Pollen dispersal distance is determined by phenology and ancillary traits but not �oral gender in an andromonoecious, y-pollinated alpine herb

Pollen-mediated gene �ow and spatial genetic structure have rarely been studied in alpine plants pollinated by Dipteran insects. Furthermore, it is not clear how different �oral traits, such as �oral gender, phenology, and ancillary traits, may affect pollen dispersal distance within a population. In this study, we conducted a paternity analysis to track pollen �ow in a population of Pulsatilla alpina, an andromonoecious alpine herb producing male and bisexual �owers. We found that the pollen was dispersed over short distances (mean = 3.16 meters) with a dispersal kernel of Weibull distribution. Nonetheless, spatial genetic structure was weak in the population (Sp statistic = 0.013), pointing to effective seed dispersal. The pollen dispersal distance was independent of the gender of the �ower of origin but depended positively on �oral stalk height and negatively on �owering date and tepal length. Although male siring success did not correlate with pollen dispersal distance, selection may favor traits increasing pollen dispersal distance as a result of reduced bi-parental inbreeding. Our study has not only provided new insights into the nature of pollen dispersal, especially of alpine plants, but has also revealed the effects of �oral traits on an important component of male reproductive success.


Introduction
Gene ow in sessile plants is largely mediated by dispersal of pollen and seeds, which together determine the spatial genetic structure within and among populations.The dispersal distance should depend on not only dispersal vectors but also on plant traits that attract and manipulate the position and behaviour of the pollinators.There has been substantial progress in investigating traits that affect seed dispersal (Vittoz and  Around 90 percent of owering plants rely on a diverse group of animals as their pollination vector (Ollerton et al. 2011).Nonetheless, our knowledge is still limited about how pollination vectors with different attributes, especially small insects, affect pollen dispersal (Ashley 2010).Although it is generally thought that animals with a larger body size lead to greater pollen dispersal distance and thus reduce genetic structures, such conclusions are predominantly drawn from comparisons between plants pollinated by birds and bees (especially hummingbirds and bumblebees) (Krauss et al. 2017;Wessinger 2021).In contrast, it remains unclear how small insects such as ies affect pollen dispersal distances, despite the fact that they are among the most important pollinators in alpine, arctic, and agricultural ecosystems (Inouye et al., 2015; see Table 1

for a summary table of studies on pollen dispersal distance in alpine plants).
A number of oral traits likely determine the foraging behavior of pollinators within a population (Waser 1983; Chittka and Thomson 2001;Ishii et al. 2008) and thus affect pollen dispersal distances.For example, in Delphinium virescens, bumblebee pollinators ew longer distances after visiting owers with a low quantity of nectar, which would likely lead to longer pollen dispersal distances for the plant (Waddington 1981).Furthermore, in hummingbird-pollinated Ipomopsis aggregata, mean pollen dispersal distance, estimated using pollen dyes, depended positively on the variance but negatively on the mean stamen length, whereas it was independent of owering date, number of owers, and corolla size (Campbell and Waser 1989).To our knowledge, few studies have evaluated the effect of oral traits on pollen dispersal at the individual level using paternity analyses to measure realized gene ow (e.g., Tomaszewski et al. 2018;Barbot et al. 2022), despite its importance as a component of male reproductive success and its potential implications on spatial genetic structure (but see studies focusing It is generally assumed that individuals dispersing their pollen over greater distances should also enjoy higher male reproductive success in terms of the total numbers of progeny sired, largely because of a supposed increase in mate availability (Harder and Prusinkiewicz 2013) and/or because of a reduced chance of mating with relatives (Price and Waser 1979).As a result, traits that facilitate pollen dispersal should be favored by selection via male reproductive success (Harder and Prusinkiewicz 2013).In windpollinated Mercurialis annua (Tonnabel et al. 2019), for instance, taller plants had a higher male siring success because they dispersed their pollen further, and the pollen dispersal distance correlated positively with male siring success.Surprisingly, in insect-pollinated species, although mating between distant individuals usually led to increased performance in components of male reproductive success (Waser and Price 1991;Souto et al. 2002), few empirical studies have tested the positive correlation between pollen dispersal distance and male siring success.
In this study, we asked how oral traits affect pollen dispersal distance in a population of the andromonoecious alpine herb Pulsatilla alpina, which relies on dipteran insects as pollinators.To this end, we selected a population comprising mostly single-owered individuals, i.e., with a single male or bisexual ower, so that we could explicitly evaluate the effect of traits at the ower level on dispersal distances.We exhaustively sampled all the owering individuals in the study population and conducted a paternity analysis using microsatellite markers.We then addressed the following questions: 1) What is the pollen dispersal kernel of P. alpina, and does it differ between male and bisexual owers?2) What is the spatial genetic structure of the P. alpina population? 3) How does pollen dispersal distance depend on oral traits, speci cally oral gender, phenology, tepal length, and stalk height?And 4) Does mating distance correlate positively with male siring success?

