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Sex Plant Reprod (1997) 10:101–106

© Springer-Verlag 1997

O R I G I N A L PA P E R

&roles:Richard A. Niesenbaum · Sheila K. Schueller

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

&misc:Received: 28 October 1996 / Revision accepted: 24 January 1997

&p.1:Abstract We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. &kwd:Key words Pollen · Pollen competition · Pollen performance · Microgametophyte&bdy:

Introduction In angiosperm reproduction, if the number of pollen grains deposited on the stigma exceeds the number of ovules in the ovary, then there may be competition among pollen tubes for access to ovules, resulting in fertilization by the fastest-growing pollen tubes (Mulcahy and Mulcahy 1975; McKenna 1986; Schlichting et al. 1987; Niesenbaum and Casper 1994). If pollen tube R.A. Niesenbaum (✉) Department of Biology, Muhlenberg College, Allentown, PA 18104-5586, USA Fax 610-821-3546, e-mail [email protected] S.K. Schueller1 Department of Biology, Swarthmore College, Swarthmore, PA 18081-1397, USA Present address: 1 Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA&/fn-block:

growth rate and offspring quality are correlated, then more intense competition will result in the production of higher quality offspring (Mulcahy and Mulcahy 1975; Janzen 1977; Mckenna 1986; Schlichting et al. 1987; Quesada et al. 1991). This is consistent with empirical evidence that large or mixed pollen loads result in more vigorous progeny (Mulcahy and Mulcahy 1975; Stephenson et al. 1986; Davis et al. 1987; Schlichting et al. 1987; Winsor et al. 1987; but see Snow 1990), and that flowers with greater stylar pollen tube numbers are more likely to produce mature fruits (Niesenbaum and Casper 1994; Niesenbaum 1996). A correlation between pollen tube growth rate and offspring quality implies that pollen tube growth rate is at least in part genetically determined (Tanksley et al. 1981; Willing et al. 1988; Ottaviano and Mulcahy 1989), and may be under strong selection (Walsh and Charlesworth 1992). Over 20000 genes may control pollen tube growth rate; however, recent studies show that environmental factors such as temperature, nutrients, and pH also may strongly influence pollen tube growth rate (Stephenson et al. 1992). Another environmental factor that could influence pollen performance is the competitive environment among pollen tubes on a stigma and within the style. The number of competing pollen tubes could potentially facilitate or inhibit germination of individual pollen grains or the growth rate of their pollen tubes (Brewbaker and Majumder 1961; Jennings and Topham 1971). Despite the large amount of theoretical and empirical evidence suggesting that pollen tube competition is a potent evolutionary force in plants, we know very little about how the environmental or non-genetic influence of the number of competing pollen tubes within a pistil influences pollen performance and sporophyte reproductive success. In this study, we examined how the competitive environment within a pistil influences overall pollen performance. We used the number of competing pollen grains and the number of pollen tubes as measures of the level of competition, and examined how the level of competition influences pollen germination, pollen tube

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penetration into the style, and pollen tube growth rate as measures of pollen performance.

Materials and methods Mirabilis jalapa L. (Nyctaginaceae) is a tropical American herb that is commonly cultivated in North America. It is perennial in the south and warm west, and annual in the north. Its tubular flowers are fragrant and vary in color within and among plants. The self-compatible, perfect flowers each have five to six stamens and a single-ovulate ovary. An individual flower opens for one night in early evening, the exact time depending on temperature and relative humidity, and closes early the next morning. As a flower begins to close, the anthers brush across the stigma, thereby facilitating self-pollination. In natural and cultivated populations, self-pollination is the primary means of reproduction in this species (Cruden 1973), and in some natural populations where the hawk-moth pollinator is nearly extinct, self-pollination is the sole means of reproduction (S. O. Garcia, Pedregal Reserve, Mexico, personal communication). There are some populations with individuals that produce cleistogmaous flowers which exhibit the same seed set as outcrossed and self-pollinated chasmogomous flowers (Cruden 1973). Preliminary data from the greenhouse population used in this study indicate that self-pollinated and outcrossed flowers exhibit the same seed set, and that rates of pollen germination and pollen tube growth are independent of whether pollen is outcrossed or selfed (R.A. Niesenbaum, unpublished data). M. jalapa provides useful traits for studying in vivo pollen competition. The long style (3–4 cm) provides ample opportunity for competition and for sorting among pollen tubes with different growth rates, and is advantageous for laboratory analysis of pollen tube growth. The pollen grains are large enough to be viewed with a ×10 hand lens so that exact numbers of pollen grains, including single grains, can be applied to the stigma. Individual seeds obtained from Henry Field Seed and Nursery (Lot B) were planted in five separate 6-inch pots with Metro-Mix 510 growing medium. The five pots were placed in the Swarthmore College greenhouse which is screened and effectively excludes potential insect pollinators. All plants were systematically rotated to average out local greenhouse environmental effects and all were watered twice daily. Experiments were performed from May to July on the majority of flowers on the five plants. Pollination and monitoring pollen performance Only self-pollen was used to determine how the number of pollen grains affects pollen performance. Self-pollen was used because this is the primary mode of pollination in this species, because there is no incompatibility system or evidence of cryptic incompatibility, and to minimize the genetic diversity of the pollen load. The three experimental treatments included: (1) a small pollen load with a single pollen grain, (2) an intermediate pollen load of five pollen grains, and (3) a large pollen load of approximately 30 pollen grains. These pollen loads are comparable to the size of pollen loads reported in natural populations of this species where the majority of stigmas of pollinated flowers are reported to have from one to ten pollen grains (Cruden 1973; del Rio and Burquez 1986). Prior to pollination, open flowers were emasculated to prevent pre-mature selfing, and anthers were collected in a petri dish. Treatments were randomly assigned to each open flower on a given night. Randomly selected pollen grains from at least three anthers per flower were individually applied with a toothpick for small (single) and intermediate pollen loads, and the actual number of pollen grains deposited on each stigma was verified with a ×10 hand lens. Large pollen loads were generated by sweeping at least three anthers per flower over the receptive stigma, and the number of pollen grains applied was estimated by averaging three counts of the number of pollen grains on the stigma with a ×10

