Species can differ in social communication behavior due to different call types used (Blumstein and Armitage, 1997; McComb and Semple, 2005), different rates of call production (Kalcounis-Rueppell et al., 2018), species specific differences in call characteristics (Insley, 1992; Rendell et al., 1999; Musolf et al., 2015), and differences in information encoded (Medvin et al., 1993; Pollard, 2011). The social complexity hypothesis posits that species-specific differences in communication systems can arise due to differences in social systems (Freeberg et al., 2012). Across species, repertoire size has been positively correlated with social group complexity (Blumstein and Armitage, 1997; McComb and Semple, 2005). Group size is often correlated with call complexity, as more complex calls can encode more information about individual identity (Medvin et al., 1993; Wilkinson, 2003; Freeberg, 2006; Pollard, 2011).

Bats exhibit an extensive range in social group size (Davis et al., 1962; Constantine, 1966), social group organization (Wilkinson, 1984; Barclay et al., 1988; Willis and Brigham, 2004; Garroway and Broders, 2007; Patriquin et al., 2010), and mating systems (Bradbury, 1977; Barclay et al., 1979; Thomas et al., 1979; Vaughan and Vaughan, 1986; Heckel and von Helversen, 2002; Keeley and Keeley, 2004). Vocal learning has been observed in some species of bats (Knörnschild et al., 2012; Vernes and Wilkinson, in press). However, knowledge of bat social calls has been limited, as bats are nocturnal, volant, and produce predominantly ultrasonic vocalizations. Most of what is known about bat social calls pertains to those emitted in the roost. Social calls in the roost have been observed to mediate short-term agonistic encounters (Barclay et al., 1979), long-term territoriality (Bradbury, 1977; Behr and von Helversen, 2004; Davidson and Wilkinson, 2004; Bohn et al., 2008), mate attraction (Bradbury, 1977; Voigt and von Helversen, 1999; Davidson and Wilkinson, 2002, 2004; Behr and von Helversen, 2004; Bohn et al., 2008; Knörnschild and Tschapka, 2012), and parental care (Barclay et al., 1979; Matsumura, 1979; Scherrer and Wilkinson, 1993; Pfalzer and Kusch, 2003; Wilkinson, 2003; Bohn et al., 2008; Knörnschild and Von Helversen, 2008; Knörnschild et al., 2012).

While the roost is likely where the majority of a bat's social interactions occur, on account of higher densities, there are behavioral contexts that occur predominantly, if not solely in flight, that may be associated with social calls. Social calls associated with foraging competition have been observed in free living T. brasiliensis and Pipistrellus spp. as well as Eptesicus fuscus in the laboratory (Barlow and Jones, 1997; Corcoran and Conner, 2014; Wright et al., 2014). Cooperative foraging has been observed in Noctilio albiventris, Phyllostomus hastatus, and Nycticeius humeralis (Wilkinson, 1992; Wilkinson and Boughman, 1998; Dechmann et al., 2009). While foraging, Noctilio leporinus produce social calls to avoid collision (Suthers, 1965). In some species, when young are learning to fly, mother-pup pairs fly together and produce social calls to maintain cohesion (Pfalzer and Kusch, 2003). Thyroptera tricolor in flight emit inquiry calls to locate roosting group mates (Gillam and Chaverri, 2012). Bats in flight are exposed to predation risk, and their distress calls induce predator mobbing behaviors in conspecifics and heterospecifics (Russ et al., 2004; Knörnschild and Tschapka, 2012). While socially hibernating species may mate in the hibernaculum (Barclay et al., 1979; Thomas et al., 1979), the use of in-flight social calls to attract mates has been observed in Pipistrellus pipistrellus (Lundberg and Gerell, 1986).

Challenges in comparing the communication behaviors of different bat species arise from there having been few studies to develop a system for classifying bat social calls, and disagreement between the existing classification schemes. The classification scheme of Pfalzer and Kusch (2003) used observed contexts to classify calls, and they found correlation between context and spectrogram shape. Type A calls were noisy and used in agonistic contexts, type B were repeating trills used in distress, type C were single pulses used for group cohesion, and type D were composed of different pulse types used for mate attraction and territoriality (Pfalzer and Kusch, 2003). Studies classifying social calls by spectrogram shape have often further broken single pulse calls into different groups, though have still disagreed on the number of call types (Melendez et al., 2006; Wright et al., 2013; Middleton et al., 2014). Lack of correspondence between different classification systems highlight the diversity of bat repertoires as well as the utility of developing more standardized cross-species classification systems for researchers to better study and communicate about the social calls of different species.