Materials And Methods
Study species and study sites Pulsatilla alpina (L.) Delarbre (Ranunculaceae) is a perennial hemicryptophyte growing in sub-alpine to alpine habitats in central Europe (Lauber et al. 2018).Several vegetative and/or reproductive shoots emerge from a perennial underground rhizome soon after the snowmelt, from early May to July.
Depending on their size and age, individuals produce between zero and ~ 20 owers with around six white tepals (the sepals and petals are not distinguishable), each on its own reproductive shoot.The species is andromonoecious: unisexual male owers bear only stamens, whereas protogynous bisexual owers bear stamens and one to a few hundred uni-ovulate pistils.The sex allocation of the species is size-dependent, with larger plants allocating absolutely and proportionally more resources to their female function (Chen and Pannell, 2023).Furthermore, small individuals may produce only a single male ower and thus function as pure males in the respective owering season (Chen and Pannell, 2023).Both male and bisexual owers are predominantly visited by ies, including house ies and syrphid ies (Chen and Pannell, 2022).Ripe fruits (technically achenes) with elongated pappus hair are dispersed by wind in early autumn (Vittoz and Engler 2007).After fruit dispersal, above-ground vegetative parts senesce, but individuals persist underground over winter.
The study was conducted during the owering season of 2022 in a single population of P. alpina, located at Solalex in the pre-Alps of Vaud canton, Switzerland ('Population S1+'; latitude: 46°17′42″N, longitude: 7°09′09″E; elevation: 1758 a.s.l.).The population was located on an open slope of sub-alpine grassland and covering an area with dimensions of about 20 m x 20 m and comprising about 150 mainly small and probably young individuals (following recent establishment after avalanche disturbances and/or herbivory by cattle).The individuals typically produced only a single male or bisexual ower during the season.We set up a 10 m x 15 m temporally fenced plot within the population, enclosing 135 owering individuals, and removed all the oral buds (i.e., around 10 buds) outside the plot at the very beginning of the owering season to prevent nearby individuals outside the plot from siring progeny in the plot (thereby improving our ability to assign paternity).

Flowering phenology and ancillary traits
We recorded the owering state of all the individuals in the population every three to four days throughout the owering season, from late May to late June 2022, noting the number of owers, number of stalks, height of the tallest foliar stalk, gender, and position of the owering individuals.On each census day, we recorded the sexual stage of all the owers in terms of seven and ve categories for bisexual and male owers, respectively (see Chen and Pannell (2023b) for a detailed description of the categories).We also photographed each ower and later counted the number of stamens based on the photographs (see details below).The onset of the male stage ( owering date) was calculated as the rst date on which anthers were seen to have dehisced (M 1 stage) for both male and bisexual owers.The height of oral stalks and the length of tepals were measured at the end of the male stage (M 2 stage), following the methods used by Chen and Pannell (2022).Around three weeks after the end of the owering season, all the owers with developing fruits were enclosed in a paper bag until the end of the growing season (early August), at which point all the seeds were collected.

Paternity analysis and estimates of pollen dispersal
To estimate pollen dispersal distance, we used variation at ten microsatellite markers to assign paternity to mature seeds (for details, see Chen and Pannell, 2023b).Leaf samples of all owering individuals were collected in July 2022 at the end of the owering season and dried in silica gel before DNA extraction.Up to ten mature seeds for all seed families were arbitrarily selected for each sampled ower for DNA extraction.Total DNA was extracted from the leaves and seed samples using the BioSprint 96 DNA Plant Kit (Qiagen, Germany).
PCR ampli cation was carried out in a nal volume of 10 µl, including 5 µl of 2× Multiplex PCR Master Mix (Qiagen, Germany), 2 µl of diluted DNA, 1 µl of distilled water, and 2 µl of multiplex containing variable primer concentrations (Chen and Pannell 2023b).Thermal cycling was performed in a TProfessional Standard Thermocycler (Biometra GmbH, Göttingen, Germany) as follows: 95°C for 15 min; 36 and 41 cycles for leaf and seed samples, respectively, at a temperature of 94°C for 30 s, 60°C for 45 s, and 72°C for 45 s; and a nal step at 72°C for 30 min before cooling down to 4°C.PCR products were analyzed by capillary electrophoresis on an ABI3100 Genetic Analyzer (Applied Biosystems).
Sires (fathers) were assigned from among the 135 owering individuals to all mature seeds for which more than ve loci were genotyped with Cervus v 3.0.7,assuming a con dence level of 80% and an error rate of 0.018 (Kalinowski et al. 2007).Pollen dispersal distance was estimated by calculating the distance between the dam (mother) and the most likely sire for each of the successfully genotyped seeds.Although the species is self-compatible with an average sel ng rate estimated to be 0.45, the selfed progeny ultimately contributes little to the next generation due to very high (0.93) inbreeding depression in the study population (unpublished data).Thus, in this study, we considered pollen dispersal only for outcrossing mating events.Male outcrossing siring success was calculated following Chen and Pannell (2023b).

