Journal of
Plant Ecology
Volume 8, Number 2,
Pages 166–172
April 2015
doi:10.1093/jpe/rtv018
available online at
www.jpe.oxfordjournals.org
The functional significance of a
stigma color polymorphism in Acer
pictum subsp. mono (Aceraceae)
Zhi-Qun Yang1,2, Da-Yong Zhang1 and Wei-Ning Bai1,*
1
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing
Normal University, Beijing 100875, China
2
State Key Laboratory of Systematic and Evolution, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
*Correspondence address. Ministry of Education Key Laboratory for Biodiversity Science and Ecological
Engineering, College of Life Sciences, Beijing Normal University, No. 19 XinJieKouWai St., HaiDian District,
Beijing 100875, China. Tel: +86-10-58804807; Fax: +86-10-58807721; E-mail: [email protected]
Abstract
Aims
Exploring the characteristics and function of a flower color polymorphism contributes to our understanding of floral evolution in
angiosperms. However, little information is available on stigma
color polymorphisms in flowering plants despite their important
functional role in plant reproduction.
Methods
We studied a stigma color polymorphism at the individual level in
Acer pictum subsp. mono (aka A. mono) by investigating stigma-color
morph proportion and comparing stigma performance and components of female fitness (pollen adhesion, pollen germination, fruit set,
seed set and fruit weight) between the two morphs (red and greenishyellow stigma) within natural populations at Dongling Mountain in
the north of China. In the flowering period, we conducted hand pollination in natural populations and then made microscopic observations using the aniline blue fluorescence method.
Important Findings
Individuals in the studied populations did not change their stigma
color between years, and flowers produced by a single tree were
uniform in their stigma color. This strongly suggests that stigma
color variation is genetically controlled. The percentage of the red
stigma flowers with germinated pollen grains was significantly
higher than that of the greenish-yellow stigma flowers when hand
pollination was conducted in the early flowering period, but not
so in the late flowering period. There was no significant difference
in the percentage of flowers with pollen adhesion to the stigma
between the two morphs. Fruit set of the red morph was significantly higher than that of the greenish-yellow morph. Our findings
suggest that the red morph may be more advantageous for pollen
germination and fruit set than the greenish-yellow morph, which
may provide a functional explanation for the high incidence of the
red stigma morph in the studied populations. Alternative explanations for the stigma color polymorphism are discussed to stimulate
further work.
Keywords: Acer, female fitness, stigma color polymorphism, stigma
performance
Received: 14 January 2015, Accepted: 24 January 2015
Introduction
Flowers are the most diverse structures exhibited by angiosperms. Many cases regarding the variation of floral traits
have focused on size, shape, scent and, most commonly, color
(Arista et al. 2013; Clegg and Durbin 2000; Gómez 2003;
Imbert et al. 2014; Peter and Johnson 2014; Schlumpberger
et al. 2009; Song et al. 2015; Zhang et al. 2015). Flower color
polymorphism, in which two or more different flower color
morphs coexist in the same population, has been intensively
studied for exploring flower evolution in plants (Huang and
Tang 2008). In general, pollinator preference is regarded as
the most likely explanation for flower color polymorphisms
(Gigord et al. 2001; Malerba and Nattero 2012; Waser and
Price 1981), in part because pollinators tend to promote assortative mating. However, some studies have showed that pollinators do not exhibit any discrimination between various
color types (Schemske and Bierzychudek 2001; Wolfe 1993).
Intraspecific variation in petal color is the most conspicuous
color polymorphism (Warren and Mackenzie 2001), but there
are also other forms of flower color polymorphisms, including pollen, gynoecium and inflorescence color variations in
© 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.
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Yang et al. | The functional significance of a stigma color polymorphism 167
natural populations. For example, two subspecies of Nigella
degenii have a dimorphism in pollen color (violet and yellow;
Jorgensen et al. 2006), Butomus umbellatus possesses a gynoecium color polymorphism (pink and white; Huang and Tang
2008), and the genus Protea exhibit an inflorescence color
polymorphism (pink and white; Carlson and Holsinger 2010).
Compared with other color polymorphisms of flower organs,
there are very few studies of stigma color polymorphisms; to
our knowledge, the only exception is Rafinski (1979), who
found two stigma color morphs (orange and white) coexist
within Crocus scepusiensis populations. However, this study
only focused on the geographic variation of the color polymorphism and did not explore the function, if any, of the
stigma color variation.
