Introduction

“Endless forms most beautiful” have motivated biologists for centuries (Darwin 1859; Carol 2005), and the remarkable floral diversity of angiosperms is one prime example. Floral diversification in many angiosperm clades is linked to interactions with animal pollinators, given that most angiosperms (~88%) are animal pollinated—a number that rises to 94% within tropical plant communities (Ollerton et al. 2011). Pollinators have behavioral preferences for different rewards, forms, and colors of flowers, which has contributed to a remarkable range of floral diversity (Sargent 2004; Waser and Ollerton 2006; Chittka and Raine 2006; Tripp and Manos 2008; Johnson 2010; Dudash et al. 2011; Van der Niet & Johnson 2012; Gervasi and Schiestl 2017; Smith and Kriebel 2017). When close relatives within a lineage occur in sympatry and are adapted to similar functional groups of pollinators, pollinator competition can arise and negatively impact fitness of one or both plant species (Caruso 2000; Grossenbacher and Stanton 2014; Muchhala et al. 2014; Sletvold et al. 2016). In such instances, selection for floral divergence in sympatry can arise, which has been documented in numerous groups of flowering plants, especially in temperate angiosperms (Sletvold et al. 2016). Pollinator competition can thus lead to greater floral divergence in sympatry compared to allopatry, or reproductive character displacement (RCD; Grossenbacher and Stanton 2014), which represents an important mode of ecological character displacement sensu the classical definition (MacArthur and Levins 1967).
Other mechanisms in addition to pollinator competition can lead to reproductive character displacement, although only some can be attributed to direct selection for character displacement. One such mechanism—reinforcement—results from direct selection to reduce gene flow between sympatric, diverging (or already divergent) lineages (Wallace 1889; Coyne and Orr 1989; Matute 2010; Hudson and Price 2014; Hopkins and Rausher 2012). Reinforcement, which has been documented in a limited number of plant lineages, these primarily in temperate regions, describes the process whereby previously allopatric lineages experience selection to avoid costly hybridization after coming into sympatry. In angiosperms, reinforcing selection often operates on floral morphology, thus driving the evolution of morphological divergence in floral traits (Grant 1966; Moyle et al. 2004; Silvertown et al. 2005; Kay and Schemske 2008; Hopkins and Rausher 2012). The underlying assumption of reinforcement is that hybridization is costly because it fails to yield offspring, or offspring have reduced fitness compared to non-hybrid offspring. Reinforcement is often thought to ‘complete’ the speciation process that begins when populations of species become isolated in allopatry but then later come into contact. While many classic studies of Drosphila and other animals support the concept of reinforcement, it has remained more controversial and less well-documented in plant evolutionary biology (reviewed in Hopkins 2013). Reinforcing selection, if common, is thought to act quickly such that natural hybrids are rarely observed.
Distinguishing between pollinator competition and reinforcement as primary drivers for RCD remains difficult despite the importance of understanding mechanisms that drive plant species divergence and floral diversification. In this study, we propose a two-step approach to help distinguish between these two processes, and then apply this approach to understand floral divergence in sympatry in a species-rich lineage of Neotropical angiosperms (Ruellia L.: Wild Petunias; Fig. 1). The first step involves emphasis on the floral characters themselves that underlie RCD. Pollinators typically select flowers based on visual and olfactory cues that signal reward (nectar and pollen, primarily) and thus divergence in these and associated characters, i.e., color, tube length, and tube width, which frequently co-vary with reward, may signal pollinator competition (Ornelas et al. 2007; Benetiz-Vieyra et al. 2014; Knauer and Schiestl 2014). In contrast, under reinforcement, selection may include traits related to pollinator preference, as above, but is likely to involve additional mechanical forms of isolation or structural incompatibilities that prevent cross fertilization (Kay and Schemske 2008; Hopkins 2013). Thus, divergence in other traits not typically associated with pollinator preference, such as style length or pollen tube length, lends support to hypotheses of reinforcement over pollinator competition.
As a second step, artificial cross pollinations and resultant data on reproductive incompatibility (RI) can be employed to help further distinguish pollinator competition from reinforcement. Under pollinator competition alone as the primary driver for RCD, selection should act to reduce visitation of a given pollinator to different plant lineages (species or incipient species), but other mechanisms to prevent hybridization such as mechanical or intrinsic isolating factors are not expected to manifest between plant lineages. In contrast, under reinforcement, plant lineages divergent in floral morphology should be recalcitrant to artificial hybridization because of mechanical incompatibilities that arise to prevent further, maladaptive hybridization. Hand pollinations bypass the action of pollinators and therefore offer additional means to distinguish between reinforcement and competition for pollinators. If hand pollinations between lineages with dissimilar flowers consistently yield non-viable offspring, reinforcement may be a primary driver of RCD. In contrast, if such hand pollinations do consistently yield viable offspring, then pollinator competition may instead be a primary driver of RCD.
In this study, we examine a species-rich and florally diverse lineage of tropical angiosperms to (1) test for RCD between species pairs and then (2) evaluate evidence in support of two different mechanisms that contribute to RCD: pollinator competition and reinforcement. We first determine which characters show the strongest pattern of RCD between species pairs. Second, we use hand pollinations in a carefully controlled glasshouse environment to test whether floral dissimilarity is correlated with RI. Finally, we assess if species pairs show greater post-pollination RI when in sympatry compared to allopatry by incorporating geographical range overlap as well as other potential effects, specifically phylogenetic relatedness. Finding that dissimilarity in floral traits (that are unlikely selected for by pollinators) is correlated with RI and that sympatric species cannot produce viable offspring is here taken as evidence in support of reinforcement, whereas finding that floral dissimilarity is unrelated to RI is taken as evidence in support of pollinator competition. The results from this study have implications for understanding the relative contribution of RCD to floral diversification, especially given few examples are known from the tropics (but see Kay and Schemske 2008; Muchhala et al. 2014), and serve as steps towards disentangling the underlying drivers of RCD.