Journal of
Plant Ecology
Volume 8, Number 2,
Pages 213–223
April 2015
doi:10.1093/jpe/rtv021
available online at
www.jpe.oxfordjournals.org
High ratio of illegitimate
visitation by small bees severely
weakens the potential function of
heteranthery
Jiao-Kun Li1,2, Yun-Peng Song3, Hui Xu1, Yan-Wen Zhang4,
Jian-Yu Zhu1 and Lu-Lu Tang3,*
1
School of Minerals Processing and Bioengineering, Central South University, No. 932, South lushan Road, Changsha
410083, China
2
School of Metallurgy and Environment, Central South University, No. 932, South lushan Road, Changsha 410083, China
3
School of Life Sciences, Central South University, No. 172, Tongzipo Road, Changsha 410013, China
4
Department of Biology, Changchun Normal University, No. 3291, North Changji Road, Changchun 130032, China
*Correspondence address. School of Life Sciences, Central South University, No. 172, Tongzipo Road, Changsha
410013, China. Tel: +86-13755137002; Fax: +86-731-82650230; E-mail: [email protected]
Abstract
Aims
In heterantherous plants, ‘division of labor’ among structurally different stamens, i.e. pollinating and feeding functions, has been
thought to reduce the evolutionary conflict of relying on pollen both
as the carrier of male gametes and as the food for pollinators. The
key to successful division of labor among different sets of stamens is
the size match between stamens and legitimate pollinators, which
results in the precise deposition of pollen onto specific locations
on pollinator’s body and facilitates cross pollination. However, the
potential impact of small illegitimate insects that are ubiquitous during the pollination process on the plant reproduction in heterantherous species has been largely neglected in previous studies and
never been demonstrated experimentally.
Methods
Here, we investigated the functions of three different types of stamens in Commelina communis. The pollinator visitation, pollen
removal and deposition were compared among flowers with different types of anthers emasculated at two natural populations.
Moreover, the mating systems of C. communis in wild populations
were estimated using microsatellite markers.
Important Findings
Our data showed that the main floral visitors for C. communis at
the two studied populations were small illegitimate bees rather than
legitimate pollinators, accounting for 77.5 and 92.2% of total flower
visits, respectively. Flower manipulations in C. communis demonstrated that the two types of brightly yellow stamens separately functioned as ‘deceptive attraction’ and ‘feeding’ functions. Although
the brown inconspicuous stamens of C. communis with the largest
amount of fertile pollen had the potential function in offering pollen
for cross pollination, the high ratio of illegitimate visitation by small
bees significantly affected the dispersal and deposition of pollen
from the pollinating anthers, and subsequently decreased the levels
of outcrossing (tm = 0.23–0.32) in wild populations.
Our work further confirmed that the size match between pollinators
and the floral morphology is the prerequisite to successfully fulfill the
functional differentiation among different sets of stamens in heterantherous plants. Local high ratio of illegitimate visitation by size
unmatched insects could significantly weaken the potential functions
of heteranthery, affecting the dispersal and deposition of functional
pollen in heterantherous plants and further the whole mating systems.
Keywords: Commelina communis, division of labor, heteranthery,
illegitimate visitation, mating system
Received: 14 January 2015, Accepted: 24 January 2015
Introduction
In flowering plants, there are approximately 20 000 plant
species that offer pollen as the only reward for pollinators
(Buchmann 1983). However, if the pollen grains, the vehicle
for the transport of male gametes, are consumed as food by
pollinators, it will inevitably reduce the number of male gametes for cross fertilization and subsequently reduce the plant
© The Author 2015. Published by Oxford University Press on behalf of the Institute of Botany, Chinese Academy of Sciences and the Botanical Society of China.
All rights reserved. For permissions, please email: [email protected]
214
fitness. Thus, it is an evolutionary dilemma for these plants
that they need to protect their precious pollen from the pollinators while maintaining enough attractiveness to pollinators.
Heteranthery, the occurrence of two or more distinct types of
stamens in the same flower, has been thought to reconcile
the evolutionary conflict by allowing structurally different
stamens to specialize in ‘pollinating’ and ‘feeding’ functions,
respectively (‘division of labor’ hypothesis) (Darwin 1899;
Müller 1881, 1882, 1883). The ‘feeding’ stamens mainly satisfy the pollinator’s demand for pollen as food, whereas the
‘pollinating’ stamens are directly involved in cross pollination
and satisfy the plant’s need for safe transport.
Heteranthery is commonly associated with bee-pollinated,
nectarless flowers (Buchmann 1983; Endress 1994; Jesson
and Barrett 2003; Vallejo-Marín et al. 2010; Vogel 1978).
