Journal of Plant EcologyVolumE 8, NumbEr 2, PagEs 166–172

aPril 2015

doi:10.1093/jpe/rtv018

available online at www.jpe.oxfordjournals.org

© 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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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: baiwn@bnu.edu.cn

Abstract

AimsExploring the characteristics and function of a flower color poly-morphism 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.

MethodsWe 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 compo-nents of female fitness (pollen adhesion, pollen germination, fruit set, seed set and fruit weight) between the two morphs (red and greenish-yellow stigma) within natural populations at Dongling mountain in the north of China. in the flowering period, we conducted hand pol-lination in natural populations and then made microscopic observa-tions using the aniline blue fluorescence method.

Important Findingsindividuals 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 signifi-cantly 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 explana-tions 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

iNTroDuCTioNFlowers are the most diverse structures exhibited by angio-sperms. 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 assor-tative mating. However, some studies have showed that pol-linators 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, includ-ing pollen, gynoecium and inflorescence color variations in

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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 gynoe-cium 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 poly-morphism 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 decidu-ous tree, native to China, Japan, Korea, Mongolia and east-ern 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 popu-lations. 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 physiol-ogy 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 papil-late) 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 popu-lations of Dongling Mountain? (ii) Are there significant differ-ences 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 mETHoDsStudy 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, per-mitting 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.

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The annual average temperature is 4–6°C. The study popula-tions of A. pictum subsp. mono co-occur with Juglans mandshu-rica 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 tem-perature 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 fre-quency of red versus greenish-yellow morph in each pop-ulation. Besides, in the early flowering period of 2012, we identified 10 red individuals and 10 greenish-yellow indi-viduals in population Goushicao, Meiyaoyu, Niulanyu and Nangou as female parents. We also chose 9 red individu-als and 10 greenish-yellow individuals in the late flower-ing 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 fab-ric 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, sof-tened 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 pollina-tion 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.

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 adhe-sion 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).

rEsulTsMorph ratios in six populations

The ratio of two stigma morphs did not significantly devi-ate 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 differ-ence 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 ger-mination 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 sig-nificant 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 polli-nation (P = 0.537, Fig. 3c), but the seed set of the greenish-yel-low 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-yel-low 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.

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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.

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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 dif-ferences between the two morphs (Table 2).

DisCussioNFlower 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 evi-dence for stigma color polymorphisms in angiosperms. Two years of field sampling and observations showed that indi-viduals 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 abil-ity 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 greenish-yellow 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 tem-perature of the early flowering period was significantly lower than that of the late flowering period (Fig. S1, see online sup-plementary 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 polymor-phism might include genetic drift, pleiotropic effects, selec-tion 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 selec-tion or through pleiotropic effects. A.  pictum subsp. mono, is a species with sexual polymorphism in which there are three sexual morphs, duodichogamy, protandry and pro-togyny. Negative frequency-dependent selection through disassortative mating among the sex morphs can main-tain 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 lin-eages meet during postglacial expansion from their respec-tive refugium. Thus, the observed stigma color polymorphism may be simply a result of post-glacial admixture if the north-ern 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

% Flowers (adhesion)

% Flowers (germination)

Fruit set Seed set Fruit weight

HP NP HP NP HP NP

P value (2012) 0.237 0.465 — — —

P value (2014) 0.575 0.249 0.676 0.160 0.406 0.795 0.505 0.898

Abbreviations: HP = hand pollination, NP = natural pollination.

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Yang et al. | The functional significance of a stigma color polymorphism 171

and more populations in the whole geographic range of A. pic-tum 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.

suPPlEmENTary DaTaSupplementary data is available at Journal of Plant Ecology online.

FuNDiNgNational Natural Science Foundation of China (41371073 and 31421063).

aCkNoWlEDgEmENTsWe are grateful to SCH Barrett, VL Sork and WJ Liao for their con-structive 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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