CYP2J19locus displaying more reddish forecrowns, we would also expect red
plumage to rapidly introgress across the population. Further work is
needed to determine the extent to which species boundaries are
maintained and the rate of introgression of red plumage across the
contact zone.
Both quantitative (hue) and qualitative (crown score) metrics recovered
the same major-effect locus. This might not be surprising if they are
both measures of the same trait, but each method has its own inherent
biases. Scoring from photographs is subjective and affected by each
observer’s perspective of the effects of ambient light. While
correlated, there was a resultant variation in their respective scores,
hence the number of individuals with intermediate scores such as
reddish, which resulted from one observer scoring the forecrown red and
the other scoring it orange. Likewise, reflectance spectrometry is also
susceptible to variation in feather and probe placement, though we did
attempt to control for this with measurements in two orientations.
Despite these potential biases in qualitative and quantitative metrics,
the same major-effect locus was recovered. Furthermore, we showed that
admixture mapping is feasible using RADseq with a sample size
< 50, as was shown for forecrown hue, but only for
major-effect loci. This was possible in spite of no prior genomic
resources available for Pogoniulus tinkerbirds or any
closely-related genera.