Density-dependent and frequency-dependent selection by bumblebees Bornbus tmestris (L.) (Hymenoptera: Apidae)

Transcription

1 BwlogicalJournal ofthe Linnean S+v (1997), 60: With 2 figures Density-dependent and frequency-dependent selection by bumblebees Bornbus tmestris (L.) (Hymenoptera: Apidae) ANN SMITHSON AND MARK R. MACNAIR Dearhnent of Biological Sciences, Univerdg of Exeter, Hahh Laboratories, Prince of Wales Road, Exeter M4 4PS Receizud 5 Januay 1995, accepd fm publicaria 24 July 1996 The behaviour of bumblebee workers foraging on arrays of artificial flowers of two colour morphs was observed. Experiments were conducted on arrays of varying morph frequencies and at three Merent total flower densities. Bumblebees consistently showed a preference for the commonest colour morph, and this behaviour was not significantly affected by changing density. In contrast, frequency-independent preferences changed significantly with density. At low densities, there was a strong bias towards the more conspicuous colour, whereas at higher densities there was no overd colour bias. Flight distances between flowers decreased significantly at high density. Bumblebees also visited flowers of similar colours sequentially, but this behaviour was not density-dependent. It is suggested that as densities increase, there is an increased probability that bumblebees detect yellow flowers, which were probably less conspicuous compared with blue flowers, and that this might be caused by changes in tlight speed with flight distance. Where there is a positive relationship between pollinator visitation and the relative fimess of a floral morph, the observed behaviour would induce positive frequencydependent selection on a plant population with two corolla colour morphs on which the bumblebees were foraging, which would result in stabilizing selection for a single corolla colour, irrespective of density. There was no indication that rare colour morphs would be preferred at high density. The probability of Merent corolla colour morphs going to fixation would, however, be affected by density The Linnean Society of London ADDITIONAL KEY WORDS:-orolla - learning. Introduction Methods Experimental... Dataanalysis... Results Frequency-dependence Order of flower visits Flight distances Discussion..... Acknowledgements... References colour - directional selection - conspicuousness - fight path CONTENTS /97/ $25.00/0/bj The Linnean Society of London

2 402 A. SMITHSON AND M. R. MACNAIR INTRODUCTION Previous studies (Smithson & Macnair, 1997; Real, 1990) have shown that bumblebees (Bombus tamhis) foraging on artificial flowers of two colour morphs select flowers in a frequency-dependent way, preferring the commonest morph. If common morph preference were shown by pollinators foraging on a real plant population variable in corolla colour, then rare corolla colour morphs would receive fewer pollinator visits. Increased pollinator visitation can result in differential morph fitness either through increasing maternal (seed set; Waser & Price, 198 1) or paternal (outcross seed paternity; Stanton et al., 1989) reproductive success. There is now considerable evidence that intraspecific variation in floral traits influences both pollinator visitation and relative reproductive success (Willson, 1994), and increased pollinator visitation may increase paternal function even where seed set is resourcelimited (Stanton et al., 1989). Where relative fitness is a positive function of the number of pollinator visits received, rare morphs will thus be at a selective disadvantage in the population through positive frequency-dependent selection (FDS) on the reproductive component of fitness. In self-compatible plant species, differential pollinator visitation may result in compensatory higher levels of sehg in rare morphs, which will thus be at a reproductive disadvantage through inbreeding depression on maternal function and reduced paternal function (Levin, 1972; Epperson & Clegg, 1987). Positive FDS will result eventually in monomorphism for the trait under selection (Thornson, 1984; Endler, 1988). There is some evidence (Levin & Kerster, 1970; Levin, 1972) for rare corolla morph disadvantage in dimorphic populations of a butterfly-pollinated plant (Phlox) through reduced seed set. There is considerable evidence that many types of predators show significant preferences for common prey morphs in populations (reviewed by Allen, 1988); this has been called apostatic selection, and would exert negative FDS on prey populations Clarke, 1962). The proximate cause of apostatic selection has often been discussed in terms of the search image (Tinbergen, 1960; Dawkins, 197 l), which suggests that perceptual changes in predators increase their ability to detect cryptic prey as a result of recent experience with similar cryptic prey. The probability of search image formation is believed to increase with increasing density up to a maximum at intermediate density (Clarke, 1962; Greenwood, 1984; Allen, 1988). Alternative hypotheses to explain apostatic selection exist (Greenwood, 1984; Gendron, 1987; Endler, 199 1). Excluding those mechanisms that result from differences in predator foraging history or location of food items (Lawrence & Allen, 1983; Gendron, 1987)) the main possibility is that apostatic selection is an indirect result of a trade-off between searching and the probability of detecting cryptic prey (reviewed in Endler, 199 1). The search rate hypothesis does not predict that predator preferences should change with prey density (Gendron & Staddon, 1983). If foraging pollinators prefer common corolla colours and exert positive FDS on plant populations, how may the variable corolla colours seen in many plant pollinators (Kay, 1978) be retained? It could be argued that rare corolla colour morphs might be at a selective advantage in a population if the increased quality of visitation that a rarely-visited individual receives results in greater maternal or paternal fecundity than that of a frequently-visited one. This could occur if the more frequently: visited common morphs became depleted of nectar, and a pollinator thus spent comparatively more time feeding on each individual flower of the rarer morphs,

