Gene flow of small mammals is inhibited by highways and the urbanized habitat matrix in a large urban forest fragment

Abstract Urbanization dramatically modifies landscapes, fragments habitats, and is spreading rapidly. Urbanization can inhibit gene flow in a variety of species, but identifying the impact of specific landscape features on gene flow is difficult without prior knowledge of which landscape features may represent barriers to dispersal. We employed a landscape genetics approach to investigate the effects of urbanization on dispersal and gene flow in the swamp rabbit ( Sylvilagus aquaticus ), a forest‐obligate species, within the largest urban forest fragment in the United States, the Great Trinity Forest (GTF) in Dallas, TX. We employed a range of spatially explicit genetic analyses to investigate how landscape features, such as roads and the urban matrix, influence dispersal. We used noninvasive techniques to obtain 367 swamp rabbit fecal DNA samples representing 80 individuals from 2019 to 2021. We used traditional population genetics approaches, including STRUCTURE, F ST , and genetic spatial autocorrelation, to quantify genetic differentiation within our study population. We then employed a resistance to dispersal mapping landscape genetics approach to link observed genetic differentiation in swamp rabbits to specific landscape features. Global F ' ST (0.182) showed significant subdivision within the population, and spatial autocorrelation analysis showed that individuals were genetically more similar than expected at distances under 3000 m and less similar than expected at distances over 7000 m. An examination of the spatial relationships between isolation by distance (IBD) residuals and urban landscape features revealed that the urban matrix inhibited gene flow, and groups of individuals separated by large highways were less related than expected based on the population's IBD trend. Swamp rabbits in the GTF, a single large and continuous forest fragment, showed equivalent genetic differentiation to other small mammals in widely disparate habitat fragments, revealing that even the largest urban habitat fragments may be unable to conserve urban‐avoiding species when they are bisected by highways and surrounded by a dense urban matrix.