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Two Orthotrichum species of the subgenus Orthophyllum were compared with other representatives of this genus using internally transcribed spacer regions 1 and 2, the chloroplast trn H-psbA region, and inter-simple sequence repeat (ISSR) and intron-exon splice conjunction (ISJ) markers. The ISSR and ISJ markers used revealed many bands and mutations specific only to O. Gymnostomum and O. Obtusifolium. Phylogenetic analysis clearly supported previous concepts postulating that species of the subgenus Orthophyllum should be recognized as the separate genus Nyholmiella.