Discussion
The contribution of RCD to Floral Diversity. In plants, a pattern of increased floral divergence among sympatric compared to allopatric populations of species is well-documented (Whalen 1978; Armbruster et al. 1994; Fishman and Wyatt 1999; Coyne and Orr 2004; Muchhala and Potts 2007; Norton et al. 2015; Gögler et al. 2015; Koski and Ashman 2016; Lagomarsino and Muchhala 2019). In this study, we document clear patterns of reproductive character displacement (RCD) in sympatric species of a diverse lineage of Neotropical plants. This adds to a growing body of evidence for pervasiveness of the pattern in plant communities (Grossenbacher and Whittall 2014; Grossenbacher and Stanton 2014; Norton et al. 2015; Koski and Ashman 2016), extending significance of the phenomenon documented in tropical flowering plants. Our data in tandem with results from numerous prior studies suggests that RCD may be a major factor underlying the great floral diversity of angiosperms. The import of RCD is likely intensified among tropical latitudes given the density and diversity of plant-pollinator interactions.
Evidence in Support of Reinforcement . Two principle mechanisms help explain patterns of RCD: pollinator competition (Muchhala et al. 2014) and reinforcement (Kay and Schemske 2008; Hopkins 2013). Character displacement of floral traits can, specifically, arise from competition for pollinators in sympatry between species that are already reproductively isolated (Muchhala et al. 2014). However, our assessment of which flower characters diverge more between sympatric species pairs than allopatric species pairs combined with results from our artificial crossing experiments suggest a role for reinforcement in Ruellia . Using hand pollinations in a controlled glasshouse environment, we found exceptionally high prezygotic isolation between sympatric species pairs: only one of 95 crossing attempts across five sympatric species pairs produced any viable seeds. In contrast, crosses between allopatric species pairs (n = 28 pairs) were 8x more likely to be successful (9% success rate over all crosses; 30% of crossed allopatric species pairs were successful at least once). We assessed how similar crossed species pairs were in the shape and color of their flowers, and our results suggest that divergence in floral shape, especially style length, may be one underlying mechanism that results in strong barriers to reproductive compatibility between sympatric species. Both results – strong reduction in crossability between sympatric compared to allopatric species pairs and divergence in morphological features not likely under selection by pollinators – better fit a model of reinforcement over pollinator competition. In this study, we also found that species with differently colored flowers also have greatly reduced crossing success, but sympatric species pairs were no more likely to have similar or differently colored flowers than allopatric species pairs. It may be that reinforcing selection acts more strongly on style length than it does on flower color.
Pollinators likely select flowers based on their overall reward, shape, and color. In contrast, it is less likely that they select flowers based primarily on style length. Yet, we have shown that style length diverges more between sympatric species pairs relative to allopatric species pairs than any other floral trait measured (Fig. 3). All sympatric species differ in style length by at least 21.8 mm, while 23 of 28 allopatric species pairs differ in style length by lesser amounts (Fig. 3). If competition for pollinators, absent any involvement of reinforcing processes, were driving patterns recovered in our dataset, we would expect all or most floral traits to show divergence in sympatry instead of the highly variable divergence among characters we recovered (Fig. 3). Further, style length is a key character that may underlie potential incompatibilities (Kay and Schemske 2008), and selection on style length would serve to generate a pre-zygotic barrier between species where hybridization is maladaptive. Nonetheless, we recognize that reinforcing selection and pollinator competition are not always mutually exclusive: severe costs of pollen transfer between related species in sympatry may involve both types of processes when reproductive isolation is not fully complete and species boundaries remain semipermeable (Grossenbacher and Stanton 2014; Harrison and Larson 2014; Muchhala et al. 2014).
Two important criteria for reinforcing selection to occur are (i) that hybridization between species pairs is possible such that it can be the subject of selection, and (ii) that hybridization is maladaptive and selected against (Kay and Schemske 2008; Hopkins 2013). We have shown that crosses between 30% of allopatric species pairs are capable of producing viable seeds. Thus, hybridization is at least possible, as was earlier demonstrated by Long (1975). We do not have experimental evidence for the second criterion, but we note that hybrids inRuellia are rare in natural environments despite extensive geographical range overlap and contemporaneous flowering periods among numerous sets of species, suggesting it is maladaptive to be a hybrid (or that hybridization is otherwise rare to begin with). The first author (E. Tripp) has seen and studied nearly 200 of 300 Neotropical species in their native habitats (www.trippreport.com/ruellia-pages) and in only one instance has a natural hybrid been encountered (i.e., between Ruellia brevifolia and R. puri ; Bolivia, E. Tripp et al. 5971 & 5977 [COLO Herbarium]; only three putative, additional natural hybrids have been reported by other authors in the literature: Daniel 1990 [Ruellia amoena & R. foetida ]; Ezcurra 1993 [Ruellia brevifolia & R. longipedunculata ; R. brevicaulis & R. coerulea ]).
