Acoustic divergence is a fundamental component of signal-based communication in animals, including humans, and has major implications for individual recognition, mate choice, and speciation. Bats have recently become an important model system for investigating acoustic communication due to their structurally complex vocal repertoires. This study explored communication calls of greater horseshoe bats ( Rhinolophus nippon) to determine whether geographically structured dialects occur and to identify factors associated with dialect formation. Communication calls from nine populations distributed across three regions were analyzed, and 12 syllable types shared among populations were identified. The associations of dialect differentiation with acoustic, climatic, morphological, and genetic distances, together with the influence of geographic barriers, were investigated. Results demonstrated significant acoustic variation among populations and regions for each syllable type, and this divergence was driven by diverse factors. As bats typically emit syllables in combination, all shared syllable types were subsequently analyzed as an integrated dataset. At this broader level, acoustic distance showed significant correlations with both geographic and genetic distance. Causal modeling further revealed that geographic distance and geographic barriers exerted direct effects on dialect differentiation. The sampled populations were classified into three dialect regions: Northeast, Central-East, and Southwest. The Qinling Mountains formed the boundary between the Central-East and Southwest regions. These findings provide clear evidence for the existence of dialect structure in communication calls of greater horseshoe bats and identify geographic isolation as a major force in the formation and preservation of vocal divergence. This study advances current understanding of animal acoustic evolution and offers insight into mechanisms linking vocal diversification with the generation of biological diversity.

Acquiring clear acoustic signals is critical for the analysis of animal vocalizations. Bioacoustics studies commonly face the problem of overlapping signals, which can impede the structural identification of vocal units, but there is currently no satisfactory solution. This study presents a bi-directional long short-term memory network to separate overlapping echolocation-communication calls of 6 different bat species and reconstruct waveforms. The separation quality was evaluated using 7 temporal-spectrum parameters. All the echolocation pulses and syllables of communication calls in the overlapping signals were separated and parameter comparisons showed no significant difference and negligible deviation between the extracted and original calls. Clustering analysis was conducted with separated echolocation calls from each bat species to provide an example of practical application of the separated and reconstructed calls. The result of clustering analysis showed high corrected rand index (82.79%), suggesting the reconstructed waveforms could be reliably used for species classification. These results demonstrate a convenient and automated approach for separating overlapping calls. The study extends the application of deep neural networks to separate overlapping animal sounds.

Different categories of social calls may be used in a variety of behavioral contexts and may have different functions. Here, we show that Great Himalayan leaf-nosed bats emit two types of calls: bent upward frequency modulated (bUFM) calls emitted during agonistic interactions between two bats that have perched close to each other and stepped upward frequency modulated (sUFM) calls emitted by a roosting individual when a free-flying bat approached. There were significant differences in acoustic parameters between bUFM calls and sUFM calls. Moreover, we tested different choices of Hipposideros armiger to playback of bUFM calls and sUFM calls. The bats avoided the location of bUFM call playbacks independent of the simultaneous playback of echolocation pulses, silence, or sUFM calls. The bats also avoided approaching the side of the flight room where sUFM calls were broadcast when also presented with playbacks of echolocation pulses, but not when simultaneously presented with silence. The results suggest that bUFM calls may serve the function of territorial defense, while sUFM calls may convey warning information. Our results, therefore, imply that the two types of calls have different functions depending on behavioral context. These differences in function suggest that bats can detect context-dependent modifications of acoustic cues across the two types of social calls.

Previously, two studies have provided evidence that bats can use magnetic field cues for homing or roosting. For insectivorous bats, it is well established that foraging represents one of the most fundamental behaviors in animals relies on their ability to echolocate. Whether echolocating bats can also use magnetic cues during foraging remains unknown, however. Here, we tested the orientation behavior of Chinese noctules (Nyctalus plancyi) during foraging in a plus-shaped, 4-channel apparatus under different magnetic field conditions. To minimize the effects of spatial memory on orientation from repeated experiments, naïve bats were tested only once in each experimental condition. As expected, under geomagnetic field and a food resource offered conditions, the bats significantly preferred to enter the channel containing food, indicating that they primarily relied on direct sensory signals unrelated to magnetic cues. In contrast, when we offered food simultaneously in all four channels and minimized any differences in all other sensory signals available, the bats exhibited a clear directional preference to forage along the magnetic field direction under either geomagnetic field or a magnetic field in which the horizontal component was rotated by 90°. Our study offers a novel evidence for the importance of a geomagnetic field during foraging.

