Comparison of the Population Genetic Structures of Borrelia Virulence Gene ospC and a Putatively Neutral Intergenic Spacer

ABSTRACT Borrelia afzelii , a major causative agent of Lyme borreliosis in Europe, exhibits high polymorphism of the outer surface protein C ( ospC ) gene, which facilitates evasion of the vertebrate immune response. Its extensive polymorphism has been hypothesized to result from balancing selection imposed by host immunity, which predicts reduced population genetic structure at ospC relative to neutrally evolving genomic regions. To test this prediction, we genotyped ospC and the rrs–rrl intergenic spacer (IGS) in B. afzelii samples from questing Ixodes ricinus ticks and bank voles ( Clethrionomys glareolus ) sampled across nine populations in Poland between 2020 and 2024. Among‐population differentiation was consistently lower at ospC than at IGS, and while IGS exhibited a significant isolation‐by‐distance pattern, no such signal was detected for ospC . Collectively, these findings support the hypothesis that balancing selection maintains ospC polymorphism and counteracts geographic differentiation, with implications for the evolutionary epidemiology of Lyme borreliosis in Europe.