Background: Plastomes of heterotrophic plants have been greatly altered in structure and gene content, owing to the relaxation of selection on photosynthesis-related genes. The orchid tribe Gastrodieae is the largest and probably the oldest mycoheterotrophic clade of the extant family Orchidaceae. To characterize plastome evolution across members of this key important mycoheterotrophic lineage, we sequenced and analyzed the plastomes of eleven Gastrodieae members, including representative species of two genera, as well as members of the sister group Nervilieae.Results: The plastomes of Gastrodieae members contain 20 protein-coding, four rRNA and five tRNA genes. Evolutionary analysis indicated that allrrngenes were transferred laterally and together, forming anrrnblock in the plastomes of Gastrodieae. The plastome GC content ofGastrodiaspecies ranged from 23.10% (G. flexistyla) to 25.79% (G. javanica).The plastome ofDidymoplexis pallenscontains two copies each ofycf1andycf2. The synonymous and nonsynonymous substitution rates were very high in the plastomes of Gastrodieae among mycoheterotrophic species in Orchidaceae and varied between genes.Conclusions: The plastomes ofGastrodieaeare greatly reduced and characterized by low GC content,rrnblock formation, lineage-specific reconfiguration and gene content, which might be positively selected. Overall, the plastomes of Gastrodieae not only serve as an excellent model for illustrating the evolution of plastomes but also provide new insights into plastome evolution in parasitic plants.

Habenaria is one of the largest terrestrial genera in the family Orchidaceae. Most field studies on Habenaria species with greenish–white and nocturnal scented flowers are pollinated by nocturnal hawkmoths and settling moths. However, H. rhodocheila presents reddish flowers lacking a detectable scent and fails to fit the moth pollination syndrome. We investigated the pollinators, breeding system, and functional traits of H. rhodocheila in South China and found that two diurnal swallowtail butterflies Papilio helenus and Papilio nephelus (Papilionidae) were the effective pollinators. When butterflies foraged for nectar in the spur, the pollinia became attached between the palpi. A triangular projected median rostellar lobe was found at the entrance (sinus) of the spur of H. rhodocheila. This lobe divided the spur opening into two entrances forcing butterflies to enter their proboscides through the left or right side. When the projection of median rostellar lobe was removed, the site of pollinium attachment changed to the eyes of the butterflies, leading to a higher rate of pollinium removal but lower rate of pollinium deposition. Our quartz glass cylinder choice experiment suggested that visual rather than olfactory cues provided the major stimuli for butterflies to locate these flowers. Hand pollination experiments suggested this species was self‐compatible but pollinator‐dependent. However, the proportion of seeds with large embryos produced in self‐pollinated fruits was significantly lower than in cross‐pollinated fruits, indicating a significant inbreeding depression. Unlike many other orchid species, fruit set was higher than rates of pollinium removal, indicating a high level of pollination efficiency in a species with friable pollinia. Shifts from moth to butterfly pollination in the genus Habenaria parallel other orchid lineages providing insights into the potential for pollinator‐mediated floral trait selection.

In this study, Anoectochilus formosanus polysaccharide (AFP) was acquired a via water extraction and alcohol precipitation method. The immunoregulatory activity of AFP was first evaluated on cyclophosphamide (Cy)-treated mice. Galacturonic acid, glucose and galactose were confirmed to be the main components of AFP. AFP demonstrated the ability to stimulate the production of TNF-α and IL-6 in RAW 264.7 macrophages. Not surprisingly, the activation of the NF-κB signaling pathway by AFP was validated via Western blot analysis. Furthermore, AFP could alleviate Cy-induced immunosuppression, and significantly enhance the immunity of mice via increasing the thymus index and body weight, stimulating the production of cytokines (IgA, IgG, SIgA, IL-2, IL-6 and IFN-γ). The improvement in the intestinal morphology of immunosuppressed mice showed that AFP could alleviate Cy-induced immune toxicity. These results have raised the possibility that AFP may act as a natural immunomodulator. Overall, the study of AFP was innovative and of great significance for AFP’s further application and utilization.