Introduction
Coloration is arguably one of the most fascinating traits in
evolutionary biology (Salis, Lorin, Laudet, & Frédérich, 2019).
Maintenance and evolution of colour polymorphism is linked to pigment
availability, pigment distribution and cell types that either produce,
contain or transport pigments across cell layers, such as keratinocytes,
melanophores, xanthophores and iridophores (Hofreiter & Schöneberg,
2010). The molecular basis of coloration has been extensively studied
across taxa, revealing a multitude of loci involved in the production of
colour derived from a) the interplay between pigmentation
availability-distribution and b) existence of taxa-specific molecular
pathways for colour production (Hofreiter & Schöneberg, 2010). For
instances, while mammals owe their colour of hair or skin to a
deposition balance between eumelanin and pheomelanin, which are forms of
melanin, other taxa might manifest coloration via melanin deposition,
carotenoid deposition and structural arrangement of cell layers, i.e.
structural colour (Hofreiter & Schöneberg, 2010).
Melanin-based pigmentation is the most ubiquitous colour-producing
pigment in extant birds (Roulin, 2014). The melanin production pathway
is complex and evolutionarily conserved but centered on the POMCgene (which encodes for the protein proopiomelanocortin) (Ducrest,
Keller, & Roulin, 2008). This protein is cleaved into several peptides
– melanocortins – that bind to up to 5 melanocortin receptors
(MC1R-5R ) each involved in specific physiological functions,
including melanin-production (MC1R ) (D’Alba & Shawkey, 2019;
Ducrest et al., 2008). Though the pleiotropic nature of the POMCgene has been posited to explain associations between melanin-based
pigmentation and other phenotypic traits, i.e., melanin-associated
phenotypes, molecular evidence is largely absent (McNamara et al., 2021;
Luis M San-Jose & Roulin, 2018). What has been well described is
structural genetic variation at MC1R and respective regulatory
regions affecting melanin-based pigmentation in several bird species
including domestic breeds (Kerje, Lind, Schütz, Jensen, & Andersson,
2003; Liu et al., 2023; Mundy, 2005).However, lack of associations has
also been reported particularly after correcting for the effect of
population structure (Avilés et al., 2019; Hoffman, Krause, Lehmann, &
Krüger, 2014; MacDougall-Shackleton, Blanchard, & Gibbs, 2003).
Arguably, while most colour polymorphisms identified in birds tend to be
protein-coding changes, the possibility for a wide variety of more
quantitative loci defining colour do exist (Johnsson, Jonsson,
Andersson, Jensen, & Wright, 2016; Roulin & Ducrest, 2013). The array
of quantitative colour-loci is evident among bird taxa, because in those
organisms coloration is linked to plumage. Feathers themselves have a
variety of different functions that first emerged duringSauropsida evolution, such as flight, protection, and mechanical
insulation via keratin-based micro-structures:(barbules, micro-barbules
and hooklets) (Benton, Dhouailly, Jiang, & McNamara, 2019). Melanin
deposition further enhances a feather’s properties: melanin-packed
melanosomes (specialized organelles in animal’s cells where melanin
synthesis and storage occur) grants thickness and resilience to
torsion/tension, provides chemical defences by oxidizing or reducing
free oxygen radicals and participate in thermoregulation by absorption
of light and conversion into heat (D’Alba & Shawkey, 2019; Field et
al., 2013). Given the multiple functionalities of the feather and
melanin, it is plausible to expect the emergence of evolutionary
trade-offs when environmental conditions favour divergent phenotypes
(Shoval et al., 2012; Terrill & Shultz, 2023). Not surprisingly,
trade-offs related to feather/melanin functions have been shown to occur
in birds in signalling and social recognition (Sheehan & Bergman,
2016), thermoregulation (Galván, Rodríguez‐Martínez, & Carrascal,
2018), and resource allocation particularly in the production of the
costly pheomelanin pigment (McNamara et al., 2021). From a molecular
point of view, putative trade-offs would translate in co-variation
between melanin-based phenotypes and loci involved in functions that
mitigate the colour effect.
About one-third of owl species are colour polymorphic and typically
harbor a higher ratio of the light colour morph in higher latitudes
consistent with Gloger’s rule (Galeotti, Rubolini, Dunn, & Fasola,
2003; Passarotto, Rodríguez‐Caballero, Cruz‐Miralles, & Avilés, 2022).
Particularly, the tawny owl is a nocturnal bird of prey exhibiting a
melanin-based colour polymorphism ranging between pale-grey and a
pheomelanin-dominated reddish-brown (Brommer, Ahola, & Karstinen,
2005). The inheritance pattern of colour polymorphism in this species is
apparently consistent with a Mendelian one locus-two allele system, is
independent of sex and age and is mediated by the irreversible
accumulation of melanin on plumage feathers during feather development
(Brommer et al., 2005). On a large geographical scale, the grey morph is
more prevalent than the brown in environments with colder and less rainy
winters and in the northern boreal zone. The high prevalence of the grey
tawny owl morph in northern latitudes may reflect a selective advantage
via background matching (crypsis) under snowy conditions to increase
protection against intra-guild predation and anti-predator mobbing
(Koskenpato, Lehikoinen, Lindstedt, & Karell, 2020). Indeed, morph
frequencies over ecological timescales were shown to be skewed towards
grey as a function of severe, snow-heavy winter conditions in higher
latitudes (Brommer et al., 2005; Karell, Ahola, Karstinen, Valkama, &
Brommer, 2011). Snow-covered environments might pose two potential costs
for the maintenance of colour polymorphism in tawny owls. The first
derives from the fact that reduced melanin deposition confers a putative
selective advantage in the form of crypsis, decreased pigment-based
thermoregulation is plausible since melanin-rich morphs are expected to
absorb more sunlight and thus reduce the cost of homeothermy – though
daylight perching might also occur in the shade (Galván et al., 2018;
Roulin, 2014). The second derives from the scarcity of resources during
extreme winters which might hinder the production of pheomelanin in the
spring breeding season due to the unavailability of protein (Britton &
Davidowitz, 2023). In relation to the former, Koskenpato et al (2016)
found a higher density of insulative barbules in feathers of grey (no
pheomelanin) tawny owls (Koskenpato, Ahola, Karstinen, & Karell, 2016),
which to the best of our knowledge remains the only evidence suggesting
a trade-off between form (colour) and function of feathers.
In this study, we present the first pair of draft assemblies of the
tawny owl genome coupled with a holistic genome screening approach to
identify genome-wide structural variation associated with colour
phenotypes. We capitalized on a decade of data collection from a tawny
owl population comprised of 96 progenitors and their respective
offspring (for a total of 370 individuals,) all of which have been
individually phenotyped for colour. Pedigree availability allowed us to
undertake a series of genome-wide association studies while controlling
for heritability and population structure. Our objectives were (1) to
identify the genetic basis of colour polymorphism in tawny owls and (2)
to inspect the molecular basis of putative melanin-phenotype
associations by associating the genomic information with the extensive
phenotypic ecological data available for this study system in southern
Finland. Overall, identifying the genomic basis of colour and
colour-associated phenotypic traits is fundamental to understand
evolution and maintenance of colour polymorphism.