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- Materials and methods
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Theory predicts that inbreeding depression (ID) should decline via purging in self-fertilizing populations. Yet, intraspecific comparisons between selfing and outcrossing populations are few and provide only mixed support for this key evolutionary process. We estimated ID for large-flowered (LF), predominantly outcrossing vs. small-flowered (SF), predominantly selfing populations of the dune endemic Camissoniopsis cheiranthifolia by comparing selfed and crossed progeny in glasshouse environments differing in soil moisture, and by comparing allozyme-based estimates of the proportion of seeds selfed and inbreeding coefficient of mature plants. Based on lifetime measures of dry mass and flower production, ID was stronger in nine LF populations [mean δ = 1−(fitness of selfed seed/fitness of outcrossed seed) = 0.39] than 16 SF populations (mean δ = 0.03). However, predispersal ID during seed maturation was not stronger for LF populations, and ID was not more pronounced under simulated drought, a pervasive stress in sand dune habitat. Genetic estimates of δ were also higher for four LF (δ = 1.23) than five SF (δ = 0.66) populations; however, broad confidence intervals around these estimates overlapped. These results are consistent with purging, but selective interference among loci may be required to maintain strong ID in partially selfing LF populations, and trade-offs between selfed and outcrossed fitness are likely required to maintain outcrossing in SF populations.
- Top of page
- Materials and methods
- Supporting Information
Mating system evolution depends on a dynamic interplay between ecological and genetic factors. Whether self-fertilization is favoured over outcrossing will depend on aspects of pollination ecology that determine the prospects of gaining male and female fitness through self- vs. outcross-pollination (Lloyd, 1992), as well as the extent to which the expression of genetic load reduces the fitness of progeny produced through self- compared with outcross-fertilization (inbreeding depression, Charlesworth & Charlesworth, 1987). Self-fertilization is potentially advantageous because it increases the maternal plant's genetic representation in its offspring and because it may allow offspring production when pollinators and/or mates are scarce (reproductive assurance, Eckert et al., 2006). Given that these advantages can be substantial, a strong selective force is likely required to prevent the spread of selfing. Hence, inbreeding depression (hereafter ID), because it is ubiquitous and usually quite strong, is generally viewed as the major selective factor maintaining outcrossing. However, ID is not a static selective force because it is expected to covary with selfing (Lande & Schemske, 1985).
Inbreeding depression is primarily caused by the expression of at least partially recessive deleterious mutations in the highly homozygous progeny produced by selfing (Charlesworth & Charlesworth, 1987; Charl-esworth & Willis, 2009). As homozygosity increases through selfing recessive mutations should be exposed to selection and ‘purged’ from populations, thereby reducing the magnitude of ID. Early theoretical work modelling joint evolution of the mating system and ID predicted that purging should destabilize mixed mating systems thereby yielding two evolutionarily stable end points: predominant selfing associated with weak ID and predominant outcrossing associated with strong ID (Lande & Schemske, 1985; Charlesworth et al., 1990). However, individuals engage in substantial amounts of both selfing and outcrossing (mixed mating) in > 40% of plant species (Goodwillie et al., 2005). Moreover, evidence for purging derived from experimental studies comparing the performance of self-fertilized vs. outcrossed progeny is mixed. Husband & Schemske (1996) demonstrated the expected negative correlation between ID and the proportion of seeds self-fertilized (s) among plant species, although the relation was not strong (r2 = 0.18 among all 44 species; r2 = 0.14 among 35 angiosperms species). Byers & Waller (1999) reviewed 52 studies that compared ID between species, populations and/or lineages that differed in inbreeding history and concluded that purging is inconsistent and rarely efficient enough to reduce ID. A recent comprehensive meta-analysis of experimental results from 100 populations of 58 species (Winn et al., 2011) found only a weak and nonsignificant negative correlation between s and ID measured across the life cycle (r2 = 0.032, n = 56 populations). Moreover, ID measured as δ = 1−(ωs/ωx), where ωs and ωx represent mean fitness of selfed and outcrossed progeny, respectively, was just as strong in populations that engaged in mixed mating (0.2 < s < 0.8, mean δ = 0.58) as in predominantly outcrossing populations (s < 0.2, mean δ = 0.54). Only populations that engaged in very high levels of selfing (s > 0.8) exhibited relatively weak ID (mean δ = 0.26).
In addition to experimental comparisons of selfed vs. outcrossed progeny, ID can be inferred from the relation between s and the inbreeding coefficient (F) of reproductively mature plants estimated using genetic markers (Ritland, 1990). In the absence of ID (δ = 0), F at equilibrium should be a simple function of s [Fe = s/(2−s)]. However, data for a large sample of plant species show that, in general, F << Fe, indicating strong ID even for species that exhibit high s (Goodwillie et al., 2005; Scofield & Schultz, 2006). Here again, evidence for purging is weak, especially for large and/or long-lived species (Scofield & Schultz, 2006). There also appears to be a high threshold level of selfing (s ~ 0.80) below which ID is not effectively purged (Lande et al., 1994; Scofield & Schultz, 2006).
