How a generalist bee achieves high efficiency of pollen collection on diverse floral resources

Abstract

Bees foraging for floral rewards are one of our most thoroughly studied examples of generalist foraging ecology. Generalist bees rely considerably on instrumental (associative) learning to acquire routines that allow them to collect nectar efficiently from diverse plant species. Although such bees must also collect pollen from diverse species, few studies have examined if and how high efficiency is achieved. We characterized how generalist bumble bees (Bombus impatiens) foraged effectively for pollen from diverse floral resources, by manipulating the presence of pollen and anther cues, in a series of experiments using pollen-bearing live flowers, flowers of a sterile pollen-less horticultural hybrid, and artificial flowers. We show that generalist bumble bees exhibit flexible and effective pollen collection by switching between 2 routines: " scrabbling " when pollen is abundant and " sonicating " when pollen is scarce. Efficient switching between these behaviors is regulated by the interplay of 2 ubiquitous floral cues: chemical anther cues stimulating pollen collection behavior and mechanical pollen cues suppressing sonication (and eliciting scrabbling). Flexible pollen collection behavior is functional: When pollen on anthers was scarce, bees collected it at a greater rate by sonicating than scrabbling. This mechanism of behavioral flexibility likely allows generalist bees to handle diverse anther morphologies efficiently and may have facilitated the recurrent evolution of plant species that conceal pollen rewards via pored floral morphology. Whereas effective nectar foraging relies heavily on associative learning of unique routines for each flower type, a weighing of 2 types of cues regulates the flexible pollen collection mechanism we describe.

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Behavioral
Ecology
Original Article
How a generalist bee achieves high eciency
of pollen collection on diverse floral resources
Avery L.Russell,a,b Stephen L.Buchmann,b and Daniel R.Papajb
aGraduate Interdisciplinary Program in Entomology and Insect Science, University of Arizona, 1140
East Lowell Street, Tucson, AZ 85721, USA and bDepartment of Ecology and Evolutionary Biology,
University of Arizona, 1140 East Lowell Street, Tucson, AZ 85721, USA
Received 24 December 2016; revised 6 March 2017; editorial decision 16 March 2017; accepted 27 March 2017.
Bees foraging for floral rewards are one of our most thoroughly studied examples of generalist foraging ecology. Generalist bees rely con-
siderably on instrumental (associative) learning to acquire routines that allow them to collect nectar efficiently from diverse plant species.
Although such bees must also collect pollen from diverse species, few studies have examined if and how high efficiency is achieved. We
characterized how generalist bumble bees (Bombus impatiens) foraged effectively for pollen from diverse floral resources, by manipu-
lating the presence of pollen and anther cues, in a series of experiments using pollen-bearing live flowers, flowers of a sterile pollen-
less horticultural hybrid, and artificial flowers. We show that generalist bumble bees exhibit flexible and effective pollen collection by
switching between 2 routines: “scrabbling” when pollen is abundant and “sonicating” when pollen is scarce. Efficient switching between
these behaviors is regulated by the interplay of 2 ubiquitous floral cues: chemical anther cues stimulating pollen collection behavior and
mechanical pollen cues suppressing sonication (and eliciting scrabbling). Flexible pollen collection behavior is functional: When pollen on
anthers was scarce, bees collected it at a greater rate by sonicating than scrabbling. This mechanism of behavioral flexibility likely allows
generalist bees to handle diverse anther morphologies efficiently and may have facilitated the recurrent evolution of plant species that
conceal pollen rewards via pored floral morphology. Whereas effective nectar foraging relies heavily on associative learning of unique
routines for each flower type, a weighing of 2 types of cues regulates the flexible pollen collection mechanism we describe.
Key words: behavioral flexibility, floral sonication, floral evolution, learning, pollen foraging, specialization.
“It is not obvious what characteristics the flowers […] might
have that may assist in the acquisition of buzzing behavior by
bees …” (Michener 1962)
INTRODUCTION
A fundamental question in behavioral ecology is how general-
ist animals forage eciently on diverse resources (Loeuille 2010;
Wright etal. 2010; Baudrot et al. 2016). This question has been
extensively addressed for generalist pollinators foraging on diverse
plant species that vary greatly in floral morphology. For instance,
generalist pollinators use instrumental (associative) learning to
acquire routines specific to each flower type to extract nectar e-
ciently (Lewis 1993; Laverty 1994; Gegear and Laverty 1995). Such
learning allows a generalist pollinator to use novel species it may
never have encountered in its evolutionary history, which is par-
ticularly relevant given how humans have increased the frequency
of such encounters (Harmon-Threatt et al. 2009; Bartomeus etal.
2016). Moreover, pollinator learning may have facilitated the diver-
sification of floral form (Schiestl and Johnson 2013). Yet nectar is
not the only floral reward and instrumental learning may not be
the only means by which flexible behavior is achieved. Pollen is in
fact oered by hundreds of thousands of plant species to pollina-
tors such as bees, beetles, flies, and some butterflies (Simpson and
Ne 1981; Kevan and Baker 1983). Bees in particular must collect
pollen, which is their primary source of protein (Nicolson 2011).
Although it is commonly assumed that bees adjust their behavior to
collect pollen eectively from a range of plant species, the nature
of this assumed flexibility and its implications for floral evolution
have scarcely been examined in comparison to nectar collection.
One obvious place to look for flexibility is in the 2 major means
by which pollen is collected from biotically pollinated flowers of
varying morphology. The pollen of most angiosperm flowers is
exposed, to varying degrees, on the anthers (Figure 1g–i). Bees
use their legs and mandibles to knock this pollen free via a behav-
ior called scrabbling (Thorp 2000; see Russell and Papaj 2016
for a video). Six to ten percent of plant species (>22 000 species
across >72 angiosperm families; Buchmann 1983; De Luca and
Address correspondence to A.L. Russell. E-mail: averyrussell@email.
arizona.edu.
Behavioral Ecology (2017), 00(00), 1–13. doi:10.1093/beheco/arx058

Behavioral Ecology
Vallejo-Marín 2013) entirely conceal their pollen within special-
ized tube-like anthers or, less commonly, corollas (e.g., Houston
and Ladd 2002; De Luca and Vallejo-Marín 2013; Corbet and
Huang 2014; Figure 1a–c). Species with this so-called poricidal
morphology are pollinated nearly exclusively by bees that extract
the concealed pollen via a complex behavior termed floral soni-
cation (the buzz pollination syndrome; Buchmann 1983; Russell,
Leonard, etal. 2016). By rapidly contracting indirect flight mus-
cles to produce powerful vibrations (i.e., buzzing) while biting the
anthers or corolla, pollen is expelled onto the bee’s body, where
it can be collected (Michener 1962; Macior 1968; Buchmann
and Cane 1989; Russell, Leonard, et al. 2016). Although bees
must sonicate to collect pollen from poricidal species, scrabbling
is observed only on nonporicidal species and never on poricidal
species (Thorp 2000) and is inecient for removing and transfer-
ring pollen from flowers that partially conceal pollen (e.g., King
and Ferguson 1994; Javorek et al. 2002; Pomeroy and Fisher
2002; Figure1d–f). We might therefore expect that bees are able
to adjust their foraging behavior to collect pollen eectively from
these types of flowers.