3 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 403 thus potentially increasing pollen removal or deposition. Experimental evidence suggests that the time a pollinator spends feeding on a flower can increase pollen export (Galen & Stanton, 1989), but the influence of probe time on pollination performance per flower is minimal through both male and female function when compared to the probability of visitation per se (Cresswell & Galen, 1991). If it is assumed that there is only one type of pollinator exhibiting similar foraging patterns, then the simplest explanations for the observed corolla colour polymorphisms in plant populations are that they are either transient, or result from a balance between selection and mutation. Alternatively, there may be circumstances in which pollinators show a preference for rare corolla colours, thus causing negative FDS which will result in stabilizing selection and polymorphism for that trait (Clarke & O Donald, 1964). At high densities of prey, predators may select the rarest, as opposed to the commonest morph: this has been demonstrated experimentally both for birds (Allen, 1972; Horsley et al., 1979; Allen & Anderson, 1984) and for mammals (Greenwood, Johnston & Thomas, 1984; Greenwood, Blow & Thomas, 1984; Church, Allen & Bradshaw, 1994). This behaviour has been called anti-apostatic selection. Several mechanisms have been used to explain the preference for rare morphs at high densities, most commonly that at high densities predators can locate rare morphs more easily because they appear conspicuous against a background of common morphs (Allen, 1972; Greenwood, 1984). In previous studies (Smithson & Macnair, 1997), it was found that bumblebees foraging on arrays of blue and yellow flower morphs show an overall preference for blue flowers in addition to preference for the commonest morph, but that the bias to blue flowers decreased when rewards given by all flowers increased. The spectral reflectance characteristics of the flowers over the range of wavelengths perceived by pollinators was recorded and analysed, and it was shown that these blue flowers probably looked more conspicuous to the eyes of bumblebees than yellow flowers when compared to the background colour, which could explain the overall bias towards blue flowers. There was a decrease in the distances travelled between flowers as overall rewards increased. Consequently the speed of flight may have slowed which may have increased the probability of yellow flowers being noticed, potentially explaining the change in bias towards blue flowers. These ideas were consistent with hypotheses which predict a trade-off between searching and the probability of detecting prey differing in conspicuousness (Gendron & Staddon, 1983; Guilford & Dawkins, 1987). The relationship between flower density in monomorphic populations and pollinator behaviour has been investigated and shown to be densitydependent both in experimental (Waddington, 1980) and field (Levin & Kerster, 1969a, b) situations. Waddington (1980) found that as plant density increased, the flight distance made by pollinators to the next visited inflorescence decreased, and Schall (1978) showed that the probability of visiting the nearest inflorescence increased as density increased. These results may be interpreted from an optimal foraging viewpoint. Foraging pollinators can maximize net energy intake if flight distances from plant to plant are determined by available nectar rewards since flight costs and probabilities of revisiting flowers are minimized (Pyke, 1978). If the hypothesis that flight distance affects flight speed and therefore the likelihood of recognizing less conspicuous flowers, then preference for less conspicuous morphs should increase with increasing density. Despite many optimal foraging studies focusing on bumblebee learning of preferences for colour morphs where morphs differ in nectar content (Heinrich, Mudge & Deringis, 1977; Real, 1990) or variability

4 404 A. SMITHSON AND M. R. MACNAIR of nectar content (Real, 1981; Real, Ott & Silverfine, 1982), we are aware of no previous study which has looked at the effects of density on pollinator preferences for colour morphs. It was therefore predicted that increasing the density of flowers made available to pollinators would affect both the preference for common morphs and the bias towards Merent corolla colour morphs. Previous experiments (Smithson & Macnair, 1997) also showed a tendency for individual bumblebees to visit corolla colour morphs of the same colour sequentially, and this visitation pattern will cause assortative mating for corolla colour. The hypothesis that density may mow frequency-dependent selection, frequency-independent (directional) selection, assortative mating and flight distances travelled between flowers was investigated, using bumblebees as experimental subjects. METHODS Experimental The methodology of these experiments was identical to that used by Smithson & Macnair (1997), and is similar to that used by other workers studying the foraging preferences of bumblebees (e.g. Real, 1981, 1990), although we have conducted experiments in the laboratory using captive-reared bumblebee colonies so that individual bees had no prior foraging biases. Experiments utilized colonies of bumblebees (Bombus tmwt@ which foraged on an array of artificial flowers of two colours. The colonies were obtained from a commercial supplier (Bunting Biological Control, Colchester, Essex), are used commercially for pollinating glasshouse crops, and contain approximately 200 worker bees. The bumblebees had not fed outside their boxes prior to experiments, and were only allowed to forage under experimental conditions. Colonies had access to pollen inside the colony box at all times, and were fed sucrose solution overnight during experiments to maintain positive colony energy budgets. AU experiments were conducted inside a fine-netted cage (0.9mx0.9mx0.9m), to which access was provided via a gated tube from the colony box. During experiments, worker bumblebees foraged on an array of artificial flowers presented on the base of the cage. The array consisted of a transparent perspex board (775 mm x 775 mm x 6 mm) into which a grid of 30 x 30 wells 25 mm apart had been drilled. Well sizes were 2.5 mm diameter and 3 mm deep. Coloured discs of card, 16mm diameter, were placed under selected wells to act as.flowers. The wells above flowers could then be filled with a selected quantity of 4oYo (w/v) sucrose solution, dispensed with a Gilson micropipette, to act as a reward on which the worker bees could feed. The two flower colours used in these experiments were yellow and blue, which were placed on a green base-board. Flower and background colours were identical to those used previously (Smithson & Macnair, 1997). Worker bumblebees were trained to forage from the array using 50 yellow and 50 blue flowers, which were placed randomly on the array, and filled completely with approximately 20@ sucrose solution to ensure food was always available in excess during training. Sucrose solution was initially mixed with honey to provide an additional olfactory cue to assist in reward location. Once worker bees had commenced active foraging from both flower colours and were consuming sucrose

