Centurio senex is an iconic bat characterized by a facial morphology deviating far from all other New World Leaf Nosed Bats (Phyllostomidae). The species has a bizarrely wrinkled face and lacks the characteristic nose leaf. Throughout its distribution from Mexico to Northern South America the species is most of the time rarely captured and only scarce information on its behavior and natural history is available. Centurio senex is frugivorous and one of the few bats documented to consume also hard seeds. Interestingly, the species shows a distinct sexual dimorphism: Adult males have more pronounced facial wrinkles than females and a fold of skin under the chin that can be raised in style of a face mask. We report the first observations on echolocation and mating behavior of Centurio senex, including synchronized audio and video recordings from an aggregation of males in Costa Rica. Over a period of 6 weeks we located a total of 53 perches, where during the first half of the night males were hanging with raised facial masks at a mean height of 2.35 m. Most of the time, the males moved just their wing tips, and spontaneously vocalized in the ultrasound range. Approaches of other individuals resulted in the perching male beating its wings and emitting a very loud, low frequency whistling call. Following such an encounter we recorded a copulation event. The observed aggregation of adult C. senex males is consistent with lek courtship, a behavior described from only few other bat species.
Centurio senex is an iconic bat characterized by a facial morphology deviating far from all other New World Leaf Nosed Bats (Phyllostomidae). The species has a bizarrely wrinkled face and lacks the characteristic nose leaf. Throughout its distribution from Mexico to Northern South America the species is most of the time rarely captured and only scarce information on its behavior and natural history is available. Centurio senex is frugivorous and one of the few bats documented to consume also hard seeds. Interestingly, the species shows a distinct sexual dimorphism: Adult males have more pronounced facial wrinkles than females and a fold of skin under the chin that can be raised in style of a face mask. We report the first observations on echolocation and mating behavior of Centurio senex, including synchronized audio and video recordings from an aggregation of males in Costa Rica. Over a period of 6 weeks we located a total of 53 perches, where during the first half of the night males were hanging with raised facial masks at a mean height of 2.35 m. Most of the time, the males moved just their wing tips, and spontaneously vocalized in the ultrasound range. Approaches of other individuals resulted in the perching male beating its wings and emitting a very loud, low frequency whistling call. Following such an encounter we recorded a copulation event. The observed aggregation of adult C. senex males is consistent with lek courtship, a behavior described from only few other bat species.
Mating systems of bats remain a rather poorly understood topic. A review of McCracken and Wilkinson [1] presented information for only 6.9% of the bat species described at that time (963 spp). This percentage probably has not increased at the same rate as the number of described bats, which currently is at 1406 species (Simmons, pers. comm. 2019). One of the reasons for the general scarceness of information is that, in addition to their nocturnal habits, bats are relatively small and highly mobile, and only over the last decades advanced technology for the nocturnal observation of bats, such as infrared cameras and ultrasound recorders, became more readily available. Additionally, variables such as time and place where bats will show courtship behavior are for many species extremely difficult or impossible to predict and to access. Mainly for this reason, most of the information on bat reproduction available is based on mist net captures, when individuals in hand can be examined for signs of pregnancy and lactation of females and testicle state in males [2]. This may yield information on sex ratio, on the reproductive cycles, however, these morphological diagnoses yield no information on the actual courtship and mating behavior.Nevertheless, the speciose order Chiroptera is known to have a high variety of mating systems. Observations indicate the existence of promiscuous (Myotis lucifugus, [3]) and monogamous (Vampyrum spectrum, [4]) mating systems; there are examples of polygyny with defense of females (Phyllostomus hastatus, [5]; Pipistrellus kuhli, [6]) as well as of polygyny with defense of resources (Artibeus jamaicensis, [7, 8], Desmodus rotundus, [9]), and there are also rare reports on the existence of lek mating in bats (Hypsignathus monstrosus, [10]).Among all mating systems known from bats, "lek" is the rarest recorded. Leks are clusters of sexually displaying territorial males at a common display ground, where female mate choice [11] causes intense competition among males, thus potentially reducing reproductive chances for individual males. Males offering a better display have in general a higher chance of being chosen by a female [11, 12]. Lek courtship behavior has been first described in birds [13]. The classic lek definition includes at least five ecological and physiological prerequisites [10, 11, 14]: 1) existence of a mating arena, 2) male territories contain no resources (food, water, protection) other than access to males, 3) females have the opportunity to select a male for mating, 4) absence of male parental care, and finally, 5) internal fertilization. There are two categories of leks, the “classic” lek, where males display in close proximity to each other, and “exploded” leks, where males are distributed over a larger area [14]. Lekking behavior includes the transfer of information between the sexes, e.g., in form of visual signals or through vocalizations. Independently of lek systems, such social communication is documented for many bat species and may include visual, tactile, olfactory and acoustic signals. In a few species, males show even sophisticated songs during courtship [15, 16].Lekking behavior occurs in a wide range of vertebrates and invertebrates but is not common. There are well-documented lek systems in insects, such as in drosophilid and tephritid fruit flies (for a complete list see [17]). In vertebrates, lek behavior is mostly reported from birds, where it has been documented from at least 148 species [18], while occasionally the behavior occurs also in amphibians (e.g., [19]), fish (e.g., [20]) and reptiles (e.g., [21]). In mammals, classic lek behavior appears to be rare and is found only in 12 species, mainly ungulates and pinnipeds, while 14 other species show a lek-like behavior that not fully matches the criteria mentioned above [22].Centurio senex GRAY 1842 is an extraordinary phyllostomid species distributed from Mexico throughout Central America to Venezuela and Trinidad and Tobago [23]. Throughout this wide range, it is in general only rarely captured, which accounts for the scarce information available. Centurio senex is predominantly frugivorous, but is also one of the few neotropical bats documented to consume hard seeds [24, 25]. The species shows a high degree of–still unexplained–facial modifications that deviate far from the general morphology of New World Leaf Nosed Bats (Phyllostomidae). Centurio senex has a uniquely wrinkled face, large greenish eyes and the characteristic nose leaf of phyllostomid bats is absent. Most interestingly, there is a strong sexual dimorphism: Males not only have more pronounced facial wrinkles than females, but show also a unique fold of skin under the chin that can be raised to cover the lower part of the face like a mask [26]. The distinct sexual dimorphism suggests a use of the face mask in reproduction. However, so far, no information on the reproductive behavior of this elusive species was available.Here, we report on our recent discovery of an aggregation of Centurio senex males in Costa Rica, which provided the first opportunity for behavioral observations on this species in its natural habitat. Goal of this paper is to describe the mating behavior and the basic acoustic repertoire of C. senex, thus providing information on one of the most iconic neotropical bat species. Based on the distinct sexual dimorphism we hypothesized that the unique face mask of the Centurio senex males is employed during courtship behavior.
Materials and methods
Study site
The study was carried out at the Biological Station of the Villa Blanca Hotel (10° 12'14N, 84°29'04" W, 1100 masl), located in San Ramón, Alajuela Province, Costa Rica. The predominant habitat is the Very Humid Tropical Forest [27] and the area combines patches of secondary forest with mature continuous forest at an average annual rainfall of ca. 2500 mm per year.Centurio senex males were found along a trail in a small forest patch around an artificial pond. The vegetation combined tall canopy trees with a moderately dense understory layer of small trees. Encountered perches of male Centurio senex were marked at night close to the hanging individual with reflective tape. The next morning, perch height above the ground was measured using a 3 m self-retracting metal tape measure (Stanley). In addition, we identified the family of the plants the bats were perched on. As bat perches were spread only over a relatively small area under a closed canopy and some perches were very close to each other (≤ 1m), an attempted GPS—mapping of the individual perches was rather inaccurate but helpful for assessing total lek area size.
