Plant Ecol (2021) 222:1325–1334

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Association of flower color with pollen reward may explain

increased bumblebee visitation to the Scotch broom yellow
morph
Andrés M. Devegili . Alejandro G. Farji-Brener

Received: 10 December 2019 / Accepted: 5 September 2021 / Published online: 12 September 2021
Ó The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract Given that pollinators usually visit flowers preferences of bumblebees can explain why the yellow
for hidden rewards, they need to rely on floral traits morph is more visited, pollinated, and abundant, while
that indicate reward status (‘‘honest signals’’). How- the other morphs are maintained at a lower frequency.
ever, the relationship between pollination, honest This is one of the few field works that shows that
signals, and floral rewards is little documented in variation in intra-specific floral traits is associated with
natural conditions. The Scotch broom (Cytisus sco- variation in floral reward and pollinator visitation rate,
parius) is an invasive shrub with polymorphism in the helping to understand the foraging preferences of
color of its flowers that can be yellow, orange, or red. pollinators and the coexistence of floral morphs in
In three areas dominated by the Scotch broom, we nature.
described the abundance of the floral morphs and Clinical trials registration: Not applicable.
estimated bumblebee (Bombus terrestris) visitation
rate. We examined whether bumblebee visitation to Keywords Scotch broom  Floral polymorphism 
the floral morphs was related to pollen reward. We Honest signal  Patagonia  Pollination
collected flowers and classified their stamens accord-
ing to their function: reward or pollen export. Then, we
measured anther size and estimated pollen quantity.
The yellow morph was more abundant and more Introduction
visited by bumblebees than the orange and red morphs.
The yellow flowers did indeed offer more pollen than Pollinators often visit flowers in search of hidden
the other morphs and this occurred only for rewarding rewards, therefore, they must rely on floral traits that
anthers, suggesting that bumblebees could use yellow indicate reward status, commonly named ‘‘honest
color as an honest signal to visit the most rewarding signals’’ (Knauer and Schiestl 2015). To attract
flowers. We discuss whether innate and/or learned pollinators, flowering plants use rewards (e.g., nectar
and pollen) and/or attractants (e.g., flower color, size,
symmetry) (Wester and Lunau 2017). Attractants are
Communicated by Shayne Martin Jacobs. easily detectable by pollinators and serve as a guide to
the rewards (Wester and Lunau 2017). When using
A. M. Devegili (&)  A. G. Farji-Brener attractants, plants can benefit by sending dishonest or
LIHO (Lab. de Investigaciones en Hormigas), INIBIOMA
honest signals. Dishonest signals are highly beneficial
(CONICET - UNCOMA), Pasaje Gutiérrez 1125,
Bariloche, Argentina to plants, as flowers are pollinated while pollinators
e-mail: [email protected] are not rewarded (Urru et al. 2011; Lunau and Wester

