Folivory has been reported in eight species of phyllostomid bats from the genera Artibeus (Leach 1821), Platyrrhinus (Saussure 1860) and Carollia (Gray 1838) (Greenhall 1957, Van der Pijl 1957, Zortéa and Mendes 1993, Kunz and Ingalls 1994, Kunz and Díaz 1995, Zortéa 1996, Bernard 1997, Esberard et al. 1998, Aguiar 2005, Acosta and Aguanta 2006, Novaes and Nobre 2009, Bobrowiec and Cunha 2010, Ruiz-Ramoni et al. 2011, Cordero-Schmidt et al. 2016, da Rocha et al. 2016, Pereira et al. 2017). Most observations of leaf consumption document bats chewing small portions of the leaf, extracting the leaf liquids and discarding the remaining fibers (Kunz and Díaz 1995), with the exception of one case [Carollia perspicillata (Linnaeus 1758)] of young leaves which were consumed whole, presumably because they were less fibrous and more digestible (Pereira et al. 2017). Folivory is seen as a strategy that might provide vitamins and micronutrients not always available in fruits, and/or proteins that are particularly important during pregnancy and lactation but usually scarce in fruits. Moreover, secondary metabolites (hormonal precursors) that stimulate/inhibit reproductive processes could also be obtained from leaves (Kunz and Díaz 1995). Finally, folivory has been recently postulated as a phenomenon to obtain water in arid environments (Cordero-Schmidt et al. 2016).
During the examination of chewed leaves for folivory studies (Duque-Marquez 2011, Ruiz-Ramoni et al. 2011) in two Neotropical bats [Artibeus amplus (Handley 1987) and Artibeus lituratus (Olfers 1818)], we noticed that completely intact leaves were dropped below the roost sites of both species, living in different environments. To confirm it, we decided to systematically study this unexpected finding, and wondered how many intact leaves do bats regularly drop below their roosting sites, which plant species do bats bring, and ultimately, why? To answer these questions, we visited the cave of Parque Las Escaleras [08°00′ N; 71°43′ W; 1320 m above sea level (asl), Pregonero, Venezuela] to collect leaves found below the roosting site of the colony of A. amplus (ca. 50 individuals) once a month from December 2008 to November 2009. One collection per month was sufficient, as the leaf remains were protected within the cave. The cave has only one small entrance and is therefore completely dark, and is not ventilated. Individuals of A. amplus clustered 20 m away from the entrance and 2 m above the ground. The colony of A. lituratus (ca. 14 individuals) was located in a residential area called “La Hacienda” [08°36′ N, 71°11′ W, 1400 m asl, Mérida, Venezuela], and leaves found below the roosting site were collected 4 times a month from November 2008 to October 2009. We collected leaves more frequently than for the previous species, because there was more human disturbance and potential leaf removal. Individuals of A. lituratus clustered beneath the palm leaf (Genus Washingtonia Wendland 1879) in a ventilated space (see Muñoz- Romo et al. 2008) and 4 to 8 m above the ground.
Our visits to the cave allowed us to recover samples of fresh (i.e. green) and old (i.e. brown) leaves that presumably corresponded to the accumulation for a month (Figure 1A). We validated our leaf sampling at the cave by occasionally placing a 4×4 m black plastic sheet just below the roost, immediately after bat emergence, and recover the sample of leaves next morning at 7:00 h (Figure 1B). Intact leaves were invariably recovered during all sampling events (Figure 1C and D). We explored the potential relationship between frequencies of intact leaves and precipitation patterns to test whether bats dropped more leaves during the rainy season, using precipitation information provided by the Climate Hazards group Infrared Precipitation with Stations (CHIRPS, http://chg.geog.ucsb.edu/data/index.html) V2.0.
