Immigrant and extrinsic hybrid seed inviability contribute to reproductive isolation between forest and dune ecotypes of Epipactis helleborine (Orchidaceae)

Reproductive isolation caused by divergent natural selection arising from differences between ecological environments or ecological interactions represents a major mechanism contributing to speciation, but its relative importance is poorly known. In this study, controlled reciprocal crossings and seed germination experiments were combined with genetic and morphometric analyses to test the hypothesis that previously described differences in mycorrhizal communities between forest and dune ecotypes of Epipactis helleborine were sufficiently strong to create complete reproductive isolation between the two ecotypes. Molecular analyses using 770 SNP markers showed that the two ecotypes were genetically distinct and that populations of the dune ecotype were genetically impoverished compared to populations of the forest ecotype. Morphologically, the two ecotypes were also significantly different, with plants of the dune ecotype generally being smaller than plants of the forest ecotype. The results further showed that immigrant seeds had a significantly lower probability of protocorm formation than native seeds, indicating strong immigrant inviability. Although both ecotypes were able to cross easily and to produce a large number of viable seeds, hybrid seeds showed significantly lower protocorm formation than pure seeds, further contributing to reproductive isolation. Overall, these results indicate that interfertile populations of a widespread orchid adapting to contrasting environments diverge as a consequence of concurrent selection acting against immigrants and hybrids and suggest that mycorrhizal fungi can play a role in the early stages of plant speciation.