The painted maple, Acer pictum subsp. mono (aka A. mono),
is a heterodichogamous, bee-pollinated, temperate deciduous tree, native to China, Japan, Korea, Mongolia and eastern Russia (Xu et al. 2010). Stigmas within an individual are
either all red (Fig. 1a) or all greenish-yellow (Fig. 1b), and
both morphs of individuals may co-occur in natural populations. This provides us with an excellent opportunity to
study the function of stigma color polymorphism (Fig. 1). It
is well known that the morphology, structure and physiology of stigma can affect pollen performance (Dulberger 1974;
Guo and Huang 1999; Hedhly et al. 2003). For instance, in
Limonium meyeri, structural stigma dimorphism (cob and papillate) affected pollen adhesion to stigmas (Dulberger 1975).
Here, we aim to test whether there is a difference of stigma
performance and female fitness components between the two
stigma color morphs in A. pictum subsp. mono. Specially, our
questions are: (i) What are the morph ratios in natural populations of Dongling Mountain? (ii) Are there significant differences between red stigma and greenish-yellow stigma morphs
in (a) the percentage of flowers with pollen adhesion to the
stigma, (b) the percentage of flowers with pollen germination
on the stigma and (c) fruit set, seed set and fruit weight?
MATERIALS AND METHODS
Study species and sites
Greenish-yellow flowers of A. pictum subsp. mono appear from
the middle of April to the middle of May in upright clusters
(corymbs). Flowers are functionally unisexual due to either
abortion of the pistil or failure of the anthers to dehisce, permitting the hand pollination of flowers without emasculation
(Shang et al. 2012). One flower generally lasts 4 or 5 days.
Flowers consist of five sepals, five petals, eight stamens and
a bicarpellate ovary. Stigmas within an individual are either
all red (red morph) or all greenish-yellow (greenish-yellow
morph). Each locule has two ovules, but only one develops
after fertilization (de Jong 1976). Fruit is a samara.
Our study site is located at the Beijing Forest Ecosystem
Research Station in Dongling Mountain, 114 km west of
Beijing, China (115°26′E, 39°58′N; 1100 m above sea level).
Figure 1: stigma color polymorphism in Acer pictum subsp. mono. One morph has red stigma (a, c) and one has greenish-yellow stigma (b, d).
Pollen germination (e) and fertilization (f). ov, ovule; po, pollen; pt, pollen tube.
168
The annual average temperature is 4–6°C. The study populations of A. pictum subsp. mono co-occur with Juglans mandshurica and Quercus liaotungensis.
Sampling
We divided flowering period (mid April to mid May) into
two equal parts (an early period and a late period). The temperature in the late flowering period was significantly higher
than the temperature in the early flowering period in 2012
(P = 0.01, Fig. S1, see online supplementary material; data
sourced from Beijing Forest Ecosystem Research Station).
Six populations were surveyed in order to determine the frequency of red versus greenish-yellow morph in each population. Besides, in the early flowering period of 2012, we
identified 10 red individuals and 10 greenish-yellow individuals in population Goushicao, Meiyaoyu, Niulanyu and
Nangou as female parents. We also chose 9 red individuals and 10 greenish-yellow individuals in the late flowering period in population Goushicao, Meiyaoyu, Niulanyu
and Nangou. In 2014, we chose 12 red individuals and 13
greenish-yellow individuals in the early flowering period and
9 red individuals and 15 greenish-yellow individuals in the
late flowering period in population Goushicao, Meiyaoyu,
Niulanyu and Nangou.
Stigma performance
Female flowers were tagged and bagged by bridal veil fabric before anthesis for the purpose of avoiding natural cross
pollination. Anthers from the male flowers of three nearby
individuals were cut and left to dry in a petri dish with a small
amount of silica gel. Pollen was brushed at the tip of stigma on
the day of anthesis. One to three female flowers were picked
for hand pollination at 1, 2, 4, 8, 12, 24 and 48 h in 2012 (0, 2,
4, 6, 8, 10, 24, 34 and 48 h in 2014), which were fixed for 24 h
in FAA (formalin–acetic acid–alcohol) and then stored in 70%
ethanol (n = 806 flowers in 2012; n = 1134 flowers in 2014).
Specimens were washed three times with distilled water, softened for 16 h in 8 mol−1 NaOH solution at room temperature,
rinsed for 1 h in distilled water and then stained for 1 h with
aniline blue (0.1% in 1/30 mol l−1 K3PO4) (Wang et al. 2002).
Pistils were carefully hand-sectioned and examined under a
fluorescence microscope (ZEISS Imager M1, Germany). The
percentage of flowers with pollen adhesion to the stigma and
the percentage of flowers with pollen germination on the
stigma were used as criteria for judging the performance of
different color morphs (Hedhly et al. 2003).
Female fitness components
In 2014, we randomly chose three corymbs for hand pollination and three corymbs for natural pollination as a control
from each of the 49 individuals. We recorded the number of
female flowers on every corymb and collected mature fruits
in the middle of September (average five female flowers per
corymb). All fruits were oven-dried at 75ºC for 48 h to ensure
a constant weight.