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2017). Conversely, honest signals are beneficial to flower color polymorphism are excellent models for
both plants and pollinators, as flowers are pollinated studying the importance of flower color in plant
and pollinators are rewarded (Knauer and Schiestl pollination.
2015). The validity of the honest signals depends on Understanding why pollinators show a preference
the variability of flower signals and rewards, the for certain flower colors is relevant to understand both
association of floral signals with the quality or quantity the evolution of flower traits and the maintenance of
of rewards, and the accuracy by which pollinators can the flower color polymorphism (Waser and Price
detect the signals (McLinn and Stephens 2006). Many 1981; Nuttman et al. 2006). Pollinator preferences for
works documented the variability of floral traits and floral traits can be summarized in two mechanisms:
rewards and the diversity of floral visitors associated innate and learned preferences (Lunau and Maier
with floral traits (Pleasants and Chaplin 1983; Rosas- 1995; Haverkamp et al. 2016). On the one hand, innate
Guerrero et al. 2014; Chartier et al. 2016). There is preferences are phylogenetic adaptations of pollina-
also evidence that pollinators have the sensory ability tors that operate before the individual experience with
to perceive the variability of floral traits (Chittka flowers. In this regard, many insects use their visual
1998). However, few studies have tested whether the system to locate flowers (Chittka and Menzel 1992)
floral signals selected by pollinators are honest and show an innate preference for certain flower colors
indicators of reward status in field situations (Ben- (Lunau and Maier 1995; Kelber 1997; Goyret et al.
itez-Vieyra et al. 2010). Knowing which floral signals 2008; Rohde et al. 2013). This innate bias to flower
can honestly indicate reward status and how they are colors is important for inexperienced foragers to locate
used by pollinators in nature is key to understanding their first food reward (Giurfa et al. 1995; Kelber 1997;
the ecology and evolution of plant–pollinator interac- Ings et al. 2009). However, the innate preference of
tions (Knauer and Schiestl 2015). pollinators to particular floral traits is independent of
Floral traits such as size, scent, symmetry, and color the actual reward conditions (Lunau and Maier 1995).
can function as honest signals. For example, the floral On the other hand, learned preferences are temporary
visitors of the wild radish (Raphanus sativus L.) prefer specializations of pollinators toward particular floral
to visit large flowers because this floral trait is traits, and these preferences are dependent on the
positively associated with a greater amount of nectar learning abilities of the species and the reward
and pollen reward (Stanton and Preston 1988). A conditions of food sources (Russell et al. 2016; Giurfa
similar pattern was found in Turnera ulmifolia L. and 2007 and reference therein). In a system comprising
some Salvia spp., where bigger flowers are preferred plants with flower color polymorphism associated
by floral visitors because they offer more nectar with variation in floral reward, the maintenance of the
(Benitez-Vieyra et al. 2010, 2014). Pollinators also floral color polymorphism could be explained by a
show a preference for flowers with scents that indicate learned preference. Although experienced pollinators
better rewards (Knauer and Schiestl 2015). Also, the should target the color morph with the highest reward,
symmetrical flowers of Epilobium angustifolium L., which would cause its prevalence in the population,
which produce more nectar than asymmetrical ones, naive and inexperienced individuals may initially
were preferred by bumblebees (Møller 1995). Among pollinate flowers of all colors. Therefore, the contin-
these traits, intra-specific variation in flower color uous production of new individuals would ensure the
(hereafter flower color polymorphism) is an ideal presence of the less rewarding color morphs in the
scenario to study whether color can function as an population. Furthermore, as memorization is not
honest signal for pollinators. First, flower color is always perfect in pollinators (Keasar et al. 1996),
easily detected by pollinators and acts as a selective experienced individuals may still contribute to ensur-
target for floral visitors (Nuttman et al. 2006; Papiorek ing that less rewarding morphs do not disappear from
et al. 2013). Second, color variation occurs within a the population. Apart from pollination-related mech-
population, so there is no need for phylogenetic anisms, other factors can also be relevant to explain
adjustment. Third, floral morphs are usually found the maintenance of flower color polymorphism in
growing very close sharing similar abiotic and biotic nature, such as edaphic factors (Horovitz 1976),
conditions. Thus, the potential effect of confounding moisture availability (Godoy et al. 1975), shade (Zhao
factors is reduced. For all these reasons, plants with et al. 2012), herbivore pressure (Irwin et al. 2003), and

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mutations affecting pigment biosynthesis (Clegg and floral signal, and (iii) there must be consistency
Durbin 2003). between floral traits and reward. Condition (i) was
Cytisus scoparius (L.) Link (Scotch broom) is a tested in previous research in which flower color was
shrub that presents yellow, orange, or red flowers manipulated evidencing that bumblebees are attracted
(hereafter floral morphs; Fig. 1A, B). In northwestern to the yellow morph because of its color and not
Patagonia (Argentina), these floral morphs show a because of its higher abundance relative to the other
clear pattern in their relative abundances and pollina- morphs (Gavini and Farji-Brener 2015). Conditions
tor visitation rates: (i) the yellow morph is more (ii) and (iii) remain unknown in this system. If our
abundant than the orange and red morphs and (ii) the hypothesis is valid, we expect to find higher pollen
yellow morph is the most visited by the bumblebee rewards in the yellow flowers compared to the orange
Bombus terrestris, the main C. scoparius pollinator in and red morphs [i.e., support for condition (ii)].
the area (Gavini and Farji-Brener 2015). C. scoparius Further, since C. scoparius flowers present division of
flowers only provide pollen as a reward to floral labor in their male sexual parts, with rewarding and
visitors (Paulino et al. 2016). We hypothesize that pollen-export stamens (Paulino et al. 2016), we expect
yellow flowers are more visited than orange and red to find greater pollen content solely in the rewarding
flowers because the yellow color acts as an honest stamens of the yellow morph [i.e., support for
signal for a greater pollen reward. The validity of an condition (iii)].
honest floral signal depends on three conditions:
(i) pollinators must be able to detect the floral signal,
(ii) the floral reward should differ according to the