The stamens of heterantherous species usually differ in position, color, size and/or shape within a flower, with two types
being most common (Barrett 2010; Vallejo-Marín et al. 2010).
The first type of stamens (‘feeding’ stamens) is located at the
center in the flower and composed of a few brightly colored
anthers that are easily accessible to pollen-collecting visitors.
The other stamen (s) (‘pollinating’ stamens) with cryptically
colored anther (s) usually displaced away from the central
axis of the flower and is primarily involved in promoting cross
pollination. In addition, some species also possess a third type
of stamens in the floral center, which are slightly larger or staminodes (Luo et al. 2009; Whalen 1979). Despite the ‘division
of labor’ hypothesis has been substantiated in some species
with dimorphic stamens and considered as the most reasonable explanation for the evolutionary significance of heteranthery (Graham and Barrett 1995; Jesson and Barrett 2003;
Luo et al. 2008; Marazzi et al. 2007; Tang and Huang 2007;
Vallejo-Marín et al. 2009), few empirical tests have been performed in species with trimorphic stamens. The function and
evolutionary significance for the third type of stamens is still
unclear.
In addition, compared with other floral rewards, such as
nectar (Cresswell 2001; Klinkhamer et al. 2001; Mitchell and
Waser 1992 ) or heat (Li and Huang 2009; Seymour et al.
2003), it is much more costly and risky to use pollen as the
only reward in heterantherous plants. If the flower visitors are
legitimate pollinators that consume pollen but also transport
it to stigmas of other plants, pollen consumption represents
payment for services rendered and may not actually reduce
the pollination efficiency. By contrast, if the pollen is mainly
collected by illegitimate visitors without pollinating, pollen
consumption can significantly affect the plant reproduction
by reducing siring opportunity directly, or causing pollen limitation at the population level (Hargreaves et al. 2009, 2010).
Unlike some flowers in Lecythidaceae (Mori et al. 1980),
Fabaceae (Westerkamp 1997; Yeo 1993) and Polygalaceae
(Westerkamp 1999) which can use hood-like petals or other
protective structures to prevent access by small visitors, there
is no special mechanism in flowers of heterantherous plants
to avoid the illegitimate insects to access to the pollinating
Journal of Plant Ecology
anthers. In heterantherous species studied before, some small
illegitimate bees has been found to land directly on pollinating anthers (Gross and Kukuk 2001), buzz only individual
anthers and so do not contact stigmas (Renner 1983). Trigona
bees have been found to chew poricidal anthers to access pollen, acting as pollen robbers in many Melastomataceae (Gross
1993; Renner 1983; Snow and Roubik 1987). In contrast to
the extensive studies on the mutually beneficial relationship
between legitimate pollinators and heterantherous plants
(Luo et al. 2008; Tang and Huang 2007; Vallejo-Marín et al.
2009), the potential impact from illegitimate visitors on plant
reproduction have been largely neglected and seldom demonstrated in heterantherous species.
Here, we test the ‘division of labor’ hypothesis in Commelina
communis, a species with trimorphic stamens, including
two long stamens (L-anther), one medium length stamen
(M-anther) and three short stamens (S-anther, staminodes)
(Fig. 1a; Ushimaru et al. 2003a, 2003b, 2007). The flowers
of C. communis do not produce nectar, and pollen is the only
reward for floral visitors (Faden 1992). Unlike buzz-pollinated species that require vibration by bees to release pollen
from poricidal anthers, the anthers of C. communis are longitudinal dehiscence, and therefore generalist insects, involving bees and flies have been reported as the main pollinators
for C. communis (Faden 1992, 2000; Ushimaru et al. 2007).
In experimental arrays, we systematically removed different
types of stamens in flowers of C. communis and quantified the
attractive roles of the three types of stamens through documenting pollinator responses in our experimental arrays. The
subsequent pollen receipt and removal in flowers with different treatments were measured to test the effects of pollinator
visits on male and female reproductive success. In addition,
the variation in pollen viability among structurally different
anthers of C. communis was investigated in our study. Taking
advantage of recently developed microsatellite markers for
C. communis (Table S1, see online supplementary material), we
further estimated the outcrossing rates of C. communis in two
wild populations based on multilocus genotypes in progeny
arrays (Ritland 2002) and addressed the following questions:
(i) Whether morphological differentiation among the three
types of stamens in C. communis is accompanied by division
of function? (ii) Whether illegitimate visitor exists during the
pollination process and if so, how does it affect the mating
system of C. communis?