Nocturnal censuses
Following the first observation of perching bats on September 15th, 2018, a total of 13 visits were made between September 27th and October 31st, when no more perched animals were present. During each visit, perched individuals were hourly monitored from 18:00 to 24:00 hours. As we observed that perches were used day after day, we marked them on October 3rd individually in order to have a reference for the following visual observations and video/sound recordings. From this date onwards, we monitored the number of bats present and recorded the use of the marked perches.For testing whether the frequency of use of the perches differed among perches we performed a G-test, using data from 8 census nights and 44 of the 53 identified perches, which had at least one observation between October 3rd and October 28th, the last day of sampling before the individuals had left the study site.For determining whether the frequency of use of the perches depended on height above ground we used a Spearman correlation. In order to check for a temporary pattern regarding the use of specific perches over time, we calculated a linear regression model, in which sampling date and time were used as predictive variables for the number of perches with individuals encountered in the entire lek.
Video and ultrasound recording
We recorded the behavior of bats, using an infrared-sensitive video camera (Sony FDRAX53/B 4K HD) that was shifted each night between several perches, depending on occupancy of the perch. Whenever possible we recorded synchronously audio sequences at a sampling rate of 500 kHz with an CM16/CMPA microphone and an Ultra Sound Gate 116Hm interface (Avisoft Bioacoustics, Berlin, Germany), connected to a laptop computer. We recorded manually and continuously, resulting in sequences of separate wav-files of 1 min length each. In order to get only sounds from the perched bat, we placed the microphone as close as possible (ca. 0.5–1 m, depending on the sensitivity of the individual to the interference and on the characteristics of the respective perch) without disturbing the perched bat. In total, we accumulated during eight nights at 14 perches a total of 20 hours, 43 minutes and 51 seconds of video recordings. Additionally, we recorded also echolocation calls from bats flying in the understory of the study area, focusing on bats in the size range of C. senex that were visible when walking the trails.
Audio analysis
For a description of the vocalizations of Centurio senex we selected recordings from males from 7 different perches, recorded over 6 days. Sequences for analyses were selected based on completeness and quality of the sound recordings and of the synchronous video recordings. Vocalizations were analyzed using the custom-made program Selena (University of Tübingen, Germany). We identified commonly observed vocalizations and measured basic spectral (peak frequency, start and end frequency at -15 dB) and temporal parameters (pulse interval, duration at– 15 dB), and calculated syllable and element durations. All measurements were performed using an FFT of 512 and an overlap of 91.14%, resulting in a frequency resolution after auto padding and interpolation of 981 Hz and a time resolution of 0.09 ms with a dynamic range of 80 dB (Blackman window). Spectrograms were made with Avisoft-Sas Lab Pro (Avisoft Bioacoustics), using a FFT of 512 and an overlap of 75%, resulting in a frequency resolution of 977 Hz and a time resolution of 0.256 ms.To test whether echolocation parameters of calls emitted while flying were different from those emitted at the perches, we performed Wilcoxon rank sum tests for non-parametric data. For parametric data with equal variances we used a Two Sample t-test. If variances were not equal, we used Welch Two Sample t-tests.
Results
Seasonal and daily phenology
On September 15th, 2018, in the early evening hours, bats were observed for the first time at the study site. The animals were hanging exposed and rather calm on small branches in the vegetation. Based on the striking facial structures and the unique and clearly visible facial masks they were identified as male Centurio senex. The animals were–compared to other phyllostomid bats—rather tolerant to the cautious approach of an observer, and appeared to be relatively reluctant to leave their perches. The exact same spots with extremely little changes (< 5cm) were occupied over different nights. Perching bats were observed in the study area until October 31st, for a total of 46 days. During this period, we were able to collect data during 13 nights.Perches of males were distributed over a rather small, ellipsoidal area of approximately 0.13 ha. Perches were found on plants belonging to a variety of families: Siparunaceae, Piperaceae, Heliconiaceae, Myrtaceae, Araceae, Acanthaceae, Melastomataceae, Clusiaceae, and Cyatheaceae.A first count on September 17th revealed the presence of 17 perched males on site. Over the next weeks this number increased to a maximum of 30 perching males during the same night (October 2nd) before starting to decrease during the first week of October (Fig 1A). Following this decline we did not find any more Centurio senex males at the site after October 31st, 2018 and assume that this dissolving of the lek marked the end of the mating period. The total number of perches recorded at the site during the entire study period was 56.
Fig 1
Phenology of a Centurio senex male aggregation.
a) Maximum number of perches occupied by male C. senex per night over the entire sampling period. The bats were first observed on September 15th, 2018. b) Development of the number of perching males over the night. Black dots refer to the first half of the study period until October 10th, 2018, white dots refer to the second half.
Phenology of a Centurio senex male aggregation.
a) Maximum number of perches occupied by male C. senex per night over the entire sampling period. The bats were first observed on September 15th, 2018. b) Development of the number of perching males over the night. Black dots refer to the first half of the study period until October 10th, 2018, white dots refer to the second half.Some perches were used more frequently than others (G Test; G = 66.34; d.f. = 43; p = 0.013). Especially at the beginning of the monitoring perches were occupied quite regularly, but perch use decreased subsequently. 3 perches were used over the entire monitoring period (S1 Fig). The average height of the perches was 2.84 ±1.26m (mean ±SD; n = 53). Frequency of use observed for a particular perch was not correlated with perch height (Spearman correlation; Rho = -0.24; n = 434; p = 0.11).No bats were observed at the site during the day, but the animals re-appeared every evening after dusk, around 18:00 hrs. It was common to observe the highest numbers of individuals in the first hours of the night, while after 23:00 hrs we noted more bats in flight and a progressive decrease of the perched individuals (Fig 1B). Around midnight commonly all bats had vacated the perches.Both, observation date (p <0.001; Fig 1A), and night time (p <0.001; Fig 1B) explain approximately 50% of the variability in the number of perches used throughout study period (F = 23.44; df = 46; p <0.001). In addition, the interaction between these two variables also was significant (p <0.001). In the first half of the study period the number of perching bats decreased markedly towards midnight, while in the second half the (much fewer) bats showed no obvious trend (Fig 1B). Bats were also present at perches during nights with moderate rain, but were rather inactive.
Vocalizations
Centurio senex showed a limited number of stereotypic behavior types that were accompanied by distinct vocalizations. In the following, we describe first these vocalizations before presenting the complete behavioral repertoire composed of the observed movements and the associated acoustic emissions.
Echolocation behavior
On all days we were able to record echolocation call sequences of flying Centurio senex. When flying along the small trails of the study site the bats emitted very short (1.3ms +/- 0.19 SD) FM echolocation calls with a mean peak frequency of 88.6 kHz +/- 1.77 SD (1st harmonic) and a mean bandwidth of 36.6 kHz +/- 4.26 SD (45 calls from 4 passes) (Table 1). Occasionally, echolocation calls contained during the onset a short, low-intensity QCF- component. While we recorded along the trails in the understory also other, similar-sized phyllostomids, vocalizations of Centurio senex differed distinctly, due to the lack of harmonics, both in echolocation calls, as well as in the other vocalizations of the bats: Our recordings of echolocation calls showed consistently only a prominent 1st harmonic, and only extremely rarely we observed a very weak 2nd harmonic.
Table 1
Analysis of vocalizations of free-flying and perching bats.