Although pollination via the buzz mechanism is generally consid-
ered a relatively specialized interaction (Buchmann 1983; De Luca
and Vallejo-Marín 2013), reports of bees sonicating on flowers
with nonporicidal morphology are found in the literature (Table1).
We have recently made many more observations of the same kind
(Table1). Rather than exclusively sonicating poricidal species, this
behavior is performed on both poricidal and nonporicidal species,
albeit to dierent degrees. On poricidal species, sonication is con-
sistently performed whereas on nonporicidal species, sonication is
variable in its expression. This variability is expressed at multiple
scales. Not only might one bee scrabble on a nonporicidal species
while another sonicates but individual bees have been observed
sonicating some flowers and scrabbling on others of the same spe-
cies (e.g., Buchmann 1983, 1985; Raine and Chittka 2007). Taken
together, these observations suggest that generalist bees may be
able to adjust when they use floral sonication versus scrabbling to
eectively collect pollen from plant species with flowers of diverse
morphologies (Buchmann 1985; Raine and Chittka 2007).
We devised laboratory experiments to examine the floral cues
that regulate the flexibility of pollen foraging behavior (sonication
and scrabbling) in the generalist bumble bee (Bombus impatiens) on
nonporicidal species. We first tested how the amount of exposed
pollen on the anthers aects expression of sonication. We then
characterized 2 sets of cues, anther-based chemical cues, and pol-
len-based mechanical cues, which together account for the variable
expression of, and switching between, sonication and scrabbling.
Finally, we assessed whether the conditional expression of floral
sonication on nonporicidal species resulted in eective pollen col-
lection, thereby providing a mechanism by which bees might eec-
tively collect pollen from plant species that partially conceal their
pollen (e.g., Figure1d–f). Our findings indicate that bee pollen for-
aging, like nectar foraging, is flexible. However, the flexibility we
describe diers in key respects from that of nectar foraging and at
least some flexibility in this pollen collection mechanism involves
innately specified responses. We also discuss how this flexibility may
facilitate the evolution of taxa with poricidal floral morphology
from nonporicidal ancestors.
METHODS
Bees
We used 154 workers from 8 commercially obtained (Koppert
Biological Systems, Howell, MI) colonies of the bumble bee Bombus
impatiens Cresson in laboratory experiments conducted between
June 2014 and April 2016. Bumble bees are globally distributed
generalist pollinators and species such as B. impatiens forage from
hundreds of angiosperm species across its range (Plowright and
Laverty 1984). We used approximately equal numbers of bees from
each colony for each treatment. We allowed bees to forage daily
for sucrose and pollen in arenas constructed of plywood (L×W×H,
82×60×60cm). The arenas were lit from above by 40W 4400
lumen LED lights (2 × 2 LED Ultra Thin Panel; 5000K Cool
(a)
(f) (g) (h) (i)
(b) (c) (d) (e)
Figure1
Examples of poricidal and nonporicidal flowers. Three species conceal their pollen either within poricidal anthers: (a) Senna covesii and (b) Solanum elaeagnifolium;
or a poricidal corolla: (c) Pedicularis groenlandica. Three species that partially conceal their pollen, to varying degrees: (d) Dianella revolute, (e) Dodonaea microzyga, (f)
Asterolasia grandiflora. Three nonporicidal species display pollen openly on anthers: (g) Deppea splendens, (h) Aloe cryptopoda, (i) Phacelia campanularia. Photographs:
(a, b, g–i): Avery Russell; (c): Walter Siegmund, licensed by CC BY-SA 3.0; (d–f): Kevin Thiele, licensed by CC BY 2.0.
Page 2 of 13

Russell etal. • Flexible pollen foraging bybees
Table1
Nonporicidal species sonicated by bees
Family Genus
Anthers directly
accessible? Sonicating bee(s)
Bees sonicated
stamens? Reference
Asteraceae Senecio vulgaris Ye s Bombus edwardsii Ye s Buchmann (1978, 1983)
Begoniaceae Begonia cucullata Ye s Bombus impatiens Ye s A. Russell, personal observation
Begoniaceae Begonia descoleana Ye s Bombus impatiens Ye s This manuscript
Begoniaceae Begonia “Angel Wing”Ye s Bombus impatiens Ye s A. Russell, personal observation
Begoniaceae Begonia “Dragon Wing”Ye s Bombus impatiens Ye s This manuscript
Begoniaceae Begonia odorata Ye s Bombus impatiens Ye s A. Russell, personal observation
Bignoniaceae Tecoma alata Ye s Bombus impatiens Ye s A. Russell and D.Papaj, personal
observation
Bignoniaceae Tecoma stans Ye s Bombus sonorus Ye s D. Papaj and K.Mauerman, personal
communication
Boraginaceae Alkanna orientalis Ye s Anthophora pauperata Ye s Stone etal. (1999)
Boraginaceae Mertensia ciliata Ye s Bombus bifarius,
B.flavifrons, B.mixtus
Ye s D. Papaj and K.Mauerman, personal
communication
Boraginaceae Mertensia paniculata Ye s Bombus mixtus,
B.frigidus, Bombus spp.
Ye s Morris (1996), A.Russell, personal
observation
Boraginaceae Symphytum ocinale Ye s Bombus pascuorum Ye s Corbet etal. (1988)
Boraginaceae Phacelia tanacetifolia Yes Anthophora urbana UnknownaBuchmann (1983, 1985)
Calophyllaceae Kielmeyera coriacea Ye s Augochloropsis
spp., Exomalopsis
fulvofasciata, Xylocopa
frontalis, X.hirsutissima
Ye s Oliveira and Sazima (1990)
Calophyllaceae Kielmeyera speciosa Ye s Augochloropsis
spp., Exomalopsis
fulvofasciata, Xylocopa
frontalis, X.hirsutissima
Ye s Oliveira and Sazima (1990)
Clusiaceae Clusia spp. Ye s Augocloropsis spp. Ye s Kaminski and Absy (2006)
Commelinaceae Tradescantia pallida Yes Anthophora spp. Yes A. Russell, personal observation
Cucurbitaceae Cucurbita foetidissima Ye s Xenoglossa angustior Ye s Buchmann (1985)
Ericaceae Diospyros virginiana No Bombus impatiens,
B.vagans
UnknownaC. Switzer, personal communication
Fabaceae Astragalus spp. Ye s Eucera spp. Ye s Z. Portman, personal communication
Fabaceae Desmanthus cooleyi Yes Protoxaea gloriosa Unknown Buchmann (1985)
Fabaceae Lupinus spp. No Bombus spp. Ye s cK. Mauerman, personal communication
Fabaceae Lupinus lepidus var. sellus No Bombus vosnesenskii Ye s cJ. Francis, personal communication
Fabaceae Trifolium spp. Ye s Bombus impatiens UnknownaC. Switzer, personal communication
Fabaceae Vicia spp. Ye s Bombus impatiens UnknownaC. Switzer, personal communication
Fabaceae Coronilla varia No Bombus impatiens UnknownaC. Switzer, personal communication
Fabaceae Swartzia apetala Ye s Bombus spp. Ye s Chiara Moço and Pinheiro (1999)
Fabaceae Swartzia pickelii Ye s Bombus spp. Ye s Machado and Lopes (2004)
Fabaceae Lespedeza bicolor “Natob Strain” No Bombus bimaculatus UnknownaC. Switzer, personal communication
Hypericaceae Hypericum “Hidcote”Ye s Bombus anis,
B.bimaculatus,
B.griseocollis,
B.impatiens,
B.perplexus
Ye s C. Switzer, personal communication
Lamiaceae Stachys recta Ye s Anthophora furcata,
A.quadrimaculata,
Bombus pascuorum,
B.terrestris, Rophites
algirus
Ye s Müller (1996)
Liliaceae Polygonatum x hybridum Yes Bombus pascuorum Ye s Corbet etal. (1988)
Loasaceae Mentzelia pumila Ye s Bombus sonorus Ye s Linsley and Cazier (1963)
Onagraceae Oenothera speciosa Ye s Bombus impatiens Ye s A. Russell, personal observation
Orchidaceae Thelymitra antenniferabYe s Lasioglossum spp. Yes Bernhardt and Burns-Balogh (1986)
Orchidaceae Thelymitra aristatabYe s Lasioglossum spp. Yes Bernhardt and Burns-Balogh (1986)
Orchidaceae Thelymitra nudabYe s Lasioglossum spp. Yes Bernhardt and Burns-Balogh (1986)
Orobanchaceae Castelleja spp. Ye s Bombus sps Ye s D. Papaj and K.Mauerman, personal
communication
Orobanchaceae Melampyrum pratense Ye s Bombus lucorum,
Megachile willughbiella
Ye s Meidell (1944)
Paeoniaceae Paeonia spp. Ye s Bombus spp. Ye s A. Russell, personal observation
Papaveraceae Papaver rhoeas Ye s Bombus terrestris Ye s Raine and Chittka (2007)
Papaveraceae Argemone arizonica Yes Xylocopa californica Ye s Buchmann (1985)
Papaveraceae Argemone spp. Yes Bombus sonorus,
Xylocopa spp.
