Estimation of divergence time and comparative plastid genomics of Orchis species (Orchidaceae).

Low-coverage sequencing in plants allows whole plastomes to be obtained that can be used to investigate phylogenetic relationships among groups. The genus Orchis (c. 20 species), is usually divided into Orchis subgenera Orchis and Masculae. These subgenera are composed of three (Anthropophorae, Italicae, and Orchis) and four (Masculae, Provinciales, Pusillae, and Robustocalcare) sections, respectively. In this study, we used genome-skimming data to assemble the plastid genomes of 11 species (15 accessions) of Orchis, representing six out of the seven sections, from which we constructed a dated phylogenetic tree. Results suggest that the divergence between the subgenera occurred c. 10.53 Mya, whereas the main separation of the sections is dated between 6.53 and 3.48 Mya. Furthermore, we found 206 (in O. anthropophora) to 230 (in O. provincialis) microsatellite regions in the assembled plastomes, which could be used to design specific primers for further population genetics and phylogenetic studies and, ultimately, inform conservation efforts. The plastome data here presented represent a new contribution to the molecular systematics of the genus, and they can be used to further explore infrageneric and infrascpecific molecular variation in Orchis.