Cities as genetic refugia: rural selection maintains Mc1R pigmentation cline in squirrels despite an urban bottleneck

Abstract How adaptive and non-adaptive processes interact to shape urban–rural clines remains a fundamental question in urban evolution. Here, we integrate whole-genome sequencing, demographic modelling and forward-in-time simulations to test how natural selection in urban and rural environments interacts with non-adaptive forces to maintain a pigmentation cline in eastern grey squirrels (Sciurus carolinensis). Coat colour polymorphism (grey versus melanic) is determined by a 24 bp deletion in the melanocortin-1 receptor gene (Mc1R). In Syracuse, New York (USA), squirrel melanism decreases from 50% in the city to less than 10% in rural forests. Our analyses revealed a strong urban bottleneck, yet urban–rural divergence at Mc1R was far greater than the genomic background, suggesting the pigmentation cline is maintained by selection. To estimate habitat-specific selection coefficients at Mc1R, we used forward simulation models using data on individual fitness and allele frequencies. Both models produce a surprising, yet consistent finding: strong selection against the melanic morph in rural forests and near neutrality in the city. We also detected elevated divergence between colour morphs at SMYD3—a gene implicated in adaptive thermogenesis—raising a possible link between pigmentation and thermal performance. Overall, our findings demonstrate that selection outside the city can be sufficient to maintain urban–rural clines amid urban bottlenecks, suggesting cities can preserve genetic diversity that would otherwise be lost in rural landscapes.