Functional Ecology 2011, 25, 1–9 doi: 10.1111/j.1365-2435.2011.01885.x

1 PLANT-ANIMAL INTERACTIONS 2 3 ‘X’ marks the spot: The possible benefits of nectar 4 5 1 guides to bees and plants 6 7 Anne S. Leonard* and Daniel R. Papaj 8 9 Department of Ecology and Evolutionary Biology, Center for Science, University of Arizona, 1041 East Lowell 10 Street, Tucson, Arizona 85721, USA 11 12 13 14 Summary 15 1. Many floral displays are visually complex, transmitting multi-coloured patterns that are thought to 16 17 direct to nectar rewards. These ‘nectar guides’ may be mutually beneficial, if they reduce 18 pollinators’ handling time, leading to an increased visitation rate and promoting transfer. Yet, 19 many details regarding how floral patterns influence foraging efficiency are unknown, as is the poten- 20 tial for learning to alter this relationship. 21 2. We compared the responses of bumblebee (Bombus impatiens Cresson) foragers to artificial flowers 22 that either possessed or lacked star-like patterns. By presenting each bee with two different foraging 23 scenarios (patterned flowers rewarding ⁄ plain flowers unrewarding, plain flowers rewarding ⁄ patterned 24 flowers unrewarding) on different days, we were able to assess both short- and long-term effects of pat- 25 terns on bee foraging behaviour. 26 3. Bees discovered rewards more quickly on patterned flowers and were less likely to miss the reward, 27 regardless of whether corollas were circular or had . Nectar guides’ effect on nectar discovery 28 was immediate (innate) and persisted even after experience, although nectar discovery itself also had a 29 30 learned component. We also found that bees departed patterned flowers sooner after feeding. Finally, 31 when conditions changed such that flowers no longer provided a reward, bees visited the now-unre- 32 warding flowers more persistently when they were patterned. 33 4. On the time-scale of a single foraging bout, our results provide some of the first data on how pollina- 34 tors learn to forage efficiently using this common floral trait. Our bees’ persistent response to patterned 35 flowers even after rewards ceased suggests that, rather than being consistently mutually beneficial to 36 plant and pollinator, nectar guide patterns can at times promote pollen transfer for the plant at the 37 expense of a bee’s foraging success. 38 39 Key-words: Bombus, constancy, efficiency, foraging, handling time, learning, nectar discovery, 40 pattern 41 42 43 rotation, and configuration: Giurfa, Eichmann & Menzel 1996; Introduction 44 Horridge 2000; Plowright et al. 2001; Avargue`s-Weber et al. 45 display a remarkable variety of colourful patterns (Dafni, 2010), surprisingly few studies address whether and how the 46 Lehrer & Kevan 1997; Fig. 1a–d). Theories on the function of presence of a nectar guide benefits plants and ⁄ or pollinators. 47 this visual complexity date at least to the time of Sprengel Often, it is assumed that floral patterns are mutually benefi- 48 (1793), who proposed that Saftmale (a German word translating cial to both plant and pollinator. Optimal diet theory (Pyke, Pul- 49 as ‘juice marks’) guide pollinators towards the flower’s nectary. liam & Charnov 1977; Sih & Christensen 2001) predicts that 50 Despite the ubiquity of such floral patterns (Kugler 1966; Penny foragers should be sensitive to the costs of acquiring particular 51 1983; Chittka et al. 1994), a good understanding of what fea- food items (nectar sources), adjusting their choices based not 52 tures make them attractive (Knoll 1926; Daumer 1956, 1958; only upon energetic rewards, but also on the time or energy it 53 Manning 1956; Free 1970; Jones & Buchmann 1974; Lunau takes to access those rewards. If pollinator fitness depends upon 54 et al. 2006; Shang et al. 2010) and evidence that pollinators maximizing the rate of nectar collection (as in social bees: Pelle- 55 perceive quite subtle aspects of their form (such as symmetry, tier & McNeil 2003; Burns 2005), then all else being equal, they 56 should select flowers that have shorter handling times. Pollina- *Correspondence author. E-mail: [email protected] tors tend to spend less time using flowers that possess nectar

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society FEC 1885 Dispatch: 20.6.11 Journal: FEC CE: Vijay Journal Name Manuscript No. B Author Received: No. of pages: 9 PE: Ulagammal 2 A. S. Leonard & D. R. Papaj

