Diversity and composition of root-associated fungal communities in critically small population of Cypripedium subtropicum

Cypripedium subtropicum is a critically small population wild orchid. Understanding the composition and diversity of its root- associated fungal communities is essential for its conservation and artificial propagation. Illumina Novaseq high- throughput sequencing was used to analyze the diversity and composition of fungal communities in the root-endosphere and rhizosphere soil of C. subtropicum collected from different regions, habitats, and population types in Yunnan Province. The number of amplicon sequence variants (ASVs) differed by only 0.33% between root-endosphere and rhizosphere soil samples. A total of 34,564 ASVs were detected in root-associated samples from Wenshan Prefecture (Maguan, Malipo, Xichou), accounting for 87.76% of the total ASVs across all samples. Fungal richness of root-associated samples in C. subtropicum was highest in Wenshan, followed by Baoshan, Nujiang, and Kunming. Three fungal ASVs were shared between root-endosphere and rhizosphere soil across 120 samples from 12 habitats. Fungal community composition and diversity differed significantly between root-endosphere and rhizosphere soil samples. Samples from Wenshan showed the highest species richness, evenness, and diversity, whereas those from Kunming exhibited the lowest. In Malipo County, fungal abundance and diversity did not differ significantly between wild and introduced populations, but both were significantly higher than in seed-propagated populations in Kunming. Notable regional variations in dominant fungi were observed: Mortierella dominated Wenshan samples, Archaeorhizomyces dominated Nujiang samples, Ilyonectria dominated Baoshan samples, and Fusarium dominated Kunming cultivated samples. NMDS analysis showed Wenshan samples aggregated closely, while other regions’ samples were dispersed. Core fungal species including Mortierella sp., Fusarium solani , and Sebacina sp ., which were distributed across all regions. This study reveals clear regional and habitat- driven patterns in root- associated fungal communities of C. subtropicum . The findings provide a reliable scientific basis for the conservation and artificial propagation of this endangered orchid.