MATERIALS AND METHODS
Study species and sites
Commelina communis L. (Commelinaceae) is an annual,
andromonoecious weed that often occurs around rice fields and
along roads in temperate northeast Asia. Each plant usually has
many inflorescences that contain both perfect and staminate
flowers. The number of perfect and staminate flowers is equal
and the flowers of both types open at sunrise each day and
last only part of a day before fading (therefore their common
Li et al. | Small bees weaken the function of heteranthery215
Figure 1: artificially manipulated flowers exposed to natural visitors. The abbreviated letter S, M and L in (a) indicate the three different stamens in Commelina communis, representing short, medium and long anther, respectively.
name ‘dayflowers’) (Morita and Nigorikawa 1999; Ushimaru
et al. 2003a, 2003b). The flowers of C. communis show bilateral
symmetry and contain three types of stamens: two long, one
medium and three short stamens. Long and medium anthers
(L-and M-anther) produce fertile pollen, whereas short anthers
(S-anther) produce only a small amount of sterile pollen
(Hardy et al. 2009; Ushimaru et al. 2003a, 2003b). Meanwhile,
C. communis is self-compatible and thought to exhibit delayed
autogamy if the entomophilous pollination is not success at the
end of anthesis (Morita and Nigorikawa 1999).
Field observations and experiments were carried out from
10 to 20 July in 2011 at ‘Caizhuan’ population located in
Changsha, Hunan Province, China (28°12′N, 112°55′E) and
from 15 to 25 August in 2012 at ‘Shenghua’ population,
about 30 km apart away from ‘Caizhuan’ population.
Floral characteristics and sex allocation
To explore the degree of size matching between pollinators
and floral organs, 20 perfect flowers of C. communis were
randomly collected from each population and the lengths of
S-, M- and L-filaments were measured using digital calipers.
Filament length was recorded as the distance between the
pedicel–receptacle junction and the anther–filament junction
(Ushimaru and Nakata 2002); style length was determined in
a similar manner, from the pedicel–receptacle junction to the
stigma. Meanwhile, the linear distances between stigmas and
the three types of anthers were also measured, respectively
(Fig. 1a).
To examine the floral sex allocation, 25 individuals of
C. communis were randomly selected in the ‘Caizhuan’ population. Perfect and staminate flowers in C. communis produce
similar amounts of pollen (Morita and Nigorikawa 1999),
and thus only perfect flowers were studied in this study.
One perfect flower for each individual was collected before
anther dehiscence in the early morning and stored in FAA
solution (formal + acetic acid + 70% ethanol, at 5: 5: 90 v/v)
until examination in the laboratory. The numbers of ovules
and pollen grains per anther type were calculated. Each one
type of anther per flower was split and pollen grains were
suspended in 1 ml of water. The number of pollen grains
in 0.05 ml pollen suspension sample was counted and nine
repeat counts were performed under a microscope. For each
flower, the total number of pollen grains per anther equaled
the average number of pollen grains in 0.05 ml suspension
sample multiplied by 20. Ovaries from all collected flowers
were dissected to determine ovule number per flower with
the aid of a stereomicroscope.
Pollen viability of different types of stamens
To compare the viability of pollen from different types of stamens in C. communis, we measured the pollen germination
rate in vivo rather than in vitro and the pollen tube length after
2 h postpollination. Preliminary studies showed that the short
anthers of C. communis produced only few malformed sterile
pollen grains that are poor contributors for the siring of seeds
(Hardy et al. 2009; Morita and Nigorikawa 1999; Ushimaru
216
et al. 2007). Therefore, only the pollen grains from M- and
L-anthers were tested for this study. Both mature pollen
grains of M- and L-anthers from 20 randomly chosen perfect
flowers in ‘Shenghua’ population were separately hand pollinated onto the receptive stigmas of female flowers, which
were emasculated all stamens and bagged before hand pollination. Pollinated stigmas were cut after 2 h and fixed in FAA
solution for later microscopic observation. Pollen tubes were
measured following the aniline blue method as described by
Dafni et al. (2005).
Pollinator preferences for heteranthery
To explore the function of three different types of stamens
in pollinator attraction in C. communis, we carried out experiments in two populations with > 100 individuals in 2011 and
2012, respectively. Four different types of flowers of C. communis were prepared artificially and exposed to natural visitors (Fig. 1): intact flowers as controls; S-removed flowers
(flowers without S-anthers); M-removed flowers; L-removed
flowers. Treatments were performed on five sunny days when
C. communis was at peak flowering. Two arrays were established each day at c. 10-m intervals. In each array, eight perfect
flowers were randomly subjected to the four treatments. All
floral treatments were carried out in the early morning before
anther dehiscence, and then all treated flowers were bagged
until insects became active at c. 06:00 h. We tested responses
of visitors to these different types of flowers by: (i) recording
the flower type visited; (ii) recording the visitor behavior and
the number of total visitor landings for each type of flower.
