4. Discussion
Pollen limitation is generally considered a common phenomenon and many comparative studies report relatively high occurrence (62–73%) in various habitats (Burd 1994, Ashman et al. 2004). However, we only recorded significant pollen limitation for approximately 41% of species (9 out of 22 surveyed) in our wet meadow community. Our findings are in concordance with a similar unusually low occurrence of pollen limitation in a temperate grassland community in western Norway (Hegland and Totland 2008). This study focused on pollen limitation and its relationship to plant species visitation rates and specialisation levels and revealed only two out of eleven (~18%) studied plant species to be significantly pollen limited. Moreover, Bennett et al. (2018) even documented no pollen limitation in investigated study of nine species in a Romanian meadow community. It might seem that the low levels of pollen limitation revealed in the pollen supplementation experiments are in agreement with the assumptions from the model by Haig and Westoby (1988), which stipulates that seed set in flowering plants should be equally limited by both pollen and resource availability. It further suggests that pollen supplementation should not increase seed set in populations at their evolutionary equilibrium, because resources should be unavailable for maturation of their additional fertilized ovules. However, Burd (2008) adjusted this model for stochastic variation in both ovule fertilization and resource availability, which made the model broadly in accordance with the recent meta-analysis (Burd 1994, Ashman et al. 2004), in which pollen limitation is found in most surveyed species.
The reported inconsistencies in the magnitude of pollen limitation could stem from several non-mutually exclusive reasons:
(1) Effect of sampling size and experimental design. Using power tests (via simulation) for pollen supplementation experiments, Thomson (2001) illustrated that moderate pollination deficits of up to 15% will usually not be detected with sample sizes of 20 individuals, and even 40 are insufficient for minor deficits. But, unfortunately, lower sampling effort (such as 20-30 individuals in our study) is an inevitable result of various logistic constrains and trade-offs between the data quantity and quality in most community studies (Hegland and Totland 2008, Wolowski et al. 2013, Lázaro et al. 2015, Bennett et al. 2018).
(2) Publication bias . The community approach, where multiple plant species are studied simultaneously, may lead to a better understanding of patterns in pollen limitation. It is because environmental characteristics, such as nutrient levels within a given community, are relatively homogenous in such studies and the role of plant traits in pollen limitation can, therefore, be better assessed. Nevertheless, there have been few studies focused on the relationships between plant traits and pollen limitation across whole communities (Motten 1986, Hegland and Totland 2008, González and Pérez 2010, Wolowski et al. 2013, Lázaro et al. 2015). All these studies recorded lower levels of pollen limitation in natural systems compared to the pollen limitation documented in comprehensive reviews that are mostly based on single-species studies (Burd 1994, Ashman et al. 2004). Therefore, the publication bias, favouring statistically significant responses which then become available for further studies, together with the omission of ‘grey literature’ and studies not written in English (Auger 2017), complicates our understanding of pollen limitation (Knight et al. 2006).
(3) Effect of pollinator abundance. Hegland and Totland (2008) discussed their results of low pollen limitation in the context of a possible higher pollinator abundance in the studied community, which could substantially reduce the quantitative pollen limitation. A partial cause of low pollen limitation in our study could be that the targeted semi-natural locality is situated in a relatively well-preserved and mosaic-like landscape with a limited influence of intensive agriculture. Such semi-natural, diverse, and heterogeneous environments support pollination services (Steffan‐Dewenter and Westphal 2008, Viana et al. 2012) and thus increase the plant reproductive success, as suggested by Bennett et al. (2018) in their Romanian meadow community.
(4) Effect of plant community composition and study species selection . In our investigated community, only a few plant species with morphologically highly specialized flowers, which are expected to be more prone to pollen limitation, were present. Therefore, this community may have a lower pollen limitation than communities with a greater proportion of specialized flowers.
(5) Choice of the pollen limitation measure . An important factor determining the recorded magnitude of pollen limitation may also be the choice of its measure. Knight et al. (2006) compared 263 studies working with different measurements of the production component of reproduction and revealed the largest effect for relative fruit set, and the lowest effect for production of seeds/flower and seeds/fruit. However, because the magnitude of pollen limitation was inter-correlated among these response variables, Knight et al. (2006) assumed that pollen limitation occurs simultaneously at different stages of the plant reproduction, but with varying intensity. Also in our study the numbers of pollen-limited species varied substantially between the two applied measures, seed production and seed mass. Furthermore, Hegland and Totland (2008) pointed out that the two main components of plant reproductive success, seed production and seed mass, are often not included in the same studies.
In our studied community, two species showed significant positive seed weight response after pollen supplementation, though we expected the negative relationship. Several studies demonstrated that seed mass decreases with pollen availability due to seed size–number trade-off (Ashman et al. 2004). For example, Ågren et al. (2006) recorded reduced mean seed size in hand-supplemented Primula farinosa by about 12%, but a larger total mass of seeds than in naturally-pollinated plants. The opposite effect, i.e. increased seed weight after pollen supplementation could be explained by the increased pollen quality (Hegland and Totland 2008). Aizen and Harder (2007) suggested that the cross-pollen used for supplementation may have higher quality than the mixture of self- and cross-pollen available under the natural pollination. It seems evident that the magnitude of pollen limitation is dependent on the treatment level, e.g. whether the experimental design is applied only on a fraction of the plant’s flowers or on the whole plant (Zimmerman and Pyke 1988). Unfortunately, because we treated only flower pairs, we can only speculate on the proportion of resource allocation in our study (Wesselingh 2007). The low differences in the seed weight between the treatments could be caused by the ability of the plant to compensate for any possible higher cost of an additional seed production induced by the supplemental pollination in only one flower.
Despite analyses of several floral and life history traits connected to plant reproduction, we only found the significant relationship of PL to the number of pollinator functional groups. This finding is in accordance with the meta-analysis of pollen limitation in different world regions (Vamosi 2006, Wolowski et al. 2014, Rodger and Ellis 2016), where the more pollinator-specialized plant species were also more pollen limited (but see Hegland and Totland 2008). However, Lázaro et al. (2015) pointed out that this relationship is not entirely clear and it is very important to distinguish between morphological (based on floral shape) and ecological (based on realized interactions) specialization. They found a strong negative relationship between pollen limitation and ecological generalization, but only for species with the morphologically specialized flowers. As a possible explanation they suggested that the morphologically specialized flowers benefit more from generalizing their pollination system in the lack of a primary pollinator (Lázaro et al. 2015). The high ecological generalization may however result in the stronger pollen limitation because of lower flower-visitor diversity with abundant low-efficiency pollinators transporting high loads of incompatible pollen (Gómez et al. 2010). Accordingly, we recorded stronger pollen limitation in the species with specialized, as well as highly generalized pollination systems. This supports the prediction that many mutual relationships between plants and visitors should be non-linear (Young 1988, Morris et al. 2010).