Bumble bee preference for flowers arranged on

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1 Functional Ecology 2008, 22, doi: /j x Bumble bee preference for flowers arranged on Blackwell Publishing Ltd a horizontal plane versus inclined planes T. T. Makino* Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, , Japan Summary 1. Determining factors affecting pollinator visitation is the key to understanding the reproductive success of animal-pollinated plants. The inclination of the ground where plants grow, which has been little studied, could be one such factor. There may be differences in foraging performance when visiting flowers on horizontal planes compared with flowers on inclines planes. And if this is the case, pollinators may have a preference for horizontal or inclined planes. To test these possibilities, a series of laboratory experiments with bumble bees and arrays of artificial flowers were conducted. 2. In the first experiment, bees were presented with a pair of floral arrays, one on a horizontal plane and one sloping. The bees preferred visiting flowers on the horizontal array. 3. In the second experiment, bees were allowed to forage on a flower array tilted at various angles ranging from 0 (horizontal) to 90 (vertical). It was found that their foraging performance decreased with increasing angles, showing a 9 1% reduction from 0 to 90 arrays. This reduction was caused by an increased travel time between flowers, when moving upslope or downslope. 4. These results suggest that plants growing on steep slopes may be less preferred by pollinators. Future studies are needed to clarify how slopes affect pollinator behaviour in field conditions and the ecological influences on plant reproduction. Key-words: bumble bee, foraging rate, inclination of the ground, pollination, sloping habitats Functional Ecology (2008) xx, Introduction In animal-pollinated plants, the number of pollinator visits to a plant is an important determinant of reproductive success. Plants that receive more visits usually achieve more pollen transfer, both in terms of receipt and removal (e.g. Galen & Stanton 1989; Wilson & Thomson 1991; Jones & Reithel 2001; Engel & Irwin 2003). The frequency of pollinator visits among plants has been shown to vary depending on many factors such as inflorescence size (e.g. Ohashi & Yahara 1998; Mitchell et al. 2004; Grindeland, Sletvold & Ims 2005; Makino, Ohashi & Sakai 2007), reward amount (e.g. Pleasants 1981; Thomson 1988; Cartar 2004; Makino & Sakai 2007), or the presence of co-flowering species (e.g. Rathcke 1988; Laverty 1992; O Neil 1999; Johnson et al. 2003). All of these factors and many others cause fitness differences among plants, thus offering insight into the evolution of floral traits. Hence, many researchers have examined factors affecting pollinator visitation. The inclination of the ground where plants grow, which has been little studied, may be one factor affecting pollinator visitation; pollinators may take longer to ascend or descend a *Correspondence author. makinott@pe.ies.life.tsukuba.ac.jp slope, which could reduce their foraging performance. Indeed, some birds have been observed to take longer in ascending flight (Tucker Schmidt-Koenig 1971; Hendenstrom 1995 reviewed by Irschick & Garland 2001). If the inclination of the ground reduces foraging performance, pollinators will prefer visiting plants growing on a flat land. To examine these possibilities, a series of laboratory experiments using bumble bees (Bombus ignitus) and arrays of artificial flowers were conducted. In the first experiment, bees were presented with a pair of arrays, one horizontal and one sloping, to see whether they preferred visiting flowers in the horizontal array. In the second experiment, bees were allowed to forage on an array tilted at various angles ranging from 0 to 90, and changes in foraging performance (the amount of nectar intake per unit of time) were estimated. Changes in travel time between flowers, which would affect foraging performance, were also measured. Based on the results obtained, the effects of ground inclination on pollinator visitation will be discussed. Methods Experiments were carried out in a flight cage made of wire mesh that measured m, set up indoors (University of Tsukuba, 2008 The Author. Journal compilation 2008 British Ecological Society

2 1028 T. T. Makino Fig. 1. Schematic views of artificial flowers and an example of the setup for experiment 1. (a) A bumble bee probing a flower on a slope. (b) An artificial flower and a nectar feeder inside it. Nectar inside the feeder was conveyed through a silk thread (the vertical line insider the feeder), and gradually accumulated in silk loops (indicated by a thick arrow), which a bee licks to retrieve the nectar. (c) A top view of the cage in experiment 1. Bees were presented with both a horizontal array and a sloping array (at a 45 slope angle in this case). Ibaraki, Japan). The temperature ranged from 23 C to 26 C so that the bees would be active, and the relative humidity was kept above 70% to prevent the artificial flowers from drying. Experiment 1 was conducted in October 2007, and experiment 2 in