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. 2010 Feb 10;105(4):595–605. doi: 10.1093/aob/mcq013

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Benjamin R Montgomery 1,*, Deanna M Soper 1, Lynda F Delph 1
PMCID: PMC2850797  PMID: 20147372

Abstract

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.

Keywords: Conspecific pollen precedence, hybridization, pollen tubes, reproductive isolation, Silene dioica, Silene latifolia

INTRODUCTION

Many plant species maintain distinct traits in zones of sympatry despite being interfertile. In such instances, it is of interest to understand what pre- and postzygotic isolating mechanisms contribute to the maintenance of species boundaries (Arnold, 1994; Ramsey et al., 2003). Prezygotic isolating mechanisms include differences in ecogeographic distributions (Ramsey et al., 2003), flowering phenology and periodicity (Levin, 1978; Arnold, 1994), reliance on different pollinators (Grant, 1949; Meléndez-Ackerman and Campbell, 1998) and spatial structure minimizing pollen exchange (Wesselingh and Arnold, 2000). However, species that overlap in flowering time and share pollinators are likely to receive mixed pollen loads on their stigmas, in which case conspecific pollen precedence may be another important isolating mechanism (Darwin, 1859; Mangelsdorf and Jones, 1926; Smith, 1970; Howard, 1999).

Conspecific pollen precedence occurs when conspecific pollen achieves fertilization of ovules more successfully than heterospecific pollen because of faster pollen germination, faster pollen-tube elongation or an increased ability to reach the micropyle and effect fertilization (Smith and Clarkson, 1956; Grant, 1963; Chen and Gibson, 1972; Howard, 1999). This mechanism and its consequences closely resemble conspecific sperm precedence, which has been detected in a variety of animals (Howard, 1999; Dean and Nachman, 2009). Faster conspecific pollen-tube elongation has been observed or inferred in multiple studies (Smith and Clarkson, 1956; Ascher and Peloquin, 1968; Jaynes, 1968; Chen and Gibson, 1972; Song et al., 2002), although other studies have not consistently detected differences (Kearney, 1932; Rieseberg et al., 1995; Diaz and Macnair, 1999), or have found faster heterospecific elongation in at least some cases (Buchholz et al., 1935; Carney et al., 1994; Emms et al., 1996; Husband et al., 2002). Similarly, several studies with mixed pollen treatments have detected disproportionate siring of seeds by conspecific pollen (Smith, 1968, 1970; Carney et al., 1994; Rieseberg et al., 1995; Emms et al., 1996; Klips, 1999; Song et al., 2002; Campbell et al., 2003), but other studies have detected no difference (Alarcón and Campbell, 2000), conspecific advantage for only a subset of species (Hauser et al., 1997; Diaz and Macnair, 1999; Wolf et al., 2001; Ramsey et al., 2003; Figueroa-Castro and Holtsford, 2009), or a heterospecific advantage for some species (Carney and Arnold, 1997; Wang and Cruzan, 1998).

The proportion of hybrid seeds produced after application of different quantities of pollen mixtures can be used to disentangle the mechanisms that cause disproportionate seed siring (Cruzan and Barrett, 1996; Wang and Cruzan, 1998). Higher attrition rates of heterospecific pollen tubes, faster pollen-tube elongation, and higher abortion rates of heterospecifically fertilized ovules can all lead to conspecific pollen siring disproportionately more seeds. However, effects of application of different pollen quantities on disproportionate seed siring differ depending on whether the advantage results from mechanisms based on speed at which fertilization is achieved (time-variant), or mechanisms unrelated to the timing of events (time-invariant; Cruzan and Barrett 1996). For time-variant mechanisms, such as differential pollen-tube elongation, receipt of larger mixed pollen loads allows conspecific pollen tubes to fertilize a greater number of ovules before heterospecific pollen tubes reach ovules, thereby resulting in disproportionately low hybridization rates compared with smaller pollen loads of similar composition. In contrast, for time-invariant mechanisms, such as different frequencies of pollen-tube attrition or different post-fertilization abortion frequencies, then a similar proportion of ovules is expected to be fertilized by conspecific pollen regardless of the pollen load. Finally, in contrast to other mechanisms, abortion of hybrid embryos caused by incompatibilities may result in reduced seed set with increased proportions of heterospecific pollen. However, abortion of hybrid embryos because of resource allocation to nonhybrid embryos would not necessarily lead to a change in seed set. These predictions are summarized in Table 1.

Table 1.

For flowers pollinated with conspecific pollen, mixed pollen or heterospecific pollen, predictions for (A) change in proportions of seeds sired by more successful pollen upon increasing the total pollen application, and (B) change in seed set with increased proportions of the more successful pollen type applied to stigma

Mechanism (A) Proportion of seeds sired by more successful pollen (B) Seed set
Prezygotic, time-variant Increased proportions No change
Prezygotic, time-invariant or post-zygotic resource reallocation No change in proportions No change
Differential post-zygotic abortion due to inviability No change in proportions Increased seed set
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Post-pollination differences in seed siring ability may contribute to maintenance of the species boundary between two Silene species, S. dioica and S. latifolia. These species are closely related (Desfeux and Lejeune, 1996), and both are dioecious, with homogametic females (XX) and heterogametic (XY) males (Grant et al., 1994). They have broadly overlapping ranges across much of Europe and are interfertile (Baker, 1947a, b; Prentice, 1978) with no reduction in seed set for interspecific pollinations (Goulson and Jerrim, 1997). Rahmé et al. (2009) found evidence of a post-pollination barrier to hybridization acting against S. dioica, but not S. latifolia pollen in interspecific crosses, but their study did not examine the mechanisms responsible for this pattern.

