Bat pollination is a vast and fascinating topic that still offers many opportunities for new discoveries, and this is especially true in the wide expanses of lowland tropical rain forests. Our knowledge of bat pollination includes detailed descriptions of the general syndrome (Bawa 1990; Helversen 1993; Tschapka and Dressler 2002) as well as descriptions of the pollination biology of individual plant species(Baker 1970; Fischer, Jimenez et al. 1992; Fleming 2000; Sazima, Fabian et al. 1982; Sazima and Sazima 1978; Tschapka and Helversen 1999a). Yet experimental studies at the population level are very rare (Muchhala and Jarrín-V. 2002) and the importance of bats as pollinators in rainforest is still relatively unknown. The next step in broadening our understanding of bat pollination is to identify what plants are pollinated by what bats and what bats pollinates plants in the lowland forests of French Guiana and these are the major goals of our research. Reaching an understanding of bat/plant interactions is technically difficult, hence, studying bat pollination is somewhat challenging. Bats are active at night and are exceptional flyers. In the primary rainforests of central French Guiana, most bat-pollinated plants are either emergent trees or epiphytes living in those trees so flowers visited by bats are commonly located at 40-50 meters above the ground. Bat flowers usually open and begin to produce scent and nectar after nightfall. Those same flowers often fall to the ground before daybreak.
Our study of bat pollination in French Guiana is structured in four steps:
- generate a list of plants that rely on bats for pollination
- refine the bat pollination syndrome
- create an online database of bat/plant interaction in pollination
- determine the threats that bat/plant interactions face as a result of habitat disturbance.
GENERATE A LIST OF PLANTS THAT RELY ON BATS FOR POLLINATION
A preliminary list of potentially bat-pollinated species of plant species was prepared by comparing the database of bat/plant interactions with the checklists of plants from central French Guiana (Belbenoit et al., 2001; Mori et al., 2002). This resulted in a list of 22 species (4 of which are introduced in the flora of central French Guiana) that have been documented in the literature to be bat pollinated. In addition, another 39 species suspected to be bat pollinated were added to this list based on their similarities with plants reported to be pollinated by bats as well as their possession of features commonly considered to be characteristic of bat-pollinated plants. As a rule, plant species with white or greenish flowers, nocturnal anthesis, inflorescences protruding or hanging from the crown or arising from the trunk or branches, and slightly unpleasant aromas, were considered to be potentially bat pollinated. Species exhibiting at least some of these characters have been included as well. Using these criteria, a list of 61 species potentially bat pollinated and found in the flora of central French Guiana was produced. Additional species will almost certainly be added to the list and some will be removed as the study progresses. In order to confirm a species as bat pollinated plant, we will use the following approaches:
Identifying species of plants visited by plants from pollen on the fur of bats.
A large part of this study will consist of capturing nectarivorous bats with mist nets, removing pollen from their fur and collect feces samples to indirectly determine what species of plants they have been visiting. During this process, all nectar-feeding bats will be considered but particular attention will be paid to Anoura geoffroyi, a nectarivorous bat that roosts in colonies in caves from Mexico to southeastern Brazil and northwestern Argentina. A population of hundreds of individuals of A. geoffroyi is known to roost in a cave at the Nouragues Nature Reserve in central French Guiana. Over the next several years,
Setting of 4 crossed mist nets for night trapping
we will capture individuals of this species throughout the year and determine what species of plants they visit. Bats will be captured with mist nets or harp traps as they return to their roosts after foraging flights. Pollen will be collected from their fur immediately after they have been captured. The bats will be kept in bags until they have defecated and then they will be released unharmed. Pollen and feces will be collected on month-long expeditions at least four times a year in order to cover all seasons. Pollen identification will be made by comparing the pollen gathered from the bats with the pollen described in the pollen atlas.
Pollen is collected directly from the bat’s fur onto commercially available double sided adhesive tape placed on SEM aluminium mounting stubs justbefore sampling. The pollen is removed as soon as possible after the bat has become entangled in the mist net by gently applying the SEM stubs to different parts of the bat's body. After collecting, the stubs are stored in clean stub holder plastic boxes with silica gel until they can be sputter coated for SEM examination. The advantages of this method are 1) minimal contamination of samples with pollen from different bats, 2)light materials needed are light and do not occupy much space, 3) rapid handling and releasing of the bats after feces have been collected, and 4) safe storage of the pollen thereby reducing contamination or deterioration through fungal and bacterial action.