December 2006 and February Four colonies of bumble bees (referred to as Colony A, B, C and D) provided by Arysta LifeScience (Tokyo, Japan) were used. ARTIFICIAL FLOWERS Figure 1 illustrates the artificial flowers used in the experiments. Each flower was made of a plastic tube embedded in a styrene foam sphere (ø = 50 mm), which appeared as the same shape from all directions. The styrene spheres were painted yellow. The plastic tubes functioned as nectar feeders, and gradually secreted a 20% (wt/wt) sucrose solution (hereafter, nectar) (for details, see Makino & Sakai 2007). Before the experiments, 0 35 ml of nectar was put into each tube. The nectar inside the tubes was conveyed upward with a silk thread, and accumulated in silk loops located at the top (Fig. 1b). After the accumulated nectar was consumed by bees, it replenished at the rate of about 0 4 µl min 1 until reaching roughly 1 µl (Makino & Sakai 2007), taking about 150 s. FLOWER ARRAY As shown in Fig. 1c, a flower array was made with 36 artificial flowers arranged in a cm Styrofoam board (3 cm thick) in a 6 6 grid (distance between nearest flowers = 32 cm). The board was painted green. To tilt the board, one end was hooked to adjustable nylon strings hanging down from the ceiling. The angle of each flower was adjusted so that its entrance faced upward. This adjustment was made to keep the posture of bees constant while extracting nectar, irrespective of the slope of the array. EXPERIMENT 1 To examine whether bees prefer visiting flowers on flat land to sloped planes, bees were presented with a pair of arrays, one horizontal and one sloping. To train the bees to forage on artificial flowers, both the horizontal and sloping arrays were set up in the cage (Fig. 1c). The

3 Bumble bee responses to slopes 1029 gate of the nest was then opened to allow bees free access to the arrays for hours. Throughout the training period, the locations of the arrays were alternated at 10-min intervals, so that the bees experienced both combinations of array type and location (i.e. horizontal-sloping and sloping-horizontal). Individual bees that were observed to learn how to probe flowers and visit flowers in both arrays were identified and marked with numbered tags glued to their thoraxes. These tagged bees were used for the experiment on the next day (or on the same day in a few cases). The angle of the sloping array was either 45 or The bees were allowed to experience only one of the two angles throughout the experiment, so that they never experienced both. In the experiment, a pair of arrays (0 vs. 45, or 0 vs arrays) was set, and the initial position of the sloping array (left or right) was assigned randomly. A trained bee was then allowed to forage alone on these arrays for five trips. Between foraging trips, the locations of the arrays were alternated before the bee came out into the cage again. While the bee was foraging, the experimenter was outside the cage and recorded the sequences of flowers visited by the bee. This experiment was repeated for 13 bees (eight from Colony A, and five from Colony B) for each pair of arrays (0 vs. 45, and 0 vs ). DATA ANALYSIS To examine any relative preference for horizontal or sloping arrays, the following three variables were analyzed: (i) The mean number of visits to flowers in each array per trip. The means for each bee were calculated, and then Wilcoxon signed ranks tests were performed to compare the results for horizontal and sloping arrays; (ii) The number of consecutive visits within an array. During a foraging trip, bees repeatedly switched between the horizontal and sloping arrays, making consecutive flower visits within an array before switching to the other. For each bee, the mean number of consecutive visits in each array was calculated, and Wilcoxon signed ranks tests were performed to compare the arrays. This variable was analyzed because the number of consecutive visits was independent of the appearance of flowers on arrays from the nest gate; (iii) The ratio of the number of flower visits in the horizontal array during a foraging trip to that in the sloping array. To examine whether a steeper slope had a greater effect, the ratios between the 0 vs. 45 and 0 vs experiments were compared using a simple t-test. Data were pooled across colonies because each colony showed the same trends in these variables, [visits per trip: 0 vs. 45, P A (probability for colony A) = 0 016, P B (probability for colony B) = 0 063; 0 vs. 67 5, P A = , P B = 0 063; visits within an array: 0 vs. 45, P A = , P B = 0 063; 0 vs. 67 5, P A = , P B = 0 063; the ratio of the number of visits: t = 1 9, P A = 0 079; t = 1 9, P B = 0 099]. Note that bees did not show any side preferences; no significant differences were found between left and right arrays (visits per trip: 0 vs. 45, P = 0 29; 0 vs. 67 5, P = 0 62; visits within an array: 0 vs. 45, P = 0 19; 0 vs. 67 5, P = 0 73; Wilcoxon signed ranks tests, n = 13 bees each). EXPERIMENT 2 To examine whether and how the inclination of the ground affects their foraging performance (nectar intake per second) and travel time between flowers, bees were allowed to forage on an array tilted at