A total of 517 intact leaves were recovered during a year below the roost of Artibeus amplus, whereas 122 were recovered below the roost of Artibeus lituratus. All recovered leaves were mature. Aspidosperma cruentum (Woodson 1935, Apocynaceae) and Tapura amazonica (Poepp. 1843, Dichapetalaceae) were the most common leaves recovered intact below the roost of A. amplus, followed by Erythrina poeppigiana [(Walpers) O.F. Cook 1901, Fabaceae; Table 1]. Most leaves of Brosimum sp. (Swartz 1788, Moraceae; ca. 80%) were brought to the roost and dropped intact by bats, and just a few were observed partially consumed (Table 1). A half of the leaves of Ficus insipida (Willdenow 1806, Moraceae) were partially eaten, and the other half was kept intact by bats (Table 1). Artibeus amplus brought an average number of 14 intact leaves of A. cruentum and 12 of T. amazonica every month, whereas less than 10 intact leaves of E. poeppigiana, Brosimum sp. and F. insipida (Figure 2). The fact that leaves of Brosimum sp. were more commonly recovered intact than consumed (Table 1) would support a stricter, non-nutritional use for this plant species used throughout the year (Figure 2). Leaves of E. poeppigiana were the most common and almost the only species recovered intact below the roost of A. lituratus (Table 1). Similar average numbers of intact leaves of E. poeppigiana were brought by both species of bats to their roosting sites (Figure 2), and the largest amount of leaves of E. poeppigiana was brought in October 2009 by both bat species. Aspidosperma cruentum (linear leaves), T. amazonica (elliptic leaves), Ficus insipida (elliptic leaves) and Brosimum sp. (obtuse leaves) all have waxy surfaces, whereas E. poeppigiana (deltoid leaflets) and Solanum sp. (Linnaeus 1753) (obtuse leaves) both have rough surfaces. None of them is sticky, has spines or is even pubescent. All of them have entire margins. We did not find clear relationships between the frequencies of intact leaves and precipitation patterns; thus leaves are used regardless of the season.
Total number of consumed and intact leaves recovered in a year at both study sites and their relative importance (%).
Roosting sites of other frugivorous and insectivorous bat species have been observed with intact leaves on the floor. For example, the only leaves of Piper amalago, found below the roost of Carollia perspicillata in Laranjeiras (Brazil), were completely intact, and the authors suggested that these might have been carried to the cave accidentally by bats (Pereira et al. 2017). These authors also showed a picture of an intact composite leaf of Senna georgica (H.S. Irwin and Barneby) found below the roost site in the cave. Wilson (1971) also found unidentified leaves below the roosting site of the insectivorous bat, Micronycteris hirsuta (Peters 1869), and Ross (1967) frequently found leaves below the roosting site of the also insectivorous species, Macrotus waterhousii (Gray 1843). Both Ross (1967) and Wilson (1971) suggested that leaves are carried back accidentally along with the insects, but this has never been observed and confirmed. It is likely that other species of bats can display this behavior, but this has been unnoticed until now.
Why bats would bring intact leaves, investing energy in costly (Alexander 2002) round trips, and drop them on the floor, just below their roosting sites? The answer seems to clearly be related to the chemical properties of the leaves. There are numerous pharmacological properties of leaves, although the specific chemical profiles of each leaf species (i.e. plant) are not always available in the literature. Dropping leaves on nests has been postulated to contribute to drive away parasites and bacteria in many bird species (Collias and Collias 1984). Antimicrobial properties of leaves are common (Ovington 1956, Cowan 1999, Hussein and El-Anssary 2018), but the specific effect against microorganisms would need specific testing experiences. The nest-protection hypothesis (Clark 1991) suggests that some of the fresh plants brought to the nest contain secondary compounds that repel parasites or mask the chemical cues that parasites use to find the host. Berkunsky et al. (2017) observed green leaves inside the nests of the turquoise-fronted parrot (Amazona aestiva Linnaeus 1758), and indicated that secondary compounds present in green leaves could reduce the presence of ectoparasites (Bucher 1988, Aramburuú et al. 2002). Secondary compounds were presumably used by coatis in Panama to deter ectoparasites, as they were observed grooming Trattinnickia aspera (Standl.) Swart 1942 resin vigorously against the fur (Gompper and Hoylman 1993). Morever, Lambrechts and Dos Santos (2000) hypothesized that even more effective would be using a mixture of plants (i.e. “potpourri effect”) that provides even more benefits than using a single plant species in the nest. Lafuma et al. (2001) performed experiments to test the potential repellent effect of aromatic plant species against the mosquito Culex pipiens (Linnaeus 1758), and found that the mixture of aromatic plants was the most efficient in repelling mosquitos, although some aromatic plants also had significant effects when used individually.
