19In many species, territory defense is thought to be one of the primary functions of acoustic 20 communication. North American red squirrels are a territorial species in which 'rattles' have long 21 been thought to be the principal signal communicating territory ownership. These vocalizations 22 have been assumed to deter intruders, thus reducing energy costs and the risk of injury associated 23 with direct aggressive interactions. However, this hypothesis has not been directly tested. Here we 24 used a speaker occupation experiment to test whether red squirrel rattles function to deter 25 conspecific rivals. We studied 29 male squirrels and removed each individual from his territory 26 twice in a paired design. During the experimental treatment we simulated the owner's presence after 27 its removal by broadcasting the owner's rattle from a loudspeaker at the center of the territory once 28 every seven minutes. During the control treatment the territory was left in silence after the 29 temporary removal of the owner. We found that the presence of a speaker replacement reduced the 30 probability of intrusion by 34% and increased the latency to first intrusion by 7%, providing support 31 for the hypothesis that rattles play an active role in reducing intrusion risk. However, intrusions 32 were not completely averted by the speaker replacement, indicating that vocalizations alone are not 33 sufficient without other cues of the territory owner. 34 2 35 Keywords 36 mammalian vocalizations, acoustic communication, territoriality, speaker occupation, red squirrel 37 38 39 Vocal communication is thought to have several principle functions, including territorial 40 defence (Catchpole, 1982; Catchpole & Slater, 1995; Bradbury & Vehrencamp, 2011). While the 41 role of vocalizations in repelling conspecific rivals is typically well accepted, this function has 42 rarely been directly demonstrated. Evidence supporting the functionality of vocalizations as a 43 deterrent for intruders has come mostly through indirect means, via observational and correlational 44 studies in the field, rather than direct experimental tests of functionality. For example, vocalizations 45 commonly observed in association with intrusion events or aggressive interactions among 46 61 2008). By inducing an aggressive reaction in territory owners, the use of playbacks can effectively 62 demonstrate that vocalizations function in immediate territorial confrontations. However, by 63measuring the response of owners, rather than intruders, these studies fail to clarify whether 64 vocalizations induce avoidance and function to keep conspecifics off the territory, even when 65 confrontations are not imminent.
66Muting and speaker occupation are two experimental designs that have been used in 67 songbirds to test the hypothesis that acoustic signals function to deter territory intrusions. In muting 68 349 long-term access to her trapline. A special thank you to J. Robertson and S. Sonnega for assistance