STIGMA RECEPTIVITY AND EFFECTS OF PRIOR SELF-

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1 American Journal of Botany 93(1): STIGMA RECEPTIVITY AND EFFECTS OF PRIOR SELF- POLLINATION ON SEED SET IN TRISTYLOUS LYTHRUM SALICARIA (LYTHRACEAE) 1 ANNA R. WAITES 2 AND JON ÅGREN 3,4 2 Department of Ecology and Environmental Science, Umeå University, SE Umea, Sweden; and 3 Department of Plant Ecology, Evolutionary Biology Centre, Uppsala University, Villavägen 14, SE Uppsala, Sweden Stigma position is a key aspect of flower morphology that may influence pollination success and seed production. In the selfincompatible, tristylous herb, Lythrum salicaria, the stigma is positioned inside the corolla tube in the short-styled morph, but outside the corolla in the mid- and long-styled morphs. We performed controlled pollinations to determine whether style morphs differ in duration of stigma receptivity and in interference caused by self-pollen deposition. As predicted, flowers of the short-styled morph remained receptive for longer if not pollinated than did flowers of the other two style morphs. When deposition of self-pollen preceded compatible cross-pollination, seed set was higher in the mid-styled morph than in the other style morphs. Interference caused by selfpollen deposition was overall strongest in the short-styled morph, but the magnitude of differences among style morphs was contingent on the relative timing of self and compatible pollen receipt. The results indicate that both differences in duration of stigma receptivity and interference from self-pollen deposition may contribute to observed variation in seed production and pollen limitation among style morphs in natural populations of L. salicaria. An association between stigma position and duration of stigma receptivity should constrain the evolution of flower morphology. Key words: flower morphology; heterostyly; Lythrum salicaria; pollen-pistil interference; self-incompatibility; self-pollination; stigma receptivity; tristyly. Identification of the functional and adaptive significance of variation in flower morphology is fundamental to our understanding of the processes that shape patterns of seed production and floral evolution (e.g., Nilsson, 1988; Campbell, 1991; Johnston, 1991; Schemske and Ågren, 1995; Galen and Cuba, 2001; Aigner, 2004). The position of the stigma within the flower is a key aspect of flower morphology, which influences the efficiency of pollen transfer (Campbell et al., 1996; Cresswell, 2000; Nishihiro et al., 2000) and the likelihood of withinflower self-pollination (Karron et al., 1997; Motten and Stone, 2000; Elle and Hare, 2002; but see Medrano et al., 2005). Less is known about the associations between stigma position and duration of stigma receptivity and pollen pistil interactions. However, a stigma concealed inside a corolla tube is less exposed to sun, wind, and rain, and may therefore remain receptive for longer than a stigma protruding out of the corolla. Moreover, intraspecific variation in stigma position has been found to be associated with differences in interference caused by self-pollen in some species (Cesaro et al., 2004). Heterostylous plants provide useful experimental systems for investigating how discrete variation in flower morphology and pollen pistil interactions affect pollen transfer and seed production (e.g., Ganders, 1979; Barrett and Glover, 1985; Kohn and Barrett, 1992; Harder and Barrett, 1993; Pailler et al., 2002; Barrett et al., 2004; Ornelas et al., 2004). The style morphs of heterostylous plants differ in the lengths of their stamens and styles and may also differ in the strength of the self-incompatibility reaction (Darwin, 1877; Dulberger, 1992). Morph-specific differences in the positioning of the stigma 1 Received for publication 23 February 2005; revision accepted 7 October The authors thank two anonymous reviewers for helpful comments on the manuscript. This study was financially supported by grants from the Swedish Research Council to J.