Although this study focuses on post-pollination barriers to hybridization between S. dioica and S. latifolia, reproductive isolation also results from differences in habitat, timing of flowering, and floral syndromes. Silene dioica tends to occur in partly shaded environments (Baker, 1947b) whereas S. latifolia typically occurs in unshaded habitats with drier soils, and more disturbance, including pastures and fallow fields (Baker, 1947b; Willmot and Moore, 1973; Prentice, 1979; Karrenberg and Favre, 2008). However, the species grow together where their habitats meet and in intermediate habitats (Baker, 1947b). Flowering phenology overlaps incompletely between the species, with peak flowering for S. dioica occurring earlier in the season than for S. latifolia (Baker, 1947a, b; Bopp and Gottsberger, 2004). Additionally, the periodicity of flowering differs, with S. latifolia flowers primarily opening and being visited at night in contrast to S. dioica flowers, which open and are visited more diurnally (Jurgens et al., 1996; Goulson and Jerrim, 1997; van Putten et al., 2007). Finally, S. dioica flowers, which are pink and tubular, are most often visited by bumblebees whereas S. latifolia flowers, which are white and tubular, are most often visited by moths (Baker, 1947a, b; Jurgens et al., 1996; Goulson and Jerrim, 1997; van Putten et al., 2007).

Nonetheless, direct and indirect evidence indicates that S. dioica and S. latifolia share pollen. In mixed plantings pollinators have been found to move pollen analogues between species, albeit less often than to conspecific flowers (Goulson and Jerrim, 1997; van Putten et al., 2007). Additionally, in contact zones, widespread introgression has been inferred from the rarity of species-specific markers in studies of allozymes (Goulson and Jerrim, 1997) and AFLPs (Minder et al., 2007; Karrenberg and Favre, 2008). Early generation hybrids are rarely observed in these same studies, suggesting a pattern of prolonged contact with occasional hybridization events followed by assortative backcrossing toward one parental species. Conspecific pollen precedence is one hypothesis proposed for the rarity of early generation hybrids (Minder et al., 2007; Karrenberg and Favre, 2008).

This study was an investigation into whether interactions occurring between pollination and seed germination limit hybridization between these two Silene species. This was done by measuring if conspecific pollen tubes elongate further than heterospecific pollen tubes in a set time period in styles of both species and by determining whether conspecific pollen sires a disproportionate share of seeds in flowers pollinated with different quantities of pure or mixed pollen.

MATERIALS AND METHODS

Seeds for Silene dioica (L.) Clairv. (red campion) and S. latifolia Poir. (white campion) were collected in the field near Alençon, France, from allopatric populations of each species. Neither field population showed morphological evidence of introgression. Plants were grown in a mix of commercial potting medium, compost and mineral soil, with supplemental lighting, weekly fertilization, and occasional chemical pest-control for spider mites in a greenhouse that excluded pollinators. Male and female plants were spatially separated.

Pollen tubes

Pollen tubes of both species were measured in styles of nine S. dioica and nine S. latifolia plants. To select flowers to pollinate, newly opened flowers were marked the preceding day and one treatment flower per plant was selected from among the marked flowers. Flowers have five styles each. Two non-adjacent styles were selected per flower to ensure no cross-contamination occurred; one selected style was randomly assigned to receive S. dioica pollen and the other S. latifolia pollen. Pollinations were performed on the same plants on each of three days, and plants lacking appropriately aged flowers on a treatment day were omitted from that round of treatments. Four pollen mixes were made per species on each day, with three male plants randomly selected for each pollen mix from among 14 S. dioica and ten S. latifolia males. Pollen was collected from equal numbers of flowers from each male and mixed on a glass slide. Silene dioica and S. latifolia pollen mixes were paired, and each pair of mixes was used to pollinate up to two flowers of each species, each on a different plant. Pollen was applied to the apical tip of the style using a new single horse-hair bristle. Styles were labelled by noting paternal identity on the adjacent petal lobe with a marker. Stigmas were collected at their base and fixed in 70 % ethanol after 3 h, which preliminary studies indicated was sufficient time for pollen tubes to elongate through much of the style without yet reaching the ovary.

To view pollen tubes, excised styles were soaked in 4 m NaOH for several hours to overnight, then stained overnight in a 0·1 n K2HPO4 solution with 0·1 % aniline blue dye. Styles were viewed and photographed at ×40 power using fluorescent microscopy with a DAPI excitation filter. Multiple photographs were required to record each style, and photographs were later reassembled by matching landmarks using ImageJ (Rasband, 1997–2007). The length of the style, as well as the lengths of the three longest pollen tubes, was measured using the freehand tool in ImageJ, and it was noted whether each reached the base of the style.

The average length of the two hand-pollinated styles was determined for each flower and was compared between species with ANOVA with individual plants nested as a random effect within species. The lengths of the longest three pollen tubes were averaged for each style, excluding styles with fewer than three pollen tubes. The average lengths of conspecific pollen tubes were then compared with heterospecific pollen tubes independently for each species using ANOVA with random effects of individual flowers nested within plant and plants nested within species. ANOVA was used to test for an interaction between donor and recipient species on average pollen-tube length, including independent effects of donor species, recipient species, and their interaction, with random effects of individual flowers nested within plants and plants nested within species. Finally, to determine whether the difference in conspecific and heterospecific pollen-tube lengths differed between species, the log-response ratio of average conspecific versus heterospecific length was calculated for each flower, and compared between species with ANOVA, including a random effect of plant nested within species. The log-response ratio was selected for this comparison because of its favourable statistical properties (Hedges et al., 1999).