Fig. 1 A Scotch broom shrub [C. scoparius (L.) Link, morphs; s = banner, w = wing, and k = keel petals; e = pollen-
Fabaceae] and B flower color morphs. C Triggered flower export stamens and r = rewarding stamens. Scale bars: B,
showing the position of stamens. D Comparison of reward and C = 1 cm; D = 0.5 cm. (Color figure online)
pollen-export stamens. a = yellow, b = red, and c = orange

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Methods terrestris has rapidly increased in abundance since

1997 (Aizen et al. 2018) and it is highly successful in
Fieldwork was conducted in three sites (* 1000 m2 opening C. scoparius’ flowers (Stout 2000; Gavini and
each) dominated by C. scoparius in Bariloche, Farji-Brener 2015). The importance of B. terrestris in
Argentina (71° 180 000 W, 41° 90 000 S). Sites were at C. scoparius pollination was evidenced after a
least 500 m from each other. The annual rainfall in the volcanic eruption in 2011 that led to a short-term drop
area is * 1800 mm and the average temperature of B. terrestris and resulted in a reduction in the
ranges from 2 °C in winter to 13 °C in summer. proportion of C. scoparius’ triggered flowers (Morales
C. scoparius is a European invasive plant that is et al. 2014). The explosive mechanism of pollen
widely distributed in northwestern Patagonia (Ar- release means that a closed keel is indicative of an
gentina). Its flowers are zygomorphic and exhibit unvisited flower, while an open keel can be used as a
flower color polymorphism (Fig. 1B, C). Since all the proxy of successful floral visitation (Parker 1997).
flowers of an individual plant corresponded to a Unopened flowers produce no fruit, implying that
unique color morph, hereafter, when we speak of autonomous selfing and apomixis do not occur and
yellow, orange, or red flowers, we are referring to that flower opening by bees is critical to reproductive
plants with that color morph. The floral morphs grow success (Suzuki 2003; Simpson et al. 2005).
intermingled throughout the study area. Flowers have We first corroborated the floral morph abundance
ten stamens, of which (a) four have a long filament and and the floral visit patterns described by Gavini and
large anther, (b) five have a short filament and small Farji-Brener (2015). To estimate the abundance of the
anther, and (c) one has an intermediate-length filament floral morphs in the population, in each of the three
(respect to the others) and a small anther. Flowers only patches we randomly located 3 plots of 10 m2 and
produce pollen as a reward for floral visitors. There is counted all the individuals of each floral morph. To
strong evidence that C. scoparius flowers have a estimate the floral visitation rate of bumblebees, we
division of labor in the male sexual parts: pollen from marked branches with closed flowers of the three floral
long-filament stamens serves male function while morphs (yellow: N = 35, orange: N = 33, and red:
pollen from short-filament stamens serves as a reward N = 35). We waited a week for the pollinators to visit
(Paulino et al. 2016). Therefore, we grouped anthers the flowers and then we selected the first 25 flowers
into two categories: (i) pollen-export anthers (from the from the apex and counted the number of open flowers.
four long-filament stamens) and (ii) rewarding anthers Branches from different plants were located at similar
(from the five short- ? intermediate-filament sta- heights from the ground (* 1.5 m) and freely
mens) (Fig. 1C, D). exposed to potential visits (i.e., they were not hidden
The mechanism of pollen dehiscence in C. scopar- behind the vegetation). Plants with flowers of each
ius is explosive (Suzuki 2003). Initially, the stamens color morph were interspersed in space. The floral visit
and pistil remain subjected to great tension inside the rate was expressed as the percentage of open flowers
keel, and when a pollinator lands on a flower its weight on a branch:
and buzz cause the keel to open, releasing the stamens
Open flowers
and pistil which impact the body of the animal. Floral visit rate ¼  100:
25
Bombus dahlbomii (native), Apis mellifera (exotic),
and, more recently, B. terrestris (exotic) have been To compare the amount of pollen between the floral
reported as effective C. scoparius pollinators (Morales morphs and stamen types, we used the anther area
and Aizen 2002; Aizen et al. 2008; Gavini and Farji- which is a character strongly associated with the
Brener 2015). Many bees (Halictidae, Colletidae), number of pollen grains (see Results). We selected 15
Diptera (Syrphidae, Nemestrinidae), and Coleoptera plants per floral morph and collected 3 flowers per
have been also reported as frequent flower visitors plant (i.e., N = 45 flowers per floral morph). We
(Aizen et al. 2008). In the three study sites (surround- separated and categorized the stamens and attached
ings of S. C. de Bariloche city), B. dahlbomii and A. their anthers with scotch tape to glass slides (N = 450
mellifera were not present, while the invasive B. anthers per floral morph, of which 270 were rewarding
terrestris was highly abundant (A. M. Devegili pers. anthers and 180 pollen-export anthers); the anther area
observations). In NW Patagonia (Argentina), B. was then measured using ImageJ Software (Schneider

