Abstract: The orchid Dactylorhiza cantabrica H.A. Pedersen is a narrow endemic occurring at western Cantabrian Mountains in NW Spain. Previous allozyme and morphological studies suggest that it might result from the hybridization of two widespread congeners: the triploid D. insularis and the diploid D. sambucina. However, this hypothesis has not been tested using multiple genetic markers, necessary to analyse phylogenies in complex genera such as Dactylorhiza. In this study, the Hyb-Seq technique is applied together with the universal Angiosperms353 probe kit to sequence multiple plastid and low-copy nuclear genes. The phylogenetic relationships between the three species, estimated based on 269 and 266 nuclear genes under concatenation and coalescent-based approaches, respectively, revealed highly-supported clades containing each putative parent, D. insularis and D. sambucina. The position of D. cantabrica was not well resolved, suggesting the existence of mixed inheritance, where different genes come from each parent. Phylogenetic networks, used for visualizing the conflict between nuclear gene trees, placed D. cantabrica between the two parents and revealed high levels of reticulation. In addition, nuclear genetic variation within and among species was explored with allele frequencies-based tools further supporting the intermediate position of D. cantabrica and the hypothesis of a recent hybrid origin. Finally, 75 plastid genes revealed that D. insularis might be the maternal donor. Altogether, our results point to the allopolyploid origin of D. cantabrica from D. insularis and D. sambucina, as well as to the clear genetic differentiation of the two parental species.

Key words: allopolyploid, hybridization, Hyb-Seq, orchids, phylogeny, reticulation