Ye s D. Papaj, personal communication;
A.Russell, personal observation
Plantaginaceae Chelone Glabra Ye s Bombus vagans, Hylaeus
annulatus
Ye s Richardson and Irwin (2015)
Plantaginaceae Penstemon cyananthus Ye s Osmia brevis Ye s Cane (2014)
Page 3 of 13

Behavioral Ecology
White, James Industry) set to a 14 h:10h light: dark cycle. Colonies
had access to ad libitum 2M sucrose solution and pulverized honey
bee-collected pollen (Koppert Biological Systems) within the forag-
ing arena. Sucrose solution was dispensed via braided cotton wicks
that extended into vials. Pollen was dispensed via custom-made
feeders constructed of chenille fibers glued within 40-dram vials
(Russell and Papaj 2016). Bees always scrabbled for this pollen; fur-
ther, of bees naive to pollen foraging that were observed on their
first few visits to feeders, none sonicated. Bees were also naive to
the pure (i.e., hand-collected) pollen used in experiments: These
pure pollen types were not present in the honey bee-collected pol-
len (Kim Skyrm, Koppert Biological Systems, personal communi-
cation), which moreover does not resemble pure pollen, as honey
bee-collected pollen is adulterated with up to 60% by mass regurgi-
tated crop sugars (Russell and Papaj 2016).
Plants and flowers
We used freshly clipped flowers with mature anthers from 8 poricidal
Solanum houstonii Martyn (synonym S. tridynamum) plants, 2 nonpori-
cidal Begonia x hybrida (“Dragonwing”), and 10 nonporicidal Begonia
descoleana L. B. Sm. and B. G. Schub plants raised in a university
greenhouse and fertilized weekly (Miracle Gro, NPK = 15-30-15).
The anthers of B. hybrida are sterile (pollenless) and made it pos-
sible to precisely control pollen cues presented to bees. Conversely,
B.descoleana, one of the species crossed to make the B.hybrida hybrid,
is fertile and presents pollen on its anthers (its only floral reward).
Likewise, S.houstonii only oers pollen to its pollinators. Agiven trial
used an approximately equal number of flowers from each plant. We
used approximately 2000 flowers in experiments.
General experimental protocol
All trials took place in a foraging arena (L×W×H, 82×60×60cm)
painted gray on floor and sides. In trials, freshly clipped flowers
were displayed horizontally (their natural orientation) on custom-
built water tubes (see Russell, Golden, etal. 2016), to prevent desic-
cation. The water tubes were Velcro-mounted on the arena wall,
facing the flight chamber’s nest entrance. Flowers were arranged on
the wall in a 3×3 Cartesian grid with each water tube spaced 7cm
apart in the horizontal and vertical axes of the grid. Fresh flowers
were used at the start of every trial and for each bee. Flowers were
never reused across trials. We systematically alternated treatments
that belonged to a given experiment (for experiment 1, the subex-
periments) in time to control for eects of day and time of day on
behavior.
Family Genus
Anthers directly
accessible? Sonicating bee(s)
Bees sonicated
stamens? Reference
Plantaginaceae Penstemon radicosus Ye s Osmia brevis Cane (2014)
Plantaginaceae Penstemon strictus Ye s Bombus nevadensis,
Osmia brevis
Ye s Cane (2014)
Ranunculaceae Delphinium spp. Ye s Bombus spp. UnknownaD. Papaj and K.Mauerman, personal
communication
Ranunculaceae Aconitum spp. Yes Bombus spp. Ye s K. Mauerman, personal communication
Ranunculaceae Aquilegia caerulea Ye s Bombus spp. D. Papaj, personal communication
Ranunculaceae Aquilegia chrysantha Yes Bombus occidentalis Unknown Pellmyr (1985)
Ranunculaceae Aquilegia formosa Ye s Bombus spp. Ye s A. Russell, personal observation
Ranunculaceae Cimicifuga arizonica Ye s Bombus huntii, B.
occidentalis
Ye s Pellmyr (1985)
Rosaceae Potentilla recta Ye s Bombus ternarius,
B.terricola
Ye s Heinrich (1976)
Rosaceae Potentilla gracilis Ye s Bombus vosnesenskii Ye s J. Francis, personal communication
Rosaceae Prunus dulcis Ye s Bombus spp. Ye s Thomson and Goodell (2001)
Rosaceae Fallugia paradoxa Ye s Bombus pennsylvanicus Ye s Buchmann (1985)
Rosaceae Rosa “Bucbi”Yes Bombus bimaculatus,
B.impatiens
Ye s C. Switzer, personal communication
Rosaceae Rosa californica Yes Bombus edwardsii,
B.vosnenskii
Ye s Buchmann (1978, 1983)
Rosaceae Rosa multiflora Ye s Bombus bimaculatus,
B.impatiens
Ye s C. Switzer pers. comm.
