Animal Behaviour 83 (2012) 905e913. Contents lists available at SciVerse ScienceDirect. Animal Behaviour

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1 Animl Behviour 83 (212) 95e913 Contents lists ville t SciVerse ScienceDirect Animl Behviour journl homepge: Florl signl complexity s possile dpttion to environmentl vriility: test using nectr-forging umleees, Bomus imptiens Rinee L. Kczorowski *, Anne S. Leonrd, Ann Dornhus, Dniel R. Ppj Deprtment of Ecology nd Evolutionry Biology, University of Arizon, Tucson, U.S.A. rticle info Article history: Received 12 Septemer 211 Initil cceptnce 24 Octoer 211 Finl cceptnce 7 Decemer 211 Aville online 2 Ferury 212 MS. numer: A Keywords: ccurcy Bomus imptiens colour efficcy ckup hypothesis lerning light intensity multimodl signl nectr forging scent Florl signls re typiclly emitted cross multiple sensory modlities, lthough why they re multimodl is uncler. One possile explntion is tht multimodl signlling ensures tht t lest one signl component will e trnsmitted effectively under vrying environmentl conditions (the efficcy ckup hypothesis). For exmple, y trnsmitting oth component A nd B, signller cn communicte under environmentl conditions where trnsmission of component A is reduced; component B cks up A. To test this hypothesis, we determined whether florl scent could ck up florl colour signl when light levels were low. We trined nectr-forging umleees to discriminte rewrding nd unrewrding trgets tht differed in colour, scent, or oth colour nd scent, nd then presented the trgets t different levels of illumintion. We mesured ees ccurcy t distinguishing the two trgets nd their rte of visits to the trined trget. Performnce on oth mesures declined under low light when trgets were unscented. The presence of scent reduced the loss of ccurcy under low light, supporting the efficcy ckup hypothesis, ut this effect depended upon the colour of the previously rewrded trget. In contrst, the presence of scent did not ffect the overll rte of correct visits under low light (correct visits/forging time). A ckup mechnism tht mintins ccurcy, ut not rte of nectr collection, does not necessrily enefit the pollintor. However, it most likely enefits the plnt through reduced pollen wstge. In short, multimodl florl signls my enefit the plnt y improving pollen trnsfer, while not enefiting the pollintor. Ó 212 The Assocition for the Study of Animl Behviour. Pulished y Elsevier Ltd. All rights reserved. Flowers use vriety of signls to dvertise rewrds to their pollintors. These signls re often trnsmitted simultneously cross multiple sensory modlities, including visul, olfctory, gusttory, tctile, nd even coustic modlities (reviewed in Rguso 24). While much is known out signl function within prticulr modlities, such s vision nd olfction, reltively little is known out why florl displys simultneously emit signls in multiple modlities, despite potentil production costs (e.g. metolic costs of florl disply components: Helsper et l. 1998; Glen 1999) nd ecologicl costs (e.g. risk of ttrcting ntgonists: Theis 26). Multimodl signlling in flowers hs een shown to enhnce umleee nectr forging (Kulhci et l. 28; Leonrd et l. 211), yet why it does so remins n open question (reviewed in Leonrd et l. 211, c). A numer of hypotheses exist for why signls in nture re generlly multimodl (Guilford & Dwkins 1991; Rowe 1999; * Correspondence: R. Kczorowski, Deprtment of Ecology nd Evolutionry Biology, BSW 31, 141 E. Lowell Street, University of Arizon, Tucson, AZ 85721, U.S.A. E-mil ddress: rineek@gmil.com (R. L. Kczorowski). Cndolin 23; Heets & Ppj 25). One set of hypotheses is efficcy sed, pertining to how well signl is trnsmitted or detected nd processed y the receiver (Guilford & Dwkins 1991; Heets & Ppj 25). For instnce, the efficcy ckup hypothesis (Heets & Ppj 25) focuses on the potentil for environmentl conditions to oscure signl trnsmission (Brdury & Vehrencmp 1998). Overcst skies might reduce the efficcy of visul signls; similrly, windy conditions might reduce the efficcy of olfctory signls. The efficcy ckup hypothesis sttes tht components of multimodl signl provide functionl redundncy in the fce of unpredictle environmentl chnge. On windy dys, when scents re less loclizle, visul stimuli my llow ees to locte nd identify flowers; on overcst dys or in deep shde, when visul stimuli re difficult to discern, scent my e more useful. The ckup hypothesis is plusile explntion for multimodl signlling for severl resons. Environmentl conditions re known to oscure signl trnsmission nd detection (Brdury & Vehrencmp 1998; Cndolin 23). Additionlly, such conditions chnge in wys tht cn e difficult, if not impossile, for orgnisms to predict. Finlly, mny nimls possess the cpcity to use different sensory modlities under different environmentl conditions (e.g. Ale 1991; Chittk et l. 1999; Eklöf et l. 22; Kroder /$38. Ó 212 The Assocition for the Study of Animl Behviour. Pulished y Elsevier Ltd. All rights reserved. doi:1.116/j.nehv

