Diet-Associated Gut Microbiome Divergence Contributes to Phenotypic Differentiation Among Geographically Structured Populations of Rana dybowskii

Geographic phenotypic variation is a widespread feature of natural populations, often driven by local environmental heterogeneity and trophic interactions. While the gut microbiome is recognized as a key mediator between environmental resources and host physiology, its role in the adaptive differentiation of amphibians remains insufficiently explored. In this study, we employed an integrative approach—combining morphological measurements, DNA metabarcoding for dietary analysis, and 16S rRNA sequencing for gut microbial profiling—to investigate three geographically structured populations (Dandong [DD], Fushun [FS], and Benxi [BX]) of the Dybowski’s frog (Rana dybowskii). The results showed that the morphological characteristics (body weight, head-body length, hindlimb length, etc.) of DD were significantly higher than those of FS and BX. Dietary analysis via gut content DNA metabarcoding identified distinct food structures; notably, the DD population consumed a higher proportion of Dipteran prey (e.g., Smittia). Correspondingly, the gut microbiota showed significant population-level clustering. We identified an enrichment of specific functional bacteria in the DD population, such as Arthrobacter, which is known for its chitin-degrading capabilities, aligning with the consumption of chitin-rich insect prey. Mantel tests further confirmed a strong correlation between dietary composition, microbial community structure, and host morphological traits. These findings suggest that diet-driven gut microbiome variation is consistent with a contributing role in the phenotypic differentiation of R. dybowskii across different habitats, although causal relationships remain to be tested directly. By integrating trophic ecology with microbial landscapes, this study provides new insights into the adaptive evolution and resource management of amphibians in heterogeneous environments.