Is the magnitude of pollen limitation in a plant community affected by pollinator visitation and plant species specialisation levels?

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1 Oikos 117: , 2008 doi: /j x, # 2008 The Authors. Journal compilation # 2008 Oikos Subject Editor: Diego Vázguez, Accepted 28 January 2008 Is the magnitude of pollen limitation in a plant community affected by pollinator visitation and plant species specialisation levels? Stein Joar Hegland and Ørjan Totland S. J. Hegland (stein.hegland@umb.no) and Ø. Totland, Dept of Ecology and Natural Resource Management, P.O. Box 5003 (UR), Norwegian Univ. of Life Sciences, NO-1432 Ås, Norway. Pollen limitation on a plant community level has received little attention, although it might show which pollinationrelated traits may cause pollen limitation to vary among species. To address several central questions in plant reproductive biology, we investigated pollen limitation in 11 plant species, including visitation and specialisation levels of all species. The female reproductive success of most species within the studied plant community was not pollen limited, but a general tradeoff between seed production and seed weight occurred as a response to supplemental pollination. In contrast to general notion, we did not find that less visited species were most pollen limited. Instead, it appears that species with high visitation rates were most pollen limited. Our study provided conflicting evidence to whether specialisation levels may affect the degree of pollen limitation within the study community. We discuss these findings in the context of recent reviews on the occurrence, causes and consequences of pollen limitation in plants. In particular, we propose that, although pollen limitation is an important phenomenon, 1) the majority of species within a plant community may not experience pollen limitation at a given moment, 2) that common notions of which plant species should experience pollen limited reproductive success do not hold true in the studied plant community, and 3) that offspring quality is as likely affected by surplus pollen loads as is the number of offspring. Pollen limitation is a widespread phenomenon (Burd 1994, Ashman et al. 2004), and several recent reviews have examined the frequency, the causes and the consequences of pollen limitation (Larson and Barrett 2000, Ashman et al. 2004, Knight et al. 2005, Knight et al. 2006, Vamosi et al. 2006). It has been estimated that 6273% of all biotic pollinated species studied experience pollen limitation (Burd 1994, Ashman et al. 2004). However, Aizen and Harder (2007) recently suggested that the frequency of pollen limitation may be overestimated because the treatment used to assess it (surplus addition of outcrossed pollen) often confounds pollen quality with quantity making it impossible to distinguish between these two effects on pollen limitation. Nevertheless, the findings in these reviews can help answer both evolutionary and management-related questions pertaining to pollination and plant reproduction. For example, Vamosi et al. (2006) found that on a global scale plant diversity was negatively related to plants reproductive success (i.e. increased pollen limitation), possibly due to higher interspecific competition among plants for pollinators in species-rich areas or because species-rich areas are most degraded by habitat fragmentation. This implies that pollinator abundance or diversity may substantially affect the occurrence of pollen limitation in plant species (Motten 1986, Ghazoul 2005), and previous reviews have suggested that plant species receiving low visitation and/or being highly specialised experience most pollen limited reproductive success (Bond 1994, Kearns and Inouye 1997, Wilcock and Neiland 2002, Knight et al. 2006). Along the same line of reasoning, Ashman et al. (2004) proposed that ecological perturbations, such as alien invasions, reductions in plant population sizes, or pollinator loss may lead to increased pollen limitation on reproduction. An examination of the correlates of pollen limitation showed that plant species with small flowers, self-incompatibility, or with many ovules per flower often are pollen limited, but there were no differences in the degree of pollen limitation between species with specialised or generalised flowers (Knight et al. 2006, see also Burd 1994, Larson and Barrett 2000). Few studies on pollen limitation include measurements on both seed production and seed mass (Ashman et al. 2004, Knight et al. 2005), and this could weaken statements about the importance of pollen limitation for the reproductive success of plants and whether the two components of plant reproductive success respond differently to inadequate pollen deposition. Tradeoffs between seed production and seed weight are also possible (Garwood and Horvitz 1985, Hainsworth et al. 1985, Primack 1987, Gorchov 1988, Ågren 1989, Marshall and Ellstrand 1988), which is 883