Journal of Plant Ecology
Statistical analysis
A G-test for goodness-of-fit was used to test whether the
observed morph ratio significantly deviated from 1:1 (Sokal
and Rohlf 1995). The percentage of flowers with pollen adhesion to the stigma and the percentage of flowers with pollen
germination on the stigma were both subjected to Wilcoxon
signed rank tests. To compare the fruit set, seed set and fruit
weight between red morph and greenish-yellow morph,
Mann–Whitney U-test was used. All of the statistical analyses
were performed with SPSS v.20 (IBM Statistics).
RESULTS
Morph ratios in six populations
The ratio of two stigma morphs did not significantly deviate from 1:1 in four of six populations surveyed (Shuigou,
Paozigou, Nangou and Niulanyu), but in population Goushicao
and Meiyaoyu, the ratios were significantly different from
1:1 (Table 1). The frequency of red morph was significantly
higher than that of the greenish-yellow morph in Goushicao,
but the opposite trend was observed in the Meiyaoyu.
Stigma performance and female fitness
components in the early flowering period
In the early flowering period of 2012 and 2014, no difference between the two morphs was found in the percentage of
flowers with pollen adhesion to the stigma (P = 0.310, 0.575,
Fig. 2a and b). The percentage of flowers with pollen germination on the red stigma in the early flowering period of
2012 was marginally higher than the greenish-yellow morph
(P = 0.08, Fig. 2c); a larger significant difference between the
two morphs was detected in 2014 (P = 0.017, Fig. 2d).
In the early flowering period of 2014, fruit set of the red
morph was marginally higher than that of the greenish-yellow
morph under hand pollination (P = 0.093, Fig. 3a); a larger significant difference between two morphs by natural pollination
was observed (P = 0.023, Fig. 3b). No significant difference in
seed set between the two morphs was found under hand pollination (P = 0.537, Fig. 3c), but the seed set of the greenish-yellow morph was significantly higher than that of the red morph
under natural pollination (P = 0.067, Fig. 3d). Fruit weight of
the red morph was significantly higher than the greenish-yellow morph under hand pollination (P = 0.019, Fig. 3e), but no
Table 1 : stigma color polymorphisms in six populations
Population Red individual Greenish-yellow individual G value P value
Shuigou
35
29
0.563
0.453
Paozigou
33
21
2.689
0.101
Nangou
12
20
2.021
0.155
Goushicao 25
12
4.667
0.031*
Niulanyu
33
46
2.149
0.143
Meiyaoyu
11
27
6.952
0.008*
*P < 0.05.
Yang et al. | The functional significance of a stigma color polymorphism 169
Figure 2: stigma performance for two morphs in the early flowering period expressed as the percentage of flowers with pollen adhesion to the
stigma (a, b) and the percentage of flowers with pollen germination on the stigma (c, d).
Figure 3: in the early flowering period, the comparison of fruit set (a, b), seed set (c, d) and fruit weight (e, f) between two morphs under
hand pollination and natural pollination, respectively.
170
Journal of Plant Ecology
difference between the two morphs was found with natural
pollination (P = 0.834, Fig. 3f).
Stigma performance and female fitness
components in the late flowering period
The various measurements of pollen performance on stigmas
(the percentage of flowers with pollen adhesion to the stigma
and the percentage of flowers with pollen germination on the
stigma) and female fitness components (fruit set, seed set, and
fruit weight) in the late flowering period all indicated no differences between the two morphs (Table 2).
Discussion
Flower color polymorphisms have been reported in many
plant species (Frey et al. 2011; Joseph and Siril 2013; Volkova
et al. 2013). The most notable flower color polymorphism is
the variation in petal color, but other parts of flowers also
exhibit color polymorphism. Our study provides new evidence for stigma color polymorphisms in angiosperms. Two
years of field sampling and observations showed that individuals in six populations of a local area near Beijing did not
change their stigma color between years, which means that
the stigma color variation in A. pictum subsp. mono is likely
to be under genetic control, rather than a developmental
change.
In our report, the two color morphs have the same ability to receive pollen, but the red morph was superior to the
greenish-yellow morph in terms of pollen germination during
the early flowering period. Moreover, the fruit set of the red
morph were significantly higher than that of the greenishyellow morph when hand pollination and natural pollination
were conducted in the early flowering period, but not so in
the late flowering period. We speculate that these results may
be related to the fluctuation of temperature in the early spring
of this temperate region. In our study site, daily average temperature of the early flowering period was significantly lower
than that of the late flowering period (Fig. S1, see online supplementary material). Temperature has been proven to have
a clear effect on pollen germination and pollen tube growth
(Hedhly et al. 2004). Consequently, temperature could be
an important constraint factor for pollination in early spring
flowering species. Generally, red color traps more heat than
greenish-yellow. Therefore, red stigma may provide more
energy for pollen germination than greenish-yellow stigma in
the early flowering period when temperature is still low, but
in the late flowering period with increased temperature, red
stigma will presumably confer little advantage.
