The evolution of sensory systems has always intrigued scientists: how does a rudimentary sense evolve into a more sophisticated one []? Echolocation, the ability to perceive the world acoustically by emitting sound pulses and analyzing the returning echoes, has evolved in at least three different animal groups (birds [], cetaceans [], and bats []), and possibly also other mammals such as in shrews [] and tenrecs [] (see Table 1 ). There is an ongoing debate as to whether bat echolocation evolved once or twice []. It is, however, agreed that more than 1,200 bat species [] can be divided into ∼85% echolocating species and ∼15% Old World fruit bats (family Pteropodidae) that are currently classified as nonecholocating [], with the exception of a few species in the genus Rousettus that use lingual echolocation []. Being nocturnal mammals, “nonecholocating” Old World fruit bats often fly at very low light levels. In 1988, Gould showed that one species of Old World fruit bat (Eonycteris spelaea) produces click sounds in the dark by wing clapping, but this study was not able to prove the functionality of the clicks []. Moreover, all previous studies have shown that Old World fruit bats lack all of the genetic and physiological adaptations that are typically associated with echolocation []. We were therefore compelled to investigate how these bats orient in darkness.

Evolutionary origins of ultrasonic hearing and laryngeal echolocation in bats inferred from morphological analyses of the inner ear.

From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats.

From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats.

We recorded individuals from three subfamilies that are widely spread within the Old World fruit bat phylogenetic tree [] (eight Eonycteris spelaea, eight Cynopterus brachyotis, and three Macroglossus sobrinus) while they were flying in “complete darkness” (< 5 × 10lux), which precluded the use of vision, as we confirmed experimentally.

When flying in the dark, all 19 wild individuals of all three species emitted brief sound signals (300–350 μs) with a click-like structure (only three to eight wave periods) that were audible to the human ear ( Figure 1 ). The average peak frequency of the signals was 17 ± 1 kHz (cross-species mean ± SD), which is lower than that of Rousettus clicks [] but much higher than the click frequency of echolocating birds []. Moreover, clicks were wideband, with energy in high frequencies and a bandwidth of 55 ± 2 kHz. At least one of our three species (Cynopterus brachyotis) has been shown to hear frequencies up to ∼70 kHz []. The time interval between adjacent clicks in Eonycteris and Cynopterus was distributed around 100 ms, whereas in Macroglossus it was shorter (∼80 ms; Figure S1 available online).

(D) Average click spectrum for each species (the average of approximately 200, 1,000, and 2,000 click spectra for Macroglossus, Eonycteris, and Cynopterus, respectively, with at least 60 clicks for each individual bat). Notice the considerable energy content in high frequencies—up to at least 50 kHz. Spectra were calculated using the Fourier transform (256 sample window). Because this analysis is not always suitable for such brief clicks, we also analyzed the clicks using the instantaneous frequency method. With this method, the average peak frequency was 12 ± 1 kHz, and the bandwidth was 56 ± 2 kHz (mean ± SD).

(A) Waveform example of a clicking sequence for each of the three fruit bat species (the sampling rate was 250 kHz). Such continuous clicking sequences for Macroglossus were rare.

To further prove functionality, we also tried to disrupt the bats’ ability to echolocate by playing loud white noise when they were performing the discrimination task. However, the bats refused to fly under these conditions. Plugging the ears of our bats proved very difficult. Moreover, this manipulation will mostly not completely eliminate hearing and might stress the animal, thus disrupting its behavior [].

Our goal was to test whether bats can use these emitted clicks functionally as echolocation. We therefore ceased training after observing that all 12 bats quickly learned the task and that each of them landed on the correct target in at least 70% of the trials (see nights five to six in Figure 2 C). However, only nine bats flew enough for us to assess their performance statistically ( Supplemental Experimental Procedures ). Seven of these nine bats (four Eonycteris and three Cynopterus) performed significantly above chance level (p < 0.05 for each bat, binomial test). The ability of the bats to perform this task proves that they are capable of using biosonar to detect, localize, and discriminate large objects. Interestingly, many researchers have suggested that bats first evolved echolocation in order to detect large objects such as cave walls (see [] for a review). Echolocation tasks that require fine detection and accurate localization, such as the wire avoidance task that we tested (above), are too difficult for these bats to solve. This is probably why Gould [], who had Eonycteris bats flying through multiple bars, could not show that clicking was functional.

