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ORIGINAL RESEARCH
published: 22 May 2015
doi: 10.3389/fpls.2015.00362
Edited by:
Lawren Sack,
University of California, Los Angeles,
USA
Reviewed by:
Martin Karl-Friedrich Bader,
New Zealand Forest Research
Institute, New Zealand
Adrian G. Dyer,
Royal Melbourne Institute
of Technology University, Australia
Isabela Galarda Varassin,
Universidade Federal do Paraná,
Brazil
*Correspondence:
Vinícius L. G. Brito,
Programa de Pós Graduação em
Biologia Vegetal, Laboratório
de Biossistemática, Department
of Plant Biology, Institute of Biology,
State University of Campinas,
P.O. Box 6109, 13083-970
Campinas, São Paulo, Brazil
viniciusduartina@gmail.com
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This article was submitted to
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Received: 06 October 2014
Paper pending published:
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Accepted: 06 May 2015
Published: 22 May 2015
Citation:
Brito VLG, Weynans K, Sazima M
and Lunau K (2015) Trees as huge
flowers and flowers as oversized floral
guides: the role of floral color change
and retention of old flowers
in Tibouchina pulchra.
Front. Plant Sci. 6:362.
doi: 10.3389/fpls.2015.00362
Trees as huge flowers and flowers as
oversized floral guides: the role of
floral color change and retention of
old flowers in Tibouchina pulchra
Vinícius L. G. Brito1,2*, Kevin Weynans3,4, Marlies Sazima1and Klaus Lunau3
1Programa de Pós Graduação em Biologia Vegetal, Laboratório de Biossistemática, Department of Plant Biology, Institute of
Biology, State University of Campinas, Campinas, Brazil, 2Instituto de Biologia, Universidade Federal de Uberlândia, Minas
Gerais, Brazil, 3Institut für Sinnesökologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany, 4Institute of
Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn, Bonn, Germany
Floral color changes and retention of old flowers are frequently combined phenomena
restricted to the floral guide or single flowers in few-flowered inflorescences. They
are thought to increase the attractiveness over long distances and to direct nearby
pollinators toward the rewarding flowers. In Tibouchina pulchra, a massively flowering
tree, the whole flower changes its color during anthesis. On the first day, the flowers
are white and on the next 3 days, they change to pink. This creates a new large-scale
color pattern in which the white pre-changed flowers contrast against the pink post-
changed ones over the entire tree. We describe the spectral characteristics of floral
colors of T. p u lc h ra and test bumblebees’ response to this color pattern when viewed
at different angles (simulating long and short distances). The results indicated the role of
different color components in bumblebee attraction and the possible scenario in which
this flower color pattern has evolved. We tested bumblebees’ preference for simulated
trees with 75% pink and 25% white flowers resembling the color patterns of T. p ul c h r a ,
and trees with green leaves and pink flowers (control) in long-distance approach. We
also compared an artificial setting with three pink flowers and one white flower (T. p u lc h ra
model) against four pink flowers with white floral guides (control) in short-distance
approach. Bumblebees spontaneously preferred the simulated T. p u l c h r a patterns in
both approaches despite similar reward. Moreover, in short distances, pollinator visits
to peripheral, non-rewarding flowers occurred only half as frequently in the simulated
T. pu l c h ra when compared to the control. Thefore, this exceptional floral color change
and the retention of old flowers in T. p u l ch r a favors the attraction of pollinators over long
distances in a deception process while it honestly directs them toward the rewarding
flowers at short distances possibly exploring their innate color preferences.
Keywords: Atlantic rainforest, attractiveness, bumblebee, color preference, flower–pollinator interaction, mass
flowering
Introduction
Floral traits and their patterns in time and space are major keys to understanding plant–
pollinator interactions and the diversification of angiosperms (Weiss, 1991a;Lunau, 2003;
Frontiers in Plant Science | www.frontiersin.org 1May 2015 | Volume 6 | Article 362
Brito et al. Floral color change in Tibouchina pulchra
Schiestl and Johnson, 2013). Traits like color, scent, size, and
shape mediate these interactions, advertising to the pollinators
the amount and quality of resources and influencing their
behavior (Schemske and Bradshaw, 1999;Handelman and Kohn,
2014). Particularly, flower color patterns are important to
attract pollinators which are visually oriented at long and short
distances, and may affect their flower constancy and preferences
(Lunau et al., 1996;Chittka et al., 1999).
Flower color changes during anthesis associated with
retention of old flowers is a very common and widespread
phenomenon in angiosperms. It occurs in at least 33 orders,
78 families, and 253 genera (Weiss and Lamont, 1997;Suzuki
and Ohashi, 2014). Previous studies have shown that this
phenomenon creates new attractive units that directly influence
the movement of pollinators, favoring both the optimization of
the foraging behavior and plant reproduction (Delph and Lively,
1989;Weiss, 1991b). In general, there are two non-exclusive
concerted hypotheses to explain flower color changes and the
retention of old flowers in angiosperms. For pollinators at long
distances, the size of the total floral display will be increased and,
so will the number of pollinator visits (Gori, 1983;Oberrath and
Böhning-Gaese, 1999). For pollinators at short distances, it is
thought that their foraging efficiency will be improved, so that the
number of superfluous visits decreases (Weiss, 1995), because the
floral color change honestly indicates rewarding flowers (Schaefer
et al., 2004).
However, if the pollinators can discriminate the colors of new
and old flowers at long distances, the effect of the increased
floral display would be worthless, because they can learn to
associate floral traits with the amount and quality of reward
(Lunau et al., 1996;Raine and Chittka, 2007). Without attraction
at long distances, the effects of floral color change at short
distance could be achieved without the costs of the retention of
old flowers because the flower visitors would not need to probe
more flowers when their intention is to visit exclusively pre-
changed flowers. Therefore, the old flowers should be similar to
new flowers at long distances, while the different floral colors
should be discriminable to pollinators at short distances. If this
is true, the effect of floral color change and retention of old
flowers at long distances can be understood as a deception, while
at short distances the same phenomena are honestly signaling
the reward to the visitors. Such an effect should be more evident
in bee-pollinated flowers, because the spatial resolution of insect
compound eyes is very poor and bees will recognize much less
details of flower color patterns and see a rough color pattern
or even a single mixed color dependent on distance (Vorobyev
et al., 1997). Moreover, bees use different sets of photoreceptor
inputs at long and short distances depending on the visual
angleinwhichthetargetisviewed(Giurfa et al., 1996, 1997;
Dyer, 2006;Dyer et al., 2008). In this sense, different color
attributes should also be important in the visual communication
between flowers and pollinators in these plants when we consider
long and short distances (Casper and La Pine, 1984;Lamont,
1985). Thus, we expect that green contrast (Chittka, 1992)
and spectral purity (Lunau et al., 1996), play different roles
in the communication at long and short distances between
plants retaining old flowers with an altered color and their
pollinators.
Flower color change may be the consequence of a pollination
event or be related to the natural senescence of the flower (Gori,
1983, 1989;Pohl et al., 2008). In most of angiosperms, and in
several species of Melastomataceae this change occurs just in
some small parts of the flowers, e.g., the base of the petals, floral
guides, filaments, or ovaries, whereas color change in the entire
corolla is more rare (Weiss, 1991b). In Tibouchina pulchra Cogn.,
a common massively flowering tree of the Atlantic Rainforest, the
color of the whole flower, including petals, stamens, and style,
changes from white on the first day of anthesis, when flowers are
rewarding, to pink on the second to the fourth day, when they
receive few or no visits (Pereira et al., 2011;Brito and Sazima,
2012). This creates a color pattern that covers the entire crown of
the tree, in which the newly opened white flowers are presented
in a pink background of old flowers (Figures 1A,B). On the other
hand, other closely related Tib ouch i na species from the same
phylogenetic clade (Michelangeli et al., 2013) change their color
from white to red in a very small area at the center of the purple
corolla and these flowers are presented in inflorescences scattered
in the green foliage. These differences among T. pulchra and its
FIGURE 1 | Floral color change and retention of old flowers in Tibouchina pulchra.(A)AT. pu l c h r a tree in flowering peak, covered by old pink flowers, and
new white flowers. (B) Close-up showing the pink background made by old pink flowers spotted with new white flowers.
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Brito et al. Floral color change in Tibouchina pulchra
congeners highlights this system as a good model to understand
the evolutionary meanings of flower color change and retention
of old flowers using an experimental design based on a natural
condition.
Here we describe T. pulchra color patterns considering
pollinator’s visual abilities, and add new insights on the
phenomenon of floral color change and retention of old flowers
in angiosperms. Our main goal is to describe the spectral
characteristics of floral colors of T. pulchra, to test flower-visiting
bumblebees’ response to this color pattern at different visual
angles (as a proxy for distance), and to discuss the possible
scenario in which this pattern has evolved. We specifically
address the following questions: (i) how is the color change
viewed when we consider the bee visual system? (ii) is this
floral color change discriminable by the bee visual system? (iii)
how do the relative spectral purity and the green contrast vary
during anthesis? (iv) do these changes in color attributes favor
the attraction of bees at long and short distances? (v) do naive
bees prefer color patterns produced by T. pulchra viewed at long
and short distance over the color patterns shown by congeneric
species?
Materials and Methods
Study Species and Site
Tibouchina pulchra is a common hermaphroditic flowering tree
that occurs in disturbed sites and secondary forests in the Atlantic
Rainforest of Brazil (Leitão-Filho et al., 1993). The large flowers
are heterantherous and herkogamous, produce a weak scent and
interact with bumblebees able to buzz the poricidal anthers and
transfer the pollen to the stigmas of conspecific self-compatible
flowers (Pereira et al., 2011;Brito and Sazima, 2012). This
pioneer species produces many gravity-dispersed seeds with high
germination rates and often quickly colonizes disturbed areas
(Zaia and Takaki, 1998).
The data collection of flower colors was made in the summer
of 2012 at the Núcleo Picinguaba, Serra do Mar State Park,
Ubatuba municipality, located on the northern coast of São Paulo
state, Brazil (23◦20 S, 44◦50 W). The climate is tropical and rainy,
with a super-humid season from October to April (Morellato
et al., 2000). The mean monthly temperature was 21.2◦Candthe
mean monthly precipitation was 174.7 mm between 2011 and
2013 (CPTEC, 2014).
Floral Colors
We took normal color and UV photographs of flowers in each
of the 4 days of anthesis. Afterward, we excluded the red
information provided by normal photography and included the
UV information as follows: we split the three color channels
of normal photographs (blue, green, and red) and replaced
the blue channel by the red channel of UV photography (as
the red sensor is UV-sensitive), the green channel by the blue
channel of the normal photography and the red channel by
the green channel of the normal photography. By this means,
we could discern the floral color patterns of new and old
flowers perceived by the visual system of a bumblebee. All
this procedure employed the sofware Jasc Paint Shop Pro 9.
The analysis of photographs in order to visualize bee-subjective
colors has recently been advanced by Garcia et al. (2014)using
digital images for representation of the spatio-chromatic signal
variability. We also measured the spectral reflection of the bases
andtipsofpetalsfromthefirst,second,third,andfourthdays
flowers from 15 trees. These measurements were made using
a USB4000 spectrophotometer (Ocean Optics, Inc.) coupled
with a deuterium–halogen light source (D2H; World Precision
Instruments, Sarasota, FL, USA) able to emit light between 215
and 1700 nm. All the measurements were taken at an angle of
45◦to the petal surface and at the same direction relative to
the petal (Chittka and Kevan, 2005). We used barium sulfate
as white standard and a black film can as black standard for
recordings of the spectral reflection (Lunau et al., 2011). We
used a standard background of green leaves and a standard
daylight illumination (D65, Wyszecki and Stiles, 1982), as well
as the spectral sensitivity functions of bumblebee (Bombus
terrestris) photoreceptors, to calculate the color locus of each
measurement using the color hexagon, a model to understand
the bee-subjective view of the flowers (Chittka, 1992;Lunau et al.,
2011).
To estimate the ability of bumblebees to discriminate
flower colors, we performed a multivariate analysis of variance
(MANOVA) using position of each flower color measurement
(base or tip) and days of anthesis as fixed factors and the values
of xand yaxis of the color hexagon as the response variable. We
calculated the mean euclidian distance in hexagon units between
the new white flowers and old pink flowers, and also among pink
flowers of different age. As reference, bumblebees can distinguish
correctly by 60% between colors with 0.09 hexagon units of
perceptual distance (Dyer, 2006). From these color loci we also
calculated the green contrast and the relative spectral purity of
the base and tip of flowers from the first to the fourth days
using the same color hexagon model (Chittka et al., 1994;Lunau
et al., 2011). The green contrast is measured as the distance
between the target color locus and the central point of the color
hexagon representing the locus of the standard green background
(Chittka, 1992). On the other hand, the relative spectral purity
is calculated as the proportion between the distance of the color
locus from the center of the hexagon and the distance of the
corresponding spectral locus representing the maximal spectral
purity considering bumblebees’ photoreceptor excitation from
the same point (Lunau et al., 1996). We built generalized least-
squares models considering the flower’s day of anthesis and the
positions of measurement as factors, to explore the differences
in these components of floral color. We visually checked the
standardized residuals vs. the fitted values plot to conclude for
the unnecessity of variance heterogeneity control in such models.
The results of this analysis were compared a posteriori using the
day of anthesis as factor in a pairwise t-test with false discovery
rate (FDR)-controlling procedures (Benjamini and Hochberg,
1995).
Bee Preference Experiments
We performed bumblebee preference tests using a Y-maze
chamber in the laboratory for long- and short-distance color
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Brito et al. Floral color change in Tibouchina pulchra
patterns using artificial paper trees and flowers built with colors
simulating the actual colors of T. pulchra flowers. Each arm
of the Y-maze was 140 cm long and the visual perception
angle in each experiment was adjusted by moving the attraction
units (artificial trees or flowers) back and forward in each
arm (Giurfa et al., 1996). We used two neon tubes (OSRAM
L58W/72-965 run with 30 kHz, providing about 2000 lux) above
each arm of the Y-maze. As white flowers are kept on the
tree during 1 day and pink flowers are kept during 3 days,
we defined a color proportion of 25% white and 75% pink
in all simulations of the color patterns of T. pulchra.Allthe
artificial trees and central flowers used were provided with a 50%
sucrose solution droplet at their center to serve as a reward.
As control treatment we used a common color pattern that
occurs in several congeneric Ti bouch ina species belonging to the
same clade in Melastomae tribe phylogeny (clade J – Eartern
Brazil, sensu Michelangeli et al., 2013)inwhichthebaseof
the petals is white and the tip of the petals is purple (e. g.,
T. heteromalla, T. fothergillae,T. clavata,T. cf. langsdorffiana).
In these species, the flowers change the color just in a very
small area in the center, from white to red, and, despite the
retentionofoldflowers,theydonotflowersomassivelyas
T. pulchra and present a number of discrete inflorescences among
extensive green leaves. Thus we could test whether bees prefer
the color pattern of T. pulchra produced by the massive flowering
and retention of old post-change flowers to a common pattern
produced by flowers with minimal (negligible) color change
and no association to massive flowering. When developing the
experimental setup we considered three behavioral responses
of the bees toward the Ti b ouch i na color patterns. Naive bees
respond due to their innate preferences, but they lose their
naivety as soon as they are rewarded; getting no reward is not
regarded as a punishment for the bees since empty flowers
arecommoninnature(Lunau et al., 1996). Experienced bees
have learnt differences between rewarding and non-rewarding
flowers and are supposed to change their preference accordingly
(Spaethe et al., 2001;Niggebrügge and Hempel de Ibarra,
2003). A fundamental study about trained bees’ response to
novel color stimuli showed that the bees chose novel colors
according to their similarity to the trained color. Only if the
tested colors were so different from the trained color that no
generalization took place, choice behavior was not affected by the
trained color but reflected innate preferences (Gumbert, 2000).
Recently it was demonstrated that trained bees show spontaneous
color preferences only for disctint color attributes, e.g., color
purity, overriding learnt preferences for trained color stimuli,
but not for other color attributes, e.g., dominat wavelength
(Papiorek et al., 2013;Rohde et al., 2013).Hereweassumed
that these spontaneous preferences might be important for bees
in guiding them to rewarding trees as well as to rewarding
flowers irrespective of their experience and the amount of
reward.
In the long-distance experiment, we used a visual perception
angle of 3◦(one paper square of 3 cm ×3 cm at 57.28 cm from
the decision point). The artificial trees were built with 75% of
a pink background representing the old, changed flowers. This
pink background was spotted with 25% of white representing the
new flowers. The control trees were built with 75% of green leaf
background spotted with 25% of pink flowers. In each chamber
arm, the trees were presented against a green background
FIGURE 2 | Experimental settings of long-distance experiment (A) and short-distance experiment (B) to test the bee preferences in a Y-maze
chamber.
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Brito et al. Floral color change in Tibouchina pulchra
simulating the forest where T. pulchra occurs (Figure 2). With the
visual angle of 3◦we simulated the crown of a T. pulchra tree with
5 m diameter viewed from a distance of 95.5 m. For comparison,
a single flower, 5 cm in diameter, can be viewed from a distance
of 96 cm under a visual angle of 3◦.
We used the same scenario to perform the short-distance
experiment with a visual perception angle of 7◦(3 cm diameter
flowers at 24.52 cm from the decision point). Four T. pulchra
flowers, one white central flower circled by three pink peripheral
flowers, were presented against a green background simulating
the green leaves. As control we used four identical flowers
with 75% pink on the periphery and 25% white on the center
(Figure 2). Thus we kept the same color proportion in the
simulation of T. pulchra flowers and in the control treatment. In
this setting, we also tested the number of approaches to peripheral
flowers, because only the central flowers were rewarded with
sugar solution in both treatments.
We made 12 consecutive trials with 10 naive workers of
Bombus terrestris, trained once for each trial in the simulation or
in the control and in the left or right side of the Y-maze chamber,
totalling 120 approaches in long- and short-distance experiments.
Therefore we used the treatment, the training simulation and
the training side of the chamber as fixed factors and the bee
identity as a random factor in generalized linear mixed-effects
models with binomial error distribution. All the statistical tests
were performed using the R 2.15.0 software using the packages
stats,nlme, and lme4 (http://www.r-project.org/).
Results
Floral Colors
Tibouchina pulchra flowers change their color from white to pink
during the 4 days of anthesis (Figure 3). When we considered
the bee-perceivable color spectrum, there was a decrease in
the reflection of green and the flowers become more bee-blue
(without UV-reflection; Figure 3). This change was abrupt and
occured simultaneously in the tip and the base of the petals.
Although there was no difference between the color of the petals’
tip and base (MANOVA, F=0.75, p=0.47), the color of
flowers of different days was different (MANOVA, F=20.24,
p<0.01). The color of these petal parts on the first day occupied a
different locus in the color hexagon when compared to the colors
on the next days of the same petal parts (Figure 4). The mean
distance between the new white flowers and old pink flowers was
0.118 ±0.032 color hexagon units, while the distance among pink
flowers on different days was 0.065 ±0.022 color hexagon units.
There was an interaction between the day of anthesis and
the base and tip of petals when explaining the green contrast
component of flower color (F=9.98, p<0.01; Figure 5A).
FIGURE 3 | Color patterns of T. pulchra flowers from the first to
the fourth days of anthesis. Photos are shown in conventional
photography (red, blue, and green channels) and replacing the blue
channel by the red channel of the UV photography (as the red sensor
is UV-sensitive), the green channel by the blue channel of the normal
photography and the red channel by the green channel of the normal
photography in order to reveal the bumblebee color vision perception.
Average reflectance curves of the base (solid line) and tip (dotted line)
are given for each day of anthesis. Shadow indicates the standard
deviation. N=15 individuals.
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Brito et al. Floral color change in Tibouchina pulchra
FIGURE 4 | Color hexagon coordinates showing the locus of base (B) and tip (T) colors of petals from T. pulchra flowers. Color measurements were
taken on the first (1), second (2), third (3), and fourth (4) days of anthesis (graph model inspired by Suzuki and Ohashi, 2014).
The green contrast decreased during anthesis and the change was
more pronounced in the base of petals. There was also a decrease
in the relative spectral purity of flowers during anthesis (F=4.09,
p<0.01),buttherewasnodifferencebetweenthebaseandthe
tip of petals (ANOVA, F=3.71, p>0.05) and no interaction
between these factors occured (ANOVA, F=0.38, p>0.05;
Figure 5B).
Bee Preference Experiments
The naive bumblebees spontaneously preferred the simulated
artificial tree with original colors of T. pulchra over the tree
simulating the congeneric species (control) in the long distance
experiments (86 approaches, z=2.50, p<0.05; Figure 6A).
This result was not influenced by the tree to which the bee was
trained (54 approaches to the same training tree, z=−1.38,
p>0.05)orthesideoftheY-mazechamberonwhichthebee
was trained (58 approaches to the same training side, z=1.64,
p>0.05). We found a similar pattern for the short-distance
experiments: the naive bumblebee preferred the simulation of
T. pulchra flowers, in which one white flower was circled by three
pink flowers, over the congeneric control flowers (78 approaches,
z=−2.12, p<0.05; Figure 6B). This result was not influenced by
the training flowers (60 approaches to the same training flowers,
z=0.01, p>0.05) or the the training side of the Y maze chamber
(65 approaches to the same training side, z=0.33, p>0.05).
Moreover, the bees approached only 28 peripheral flowers in
the simulated setting of short distance experiments, while they
approached 55 peripheral flowers in the control setting (t-test,
t=−2.12, p<0.05).
Discussion
The floral color change in T. pulchra occurs during the 4 days of
the anthesis and the whole flower changes its color from white
to pink in the human visual system. However, when we consider
the bee visual system the change occurs mostly at the base of
the petals in the green range of wavelength, which becomes less
prevalent in the flower color composition during the anthesis.
The bees cannot easily discriminate old flowers from different
days of anthesis due to the small perceptual distance between
their color loci. However, the new white flowers are distinct
from the old pink flowers when we consider the distance of 0.09
hexagon units as a bee discrimination threshold (Dyer, 2006).
This result indicates that T. pulchra post-changed flowers, when
retained at the treetop, create a background different from that
of green leaves, covering the whole tree where the new flowers
are exposed to pollinators. This pattern should be even more
conspicuous for the bees once the production of green leaves
decreases during the flowering time in T. pulchra and the treetop
is covered almost exclusively by the new and old flowers (Brito
and Sazima, 2012).
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Brito et al. Floral color change in Tibouchina pulchra
FIGURE 5 | Green contrast (A) and relative spectral purity (B) variation
along the 4 days of anthesis in base and tip of petals of T. pulchra
flowers. Bars indicate SE. Letters indicate significant differences (p<0.05)
between the days after pairwise t-test with FRD-controlling procedures.
In general, the retention of old flowers has been suggested
to be a strategy to attract more pollinators at long distances by
increasing the floral display size (Gori, 1983;Weiss, 1991b;Kudo
et al., 2007). On the other hand, bees are unable to resolve the
distinct parts of the color pattern at long distances, which limits
their capacity to discriminate fine color patterns from a uniformly
colored area at long distances (Dafni et al., 1997;Hempel de
Ibarra et al., 2014). Thus, it should be assumed that at long
distances the bees see a single mixed color composed by the
colors of the color pattern and their ratio. The results of the long-
distance experiment indicate that the bees prefer the T. pulchra
over the hypothetical ancestral tree. In this sense, it is noteworthy
that T. pulchra does not display red (or subjective bee black)
as a post-change color as many other flowers including other
Tib o uchi n a species do (Weiss, 1991a; Weiss and Lamont, 1997),
because bees have a very limited ability to see red (Lunau and
Maier, 1995). Since all colors involved absorb ultraviolet light,
white as well as pink is spectrally pure bee-bluegreen, whereas red
would appear similar to the green background color to which the
eyes of the bees are adapted. The spectral reflectance properties of
white and (pale) pink are more similar than that of white and red,
and this should increase the overall attractiveness of the mixed
colors of the tree in the forest gaps. However, it is still no trivial
question whether the progress in attractiveness was mediated by
the color mixed of one quarter white and three quarters pink
of T. pulchra over the hypothetical ancestral color mixed of one
quarter pink and three quarters green or the increased display size
of a colored target object or even an interaction of these factors.
The short-distance experiment also showed that bees prefer
the simulated T. pulchra floral composition to the control, in
which the same amount of white and pink colors was presented.
Moreover, in this experiment the bees made fewer mistakes
(approaches to peripheral flowers) in T. pulchra simulated
flowers, as was foreseen by the short distance hypothesis
to explain floral color change (Weiss, 1995). When floral
color change is associated with retention of old flowers, the
color differences at short distances should be associated with
differences in floral resources to encourage pollinator visits
(Lamont, 1985). Moreover, such color pattern associated with
differences in reward, should be strengthened by the differences
in scent between new and old flowers (Pereira et al., 2011). In
the nectarless T. pulchra flowers the pollen is almost depleted
during visits to the new white flowers, and therefore, visits to old
pink flowers are rewardless and thus rare or non-existent (Pereira
et al., 2011;Brito and Sazima, 2012). When pollinators restrict
their visits to newly opened white flowers, they increase their
efficiency by getting more pollen per visit, besides promoting
pollination and avoiding pollen wastage (Weiss, 1995). Moreover,
the color pattern of T. pulchra should also favor the movement of
bees to longer distances, promoting outcrossing among different
trees (Harder and Barrett, 1995;Sun et al., 2005).
The phenomenon of floral color change in plants is closely
linked to the pollinators’ ability to learn and associate color with
the amount and quality of reward (Lunau et al., 1996;Raine
and Chittka, 2007;Pohl et al., 2008). In T. pulchra,aspecies
strictly dependent on large bees to set fruits (Brito and Sazima,
2012), the floral color attributes should also be important for
the functioning of the strategy of floral color change considering
long and short distances. In addition to the low spatial visual
resolution, bumblees are unable to distinguish colors at long
distances, because they use only the information from green
photoreceptors when the visual angles are lower than 2.7◦(Dyer
et al., 2008). Other bee species might use a deviant critical visual
angle, e.g., for the Western honeybee the critical visual angle
is 15◦(Dyer et al., 2008), which means that a bumblebee is
able to detect a T. pulchra tree from a distance that is more
than five times larger than that of a honeybee. Bumblebees
detect stimuli containing both green-receptor-contrast and color
contrast at a visual angle of approximately 2.3◦, whilst stimuli
that contain only color contrast are only detected at a visual
angle of 2.7◦(Dyer et al., 2008). On the other hand, the
respective viewing angles for honeybees amount to 5◦and 15◦.
The maximal detection distance for a T. pulchra tree possessing
a crown of 5 m diameter for bumblebees via green contrast
amounts 125 and 106 m via color contrast, whereas honeybee
have to approch to 57 m to detect the tree via green contrast
and up to 19 m to detect it via color contrast. Therefore, the
calculation of the maximal detection distance for T. pulchra
trees presumes that bees would detect the grouped flowers
better than single flowers. The grouping of flowers into patches,
experimentally simulated by three spatially separated disks –
similar to the the experimental design in our study – ascompared
Frontiers in Plant Science | www.frontiersin.org 7May 2015 | Volume 6 | Article 362
Brito et al. Floral color change in Tibouchina pulchra
FIGURE 6 | Number of bee approaches in (A) long-distance experiment and (B) short-distance experiment simulating color patterns of T. pulchra.
∗∗∗ <0.005 significance using a logistic regression model. NS, non-significant.
to a single disk improved their detectability by bees and such
improvement of detectability should be stronger for bumblebees
than for honeybees (Wertlen et al., 2008). Thus, in a long-distance
perspective the pattern composed by the new and old flowers
in T. pulchra,aswellastheretentionoftheseflowersinthe
tree as a whole, provides a large, attractive and deceptive object
in the gaps of the forest for the bees. The attractiveness of the
whole tree should be given by the high spectral purity values
of new and old flowers, which also favors the discrimination
of the trees in the green forest background while it does not
indicate the differences between new and old flowers. In fact,
experienced bumblebees exhibit a preference for spectrally purer
colors over trained colors even if the perceptual color distance
is small (Rohde et al., 2013). When this stimulus is perceived at
long distance and an approach is made, the bee vision changes
automatically to a color vision in which all the photoreceptors
are used (Giurfa et al., 1996, 1997;Giurfa and Lehrer, 2001;Dyer
et al., 2008). In this context, the new rewarding and the old
rewardless flowers of T. pulchra can be honestly discriminated
and dominant wavelength, associated with the differences in
the green contrast among flowers from different days, should
be the major mediator of the bee attraction process at short
distances.
In general, bees have an innate preference for high spectrally
pure and contrasting colors and this may explain the color
patterns of flower structures in angiosperms (Lunau and Maier,
1995;Lunau et al., 1996). Mostly, the floral guides display large
visual contrast against the corolla and higher spectral purity than
the corollas. This set is more spectrally pure than the green leaves,
creating a unidirectional color pattern of increase in spectral
purity that may direct the pollinators to the rewarding sites and
reproductive structures of the flower (Lunau, 1996). Floral color
change is also present in other Tibouchina species, in which this
change occurs only in the white base of the petals, while the
periphery remains with the same color, often purple (e. g., T.
heteromalla, T. fothergillae,T. clavata,T. cf. langsdorffiana). This
color pattern creates the unidirectional floral color disposition
in new flowers that is suspended in the old ones. As this color
pattern is very common along the Ti b ouchi na Brazilian clade
(clade J – Eastern Brazil, sensu Michelangeli et al., 2013), we stated
that, in T. pulchra, the retention of old pink flowers together
with massive flowering favored an enlargement of the previous
floral guide to the whole periphery of the corolla. In fact, some
individuals do present a pink color in the petals’ tip of their new
flowers, probably a vestige of a pink corolla. Therefore, T. pulchra
trees completely covered by flowers function as a huge flower
in forest gaps and the new white flowers function as oversized
large floral guides, guiding the pollinators to the reward and the
reproductive floral structures.
The floral color change of T. pulchra is exceptional in multiple
aspects. Floral color change in most flowers is restricted to
floral guides or small flowers in many-flowered inflorescences
(Weiss, 1991b), whereas T. pulchra has large flowers which
change their color in the entire visually signaling apparatus. In
some plants, the floral color change is triggered by pollination
(Gori, 1983;Van Doorn, 2002), but in T. pulchra color change
indicates senescence and is associated with flower duration.
Moreover, flowers dominate the visual display of the entire
plant of T. pulchra, whereas green leaves normally dominate
it in other plants. This has enabled us to use T. pulchra
as a model plant to test experimentally the sustainability
of long distance attractiveness hypothesis for the first time.
Frontiers in Plant Science | www.frontiersin.org 8May 2015 | Volume 6 | Article 362
Brito et al. Floral color change in Tibouchina pulchra
However, it remains open whether the color pattern, the display
size or a synergetic effect of both is responsible for this bee
preference at long distances, because these parameters were
combined in our experimental setup. Future experiments might
disentangle which of these parameters have been important
for the evolution of flower color change and retention of old
flowers. Future studies also might show whether the special
floral color change of T. pulchra was favored by the scattered
distribution of the plants in rare disturbed areas, once it would
favor the attraction of the pollinators from very large distances
matching the average distances between single trees. Since
the visual attention in a complex search task differs between
honeybees and bumblebees (Morawetz and Spaethe, 2012), the
long distance signaling of flowering T. pulchra trees might
represent a strategy to selectively address bumblebees, solitary
foragers that largely rely on their own experience, instead of
mass-recruiting honeybees and stingless bees. Recent studies
have highlighted different search strategies in bees depending
of environmental consitions and intracolony-communication
(Morawetz and Spaethe, 2012;Bukovac et al., 2013); however, it
is still unknown whether tropical bumblebees, the most frequent
flower-visitors and pollinators, possess a distinct search strategy.
In this regard it seems noteworthy that the nectarless flowers
of T. pulchra are not attractive to honeybees that are not
capable of buzzing the flowers for pollen reward. T. pulchra thus
might benefit from adjusting their visual display for pollinating
bumblebees.
In this study, the long-distance experiment demonstrated
that naive bumblebees prefer the simulating trees with color
change over the control model in which there was no color
change or massive flowering. The same preference was found
in the short-distance experiment demonstrating that both long-
and short-distance hypotheses can explain this phenomenon in
angiosperms, as previous studies have shown (Casper and La
Pine, 1984;Delph and Lively, 1989;Weiss, 1991b;Weiss and
Lamont, 1997; Ida and Kudo, 2003). Furthermore, we suggest
that different attributes of floral colors play different roles in
long and short distances regarding the attraction and direction
of pollinators among flowers. Moreover, because we performed
our experiments with naive bumblebees, the results reinforce the
idea that floral color change creates color a patterns that increase
unidirectionally the attractiveness from the greens leaves to the
new flowers. In this sense, floral color change when associated
with retention of old flowers, could be favored in angiosperms by
exploring innate preferences of bees (Lunau, 1996; Papiorek et al.,
2013).
Acknowledgments
We thank the Instituto Florestal (Parque Estadual da Serra do
Mar, Núcleo Santa Virginia and Núcleo Picinguaba) for permits
to study in protected public land (license COTEC/SMA no.
260108-011-806/2010). We thank Sarah Papiorek for the flower
photos, Leonardo R. Jorge and Carlos H. Tonhatti for statistical
support, Pedro J. Bergamo, Ann Thorson, and Prof. Kazuharu
Ohashi for kindly review and improvement of previous versions
of the manuscript. VLGB and MS received grants from FAPESP
(2010/51494-5 and 2012/50425-5) and the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (303084/2011-1).
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Conflict of Interest Statement: The reviewer Adrian G. Dyer declares that, despite
having collaborated with the author Klaus Lunau, the review process was handled
objectively and no conflict of interest exists. The authors declare that the research
was conducted in the absence of any commercial or financial relationships that
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