2 96 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e913 et l. 27). Despite the plusiility of the ckup hypothesis, it hs rrely een tested. Of notle exception re recent studies of visul nd virtory signl components in wolf spider courtship displys (Rundus et l. 21; Wilgers & Heets 211). If multimodl signls serve ckup function, such signls should mintin high levels of efficcy cross rnge of vrition in the environment. For florl signls, vrition in the environment my e due to physicl fctors (e.g. vrition in light, wind, temperture, humidity, etc.) or iotic fctors (e.g. vrition in florl species composition, degree of vegettive cover, etc.). Although pollintors responses to multimodl signls hve een explored in numer of studies (reviewed in Leonrd et l. 211, c), no study hs ssessed the reltive effectiveness of multimodl nd unimodl signls under vrile environmentl conditions. The im of the present study ws to test the efficcy ckup hypothesis in plntepollintor context with the gol of providing insight into the dptive enefit of multimodl florl signls. We specificlly sked whether the olfctory component of florl disply cn compenste for the loss of visul informtion under low illumintion, s might occur t dwn or dusk, in deep shde, or under overcst skies. Reduced illumintion hs een found to increse serch times for smll flowers in ees (Chittk & Spethe 27), suggesting tht the efficcy of visul signls is indeed reduced t low light levels. We first trined nectr-forging umleees to discriminte rewrding nd unrewrding trgets tht differed in colour, scent, or oth colour nd scent. We predicted tht visul signls would lose efficcy s light levels declined, resulting in reduced performnce, ut tht this decline in performnce would e mitigted y the ddition of scent. Our mesures of ee performnce included ccurcy in lnding on the previously rewrded trget type (i.e. correct lndings over totl lndings) nd rte of visits to the previously rewrded type (i.e. correct lndings per forging time). These performnce mesures not only hve implictions for pollintor fitness (rte of energy intke), ut lso for plnt fitness (rte of pollen trnsfer to or from conspecifics). Our design thus llowed us to consider the enefit of multimodl signl from the perspectives of oth the sender (plnt) nd receiver (pollintor). METHODS Two commercilly otined colonies of the common estern umleee, Bomus imptiens (Koppert Biologicl Systems, Romulus, MI, U.S.A.), were used (N ¼ 63 nd 33 from the different colonies). During the experiment, single colony ws connected to forging ren ( cm), with plstic nd mesh tuing. Gtes in the tuing permitted regultion of ee trffic to the ren. Colonies were given full ccess to the forging ren when trils were not occurring, where ees were fed dily 3% (wt/wt) sucrose solution from multiwell feeder. Pretrining We rn weekly pretrining sessions to identify individul ees willing to feed from the rtificil flower rry. During pretrining, ll ees hd ccess to horizontl rry of 6 rtificil flowers (in 1 6 grid) spced t 8 cm intervls, n rrngement tht mtched rrys in experimentl trils. All pretrining flowers were light lue, mtte-lminted pper disks (25 mm dimeter), ttched to reservoir tue contining thin cotton wick tht protruded just ove the centre of the disk. To provide sitution similr to experimentl trils, hlf of the pretrining flowers were rewrding (wick provided 3% sucrose solution), while the other hlf were unrewrding (dry). Rewrding nd unrewrding flowers were positioned rndomly into the rry. Bees tht fed from rtificil flowers during pretrining were tgged with n identifying numer (E. H. Thorne Ltd, Wrgy, U.K.) nd used in further experiments. Trining To test the ckup efficcy hypothesis, ees were trined in one of two discrimintion tsks: visul only (trgets differed in colour only) nd imodl (trgets differed oth in colour nd in scent). To determine the efficcy of the odours used in the imodl tretment, third group of ees were trined in n olfctory-only discrimintion tsk (trgets differed in scent only). For the visul-only tsk, we used two colours tht differed slightly in lue to green rtio (one with n equl rtio, Blue 1; the other with slightly more green, Blue 2). Very similr colours were chosen to mke the discrimintion tsk more chllenging, incresing the chnces tht n dditionl cue would enhnce discrimintion. Colours were printed on wterproof pper (Ntionl Geogrphic, Mrgte, FL, U.S.A.) using Cnon Pixm MX86 inkjet printer, nd lminted (Xyron mtte lminte, Scottsdle, AZ, U.S.A.). We mesured the reflectnce of these colours (Fig. 1) nd determined tht the difference in chromtic contrst (colour distnce) ws reltively low (pproximtely.3; using spectrl sensitivity functions (Peitsch et l. 1992; Stveng et l. 1993) sed on B. imptiens photoreceptor pek sensitivities (Skorupski & Chittk 211) nd spectrometric nlysis softwre (AVICOL 4.; Gomez 26) sed on clcultions from Chittk 1992). A colour distnce of.62 is reltively difficult for umleees (B. terrestris) to discriminte (Dyer & Chittk 24). For the imodl tsk, we used the sme colours ut dded linlool nd gerniol, two structurlly similr monoterpenes common in florl scents (Schiestl 21). We pipetted 2 ml of 1:1 (1 M) solution of the compound in minerl oil onto cotton sw plced inside of the pipette tip serving s the se of the trget. The scents percolted through smll holes in the lminted pper trget. Blue 1 ws lwys pired with linlool nd Blue 2 ws lwys pired with gerniol. For the olfctory-only tsk, we gin used linlool nd gerniol, ut ll trgets were Blue 1. In trining sessions, ees were relesed individully into the ren contining 6-trget horizontl rry. For ech ee, one trget type ws rewrding (5 ml of 5% sucrose) nd one ws unrewrding (5 ml of wter). This reltionship ws lnced cross ees. Reflectnce (%) 5 Blue 1 Blue 2 Bckground Wvelength (nm) 6 7 Figure 1. Percentge reflectnce cross wvelengths for the two colours used to distinguish florl types, Blue 1 nd Blue 2, nd the ckground. Rw dt spectr were interpolted nd smoothed in Avicol 4..