various angles. To train bees to forage on artificial flowers, the flower array was set horizontally at the centre of the cage. Bees were then allowed free access to the array for about 3 to 5 h. During this period, individual bees that learned how to probe artificial flowers were identified and marked with numbered tags. These tagged bees were used for the experiment. Each experiment day, the bees were allowed to forage freely for about min on an array that was set horizontally in the middle of the cage floor. During this period, one active forager was chosen as a target bee (when there were multiple active foragers, a target bee was chosen at random). When the bee returned to the nest, the array was moved to a corner of the cage to minimize the effects of any spatial memory developed during the training period [although some area of the array in the training phase covered an area of the array in the test phase, the number of visits to flowers in the overlapped area did not significantly differ from that in the nonoverlapped area during the first trip (P = 0 44, n = 16 bees, Wilcoxon signed ranks test)]. The target bee was then allowed to forage alone while the experimenter observed. The target bee would make about 360 visits to flowers before returning to the nest, and it took roughly 15 min for one foraging trip. Each time the target bee made two trips, the slope of the array was increased at intervals of 22 5, to a maximum angle of 90. Accordingly, the bee experienced slopes of 0 (horizontal), 22 5, 45, 67 5 and 90, in that order. After the bee had completed two trips to the 90 array, the array was set back to the horizontal position on the floor, and the bee was allowed to make two additional trips. This observation was conducted to confirm that results were not affected by experience. For example, if a variable increased with increasing slope angle, and continued increasing when the angle returned to 0, we would have to conclude that the change was correlated not with slope angle, but with experience. While the bee was foraging, the experimenter was outside the cage recording the sequence of flowers visited by the bee. To measure probing time per flower and travel time between flowers, each sequence was recorded with a digital video camera (Handycam DCR-SR100, Sony, Japan) positioned at one corner of the cage. This experiment was repeated for 16 bees (eight from both Colony C and D). DATA ANALYSIS From recorded images of each foraging trip, thirty consecutive flower visits were taken from around the middle of each trip, and measured for probing time and travel time for each flower visit to 1/30 s. In this study, probing time corresponds the time during which a bee stayed still on a flower, inserting its head into the flower entrance and extending its tongue to consume nectar inside. Travel time is the time spent moving between flowers, which corresponds to the interval between successive probes. Note that preliminary measurements showed that both travel and probing time did not significantly changed during a trip, indicating that the results in this study were not altered by the phase chosen for measurements; I took each of thirty consecutive visits from the beginning, middle, and end of the second trip in the 90 array, and compared mean time calculated for each bee among phases with anovas (travel time: F 2,30 = 1 1, P = 0 35; probing time: F 2,30 = 1 8, P = 0 19; n = 16 bees). The gross rate of nectar intake (µl sec 1 ; hereafter, foraging rate) for each bee was estimated in the following way: First, for every revisit to flowers during a foraging trip, the number of flower visits a bee made before returning to a flower (return cycle) was counted. Second, for each revisit to a flower, the time elapsed since the last visit to the flower (return interval) was estimated by multiplying the return cycle by the mean time taken to visit one flower (i.e. the mean travel plus probing times, which were measured from the video). Third, based on the return intervals, the amount of nectar available at each flower visit was calculated and summed for the entire trip,

4 1030 T. T. Makino Fig. 2. Box-and-whisker plot of (a) the number of flower visits made during a foraging trip, and (b) the number of consecutive flower visits made before switching to the other array. Median, quantiles and extreme values are shown. For each pair of arrays, the P values obtained from Wilcoxon signed ranks tests for the differences are also shown (n = 13 bees for both 0 vs. 45 and 0 vs experiments). Fig. 3. The changes with slope angle in (a) foraging rate and (b) travel time between flowers. The horizontal axes indicate slope angles ordered in time. Symbols represent individual bees (n = 16 bees). Different letters indicate significant difference between slope angles (P < 0 05, paired t-tests with sequential Bonferroni corrections). assuming that (i) nectar accumulated in flowers at a constant rate (0 4 µl min 1 ) until it reached 1 0 µl, at which time replenishment stopped, (ii) all the accumulated nectar was drained at one visit, and (iii) nectar secreted during probing time was also taken by the bee. [Assumption (i) is based