2 evident in fruit crop production through thinning-effects (Stephenson 1981). Therefore, increased seed production after pollen supplementation may be a poor descriptor of pollen limitation, if offspring quality simultaneously is negatively affected due to the tradeoff. On the other hand, excess levels of pollen on stigmas can directly positively affect offspring quality, although seed production is unaffected (Totland 1994, Hegland and Totland 2007) through either male competition (Snow 1986, Snow and Spira 1991) or female choice (Delph et al. 1998, Marshall and Ellstrand 1988). Another concern is that different measurements of the production component of reproduction (e.g. seed number vs fruit number vs fruit set) used to address pollen limitation could give different results, and are often not reported within the same study (Knight et al. 2006). We obtained a multi-species data-set on pollen limitation and plant species visitation rates and specialisation levels of 11 herb species from a single temperate grassland community. We used direct measurements of plant species visitation rates and specialisation level in addition to ecological surrogates of these (i.e. plant traits), such that we could assess if some of the main findings from recent meta-analyses on pollen limitation (Larson and Barrett 2000, Ashman et al. 2004, Knight et al. 2005, 2006) occur at the within-community level. Moreover, by including different measures of pollen limitation on several species, an evaluation of potential resource allocations and the potential tradeoffs between different variables became possible. We asked three main questions: 1) What is the frequency of species experiencing pollen limitation on offspring production or weight at a plant community scale? 2) Are there relationships between components of pollen limitation, and is there a general tradeoff between offspring production and weight in plant species within this plant community? 3) Is pollen limitation of plant species related to their flower visitation rates or their pollinator specialisation level? Material and methods Study community and study species Our study community was situated in the temperate region on the border between the boreo-nemoral and the south-boreal zone close to the shore of the Sognefjord at Kaupanger in western Norway. The study site was a ca m dry meadow on a steep slope of the species-rich grassland complex Rudsviki. About 35 insect-pollinated flowering plant species occurred on the site during the study seasons. The pollinator assemblage was dominated by various species of bumblebees (Bombus), flies (Diptera), and a few beetle (Coleoptera) species (see also Table 1 in Hegland and Totland 2005 and Hegland and Boeke 2006 for more information on the plant and pollinator community in Rudsviki). In 2003 and 2004, we collected data on 11 abundant insect-pollinated plant species (Table 1), i.e. about onethird of the insect-pollinated species in the community. These species display diverse floral traits and host various pollinators. Experimental manipulations to test for pollen limitation To quantify pollen limitation, we generally added conspecific cross-pollen from two to three anthers on a plant situated one to three meters away from the pollen recipient. We supplementary pollinated a single flower or an inflorescence of 1520 pairs of individuals in each species. One pairmember received supplemental pollen; the other (growing ca 1 m away from the supplementary pollinated plant) served as a control that received only natural levels of pollination (Table 1). The procedure was repeated on two consecutive days. For the three umbelliferous or composite flowering species (Carum carvi, Pimpinella saxifraga and Centaurea jacea), we conducted supplemental pollination by carefully daubing a whole pollen-donor inflorescence onto the pollenrecipient inflorescence. This procedure possibly mimics the generalist pollination strategy of these species and was performed with new donor inflorescences for three consecutive days. The relatively short distance (compared to many other studies; Aizen and Harder 2007) between the pollen donor and recipient plants possibly contributed to minimising the effects of differences in pollen quality experienced by supplemental pollinated and control flowers. However, it is still possible that a larger fraction of self-pollen was deposited on the hand-pollinated stigmas than naturally Table 1. Basic characteristics of the 11 study species from the temperate grassland