PF [kHz]
SF [kHz]
EF [kHz]
BW [kHz]
PD [ms]
PI [ms]
Rep. rate
FREE-FLYING BATS
Echolocation calls
88.6 (4)
± 1.77
112.7 (4)
±3.04
76.1 (4)
±1.35
36.6 (4)
± 4.26
1.3 (4)
± 0.19
54.2 (4)
±19.79
23.1 (4)
±9.02
PERCHING BATS
Echolocation calls
91.2 (7)
± 4.59
116.5 (7)
± 12.17
76.4 (7)
± 3.27
40.1 (7)
± 9.61
1.3 (7)
± 0.42
52.0 (7)
±12.74
26.2 (7)
± 2.81
Trills
82.9 (7)
± 6.67
102.3 (6)
± 3.50
104.0 (6)
± 4.06
24.5 (6)
± 9.62
1.7 (6)
± 0.21
4.0 (7)
± 0.28
257.1 (7)
± 9.65
Wing beats1
-
-
-
-
-
85.5 (7)
± 6.51
14.2 (7)
± 6.98
Wing beat calls
81.7 (7)
± 3.05
97.9 (5)
± 5.44
64.0 (5)
± 3.46
33.9 (5)
± 2.97
2.4 (5)
± 0.11
86.0 (7)
± 5.96
12.3 (7)
± 1.99
High frequency calls
159.5 (7)
± 4.59
159.2 (7)
± 3.39
165.5 (7)
± 4.14
6.9 (7)
± 4.97
6.9 (7)
± 0.69
-
-
Downward modulated calls
88.2 (7)
± 1.96
103.4 (7)
± 4.90
90.5 (7)
± 1.76
13.2 (7)
± 4.38
28.9 (7)
± 4.97
-
-
Whistles2
28.36 (7)
± 5.90
-
-
-
-
-
-
End sweeps
10.1 (7)
± 2.24
16.7 (7)
± 1.39
5.1 (7)
± 0.69
11.6 (7)
± 1.26
10.6 (7)
± 1.46
-
-
Mean values ± SD of peak frequency (PF), start frequency (SF), end frequency (EF) and bandwidth (WB) in kilo hertz [kHz]; pulse duration (PD) and pulse interval (PI) in milli seconds [ms]; repetition rate (Rep. rate) in calls per second [calls * s -1]; (n) number of sequences analyzed for each parameter. 1Wing beats are not vocalizations with distinct measurable sound parameters, but noise pulses generated by the bat beating the wings. 2Reliable measurements of whistle start, end frequency and bandwidth are not possible, because of the inevitably overloaded recordings of this element.
Mean values ± SD of peak frequency (PF), start frequency (SF), end frequency (EF) and bandwidth (WB) in kilo hertz [kHz]; pulse duration (PD) and pulse interval (PI) in milli seconds [ms]; repetition rate (Rep. rate) in calls per second [calls * s -1]; (n) number of sequences analyzed for each parameter. 1Wing beats are not vocalizations with distinct measurable sound parameters, but noise pulses generated by the bat beating the wings. 2Reliable measurements of whistle start, end frequency and bandwidth are not possible, because of the inevitably overloaded recordings of this element.
Social vocalizations
Bats were vocalizing spontaneously while perching alone but reacted also to the approach of another individual. A typical audio sequence contained 4 different elements, that were emitted in a distinct order (Figs 2 and 3). The sequence proceeded mostly in the order indicated below, however, elements could also be repeated and the sequence could be interrupted at any point. Courtship songs generally started with an echolocation sequence (ES), followed by a trill call (TC). The approach of another bat often triggered a wing beat sequence (WBS), accompanied by synchronized echolocation calls with reduced bandwidth. Finally, we recorded a complex whistle sequence (WS), consisting of several calls in the high ultrasound range followed by a lower, extremely loud whistling component and an even lower, for humans audible low frequency end sweep (S1 and S2 Audios).
Fig 2
Example of a typical courtship song of Centurio senex.
The four major elements are indicated by color bars: echolocation sequences (ES, green bars), trill calls (TC, yellow bars), wing beat sequences (WBS, pink bar), whistle sequence (WS, red bar. Note harmonic artefacts caused by the inevitably overloaded recording of this always relatively loud element.). The repetition of echolocation and trill sequences resulted in this example in an 8 element song. (Parameters for all spectrograms: Blackman Window, FFT 512, frame size 100%, overlap 75%, resulting in a spectral resolution of 977 Hz and a temporal resolution of 0.256 ms).
Fig 3
Song elements.
A complete courtship song is composed of at least four elements: A) echolocation sequence, B) trill call, C) wing beat sequence, D) whistle sequence (Note harmonic artefacts caused by the overloaded recording of this relatively loud element).
Example of a typical courtship song of Centurio senex.
The four major elements are indicated by color bars: echolocation sequences (ES, green bars), trill calls (TC, yellow bars), wing beat sequences (WBS, pink bar), whistle sequence (WS, red bar. Note harmonic artefacts caused by the inevitably overloaded recording of this always relatively loud element.). The repetition of echolocation and trill sequences resulted in this example in an 8 element song. (Parameters for all spectrograms: Blackman Window, FFT 512, frame size 100%, overlap 75%, resulting in a spectral resolution of 977 Hz and a temporal resolution of 0.256 ms).
Song elements.
A complete courtship song is composed of at least four elements: A) echolocation sequence, B) trill call, C) wing beat sequence, D) whistle sequence (Note harmonic artefacts caused by the overloaded recording of this relatively loud element).Within these four main elements we identified 8 different syllables (Fig 4, Table 1):
Fig 4
Syllables in the vocal repertoire of Centurio senex: Echolocation Call (EC), trill (TS), Wing Beat (WB) and associated Wing Beat Call (WBC), high frequency call (HF), downward modulated call (DM), Whistle Call (WC) and an end sweep (E).
Few to many echolocation calls (EC) were contained within echolocation sequences. Echolocation calls of perched bats did not differ significantly from those emitted by flying animals (S1 Table). Echolocation calls were 1.3 ms long, steeply frequency modulated with a peak frequency around 91 kHz, and a high bandwidth of 40 kHz (Figs 3A and 4).Trill syllables (TS) consisted of a down sweep / upsweep connected to the next TS syllable, thus forming a continuous, sinusoid shaped call. Peak frequency was consistently located at the minimum frequency of these elements, while the maximum frequency of each syllable was very much quieter and for that reason very often not visible in the records. Entire trill sequence contained in general more than 20 TS, were between 80 ms and 130 ms long and were characterized by decreasing peak frequencies of the elements from ca. 120 kHz to 70 kHz (Figs 3B and 4, S2 Table).Wing beat sequences differed from all other sounds produced by Centurio in so far as one part is not a vocalization, but a low intensity, yet distinct noise pulse (WB) ranging up to more than 50 kHz that probably was produced by the bat beating its wings at a frequency of ca 14 beats per second (mean number of wing beats in WBS: 7; ±1.0 SD; n = 7, S2 Table). Additionally, each wing beat was ended with a wing beat call (WBC), that seemed to be a lower bandwidth version of an echolocation call EC (Figs 3C and 4).The final vocalization contained a highly stereotypic sequence of four distinct elements. First, an extremely high, 7 ms short call (HF) with a peak frequency of 160 kHz, followed by a longer (29 ms) downward modulated call (DM) descending from 104 kHz to 90 kHz. The following whistle call (WC), a ca. 20 ms long call at ca. 28 kHz was by far the loudest part of the Centurio senex call repertoire. The high amplitude of this syllable caused in many recordings a distinct echo reverberating from the surroundings. Last followed an end sweep (E) below 10 kHz that is clearly audible to humans. The extreme dynamic differences among the syllables in this final sequence made it basically impossible to obtain good quality recordings of both the low intensity high-frequency syllables (HF, DM) and the extremely loud low-frequency whistle call (WC) of a sequence, so recordings of the latter were mostly overloaded, with the sonograms showing false harmonic artefacts (Figs 3D and 4).Within the song elements, syllables could be repeated at sometimes quite variable number. Particularly the echolocation and trill sequences differed both within and among recordings in number of syllables, resulting in different durations of song elements (S2 Table).
Behavioral observations
Display behavior
From sunset to ca. midnight males spent most of the time on the perches and consistently showed a rather stereotypic behavior. We did not observe anything resembling physical aggression between bats at the perches. Most of the time bats perched with raised skin mask, and lowered it only occasionally. For both raising and lowering of the mask the bats used their thumbs (Fig 5A and 5B).
