Study 1: Is the cloaca a uniquely specialized region of the gastrointestinal and reproductive tracts?
Sceloporus virgatus lizards are small insectivorous animals found in the Madrean Sky Islands of the southwestern United States and south into Mexico (Stebbins, 2003). Our study population is at the northern reach of their range, in Cochise County, Arizona near the American Museum of Natural History’s Southwestern Research Station (SWRS). To address the adaptive hypothesis that the cloacal microbiome will be unique from that of the upper GI and reproductive tracts, we collected gravid Sceloporus virgatusfemales (n = 8) between 28 June and 01 July 2019 using a loop of fishing line tied to a retractable fishing pole. Female cloacae were swabbed immediately following capture. Females were then kept in large outdoor enclosures at SWRS with access to water bowls and naturally occurring prey for 1-4 d until being shipped overnight to the University of Puget Sound in individual plastic containers on ice packs. Upon arrival, we euthanized females using buffered MS-222 (Conroy et al., 2009). We collected transverse sections of tissue (~2-4 mm long) from the cloaca (expanded tissue immediately above the vent), lower intestine (~10 mm above cloaca), upper intestine (~5 mm below stomach), and oviduct (at the position of the lowest egg on the left side) using heat sterilized instruments, and did not attempt to separate or isolate mucosal vs. luminal contents. Microbes were eluted from swabs into an Amies solution by manual shaking and were stored at -80°C, as were tissue samples, until DNA extraction.