hand lens. Each night, a sub-sample of all pollen used was tested for viability using the fluorochromatic chain reaction with fluorescein diacetate (Heslop-Harrison and Heslop-Harrison 1970; Kearns and Inouye 1993). To determine if our methods of pollination damaged the pollen, viability tests were conducted with pollen applied to a microscope slide using both pollination techniques. Preliminary experiments indicated that pollen tubes first reach the base of the style within 60 min. To examine variation in pollen tube growth rates during this initial period, styles were removed from the ovary with fine forceps 45 min after pollination and fixed in a 70% solution of ethanol for 24 h. After rinsing in distilled water, the styles were softened and cleared in 8 N NaOH for 48–72 h. The softened styles were placed in distilled water for at least 1 hour before staining with a 0.1% solution of aniline blue in 0.1 M K3PO4 for 4 h. Each style was then examined at ×100 under UV light. The number of germinated pollen grains with pollen tubes penetrating the stigmatic surface, and the number of pollen tubes that penetrated the stigma and entered the style were counted. The length from the stigmatic surface to the tip of every pollen tube was measured using an ocular reticle, and pollen tube growth rates were calculated based on the 45 min growth period. Some ungerminated pollen grains or partially germinated pollen grains that did not have tubes penetrating the stigmatic surface may have been washed off in this process, but careful treatment of the styles and examination of staining dishes indicated that loss of pollen was minimal. Analyses The proportion of pollen grains that germinated per style and the proportion that had pollen tubes penetrate the stigma and enter the style were compared among treatments using a non-parametric Kruskal-Wallis χ2 approximation (SAS Institute 1987). Because most pollen tubes of germinated pollen grains do not penetrate the stigma and enter the style, a better estimate of competitive environment within the style is the actual number of pollen tubes rather than the initial treatment. A mixed model analysis of variance of pollen tube growth rate (mm/45 min) was conducted with the number of pollen tubes per style, individual plant, and their interaction as the classification variables. Pollen tube number was tested over the interaction and individual plant was tested over the error term. An F-max test (Berenson et al. 1983) was used to test the assumption of homogeneity of variance, and to examine the trend in variation of pollen tube growth rate with tube number per style. Variances were calculated using growth rates of individual pollen tubes in all styles with a particular number of tubes. Because the data were not homoscedastic, they were log(x+1)-transformed prior to the analysis of variance (Sokal and Rohlf 1981). To control for the degree of pollen selection at the stigma, a one-way ANOVA of the effects of pollen tube number on tube growth rate was done for treatment 3 only, the subset of data that represented the greatest degree of selection. Also, pollen tube growth rates of single pollen tubes were compared for styles under treatment 1 (no selection) and treatment 3 (strong selection).

Results The number of pollen grains deposited on stigmas designated for small load and intermediate load treatments were always exactly one and five, respectively. The mean number of pollen grains deposited on stigmas of flowers designated for large load treatment was 30.0±1.9 (x±1 standard error, n=78), and always greater than 25. This treatment did not completely cover the stigmatic surface with pollen grains, as ascertained with a ×10 hand lens.

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Fig. 1 Mean percentage of pollen grains that germinated per stigma for small (one grain), intermediate (five grains), and large (>25 grains) pollen loads. Error bars represent 1 SD&ig.c:/f

Fig. 2 Mean percentage of pollen grains that had pollen tubes penetrate the stigma for small (one grain), intermediate (five grains), and large (>25 grains) pollen loads. Error bars represent 1 SD&ig.c:/f

Tests for pollen viability revealed that on most nights greater than 98% of the examined pollen grains fluoresced brightly, and were thus deemed viable. On five individual nights, significantly fewer than 90% (35–85%) of individual pollen grains were classified as highly viable.These nights were unusually hot and humid, and anther dehiscence was delayed. Data from these nights were not used in the subsequent analyses. Tests with pollen applied to a microscope slide using the two pollination techniques, application with a toothpick and direct application with the anther, showed no loss of viability or differences between techniques. The number of pollen grains deposited on a stigma significantly influenced both the percentage of pollen

Fig. 3 Number of pollen tubes per style versus mean pollen tube growth rate (mm/45 min). Error bars represent 2 SD&ig.c:/f

grains that germinated and the percentage of pollen grains whose pollen tubes penetrated the stigma (Fig. 1 and Fig. 2, respectively). The percentage of pollen grains that germinated was significantly greater at intermediate pollen loads than at large and small loads (Kruskal-Wallis χ2 approximation=19.9, df=2, P