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

Abstract

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.

Keywords: MIG-seq; chromosome; cryptic species; integrative taxonomy; phylogeny; reproductive biology; species complex.

Figure 1.

Goodyeracrassifolia in its natural habitat A flowering individual B flowers C fruiting individual D leaves. Scale bars: 30 mm.

Figure 2.

Goodyeracrassifolia from Kami City, Kochi Prefecture (Hisanori Takeuchi G161-1, KYO) A dorsal sepal (abaxial view) B lateral sepals (left: abaxial view, right: adaxial view) C lateral petals (left: abaxial view, right: adaxial view) D lip and column (dorsal view) E lip (left: adaxial view, right: lateral view) F column (left: obliquely dorsal view, right: ventral view) G column (left: ventral view, right: lateral view) H lateral appendages removed from column (left: dorsal view, right: ventral view) I lateral appendages removed from column (both: dorsal view) J pollinarium (left: dorsal view, right: ventral view) K anther cap (left: dorsal view, right: ventral view). Arrows indicate the conspicuous lateral appendages. Photographs except G and I are derived from the same flower. G and I are used to show morphological variation of column within the same individual. Scale bars: 3 mm.

Figure 3.

Goodyeracrassifolia from Higashimuro County, Wakayama Prefecture (Yasuo Takada s.n., KYO) F, G column The conspicuous lateral appendages are indicated by arrows H column removing lateral appendages I, J lateral appendages removed from column K pollinarium L anther cap and pollinarium A dorsal sepal (abaxial view) B lateral sepals (left: abaxial view, right: adaxial view) C lateral petals (left: abaxial view, right: adaxial view) D lip and column (dorsal view) E lip (left: adaxial view, right: lateral view) F column (left: dorsal view, right: ventral view) G column (obliquely lateral view) H column removing lateral appendages (ventral view) I lateral appendages removed from column (left: dorsal view, right: ventral view J lateral appendages removed from column (ventral view) K pollinarium (ventral view) L anther cap and pollinarium (left: dorsal view, right: ventral view). Arrows indicate the conspicuous lateral appendages. Photographs except G, H, J, K are derived from the same flower G, H, J show the variation of column morphology within the same individual, while K is used because pollinaria were detached from anther cap of a flower that was mainly used. Scale bars: 3 mm.

Figure 4.

Goodyeracrassifolia (Koji Tanaka KS209, KYO; photographed after immersion in 50 percent ethanol) A flower (lateral view) B flower (dorsal view) C dorsal sepal (abaxial view) D lateral sepals (left: abaxial view, right: adaxial view) E lateral petals (left: abaxial view, right: adaxial view) F lip and column (lateral view) G lip (left: adaxial view, middle: lateral view, right: abaxial view) H column (left: dorsal view, right: obliquely ventral view) I column with partially detached lateral appendages (left: ventral view, right: lateral view) J lateral appendages removed from column (ventral view) K anther cap and pollinarium (ventral view). Arrows indicate the conspicuous lateral appendages. All photographs are derived from the same flower. Scale bars: 3 mm.

Figure 5.

Goodyeracrassifolia (Hisanori Takeuchi & Kenji Suetsugu KS208, KYO) A dorsal sepal (adaxial view) B lateral sepal (adaxial view) C lateral petal (adaxial view) D lip and column (dorsal view) E lip (left: adaxial view, right: lateral view) F longitudinal section of lip (adaxial view) G column and anther (left: top view, right: lateral view) H column (left: dorsal view, middle: lateral view, right: ventral view) I pollinarium (left: dorsal view, right: ventral view) J anther cap (dorsal view). Arrows indicate the conspicuous lateral appendages. All photographs are derived from the same flower. Scale bars: 3 mm.

Figure 6.

Somatic chromosomes (A–C) and their explanatory drawings (D–F) of Goodyeracrassifolia and its closely related taxa A, DG.crassifoliaB, EG.schlechtendalianaC, FG.schlechtendalianavar.yakushimensis. Scale bars: 10 μm.

Figure 7.

Phylogenetic tree of Goodyeracrassifolia and its closely related taxa reconstructed using MIG-seq data. Bootstrap values within species, and those less than 50%, are not shown. Branch length represents the average number of substitutions per site.

Figure 8.

Neighbor-Net network for Goodyeracrassifolia and its closely related taxa, based on uncorrected P distances calculated from 4790 SNPs.

Figure 9.

Population structure of Goodyeracrassifolia and its closely related taxa, inferred with STRUCTURE 2.3.4. Using K = 2 and K = 3 generated the largest and second-largest delta K, indicating that they were the most and second most optimal, respectively. Species and populations are separated by broad and narrow vertical black lines, respectively.