Discussion

Surprisingly, in our study heterospecific pollen interference did not affect seed set nor seed number, i.e. it did not affect whether a flower would produce or not at least one seed nor the amount of seeds produced. Rare species had a tendency to produce more seeds, but this trend was not significant. Breeding system did affect seed set, but not in relationship with conspecific or heterospecific pollen treatments, with self-incompatible species less likely to set seed compared to self-compatible species. Lastly, we could show that for rare recipients treated with pollen from rare donors, more distantly related recipient-donor species pairs had a lower reduction in seed number compared to closely related recipient-donor species pairs. Hereafter we discuss these results as well as potential ecological and evolutionary implication.
In a co-flowering community, we can expect common species to receive pollen from a rare species unfrequently, thus an adaptation to that type of pollen is unlikely. On the other hand, a rare species is likely to receive frequently pollen from common species, thus making an adaptation to heterospecific pollen receipt more likely, like predicted by the tolerance-hypothesis (Hao et al., 2023). Indeed, in a study by Arceo-Gomez and colleagues (Arceo-Gómez et al., 2016) the authors showed how HP tolerance for a Clarkia species did depend on previous exposure of the population to HP, but rather than acting on the recipient individual, would act on the donor individual, by improving CP performance. On the other hand, such adaptation was not observed for a congeneric Clarkia species, suggesting that adaptation is context- and species-specific. Adaptation could explain the low effect of HP overall in our study species. On the other hand, despite our study species do co-occur and co-flower in nature, the seed-material did not consistently originate from populations co-occurring at local scale, but co-occurring only at regional scale, thus missing potential adaptations at the population level. Further, the (co-occurrence) history of the populations from which the seed material was collected is unknown.
Another factor we analyzed is the evolutionary relatedness, measured as phylogenetic distance, between recipient and donor species in interaction with recipient and donor status. While overall no pattern emerged, we could show a decrease of HPI for more distantly related recipient-donor pairs when both were rare. The likely reason for the absence of these patterns for common recipients and rare recipients with common donors could be the lack of close relatives for these groups in our study species set. Indeed, the range for these groups included only phylogenetic distances larger than 189 * 106 years. For closely related recipient-donor pairs, a stronger HPI could be caused by similar recognition systems between recipient stigma and donor pollen grains. For a better understanding of these patterns, a study species set with a broader range should be used.
In this study we looked only at pairwise HP interactions, while in a plant community it is likely to have multi-species mixes of HP that are transferred between flowers. Arceo-Gomez (T. Ashman & Arceo-Gómez, 2011) performed HP hand-pollinations with mixes up to three species and showed that HPI increased with the number of heterospecific pollen donors. Further, the strength depended on specific species composition. Indeed, in our study, HPI varied considerably among different species-pairs. Some species are known to produce strongly allelopathic pollen (Kanchan & Chandra, 1980), but in our study it did not look like a specific species had a consistent negative effect on all other species in terms of HPI (see Figure S 9).
For our species, self-incompatible species showed an overall lower seed set, while breeding system did not affect seed number or HPI in any way. Self-compatibility might play a role especially in a natural community, where HP can act through the mentor effect (de Nettancourt, 1997), and allowing for self-fertilization even in self-incompatible flowers, with consequent ovule abortion (Lynn, Sullivan, & Galen, 2022). In our study we showed how self-incompatible species are less likely to produce seeds even when enough pollen is present. We emasculated our recipient species whenever possible prior to treatment, but due to the small flower size, three out of eight species were left with their anthers to avoid complete flower abortion. These three species (Bupleurum rotundifolium , Fallopia convolvulus and Myosotis arvensis ) are all self-compatible. Thus, one explanation could be that seed set induced by selfing is more secure compared to seed set from outcrossed pollen, despite the genetic advantages of outbreeding.
Specific flower morphology and in particular a smaller stigma size in restrictive flowers (i.e. flowers with a reduced access to the flower interior) have been shown to reduce HP deposition, while at the same time increasing CP deposition (Montgomery & Rathcke, 2012). In our study, we did not analyze the effect of flower morphology or flower traits, since due to our small sample size in species number (eight species in total), species and trait would be confounded. In a natural community, flower morphology would also play an important role in terms of pollinator sharing and flower constancy (the tendency of pollinators to forage on the same flower type (Waser, 1986)) since some flowers are adapted to specific groups of pollinators and thus sharing among these species is more likely. For example, both Ajuga chamaepitys andFallopia convolvulus , being lip flowers, rely on bumblebees as their most common pollinators (Kuehn et al., 2004).
While in our study we did not find any strong effect of HP on seed set and seed number, HPI remains an important aspect of co-flowering communities (T. L. Ashman & Arceo-Gómez, 2013), since it allows species to affect other species without direct competition and at a distance above the direct interactions. In a co-flowering community; we can expect a variable and complex pollination landscape that could promote evolution of flower morphology and avoidance mechanism also due to HPI. It seems that while mechanisms as HPI and adaptations to it do play a role in shaping plant communities, this patterns are highly variable depending on the context and on the species observed. We conclude that heterospecific pollen interference plays a minor role for rare plant species. Rather, other factors, like pollen limitation mediated by low pollinator visitation rates, are likely to affect rare plant species at the level of interactions. Adaptation and species-specific interactions may explain the low overall effect of HPI in our study. The complexity of multi-species interactions and the specific composition of heterospecific pollen mixes may further influence the strength of HPI. Additional research is needed to explore these factors and their implications for both in-sit and ex-situ conservation strategies.