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- Materials and Methods
Plants are immobile and consequently depend on abiotic and biotic vectors to transport their pollen for sexual reproduction. Under field conditions, however, the pollen grains deposited on female flowers are not always adequate for seed production (Wilcock & Neiland, 2002), such as when self-pollination or pollination from closely related neighbouring plants occurs, which can reduce the performance of the next generation through inbreeding depression (Heywood, 1993; Nason & Ellstrand, 1995; Herlihy & Eckert, 2004). In many angiosperms, pollen selection mechanisms that reduce inbreeding depression and promote outcrossing have evolved. These mechanisms include self-incompatibility, in which only outcross-pollen grains are used for seed production (Seavey & Bawa, 1986; de Nettancourt, 1997), and inbreeding avoidance, in which pollen grains from more distant and nonrelated parents are more likely to sire seeds (Waser & Price, 1993; Souto et al., 2002; Glaettli et al., 2006). In these systems, outcross-pollen grains typically have a much greater reproductive advantage relative to self- or related-pollen grains at pre-zygotic stages, such as pollen germination (de Nettancourt, 1997) or pollen tube growth (cryptic self-incompatibility, Bateman, 1956; inbreeding avoidance, Waser & Price, 1993; Souto et al., 2002; Glaettli et al., 2006). Ovarian inhibition of self-pollen tube or self-embryo abortion (late-acting self-incompatibility; Seavey & Bawa, 1986) and early-acting inbreeding depression also enhance the outcrossing rate. As a result, these traits may cause a difference in pollen donor composition between the pollination stage (i.e. reach stigmas) and seed stage (i.e. sire seeds). In addition, the difference may also be a result of mate quality factors, such as pollen tube competition (Mulcahy, 1979), and the different growth rates of fertilized ovules (Korbecka et al., 2002).
To demonstrate experimentally the effectiveness and evolution of pollen selection mechanisms, it is important to evaluate the extent of self- and related-pollen grain depositions in plant populations exhibiting fine-scale genetic structure. Under natural conditions, individual female flowers may receive a greater proportion of pollen grains from neighbouring plants relative to distant plants. If closely related individual plants (i.e. with fine-scale genetic structure) are distributed in a spatially aggregated manner, most of their seeds are likely to derive from the relatively unfavourable outcross-pollen grains from neighbouring plants. Furthermore, pollen limitation is frequently observed in natural populations (Burd, 1994; Wilcock & Neiland, 2002; Ashman et al., 2004), especially in self-incompatible tree species (Larson & Barrett, 2000) in which, as a result, outcross-pollen grains from related neighbouring plants may compensate for the limitation of more favourable pollen grains from more distant plants.
In this study, we identified individual pollen donors at both the pollination and seed stages using DNA amplification and paternity analysis based on microsatellite genotyping of individual pollen grains and seeds. DNA amplification from a single pollen grain or pollinarium (i.e. a pollen package) has been reported in several recent studies (Petersen et al., 1996; Suyama et al., 1996; Ziegenhagen et al., 1996; Aziz et al., 1999; Matsunaga et al., 1999; Widmer et al., 2000; Cozzolino et al., 2005; Parducci et al., 2005; Matsuki et al., 2007, 2008; Paffetti et al., 2007; Zhou et al., 2007; Aziz & Sauve, 2008; Chen et al., 2008; Ito et al., 2008). Multiplex PCR techniques that amplify some microsatellite regions in a single reaction allow for easy paternity analysis from a single pollen grain (Matsuki et al., 2007, 2008). However, the pollen donor composition at the pollination stage has not been investigated under natural conditions.
Therefore, in this study, we evaluated the process of pollen selection in the early phases of reproduction, and analysed the fine-scale genetic structure using microsatellite genotyping of pollen grains, seeds and potential paternal trees in the self-incompatible monoecious tree species Castanea crenata. We did not investigate inbreeding depression in C. crenata; however, Quercus crispula, belonging to the same family (Fagaceae), exhibits biparental inbreeding depression at both the seed germination and seedling stage (Ubukata et al., 1999). In addition, inbreeding depression is thought to be strong in long-lived perennial tree species (e.g. Koelewijn et al., 1999; Ishida, 2006), probably as a result of the high rate of genomic mutation per generation (Morgan, 2001).
Here, we specifically addressed the following questions:
Does pollen donor composition differ between the pollination and seed stages?
If so, which pollen donors (i.e. self, related or nonrelated individuals) are more likely to sire seeds in a natural population of C. crenata?
Is there genetic structure?
If so, are the outcross-pollen grains of related individuals used for seed production in the self-incompatible tree species C. crenata?