It remains to be tested whether leaves found here may have any effect against bacteria, fungi and/or specific life cycle stages of endo/ectoparasites. In a previous study, sampling of bacteria around the roosting site in the same cave (from Parque Las Escaleras) showed that all but one sample belonged to Proteobacteria (Mérida-León et al. 2016) and these have been reported to be potentially pathogenic to humans (Rizzatti et al. 2017) and, presumably, other living organisms. It remains to be determined whether a particular combination of leaves would be functionally important for bats, as mixing leaves might increase specific effects or become functionally effective (Lafuma et al. 2001). Diverse biological effects (e.g. controlling, repellant) against several organisms that include fungus, bacteria, protozoans, helminths, insects and/or mites have been reported for species of Aspidosperma Martius & Zuccarini (Aguiar et al. 2015, de Almeida et al. 2019), Tapura Aublet (Taljaard 2014), Erythrina Linnaeus (Sato et al. 2003, Kumari et al. 2017), Brosimum Swartz (Coqueiro et al. 2014, Borges et al. 2017) and Ficus Linnaeus (de Amorin et al. 1999), Romeh 2013, Wan et al. 2017). Plant volatiles from leaves are extremely common and very diverse (Pichersky et al. 2006, Courtois et al. 2009). Green leaf volatiles (GLVs), which are present in almost every green plant, consist of a family of C6 compounds, including aldehydes, alcohols and esters, and their fragrance sometimes is easily perceived by humans (Scala et al. 2013). Some of these volatile compounds produced by leaves may act as defensive molecules against arthropods, affecting insect fecundity (Hildebrand et al. 1993). For example, volatile six-carbon compounds in plant tissues reduced tobacco aphid fecundity at certain concentrations when added to headspace vapor to which aphids were exposed (Hildebrand et al. 1993). Repelling organisms from the floor could increase pup survival by driving away potentially dangerous organisms that could negatively affect non-volant pups that could fall from roost sites to the cave floor where they can die unless retrieved by an adult (Bohn et al. 2009). For example, specific chemicals from leaves could increase the survival of fallen pups by keeping away spiders, well known as predator of bats (Nyffeler and Knörnschild 2013), by allowing time for mothers to come and grab them. Specific plant volatiles from leaves brought by bats need to be identified and their effects especially tested against diverse organisms.
Our observations provide strong evidence of a new behavior that requires further research and experiments to be fully understood. Reporting this behavior for the first time in bats reinforces the need for further detailed investigation: Does dropping leaves involve other bats species regardless of their trophic preferences? Are coexisting bat species using the same available plants as biological controllers of pathogens? Are females and males equally dropping leaves?
For the valuable field and laboratory assistance, we thank D. Ruiz-Ramoni, A. Duque, G. Bianchi-Pérez and L.M. Otero. We are grateful to L.E. Gámez for his help in identifying some plant species, and Marcelo Passamani for his recommended reference. We are grateful to Associate Editor Stéphane Aulagnier and two anonymous reviewers for their valuable comments and recommendations to improve the revised version of this manuscript. MMR dedicates this work to Keanu Reeves. This research was supported by Consejo de Desarrollo Científico, Humanístico y Tecnológico (CDCHT, Universidad de Los Andes), project C-1649-09-01-B, and Laboratorio de Zoología Aplicada, Universidad de Los Andes.
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