Discussion
In our study heterospecific pollen interference did not affect seed set
or seed number, i.e. it did not affect whether or not a flower would
produce at least one seed or 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 to conspecific or heterospecific pollen treatments, with
self-incompatible species less likely to set seed compared to
self-compatible species. Lastly, for rare recipients treated with pollen
from rare donors, more distantly related recipient-donor species pairs
tended to have 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 infrequently, 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, as predicted by the
tolerance-hypothesis (Hao et al. 2023). Indeed, in a study by
Arceo-Gomez and colleagues (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, even though 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 (Moyle and Nakazato 2010; Moyle, Olson, and Tiffin 2004;
Scopece, Widmer, and Cozzolino 2008). 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. Ashman and Arceo-Gomez (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 Some species are known
to produce strongly allelopathic pollen (Kanchan and Chanra 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 seed number (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, and 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
(Goodwillie and Weber 2018).
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 and 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). Morphological
differences and pollinator type can also drive the direction (for
example, from species A to species B but not from species B to species
A) and amount of pollen transferred (Fang and Huang 2013). In our study,
we used all species both as donors and as recipient, and used a
saturated amount of pollen, but this could differ in natural conditions,
where pollen limitation is common(Burd 1994; Knight et al. 2005; Larson
and Barrett 2000).
While interspecific pollen transfer seems to be widespread, the
magnitude and the effect on reproductive output are highly context
dependent. In line with our findings, several studies showed limited or
no effect of HP deposition on seed production (see Morales and Traveset
2008 and references within), while conspecific pollen loss seems to be
the more important component of interspecific pollen transfer (Mitchell
et al. 2009). Further, the impact of HP deposition has been shown to
depend on timing of HP application, with seed set more affected when
heterospecific pollen is applied prior to conspecific pollen, compared
to a simultaneous mixed application (see Morales and Traveset 2008 and
references within) as in this study.
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 (Ashman and Arceo-Gómez 2013), since it allows species to
affect other species without direct competition and at a distance above
the direct interactions. In our study we could do a full recipient-donor
combination treatment for a total of eight species, which could mean a
reduced statistical power when comparing groups within. While using a
multi-species approach can be labour-intensive, we suggest to increase
the number of species for further studies, or to perform multiple
experiments to join the data in a meta-analysis, in order to increase
statistical power. 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. At the same time,
the lack of stable conditions and the variability of the direction of
selection between years and generations (Feinsinger 1987) could hamper
adaptation to HP deposition, preventing the emergence of clear patterns.
It seems that while mechanisms such as HPI and adaptations to it play a
role in shaping plant communities, these 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 in our study system. Rather, other factors, like pollen
limitation mediated by low pollinator visitation rates, or conspecific
pollen loss to heterospecific recipient flowers, are likely to affect
rare plant species. 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.