Å. and from J. C. Kempes Minnes Stipendiefond to A.R.W. 4 Author for correspondence ( jon.agren@ebc.uu.se) 142 should affect pollen receipt because stigma position may influence both the duration of stigma receptivity and the efficiency of pollen transfer during pollinator visits. The relative efficiency of pollen transfer to stigmas positioned at different heights in the flower can be expected to vary with pollinator morphology and behavior (Wolfe and Barrett, 1989; Arroyo and Dafni, 1995). In natural populations of several heterostylous plants, both the total number of pollen grains received per flower and the proportion of compatible to incompatible pollen grains received vary among style morphs (e.g., Ganders, 1974, 1979; Ornduff, 1975; Barrett and Glover, 1985; Wolfe and Barrett, 1989; Nishihiro et al., 2000). However, the extent to which this variation reflects differences among style morphs in the duration of stigma receptivity, visitation rate, or efficiency of pollen transfer during individual visits is poorly understood (Wolfe and Barrett, 1989; Arroyo and Dafni, 1995). In particular, there is no quantitative information on the relative duration of stigma receptivity in the style morphs of heterostylous plants. Differences among style morphs in the extent to which selfpollen deposition interferes with the performance of compatible pollen could contribute to morph-specific differences in seed output. In tristylous Pontederia sagitatta, self-pollen deposition reduced seed set in the long- and mid-styled morph, but not in the short-styled morph (Scribalio and Barrett, 1994). Similarly, only the long-styled morph had reduced seed set after self-pollen deposition in distylous Turnera ulmifolia (Shore and Barrett, 1984), and the long-styled morph was more sensitive to self-pollen deposition than the short-styled morph in dimorphic Narcissus assoanus (Cesaro et al., 2004). However, it is not known whether such differences are common among heterostylous plants. In Swedish populations of the tristylous, self-incompatible herb, Lythrum salicaria, the long-styled morph tends to produce fewer seeds per flower than the mid- and short-styled

2 January 2006] WAITES AND ÅGREN STIGMA RECEPTIVITY AND SELF-POLLEN DEPOSITION 143 morphs because of differences in pollen limitation (Ågren, 1996; Ågren and Ericson, 1996; Waites and Ågren, 2004). Moreover, stigmas of the long-styled morph receive considerable amounts of incompatible pollen (Waites and Ågren, 2004). Stigma position differs among style morphs. The stigma is positioned inside the corolla tube in the flowers of the short-styled morph, but is exposed outside the corolla in the flowers of the mid- and long-styled morphs. In this study, we conducted controlled crosses to determine whether morph-specific differences in the duration of stigma receptivity and interference from self-pollen may contribute to the observed variation in seed output and pollen limitation. More specifically, we (1) tested the hypothesis that the duration of stigma receptivity is longer in the short-styled morph than in the mid- and long-styled morphs and (2) determined whether interference of self-pollen deposition with the performance of legitimate cross-pollen varies among style morphs of L. salicaria. MATERIALS AND METHODS The plant Purple loosestrife, Lythrum salicaria L. (Lythraceae), is an insect-pollinated, self-incompatible, tristylous, perennial herb, which is native to Eurasia and an invasive species in North America (Hultén and Fries, 1986; Thompson et al., 1987). Populations of L. salicaria include three morphologically distinct mating types: plants with long-styled (L), mid-styled (M), or short-styled (S) flowers. Each flower of a given morph produces two anther whorls at levels corresponding to the positions of stigmas in flowers of the other two morphs (reciprocal herkogamy). Because of physiological intramorph incompatibility, full seed set requires that pollen from anthers of corresponding height is deposited on the stigma (Darwin, 1877; O Neil, 1994; Mal et al., 1999). Experimental design Plants were derived from three large L. salicaria populations in the Skeppsvik archipelago on the coast outside Umeå, N. Sweden (populations 1, 3, and 14 in Ågren, 1996; Waites and Ågren, 2004). The experiment included 54 plants raised in the greenhouse from seeds collected in the field, that is, six plants of each style-morph from each of the three populations (each plant from a different maternal family). We conducted controlled pollinations to determine whether the style morphs differed in duration of stigma receptivity, seed set after self-pollination, and the interference of self-pollination with seed production after legitimate crosspollination. During the experiment, plants were checked daily, and mature flower buds were selected and marked prior to anthesis. Each morning between 0800 and 0900 hours, we identified newly opened flowers and assigned them to one of 12 treatments. To determine the duration of stigma receptivity, we pollinated individual flowers with compatible pollen early during the morning of the first day of anthesis (between 0800 and 0900 hours), or 1 h, 2 h, 4 h, 8 h, or 24 h later. Each experimental flower was pollinated with compatible pollen from two randomly chosen pollen donors (one of each style morph) from the same population as the pollen recipient. Pollen was transferred by brushing