Ecological Boundaries Drive Microbial Diversity and Spillover Risk in Mixed Pastoral Systems of Inner Mongolia

Background Over 60% of human infectious diseases originate from animals, with risks amplified at wildlife–livestock–human interfaces, particularly in mixed pastoral systems. In the grassland regions of Inner Mongolia, interactions among free-ranging livestock, wild fauna, and human activity create hotspots for pathogen exchange. However, the ecological drivers and transmission patterns of microbial communities in these settings remain poorly understood. Results We conducted extensive field surveys and collected 1,527 individuals across 20 sites spanning pastures and natural grasslands. Metagenomic and metatranscriptomic sequencing yielded over 30.5 million contigs, identifying 41 bacterial, 24 fungal, 28 parasitic, and 174 viral genera or species. Arthropod vectors and wild birds harbored the highest viral diversity, including eleven novel species. Ecological network analyses revealed substantial microbial sharing among phylogenetically distant hosts, particularly mediated by arthropod vectors and resident birds. Generalized linear and additive models identified livestock density, host body size, phylogenetic distance, and habitat overlap as key predictors of microbial richness, abundance, and cross-species transmission. Random forest models highlighted mosquitoes, Eurasian tree sparrows, and rock pigeons as high-risk microbial spreaders. Conclusions This study provides a system-wide view of microbial diversity and spillover dynamics in Inner Mongolia’s pastoral landscapes. Arthropod vectors and resident birds act as ecological boundaries linking hosts across habitats, while different microbial groups display distinct network and model patterns. Analyses restricted to a single microbe type risk misrepresenting transmission dynamic, underscoring the value of multi-taxa, trait-based surveillance to improve spillover prediction and control in agro-ecological systems.