Reproductive success and hybrid status

To investigate the effects of pollen competition on hybridization rates between S. dioica and S. latifolia, hand-pollinations were performed with varied pollen composition and density on flowers of greenhouse-grown individuals. For each species, five female plants with abundant flowers were selected. For pollen donors, three male S. dioica and four male S. latifolia plants were selected. Pollen from flowers of all three S. dioica donors were always included together. For S. latifolia donors, pollen from three plants was also included in each mix, but which donors were included was varied so as to exclude male siblings of the female being pollinated.

Pollinations were performed with four different compositions at two quantities. Pollen mixes were made to the following four ratios by mass (S. dioica:S. latifolia): 0 : 1; 1 : 3; 3 : 1; 1 : 0. The 1 : 3 and 3 : 1 ratios were chosen with the goal of allowing each species to be well represented, representing a situation in which an indiscriminate pollinator delivers pollen of both species. Each species was made more abundant in one mix because pollen relative abundance may affect hybridization rates (Carney et al., 1994), though it does not necessarily do so (Rieseberg et al. 1995; Alarcón and Campbell, 2000). To determine the relative abundance of pollen grains in mixed-pollen treatments, a mix of pollen from several flowers was collected separately and weighed for each of seven S. latifolia and six S. dioica males, including most individuals whose pollen was included in pollen mixes. Subsequently, to determine pollen density, pollen counts and size distributions were determined for each sample of known mass with a particle-size analyser (Micromeritics Elzone 2, Norcross, GA, USA). Pollinations were performed in two replicates per plant with each set corresponding to the eight combinations of composition (four treatments) and quantity (two treatments), with each set initiated on different days. Two flowers for each pollen composition by quantity combination were pollinated on each female plant, and five females were treated per species, for a total of 160 flowers (four compositions × two quantities × two replicates × five plants × two species). To ensure that each pollen mixture was applied to the same numbers of flowers of each species, S. dioica and S. latifolia females were paired, and initially the same pollen mix was used for each member of the pair. To create pollen mixes, pollen was collected into separate microcentrifuge tubes for each species from an equal number of flowers from each of three donor plants. The pure pollen loads were subsequently weighed within 10 µg on a Sartorius micro scale. Pollen was then combined as required and mixed on a glass slide with a needle. Pollen mixes were stored on slides in the shade in plastic containers containing moistened filter paper to maintain humidity. Pollen was initially collected in mid-morning (0900–1030 h), and pollinations were completed by mid-afternoon (1430 h).

Treatments within a replicate were randomly assigned among open flowers with elongated styles, indicating that the flowers had been open for at least 1 d. If at least eight flowers were available simultaneously, all treatments within a replicate were performed on the same day. Otherwise, treatments were performed over multiple days with new pollen mixes from the same males on each day. Treatments were repeated for aborted flowers, but the fruit set of replacement flowers was excluded when analysing fruit set.

To perform pollinations, pollen mixes were applied to each stigma within a flower on the tip of a new single horse-hair bristle. Low quantity treatments were intended to be low enough that pollen competition would be weak, and high quantity treatments were intended to supply excess pollen, such that pollen competition would be important. For low-quantity treatments, the bristle's tip was tapped perpendicularly into the pollen on the slide's surface. For high-quantity treatments, 1 mm of the bristle's tip was pulled along the slide, parallel to the surface. Then, regardless of treatment, the bristle was rubbed against the apical end of the stigma, which in both species extends along the inner side of style over much of its length. To test whether these treatments succeeded in delivering different pollen loads, one of the five styles was collected from most flowers (n = 145) into 70 % ethyl alcohol 1 d later. Preliminary experiments demonstrated that pollen tubes reached the base of the style within several hours, so removal of one style after this longer time period is unlikely to affect fertilization. Pollen grains adhering to each style were subsequently stained with Alexander's stain then counted, and dislodged pollen was counted by centrifuging the collection tubes then mounting the collected pollen in basic fuchsin gel (Kearns and Inouye, 1993). Similarities in pollen morphology and overlapping size distributions between species precluded identifying pollen to species.

Fruiting status was monitored and fruits were collected at maturity. Silene dioica retain seeds in the upright capsule following maturation, so fruits were collected upon opening. Silene latifolia fruits were not always upright at maturation so, prior to opening, fruits were partially enclosed in waxed bags. Fruits were stored in coin envelopes at room temperature.

To determine seed germination rates and hybrid status, a subset of seeds were planted in several rounds in the greenhouse. For fruits from the first replicate of pollinations, six seeds were planted in the initial round and four seeds were planted in the next round. For the second replicate, four seeds were planted in each of two rounds. Within each replicate, combined data from the two rounds of plantings were used to estimate seed germination rates. Additional seeds were later planted for those fruits whose initial plantings yielded less than six seedlings from which DNA could be extracted, but these additional plantings were excluded from estimations of seed germination rates because not all treatments were equally represented. Seeds were planted into a sterilized mix of commercial potting medium, compost and mineral soil. Germination, as indicated by emergence of cotyledons, was monitored and final germination was tallied approx. 6 weeks after planting.