Special attention was paid to whether visitors contacted the
stigmas. Observations were continuously monitored for about
4 h every day and ended c.10:00 h, when no insect came to
visit. All visitors were photographed and sample specimens
were captured for identification.
To examine the effect of pollinator visits on male and
female reproductive success, pollen removal from brown
L-anthers and yellow M-anthers and pollen receipt per stigma
in flowers with different treatments were measured. After
visitor activity ceased for 2 h, we collected the flowers that
had been subjected to the above treatments. Brown L-anthers
and yellow M-anthers were stored separately in FAA solution
in centrifuge tubes and brought back to the laboratory. The
number of pollen removed was equal to the mean number
of pollen grains produced by L- and M-anthers (see ‘floral
sex allocation’) minus the corresponding remaining pollen
number. The pistil of each flower with S-, M- and L-removed
was separately fixed in FAA solution and stained with aniline blue. Pollen grains deposited on the stigma were counted
under a fluorescence microscope.
Mating system
Sampling design
The fruit of C. communis is a dehiscent, ellipsoid capsule with
two locules each containing two seeds (Hong and DeFillipps
2000).The mature seeds easily fell down to the ground from
Journal of Plant Ecology
the dehiscent fruits and were difficult to collect all seeds from
per individual, therefore, we tried to sample four to seven
mature seeds per plant from up to 20 randomly chosen individuals per population, with at least 10 m apart from each
other. A total of 194 progeny plants and 40 maternal plans
were included in the experiment. All seeds belonging to a
mother plant were grouped as a family. Seeds from the maternal plants were geminated in soil in the glasshouse at Central
South University. Fresh leaves sampled from maternal plants
in the field and from young seedlings (2–3 weeks post germination) in the glasshouse were dried in silica gel. Total
genomic DNA was extracted using a modified CTAB protocol
(Doyle and Doyle 1990).
Simple sequence repeat analysis
For mating system analysis, 40 polymorphic microsatellite loci
were developed as described by Zane et al. (2002). Of those 40
microsatellites, six were polymorphic and selected to use in
this study (Table S1, see online supplementary material). PCR
amplification conditions for all loci were as follows: A denaturing period of 5 min at 94°C, followed by 35 cycles of 30 s at
94°C, 45 s at 53–57°C (the melting temperature was specific
for different markers), 45 s at 72°C, then 12 min at 72°C for
final extension. The PCR reactions were performed in 20 μl
of reaction volume containing 0.25 mM of dNTPs, 2 μl of
10 × Taq buffer (10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2 and
50 mM KCl), 0.2 μM of each primer, 2 units of Taq polymerase (TaKaRa Inc.) and 50 ng of genomic DNA. We checked
PCR products with a 3% agarose × TBE electrophoresis, and
sent them to DNA sequencing and services (Beijing Bio-ulab
Co., Ltd, China) for fragment analysis in an ABI 3730xl capillary sequencer with a GeneScan 500 LIZ internal size standard (Applied Biosystem).
Data analysis
To analyze the effect of different types of stamens on pollinator visitation, pollen removal and pollen receipt, one-way
analysis of variance (ANOVA) was performed. The differences
in pollen viability and pollen tube length between the two
types of stamens were compared using Student’s t-tests. All
statistical analyses were performed using IBM SPSS Statistics
Version 21.0 (IBM Corp., Armonk, NY, USA). Throughout this
report, all means are accompanied by their standard deviation
unless otherwise indicated.
MICRO-CHECKER version 2.2.3 (van Oosterhout et al.
2004) was used to estimate the frequencies of null alleles in the
microsatellite markers. The number of alleles per locus (Na),
observed heterozygosity (Ho) and expected heterozygosity (He)
were estimated in GENEPOP version 4.0.10 (Rousset 2008).
The mating system was analyzed according to the mixed
mating model of Ritland and Jain (1981) using MLTR software (Ritland 2002). The following parameters at population level were calculated: (i) multilocus outcrossing rates
(tm); (ii) single-locus outcrossing rates (ts); (iii) the difference
between multilocus and single-locus estimates of outcrossing
Li et al. | Small bees weaken the function of heteranthery217
(tm − ts), that is often used to characterize the level of biparental inbreeding (the outcrossing resulted between closely
related individuals) rates (Ritland 2002); and (iv) the correlation of outcrossed paternity within progeny (rp). In addition,
the number of pollen donors contributing to each family was
estimated as 1/rp (Ritland 1989). The inbreeding coefficient
of maternal parents (Fm) was also calculated using the MLTR
software (Ritland 2002). The maximum likelihood was used
to estimate all the parameters with 1000 bootstraps.