5 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 405 solution from the flowers, the proportion of honey present in the wells was decreased to zero so that only visual cues could be used to locate flowers during experiments. Active foragers were then marked on the thorax with spot combinations of solventfree correction fluid to allow individual identification. Thus during training each forager gained equal experience of both flower colours, and also learned to associate both flower colours with a reward, but had no experience of experimental densities, morph frequencies, or reward schedules. Each trained and individually-marked bumblebee was only used in one experiment, and was allowed access to the experimental array five times sequentially at one morph frequency. During each trip to the array (defined as a bout) a bee typically visited and consumed the rewards from approximately 50 flowers, after which it returned to the colony box. After five bouts had been recorded, the bee was removed from the colony to ensure individuals were only used in one experiment. During experiments only one bumblebee at a time was allowed access to the array. Between bouts the array was washed with water, the wells were rinsed with water, the array dried, the distributions of flowers were rerandomized (using random number tables), and then flowers refilled. All bumblebees flew from one flower to the next whilst foraging irrespective of density, walking between flowers was rarely observed. bumblebee orientation to the flowers was accurate, they did not land on the background component of the array, and they landed without difficulty. Previous experiments showed that revisitation to previously emptied flowers was low (10%; Smithson & Macnair, 1997) and thus unlikely to influence bumblebee preferences. It is not known, for pollinators, exactly what densities of flowers correspond to the intermediate and high densities that cause apostatic and anti-apostatic selection respectively for predators. Densities of 2 m-2 (Allen, 1976) have produced apostatic selection for birds feeding on pastry baits, and densities of 800m-2 (Horsley et al., 1979) to mw2 (Allen& Anderson, 1984) have generated anti-apostatic selection for a similar system. The flower densities used in these experiments were constrained at the lower end by a minimum number of flowers that had to be present on the array in order to fill the crop of a bumblebee on one foraging trip to the array, and at the upper end by the maximum number of dispenses of sucrose solution that could be made into flowers on the array and offered to a bumblebee worker before the dispensed quantities of sucrose solution evaporated. Three experiments were carried out as follows: (1) 150 flowers per array, density 248 m-2, flowers cover 5% of board, reward 2 pl in all flowers. (2) 200 flowers per array, density 331 m-2, flowers cover 6.7% of board, reward 2 pl in all flowers. (3) 300 flowers per array, density 497m-2, flowers cover 10% of board, reward 2 pl in all flowers. Experiment 1 is identical to an experiment previously carried out (Smithson & Macnair, 1997)) where preference for common morphs was found. The experiment has not been repeated and results found previously have been reanalysed here. Although the previous experiment used a different bumblebee colony to obtain data, it has been found that consistent results were obtained between colonies when studying frequency-dependent and directional selection (Smithson, 1995). For each experiment nine different morph frequencies were used, from 10% to 90% yellow flowers, and replicates of 4-5 different bumblebees were tested at each

6 406 A. SMITHSON AND M. R. MACNAIR frequency for each experiment. For each visit to the array by an individual bumblebee, the number of flowers of each colour probed were recorded (i.e. a flower was defined as visited when the bee inserted its proboscis into the well), along with the location of flowers probed and the order of flower visits. The proportions of yellow flowers visited were calculated for each bee after combining all five visits to the array. For each experiment, the proportions of yellow flowers visited.were compared to those available on the array using the methods of Greenwood & Elton (1979), which fits a sigmoidal function to the data from the equation: where F= proportion of yellow flowers visited. A, =proportion of yellow flowers available in the array. A2 =proportion of blue flowers available in the array. b = coefficient measuring the degree of frequency-dependence. When b > 1 the curve represents preference for common colours, when b = 1 random selection, and when b <1 preference for rare colours. V= coefficient measuring frequency-independent or directional selection, the preference for yellow flowers compared with blue flowers. When V >1 yellow flowers are preferred irrespective of frequency, and when V < 1 blue flowers are preferred. Data were fitted to the logarithmic transformation of equation (1) by linear leastsquares regression, after logarithmic transformation of the data (Greenwood & Elton, 1979). Any data points in which no flowers of one colour were visited were transformed after assigning nominal values of half a flower visited to that colour. Parameter standard errors were estimated using a bootstrap procedure (Efron & Tibshirani, 1986), qving estimates robust to any points exerting high leverage on the data set as descnbed in Smithson & Macnair (1997). Confidence limits for fitted parameters were calculated from distributions of bootstrap parameters using the percentiles method (Efion & Tibshirani, 1986). Fitted parameters were compared over experiments using the methods described in Greenwood & Elton (1979) and Elton & Greenwood (1987); log-transformed values of Vwere used in all comparisons. 01, the morph frequency at which equal overall preference was shown for each of the flower colours, was estimated for each experiment by interpolation on the fitted curve as determined by K Departures from non-randomness of the order in which flowers were visited were tested by quantifying the numbers of times each bee moved between each colour combination (blue +blue, blue -+yellow, yellow -+blue, yellow+ yellow) from each flower to the second, third and fourth flowers occurring next in the sequence (henceforth referred to as positions 1, 2 and 3). Transition matrices could then be computed for each bee from counts of moves in each category. Counts were

