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To elucidate the molecular phylogeny of Neottia japonica, which is a terrestrial orchid distributed in East Asia, the internal transcribed spacer (ITS) of nuclear DNA and the matK of chloroplast DNA were used. A total 22 species of 69 accessions for ITS and 21 species of 114 accessions for matK phylogeny were analyzed with the maximum parsimony and Bayesian methods. In addition, we sought to establish a correlation between the distribution, morphology of the auricles and genetic association of N. japonica with phylogenetic data. The phylogenetic results suggest that N. japonica is monophyletic and a sister to N. suzukii in terms of the ITS phylogeny, while it is paraphyletic with N. suzukii in terms of the matK phylogeny. N. japonica and N. suzukii show similar morphologies of the lip and column, they both flower in April, and they are both distributed sympatrically in Taiwan. Therefore, it appears to be clear that N. japonica and N. suzukii are close taxa within Neottia, although there is incongruence between the nrDNA and cpDNA phylogenies of N. japonica. The incongruence between the two datasets may have various causes, meaning that further studies are needed to confirm the evolutionary process of N. japonica. The phylogenetic status of N. kiusiana, which was not included in previous studies, was as a sister to N. nidus-avis. Meanwhile, the ITS and matK phylogenies are unsuitable for identifying genetic associations with the characteristic of auricles. The phylogenetic topologies of Korean, Taiwanese and mainland Chinese individuals suggest that the populations of N. japonica in Korea originated from China’s mainland and island areas. The characterization of regional gene differences could provide useful preliminary data for future studies.
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