Collection of pollen on stub from a bat trapped in a mist net
By collecting pollen while bats are still in the net we hope to eliminate contamination from excessive manipulation of different bats with the same gloves. Another source of contamination may come from the net itself if it has been previously occupied by bats covered with pollen but this may be negligible. Another problem is the oxidation of SEM stubs. These need to be stored in dry boxes even before they are used because stubs that have become Stubs stored in stub holders
oxidized are more resistant to sputter coating.
Cage experiments
Flowering branches of putatively bat pollinated plants that will open on a given evening will be cut, placed in a bucket of water, and the stem cut under water. We have learned that flowers on branches treated this way almost always open, produce nectar, and emit aromas in the same way that they do in the canopy. It appears that all of the nutrients and hormones needed for a given flower to open are in or close to the flower after 17:00 h. This method does not work if the branches are cut too early in the afternoon. In addition, buds that would have opened on subsequent days will not open. Nectarivorous bats captured the same night are put into a cage with the flowering branches and observations are made on flower visitation by the bats. When bats are placed in cages with bat flowers, the bat, after a short period of adaptation, visits the flower for nectar or pollen. In our January 2003 expedition to Nouragues, Brian Keeley obtained excellent pictures of Lionycteris spurellii visiting the flowers of L. poiteaui.
This technique does not substitute for actual observations of bats visiting flowers, but it does give valuable data on how the bat approaches and enters the flowers. In addition, if a bat that normally visits flowers in cages does not visit a given species we will then know to search forvariations of the flower syndrome that account for this lack of interest.
REFINE THE BAT POLLINATION SYNDROME
The bats that pollinate flowers fall into two main groups, those that hover at the flower (glossophagines) and those that land on the flower when they take nectar and/or pollen (carolliines, phyllostomines, and stenodermatines). The bat/plant pollination syndrome was developed considering these two groups of bats as equivalents, and this may have resulted in a too broadly defined bat pollination syndrome. This is tantamount to considering perching birds as the equivalent of hummingbirds, a mistake that was not made by biologists, probably because of the ease in observing the behavioral differences between the perching birds and hummingbirds. The recognition of the behavioral differences between bats that hover and bats that land on flowers only became apparent after the bat/plant pollination syndrome had been developed; the syndrome is, therefore, in need of refinement.
Flower morphology
This part of the study will be based on a literature review, examination of species in herbaria, and field observations. For each species of bat-pollinated plant, I will record information on flower/inflorescence type and position in the tree, flower size and shape, flower color, stamens and pistil position, anther attachment, the presence or absence of pollen kit, the size of the pollen, and the surface of the pollen exine. This information will be placed in a database that will be linked to the database in which bat visitations to flowers have been recorded.
Floral aroma
The floral aroma of bat pollinated flowers is collected by using headspace trapping techniques. The flower or the entire inflorescence is enclosed in plastic bags and the air surrounding the flower is pumped through a cartridge containing an absorbent (Tenax) at a flow rate of 100-150ml/min for 1/2 hour. The flow rate and time of collection depend upon the intensity of the aroma emitted. The molecules composing the aroma are trapped on the adsorbent. Each collection should be accompanied by a "blank" collection so environmental contaminants can be detected
Scott Mori and Vanessa Hequet collecting aroma samples of Lecythis poiteaui from the ground
and subtracted from the studied aroma. Even in the most remote environments there is often contamination from petroleum derived products. Once collected, the cartridges should be kept dry and sent to the laboratory as soon as possible. We have, however, kept cartridges for several weeks without subsequent problems in the analyzes.
The floral aroma analysis is being done in collaboration with Mr. Kenneth Purzycki (former scientist and now consultant to Givaudan, a creator and supplier of fragrances and flavors). In the laboratory, the fragrant molecules are eluted from the cartridge with an automatic thermal desorption system and injected on a GC-MS system for separation and analysis. Mr. Purzycki possess an electronic library of floral scent composed of thousands of chemicals reported from the aromas of flowers. The data is reported in graphs showing the molecular weight of each of the chemicals and in a table showing the name and concentrations of the chemicals (Fig. 1, Table 1).
