Limited evidence for genetic differentiation or adaptation in two amphibian species across replicated rural-urban gradients.

Urbanization leads to complex environmental changes and poses multiple challenges to organisms. Amphibians are highly susceptible to the effects of urbanization, with land use conversion, habitat destruction, and degradation ranked as the most significant threats. Consequently, amphibians are declining in urban areas, in both population numbers and abundance, however, the effect of urbanization on population genetic parameters remains unclear. Here, we studied the genomic response to urbanization in two widespread European species, the common toad (26 localities, 480 individuals), and the smooth newt (30 localities, 516 individuals) in three geographic regions: southern and northern Poland and southern Norway. We assessed genome-wide SNP variation using RADseq (ca. 42 and 552 thousand SNPs in toads and newts, respectively) and adaptively relevant major histocompatibility complex (MHC) class I and II genes. The results linked most of the genetic differentiation in both marker types to regional (latitudinal) effects, which also correspond to historical biogeography. Further, we did not find any association between genetic differentiation and level of urbanization at local scales for either species. However, urban smooth newts, but not toads, have lower levels of within-population genome-wide diversity, suggesting higher susceptibility to the negative effects of urbanization. A decreasing level of genetic diversity linked to increasing urbanization was also found for MHC II in smooth newts, while the relationship between MHC class I diversity and urbanization differed between geographic regions. We did not find any effects of urbanization on MHC diversity in the toad populations. Although two genetic environment association analyses of genome-wide data, LFMM and BayPass, revealed numerous (219 in and 7040 in ) SNPs statistically associated with urbanization, we found a marked lack of repeatability between geographic regions, suggesting a complex and multifaceted response to natural selection elicited by life in the city.