Fig 5
Field photographs a) Centurio senex male with raised facial mask; b) male with lowered facial mask; c) perching male being approached by another individual; d) detail of same perching male during the approach, just before emitting a whistle sequence.
Field photographs a) Centurio senex male with raised facial mask; b) male with lowered facial mask; c) perching male being approached by another individual; d) detail of same perching male during the approach, just before emitting a whistle sequence.Phase 1) Over long time periods perched males were just rubbing the wing tips subtly against each other (85.9 ± 8.6% of total observation time), while often moving their head and ears, apparently in a quiet state of alert (S1 Video). Bats emitted in this situation occasionally echolocation sequences (ES), but also the conspicuous descending trill calls (TC).Phase 2) When another bat approached the perching bat its general alertness increased, indicated by more pronounced ear movements and by directing the whole body towards the visitor. As the visiting bat approached closely, sometimes in a short hovering flight, the perched male began a wing beat sequence (WBS), with each wing beat (WB) followed by a wing beat call (WBC). The encounter escalated with the perched male making a quick, thrusting motion with the entire body towards the visitor and, in most cases (77.9%, n = 181), emitting the very loud and to humans partially audible whistle sequence (WS), whereupon the visitor left (S2 Video). This thrust was accompanied by the perching bat diving deeper into the facial mask, so that both ears were pointing downwards, covering the eyes, and thus minimizing the facial area exposed to the approaching bat (Fig 5C and 5D). The entire phase–from a visitor approaching the perched male to leaving following a whistle call–was in general very short and lasted less than one second. Following the departure of the visitor, the perched male quickly returned to Phase 1) behavior. Unfortunately, the quality of the video did not allow to distinguish reliably between males and females in the visitors. The fast-moving visitors were inevitably blurred; additionally we had to focus on the perched individual, so the hovering individuals were mostly out of focus and it was only rarely possible to see whether a folded face mask was present or not.In addition to the rather stereotypic response of perching males to visits, we observed the animals occasionally also removing smaller leaves in the immediate surroundings of their perch, and others rubbing their body against branches (n = 10). During the entire recording period, we also observed males on 9 perches grooming themselves 43 times.
Copulation
On October 10th at 19:40 hrs, we observed an approach to a perching male that ended with the usual whistle sequence and the departure of the visitor. Just five seconds later the perched bat was visited again and this time the visitor landed immediately, very close and almost on the back of the perching male. The visitor was a female, clearly recognizable by the lack of a face mask. The male immediately lowered his mask and quickly placed himself in a face-to-face position with the female. Five seconds later he changed his position, moved around the female and pressed his face onto the middle of her back, in a position typical for mating in bats. The visibly vibrating male held the female, using thumb claws and wings, and seemed also to bite into the fur on the lower back of the female, presumably while penetrating. Both bats remained in this position for ca. 30 seconds, and then the female got restless and departed. The male immediately raised the mask again and returned to phase 1) behavior (S3 Video).In the minute prior to the observed copulation the perching male had been singing several times. A first courtship song (t = - 10 sec before the landing of the female) consisted of an echolocation sequence followed by a trill. The second song (t = - 5 sec) was a complete song with an echolocation sequence, trill, wing beat, and whistle. At the end of the second song a bat approached and got so close to the perching male that the recording shows its echolocation signals during the wing beat sequence, before leaving. The third song (t = -1 sec) was an incomplete song with an echolocation sequence, trill and wing beat sequence, which stopped at the landing of the female (t = 0). During most of the following copulation we recorded no vocalizations, however, close to the end of the act, when the female started to get visibly restless (t = + 33 sec), the bat resumed to performing rather enthusiastically several trilling sequences and continued to do so until the female had left (t = + 38 sec) (S3–S5 Audios).
Discussion
We observed an accumulation of Centurion senex males over a period of several weeks until the animals disappeared. As such an event had never been observed before we refrained from all manipulations of the animals in order not to produce a disturbance that might cause them to abandon the area, which clearly set distinct limitations to our study. Nevertheless, our study provides the first information on echolocation and courtship behavior of a rare and highly interesting frugivorous bat.
Echolocation
In general, echolocation of phyllostomid bats is characterized by short, frequency-modulated (FM) calls with distinct harmonics [28]. These calls are generally seen as an adaptation to the orientation by echolocation within cluttered space [29], as short and broadband echolocation calls allow a precise spatial resolution [30]. In the temporal domain, the echolocation calls of C. senex match this general pattern, however, the frequency domain shows distinct deviations from the family. While the calls of most phyllostomids show highest energy in the 3rd harmonic [28], our recordings from Centurio senex showed consistently just the single 1st harmonic and only rarely we observed an extremely weak 2nd harmonic. Emission of echolocation calls with the main energy in the first harmonic is extremely rare in the family and has been observed only in few species, e.g., in the nectarivorous Phyllonycteris poeyi [31] or Leptonycteris yerbabuenae (Gonzalez-Terrazas, pers. comm.). The single 1st harmonic of Centurio senex covers a relatively high bandwidth compared to similar-sized phyllostomids [32, 33], so functionally the species should have access to the same echo information as species covering a larger frequency range by using several harmonics. Harmonics are a common phenomenon in vocalizations, and their absence can be explained by filtering characteristics, e.g., of the vocal tract [34]. Due to its ability to consume hard seeds [25], Centurio senex has the relatively shortest skull of all phyllostomid bats and so the vocal tract seems not to be overly long and distinctly limiting the emission of harmonics. However, the relation between skull morphology and sound production in bats is complex and clearly needs more research [35]. The lack of harmonics is also not explained by the facial mask of the perched males, as the echolocation calls of masked perched bats did not differ significantly from those of flying individuals, when Centurio senex males usually did not wear the mask.
Lek mating
Our observations suggest that Centurio senex employs a lek mating strategy, as our observations match many characteristics of a “classic” lek [14, 22]: (1) Males aggregate on a relatively small area and show a unique displaying behavior. With their masks raised, male Centurio move over long time periods just their wing tips and vocalize. (2) In the interaction with males, females apparently do not gain more than genetic material. Males do not offer the females access to a particular and limited resource. (3) Although we do not have direct observational data, it is highly unlikely that Centurio males contribute to parental care, as this is unknown from the majority of bat species. The sex ratio of Centurio is reported to be biased in favor of females (2:1) [36]. (4) Females are free to select and fly to any of the perching males. In addition, C. senex shows also a distinct sexual dimorphism, internal fertilization, and high mobility of females that allows them easily to choose between males. We observed the animals until the aggregation of males dissolved. As we were very careful not to disturb the perched animals, we believe that the disappearance of the animals reflected the end of the natural mating period.Lek courtship behavior seems to be rare in bats, with only few species showing true leks or lek-like behavior [20]. So far, the hammer-headed bat, Hypsignathus monstrosus (Pteropodidae) is the only bat reported to form classic leks [10]. Here, males congregate for calling in a specific area, and males use every night the same spot on a branch for calling. The visit of a female to a chosen male is immediately followed by copulation, lasting for 30–60 seconds. Female selection seems to be rather strong, with only 6% of the males obtaining 79% of the observed copulations. Seven more bat species show a lek-like behavior, that either does not fully correspond to the classic lek definition or remains to be confirmed as such, due to the lack of behavioral data [22].Clearly, more information on Centurio senex would be desirable, but based on the available data its behavior resembles the classic lek mating pattern of Hypsignathus monstrosus, with calling males relatively close to each other within a relatively small area and females visiting perched males for copulating. Open questions are, e.g., the relative density of males and females in the area, and whether the visitors to the perching males are all females. Also, we recorded just a single copulation, but as we had only a single camera to shift between the perches, we cannot say whether this low number reflects actually a low mating success of the males, or is just the result of our limited sampling.