Phylogenetic relationships of Icaronycteris, Archaeonycteris, Hassianycteris, and Palaeochiropteryx to extant bat lineages, with comments on the evolution of echolocation and foraging strategies in Microchiroptera.

These spontaneous adjustments in click rate in wild untrained bats strongly imply that clicking is a natural behavior for these bats. Are these clicks used for sensing in the dark? Because Macroglossus bats did not increase clicking significantly, we focused on the other two species. We first tested the bats with the classical wire avoidance paradigm. Here, 12 thick (20 mm diameter) cables were hung across the flight room. Although these cables were ∼100 times thicker than echolocating bats are able to detect [], our bats failed to detect them and constantly crashed into them. We therefore tested the bats in a more basic detection task. We trained 12 bats (six Eonycteris and six Cynopterus) to acoustically discriminate between two large (90 × 90 cm) objects: a highly acoustically reflective black board and a sheet of cloth that was identical in size, color, and odor but was 19 dB less reflective ( Supplemental Experimental Procedures ). The bats had to detect the objects, to discriminate between them, and then to land on the more acoustically reflective object in a completely dark (<5 × 10lux), large anechoic room. The objects were randomly repositioned in the room after each landing (or touching; Supplemental Experimental Procedures ).

The role of pinna movement for the localization of vertical and horizontal wire obstacles in the greater horseshoe bat, Rhinolopus ferrumequinum.

Moreover, in a second experiment in which two of the species flew in a large space in an attempt to discriminate between two objects (see details below), Eonycteris and Cynopterus bats increased their click rate in the dark even more dramatically, with Eonycteris clicking as much as 7.5 clicks per second—the click rate of the echolocating bat Rousettus [] ( Figure 2 B).

We next aimed to reveal whether these clicks are used in a functional manner for sensing in the dark. We flew bats in the same tunnel as before, this time under light (∼5 lux). For each individual, we ran four randomly ordered sessions: two in the dark and two in the light, with at least six trials per bat at each session ( Supplemental Experimental Procedures ). All Eonycteris and Cynopterus bats clicked significantly more in the dark. These bats significantly increased their click rate in the dark, by 3.5- to 5-fold on average ( Figure 2 A). Macroglossus bats also increased their click rate in the dark, but they clicked less than the other two species, and their increase was not significant ( Figure 2 A).

(C) Landing performance (proportion of correct landings) in the object discrimination task. In total we monitored 433 landing events for all 12 bats. The mean and SD for all (six) individuals of each species are presented. Horizontal dashed lines depict the chance level (0.5) and learning criterion (0.7).

(A and B) Clicking in light versus dark conditions (A) in the confined tunnel and (B) during the discrimination task in a large flight room. In both experiments, the average number of clicks per second was computed ( Supplemental Experimental Procedures ). In the tunnel (A), Eonycteris increased clicking from 1.2 ± 1.1 to 5.1 ± 2.8 and Cynopterus from 1.3 ± 1.0 to 3.5 ± 2.4 clicks per second (mean ± SD for all eight bats of each species; p = 0.003 and p = 0.02, respectively). Macroglossus increased clicking from 0.3 ± 0.4 to 2.1 ± 1.3 clicks per second (n = 3 bats, p = 0.25). In the discrimination task (B), Eonycteris increased clicking from 1.1 ± 0.7 to 7.5 ± 3.8 and Cynopterus from 1.2 ± 0.8 to 4.5 ± 3.8 clicks per second (n = 6 bats for both species; p = 0.002 and p = 0.004, respectively). In both (A) and (B), we ran the nonparametric Wilcoxon ranked-sum test for paired sampled data.p < 0.05,p < 0.01.