It is preferable that aromas be gathered from flowers still attached to the plant, but our preliminary experiments demonstrate that the chemicalcomposition of the aroma of Lecythis poiteaui captured from flowers on cut branches is similar to that captured from flowers in the tree. Additional experiments will be carried out in order to test the hypothesis that "Flowers have the same aroma composition regardless of whether the aromas are collected from flowers attached to the plant or from cut branches sampled on the ground." This hypothesis assumes that aroma sampling on the ground is made on a flower that was cut after 17:00 pm and was bearing Ken Purzycki and the GC-MS setting
buds that would have opened that night. Our initial studies of L. poiteaui not only indicate that floral aroma collected from the canopy and from the ground are the same (Fig. 1), but that the flowers open at the same time. From this preliminary experiment, we have concluded that a bud destined to open on a given night has the nutrients and hormones need for anthesis, either in the bud or in the nearby leaves and stems, several hours before anthesis. Other species need to be studied in order to determine how widely the cut branch technique can be applied for sampling floral aromas and nectar from flowers on cut branches. It is important that the validity of the "cut branch" sampling method be established because of the difficulty in sampling flowers in the canopy as compared to sampling them from flowers on cut branches on the ground.
Based on previous observations, we know that L. poiteaui opens it flowers shortly after dusk and the flowers fall sometime between 0330 and 0430 h. We do not know, however, when peak flower visitation by bats takes place nor do we understand variation in floral aroma throughout the flowering cycle. To answer these questions we will undergo sequential sampling during which aromas will be collected every two hours overnight.
Once we have learned more about the chemical composition of species of bat-pollinated flowers, we will compare floral chemistry with the known pollinators to determine if different bats are attracted by different chemicals. Although earlier work has suggested that the floral aromas of bat pollinated plants are dominated by sulfur-bearing compounds (Knudsen and Tollsten 1995), the work of Bestman et al. (1997) didn’t confirm this hypothesis. More detailed study is thus required to shed light on the characteristics of the aromas of bat flowers and determine id different aromas attract different bats.
Nectar
Most species of bat-pollinated plants possess nectar low nectar concentrations in the order of 17%. Nectar composition of all bat-pollinated flowers studied in the field will be recorded with a refractometer. In addition, we will examine the possibility of making more sophisticated analyzes of nectar in order to determine what other nectar constituents, such as amino acids are present.
Pollen
A detailed pollen atlas has been developed to facilitate the indirect determination of plants visited by bats. As part of this study, pollen size and exine ornamentation is being recorded. This information will be used to determine if bat-pollinated plants are characterised by specific pollen size or ornamentation. This issue have been discussed by several authors (Chavez 1975; Luckow and Hopkins 1995; Stroo 2000) but their conclusions have been contradictory.
Phenology
The timing of flowering and its relationship to environmental factors is important to flower-visiting bats because of their dependence on flowers for pollen and nectar as a source of food. The flowering times of bat-pollinated plants will be established based on records in the herbarium of The New York Botanical Garden (NY) and the Institut de Recherche pour le Développement in Cayenne (CAY). Those data will be complemented by observations made during our field work. Knowledge of the flowering season of bat pollinated plants will also give useful hints for identifying pollen found on bat fur and in bat feces depending on what time of the year it has been collected.
The refinement of the bat/plant pollination syndrome will be based on data gathered from bat/plant visitation observations, floral morphology, floral aroma, nectar features, pollen features, and phenology. In addition, I will prepare a less extensive database on the features of bats that adapt them for taking nectar and pollen from flowers. This information will be placed in three linked databases:
1) a database in which bat/plant interactions reported in the literature and on our bat/plant expeditions are recorded,
2) a plant database in which all of the features of plants that serve to attract bats to them are recorded, and
3) a bat database in which all of the features that adapt plants for pollination are recorded.
The bat/plant database
This database includes reports of relationships between plants pollinated and dispersed by bats and is currently available for consultation at www.botanypages.org/.
The plant database
This database will include records for each known or suspected species of plant pollinated by bats, and will have fields for plant family, plant genus, plant species epithet, bat pollinator (s), plant life form, plant phenology, inflorescence type, inflorescence position, flower size, flower color, pollen type, nectar composition, and floral aroma chemistry.