The use of passive acoustic monitoring to study the ecology and conservation of bats has increased in recent years due to threats, such as white nose syndrome and wind turbines, that require long term monitoring (Ford et al., 2011; Loeb et al., 2015). Based on species specific differences in echolocation call characteristics, researchers can use recordings of echolocation calls to measure bat species presence and abundance (Britzke et al., 2013; Li and Kalcounis-Rueppell, 2018; Schimpp et al., 2018). The recordings are associated with time of night, time of year, and location, allowing for the analyses of spatial and temporal patterns (Li and Kalcounis-Rueppell, 2018; Schimpp et al., 2018). As bats produce foraging buzzes with distinct spectral and temporal characteristics, passive acoustic monitoring also provides a measure of foraging behavior (Kalcounis-Rueppell et al., 2013; Grider et al., 2016). Bat social calls are sometimes present in the recordings generated during passive acoustic monitoring (Bohn and Gillam, 2018). It may be possible to use information from the surrounding echolocation pulses and the spatial and temporal data about the recording to study bat in-flight social calls (Bohn and Gillam, 2018).

Most information on species-specific calling behavior in bats pertains to bat echolocation calls (reviewed by Jones and Siemers, 2011). By using the species-specific characteristics of echolocation pulses surrounding social calls, it is possible to assign social calls to species. Number of social calls per species can be related to bat activity to test if species differ in how often they produce social calls. After classifying calls to type, species repertoires can be compared. Social call spectral and temporal characteristics can be measured to test for species-specific signatures. Species-specific differences in the spectral and temporal characteristics of social calls have been observed (Pfalzer and Kusch, 2003; Russ et al., 2004; Luo et al., 2017). Analysis of the screech calls from 31 species from Emballonuridae, Rhinolophidae, Hipposideridae, Miniopteridae, and Vespertilionidae showed that call characteristics varied according to phylogeny, morphology, and social group size (Luo et al., 2017).

Several variables that can be measured using passive acoustic monitoring could be relevant to the production of in-flight social calls. The production of agonistic social calls by P. pipistrellus and cohesive calls by P. hastatus are correlated with increased bat abundance (Wilkinson and Boughman, 1998; Bartonička et al., 2007; Budenz et al., 2009). The number of species present may affect which types of social calls are produced. Some social calls, such as those used by Pipistrellus spp. for foraging competition appear to be only used for conspecific communication (Barlow and Jones, 1997; Barratt et al., 1997). Other call types, such as the distress calls of Pipistrellus spp. are used to communicate with conspecifics and heterospecifics (Russ et al., 2004). Laboratory studies of E. fuscus show that calls used for competing over prey items are frequently followed by a terminal buzz by the emitter, while other call types are not (Wright et al., 2013, 2014). The sonar jamming calls used by T. brasiliensis occur simultaneously to the foraging buzz of the intended receiver (Corcoran and Conner, 2014). Therefore, it would be expected that social calls used for interactions while foraging would be more associated with foraging buzzes than call types with other functions. Social calls associated with maintaining group cohesion when exiting the roost would be expected to occur mostly at the start of the night. Social calls associated with locating the roost would be expected to occur mostly at the end of the night.

Social call production may vary throughout the year, as the reproduction and social organization of bats, particularly in temperate climates, exhibit seasonality. The social organization of spring and summer maternity and bachelor colonies differs from that of winter hibernacula (Bradshaw, 1962; Senior et al., 2005; Perry and Thill, 2008; Hein et al., 2009). Tandem flights of mothers and young of the year occur in late summer (Pfalzer and Kusch, 2003). Bats mate predominately in the fall, and in many species, this is associated with swarming to hibernacula (Bradshaw, 1962; Lundberg and Gerell, 1986; Senior et al., 2005; Burns and Broders, 2015). Middleton (2006) observed seasonality in the complex social calls of common pipistrelles, used for territoriality and mate attraction, finding that call production peaked in April-May and again in September.