DNA extractions were performed on true leaves of seedlings using Qiagen DNeasy Plant Mini kits. Markers used to determine paternity depended on the sex of the seedling and the species of the mother. To identify the sex of seedlings, PCR amplification for the Y-linked SlAP3Y MADS box gene was performed using the forward primer 5′-GGCATGGAGATCTCCTCATGGATC-3′ and reverse primer 5′-TATATTCGAGACAACATGGCCTGG-3′ (Matsunaga et al., 2003). Preliminary amplifications confirmed that these primers amplified a product in males but not in females of both species. For crosses with S. dioica mothers, DNA from female seedlings was amplified with PCR for the MADS box gene SlAP3A, using primers 5′-GGCATGGAGATCTCCTCATGGATC-3′ and 5′-ATACTGGAGATAACACAGCCTAGT-3′. Preliminary PCR indicated that these primers amplified similarly sized products in parental plants of both sexes of S. latifolia but not S. dioica. Although Matsunaga et al. (2003) report SlAP3A to be autosomal, the marker in this study was present in female but not male F1 hybrids resulting from female S. dioica by male S. latifolia crosses, indicating X-linkage. Consequently, a different marker was used for male seedlings involving S. dioica seed parents. Species paternity for these crosses was determined by amplification with PCR for the Y-linked SCAR marker SCQ14 using the primers 5′-GGACGCTTCATGACCCAT-3′and 5′-GGACGCTTCAGCGGGCGG-3′. This marker amplifies a Y-linked band in S. latifolia but not S. dioica males (Zhang et al., 1998).

For crosses with S. latifolia mothers, DNA from female seedlings was amplified with PCR for SlAP3A's homologue in S. dioica, SdAP3A, using the same forward primer as for SlAP3A and the reverse primer 5′ -GGTCGCAAACCACTAGTTTATACTC-3′ (Matsunaga et al., 2003). This primer combination was found to amplify a band in parental plants of both sexes for S. dioica but not S. latifolia. As for SlAP3A, X-linkage of SdAP3A was inferred from successful amplification of the marker for F1 female but not male hybrids with S. dioica seed parents. DNA from male seedlings was amplified with PCR for the MADS box gene SdAP3Y, using the forward primer 5′-CGAACAGAGGAACTACGGCGG-3′ and the reverse primer 5′-GGCCATCACGAACTAATCACA-3′. Preliminary PCRs indicated that SdAP3Y amplified a band in males of S. dioica but not S. latifolia.

PCR was performed with Taq DNA polymerase from Bioline or with Promega Green Master Mix. For reactions with Bioline Taq polymerase, the following conditions were used: 1·0 µL 10× KCl buffer (500 nm KCl, 100 mm Tris–HCl pH 8·8, 15 mm MgCl2, 1 % Triton 100), 0·4 µL of dNTP (2·5 mm each of dATP, dCTP, dGTP and dTTP), 0·2 µL each of forward and reverse primer (10 µm), 0·52 µL MgCl2 (15 mm), 0·08 µL Taq DNA polymerase (5 U μL−1), 1·0 µL of DNA and 6·6 µL of water. For reactions with Promega Green Master Mix, the following conditions were used: 5 µL 2× master mix (Taq DNA polymerase, 400 µL each of dATP, dCTP, dGTP and dTTP, 3 mm MgCl2), 3·0 µL water, and the same volumes of primers, MgCl2, and DNA as for reactions with Bioline Taq polymerase. Cycling parameters were one cycle of 94 °C for 1 min; 30 cycles of 94 °C for 1 min; 63 °C for 1 min; and 72 °C for 2 min; and one cycle of 72 °C for 5 min. PCR products were run on 1 % agarose gels and stained with ethidium bromide.

PCR runs included seedlings from pure heterospecific and pure conspecific pollinations, as well as seedlings from mixed pollinations, in order to act as positive and negative controls and allow assessment of error rates in determining hybrid status. In total, 852 seedlings were genotyped.

Data analysis

To determine whether the numerical ratio of pollen in pollen mixes of the two species differed from the ratio of pollen masses, pollen diameter and density (counts per milligram) from anther-collected pollen were compared between species with two-tailed t-tests. To determine whether more pollen was in fact received in the high-quantity treatment, stigma pollen counts were square-root transformed to improve normality, then tested with ANOVA, including a random effect of plant within species, pollen quantity, and an interaction between species and pollen quantity. One positive outlier was excluded from the large quantity treatment, which served to reduce the difference between treatments, thus providing a conservative estimate of differences.

To determine if pollination with heterospecific pollen decreased reproductive success relative to conspecific pollen, fruit and seed set and seed germination rates were analysed across treatments. Fruit set, excluding replacement flowers for initial unsuccessful pollinations, was analysed in S-Plus 7·0 using generalized estimating equations (GEE) with an independent correlation structure to account for correlated responses among flowers within a plant, and a binomial distribution. The statistical model included independent effects of species, proportion of conspecific pollen, pollen quantity (large or small) and interactions between species and pollen type and between proportion of conspecific pollen and pollen quantity (other interactions could not be fit as a result of a lack of variation within or between some treatment combinations). Seed set, square-root transformed, was analysed with ANOVA including a random effect of plant within species, as well as pollen type, proportion of conspecific pollen, and interactions among all three variables. Seed germination was compared between pure heterospecific and conspecific pollinations with a GEE with a binomial distribution. The numbers of germinated and ungerminated seeds per flower were included as a two-vector response variable, and maternal species as well as pollen type (heterospecific or conspecific) were included as independent variables with an interaction term.

Following Hauser et al. (1997), a statistical model was constructed to investigate whether conspecific pollen was disproportionately successful in siring seeds, after accounting for differences in seed germination rates and seedling-identification success. The data for the model included counts of the number of seedlings identified as hybrid or conspecific for each treated flower. For pollen mixes, the relative abundance of pollen from each species was estimated based on the relative mass of each species' pollen and the different pollen densities.