dehiscing anthers with compatible pollen across the stigma. To quantify the extent to which prior self-pollination interferes with seed production, we pollinated flowers with self-pollen during the morning of the first day of anthesis (between 0800 and 0900 hours) and then with compatible cross-pollen either immediately following self-pollination, or 1 h, 2 h, 4 h, 8 h, or 24 h later, as above. Self-pollen was applied by gently brushing one dehiscing anther from each anther whorl of the experimental flower across the stigma. To quantify seed production after self-pollination only, we selfpollinated flowers during the morning of the first day of anthesis as described. To assess uncontrolled pollen transfer in the greenhouse, control flowers were marked but left unpollinated. Each treatment was replicated twice on each experimental plant. No unpollinated flowers produced seeds, suggesting that uncontrolled pollen transfer was not a problem in the greenhouse. Fruits were collected at maturation and the number of filled seeds was counted under a stereomicroscope. All statistical analyses of variation in number of seeds produced per flower were based on plant means. To quantify the interference of prior deposition of self-pollen with the performance of compatible pollen, we calculated the difference in seed output between flowers receiving and not receiving self-pollen prior to the receipt of compatible pollen at a given interval after flower opening for each plant. Pollen production data and observations of stigmatic pollen loads in the field suggest that the experimental flowers received more compatible pollen grains, but similar amounts of incompatible pollen grains in comparison to flowers in the source populations. Pollen production data obtained in the field (population 1) and from plants grown in a common garden experiment (population 14) indicate that up to about 4000 compatible and 7000 incompatible pollen grains may have been transferred to stigmas on long-styled plants in the cross- and self-pollination treatments, respectively. The corresponding figures for mid-styled plants reached 6000 compatible and 4000 incompatible pollen grains, and for short-styled plants reached 5000 compatible and 4000 incompatible pollen grains (J. Ågren and A. Waites, unpublished data). These pollen loads greatly exceed the maximum 200 ovules produced per flower and the stigmatic pollen loads observed in the often pollen-limited populations in the Skeppsvik archipelago (Waites and Ågren, 2004; A. Waites and J. Ågren, unpublished data). However, the number of incompatible pollen grains applied in the self-pollination treatment was of the same order as observed on long-styled plants in the field (mean 1000 incompatible pollen grains per stigma in 13 and 11 populations in the Skeppsvik archipelago during two different years; Waites and Ågren, 2004). Statistical analysis We analyzed variation in (1) seed output per flower after legitimate cross-pollination only, (2) seed output per flower when selfpollination preceded compatible pollination, and (3) interference with repeated-measure ANOVA models using PROC MIXED in SAS (2002). In these models, time of pollination was the within-subject factor, and population and style morph between-subject factors. In preliminary analyses, we used Akaike s information criterion to identify the covariance structure that best fit the data (Littell et al., 1996). To explore differences among style morphs in the relationships between stigma receptivity (seed set after legitimate pollination only) and time of pollination, and between interference and interval between self- and cross-pollination, we used PROC MIXED to analyze models that treated time of pollination as a continuous variable (Littell et al., 1996). These models included data only from the day of anthesis, because on the following day seed set was very low in all treatment combinations (see Results). Initial models included both linear and quadratic terms. However, because quadratic terms and interactions with quadratic terms were not statistically significant, they were excluded from the final models. When ANOVA models indicated significant time-of-pollination stylemorph interactions or a significant main effect of style morph, we used contrasts to explore these differences further. In addition, we used one-tailed t tests to determine for which time-of-pollination style-morph combinations interference differed significantly from zero. The sequential Bonferroni test was applied to control the experimentwise type I error rate (Holm, 1979). Because few flowers produced seed following self-pollination only, we compared seed output of the three morphs in this treatment using the