Rudsviki, Kaupanger in western Norway. Study species Study year Main visitor Breeding system Pollinated unit Fruit unit Carum carvi 2003 Coleoptera Little selfing 1 Inflorescence Infructescence Centaurea jacea 2003 Bombus Little selfing 2 Inflorescence Infructescence Clinopodium vulgare 2004 Bombus Little selfing 3 Flower Infructescence Geum rivale 2003 Bombus Substantial selfing 4 Flower Infructescence Hypericum maculatum 2004 Diptera Substantial selfing 5 Flower Infructescence Lotus corniculatus 2004 Bombus Substantial selfing 6 Flower Fruit Pimpinella saxifraga 2004 Coleoptera No record 7 Inflorescence Fruit Potentilla erecta 2003 Diptera No selfing 8 Flower Fruit Prunella vulgaris 2004 Bombus Substantial selfing 9 Flower Fruit Ranunculus acris 2003 Diptera No selfing 10 Inflorescence Infructescence Trifolium repens 2003 Bombus Little selfing 11 Flower Infructescence Note: Main visitor is based on transect walks in the year of study. Breeding system is as reported in 1) Bouwmeester and Smid 1995; 2) Hardy et al. 2001; 3) Ecological Flora of British Isles 2007, information on several close relatives; 4) Taylor 1997; 5) Ecological Flora of British Isles 2007, information on several close relatives; 6) Jones and Turkington 1986; 7) no record of breeding system was found on Pimpinella saxifraga; 8) Watson 1969; 9) Winn and Werner 1987; 10) Totland 1994; 11) Burdon Fruit unit is the reproductive unit collected. 884

3 pollinated stigmas, and consequently we are not fully able to distinguish between a pollen quantity versus quality effect in our study (Aizen and Harder 2007). For most of the 11 species, we only supplementary hand-pollinated one flower/inflorescence, but for two species, Ranunculus acris and Geum rivale, we supplementary hand-pollinated all flowers of each individual. The data for R. acris is also presented elsewhere (Hegland and Totland 2007). For eight of those nine species where only one flower/inflorescence was supplementary hand-pollinated, we also measured seed number and weight in a simultaneously blooming openpollinated flower on the hand-pollinated individual. These internal control flowers were used to test for resource reallocation effects within the plants by comparing their reproductive success with that of flowers on the control plants (Totland and Sottocornola 2001). This procedure may not have adequately addressed translocation issues since only one flower was supplementary pollinated, but by using an internal control flower closely situated to the hand-pollinated flower we were more likely to detect potential resource reallocation effects (Knight et al. 2006). Six species were hand-pollinated in 2003 and five in 2004 (Table 1). We collected fruits or infructescences (Table 1) from the handpollinated flowers, the internal control flowers, and the flowers on the control plant of all study species and measured 1) the number of viable seeds or fruits per flower or inflorescence (hereafter, seed production), 2) the number of viable seeds per fruit; 3) the proportion of developed fruits per flower or inflorescence; and 4) weighed all the seeds per flower collectively (after drying at room temperature for one year). To avoid loss of seeds, we collected fruits just before complete maturation. The measurements were used to calculate mean values per species for each of the four response variables for use in statistical analysis. For most analyses, we only applied seed production and seed weight as response variables. The proportion of developed fruits is often used as a production response variable in pollen limitation studies, but because we did not sample more than one fruit with a single seed for two of the species, we could not estimate this variable for all species. Consequently, this variable was only used in some of our analyses (Table 3). Data collection and variable calculation on pollinator visitation and specialisation level In both years, we collected data on the specialisation level and visitation rate of species at the same time that we performed supplemental hand-pollination experiments. We obtained data on the specialisation level of plant species by transect walks. Three 47 to 59 m long transects were evenly distributed (ca 13 m apart) along the horizontal axis of the study area. The transects were walked at a slow pace every 3-4 days between 08:00 and 19:00 h during the main part of the flowering season, which lasted from 26 May to 16 August in 2003 and from 27 May to 6 August in Walking pace was kept constant to ensure that all parts of transects were sampled with equal intensity. Transects were walked during periods with no rain and little wind. We walked transects in the same direction every time to avoid shading by the collector, which may affect