The bat database
This species will include data on each of the bat species known to pollinate plants in central French Guiana. This database have fields for bat family, bat subfamily, bat genus, bat species, size in grams, forearm length, mussel length, mussel shape, tongue length, tongue modification, dentition, foraging mode (hovering or not hovering), and diet.
DETERMINE THE THREATS THAT BAT/PLANT INTERACTIONS FACE AS A RESULT OF HABITAT DISTURBANCE
The rain forest of French Guiana is part of one of the last, relatively untouched wilderness areas in the world. My goal is to contribute to the preservation of large expanses of this area by demonstrating how complex and susceptible to disruption this ecosystem is. I will use one of the most finely tuned relationships in this rain forests, that of pollination by bats to demonstrate how important it is to conserve large expenses of untouched rain forest in order to preserve its biocomplexity of this ecosystem. In particular, I feel that the relationship of glossophagine bats and plants of undisturbed habitats is one of the most highly evolved in the New World tropics. Take away the plants that the bats visit and the bats will not survive; on the other hand, take away the bats and the plants they pollinate will not continue produce seed.
View of Rainforest in Nouragues
By studying bat pollination in the undisturbed rain forest of French Guiana, I will reveal the details of this highly evolved relationship. Understanding this interaction is the first step toward elaborating a strategy for conserving all of the elements of lowland rain forest in the New World.
The list of plant pollinated by bats generated in this study will be used for estimating the vulnerability of plants pollinated by bats, and especially the vulnerability of bats that rely on flowers for part of their diet. Trees, for example, that depend on bats for pollination, may persist for many years after bats have been eliminated. On the other hand, bats that need trees for survival will disappear immediately after the trees have been destroyed. Hence, I will demonstrate that monitoring glossophagine bat populations may be one of the most efficient ways of determining the degree of rain forest disturbance throughout the Neotropics.
Literature
Baker HG (1970) Two cases of bat pollination in Central America. Revista Biol. Trop. 17, 187-197.
Bawa KS (1990) Plant-pollinator interactions in tropical rain forests. Annu. Rev. Ecol. Syst. 21, 399-422.
Chavez RP (1975) Observaciones en el polen de plantas con probable polinizacion quiropterofila. An. Esc. Nac. Cienc. Biol. 21, 115-143.
Fischer EA, Jimenez FA, Sazima M (1992) Polinização por morcêgos em duas especies de Bombacaceae na estação Ecologica da Jureia, São Paulo. Revista Brasil. Bot. 15, 67-72.
Fleming TH (2000) Pollination of cacti in the Sonoran Desert. Amer. Sci. 88, 432-439.
Helversen Ov (1993) Adaptations of flowers to the pollination by glossophagine bats. In 'Animal-plant interactions in tropical environments'. (Eds W Barthlott, CM Naumann, K Schmidt-Loske and KL Schuchmann) pp. 41-59. (Zoologisches Forschungsinstitut und Museum Alexander Koenig: Bonn, Germany)
Luckow M, Hopkins HCF (1995) A cladistic analysis of Parkia (Leguminosae: Mimosoideae). Amer. J. Bot. 82, 1300-1320.
Muchhala N, Jarrín-V. P (2002) Flower visitation by bats in cloud forests of western Ecuador. Biotropica 34, 387-395.
Sazima M, Fabian ME, Sazima I (1982) Polinização de Luehea speciosa (Tiliaceae) por Glossophaga soricina (Chiroptera, Phyllostomidae). Revista Brasil. Biol. 42, 505-513.
Sazima M, Sazima I (1978) Bat pollination of the passion flower, Passiflora mucronata, in southeastern Brazil. Biotropica 10, 100-109.
Stroo A (2000) Pollen morphological evolution in bat pollinated plants. Plant Syst. Evol. 222, 225-242.
Tschapka M, Dressler S (2002) Chiropterophily: on bat-flowers and flower bats. Bot. Mag. 19, 114-125.
Tschapka M, Helversen Ov (1999a) Pollinators of syntopic Marcgravia species in a Costa Rican lowland rain forest: bats and opossums. Plant Biol. 1, 382-388.
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