Rosaceae Rosa nitida Ye s Bombus ternarius Ye s Heinrich (1976)
Rosaceae Rosa rugosa Ye s Bombus terrestris Ye s Dobson etal. (1999)
Rosaceae Rosa virginiana Ye s Bombus pennsylvanicus Ye s Buchmann (1983)
Rosaceae Rubus odoratus Yes Bombus bimaculatus,
B.impatiens
Ye s C. Switzer, personal communication
Rosaceae Rubus parviflorus Ye s Bombus occidentalis Ye s Buchmann (1983)
Solanaceae Physalis philadelphica Ye s Bombus impatiens Ye s C. Switzer, personal communication
Solanaceae Physalis longifolia Ye s Colletes latitarsis Ye s Paine and Roulston (2012)
Theaceae Stewartia sinensis Ye s Bombus perplexus Ye s C. Switzer, personal communication
Verbenaceae Callicarpa cathayana Ye s Bombus impatiens Ye s C. Switzer, personal communication
Verbenaceae Callicarpa dichotoma Ye s Bombus bimaculatus,
B.impatiens
Ye s C. Switzer, personal communication
Verbenaceae Callicarpa japonica Yes Bombus impatiens Yes C. Switzer, personal communication
Zygophyllaceae Kallstroemia grandiflora Ye s Bombus impatiens Ye s A. Russell, personal observation
Zygophyllaceae Guaiacum coulteri Ye s Anthophora spp. Ye s A. Russell, personal observation
List comprises 26 families, 47 genera, and 73 species of angiosperms.
aPossible that sonicates to push deeper into the flower to access nectar?. bRewardless. cPushes open banner petal and then sonicates anthers.
Table1
Continued
Page 4 of 13

Russell etal. • Flexible pollen foraging bybees
To initiate a trial, a single flower-naive individual was intro-
duced into the arena. Bees readily visited all types of flowers, live
and artificial. We recorded landings made by the bee on the flow-
ers. Alanding was defined as taking place when a bee touched the
flower with at least 3 legs simultaneously. Three types of landings
were noted: landings with sonication buzzes, landings with only
scrabbling, and landings without scrabbling or sonication buzzes.
On rare occasions, in experiment 1 and 3 only, bees switched from
scrabbling to sonicating on the same landing: These rare landings
were also classified as landings with sonication buzzes. Sonication
buzzes were identified by their distinctive sound and the stereo-
typed posture of the bees on the flowers (see Russell etal. 2016 for
extended description) and occurred only after a bee had landed.
Scrabbling involved the bee manipulating the anthers with the
mandibles and legs (see Russell and Papaj 2016 for videos and
extended description). Virtually all flower visits where flowers pre-
sented pollen, small 20 μm diameter cellulose powder, or 20μm
diameter plastic microspheres (Supplementary Figure S2) involved
collection attempts (either by sonicating or scrabbling). Bees never
scrabbled when the anthers (surrogate or live) were bare or pre-
sented large 180 μm diameter cellulose powder. We tracked
whether bees landed on previously unvisited flowers or on previ-
ously visited flowers to allow comparison of behavior between these
2 categories. Bees nearly always visited all flowers in an array at
least once. Bees were allowed to make up to 20, 30, or 40 landings
in an array (depending on experiment), after which the trial was
terminated. Atrial was sometimes terminated before the maximum
number of landings if the bee did not forage for a period of 5min.
Most bees (71%) made the maximum number of allowed landings
(bees visiting unrewarding arrays tended not to complete the maxi-
mum number of allowed landings) and all bees were included in
analyses. We euthanized each bee and all its flowers after the bee
completed its trial: A given bee was tested only once in a single
foraging bout (i.e., a single trial).
To facilitate recording of behavior, video for all trials was cap-
tured at 30fps with a high-definition digital camcorder (Canon
VIXIA HF R400) positioned in front of the array. Audio was input
to the camcorder using an external microphone (33–3013 Lavaliere
Microphone, RadioShack) attached to the center of floral arrays.
A Zoom H2 Handy Recorder (ZOOM Corporation) was used to
amplify and verify sonication buzzes in ongoing trials.
Experiments
Experiment1
Here, we sought to determine whether bees buzzed the flowers of
nonporicidal species and the role of pollen availability in mediat-
ing this response. This experiment (composed of 3 subexperiments)
used 54 bees from 7 colonies and each bee was presented with an
array of 9 flowers. In experiment 1a, we used the natural species,
B. descoleana to characterize the normal pollen collection behavior
of bees; in experiments 1b and 1c, we used the pollenless hybrid,
B.hybrida, supplemented with controlled amounts of pollen to pre-
cisely determine how pollen availability aected pollen collection
behavior.
In experiment 1a, bees were each allowed 40 landings. We split
bees in experiment 1b into 2 treatments and allowed each bee 20
landings. In one treatment, B. hybrida flowers were each supple-
mented with 1.5mg pistachio pollen (Pistacia vera; Pollen Collection
and Sales; Lemon Cove, CA) added to their anthers (mean ± SE:
1.52 ± 0.02). Pollen was stored at −18 °C and weighed using a
Sartorius Analytic Balance (Data Weighing Systems, Inc.) to the
nearest 0.1mg. We used pistachio pollen in experiment 1b, because
it was available in large quantities, unlike Begonia pollen. In the
other treatment, B. hybrida flowers were pollenless (their natural
state). In experiment 1c, bees were split into 3 treatments and each
bee allowed 40 landings. Each treatment varied by the quantity of
added pollen: flowers presented 1, 2, or 4 mg of pollen on their
anthers (mean ± SE: 1.0±0.01; 2.0±0.01; 4.0 ± 0.01). We dis-
carded one bee that collected pollen only 5 times. The amount of
pollen added to flowers was within the range that live flowers con-
tain (see Russell and Papaj 2016).
Experiment2
Here, we wanted to determine whether chemical anther cues of
nonporicidal B.hybrida and poricidal S.houstonii elicited sonication
by bees. To this end, we used surrogate flowers made with real
corollas and surrogate foam anthers. Use of the live flower’s corolla
allowed us to assess whether an anther extract applied to the sur-
rogate anthers alone elicited sonication. This experiment used 22
bees from 5 colonies.
We made surrogate flowers by cutting o and discarding the
stamens from the corollas of flowers (Figure 4a, b). Pure pen-
tane or a pentane anther extract was applied to Yellow Fibrecraft
Foam (Jo-Ann Stores, LLC.), cut into cuboids (L×W×H,
1.4×0.2×0.2cm). These surrogate anthers were hot-glued to the
corollas. Surrogate flowers were arranged in a 3× 3 grid without
a central flower (8 total targets), with pentane control and extract-
treated targets alternated by position. See Supplementary Material
for details. We split bees into 2 treatments and allowed each bee
30 landings. Arrays and extracts were made from B.hybrida in one
treatment and from S.houstonii in the other treatment.
Experiment3
We investigated whether mechanical stimulation by odorless par-
ticles, similar in size to pollen, mediated pollen collection behavior
similarly to pollen. This experiment (composed of 2 subexperi-
ments) used B.hybrida and 38 bees from 2 colonies; each bee was
presented with an array of 9 flowers.
In experiment 3a, bees were allocated to each of 3 treatment
groups and each bee allowed 40 landings. Each treatment varied
by the quantity of supplemental small cellulose powder (20 µm
Cellulose Microcrystalline Powder; Sigma Aldrich, St. Louis, MO):
flowers presented 1, 2, or 4mg of cellulose powder on their anthers
(mean ± SE: 0.98±0.03; 2.0 ± 0.02; 3.99 ±0.02). We discarded
one bee that landed on flowers but did not collect any cellulose.