nonparametric Kruskal Wallis test with a correction of H for ties (H c ; Zar, 1984). RESULTS Seed set after self-pollination Seed set was very low after pollination with self-pollen, but was somewhat higher in the M than in the L and S morphs (seed number per flower, median, first quartile, and third quartile, L 0, 0, 46.8; M 3.3, 0, 49; S 0, 0, 0; Kruskal Wallis H c 7.7, P 0.02). Only 50% of mid-styled plants, 28% of the long-styled plants, and 11% of the short-styled plants (N 18) produced any seeds after self-pollination. Duration of stigma receptivity The duration of stigma receptivity varied among style morphs as indicated by a significant time-of-pollination style-morph interaction in the anal-

3 144 AMERICAN JOURNAL OF BOTANY [Vol. 93 TABLE 1. Effects of population of origin, style morph, and time of legitimate cross pollination (between 0800 and 0900 hours on the day the flower opened, or 1 h, 2 h, 4 h, 8 h, or 24 h later) on seed output per flower after cross-pollination only and on the degree to which prior selfpollination reduced seed set per flower in tristylous Lythrum salicaria analyzed with repeated-measure ANOVA. Interference was quantified as the difference in number of seeds produced when legitimate cross-pollination was preceded by self-pollen deposition between 0800 and 0900 hours on the day the flower opened and number of seeds produced when flowers were pollinated with compatible pollen only. The analyses were conducted on plant means, and the final models were based on unstructured covariance (legitimate cross-pollination only) and autoregressive covariance of order one (interference). Source Num df Seed production after legitimate cross pollination only Den df F P Interference from prior self-pollination Den df F P Population (pop) Style morph Pop style morph Time Time population Time style morph Time pop style morph Note: Num df numerator degrees of freedom, Den df denominator degrees of freedom. ysis of seed output of flowers receiving only legitimate crosspollination (Table 1). During the day of anthesis, stigma receptivity declined more slowly for the S morph than the L and M morphs (regression coefficient SE, extracted from the model that treated time as a continuous variable; L, , df 50, t 3.8, P ; M, , df 50, t 5.5, P ; S, , df 50, t 1.6, P 0.12; Fig. 1A). Compared to flowers pollinated with compatible pollen during early morning (between 0800 and 0900 hours on the first day of flowering), mean seed set was 15 29% lower when flowers were pollinated 4 h later, and 17 41% lower when flowers were pollinated 8 h later (statistically significant decrease in the L and M morphs, but not in the S morph; Fig. 1A). Seed set was 1.6 times higher in the S morph than in the L morph when pollination with compatible pollen was delayed by 8 h (contrast L vs. S, F 1,44 7.5, single-test significance level, P 0.009, not statistically significant at the experimentwise 0.05 level after sequential Bonferroni correction; Fig. 1A). All three style morphs set few seeds when flowers were pollinated one day after flower opening (Fig. 1A). Seed set when pollination with compatible pollen was preceded by the deposition of self-pollen Flowers of the M morph that received self-pollen prior to compatible pollen produced more seeds than those of the other style morphs (Fig. 1B; repeated measures ANOVA model with unstructured covariance, F 2,44 3.6, P 0.04; contrast, M vs. L, F 1, , P 0.026; M vs. S, F 1, , P 0.026; L vs. S, F 1, , P 0.98). Overall, seed production decreased with the interval between self- and cross-pollination (F 5, , P ), and this effect did not differ among style morphs or populations (P 0.05). Seed set did not differ among populations (F 2,44 1.2, P 0.30). Interference of self-pollen Prior self-pollination reduced seed set more strongly in the S morph than in the L and M morphs, but the magnitude of this difference varied with the interval between self and legitimate cross pollination as indicated by a significant time-of-pollination style-morph interaction (Table 1, Fig. 1C). In the short-styled morph, prior selfpollination reduced seed set when 1 h or more had passed between self-pollen deposition and pollination with compatible pollen, and the reduction in seed production caused by prior self-pollen deposition increased significantly with the interval between self- and legitimate cross-pollination on the day of anthesis (interference regressed on interval between self- and legitimate pollination, regression coefficient SE, , df 209, t 6.7, P ; Fig. 1C). In contrast, in the long-styled morph, prior self-pollination reduced seed number per flower only when at least 2 h had passed between self-pollen deposition and pollination with