7 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 407 combined over all bees at each morph frequency and position used for each experiment, and then tested for randomness of visit order using the G-test (Sokal & RON, 1981). To find whether significant differences from random visit order were due to similar colours being visited sequentially, an index of asso&vity was computed from each transition ma& to - 1 and is positive when and negative when between for each- individual bee. This index extends from + 1 more movements are made between the same colour, different colours. The index was computed as: (BBX rr)-(byx YB) I= (BB+Bz) X (TB+ rr) where BB= observed number of blue to blue movements. rt= observed number of yellow to yellow movements. BT= observed number of blue to yellow movements. YB= observed number of yellow to blue movements. The index was compared between experiments and between colonies by analysis of covariance on the arcsine-transformed index, using morph frequency as the covariate. Flight distances found at each density were estimated as the shortest distance between successive pairs of flowers visited by each worker bee using the array coordinates of each flower position. Data from the fist and fifth bouts were analysed and the mean flight distances found for each bee. Flight distances were pooled over all bees tested at each frequency for the three experiments. RESULTS The data are plotted in Figure 1, along with the sigmoidal switching curve fitted using least-squares regression. Figure 1D compares the fitted lines for each experiment. The fitted parameters and standard errors are given in Table 1. To test for significant differences in frequency-dependent selection with increasing density, the fitted values of b were tested for heterogeneity using the methods of Elton & Greenwood (1987). No significant differences were found (F2,70 = , 0.1 >P>0.05), although there was a trend towards increasing frequency-dependence with increasing density. The pooled estimate of b was f ( f 1 SE), and this is significantly greater than 1 (t=8.053, P<O.OOl), thus there was a preference for common colours in all experiments. Differences amongst fitted values of V were tested in a similar way, and in this case significant differences were found (F2,70 = ; P<O.OOl), showing that changes in density caused differences in the frequency-independent component of choice. From Table 1 and Figure 1 it can be seen that at low density (experiment 1) there is strong preference for blue flowers, whereas at high density (experiment 3) there is no overall preference for either colour and the fitted curve cuts the line of equal preference at approximately 50% yellow flowers (Table 1).

8 408 A. SMITHSON AND M. R. MACNAIR B 1 :? 0 E !a Proportion of yellow flowers available Proportion of yellow flowers available Figure 1. Results of wrperiments studying the effects of varying morph frequency and density on bumblebee choice behaviour. Data from each experiment are shown, along with curves fitted to data from equation (1) using the methods described in the text. A, experiment 1 (density 150 flowers), B, experiment 2 (density 200 flowers), C, experiment 3 (density 300 flowers), D, combined plot of curves fitted to each density tested. Density: (-) 300 flowers; (---) 200 flowers; (---) 150 flowers. Overlapping points are shown slightly displaced for clarity. TABLE 1. Results of bumblebee choice experiments on arrays of yellow and blue artificial flowers with varying frequency and density. Data were totalled over all five bouts for individual bees. The table shows parameters and standard errors from fitting results of three experiments to equation (1) by linear regression on log-transformed data. Standard errors were found from 1000 bootstrap estimates of regression parameters (Efron Br Tibshirani, 1986). a represents the frequency of yellow flowers at which equal preference is shown for both colour morphs b Mean bout length (fkequency- V Sample size (number of flowers dependent (directional Experiment (number of bees) visited) selection) selection) U f f f f f k f f I.043 f

9 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 409 TABLE 2. Results of bumblebee choice experiments on arrays of yellow and blue artificial flowers with varying frequency and density, with data grouped to show the effects of increasing experience on bumblebee behaviour. Data from individual bees were divided sequentially into sets of 50 flowers visited. The table shows parameters and standard errors from fitting data from each experiment to equation (1) by linear regression on log-transformed data. Standard errors were found from 1000 bootstrap estimates of regression parameters (Efron & Tibshirani, 1986) 6 Number of (frequency-dependent V Experiment flowers visited selection) (directional selection) f f f f f f f f f f f fO f f f f f f0.283 It can also be seen from Table 1 that there is a small increase in mean bout length with increasing density, and these differences proved to be significant (F2,551 = ; P<O.OOl). It could be argued that the observed experimental effects might have been caused by differences in bout length, since increase in bout length may reflect increasing experience of the array which in turn might cause differences in behaviour. Therefore data sets for each experiment were reanalysed by dividing data for each bee into sequential sets of 50 flowers visited, to a maximum of 150 flowers. Data were fitted to equation (1) as described above, and the results are given in Table 2. Within each experiment, there is a significant increase in b with increasing experience (experiment 1: F2,72 = ; P<O.OOl; experiment 2: (F2,76 = 4.559; ; experiment 3: F2,76= ; P<O.oOl). There were no changes in Vwith increasing experience in any experiment (experiment 1: F2,68= 2.028; ; experiment 2: F2,76 = 1.210; ; experiment 3: F2,75 = 0.507; ). Comparing results between experiments at 150 flowers, there were highly significant differences in V (F2,75=71.367; P<O.OOl), but no simcant differences in b (F2,76=0.570; ), and results are thus completely consistent with those found from analysing total data. It should be noted that, although there were no significant differences in b after the effects of numbers of flowers visited has been removed, there is still a weak trend for increasing frequency-dependence with increasing density (Table 2). Results of testing transition matrices of sequences of movements are shown in Table 3, and it can be seen that nearly all matrices give significant differences from random visit orders. Mean assortivity indices plotted according to experiment and morph frequency are given in Figure 2. Nearly all indices were significantly positive indicating preference for visiting flowers of similar colours sequentially irrespective of overall proportions of flower colours visited. Overall mean assortivity indices are given in Table 4. It can be seen from Table 4 and Figure 2 that there are no