3 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e Trining trgets were 16 mm in dimeter (the pproximte size ssocited with incresed serch times under low light; Chittk & Spethe 27) nd lcked wick; solutions were pipetted directly onto the trget. On ech trining tril, ees were llowed to forge in the ren for t lest 1 min, or until they returned to the nest or stopped forging for longer thn 2 min, whichever occurred erlier. After ech tril the ee ws returned to the nest. Ech ee ws given t lest two trining trils, nd s mny dditionl trils s ws necessry for the ee to rech 15 visits (men SE numer of trining trils: imodl tretment: 3.1.2; visul-only tretment: 2.6.1; olfctory-only tretment: 2.9.2). Between trils, we clened trgets with 3% ethnol to remove ny scent mrks nd redistriuted their positions using one of three rndom ptterns to prevent lerning of sptil cues. All trils were conducted under moderte light intensity (see elow) nd videorecorded. Testing After trining (t lest two trils nd >15 flower visits), the forging success of ech ee ws immeditely ssessed upon its return to the ren using n rry of unrewrded (wter-only) trgets of the sme types to which it hd een trined. Wheres trining occurred for ll ees under moderte light intensity, ee ws tested under one of three levels of light intensity (high, moderte, or low). Light intensity ws mnipulted y mens of neutrl density filters (Rosco E-colour no. 29, Stmford, CT, U.S.A.) fitted cross four 25 W hlogen light sources. Ech filter ws of opticl density equl to.3, with pproximtely 5% trnsmittnce; we lyered filters to crete different light levels (no filter for high, one filter for moderte nd four filters for low light intensity; irrdince spectr in Fig. 2). These filter comintions produced grdient of light levels (men of multiple redings in five res of ren SE: low ¼ 62 3 lx, moderte ¼ lx, high ¼ lx). The low level ws equivlent to deep shde or thick cloud cover (round 1 lx; Chittk & Spethe 27); this is ove those light levels t which ees switch to chromtic vision (Menzel 1981; Rose & Menzel 1981). The high light level ws equivlent to light shde or slightly overcst conditions (Johnsen et l. 26). The irrdince differences only slightly ltered the position of trgets in umleee colour spce (Fig. 3) nd chromtic contrst etween the two colours vried little with light intensity (high:.35; moderte:.38; low:.31). Temperture with testing lights on remined within 2 C from strting temperture in ll tretments. Twelve ees were tested per light intensity in the visul-only nd imodl tretments, while eight ees were tested per light Irrdince (mmol photons/nm) High Moderte Low Wvelength (nm) 6 7 Figure 2. Totl irrdince (mmol photons/nm) cross wvelengths for the different light intensities. intensity in the olfctory-only tretment, for totl of 96 ees. Test trils lsted for 1 min or until the ee returned to the nest (men SE numer of visits per test tril ¼ ). We clculted ccurcy (proportion of visits to previously rewrded trget type (numer of correct visits/numer of totl visits)) nd the rte of correct visits (numer of visits to previously rewrded trget type per time spent forging (numer of correct visits/s)). Test ees were lter scrificed for mesurements of ody size (thorx width), which is known to ffect umleee forging performnce (Spethe & Weidenmüller 22), prticulrly under different light conditions (Kpustjnskij et l. 27). Anlysis of Trining Performnce We were lso interested in compring how redily ees lerned to discriminte the two trget types depending on the colours or odours ville during trining. As n index of lerning performnce, we estimted the numer of visits it took ech ee to rech criterion of 8 out of 1 consecutive visits to the rewrding flower type. We nlysed the numer of visits needed to rech this criterion (squre root trnsformed) using multiple generl liner models (GLM; JMP 8., SAS Institute, Cry, NC, U.S.A.) nd Tukey s HSD for post hoc tests ( ¼.5). We lso investigted whether there ws n initil is for ees to lnd on prticulr colour (in visul only nd imodl) or odour (in olfctory only) on their first lnding of the first trining tril (chi-squre tests; Microsoft Excel 27). Anlysis of Testing Performnce Our centrl question of whether olfctory signls cn compenste for the loss of visul informtion ws ssessed y compring ee performnce in the visul-only nd imodl tretments. The olfctory-only tretment only served s control to determine whether the efficcy of n olfctory signl might itself chnge under different light