Statistical analysis
We conducted the following analysis within the R statistical framework v 4.0.3(R Core Team 2021).To quantify the pollen dispersal kernel of P. alpina, speci cally, to compare that of bisexual and male owers, we used the R package disp t (Proença-Ferreira et al. 2023).We tted the pollen dispersal distance with three separate models for all the owering individuals, only individuals with a bisexual ower, and only individuals with a male ower, respectively.For each model, we used AIC values to compare the ts of Weibull, geometric, 2Dt, and exponential distributions (for the formula of each distribution, see Proença-Ferreira et al., 2023), i.e., four of the most common distributions for describing pollen dispersal (Austerlitz et al. 2004).The best-tted distribution was then used in the following analysis to evaluate the effects of traits on pollen dispersal distance.
To assess the spatial genetic structure of the owering individuals in the population, we used the software SPAGeDi version 1.5 (Hardy and Vekemans 2002), following the procedure described by Vekemans and Hardy (2004) based on pairwise kinship coe cients between individuals.We conducted Nason's estimator of kinship coe cient (F (r) ) (Loiselle et al. 1995).The average relationship coe cients of the ten microsatellite markers per distance class were estimated and their signi cance per class was tested with 1000 permutations.We used Sp to evaluate the extent of spatial genetic structure, which is de ned as: , where β is the regression slope of F (r) on ln(spatial distance), and F (1) is the mean of F (r) among individuals for the rst distance class (Vekemans and Hardy 2004).
To evaluate how oral gender, phenology, and ancillary traits affect pollen dispersal distances in P. alpina, we used a generalized additive model with a Weibull distribution (gamlss package; Stasinopoulos et al., 2018).The pollen dispersal distance of sires with a single bisexual or male ower was set as the response variable and oral gender (i.e., bisexual or male), owering date, tepal length, and stalk height as explanatory variables.We included only the single-owered sires with a complete set of measurements of the traits.We set the identity of the sires as a random variable because the mating events from the same ower share the same oral phenotype.We evaluated the residuals of the model using the plot function in the gamlss package (Figure S1; Stasinopoulos et al., 2018).
To investigate the relationship between pollen dispersal distance and oral male siring success, we used a generalized linear model (glmer function in the lme4 package; Bates et al. 2015) with a Poisson distribution.We set the mean pollen dispersal distance of each single-owered individual as an explanatory variable.We included an observation-level random variable to account for overdispersion (Harrison 2014).We evaluated the residuals of the model using the package DHARMa (Hartig 2019)

Pollen dispersal kernel
We identi ed 513 outcross mating events for 854 genotyped seeds.Pollen dispersal distances for outcrossing were generally short, with an average of 3.16 m separating sire from the dam and 25%, 50%, and 75% of seeds sired by males < 1.0 m, < 2.15 m, and < 4.36 m away from the corresponding dam (Fig. 1A).Pollen dispersal of P. alpina was best tted by a Weibull distribution for all three types of sires (Table S1).We found that the parameter b was close to 1 in all three cases, indicating a mostly fat-tailed distribution (Table 2; Fig. 1).The values of the parameters used in the Weibull distribution along with the mean dispersal distance, skewness, and kurtosis of the kernel can be found in Table 2.

Spatial genetic structure
Spatial genetic structure analysis among the 135 owering individuals showed a signi cantly negative β value (β = −0.013,P < 0.001).The Sp statistic value of the owering individuals was 0.013.Mean kinship coe cients ( ) across all distance classes was − 0.0001.Analysis of ne-scale genetic structure indicates a signi cant positive autocorrelation among individuals located up to around 3 meters apart (Fig. 1D).
Pollen dispersal distance and male siring success Male outcross siring success did not depend on the mean pollen dispersal distance of the single-owered individual (N = 72; Fig. 3; P > 0.05).

