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1. Unreliable pollinator service is thought to promote the evolution of self-compatible plant breeding systems, because selfing may provide reproductive assurance when outcrossing opportunity is limited. The recurrent evolution of self-compatible homostyly from obligately outcrossing heterostylous species has been regarded as a classic example of evolutionary response to lack of pollinators or mates, as homostyly frequently occurs in pollinator-limited or marginal environments. However, male and female sexual organs of homostylous species may display spatial separation (herkogamy), an arrangement presumed to promote outcrossing. It is largely unknown to what extent variation in herkogamy affects opportunities for autonomous selfing and reproductive assurance in self-compatible, homostylous species.
2. Using the homostylous Primula halleri, restricted to alpine environments, we investigated whether herkogamy occurs and varies during anthesis, among individuals, and populations, and compared the effects of herkogamy on seed set among three experimental treatments, to elucidate how herkogamy affects reproductive strategies in a homostylous species.
3. Herkogamy decreases during anthesis, but the ultimate expression of herkogamy in mature flowers differs among individuals and populations. Caging caging experiments indicate that herkogamy reduces a plant's potential for autonomous selfing, and emasculation and open-pollination treatments demonstrate that herkogamy markedly decreases total seed set and the potential for reproductive assurance.
4. Herkogamy early in anthesis may enhance outcrossing potential, while its decrease later could enable reproductive assurance via delayed autonomous selfing in some, but not all plants. Conversely, pronounced herkogamy in older flowers comes at the cost of reduced total reproductive output and imposes pollinator dependence for reproduction, but may promote the genetic diversity of populations.
5. Our study suggests that even small amounts of herkogamy can have large effects on the reproductive strategy of homostylous species, by enabling more outcrossing than generally thought to be typical of homostyly.
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Scarcity of pollinator services has major consequences for plant reproduction and evolutionary processes, as recognized by early naturalists (e.g. Müller 1881; Schröter 1926). The pollinator fauna of alpine (i.e. above tree line) environments is generally depauperate, in terms of number of species and individuals, as compared to that of lower altitudes (e.g. Arroyo, Primack & Armesto 1982; Warren, Harper & Booth 1988). Moreover, the short flowering season and fluctuating weather conditions typical of alpine ecosystems further impair the reliability of pollinator services (Totland 1994; Bergman, Molau & Holmgren 1996; Körner 2003). Similar trends in pollination conditions occur with increasing latitude towards the poles (Hocking 1968; Kevan 1972).
When lack of pollinators limits outcrossing opportunity and reproductive output (García-Camacho & Totland 2009), autonomous selfing (i.e. autogamy unaided by pollinators) may boost total seed production (i.e. reproductive assurance; Eckert, Samis & Dart 2006). Similarly, autonomous selfing may be beneficial when mates are scarce, as during colonization processes (Baker 1955), and in geographically or ecologically marginal habitats (Lloyd 1980). Reproductive assurance has often been invoked as an explanation for the evolution of selfing from primarily outcrossing ancestors (Darwin 1876; Fausto, Eckhart & Geber 2001; Kalisz, Vogler & Hanley 2004; Eckert, Samis & Dart 2006; Moeller 2006), widely recognized as one of the most frequent evolutionary transitions in flowering plants (Stebbins 1950; Grant 1981). Despite the ecological and evolutionary importance of reproductive assurance, experimental demonstrations remain scarce (reviewed by Eckert, Samis & Dart 2006).
Selfing is often associated with negative fitness effects because of reduced survival and fertility of the offspring (inbreeding depression; Charlesworth & Charlesworth 1987; Charlesworth & Willis 2009), as well as with long-term negative consequences on the genetic variability and viability of populations, potentially representing an evolutionary dead end (reviewed by Takebayashi & Morrell 2001). Therefore, autogamy may decrease fitness when ovules and pollen that could otherwise be outcrossed are self-fertilized (i.e. gamete discounting; Herlihy & Eckert 2002; Eckert & Herlihy 2004). Discounting costs can be incurred when autonomous selfing takes place prior to or competing with outcrossing (Lloyd 1992), or when pollinators mediate selfing concurrently with outcrossing by foraging within flowers (facilitated selfing) or between flowers of the same plant (geitonogamy; Vaughton & Ramsey 2010). However, if autonomous selfing occurs at the end of floral life after opportunities for outcrossing have been exhausted (i.e. delayed autonomous selfing; Lloyd 1992), it affords the benefits of autogamy, while avoiding discounting costs, a ‘best-of-both-worlds’ scenario that seems ideally adaptive in alpine/arctic habitats (Kalisz & Vogler 2003; Moeller 2006; Duan et al. 2010; Vaughton & Ramsey 2010). The relative timing and mode of selfing and outcrossing events may thus be important for overall reproductive fitness and long-term evolutionary survival (Lloyd 1992; Eckert, Samis & Dart 2006; Vaughton & Ramsey 2010).
