Where is the stigma on a plant

The positions of stigma and stamen were dissimilar in different flower development stages. The pollen viability, stigma receptivity, pollen removal and pollen deposition on stigma were investigated at different phases.

As the flower developed, the anthers moved upwards, making way for pollen deposition during the female phase. Meanwhile, the stigma becomes receptive by moving into the way and consequently was deposited with sufficient pollen. The position exchange of the stamen and stigma created a dynamic herkogamy at the floral phase with different sexual functions.

This floral mechanism effectively avoided sexual interference and maintained pollination accuracy. In Ajuga , the movement herkogamy might be of adaptive significance in response to the changes in the pollination environment.

Herkogamy and dichogamy are mechanisms of hermaphroditic angiosperms to spatially and temporally separate pollen presentation and stigma receptivity. A dichogamous flower can effectively avoid self-pollination and enhance outcross mating due to the separation of the male and female function within the flower Lloyd and Webb ; Bertin and Newman ; Barrett a.

For homostylous flowers without assortative mating mechanism such as, heterostylous flower , herkogamy is mainly thought to reduce interference between male and female functions Lloyd and Webb ; Webb and Lloyd ; Barrett a , b ; Li et al.

Lloyd and Webb recognized that herkogamy might effectively avoid sexual interference but it might also result in a conflict due to the spatial separation of male and female functions, which can minimize the pollination accuracy in hermaphroditic plants. Pollen may be placed in a site on the pollinator different from the point of stigma contact. Armbruster et al. Floral mechanisms through interactions of herkogamy and dichogamy may decrease the interference between pollen removal and pollen receipt, thus maintaining the pollination accuracy Medan and Ponessa ; Armbruster et al.

An ideal floral mechanism to resolve herkogamy dilemma is that male and female functions separate in time and moreover, stamen and stigma sequentially occupy the same position for pollination at male and female phases.

For example, the stamen locates a spot with high pollination accuracy during the male phase, while the stigma remains away from the position to make way for pollen export. During the female phase, the stigma and stamen exchange their positions in order to place the stigma in the accurate point for pollen deposition.

This mechanism of movement herkogamy could be achieved by successive stamen movement Ren and Tang ; Armbruster et al. In this study, we reported a floral mechanism of movement herkogamy in a protandrous herb, Ajuga decumbens , where the stamen and stigma of a flower exchanged their position by movement in opposite directions with floral development.

To detect whether and how this floral mechanism avoided sexual interference, and maintained the pollination accuracy, the reproductive ecology of A. Ajuga decumbens is an annual or biennial herb endemic to China. It inhabits streamsides, roadsides and wet areas. Individual plants produce multiple verticillasters Flowers are zygomorphic and nectar-rich.

The calyx consists of five nearly equal lobes with densely pilose, while the corolla tube is nearly twice as long as the calyx with sparsely pilose. The corolla has a very short two-lobed upper lip and a three-lobed lower lip Fig.

Each flower produces four epipetalous stamens arranged in two rows, four ovules and a bifid stigma Wu and Li ; Fig. The anthers dehisce longitudinally. The flowering season of this species in China is usually from March to June. Floral morphology and inflorescence trait of Ajuga decumbens. A to D indicate male phase, middle phase, female phase and witling phase, respectively, while E shows the inflorescence trait. All experiments were conducted in a natural population of A. Before flower anthesis, plants from the natural population were randomly selected and transplanted into pots on 15 February Each individual was planted in a cm pot filled with the original surface soil from the natural population area.

All the plants were managed under the same conditions, for example, with the same soil and water source. They were initially bagged with nylon net mesh to keep them away from any kind of insects by the side of the natural population. The transplanted plants were watered once every 3 days during our investigations. The transplanted plants were selected for further investigations if they showed similar performance as those of natural population in plant height, number of inflorescences per plant and number of flowers per inflorescence.

To explore the pollinator composition of A. On sunny days, i. Pollination observation was carried out during a period of 10 min. A total of 24 observation periods were conducted eight periods on each observation day.

The pollinators whose bodies contacted the reproductive organs stigma or anthers were recorded and the number of flowers visited by each pollinator in the plot was also noted.

We traced the pollinator that visited a plot to record the number of flowers visited on each plant and each inflorescence. For each pollinator, the visited flowers per plant and inflorescence within a single foraging bout were calculated.

