Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls

RESEARCH ARTICLE Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls Kerstin Musolf1,2*, Stefanie Meindl1, Angela L. Larsen3, Matina C. Kalcounis-Rueppell3, Dustin J. Penn1 1 Department of Integrative Biology and Evolution, Konrad Lorenz Institute of Ethology, University of Veterinary Medicine, Vienna, Austria, 2 Department of Biology, Brooklyn College, Brooklyn, New York, United States of America, 3 Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina, United States of America * Abstract OPEN ACCESS Citation: Musolf K, Meindl S, Larsen AL, KalcounisRueppell MC, Penn DJ (2015) Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls. PLoS ONE 10(8): e0134123. doi:10.1371/ journal.pone.0134123 Editor: William J. Etges, University of Arkansas, UNITED STATES Received: January 26, 2015 Accepted: July 6, 2015 Published: August 26, 2015 Copyright: © 2015 Musolf et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The research was supported by a grant to Dustin J. Penn from the Austrian Science Fund (FWF P 28141-B25). Angela L. Larsen developed this publication under STAR Fellowship Assistance Agreement no. FP-91763501-0 awarded by the U.S. Environmental Protection Agency (EPA). The study has not been formally reviewed by EPA, and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views expressed in this Male house mice (Mus musculus) emit ultrasonic vocalizations (USVs) during courtship, which attract females, and we aimed to test whether females use these vocalizations for species or subspecies recognition of potential mates. We recorded courtship USVs of males from different Mus species, Mus musculus subspecies, and populations (F1 offspring of wild-caught Mus musculus musculus, Mus musculus domesticus (and F1 hybrid crosses), and Mus spicilegus), and we conducted playback experiments to measure female preferences for male USVs. Male vocalizations contained at least seven distinct syllable types, whose frequency of occurrence varied among species, subspecies, and populations. Detailed analyses of multiple common syllable types indicated that Mus musculus and Mus spicilegus could be discriminated based on spectral and temporal characteristics of their vocalizations, and populations of Mus musculus were also distinctive regardless of the classification model used. Females were able to discriminate USVs from different species, and showed assortative preferences for conspecific males. We found no evidence that females discriminate USVs of males from a different subspecies or separate populations of the same species, even though our spectral analyses identified acoustic features that differ between species, subspecies, and populations of the same species. Our results provide the first comparison of USVs between Mus species or between Mus musculus subspecies, and the first evidence that male USVs potentially facilitate species recognition. Introduction Ultrasonic vocalizations (USVs) in rodents have been recognized for more than 100 years (in theory: [1], first recording [2]), and recent analyses of the spectrographic features of the USVs of male laboratory house mice (Mus musculus) have revealed surprising complexity [3]. Male house mice emit USVs during courtship, and their vocalizations may facilitate mating [4]. PLOS ONE | DOI:10.1371/journal.pone.0134123 August 26, 2015 1 / 24 Female Preferences for Species-Specific Male USVs publication are solely those of the authors, and EPA does not endorse any products or commercial services mentioned in this publication. Competing Interests: The authors have declared that no competing interests exist. Male USVs are attractive to females (wild [5] and laboratory mice [6]), and it has been suggested that females use USVs to obtain information about male quality and compatibility [7]. For example, females may use male USVs for individual recognition, kin recognition, and inbreeding avoidance. In wild-derived house mice, several features of male USVs are more similar between brothers than among non-siblings [8], and females show preferences for playbacks of USVs from unrelated males compared to their siblings [5]. Similarly, females may also use male USVs to avoid interspecific or subspecific hybridization [7, 9]. Courtship vocalizations in many species have been found to contain species-specific calls and are used to avoid hybridization [10]. Recent evidence suggests that vocalizations facilitate species recognition in Neotropical singing mice (Scotinomys spp. [11, 12]). Therefore, we aimed to test whether female house mice (M. m. musculus) recognize and show assortative preferences for the USVs of males from their own species compared to other mice—including a closely related species, subspecies, and hybrids, and whether spectral and temporal features of the USVs of these groups differ. Previous studies on house mice suggest that male courtship USVs function to coordinate mating behavior (reviewed in [7]). USVs appear to signal male sexual arousal [13], help to keep females in close proximity during courtship [4], and facilitate copulation behavior [14]. Females potentially use male USVs to recognize male social status, as dominant males call at higher rates than subordinates [15, 16] and social defeat leads to reduced calling [17]. The USVs of wild male house mice also contain signatures of individuality [8] and females prefer the calls of unrelated males versus siblings (kin recognition) [5]. Male USVs contain strainspecific features, even when cross-fostered [18–20], and females learn the USVs of their parents and prefer songs that differ from their parents’ (classical negative imprinting) [21]. Thus, female mice may use male USVs, in addition to chemical signals (e.g., [22–24]), to select their mates. However, there has been no test of whether female house mice use male USVs for species or subspecies recognition or whether they show assortative preferences (differential attraction to calls of conspecific males) [7, 9]. Furthermore, USV studies have primarily been conducted on inbred laboratory strains and there have been no comparisons of USVs among Mus species. More studies on wild rodents are needed because their vocalizations show more complexity than their laboratory counterparts (Peromyscus californicus [25], M. m. musculus [26]). There are more than 23 species in the Mus genus and the phylogeny of the group is well resolved [27, 28]. M. musculus, the species from which laboratory (...truncated)