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Propagule movement determines the scale and magnitude of ecological and evolutionary interactions among spatially structured plant populations. Seed dispersal is the vector of demographic connectivity between populations, regulating extinction–colonization processes, source–sink dynamics and species’ coexistence (Nathan & Muller-Landau, 2000); pollen flow defines reproductive assurance, mating patterns and the degree of reproductive isolation between plant demes (Ellstrand, 1992). Both seed and pollen dispersal determine the rate of among-population gene flow, and thus influence genetic population divergence (Wright, 1931), effective metapopulation size, probabilities and times of fixation of new alleles (Whitlock, 2003), and the evolution of local adaptation in heterogeneous environments (Lopez et al., 2008). In the current global warming scenario, the seed dispersal range will place maximum limits on the ability of species to track suitable ecological niches via migration, and both pollen and seed dispersal ranges will establish bounds for the potentially adaptive flow of genotypes from warmer to colder regions (Davis & Shaw, 2001).
Ecological and evolutionary implications of propagule movement, however, depend on the chances of effective pollination and effective seedling establishment, and the scale of potential and effective dispersal need not coincide. For wind-pollinated trees, it has long been known that airborne pollen can flow tens to thousands of kilometres, based on pollen samples collected over the sea, inland beyond the tree line or before local pollen shedding (Sarvas, 1962; Koski, 1970; Nichols et al., 1978; Campbell et al., 1999). It is also recognized that a large proportion of pollen remains viable after such long-distance transport (Lindgren et al., 1995; Varis et al., 2009; Williams, 2010), provided that atmospheric conditions are not very wet or cold (Pulkkinen & Rantio-Lehtimäki, 1995; Bohrerova et al., 2009). Yet, there is a gap between the order of magnitude of maximum documented distances of airborne tree pollen transport (up to 102–103 km) and effective pollination (up to 101 km; see table 2 by Petit & Hampe, 2006; but see Ahmed et al., 2009 for longer distance insect pollination).
An evident discrepancy of this kind is seen for Scots pine (Pinus sylvestris), a monoecious, wind-pollinated, predominantly outcrossing conifer with a wide geographical distribution. Substantial amounts of viable Scots pine pollen have been found to move hundreds of kilometres across northern Europe (Koski, 1970; Lindgren et al., 1995; Varis et al., 2009), and it is presumed that efficient pollen gene flow contributes to the large effective population size of the species (Muona & Harju, 1989). Nonetheless, molecular studies in the species have been unable to identify effective pollen donors beyond a few hundred metres from mother trees (Yazdani et al., 1989; Robledo-Arnuncio & Gil, 2005). Although background pollination from unknown sources into isolated stands is likely to be the result of longer distance dispersal events (e.g. Harju & Muona, 1989; Robledo-Arnuncio & Gil, 2005), experimental constraints have prevented precise measurements of the actual scale of this potentially long-distance pollen immigration process. It is actually unclear whether discrepancies between maximum documented scales of potential and effective pollen dispersal largely derive from obstacles to successful long-distance mating (such as phenological asynchronies, competition with local pollen and genetic incompatibilities) or, rather, from greater difficulties in assessing effective dispersal over broad areas.
The characterization of long-distance pollination is, indeed, a great challenge, as it involves not only the sampling of presumably rare mating events, but also ascertaining, among potentially many distant sources, the origin of the corresponding effective male gametes. In addition, it is not simply the detection of effective long-distance dispersal events that should be targeted, but rather the unbiased estimation of long-distance migration rates, the precise magnitude of which strongly influences the expected adaptive outcome of pollen gene flow (Lenormand, 2002). Genetic parentage analysis provides an efficient means of characterizing effective dispersal (e.g. Oddou-Muratorio et al., 2005; Burczyk et al., 2006), but requires exhaustive genotyping of all potential candidate parents, and thus becomes unfeasible over large scales, unless the population density is extremely low (Ahmed et al., 2009). Genetic assignment methods allow the establishment of the population origin of every individual in a sample without exhaustive genotyping of candidate parents (Manel et al., 2005), but suffer low power when genetic differentiation among source populations is modest, and are not efficient for the estimation of unbiased migration rates (Cornuet et al., 1999; Paetkau et al., 2004). Alternative Bayesian methods use gametic disequilibrium information in population samples of multilocus genotypes to jointly estimate contemporary migration rates between populations, the individual origin of every individual, genotypic frequencies before dispersal and population inbreeding coefficients, assuming either low migration (Wilson & Rannala, 2003) or migration–drift equilibrium (Faubet & Gaggiotti, 2008). The latter general procedures are promising for the estimation of long-distance contemporary dispersal rates in different kinds of organism, although their accuracy and convergence properties can be sensitive to model assumption violations (Faubet et al., 2007; Faubet & Gaggiotti, 2008).