The expected proportion of hybrid seeds was calculated as

graphic file with name mcq013eqn1.jpg 1

where pij is the proportion of heterospecific pollen in pollen mix j being applied to a flower of species i, and sijk is the relative competitive ability of heterospecific pollen when applied in quantity k (high or low dose), with the competitive ability of conspecific pollen defined to equal one.

To account for different seed germination rates, the expected proportion of hybrid seedlings was calculated as

graphic file with name mcq013eqn2.jpg 2

where gi.hyb and gi.con are the germination rates of hybrid and conspecific seeds, respectively, for maternal species i. Finally, to account for the probability of misidentifying hybrid or conspecific seedlings, the proportions of hybrid seedlings expected to be observed were calculated as

graphic file with name mcq013eqn3.jpg 3

where di.hyp and di.con are the probabilities of misidentifying the paternity of a hybrid or conspecific seedling, respectively, of maternal species i.

Consequently, the probability of observing nijk hybrid seedlings out of Nijk seedlings scored in total for pollen mix j applied at quantity k to a flower l of species i is the binomial probability with parameter rijk. This results in a likelihood function with a logarithmic transformation of

graphic file with name mcq013eqn4.jpg 4

The parameters for seed germination rates, gi.hyb and gi.con, and for seedling misidentification, di.hyp and di.con were estimated from the pure heterospecific and pure conspecific treatments for each maternal species. The parameter sijk was estimated by maximum likelihood estimation with the Solver Add-in of Microsoft Excel version 11·5. Because the parameter sijk was estimated after accounting for differences in seed germination and misidentification rates, as determined by pure conspecific and heterospecific crosses, resulting differences in the value of sijk reflect differences in pre-germination processes. The differences in sijk between maternal species, between majority S. dioica and majority S. latifolia mixes, and between high and low pollen quantities were tested for significance with the likelihood ratio test: D = –2 (loge Lsimplified – loge Lsaturated), with Lsaturated referring to the full model and Lsimplified referring to models in which sijk was variously constrained to not differ between pollen quantities, pollen compositions, or maternal species. D approximately follows a chi-square distribution with degrees of freedom equal to the difference in degrees of freedom between the models being compared (Hosmer et al., 1989).

RESULTS

Pollen tubes

Average style length was significantly longer for S. latifolia than S. dioica (F1,16 = 80·30, P < 0·001), with averages (± s.e.) of 9·0 ± 0·2 mm for S. dioica and 15·8 ± 0·5 mm for S. latifolia. The average length of the longest three pollen tubes was greater for conspecific than heterospecific pollen tubes for both S. dioica (F1,14 = 12·71, P = 0·003) and S. latifolia (F1,14 = 8·29, P = 0·017; Fig. 1). In the analysis including both species, pollen tubes were significantly longer in S. latifolia than S. dioica styles, and there was a significant interaction between the species identity of the pollen and style (Table 2). The interaction is attributable to the length of S. latifolia pollen tubes responding more strongly than that of S. dioica to the species identity of the style (Fig. 1). For S. dioica, pollen tubes reached similar lengths regardless of the style's identity, but S. latifolia pollen tubes were longer in S. latifolia than S. dioica styles. The relative length of conspecific versus heterospecific pollen tubes did not differ significantly between species (F1,16 = 1·11, P = 0·31). However, the longest pollen tube was not always conspecific in either species. In S. dioica styles, the longest pollen tube was not conspecific more often than expected by chance (eight of 15 trials, binomial test, P = 1), whereas in S. latifolia the longest pollen tube was conspecific more often than expected by chance (13 of 15 trials, binomial test, P = 0·007).

Fig. 1.

Fig. 1.

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Length of S. dioica and S. latifolia pollen tubes in styles of S. dioica and S. latifolia. Error bars ± 1 s.e.

Table 2.

ANOVA testing effects of (A) flower species on style length and (B) flower species, pollen species and their interaction on pollen-tube lengths

Source d.f. SS F P
(A) Style length
 Flower species 1 454·1 80·30 <0·001
 Plant (flower species) 16 90·48
 Error 21 10·11
(B) Pollen-tube lengths
 Flower species 1 513950 8·164 0·01
 Plant (flower species) 16 1007312
 Pollen species 1 3644·7 0·182 0·67
 Flower species × pollen species 1 382125 19·106 < 0·001
 Error 28 560017
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Reproductive success and hybrid status

Average pollen diameter was significantly smaller for S. dioica than S. latifolia pollen (two-tailed t-test, t12 = 9·95, P << 0·01), with an average diameter (± s.e.) of 35·7 ± 0·4 µm for S. dioica and 41·8 ± 0·5 µm for S. latifolia. There were significantly more pollen grains per milligram for S. dioica than S. latifolia pollen (t12 = 2·8, P = 0·02), with an average of 8·1 ± 0·7 pollen grains × 105 mg−1 for S. dioica and 5·8 ± 0·3 pollen grains × 105 mg−1 for S. latifolia. Based on this, pollen mixes with 25 % and 75 % S. dioica pollen by mass correspond to 31·8 % and 80·7 % by quantity, respectively. Counts of pollen applied to styles differed significantly between high and low pollen quantities (quantity term from ANOVA: F1,131 = 323, P ≪ 0·001), and neither the maternal species nor the interaction was significant (both P ≫ 0·10). Single styles in the high and low treatments received an average (±s.e.) of 344 (±14) and 95 (± 5) pollen grains, respectively.

Among flowers pollinated with pure heterospecific or pure conspecific pollen, there was a trend toward lower fruit set for flowers of S. latifolia than S. dioica, and fruit set did not vary significantly with pollen type, pollen quantity, or any measured interactions (Fig. 2A and Table 3). Seed set was significantly higher for large than small pollen quantities and tended to increase with increasing proportions of conspecific pollen, but did not significantly differ between species or with any interactions (Fig. 2B and Table 4).

