Pollinator-relevant floral traits underlie bidirectional hybridisation in the orchid genus Gymnadenia

Speciation through the emergence of reproductive isolation barriers is a fundamental concept of evolutionary biology. Here we use two closely related but phenotypically distinct Gymnadenia orchid species and their proposed hybrids to study the barriers associated with this phenomenon. We analysed the potential pre– and post-zygotic barriers to hybridisation and conducted the first comprehensive analysis of pollinator-influenced phenotypic traits in both parents and their hybrids. We also uncover the previously uncharacterised, but much hypothesised, genetic origins of the hybrids and use both existing literature and novel field observations to predict how the hybrids may have emerged in their respective populations. We demonstrate that all hybrids are F1s, despite their capacity to produce viable seed. We find the hybrids to be intermediate in most phenotypic traits except for their anthocyanin profile, which corresponds to one of the parent species. We use known anthocyanin biosynthesis genes in this system to study the origin of the hybrids’ distinct pigment phenotype and model how the resulting floral colours may be perceived by different pollinator guilds. Interestingly, we find significant variation in the directionality of hybrid formation, in which the hybrid populations of two widely separated sample sites have inverse patterns of maternal parent species. This study progresses our understanding of the evolution and establishment of distinct pollinator-mediated floral phenotypes in groups of closely related species. Moreover, this study provides an insight into the influence of spatially distinct pollinator interactions in populations of species increasingly at risk as global climates continue to change and influence ecological processes.