It is extremely difficult to definitely prove that a bat cannot estimate range, because every experiment showing that an animal cannot perform a task might be a result of an inadequate experimental design. However, the bats’ behavior strongly suggested that these fruit bats cannot estimate the accurate range of an object, based on the time delay between sound emission and echo reception, as all other bats can []. The ability to detect and discriminate objects in the dark, but the inability to smoothly land on them, strongly implies that clicking is a functional, yet rudimentary form of echolocation. Estimating range is considered a hallmark of bat echolocation. The presumed inability of our bats to accurately estimate range therefore suggests that they represent a less sophisticated stage of echolocation.

Although bats learned the task and were able to detect the objects and discriminate between them, in certain sensory aspects, mainly localization, their performance was poor in comparison to other echolocating bats. Various species of echolocating bats (including Rousettus; see Movie S1 ) have been shown to smoothly land on small targets while relying on echolocation []. Despite the target being very large, our bats mostly required several attempts in order to land, often crashing into the target in an uncontrolled manner ( Movie S2 ). In contrast, under light (even very dim light, <1lux), bats always landed smoothly on the landing platform on the first attempt ( Movie S3 ).

How is it possible that these clicks were overlooked during so many years of studying echolocation? The acoustic characteristics of such brief clicks make them very hard to record without an extremely quiet and anechoic environment and without highly sensitive equipment. The signals are much weaker than those of lingual or laryngeal echolocating bats [], and their click-like characteristics with energy that spreads into low frequencies make them difficult to distinguish from noise and easy to overlook by echolocation researchers who are used to tonal signals. Moreover, unlike laryngeal emitters, which always echolocate, Old World fruit bats probably click only in specific situations, such as in extreme darkness.

Gould [] hypothesized that Eonycteris clicks were produced by wing clapping. Even though we prove that clicks are generated by the wings, we are not convinced that the act of the wings touching each other is the mechanism. Gould showed that the wings touch each other during flight; however, he did not establish a causal link between wing touching and clicking by manipulating the first to alter the latter. Moreover, he mentioned that touching occurred in the dark but sometimes also in the light. We analyzed high-speed videos of flying bats but were unable to find a clear difference between the wing cycle in dark versus light—the wings seemed to touch in both cases. Moreover, when we padded the forearms of (two Eonycteris and two Cynopterus) bats by taping foam on both sides, the clicking did not stop. There are many mechanisms to produce clicks by the wings, including (1) two parts of one wing touching each other, (2) a wing touching the body, or (3) snapping of bones. At least two of these mechanisms were shown to occur in birds []. More research is necessary in order to determine the exact sound production mechanism.

We confirmed that the bats’ wingbeat rate did not differ in light versus dark, negating the possibility that the differences in click rate merely reflected a change in the wingbeat rate (Wilcoxon test, p > 0.05 for all three species; Supplemental Experimental Procedures ).

We found that clicks were generated by the wings and not by the larynx or tongue as in all other echolocating bats. Several findings support this conclusion. (1) High-speed video of flying bats revealed that their mouth remained closed during sound production. In orally echolocating bats (including Rousettus), the mouth is open while emitting ( Figure S2 A). (2) Clicking was perfectly synchronized with wingbeat ( Figure S2 B). (3) The clicks’ temporal structure varied greatly, suggesting a noisy sound production mechanism in contrast to laryngeal or lingual emission ( Figure S2 C). (4) Sealing the bats’ mouths (two Eonycteris and one Cynopterus; Supplemental Experimental Procedures ) did not stop clicking. (5) To further confirm that sound was produced by the wings, we impaired the right wing of the two best clickers (one Eonycteris and one Cynopterus) by taping three layers of insulating tape onto the leading edge of their right wing forearms. We flew these two bats in complete darkness. Both bats could fly in this situation, but the extra weight on their right wing made the wingbeat nonsymmetric, with the impaired wing lagging behind the other one. Both bats completely stopped clicking in this situation.