Fig. 2.

Fig. 2.

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(A) Fruit set and (B) seed set for pollinations with pure heterospecific or conspecific pollen, or pollen mixes composed of majority S. dioica or S. latifolia pollen, applied to flowers of S. latifolia (squares) or S. dioica (circles), with pollen applied in large quantities (open symbols) or small quantities (closed symbols); error bars ± 1 s.e. Curve fits shown separately for large and small pollen quantities irrespective of species.

Table 3.

Regression coefficients of GEE with binomial distribution testing effects of species, as well as proportion of heterospecific pollen, pollen quantity (high or low) and their interaction on fruit set

Source Estimate s.e. Z P
Intercept 2·365 0·927 2·55 0·01
Species –1·584 0·970 –1·63 0·10
Proportion of heterospecific pollen 0·401 0·939 0·43 0·67
Quantity –0·316 0·828 –0·38 0·70
Species × proportion of heterospecific pollen –0·522 1·102 –0·47 0·64
Proportion of heterospecific pollen × pollen quantity 0·707 0·782 0·9 0·37
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Table 4.

ANOVA of effects of maternal species, proportion of heterospecific pollen, pollen quantity and interactions on seed set

Source d.f. SS F P
Species 1 12·60 0·826 0·39
Plant (species) 8 122·10
Proportion of heterospecific pollen 1 20·64 3·535 0·06
Pollen quantity 1 35·48 6·074 0·02
Species × proportion of heterospecific pollen 1 1·37 0·235 0·63
Species × pollen quantity 1 4·00 0·685 0·41
Proportion of heterospecific pollen × pollen quantity 1 0·64 0·110 0·74
Species × proportion of heterospecific pollen × pollen quantity 1 13·11 2·245 0·14
Residuals 64 373·82
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Germination rates were significantly higher for hybrid than conspecific seeds over both maternal species; however, germination rates did not vary significantly among maternal species, and there was not a significant interaction between seed hybridity and maternal species (Tables 5 and 6). Identification of seedlings resulting from pollinations with only conspecific or heterospecific pollen revealed error rates in assigning species paternity of between 2–6 %, depending on maternal and paternal species (Table 5). Error was largely attributable to PCR failure, as indicated by subsequent successful amplification of a relevant hybrid marker in most reamplifications of putative hybrids that initially failed to amplify either marker (because only putative hybrids were retested, results were not modified to reflect retrial results). Because the estimates of error were incorporated in calculating relative competitive ability, the estimates of relative competitive ability should not be affected by the error rate.

Table 5.

Parameter estimates (means) for pollen competition model, including seed germination rates (g), misidentification rates (d) for both maternal species and for conspecific and heterospecific pollen, and relative competitive ability (s) of heterospecific pollen on maternal species indicated for majority S. latifolia or S. dioica pollen mixes and large or small pollen quantities

Variable S. latifolia S. dioica
Seed germination rate (%) (s.e. +/–)†
 Conspecific 78·4 (4·3/5·1) 75·7 (3·4/3·8)
 Heterospecific 89·2 (3·6/5·2) 85·0 (3·5/4·4)
Misidentification rate (%)
 Conspecific 2·7 1·7
 Heterospecific 6·0 6·0
Heterospecific pollen fitness (s)
 Overall‡ 0·22** 1·26 n.s.
 Majority S. latifolia mix
  Large 0·28 1·15
  Small 0·28 0·71
 Majority S. dioica mix
  Large 0·26 2·41
  Small 0·14 1·19
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† Germination means and standard errors back-transformed from binomial regression with logit link.

‡ Significance levels: n.s., result does not significantly differ from one; ** P < 0·001.

Table 6.

Binomial generalized linear model of effects of maternal species, hybrid status (pure species or hybrid) and their interaction on germination rates

Source Num. d.f. Dev. exp. Den. d.f. Resid. dev. P
Intercept 79 166·69
Species 1 1·12 78 165·57 0·29
Hybrid status 1 10·12 77 155·45 0·001
Species × hybrid status 1 0·24 76 155·21 0·62
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In mixed pollinations, S. latifolia generally sired seeds on S. dioica flowers in proportion to its pollen's relative abundance, while S. dioica sired disproportionately few seeds relative to its pollen's relative abundance on S. latifolia flowers (Fig. 3). The log-transformed maximum likelihood function was evaluated for several models to determine whether competitive ability differed between quantities and between species. The model fit was significantly improved by allowing the relative competitive ability, s, of S. dioica pollen on S. latifolia flowers to differ from 1·0, a value indicative of no competitive difference (χ2 = 56·3, P < 0·001). Furthermore, the model fit was significantly better when s was allowed to vary between maternal species compared with a model in which s was constrained to be the same for both maternal species (χ2 = 43·8, P < 0·001). These results indicate, respectively, that S. dioica pollen is significantly outperformed by S. latifolia pollen in siring seeds on S. latifolia and that S. dioica pollen is significantly less competitive at siring seeds on S. latifolia than S. latifolia is on S. dioica (Table 5). For pollen of S. dioica applied to S. latifolia, the model fit was not significantly improved either by allowing s to differ between the majority S. dioica and majority S. latifolia pollen mixes (χ2 = 0·56, P = 0·45), or by allowing s to differ between high and low quantities (χ2 = 1·17, P = 0·28).

Fig. 3.

Fig. 3.

