Results
Our results show reproductive character displacement (RCD) between sympatric species of Ruellia relative to allopatric species (Fig. 3). Overall floral form is significantly more different between sympatric species pairs than between allopatric species pairs (Ranosim = 0.63, p = 0.017). Sympatric species were not significantly more different for overall leaf form (Ranosim = 0.38, p = 0.121). When examining individual floral characters, four of these showed a significantly greater difference between sympatric species pairs (Fig. 3), with style length showing the most pronounced difference (Fig. 3;Ranosim = 0.85, p < 0.001). In contrast, only one leaf character appears to show significantly greater differences between sympatric species pairs relative to allopatric species pairs (leaf length; Ranosim = 0.54, p = 0.036). If conservative Bonferroni corrections are applied to these multiple tests of significance, only style length shows a significantly greater difference between sympatric species pairs relative to allopatric species pairs.
Among 95 total crossing attempts across five sympatric species pairs, only one instance was successful and yielded mature, viable seeds (a single cross between Ruellia conzattii and R. hirsuto-glandulosa ). In contrast, 63 of 730 crossing attempts between allopatric species pairs yielded mature viable seeds (Supplementary Table 2). Statistical analyses using generalized linear mixed models to control for the non-independence of data points involving the same species (following Tobias et al. 2014) showed that occurrence in sympatry significantly reduced crossing success of species pairs (Table 1; likelihood ratio-test, Χ2 = 5.00, d.f. = 1, p = 0.025). This result is supported by a non-parametric Wilcoxon test on a phylogenetically corrected dataset (W = 6, p = 0.032).
Using the same generalized linear mixed model approach, we found that pairs of species with similarly shaped and similarly colored flowers had significantly higher crossing success (Table 1; Fig. 4; flower color: Χ2 = 18.94, d.f. = 1, p < 0.001; flower shape: Χ2 = 5.76, d.f. = 1, p = 0.016; multivariate depiction of floral morphospace provided in Supplementary Fig. 2). Meanwhile, similarity in vegetative morphology did not significantly influence crossing success (Table 1; Χ2 = 2.91, d.f. = 1, p = 0.089). We also found that more distantly related species pairs, quantified as interspecific genetic distance in a maximum likelihood phylogeny, had significantly lower crossing success (Table 1; Fig. 4; Χ2 = 4.29, d.f. = 1, p = 0.038). Similar results with respect to significance of fixed effects were obtained when testing the effect of time since divergence in a temporally calibrated phylogeny (Supplementary Table 3). In a multivariate analysis of drivers of crossing success across all species pairs, we found the same direction for our fixed effects as in univariate analyses, but floral shape similarity and geographical range overlap of species pairs did not significantly modulate crossing success (Table 1). An assessment of our model showed these two fixed effects to be highly correlated (r = 0.37) whereas none of the other fixed effects were highly correlated with each other (r < 0.17). If either geographical range overlap of species pairs or similarity in floral shape was removed from the model, the model performed better in explaining crossing success (Table 1; ΔAICc after removing geography = 1.3, ΔAICc after removing floral shape similarity = 0.9), but if both were removed, the model performed worse (ΔAICc = -2.5). This may be expected given the documented reproductive character displacement in sympatric species pairs.