Dietary ecology structures gut microbiome assembly in sympatric wild mammals from Kruger National Park, South Africa

The gut microbiome plays a critical role in host health, yet its composition in African wild mammals remains underrepresented. We investigated how dietary groups (carnivores, non-ruminant, and ruminant herbivores) influence gut microbiota composition and diversity in coexisting wild mammals from the Kruger National Park, South Africa. After quality filtering, 89 samples (carnivores: n = 31; non-ruminants: n = 37; ruminants: n = 21) were included. Gut microbial communities clustered strongly by dietary group (weighted: pseudo-F = 27.76, p = 0.0001) with smaller but significant species-level differences within groups. Random Forest classification accurately distinguished dietary groups (93%), with six samples showing atypical clustering in beta-diversity analyses. Non-ruminants showed the highest alpha diversity (Shannon: median 6.89 [IQR 6.53–7.08]; Faith’s PD: 18.02 [15.25–20.43]), followed by ruminants and carnivores (both p < 0.001). Taxonomic profiling revealed group-specific signatures. Carnivores were dominated by Fusobacteriaceae, Clostridiaceae and Peptostreptococcaceae. Both ruminants and non-ruminants showed high levels of fibre-degrading bacteria, such as Rikenellaceae and Oscillospiraceae, while non-ruminants were additionally enriched in Spirochaetaceae and p -251-o5, reflecting hindgut fermentation strategies. Predicted functional profiling using PICRUSt2 revealed metabolic pathway enrichments, with carnivores enriched in amino acid biosynthesis and degradation pathways. In contrast, ruminants showed enrichment in branched amino acid and tryptophan biosynthesis, while non-ruminants were enriched in aerobic respiration, TCA cycle, and L-leucine degradation pathways. Focal species comparisons distinguished black from white rhinoceros and spotted hyaena from other carnivores, consistent with diet-driven metabolic specialisation. Species-level differences were evident at family and genus levels, with variable importance analyses identifying key predictive taxa. Diet strongly influenced gut microbiota composition and diversity, with herbivores exhibiting richer, more complex communities compared to carnivores. Among herbivores, non-ruminants harboured higher diversity than ruminants, likely due to their diverse, less specialised hindgut fermentation. These findings advance our understanding of wildlife microbiomes and provide a foundation for future conservation and health monitoring in African ecosystems.