Host-Mediated Selection Shapes Conserved Root Bacterial Microbiomes Across Geographically Separated Thismia Species

Thismia species are non-photosynthetic plants entirely dependent on fungal partners for carbon and nutrients. While their arbuscular mycorrhizal associations are well-documented, bacterial symbiont roles remain unexplored. Using 16S rRNA gene amplicon sequencing, we investigated endophytic bacterial communities in T. gardneriana, T. javanica, and T. mirabilis from geographically distinct locations in Thailand. Despite geographic separation, Thismia spp. consistently harbored bacterial compositions taxonomically and functionally distinct from surrounding soil microbiomes. Root endospheres were significantly enriched in Pseudomonadota and Bacteroidota, particularly Puia, while showing reduced compositional dynamics of Acidobacteriota and Planctomycetota. Bacterial communities in Thismia roots were markedly distinct from surrounding soil, while root endosphere communities from geographically distinct habitats clustered together regardless of spatial separation. Mantel and partial Mantel tests confirmed that host species identity, not geographical location, was the primary predictor of root bacterial community structure. Functional prediction analyses suggested root-associated communities were enriched for nitrogen cycling pathways, particularly nitrogen fixation and nitrate reduction. The selective enrichment of Bacteroidota, known for nitrogen fixation and phosphate mobilization, suggests these bacteria provide critical nutritional support in nutrient-poor forest floor environments. Isolated root strains belonged exclusively to Bacillota, including Neobacillus with plant growth-promoting traits. Our findings highlight the importance of tripartite plant-fungal-bacterial interactions in Thismia nutritional ecology.