Several inferential advantages inherent in plants allow for a more straightforward approach to contemporary long-distance effective dispersal estimation (see Robledo-Arnuncio et al., 2009). First, seed and pollen dispersal typically take place during discrete synchronized periods, before which gene frequencies of candidate source populations can be estimated independently of migration rates. Second, paternally and maternally inherited DNA markers are available for many species, making possible the estimation of male and/or female gametic migration separately, without requiring Hardy–Weinberg equilibrium assumptions or joint estimation of population inbreeding coefficients. It is then possible to estimate contemporary migration rates independently of other parameters, using an analogue of classical mixture analysis for fish stock identification (Milner et al., 1981; Manel et al., 2005), but based on population haplotypic samples of adults and seeds collected before and after dispersal, respectively (Robledo-Arnuncio et al., 2009; see also Broquet et al., 2009).
Based on a multiple population extension of the maximum likelihood approach used in Robledo-Arnuncio et al. (2009) and intensive Monte Carlo significance testing, this study aims to demonstrate the ability of airborne tree pollen for effective pollination over mesoscale distances. The origin of effective pollen immigrants into a small Iberian Pinus sylvestris L. remnant was investigated, revealing significant effective pollen flow (up to 4.4%) from a large conspecific population located c. 100 km away. Results of this study suggest that the well-known mesoscale airborne transport of viable pine pollen clouds can result in successful pollination.
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The results of this study indicate that airborne tree pollen transport can result in effective pollination over a larger scale than documented to date for wind-pollinated species, as shown by the 6.7% of effective male gamete migrants into a strongly isolated Scots pine remnant. Notably, effective pollen migration from a large population 100 km away from the remnant was estimated at 4.4%. Extensive numerical analysis showed that the latter estimate remains significant even after controlling for small adult reference samples, unequal migration rates and the presence of unsampled populations, all of which were a minimum of 50 km away from the recipient population in the study system. Pollen migration from a more nearby (c. 30 km away), but small, woodland was null (Fig. 1; Table 2), consistent with mass-action models, which predict increasing immigration for larger source to sink population size ratios (Holsinger, 1991; Ellstrand & Elam, 1993). Moreover, the proximity advantage of discrete source populations is expected to diminish under leptokurtic propagule movement (Klein et al., 2006), and the pattern of pollen dispersal has been shown to be markedly fat-tailed in Scots pine (Robledo-Arnuncio & Gil, 2005).
Pollen migration from the largest sampled population (F) appeared, however, to be null, and that from population C was nonsignificant and lower than that from the smaller population E. Two additional factors could explain the variation in migration rates: atmospheric conditions and flowering phenology. Turbulent updrafts within and above the canopy during pollen release, wind conditions during transport, stability during deposition, and other microscale and synoptic atmospheric processes driving long-distance dispersal are highly stochastic (Kuparinen et al., 2007; Nathan et al., 2008), and we lack related information in the study region that might help to approximate the expected migration rate distribution. It seems clear, however, that the sole effects of interpopulation distance and relative population size are unlikely to explain long-distance pollen movement across broad heterogeneous regions. With regard to phenology, it is well known that Scots pine flowering overlaps over large climatic gradients, even over 500–1000-km latitudinal clines with strong temperature differences, but also that earlier pollen is expected to have a competitive advantage (Sarvas, 1962; Pessi & Pulkkinen, 1994; Lindgren et al., 1995; Pulkkinen & Rantio-Lehtimäki, 1995). This advantage could have favoured effective pollen dispersal from population E, which is c. 1400 m in elevation, an intermediate altitude between recipient population A (800 m) and population C, which grows up to the treeline (1500–1800 m) and thus presumably starts to shed pollen later.
This study establishes the possibility of effective wind pollination over mesoscale distances for Scots pine, but the observed distribution of pollen migration rates should not be generalized directly to other species or to different demographic settings. For comparatively larger recipient populations, the dilution effect associated with more abundant local pollen shedding might reduce the proportion of long-distance pollination. For species with pollen grains not as buoyant as those of Pinus (Williams, 2008), as another example, mechanistic models would predict relatively shorter range dispersal. The present findings represent, however, a benchmark for future studies of different biological and demographic systems.
Ecological and evolutionary implications
An order of magnitude increase in the expected range of effective pollen dispersal would have an impact on the assessment of many ecological and evolutionary processes. Strongly geographically isolated tree fragments, either remnants or founders, may be within mating distance of central populations more often than presumed. A yearly pollen immigration rate of c. 5% might translate into an even larger per generation rate, as opportunities for long-distance dispersal accumulate over many decades of tree maturity. Indeed, in the same way that the reproductive assurance brought by longevity is considered to be a potential ultimate cause of the predominantly outcrossed mating system of trees (Ashman et al., 2004; Petit & Hampe, 2006), the large mating neighbourhood granted by long-distance pollination may represent a spatial component of reproductive assurance as important as the temporal one conferred by longevity.
