Diverse species assemblages theoretically partition along multiple resource axes to maintain niche separation between all species. Temporal partitioning has received less attention than spatial or dietary partitioning but may facilitate niche separation when species overlap along other resource axes. We conducted a broad-scale acoustic study of the diverse and heterogeneous Great Smoky Mountains National Park in the Appalachian Mountains. Between 2015 and 2016, we deployed acoustic bat detectors at 50 sites (for a total of 322 survey nights). We examined spatiotemporal patterns of bat activity (by phonic group: Low, Mid, and Myotis) to test the hypothesis that bats partition both space and time. Myotis and Low bats were the most spatially and temporally dissimilar, while Mid bats were more general in their resource use. Low bats were active in early successional openings or low-elevation forests, near water, and early in the evening. Mid bats were similarly active in all land cover classes, regardless of distance from water, throughout the night. Myotis avoided early successional openings and were active in forested land cover classes, near water, and throughout the night. Myotis and Mid bats did not alter their spatial activity patterns from 2015 to 2016, while Low bats did.

We observed disparate temporal activity peaks between phonic groups that varied between years and by land cover class. The temporal separation between phonic groups relaxed from 2015 to 2016, possibly related to changes in the relative abundance of bats or changes in insect abundance or diversity. Temporal separation was more pronounced in the land cover classes that saw greater overall bat activity. These findings support the hypothesis that niche separation in diverse assemblages may occur along multiple resource axes and adds to the growing body of evidence that bats partition their temporal activity.