compatible pollen, and in the mid-styled morph, no statistically significant interference was detected (Fig. 1C). Moreover, the relationship between interference and pollination interval was weaker and not statistically significant in the L and M morphs (L, , df 209, t 1.8, P 0.07; M, , df 209, t 0.7, P 0.47; Fig. 1C). DISCUSSION This study has demonstrated experimentally that both the duration of stigma receptivity and the effects of self-pollen deposition on seed set vary among style morphs in tristylous Lythrum salicaria. Moreover, the magnitude of the morph-specific effects of prior self-pollination on seed set was contingent on the relative timing of the deposition of self and compatible pollen. The results demonstrate that duration of stigma receptivity and interference from incompatible pollen can vary with stigma position and should therefore be considered when mechanisms underlying floral evolution are explored. More specifically, they indicate that the relative seed output of the three style morphs of L. salicaria will be influenced by the rate at which compatible pollen is received, and by the relative timing of self and legitimate cross pollination. They thus identify mechanisms that could explain previously documented differences in pollen limitation and seed production among style morphs in natural populations of L. salicaria. As predicted, flowers of the short-styled morph remained receptive for longer if not pollinated than flowers of the other two style morphs (Fig. 1A). This result is consistent with the concealed stigma of the short-styled morph drying more slowly than a stigma that is exposed outside the corolla. To explore this hypothesis further, it would be interesting to determine the effects of corolla removal on duration of stigma receptivity in the three style morphs. Long stigma receptivity should be advantageous when pollinator visitation is low. Moreover, the probability of pollen transfer to a stigma inside the corolla tube should be lower than to stigmas that are positioned at or outside the mouth of the corolla tube (cf. Wolfe and Barrett, 1989; Campbell, 1991;

4 January 2006] WAITES AND ÅGREN STIGMA RECEPTIVITY AND SELF-POLLEN DEPOSITION 145 Fig. 1. Seed set and degree of interference from prior self-pollination in the long- (L), mid- (M), and short-styled (S) morphs of Lythrum salicaria (N 18 plants per style morph). Means SE (based on plant means) are given. (A) Seed number per flower after pollination with compatible pollen between 0800 and 0900 hours on the day of flower opening, or 1 h, 2 h, 4 h, 8 h, or 24 h later. Means that differ significantly from that observed after pollination at time 0 (between 0800 and 0900 hours on the first day of flowering) are indicated with filled symbols (contrasts evaluated separately by morph, experimentwise P 0.05 determined with sequential Bonferroni correction). (B) Seed number per flower when self-pollination at time 0 h preceded legitimate cross-pollination at different times. (C) Interference from prior selfpollen deposition (difference in number of seeds produced when self-pollination at time 0 h preceded legitimate cross-pollination and number of seeds produced when flowers were pollinated with compatible pollen only). Values that are significantly different from zero are indicated with filled symbols (one-tailed t tests, experimentwise error rate P 0.05). Nishihiro et al., 2000), and longer stigma receptivity may compensate partially for this. To our knowledge, this is the first study comparing quantitatively the duration of stigma receptivity in the different style morphs of a heterostylous plant. However, because style morphs differ considerably in the extent to which the stigma is exposed to desiccating wind and sun in many heterostylous species, we expect morph-specific differences in duration of stigma receptivity to be widespread at low pollination levels, i.e., when high visitation rates do not induce rapid wilting of flowers in all style morphs. More generally, the results suggest that effects on duration of stigma receptivity may constrain the evolution of flower morphology, in particular in environments where pollinator visitation is low. Morph-specific differences in effects of prior self-pollination on seed set vary among tristylous species. In the present study, the negative effect of prior self-pollen deposition on seed set was stronger in the short-styled morph than in the other style morphs, although the magnitude of this difference was contingent on the relative timing of the deposition of self and legitimate cross pollen (Fig. 1C). In contrast, prior selfpollen deposition reduced seed set in the long- and mid-styled morphs, but not in the short-styled