Comparative analysis of two nonlethal methods for the study of the gut bacterial communities in wild lizards

Fecal samples or cloacal swabs are preferred over lethal dissections to study vertebrate gut microbiota for ethical reasons,but it remains unclear which nonlethal methods provide more accurate information about gut microbiota.We compared the bacterial communities of three gastrointestinal tract(GIT)segments,that is,stomach,small intestine(midgut),and rectum(hindgut)with the bacterial communities of the cloaca and feces in the mesquite lizard Sceloporus grammicus.The hindgut had the highest taxonomic and functional alpha diversity,followed by midgut and feces,whereas the stomach and cloaca showed the lowest diversities.The taxonomic assemblages of the GIT segments at the phylum level were strongly correlated with those retrieved from feces and cloacal swabs(rs>0.84 in all cases).The turnover ratio of Amplicon Sequence Variants(ASVs)between midgut and hindgut and the feces was lower than the ratio between these segments and the cloaca.More than half of the core-ASVs in the midgut(24 of 32)and hindgut(58 of 97)were also found in feces,while less than 5 were found in the cloaca.At the ASVs level,however,the structure of the bacterial communities of the midgut and hindgut were similar to those detected in feces and cloaca.Our findings suggest that fecal samples and cloacal swabs of spiny lizards provide a good approximation of the taxonomic assemblages and beta diversity of midgut and hindgut microbiota,while feces better represent the bacterial communities of the intestinal segments at a single nucleotide variation level than cloacal swabs.