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Proportion of hybrid seeds observed for pollinations with pure heterospecific or conspecific pollen, or pollen mixes composed of majority S. dioica or S. latifolia pollen, applied to flowers of S. latifolia (squares) or S. dioica (circles), with pollen applied in large quantities (open symbols) or small quantities (closed symbols). Line represents best fit for observed proportion of hybrid seeds for treatments with pure heterospecific or conspecific pollen only. The best-fit line was combined between species, and the slight deviation from the origin and 1 : 1 slope results from the misidentification rate of pure conspecific and heterospecific seedlings. For mixed pollen loads, deviations from the best-fit line indicate an excess or lack of hybrids; error bars ± 1 s.e.

The overall siring success of S. latifolia pollen applied to S. dioica flowers did not differ significantly from 1·0 (χ2 = 1·88, P = 0·17). However, irrespective of pollen quantity, the relative competitive ability of S. latifolia pollen in majority S. dioica mixes (s = 1·76) was significantly higher than in majority S. latifolia mixes (s = 0·89; χ2 = 4·51, P = 0·03). Additionally, irrespective of pollen composition, the relative competitive ability of S. latifolia in large quantities (s = 1·88) was significantly greater than in small quantities (s = 0·88; χ2 = 4·37, P = 0·04).

DISCUSSION

In this study, it was found that S. latifolia pollen sires seeds in proportion to its pollen's relative abundance on S. dioica flowers, but S. dioica pollen sires disproportionately few seeds on S. latifolia flowers. This result indicates that mechanisms operating between pollination and seed set limit of introgression of S. dioica into S. latifolia, while not limiting introgression of S. latifolia into S. dioica. Our methods differed from the related study of Rahmé et al. (2009) in several respects: most notably, our study included majority S. dioica or majority S. latifolia mixes, whereas theirs included an equal pollen mix; and our study included two pollen quantities, the larger of which was about 10 % smaller than the quantity they applied. Despite these differences, both studies found an asymmetrical barrier to hybridization of similar strength operating against S. dioica pollen, suggesting that this is a general result across different populations and conditions.

Although only S. latifolia exhibited a conspecific seed-siring advantage, both species exhibited conspecific advantage in our measures of pollen-tube growth. Conspecific advantage in pollen-tube growth is unlikely to result from heterospecific pollen-tube attrition because tubes of both species in heterospecific styles were on average shorter than conspecific styles, indicating that pollen tubes had not yet achieved the length necessary for fertilization of conspecific flowers. Interactions between pollen tubes and stylar tissue are known to affect pollen-tube elongation, although the mechanisms involved and the role of these interactions in mediating interspecific fertilizations is a matter of ongoing research (Wheeler et al., 2001). The findings that conspecific pollen tubes were longer in both crosses and that S. latifolia pollen tubes elongated further in conspecific than heterospecific stigmas suggest that pollen–pistil interactions affected growth rates. The lack of a conspecific siring advantage for S. dioica despite its conspecific advantage in pollen-tube elongation could indicate that other mechanisms, such as differential pollen attrition over longer time intervals, were more important in determining paternity.

The conflict between the pollen-tube growth and hybridization results may have arisen because the pollen-tube study measures the performances of only the few longest pollen tubes, while the hybridization study assesses performance over all pollen grains that sire viable seeds. For example, pollen-tube elongation is influenced by interactions between stylar tissue and nearby pollen tubes (Cruzan, 1990). Thus, conspecific pollen tubes may initially grow faster through stylar tissue, but such growth could facilitate the passage of later tubes (Visser and Verhaegh, 1980), potentially reducing or eliminating conspecific advantage. In this study, measurements were restricted to the longest few pollen tubes because shorter pollen tubes were not discernable. Additionally, the discrepancy between pollen-tube lengths and paternity may have occurred because pollen tubes were measured at only one time interval, and growth rates may vary with location in the style (Mulcahy and Mulcahy, 1983, Douglas and Cruden, 1994). By virtue of its larger pollen-grain size S. latifolia may have maintained a faster growth rate than S. dioica in S. dioica styles after the 3-h time period, thereby eliminating S. dioica's initial advantage in conspecific styles. Measures of pollen tubes may have more accurately predicted seed-siring success for S. dioica, the shorter-styled species, because pollen tubes were closer to the style's base in this species at the time of measurement.

Other studies investigating pollen-tube growth and seed hybridity have also found conflicting results. Studies by Emms et al. (1996) and Carney et al. (1994) both found cases in irises of a conspecific seed-siring advantage despite a heterospecific pollen-tube growth advantage. Rieseberg et al. (1995) did not detect conspecific pollen-tube growth advantage in sunflowers, but they did detect conspecific advantage in siring seeds. In contrast, Husband et al. (2002) and Song et al. (2002) found agreement between patterns of tube elongation and seed hybridity in fireweed and rice, respectively. These results indicate that studies of pollen-tube growth rates are valuable in investigating one mechanism of conspecific pollen precedence, but that such studies alone are not sufficient to predict conspecific pollen precedence overall.

Previous studies have found that when differential pollen-tube elongation or pollen-tube attrition occurs, pollen from the longer-styled species may perform better in interspecific crosses than pollen from the shorter-styled species in reciprocal crosses (Buchholz et al., 1935; Smith, 1970; Carney et al., 1996; but see Emms et al., 1996; Diaz and Macnair, 1999; Wolf et al., 2001). Silene latifolia generally has larger flowers and pollen than S. dioica (Baker, 1947b; Prentice, 1978), and styles were longer for S. latifolia than S. dioica at the time of pollination in our pollen-tube study. It has been hypothesized that the longer-styled species may generally be less disadvantaged in interspecific crosses because its pollen tends to be larger (Diaz and Macnair, 1999) and its pollen tubes are adapted to elongate faster and to greater lengths (Smith, 1970, Carney et al., 1996). The present paternity results are consistent with this hypothesis, although the pollen-tube results are not. However, for aforementioned reasons, paternity success is a better indicator of overall success in this study.