Male display behavior
What are the cues that attract females and can be the base for female mate choice and sexual selection in Centurio senex? We suspect that the male display behavior might utilize several sensory canals, some effective at distance and others only at close range.
Acoustics
Acoustic signals seem to be important since these are presented throughout the time and may advertise the presence of a male also over larger distances. Conspicuous elements are the trilling calls emitted spontaneously during phase 1), which is characterized by the bat moving subtly the fingers against each other, even without another individual close by. Once the perched male is approached by a visitor in phase 2), the acoustic behavior intensifies. The agitated bat starts to beat its wings, which produces broadband noise pulses, and in most cases, the encounter ends with the extremely loud whistle call, upon which the visitor leaves. The impressive volume of this last element—that seems to be rather excessive as it is only emitted once the visitor is very close—might provide information on the physical condition of the perch owner. While the comparably quiet and high frequency trilling calls probably can be heard only over a rather limited range, the final whistle call is not only extremely loud but contains also predominantly lower frequencies, thus it suffers much lower levels of atmospheric attenuation and therefore may be perceived over a larger distance [30]. A close-range function of the trilling calls is also supported by their continued use during the observed copulation. Audio recordings from a single perched male showed rarely trilling calls from other males, while often there were whistle calls at various levels of intensity, indicating the presence of other males at different distances (see also S2 Video).The acoustic channel is rather commonly used in bat courtship [e.g., 10, 37–39]. The acoustic repertoire of Centurio senex is relatively limited, especially compared with particularly vocal species, such as the emballonurid Saccopteryx bilineata [40, 41]. Nevertheless, male quality or even identity could be coded in call intensity as well as in call rate, i.e. the repetition of certain syllables, e.g., in the duration of trill sequences [42]. For future investigations on Centurio senex, it might be interesting to measure absolute sound pressure levels, especially of the loud whistle calls and relate calling behavior to body size parameters of the advertising males and their position within the lek site.Interestingly, although the behavior of Centurio males involves distinct vocalization both in the display phase and during the encounters, vocalizations appear to be emitted while the skin mask totally covers the mouth, which could result in a slightly reduced emission volume and a modified frequency spectrum. However, nostrils are mostly uncovered by the raised mask, suggesting at least a partially nasal emission of the observed social vocalizations. Perhaps the unique facial wrinkles which are in males more pronounced than in females, might assist in directing the sonar beam of these calls.The audible wing beats, representing noise pulses generated through friction, are audible at least on close range. Similar non-vocal sound-production through wing movements has been observed in Old World Fruit bats, although not in a social context but in a rudimentary form of echolocation [43]. In C. senex, this behavior might be connected to another morphological peculiarity: Wings of the bat show a distinct pattern of parallel folds between the 5th and the 4th digit [23] that might support the observed sound production. Questions remain, however, as this character is also present in females and seems to not be sexually dimorphic (pers. obs, BRH). Non-vocal sound production appears to be very unusual in bats.The combined acoustic activity of all males within the relatively small lek area, especially of the whistle calls (S2 Video), and to a lower degree also the trilling calls, might create an acoustic beacon that guides females into the display area [44]. In this context perch position within the lek might be essential, as an orientation of females towards a high acoustic activity would lead them to the center of the lek, resulting in increased mating chances for males occupying center positions. For several bird species, position of the perch has been documented to be a decisive factor [45]. Our results show that not all perches were used equally, with some being occupied over longer periods while others were occupied only occasionally.
Vision
While echolocation is clearly the predominant sensory channel for the nocturnal bats, vision may be also used for obtaining information about the surroundings [46, 47]. Within the phyllostomid family, eye size is particularly variable, indicating species-specific differences in the use of visual information (e.g., [48]). The conspicuously white color of Centurio senex males´ facial mask and the white spot near the ears might be visual signals that could improve the visibility. The white color provides a distinct contrast to the darker background vegetation. As the males were displaying at the lek site over 46 days, the perching bats were present during all phases of the moon, including two full moons (25.9.2018, 24.10.2018), so at least on some nights the light available in the understory might be sufficient, at least on close distance. As Centurio senex males and females have relatively large eyes (Fig 5B), vision seems to be an important sense for the species, perhaps for landmark orientation during commuting flight, but on close distance in the understory, this could also support mate choice. The extension and shade of the white color of the mask might perhaps even transmit visual information on male quality to interested female vistors. In this context, the observed perch cleaning behavior could improve visibility of a perching male to passing females.
Olfaction
Although our strictly observational approach did not allow to collect olfactory data, chemical cues could also be essential elements in male display behavior. Olfaction is used by bats for various purposes, ranging from foraging to social interactions [e.g., 49–51]. The use of olfactory cues by Centurio senex is supported by our observation of males rubbing their bodies on the branch, which could represent a scent marking behavior. Early on was reported that male Centurio senex have a strong odor in the chin area [26]. Our photos of the naked facial skin indicate a moist surface, suggesting secretion of fluids in the area of the skin pouch. The pouch might even offer a possibility for controlling the release of olfactory signals: As long as the mask is raised there is only limited exposure of the facial skin to the air, while a lowered mask could allow free dissipation of locally produced olfactory volatiles. A similar odor-releasing system has been described from the Greater sac-winged bat, Saccopteryx bilineata (Emballonuridae), where males prepare a cocktail of body fluids of different origin in their wing sacs and fan the resulting odour during hovering displays towards the perched female [50]. Images from our study in comparison to males observed on other occasions give the impression that the skin pouch is slightly inflated. It would be interesting to compare the morphological and physical conditions of the skin mask between a displaying male captured from its perch to that of a male captured at another time of the year in a non-reproductive context. Seasonal changes in sexually dimorphic structures, other than in the primary reproductive organs, are uncommon in bats, but are reported for a close relative of Centurio senex. Pygoderma bilabiatum males show sexually dimorphic forelimb swellings that seem to vary over the reproductive cycle [52]. Further observations on C. senex should, therefore, combine a histological study of the skin mask with the collection of scent-samples from reproductive and non-reproductive males and females.
Facial mask
Most of the time males perched with raised face mask, even when singing. While this confirms our initial hypothesis, the actual function of the mask for Centurio senex males remains still rather unclear. A possible role might be indicated by our single visual record of an approaching visitor, when the perched bat’s mask is fully raised (Fig 5D) and exposure of the face is minimized, suggesting a protective function, perhaps for the large eyes, although we never observed any obvious aggressive interactions. Slow motion video recordings would be highly useful to see whether such a protective behavior is a common element of all encounters. An important question in this context would be also whether all visitors are females or whether perching males receive also visits from other males, that are recognizable by their mask. Our observations on the lek courtship behavior of Centurio senex males might also have some implications for related species: Besides Centurio senex there are also several other species within the subtribe Stenodermatina (short-faced or white-shouldered bats, [53]) that show a similar sexual dimorphism. Besides the already mentioned Pygoderma males with its temporal glandular swellings on the wings [52], there is also Sphaeronycteris toxophyllum, where males also have a retractable face mask and additionally develop with sexual maturity a permanent, visor-like structure on the forehead. Perhaps also this structure has a potential protective function during a similar courtship behavior as here described for Centurio senex. Clearly, we are still far from a full understanding of these unique sexual dimorphic structures.
Costs of lek mating
Lek mating involves a significant investment for Centurio senex males. As perches were occupied between nightfall and ca. midnight, males spent at least 50% of their potential foraging time at the lek site without eating. Most of this time is spent motionless or with subtle finger movements, complemented by singing and the occasional behavioral and acoustic reactions to a visitor. Besides the cost for the vocalizations [54], additional energy is invested in perch maintenance behavior, such as removing smaller leaves and in the presumed scent marking of the perch. Modification of plants into day roosts is a potentially costly common behavior employed by male and female stenodermatine tent-making bat species [55], however, there are no reports on simple modifications of night roosts such as the small branches used by C. senex. Incidentally, the use of an over several days stable and thus predictable perch, while persistently vocalizing might also increase distinctly the predation risk for Centurio senex males. This stationary and acoustically conspicuous behavior has the potential to make them a target for acoustically in the rain forest understory hunting predators such as various species of owls.
