Stochasticity Prevails but Differs: Tissue-Specific Assembly of Gut Microbiomes Across Seasons in an Amphibian Model

Gut microbiota generally undergoes dynamic remodeling in concert with multifaceted self-regulation of amphibian hosts during key life stages, such as metamorphosis and hibernation. However, the spatiotemporal dynamics of amphibian gut microbiomes across the lifecycle remain poorly understood. In this study, we applied 16S rRNA gene amplicon sequencing to characterize the gut microbiomes of cultivated Black-spotted frog (Pelophylax nigromaculatus) across seasons. The gut microbiomes exhibited tissue-specific succession, and structural discrepancies between gut regions fluctuated temporally. Both small- and large-intestine microbiomes showed temporal decay patterns in abundance-unweighted intercommunity indices, but not in abundance-weighted indices. Compared with large-intestine microbiomes, small-intestine microbiomes were more randomized yet more centralized in terms of amplicon sequence variants, particularly within Proteobacteria (especially Pseudomonas). The alpha diversity of small-intestine microbiomes was comparatively lower, and their taxonomic composition was more stable over time. We further elucidated the assembly mechanisms of gut microbiomes by systematically analyzing dominant driving factors, ecological processes, phylogenetic traits, source-sink relationships, and co-occurrence networks. Stochastic processes played a dominant role in gut microbiome assembly, while deterministic processes (e.g., habitat filtering and microbial interaction) contributed more strongly to large gut microbiomes than to small gut microbiomes. Overall, this study provides insights into the ecological dynamics and assembly mechanisms of amphibian gut microbiomes across the lifecycle and may inform targeted microbiome modification for amphibian breeding and conservation.