10 410 A. SMITHSON AND M. R. MACNAIR TABLE 3. Results of analysis of sequence of flower visits for position 1. Counts are numbers of movements between blue and yellow flowers from one flower to the next in the sequence over all bouts for each bee. Results were totalled over all bees tested at each proportion of yellow flowers available, and tested for non-random visit order using a G-test (Sokal& Rohlf, 1981). Results of G- tests are given along with their significance after Bonferroni correction (*RO.O5, **RO.Ol, *** RO.001, with correction) Proportion of Number of movements made between flower colours Expen- yellow flowers ment available Blue+Blue Blue+YeUow Yellow+Blue Yellow+Yellow Total G, *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * *** *** *** *** *** *** obvious differences in the tendency to visit flowers of similar colours sequentially between experiments, and an analysis of covariance on arcsine-transformed assortivity indices failed to reveal any significant effects of experiment on assortivity index (F2,10, =2.388; P>0.05). Flkht dktuwes The statistics of flight distance data for each experiment for the first and fifth bouts were calculated, and are shown in Table 5a. An analysis of variance was carried out on logarithmically-transformed data to test for significant effects of density and bout on flight distance. Results (Table 5b) indicated that there was a significant decrease in mean flight distances as flower densities increased (F7,823; p<o.ool), but that there was no effect of bout number on flight distance.

11 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 41 1 Assortivity index Density flowers c Density - o.o 200 flowers Proportion of yellow flowers available Figure 2. Mean assortivity indices f 1 standard error for individual bumblebees plotted according to experiment 2 (H) and 3 (U) are shown (see text). and morph frequency. Positions 1 (a), TABLE 4. Mean assortivity indices over all morph frequencies for each experiment, 1 standard error, for positions 1, 2 and 3 in the sequence of flower visits (see text) ~~ Experiment Position 1 Position 2 Position f * f f f f f f f0.076 DISCUSSION In these experiments, increasing density did not result in a change in frequencydependent behaviour, bumblebees preferring common colours in all experiments. No tendency to prefer rare morphs at high density was found. Increasing experience resulted in increased preference for common colours as was found in previous experiment (Smithson & Macnair, 1997), the only observed trend in frequencydependence over experiments was a weak and non-sigdicant tendency for common colours to be increasingly preferred with increasing density. How may these results be explained? Since it has been hypothesized that anti-apostatic selection at high density is caused by rare morphs appearing more conspicuous against a background of common morphs (Allen, 1972; Greenwood, 1984), the simplest explanation is

12 412 A. SMITHSON AND M. R MACNAIR Tmm 5. Results of analysis of flight distances at each density (a) Distances between successively Visited flowers were calculated for the first and fifth bouts and the mean distance found for each bee. Results combined over frequencies for each density. Mean tlight distances (mm) and standard errom given for each bout. Flight distance, Flight distance, Experiment Sample size bout 1 bout f f f f f f Results of two-way analysis of variance on logarithmically-transformed flight distance data, to see the effects of experiment and bout on flight distances travelled. Source S.S. df F P Experiment <o.oo I Bout n.s. Experiment x n.s. Bout Error that the densities of flowers used in these experiments were insufficient to induce rare morph preference, because bumblebees always perceived all morphs against a green background. However, Horsley et al. (1979) found that birds foraging on pastry baits showed anti-apostatic selection at densities of 800 m-2, when only 4% of the background was covered by the baits. Cook & Miller (1977) found that quail forage apostatically at a bait density of 1.25 m-* (0.006% background covered), but showed no frequency-dependent selection at a higher bait density of 7.5 m-2 (0.037% background covered). Clearly, the ranges of flower densities and proportions of backgrounds covered in these experiments with bumblebees overlap with those used in experiments with birds where distinct differences in frequency-dependent behaviour were found. The ultimate cause of apostatic selection is believed to be that foragers maximize net energy consumption by concentrating on common morphs, because this allows increased rates of foraging (Allen, 1988). Preferences for commonest morphs can also be predicted from optimal foraging models if prey types differ in profitability (Hubbard et al., 1982), but since the flower morphs used in these experiments contained identical sucrose rewards, were identical in all ways other than colour and no differences in handling times between the morphs was observed, there is no difference in profitability in these experiments. It has been suggested that preference for rare morphs at high density might alternatively be because at high density predators are confused by large numbers of similar morphs (Greenwood, 1984). Preference for rare morphs have been demonstrated for predators chasing high densities of live and highly mobile prey (reviewed in Allen, 1988; Wilson, Allen & Anderson, 1990). Flowers, however, are not mobile or difficult to capture, indeed it is advantageous for a flower to be captured, increasing the potential for pollen export and import. Even at high density, it seems unlikely that bumblebees would be confused by large numbers of a stationary, conspicuous corolla colour morph, thus it may be unlikely for rare morph preferences to be displayed in this way. Rare morph preferences by bumblebees at high flower densities would increase net energy

