Comparative analysis of cloacal microbiota in Henophidia (non-venomous) and Caenophidia (venomous) snakes

The evolutionary divergence between Henophidia (non-venomous) and Caenophidia (venomous) snakes has produced distinct cranial morphologies, digestive strategies, and presence of specialised venom systems in Caenophidia , yet the extent to which these long-standing diverging trajectories have shaped cloacal microbiota assembly remains poorly understood. We characterised cloacal microbiota in 70 captive snakes (52 Caenophidia , 18 Henophidia ) by 16S rRNA amplicon sequencing. Beta diversity was tested by PERMANOVA, differential abundance by ANCOM-BC2, community types by Dirichlet Multinomial Mixture modelling (DMM), and microbial interactions by SparCC co-occurrence networks. Predicted functional potential (PICRUSt2) was analysed by ALDEx2 differential abundance testing and elastic net feature selection. Henophidia exhibited significantly higher bacterial richness and greater compositional variability than Caenophidia . Community composition showed clade-associated differences (PERMANOVA) and partitioned into two distinct DMM community types. The Henophidia network was 11.9-fold denser and more modular, with Burkholderiaceae as a keystone hub, whereas the Caenophidia network was sparse. Henophidia showed predicted enrichment in C1 metabolic pathways (ethylmalonyl-CoA, formaldehyde assimilation I, glycine betaine degradation I, methylaspartate cycle), aromatic compound catabolism, and nitrogen recycling, whilst Caenophidia showed enrichment in allantoin and glucuronate degradation. This multi-method analysis suggests Burkholderiaceae as a candidate keystone taxon in Henophidia and indicates that phylogenetic clade is a major contributor to cloacal microbiota structure. The lower richness in Caenophidia raises a testable hypothesis that broad-spectrum antimicrobial activity of their venom components may selectively filter susceptible microbial lineages, motivating future shotgun metagenomic studies in wild populations of snakes.