INTRODUCTION
For centuries, species have been delineated based on their physical
appearance. Early naturalists differentiated between species based on
functional traits such as jaw and limb shape, and beak size, or
otherwise on traits that function in social selection, such as fur
pattern and plumage coloration (Birkhead, 2008; Leroi, 2014). Such
phenotypic characters, representing clear visual differences to the
human eye, have been thought to aid the species themselves in
recognising their own kind and finding potential mates. It has long been
of interest to identify which traits mediate reproductive isolation
between related species (Mayr, 1942), and more recently, which genes
underpin those traits; in other words, the genes that mediate speciation
(Nosil & Schluter, 2011). Hybrid zones between related species provide
an unparalleled opportunity to identify genes associated with phenotypic
characters (Delmore et al., 2016; Brelsford et al., 2017), including
those that play an important role in mate choice and species recognition
(Harrison & Larson, 2016). In some such cases, it is just a small set
of genes that maintain phenotypic differences between hybridising taxa,
especially those functioning in coloration and pattern differences
(Poelstra et al., 2014; Toews et al., 2016). And evidence is starting to
mount that the extent of divergence in plumage coloration between
populations affects assortative mating (Haas et al., 2009; Scordato et
al., 2017; Billerman et al., 2019).
Genes involved in coloration and patterning pathways have received a lot
of attention (Mundy, 2005; Hoekstra, 2006), with a number of recent
studies identifying genes associated with the melanin pigmentation
pathway as a result of research across hybrid zones (e.g., (Delmore et
al., 2016; Toews et al., 2016; Walsh et al., 2016; Campagna et al.,
2017). Carotenoid-based coloration, incorporating the reds, oranges and
yellows, has received far less attention (Toews et al., 2017), because
carotenoid uptake is dependent on diet, meaning that identifying the
genes involved in carotenoid processing is especially challenging
(Campagna et al., 2017). Nevertheless, much progress has been made
recently with the scavenger receptor B1 (SCARB1 ) identified as a
mediator of carotenoid-colour expression in birds (Toomey et al., 2017),
while a region of the Z chromosome has been identified as a potential
regulatory region for follistatin and is associated with
sex-linked red colour polymorphism in Gouldian finch Erithrura
gouldiae (Kim et al., 2019). Previous work has also identified
candidate genes to explain differences between red and yellow feather
coloration through lab crosses of yellow canaries with red siskins
(Lopes et al., 2016) and red and orange billed individuals of zebra
finches (Mundy et al., 2016). Both studies specifically identified the
gene CYP2J19 as the one that encodes the ketolases that catalyze
the conversion of yellow dietary carotenoids to red C4-ketocarotenoids.
Both studies were focused on laboratory-based crosses involving
artificially selected forms of species developed to express certain
traits (“red factor” canaries and yellowbeak zebra finches
respectively). A field-based study in long-tailed finch Poephila
acuticauda provided further support for the role of CYP2J19 in
carotenoid-based bill colour across a natural hybrid zone (Hooper et
al., 2019), while CYP2J19 gene expression has also been
associated with red coloration in weaverbirds (Twyman et al., 2018b).
But evidence that it explains variation between red and yellow feather
coloration in wild populations of birds is still equivocal.
Red-fronted (Pogoniulus pusillus, Dumont, 1816) and
yellow-fronted (P. chrysoconus, Temminck, 1832) tinkerbirds
provide such an opportunity to investigate the genes involved in
carotenoid processing because they hybridise extensively at a contact
zone in southern Africa (Nwankwo et al., 2019). They differ
phenotypically most noticeably in their forecrown coloration; hence
their vernacular names (Fig. 1). Their other plumage differences are
also in carotenoid-based colours, but those are more subtle differences,
to the human eye at least, between shades of yellow and white that
characterize the colour of their underparts, rump, wing bar, and
supercilium (Hockey et al., 2005). The two species have been shown to
hybridise extensively with asymmetric introgression of red forecrown
plumage into the P. chrysoconus genomic background, in spite of
phylogenetic analysis on mitochondrial DNA revealing the two species
have been diverging for over four million years (Nwankwo et al., 2019).
Despite the deep divergence between the species, the extent of
introgression indicates that hybrids are viable and fertile, though some
reduced hybrid fitness might maintain a tension zone and inhibit the two
species collapsing into one (e.g., Kearns et al., 2018). Thus, if at
least one species continues to differentiate the two phenotypes and
mates assortatively, the plumage trait and the genes underlying it may
play an important role in maintaining species divergence.
Here we investigate the genomic regions associated with the red and
yellow feathers in red-fronted and yellow-fronted tinkerbirds that could
play an important role in reproductive isolation. We sampled 79
individuals across the hybrid zone in southern Africa, collecting
forecrown feathers for spectrophotometric analysis (Nwankwo et al.,
2019). We performed a genome-wide association study (GWAS) to identify
single nucleotide polymorphisms (SNPs) associated with plumage
coloration. The study included assembly of a short-read genome of an
individual Pogoniulus pusillus and double-digest
restriction association DNA sequencing (ddRAD). Our aim was to identify
the genes associated with carotenoid plumage coloration across a natural
contact zone, and specifically which regions of the genome explain
differences between red and yellow plumage. We further tested whether
those genes, or SNPs at other loci, might be associated with the shade
of yellow to white or extent of yellow versus black plumage in other
feather patches, while also testing for any differences in plumage
attributable to sex. We then compared alleles at loci associated with
forecrown colour with the hybrid index across autosomes and the Z
chromosome, and interspecific heterozygosity of individuals, to
elucidate the patterns of interbreeding across genotypes that explain
asymmetric introgression between species that have been diverging for
over 4 million years (Nwankwo et al., 2019).