1 (a) (b) (c) (d) 2 3

4 COLOR 5 6 7 8 9 (e) (f) (g) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Fig. 1. Animal-pollinated plants display a variety of floral patterns, which often function as nectar guides (a) Alstroemeria sp. with honey bee, Apis 25 mellifera (b) Ipomoea ternifolia, (c) Lamiaceae, (d) Viola sp., (e) Circular and (f) Petaloid artificial flowers and patterns used in experiment. (g) Reflec- tance spectra for components of artificial flowers used in experiment; our colours mimic a natural situation in which foliage provides a grey-green 26 background, flower petals are blue and nectar guide patterns are UV-blue. Photographs: A.S. Leonard. 27 28 29 guides, suggesting that these patterns are indeed associated with of a single foraging trip, where a pattern is always associated 30 foraging efficiency (Waser & Price 1983, 1985; Dinkel & Lunau with a reward. Because nectar availability of any one plant 31 2001; but see West & Laverty 1998). species can fluctuate (because of temporal changes in produc- 32 Yet, we lack many basic details regarding how floral patterns tion or depletion by floral visitors: Heinrich 1979a), our multi- 33 influence pollinator behaviour. For example, rather than explor- day experiment allowed us to examine how a floral pattern 34 ing how patterns affect the sequence of behaviours on the affected the capacity of bees to switch floral types when a pre- 35 flower, previous studies have focused on overall flower han- viously rewarding type becomes unrewarding. In this scenario, 36 dling time, or even travel time between flowers. Additionally, the interests of the bee are in potential conflict with those of 37 the long-term benefits of a guide remain unclear, because pollin- the plant. The bee stands to benefit if the nectar guide on the 38 ators may acquire handling skills (e.g. Laverty 1994a) that once-rewarding floral type facilitates switching to a novel type, 39 allow them to eventually forage equally efficiently without a whereas the plant might benefit if the nectar guide impedes 40 guide. Although a naı¨ve bee’s preference for landing on pat- switching. Does experience using a nectar guide to locate a 41 2 terned flowers is well-established (e.g. Lehrer et al. 1995; Dafni, reward thus facilitate – or hinder – foraging on flowers that 42 Lehrer & Kevan 1997; Simonds & Plowright 2004), the effects lack guides? 43 of patterns on foraging efficiency have not been assessed Our study explored two general aspects of the relationship 44 beyond the timeline of a single foraging bout. Perhaps not sur- between floral patterns and bumblebee (Bombus impatiens) nec- 45 prisingly, nothing has been reported about how experience tar foraging. First, we focused on possible handling time bene- 46 shapes the guiding function of floral patterns, if at all. fits of a pattern. We examined the effects of a floral pattern not 47 Understanding how the on-flower effects of nectar guides only on nectar discovery but also on a bee’s decision to leave 48 change with experience would provide a more precise descrip- the flower. Secondly, we also determined how pollinator experi- 49 tion of the function(s) of this common floral trait. It might also ence affected nectar discovery in the context of floral patterns. 50 yield a more informed perspective on the possible benefits of Whereas we hypothesized that a nectar guide would facilitate 51 guides to plant and pollinator. In general, nectar guides are naı¨ve bees’ nectar discovery, we developed two opposing pre- 52 presumed to benefit both plant and pollinator. A reduction in dictions for how the benefit of a guide might change with expe- 53 nectar discovery time, for instance, might increase the rate at rience. If nectar guides aid bees in learning how to locate nectar 54 which a bee acquires energy in the form of nectar (of benefit more effectively, then the relative time savings of a nectar guide 55 to the bee) and simultaneously improve the rate at which the should grow over the course of a foraging trip. Alternatively, if 56 bee transfers pollen (of benefit to the plant). Yet, most studies guides primarily benefit naı¨ve bees, the relative benefit of a examine pollinator responses to nectar guides on the time-scale guide should decrease.

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 Nectar guides and bumblebee foraging 3