RESULTS
Floral traits and sex allocation
In C. communis, the filament of L-stamen was nearly as long as the
style and was longer than that of the central S- and M-stamens
(Fig. 1a; Table S2, see online supplementary material). Compared
with the two types of yellow anthers, the lateral L-anther was
much closer to the stigma (Fig. 1a; Table S2 , see online supplementary material). There was no significant difference on the number
of pollen grains produced by per L- (1416.4 ± 256.2) and M-anther
(1291.2 ± 288.2). However, the S-anther contained significantly
less pollen grains (33.8 ± 22.6) than did both L-and M-anther (F2,
72 = 293.88, P < 0.0001). The number of ovules was four in each
flower, and the pollen/ovule ratio therefore was 1031.0 ± 194.1.
In vivo pollen germination rate and pollen
tube growth
The pollen grains from both M- and L-anthers have germinated on the receptive stigmas in C. communis, even only 2 h
after pollination (Fig. 2a and b). The mean rates of germination for pollen grains of M- and L-anthers were 74.1 ± 23.1 and
74.9 ± 12.9%, respectively. There was no significant difference
in germination rate between these two types of pollen grains
(t = −0.14, P = 0.89; Fig. 2c). In addition, the mean length of
pollen tubes at 2 h after pollination was 87.5 ± 15.9 μm for
M-anthers and 86.0 ± 26.0 μm for L-anthers, respectively, and
no significant difference was observed between these two figures (t = 0.22, P = 0.83; Fig. 2d).
Pollinator behavior on different types of anthers
Three types of insects, including Lasioglossum sp., Halictus
sp. and syrphid flies were observed to visit the flowers of
Figure 2: in vivo pollen germination rate and pollen tube growth on stigmas of Commelina communis after 2 h hand pollination. (a) pollen
grains from M-anthers germinated on the receptive stigmas (M-stigma). (b) pollen grains from L-anthers germinated on the receptive stigmas (L-stigma). Values (mean ± SD) with the same superscript letters in (c) and (d) indicate that differences are not significant (P > 0.05).
Abbreviations: pg (pollen grain) and pt (pollen tube). Scale bars 100 μm.
218
C. communis at both sites. Lasioglossum sp. was the most frequent visitor in our experiment, accounting for 77.5% (310)
of total flower visits (400) in ‘Caizhuan’ and 92.2% (368)
of total flower visits (399) in ‘Shenghua’. Flower visits by
Halictus sp. and syrphid flies were much less frequent compared with Lasioglossum sp., accounting for 16.0 and 6.5% in
‘Caizhuan’ and for 3.5 and 4.3% in ‘Shenghua’. The straight
body length (from the head to the tail) of Lasioglossum sp. was
6.27 ± 0.11 mm (n = 15), which was shorter than the distance
between S- or M-anther and stigma (Table S2, see online
supplementary material). Although Lasioglossum sp. usually
alighted on the filaments of M-stamens and collected pollen
grains from M-anthers, they rarely touched the stigmas of
C. communis due to their small body size (Fig. 1b). Meanwhile,
Lasioglossum sp. was also observed to directly collect pollen
grains from the L-anthers (Fig. 1c).
The visitation rates were similar in ‘Caizhuan’ and
‘Shenghua’ during 2011 and 2012 (Fig. 3a and b). Compared
with intact flowers (Control), both S- and M- removed flowers received significantly fewer visits. By contrast, L-removed
flowers received as many visits and were as attractive to pollinators as intact flowers (Fig. 3a and b; F3, 76 = 10.85, P < 0.01
for ‘Caizhuan’; F3, 76 = 3.42, P = 0.02 for ‘Shenghua’). There
was no significant difference on pollinator preference between
S- and M-removed flowers at both sites (Fig. 3a and b).
Pollen deposition and removal
In both populations, flowers with L-anther removed received
significantly fewer pollen grains than did intact flowers,
while removal of S- and M-anther had no effect on pollen
receipt (Fig. 3c and d; F3, 76 = 7.11, P < 0.01 for ‘Caizhuan’; F3,
76 = 3.24, P < 0.01 for ‘Shenghua’). Moreover, almost all of pollen grains in both M- and L-anthers were removed by pollinators in two populations (Fig. 3e and f). The number of pollen
grains removed from L-anthers was not significantly different
between intact flowers and M- or S-anther removed flowers
in two populations (Fig. 3e and f; F2, 57 = 0.54, P = 0.58 for
‘Caizhuan’; F2, 57 = 1.92, P = 0.16 for ‘Shenghua’). Similarly,
there was no significant difference between the intact flowers and flowers with L- or S-removed in pollen removal from
M-anthers in population of ‘Caizhuan’ (F2, 57 = 1.00, P = 0.37)
and ‘Shenghua’ (F2, 57 = 1.90, P = 0.16).