intensities. Therefore, we rn two seprte, trgeted nlyses (GLM; JMP 8.), one for visul versus imodl tretments to specificlly test the efficcy ckup hypothesis, nd one for the olfctory tretment only; in ech cse, the outcome vriles were ccurcy (rcsine root trnsformed) nd the rte of correct visits. The nlysis of visul-only nd imodl ssys included tretment, light intensity nd colour of the previously rewrded trget type (nd ll possile interctions) s fctors in the model. The nlysis of the olfctory-only ssys included light intensity nd odour of the previously rewrded trget type (nd their interction) s fctors in the model. There were no significnt effects of colony (ccurcy: F 1,94 ¼.9, P ¼.76; rte of correct visits: F 1,94 ¼.965, P ¼.328) or ody size (ccurcy: F 1,93 ¼.8, P ¼.783; rte of correct visits: F 1,93 ¼.715, P ¼.4), so these fctors were not included in the model. Post hoc tests (Tukey HSD t ¼.5) were used for comprisons of interest. Becuse there were significnt colour effects in oth trining nd testing (see Results elow), we renlysed test performnce (ccurcy nd rte of correct visits) for the visul-only versus imodl tretments, ut with dt seprted y the colour of the previously rewrded trget type. Modlity tretment, light intensity nd their interction were the fctors included in the seprte models (GLM; JMP 8.). RESULTS Trining Performnce Bees lerned to discriminte etween trgets more quickly when they differed in oth colour nd scent thn when they differed in colour lone (Fig. 4). Over the course of the trining

4 98 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e913 E(Blue) Blue UV-lue Blue-green E(UV) UV Green E(Green) Blue 1 low Blue 2 low Blue 1 moderte Blue 2 moderte Blue 1 high Blue 2 high Pretrining UV-green Figure 3. Loction in ee colour spce of ech of the two experimentl colours, Blue 1 nd Blue 2, under three light intensities. The colour of the pretrining trget under the light conditions used is lso included. Inset shows close-up of the dt points. Blue 1 is denoted y white symols, Blue 2 y lck symols, nd pretrining y grey symol. The two colours under the sme light intensity hve the sme symol shpe. The centre of the hexgon represents the ckground. Visits to criterion (8 in 1 correct visits) % Correct in 1 visits () () Trining visits ** * NS Blue 1 Blue 2 Visul 1 Bimodl-Blue1 Bimodl-Blue2 Visul-Blue1 Visul-Blue2 Blue 1 Blue 2 Bimodl Figure 4. Performnce of ees during trining. () Lerning cquisition ssocited with ech trining colour for visul nd imodl tretments. Ech point represents the proportion of correct visits in 1 consecutive trining visits (men SE, N ¼ 18 per line). Filled symols nd solid lines denote Blue 1, unfilled symols nd dshed lines denote Blue 2. The grey dotted line denotes rndom choice. A four-prmeter logistic curve ws used to fit the dt. () Distriution of the numer of visits it took for ees to rech criterion of 8 correct visits in 1 consecutive visits when either Blue 1 or Blue 2 ws the rewrding colour in oth visul nd imodl tretments (N ¼ 18 ees per ox plot). *P <.5; **P <.1. sessions, verge ee ccurcy improved fster nd remined higher in the imodl thn in the visul-only tretment (Fig. 4). Bees lso required significntly more visits to rech criterion of 8 in 1 correct lndings when signls were visul only thn when signls were imodl (GLM: tretment effect: F 1,68 ¼ 62.14, P <.1). There ws significnt effect of trining colour: ees required significntly fewer visits to rech this criterion when the colour of the rewrding trget type ws Blue 1 compred to Blue 2 (GLM: colour: F 1,68 ¼ 9.82, P ¼.3). However, the trining colour effect ws only significnt (P <.5) in the visul-only tretment, not the imodl tretment (Fig. 4), lthough there ws no significnt interction effect (GLM: tretment*colour: F 1,68 ¼ 2.26, P ¼.137). There ws lso significnt first-choice is to lnd on Blue 1-coloured trgets in oth tretments (chi-squre test: P <.1; visul only: c 2 1 ¼ 13.4; imodl: c2 1 ¼ 7.1). Testing Performnce: Visul Only versus Bimodl An initil nlysis tht pooled ll dt from the visul-only nd imodl tretments determined tht there were significnt colour effects for oth performnce mesures (GLM: effect of colour on ccurcy: F 1,6 ¼ 13.3, P <.1; effect of colour on correct visit rte: F 1,6 ¼ 5.95, P ¼.18): performnce ws significntly etter when the trining colour ws Blue 1. Given tht these colour effects were lso present during trining (s noted ove), we proceeded with seprte nlyses, compring the Blue 1 visul-only group with the Blue 1/linlool imodl group nd the Blue 2 visul-only group with the Blue 2/gerniol imodl group. Accurcy Bee ccurcy ws high for oth modlity tretments nd cross ll light levels, regrdless of trining colour (ll ees performed significntly etter thn rndom choice,.5, P <.1, except in the visul-only tretment with Blue 2 s the rewrding trget under low light, P ¼.56; one smple, two-tiled t tests; see Fig. 5). Light intensity significntly ffected ee ccurcy with oth trining colours (GLM: Blue 1: F 2,3 ¼ 6.92, P ¼.3; Blue 2: F 2,3 ¼ 7.59, P ¼.2), lthough significnt increses in ccurcy with incresing light intensity were found only in the visul-only