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et al. 2012). To examine whether the anther area is a were performed with glm.nb, lmer, and lsmeans
good predictor of the number of pollen grains in a function from MASS, lme4, and lsmeans packages,
subset of anthers we estimated the number of pollen respectively (Bates et al. 2007; Ripley et al. 2013;
grains following Paulino et al. (2016). We collected Lenth 2015). All the analyses were done in R (R Core
ten flowers of each floral morph and from different Team 2015).
plants and then we carefully dissected the petals
(without triggering the anthers) and cut one pollen
export and one rewarding anther from each flower. We Results
took photographs of the anthers and measured their
size using ImageJ Software. To release the pollen The yellow morph was more abundant than the orange
grains from the anther, we put each anther in an and red morphs (Fig. 2A). Field observations allowed
Eppendorf tube with 200 lL of water ? 1 lL of us to confirm that the opening of the flowers was
detergent (surfactant that helps to release pollen) and actively carried out by the bumblebee B. terrestris and
then we vortexed the mixture during 60 s. We placed a that these pollinators rarely visit open flowers. Con-
droplet of 10 lL of the mixture on a glass slide and, sequently, the percentage of open flowers in a branch
using a stereoscopic loupe, we took a photograph of is a reliable estimator of the bumblebee visitation rate
the droplet. We repeated this last procedure three to the floral morphs. Bumblebee visitation rates
times for each mixture and then counted the pollen differed among floral morphs and this difference did
grains in the three droplets using ImageJ. Finally, to not vary across the study sites (GLM, floral morphs: v2
estimate the total number of pollen grains within the = 17.19, df = 2, p \ 0.001; sites: v2 = 1.16, df = 2,
anther, we multiplied the average number of pollen p = 0.56); the yellow morph showed 59 ± 5%
grains in the droplets by its dilution factor. (mean ± SE) of flowers visited, the orange morph
Since flower number, flower size, and plant size 19 ± 4%, and the red morph 21 ± 4% (Fig. 2B).
could influence floral rewards and then floral visitation The number of flowers per plant, flower size, plant
rates (Pleasants and Chaplin 1983), we estimated and size, and the study sites did not influence the area of
included these co-variables in the analyses. To the rewarding anthers (GLMM, v2 = 2.45, df = 2,
estimate the number of flowers per plant, we selected p = 0.29) and pollen-export anthers (GLMM, v2 =
three branches and counted the flowers between the 1.78, df = 2, p = 0.62). The area of the rewarding
apex and 50 cm below. For the flower size, we anthers differed among the floral morphs (GLMM, v2
measured flower height (distance between standard = 18.26, df = 5, p = 0.003; Fig. 3A); with yellow
and keel) and width (distance between wings). Finally, flowers having a bigger rewarding anther area than the
to estimate plant size, we measured plant height and other morphs. The areas of the pollen-export anthers
width (average from three measures) and used the did not vary between the morphs (GLMM, v2 = 0.25,
formula of a cylinder

