Roadkill is a serious threat to biodiversity conservation especially when roads are near natural habitats of wildlife, such as the Emas National Park (ENP), a Protected Area in Mid-west Brazil in the Cerrado Biome. We aimed to identify the species killed on a stretch of the GO-341 highway that is tangent to the ENP. We investigated if roadkills were influenced by seasonality, sugar cane harvest and by the activity pattern of the animals (diurnal/nocturnal). We also analyzed if roadkills were aggregated in space, where the roadkill hotspots were located, if they were influenced by seasonality, and if they were different for the most abundant species. The highway was monitored with a vehicle, in the morning, at a speed of 40 to 60 km/hour, daily, with two observers. We covered a total of 4,230 km during the 90-day monitoring period, which included dry and rainy seasons. We recorded 132 wild animals’ roadkills: 67 birds (51 %), 60 mammals (45 %) and 5 reptiles (4 %). We identified 22 vertebrate species, including 13 birds, six mammals and three reptiles. The roadkill rate was 0.03 animals/km/day. During the dry season, the blue-and-yellow macaw (Ara ararauna) had higher roadkill rates. The crab-eating fox (Cerdocyon thous) was the only species with higher roadkill rates during the sugarcane harvest. Most birds killed had diurnal habits, for mammals, no difference was observed concerning the activity pattern. Bird roadkills were aggregated considering the entire data set, as well as for the dry and rainy seasons separately. Mammal roadkills were aggregated only when considering the entire data set. The location of bird roadkill hotspots differed between seasons. The roadkill hotspot location of birds and mammals highly overlapped those observed for the two most abundant species of these taxa, respectively, Ara ararauna and Euphractus sexcinctus.
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