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ABSTRACT Cities impose unique selection pressures on wildlife and generate clines in phenotypic traits along urban–rural gradients. Roads are a widespread feature of human‐dominated landscapes and are known to cause direct wildlife mortality; however, whether they act as a selective force influencing phenotypic trait variation along urban–rural gradients remains unclear. This study tested the hypothesis that roads influence natural selection of coat color in the eastern gray squirrel (Sciurus carolinensis), a species with two distinct coat colors: a gray morph that is common in all areas and a melanic morph more prevalent in urban areas than in rural ones. Vehicular collisions are a significant cause of mortality in eastern gray squirrels, with the melanic morph more visually conspicuous on roads and more easily detected and avoided by drivers than the gray morph. Standardized road cruise surveys along an urbanization gradient in Syracuse, New York, USA, revealed that the prevalence of melanism among living squirrels in Syracuse was negatively related to distance from the city center, whereas there was no urban–rural cline in melanism among road‐killed individuals, with the melanic morph underrepresented among road‐killed squirrels by up to 30% along the urbanization gradient. An examination of the prevalence of each color morph on and off road surfaces in a range‐wide compilation of > 100,000 photographs ofS. carolinensisalso indicated that the melanic morph was underrepresented among road‐killed squirrels imaged. Our study highlights vehicular collisions as an important source of natural selection on phenotypic traits, suggesting a potential role in shaping patterns of urban evolution and contributing to the maintenance of urban–rural clines. 

Abstract Urbanization is a persistent and widespread driver of global environmental change, potentially shaping evolutionary processes due to genetic drift and reduced gene flow in cities induced by habitat fragmentation and small population sizes. We tested this prediction for the eastern grey squirrel (Sciurus carolinensis), a common and conspicuous forest‐dwelling rodent, by obtaining 44K SNPs using reduced representation sequencing (ddRAD) for 403 individuals sampled across the species' native range in eastern North America. We observed moderate levels of genetic diversity, low levels of inbreeding, and only a modest signal of isolation‐by‐distance. Clustering and migration analyses show that estimated levels of migration and genetic connectivity were higher than expected across cities and forested areas, specifically within the eastern portion of the species' range dominated by urbanization, and genetic connectivity was less than expected within the western range where the landscape is fragmented by agriculture. Landscape genetic methods revealed greater gene flow among individual squirrels in forested regions, which likely provide abundant food and shelter for squirrels. Although gene flow appears to be higher in areas with more tree cover, only slight discontinuities in gene flow suggest eastern grey squirrels have maintained connected populations across urban areas in all but the most heavily fragmented agricultural landscapes. Our results suggest urbanization shapes biological evolution in wildlife species depending strongly on the composition and habitability of the landscape matrix surrounding urban areas.