The pollinators were collected and identified by the Institute of Zoology, Chinese Academy of Sciences. To reveal the mating system of A. Ten inflorescences for each treatment were selected from 10 plants 30 inflorescences were selected in total ; five flowers per inflorescence were used for hand-pollination with self- or cross-pollen in treatment i and ii , all flowers of the inflorescence were used to test autonomous selfing in treatment iii.

To ensure sufficient pollination, all the flowers were hand-pollinated twice each day on two consecutive days during the female phase. In addition, 10 plants were placed into the natural population for open pollination and this served as a control one inflorescence per plant was selected for calculating fruit set and seed set.

The average total number of flowers per inflorescence in the treatments for testing autonomous selfing and open pollination were Fruit set was calculated as the fruit number divided by the number of flowers.

A generalized linear model with binomial error structure was used to assess the effects of pollination treatment on the fruit set. A Poisson generalized linear model was used to test the effects of pollination treatment on the seed number per fruit in a fixed model. To detect the dynamic changes of A.

The flower status was monitored twice every day from open to wilt, especially on the position of stigma and stamens, the anther dehiscence and the status of stigma lobes. According to the relative position of the stigma and stamens, the floral development was divided into four phases: i male phase, indicating that the anthers are dehiscing meanwhile the stigma is higher than the stamens, the stigma lobes are close; ii middle phase, indicating that the stigma and stamens are at the same height and the stigma lobes are ready to spread; iii female phase, indicating that the stigma lobes are fully opened and lower than the anthers; and iv wilting phase, indicated by the wilted corolla and withered anthers and stigma.

During this process, the stamen and stigma move in the opposite direction and exchange their positions. To precisely evaluate the position of the stamen and stigma, the distance from the stamen and the stigma to the lower lip was measured at the male, middle and female phases, respectively. We set the lowest point of the lowest anther and the lower lobe of stigma as the starting point, respectively.

The symmetry line on the lower lip surface was set as the ending line and the shortest distance from the starting point to this line on the lower lip surface was measured by an electronic Vernier caliper mm; see also Fig. Thirty flowers at each phase were picked for measurement of such distance, respectively. Independent T -test was used to detect the differences in the distance from the stamen and the stigma to the lower lip among the three phases.

To test the changes in male and female functions and their relation to different floral phases, we tested stigma receptivity and pollen viability using the flowers from bagged individuals.

In each floral phase, the stigmas of 15 flowers from different plants were collected to test the receptivity. The stigmas were stained by MTT 3- 4,5-dimethylthiazolyl -2,5-diphenyltetrazolium bromide. Afterwards, the dark or brown spots on the stigma revealed the presence of dehydrogenase, which in turn was used as an indicator for stigma receptivity Dafni The ratio of stigma receptivity was calculated as the number of stained stigmas divided by the number of the total checked stigmas.

In each floral phase, the pollen grains of six flowers from different plants were collected to evaluate the pollen viability. The pollen germination ratio was then checked by an epifluorescence microscope Nikon E The ratio of the pollen grains with pollen tubes out of the total pollen grains showed the pollen viability.

One-way ANOVA was conducted to detect the differences in the ratio of pollen viability among the four floral phases. To satisfy the normal distribution, pollen germination ratio was transformed by arcsine. Student—Newman—Keuls was used for multiple comparisons. To investigate the dynamic pollen removal across the floral phases, we examined the remaining pollen before and after the style defluxion under open pollination.

A total of buds were randomly selected from 15 plants eight from each plant and divided into four groups 30 flowers for each group according to the floral development phase, namely, bud phase, male phase, middle phase and female phase.

The anthers of the labelled flowers from each group were collected at the end of the corresponding floral phase. The 15 plants were placed into the natural population during the experimental period. One-way ANOVA was conducted to detect the differences in the remaining pollen among the four phases, and Student—Newman—Keuls was used for multiple comparisons. To investigate the dynamic pollen deposition across the floral phases, we compared the pollen deposition on the stigma of emasculated flowers and intact flowers under open pollination.

Emasculation was done by clipping off all the anthers prior to the opening of the flowers, and intact flowers were set as control lines. A total of flowers from 30 plants eight from each plant were arranged into two sections: were emasculated and another were not emasculated. The emasculated and intact flowers were divided into four groups 30 in each group according to the four floral phases.

The 30 plants were placed into the natural population during the experimental period. The style leads to the ovary that contains the female egg cells called ovules. The male parts are called stamens and usually surround the pistil. The stamen is made up of two parts: the anther and filament. The anther produces pollen male reproductive cells. The filament holds the anther up. During the process of fertilization , pollen lands on the stigma, a tube grows down the style and enters the ovary.

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