Roadkill is one of the main threats associated with highways, directly contributing to the depletion of natural populations. Here we establish the effect of landscape, seasonality and climatic variables on roadkill of wild vertebrates on two highways associated with Pedra Azul State Park (PEPAZ), state of Espírito Santo, southeastern Brazil. The highways BR-262 and ES-164 (20 km stretch in each road) were sampled monthly, between January and December 2015, totaling 60 days sampled/stretch. A total of 355 roadkilled vertebrates (Amphibia = 10; Reptilia = 40; Aves = 155; Mammalia = 149; undetermined = 1) distributed among at least 82 taxa were recorded (Amphibia = 4; Reptilia = 11; Aves = 47; Mammalia = 20). The roadkill rate was equivalent between highways, but birds were the most affected group on the ES-164 and mammals on the BR-262. A greater number of records were obtained during the rainy season for both highways. Temperature had a positive influence on the monthly roadkill rate, but no influence was observed from precipitation. The distance from water bodies and the type of land use and occupation influenced the roadkill records distribution. Roadkill of mammals was associated with areas of overgrown vegetation; birds with agricultural crops and built-up areas; and reptiles marginally with pasture areas. The effect of the highways was complementary because they affected the vertebrate classes differently. This could have severe impacts on biodiversity, highlighting that PEPAZ is part of an important ecological corridor of the Atlantic Forest in Espírito Santo. Speed control and installation of warning signs and wildlife crossing mechanisms are recommended to promote the long-term conservation of the regional biota.
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