We examined pollen and cellulose powder under a compound
microscope to determine whether cellulose powder physically
resembled pollen and thus was a good proxy. We found that unlike
pistachio, cherry (Prunus avium; Pollen Collection and Sales; Lemon
Cove, CA), or Begonia pollen, 20 µm diameter cellulose powder
(180 µm diameter microcrystalline cellulose powder less so) were
not of uniform size or shape (Supplementary Figure S2). However,
in experiment 3b, assays comparing foraging behavior on arrays
of flowers oering either 2 mg 20 µm cellulose powder (results
from experiment 3a, which were of bees that had been concur-
rently tested) or 2 mg uniformly shaped and sized 20 µm diam-
eter polystyrene microspheres (Supplementary Figure S2c; 20µm
Polystyrene DVB Microspheres; Thermo Scientific, Fremont,
CA) confirmed using cellulose did not aect patterns of behavior
(Figure 5, Supplementary Figure S3d; Welch 2 sample t-test: pro-
portion of visits that involved sonication, 20µm cellulose × 20µm
Page 5 of 13

Behavioral Ecology
microspheres, t11.991 = −0.0428, P= 0.967). We discarded 2 bees
which landed on flowers but did not collect microspheres or cel-
lulose, respectively.
Experiment4
Here, we wanted to find out whether pollen collection behavior
was mediated by pollen-sized particles specifically. This experiment
used B.hybrida and 17 bees from 2 colonies; each bee was presented
with an array of 9 flowers.
Bees were allocated into each of 2 treatments and each bee
allowed 40 landings. In one treatment, flowers each had 2mg of
180 µm diameter microcrystalline cellulose (Avicel PH-200 NF;
FMC Corporation, Philadelphia, PA) added to their anthers (mean
± SE: 2.0± 0.02). These cellulose particles are larger than typical
pollen oered by biotically pollinated plants and collected by bees
(mean diameter: 34µm; Roberts and Vallespir 1978). In the other
treatment, flowers were bare. We discarded one bee that made only
2 landings.
Experiment5
Here, we investigated whether bees that collected pollen from non-
poricidal flowers by sonicating were able to collect it at a faster rate
than those that collected pollen by scrabbling. This experiment
used artificial flowers and 23 bees from 2 colonies. We used artifi-
cial flowers to control the amount of pollen presented and whether
bees would sonicate or scrabble.
Bees were allocated into each of 2 treatments: in one treatment
bees only scrabbled for pollen and in the other treatment bees only
sonicated for pollen. Each bee was allowed 2 landings to each arti-
ficial flower (8 pollen collecting landings per bee), whereupon, the
flower was removed from the foraging arena. While removing the
flower, the bee continued to visit flowers and did not exhibit signs
of being threatened by our activity, such as aggressive behavior or
attempts to escape from thearena.
The artificial flower’s corolla (diameter: 2.8 cm) was made
from purple Fibrecraft Foam, cut with a puncher (Medium Plum
Blossom; Punch Bunch Inc., CO). The surrogate anthers were of
the same design used in experiment 2 and were hot-glued onto the
center of the corolla, such that 2 of the rectangular surfaces faced
up at an angle simultaneously (Figure6a). Cherry pollen (mean mg
per anther ± SE: 0.63±0.05, N=15 measurements) was spread
evenly onto those 2 surfaces.
Flower-naive bees scrabbled for pure cherry pollen on artificial
flowers and never sonicated. To ensure that bees in the comparison
treatment only ever sonicated on artificial flowers, we trained bees
to sonicate in response to a floral odor, prior to visiting artificial
flowers. In this training, bees were allowed to make 8 rewarding
visits to an array of 4 poricidal S. houstonii flowers (pollen is their
only floral reward); sonication is the only behavior these bees
use to extract pollen from the poricidal anthers. After this train-
ing, bees were labeled with unique color combinations of acrylic
paint (painting does not appear to aect pollen collection behav-
ior; Switzer and Combes 2016) and returned to their colony. After
depositing its pollen loads the bee was tested by allowing it to col-
lect cherry pollen from artificial flowers treated with S. houstonii
anther extract. Bees tested in this way always sonicated to collect
pollen. We discarded one bee that completed the training phase
but did not visit the surrogate flowers in the testing phase. Training
compelled bees to sonicate but should not have made bees more
eective sonicators (and thus more ecient pollen collectors than
if they had never sonicated previously): Sonication is fully eective
at first expression and modified little with experience (see Russell
et al. 2016 for details). Bees had extensive experience scrabbling,
but both set of bees were naive to foraging on surrogate anthers
and pure pollen.
To calculate the rate of pollen collection per bee, we divided the
amount of cherry pollen collected by the total time on the flower
(defined by the start of the flower visit until the end of a visit,
summed across all 8 bee landings). Total time on flower was esti-
mated from video footage viewed frame-by-frame with Avidemux
software (fixounet@free.fr). The end of a visit was defined as the
first video frame in which the bee no longer contacted the flower
with its legs. To determine the amount of pollen collected, we
euthanized each bee immediately after it completed its trial and
removed and weighed its pollen load. Because the rate of pollen
collection depends on its individual currency components (i.e., the
amount of cherry pollen collected and time on the flower), we also
analyze these components for dierences among treatments.
Data analyses
All data were analyzed using R v.3.2.0 (R Development Core Team
2010).
Variables being analyzed were a composite of each bee’s
responses; specifically they were proportion variables. We analyzed
dierences in the proportion of landings that included sonica-
tions, in the proportion of landings with sonication per flower type,
and in the rate of cherry collection. When analyzing dierences
between 2 treatments (or 2 flower states) we used t-tests if assump-
tions of normality and equal variance were met (using Shapiro–
Wilk and F-tests, respectively, in the mgcv package: Wood 2016) or,
otherwise, Wilcoxon-signed rank tests. Where we were interested in
patterns across 3 treatments, we used one-way analysis of variance
(Anova) using the aov() function in R.In cases of significant eects,
we ran Tukey’s post hoc test, using the TukeyHSD() function in R,
to determine which pairs were significant.
RESULTS
Experiment 1: bumble bees sonicate the anthers
of nonporicidal flowers but pollen suppresses this
response
Foraging bumble bees flexibly switched between sonication and
scrabbling between floral visits when foraging from nonporicidal
flowers (Figure 2). Bees rarely sonicated previously unvisited flow-
ers of B.descoleana, a nonporicidal species that oers only pollen as
a reward but readily sonicated flowers that had been depleted of
pollen during previous visits (Figure3a, b; Wilcoxon matched-pairs
rank sum tests: first landings vs. repeated, V=36, P<0.0143).