capture rates of some types of insects (unpubl.). The first insect observed visiting a flower within a 1.7 m strip was caught with a net and determined to species level immediately or collected for later determination. Most insects could not be classified to species level immediately and were sent to experts or identified by us in the lab. We related the magnitude of pollen limitation to specialisation level, which we measured as either the total number of insect species (species richness) or the total number of insect families (family richness) visiting each plant species in the year we supplementary hand pollinated the species. We obtained data on visitation rates of plant species supplementary pollinated in 2003 as described in Hegland and Totland (2005). Visitation was observed for 10 min periods for all species in 20 randomly placed plots ( m) during the whole season (in total 201 observation periods). The visitation rates of hand-pollinated plant species in 2004 were attained during 5 min observation periods in 10 circular plots with a 1 m radius, which were originally designed as control plots in a plantplant interaction study (in total 121 observation periods, unpubl.). The number of flowers or inflorescences of all species were counted after each observation period in both years to enable calculations of visitation rates per flower or inflorescence. All visitation frequency data were obtained during periods of no rain and little wind. We used the standardised visitation rate for each species in the year of study; calculated as the mean number of visits per minute per plot divided by the mean number of flowers or inflorescences per plot. Because the standardised specialisation level variable (the number of species or families divided by the number of transect walks conducted) was highly correlated (r 0.99) with the absolute number of observed species or families, we used the latter as a measure of specialisation level because it is easier to interpret when inspecting a chart. Data analysis We used two-tailed paired t-tests (using SPSS ) to examine whether supplemental hand-pollination affected reproduction (seed production or seed weight) of handpollinated flowers of individual plant species. To test for correlations among the reproductive success variables and relationships between the reproductive success variables and the visitation rate or specialisation level of the study species, we first calculated effect sizes for the variables of reproduction (14 above) of all species in the supplemental pollination experiment. We used the Hedges d standardised effect size as calculated with MetaWin ver. 2 (Rosenberg et al. 2000). Positive effect size (d) implies positive impact of increased pollen availability on the reproductive response. Correlations among effect sizes were examined with Pearson correlations, and the relationships between the effect sizes (responses) and the specialisation levels or standardised visitation rates (predictors) were done using the curve estimation procedure in SPSS With this procedure, we could test whether relationships between effect sizes and specialisation levels or visitation rates increased or decreased steadily (i.e. linear relationships) or whether they levelled off (i.e. logarithmic relationships). 885

4 Results Frequency of pollen limitation at a plant community level Only one of 11 species showed a statistically significant positive production response to our supplemental hand pollination (see Table 2 for all means); Centaurea jacea had a higher seed production (i.e. mean number of viable seeds per flower) when supplied with surplus outcross pollen (paired one-tailed t-test: t 2.45, DF 18, p 0.012). There was a trend towards higher seed production in handpollinated flowers of Geum rivale (t1.54, DF19, p 0.07) and Prunella vulgaris (t 1.44, DF 15, p 0.085), but significantly lower in hand-pollinated flowers of Lotus corniculatus (t 2.55, DF 15, p 0.011), and marginal lower in Trifolium repens (t1.45, DF19, p 0.08). The seed mass (i.e. mean seed weight per flower) of hand-pollinated flowers of Ranunculus acris was higher than in control flowers (t 1.72, DF 19, p0.05) indicating an effect of supplemental pollination on offspring quality. The seed production and seed weight for all the other species did not respond to supplemental hand-pollination (all p-values 0.13, Table 2). The resource reallocation test showed that none of the species had significantly lower seed production or seed weight in the internal control flowers on supplementarypollinated plants compared to the control flower on the naturally pollinated plant, whereas one species had higher seed production in these internal control flowers (Lotus corniculatus: t2.55, DF 15, p0.022). Relationships