1 Methods 15Æ75 cm2; perimeter of circle: 15Æ70 cm; petaloid: 23Æ80 cm; measured 2 with Adobe Photoshop CS3, Adobe Systems, San Jose CA, USA). 3 SUBJECTS AND PRE-TRAINING Shape differences may have altered the detectability of flower targets, as 4 honey bees detect circular targets at a longer distance than petaloid tar- 5 We used 30 B. impatiens workers as subjects, selected from a colony gets (Ne’eman & Kevan 2001). On any given foraging trip, six of these obtained from Koppert Biological Systems (Romulus, MI, USA). The 6 flowers were plain, and six had a light (human white; bee UV-blue), colony was provided with pollen ad libitum and housed in a plastic box 7 radially symmetrical, nectar guide pattern. The pattern was identical for (L · W · H: 22Æ0 · 24Æ0 · 12Æ0 cm). Experiments occurred in a room- both circular and petaloid flowers. Although our artificial flowers were 8 sized experimental chamber (L · W · H: 3Æ05 · 1Æ92 · 1Æ55 m), fitted not modelled on a specific species, many bee-pollinated flowers present 9 with a screen door to permit observation. The experimental chamber was a blue corolla with light, UV-reflective radiating lines (e.g. Iris, Salvia, 10 connected to the colony via a gated buffer box (L · W · H: Ipomoea). Generally, chromatic contrast between pattern and corolla is 11 35Æ0 · 22Æ0 · 15Æ0 cm) that allowed us to release individual foragers, fit- important in guiding bumblebees’ orientation towards flowers (e.g. Lunau, 12 ted on the thorax with numbered tags (E.H. Thorne Ltd., Wragby, Wacht & Chittka 1996; Lunau et al. 2006). Likewise, both circular- and 13 Lincolnshire, UK) for identification. The chamber was illuminated by star-shaped flowers commonly display star-shaped guides (Dafni & 14 fluorescent lighting (see Fig. S1a, Supporting Information; Sylvania Cool Kevan 1996). 15 3 White 34 Watt bulbs, # F40CW1SS, 560 lux measured at centre of array). Each bee was videotaped (30 frames ⁄ s; Sony DVM-60PR Mini DV 16 In order to train bees to visit the experimental chamber and forage at cassettes) during two foraging trips, occurring 2 days apart. During each the floral array, we allowed the colony free overnight access to a pre- 17 trip, the bee encountered both plain and patterned flowers; one type was training array. The pre-training array offered nine feeders, each provid- 18 rewarding and one type was unrewarding, but this relationship was ing 10 mL of 30% w ⁄ w sucrose through a cotton wick. The pre-training switched for each individual bee across foraging trips. Repeatedly 19 array was similar to the experimental array, and it consisted of a green assaying the same forager allowed us to determine whether handling 20 horizontal board (60 · 45 cm, DecoArt acrylic paint, ‘Avocado’ times were shorter when rewarding flowers had patterns, even if individ- 21 #DA052) with feeders spaced 10 cm apart. Each white cylindrical fee- ual bees varied in their foraging speed. On Day 1, 14 bees had plain 22 der base (height: 5Æ5 cm; diameter: 1Æ7 cm) was topped with a light grey flowers rewarding ⁄ patterned flowers unrewarding, followed by pat- 23 artificial flower, the same size (5Æ0 cm diameter) and shape (circle, terned flowers rewarding ⁄ plain flowers unrewarding on Day 3. For 24 N = 4 or petaloid, N = 5) as the flowers used in foraging experiments. seven of these bees, all flowers were circular, and for seven of these 25 No patterns were present on pre-training flowers. We varied the wick’s bees, all flowers were petaloid. Conversely, 16 bees on Day 1 had pat- 26 position relative to the centre or edges on each of the nine flower sur- terned flowers rewarding ⁄ plain flowers unrewarding, followed by plain 27 faces to prevent bees from learning to feed in a particular flower region. flowers rewarding ⁄ patterned flowers unrewarding on Day 3. For nine of The floral arrays used in pre-training and experiments were positioned 28 these bees, all flowers were circular, and for seven of these bees, all on a stool in the centre of the experimental chamber, at a height of 29 flowers were petaloid. Between foraging trips (Day 2), bees were pro- 50 cm above the ground. vided access to the pre-training array described above. During a forag- 30 ing trip, bees were allowed to visit flowers until they had drained all six 31 of the rewarding flowers, or until they did not visit the array for 3 min, FLORAL ARRAY 32 at which point they were collected and returned to the colony. After each 33 The floral array held 12 artificial flowers (Fig. 1e,f), arranged at 10cm foraging trip, flowers were cleaned with 30% ethanol to remove any 34 intervals in a 3 · 4 grid. Flowers consisted of a white cylindrical base scent marks deposited by foragers. 35 (height: 5Æ5 cm; diameter: 1Æ7 cm) connected to a flower top (5Æ0cm We used iMovie 8Æ0Æ6 (Apple Computer Inc., California, USA) to 36 diameter). tops were light blue, printed on water-resistant paper record the sequence of landings and measure the frame-by-frame details 37 (Adventure Paper, National Geographic, Margate, FL, USA), using a of initial landings on up to 12 flowers (six rewarding, six unrewarding) 38 Canon Pixma MX860 inkjet printer, and laminated (Xyron matte lami- within a foraging trip. If a bee departed its first visit to a rewarding 39 nate, Scottsdale, AZ, USA). Figure 1g shows the reflectance spectra for flower without having located the reward, we scored the visit as a ‘miss’ 40 colours present on the floral array. Because humans and bees are sensi- and used data from its first successful revisit to that flower. During a typ- ical visit to a rewarding flower, a bee landed, searched on the surface of 41 tive to different wavelengths of light, these colours can be represented in bee colour space (Chittka 1992) using the reflectance data, informa- the flower, located the reward, consumed all the reward and then 42 tion about the lighting conditions, as well as the sensitivity of B. impa- searched again on the surface of the flower before leaving. We measured 43 tiens’ three photoreceptors (Skorupski & Chittka 2010) (Fig. S1, the time spent searching for the reward after landing, as well as the time 44 Supporting Information). Using this model of bee colour space, we spent searching on the flower after feeding. Our sample sizes for certain 45 determined that our flower colours offered substantial green contrast comparisons were <30 because some bees did not feed on their Day 3 46 against the background, as well as green contrast between corolla and rewarding flower type (N = 2). Sample sizes for comparisons of post- 47 guide (Fig. S1c, Supporting Information). Green contrast is critical for feeding search time on the final flower visited were similarly reduced 48 bees’ long-range detection of flowers (Giurfa et al. 1996; Kevan & because a few bees (N = 3) remained motionless for longer than 30 s 49 Backhaus 1998; Spaethe, Tautz & Chittka 2001). The flower top had a and were therefore excluded from this analysis. 50 small central hole (d: 1Æ5 mm) through which bees accessed a well hid- 51 den in the white plastic base. Depending on treatment, this well con- ⁄ Results 52 tained either 10 lL of 50% w w sucrose (rewarding flowers) or 10 lL of deionized water (unrewarding flowers). Rewarding and unrewarding 53 flowers were distributed haphazardly across the grid, and their position PATTERNS REDUCED SEVERAL COMPONENTS OF 54 was changed between foraging trips. HANDLING TIME 55 Flowers were either circular (N = 16 bees) or petaloid (N = 14 bees). Comparing the mean time bees took to locate the reward on the 56 The petaloid flower had 19Æ8% less surface area, but 51Æ6% more perim- eter than the circle (surface area of circle: 19Æ63 cm2; petaloid: six rewarding flowers available in a given trip (Fig. 2a), reward

