Cloaca

Snakes were sampled from two sites in Winston County (one individual, N32.98463 X W088.9980) and Lafayette County (seven individuals, N34.427238 X W089.38631), Mississippi, in spring of 2011 and spring of 2012 (Table 1). All snakes sampled were encountered during nighttime surveys along small streams leading to larger bodies of water. Snakes were collected by hand and safely restrained with clear plastic tubing placed over the head during sample collection. Once restrained, snakes were palpated to evaluate whether prey items were present in the GIT then the exterior cloaca of the snake was cleaned using a sterile alcohol pad. This sterilization step was to ensure that the cloacal sample primarily included cloaca associated microbes rather than environmental or transient microbes. Following cleaning, cloacal swabs were collected by inserting a sterile polyester-tipped applicator (Fisher Cat# 23-400-122) into the cloaca, taking care not to insert beyond the coprodeum and into the large intestine, then turning the swab several times before withdrawing. Once withdrawn the applicator was immediately placed into a sterile 2 ml tube and placed on ice before being transferred to a -20°C freezer prior to DNA extraction.
Three individuals (samples 103, 110, and 111) were sampled in more detail to determine the bacterial communities of their small and large intestines. These snakes were transported to the Department of Biology at the University of Mississippi where they were humanely euthanized. Immediately following death, a mid-ventral incision was made to expose the GIT, which was then removed. None of the individuals had identifiable prey items present in the GIT. Incisions were made in proximal and distal ends of both the small and large intestines, which were then swabbed with sterile polyester-tipped applicators that were immediately placed in sterile 2 ml collection tubes and frozen (-20°C) until DNA extraction. The remainder of the snake was preserved in 10% buffered formalin and whole specimens were deposited at the Sam Noble Oklahoma Museum of Natural History (Table 1).

Handling, imaging, and spectrometer analyses of animals in this study were permitted by and conducted in accordance with all stakeholders involved including the United States National Park Service (Permit #: GRSM-2022-SCI-2173), the University of Tennessee, Knoxville Institute of Animal Care and Use Committee (Protocol #: 2899-0522), Tennessee Wildlife Resources Agency (License #: 5338), and North Carolina Wildlife Resources Commission (License #: 22-SC01508). This study was also conducted in accordance with ARRIVE guidelines¹⁵ . As part of an ongoing study researching southern Appalachian montane salamander biofluorescence, we captured several P. metcalfi individuals with biofluorescent vibrant green speckling along their venters. This speckling pattern varies in density and intensity but begins just posterior to the gular fold and extends across the venter to the apex of the tail (Fig. 1). Occasionally, this pattern extends into the dorso-lateral region particularly around the base of the tail and the cheeks but is almost never seen dorsally. Throughout the course of this study, we have seen this pattern displayed by salamanders within the P. jordani and glutinosus complexes. Interestingly, this pattern was not consistent across every P. metcalfi individual that we sampled. We therefore hypothesized that this ventral biofluorescent speckling was sex-determined.Figure 1

Images of the biofluorescent patterns of P. metcalfi filtered with a 500 nm longpass filter while exposed to blue light. (A) Ventral view of a male displaying the biofluorescent speckles. (B) Dorsal view of a male displaying the speckling down each digit with fluorescing bones. (C) Ventral view of a large female displaying post-cloacal speckling and fluorescent fat stores. (D) Male displaying the ventral trunk fluorescent speckles. (E) The one large female displaying less intense full ventral trunk speckling.

Sampling occurred within Great Smoky Mountains National Park in Haywood County, NC, June and July 2022 at ~ 5000 ft in elevation. We conducted both diurnal and nocturnal visual encounter and natural cover object surveys capturing all P. metcalfi encountered and placed them in clean plastic bags. Clean gloves were worn while handling each individual and equipment was sterilized with 0.7% Chlorohexidine gluconate if it came into contact with any salamander. Each individual was measured for SVL and examined for the presence of a mental gland. Every individual was then photographed dorsally and ventrally in a dark environment while exposed to blue excitation light (Nightsea Xite Flashlight, 440–460 nm) using a digital Olympus Tough TG-6 and/or a DSLR Canon EOS Rebel T8i camera with 500 nm longpass filters. Both cameras were set to factory macrophotography presets and exposure compensation was adjusted (− 1–0) to account for ambient light. Photographs were taken while the animals were in a plastic bag either in the field or lab depending on the proximity of their capture to the lab space. We used an OceanInsight FLAME-S-VIS–NIR–ES spectrometer equipped with a 600 nm UV/VIS fiber optic probe and linear variable longpass filter set to 500 nm to collect spectral data on each salamander across four anatomical regions in a dark environment while exposed to blue excitation light^{5 (link),8 (link)}. The sampled anatomical regions included the base of the tail just posterior to the cloaca ventrally and dorsally, and any two areas which exhibited the most intense biofluorescence, ventrally and dorsally. In one case, we opportunistically analyzed the spectral emissions of the mucous of a female individual.
We used the Wilson formula¹⁶ to calculate 95% confidence intervals (CI) using our observed probabilities of ventral speckling (P; n = 26). To compare the probabilities of ventral speckling between presumed adult males and females (n = 26) we ran contingency tables to test the null hypothesis that males and females are equally likely to have ventral speckling of any kind (α = 0.05). We then conducted a binomial regression to test the null hypothesis that SVL had no effect on presence of female ventral fluorescent speckling (n = 19). All analyses were conducted, and figures were created, in R Studio (v. 2022.02.3+492).