The objective of this study was to use passive acoustic monitoring to test the hypothesis that bats use dedicated social calls to mediate different types of social interactions while flying. We predicted that if bats use social calls for multiple functions in-flight, bats would produce different types of social calls with distinct spectral and temporal parameters, consistent with types produced by bats in other regions. As bat species differ in social group organization, we predicted species will differ in how often they produce social calls, and proportional call type usage. We predicted that shared called types exhibit greater between species variation than within species variation in spectral and temporal characteristics, which could allow for species recognition. We predicted that the production of social calls is related to behaviorally relevant factors such as bat activity, whether multiple species are present, foraging activity, time of night, and time of year.

The social call types detected using passive acoustic monitoring are broadly consistent with those from laboratory studies and studies of European species (Pfalzer and Kusch, 2003; Melendez et al., 2006; Wright et al., 2013; Middleton et al., 2014). Most of the call types we recorded for E. fuscus are consistent with the laboratory studies of Wright et al. (2013), however they did not observe the oscillating call observed herein, and we did not observe the short frequency modulated call they reported. To our knowledge this is the first time these social calls have been observed for E. fuscus in the field, as well as for most of the N. humeralis social calls in any context. The one N. humeralis call type which appears to have been described before was the oscillating call, which functions as a pup-isolation call in the roost (Scherrer and Wilkinson, 1993). The oscillating call of N. humeralis greatly resembles mother-pup calls used by T. brasiliensis in the roost (Bohn et al., 2008). The oscillating calls produced by T. brasiliensis in-flight did not resemble the N. humeralis oscillating call, but rather the sonar jamming oscillating calls reported by Corcoran and Conner (2014) or individual pulses from the complex calls. The song-like, complex calls of T. brasiliensis have mostly been described from studies in the roost or laboratory, however they have also been observed in flight (Bohn et al., 2008; Bohn and Gillam, 2018).

Social calls were differentiated from echolocation calls through visual examination of spectrogram shape, and this difference was supported by DFA. While downsweep social calls and echolocation calls both have frequency solely decreasing through the call, downsweep social calls are not likely to be abnormal echolocation calls. Within species, mean duration of downsweeps exceeded twice the duration of echolocation calls and frequency characteristics for downsweeps were consistently lower than corresponding frequency characteristics for echolocation calls. While T. brasiliensis echolocation calls do increase in duration when flying at high altitudes, the bandwidth decreases to near constant frequency, making it unlikely that the broadband downsweep calls are actually isolated calls from distant and high-altitude T. brasiliensis (Gillam et al., 2009). The echolocation calls of high-altitude T. brasiliensis still have a peak frequency of ~25 kHz, therefore it is unlikely that the higher frequency QCF calls were high altitude T. brasiliensis echolocation calls mistaken for social calls (Gillam et al., 2009).

We found species specific differences in the temporal and spectral characteristics of all call types tested. Analysis of the screech calls of 31 species of bats from 5 families in China showed species specific differences in social call characteristics correlated with phylogeny, morphology, and social group size (Luo et al., 2017). While three species are insufficient for a phylogenetic analysis of the causes of variation, it is interesting to note that the direction of species-specific differences between two species were not consistent across call types. Eptesicus fuscus upsweeps were higher bandwidth than N. humeralis upsweeps, while E. fuscus downsweeps were lower bandwidth than N. humeralis downsweeps, suggesting that there may not be a consistent phylogenetic signal across multiple call types.

The presence of species-specific signatures in social calls suggests the possibility that a bat listening to the call would be able to discern emitter species. However, playback experiments would be needed to determine if bats actually attend to these differences in social call characteristics. Playback studies have yielded mixed results as to whether bats attend to species-specific signatures (Russ et al., 2004; Schöner et al., 2010). Whether species discrimination occurs may depend on call function. Entering a roost of heterospecifics would likely be disadvantageous, and therefore species discrimination would be expected, while deterring a predator may be advantageous regardless of the species of the predator's immediate prey, and therefore species discrimination would not occur (Russ et al., 2004; Schöner et al., 2010). We found that social calls were often produced in multiple species bat passes, possibly suggesting some types may function for communicating with heterospecifics.