The siring success of Silene dioica pollen on S. latifolia flowers was similar, whether pollen mixes were applied in small or large quantities. This outcome suggests that conspecific pollen on S. latifolia flowers has a pre-zygotic time-invariant advantage, such as lower rates of pollen-tube attrition, which could result from the longer style length in S. latifolia. Seed set tended to be higher for flowers pollinated with conspecific than heterospecific pollen, indicating that hybrid inviability may also contribute to S. dioica's seed siring disadvantage (Table 1). Differences in seed germination rates were controlled for in the analysis, so do not influence the calculated differences in siring success. Thus, a combination of pollen-tube attrition and post-fertilization abortion appears the most likely explanation for the conspecific pollen advantage in S. latifolia.

This outcome suggesting the lack of a time-variant advantage for conspecific pollen in S. latifolia flowers is surprising because conspecific pollen tubes were longer, suggesting competitive superiority, and the difference in pollen quantity was large, being 3-fold higher than in the low quantity treatment. One explanation for this pattern could be that pollen limited seed set at both small and large quantities, such that pollen-tube competition was unimportant and most pollen tubes reaching the ovary succeeded in fertilization. On average, total pollen receipt ranged from nearly 500 pollen grains per flower (95 grains per style × 5 styles) in the low treatment to >1700 grains (344 grains per style) in the high treatment. Flowers of greenhouse grown S. dioica and S. latifolia flowers from the present study populations contain nearly 300 and 400 ovules, respectively (L. F. Delph, unpubl. res.). Thus, flowers in the high pollination treatment received on average at least 4-fold more pollen grains than they had ovules. Nonetheless, average seed set was considerably lower than ovule counts for both pollen quantities, and higher seed set in the high- than low-pollen quantity treatment indicates pollen limitation for the low-quantity treatment. Thus, it is possible that the excess pollen was not enough for competition to substantially impact the results.

The siring success of S. latifolia was greater when mixes were applied to S. dioica flowers in large quantities, suggesting a time-variant heterospecific advantage for S. latifolia pollen (Table 1). However, the lack of an overall statistically significant advantage to S. latifolia pollen on S. dioica flowers indicates that the advantage is small or compensated for by a time-invariant disadvantage. The decreased siring success of S. latifolia in majority S. latifolia pollen mixes relative to majority S. dioica mixes may have resulted from the increased importance of intraspecific relative to interspecific competition among pollen grains when S. latifolia pollen was common. Increased intraspecific competition could occur if the two species of pollen tend to reach ovules in different parts of the ovary, as has been inferred in other studies (Manglesdorf and Jones, 1926; Smith and Clarkson, 1956; Carney et al., 1996; but see Kearney, 1932). The same pattern could also be created if the rate of maternal provisioning to hybrid ovules decreased as the numbers of such ovules increased.

Patterns of chloroplast and nuclear markers in the field suggest that S. latifolia is usually the seed parent when introgression occurs, an outcome that could result from S. latifolia producing more seeds than S. dioica or from S. dioica pollen achieving more fertilizations on S. latifolia flowers than S. latifolia pollen on S. dioica flowers (Minder et al. 2007). The data indicate that S. dioica pollen is not more competitive than S. latifolia in siring viable seeds on the other species' flowers; thus, the pattern of introgression is more likely explained by either different levels of seed production or by S. dioica pollen reaching S. latifolia flowers at a higher rate than S. latifolia pollen reaching S. dioica flowers. Consistent with the latter hypothesis, Goulson and Jerrim (1997) found that for arrays with equal numbers of both species, a higher proportion of S. latifolia flowers received S. dioica pollen than vice versa. The pattern could also result from higher survival under field conditions of hybrids with S. latifolia than S. dioica seed parents. Karrenberg and Favre (2008) argue that differential introgression from S. dioica to S. latifolia may occur because contact sites tend to be located in conditions better suited for S. latifolia than S. dioica.

Parsing the relative contributions of various barriers to hybridization is a challenge. In a study designed to estimate the sequential effects of multiple factors contributing to reproductive isolation between two Mimulus species, Ramsey et al. (2003) found that, despite strong conspecific pollen precedence, this mechanism accounted for only a small proportion of total isolation. As with Mimulus, this and other studies of these Silene species suggest that multiple barriers contribute to isolation and the strength of each barrier is not necessarily reciprocal for both species (Goulson and Jerrim, 1997; Minder et al., 2007, Karrenberg and Favre, 2008).

Conclusions

This research demonstrates that conspecific pollen tubes elongate farther in the measured time period in both species. However, conspecific pollen was only more successful than heterospecific pollen in siring viable seeds in one species, S. latifolia. Our finding in S. dioica of a conspecific pollen-tube advantage at an intermediate point in the style but no conspecific advantage in seed siring suggests that pollen-tube growth rates may differ with location in the style. The siring advantage of S. latifolia pollen in its own styles may result from increased time-invariant pre-fertilization advantages or post-hybridization advantages in viability or sequestering of resources. Overall, these results indicate that net barriers to hybridization operating between pollination and seed germination contribute to reproductive isolation for S. latifolia but not S. dioica seed parents.

ACKNOWLEDGEMENTS

We thank C. Herlihy and S. Scarpino for provision of seeds. Comments of anonymous reviewers improved this manuscript. This work was supported by National Science Foundation grant DEB 0210971 to L.F.D.

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