Introduction
Coloration is arguably one of the most fascinating traits in
evolutionary biology (Salis, Lorin, Laudet, & Frédérich, 2019). The
(absolute) fitness value attributed to colour-based phenotypes are more
than often easily quantified, rendering these types of biological
systems extremely enticing to those who wish to unveil the mechanisms of
adaptive evolution. Maintenance and evolution of colour polymorphism is
unequivocally 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 certainly 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
hair or skin to a deposition balance between eumelanin and pheomelanin,
which are forms of a single pigment type - 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, recently coined as
melanin-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), though 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).
Indeed, the array of quantitative colour-loci is particularly evident in
birds, organisms where coloration is unequivocally linked to the
structure on which it manifests: the feather. The feather itself has a
variety of different functions that first emerged duringSauropsida evolution, such as flight, protection, and mechanical
insulation via keratin-based micro-structures such as 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, the provision of chemical defences to the
organism 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). Indeed, 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
functions that mitigate the colour effect.
Owls are the most color polymorphic avian taxa (Galeotti, Rubolini,
Dunn, & Fasola, 2003). About one-third of owl species are color
polymorphic and typically harbor a higher ratio of the light colour
morph in higher latitudes consistent with Gloger’s rule (Passarotto,
Rodríguez‐Caballero, Cruz‐Miralles, & Avilés, 2022). The tawny owl is a
nocturnal bird of prey whose populations exhibit a remarkable
melanin-based colour polymorphism ranging between pale-grey and a
pheomelanin-dominated reddish-brown (Brommer, Ahola, & Karstinen,
2005). The inheritance pattern of color polymorphism in this species is
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 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 expected since melanin-rich morphs are expected to
absorb more sunlight and thus reduce the cost of homeothermy (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 (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 objective is primarily to
identify the genetic basis for colour polymorphism in tawny owls. Since
our study system in southern Finland is prone to extreme winter
conditions, we also predict we will identify genomic regions of
biological function related to adaptations to a cold environment, and
that these will co-vary with melanin-based phenotypes. Overall,
identifying the genomic basis of colour and colour-associated traits is
fundamental to our understanding not only of evolution and the
maintenance of colour polymorphism but will also shed light on
evolutionary trade-offs related to melanin phenotypes and fitness
traits.