Many studies based on acute short-term noise exposure have demonstrated that animals can adjust their vocalizations in response to ambient noise. However, the effects of chronic noise over a relatively long time scale of multiple days remain largely unclear. Bats rely mainly on acoustic signals for perception of environmental and social communication. Nearly all previous studies on noise-induced vocal adjustments have focused on echolocation pulse sounds. Relatively little is known regarding the effects of noise on social communication calls. Here, we examined the dynamic changes in the temporal parameters of echolocation and communication vocalizations of Vespertilio sinensis when exposed to traffic noise over multiple days. We found that the bats started to modify their echolocation vocalizations on the fourth day of noise exposure, with an increase of 42-91% in the total number of pulse sequences per day. Under noisy conditions, the number of pulses within a pulse sequence decreased by an average of 17.2%, resulting in a significantly slower number of pulses/sequence (P < 0.001). However, there was little change in the duration of a pulse sequence. These parameters were not significantly adjusted in most communication vocalizations under the noise condition (all P > 0.05), except that the duration decreased and the number of syllables/sequences increased in 1 type of communicative vocalization (P < 0.05). This study suggests that bats routinely adjust temporal parameters of echolocation but rarely of communication vocalizations in response to noise condition.

Acoustic signals play a crucial role in transmitting information and maintaining social stability in gregarious animals, especially in echolocating bats, which rely primarily on biological sonar for navigating in the dark. In the context of foraging without relying on tactile, visual or olfactory cues, acoustic signals convey information not only on food but also on ownership and defense of resources. However, studies on such information remain fragmentary. In the present study, we aim to document the social vocal repertoire of Myotis macrodactylus at natural foraging sites. Multiple acoustic analyses and spectrographic classification revealed a rich foraging vocal repertoire comprising 6 simple syllables and 2 composites. Discriminant function analyses associated with a subset-validation procedure provided an optimal method to spectrographically classify all recorded sounds into different syllable types. Multidimensional scaling of median values of multiple parameters further confirmed notable differences among these syllables in a 3-D space. In addition, Euclidean distance analysis showed that there were some spectral similarities between specific social vocal syllables and feeding buzzes, which implied a potential jamming role. Altogether, the data indicate that bats at foraging sites under natural conditions used variant social vocalizations with different functions in addition to echolocation calls, providing supporting evidence for further work on the function and vocal mechanisms of acoustic communication in mammals.

For analysis of vocal syntax, accurate classification of call sequence structures in different behavioural contexts is essential. However, an effective, intelligent program for classifying call sequences from numerous recorded sound files is still lacking. Here, we employed three machine learning algorithms (logistic regression, support vector machine and decision trees) to classify call sequences of social vocalizations of greater horseshoe bats (Rhinolophus ferrumequinum) in aggressive and distress contexts. The three machine learning algorithms obtained highly accurate classification rates (logistic regression 98%, support vector machine 97% and decision trees 96%). The algorithms also extracted three of the most important features for the classification: the transition between two adjacent syllables, the probability of occurrences of syllables in each position of a sequence, and the characteristics of a sequence. The results of statistical analysis also supported the classification of the algorithms. The study provides the first efficient method for data mining of call sequences and the possibility of linguistic parameters in animal communication. It suggests the presence of song-like syntax in the social vocalizations emitted within a non-breeding context in a bat species.

Summary statement This study reveals the impact of anthropogenic noise on spectrally distinct vocalizations and the limitations of the acoustic masking hypothesis to explain the vocal response of bats to chronic noise. Abstract The acoustic masking hypothesis states that auditory masking may occur if the target sound and interfering sounds overlap spectrally, and it suggests that animals exposed to noise will modify their acoustic signals to increase signal detectability. However, it is unclear if animals will put more effort into changing their signals that spectrally overlap more with the interfering sounds than when the signals overlap less. We examined the dynamic changes in the temporal features of echolocation and communication vocalizations of the Asian particolored bat ( Vespertilio sinensis ) when exposed to traffic noise. We hypothesized that traffic noise has a greater impact on communication vocalizations than on echolocation vocalizations and predicted that communication vocalization change would be greater than echolocation. The bats started to adjust echolocation vocalizations on the fourth day of noise exposure, including an increased number of call sequences, decreased number of calls, and vocal rate within a call sequence. However, there was little change in the duration of the call sequence. In contrast, these communication vocalization features were not significantly adjusted under noise conditions. These findings suggest that the degree of spectral overlap between noise and animal acoustic signals does not predict the level of temporal vocal response to the noise.

