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

Reproductive isolation is mediated by ecological, phenotypic, and genetic barriers. Where such barriers are not upheld, hybridisation can occur, revealing insight about the formation of species. Here, we provide an integrative investigation of hybrid formation between the orchids Gymnadenia conopsea and G. rhellicani and potential isolating barriers in zones of overlap in the European Alps. We examine opportunities for hybridisation by quantifying hybrid frequency and pollinator assemblages of parent species. Next, we characterise floral phenotypes and genetic parentage of hybrids to examine how genetic inheritance of traits shapes ecological interactions, and thus fitness, of hybrids. We find that pollinator assemblages of parents overlap partially, consistent with observed rare hybrids (<1% of the population). Phenotypically, hybrids were intermediate across floral traits, consistent with all hybrids being identified as F1s, resulting from a primary hybridisation event between G. conopsea and G. rhellicani. No evidence of backcrossing or introgression was found, but direction of pollen transfer varied across sites. Additionally, hybrids were heterozygous for parental alleles at two loci impacting anthocyanin production, suggesting hybrids intermediate floral colour reflects inheritance of these alleles. Finally, we find little support that ploidy variation or hybrid seed infertility explains the absence of advanced generations of hybrids. Thus, we suggest that floral isolation represents an important isolating mechanism between G. conopsea and G. rhellicani in limiting the formation of uncommon, unfit hybrids and shaping species boundaries.