13 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 413 consumption by reducing foraging rate through increasing travel times between flowers, and would probably be energetically disadvantageous. Another alternative suggestion for anti-apostatic selection at high density is that rare morph preference occurs due to initial sampling behaviour (Greenwood et al., 1984a,b), but such behaviour was not apparent in these experiments. A third alternative, due to Tinbergen (1960), is that a mixed diet is beneficial for individual predators, thus at high densities rare morphs are selected to increase the diversity of the diet (Greenwood, 1984; Church et az., 1994). Although bumblebee colonies as a whole are observed to receive food in the form of pollen and nectar from a wide variety of flower species (Heinrich, 1979), individually foraging worker bees are constant to a limited number of plant species ( majoring and minoring behaviour; Heinrich, 1976), thus colony dietary diversity is ensured through having a pool of worker bees with preferences for different flower species. Therefore it is possible that frequencydependence did not decrease with increasing density as predicted because the response of the foraging strategies of bumblebee workers to density are determined by their sociality and nectivorous habits, and may thus be totally different when compared to the response of vertebrate predators. Further experiments are required using different frequencies of floral morphs at very high densities to test this suggestion, although this might prove difficult in practical terms. In nature, however, it is likely that flower corollas would almost always be viewed against a background of sepals, leaves, etc., and it seems unlikely that the high corolla densities necessary to obscure background would be found except under artificial conditions. It has been demonstrated that as density increases, bumblebees showed a decreasing bias towards blue flowers, until at the highest density used there is no frequencyindependent component of preference. Two other studies testing for frequencydependent selection over different prey densities have also suggested changes in frequency-independent components of selection, one using chicks feeding on coloured crumbs (Willis et az.; 1980) and the other human predators selecting computergenerated prey images (Tucker & Allen, 199 l), but neither study found frequencydependent selection acting concomitantly. Tucker & Allen (1 99 1) suggested that, in their experiments, the frequency-independent component of selection also decreased as density increased. However, Sherratt & Harvey s (1989) work on predation of tadpoles by Odonata larvae found weak, non-significant, increases in the frequencyindependent component of predation as density increased. As flower density was increased in these experiments, distances flown by bumblebees from flower to flower decreased as was predicted due to preferences for nearestneighbour visitation. Flight distances did not change with increasing experience of the array. The results of these experiments are consistent with previous experiments using bumblebees (Smithson & Macnair, 1997), whereby increasing rewards in both corolla colour morphs also decreased both bias towards blue flowers and mean flight distances between flowers. In previous experiments, frequency-dependence also decreased slightly as frequency-independent preferences decreased, but in these experiments frequency-dependent preferences weakly increased, not indicating any interactions between frequency-independent and frequency-dependent preferences. The training protocol used before experiments ensured that each bee had received equal experience of both flower colours which always provided the same amount of sucrose solution, so these changes in frequency-independent preferences cannot be interpreted as novelty effects or learned biases to a flower colour before experiments commenced. Each colour morph offered the same reward and had similar handling

14 414 A. SMITHSON AND M. R. MACNAIR times, thus optimal foraging theory cannot explain changes in preference with changing total density. Because an extensive analysis of flower and background colours used in experiments (Smithson & Macnair, 1997) revealed that the blue flowers would look more conspicuous relative to the background compared to yellow flowers, it is suggested therefore that as the densities of flowers increased, the probability that a bumblebee would detect and visit less conspicuous yellow flowers increased on initial visits to the array, until at the highest density used there was equal preference for yellow and blue flowers: subsequent learned preference for the commonest morph allowed the observed hal frequency-dependent preferences to be displayed. The experimental work carried out by Mayberry (1987) on bumblebee flight showed that, as the distances travelled between successively-visited flowers increased, mean flight speeds increased, and the height of the flight path also increased, due to the mechanics of bumblebee flight. We suggest therefore that the cause of the observed changes in the probability of detection of the less conspicuous yellow flowers was the change in flight speed and height of flight path resulting from changes in inter-flower flight distances with density. There is evidence from experiments using bobwhite quail that there was a decrease in search speed as prey became more cryptic, and that the probability of prey detection decreases as search speed increases (Gendron, 1986). Trade-offs between the speed of predator search and the accuracy of prey detection have been widely discussed, often as an alternative mechanism to explain frequency-dependent preferences by foragers (Gendron & Staddon, 1983; Staddon & Gendron, 1983; Guilford & Dawkins, 1987). Similarly, Guilford (1 986) has proposed that more conspicuous prey may be more rapidly identified from a greater distance than more cryptic prey, and has presented evidence from experiments using chicks and great tits to support this hypothesis (Guilford, 1991). The hypothesis that there are trade-offs between flight speed, height above a flower and probability of recognizing a flower in pollinating insects is therefore not at variance with experimental data from birds. However, these experiments were not designed to distinguish the behavioural mechanisms hypothesized to cause colour preferences in bumblebees, in particular we cannot distinguish when flowers were perceived but rejected or when not perceived: further and more detailed experiments would be required to test these hypotheses. In terms of the impact of the observed bumblebee behaviour on the evolutionary dynamics of corolla colour variation in plant populations, a number of predictions may be made. At the range of densities of flowers investigated, bumblebees always showed a preference for commonest corolla colour morphs irrespective of density. Thus common corolla colours would be at a selective advantage in plant populations if there is a positive relationship between the number of pollinator visits received and reproductive fitness, thus causing positive FDS on corolla colour. It could be argued that, in plant populations where pollinators are abundant, pollinator preference would result in depleted nectar levels being present in the flowers of the most frequent morph, and thus pollinators would subsequently learn a preference for rare morphs with more abundant nectar. This might be the case particularly in higher density populations, if high density resulted in disproportionately higher numbers of pollinators being attracted to the population. However, data from other experiments similar to those presented here (Smithson, 1995) showed that the effects of large (up to 5 pl:l pl) differences in the quantities of reward present in the two corolla colour morphs resulted in small changes in bumblebee foraging preferences

