The loss of self-incompatibility (i.e., postpollination, prezygotic mechanisms that prevent self-fertilization; Igic et al. 2008) is widely acknowledged as one of the most frequent transitions in plant evolution (Stebbins 1950, 1970). Furthermore, it has important implications for microevolutionary processes (Igic et al. 2008) and macroevolutionary patterns of clade diversification (Takebayashi and Morell 2001; Goldberg et al. 2010; Ferrer and Good 2012; J. M. de Vos et al., unpubl. ms., 2013). Much of the evolutionary significance of the loss of self-incompatibility relates to the notion that it is necessary for the transition from allogamous (outcrossing) to predominantly autogamous (selfing) mating (Stebbins 1970; Barrett 2002; Busch and Schoen 2008; Wright et al. 2008; Karron et al. 2012). Although self-incompatible flowers are necessarily outcrossing, self-compatible flowers can either outcross self or have an intermediate selfing rate, but high rather than low selfing is more common for self-compatible taxa (Raduski et al. 2012). Early works suggested that transitions toward selfing after the loss of self-incompatibility are associated with a suite of changes in morphological and reproductive floral characters (Darwin 1876; Ornduff 1969; Stebbins 1970), including decreased floral display, reduced pollen-to-ovule-number ratio, smaller distance between male and female organs within flowers (i.e., less herkogamy), and general reduction of floral size, collectively termed the “selfing syndrome” (see table 1 in Ornduff 1969; Cruden 1977; Ritland and Ritland 1989; Goodwillie et al. 2010; Sicard and Lenhard 2011).
The selfing syndrome is considered a common phenomenon; transitions from outcrossing to selfing are thought to be “in most cases” (Sicard and Lenhard 2011, p. 1433) if not “almost universally” (Foxe et al. 2009, p. 5241) associated with the selfing syndrome. Stebbins (1970, p. 310) stated in an early discussion that “in all self-fertilizers, flower size diminishes below that found in their cross-fertilizing ancestors,” suggesting that evolution toward a selfing syndrome upon the loss of self-incompatibility is a deterministic evolutionary trend. Most of our understanding of the evolution of floral traits in response to shifts toward increased selfing stems from analyses performed on a few taxa (e.g., in Capsella, Slotte et al. 2010; Eichhornia, Vallejo-Marín and Barrett 2009; Leavenworthia, Busch and Urban 2011; Mimulus, Ritland and Ritland 1989), or from informal interpretations of data on large numbers of species (e.g., Darwin 1876; Ornduff 1969; Stebbins 1970). Few comparative studies involving a larger number of species in an explicit phylogenetic framework have been conducted (but, see Goodwillie et al. 2010, for an angiosperm-wide analysis of floral display in inflorescences and selfing rates). Specifically, although multiple, independent losses of self-incompatibility are documented in several clades (e.g., Linanthus section Leptosiphon [Polemoniaceae], Goodwillie 1999; Solanaceae, Goldberg et al. 2010; Triticeae [Poaceae], Escobar et al. 2010), it is unclear whether, or to what extent, replicate transitions in different species within a clade lead to similar patterns of phenotypic change (i.e., similar evolutionary trajectories). Are the floral displays of self-compatible species always smaller than those of their self-incompatible relatives, as Stebbins (1970) suggested? Do individual floral traits respond differently to the shift from outcrossing to selfing? Do different floral traits evolve synchronously or asynchronously with the loss of self-incompatibility? These questions were identified as major gaps in our understanding of the transition to self-fertilization (Karron et al. 2012) and are addressed in this study.
