Int. J. Plant Sci. 170(9):1103–1108. 2009. Ó 2009 by The University of Chicago. All rights reserved. 1058-5893/2009/17009-0001$15.00 DOI: 10.1086/605872
SELECTION LINES OF SILENE LATIFOLIA (CARYOPHYLLACEAE) DIFFER IN HOW STRESS AFFECTS POLLEN PRODUCTION Christopher R. Herlihy1 and Lynda F. Delph2 Department of Biology, 1001 East Third Street, Indiana University, Bloomington, Indiana 47405, U.S.A.
Mating displays can incur costs in stressful environments. We examined whether males from artificialselection lines differing in floral display (many vs. few flowers) responded differently to stress in the sexually dimorphic dioecious plant Silene latifolia. Males produce more flowers than females, presumably as a consequence of sexual selection for large floral displays. However, ecophysiological traits are genetically correlated with flower production, which may negatively affect many-flower-producing males under stressful conditions. To test this premise, we varied water and nutrients and measured flower production, petal size, and pollen production in males. We also measured ovule production in females. Most male traits responded negatively to one or both of the stress treatments. Under high nutrients, pollen production per plant was higher in the many-flower than in the few-flower line, whereas under low nutrients the reverse was true, leading to a significant interaction effect. Moreover, the reduction in pollen production per flower caused by stress was greater than the reduction in ovule production per flower, but only in the many-flower line. Our results show that pollen production for the two selection lines is differently plastic, which may lead to among-population differences in both trait means and sexual dimorphism. Keywords: artificial selection, flower number, petal size, plasticity, sexual dimorphism.
Introduction
that varied greatly in flower number in the dioecious plant Silene latifolia (Delph et al. 2004). This approach is useful, given that significant genetic correlations among floral, leaf, and ecophysiological traits have been shown to occur both within and between the sexes in this species (Gehring and Monson 1994) and specifically within these selection lines (Delph et al. 2004, 2005), such that plants producing more flowers make thinner leaves and have higher rates of photosynthesis and transpiration; in other words, floral-display size cannot evolve independently of leaf and physiological traits (Delph et al. 2002, 2004, 2005; Delph 2007; Steven et al. 2007). Moreover, the species is sexually dimorphic, with males producing many more flowers than females (Carroll and Delph 1996). The size of the floral display in males is likely to have been under sexual selection, because elaborate displays attract more pollinators than do moderate displays in this species (Shykoff and Bucheli 1995). Relative to females, males also produce more nectar per plant, more branches, and thinner leaves, have higher rates of gas exchange and lower water-use efficiency, and are less tolerant to competition (Lovett Doust et al. 1987; Meagher 1992; Gehring and Monson 1994; Delph and Meagher 1995; Laporte and Delph 1996; Gehring et al. 2004; Delph et al. 2005). Hence, selection favoring males that make more flowers may be constrained by physiological costs associated with making many flowers (Delph et al. 2005). Among populations of S. latifolia, there is variation in the means of floral, vegetative, and physiological traits as well as the degree of sexual dimorphism for some of these traits (Delph et al. 2002; Delph and Bell 2008), suggesting that abiotic conditions may play a role in shaping both. For example, a commongarden experiment revealed an almost threefold difference in
Sexual selection may often favor elaborate mating displays to increase mating opportunities, but such displays may be constrained by environmental variation, such that the optimal display is produced or selected for only in the highest-quality environments (Andersson 1994). In plants, large floral displays often increase pollinator attraction and may thereby increase pollen export and siring success (Stephenson and Bertin 1983; Bell 1985; Stanton 1994). However, the production and maintenance of floral displays containing many flowers may impose large resource costs that incur life-history consequences. If this occurs, then less elaborate floral displays would be predicted to occur in low-resource environments, as a result of either plasticity or selection to reduce floral display. Indeed, floral traits often vary within and among populations of plants in ways that suggest that variation in abiotic factors can influence both the plastic expression of these traits and, ultimately, how selection acts on them (Frazee and Marquis 1994; Galen 1999, 2005; Carroll et al. 2001; Strauss and Whittall 2006; Delph and Bell 2008). We used artificial-selection lines of a dioecious plant that differ in their floral-display size to determine whether males producing large displays are differentially affected by stressful, low-resource conditions, as compared to males producing relatively small displays. Specifically, we used selection lines 1 Current address: Department of Biology, Davis Science 128, P.O. Box 60, Middle Tennessee State University, Murfreesboro, Tennessee 37132, U.S.A. 2 Author for correspondence; e-mail:
[email protected].
Manuscript received April 2009; revised manuscript received June 2009.