Outcrossing rate and mating system parameters
The populations of C. communis studied here showed relatively low outcrossing rates (Table 1). The average multilocus outcrossing rate (tm) was 0.281 ± 0.066, ranging from
0.234 ± 0.065 in ‘Shenghua’ to 0.318 ± 0.112 in ‘Caizhuan’,
indicating a mixed mating system with a predominance of
self-fertilization in C. communis (Table 1). The estimates of tm
− ts values were positive for the species (0.165 ± 0.039) and
populations (0.141 ± 0.041 and 0.179 ± 0.060, respectively, for
‘Shenghua’ and ‘Caizhan’), indicating that biparental inbreeding contributed to the selfing rate of the C. communis populations (Table 1). The maternal inbreeding coefficient (Fm)
Journal of Plant Ecology
agreed with the other estimates indicating the presence of a
relatively high inbreeding level in the species (0.365 ± 0.028)
(Table 1). In addition, the correlation of outcrossed paternity
within progeny (rp) in the two populations was very high
(0.999 ± 0.738 and 0.837 ± 0.340, respectively for ‘Shenghua’
and ‘Caizhuan’), indicating the populations studied here possessed a low number of pollen donator parents (Table 1).
Discussion
Our data indicated that the three different types of stamens
in C. communis were responsible for different functions and
played different roles during the pollination process. The
brightly yellow S-anthers with little sterile pollen mainly
functioned as ‘deceptive attraction’ for pollinators (Hardy
et al. 2009; Hrycan and Davis 2005) and the M-anthers with a
large number of high viability pollen grains were considered
as feeding anthers, providing pollen as ‘food’ for pollinators
(Ushimaru et al. 2007). Although the brown inconspicuous
L-anthers produced the largest amount of fertile pollen and
had the potential role in offering pollen for cross pollination,
the high ratio of illegitimate visitation by small bees could significantly affect the dispersal and deposition of pollen from the
L-anthers in C. communis, decreasing the levels of outcrossing
(tm = 0.23–0.32) in wild populations and severely weakening
the potential functional significance of heteranthery.
Functional differences among the three types of
anthers
The similar pollinator visiting frequencies between intact
flowers and flowers without L-anthers in C. communis at two
populations demonstrated that the brown L-anthers did not
play a direct role in attracting the pollinators (Fig. 3a and b).
By contrast, the brightly yellow anthers (S- or M-anthers)
were the main factor to affect the pollinator visitation and
functioned as flower attraction for pollinators (Fig. 3a and
b). Although staminodes have been shown to have nutritive function in some species, offering food for pollinators
(Ronse Decrane and Smets 2001; Vogel 1978; Walker-Larsen
and Harder 2000), the total number of pollen grains produced by three S-anthers was very small in C. communis, only
about one-thirteenth of that produced by one M-anther and
no insects were observed to forage pollen from the S-anthers
in our study. Because of the shorter filaments and nonviable pollen (Hardy et al. 2009; Morita and Nigorikawa 1999;
Ushimaru et al. 2007), the S-anthers of C. communis were also
unlikely to play a role in the pollination (Kaul and Koul 2008,
2012).Therefore, the nonrewarding S-anthers that were striking against the blue background of the corolla (Fig. 1) might
mainly serve as ‘deceptive attraction’ to mimic large quantities of pollen and lead potential pollinators to the central
anther where the food reward was (Hrycan and Davis 2005;
Hardy et al. 2009).
For Commelina flowers, lack of nectar is one of the most outstanding features, which significantly affects their reproductive
Li et al. | Small bees weaken the function of heteranthery219
Figure 3: effects of three types of stamens on visitor responses (a, b), consequent pollen receipt (c, d) and pollen removal (e, f) in the two field
populations of Commelina communis. The black and blank bars in (e) and (f) represent the number of pollen removal from L-and M-anthers,
respectively. Values (mean ± SD) with different superscript letters indicate that differences are significant (P < 0.05) in pollinator visits, pollen
receipt and pollen removal using a Tukey post hoc comparison.
success (Morita and Nigorikawa 1999). Thus, the pollen must
serve two functions, pollinating and rewarding the pollinators. Stamen placement within a flower determines whether
pollen will be fed upon or used for pollination (Lloyd 2000;
Luo et al. 2012). McCollum et al. (1984) called the central
anther of C. erecta a feeding anther and proposed it to be analogous to nectar, serving only to provide a reward for pollinators and having very little to do with fertilization outside this
role. Like other Commelina species (Faden 1992; Hrycan and
Davis 2005; Oziegbe et al. 2013), the central M-anther occupied a similar position with S-anthers in the flower of C. communis (Fig. 1a). Meanwhile, the brightly yellow M-anthers
that were attractive for pollen-collecting visitors (Fig. 3a and
b) occupied a large number of fertile pollen grains (Fig. 2c and
d), indicating their potential role in providing a nutritional
reward for pollinators. There was no significant difference
among the total number of pollen produced by M-anthers
and that of removed pollen from M-anthers in intact flowers (t = 1.08, P = 0.30 for ‘Caizhuan’; t = −0.12, P = 0.90 for
‘Shenghua’), indicating that nearly all pollen in M-anthers
was depredated by visitors (Fig. 1b). Thus, once pollinators
were lured into a flower of C. communis, the medium stamens
might primarily function as ‘feeding’ anthers to reward the
pollinators (Ushimaru et al. 2007).
