Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossingtaxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceousspecies, such as terrestrial orchids.† Methods Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequencevariation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypesusing a parsimony network.† Key Results Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels ofsequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimonyplastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemictaxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greatergenetic divergence separated the Italian and other European accessions of E. helleborine.†Conclusions Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italianpeninsula has only recently been colonized by Epipactis, probably during the most recent phase of theQuaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage.Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could haverepresented the mechanism promoting this rapid diversification and the observed high taxonomic complexitydetected in the E. helleborine species complex.