Conclusion
Our study summarizes the first behavioral data from a lek of Centurio senex males, observed over a period of almost 6 weeks at a Costa Rican highland forest site. The exclusively male bats used stable perches and showed distinct behavioral patterns including an acoustic signaling with stereotypical elements that might serve to attract females and perhaps also to signal male quality. Perching animals were regularly approached by other individuals and in one case such an encounter was followed by a copulation. Males often spent several hours on the perch without leaving and abandoned it only after midnight. This endurance left males with only half of the night time for foraging, so the time spent on the perch is a significant energetic investment and could be a honest signal for male quality. Our study has some obvious shortcomings. While we were extremely lucky to obtain the first observations on the behavior of this interesting species we deliberately refrained from mist-netting bats in order not to scare the animals away from our study site. The downside of this cautious approach is, however, that we still lack essential data, such as the sex ratio at the site, the body condition of individual males or the identity of all visitors. In this context, it would also be highly interesting to assess potential olfactory signals of the perching males, perhaps in the facial region. Hopefully, the encounter of a future lek of Centurio senex will allow us to close some of the current gaps in the knowledge on the behavior of one of the most iconic bats of the Neotropics.
Individual perch use between October 4th and October 20th.
Dots indicate the presence of a Centurio senex male at the respective perch. Perches occupied during successive surveys are connected by a line.(TIFF)Click here for additional data file.
Echolocation parameters showed no significant differences between calls emitted in flight and those from perched animals.
(DOCX)Click here for additional data file.
Song elements: Duration and number of syllables recorded at different perches.
(Wing beat sequence: each syllable comprises a wing beat and its associated wing beat call, if present). n is the number of elements analyzed for each song.(DOCX)Click here for additional data file.
Display behavior of Centurio senex, showing a male with raised mask initially rubbing the wing tips against each other.
At t = 34 sec another animal approaches and the perched male produces an audible whistle call. After the encounter the male returns to wing tip rubbing.(MOV)Click here for additional data file.
Interaction between a perching male and a visitor (t = 5–11 sec).
Besides the main interaction, many whistle calls from other males in the lek at widely varying intensity can be heard (e.g., at t = 3, 4.5, 7.5, 9.5, 11.5, 15, 16, 17, 23, 23.5, 24, 26, 34, 36, 36.5, 37.5, 40.5 sec, etc.).(MOV)Click here for additional data file.
Copulation between perching male and a visiting female.
After the approach of a hovering individual (t = 15 sec) a female lands (t = 20 sec) and a copulation follows (t = 25–58 sec).(MOV)Click here for additional data file.
Audio file courtship song sequence from Fig 2.
This is the original file with a sampling rate of 500 kHz, that can be played with specialized sound analysis software. Note the clearly audible whistle call at the end of the recording.(WAV)Click here for additional data file.
Same as S1 Audio, just slowed down by a factor 10, so it can be played by standard audio device.
(WAV)Click here for additional data file.
Audio sequence from copulation event.
The copulation started approximately at t0 = 11.25 sec in the original audio file. Note first (t0−10 sec) and second (t0−5 sec) courtship song sequences. The silent phase after t0 covers the actual copulation. Once the female got restless (t0 + 33 sec) the male performed a large number of inspired trilling sounds until the female left (t0 + 38). This is the original file with a sampling rate of 500 kHz, that can be played with specialized sound analysis software.(WAV)Click here for additional data file.
Same as S3 Audio, just slowed down by a factor 10, so it can be played by standard audio device.
(WAV)Click here for additional data file.(WAV)Click here for additional data file.
Acoustic original data.
(XLSX)Click here for additional data file.12 Aug 2020PONE-D-20-16747The masked seducers: Lek courtship behavior in the Wrinkle-faced batCenturio senex (Phyllostomidae)PLOS ONEDear Dr. Rodriguez-Herrera,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Specifically, it would be important to address the reviewers' comments about clarifying some parts of the paper and including appropriate caveats about the results.Please submit your revised manuscript by Sep 26 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsWe look forward to receiving your revised manuscript.Kind regards,Vivek NityanandaAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Thank you for stating the following in the Competing Interests section:"The authors have declared that no competing interests exist."We note that one or more of the authors are employed by a commercial company: Hotel Villablanca.2.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.Please also include the following statement within your amended Funding Statement.“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.We will update your Data Availability statement on your behalf to reflect the information you provide.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: The authors described for the very first time Lek mating behavior in the bat Centurio senex, this is a valuable and exciting finding. Furthermore, the manuscript is expertly prepared. It is extremely rare to read an article that so thoroughly anticipates and answers questions/concerns. Both the topic, the evaluation of the data, and the quality of the writing, made this a true pleasure to review.My only very minor comments are as follows:- Even if its just observational, as the bats weren't individually marked, do the authors have an idea if the same roosts were used by the same individuals on consecutive days (that is: had some of them maybe some natural markings that made them identifiable)?- Although all 53 perches were marked, I am assuming not all were fitted with cameras & microphones, thus I would encourage the authors to acknowledge this and discuss the possibility that other copulation events might have been missed. The authors present compelling evidence that this is in fact mating behaviors but with only one recorded event I believe it necessary to address this either lack of success by the males or impossibility to survey all perches simultaneously allowing for some missed events.- A table or set of graphs that shows the statistical comparison of the features of perched and in flight echolocation would be very useful. Also, the metabolic cost of perched echolocation and potential uses in communication has been discussed for other species (for example: Front Physiol. 2013; doi: 10.3389/fphys.2013.00066. Metabolic costs of bat echolocation in a non-foraging context support a role in communication. D. Dechmann, M. Wikelski, H. van Noordwijk, C. Voigt, S. L. Voigt-Heucke.)- The authors discuss the differences in the composition of the songs in regards to the number of elements, but, were there differences in the spectro-temporal signatures of the syllables among the bats that may allow for identification of individuals? (Bohn KM, Schmidt-French B, Schwartz C, Smotherman M, Pollak GD (2009) Versatility and Stereotypy of Free-Tailed Bat Songs. PLoS ONE 4(8): e6746. https://doi.org/10.1371/journal.pone.0006746)- For the behavioral observations it would be useful to have percentages of time, for example the authors describe "94.1 ± 13.89 min of total observation time" which one needs to go back further up to check how long was the total observation time. Adding % would aid the reader to quickly understand if this is a significant amount of time.- Are all visitors female? The authors mention this question in the discussion do not explain why this is not something that can be know with the collected data. What is the limitation to answering this question with the collected data? viewing angle for the visiting bat? few perch sites being recorded and this identification being hard to do with the naked eye? Any or all of this are valid, it just would be good to explain why this is not known.- The authors mention that sound production with the wings in bats is rare and this is true, yet they might want to cite a paper that shows a species of fruit bats that use wing beat sounds for sonar (Nonecholocating Fruit Bats Produce Biosonar Clicks with Their Wings. Arjan Boonman, Sara Bumrungsri, Yossi Yovel. https://doi.org/10.1016/j.cub.2014.10.077)I would also recommend citing the two following review articles:- This one reviews auditory processing of communication in bats and points out which species until the data it was published have been described to show songs. This is relevant the introduction. (Salles, A., Bohn, K. M., & Moss, C. F. (2019). Auditory communication processing in bats: What we know and where to go. Behavioral Neuroscience, 133(3), 305–319. https://doi.org/10.1037/bne0000308)- This other one more broadly reviews social communication in bats and includes detailed descriptions of acoustic, visual, tactile and olfactory information different species obtain through these sensory modalities. (Chaverri, G., L. Ancillotto, and D. Russo. 