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

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.