Nocturnal scent in a ‘bird-fig’: A cue to attract bats as additional dispersers?

The plant genus Ficus is a keystone resource in tropical ecoystems. One of the unique features of figs is the diversity of fruit traits, which in many cases match their various dispersers, the so-called fruit syndromes. The classic example of this is the strong phenotypic differences found between figs with bat and bird dispersers (color, size, presentation, and scent). The ‘bird-fig’ Ficus colubrinae represents an exception to this trend since it attracts the small frugivorous bat species Ectophylla alba at night, but during the day it attracts bird visitors. Here we investigate day to night changes in fruit scent as a possible mechanism by which this ‘bird-fig’ could attract bats despite its fruit traits, which should appeal solely to birds. Analyses of odor bouquets from the bat- and bird-dispersal phases (i.e. day and night) differed significantly in their composition of volatiles. We observed a significant increase in relative amounts of sesquiterpene and aromatic compounds at night while relative amounts of two compounds of the fatty acid pathway were significantly higher during day. This finding raises the question whether Ficus colubrinae, a phenotypically classic ‘bird-fig’, might be able to attract bat dispersers by an olfactory signal at night. Preliminary observations from feeding experiments which indicate that Ectophylla alba is capable of finding ripe figs by scent alone point in this direction. However, additional behavioral experiments on whether bats prefer the ‘night-bouquet’ over the ‘day-bouquet’ will be needed to unequivocally answer this question.

hence considered to be mainly bird-dispersed [12,30]. However, farther north where F.  Study animal: E. alba is a frugivorous, small-bodied leaf-nosed bat species (Phyllostomidae) that 1 0 7 is distributed from northern Honduras to north-eastern Panama [31]. It modifies leaves, groups of typically four to eight individuals [29].
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The copyright holder for this preprint . http://dx.doi.org/10.1101/418970 doi: bioRxiv preprint first posted online Sep. 17, 2018; Behavioral experiments: We captured groups of E. alba from roosts in Heliconia leaves in the 1 1 1 area of TRC and selected single males for the feeding experiments in order to prevent lactating or 1 1 2 pregnant females or juveniles from isolation of the social group. All individuals that were not 1 1 3 considered for further experiments were set free immediately in close proximity to the roost. Following the capture, a single male was released into a flight tent (Eureka; ground area 4 x 4m, 1 1 5 height 2.5m) several hours before sunset. At nightfall we installed a freshly cut branch of F. colubrinae that yielded a range of fruits of different stages of maturity into the flight tent. In 1 1 7 order to adjust to the foraging situation we allowed the bat to feed on ripe fruits. After the 1 1 8 consumption of five fruits we started choice trials in order to test whether E. alba relies mainly 1 1 9 on olfaction for the short-range localization of ripe fruits or if objects similar in shape, color or 1 2 0 presentation raise a bat's attention. On one side of the branch we presented a strong olfactory cue 1 2 1 to the bat that lacked visual or echo-acoustic properties of natural figs, i.e. we presented a fully 1 2 2 opaque tissue bag that was filled with ten ripe figs (similar methods have been used to test for the 1 2 3 response of bats to olfactory cues in absence of natural fruit shape or surface structure: Kalko and colubrinae fruits in terms of form, color, and fruit presentation (in branch forks). We rated E. flights to or a landing next to the object followed by a directed movement to it occurred. In total, 1 3 0 we tested six individual bats. Every bat was tested only once in order to avoid bias caused by 1 3 1 learning effects. It was not possible to record data blind because our study involved focal 1 3 2 animals. We documented bat behavior using an infrared camera (Sony Night-Shot DCR-HC42E, 1 3 3 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418970 doi: bioRxiv preprint first posted online Sep. 17, 2018; Sony, Japan) that was connected to a video recorder (GV-D 900E, Sony, Japan). We stored 1 3 4 recordings on MiniDV video tapes (DVM60PR3, Sony, Japan).  After sampling, all sorbent tubes were eluted with 0.050 ml of 10:1 pentane/acetone. Eluted Germany), and pentane (SupraSolv, Merck). Sorbent tubes were cleaned three times with ethanol,  . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  Retention indices were calculated from a homolog series of n-alkanes. Structural assignments 1 6 7 were based on comparison of analytical data obtained with natural products and data reported in  of fruit scent compounds using SPSS 17. We used the resulting principal components (PCs) with 1 7 2 an eigenvalue above one to run a discriminant function analysis (DFA) in order to test for 1 7 3 differences in the scent composition between (1) unripe fruits during night, (2) ripe fruits during 1 7 4 day, and (3) ripe fruits during night. We used the factor loadings after varimax rotation and the 1 7 5 standardized discriminant function coefficients to assess the importance of individual compounds. Factor loading above 0.5 were considered high. Finally, we compared relative 1 7 7 amounts of single compounds of ripe fruits during day and night (groups 2 and 3) using Mann-  CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. After releasing captured bats into the flight tent, the bats performed circular inspection flights for 1 8 3 several minutes before they roosted in a corner of the flight tent until dusk. Shortly before dusk 1 8 4 we installed a natural branch of F. colubrinae with several ripe and unripe fruits. All six bat 1 8 5 individuals performed search flights that lasted between less than one minute and almost two hours (mean ± standard deviation: 32 ± 43 minutes, n = 6) until the bats approached the branch 1 8 7 for the first time. Then the bats conducted two to nine approximation flight towards the branch 1 8 8 over a period of one to 91 minutes (mean ± standard deviation: 19 ± 36 minutes, n = 6) before  minutes (mean ± standard deviation: 16 ± 21 minutes, n = 5, see Table 1) and one to five approaches the bats either landed on or right next to the bag or landed more than 5 cm away and . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418970 doi: bioRxiv preprint first posted online Sep. 17, 2018; 1 0 In the chemical analyses we registered 14 distinct peaks that were attributed to 17 individual 2 0 5 substances (3 peaks showed co-eluting substances), 13 of which were unambiguously identified 2 0 6 by mass spectrometry (Table 2). Nonanal and 1-tetradecanol contributed the largest share to the 2 0 7 overall bouquet (Fig. 1, Table S1). Three further substances could be assigned to substance sesquiterpenenes, and aromatic compounds. In three cases, two substances contributed to a single 2 1 2 peak in the GC-analysis. In those cases, the overlapping substances were represented by a single 2 1 3 value for the following analyses. Two of the identified substances, indene and anthracene, have a 2 1 4 main relevance in industrial applications and were therefore excluded from all further analyses. They were considered environmental pollutants that accumulated on the outside of the fruits over 2 1 6 time since our field site was closely located to human structures including infrastructure and 2 1 7 industry. There were no significant differences in relative amounts of indene and anthracene 2 1 8 among day and night in ripe fruits.