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mean flower production by males from nine different populations (Delph et al. 2002). In terms of the degree of sexual dimorphism, Delph and Bell (2008) examined whether differences among populations could be caused by one sex responding more plastically than the other to water stress. One measure of flower size exhibited among-population variation in the extent of sexual dimorphism. However, the two sexes did not differ in their plastic response to water stress, suggesting that sex-differential plasticity cannot account for the amongpopulation variation in sexual dimorphism in flower size (Delph and Bell 2008). The authors therefore suggested that selection on flower size could vary among populations if a trait correlated with size was sex-differentially plastic. For example, if pollen production is relatively low in individuals making small flowers only in dry sites and pollen production is more plastic than ovule production, then disruptive selection on size would likely vary between wet and dry sites (Delph and Bell 2008). Under such a scenario, we would predict that among populations, flower production, which trades off with flower size, should be more variable in males than in females. Here, we investigate how flower production, flower size, and pollen production in males are affected by variation in water and nutrients in S. latifolia, in part because water and nutrient availability are two variables that have been shown to cause both plastic responses and selection in plants. For example, soil moisture has been shown to affect the extent of selection on flower number (Caruso et al. 2003), rainfall at the time of flowering has been shown to be positively correlated with flower size (Galen 1999, 2005), and water stress, combined with nutrient limitation, has been shown to reduce corolla size and ovule number (Frazee and Marquis 1994). Water and nutrient stress have also been shown to have large effects on reproductive traits (reviewed in Delph et al. 1997) such as pollen grain size and number (Lau and Stephenson 1993, 1994), pollen competitive ability (Young and Stanton 1990; Lau and Stephenson 1993, 1994), and ovule production (Frazee and Marquis 1994). In addition, these variables are pertinent for this study because S. latifolia grows in a wide range of habitat types that vary widely for these and other variables (Baker 1948). We focused on the effect of water and nutrient stress in males because they bear a higher cost of reproduction than females (Delph et al. 2005). In addition to examining how males from the two lines respond to water and nutrient stress, we tested Delph and Bell’s (2008) suggestion that pollen production might be reduced more than ovule production in stressful conditions. Our study addressed two main questions. (1) In males, are the effects of water and/or nutrient stress more pronounced in the selection line that produces many small flowers than in the one that produces relatively few large flowers? We tested for such line-differential plasticity by looking for a significant interaction between selection line and either the water and/or nutrient treatments. (2) Is pollen production more sensitive to water and/or nutrient stress than ovule production? We tested for such differential plasticity by comparing how pollen and ovule production per flower was affected by water and/or nutrients within each selection line. We also looked at whether variation in male flower production among populations was variable and positively related to the extent of sexual dimorphism.
Methods and Material Study Species Silene latifolia (Caryophyllaceae) is a dioecious, short-lived perennial native to Europe (Baker 1948) but naturalized throughout much of North America (McNeill 1978). It produces white-petaled flowers that open in the evening and are primarily pollinated by night-flying moths (Young 2002). We produced the seeds for our experiment by crossing plants from previous artificial-selection experiments in which selection was performed to increase flower size in males and decrease flower size in females (Delph et al. 2004). Because of a strong between-sex genetic correlation for flower size, this selection increased the phenotypic range of flower size greatly in the two sexes. Moreover, because of a strong sizenumber trade-off, this selection also increased the phenotypic variation in flower production within each sex. Seeds from families of the two replicate selection experiments were grown to flowering and crossed to each other to eliminate any inbreeding effects from previous generations. This produced four independent families via crosses between the many-small-flower selection lines from each experiment and four independent families via crosses between the few-large-flower selection lines from each experiment. This pooled the two experiments into one set of families with many small flowers and one set with few large flowers, which we refer to respectively as the manyflower line and the few-flower line. Petal size was measured, and the results are reported here. Calyx width, calyx length, and flower mass measurements were also taken, and the results verified that the families were divergent in these measures of flower size in the expected direction (data not shown).