among effect sizes for pollen limitation There were several significant correlations among the effect sizes for pollen limitation. For example, all the effect sizes describing the production responses to pollen supplementation were positively correlated (Table 3). Interestingly, there was also a negative correlation between the effect size for seed production and for seed weight indicating a possible tradeoff between offspring quantity and quality (Table 3). Two-tailed t-tests showed no significant differences in effect sizes between study years (2003 vs 2004; seed production: t0.18, DF9, p0.87; seed weight: t1.14, DF 9, p 0.29). Effect sizes of pollen limitation did not differ between species with contrasting breeding systems (substantial selfing vs little/no selfing reported; seed production: t0.27, DF8, p0.80; seed weight: t1.59, DF 8, p0.17) or floral shape (open vs tubular flowers; seed production: t0.45, DF 9, p0.67; seed weight: t 1.27, DF 9, p0.24). Relationship between pollen limitation and the visitation rate and specialisation level Curve estimation revealed that the effect sizes for pollen limitation on seed production for the 11 species was positively related to the mean visitation rate of the species (Fig. 1A), whereas the effect size for seed weight was negatively related to visitation rates (Fig. 1B). For both relationships, a logarithmic function explained the variation better than a linear function, but linear models were also significant (Table 4). None of the effect sizes were related to the variables describing pollinator specialisation level (all p values0.4, Fig. 1CF). The negative effect sizes for pollen limitation on seed production of some of the study species may be experimental artefacts due to reduced seed production after supplemental pollination. Moreover, the sample sizes for seed weights of hand-pollinated flowers were very low for L. corniculatus (n9 and 2) and T. repens (n13 and 8). Therefore, we performed two alternative curve estimations. When we set the negative effect size values to zero, the models were largely similar, although the relationship with visitation rates became only marginally significant (Table 4). When we excluded the species with negative effect sizes, the relationship between effect size and family richness became linearly negative and statistically significant (Table 4) and the relationship with visitation rate was no longer significant. However, it should be mentioned that the number of observations (i.e. species) was quite low in these latter analyses. There was no significant correlation between visitation rates and specialisation levels (species richness: r0.53, n11, p0.10; family richness: r0.30, n 11, p0.30), but the two variables for Table 2. Mean seed production and weight of control and supplemental hand-pollinated flowers of the 11 study species from the temperate grassland Rudsviki, Kaupanger in western Norway. Study species Seed production Seed weight (mg) n Control Hand-pollinated n Control n Hand-pollinated Carum carvi (61.8) (38.0) (0.930) (0.472) Centaurea jacea (11.6) 22.6 (12.4) (0.851) (0.545) Clinopodium vulgare (0.5) 3.8 (0.4) (0.049) (0.039) Geum rivale (16.6) 79.2 (23.4) (0.251)) (0.217) Hypericum maculatum (157.0) (109.6) (0.004) (0.004) Lotus corniculatus (2.9) 0.3 (0.9) (0.079) (0.008) Pimpinella saxifraga (98.6) (92.4) (0.148) (0.114) Potentilla erecta (3.6) 5.9 (4.5) (0.231) (0.225) Prunella vulgaris (1.4) 3.4 (1.0) (0.126) (0.112) Ranunculus acris (4.0) 14.9 (6.7) (0.261) (0.459) Trifolium repens (1.3) 1.1 (1.6) (0.086) (0.080) Note: n is the number of reproductive units used to calculate the given means. For some species, the n for seed weight differs between treatments (due to differences in survival) and is given separately. Standard deviations are reported in parentheses. 886

5 Table 3. Pearson correlations coefficients among effect sizes (d) for variables used to express effects of supplemental hand-pollination for 11 herb species in a temperate grassland. Variables d for seed production d for seeds per fruit d for developed fruits d for seeds per fruit (pb0.001) d for developed fruits (p0.042) (p0.008) d for seed weight (p0.033) (p0.068) (p0.58) Note: The significance (p) of correlations is shown in parentheses; number of observations (n) was 11 for all correlations except those including d for developed fruits where n was 9. specialisation levels were strongly correlated (r 0.80, n 11, p0.003). Two-tailed t-tests revealed no significant differences in visitation (t0.17, DF 9, p 0.87) or specialisation levels (insect richness: t0.41, DF 9, p 0.69; family richness: t 0.13, DF 9, p0.90) between the two study years. Discussion Frequency of pollen limitation at the plant community level