Ancestral and monophyletic presence of diplostigmaty in Sebaea (Gentianaceae) and its potential role as a morphological mixed mating strategy.

• Diplostigmaty, the presence of a primary (apical) stigma and secondary (mid-stylar) stigmas along the style, is only known from the genus Sebaea (Gentianaceae). Early work indicated that the secondary stigmas provide a mechanism of autogamy, suggesting that it might ensure reproductive assurance. * Here, we test the monophyly of this unique morphological trait. Using Bayesian methods, we infer a nuclear DNA phylogeny for 96 accessions, including c. 50% of the species from the genus Sebaea. With this phylogeny, we infer the distribution of ancestral states on critical nodes using parsimony and likelihood methods. * The inferred nrDNA phylogeny shows that the genus Sebaea is divided in two statistically well-supported clades, A and B, consistent with recent estimates. The most recent ancestor (MRCA) of clade A, except the most basal species (Sebaea pusilla), is resolved as diplostigmatic. No reversal to a single stigma is observed within this clade. * We suggest that diplostigmaty is evolutionarily stable through time. We also discuss why this reproductive system is not found elsewhere than in Gentianaceae and the potential advantage of diplostigmaty as a stable mixed mating strategy.