Several explanations that are not mutually exclusive have been proposed for the correlation between small flowers and selfing (Sicard and Lenhard 2011). First, small floral size may facilitate autonomous selfing and be directly targeted by selection, for instance, when selfing provides reproductive assurance under mate- or pollinator-limited conditions (Eckert et al. 2006). Second, if reproductive fitness is decoupled from the attractiveness of floral display for pollinators, as is the case in strict selfers, theory predicts that resources would not be invested in large flowers, but rather in increased reproduction (e.g., ovule production; Brunet 1992). Third, the selfing syndrome may be a pleiotropic effect of selection for small flowers driven by selection for the avoidance of herbivory (Eckert et al. 2006) or by selection for fast maturation in marginal habitats (Guerrant 1989; Aarssen 2000). These arguments suggest that after a transition toward selfing, floral size is under selection to progressively diminish in a range of scenarios.
Despite the broad acceptance of the selfing syndrome as a general phenomenon, the loss of self-compatibility does not necessarily result in small floral size. In fact, showy flowers with highly specialized pollination systems are often self-compatible and can have high selfing rates, in contrast with the prediction of the selfing syndrome (reviewed by Fenster and Martén-Rodríguez 2007). This conflict may be explained by the idea that showy, specialized flowers, relying on a small subset of the potential pollinator community, are inherently prone to reproductive failure, thus selfing may assure reproduction when outcrossing fails. This notion implies that self-compatibility and high selfing rates do not necessarily lead to small flowers, as predicted by the selfing syndrome. It is thus topical to ask whether floral traits respond to the loss of self-incompatibility consistently across different species.
In this study, we assess the extent to which the loss of self-incompatibility and the associated ability to self results in a deterministic trend of phenotypic evolution toward smaller floral size, as predicted by the selfing syndrome, using the primroses as our study system. This group of about 550 species (Primula and nested genera, Primulaceae, i.e. “/Primula,” where forward slash indicates clade name sensu Mast et al. 2001) is a classic model for the evolution of selfing, discussed in the seminal works of Ornduff (1969) and Stebbins (1970) as a prototypical example of repeated losses of self-incompatibility and associated origins of selfing, in the form of transitions from heterostyly to homostyly. Heterostyly is a form of heteromorphic incompatibility in which populations consist of two (distyly) or three (tristyly) genetic morphs that differ in the reciprocal placement of sexual organs and in mating type, so that only crosses between morphs are fully fertile (reviewed by Ernst 1962; Ganders 1979; Barrett 1992; Wedderburn and Richards 1992; Barrett and Shore 2008; Cohen 2010; Naiki 2012). Homostylous species have only one floral morph (i.e., are monomorphic), and crossing experiments established that they are self-compatible, hence, self-fertilization is possible (Ernst 1962; Richards 2003). Detailed phylogenetic studies concluded that the crown node of /Primula was heterostylous and indicated several, deeply nested losses of heterostyly within the clade (Mast et al. 2006, J. M. de Vos et al., unpubl. ms., 2013). Similar patterns occur in many of the about 28 families that include heterostylous taxa, with homostylous species evolving multiple times independently from heterostylous ancestors (e.g., in Amsinckia, Boraginaceae, Schoen et al. 1997; Narcissus, Amaryllidaceae, Graham and Barrett 2004; Nymphoides, Menyanthaceae, Tipperey and Les 2011; Pontederiaceae, Kohn et al. 1996; Turnera, Turneraceae, Truyens et al. 2005). The recurrent transition from heterostyly to homostyly is an important model for floral evolution and the evolution of selfing in angiosperms (Barrett 2003), making it an ideal system to evaluate the selfing syndrome from a quantitative, comparative perspective.
Here, we analyze a large dataset of continuous floral traits of /Primula by using a combination of recently developed comparative methods that employ explicit models of quantitative trait evolution and account for both evolutionary relationships and intraspecific variation. We assess whether floral evolution among heterostylous and homostylous lineages is congruent with predictions of a selfing syndrome, by asking: Do heterostylous and homostylous species differ in (i) overall floral size and (ii) individual floral traits? (iii) Is the pattern of phenotypic change (i.e., evolutionary trajectory) of each floral trait affected by the loss of heterostyly? By answering these questions, our study contributes to an improved understanding of the phenotypic consequences of the loss of self-incompatibility, one of the most important transitions in flowering plant evolution.