5 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e Accurcy (no. correct/no. totl visits) () () tretment with no significnt differences cross light intensities in the imodl tretment (see Fig. 5). When Blue 2 ws the previously rewrded colour, ccurcy ws significntly higher when signls were imodl compred to visul only (GLM: tretment effect: F 1,3 ¼ 19.17, P <.1; Fig. 5). However, this ws the cse cross ll light levels: there ws no significnt interction etween tretment nd light intensity (GLM: tretment*light intensity: F 2,3 ¼.71, P ¼.54) nd, thus, no sttisticl support for the efficcy ckup hypothesis. When Blue 1 ws the previously rewrded colour, ccurcy ws gin significntly higher when signls were imodl versus visul only (GLM: tretment effect: F 1,3 ¼ 15.41, P ¼.1). However, there ws lso highly significnt interction etween tretment nd light level (GLM: tretment*light intensity: F 2,3 ¼ 6.4, P ¼.6; Fig. 5). The pttern of interction provided support for the efficcy ckup hypothesis: there ws greter decline in ccurcy with decresing light intensity when signls were visul only compred to when they were imodl (compre lines in Fig. 5). In short, in terms of ccurcy there is sttisticl support for the efficcy ckup hypothesis when Blue 1 ws the previously rewrded colour ut not when Blue 2 ws the previously rewrded colour. Correct Visit Rte c Low Moderte Light intensity High Bimodl Visul Figure 5. Accurcy of choice (numer correct/numer of totl visits) cross light intensities for visul nd imodl tretments, seprted y trining colour (men SE, N ¼ 6 per dt point). The dshed line represents rndom choice. Letters represent significnce: points tht do not shre letter were significntly different from ech other t P <.5. () Bees tht hd Blue 1 s the previously rewrded trget colour. () Bees tht hd Blue 2 s the previously rewrded trget colour. For oth trining colours, the rte of visits to the previously rewrded trget type (¼ correct visits per time) ws significntly ffected y light intensity (GLM: Blue 1: F 2,3 ¼ 16.26, P <.1; c Blue 2: F 2,3 ¼ 15.11, P <.1; Fig. 6). Correct visit rte significntly incresed (P <.5) with incresing light intensity in oth the visul-only nd imodl tretments. When Blue 2 ws the previously rewrded colour, there ws no significnt difference etween the modlity tretments (GLM: F 1,3 ¼.26, P ¼.613; Fig. 6) nd no interction etween tretment nd light intensity (GLM: tretment*light intensity: F 2,3 ¼.28, P ¼.756), thus, in terms of correct visit rte, no sttisticl support for the efficcy ckup hypothesis when Blue 2 ws the previously rewrded colour. When Blue 1 ws the previously rewrded colour, there ws lso no significnt difference etween the modlity tretments overll (GLM: F 1,3 ¼.33, P ¼.568; Fig. 6), ut there ws significnt interction etween tretment nd light level (GLM: tretment*light intensity: F 2,3 ¼ 4.29, P ¼.23). However, this pttern of interction did not provide support for the efficcy ckup hypothesis. Insted, it reflects high rte of correct visits for the imodl tretment under moderte light intensity (Fig. 6, grey solid line). In other words, in terms of visit rte, there ws no indiction tht dding odour to the discrimintion tsk compensted for loss of efficcy of the visul stimulus t low light levels. Olfctory-only Assys The olfctory-only ssys served s control to determine whether the efficcy of olfctory signls ws ffected y different Rte of correct visits (no. correct visits/s) () () cd d Low cd Moderte Light intensity High Bimodl Visul Figure 6. Correct visit rte (numer of correct visits/s) cross light intensities for visul nd imodl tretments, seprted y trining colour (men SE, N ¼ 6 per dt point). Letters represent significnce: points tht do not shre letter were significntly different from ech other t P <.5. () Bees tht hd Blue 1 s the previously rewrded trget colour. () Bees tht hd Blue 2 s the previously rewrded trget colour.