15 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 415 when compared to their frequency-dependent foraging patterns-the commonest corolla colour was still preferred. Learned avoidance of common colour morphs only occurred when the common morph was emptied of nectar. Very high levels of pollinator competition for scarce rewards may also result in fecundity, through both male and female function, in both morphs being limited by the resources put into reproduction, rather than by pollinator visitation (Stanton & Preston, 1988), and thus in such cases corolla colour variation would be neutral. If fecundity through male or female function is pollinator-limited, the observed frequency-dependent pollinator preferences will cause positive FDS. As flower densities increase, however, there would be reduced directional selection on corolla colour. Thus the effects of positive FDS and directional selection on plant populations at low densities would be predicted to lead to a high probability of fixation in the population for the most preferred (conspicuous) corolla colour morph. With increasing plant density, directional selection decreases and the identity of the morph to go to fixation under FDS would be predicted to depend on the initial frequency of the colour morphs and the genetic control of these traits (Clarke & O'Donald, 1964). These effects will be in addition to the genetic effects of increased nearest-neighbour visitation by pollinators with increasing density, which will result in increased population subdivision, inbreeding and reduced gene flow (Bateman, 1947; Levin & Kerster, 1969a). Bumblebee visitation patterns would also result in assortative mating between corolla colour morphs, although this behaviour was not affected significantly by density. Overall, the resultant frequency- and density-dependent dynamics of corolla colour traits will be complex, but through the observed preference for common colours by pollinators positive FDS would be induced and would be predicted to result eventudy in monomorphism for these traits. There is no evidence that corolla colour variation could be maintained by negative FDS through the behaviour of pollinators foraging for nectar on plants at these densities. In these experiments, pollinator behaviour was modelled using a single species of bumblebee foraging on artificial flowers in the laboratory, and clear effects of both colour morph frequency and density on behaviour could be seen despite variability of behaviour between individual bees. To accurately test the effects of the observed behaviours on the evolutionary dynamics of corolla colour variation in plant populations, it would be necessary to repeat these experiments under more complex conditions in field populations of plants, and using a wider spectrum of flower visitors. ACKNOWLEDGEMENTS We are grateful to Prof. Wotjek Krzanowski for statistical advice, and to Dr Alison Cooper for drawing our attention to literature on bumblebee flight. We are grateful for the comments of Dr John Allen and two anonymous referees on an earlier version of the manuscript. AS. was in receipt of N.E.R.C. Studentship GT4/ 92/TLS/ 18 to J.E. Cresswell and M.R. Macnair. REFERENCES Allen JA Evidence for stabilizing and apostatic selection by wild blackbirds..n&n 237: Allen JA Further evidence for apostatic selection by wild passerine bwds-9:l experiments. Hen@ 36:

16 416 k SMITHSON AND M. R. MACNAIR Allen JA Frequency-dependent selection by predators. lmm$hicai fiansacrions ofthc Ryal.hie9 oflondon, Sniw B Allen JAY Anderson EEP Selection by passerine birds is anti-apostatic at high prey density. BW~gicalJoumal ofthc Lhwan So& 23: Bateman AJ Contamination in seed crops III. Relation with isolation distance. Hncdfy 1: Church SC, Allen JA, BradshowJWS Anti-apostatic food selection by the domestic cat. Animal &houiour 48: Clarke By O Donnld P Frequency-dependent selection. Hem Clarke BC Balanced polymorphism and the diversity of sympatric species. in: Nichols D, ed. T ~ mand y Gmgrafdy. London: Systematics Association, Cook LM, Miller P Density-dependent selection of polymorphic prey-mme data. Amnican Nduralist 111: Cresswell JE, Wen C Frequencydependent selection and adaptive surfaces for floral character combinations: The pollination of Polmonium viscosutn. Anmican Nialirt 138: Dawkins M Perceptual changes in chicks: Another look at the search image concept. Animal &haviour Ehn By T i W R Bootstrap methods for standard errors, confidence intervals, and other measures of statistical accuracy. shlicrical Sciancc 1: Elton RA, Greenwood JD Frequency-dependent selection by predators: comparison of parameter estimates. W s 48: Endler JA Frequencydependent predation, crypsis and aposematic coloration. Philacophicol Cransactionc of thc Ryal &ty of-, Sniw B Endler JA Interactions between predators and prey. In. KrebsJR, Davies NB, eds. Bchwioural Eology, An EvoluEimuuy +h, 3rd Edition. Oxford: Blackwell Scientific Publications, Eppason BK, Clegg MT Frequencydependent variation for outcrossing rate among flower-color morphs of&-- EuOluh41: Wen C, Stanton ML Bumblebee pollination and floral morphology: Factors inffuencing pollen dispersal in the alpine sky pilot, Pohonium uiscasuni (Polemoniaceae). AmnicrmJumal ofbatuny Gendron RP Searching for cryptic prey: Evidence for optimal search rates and the formation of search images in quail. Animal &haotour 34: Gendron RP Models and mechanisms of frequency-dependent predation. American Natudst Gendron RP, StaddonJER Searching for cryptic prey: The effect of search rate. Amnicrm Naturalirt 121: GreenwoodJD The functional basis of frequency-dependent food selection. BWlogicalJ~~mal ofhe h a n So&& 23: GreenwoodJD, Elton RA Analysmg experiments on frequencydependent selection by predators. Journal ofaw Ecologv 48: , GreenwoodJD, JohnrtonJp, Thomas GE. 1984% Mice prefer rare food. BW&alJmrmnl oftbe Linncrm socic3) Greenwood JD, Blow NC, Thomas GE. 1984b. More mice prefer rare food. Biolugual Jmal of k &man sotic3, Guilford T How do warning colours work? Conspicuousness may reduce recognition errors in experienced predators. Amhal Bdraviaa 34: Guilford T Studying warning signals in the laboratory. In: Blanchard &J, Brain PF, Blanchard DC and Parmigiani S, eds. Etk+hunfal Appmacllw to thc Study of Bthamk London: Kluwer Academic Publishers, Guilford T, Dawkins MS Search images not proven: A reappraisal of recent evidence. Animal Behibur 35: Heinrich BH The foraging specializations of individual bumblebees. Ecological Monogrqh 46: Heinrich BH Bumblch ccods. Cambridge: Harvard University Press. HeiPrich By Mudge PR, Deringis PG Laboratoxy analysis of flower constancy in foraging bumblebees: Banbus and B. &oh B h h d Ekologv rmd &bbloo Horsley D, Lynch BM, CnenwoodJD, HuQnrn By Mosely S Frequency-dependent selection by birds when the density of prey is high. 3mmal ofhimal Ecology 48: 48-90, Hubbard SF, Cook RM, Glover JG, GmenwoodJD Apostatic selection as an optimal foraging strategy. Jmmal ofanimal Ecology 51: Kay QON The role of preferential and assortative pollination in the maintenance of flower colour polymorphisms. In. Richards 41; ed. The pollination off&ws Imectr. London: Academic Press, Lawrence S, Allen JA On the term search image. 0h.s 40: Levin DA Low frequency disadvantage in the exploitation of pollinators by corolla variants in phlox. Amrican Nialut 106: Levin DA, Kcrater HW. 1969% Density-dependent gene dispersal in &. Anrnican Nafuralist Levin DA, Kcrater HW. 1969b. The dependence of bee-mediated pollen and gene dispersal upon plant density. Evolution 23: Levin DA, Kcrater HW Phenotypic dimorphism and populational fitness in phlox. Evolurion

