How mutualisms evolve or how these interactions are maintained is still not sufficiently understood [12]. Particularly, if partners regularly separate, they require species-specific mechanisms to find each other again. This is also true for the carnivorous pitcher plant Nepenthes hemsleyana (Nepenthaceae), which recently was reported to have a mutualistic interaction with the insectivorous batKerivoula hardwickii (Vespertilionidae). This bat fertilizes the plant with its feces while roosting inside the pitchers. The bat droppings enhance the nitrogen intake of N. hemsleyana by 34% on average [10]. In turn, the pitcher plants provide the bats with roosts that are free of parasites, have a stable microclimate, and offer enough roosting space for one or two bats while at the same time preventing the bats from falling into the digestive fluid due to their unique morphological shape and low fluid level [11]. Finding and identifying N. hemsleyana pitchers that grow in the dense Bornean peat swamp forests, however, is a challenging task for echolocating bats: they have to distinguish echoes of the pitchers from those of the cluttered surroundings [13, 14]. The situation is further complicated by the fact that the bats need to distinguish the rare [11, 15] N. hemsleyanapitchers from the more common and similarly shaped pitchers of sympatricNepenthes species, which are unsuitable for roosting [10].
In the Neotropics, a few bat-pollinated plants found an efficient solution to attract bats by developing floral ultrasound reflectors [7, 9], which enabled them to exploit the bats’ echolocation system. However, such reflectors have never been described for plants outside the Neotropics, probably because in the Paleotropics, chiropterophilous plants are pollinated by fruit bats (Pteropodidae) that are unlikely to use echolocation for foraging [16, 17]. We hypothesized that this phenomenon can also be found in the Paleotropics. If so, bat-dependent plants such as N. hemsleyana should have echo-reflecting structures making it easier for bats to localize and identify pitchers. Pitchers lacking such reflectors should be more difficult to find. Additionally, the bats should have echolocation calls that facilitate the finding of targets even within highly cluttered surroundings.
To test whether a certain pitcher structure serves as an effective reflector that acoustically stands out in cluttered environments and guides the bats to their target, we measured ultrasound echoes of pitchers from different angles using a biomimetic sonar head. We sampled pitchers of both N. hemsleyana and its closest relative, Nepenthes rafflesiana (Figure S1), which does not host bats, and ensonified them in the elevation plane (from −40° to 110°; each species n = 9;Figure 1) and the azimuth (horizontal circular) plane (90° on either side of the pitchers’ orifice; each species n = 8; Figure 2A).
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We analyzed the mean spectral target strength (TS), which is a measure of acoustic backscattering of an object, for the whole frequency area of 40–160 kHz. For the measurements in the elevation plane, we found a clear peak forN. hemsleyana pitchers (Figure 1) for angles where the sonar beam ensonified the exposed and prolonged inner back wall at the pitcher’s orifice. This concave structure is lacking in N. rafflesiana (Figure S1A) and other sympatric Nepenthesspecies (e.g., N. ampullaria, N. bicalcarata; Figure S1B). Consequently, within this area, N. hemsleyana pitchers have significantly higher TS than N. rafflesianapitchers (Wilcoxon signed-rank test: V = 11.0, p < 0.001; compare Figures 1A, 1B, and S3A). Interestingly, this was also the area where the bats usually approached the pitchers (0° to 30°, data not shown; Figure S2). When ensonifying the pitchers from steeper angles (>30°), the sonar beam pointed into the pitcher’s cavity, resulting in a strong decline in TS for both species due to sound energy loss by multiple reflections. As N. hemsleyana pitchers are elongated compared to those of N. rafflesiana, the TS changed more abruptly and reached much lower values above 30° angles in the former species. This pattern of a very loud reflector echo followed by a weak echo of the pitcher’s cavity can be seen as a contrast enhancement mechanism, which facilitates the recognition of the orifice.
Ensonifying N. hemsleyana’s orifice in the complete azimuth plane (180°) around the exposed inner pitcher surface showed that the TS for the area between −50° and +50° is significantly higher than in N. rafflesiana pitchers (Wilcoxon signed-rank test: V = 0.0, p < 0.001; Figures 2A and S3B). Thus, echoes fromN. hemsleyana are reflected with higher intensity across a wide angle. As a result, the catchment area, which is the area where the bats are able to detect an object by echolocation, is also significantly larger for N. hemsleyana pitchers (13.0 ± 1.5 m2, mean ± SD) than for N. rafflesiana pitchers (11.2 ± 0.6 m2; one-sided Welch two-sample t test: t = −2.98, p = 0.007). Such an increased catchment area can also be found in reflectors of bat-pollinated flowers in the Neotropics [7, 9].
One of these bat-pollinated plants (Marcgravia evenia) not only features an increased catchment area but additionally shows characteristic spectral signatures [9]. We therefore also analyzed the spectral contents of the pitchers’ echoes and found that directional spectral information of N. hemsleyana pitchers clearly differs from that of N. rafflesiana (as exemplarily shown in the spectral directional plots in Figures 2B and 2C). Sliding-window comparisons (27°) of the spectra of N. hemsleyana and N. rafflesiana pitchers (n = 8 each) revealed significant spectral differences between the species within an angular range of 20° to 25° on either side of the pitcher’s orifice, angles at which the back wall is ensonified (Figure 2D; see Supplemental Information). Thus, the bats could use the pitchers’ species-specific spectral pattern to identify them, especially during lateral approaches, while the significantly increased TS of N. hemsleyanapitchers helps the pitchers to acoustically stand out in cluttered surroundings.
Bats in the genus Kerivoula generally have relatively short, high-pitched calls [18] covering a very large bandwidth, which further increases when they approach an object [19]. Such a call design is typical for the guild of narrow-space gleaning foragers [20] as it facilitates hunting in dense vegetation [19, 20]. Calls of Kerivoula have also been proposed to facilitate detection of fluttering prey [21].