The most common call type for E. fuscus was the complex call, and the majority of complex calls were produced by E. fuscus. In a laboratory setting, E. fuscus complex calls were emitted when two bats were approaching the same insect (Wright et al., 2014). Complex calls functioned for food item defense, where the bat closer to the insect emitted the call and the second bat would alter its flight trajectory to avoid the emitter and the insect (Wright et al., 2014). We found that complex bouts were negatively correlated to heterospecific activity, suggesting this food item defense interaction occurs predominantly between conspecifics. While all species used in this study are insectivorous, they exhibit preferences for different insect taxa (Safi and Kerth, 2007). It is not surprising that a call associated with food item defense would be predominately used to communicate with conspecifics, as conspecifics present more competition for food than do heterospecifics. The use of complex calls to compete for food solely with conspecifics has also been observed in P. pipistrellus and P. pygmaeus, each of which only respond to conspecific complex calls (Barlow and Jones, 1997; Barratt et al., 1997). The social call production of E. fuscus suggests the most common in-flight social interaction for this species is to compete with conspecifics for food. Direct video observations and playbacks of complex calls to free flying E. fuscus could be used to test this hypothesis.

The complex and upsweep calls of E. fuscus exhibit seasonal patterns. While the number of complex call bouts remained fairly constant across seasons, they became proportionally more common in the winter due to the decrease in overall bat activity. As these calls appear to mediate foraging competition, it makes sense that they are relatively more common when resources are scarce. Relative production of upsweeps was highest during the spring. In a laboratory setting, upsweep calls were mostly produced by juvenile E. fuscus (Wright et al., 2013). However, use by juveniles cannot explain the prevalence of upsweep calls in the spring, as the young of the year have not yet been born (Bradshaw, 1962; Rydell, 1989).

Upsweep calls were commonly produced by E. fuscus and N. humeralis. However, upsweep calls are associated with different contexts in the two species. The upsweeps of E. fuscus exhibited an association with foraging buzzes that was not seen with the upsweeps of N. humeralis, suggesting the calls serves different functions. This is unusual, as previous across species studies on bat social calls have shown that the same call types serve the same functions across species, even when species specific signatures are present (Pfalzer and Kusch, 2003; Russ et al., 2004; Carter et al., 2012; Luo et al., 2017). Further studies on the contexts of upsweep calls in other vesper bats coupled with phylogenetic analysis could be used to test if the upsweeps of E. fuscus and N. humeralis are due to convergent call evolution, or if the function of the call changed over evolutionary history.

In other bat species, downsweep calls have been observed to function in maintaining group cohesion (Pfalzer and Kusch, 2003; Carter et al., 2012; Gillam and Chaverri, 2012). Production of downsweep, downsweep-upsweep, and upsweep bouts was found similar contexts, suggesting some redundancy in downsweep and upsweep call function. Calls to maintain group cohesion would be advantageous when commuting as a group to foraging patches, seen in Phyllostomus hastatus (Wilkinson and Boughman, 1998). Female N. humeralis cooperatively forage (Wilkinson, 1992). We found that social call production relative to bat activity for N. humeralis far exceeded that of other species, and the most common call type produced by N. humeralis was the downsweep. Maintaining group cohesion while commuting could explain why N. humeralis produces social calls, particularly downsweeps, more often than other species. However, the infrequent association between either downsweep or upsweep calls with foraging buzzes suggests that, even if used to maintain group cohesion when commuting, N. humeralis does not use them to advertise foraging patches. Bats respond to the foraging buzzes of conspecifics (Dechmann et al., 2009). It is possible that cooperatively foraging N. humeralis use social calls to maintain cohesion when commuting, and then attend to foraging buzzes as a cue when prey have been located.

Downsweep, downsweep-upsweep, and upsweep bouts were often recorded in bat passes where multiple species were present. Maintaining group cohesion in-flight would only occur with conspecifics from the same social groups. Cambell's monkeys, Cercopithecus campbelli, use contact call types that encode more individual information when in the presence of heterospecifics, likely due to the more complex acoustic environment (Coye et al., 2018). If a similar phenomenon occurs in bats, that may explain the use of contact calls when heterospecifics are present. While bats can attend to echolocation calls to maintain group cohesion (Dechmann et al., 2009; Egert-Berg et al., 2018), dedicated social calls may still be useful for group cohesion as social calls can encode more information (Gillam and Fenton, 2016) and are often louder and lower frequency, allowing for transmission over longer distances (Middleton et al., 2014).