This result is probably an eect of a dierence in pollen availabil-
ity and not of correlated factors, such as a mark left by the bee on
flowers: bees readily sonicated flowers of B.hybrida, a nonporicidal,
pollenless hybrid (Figure3c) and sonicated them on a higher pro-
portion of visits relative to visits to B.hybrida flowers whose anthers
had been supplemented with pollen (Figure3d, e; Welch 2 sample
t-test: pollen versus pollenless, t19.087 = −7.81, P < 0.0001). Even
naive bees on their initial visit only sonicated pollenless flowers and
only scrabbled pollen-supplemented flowers (Supplementary Figure
S3a). Bees sonicated pollen-supplemented flowers more frequently
after they had been depleted of added pollen during previous visits
(Figure3f; Wilcoxon matched-pairs rank sum tests: first landings vs.
repeated, V=45, P<0.0092). Additionally, bees sonicated flowers
Page 6 of 13

Russell etal. • Flexible pollen foraging bybees
more frequently that were initially enriched with smaller amounts
of pollen (Figure 3g; Anova: pollen amount eect: F2,19 = 28.18,
P < 0.0001). In all cases, bees landing on pollen-supplemented
flowers collected that pollen (Supplementary Figure S1b). Bees
never scrabbled on bare flowers.
Experiment 2: chemical extracts of anthers from
nonporicidal or poricidal species elicit sonication
by bumblebees
Bees sonicated on a greater proportion of visits to surrogate flowers
treated with a pentane extract of live anthers versus on visits to sur-
rogate flowers treated with a pentane control (Figure4). Surrogate
flowers consisted of live corollas bearing artificial foam anthers
treated with the pentane anther extract or pentane control. In both
treatments (surrogates and extract either made from nonporicidal,
rewardless B. hybrida, Figure 4a, or from poricidal Solanum housto-
nii, Figure 4b), visits to anther extract-treated surrogates resulted
in sonication significantly more often than visits to surrogates
treated with a pentane control (Wilcoxon matched-pairs rank sum
tests, extract vs. pentane: Figure4c; B.hybrida: V =60, P < 0.019;
Figure4d; S.houstonii: V=5.44, P<0.0039). All bees but one made
their first buzz on an anther extract-treated surrogate flower. Bees
never sonicated corollas and never scrabbled on surrogate flowers.
Experiment 3: pollen-like mechanosensory
stimuli suppress sonication
Patterns of behavior by bees collecting plastic microspheres and cel-
lulose powder (both 20µm diameter particles) similar in size to pol-
len were similar to patterns of bee behavior observed in experiment
1 (Figure 5). Bees were more prone to sonicate B.hybrida flowers
depleted of plastic or cellulose in previous visits (Figure5b; for both
treatments, Wilcoxon matched-pairs rank sum tests: first landings
vs. repeated: V= 28, P< 0.016) and were more prone to sonicate
flowers supplemented with less cellulose (Figure5c; Anova: amount
of cellulose treatment eect: F2,19=40.08, P< 0.0001). Bees visit-
ing flowers supplemented with 20µm plastic or cellulose particles
always collected the particles (Figure 5a, Supplementary Figure
S1c). We used cellulose because it elicited equivalent patterns of
behavior as plastic (Figure5, Supplementary Figure S3d) and much
larger cellulose particles stick to the anthers (unlike larger plastic
microspheres).
Experiment 4: suppression of sonication is
contingent on pollen or particlesize
Cellulose particles (180µm diameter) much larger than pollen typi-
cally collected by generalist bees (Roberts and Vallespir 1978) did
not suppress sonication and bees did not scrabble for these particles,
as they did for the 20µm cellulose powder, plastic microspheres, or
pollen. Specifically, the proportion of landings involving sonication
by bees visiting B.hybrida flowers that presented large cellulose par-
ticles on their anthers was not significantly dierent from that by
bees alighting on unmanipulated bare flowers (Figure5f; Wilcoxon
matched-pairs rank sum tests: 180 µm cellulose versus pollenless,
W=30, P=0.873).
Experiment 5: bees removed pollen at a higher
rate by sonicating than by scrabbling when
nonporicidal flowers presented small amounts
ofpollen
Because bees switched from scrabbling to sonicating when pollen
was depleted on live flowers, we investigated the benefit of using
one versus the other routine when pollen was depleted. Bees that
only sonicated pollen-supplemented artificial (all foam) nonpori-
cidal flowers (Figure 6a) collected pollen at a significantly higher
rate than bees that only scrabbled for pollen (Figure6b; Welch 2
sample t-test: collection rate, scrabble vs. buzz: t17.123 = 2.4693,
P<0.025; N=11 bees per treatment). The dierence corresponds
to a 52% higher pollen collection rate for sonicating bees compared
to scrabblingbees.
Neither the length of time foraging on flowers nor the amount
of pollen collected alone accounted for the significant dierence in
the higher collection rate by sonication over scrabbling. The mean
amount of time spent visiting artificial flowers supplemented with
cherry pollen did not dier between treatments where bees only son-
icated versus treatments where bees only scrabbled (Wilcoxon: time
foraging, scrabble vs. buzz: W=42, P=0.243, mean seconds ± SE:
100(a)
80
60
40
Percent transition type
(mean + SE)
20
0
To same To buzz To scrabble
Transition type
100(b)
80
60
40
20
0
To same To buzz To scrabble
Transition type
Figure2
Mean percentage of landings (± SE) by Bombus impatiens that involved bees either staying with the same collection behavior or switching between collection
routines from one to the next floral visit. Bees either switched from using sonication on one visit to using scrabbling on the next visit (“to scrabble”), from
using scrabbling on one visit to using sonication on the next visit (“to buzz”), or did not switch collection behaviors from one to the next visit (“to same”). (a)
Bees foraging on Begonia descoleana; (b) Bees foraging on B.hybrida supplemented with 1mg of pistachio pollen. N=9 and 7 bees for B.descoleana and B.hybrida
treatments, respectively; data from experiment 1.
Page 7 of 13

Behavioral Ecology
sonicated, 58.79±5.36; scrabbled, 78.50±12.55, N =11 bees per
treatment). Likewise, the amount of cherry pollen collected did not
dier for sonicating bees versus scrabbling bees (Wilcoxon signed
rank tests: amount of pollen collected, pollen collected, scrabble vs.
buzz: W= 87, P=0.0869; mean mg ± SE: sonicated, 2.15±0.30;
scrabbled, 1.47±0.15; N=11 bees per treatment).
Results summary
Our results demonstrate that bees exhibited flexible pollen collec-
tion behavior by switching between routines (floral sonication and
scrabbling). Anther chemical cues from nonporicidal B.hybrida, as
well as from poricidal S. houstonii, elicited sonication behavior in
flower-naive bumble bees. Mechanosensory pollen cues in turn
(b)
(a)
(c)
(d)
80
60
40
Percent lands with sonication
(mean + SE)
20
0
First lands Repeat lands
a
b
Flower state
(f)
50
40
30
20
Percent lands with sonication
(mean + SE)
10
0
First lands Repeat lands
a
b
Flower state
(g)
50
40
30
20
10
0
1mg 2mg 4mg
a
b
c
Amount pollen per flower
(e)
80
100
60
40
Percent lands with sonication
(mean + SE)
20
0
Pollen No pollen
a
b
Treatment
Figure3
Mean percentage landings (± SE) by Bombus impatiens resulting in sonication in treatments where the availability of pollen presented by Begonia descoleana and
B.hybrida was varied. (a) B.descoleana and (c) B.hybrida in its natural state and (d) B.hybrida supplemented with 2mg pollen. Mean percentage landings resulting
in sonication (b) of B.descoleana flowers on the first landings versus repeat landings; (e) of B. hybrida pollenless flowers or pollen-supplemented flowers; (f) of
pollen-supplemented flowers on the first landings to each flower in an array versus repeat landings; and (g) of flowers initially supplemented with 1, 2, or
4mg of pollen. (b) N=9 bees for B.descoleana treatment. (e, f) N=12, 11 bees for the B.hybrida pollenless and pollen-supplemented treatment, respectively. (g)
N=7, 7, and 8 bees for the 1, 2, and 4mg pollen treatments, respectively. Dierent letters above bars indicate significant dierences at P<0.05 according to
a Wilcoxon test, t-test, or Tukey’s post hoc test.