220
Journal of Plant Ecology
Table 1 : mating system parameters in two Commelina communis populations: multilocus outcrossing rate (tm), single-locus outcrossing
rate (ts), outcrossing rate between related individuals (tm − ts), correlation of paternity (rp), inbreeding coefficient of maternal parents (Fm)
Parameters
Number of families
Caizhuan
Shenghua
Species
20
20
40
Number of individuals
100
94
194
tm
0.318 (0.112)
0.234 (0.065)
0.281 (0.066)
ts
0.140 (0.056)
0.093 (0.027)
0.116 (0.030)
tm − ts
0.179 (0.060)
0.141 (0.041)
0.165 (0.039)
rp
0.837 (0.340)
0.999 (0.738)
0.956 (0.208)
1/rp
Fm
1.19
0.392 (0.039)
1.00
0.341 (0.041)
1.05
0.365 (0.028)
Data are means with standard errors in parentheses.
The anther color is thought as another attractant signal
for pollen-collecting insects by a virtue of being of a similar
color to pollen and reflecting comparable UV patterns (Lunau
2000). Similar with other heterantherous species, the color of
L-anthers in C. communis is more inconspicuous and cryptic
than that of both S- and M-anthers, and hence less attractive
for pollinators (Fig. 3a and b). For ‘division of labor’ hypothesis, the pollen from the less conspicuously colored stamens is
not intended as a reward for pollinators and while legitimate
pollinators are foraging in the central stamens, their abdomen
will frequently come in contact with both the pollen-laden
anthers and the stigma, facilitating outcrossing (Luo et al. 2008;
Vallejo-Marín et al. 2009; Tang and Huang 2007). However,
the main visitor of C. communis in our study is Lasioglossum
sp. (accounting for 77.5 and 92.2% of total flower visits in
‘Caizhuan’ and ‘Shenghua’), which is a relative small bees to
the flower and could not contact L-anthers or the receptive
stigma when they fed on the central M-anthers in the flower
at the same time (Fig, 1b). Thus, removal of S- or M-anthers
that sharply declined the insect visitation did not significantly
affect the pollen receipt in flowers of C. communis in our study
(Fig. 3c and d). In addition, Lasioglossum sp. was also observed
to collect the pollen from L-anthers (Fig. 1b) and there was no
significant difference on the number of pollen removal from
L-anthers between intact flowers and S- or M- removed flowers (Fig. 3e and f), indicating that Lasioglossum sp. was likely a
pollen thief rather than a legitimate pollinator. Thus, although
the inconspicuous brown L-anthers with the largest amount
of fertile pollen had the potential pollinating function, local
high ratio of illegitimate visitation by size unmatched bees
severely impaired the dispatch and receipt of functional pollen in C. communis.
The influence of illegitimate visitation on
outcrossing rate in C. communis
The ratio of pollen per ovule (1031.0 ± 194.1) in perfect flowers of C. communis was between the ranges set by Cruden
(1997) for facultative xenogamy and xenogamy (796.6 and
5859.2, respectively), suggesting that pollinator mediated outcrossing was important for this species. Although we found
that C. communis could be described as mixed-mating (0.20 ≤ t
≥ 0.80) (Schemske and Lande 1985), the average outcrossing
rates in the two wild populations as measured by six simple
sequence repeat loci were relatively low (tm = 0.23–0.32) and
fell towards the low threshold of the mixed mating spectrum,
suggesting self-pollination predominated the reproduction of
this species here.
