Embryonic inbreeding depression varies among populations and by mating system in Witheringia solanacea (Solanaceae)

The transition from genetically enforced self-incompatibility (SI) to a selfing mode of life has been traversed by many plant lineages, perhaps more often than any other evolutionary transition in flowering plants (Stebbins, 1974; Igic et al., 2008). Lineages that make this transition from SI to selfing must cope with initially high inbreeding depression. Early-acting inbreeding depression, that is, inbreeding depression operating during embryogenesis, seed maturation, and germination, is typically much higher in predominantly outcrossing than in predominantly selfing species, whereas late-acting inbreeding depression is similar for species with different breeding systems (Husband and Schemske, 1996). The increased homozygosity caused by selfing can eliminate or purge recessive lethals, reducing inbreeding depression and thereby permitting the spread of selfing variants (Lande and Schemske, 1985; Husband and Schemske, 1996; Crnokrak and Barrett, 2002; but see Byers and Waller, 1999). The ability to purge early-acting inbreeding depression is evidently key in the evolution of selfing from out-crossing (Glemin et al., 2001; Porcher and Lande, 2005), and estimates of its magnitude in SI populations are therefore of interest. For self-compatible (SC) species, early-acting inbreeding depression can be estimated simply by comparing seed production resulting from self- and outcross-pollinations. This method cannot be used for species with intact SI systems. Selfed seed can be produced by bud pollinations or other methods used to circumvent SI (de Nettancourt, 2000), but the cause of reduced seed set in these cases is due to an unknown combination of residual SI and embryonic inbreeding depression (e.g., Karkkainen et al., 1999). Inbreeding depression can be estimated by comparing inbreeding coefficients of adults to juveniles, but this method is recommended only for species with selfing rates of at least 10% (Ritland, 1990). Another approach to estimating inbreeding depression of SI species is to construct outcross matings among relatives (e.g., Vogler et al., 1999). This method has the virtue of permitting a regression of genetic load over inbreeding coefficient; however, it is infrequently used, perhaps due to labor and space requirements. A pollen chase experiment can be used to estimate the extent of embryonic inbreeding depression for some species with suspected or confirmed SI systems (Krebs and Hancock, 1990; Barrett, 2002). In a pollen chase experiment, self-pollen is applied in the bud, with outcross pollen being applied after anthesis (the SO treatment). As a control, outcross pollen is applied on a paired flower, both in the bud and after anthesis (the OO treatment). To the extent that self-pollen is successful in fertilization, but generates progeny of lowered embryonic fitness, seed set will be lowered in fruits with self-pollen precedence. The reduction in fruit and seed production for SO as opposed to OO treatments can be used as an estimate of lethal inbreeding depression acting at the embryonic stage. If the SI system involves degradation of self-pollen tubes in the style, fluorescence microscopy can be used to observe stylar pollen growth to document the success of bud pollinations in overcoming the SI response. Such data can be used to correct potential errors of interpretation that arise when partial SI persists in the bud (Fig. 1). Fig. 1 Unobserved residual self-incompatibility (SI) deflates estimates of embryonic inbreeding depression. Filled circles represent out-crossed ovules and empty circles represent selfed ovules. A line through the circle represents embryonic mortality. In both ... Estimates of inbreeding depression in species where variation in SI has been documented can test whether variation in SI corresponds with variation in inbreeding depression (e.g., Vogler et al., 1999; Busch, 2005). We have found both qualitative and quantitative variation in the SI response of Witherin-gia solanacea L’Her, a tropical shrub occurring in Central America. Most individuals are SI, but autogamous SC variants have been found in two very small isolated Costa Rican populations (Stone et al., 2006). An undescribed SC variant occurs sympatrically with a large SI population in the Monteverde region of Costa Rica and in at least one small isolated population farther west (Stone and Jenkins, 2008). In addition, two large SI populations differ quantitatively in the strength of their SI response, with the Monteverde population producing lower levels of stylar RNase and permitting more self-pollen tubes to grow to the base of the style in bud pollinations than the Las Cruces population (Stone et al., 2006). To gain insight into the mating system evolution of this species, we used a pollen chase experiment to compare the level of early-acting inbreeding depression for plants that varied in SI response. We compared fruit and seed set for SO vs. OO pollinations for SC plants and for plants from the Las Cruces and Monteverde populations that differ quantitatively in the strength of their SI response. For SI plants, we compared germination rates for seeds resulting from the two treatments. We also genotyped seedlings at five microsatellite loci to estimate selfing rate of surviving progeny, which we used in concert with previously published pollen tube data to estimate the relative fitness of selfed and outcrossed embryos. We hypothesized that selfing lineages would show decreased inbreeding depression due to purging of genetic load. We also hypothesized that the two SI populations might show differences in inbreeding depression corresponding to quantitative variation in the SI response. If the less robust SI system in Monteverde has led to a history of selfing, that population might show lower inbreeding depression due to partial purging of genetic load.