Alternatively, downsweep and upsweep calls may serve multiple functions depending on context. Phyllostomus hastatus screech calls function for anti-predator mobbing (Knörnschild and Tschapka, 2012) and cohesion when commuting (Wilkinson and Boughman, 1998). Pippistrellus spp. complex calls function in mate attraction (Lundberg and Gerell, 1986) and foraging competition (Barlow and Jones, 1997). The exchange of contact calls by group members during agonistic interactions with non-group members has also been observed in birds (Nowicki, 1983; Hopp et al., 2001). Heterospecifics would be inherently non-group members. The majority of downsweep, downsweep-upsweep, and upsweep bouts were produced in the spring, when bats are returning from hibernacula and winter-feeding grounds (Scales and Wilkins, 2007; Valdez and Cryan, 2009). Therefore, agonistic encounters with unfamiliar individuals would be highest when bats are first starting to be active on the landscape again. Male N. humeralis are solitary living in the spring and summer (Perry and Thill, 2008; Hein et al., 2009), therefore any bat a male N. humeralis encounters would be a non-group member. An increased rate of agonistic signal production when presented with unfamiliar individuals has been observed in birds and frogs (Lesbarrères and Lodé, 2002; Briefer et al., 2008). Playbacks in spring versus other seasons could also be used to test this hypothesis.

A possible function for the apparent signal redundancy across species for downsweep and upsweep calls may be that call type usage varies by signaler identity. Wright et al. (2013) found that the likelihood of E. fuscus upsweep calls were positively correlated with the number of juveniles and males in a dyad. If age or sex specific social call use exists for other species, then social calls may be useful for assessing population demographics in ecological and conservation passive acoustic monitoring studies. Sexual segregation of foraging habitats has been observed in some bat species (Senior et al., 2005; Safi et al., 2007). The use of Bayesian statistics to infer sex from echolocation call characteristics has been suggested as a method for discerning sex ratios from passive acoustic monitoring (Lehnen et al., 2018), however some bat species, including E. fuscus, do not exhibit sex-specific differences in echolocation call characteristics (Heller et al., 1989; Masters et al., 1995).

Eptesicus fuscus, N. humeralis, and T. brasiliensis differ in how often they produce social calls associated with different contexts. Nycticeius humeralis produces social calls the most often. Downsweeps and upsweeps are the main call types used by N. humeralis, and appear to be broad functioning contact calls. The higher use of contact calls may be related to females cooperatively foraging (Wilkinson, 1992), and the lower sociality of males (Perry and Thill, 2008; Hein et al., 2009), leading to increased contact with unfamiliar individuals. Eptesicus fuscus most commonly produced a call type used for foraging competition (Wright et al., 2014), used specifically to compete with conspecifics. E. fuscus also commonly produced upsweeps, which in laboratory settings is mostly produced by juvenile and male bats (Wright et al., 2013), however E. fuscus upsweep calls are associated with foraging buzzes, unlike N. humeralis upsweep calls. Tadarida brasiliensis social calls were occasionally recorded, and were primarily complex calls. Eptesicus fuscus, N. humeralis, and T. brasiliensis have different social behaviors in the roosts (Davis et al., 1962; Wilkinson, 1992; Willis and Brigham, 2004; Bohn et al., 2008; Perry and Thill, 2008; Hein et al., 2009). Differential usage of social calls suggests these species also exhibit different social behaviors while in flight.

The data used for this study have been made available at https://doi.org/10.6084/m9.figshare.8145926.v2.

The recording of bats in this study was approved by the UNCG Institutional Animal Care and Use Committee and by the North Carolina Wildlife Resources Commission.

MK-R and BS conceived of the study. BS identified bat passes with social calls, identified bat passes to species, analyzed call spectral and temporal characteristics, performed all statistical analyses, and wrote the initial draft of the manuscript. HL verified the species identification for many of the bat passes, contributed to the selection of statistical analyses, and contributed to variable selection for the context of social call production. MK-R contributed to the selection of statistical analyses and to the writing of the manuscript.