Finally, increased floral divergence among sympatric compared to allopatric populations of species is exceptionally well-documented in plants, and reinforcement against maladapted hybrids is likely to account for at least some of these instances. That reinforcing selection may sometimes involve action on only a single locus (Hopkins and Rausher 2012) suggests the potential for relatively simplistic genetic framework underlying contributions of reinforcement to the evolution of RI. Taken together, both the potential frequency and simplicity underlying reinforcement suggest that this type of selection may commonly contribute to completing the process of RI (Liou and Price 1994). However, a paucity of studies that have employed comparative data across clades to investigate RCD and its drivers has precluded understanding of whether such processes help drive lineage diversification and, if so, just how pervasive these processes are (Servedio 2004; Yukilevich 2012; Hopkins 2013). That sympatric species pairs in Ruellia are more reproductively isolated and more divergent in floral morphology than are allopatric pairs provides evidence in support of reinforcement.
Alternatively, if reproductive barriers between species in a given lineage are incomplete, sympatric species that are similar in floral morphology may interbreed due to sharing of pollinators coupled with mechanical and genetic compatibility (Templeton 1981). If gene flow between these diverged yet reproductively compatible lineages is recurrent and prolonged, such lineages may ‘fuse’, likely with the more fit or otherwise more abundant species in a given environment genetically swamping the less fit, less abundant species (Webb et al. 2011). Meanwhile, sympatric species that are highly dissimilar in floral form may be unable to interbreed and maintain distinct evolutionary lineages boundaries. Thus, ‘differential fusion’ (Templeton 1981) can yield a pattern of RCD similar to that driven by reinforcement. Although we cannot fully rule out differential fusion in this study, under such a model (and in contrast to reinforcement), natural hybrids should be commonly observed in nature between incompletely isolated lineages. However, as discussed above, natural hybrids are exceedingly rare inRuellia . Additionally, extensive evidence now documents reinforcement even in the face of gene flow (Matute 2010; Roda et al. 2017).
Additional Drivers of Variation in Crossing Success . Consistent with studies of model systems in animal speciation biology (Coyne and Orr 2001), we found that crossing success declines with increasing time of evolutionary divergence between species pairs. Whereas it is well established in animals that genetic distance is a significant predictor of interspecific fertility (Coyne and Orr 1989), there has historically been less consensus in plants (Edmands 2002; Moyle et al. 2004). Moyle and colleagues (2004) used comparative data from multiple species to demonstrate that increasing genetic distance strongly decreased crossability in one of the investigated study systems (Silene ), but not in the other two lineages they examined. Similarly, using a massive dataset on species crossability in Eucalyptus , Larcombe et al. (2015) found decreased reproductive compatibility with increasing genetic distance. Our and other studies (e.g., Scopece et al. 2007; Moyle et al. 2014; Brandvain et al. 2014) confirm a growing generality of this pattern in plants. The implications of this generality with respect to reinforcement are that reinforcement, as a process important to speciation, should scale with genetic distance. That is, at shallower levels of evolutionary divergence, reinforcement may potentially play a greater role in RI than it does at comparatively deeper evolutionary divergences. This prediction should be tested in a carefully controlled experiment focused on a series of species pairs that vary in their degree of phylogenetic relatedness but otherwise have in common other life history attributes.
Variation in Reproductive Character Displacement Across Clades and Latitudes . In addition to genetic distance, at least four factors should increase opportunities for and thus the potential impacts of both reinforcing selection and pollinator competition in driving floral divergence in sympatry: clade taxon richness, geographically wide-ranging and overlapping species, high densities of individuals within populations, and consequences of gene flow. In Ruellia , hundreds of species span one of the largest latitudinal gradients occupied by any lineage of flowering plants: ca. 80 degrees (i.e., from ~43˚N near Milwaukee, Wisconsin to ~37˚S in central Argentina). Over half of the ~300 New World species have broad geographical ranges (i.e., ranges that extend beyond the borders of a single country). Additionally, there exists widespread co-occurrence of both closely related and more distantly related species in Ruellia , and populations often consist of tens to hundreds of individuals (Tripp 2007; McDade and Tripp 2007; Tripp 2010; Tripp and Luján 2017). Species-rich lineages in which close relatives commonly encounter one another in natural environments should, on the whole, witness greater opportunity for floral diversification via either reinforcing selection or pollinator competition. If these opportunities involve maladaptive gene flow, then reinforcement is expected to be strong and fast acting to reduce energetic costs of producing unfit hybrids. If the above predictors are accurate, emergent properties associated with lineages such as total species number, degree of range overlap, and phylogenetic relatedness of co-occurring species should help predict the relative frequency and importance of underlying drivers of reproductive character displacement in natural landscapes.
We expect that these emergent characteristics of lineages associated with opportunity for reinforcing selection and/or pollinator competition should be more pronounced in tropical (compared to temperate) latitudes, where there typically exists much greater taxonomic and functional diversity of pollinators. Thus, variation in phenomena such as reinforcement across latitudes may be one mechanism contributing to latitudinal gradients in sympatric, and perhaps overall, biodiversity.Ruellia and other broadly ranging lineages (e.g.,Asclepias ) provide excellent systems in which to study whether and how processes including reinforcement and, presumably, competition vary with latitude in plants.