Plants have evolved a wide range of floral traits that may influence the dynamics of sexual reproduction, providing the morphological and physiological basis of plant reproductive strategies. The study of the function and loss of complex floral polymorphisms has supplied key model systems for understanding the evolution of selfing (Darwin 1877; Barrett 2003, 2010). A prime example is the evolution of self-compatible homostylous species from obligately outcrossing heterostylous species (Ganders 1979; Barrett 1992; Barrett & Shore 2008; Cohen 2010). Heterostyly is thought to promote cross-pollination and reduce selfing and sexual interference, via the reciprocal placement of male and female organs in different floral morphs and an incompatibility system that prevents pollen germination within the same flower or floral morph (Darwin 1877; Charlesworth & Charlesworth 1979; Ganders 1979; Barrett 1992; Barrett, Jesson & Baker 2000; Barrett 2002; Barrett & Shore 2008; Cohen 2010). Therefore, heterostylous flowers depend on pollinators and mates for sexual reproduction. Homostylous species evolved multiple times independently from heterostylous ancestors in many of the c. 28 plant families with heterostyly (e.g. in Amsinckia, Boraginaceae, Schoen et al. 1997; Houstonia, Rubiaceae, Church 2003; Narcissus, Amaryllidaceae, Graham & Barrett 2004; Pontederiaceae, Kohn et al. 1996; Primula, Primulaceae, Mast, Kelso & Conti 2006; Turnera, Turneraceae, Truyens, Arbo & Shore 2005). Homostylous species have only one floral morph (i.e. monomorphic) and are self-compatible; hence self-fertilization is possible (reviewed by Ernst 1962; Ganders 1979; Barrett 1992; Barrett & Shore 2008; Cohen 2010). Although the term homostyly is sometimes applied to any plant species with stigmatic surface and pollen presentation at the same level, we refer here exclusively to monomorphic species that evolved within the context of heterostylous groups.
Because of the advantages of selfing, homostylous species have been hypothesized to be more successful than heterostylous relatives under ecological conditions that limit pollinator abundance, visitation activity or mate density (e.g. in Amsinckia, Ganders 1975; Plumbaginaceae, Baker 1966; Primula, Kelso 1992; Richards 2003; Guggisberg et al. 2006; Psychotria, Sakai & Wright 2008; Turnera, Barrett & Shore 1987). However, spatial separation between male and female sexual organs (i.e. herkogamy; Webb & Lloyd 1986) has been reported in several homostylous species (e.g. in Primula, Ernst 1962; Al Wadi & Richards 1993; Tremayne & Richards 1993; Amsinckia, Johnston & Schoen 1996; Turnera, Barrett & Shore 1987; Narcissus, Medrano, Herrera & Barrett 2005; Larrinaga et al. 2009). Herkogamy can negatively affect the relative selfing rate (e.g. shown in Aquilegia, Brunet & Eckert 1998; Herlihy & Eckert 2007; Clarkia, Holtsford & Ellstrand 1992; Datura, Motten & Stone 2000; Mimulus, Karron et al. 1997; Nicotiana, Breese 1959; Turnera, Belaoussoff & Shore 1995; but see Medrano, Herrera & Barrett 2005 on Narcissus), as it may decrease autonomous or facilitated selfing (Webb & Lloyd 1986; Barrett 2002). Importantly, herkogamy is usually heritable and may thus respond to selection (e.g. Shore & Barrett 1990; Lennartsson et al. 2000; Herlihy & Eckert 2007; Bodbyl Roels & Kelly 2011). However, it remains unclear exactly how variation in herkogamy may influence the potential for autonomous selfing under conditions of limited pollinator availability (Moeller 2006).
In primroses (Primula), the classic model for homostyly (Scott 1865; Darwin 1877), homostylous species have been predicted to be better adapted than their heterostylous relatives to the ecological settings typical of alpine and arctic environments (e.g. Kelso 1992; Richards 2003; Guggisberg et al. 2006; Carlson, Gisler & Kelso 2008; Guggisberg, Mansion & Conti 2009). However, the potential role of herkogamy on the reproductive behaviour of homostylous primroses has never been considered: the alpine Primula halleri J.F.Gmel. (Fig. 1) provides an ideal study system to investigate it. P. halleri represents a classic example of the loss of heterostyly in alpine environments (e.g. Darwin 1877; Schröter 1926; Richards 2003), and extensive crossing experiments conclusively demonstrated that it is self-compatible (Ernst 1951). Early studies reported the occurrence of herkogamy in the species (Schröter 1926) and remarked that it may vary during floral anthesis (Ernst 1925). It is thus conceivable that this developmental variation, if sufficiently large to affect floral function, might offer contrasting mating opportunities at different stages of anthesis, including the possibility of delayed autonomous selfing. Mating opportunities that shift with the age of a flower have been mentioned in several species, although experimental evidence is generally limited (reviewed by Marshall et al. 2010).
Figure 1. Inflorescence of Primula halleri showing developmental variation in herkogamy among flowers. Several flowers were removed, and the remaining flowers were opened longitudinally to expose the position of the sexual organs (stigma: ♀; anthers: ♂). The relative ages of the remaining flowers are indicated with letters ‘a’ (first flower that opened) to ‘g’ (youngest bud); flower ‘e’ represents an incompletely opened flower, in which anthesis is about to commence and anthers are about to dehisce. Scale bar indicates 1 cm. Note that the style extends beyond the anthers more pronouncedly in younger (centre of inflorescence) than in older, open flowers (periphery of the inflorescence), illustrating the general trend that herkogamy decreases with floral age.
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The present study addresses the current gap of knowledge on how variation in herkogamy may affect pollinator dependence and opportunity for reproductive assurance in homostyly. The traditional focus on the proximity of sexual organs and self-compatibility has led to an interpretation of homostylous taxa as being primarily selfing and adapted to unreliable pollinator services, while the possible effects of herkogamy have been largely overlooked. Using P. halleri as our study system, we test whether: (i) the phenotypic expression of herkogamy changes during anthesis and variation between individuals and populations occurs and (ii) herkogamy affects seed set in open-pollinated, caged and emasculated plants. More broadly, the present study examines the effects of intraspecific and developmental variation in sexual organ distance on different components of plant reproductive success and contributes to understanding plant reproductive strategies in alpine environments.