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 4 A. S. Leonard & D. R. Papaj

1 (a) (a) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 (b) 19 (b) 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Fig. 3. Effect of foraging experience on flower handling. (a) On their 34 first landing, bees located nectar more quickly when flowers were pat- 35 Fig. 2. Mean time spent by bees on flowers. Regardless of whether terned; yet, over the course of the trial, bees showed similar decreases in 36 flowers were circular or petaloid, when flowers were patterned, bees (a) time to locate nectar when flowers were plain and patterned. Floral 37 located the reward more quickly and (b) spent less time on the flower shape did not affect either of these two measures. (b) Bees spent similar 38 after feeding. amounts of time on their first flower after feeding regardless of pattern; this post-feeding search time declined over the course of a trial, but did 39 so similarly when flowers were plain and patterned. Floral shape had no 40 discovery was faster when flowers were patterned effect on post-search time.

41 (F1, 26 =19Æ62, P <0Æ001); bees also located the reward more 42 quickly on petaloid flowers than on circular flowers P =0Æ104; interaction: F1, 26 =0Æ128, P =0Æ723). Bees were

43 (F1, 26 =6Æ65, P =0Æ016). There was no interaction between more likely to miss the reward on plain flowers (F1, 28 =7Æ639,

44 flower shape and pattern presence (F1, 26 =0Æ208, P =0Æ652), P =0Æ010); unexpectedly, misses were also more frequent on 45 suggesting that patterns enhanced foraging to a similar extent petaloid flowers (F1, 28 =5Æ907, P =0Æ022). In short, we con- 46 on circular and petaloid flowers. On circular flowers, bees clude from these results that a pattern of radiating lines func- 47 located the reward an average of 41Æ1% faster in the presence of tions as a nectar guide and does so regardless of the shape of the 48 a pattern; on petaloid flowers, bees located the reward an aver- flower itself. 49 age of 44Æ5% faster with a pattern present. Similarly, a pattern 50 reduced the time bees spent searching on the flower after feed- NECTAR GUIDES ARE INITIALLY HELPFUL, AND THEIR 51 ing (Fig. 2b: F1, 26 =4Æ26, P =0Æ049). However, flower shape BENEFIT PERSISTS EVEN AFTER EXPERIENCE 52 did not influence this post-feeding search time (F1, 26 =0Æ625, 53 P =0Æ436), nor was there a significant interaction between pat- If patterns save bees the cost of learning to handle the flower,

54 tern and shape (F1, 26 =1Æ592, P =0Æ218). It is worth noting then we expected that the benefits of a guide should be evident 55 that we found no significant differences between the time bees even on the bees’ first landing. This expectation was met for 56 spent feeding from flowers of different types (Pattern: reward discovery time (Table S1, Supporting Information;

F1, 26 =2Æ744, P =0Æ108; Flower shape: F1, 26 =2Æ834, Fig. 3a): on both their first and last landing, bees located the

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 Nectar guides and bumblebee foraging 5