The Evolution of Echolocation

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Rossiter S.J. Evolutionary origins of ultrasonic hearing and laryngeal echolocation in bats inferred from morphological analyses of the inner ear. Rudimentary echolocation does not seem to require any adaptations beyond the ability to produce sound (e.g., clicks) and analyze sound (echoes). Other mammals (e.g., shrews [] and tenrecs []; see Table 1 ) have been suggested to use echolocation, and many other mammals can probably be trained to use simple echolocation. This has been demonstrated in humans []. A main approach for studying the origin of echolocation is examining ancient bat fossils []. For instance, researchers compare the relative size of hearing-related organs (e.g., the cochlea, the stylohyal bone, etc.) in fossil bats with those of extant bat species in an attempt to infer use of echolocation by ancient bats []. Other studies use a comparative approach and study relevant genes such as Prestin [] or search for adaptations in hearing related organs such as the cochlea []. None of these suggested adaptations, however, are present in Old World fruit bats, including the clicking Rousettus bats. These adaptations therefore do not allow distinguishing between echolocating and nonecholocating bats, but rather between laryngeal echolocating bats and the remaining species. It is therefore impossible to determine whether a fossil bat used (rudimentary) echolocation based on these adaptations.

26 Almeida F.C.

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DeSalle R.

Simmons N.B. Evolutionary relationships of the old world fruit bats (Chiroptera, Pteropodidae): another star phylogeny?. 46 Schoeman M.C.

Goodman S.M. Vocalizations in the Malagasy cave-dwelling fruit-bat, Eidolon dupreanum: possible evidence of incipient echolocation?. Figure 3 The Tree of Bat Echolocation Show full caption 26 Almeida F.C.

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DeSalle R.

Simmons N.B. Evolutionary relationships of the old world fruit bats (Chiroptera, Pteropodidae): another star phylogeny?. Current knowledge about the different types of Old World fruit bat echolocation mapped on top of their phylogenetic tree. The tree was adapted with permission from Figure 1 in []. Colors and symbols represent types of sound production and echolocation in genera in the fruit bat family. These include sophisticated echolocators (e.g., Rousettus), rudimentary (wing) echolocators (e.g., Eonycteris and Cynopterus), clicking bats that have not yet been studied (e.g., Macroglossus and Eidolon), and, finally, many species for which nothing is known. Branches leading to ancestors that had a probability of more than 50% to click based on the maximum-likelihood analysis are colored blue. We suggest that comparison of closely related species with different levels of echolocation (e.g., Eonycteris and Rousettus) might provide new insights about the evolution of echolocation. Our results suggest that generating clicks with the wings is a general ability of Old World fruit bats ( Figure 3 ). The three species that we studied do not represent a monophyletic group [] within the Old World fruit bat tree, and clicking has also been recorded in another genus []. Moreover, we have recently recorded wing clicks produced by wild Rousettus aegyptiacus bats while they were flying in the field, in addition to their lingual echolocation clicks ( Figure S3 ). A maximum-likelihood analysis ( Supplemental Experimental Procedures ) suggests a probability of 10% that wing clicking was a trait exhibited by the common ancestor of all Old World fruit bats. This analysis was performed with the most conservative assumption that all Old World fruit bats except for the five discussed above do not click. This probability is thus high when the high uncertainty is taken into account, and studying more species will likely reveal many more clicking species.

Even though it seems to be a common ability, differences in the tendency to click surely exist within the Old World fruit bat family, and they probably reflect different life styles. Being a typical cave dweller, Eonycteris often has to fly in complete darkness, and in accordance, it also exhibited the highest click rate. Macroglossus, on the other hand, is a tree-dwelling species and a very slow flier, giving it ample time to sense its surroundings with a low risk of collision. Accordingly, it exhibited fewer clicks than the other two and did not significantly increase its click rate in the dark.