Assuming that all 6.7% of seeds sired by long-distance pollen were able to germinate and establish in recipient population A, and that this yearly pollen migration rate translated into an equal per generation rate, without seed immigration, the resulting gene flow rate for autosomal loci would be m = 0.067/2 = 0.033, yielding Nm = 36 × 0.033 = 1.2 migrants per generation, further assuming equal effective and census sizes. Under an island migration model (Wright, 1931), this estimate suggests that long-distance pollen flow would largely counterbalance the strong rate of drift-induced neutral genetic differentiation expected in such a small population. The consequences for local adaptation are more uncertain, as long-distance pollen movement across heterogeneous habitats may bring in gametes adapted to very different environments. It could therefore counteract divergent natural selection, hampering local adaptation and increasing genetic load (Garcia-Ramos & Kirkpatrick, 1997; Lenormand, 2002). For a given gene flow rate, selection is in fact expected to oppose maladaptive effects of pollen dispersal less efficiently than those of seed dispersal in strongly heterogeneous habitats (Lopez et al., 2008). However, long-distance pollen gene flow is also expected to increase additive genetic variance within tree populations (e.g. Yeaman & Jarvis, 2006 for Pinus), providing the necessary basis for adaptive divergence (Lenormand, 2002), especially in small populations (Alleaume-Benharira et al., 2006).
Predicting the net effect of long-distance pollen migration on adaptation is thus difficult. A critical question for the Scots pine remnant in its dry environment will be whether, in the face of climate warming, selection will overcome the gametic inflow from mountain populations that are locally adapted to colder and wetter conditions (Alía et al., 2001). Although long-distance pollen immigration might have an ultimately maladaptive effect in this southern relict, it might not be the case in populations such as northern woodlands distributed along steep latitudinal climatic gradients. South to north pollen flow has been considered to hamper local adaptation through frost tolerance disruption in northern Finnish Scots pine populations (Aho, 1994; cited in Savolainen et al., 2007). This situation might be reversed soon, however, as previously maladaptive southern gene flow could favour local adaptation of northern populations to climate warming (Davis & Shaw, 2001), presumably in a more pervasive and rapid way than migration via seed dispersal.
Two key experimental advantages in this study were the small size of the recipient stand, which allowed an accurate determination of total pollen immigration, and its strong geographical isolation from peripheral discrete populations, which facilitated the estimation of immigrant proportions from long-distance sources. The approach used can only be applied to discrete populations, relying on a good characterization of their haplotypic frequencies before dispersal at paternally inherited loci. The sampling intensity required to achieve the latter condition is a function of marker polymorphism. Scots pine chloroplast SSR haplotypes have moderate levels of variation, yielding acceptable estimation error for relatively small sample sizes, but the bias and variance may increase, and the CI coverage decrease, for higher or lower haplotypic diversity levels, especially with low levels of differentiation between populations and/or larger recipient populations (JJ Robledo-Arnuncio, unpublished data).
Given the spatial scale involved in long-distance dispersal studies, additional concerns in abundant species are unsampled populations, which will result in overestimates of migration from sampled sources (see Slatkin, 2005 in the context of historical migration), in turn yielding overestimates of dispersal range if missing sources are closer to the recipient population than sampled sources, or underestimates otherwise. Researchers should consider the minimum estimated dispersal distance with caution when unsampled sources are likely to occur within this distance, and should be advised to conduct significance tests under the assumption of incoming pollen from missing sources. If the amount of migrants is large, which will rarely be the case in long-distance plant dispersal, it might be possible to recover genotypic frequency information about unsampled sources from the seed sample, as in fish stock identification problems (Smouse et al., 1990). However, even when all individuals in a large sample are migrants (as in fish stocks), the presence of entire unsampled populations seems difficult to overcome (Smouse et al., 1990).
Finally, contemporary seed migration estimation could be undertaken in a similar way, using either maternally inherited DNA markers or biparentally inherited markers, such as nuclear microsatellites, assessed at maternal origin seed tissue (Godoy & Jordano, 2001). In the latter case, the potentially enormous number of different genotypes would enforce intensive population sampling, and Bayesian procedures jointly estimating genotypic distributions could help in tackling the problem (Pella & Masuda, 2001). A drawback of time-consuming Bayesian computations, however, is the difficulty in conducting sufficiently replicated numerical uncertainty analysis of low migration estimates. In general, if no polymorphic uniparentally inherited markers were available, or maternal origin seed tissue for analysis dealing with seed dispersal, less specific methods for multilocus diploid markers would become necessary to estimate contemporary migration rates, at the cost of additional assumptions and higher parameter dimensionality (Wilson & Rannala, 2003; Faubet & Gaggiotti, 2008; Broquet et al., 2009).
The apparent gap between the scale of airborne propagule transport and effective dispersal might be bound to disappear for many plant species as more comprehensive datasets and ad hoc statistical methods become available to unravel previously cryptic long-distance ecological processes. The present study supports the view that this gap may not reflect a biological reality for Scots pine, extending the scale of documented effective pollen dispersal range for wind-pollinated trees.