morph in Pontederia sagittata (Pontederiaceae; Scribalio and Barrett, 1994). Several factors may contribute to morph-specific differences in the effects of prior-self-pollination including: differences in the adherence of self-pollen grains to stigmas (Scribalio and Barrett, 1994), differences in pollen production by anthers at different positions in the flower and hence received by the different style morphs (Shore and Barrett, 1984; Scribalio and Barrett, 1994), and differences in the growth of self-pollen tubes in the style (Shore and Barrett, 1984). The first two mechanisms cannot explain why the short-styled morph was more sensitive to prior self-pollination in L. salicaria. In contrast to tristylous P. sagittata (Scribalio and Barrett, 1994), self-pollen adhered equally to the stigma in L. salicaria in the present study. Moreover, estimates of pollen production indicate that stigmas of the short-styled morph, which was more sensitive to prior self-pollination, did not receive more incompatible pollen grains than stigmas of the mid-styled and longstyled morphs in the self-pollination treatment (see Materials and Methods; J. Ågren and A. Waites, unpublished data). To explore whether morph-specific differences in interference caused by prior self-pollination depend on differences in how far self-pollen grow in the style (Shore and Barrett, 1984), an examination of pollen tube growth in the three style morphs of L. salicaria following self-pollination would clearly be of interest. Seed set after self-pollination varied significantly among style morphs and was highest in the mid-styled morph. This is consistent with the results of controlled crosses performed with plants from British and North American populations of L. salicaria (Darwin, 1877; O Neil, 1994; Mal et al., 1999) and suggests that the incompatibility reaction is somewhat weaker in the mid-styled morph than in the other morphs. However, the difference in seed set after self-pollination was not sufficiently large to explain by itself why the M morph consistently produced more seeds than the L and S morphs when legitimate cross pollination was preceded by self-pollen deposition (Fig. 1B). Morph-specific differences in seed output have been recorded both in the native and introduced ranges of L. salicaria and may be related to differences among style morphs in duration of stigma receptivity, interference from self-pollen, rates of pollen receipt, or a combination thereof. Long-styled plants tend to produce fewer seeds and to be more strongly pollenlimited than mid-, and short-styled plants in several Swedish populations (Ågren, 1996; Ågren and Ericson, 1996; Waites and Ågren, 2004), whereas short-styled plants produce fewer seeds and are more strongly pollen limited in two North American populations (O Neil, 1992). The results of the present study suggest that morph-specific differences in seed output may be observed even if the timing of self and compatible pollen deposition is the same in the three style morphs. When

5 146 AMERICAN JOURNAL OF BOTANY [Vol. 93 the rate of pollen deposition is low, the S morph may have an advantage because of its longer stigma receptivity. If self-pollen is regularly deposited before legitimate cross pollen, the M morph should have an advantage. In Swedish L. salicaria populations, legitimate cross pollen may often be deposited at relatively low rates as indicated by frequent pollen limitation (Ågren, 1996; Ågren and Ericson, 1996; Waites and Ågren, 2004). The likelihood that self-pollen is deposited prior to legitimate cross pollen may be related to the size of the individual plant, but also to population size and density. Geitonogamous self-pollination is likely to increase with the number of open flowers (de Jong et al., 1993; Barrett et al., 1994; Karron et al., 2004) and may be particularly high in small populations of low density (Waites and Ågren, 2004). To add complexity, the rate of pollen receipt and the composition of the pollen load is not necessarily the same in the three style morphs. In a study of two L. salicaria populations in the Skeppsvik archipelago in northern Sweden, the mid-and the long-styled morphs had larger total conspecific pollen loads than the short-styled morph in one population, whereas no morph-specific differences in pollen load were detected in a second population (A. Waites and J. Ågren, unpublished data). Field studies of the transfer of pigments used as pollen analogues in L. salicaria populations in North America indicated that both the total number of pollen grains and the proportion of compatible pollen grains received may differ among style morphs (O Neil, 1992). 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