The processes maintaining the stigma color polymorphism might include genetic drift, pleiotropic effects, selection through thermal environments by different stigma
colors, and/or an admixture of different populations
expanded post-glacially from the northern and southern
refugia. In four of six populations, the balanced morph
ratio of 1:1 suggests that the stigma color polymorphism
can be maintained by negative frequency-dependent selection or through pleiotropic effects. A. pictum subsp. mono,
is a species with sexual polymorphism in which there are
three sexual morphs, duodichogamy, protandry and protogyny. Negative frequency-dependent selection through
disassortative mating among the sex morphs can maintain the sexual polymorphism (Shang et al. 2012). If the
genes determining sexual morph have pleiotropic effects
on stigma color, then the stigma color polymorphism can
be maintained by the same negative frequency-dependent
selection. However, we have little evidence for or against
this possibility. In future, we will investigate whether there
is a correlation between stigma colors and sexual morphs or
other traits under negative frequency-dependent selection.
Acer pictum subsp. mono, as a temperate-deciduous tree, is
widely distributed from northeastern to southern China (Xu
et al. 2010). Assuming that different stigma colors created
different thermal environments for pollen germination and
growth, we predict that the frequency of red stigma morphs
should increase from south to north due to increasingly lower
temperature in the early spring when the trees blossom. Two
phylogeographic studies of the painted maple indicated the
existence of two separate glacial refugia, one in northeastern
China and another in southern subtropical China (Guo et al.
2014; Liu et al. 2014). Our studied populations are located
in the admixture zone where the southern and northern lineages meet during postglacial expansion from their respective refugium. Thus, the observed stigma color polymorphism
may be simply a result of post-glacial admixture if the northern lineage has the red stigma and the southern lineage has
the greenish-yellow stigma, which awaits further verification.
The morph ratios of two colors would be close to 1:1 under
negative frequency-dependent selection but more variable
under the admixture hypothesis. Our survey has showed
that in two of six populations the ratio deviated significantly
from 1:1, seemingly in favor of the admixture hypothesis.
However, our data are far from judging the two hypotheses
Table 2 : stigma performance and female fitness components in the late flowering period
Fruit set
% Flowers
(adhesion)
% Flowers
(germination)
P value (2012)
0.237
0.465
P value (2014)
0.575
0.249
Abbreviations: HP = hand pollination, NP = natural pollination.
Seed set
HP
NP
HP
0.160
0.406
—
0.676
Fruit weight
NP
HP
0.795
0.505
—
NP
—
0.898
Yang et al. | The functional significance of a stigma color polymorphism 171
and more populations in the whole geographic range of A. pictum subsp. mono should be sampled and studied. Geographical
investigations of morph frequencies can provide insights into
how stigma color polymorphism may have originated and is
currently maintained in populations.
Guo YH, Huang SQ (1999) Evolution of pollination system and characters of stigmas in Najadales. J Syst Evol 37:131–6.
Hedhly A, Hormaza JI, Herrero M (2003) The effect of temperature
on stigmatic receptivity in sweet cherry (Prunus avium L.). Plant Cell
Environ 26:1673–80.
Supplementary Data
Hedhly A, Hormaza JI, Herrero M (2004) Effect of temperature on
pollen tube kinetics and dynamics in sweet cherry, Prunus avium
(Rosaceae). Am J Bot 91:558–64.
Supplementary data is available at Journal of Plant Ecology
online.
Huang SQ, Tang XX (2008) Discovery of gynoecium color polymorphism in an aquatic plant. J Integr Plant Biol 50:1178–82.
Funding
Imbert E, Wang H, Conchou L, et al. (2014) Positive effect of the yellow morph on female reproductive success in the flower colour
polymorphic Iris lutescens (Iridaceae), a deceptive species. J Evol Biol
27:1965–74.
National Natural Science Foundation of China (41371073 and
31421063).
Acknowledgements
We are grateful to SCH Barrett, VL Sork and WJ Liao for their constructive suggestions and to QG Zhang, W Liu, XK Ma and H Shang
for their assistance with statistical analysis. We would also like to
thank YY Yin, G Yin and ZY Sun for their help with hand pollination
and fruit collection. Beijing Forest Ecosystem Research Station kindly
provided the temperature data.
Conflict of interest statement. None declared.
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