df = 2, p = 0.88; Fig. 3B).
volume ¼ height  P  radius2 . The areas of the rewarding and pollen-export
To compare the floral visitation rate of bumblebees anthers were positively associated with the number
to the three floral morphs and for the three study sites, of pollen grains, explaining 78% and 63% of its
we used a general linear model with negative binomial variation, respectively (Fig. 4A, B). The number of
error distribution (Zuur et al. 2009). To compare the pollen grains in rewarding anthers was much higher
anther areas between the floral morphs, we used for yellow flowers (3518 ± 253 pollen grains,
generalized linear mixed models, with the floral morph mean ± SE) than the orange and red morphs (orange:
as a fixed effect, plant size, flower size, and flower 1743 ± 325, red: 1753 ± 328). Conversely, the num-
number as co-variables and plant as a random effect ber of pollen grains in pollen-export anthers was
(Zuur et al. 2009). Both homoscedasticity and nor- similar between the morphs (yellow: 9125 ± 1001,
mality were tested. Chi-squares and p values were orange: 8286 ± 1061, red: 7597 ± 828).
obtained by likelihood ratio tests of the full model with
the effect in question against the model without the
effect. GLM, GLMMs, and multiple comparisons

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Fig. 2 A Representativeness of Scotch broom floral morphs in open flowers per 25 flowers counted from the branch apex. Sites
the three study sites. Representativeness is expressed in are not differentiated because the bumblebee visitation rate was
percentages obtained with the abundances of the morphs at not different among the sites for each floral morph. Bars and
each site. B Comparison of bumblebee visitation rate for the whiskers represent mean ± SE. Different letters depict signif-
Scotch broom color morphs. Visitation rate is the percentage of icantly different groups

Fig. 3 Comparison of the anther area of the Scotch broom floral anther area did not vary among the sites for each floral morph.
morphs. Morphs are further separated by A rewarding and Dots and whiskers represent mean ± SE. Different letters
B pollen-export stamens. Sites are not differentiated because the depict significantly different groups

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Fig. 4 Relationship between the anther area and the number of pollen grains for A rewarding stamens and B pollen-export stamens of
the Scotch broom shrub. The floral morphs are represented by colors. Asterisks depict significant linear regressions. ***p \ 0.001

Discussion pollinators can detect those traits. Here we found a

high variability of pollen supply among the floral
Floral traits that indicate reward status (‘‘honest morphs [i.e., support for condition (i)] and a consistent
signals’’) are critical to plant fitness because seed relationship between flower color and reward status
output depends on the ability of pollinators to deliver [i.e., support for condition (ii)]. Concerning condition
pollen while searching for food rewards (Knauer and (iii), it is well known that bumblebees can readily
Schiestl 2015). Evidence for a positive association distinguish the flower colors studied herein (Niesen-
between floral trait variation and associated rewards is baum et al. 1999; Dyer and Chittka 2004) and can
scarce for polymorphic flowers. We found that flower learn to associate particular colors with rewards to
color may function as an honest signal of increased discriminate flowers with different reward qualities
pollen reward for Scotch broom pollinators. The (Waddington 2001; Raine and Chittka 2007). More-
yellow flowers of the polymorphic Scotch broom give over, flower color is the most reliable attractant signal,
more pollen to pollinators than the orange and red even under variable and changing light environments
flowers. This is supported by characteristics of the (Dyer and Chittka 2004). In our study system, color
rewarding anthers, which were 18% larger and had appears to be a key signal for the bumblebee B.
101% more pollen in the yellow morph than the orange terrestris (Gavini and Farji-Brener 2015) to choose
and red morphs, while pollen-export anthers sustained among the more rewarding yellow flowers. Floral
a similar amount of pollen among the three floral scents can also vary between floral morphs (Majetic
morphs. Collectively, results suggest that flower color et al. 2007; Salzmann and Schiestl 2007) and floral
acts as an honest signal in the Scotch broom, scent variation can influence the choice of naı̈ve
explaining the higher rate of pollinator visits to yellow bumblebees such as B. terrestris (Suchet et al. 2011).
flowers and the higher abundance of the yellow morph Although it is clear that the three conditions that
in the population. validate flower color as an honest signal are met in the
In plant–pollinator interactions, the validity of an Scotch broom, it is less clear whether bumblebees
honest floral signal depends at least on three condi- have an innate preference for yellow flowers or they
tions: (i) a variable reward amount offered by flowers learn to associate yellow color with a greater pollen
that differ in certain traits, (ii) a consistency between reward. Knowing which foraging preference operates
floral traits and reward, and (iii) the accuracy by which

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in pollinators can be important for understanding the flowers of the Scotch broom may function as an honest
maintenance of color polymorphism in nature. signal to a higher pollen reward to bumblebees. The
Foraging preferences can be influenced by innate link between flower color and pollen reward may
biases, flower constancy, previous individual experi- explain the higher visitation rate of the bumblebee B.
ence, and/or social information acquired from con- terrestris to the yellow morph and the higher abun-
specifics. Bumblebees in general and our target dance of the yellow morph in the Scotch broom
species in particular do not appear to show an innate population. This is one of the few works that shows in
preference for a single color (Gumbert 2000; Tastard field conditions that variation in intra-specific floral
et al. 2008). For instance, in the study area, B. traits can be related to variation in floral reward,
terrestris is the main pollinator of Alstroemeria aurea, helping to understand the foraging preferences of
which has orange flowers (Aizen and Raffaele 1998). pollinators and the coexistence of floral morphs in
Moreover, variability of a single floral trait as in flower nature.
color polymorphism does not necessarily translate in
flower constancy in bumblebees (Gegear and Laverty Acknowledgements We thank two anonymous reviewers for
their valuable suggestions and recommendations on an earlier
2005). Alternatively, previous experience and/or
version of the manuscript. We thank MA Aizen for his
social learning may also explain the foraging prefer- suggestions on an earlier version of the manuscript. We thank
ences of bumblebees in nature (Goulson 2010). We K Huberman for her help with English. AM Devegili is
hypothesize that a learned preference of B. terrestris to supported by a doctoral fellowship from the Consejo Nacional
de Investigaciones Cientı́ficas y Técnicas of Argentina. This
the most rewarding yellow flowers can explain the research was partially funded by Agencia Nacional de
higher visitation rate to yellow flowers, the higher Promoción Cientı́fica y Tecnológica (PICT FARJI-BRENER
abundance of the yellow morph in nature, and the 2015-1319).
persistence of the orange and red morphs in lower
abundances. First, naı̈ve individuals may visit all the Author contributions AMD and AGFB conceived and
designed the experiments; AMD collected the data and
floral morphs before learning to associate flower color performed the experiments; AMD analyzed the data; AMD
with reward status (Raine and Chittka 2007). Second, and AGFB wrote the manuscript.
as memorization not always persists for the whole
lifetime of bumblebees (Keasar et al. 1996), experi- Funding This research was partially funded by Agencia
enced individuals would still occasionally visit red and Nacional de Promoción Cientı́fica y Tecnológica (PICT FARJI-
BRENER 2015–1319).
orange flowers. And third, experienced bumblebees
would still visit red and orange flowers when they are Data availability The research data are available in the
close to the yellow ones (Gavini and Farji-Brener figshare repository (https://doi.org/10.6084/m9.figshare.
2015). These mechanisms, which are related to the 16567539).
learning abilities of pollinators, could explain why the
Declarations
orange and red morphs are maintained in the Scotch
broom population. Finally, other mechanisms differ- Conflict of interest AMD and AGFB declare that they have
ent from pollination have been proposed to explain the no conflict of interest.
existence of flower color polymorphism, such as
Ethical approval Not applicable.
edaphic factors, moisture availability, sunny vs. shady
habitats, or herbivory (Hannan 1981; Irwin et al. Consent to participate AMD and AGFB participated in the
2003). However, these latter mechanisms may not be research.
relevant for explaining Scotch broom polymorphism,
Consent to publish AMD and AGFB give their consent to
as the floral morphs of C. scoparius grow very close to publish the research.
each other sharing the same abiotic and biotic
conditions. Plant reproducibility Not applicable.
Pollinators visit flowers in search of rewards and,
therefore, to maximize their foraging efficiency they
should select floral cues that indicate reward status, so
called ‘honest signals’ (Knauer and Schiestl 2015).
We presented evidence suggesting that the yellow

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