on Makino & Sakai (2007), in which the authors examined the changes in the volume of the nectar in a flower after removing accumulated nectar with a piece of filter paper. Assumption (ii) is based on a positive correlation between probing time on a flower and the time elapsed since it was last visited (see Fig. S1 in Supporting Information). Assumption (iii) was made because there is no reason to think that capillary action ceases while a bee probes nectar. Note that the result does not change when foraging rate was calculated with an assumption that capillary action stops while a bee probes a flower.] Fourth, the total nectar gain was divided by the total time spent on inter-flower movements and flower probing, to obtain the foraging rate. Two-way anovas for the repeated testing for the same individuals (Sokal & Rohlf 1995) were performed to test the effects of slope angle on foraging rate and travel time. For each dependent variable, each bee had two data points for each slope angle because it made two trips for each angle. To avoid pseudo-replication in the anovas, means for each slope angle for each bee were used, reducing the data to a single point per bee for each angle. Both the slope angle and individual bees were considered as categorical variables. The first 0 and the last 0 were treated as different categories in these analyses. For multiple comparisons among slope angles, paired t-tests with sequential Bonferroni corrections were conducted. Note that data were pooled across colonies because the two colonies were similarly affected by slope (foraging rate: F 5,35 = 15 9, P C < ; F 5,35 = 16 6, P D < ; travel time: F 5,35 = 39 6, P C < ; F 5,35 = 29 4, P D < ). Results EXPERIMENT 1 During a foraging trip, bees made significantly more visits to flowers on the horizontal array than on the sloping array (Fig. 2a). The bees also made significantly more consecutive visits on the horizontal array before switching to the sloping array (Fig. 2b). The difference between arrays was greater when the slope was steeper; the ratio of the number of visits in the horizontal array per trip to that in the sloping array was 0 86 ± 0 10 in the 0 vs. 45 experiment (mean ± SD, n = 13 bees), which was significantly higher than that in the 0 vs experiment (0 74 ± 0 11, n = 13 bees; t = 2 8, P = , simple t-test). EXPERIMENT 2 The foraging rate of the bees significantly differed among slope angles (effect of slope angle: F 5,75 = 25 9, P < ; effect of individual bees: F 15,75 = 17 2, P < ). Figure 3a shows that the foraging rate showed a 9 1% reduction from 0 to 90 arrays, and went up when the angle returned to 0. Travel time also significantly differed among slope angles (effect of

5 Bumble bee responses to slopes 1031 Fig. 4. Box-and-whisker plot of travel time in a 67 5 array for comparison among different directions (n = 16 bees). Median, quantiles and extreme values are shown. Different letters indicate significant difference between directions (P < 0 05, paired t-tests with sequential Bonferroni corrections). Note that movements to flowers other than the neighbouring flowers are excluded from the analysis. Detailed results including travel time in other direction (diagonal upslope/downslope) are provided in Fig. S2. foraging rate could cause a considerable difference in the number of reproductive individuals (new queens or males) at the end of the season. The preference of bees for flats (Fig. 2) is, therefore, considered to be reasonable. I hope that the present results will encourage future work on the ecological influences of slope. Because inclined planes are found everywhere (e.g. banks of a river, hills, mountainous regions), it is likely that bees in nature are often faced with the cost of slope. Thus it is worth examining how the terrain on which flowers are presented affects foraging behaviour of bees under field conditions. Not only foraging by worker bees, the terrain may also affect the nest site selection by queens (Nakamura & Toquenaga 2002; Suzuki, Kawaguchi & Toquenaga 2007); it might be expected that queens found their nests at sites surrounded by relatively flat areas for food economics. Moreover, from the point of view of pollination biology, it also remains to be seen whether slopes actually influence pollen flow among plants through the effects on pollinators. Future work on these issues will lead us to better understanding of plant pollinator interactions. CAUSES OF THE CHANGE IN FORAGING RATE WITH SLOPE ANGLE slope angle: F 5,75 = 65 5, P < ; effect of individual bees: F 15,75 = 14 1, P < ). Figure 3b shows that it increased with increasing slope angle, and then decreased when the angle returned to 0. At the same slope angle, lateral movements required the shortest travel time (Fig. 4), indicating that upslope and downslope movements are costly for bees (changes in travel time with slope angle in each direction are shown in Fig. S2 in Supporting Information). Discussion This study demonstrated that bees preferred visiting flowers on a flat surface when presented with the choice between horizontal and sloping arrays (Fig. 2). This suggests that they disliked the reduction in foraging