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- Materials and Methods
Gynodioecy is the coexistence of hermaphrodites and females within plant populations (Darwin, 1877; Lewis, 1941). Inbreeding depression, the fitness reduction of inbred relative to outbred individuals (Lande & Schemske, 1985; Charlesworth & Charlesworth, 1987), is the major hypothesis explaining the maintenance of females within gynodioecious populations (see review in Charlesworth, 1999). Comparative data showed that the strength of inbreeding depression varies through a plant's life cycle and that its magnitude is generally high at late stages of the life cycle (i.e. during reproduction), regardless of the mating system (Husband & Schemske, 1996). As for self-fertilizing species, experimental investigations on gynodioecious species with intermediate self-fertilization rates found higher inbreeding depression in late than in early stages of the life cycle (Sakai et al., 1997; Bailey & McCauley, 2006). These high magnitudes of late-acting inbreeding depression are thought to be the consequence of mildly deleterious alleles, which are inefficiently purged through natural selection and can result in high genetic load (Charlesworth et al., 1991; Husband & Schemske, 1996; Willis, 1999a). Despite the importance of late-acting inbreeding depression, only a handful of studies have investigated its magnitude in components of male fitness of hermaphroditic species (Willis, 1993, 1999b; Carr & Dudash, 1997; Chang & Rausher, 1999; Melser et al., 1999) and it therefore deserves more investigation, including in gynodioecious species. For this sexual polymorphism, the cost of inbreeding should affect selfing hermaphrodites most strongly, whereas the impact on outcrossing females should be lower. Hence, high magnitudes of inbreeding depression should maintain the conditions favoring the outbreeding advantage of female plants.
Sex determination of gynodioecious species generally involves epistatic interactions between cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility (Koelewijn & Van Damme, 1995a,b; Charlesworth & Laporte, 1998; Byers et al., 2005). The inheritance of male fertility restoration is generally a consequence of multiple restorer genes that are dominant rather than recessive (Koelewijn & Van Damme, 1995b; Charlesworth & Laporte, 1998). Thus, self-fertilization of hermaphrodites may not only result in offspring with reduced fitness but is also likely to increase the proportion of female progeny if hermaphrodites are heterozygous for dominant restorers. Emery & McCauley (2002) and Bailey & McCauley (2005) investigated introduced North American populations of the gynodioecious Silene vulgaris and detected a 20% increase in the proportion of females in the offspring of self-fertilized compared with cross-fertilized hermaphrodites. Their results were consistent with the action of dominant nuclear restorers. However, the magnitude of the female excess in the progeny of inbred compared with oubred hermaphrodites was larger than expected under random mating, considering a single restorer locus with one dominant allele (Emery & McCauley, 2002). A population composed of RR hermaphrodites should only produce hermaphrodites regardless of the relatedness between mates. Similarly, a population composed of Rr hermaphrodites should produce 25% females on average, whether selfed or outcrossed. Thus, the largest difference in sex ratio between selfed and outcrossed progenies would occur in a population with equal proportions of RR and Rr hermaphrodites. In this case, selfing would result in 12.5% females (0.5 × 0.25) and random mating among hermaphrodites in 6.25% females (0.5 × 0.5 × 0.25). Thus, the relative excess of females in the progeny of selfed compared with outcrossed hermaphrodites should not exceed 6.25% if sex determination is controlled by a single restorer with one dominant allele restoring the male function (Emery & McCauley, 2002; Bailey & McCauley, 2005). A comparison between native and introduced populations of S. vulgaris could identify differences in the genetic structure of sex determination. Owing to the recent colonization history in the introduced range (McCauley et al., 2003), bottlenecks could have led to an overall loss of polymorphism, and sex determination may be different from the source population(s). Furthermore, a joint estimation of the effects of selfing on sex ratio variation and male and female fertility traits would allow greater insights into the complexity of sex allocation in gynodioecious individuals.
Indeed, sex expression is variable and individuals with intermediate sex phenotypes can be found within natural populations of gynodioecious species (Assouad & Valdeyron, 1975; Dulberg & Horovitz, 1984; Shykoff, 1988; Ågren & Wilson, 1991). A partial male-sterile (PMS) individual can grow either a mixture of hermaphroditic and female flowers (i.e. gynomonoecy) or intermediate floral phenotypes displaying variability in the number of functional anthers among flowers (Koelewijn & Van Damme, 1995b, 1996; Delph & Mutikainen, 2003). This continuum of phenotypic sex expression stresses the need for a more quantitative description of gender, as suggested by Lloyd (1980). Sex expression of PMS plants can be affected by the environment (Koelewijn & Van Damme, 1996), although evidence for a genetic basis also exists (Koelewijn & Van Damme, 1995b). Using experimental crosses, Koelewijn & Van Damme (1995b) demonstrated that restoration of male fertility was the result of numerous nuclear loci and that PMS plants were a consequence of incomplete restoration whereas hermaphrodites were completely restored at male fertility loci. Moreover, the frequency of hermaphrodites in a given progeny has been shown to correlate positively with the pollen production of those hermaphrodites, in two gynodioecious species: Thymus vulgaris (Gigord et al., 1999) and Plantago coronopus (Koelewijn, 2003). Thus, if partial male sterility has a genetic basis linked to the expression of restorers, PMS individuals are more likely to occur in the progeny of self-fertilized hermaphrodites (i.e. as a result of increased homozygosity) than in the progeny of cross-fertilized hermaphrodites, and their frequency should correlate positively with the decrease in the proportion of hermaphrodites between inbred and outbred progeny.
In this study we investigate the effects of two consecutive generations of self-fertilized and cross-fertilized hermaphrodites on the qualitative and quantitative sex expression of the gynodioecious S. vulgaris. To assess the effects of inbreeding on female and male fertility as well as on progeny sex ratio, we sampled families of S. vulgaris from three different valleys in the western Swiss Alps, occurring in the native range of the species. Our investigation had four main goals: to determine the intensity of the shift in sex ratio between self- and cross-pollinated plants; to investigate whether partial male sterility is more frequent and intense in the progeny of inbred individuals; to determine the magnitude of inbreeding depression in reproductive traits, separately for female and male fitness; and to explore potential correlations between the magnitudes of inbreeding depression for reproductive traits and relative differences in the proportion of hermaphrodites between pollination treatments.