Page 8 of 13

Russell etal. • Flexible pollen foraging bybees
suppressed expression of the sonication routine. Bees more often
buzzed anthers that lacked pollen or had little pollen remaining;
these patterns were the same when pollen was substituted for
pollen-sized (but odorless) cellulose or plastic powder. Bees never
scrabbled when the anthers (surrogate or live) werebare.
Additionally, cellulose particles much larger than pollen typically
collected by generalist bees (Roberts and Vallespir 1978) did not
suppress sonication and bees never scrabbled; accordingly, simply
impeding detection of the anthers by covering them with particles
did not suppress sonication. Bees packed particles (pollen, small
plastic microspheres, small, or large cellulose particles) from B.hyb-
rida in their scopa as they would the pollen of the natural species
B.descoleana (Supplementary FigureS1).
Finally, when anthers presented small amounts of pollen, soni-
cation resulted in a greater pollen collection rate (i.e., weight har-
vested per unit time or per flower) than scrabbling behavior.
DISCUSSION
The consequences of flexibility in generalist pollinator behavior
for the evolution of plant-pollinator mutualisms have been consid-
ered extensively in the context of nectar foraging. Flexible nectar
foraging behavior via learning can facilitate floral trait evolution
(e.g., Sargent 2004; Gómez et al. 2015; Guzmán et al. 2015;
Rojas-Nossa et al. 2016) and species invasions (Harmon-Threatt
etal. 2009; Bartomeus etal. 2016), for example, by allowing polli-
nators to use floral morphologies they have not previously encoun-
tered (e.g., Chittka and Thomson 1997; Bartomeus etal. 2016).
Our findings suggest that generalist bees can also flexibly and
eectively adjust pollen-collecting behavior to make use of diverse
floral resources. These pollinators may thus exert similar impacts
on floral diversity in the context of pollen collection, particularly
as regards the form and arrangement of anthers and associated
structures. For instance, the strongly curved petals of many species
of Senna enhance pollination by sonicating bees via deflection of
expelled pollen onto pollinating spots (e.g., Wolfe and Estes 1992;
Westerkamp 2004). Similarly, bee-pollinated plant species often
present their pollen more gradually and/or divide it among “feed-
ing” and “pollinating” anther sets and thereby optimize pollen
delivery (Castellanos etal. 2006; Vallejo-Marín etal. 2010).
Our results have particularly important implications for the
widespread and repeated evolution of poricidal floral morphol-
ogy from nonporicidal ancestors (Buchmann 1983; Vallejo-Marín
etal. 2010; De Luca and Vallejo-Marín 2013), particularly if pori-
cidal morphology evolves gradually, via a series of intermediate
stages, as commonly presumed for traits (Futuyma 2013; Guzmán
et al. 2015). Under a scenario of gradual evolution, intermediate
stages will have pollen partially concealed to varying degrees (e.g.,
Figure1d–f). Scrabbling alone is ineective at removing unexposed
pollen from such flowers (King and Ferguson 1994; Javorek et al.
2002; Pomeroy and Fisher 2002) and thus such intermediates are
at a potential disadvantage. However, our findings suggest that a
combination of scrabbling and sonication might result in eec-
tive pollen collection, and thus eective pollen transfer, for plants
with anthers of these intermediate types. As pollen is progressively
concealed during this evolutionary process, sonication would be
expressed more and more during pollen collection, until eventu-
ally, when flowers are fully poricidal and pollen entirely concealed,
it would be the only behavior used to extract pollen. To test this
proposed scenario, future work should evaluate whether bees are
(c)
(a) (b)
100
80
60
a
b
40
Percent lands with sonication
(mean + SE)
20
0Begonia extract Pentane
Flower type
(d)
100
80
60
a
b
40
20
0
Solanum extract Pentane
Flower type
Figure4
Mean percentage landings (±MSE) resulting in sonication by Bombus impatiens on anther extract surrogates and on pure pentane treated surrogates made
using nonporicidal Begonia hybrida or poricidal Solanum houstonii. (a) B.hybrida surrogates. (b) S.houstonii surrogates. (c, d) Mean percentage landings (± SE) on
surrogates treated with anther extract or pure pentane resulting in sonication. N= 11 bees per treatment. Dierent letters above bars indicate significant
dierences at P<0.05 according to a Wilcoxon test.
Page 9 of 13

Behavioral Ecology
indeed able to collect and transfer pollen relatively eciently from
plant species that partially conceal their pollen, using a combina-
tion of scrabbling and sonication. Additionally, our results strongly
suggest that rather than there being a relatively tight coevolutionary
relationship between sonicating bees and poricidal floral morphol-
ogy (Macior 1964; Buchmann 1983), floral sonication probably first
evolved in the context of collecting pollen from diverse nonpori-
cidal ancestors, as a strategy for collecting pollen eciently from
partially depleted or just dehiscing anthers (see Raine and Chittka
2007). Subsequently, poricidal morphology evolved repeatedly in
diverse lineages, its evolution facilitated by the pre-existing capacity
of bees to extract pollen via sonication (see Buchmann 1985; De
(b)
(a)
(c)
40
30
20
Percent lands with sonication
(mean + SE)
10
0
First lands Repeat lands
a
b
Flower state
(e)
50
60
40
30
20
Percent lands with sonication
(mean + SE)
10
0
a
b
c
(f)
20
40
60
80
100
0
1mg 2mg 4mg Cellulose No cellulose
Treatment
aa
Amount cellulose per flower
(d)
40
30
20
Percent lands with sonication
(mean + SE)
10
0
First lands Re peat lands
Flower state
a
b
Figure5
Mean percentage landings (± SE) by Bombus impatiens resulting in sonication in treatments where the availability or size of plastic microspheres or cellulose
particles presented by Begonia hybrida was varied. (a) Representative forager that collected plastic microspheres (black arrow). (c) B.hybrida with 4 mg of
20µm size cellulose particles. Mean percentage landings (± SE) resulting in sonication (b) on flowers supplemented with 2mg 20 µm plastic microspheres
on first landings to each flower in an array versus repeat landings; (d) on flowers supplemented with 2mg 20µm cellulose particles on first landings to each
flower in an array versus repeat landings; (e) on flowers initially supplemented with 1, 2, or 4mg of 20µm cellulose particles; and (f) on bare flowers versus
flowers enriched with 180µm cellulose particles. (b) N=7 bees per treatment. (d, e) N=7 bees each for the 1, 2, and 4mg cellulose treatments. (f) N=8 per
treatment. Dierent letters above bars indicate significant dierences at P<0.05 according to a Wilcoxon test or Tukey’s post hoc test.
