Recovered microbiome of an oviparous lizard differs across gut and reproductive tissues, cloacal swabs, and faeces.

Microbial diversity and community function are related, and can be highly specialized in different gut regions. The cloacal microbiome of Sceloporus virgatus females provides antifungal protection to eggshells, a specialized function that suggests a specialized microbiome. Here, we describe the cloacal, intestinal, and oviductal microbiome from S. virgatus gravid females, adding to growing evidence of microbiome localization in reptiles and other taxa. We further assessed whether common methods for sampling gastrointestinal (GI) microbes - cloacal swabs and faeces - provide accurate representations of these microbial communities. We found that different regions of the gut had unique microbial communities. The cloacal microbiome showed extreme specialization averaging 99% Proteobacteria (Phylum) and 83% Enterobacteriacaea (Family). Enterobacteriacaea decreased up the GI and reproductive tracts. Cloacal swabs recovered communities similar to that of lower intestine and cloacal tissues. In contrast, faecal samples had much higher diversity and a distinct composition (common Phyla: 62% Firmicutes, 18% Bacteroidetes, 10% Proteobacteria; common families: 39% Lachnospiraceae, 11% Ruminococcaceae, 11% Bacteroidaceae) relative to all gut regions. The common families in faecal samples made up <1% of cloacal tissue samples, increasing to 43% at the upper intestine. Similarly, the common families in gut tissue (Enterobacteriaceae and Helicobacteraceae) made up <1% of the faecal microbiome. Further, we found that cloacal swabs taken shortly after defaecation may be contaminated with faecal matter. Our results serve as a caution against using faeces as a proxy for GI microbes, and may help explain high between-sample variation seen in some studies using cloacal swabs.