That only two (one production and one weight response) out of 11 plant species appear to be significantly pollen limited, contrasts with recent reviews of single-species studies (Burd 1994, Ashman et al. 2004, Knight et al. 2005, but see Knight et al. 2006). There are several possible explanations for these seemingly unusual results, both from our study and from other studies. Our results may be a consequence of high pollinator abundance in the study community, such that quantitative pollen limitation rarely occurs. This explanation has also been used in other areas where pollen limitation appeared to be low (Thomson 2001). Comparing the observed visitation rates with those few cases where multi-species data are easy accessible (Motten 1986, Totland 1993), it seems that the flower visitation rate (i.e. a measure of the relative pollinator density) in Rudsviki were quite high. It is also possible that earlier studies represent a biased collection of species, and that this researcher bias may have influenced their results, as suggested by Thomson (2001). Our collection of study species represents a relatively diverse array of species from the study community (Table 1). In general, our results also agree with other community studies known to us. Motten (1986) found that the few species responding to supplemental pollination within a community were those pollinated by bumblebee-queens. A specialisation on these pollinators may contribute to pollen limitation because bumblebee phenology may be affected by climatic factors and brought out of synchrony with the blooming period of the plant species. In our study, only one species was primarily visited by bumblebee-queens (Geum rivale unpubl.), and there was a trend towards pollen limitation of its reproduction. Since we conducted the pollen supplementation for most species at the flower level, as opposed to whole-plant level, we potentially overestimated the occurrence of pollen limitation due to resource allocation mechanisms (Stephenson 1981, Knight et al. 2006). It is frequently observed that plants may reallocate resources within or among years, such that a reproductive effect of increased pollen availability is reduced in later emerging flowers or the subsequent year (Stephenson 1981, Ehrlén 2002, Wesselingh 2007). Nevertheless, none of the eight species which we examined for resource reallocation showed any changes in seed production or mass after supplemental pollination. Results from the two species, where we conducted whole-plot supplemental pollination, R. acris and G. rivale, suggest that later emerging flowers produce fewer seeds, but that the drop in seed production was equal among pollen supplemented and control flowers (unpubl.). Aizen and Harder (2007) suggested several weaknesses of the usual pollen supplementation approach to study pollen limitation, which could also have affected our results. First, the pollen used for supplemental pollination may have higher quality than the pollen received from natural pollination, making it difficult to separate the pollen quantity versus quality effects on female reproductive output. Second, effects of supplemental pollination may depend on the timing of hand-pollination, and the relatively early supplementation of pollen that we used could have increased the chance of detecting pollen limitation. Thus, some of the significant effects we found and that were also reported in other studies, may be caused by a pollen quality effect rather than by a pollen quantity effect (Aizen and Harder 2007). Relationships between effect sizes for pollen limitation The positive correlations among the three different measures for seed and fruit production (effect sizes for number of viable seeds, seeds per fruit and proportion developed fruits), suggest that the variables used to describe pollen limitation on seed production provide similar results. This result corroborates with meta-studies that show clear correlations among most variables used in pollen limitation studies (Knight et al. 2005, 2006). Interestingly, the correlation between the magnitude of pollen limitation (i.e. effect size) for seed production and seed weight was negative, indicating that there might be a tradeoff between these variables at the species level. Such tradeoffs have been found for several independent plant species (Marshall and Ellstrand 1988, Ågren 1989), but not in a meta-study considering many species (Knight et al. 2006). Our study is the first to show such tradeoffs among multiple species within a community. Most studies on pollen limitation apply a seed and/or fruit number response and not a seed size response to pollen supplementation experiments, even though seed size may have profound 887