Experiment Varying Water and Nutrient Availability To investigate the effects of water and/or nutrient availability on traits, we grew plants under varying water and nutrient treatments in a greenhouse at Indiana University. We planted 72 seeds per family from our eight families in 50 : 50 soil : MetroMix. After germination, seedlings were transplanted into 4-in clay pots and randomly assigned to water and nutrient treatments. Plants were germinated and grown under a 16-h day length and were moved within the greenhouse weekly to minimize positional effects. We kept all males that flowered (n ¼ 165, mean 5.4 per family/treatment) and randomly selected three females per family/treatment, when available, from those that flowered (n ¼ 92). Our experiment was fully factorial, with the following four treatments: high water–high nutrient, low water–high nutrient, high water–low nutrient, and low water–low nutrient. Plants in the high-water treatments were watered daily, whereas plants in the low-water treatments were watered only every 2–3 d, such that the plants reached the wilting point between waterings. Plants in the high-nutrient treatments were fertilized once per week with a dilute 20 : 20 : 20 fertilizer, whereas no fertilizer was added during the course of the experiment in the low-nutrient treatments. The high-water– high-nutrient conditions were identical to the conditions used to evaluate the effect of selection in Delph et al. (2004). On each male plant, we recorded the date of first flowering, and 30 d after this date we counted the total number of flowers that had been produced over this period. In analyses
HERLIHY & DELPH—RESPONSE TO STRESS reported below, flower number was ln transformed to improve normality. We measured the length of one petal limb (the portion of the petal extending beyond the fused calyx) of the third, fourth, and fifth flowers produced on each male, in order to minimize any effect of within-plant variation on flower size. These measurements are presented as the means of the three measured flowers. We also collected an undehisced anther from each male from a single flower 30 d after first flowering. Each anther was placed in a glass vial and allowed to air dry and dehisce. Once the anther dehisced, we added 500 mL of 3 : 1 glycerol : lactic acid to each vial. To quantify pollen production, we vortexed each sample and placed two replicates of 10 mL each on a microscope slide with a drop of Alexander’s stain (Dafni et al. 2005, p. 116). We then counted the number of pollen grains in each 10-mL replicate and multiplied each count by 50 to estimate the total number of pollen grains per anther. As there are 10 anthers per flower, we then multiplied the number of pollen grains per anther by 10 to obtain pollen number per flower. We report the means of the two replicate measurements. We then multiplied estimates of pollen production per flower by the number of flowers produced in the 30 d after first flowering to estimate pollen production at the wholeplant level. In analyses reported below, pollen production per plant was ln transformed to improve normality. On females, we also recorded the date of first flowering and collected one flower per plant 30 d after this date. We preserved these flowers in 70% ethanol and later counted the number of ovules in each flower under a dissecting microscope. To evaluate the effects of water and nutrient treatments on flower number, petal size, and pollen production in males from the two divergent selection lines, we used three-way ANOVAs to examine the effects of line, water treatment, nutrient treatment, and their interactions. To test whether pollen production was more plastic than ovule production, we compared gametophyte production (pollen per flower or ovules per flower), using two-way ANOVAs, and evaluated the sex 3 treatment interaction within each selection line.
Among-Population Variation in Sexual Dimorphism We analyzed flower production data from a survey of nine populations of S. latifolia across its range (Delph et al. 2002) to test whether flower production by males is more variable than
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that by females and whether it is correlated with the degree of sexual dimorphism among populations. We calculated the magnitude of sexual dimorphism in flower production in each population by using the sexual dimorphism index of Lovich and Gibbons (1992): ðlarge mean=small meanÞ 1. We assessed among-population variation in flower production by comparing coefficients of variation for flower production in males and in females and calculated Pearson correlations between sexual dimorphism for flower production and mean flower production in each sex.
Results Flower Number and Petal Size Males from the few-flower line made significantly fewer flowers than males in the many-flower line (mean 6 1 SE ¼ 209:9 6 11:5 vs. 376:3 6 14:7), as expected. Water and nutrient stress both reduced flower number, and there was a significant interaction between water and nutrient stresses (table 1). Nutrient stress had a stronger effect on flower production than water stress (fig. 1; table 1), and the low-nutrient treatment reduced flower production in both the many-flower line (38% decrease) and the few-flower line (31% decrease); the line 3 nutrient treatment interaction was not significant. Flowers from the few-flower line had 6% longer petal limbs than plants in the many-flower line (11:7 6 0:1 vs. 11:0 6 0:2 mm), a significant difference (table 1). Water stress significantly reduced petal limb length (P ¼ 0:005), but nutrient stress did not (table 1; fig. 1). In addition, there was a marginally significant line 3 water treatment interaction for petal limb length (P ¼ 0:053), with the many-flower line reducing its petal size more than the few-flower line.
Line-Differential Pollen Production and Sex-Differential Gametophyte Production Males from the few-flower line produced flowers that contained 48% more pollen grains per flower than plants from the many-flower line (30; 980 6 1120 vs. 20; 890 6 1480), and these differences were significant (table 1). However, because males from the many-flower line made more flowers, the difference in pollen production between the many- and few-flower lines at the whole-plant level (8:2 6 0:8 million
Table 1 Effects of Selection Line (L), Water (W), and Nutrient (N) Treatments on Flower Number, Petal Limb Length, and Pollen Production per Flower and per Plant in Males of Silene latifolia
Number of flowers P Petal limb length P Pollen per flower P Pollen per plant P
Error df
L
W
N
156
76.77 (