1 reward more quickly on patterned flowers, regardless of floral (a) 2 shape (P =0Æ005). If nectar guide patterns primarily benefit 3 naı¨ve bees, we expected that the relative benefit of a guide 4 would be greatest at the start of a foraging trip. Alternately, if 5 patterns facilitate bees’ learning to locate nectar, then the rela- 6 tive benefit of a guide should be greater at the end of a trip. 7 Although bees located sucrose progressively faster over the 8 course of a trip (P =0Æ001), this decrease was independent of 9 flower pattern or shape: patterns sped reward discovery simi- 10 larly for naı¨ve and experienced bees (Table S1, Supporting 11 Information). The order in which bees experienced the two 12 kinds of foraging trips (plain flowers rewarding on first trip vs. 13 patterned flowers rewarding on first trip) did not have a signifi- 14 cant effect or interaction with these factors, although we did (b) 15 note an interaction between pattern, flower position within a trip 16 (first vs. last), flower shape and order of rewarded floral type 17 across trips that bordered on statistical significance (P =0Æ061; 18 Table S1, Supporting Information). 19 In contrast, although post-feeding search time declined 20 between the first and last flower visited (P =0Æ014, Table S1, 21 Supporting Information; Fig. 3b), initial post-feeding search 22 time was not lower on flowers with nectar guides (P =0Æ631), 23 nor was there a significant interaction between pattern pres- 24 ence and flower order (first vs. last). Likewise, floral shape 25 had no significant effect on post-feeding search time 26 (P =0Æ398). We did note a non-significant interaction between 27 pattern and shape (P =0Æ061), suggesting that differences in 28 post-feeding search time (on both first and last flowers) 29 Fig. 4. Whether bees fed from plain or patterned flowers on Day 1 between plain vs. patterned flowers may be smaller on petaloid affected foraging behaviour on Day 3. (a) Bees that collected sucrose 30 flowers. As above, the order in which bees experienced the from the first plain flower on Day 1 located the reward on the first pat- 31 different flower patterns as rewarding did not have a signifi- terned flower at a similar speed on Day 3. However, other bees that col- 32 cant effect on post-feeding search (Table S1, Supporting Infor- lected sucrose from the first patterned flower on Day 1 took longer to 33 mation). However, we did find a significant interaction locate the reward on the first plain flower on Day 3. Flower shape was 34 not a significant factor in this analysis. (b) While bees that collected between the order of rewarded floral types across trips and sucrose from plain flowers on Day 1 made a similar proportion of land- 35 flower position within a trip (first vs. last; P =0Æ036). Regard- ings on rewarding patterned flowers on Day 3, bees that collected 36 less of whether flowers were patterned or plain, bees that had sucrose from patterned flowers on Day 1 persisted in landing on (now 37 plain flowers rewarding on Day 1 ⁄ patterned flowers rewarding unrewarding) patterned flowers on Day 3. Dotted line at 0Æ50 indicates 38 on Day 3 tended to spend less time searching on their last random choice. 39 flower after feeding than on their first flower. Bees who experi- 40 enced the two trips in the opposite order (patterned flowers NECTAR GUIDES AFFECT BOTH INITIAL AND 41 rewarding on Day 1 ⁄ plain flowers rewarding on Day 3) tended LONG-TERM LANDING PREFERENCES 42 to spend a relatively similar amount of time on their first and 43 last flower after feeding. Bees showed an initial bias towards landing on flowers with pat- 44 Our analysis also shows that using a guide to locate rewards terns. On Day 1, regardless of whether patterned or plain flow- 45 had a long-term negative effect on handling of plain flowers. ers were rewarding, bees were more likely to make their first 46 Figure 4a shows the mean time bees took to locate the sucrose landing on a patterned flower (no significant effect of shape 47 reward on their first flower, for both foraging trips. Bees that on z = )0Æ276, P =0Æ784; 2-tailed binomial test on combined data: 48 Day 1 collected sucrose from plain flowers took a similar P =0Æ0014). 49 amount of time to locate their first reward on Day 3, when Interestingly, experience feeding on patterned and plain flow- 50 rewarding flowers had patterns (P =0Æ269, Table S2, Support- ers had different long-term effects on bees’ landing preferences 51 ing Information). However, bees that on Day 1 collected sucrose (Fig. 4b). We compared the relative proportion of landings on 52 from patterned flowers were slower to locate their first reward rewarding flowers, asking whether the order in which bees 53 on Day 3, when rewarding flowers were plain (P =0Æ015, experienced the two foraging trip types (Trip order as between- 54 Table S2, Supporting Information). Bees’ nectar discovery was subjects factor with 2 levels) interacted with the effect of day 55 thus less disrupted by the appearance of a novel pattern on a (Day as within-subjects measure with 2 levels: 1; 3). We found 56 rewarding flower than it was by the disappearance of a pattern a significant effect of Day (Table S3, Supporting Information; previously useful in finding nectar. P =0Æ007) but not of Trip order (P =0Æ294); the interaction of

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 6 A. S. Leonard & D. R. Papaj

