Morphological, ecological, and molecular phylogenetic approaches reveal species boundaries and evolutionary history of Goodyeracrassifolia (Orchidaceae, Orchidoideae) and its closely related taxa.

Species delimitation within the genus Goodyera is challenging among closely related species, because of phenotypic plasticity, ecological variation, and hybridization that confound identification methods based solely on morphology. In this study, we investigated the identity of Goodyeracrassifolia H.-J.Suh, S.-W.Seo, S.-H.Oh & T.Yukawa, morphologically similar to Goodyeraschlechtendaliana Rchb.f. This recently described taxon has long been known in Japan as "Oh-miyama-uzura" or "Gakunan" and considered a natural hybrid of G.schlechtendaliana and G.similis Blume (= G.velutina Maxim. ex Regel). Because the natural hybrid between G.schlechtendaliana and G.similis was described as G.×tamnaensis N.S.Lee, K.S.Lee, S.H.Yeau & C.S.Lee before the description of G.crassifolia, the latter might be a synonym of G.×tamnaensis. Consequently, we investigated species boundaries and evolutionary history of G.crassifolia and its closely related taxa based on multifaceted evidence. Consequently, morphological examination enabled us to distinguish G.crassifolia from other closely related species owing to the following characteristics: coriaceous leaf texture, laxly flowered inflorescence, long pedicellate ovary, large and weakly opened flowers, and column with lateral appendages. Ecological investigation indicates that G.crassifolia (2n = 60) is agamospermous, requiring neither pollinators nor autonomous self-pollination for fruit set, whereas G.schlechtendaliana (2n = 30) is neither autogamous nor agamospermous but is obligately pollinator-dependent. MIG-seq-based phylogenetic analysis provided no evidence of recent hybridization between G.crassifolia and its close congeners. Thus, molecular phylogeny reconstructed from MIG-seq data together with morphological, cytological, and ecological analyses support the separation of G.crassifolia as an independent species.