6 91 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e913 light intensities. We noted tht during trining, ees did not show first-choice is for prticulr odour (chi-squre test: c 2 1 ¼ 1.5, P ¼.121), nor ws their discrimintion etween trgets ffected y the identity of the previously rewrded odour: there ws no significnt difference in the numer of visits to rech criterion of 8 in 1 correct (GLM: odour effect: F 1,34 ¼.2, P ¼.659). During testing, ee ccurcy t distinguishing the previously rewrded trget type when signls were olfctory only (Fig. 7) ws not significntly ffected y light intensity (GLM: F 2,18 ¼.24, P ¼.789), odour type (GLM: F 2,18 ¼.39, P ¼.543), or their interction (GLM: F 2,18 ¼ 1.41, P ¼.269). However, the rte t which ees visited previously rewrded trgets (Fig. 7) ws significntly ffected y light intensity (GLM: F 2,18 ¼ 3.6, P ¼.49), with lower rtes overll t low light intensity thn t moderte or high intensity (lthough rtes t high nd moderte intensity were not significntly different). Correct visit rte ws not ffected y type of odour (GLM: F 2,18 ¼.53, P ¼.476) or the interction of light intensity nd odour type (GLM: F 2,18 ¼ 2.67, P ¼.97). DISCUSSION Environmentl heterogeneity is mjor, yet lrgely overlooked, fctor in plntepollintor communiction (ut see Kilkenny & Gllowy 28). Fctors such s illumintion (Jkosen & Olsen 1994; Chittk & Spethe 27; Kpustjnskij et l. 27), wind speed (Streinzer et l. 29) nd temperture (Jkosen & Olsen Accurcy (no. correct/no. totl visits) Rte of correct visits (no. correct visits/s) () () Low Moderte Light intensity High Gerniol Linlool Figure 7. Bee performnce for the two scents, gerniol nd linlool, used in the olfctory-only tretment cross different light intensities (men SE, N ¼ 8 per dt point). Letters represent significnce: points tht do not shre letter were significntly different from ech other t P <.5. () Bee ccurcy (numer correct/numer of totl visits). The dshed line represents rndom choice. () Correct visit rte (numer of correct visits/s). 1994; Sge et l. 28) cn ll ffect the trnsmission nd/or perception of florl signls. The efficcy ckup hypothesis ccounts for potentil environmentl heterogeneity, proposing tht multimodl signls ensure tht pollintors receive pproprite florl informtion despite environmentl degrdtion of signlling in one modlity. Our experiment directly ssessed how umleees ility to discriminte etween florl types tht differed in rewrd vlue depended oth upon light level nd upon the multimodl nture of the florl disply. We first estlished tht low illumintion reduced the efficcy of visul-only signls, oth in terms of lnding ccurcy (Fig. 5) nd correct visit rte (Fig. 6), regrdless of trining colour. Then we sked whether the visul signl s loss of efficcy under low light ws reduced with the ddition of n olfctory component (imodl trgets). We found support for this ide in terms of lnding ccurcy; however, the effect ws conditionl upon trining colour, occurring only when ees were rewrded for visiting the colour they initilly preferred (Blue 1). The oserved increse in ccurcy is most likely due to dditionl informtion provided y scent, given tht ees were highly ccurte in discriminting flowers sed solely on these scents lone in the olfctory-only tretment. However, nother possile mechnism could e tht the presence of scent improved colour perception (Kunze & Gumert 21; Leonrd et l. 211). Assessing the underlying mechnism ehind the contextdependent ckup effect on ccurcy is n intriguing question for future reserch. When we considered correct visit rte, mesure tht tkes into ccount not only ccurcy ut lso forging speed, we found no evidence tht n olfctory signl compensted for the loss of visul informtion: similr decrese in correct visit rte under low light occurred when the signls were visul, imodl or olfctory (Fig. 6: visul versus imodl; Fig. 7: olfctory only). In other words, ees were mking fewer correct choices per unit time when illumintion ws low, regrdless of whether n olfctory component ws present. The effect of light intensity on correct visit rte suggests tht light intensity ffects not only discrimintion of visul cues ut lso the speed t which ee forges for nectr, n effect previously demonstrted in honeyee forging (Rose & Menzel 1981). Thus, ecuse the correct visit rte remined low under low light intensity even with olfctory informtion, the presence of scent could not compenste for the loss of visul efficcy in terms of rewrd collection for the ee. In short, our results point towrds complex, conditionl reltionship mong signl complexity, pollintor ehviour nd components of plnt nd pollintor fitness. Although mny studies hve explored the effect of multimodl florl signls on ees (e.g. Couvillon & Bittermn 198, 1982, 1987; Giurf et l. 1994; Kunze & Gumert 21; Reinhrd et l. 24; Leonrd et l. 211), efficcy ckup ws not focus of these studies. Our results show tht efficcy ckup is potentil explntion for the evolution of multimodl florl signls, ut this is not mutully exclusive with other hypotheses tht propose differing explntions (Leonrd et l. 211c). Content-sed hypotheses relte to the informtion provided y the signller (plnt), wheres efficcy-sed hypotheses relte to the ility of the receiver (pollintor) to detect nd process tht informtion (Heets & Ppj 25). For exmple, the efficcy trde-off hypothesis proposes tht different modlities del with different chllenges of signl trnsmission through the environment (e.g. short-rnge versus longrnge; Streinzer et l. 29). The ttention-ltering hypothesis suggests tht signl in one modlity lters the ttention of the receiver to signl in nother modlity (e.g. the presence of scent improves colour perception; Kunze & Gumert 21; Leonrd et l. 211). Multiple explntions could e relevnt under different circumstnces or with different orgnisms.

