Discussion
The maintenance and evolution of colour polymorphism remains one of the
most appealing yet elusive evolutionary questions. Since diversity in
life history strategies and complex traits have shown to segregate with
colour-defined phenotypes, it is plausible to expect evidence of those
associations also at a molecular level. Here we report the first
assemblies of the tawny owl genome and a comprehensive
genome-wide-association study leading to the discovery of structural
variation associated with colour polymorphism – though not on the
traditional M1CR gene.
Suggestion of a molecular basis of food intake, starvation
response and energy homeostasis
The most obvious target for the genetic regulation of coloration in
melanin-based pigmentation is the coding sequence of the geneMC1R . Contrary to general expectations, comparisons between
coding sequences of grey and brown tawny owl revealed MC1R to be
100% conserved between morphs. These observations are however not
exceptional, as other avian species exhibiting equally remarkable colour
polymorphisms also show conservation of MC1R coding region
(Avilés et al., 2019; Hoffman et al., 2014; MacDougall-Shackleton et
al., 2003). Among the investigated genes that are to some extent
involved in the melanin-production pathway, only the melanin
concentration hormone receptor (MCHR ) exhibits non-synonymous
substitutions between colour morphs. A direct and functional
relationship between MCHR polymorphism and melanin pigmentation
has been found exclusively in teleost fishes, specifically trout and
salmonids (Diniz & Bittencourt, 2019). Among mammal and avian taxa
alike, the functionality of this neuromodulator is rather associated
with fasting control and energy balance, physiological traits plausible
to be under selection in food limitation conditions (Cui et al., 2017).
In the genome-wide association study (GWAS) we expanded the molecular
screen to an original dataset of 370 individuals and respective
pedigrees. Correcting for both genetic relationship and population
substructure the GWAS suggested additional evidence for a scenario where
resource-related physiological responses are colour-morph specific, as
predicted by Karell et al (2011). Associations with colour phenotypes
revealed two candidate loci, FAM135A and FTCD, whose
biological functions were shown to be linked to the lipid metabolism
pathway, fat storage mechanisms, and maintenance of homeostasis under
starvation in both pigs and chickens in captivity (Poleti et al., 2018;
Zhang et al., 2021). We propose that in our tawny owl population the
putative biological functionality of these candidate loci are better
contextualized in the harsh winter conditions in Southern Finland. At
60° N latitude, Finnish tawny owls experience snowfall periods that
usually start in December and generally peaking in January and February
with snow-coverage usually lasting until the months prior to the onset
of the tawny owl’s breeding season (Karell et al., 2011). Previous work
on the study population used here have shown that these extreme
conditions do impose a strong selective pressure against brown tawny
owls while resulting in a relatively higher survival rate of grey tawny
owls following snow-heavy winters (Karell et al., 2011). Since we find a
big effect size of these candidate loci in predicting grey coloration
(up to 100% in some genotype combinations) our findings suggest a
genetic regulatory mechanism for grey coloration in this population as
well as some of the loci underlying the expected adaptation to their
local environment.
The relevance of an adapted lipid metabolism (whether it would be
degradation, accumulation, or deposition) to survival in cold
environments has been reported multiple times across taxa (Blem, 1976;
Lucassen, Koschnick, Eckerle, & Portner, 2006). To the best of our
knowledge, this study is unique in demonstrating a genetic link between
lipid metabolism and melanin-pigmentation in a natural population,
effectively rendering the tawny owl system as the first where the
molecular basis of intraspecific melanin-associated phenotypes have been
proposed.
A brief exploration of signatures of selection – via HWE tests and
temporal shifts of candidate loci’s genotype frequencies – showed that,
when they did occur, deviations from HWE were associated with an excess
of homozygotes for the most common allele in either locus implying that
selection appears to be against grey-coated individuals. Because
ecological field studies rather reported a strong selection against
brown individuals in harsh winter conditions, putative selection against
grey morphs could be interpreted at the light of milder northern winters
associated with ongoing climate warming(Räisänen, 2021).
Lighter plumage colour and putative trade-offs in heat
absorption mechanisms
While the advantage of lighter plumage coloration in snow-covered
environments might be expected under the background matching hypothesis,
it is undeniable that less melanin pigmentation reduces the integument
capacity to transform UV radiation into heat (Margalida, Negro, &
Galván, 2008). Tawny owl genome analyses show that the architecture
surrounding the candidate loci FTCD contains both a collagen
(Col6a2 ) and a β-keratin gene (BISK1 ), both playing a key
role in integument micro-structure (Kowata et al., 2014). Of particular
relevance is the identification of BISK1 in the tawny owl genome.
This novel copy of a β-keratin gene has only been recently identified in
the chicken, and because its expression is confined to the barbule cells
of that species’ contour feathers, it is suggested to be relevant for
the formation of micro-barbules in pennaceous feathers – important
insulation structures (Kowata et al., 2014). We hypothesize that the
conservation of the Col6a2 -FTCD-B1SK1 region in tawny owls
and the identification of a candidate loci associated with plumage
colour also indicates the molecular basis of a trade-off, hypothetically
linked to the grey phenotype. While there is empirical evidence for
morphological differences between morphs where grey tawny owls have a
higher number and density of barbules than brown morphs (Koskenpato et
al., 2016), it is unknown whether this morphological difference in
feather structure translates into morph-specific net insulative
properties..