2018. Social communication in bats. Biological Reviews, 93: 1938-1954).Reviewer #2: Review for: Rodriguez-Herrera et al.: The masked seducers: Lek courtship behavior in the Wrinkle-faced batCenturio senex (Phyllostomidae)The authors report on an observational study on the bat species Centurio senex in Costa Rica. The bat is famous for its rather strange looking facial mask. Beyond, not much is known about the species. This is also true for lek courtship behavior in bats.Overall, I very much enjoyed reading the manuscript. The fact that the study is more or less purely descriptive and does not contain many hypothesis, predictions and experiments is in this case a plus. It allows the authors to tell the readers about their observations free from any bias. Moreover, especially in the discussion the authors address many questions and give ideas for future studies, which can then be conducted based on hypothesis and experiments. The manuscript thus can be seen as an important starting point for further studies on C. senex, lek behaviour in bats and more generally courtship behaviour in animals.I only have some minor points, which I will address in the following:Introduction“Additionally, variables such as time and place where bats will show courtship behavior are for many species extremely difficult or impossible to predict” … and to access, I would say.Hypothesis: The only hypothesis in the paper refers to the facial mask. However, I am missing some predictions that explain this hypothesis. What might be the function of the mask? In example: Might certain features of the mask (e.g. size) act as a sign for good genes? Might it be a protection against competing males (as the authors suggest in the discussion)?Materials and Methods“On October 3rd, perches were individually marked.” Why only on this date? Or do the authors mean: From October 3rd on, perches were individually marked?Ultrasound recordingRegarding the equipment for the USG 116Hm: Which microphone did the authors use? The authors write that the microphone was placed “as close as possible”? How close was this on average?Did the authors trigger manually, automatically or did they choose continuous recordings? If the recording was done automatically, which settings did they choose?“Additionally, we recorded also echolocation calls from bats flying in the study area.” Which bats were recorded? Does this refer to C. senex or any bat species flying in the study area?ResultsWere there any obvious reasons, why the number of perching males started to decrease in October? Could this have been the end of mating season, rainy/dry season or due to monitoring? If so, it should be discussed in the Discussion section.Average height of the perches: In the M&M section it is missing how these measures were done.Echolocation behavior: For the bandwidth the SD is missing in the text.DiscussionVision: Although this cannot be proven, I would guess that the males’ facial mask itself might be an indication that females select males based on visual information.Olfaction: This is just an idea for future tests that came to my mind. Might the facial mask have an olfactory function similar to the pouches of Saccopteryx? Secreted fluids would than be protected as long as the mask is lowered. When the mask is raised, pheromones could be spreaded through the air.Best wishesMichael Schöner**********6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Yes: Michael Schöner[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.27 Sep 2020Video 3 now is available.Comments from Dr. Vivek Nityananda Academic Editor PLOS ONEComment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.Response: The manuscript “The masked seducers: Lek courtship behavior in the Wrinkle-faced batCenturio senex (Phyllostomidae)” meets PLOS ONE's style requirements as suggested.Comment 2: "The authors have declared that no competing interests exist."We note that one or more of the authors are employed by a commercial company: Hotel Villablanca.Response: Hotel Villa Blanca did not play a role as funding source nor in the study design, data collection and analysis, decision to publish, or preparation of the manuscript, however they provided financial support in the form of salaries for two of the authors, yet not for working on this project. We have reviewed the statements relating to the author contributions, and we made sure to specifically and accurately indicate the role these authors had in the study. We have updated author roles in the Author Contributions section of the online submission form.Additionally, we have included the following statement within the amended Funding Statement “The owner of the study site, Hotel Villa Blanca, provided support in the form of salaries for authors J.V. and E.H., but did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are defined in the ‘author contributions’ section. Furthermore, we state and explain this role within the updated Funding Statement.We have also provided an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment of the mentioned authors.Within the Competing Interests Statement, we confirmed that this commercial affiliation did not alter our adherence to all PLOS ONE policies on sharing data and materials. We included the following statement as required by PLOS ONE "This does not alter our adherence to PLOS ONE policies on sharing data and materials.”Comment 3: We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly.Response: In addition to two tables provided as supplementary information we have now uploaded the data set necessary to replicate our key findings as Supporting Information entitled “S4 Original Data The masked seducers”.Comments from Reviewer #1“The authors described for the very first time Lek mating behavior in the bat Centurio senex, this is a valuable and exciting finding. Furthermore, the manuscript is expertly prepared. It is extremely rare to read an article that so thoroughly anticipates and answers questions/concerns. Both the topic, the evaluation of the data, and the quality of the writing, made this a true pleasure to review.My only very minor comments are as follows:”Comment 1: Even if it's just observational, as the bats weren't individually marked, do the authors have an idea if the same roosts were used by the same individuals on consecutive days (that is: had some of them maybe some natural markings that made them identifiable)?Response: We totally agree with the relevance of being able to identify and track individuals, however, in situ C. senex individuals did unfortunately not show any evident natural marks that permitted an individual identification. In addition, as we were anxious not to lose this unique opportunity, we decided not to perform any marking or capture activities in order not to scare the animals away from the site. We apologize but we have no further evidence or data to add, although we believe that the perches are used consistently by the same individuals. Hopefully next time…Comment 2: Although all 53 perches were marked, I am assuming not all were fitted with cameras & microphones, thus I would encourage the authors to acknowledge this and discuss the possibility that other copulation events might have been missed. The authors present compelling evidence that this is in fact mating behaviors but with only one recorded event I believe it necessary to address this either lack of success by the males or impossibility to survey all perches simultaneously allowing for some missed events..Response: We had only a single camera set up that was shifted between perches and we include this information now in the Methods. We added also a remark in the Discussion on the problems of distinguishing between a “real” low copulation success of males and low observation probability of the event, due to a single available camera for sometimes more than 30 perches.Comment 3: A table or set of graphs that shows the statistical comparison of the features of perched and in-flight echolocation would be very useful.Response: We added to the Supplementary Information a table (S2 table) containing the statistical comparison between the echolocation call parameters of calls emitted by animals in flight and from animals at perches.Also, the metabolic cost of perched echolocation and potential uses in communication has been discussed for other species (for example: Front Physiol. 2013; doi: 10.3389/fphys.2013.00066. Metabolic costs of bat echolocation in a non-foraging context support a role in communication. D. Dechmann, M. Wikelski, H. van Noordwijk, C. Voigt, S. L. Voigt-Heucke.).Response: We cite now the suggested paper in our paragraph on metabolic costs of courtship behaviour.Comment 4: The authors discuss the differences in the composition of the songs in regards to the number of elements, but, were there differences in the spectro-temporal signatures of the syllables among the bats that may allow for identification of individuals? (Bohn KM, Schmidt-French B, Schwartz C, Smotherman M, Pollak GD (2009) Versatility and Stereotypy of Free-Tailed Bat Songs. PLoS ONE 4(8): e6746. https://doi.org/10.1371/journal.pone.0006746 .Response: While we did not observe any obvious differences in spectro-temporal parameters, we can imagine that there might be differences between individuals for some of the measured parameters. However, for really attempting such an investigation it would be mandatory to have a sample of unequivocally marked individuals, preferably with additional body metrics. With the current data set we are not able to distinguish reliably between individual variability or individual signatures. While we therefore believe this approach to be outside the scope of the current manuscript, we certainly hope to conduct such a study in the future and cite the mentioned paper now in the manuscript.Comment 5: For the behavioral observations it would be useful to have percentages of time, for example the authors describe "94.1 ± 13.89 min of total observation time" which one needs to go back further up to check how long was the total observation time. Adding % would aid the reader to quickly understand if this is a significant amount of time.Response: Thanks for the suggestion. We changed in the Results the values