7 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e Conditionl Effects of Signl Types on Bee Behviour Why did the presence of n olfctory signl ck up the loss of visul informtion (in terms of lnding ccurcy) when the colour rewrded during trining ws Blue 1, ut not Blue 2? The colours used in this experiment were most likely perceived y the ees to e very similr, sed on their close loction in ee colour spce (Fig. 3). It is thus intriguing tht we found significnt initil lnding preference, s well s lerning nd performnce differences sed on trining colour. Even though hlf our sujects lerned to visit Blue 2 during trining sessions (sed upon our lerning criterion), n innte is towrds Blue 1 could hve ffected their test performnce. We noted tht Blue 1 hd greter reflectnce t lower wvelengths (lue region; Fig. 1), nd tht it ws oth initilly preferred nd lerned more redily. Perhps the ees stronger innte preference for lower wvelengths (Giurf et l. 1995; Gumert 2; Chittk et l. 21; Rine & Chittk 27) resulted in fster lerning or stronger persistence when Blue 1 ws rewrding (cf. Giurf et l. 1997; Ings et l. 29). It seems unlikely tht the is towrds Blue 1 resulted from exposure to the light lue used in pretrining trils since tht colour ppers to e closer to Blue 2 in ee colour spce (Fig. 3). Whtever the source of the is for Blue 1, its presence constrins the generlity of the ckup effect of florl scent on lnding ccurcy. For exmple, ees trined to lnd on Blue 2 still lnded on Blue 1 during tests t reltively high frequency (Fig. 5), cross ll three light levels. Thus, for Blue 2-trined ees, the reltive enefit of imodl trget did not increse s light levels declined, ut styed reltively constnt: in contrst to Blue 1-trined ees, Blue 2-trined ees enefited from imodl trget even when illumintion ws high. These findings suggest interply etween the reltive ttrctiveness of visul versus olfctory signls nd their enefit in chnging environment. Essentilly, if flower presents highly ttrctive visul signl, the enefits of multimodlity only ecome cler with the loss of visul informtion. For flowers with less ttrctive visul signls, multimodlity is evidently dvntgeous cross ll light levels. This ide could e tested y systemticlly vrying the ttrctiveness of visul nd olfctory signls, while mesuring ee forging performnce long n environmentl grdient (e.g. illumintion levels, wind speed). This study utilized oth similr colours nd similr scents known to e reltively ttrctive for the ee, ut we might expect different results with different colours, scents or colourescent comintions, especilly given the context dependence oserved with the colours used in this study. For exmple, olfctory informtion my not e very useful if the visul signl is highly divergent (Giurf et l. 1994) ecuse the colours re likely to e discriminle even under low light. In this cse, dditionl olfctory informtion would e less likely to increse ee performnce. On the other hnd, olfctory informtion my e even more useful if the scents re more divergent (or otherwise more esily distinguishle) nd more likely to increse ee performnce, especilly if the colours re more similr. Given our results, we might lso expect greter enefit of multimodlity with the presence of less fvoured stimulus. Our gol ws not to determine under which circumstnces efficcy ckup is likely to e relevnt, ut to show tht efficcy ckup cn explin evolution of multimodl signls, which is supported y our results. Potentil Effects of Signl Complexity on Bee versus Plnt Fitness We considered two relted mesures of ee forging ehviour under different light levels. The first of these ws lnding ccurcy, s discussed ove. The second ws visit rte to the previously rewrded flower type. Although we found tht ees trined nd tested with imodl trgets showed higher lnding ccurcy thn ees whose trgets were visul-only, they did not show higher rte of lnding upon the previously rewrded flower type. Accurcy is commonly used s the only outcome vrile in lerning experiments. Although it my e relevnt when ees fce significnt costs ssocited with incorrect choices (time costs (Burns 25), which my depend upon the frequency of rewrding flowers in community (Burns & Dyer 28), or nutritive costs (Adler 2), which my depend upon the frequency of unfvourle flowering species in community), it my not lwys e the most pproprite mesure of pollintor fitness (Burns 25). Insted, nectr collection rte, or relted vrile such s correct visit rte, is more likely to hve the most direct impct on ee fitness (Pelletier & McNeil 23). In contrst, ee ccurcy my hve prticulrly strong impct on plnt fitness ecuse it fcilittes pollen trnsfer mong conspecific plnts. Accurcy my e t premium