Frequently, individuals of one species respond to alarm calls produced by another species. This form of interspecies communication can be critical for survival. How animals recognize and respond to heterospecific alarm calls is, however, still poorly understood. This is especially true for bats, which are highly vocal and possess a specialized auditory system. We hypothesized that, among bat species, acoustic similarity between heterospecific and conspecific alarm calls is a key factor that underlies call recognition. However, this ability could also be acquired via associative learning from prior exposure or result from phylogenetic relatedness among caller and receiver species, as shown by others. Here we focused on distress calls of bats as representing a certain type of alarm call. To test our hypothesis, we recorded distress calls of 11 bat species from four families at five different sites. We broadcast the recorded calls to three of these species (Rhino lophus sinicus, Myotis badius and Myotis laniger). For all three species, neither prior exposure (familiarity) nor phylogenetic relatedness explained the variation in the response strength, as measured by echolocation vocalizations of receiver species in response to playback of heterospecific distress calls. In contrast, acoustic similarity, especially in peak frequency, between conspecific and heterospecific distress calls, was positively correlated with response strength in the receiver species. Thus, our findings emphasize that, at least among bats, acoustic similarity is a key factor allowing recognition of heterospecific alarm calls. It remains unclear whether evolutionary, genetic and/or neurophysiological mechanisms entrain an individual to respond selectively to a specific acoustic feature. (C) 2018 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

All bats use acoustic signals for passive listening to ambient sounds and for intra- and inter-species communication. In addition, most bats have evolved an active sonar system that uses calls for exploring the environment acoustically. In the majority of bats, the larynx produces these echolocation pulses. Echolocation is absent in the family of Old World fruit bats except for one genus, Rousettus, which "reinvented" echolocation by producing sonar pulses using tongue clicks. This chapter first discusses how sounds are produced by the bat larynx by outlining its characteristic morphological features and detailing general sound production mechanisms, including non-linear features that play a key role in enabling echolocating bats to switch between echolocation and communication sounds. Subsequently, details are presented on the neuronal basis for sound production in echolocating bats by briefly discussing the neural innervation pattern of the larynx and how this affects spectral and temporal features of sounds, followed by a summary of the neuronal connection patterns and mechanisms within the brain stem as well as in higher-order brain structures. The chapter then explores the role of various forms of sensory feedback in sound production, such as auditory and somatosensory stimulation, and highlights how sound production is intertwined with other motor patterns, such as flying. The chapter also briefly explores the diversity in the shape of nose leafs and pinnae in different species of echolocating bats and the role that dynamic changes of such facial characteristics may play in echolocation. Finally, the tongue-click echolocation in Rousettus is discussed.

Egyptian fruit bats have good vision but can also accurately echolocate relying on their well-developed auditory system. We asked two questions: 1. Can these bats acquire a mental image of an object using vision (or hearing) alone? 2. If such a mental image has been formed, can it be transferred from one sensory modality (e.g., vision) to another (e.g., hearing)? We trained the bats to first localize a rewarded object (an X) that was presented pseudo-randomly at either the left or right side using either vision (group 1) or hearing (group 2) alone. After only a few weeks, all experimental bats were able to perform these tasks. We then had the bats discriminate between a rewarded object (the X) and an unrewarded object (a circle) using either vision (group 1) or hearing (group 2) alone. Again, all bats mastered this task after only a few weeks of training. Finally, we tested if the same bats could also discriminate the objects using the other sensory modality (group 1: hearing; group 2: vision). After only a few sessions, the bats performed correctly suggesting that the bats can indeed transform a mental image that they obtained using one sensory modality to another modality.

How animals, including mammals, can respond to and utilize the direction and intensity of the Earth’s magnetic field for orientation and navigation is contentious. In this study, we experimentally tested whether the Chinese Noctule, Nyctalus plancyi (Vespertilionidae) can sense magnetic field strengths that were even lower than those of the present-day geomagnetic field. Such field strengths occurred during geomagnetic excursions or polarity reversals and thus may have played an important role in the evolution of a magnetic sense. We found that in a present-day local geomagnetic field, the bats showed a clear preference for positioning themselves at the magnetic north. As the field intensity decreased to only 1/5th of the natural intensity (i.e., 10 μT; the lowest field strength tested here), the bats still responded by positioning themselves at the magnetic north. When the field polarity was artificially reversed, the bats still preferred the new magnetic north, even at the lowest field strength tested (10 μT), despite the fact that the artificial field orientation was opposite to the natural geomagnetic field (P<0.05). Hence, N. plancyi is able to detect the direction of a magnetic field even at 1/5th of the present-day field strength. This high sensitivity to magnetic fields may explain how magnetic orientation could have evolved in bats even as the Earth’s magnetic field strength varied and the polarity reversed tens of times over the past fifty million years.