6 Fig. 1. The relationship between effect sizes for the mean number of viable seeds per flower or inflorescence (i.e. seed production) and the mean seed weight per flower (as response to supplemental hand-pollination), and (A) and (B) the visitation to plant species represented by the mean pollinator visitation rate (the mean number of visits per flower per minute); (C)(F) the specialisation level of plant species represented by the total number of visiting insect species (i.e. species richness: (C) and (D)) and the total number of visiting insect families (i.e. family richness: (E) and (F)). Lines represent significant relationships (see Table 4 for significance values of all relationships). effects on recruitment and establishment, and ultimately population dynamics (Ashman et al. 2004). One of the species in this study, Ranunculus acris, had significantly higher seed weight after supplemental pollination and we have shown that this effect may have subsequent effects on germination and seedling survival of this species in a natural setting (Hegland and Totland 2007). Higher pollen loads have also been shown to positively affect offspring performance for several species in greenhouse experiments (Winsor et al. 2000, Kalla and Ashman 2002, Colling 2004), which may also be a result of pollenpollen competition, or related processes. Relationship between pollen limitation and visitation rate Contrary to our expectation, we found that plant species receiving most visits also had the largest pollen limitation on seed production. There seems to be some levelling off (as seen by the logarithmic relationships) such that the increase in seed production in response to supplemental pollination amplifies more slowly among species with higher visitation rates. Even if the relationship between effect size for seed production and visitation rates is not significant when we remove the special cases where effect sizes are negative, a 888

7 Table 4. Curve estimations for relationships between Hedges d effect sizes (response) and visitation and specialisation levels (predictors) of the 11 study species. Response Predictor Model DF R 2 p d for seed production Visitation rate Linear Logarithmic Insect richness Linear Logarithmic Family richness Linear Logarithmic d for seed production Visitation rate Linear (negative values set at zero) Logarithmic Insect richness Linear Logarithmic Family richness Linear Logarithmic d for seed production Visitation rate Linear (negative values excluded) Logarithmic Insect richness Linear Logarithmic Family richness Linear Logarithmic d for seed weight Visitation rate Linear Logarithmic Insect richness Linear Logarithmic Family richness Linear Logarithmic Note: the negative values found for Hedges d for seed production may be experimental artefacts (see also low n) and instead we ran two alternative analyses (with negative values set at zero or excluded, respectively) for this particularly effect size. non-significant relationship is still different than expected. The predominant view on the causes of pollen limitation is derived from the idea that species experience pollen limited because individual plants receive inadequate amounts of pollen. Consequently, less visited species are most likely to be pollen limited. One reason for finding the contrary result could be a correlation between pollinator visitation and ovule numbers in plant species, because species with a higher number of ovules often are more pollen limited than species with fewer ovules (Knight et al. 2006). However, only one of those three species (Centaurea jacea, Prunella vulgaris and Geum rivale) experiencing significant (or marginally significant) effects of supplemental pollination have many ovules (G. rivale: ca 100 per flower). Furthermore, the seed mass of species receiving many visits seems to be less influenced by supplemental pollination than species receiving few visits, which highlight the importance of investigating seed mass in pollen limitation studies. Although there might be tradeoff mechanisms, higher offspring quality can also be caused by competition among pollen on stigmas (Snow 1986, Snow and Spira 1991), because a surplus of pollen deposited on stigma may cause gametophytic competition among pollen donors mediated by variation in pollen tube growth rates. Instead of affecting seed number, this may affect progeny vigour, if there is a positive correlation between gametophytic and sporophytic quality. Maternal mechanisms may play a larger role when excess pollen is deposited because the order of pollen appearance on stigmas may affect the quality of offspring since ovules fertilised earlier may be better provisioned by the mother (Delph et al. 1998). Alternatively, surplus pollen may affect maternal choice