Species delimitation in the Orychophragmus violaceus species complex (Brassicaceae) based on morphological distinction and reproductive isolation

Accurate species delimitation is critical for diverse biological disciplines, and most recent species delimitation studies are mainly based on genetic evidence. However, few of these focused on the examination of morphological distinctness and reproductive isolation. In this study, we investigated morphological variation and reproductive isolation within and between four potential species of the Orychophragmus violaceus species complex (Brassiaceae) based on available genetic evidence. Four distinct phenotypic clusters were confirmed based on statistical analyses of 42 morphological characters for > 300 individuals of 12 populations in an experimental garden in which ecological variation was eliminated. Reproductive isolation was clear between species as fruit set from interspecific crossing was significantly lower than that resulting from intraspecific pollinations between different populations. Based on these findings and the genetic distinctness reported before, four species, including two new species described here, are recognized in the O. violaceus complex. Our study highlights the importance of reproductive isolation in delimiting species boundaries in plants by applying the widely used genetic evidence.