for plnts when illumintion declines, s shde-growing plnts my receive fewer pollintor visits thn their counterprts growing in res of high irrdince (Herrer 1995; O Connell & Johnson 1998; Kilkenny & Gllowy 28; ut see Sánchez-Lfuente et l. 25; Hnsen & Totlnd 26). For plnts tht fce this kind of sptil vrition in light levels, differences in pollintor visits hve een ttriuted to the effects of shde on florl disply size, s well s to ccompnying decreses in temperture, relevnt for ectothermic forgers like ees (Herrer 1995,, 1997). Our experiment suggests third explntion for ees reluctnce to visit shde-growing plnts: they lso forge less effectively under reduced light levels. The oserved reduction in forging speed t low light levels occurred without significnt chnges in temperture (2 C with or without lights on), which we monitored throughout the experiment. For plnts tht fce temporl vrition in light levels (e.g. occsionl cloudy dys), our results show enefit of trnsmitting scent (t lest when flowers re visully difficult to detect). One might even expect tht plnts would increse scent production when visul cues lose efficcy. Do plnts do this? We found no evidence of this in the literture. In fct, severl studies suggest tht plnts reduce their emission of florl voltiles when illumintion is decresed (Jkosen & Olsen 1994; Jkosen et l. 1994; Underwood et l. 25). This finding suggests signlling strtegy different from n efficcy ckup strtegy: rther thn wsting energy to produce scents for pollintors tht my e reluctnt to forge under low light levels, the plnt my conserve resources for signlling until environmentl conditions re etter suited for pollintor ttrction. Finlly, vrition in light levels is only one of multiple spects of environmentl heterogeneity potentilly relevnt to florl displys. For exmple, lthough we focused on the potentil for n olfctory signl to compenste for the loss of visul informtion, the complementry reltionship is possile: perhps components of the visul disply compenste for olfctory uncertinty, which my occur when wind speed increses, when wind ecomes more turulent, or when voltiles from coflowering plnt species re more similr nd thus more difficult to discriminte. We pln to explore these possiilities in future experiments. Conclusion Although interest in understnding the function of complex multimodl signls hs grown in the pst decde (reviewed in Heets & Ppj 25), few studies hve explored this prdigm in plntepollintor system (Leonrd et l. 211). Tken s whole, our results suggest n dptive enefit of multimodl florl signls my e to mintin pollintors ccurcy under environmentl vriility. Our results show support for the efficcy ckup hypothesis in terms of ccurcy, ut not in the rte of correct visits.

8 912 R. L. Kczorowski et l. / Animl Behviour 83 (212) 95e913 Interestingly, the ckup function ppers to e context dependent, given tht ckup only occurred when one of the two colours used ws rewrding. Since plnts my enefit directly from pollintor ccurcy while pollintors enefit directly from their rte of correct visits, plnts my enefit more from ckup function of multimodl signls thn do pollintors. While we focused on the potentil enefit of multimodl florl signl under chnging environmentl conditions, mny other possiilities remin to e explored in more detil (Leonrd et l. 211c), including the potentil for multimodl florl signl to fcilitte discrimintion lerning (Kulhci et l. 28; Leonrd et l. 211), promote florl constncy (Geger 25), or fcilitte pollintors ility to locte flower t different sptil scles (Streinzer et l. 29). Acknowledgments We thnk W. Huffmn, J. Brent, S. Admo, I. Nieves, R. Corrl, N. Andrews nd T. Mzzrell for ssistnce in conducting experiments, nlysing video nd/or entering dt nd P. Mrek for ssistnce with collecting irrdince dt. We lso thnk memers of the Ppj nd Dornhus ls for helpful discussions pertining to these experiments, nd two nonymous referees for their helpful comments. This study ws supported y Ntionl Science Foundtion (NSF) Grnt IOS References Ale, K. P Common themes nd vritions in niml orienttion systems. Americn Zoologist, 31, 157e167. Adler, L. S. 2. The ecologicl significnce of toxic nectr. Oikos, 91, 49e42. Brdury, J. W. & Vehrencmp, S. L Principles of Animl Communiction. Sunderlnd, Msschusetts: Sinuer. Burns, J. G. 25. Impulsive ees forge etter: the dvntge of quick, sometimes inccurte forging decisions. Animl Behviour, 7, e1ee5. Burns, J. G. & Dyer, A. G. 28. Diversity of speedeccurcy strtegies enefits socil insects. Current Biology, 18, R953eR954. Cndolin, U. 23. The use of multiple cues in mte choice. Biologicl Reviews, 78, 575e595. 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