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Daily differences of single compounds in ripe fruits: All scent compounds analyzed were present 2 3 3 in diurnal and nocturnal scents. In general, fatty acid pathway compounds dominated both diurnal 2 3 4 and nocturnal scents (Fig. 1). However, relative amounts of sesquiterpene compounds increased 2 3 5 at night and FAPCs decreased, except the two long-chain alcohols (Table 2). Six out of twelve  (Table 2). Our study shows that scent seems to be an important cue for E. alba to find ripe fruits of F. reported in other fruits consumed by small phyllostomid bats. We suggest that our findings show 2 5 2 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418970 doi: bioRxiv preprint first posted online Sep. 17, 2018; 1 2 initial signs of an adaptation of F. colubrinae towards dispersal by small bats such as E. alba, but 2 5 3 further behavioral trials are required to unequivocally demonstrate this relationship.

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Semi-natural feeding trials showed that phyllostomid bats locate fruits by echolocation 2 5 5 [15] or olfaction [6,13,14] as the primary sensory cues. Our results from the feeding showed strong responses to the tissue bag that gave a strong olfactory cue but lacked natural 2 5 8 texture, shape, size, or presentation of figs that might be of importance for detection by 2 5 9 echolocation. Therefore, we assume that echolocation may not play such a dominant role for E. frequency modulated signals which are typical for phyllostomid bats is possible but largely 2 6 7 depends on the fruit presentation and the complexity of the surrounding clutter. However, F.  Olfactory cues enable plants to signal the readiness of fruits for dispersal. Accordingly, 2 7 3 temporal changes in the volatile profile of fruits are common during the process of ripening (e.g. . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418970 doi: bioRxiv preprint first posted online Sep. 17, 2018; 1 3 ripening. Additionally, we observed significant changes among day and night, caused by day-2 7 7 time specific scent production. Circadian changes in the volatile profile of fruits have been more figs' to additionally attract certain bat species as dispersers and hence allow for dispersal during 2 8 7 the daytime and at nighttime. To achieve seed dispersal by distinct animal taxa may result in 2 8 8 multiple benefits to a reproducing plant. The contribution to overall seed rain by birds or bats, 2 8 9 respectively, may vary quantitatively across seasons [4]. Microhabitat deposition also strongly 2 9 0 depends on the disperser since birds tend to disseminate seeds when perched while bats usually 2 9 1 defecate seeds during flight. The resulting seed rain can be dominated by chiropterochorously and birds. This way the plant may maximize dispersal rates of the year-round produced fruits and 2 9 6 seeds may arrive in a more heterogeneous range of microhabitats for germination. All unambiguously identified compounds except 1-dodecanol, 1-tetradecanol, and (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. surprising since feeding trials showed that fruit scents, which were dominated by monoterpenes 3 0 6 were highly attractive to the phyllostomid bat Artibeus jamaicensis [6]. Instead, in our samples 3 0 7 sesquiterpenes increased throughout and in parts significantly during night, while fruit scents that  Taking the results from behavioral trials and chemical analyses together, our study cannot finally by altered scent production, but it provides evidence that this might indeed be a strategy in the 3 2 3 genus Ficus. Daily variation in the volatile profile of fruits may be more common than previously 3 2 4 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.