1 interest between these two factors was, however, significant on-flower behaviour (e.g. nectar concentration and volume: 2 (P <0Æ001). Note this analysis did not find a significant effect Thomson 1986; corolla depth: Harder 1983; structural complex- 3 of shape (Table S3, Supporting Information). We did note a ity: Laverty 1980), nectar accessibility may also allow the plant 4 trend towards statistical significance for an interaction between to optimize the rate of pollen transfer. Our discovery that bees 5 Shape, Day and Trip order (P =0Æ062): bees that fed from plain spend less time on flowers with nectar guides after feeding sug- 6 circular flowers on Day 1 tended to make a higher proportion of gests that floral patterns might help to regulate on-flower time, 7 landings on patterned circular flowers on Day 3, whereas bees thus minimizing pollen spillage, or simply controlling the 8 whose flowers were petaloid showed a more similar proportion amount of pollen distributed to individual visitors (e.g. Harder 9 of landings on rewarding types across both days. In general, & Thomson 1989). If time spent by the bee searching on the 10 these results show that although bees that on Day 1 fed from flower increases the probability of self-fertilization, then the 11 plain flowers readily switched to feeding from patterned flowers reduction in post-feeding time could be particularly beneficial to 12 on Day 3, bees that on Day 1 fed from patterned flowers per- self-compatible species. 13 sisted in landing on these flowers on Day 3, when they were Why bees should spend less time after feeding on patterned 14 unrewarding. flowers is an open question. Post-feeding behaviour generally 15 consisted of searching on the flower surface, presumably for 16 other nectaries or pollen sources, a strategy that may reflect the Discussion 17 variety of floral architectures bees are likely to encounter (e.g. 18 Floral patterns are often assumed to benefit both plant and polli- isolated flowers, inflorescences, composite flowers). Our results 19 nator by efficiently guiding foragers to nectar. On the time-scale raise the possibility that a pattern facilitates the bee’s learning 20 of a single foraging trip, our experiment supports this view: about the number of rewards available per landing more effec- 21 when flowers had star-shaped patterns, bumblebees not only tively than a plain flower surface. 22 located rewards more quickly but also were less likely to miss We also noted that bees showed a lower frequency of 23 the reward and to linger on the flower after feeding. Interest- ‘misses’ when flowers had guides. For the plant, such misses 24 ingly, although bees generally foraged more quickly on petaloid are at best neutral. However, they may be costly if bees subse- 25 flowers than on circular flowers (perhaps because petals them- quently avoid visiting floral types that have been unrewarding. 26 selves provide spatial information about the position of the nectar Although visits where bees failed to locate sucrose were gener- 27 well), patterns were similarly beneficial on both corolla shapes. ally brief in our experiment (mean duration: 2Æ12 ± 0Æ50 s), 28 We established that these benefits are evident on the first flower Laverty’s (1980) study suggests that under natural conditions, 29 visited; nevertheless, speed in using a pattern to locate rewards misses may have a larger impact on foraging efficiency, as bum- 30 improved across a foraging trip. Our experiment is the first, to blebees were observed to spend several minutes unsuccessfully 31 our knowledge, to show that the nectar discovery benefit of a attempting to access nectar from various plant species. 32 nectar guide persists even after bees gain experience in handling Flower handling will likely be influenced by multiple plant 33 a flower. traits, yet how they interact to influence pollinator behaviour is 34 We also found that experience using nectar guides to locate largely unexplored (Fenster et al. 2004). To date, synergistic 35 sucrose had long-term effects on foraging behaviour. For exam- effects of patterns have been largely considered from the per- 36 ple, when we compared how long it took bees to locate the spective of colour values and symmetry: perhaps not surpris- 37 reward on the first flower of each trip, we found that, 2 days ingly, the effectiveness of a guide depends not only on pattern, 38 after collecting sucrose from patterned flowers, bees took much but also on the degree of chromatic contrast between guide and 39 longer to locate a reward on a plain flower. In contrast, bees that petals (e.g. Lunau, Wacht & Chittka 1996; Dinkel & Lunau 40 initially found rewards in plain flowers located the reward on 2001). Additionally, noting a widespread correspondence 41 their first patterned flower after a similar amount of time. Addi- between both internal (nectar guide) and external (corolla) floral 42 tionally, bees that collected sucrose from patterned flowers on contours, Dafni and collaborators (Dafni & Kevan 1996; Dafni, 43 Day 1 continued to visit these flowers on Day 3 even when they Lehrer & Kevan 1997) suggested that symmetry complementar- 44 no longer offered a reward. As a whole, these results raise the ity may itself be an important determinant of handling effective- 45 possibility that the long-term consequences of a nectar guide ness. Because bees track the contours of flowers as they fly 46 may be tilted in favour of the plant, which merely by producing towards them (Lehrer, Wehner & Srinivasan 1985), any shape 47 a pattern can gain pollinator visits, regardless of reward status. mismatch between corolla and guide might slow nectar discovery. 48 Having matched radially symmetrical guides with radially 49 symmetrical corollas, our experiment allowed us to determine HANDLING TIME BENEFITS OF NECTAR GUIDE 50 whether guide and corolla shapes interact to influence handling PATTERNS TO PLANT AND POLLINATOR 51 time. We anticipated that the guide might be either more effec- 52 Making a flower easy to handle may promote a plant’s repro- tive on petaloid flowers (drawing the bee away from 53 ductive success in several ways. Apart from promoting visita- edges, which bees tend to follow and probe: Laverty 1980; 54 tion of a particular pollinator species via reduced handling time Lehrer & Srinivasan 1993; Lehrer, Wehner & Srinivasan 1985) 55 (e.g. bees, as documented in this experiment), easily accessed or less effective on petaloid flowers (because visual or tactile 56 nectar might also broaden the range of potential pollinators (Ol- information provided by petals might direct pollinators towards lerton et al. 2007). Further, like other floral traits that influence the nectary: Goyret & Raguso 2006). In fact, we found that the

