Experimental sympatry suggests geographic isolation as an essential reproductive barrier between two sister species of Pedicularis

Although mechanical isolation mediated by shared pollinators has been considered as a classic model of pollinator‐mediated floral isolation in Pedicularis, a superdiverse genus in Hengduan Mountains, southwest China, there has been no empirical study of interspecific pollen flow between closely related species. We examined reproductive barriers at six different stages between Pedicularis cranolopha and Pedicularis tricolor, two sister species. The two sister species were geographically isolated from each other based on our field survey and the records of herbarium specimens. Translocation experiments showed that flowering phenology partly overlapped and bumblebee pollinators did not discriminate between flowers of the two species. Bumblebee interspecific moves could mediate interspecific pollination as traced using fluorescent powder, in which pollen analogs placed on one species were transferred to the stigmas of the other species in experimental plots containing both species. Heterospecific pollen tubes grew in the style as well as conspecific pollen in hand‐pollination experiments. Reciprocal hybridization between the two species could produce (partially) viable seeds, suggesting weak post‐pollination barriers. Our results showed that geographic isolation was an important barrier of two species, and the total reproductive isolation between two species was incomplete when without geographical isolation. The formation of Big Snow Mountains could introduce an important pre‐zygotic reproductive barrier between the two sister species of Pedicularis; such geographical isolation could be responsible for allopatric speciation, giving a clue to understanding the rapid radiation on mountain areas. The relative contributions of reproductive barriers at six stages to total isolation including all barriers (A), and in sympatry (B) without geographic isolation between Pedicularis cranolopha (white bars) and Pedicularis tricolor (gray bars), illustrating that except the geographic isolation alone could contribute complete reproductive isolation between the two species, the other five barriers could not. Flowering phenology partly overlapped and bumblebee pollinators did not discriminate between flowers of the two species. Bumblebee interspecific moves could mediate interspecific pollination and reciprocal hybridization between the two species could produce (partially) viable seeds, suggesting that post‐pollination barriers were weak.