performance that occurred on slopes (Fig. 3a). They might also avoid energetic costs found in some animals walking upslope or downslope (cockroach: Full & Tullis 1990; gecko: Farley & Emshwiller 1996; ant: Lipp, Wolf & Lehmann 2005). The preference of bees for flowers on a flat surface indicates that plants growing in sloping habitats may be less preferred by pollinators. These findings are, to my knowledge, the first to demonstrate the importance of the inclination of the ground in pollinator visitation to flowering plants. The reduction in foraging rate indicates that foraging on slopes is costly for bees. Figure 3a shows that, during the same length of time (e.g. daytime), bees foraging on slopes will experience up to about 10% reduction in food income compared with those foraging on flats. This percentage might seem small, but could result in a large reduction at colony-level foraging; i.e. in a colony of bees, foods collected by workers is used for raising new workers, so that the colony size often shows exponential growth, indicating that even a slight difference in The reduction in foraging rate with increasing slope angle (Fig. 3a) was caused by the increased travel time (Fig. 3b). To efficiently harvest floral resources that renew in a decelerating way, bees must keep returning to each flower before it reaches saturation. This is because a return visit after saturation increases the time that a plant spends not renewing, as shown by the models of Possingham (1989) and Ohashi & Thomson (2005). Indeed, the proportion of such returns after saturation increased with slope angle in this study (see Fig. S4), and this increase was considered to be caused by the increased travel time on slopes, i.e. bees took longer to return to flowers on slopes because of the increase in travel time. The increased travel time on a slope could be explained by several factors. (i) Locomotor constraints on upslope/downslope movements. Observed bees took longer to ascend and descend a slope than to move laterally (Fig. 4), indicating locomotor constraints associated with gravity. To ascend a slope, bees may have to work harder to resist gravity than when moving in the other directions. To descend a slope, bees may have to make greater efforts for braking to avoid the excessive acceleration that makes landing on a flower difficult. (ii) Masking effect of flowers. On a steep slope, bees positioned on flower entrances had a limited view of flowers positioned below (Fig. 1a), which might prevent bees from moving directly to those flowers. This masking effect probably reduced downslope movements by bees and instead increased diagonal-downslope movements (see Fig. S3). This result indicates that the bees were forced to fly to farther flowers on slopes, consequently spending more time travelling. (iii) Constraints on performing different tasks. In contrast to a flat surface, where bees conducted lateral movements only, on a sloping plane, bees had to perform different tasks depending on the direction of flight. Chittka & Thomson (1997) showed

6 1032 T. T. Makino that bees took longer when they handled flowers of varying morphs requiring different tasks, than when they constantly handled flowers requiring a single task. Although the kinds of tasks performed are different (handling flowers with morphs, or flying in different directions), similar constraints might be responsible for the increased travel time in a sloping array. (iv) Counteractive wind pressure from the ground when bees take off. This wind pressure acts as a force to lift bees taking off from a horizontal surface, but it may become a nuisance on a slope because the pressure acts sideways, blowing them away from the slope surface. These explanations are not mutually exclusive, but their relative importance may vary depending on pollinator or plant species. For example, wind pressure does not matter when flowers are located high above the slope surface. In contrast, the masking effect of flowers (and also that of leaves) seems more general. Further research is needed to determine how common these factors are. Acknowledgments M. Mitsuhata provided a facility of bumble bee colonies. Useful discussion and invaluable help have been contributed by K. Ohashi, S. Sakai and J. Cresswell. Helpful comments from two anonymous reviewers improved the manuscript. The work was supported by a fellowship of the Japan Society for the Promotion of Science to TTM. References Cartar, R.V. (2004) Resource tracking by bumble bees: response to plant-level differences in quality. Ecology, 85, Chittka, L. & Thomson, J.D. (1997) Sensori-motor learning and its relevance for task specialization in bumble bees Behavioral Ecology & Sociobiology, 41, Engel, E.C. & Irwin, R.E. (2003) Linking pollinator visitation rate and pollen receipt. American Journal of Botany, 90, Farley, C.T. & Emshwiller, M. (1996) Efficiency of uphill locomotion in nocturnal and diurnal lizards. Journal of Experimental Biology, 199, Full, R.J. & Tullis, A. 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Return cycle, its CV, and the proportion of returns after saturation. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.