mentioned for the percentage of time spent by bats in phase 1.Comment 6: Are all visitors female? The authors mention this question in the discussion do not explain why this is not something that can be know with the collected data. What is the limitation to answering this question with the collected data? viewing angle for the visitingbat? few perch sites being recorded and this identification being hard to do with the naked eye? Any or all of this are valid, it just would be good to explain why this is not known.Response: Live observations are not possible in these quickly moving, nocturnal and small animals and unfortunately also the video-recordings do not help very much to obtain the required details. The fast-moving visiting bats are inevitably blurred due to the frame rate / time resolution of the video camera, and additionally we could only focus on the perched individual, so the hovering individuals were mostly out of focus. This results in poor quality that did not allow a reliable assessment of the visiting animals through the absence or presence of the mask. We included a short statement in the Results explaining the problem.Comment 7: The authors mention that sound production with the wings in bats is rare and this is true, yet they might want to cite a paper that shows a species of fruit bats that use wing beat sounds for sonar (Nonecholocating Fruit Bats Produce Biosonar Clicks with Their Wings. Arjan Boonman, Sara Bumrungsri, Yossi Yovel. https://doi.org/10.1016/j.cub.2014.10.077)Response: Thanks for the suggestion, we cite this paper now in the Discussion.Comment 8: I would also recommend citing the two following review articles:- This one reviews auditory processing of communication in bats and points out which species until the data it was published have been described to show songs. This is relevant the introduction. (Salles, A., Bohn, K. M., & Moss, C. F. (2019). Auditory communication processing in bats: What we know and where to go. Behavioral Neuroscience, 133(3), 305–319. https://doi.org/10.1037/bne0000308)- This other one more broadly reviews social communication in bats and includes detailed descriptions of acoustic, visual, tactile and olfactory information different species obtain through these sensory modalities. (Chaverri, G., L. Ancillotto, and D. Russo. 2018. Social communication in bats. Biological Reviews, 93: 1938-1954).Response: Both references are now included in the Introduction.Comments from Reviewer #2“Overall, I very much enjoyed reading the manuscript. The fact that the study is more or less purely descriptive and does not contain many hypothesis, predictions and experiments is in this case a plus. It allows the authors to tell the readers about their observations free from any bias. Moreover, especially in the discussion the authors address many questions and give ideas for future studies, which can then be conducted based on hypothesis and experiments. The manuscript thus can be seen as an important starting point for further studies on C. senex, lek behaviour in bats and more generally courtship behaviour in animals.I only have some minor points, which I will address in the following:Comment 1: Introduction“Additionally, variables such as time and place where bats will show courtship behavior are for many species extremely difficult or impossible to predict” … and to access, I would say.Response: Thank you for the suggestion. We have modified the first sentence as follows “Additionally, variables such as time and place where bats will show courtship behavior are for many species extremely difficult or impossible to predict or access.”Hypothesis: The only hypothesis in the paper refers to the facial mask. However, I am missing some predictions that explain this hypothesis. What might be the function of the mask? In example: Might certain features of the mask (e.g. size) act as a sign for good genes? Might it be a protection against competing males (as the authors suggest in the discussion)?Response: Our problem was to set up a hypothesis that is really testable. As our study was totally hands-off and purely observational with no information on individual animals, our options were extremely limited. For that reason, unfortunately, none of the suggested predictions would be really verifiable, as the available data would not allow testing for a correlation of good genes or even just of reproductive success with features of the mask. Similarly, although we really like the idea of the mask serving as a physical protection against competing males, we cannot even reliably distinguish between male and female visitors and the details of the interaction between perched and visiting individuals are rather unclear. With a lot of luck we managed to get a single acceptable photo of a visit (Fig. 5c), but for really obtaining data we would have needed a high-speed camera, which was not possible in the short time available. With all due respect we would therefore prefer to stay with our very simple, yet verifiable hypothesis, that is based on the distinct sexual dimorphism known already before our study, and then discuss, based on our findings and observations, potential functions of the facial mask, including also the protective function, in the Discussion.Comment 2: Materials and Methods“On October 3rd, perches were individually marked.” Why only on this date? Or do the authors mean: From October 3rd on, perches were individually marked?Response: To clarify this comment we have rewritten this section in the MM section to make our statement clearer. Also, we describe in the Results this point in detail as follows “The animals were – compared to other phyllostomid bats - rather tolerant to the cautious approach of an observer, and appeared to be relatively reluctant to leave their perches. The exact same spots with extremely little changes (< 5cm) were occupied over different nights. Perching bats were observed in the study area until October 31st, for a total of 46 days. During this period, we were able to collect data during 13 nights.”Comment 3: Ultrasound recordingRegarding the equipment for the USG 116Hm: Which microphone did the authors use? The authors write that the microphone was placed “as close as possible”? How close was this on average? Did the authors trigger manually, automatically or did they choose continuous recordings? If the recording was done automatically, which settings did they choose?“Additionally, we recorded also echolocation calls from bats flying in the study area.” Which bats were recorded? Does this refer to C. senex or any bat species flying in the study area?Response: We added the missing information on the microphone, microphone placement and on our recording strategy at the porches and along the trails in the understory. We recorded only phyllostomid bats along the trails, and while the echolocation calls are rather generic within phyllostomids in general, those of Centurio senex are different, based on harmonic structure. We explain this in the Results.Comment 4: ResultsWere there any obvious reasons, why the number of perching males started to decrease in October? Could this have been the end of mating season, rainy/dry season or due to monitoring? If so, it should be discussed in the Discussion section.Response: As we were really careful not to disturb the animals we believe that the disappearance of the animals was due to the end of the mating period. We added a sentence in the Results section to clarify this statement and also in the Discussion.Average height of the perches: In the M&M section it is missing how these measures were done.Response: We describe now in MM how the average height of perches was measured.Echolocation behavior: For the bandwidth the SD is missing in the text.Response: SD of the bandwidth was added.Comment 5: DiscussionVision: Although this cannot be proven, I would guess that the males’ facial mask itself might be an indication that females select males based on visual information.Olfaction: This is just an idea for future tests that came to my mind. Might the facial mask have an olfactory function similar to the pouches of Saccopteryx? Secreted fluids would then be protected as long as the mask is lowered. When the mask is raised, pheromones could be spreaded through the air.Response: Fascinating idea, thank you! Indeed, this would be very interesting to investigate in the future! Currently, many raising questions cannot be answered. We added the point about the facial mask per se providing optical information to interested females, as well as the possibility of the mask aiding in dispensing olfactory signals in the Discussion.Submitted filename: Response to Reviewers2.docxClick here for additional data file.8 Oct 2020The masked seducers: Lek courtship behavior in the Wrinkle-faced batCenturio senex (Phyllostomidae)PONE-D-20-16747R1Dear Dr. Rodriguez-Herrera,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.Kind regards,Vivek NityanandaAcademic EditorPLOS ONEAdditional Editor Comments (optional):Reviewers' comments:19 Oct 2020PONE-D-20-16747R1The masked seducers: Lek courtship behavior in the Wrinkle-faced batCenturio senex (Phyllostomidae)Dear Dr. Rodriguez-Herrera:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.If we can help with anything else, please email us at plosone@plos.org.Thank you for submitting your work to PLOS ONE and supporting open access.Kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Vivek NityanandaAcademic EditorPLOS ONE
Authors: Signe Brinkløv; Lasse Jakobsen; John M Ratcliffe; Elisabeth K V Kalko; Annemarie Surlykke Journal: J Acoust Soc Am Date: 2011-01 Impact factor: 1.840
Authors: Tania P Gonzalez-Terrazas; Carlos Martel; Paulo Milet-Pinheiro; Manfred Ayasse; Elisabeth K V Kalko; Marco Tschapka Journal: R Soc Open Sci Date: 2016-08-10 Impact factor: 2.963
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5