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- Materials and methods
- Supporting Information
An evolutionary transition from a reproductive system involving mating among unrelated individuals (outcrossing) to predominant self-fertilization has occurred thousands of times in plants. The most widely held hypothesis for why selfing evolves is that it provides reproductive assurance (RA) when pollinators and/or mates are scarce (Eckert, Samis & Dart 2006; Cheptou & Schoen 2007; Harder & Aizen 2010). The RA hypothesis can be tested experimentally by manipulating the capacity of flowers for self-pollination, usually by emasculation (anther removal) and then measuring the effect on seed production and the mating system (Schoen & Lloyd 1992; Schoen, Morgan & Bataillon 1996). The RA hypothesis is supported if seed production (F) and the proportion of seeds self-fertilized (s) are higher for intact flowers (I) capable of self-pollination than emasculated flowers (E) no longer capable of self-pollination (Eckert et al. 2010). Almost all of what we know about the selective benefits of RA in natural plant populations has come from these emasculation experiments, which reveal that selfing often provides RA in nature (Eckert, Samis & Dart 2006).
Emasculations can be performed at different stages of floral development to estimate the timing of self-pollination, which may strongly influence the costs and benefits of selfing (Lloyd 1992; Schoen & Lloyd 1992; Schoen, Morgan & Bataillon 1996). For instance, selfing that occurs at the end of floral life, after opportunities for outcrossing have passed (‘delayed selfing’), may provide RA without compromising the production of high viability outcrossed seed or outcross siring success through pollen export. In contrast, selfing occurring before opportunities for outcrossing (‘prior selfing’) or simultaneously with outcrossing (‘competing selfing’) may also increase total seed production (i.e. provide RA) but at the cost of reducing the number of outcrossed seeds produced (seed discounting) or seed sired on other plants (pollen discounting). However, few studies have varied the timing of emasculation to estimate the timing of selfing (Leclerc-Potvin & Ritland 1994; Griffin, Mavraganis & Eckert 2000; Zhang & Li 2008).
Using floral emasculation to estimate RA (i.e. RA = FI − FE) is based on the critical assumption that removing anthers from flowers only reduces the capacity of flowers for within-flower (autogamous) self-pollination. However, emasculation may also reduce seed production if it damages flowers, thereby reducing their capacity to mature seeds. It may also reduce seed production by diminishing opportunities for outcrossing by shortening floral life span via a wound-induced hastening of floral senescence (O'Neill 1997). The absence of anthers may also reduce visitation by pollinators if they avoid emasculated flowers because they seek pollen as a reward and/or because anthers are involved in pollinator attraction (Griffin, Mavraganis & Eckert 2000; Eckert & Herlihy 2004). Violations of this critical assumption overestimate RA but can be detected by comparing I vs. E flowers in terms of seed production after hand-pollination, floral life span and pollinator visitation. The later might be problematic because visitation rates are likely to be low under ecological conditions that might select for RA. However, the effect of emasculation on outcross-pollination can be inferred by comparing the proportion of seeds outcrossed (t) vs. selfed (s, s + t = 1) estimated using genetic markers. The production of more outcrossed seeds by I flowers than E flowers (tIFI > tEFE) indicates that emasculation compromises opportunities for outcrossing. Few experimental studies that have used emasculation have addressed these assumptions (see Table S1, Supporting information).
In this study, we emasculate flowers to manipulate self-pollination and its timing, and then measure the effects on seed production and the mating system in Camissoniopsis cheiranthifolia (Onagraceae, Fig. 1). In doing so, we test for the potential side effects of emasculation that may complicate interpretation of experimental results. This species is a short-lived, herbaceous endemic of Pacific coastal dunes of western North America, from southern Baja California Mexico, through California to southern Oregon USA, and exhibits striking co-variation between floral morphology and the mating system across its geographic range (Raven 1969; Dart et al. 2012). Plants from populations in San Diego County California produce large, self-incompatible (SI) flowers (large-flowered, self-incompatible = LF-SI) that are primarily outcrossing (mean t = 0·80, range = 0·62–0·99, n = 3 populations, Dart et al. 2012). Further north along the mainland coast to Point Conception in northern Santa Barbara County California, plants produce large flowers that are fully self-compatible (LF-SC) and engage in a variable mixture of outcrossing and selfing (mean t = 0·74, range = 0·47–0·96, n = 9). North of Point Conception to the range limit in southern Oregon, in Baja California and on the Channel Islands, plants produce small, self-compatible flowers (SF-SC) that are predominantly self-fertilizing (mean t = 0·24, range = 0·001–0·57, n = 10).