and lead to selective abortion of developing seeds (Marshall and Ellstrand 1988). Finally, it has been shown that differences in pollen quality between supplementary pollinated and control flowers may determine some of the effects on seed production (Aizen and Harder 2007), and it remains to be determined how this affects the quality of progeny (Hegland and Totland 2007). Relationship between pollen limitation and specialisation level Theory predicts that plant species characterised by a high degree of pollinator specialisation will be more prone to experiencing pollen limitation on their reproductive success (Bond 1994, Aguilar et al. 2006), perhaps due to increased vulnerability caused by fluctuating abundances of their main pollinators. Higher diversity of pollinators has for example been shown to increase fruit production in coffee (Klein et al. 2003) and an effect of pollinator specialisation may also be expected on offspring quality (Herrera 2000). We found no effect of pollinator diversity on the magnitude of pollen limitation, which confirms the results from studies employing surrogates of specialisation levels, such as floral shape or morphology (Knight et al. 2006). Similar conclusions were obtained in a meta-analysis on the effects of habitat fragmentation (thus potentially limited pollen availability), which showed that the decrease in reproductive success due to fragmentation was equal in generalist and specialist plant species (Aguilar et al. 2006). Our results might also be explained by a situation where most species have a rather generalised visitor assemblage (Fig. 1CF), and few of our study species represent the highly specialised range of the continuum from generalisation to specialisation. When excluding the negative effect sizes from the analyses, it appears that species visited by few insect families have a 889

8 higher degree of pollen limitation than species visited by many insect families. Thus, our results offer conflicting evidence to the general notion that specialised plants should be more prone to pollen limitation. Future community level studies of pollen limitation can test this idea more thoroughly. The results presented here offer limited opportunities to determine species vulnerability to decreased pollen availability based on the degree of pollinator specialisation or other plant traits (e.g. plant breeding system) that are expected to affect the magnitude of pollen limitation in plant species (Ashman et al. 2004, Knight et al. 2006, Vamosi et al. 2006). Therefore, we still do not have sufficient information on how to a priori identify plant species vulnerable to decreased pollinator abundance and diversity. Conclusions By using different types of data from a temperate plant community, we have studied the phenomenon of pollen limitation in many plant species simultaneously. Although we advise against drawing too strong conclusions based on data of pollen limitation in 1520 pairs of individuals in 11 plant species, our results pinpoint several important issues which can be addressed in future studies. First, more studies should be performed using multiple species within a plant community or a landscape to assess whether the prevalence of pollen limitation in published studies are not artefacts of publication bias or researcher bias. Second, several of the variables potentially describing pollen limitation appear to yield similar results, implying that it may not be necessary to standardise the use of variables. Third, the tradeoff between the production and weight components of reproduction was substantial, and we encourage future studies to include both types of measurements to better understand how pollen limitation may affect plant reproductive success. Lastly, more studies are required to establish whether the positive relationships between the magnitude of pollen limitation and pollinator visitation in the plant species in our study community was more than a coincidence. Acknowledgements The authors wish to thank the enthusiastic assistants: John Wirkola Dirksen, Sondre Eikås, Vegard Eldholm, Peter Greve, Stian Heid, Maria Collett Knagenhjelm, Torkjel Solbraa, Knut Fredrik Øi and Håvard Øyrehagen. Most insects could not be identified to species level immediately and we leaned on the expertise of Adrian Pont, Tore Nielsen, Frode Ødegaard, Knut Rognes, Bradley Sinclair, Michael Ackland, Stig Andersen, Henry Disney, Jens-Herman Stuke, Christian Kehlmeier, Stephan Lehmann, Atle Mjelde, Øistein Berg, Sigmund Hågvar, Vidar Selås, Tom Pedersen and Fred Midtgaard to whom we are very grateful. Rebekka Lundgren kindly commented on an earlier draft, and Vilma Bischof reviewed the use of English language. 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