One of the most efficient mechanisms to optimize signal-to-noise ratios is the Lombard effect - an involuntary rise in call amplitude due to ambient noise. It is often accompanied by changes in the spectro-temporal composition of calls. We examined the effects of broadband-filtered noise on the spectro-temporal composition of horseshoe bat echolocation calls, which consist of a constant-frequency component and initial and terminal frequency-modulated components. We found that the frequency-modulated components became larger for almost all noise conditions, whereas the bandwidth of the constant-frequency component increased only when broadband-filtered noise was centered on or above the calls' dominant or fundamental frequency. This indicates that ambient noise independently modifies the associated acoustic parameters of the Lombard effect, such as spectro-temporal features, and could significantly affect the bat's ability to detect and locate targets. Our findings may be of significance in evaluating the impact of environmental noise on echolocation behavior in bats.

Mutual recognition is the product of species coexistence, and has direct effects on survival and reproduction of animals. Bats are able to discriminate between sympatric different heterospecifics based on their echolocation calls, which has been shown both in free-flying and captive bats. To date, however, the factors that may determine the behavioral responses of bats to echolocation calls from sympatric heterospecifics have rarely been tested, especially under well-controlled conditions in captive bats. Hence, we aimed at tackling this question by performing playback experiments (habituation–dishabituation) with three horseshoe bat species within the constant-frequency bat guild, which included big-eared horseshoe bats ( Rhinolophus macrotis ), Blyth’s horseshoe bats ( Rhinolophus lepidus ), and Chinese horseshoe bats ( Rhinolophus sinicus ). We studied the behavioral responses of these three species to echolocation calls of conspecifics, to other two species, and to another heterospecifics bat, Stoliczka’s trident bat ( Asellisus stoliczkanus ), which also belongs to this guild. We found that the three rhinolophid species displayed a series of distinct behaviors to heterospecific echolocation but few to conspecific calls after habituation, suggesting that they may have been able to discriminate sympatric heterospecific echolocation calls from those of conspecifics. Interestingly, the behavioral responses to heterospecific calls were positively correlated with the interspecific overlap index in trophic niche, whereas call design had only a minor effect. This implies that the behavioral responses of these bats to heterospecific echolocation calls may be related to the degree of interspecific food competition.

Auditory feedback from the animal's own voice is essential during bat echolocation: to optimize signal detection, bats continuously adjust various call parameters in response to changing echo signals. Auditory feedback seems also necessary for controlling many bat communication calls, although it remains unclear how auditory feedback control differs in echolocation and communication. We tackled this question by analyzing echolocation and communication in greater horseshoe bats, whose echolocation pulses are dominated by a constant frequency component that matches the frequency range they hear best. To maintain echoes within this “auditory fovea”, horseshoe bats constantly adjust their echolocation call frequency depending on the frequency of the returning echo signal. This Doppler-shift compensation (DSC) behavior represents one of the most precise forms of sensory-motor feedback known. We examined the variability of echolocation pulses emitted at rest (resting frequencies, RFs) and one type of communication signal which resembles an echolocation pulse but is much shorter (short constant frequency communication calls, SCFs) and produced only during social interactions. We found that while RFs varied from day to day, corroborating earlier studies in other constant frequency bats, SCF-frequencies remained unchanged. In addition, RFs overlapped for some bats whereas SCF-frequencies were always distinctly different. This indicates that auditory feedback during echolocation changed with varying RFs but remained constant or may have been absent during emission of SCF calls for communication. This fundamentally different feedback mechanism for echolocation and communication may have enabled these bats to use SCF calls for individual recognition whereas they adjusted RF calls to accommodate the daily shifts of their auditory fovea.