Figure 1. Flower of Camissoniopsis cheiranthifolia (Onagraceae). This flower is on a plant raised in the glasshouse from seed collected from a large-flowered population. Small flowers are very similar in morphology but half the diameter with little spatial separation between the stigma and dehiscing anthers. Emasculation involved removing the anthers but leaving the filaments.
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Species like C. cheiranthifolia with wide mating system variation provide excellent opportunities to test predictions concerning the fitness costs and benefits of selfing, especially with respect to the RA hypothesis. Yet these species are underexploited in this regard (but see Elle & Carney 2003; Kennedy & Elle 2008). There seems to be at least two distinct mating system transitions in C. cheiranthifolia among populations along the mainland coast of California (Raven 1969). There is a transition from predominant outcrossing promoted by large SI flowers (LF-SI) in San Diego County to large but self-compatible flowers (LF-SC) and mixed mating (outcrossing + selfing) in somewhat more northerly populations. Theory suggests that the loss of SI but the retention of large flowers is likely via selection for RA (Porcher & Lande 2005). Although LF-SC flowers are self-compatible, dehiscing anthers are held well away from receptive stigmas (mean = 3·27 mm of herkogamy) throughout floral anthesis, which likely limits self-pollination before and during opportunities for outcross-pollination. However, flowers in these populations typically open for two consecutive days, and anthers and stigmas often come into contact when flowers close for the evening at the end of each day. The extent to which this occurs correlates positively with the proportion of seeds selfed (Dart et al. 2012). This potential mechanism of delayed selfing provides RA, while possibly limiting the costs of seed and pollen discounting (Lloyd 1992).
Populations further north along the California coast (north of Point Conception) have progressed further along the transition to higher s. Linsley et al. (1973) report that flowers of C. cheiranthifolia are primarily visited by pollen-collecting females and nectar-collecting males of several oligolectic species of Andrenid bees but that visitation to flowers in SF-SC populations is infrequent largely because heavy morning fog characteristic of this region impedes pollinator visitation. Hence, higher levels of selfing may have evolved to provide RA. SF-SC flowers seem to differ from LF-SC flowers in the timing of self-pollination. SF-SC flowers bear stigmas and anthers in close proximity throughout floral life such that self-pollen, whether it is shed on to stigmas autonomously or through the actions of visiting pollinators, likely competes with outcross pollen for fertilizations (competing selfing, sensu Lloyd 1992). SF-SC flowers also experience autogamous pollination before flowers open (prior selfing) because anthers dehisce shed pollen especially on the underside of the globular stigma while still in bud. This temporal shift in self-pollination to earlier in floral life may be a response to chronic outcross pollen limitation in dune habitats north of Point Conception (Lloyd 1992; Eckert et al. 2010).
We discovered two populations of C. cheiranthifolia in the transition zone just north of Point Conception that contain both LF-SC and SF-SC phenotypes that differ in floral traits and genetic estimates of s to about the same extent as LF-SC and SF-SC phenotypes in segregated populations (Dart et al. 2012). This within-population variation allowed us to use emasculations to investigate differences in the timing and fitness consequences of selfing when divergent phenotypes experience the same pollination environment.
This study compares LF-SC (hereafter LF) populations south of Point Conception to SF-SC (hereafter SF) populations to the north to address the following questions: (i) Is prior selfing that occurs before flowers open more prevalent in SF than LF populations due to self-pollination in the bud? (ii) Does autogamy occurring after flowers open provide RA by increasing seed production and s, and are these effects greater in LF populations where autogamy occurs after flowers open than in SF populations where self-pollination is expected to occur before flowers open? (iii) Does selfing occurring when flowers close increase seed production, and if so, are these effects greater in LF than SF populations? (iv) Do the differences between LF and SF populations in the timing and fitness consequences of selfing also occur between LF and SF phenotypes within populations? Addressing this question will indicate the extent to which differences are due to floral morphology and development as opposed to variation in the pollination environment and/or other extrinsic ecological factors that may differ between pure LF and SF populations. (v) Are there unintended side effects of emasculation that could complicate the interpretation of the results? In particular, we determine whether emasculation damages flowers and reduces their capacity to produce seed and reduces floral longevity and thus opportunities for outcrossing. We also use genetic estimates of self-fertilization and outcrossing to infer whether emasculation decreases cross-pollination through reduced pollinator visitation.