Page 10 of 13

Russell etal. • Flexible pollen foraging bybees
Luca and Vallejo-Marín 2013) and by the pre-existing occurrence
of sonication elicitor chemistry in emerging poricidalforms.
Our results suggest that flexibility in pollen collection involves
a dierent mechanism than that commonly studied in nectar col-
lection. Whereas bees use instrumental learning to acquire and
refine unique routines specific to particular flower types to extract
nectar (e.g., Lewis 1993; Laverty 1994; Gegear and Laverty 1995),
the flexibility in pollen collection described here involved switch-
ing between 2 seemingly stereotyped motor routines, floral soni-
cation and scrabbling. Switching is regulated by the interacting
eects of 2 floral cues: chemosensory anther cues and mechano-
sensory pollen cues. Importantly, chemosensory anther cues elicited
sonication and mechanosensory pollen cues suppressed sonication
(and elicited scrabbling) by even naive bees on their first floral visit
(Supplementary Figure S3a–c), suggesting that use of either routine
is innately specified.
Although switching did not appear to require instrumen-
tal learning in our study, the eectiveness of collection routines
might nevertheless be altered with experience. To improve pol-
len collection eciency, bees may learn when to switch between
scrabbling and sonicating or adjust subtle characteristics of the 2
routines themselves (e.g., Morgan et al. 2016; Russell etal. 2016).
Sonication has a strong innate component and is aected little by
experience (Russell etal. 2016); however, scant work has exam-
ined whether bees learn to scrabble (see Raine and Chittka 2007).
Furthermore, experience could influence how bees collect pollen
in response to naturally varying floral features other than pol-
len presentation. Across taxa, pollen varies greatly in stickiness,
clumping, size, and surface structure (Roberts and Vallespir 1978;
Hesse 1981; Vaissière and Vinson 1994; Pacini and Hesse 2005;
Lunau etal. 2015). Finally, pollen-foraging bees likely use instru-
mental learning when they must gain access to anthers concealed
within a corolla (e.g., keel flowers; Reinhardt 1952; Westerkamp
and Weber 1999). Bees probably also use appetitive learning in
response to pollen guides, analogous to the role of learning in
nectar guide use (Leonard and Papaj 2011), as well as other flo-
ral features such as color, pattern and scent. However, studies on
instrumental and appetitive learning in pollen foraging are rare,
compared to studies of these processes in nectar foraging (Muth
et al. 2015; Muth et al. 2016; Nicholls and Hempel de Ibarra
2016; Russell, Golden, etal. 2016; Russell, Leonard, etal. 2016).
It is somewhat surprising that sonication results in a higher rate
of pollen removal than scrabbling from nonporicidal flowers given
that scrabbling is the preferred strategy for nonporicidal flowers.
However, we are relatively ignorant of the relative costs of scrab-
bling and sonication. Although we have shown that sonication
results in higher rates of collection of exposed pollen at low lev-
els (when bees would switch from using scrabbling to using soni-
cation on live flowers), we have not assessed what costs might be
involved in achieving these higher rates. For example, sonication is
likely energetically expensive relative to scrabbling: Flight (which,
like sonication, uses indirect flight muscles) is on average 50% more
energetically expensive than walking locomotion (which, like scrab-
bling, involves leg motion) (Balfour etal. 2015). We have also not
demonstrated whether sonication results in higher rates of collec-
tion of exposed pollen at high levels. It would be useful to evaluate
the extent to which energetic and other costs influence the relative
use of sonication and scrabbling, especially on dierent anther
morphologies. Likewise, each component of pollen collection rate
(amount of pollen collected and time spent collecting that pollen)
might conceivably vary on flowering plant species with diering
anther morphology and amounts of available exposed pollen.
Studies of the common floral cues involved in the collection of
nectar have been important in elucidating how shifts from nectar
rewards to deception may evolve (e.g., Raguso 2004; Jersáková
et al. 2006; Pohl et al. 2008). Our results likewise shed light on
repeated and common shifts from pollen rewards to deception and
the evolution of pollen and anther mimicry generally (e.g., Vogel
1978; Schemske and Agren 1995; Lunau 2007). Our evidence
suggests that the floral cues mediating pollen collection behavior
by generalist bees may be simple. These cues may also be widely
shared among angiosperms (e.g., chemical anther cues from at least
2 distantly related species elicited sonication and we have since suc-
cessfully tested many more (A. Russell and D.Papaj, unpublished
data; see also Dobson et al. 1999). Mimicry of anthers by stami-
nodes and pistils (Schemske and Agren 1995; Lunau 2007) may
only require emulation of chemical cues. Similarly, mechanical cues
alone may be sucient to generate pollen mimicry. Consistent with
(a) (b) 0.05
0.04
0.03
0.02
0.01
0.00
Treatment
Scrabble
a
b
Buzz
P
ollen collection rate in mg/sec
(mean + SE)
Figure6
Pollen collection rate for Bombus impatiens using scrabbling versus sonication behaviors to forage from artificial flowers. (a) Nonporicidal artificial foam flower
(pollen is exposed). (b) Pollen collection rate for bees that scrabbled and bees that sonicated. N=11 bees in each treatment. Letters above bars within a panel
indicate significant dierences at P<0.05 according to a t-test.
Page 11 of 13

Behavioral Ecology
this, generalist bees collect pollen-sized particles from flowers, such
as pseudopollen and even fungal spores (e.g., Simpson and Ne
1981; Bernhardt and Burns-Balogh 1986; Eltz et al. 2002; Davies
and Turner 2004).
In closing, examples of learning in mediating flexibility of forag-
ing behavior by animals are common (Kamil and Roitblat 1985;
Krebs and Inman 1992; Papaj and Lewis 1993). However, there is
comparatively little research on mechanisms of flexibility that may
not rely on learning, especially with respect to pollinator behavior.
In this study, we found that pollen foraging generalist bumble bees
were able to cope eectively with variability in their floral resources
by switching between their 2 pollen collection behaviors. The
switching mechanism involved a weighing of 2 types of general
floral cues. Although this mechanism is dierent from learning, it
is analogous in the sense that it prepares generalist bees to forage
even from floral forms they may never have encountered in their
evolutionary history. Bees foraging from flowers constitute a model
system for examining how generalists manage collection of dier-
ent food types on dierent resources. Accordingly, we expect that
generalist taxa, including other invertebrates, as well as vertebrates
that forage on a wide range of biotic resources may also exhibit
flexible foraging not dependent on learning.
SUPPLEMENTARY MATERIAL
Supplementary data are available at Behavioral Ecology online.
FUNDING
This work was supported by the Graduate & Professional Student
Council and the National Science Foundation (IOS-1257762).
The authors are grateful to 2 anonymous reviewers, Judith Bronstein, and
Shayla Salzman for helpful comments, Pollen Collection and Sales (Lemon
Cove, CA) for pollen, to the FMC Corporation for Avicel PH-200 NF, to
Andrew Walzer at Thermo Scientific for microspheres, to Abreeza Zegeer
for greenhouse care, to China Rae Newman and Eleni Moschonas for assis-
tance in running experimental trials, and to Wulfila Gronenberg for access
to microscopes and technical support.
Data accessibility: Analyses reported in this article can be reproduced using
the data provided by Russell etal. (2017).
Handling editor: David Stephens
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