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

Abstract

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.

Fig 1. Spectrograms of the seven ultrasonic syllable types.

Syllable types are classified according to spectrographic parameters, e.g., start, end, and center frequency, frequency at peak energy and duration (details in Methods). (A) Frequency Upsweep (B) Frequency Downsweep (C) Constant Modulated (D) U-Shaped (E) U-Shaped Inverted (F) 1-Frequency-Step (G) 2-Frequency-Step

Fig 2. Latency to vocalize of males of different Mus populations.

N = 10, except for Hybrid 1 = 5, Hybrid 2 = 7 and M.m.d. = 4. Significant differences were observed among populations (p = 0.026). The box represents the interquartile range that contains the middle 50% of values. The thick line across the box indicates the median. Upper and lower whiskers limits are set to 1.5 x interquartile range above and below the third and first quartile. Outliers shown as circles. The asterisk represents significance at a level of p < 0.05.

Fig 3. USV repertoires of different Mus populations based on the first 100 syllables.

N = 10, except for Hybrid 1 = 5, Hybrid 2 = 7 and M.m.d. = 4. Syllable type ‘Frequency Upsweep’ is the most common type with a proportion of on average 56.6% (± 21.35 SD) of the repertoire. Syllable types ‘U-shaped’, ‘U-shaped Inverted’ and ‘2-Frequency Step’ were not emitted by all populations.

Fig 4. Classification plot of the first two linear discriminant axes for DFA on reduced data set (6 PC variables and duration) to classify all 8 populations.

For interpretation, the proportion of the data set explained by each axis is given and the standardized co-efficient with the highest absolute values are presented.

Fig 5. Female preferences for male USV playbacks.

Time (s) females (N = 20 except trial 2: M.m.m.1 vs M.m.m.2, N = 19) spent at fence / speaker zone in proximity to male USV playbacks. The box represents the interquartile range that contains the middle 50% of values. The thick line across the box indicates the median. Upper and lower whiskers limits are set to 1.5 x interquartile range above and below the third and first quartile. Outliers shown as circles. The asterisk represents significance at a level of p < 0.05.