The strength of ID acting at different stages of the life cycle should coevolve with the mating system. Strongly, deleterious mutation can build up in outcrossing populations if they are highly recessive, and these mutations, when made homozygous by inbreeding, are expected to cause marked declines in fitness (lethal or nearly so) very early in the life cycle. Hence, they are readily purged from partially selfing populations. In contrast, mildly deleterious mutations can build up even if only partly recessive, may often cause inbreeding depression expressed later in the life cycle, and remain recalcitrant to purging in the face of chronic selfing (Lande & Schemske, 1985; Charlesworth & Charlesworth, 1987; Charlesworth et al., 1990; Lande et al., 1994). Several theoretical models have shown that ID being expressed more strongly ‘predispersal’ (during seed maturation) than ‘post-dispersal’ (from germination through reproduction) might set the stage for the evolutionary maintenance of mixed mating (Porcher & Lande, 2005a; Harder & Routley, 2006; Aizen & Harder, 2007; Harder et al., 2008). However, empirical support for this assumed contrast between pre- and post-dispersal ID is mixed. Husband & Schemske (1996) found that, as predicted, predispersal ID was strong for outcrossing populations and weak for selfing populations, whereas inbreeding depression expressed later in the life cycle was wither relatively weak (germination and juvenile survival) or did not differ between outcrossing and selfing populations (growth/reproduction). In contrast, a more recent meta-analysis of data from a larger number of species (Winn et al., 2011) found that the difference in predispersal ID between outcrossing and selfing taxa was the result of extremely strong ID for seed set among gymnosperms. Predispersal ID did not covary with the mating system among angiosperms.
Broad interspecific surveys have failed to consistently support some of the fundamental predictions concerning how ID should coevolve with the mating system. More direct tests of theory are provided by species that exhibit wide variation in s among populations (e.g. Busch, 2005). In these species, variation in the mating system is less likely to be confounded with differences in life history and long-term demography that might also affect the magnitude of ID independently of the mating system (Bataillon & Kirkpatrick, 2000; Scofield & Schultz, 2006). However, taking together the relatively small sample of species (n = 9) for which both ID and s have been directly estimated for more than one population does not support purging (Winn et al., 2011). ID correlates negatively with s for some species (Clarkia tembloriensis – Holtsford & Ellstrand, 1990; Eichhornia paniculata – Toppings, 1989; Barrett & Husband, 1990; Leptosiphon jepsonii – Goodwillie, 2000; Goodwillie & Knight, 2006) but not others (Lupinus perennis – Shi, 2004; Michaels et al., 2008; Salvia pratensis – van Treuren et al., 1993; Ouborg & van Treuren, 1994). However, firm conclusions are not possible because relatively few studies have estimated these parameters for a large sample of populations that vary widely in the mating system. Even fewer studies have inferred ID from the relation between F and s among populations, and the scant results do not suggest reduced ID in chronically selfing populations (Eckert & Herlihy, 2004).
In this study, we quantify variation in the magnitude and timing of ID among populations of Camissoniopsis cheiranthifolia (Hornemann ex Sprengel) W.L. Wagner & Hoch (Onagraceae), a near-annual species that exhibits broad variation in the mating system across its geographical range along the Pacific coastal dunes of western North America (Raven, 1969; Samis & Eckert, 2007; Dart et al., 2012). Based on previous analyses of floral variation and outcrossing estimated using genetic markers, populations can be organized into three groups: (1) large-flowered, gametophytically self-incompatible populations that occur exclusively in San Diego County, California (LF-SI, mean corolla width = 29.6 mm, mean proportion of seeds self-fertilized (s) = 0.20, range = 0.01–0.38); (2) large-flowered, self-compatible populations that occur north of San Diego County to Point Conception in northern Santa Barbara County, California (LF-SC, mean corolla width = 27.7 mm, mean s = 0.26, range = 0.04–0.53); and (3) small-flowered, self-compatible populations (SF-SC, mean corolla width = 15.6 mm, mean s = 0.86, range = 0.43–0.99) that occur north of Point Conception to the northern range limit in Coos Bay Oregon, on the Channel Islands, and in Baja California. Based on results from common-garden experiments in the glasshouse and field observations over several years, there is no appreciable variation in life history or population dynamics associated with striking differentiation in the mating system (C.G. Eckert & S. Dart, unpublished).
We compare the fitness of selfed and outcrossed progeny produced by hand pollination for a large sample of populations to test the predictions that small-flowered (SF), predominantly selfing populations should exhibit weaker ID than large-flowered (LF), predominantly outcrossing populations. We also test the prediction that the difference in ID between LF and SF populations should be most pronounced early in the life cycle during seed maturation (predispersal) than later in life after seed dispersal because this is a key assumption in several theoretical models of mating system evolution.
In addition, we compare the performance of selfed vs. outcrossed progeny in two glasshouse environments that differ strongly in soil moisture. It is generally expected that ID will be expressed more strongly in more challenging environments, and the dependence of ID on the environment might significantly affect the outcome of mating system evolution (Lloyd, 1980; Cheptou & Donohue, 2011). However, this prediction has received only mixed support (Armbruster & Reed, 2005; Willi et al., 2007; Waller et al., 2008; Fox & Reed, 2011). As is typical of the Mediterranean biomes, there is strong seasonal variation in precipitation across the geographical range of C. cheiranthifolia, and the species exhibits classic adaptations to seasonal drought (e.g. deep tap roots, densely pubescent leaves). Rainfall occurs from November to March, followed by a prolonged dry season during which soil moisture in the dunes can rarely be detected with standard instruments and drought stress appears to cause significant mortality and reduced growth of C. cheiranthifolia (S. Dart & C.G. Eckert, personal observation). We tested whether simulated drought enhances the expression of ID because geographical variation in drought may influence the evolution of the mating system in this species. The duration and intensity of drought increases from north to south along the Pacific coast, such that a concomitant increase in ID could favour the maintenance of large flowers and predominant outcrossing in southern California while selfing may be more likely to evolve in populations occurring in moister habitat north of Point Conception and on the Channel Islands.
Finally, we complement our experimental analysis of ID by comparing s to the inbreeding coefficient (F) of mature plants within and among populations to estimate ID following Ritland (1990). Population-genetic estimates of ID reflect processes that occur across much of the life cycle under natural conditions, yet very few studies have compared population-genetic estimates with those obtained experimentally (Eckert & Barrett, 1994; Kohn & Biardi, 1995).