17 DENSITY- AND FREQUENCY-DEPENDENT SELECTION 417 MayberqJH The energetics of foraging insects. Unpublished Ph.D. Thesis, University of Cambridge. Pyke CH Optimal foraging: patterns of bumblebee between inflorescences. 77ttmttical Populatton Biology Real LA Uncertainty and pollinator-plant interactions: The foraging behavior of bees and wasps on artificial flowers. Ecology 62: 2G26. Real LA Predator switching and the interpretation of animal choice behaviour: The case for constrained optimization. In: Hughes RN, ed. Behavioural Mechk of Fwd Schction. Heidelberg: Springer-Verlag, Real LA, On J, Silverfine E On the trade-off between the mean and the variance in foraging: Effect of spatial distribution and color preference. Ecology Schaal BA Density dependent foraging on &tni pynrostachya. Euolurion 32: Sherratt TN, Harvey IF Predation by larvae of Pantakaj7azwcens (Odonata) on tadpoles of Phyllmncdusa lriniheis and physalonus pustulok the influence of absolute and relative density of prey on predator choice. Oikos 56 17&176. Smithson A Frequency-dependent selection on floral traits through the foraging behaviour of bumblebees Bmnbuc hshr. Unpublished Ph.D. Thesis, University of Exeter. Smithson A, Macnair MR Frequency-dependent selection by pollinators: Mechanisms and consequences with regard to behaviour of bumblebees Bombus h s t n i (L.) (Hymenoptera, Apidae). Joml of Evoluw Biology: Sokal RR, Rohlfw Bimnctty, 2nd edition. New York W.H. Freeman. Staddon JER, Gendmn RP Optimal detection of cryptic prey may lead to predator switching. Amnican Naturalist 122: 84S-848. Stanton ML, Preston RE A qualitative model for evaluating the effects of flower attractiveness on male and female fitness in plants. AmniGan Journal ofbotany 75: Stanton ML, Snow AA, Handel SN, Bereczky J The impact of a flower-color polymorphism on mating patterns in experimental populations of wild radish (Raphanu raphanubum L.). Ewlulwn Thomson V Polymorphism under apostatic and aposematic selection. Here Tinbergen L The natural control of insects in pinewoods. I. Factors influencing the intensity of predation by songbirds. Adiues Neer~~cs dc ~oologic Tucker GM, Allen JA Selection by humans searching for computer-generated prey images: the effect of prey density. BwlogitalJoumal ofthe Linm.!hie9 44: Waddington KD Flight patterns of foraging bees relative to density of artificial flowers and distribution of nectar. Oecologia 44: Waser NM, Price MV Pollinator choice and stabilizing selection for flower colour in fibhinium nel~onu. Ewlurion 35: Willis AJ, McEwMJWT, GreenwoodgD, Elton RA Food selection by chicks: Effects of colour, density and frequency of food types. Anzinal &hauiour 28: Willson MF Sexual selection in plants: Perspective and overview. A&an Naturalist la S 1 SS39. Wilson SE, Allen JA, Anderson KP Fast movement of densely aggregated prey increases the strength of anti-apostatic selection by wild birds. BWlogicdJoumal ofthe Linnmn.!hie& 41: