Pollinator recognition by a keystone tropical plant
Matthew G. Betts
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a,1
, Adam S. Hadley
a
, and W. John Kress
b
a
Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR 97331; and
b
Department of Botany, National Museum of Natural
History, Smithsonian Institution, Washington, DC 20013-7012
Edited by James D. Thomson, University of Toronto, Toronto, Canada, and accepted by the Editorial Board February 10, 2015 (received for review Octobe
r
10, 2014)
Understanding the mechanisms enabling coevolution in complex
mutualistic networks remains a central challenge in evolutionary
biology. We show for the first time, to our knowledge, that a
tropical plant species has the capacity to discriminate among floral
visitors, investing in reproduction differentially across the pollina-
tor community. After we standardized pollen quality in 223 aviary
experiments, successful pollination of
Heliconia tortuosa
(mea-
sured as pollen tube abundance) occurred frequently when plants
were visited by long-distance traplining hummingbird species with
specialized bills (
x
pollen tubes
=
1.21
±
0.12 SE) but was reduced
5.7 times when visited by straight-billed territorial birds (
x
pollen
tubes
=
0.20
±
0.074 SE) or insects. Our subsequent experiments
revealed that plants use the nectar extraction capacity of tropical
hummingbirds, a positive function of bill length, as a cue to turn
on reproductively. Furthermore, we show that hummingbirds with
long bills and high nectar extraction efficiency engaged in daily
movements at broad spatial scales (
∼
1 km), but that territorial
species moved only short distances (
<
100 m). Such pollinator rec-
ognition may therefore affect mate selection and maximize receipt
of high-quality pollen from multiple parents. Although a diffuse
pollinator network is implied, because all six species of humming-
birds carry pollen of
H. tortuosa
, only two species with specialized
bills contribute meaningfully to its reproduction. We hypothesize
that this pollinator filtering behavior constitutes a crucial mecha-
nism facilitating coevolution in multispecies plant
–
pollinator net-
works. However, pollinator recognition also greatly reduces the
number of realized pollinators, thereby rendering mutualistic net-
works more vulnerable to environmental change.
coevolution
|
hummingbirds
|
pollinator networks
|
mutualism
|
specialization
T
he remarkable diversity of angiosperms has been, in part,
attributed to the evolution of complex floral structures and
the variety of strategies for sexual reproduction (1). The resulting
increase in genetic mixing facilitates adaptation to changing en-
vironmental conditions, enhances morphological diversification,
and leads eventually to speciation (2).
Much research has focused on the genetic benefits afforded by
long-distance pollen transfer among individuals, particularly in
animal-pollinated plants (3, 4). However, pollen dispersal kernels
are often short, resulting in self-pollination or gene exchange
among closely related individuals (5, 6). In response, plants have
evolved floral traits and attractants that influence the degree of
pollinator specialization (7) and act as filters against inefficient
pollinator services (8) or enhanc
e pollen transport (9). Co-
evolution occurs when evolution of pollinator morphology tracks
these floral changes, which in turn, drives reciprocal changes in
floral traits (10).
Although the potential for coevolution in the most specialized
obligate mutualisms is clear (11), the processes by which co-
evolution occurs in more complex interaction networks remains
a central question in evolutionary biology (10, 12). The striking
fit (trait matching) between morphologies of some plants and
their pollinators in many systems (7, 13) suggests a high degree
of plant
–
pollinator specialization. Such matches between flowers
and groups of pollinators are the basis of the identification of
floral syndromes (or pollination web modules), which link floral
traits with particular types of pollinators (e.g., bird-pollinated
and bee-pollinated) (14, 15). H
owever, observational data of
pollination mutualisms indicate a high degree of generality, even
within floral syndromes (16, 17); plant species may exhibit highly
specialized morphological traits (e.g., long, curved corollas) but
are visited by many pollinators, most of which lack specialized
foraging morphologies (18, 19). The importance of coevolutionary
processes in the presence of many
pollinator species, each of which
may impose conflicting selection
pressures, remains much debated
(20). This situation is exemplified by the species-rich guild of
tropical hummingbirds that shows strikingly high among-species
diversity in bill length, bill curvature, and spatial behavior (21);
however, both specialized and generalized hummingbird species
visit plants with apparently high morphological specialization
(22). How can strong morphological coevolution occur if plants
are visited by many pollinator species with high trait variation?
In this paper, we report a previously unidentified mechanism
that increases the realized specialization between a plant and its
suite of floral visitors. Through a
series of experiments in a tropical
plant
–
hummingbird system, we show that a keystone understory
herb (23),
Heliconia tortuosa
Griggs, has the capacity to discrimi-
nate among floral visitors repre
senting a wide range of morphol-
ogies within a functional group. Flowers of
H. tortuosa
recognize
specialized pollinators that potenti
ally carry high-quality pollen and
are most likely to facilitate mate selection.
Results
Testing the Pollinator Recognition Hypothesis.
Our discovery of pol-
linator recognition was the direct result of original hand-pollina-
tion experiments designed to test the degree of pollen limitation in
H. tortuosa
in relation to landscape fragmentation (23). Pollen
limitation was measured as the di
fference between hand pollination
Significance
By using structural characteristics, such as long tubular flowers,
plants are known to achieve selective visitation by certain
pollinator species. These morphological traits typically arise
over evolutionary timescales. We show for the first time, to our
knowledge, that at least one plant has also evolved the ca-
pacity to recognize pollinator species immediately after visi-
tation, thereby increasing the likelihood that a flower visitor
has delivered high-quality pollen. This novel responsiveness by
the plant leads to functional specialization in an apparently
generalized tropical plant
–
pollinator network. Such specialized
linkages likely facilitate coevolution but also, render pollina-
tion mutualisms more vulnerable to environmental change.
Author contributions: M.G.B. and A.S.H. designed research; M.G.B., A.S.H., and W.J.K.
performed research; M.G.B. and A.S.H. contributed new reagents/analytic tools; M.G.B.
analyzed data; and M.G.B., A.S.H., and W.J.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. J.D.T. is a guest editor invited by the Editorial
Board.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. Email: matt.betts@oregonstate.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1419522112/-/DCSupplemental
.
www.pnas.org/cgi/doi/10.1073/pnas.1419522112
PNAS Early Edition
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ECOLOGY
of covered flowers and those left unmanipulated with full ex-
posure to pollinators (24). We measured pollination using the
abundance of pollen tubes following methods by Kress (25).
Surprisingly, hand-pollinated flowers averaged 5.04 times [95%
confidence interval (95% CI)
=
3.65
–
7.24] fewer pollen tubes
per style than open-pollinated flowers [generalized linear
model (GLM):
t
=
8.36,
P
<
0.0001; hand:
x
=
0.21 (95% CI
=
0.15
–
0.30) tubes per style; open:
x
=
1.08 (95% CI
=
1.01
–
1.16)
tubes per style].
We hypothesized that such decreased plant reproduction after
hand pollination could have two potential causes. The quality of
pollen brought by hummingbirds could be higher (5), reflecting
either longer distance or more genetically mixed pollen transfer
by natural pollinators than in our hand-pollination experiments
(the pollen-quality hypothesis). Alternatively, the plant might dis-
tinguish pollen quality indirectly based on a physical or chemical
cue transmitted by the pollinator (the pollinator-recognition hy-
pothesis). To distinguish these hypotheses, we designed an experi-
ment in which we hand-pollinated flowers as before but introduced
pollen-free clean hummingbird pollinators under controlled aviary
conditions. Under the pollen-quality hypothesis, visitation by a
pollen-free pollinator should not increase pollen tube abundance
in flower styles. Under the pollina
tor-recognition hypothesis,
visitation alone by hummingbirds
—
even in the absence of pollen
on the bird
—
should increase the abundance of pollen tubes over
pollinator-excluded controls.
To test these hypotheses, we captured 148 individual floral
visitors representing six hummingbird species and one common
species of butterfly
—
Anartia fatima
. All species have been ob-
served visiting flowers of
H. tortuosa
. After cleaning humming-
birds of all pollen (
Materials and Methods
), they were released
individually into aviaries containing a single flower that we had hand
pollinated with pollen from 30 to 100 m away. We released but-
terflies into mesh bags covering a hand-pollinated flower. Flower
styles were collected the next day and inspected for pollen tubes
using epifluorescence microscopy (25) (
SI Materials and Methods
).
Hummingbird flower visitation strongly influenced pollen tube
abundance [generalized linear mixed model (GLMM):
F
=
4.57,
P
<
0.0001], supporting the pollinator-recognition hypothesis.
However, this effect was mediated by specific species of hum-
mingbirds (Table 1). For instance, flowers visited by violet sab-
rewings (
Campylopterus hemileucurus
) contained 3.10 times (95%
CI
=
2.33
–
6.17,
x
pollen tubes
=
1.39
±
0.22 SE) more pollen tubes
than green-crowned brilliants (
Heliodoxa jacula
;
x
pollen tubes
=
0.38
±
0.18 SE) and 28.6 times (95% CI
=
21.39
–
56.94) more
pollen tubes than rufous-tailed hummingbirds (
Amazilia tzacatl
;
x
pollen tubes
=
0.04
±
0.04 SE) (Fig. 1). Visitation by
A. fatima
resulted in no pollen tubes (0 of 21). To our knowledge, this be-
havior in
H. tortuosa
is the first evidence that a plant has the
capacity to distinguish among species of floral visitors and re-
spond by preventing (or facilitating) pollen tube growth. We term
this plant behavior pollinator recognition, because unlike previous
forms of pollinator filtering, such as morphological differences in
plant corollas (26
–
28), the mechanism that we report operates in
physiological
–
ecological time rather than on an evolutionary
timescale.
Testing the Mechanism Used in Pollinator Recognition.
Next, we
tested for the cue used by plants to distinguish among polli-
nators. Species of tropical hummingbirds differ strongly in their
capacity to extract nectar from ornithophilous plants (29), and
these differences correspond to the degree of specialization in
hummingbird bill morphology (7, 29). The capacity to distinguish
visitors that carry high-quality pollen loads from those that do
not should be adaptive (5, 30). M
orphological specialists are
more likely to be faithful to a particu
larflowerspecies(27),reducing
the risk of mixed-species pollen loads. Specialization should also
require greater movement distances among plants to acquire
necessary resources (30), thereby increasing the potential for
gene flow. We, therefore, hypothesized that capacity for nectar
extraction is the mechanism used by
H. tortuosa
to recognize the
identity of morphologically specialized floral visitors.
Three lines of evidence support this hypothesis. First, similar
to the work by Wolf et al. (29), we found strong differences among
pollinator species in their capacity to extract nectar (GLM:
F
=
81.30,
R
2
=
0.75,
P
<
0.0001) (Fig. 2
A
and
Table S1
). Bill length
directly reflected nectar extraction capacity of pollinators (
r
=
0.85,
P
=
0.008). Second, the nectar extraction capacity of each
species was correlated with mean pollen tube abundance in ex-
perimentally pollinated flowers (Fig. 2
B
)(
r
=
0.73,
P
=
0.039).
The outlier in this analysis was the scaly-breasted hummingbird
(
Phaeochroa cuvierii
) (Fig. 2
A
and
B
), which in our aviary
experiments, we have only observed robbing nectar by piercing
flower bases. Despite the fact that this species removed high
nectar amounts, the damage that it inflicts on the flower ap-
parently reduces pollen tube growth. Removing this species from
analysis resulted in a stronger correlation between pollinator
nectar extraction capacity and mean pollen tube abundance (
r
=
0.83,
P
=
0.020). Third, experimental extraction of nectar signifi-
cantly increased the abundance of pollen tubes in hand-pollinated
flowers (GLMM:
Z
=
2.53,
P
=
0.011) (
SI Materials and Methods
).
Pollen tubes were 3.67 times (95% CI
=
1.38
–
9.74) more common
when we extracted nectar than when flowers were hand-pollinated
without nectar extraction (Fig. 2
C
)(GLMM:
Z
=
2.67,
P
=
0.008).
Discussion
To our knowledge, these findings provide the first evidence of
pollinator recognition in plants.
H. tortuosa
is capable of dis-
cerning morphologically specialized hummingbird species from
those with generalized traits. Elegantly, the traits themselves
(i.e., long, curved bills) enable some hummingbird species
to extract more nectar, which in turn, is the very cue used by
the plant to become receptive. This plant behavior raises the
question of whether pollinator recognition confers any contem-
porary adaptive advantages to the plant. The fact that it is the
specialized, traplining species that tend to induce pollen tube
Table 1. Results of GLMM predicting differences in pollen tube
abundance as a function of different hummingbird pollinator
species released into aviaries with hand-pollinated
H. tortuosa
Parameter
β
LCI UCI
Z P P-FDR
Intercept: green-crowned
brilliant
−
1.10
−
2.00
−
0.20
−
2.41 0.016
—
Self-pollen
−
0.78
−
1.31
−
0.25
−
2.92 0.004
—
Green hermit* 1.06 0.13 1.99 2.22 0.027 0.049
Rufous-tailed hummingbird
−
2.23
−
4.40
−
0.06
−
2.01 0.045 0.054
Scaly-breasted hummingbird
−
0.16
−
1.90 1.58
−
0.18 0.854 0.561
Stripe-throated hermit
−
0.35
−
1.86 1.16
−
0.46 0.646 0.483
Violet sabrewing* 1.33 0.37 2.29 2.71 0.007 0.015
Intercept: rufous-tailed
hummingbird
−
3.33
−
4.34
−
2.32
−
3.28 0.001
—
Self-pollen
−
0.78
−
1.31
−
0.25
−
2.92 0.004
—
Green hermit* 3.28 2.26 4.30 3.21 0.001 0.004
Green-crowned brilliant 2.23 1.12 3.34 2.01 0.045 0.054
Scaly-breasted hummingbird 2.06 0.79 3.33 1.63 0.104 0.098
Stripe-throated hermit 1.87 0.68 3.06 1.58 0.114 0.098
Violet sabrewing* 3.56 2.53 4.59 3.46 0.001 0.003
We controlled for our two pollen quality treatments statistically by including
self-pollen vs. outcrossed pollen as an indicator variable.
P-FDR
,
P
values that
have been corrected for false discovery rates associated with multiple compar-
isons among species (
SI Materials and Methods
).LCIandUCIarelowerand
upper 95% confidence intervals, respectively.
*Species that differed significantly from the two reference species of green-
crowned brilliant and rufous-tailed hummingbirds.
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Betts et al.
growth suggests that pollinator recognition may increase the
quality of pollen receipt, thereby enhancing fitness. This advan-
tage could occur through three possible mechanisms. First, this
plant behavior could facilitate long-distance gene transfer or at
least, reduce the frequency of self-pollination.
H. tortuosa
is
a clonal herb, with plants often occurring in clumps of
>
10 indi-
viduals (25, 31). This high concentration of nectar resources often
results in defense of a single clonal clump by territorial humming-
birds, which strongly constrains pollen flow (28). However, the large
body size and specialized morphology of traplining hummingbirds
require them to move further to acquire necessary resources (21),
thus potentially facilitating gene flow and enhancing plant fitness
(32). To test this hypothesis, we assembled movement data on all
seven pollinator species (
SI Materials and Methods
). Pollen tube
abundance was positively correlated with the median movement
distance that each species moved within 1 d (Fig. 3
A
)(
F
=
48.98,
R
2
=
0.87,
P
=
0.0004). We also found a strong effect of movement
behavioral strategy on the number of pollen tubes; traplining
species, which regularly move long distances across landscapes to
acquire nectar resources (32, 33), elicited greater pollen tube
abundance than territorial species (6) (Fig. 3
B
)(GLMM:
Z
=
4.52,
P
<
0.0001).
Second, the capacity to recognize specialized pollinators could
also function to maximize the diversity of conspecific pollen re-
ceived. Because of the placement of the anthers in
H. tortuosa
and the foraging position of green hermit and violet sabrewing
(
Movie S1
), these species are more likely, on average, to carry
high pollen loads than nonspecialized species. Our data on hum-
mingbird pollen loads support this hypothesis; traplining species
carried significantly higher individual loads of
H. tortuosa
pollen
(
^
x
=
154.47; 95% CI
=
129.02
–
183.09) than territorial species (
^
x
=
28.78; 95% CI
=
14.15
–
58.55; GLM:
t
=
4.63,
P
<
0.0001). Although
H. tortuosa
ultimately requires only three grains to fertilize all seeds
in the ovary, evidence from other plant species suggests that high
pollen abundance on the stigma increases pollen tube competi-
tion (or potentially, allows greater opportunity for female choice),
thereby increasing the quality of pollen reaching the ovary (34).
Indeed, an increase in the number of pollen donors has been
shown to be positively correlated with seed weight (34).
Third, recognition of morphologically specialized pollinators
might reduce the risk of pollen allelopathy or other types of
pollen interference, whereby nonspecific pollen is deposited on
the stigma by generalist pollinators and interferes with pollen
receipt (35). In our study, both trapliners and territorial hum-
mingbird species carried substantial non-
Heliconia
pollen, and
we found no statistical differences between these groups (GLM:
t
=
1.55,
P
=
0.121). However, on average, trapliners carried lower
loads of non-
Heliconia
pollen grains (
^
x
=
12.55; 95% CI
=
9.58
–
16.44) vs. territorial hummingbird species (
^
x
=
29.96; 95%
CI
=
9.87
–
89.12).
Together, our results suggest that pollinator recognition by
H. tortuosa
facilitates mate selection and is most likely to do so
by (
i
) increased outcrossing caused by receipt of longer-distance
nonself-pollen or (
ii
) enhanced conspecific pollen diversity.
Species in the genus
Heliconia
lack two mechanisms that
promote outcrossing in many flowering plants, namely physio-
logical self-incompatibility and spatial/temporal separation of
sexual function (25). However, the often long and highly curved
flowers of
Heliconia
species, including
H. tortuosa
, are a striking
floral mechanism to screen out certain visitors. In many bird-
pollinated plant species, flower length has been proposed as an
adaptation to allow plants to exclude pollinators that are unlikely
to be carrying high-quality pollen (29). In
H. tortuosa
, the long,
curved perianth reduces but does not preclude visitation by
territorial hummingbirds, which still receive some nectar rewards
(29). Repeated deposition of pollen by such locally foraging
species of hummingbirds would clearly increase the levels of in-
breeding in the plants as well as cause a reduction in the diversity
and abundance of conspecific pollen. Because of the energetic
costs of fruit and seed development (36), a mechanism that
enables a plant to distinguish low- from high-quality pollinators
before investing in seed production would confer a considerable
adaptive advantage.
Plant recognition of pollinators may occur in other plant taxa,
particularly in relatively stable tropical systems with high polli-
nator diversity. One hypothesis is that the fitness gains afforded
by pollinator recognition in terms of increasing mutualist spe-
cialization and possibly, outcrossing rates may have provided an
initial mechanism for corolla lengthening in other plant families.
Minor microevolutionary increases in corolla length should carry
no fitness benefits until they accumulate sufficiently to induce
switches by poor-quality pollinators to alternative flower species
(30). However, if plants use minor differences in nectar extrac-
tion as a cue to indicate visitation by a high-quality pollinator,
corolla lengthening in tandem with pollinator recognition would
have immediate benefits. An alternative hypothesis is that pol-
linator recognition may have evolved to more effectively prevent
A
B
C
Fig. 1.
Results of aviary experiments, where flowers were visited by pollen-
free pollinators after being hand-pollinated under controlled conditions.
(
A
) A bract of
H. tortuosa
with a male green hermit hummingbird. (
B
) Pollen
tubes, the first step in reproduction, viewed in the style of
H. tortuosa
using
epifluorescence microscopy. (
C
) Experimental addition of hummingbirds
strongly influenced the abundance of pollen tubes, but this effect was
species-dependent. Means and SEs are shown.
P
values for contrasts are
reported in Table 1. Green represents those species stimulating pollen tube
growth, and red indicates less-effective species. Butterfly,
A. fatima
; GCBR,
green-crowned brilliant; GREH, green hermit (
Phaethornis guy
); RTAH,
rufous-tailed hummingbird; SBRH, scaly-breasted hummingbird; STHR, stripe-
throated hermit (
Phaethornis striigularis
); VISA, violet sabrewing.
Betts et al.
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ECOLOGY