Multiple barriers maintain species boundaries in sympatric brood‐site deceptive Gastrodia orchids

Societal Impact Statement Gastrodia orchids are inconspicuous forest plants that lack chlorophyll and obtain carbon from fungi. Their dull‐colored, ground‐level flowers are often hidden among leaf litter, yet they rely on small flies for pollination. We show that two co‐occurring Gastrodia species remain distinct even though they share similar pollinators. Limited fly movement and reduced reproductive success after cross‐species pollination restrict hybrid formation, while fungal requirements may impose additional barriers after seed formation. These findings show how multiple barriers can preserve species differences in cryptic forest orchids and highlight the need to conserve the insects, fungi, and forest‐floor microhabitats that support such hidden life histories. Summary Because orchids show remarkable floral diversity and often rely on specific pollinators, pollinator‐mediated isolation has traditionally been regarded as the principal reproductive barrier in this family. However, growing evidence indicates that additional mechanisms also contribute to reproductive isolation. Gastrodia confusa and Gastrodia pubilabiata attract similar drosophilid pollinators through brood‐site deception, probably by mimicking fermenting substrates. Despite habitat overlap, morphologically intermediate individuals have not been reported in the wild, suggesting that reproductive barriers other than pollinator specificity restrict natural hybrid formation. This study examined the mechanisms and strength of reproductive isolation between these two sympatric Gastrodia species by integrating field observations, pollen tube observations, controlled pollination experiments, and molecular analyses. Field observations showed that the two species shared largely overlapping drosophilid pollinator assemblages at sympatric sites, but the short‐distance movements of individual flies likely limited interspecific pollen transfer under natural spatial arrangements. Interspecific hand‐pollination reduced fruit set and the proportion of seeds with well‐developed embryos but did not cause complete reproductive failure. Molecular analyses detected no F 1 ‐like genomic profiles and provided only limited evidence of possible natural introgression between G. confusa and G. pubilabiata at sympatric sites. These findings suggest that species boundaries between the two Gastrodia species are maintained not by a single complete barrier but by the cumulative effects of multiple partial prepollination and postpollination barriers. Future studies should determine whether seeds produced by interspecific pollination can germinate, whether hybrid seedlings can establish successfully, and whether hybrids retain compatibility with the fungal partners required by either parental species.