Auditory feedback from the animal’s own voice is essential during bat echolocation: to optimize signal detection, bats continuously adjust various call parameters in response to changing echo signals. Horseshoe bats exhibit a particularly well-developed form of auditory feedback. Their echolocation pulses are dominated by a constant frequency component that matches the frequency range they hear best. To maintain echoes within this “auditory fovea,” horseshoe bats constantly adjust their echolocation call frequency depending on the frequency of the returning echo signal. This Doppler-shift compensation behavior represents one of the most precise forms of sensory-motor feedback known. When examining the Lombard effect in horseshoe bats, we found that noise had different effects on call amplitude and frequency rises indicating different neural circuits and/or mechanisms underlying these changes. Both, amplitude and frequency rises were extremely fast and occurred in the first call uttered after noise onset, suggesting that, in contrast to Doppler-shift compensation, the Lombard effect did not require any auditory feedback. Bats also possess a large repertoire of communication calls, which differ greatly from those emitted during echolocation. We compared the variability of echolocation pulses and one common type of communication signal and found fundamentally different feedback mechanisms for echolocation and communication.

Vocal communication plays an important role for individual recognition and male-female interaction during mating in greater horseshoe bats, especially in respect to mate fidelity, which ensures that the bats can maintain a stable social organization. Few studies, however, have addressed the calling behavior during copulating in bats. Here, we initially report the copulation vocalizations and behaviors of both male and female greater horseshoe bats. During copulation, the male assumed a dorsal position and arched his back, arming around the female using his feet and thumbs. The male repeatedly produced very short constant frequency (SCF) syllables with high intensity and repetition rate (male 1: 16.48±4.8 ms, male 2: 17.79±4.03 ms) when he tried to insert the penis into the female, and then long syllables (male 1: 42.08±12.67 ms, male 2: 43.02±11.44 ms) after penile insertion. The female bats sometime refused the male bats in the early phase of copulations as emitting noise bursts and broad-band vocalizations, but kept silence during actual copulation. We also found that the SCF copulation calls of one male remained stable peak frequencies on different copulation days although its echolocation call frequency varied each day. Moreover, different male individuals maintained their own "private frequency" in the SCF copulation calls. Therefore, we predicted that the SCF copulation calls may serve as an indicator for female greater horseshoe bats to recognize the mating males in order to maintain mate fidelity because horseshoe bats exhibit sexual segregation before mating. Our results stipulate further studies on mating system and copulation strategies in polygynous bats. Such work may also aid in promoting the preservation of greater horseshoe bats.

The Lombard effect, an involuntary rise in call amplitude in response to masking ambient noise, represents one of the most efficient mechanisms to optimize signal-to-noise ratio. The Lombard effect occurs in birds and mammals, including humans, and is often associated with several other vocal changes, such as call frequency and duration. Most studies, however, have focused on noise-dependent changes in call amplitude. It is therefore still largely unknown how the adaptive changes in call amplitude relate to associated vocal changes such as frequency shifts, how the underlying mechanisms are linked, and if auditory feedback from the changing vocal output is needed. Here, we examined the Lombard effect and the associated changes in call frequency in a highly vocal mammal, echolocating horseshoe bats. We analyzed how bandpass-filtered noise (BFN; bandwidth 20 kHz) affected their echolocation behavior when BFN was centered on different frequencies within their hearing range. Call amplitudes increased only when BFN was centered on the dominant frequency component of the bats' calls. In contrast, call frequencies increased for all but one BFN center frequency tested. Both amplitude and frequency rises were extremely fast and occurred in the first call uttered after noise onset, suggesting that no auditory feedback was required. The different effects that varying the BFN center frequency had on amplitude and frequency rises indicate different neural circuits and/or mechanisms underlying these changes.

We previously reported that band-pass filtered noise (BFN, bandwidth 20 kHz) affected the echolocation behavior of horseshoe bats in different ways depending on which frequencies within the bats' hearing range BFN was centered. We found that call amplitudes only increased when BFN was centered on the dominant frequency of the bats' calls. In contrast, call frequencies were shifted for all BFN stimuli centered on or below the dominant frequency of echolocation calls including when BFN was centered at 20 kHz (BFN20), which is far below the range of the bats' echolocation call frequencies. In the present study we focused on masking effects of BFN20 and used it as a model stimulus for anthropogenic noise to investigate how this noise affected call parameters of echolocation calls, such as their frequency, amplitude, duration and rate. We show that only call frequencies shifted in response to masking BFN20, whereas all other call parameters tested exhibited no noise-related changes. Our findings provide an empirical basis to quantitatively predict the impact of anthropogenic noise on echolocation behavior in bats. They also emphasize the need for a better understanding of the impact of anthropogenic noise on bioacoustic communication and orientation systems in general.