The difference between multilocus and single locus outcrossing rates (tm − ts) represents an estimate of the biparental
inbreeding degree, as in its presence ts will be smaller than tm
because outcrossing events that are not detected at a single
locus have a higher probability of being detected as more loci
are examined (Ritland 1996).This difference was significant
in both populations, being 0.141 ± 0.041 and 0.179 ± 0.060,
respectively for ‘Shenghua’ and ‘Caizhan’, which indicates
that of the fraction of multilocus outcrossing, around 14 and
18% has occurred between related individuals. The correlation of paternity gives the probability that a randomly chosen
pair of progenies from the same array were full sibs (rp). This
probability was very high for both populations of C. communis
(0.999 ± 0.738 and 0.837 ± 0.340, respectively for ‘Shenghua’
and ‘Caizhuan’), indicating that most progeny derived from
outcrossing within a family were full-siblings that shared the
same male parents. Even though the progenies were derived
from 20 maternal plants of C. communis in the two populations,
only about 1–2 plants (1/rp) contributed as pollen donors
(Table 1). Especially, the number of pollen donors contributing
to each family (1/rp) was 1 for ‘Shenghua’ population, suggesting that absolute autogamy existed in this population, with the
seed parent donating both male and female gametes (ovule
and pollen grains). As observed by the maternal inbreeding
coefficient and by the multilocus and single-locus rates, the
mating systems C. communis were significantly affected by the
limited functional pollen dispersion, which might be due to
the high ratio of visitations by smaller illegitimate visitors.
The possible reasons leading to self-pollination in
C. communis
During the legitimate visits of medium to large bees, heteranthery results in the deposition of pollen on different parts of the
Li et al. | Small bees weaken the function of heteranthery221
pollinator’s body and therefore pollinator-assisted cross pollination firstly happens (Bowers 1975; Jesson and Barrett 2005;
Lloyd and Schoen 1992; Vallejo-Marín et al. 2009). However,
autonomous selfing may occur in C. communis when cross pollination fails. Many members of Commelinaceae, including
C. communis have been reported to be self-compatible and a
similar mechanism of style coiling causing autogamy was seen
at the end of anthesis (Hrycan and Davis 2005; Oziegbe et al.
2013). In flowers of C. communis, the stigma and L-anthers
are in closest proximity (Fig. 1; Table S2, see online supplementary material). Thus, the significantly reduced number of
pollen on the stigmas in L-removed flowers may be due to the
failure of contact between L-anther and stigma (Fig. 3c and
d). In addition, the central anthers also have been considered
to be responsible for autogamy in some C. species. The styles
of perfect flowers of Commelina dianthifolia coil, brushing the
stigma against the fertile central stamen and effecting pollination (Hrycan and Davis 2005). This mechanism also has
been observed in C. erecta, C. lagosensis and C. diffusa (Oziegbe
et al. 2013). The high viability of pollen from central stamens
in C. communis, with equal viability as functional pollen, suggested that the central stamens might be also responsible for
autogamy rather than just feeding pollinators. Furthermore,
self-pollination within flower may occur when the smaller
Lasioglossum sp. and other opportunistic insects illegitimately
manipulate the flower (Renner 1989).
on the plant reproduction in heterantherous species. Our
data suggested that local high ratio of illegitimate visitation by smaller bees could significantly affect the dispersal
and deposition of functional pollen and further the whole
mating systems in C. communis, significantly weakening the
potential functions of heteranthery.
CONCLUSION
Barrett SCH (2010) Darwin’s legacy: the forms, function and
sexual diversity of flowers. Philos Trans R Soc Lond B Biol Sci
365:351–68.
Functional division of different types of stamens into
attraction and fertilization functions is one of the important ways to reconcile the dual function of pollen in heterantherous species. Evidently, the brightly colored stamens
can be employed by plants to draw the attention of pollinators away from the inconspicuous and cryptic stamens,
protecting the functional pollen from being consumed.
Heteranthery with various forms occurs in diverse taxonomic groups and is phylogenetically associated with buzz
pollination, enantiostyly and zygomorphy, indicating that
it has probably evolved as a result of pollinator-mediated selection (Endress 1994; Graham and Barrett 1995;
Jesson and Barrett 2003; Vallejo-Marín et al. 2010). The
size match between pollinators and the floral morphology
may be required to assure the precise pollen placement
on the pollinator’s body, facilitating crossing pollination.
However, pollen that provides a rich source of protein can
also attract a variety of illegitimate insects, which may act
as pollen thieves and cannot be excluded by the heterantherous plant itself. In this scenario, excess pollen consumption may be detrimental to plant fitness and thus there is
no selection for anther specialization and dimorphism. In
our study, the absence of legitimate pollinators in natural
populations of C. communis that may be due to changes of
pollination environment, provides us an opportunity to
explore the potential influence from illegitimate visitors
SUPPLEMENTARY MATERIAL
Supplementary material is available at Journal of Plant Ecology
online.
Funding
Postdoctoral Science Foundation of Central South University
(502080023); National Science Foundation of China
(31000106, 31100173).
Acknowledgements
We thank RL Guo, Y Gao, Y Zhang and L Fen for field assistance and
YB Gong for data analysis, and SQ Huang for valuable comments on
and improvements to the manuscript.
Conflict of interest statement. None declared.
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