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 Nectar guides and bumblebee foraging 7

1 pattern was equally effective on both flower types. The ability exploit a pre-existing sensory bias, such as a preference for radi- 2 of a nectar guide to reduce handling time thus appears to be ating lines that resemble a nest entrance (Biesmeijer et al. 2005; 3 robust across corolla shapes (and ⁄ or sizes). Even if a flower’s see also Naug & Arathi 2007). If pollinators’ attraction to a pat- 4 petal shape and orientation provide significant information tern is persistent enough, it may allow the plant to limit rewards. 5 about the location of the nectary, a pattern still facilitates bee For example, the presence of anther-mimicking radial lines or 6 foraging. pollen-mimicking blotches increases the attractiveness of flow- 7 ers to bees (Lunau 2000); this attraction is so strong that the 8 mere presence of a (presumably pollen-mimicking) yellow dot NECTAR GUIDES AND LEARNING 9 interferes with bumblebees’ (B. terrestris) ability to discriminate 10 Although a large body of literature has established that bees between unrewarding and rewarding artificial flower types 11 both have naı¨ve pattern preferences and can learn to discrimi- (Pohl, Watolla & Lunau 2008). This work also shows that the 12 nate between patterns that differ in particular characteristics degree to which patterns inhibit a shift to the rewarding flower 13 (e.g. Kugler 1936; Barth 1985), very little is known about the type depends upon colour and placement; because we only 14 related question of how experience with patterns affects nectar observed bees on two foraging trips, we cannot comment upon 15 discovery time. This question has remained open, despite the whether or not our bees would have eventually learned to com- 16 fact that nectar-foraging bees are known to show experience- pletely avoid unrewarding patterned flowers. Although we focus 17 based changes in several aspects of foraging (e.g. choosing on bees, it is worth mentioning that other species respond to 18 flowers on the basis of reward rather than display size: Makino nectar guides (e.g. hawkmoths: Knoll 1926; hummingbirds: 19 & Sakai 2007; increases in floral constancy: Heinrich 1979b; Waser & Price 1983; syrphid flies: Dinkel & Lunau 2001). 20 Laverty 1980; establishment of traplines: Ohashi, Thomson & Whether these distantly related species might show similar 21 D’Souza 2007). We showed that although a pattern reduces nec- experience-based effects in use of and response to nectar guides 22 tar discovery time from the very start of a foraging trip, perfor- remains an intriguing question for future research. 23 mance on flowers with guides improves with experience. Both Previously, the connection between handling time, pollinator 24 relatively naı¨ve and experienced foragers, then, benefit similarly learning and plant vs. pollinator fitness has been explored in the 25 from the presence of a floral pattern. context of Darwin’s ‘Interference Hypothesis’ regarding the 26 Interestingly, the asymmetry (Fig. 4a) in the degree to which phenomenon of floral constancy (Laverty 1994b; Gegear & 27 Day 1 experience affected bees’ initial location of nectar on Laverty 1998; Chittka, Thomson & Waser 1999). According to 28 Day 3 (when rewarding flower types were switched from plain this hypothesis, bees’ tendency to selectively visit one or two 29 to patterned or vice versa) suggests that learning to use a pattern flower species, even when equally rewarding other species are 30 to locate a reward may impair bees’ performance on other floral available, results from a limitation on their ability to learn how 31 types. Future studies could investigate whether this effect to efficiently handle multiple flower types simultaneously 32 applies similarly to bees foraging on flowers with two different (Chittka & Thomson 1997). In this scenario, a plant may actu- 33 nectar guides or is specific to a pattern’s presence vs. absence. ally transport more pollen to conspecifics by producing a diffi- 34 Previously, such ‘switching costs’ have largely been considered cult-to-handle flower, because once a bee has invested in 35 in the context of floral morphology, rather than visual signals learning how to handle it, it may be more likely to restrict its 36 (e.g. Laverty 1994b); they have also been generally documented visits to this type. With relevance to the current experiment, 37 over relatively short time-scales. Our study indicates that such one implication of our findings is that a trait that makes a flower 38 effects may be present even 48 h after a single foraging bout. easier to handle (a nectar guide), rather than more difficult, may 39 also manipulate pollinators’ propensity to visit a particular 40 flower type. 41 FORAGING EFFICIENCY VS. POLLEN TRANSFER 42 Given that the reproductive interests of partners in a mutualism Acknowledgements 43 are not always perfectly aligned (Bronstein 1994), it is possible 44 that like other floral traits (e.g. signal complexity in general: We thank R. Kaczorowski, P. Marek and J. Jandt for comments on the manuscript 45 Gegear 2005; Leonard, Dornhaus & Papaj 2011; repellants in and the Papaj and Dornhaus laboratories for discussions. This work was supported by the University of Arizona Center for Insect Science through NIH Training 46 nectar: Kessler, Gase & Baldwin 2008; pollinia that slow bee Grant # 1K12 GM000708 and by NSF Grant #IOS-0921280. 47 foraging: Morse 1981), patterns may sometimes increase the 48 plant’s reproductive success at the expense of the pollinator’s 49 foraging efficiency. Indeed, because nectar guides are signals, References 50 we expect that their effect on foraging efficiency may be more Avargue`s-Weber, A., Portelli, G., Bernard, J., Dyer, A.G. & Giurfa, M. (2010) 51 complex than morphological traits that also affect handling Configural processing enables discrimination and categorization of face-like 52 stimuli in honeybees. Journal of Experimental Biology, 213, 593–601. time, such as corolla depth (Inouye 1980) or petal microtexture Barth, F.G. (1985) and Flowers: The Biology of a Partnership. Princeton 53 (Whitney et al. 2009). This is because floral signals have the University Press, Princeton. 54 potential to deceive or exploit reward-seeking pollinators. For Biesmeijer, J.C., Giurfa, M., Koedam, D., Potts, S.G., Joel, D.M. & Dafni, A. 55 (2005) Convergent evolution: floral guides, stingless bee nest entrances, and example, rewardless flowers may produce nectar guides but not insectivorous pitchers. Naturwissenschaften, 92, 444–450. 56 nectar (Jersa´kova´, Johnson & Kindlmann 2006; Schaefer & Bronstein, J.L. (1994) Conditional outcomes in mutualistic interactions. Trends in Ruxton 2010). Even in rewarding species, floral patterns may Ecology & Evolution, 9, 214–217.

Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 8 A. S. Leonard & D. R. Papaj

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Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society, Functional Ecology, 25, 1–9 Nectar guides and bumblebee foraging 9

1 Thomson, J.D. (1986) Pollen transport and deposition by bumble bees in Erythro- Table S1. General linear models used to assess the effects of 2 nium: influences of floral nectar and bee grooming. Ecology, 74, 329–341. flower pattern, flower shape, flower order within a trip, and trip Waser, N.M. & Price, M.V. (1983) Pollinator behaviour and natural selection for 3 flower colour in Delphinium nelsonii. Nature, 302, 422–424. order on nectar discovery and post-feeding search time. 4 Waser, N.M. & Price, M.V. (1985) The effect of nectar guides on pollinator prefer- 5 ence: experimental studies with a montane herb. Oecologia, 67, 121–126. Table S2. General linear models exploring how the order in West, E.L. & Laverty, T.M. (1998) Effect of floral symmetry on flower choice and 6 foraging behaviour of bumble bees. Canadian Journal of Zoology ⁄ Revue which bees fed from plain vs. patterned flowers affected nectar 7 Canadienne de Zoologie, 76, 730–739. discovery on the first flower of a given foraging trip. 8 Whitney, H.M., Chittka, L., Bruce, T.J.A. & Glover, B.J. (2009) Conical epider- mal cells allow bees to grip flowers and increase foraging efficiency. Current 9 Biology, 19, 948–953. Table S3. General linear model used to assess how proportion 10 of landings on rewarding flower type was affected by day, 11 Received 6 March 2011; accepted 6 June 2011 flower shape, and the order in which bees fed from plain vs. pat- Handling Editor: Marc Johnson 12 terned flowers. 13 14 As a service to our authors and readers, this journal provides 15 Supporting Information supporting information supplied by the authors. Such materials 16 may be re-organized for online delivery, but are not copy-edited Additional supporting information may be found in the online 17 or typeset. Technical support issues arising from supporting version of this article. 18 information (other than missing files) should be addressed to the 19 authors. Figure S1. Colors of our flower tops and nectar guides repre- 20 sented in bee color space. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

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