In this paper, three plastomes of Mankyua chejuense, Helminthostachys zeylanica, and Botrychium ternatum in Ophioglossaceae were completely sequenced in order to investigate the plastome evolution and phylogeny of eusporangiate ferns. They were similar to each other in terms of length and the gene orders; however, six unknown open reading frames (ORFs) were found between rps4 and trnL-UAA genes in M. chejuense. Similar sequence regions of six ORFs of M. chejuense were found at the plastomes of Ophioglossum californicum and H. zeylanica, as well as the mitochondrial genome (mitogenome) of H. zeylanica, but not in B. ternatum. Interestingly, the translated amino acid sequences of three ORFs were more similar to the proteins of distantly related taxa such as algae and bacteria than they were to proteins in land plants. It is likely that the six ORFs region arose from endosymbiotic gene transfer (EGT) or horizontal gene transfer (HGT), but further study is needed to verify this. Phylogenetic analyses suggested that Mankyua was resolved as the earliest diverging lineage and that Ophioglossum was subsequently diverged in Ophioglossaceae. This result supports why the plastome of M. chejuense have contained the most ancestral six ORFs in the family.
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