To examine whether the bats’ call design is also suitable for the detection of pitchers, we recorded the echolocation calls of five K. hardwickii individuals upon their approach toward pitchers, selected the last five calls, and analyzed their starting, peak, and end frequency, bandwidth, duration, and pulse interval [19] as well as directionality [22]. The analyzed calls consisted of only the first harmonic with a very short duration, broad bandwidth, and exceptionally high starting frequencies of up to 292 kHz (Figures 3A and 3B ). To our knowledge, these are the highest frequencies ever recorded in bats. These high-pitched calls result in a very high call directionality [20, 23, 24, 25] (Figures 3A and 3C), which facilitate localization and classification of targets in cluttered surroundings as only the object of interest is ensonified while clutter echoes are blended out [23]. Thus, these calls are well suited to detect targets in highly cluttered space, including pitchers that are partially hidden in vegetation. Interestingly, other bat species interacting with plants that offer reflectors, e.g., Glossophaga soricina, have similar echolocation calls. They are also broadband and high pitched [25], except that Glossophagine calls often consist of multiple harmonics and are slightly shorter. Generally, such calls should enable the bats to get a highly resolved acoustic image of targets and facilitate recognition of floral reflectors [25, 26, 27] or, in the case of N. hemsleyana, species-specific spectral signatures of the pitchers.
To test the efficacy of the reflector of N. hemsleyana in attracting bats, we conducted a series of behavioral experiments with wild K. hardwickii in a flight tent. In the first experiment, we tested whether the reflector helps the bats to find pitchers faster in a cluttered environment. We measured the time until the bats (n = 24) approached a single pitcher hidden within shrubbery. In this experiment, the pitchers’ reflector was either unmodified or enlarged or completely removed (n = 8 individual bats per type of pitcher; Table S1A; Movie S1). Bats needed significantly less time to approach enlarged (92.4 ± 58.5 s; W = 2; p < 0.001) and unmodified (182.1 ± 111.0 s; exact Wilcoxon rank-sum test: W = 10; p = 0.02) pitchers than those with removed reflectors (408.8 ± 228.1 s; Figure 4A).
In a second experiment, we tested whether the reflector is decisive for roost identification: we simultaneously confronted a single bat (n = 18) with three types of N. hemsleyana pitchers with modified reflectors (enlarged, partly or completely removed; Table S1B) and an unmodified N. hemsleyana pitcher as control (Movie S2). Bats approached enlarged pitchers significantly more often than expected by chance (number of approaches per bat = 3.1 ± 3.6; permutation tests, p = 0.005; for explanations, see Supplemental Experimental Procedures), whereas pitchers with reduced reflectors were approached significantly less frequently than expected (1.0 ± 1.3; p = 0.03; Table S2). The number of approaches to unmodified control pitchers did not differ from random expectations (2.1 ± 2.1; p = 0.26). These results confirm that the reflector is crucial for attracting the bats to the pitchers. When it came to the final roost selection, bats predominantly entered pitchers with unmodified reflectors and avoided those that had been enlarged or reduced (p < 0.001; Figure 4B; Table S1B). These results suggest that bats are initially attracted by the enlarged reflectors but then do not identify them as N. hemsleyana, possibly because such artificial reflectors do not contain the typical N. hemsleyana spectral cues.
To assess the importance of the reflector over other structures of the pitcher in attracting bats and to exclude the possibility that the bats generally avoided roosting in modified pitchers, we conducted further choice experiments. This time, we modified lids or peristomes of N. hemsleyana pitchers but kept the reflectors intact. The bats’ roost choice was not influenced by such modifications (Table S1C), demonstrating that bats did not generally avoid roosting in modified pitchers and that other structures of the pitcher were not important compared to the reflector.
Taken together, the results of the ensonification measurements and the behavioral experiments provide strong support that the reflector ofN. hemsleyana is crucial for the bats to find, identify, and finally enter pitchers.
As predicted, we found that bats are attracted to echo-reflective structures in a Paleotropical plant. Ensonifications revealed that the exposed back wall ofN. hemsleyana efficiently reflects acoustic signals over a wide range of angles of sound incidence. Additionally, the pitchers are characterized by a species-specific spectral pattern facilitating echo-acoustic recognition of N. hemsleyanapitchers by the bats. We confirmed the importance of the reflector for the detection and identification of suitable roost pitchers with behavioral experiments. According to our predictions, bats had a high affinity to pitchers with intact reflectors. They needed more time to find pitchers where the reflector was missing, and they subsequently rejected them as roosts. Interestingly, pitchers with enlarged reflectors were found faster in the cluttered environment and were approached more often. This suggests that natural selection could act on pitchers to develop larger reflectors, leading to more bat visits and hence a higher nutrient intake. Finally, due to the narrow beam width of their calls, the bats should easily recognize N. hemsleyana pitchers with a reflector, even within the typically cluttered environment they occur.
Overall, our findings suggest that N. hemsleyana exploits the bats’ perceptual bias to attract them echo-acoustically. This helps the bats to quickly find and enter suitable day roosts and the plants to benefit from higher nitrogen intakes [10]. Our study provides the first example of a plant structure allowing bats to find it and identify it for reasons other than pollination. From an evolutionary point of view, our findings support the hypothesis that unrelated Neotropical bat-pollinated angiosperms and Asian carnivorous plants have convergent structures that specifically reflect bats’ echolocation calls. Further studies will be necessary to infer whether structures involved in such complex plant-animal interactions primarily evolved by natural selection for their current use (adaptations to the bats) or were coopted for their current use (exaptations, probably followed by secondary adaptation), either from adaptations to other functions or from non-adaptive structures [28].