Discussion
Short pollen dispersal distance with weak spatial genetic structure We found that pollen dispersal distance in the y-pollinated alpine herb P. alpina was dominated by mating over short distances.Indeed, 75% of mating events were within 5 m.Although it has been suggested that pollen dispersal distance in herbaceous species is in general short (references in Despite the short pollen dispersal distance and the mixed mating system and herbaceous growth form of P. alpina, we found the spatial genetic structure of the study population to be weak, likely as a result of e cient seed dispersal by wind.Spatial genetic structure in plants has been shown to depend on the mating system, pollination system, life form, and dispersal mechanism of the species (Vekemans and Hardy 2004), and e cient dispersal associated with any one of the factors should break down spatial patterns created by others (Meirmans et al. 2011).In general, high spatial genetic structure is expected for species pollinated by insects, especially those with predominant sel ng, and those with a herbaceous growth form and seed dispersed by gravity (Vekemans and Hardy 2004).In contrast, seed dispersal by wind in P. alpina is likely to be e cient, especially as the species produces elongated oral stalks up to 70 centimeters above ground from which the bisexual owers disperse their seeds (Chen and Pannell 2022), with a maximum dispersal distance of 80 meters (Vittoz and Engler 2007).Of course, we quanti ed only the pollen dispersal kernel in this study, so that estimates of both pollen and seed dispersal kernels together will be needed to provide a more complete picture of the mechanisms shaping the spatial genetic structure of P. alpina (e.g., García et al., 2007;Krauss et al., 2008).

Pollen dispersal distance is independent of oral gender
We did not nd any difference in the pollen dispersal pattern between male and bisexual owers in andromonoecious P. alpina, despite the fact that both types of owers differed substantially in their sex allocation, morphology, and phenology (Chen and Pannell 2022, 2023b).First, we found the pollen dispersal kernels of male and bisexual owers to be similar in terms of their shape, skewness, and kurtosis.Second, we detected no difference in mean dispersal distance, irrespective of morphology or phenology (a supplementary analysis using a univariate gamlss model with oral gender as an explanatory variable showed the same results; P > 0.05).These results conform to those of a previous study that found male and bisexual owers to have similar male siring success (Chen and Pannell 2023b).
Direct comparisons between male and bisexual owers in andromonoecious species have been made for various components of male tness (Cuevas and Polito 2004; Schlessman et al. 2004; Dai and Galloway 2012), but not for pollen dispersal distance, as far as we are aware.The study on andromonoecious Anticlea occidentalis provided some indirect assessment of the effects of bisexual and male owers on pollen dispersal distance by removing all the anthers from either bisexual or male owers.That study showed that removing stamens of male owers led to greater pollen dispersal distances compared to individuals with intact owers or with their bisexual owers emasculated, though the observed effects largely depended on both the paternal and maternal plants (Tomaszewski et al. 2018).

Phenology and ancillary traits affect pollen dispersal distance
The impact of owering phenology on different aspects of female reproductive success has been extensively studied for alpine plant populations (e.g., Kudo, 2006; Collin and Shykoff, 2010; Kameyama and Kudo, 2015; Preite et al., 2015), but we remain largely ignorant of how phenology affects the male components of reproductive success.Our present results for P. alpina indicate that pollen dispersal distance, a major component of male reproduction success, depended negatively on the owering date (onset of the male function).Pollen dispersal distance in Ipomopsis aggregata was found to be independent of phenology, based on an investigation using pollen dyes (Campbell and Waser 1989).In contrast, Hirao et al. ( 2006) using a two-generation analysis (Smouse et al. 2001), found that the number of effective pollen donors was higher in the late than the early season in Rhododendron aureum.So far, we can only conclude that the effect of phenology on the pollen dispersal distance in alpine plants is species-speci c, and any patterns that do exist will only emerge with the study of further species and populations.
The negative dependence of pollen dispersal distance on phenology in P. alpina is likely a result of an increase in owering density of co-owering species rather than a change in conspeci c owering density (Figure S2) or in potential mating distance throughout the owering season (Figure S3).According to 'optimal foraging theory' (Pyke et al. 1977), a pollinator should tend to move shorter distances between owers in more rewarding patches, leading to shorter pollen dispersal distances (Levin and Kerster 1969b; Diaz-Martin et al. 2023).In the study area, P. alpina is usually the sole owering species in its early owering season, but it co-owers with other y-pollinated species (such as Dryas octopetala and Ranunculus montanus) in the later season (KC, personal observations), such that there is a rapid increase in the density of owers in the community as the season progresses.If the dipteran insects, as generalist pollinators (Inouye et al. 2015), follow an optimal foraging strategy by assessing oral rewards at a community level, this may result in shorter pollen dispersal distance at the late owering season.
In insect-pollinated P. alpina, pollen dispersal distances were positively dependent on oral stalk height.Although a positive correlation between stalk height and pollen dispersal distance has been generally expected and found in wind-pollinated species (Okubo and Levin 1989 , it has, to our knowledge, not been reported for species relying on animals as their pollen dispersal vector.Flowers presented on taller stalks likely attract more pollinators and their pollen may be dispersed further.For instance, syrphid ies, the major pollinators of P. alpina (Chen and Pannell 2022), were found to be more likely to visit taller plants within and among species in grassland habitats (Klecka et al. 2018a, b).
It is not clear why owers with a larger tepal length had a shorter pollen dispersal distance in P. alpina than those with smaller tepals.Given that tepal length showed no correlation with stamen number (Chen and Pannell 2022), it is unlikely that the short dispersal distance is a result of pollinators staying longer in a ower for greater pollen rewards, as predicted by 'optimal foraging theory' (Pyke et al. 1977).
Alternatively, dipteran pollinators may also visit the owers for heat as a reward, which is common in the arctic, temperate, and alpine environments (Hocking and Sharplin 1965;Kudo 1995;Inouye et al. 2015).Indeed, it has been shown that the actinomorphic owers of P. alpina could be around 10 Celsius degrees warmer than the air temperature (Dietrich and Körner 2014).If larger tepals lead to warmer temperatures in the owers, pollinators may forage shorter distances around the patch and thus cause shorter pollen dispersal distances (Pyke et al. 1977).Although the actual mechanisms behind the observed patterns between pollen dispersal distance and different traits in P. alpina remain obscure, our results have nevertheless revealed how intra-speci c variation in oral traits affects an important component of plant mating.
The effects of phenology and morphological traits on pollen dispersal distances in P. alpina imply that these traits may be under selection via male reproductive success, e.g., through enhancing the quality of offspring.It is worth noting that we detected no clear dependence between mean pollen dispersal distance and the number of seeds sired, in contrast with the common assumption of a positive correlation in theory (Okubo and Levin 1989; Fromhage and Kokko 2010) and the common observation of such a correlation for wind-pollinated species (Tonnabel et al. 2019;Zeng et al. 2023).Contrary to windpollinated species in which the pollination likely follows a mass-action mechanism, pollen ow between sires and dams in animal-pollinated plants may be much more complex (Harder 1990   Partial effects of oral traits on pollen dispersal distances of single-owered individuals (N = 297 outcross mating events from 72 sires).Floral traits are all standardized.The mating events are represented by blue dots.The 95% con dence interval of the estimates is shown around the regression lines.
Engler 2007; Thomson et al. 2011; Côrtes and Uriarte 2013; Tamme et al. 2014), but we remain largely ignorant of how pollen dispersal distance varies among dispersal vectors and for different plant trait values, likely due to the di culties of tracking the gene ow by pollen movements.

Table 1
are less than 3 m apart and the high level of inbreeding depression estimated for the species (unpublished data).Summary of studies reporting direct estimates of pollen dispersal distances in alpine plants.References: a. Petrén et al. (2021) b.Buehler et al. (2012) c.Scheepens et al. (2012) d.gisdóttir et al. (2009) e. Thomson and Thomson (1989) f.Pluess and Stöcklin (2004) g.Campbell and Waser (1989) h.Chen and Pannell (2022) i.This study j.Matter, Kettle, Ghazoul and Pluess (2013) k.Matter, Kettle, Ghazoul, Hahn, Krauss et al. 20175;Krauss et al. 2017).It thus seems likely that traits that increase pollen dispersal distances in animalpollinated species may not necessarily be related to increased male reproductive success in terms of numbers of progeny sired, as we have found here (see Chen and Pannell (2023b) for the estimates of selection gradients via siring success).Nonetheless, traits facilitating pollen dispersal may still be favored as a result of enhanced quality of offspring, given the somewhat elevated relatedness of P. alpina individuals that

Table 2
Estimates of parameters for the Weibull distribution pollen dispersal kernel for all the sires (A), singleowered hermaphrodites (B), and single-owered males (C).

Table 3
Partial effects of oral gender and oral traits on pollen dispersal distances in single-owered individuals (N = 297 mating events from 72 sires).Only outcrossing mating events were included in the analysis.