*2.2. Tissue Morphology*

H&E and trichrome-stained gular tissue cross-sections are shown in Figure 3. Gular tissue dissected from frogs belonging to the genus *Xenopus* are substantially thicker and contain a higher density of muscle bundles and mucous glands. Observations of the collagen structure between the families showed marked contrasts. The collagen structure in the male *EC* had a continuous and crimp structure, in stark contrast to the female *EC*, *XL*, and *XM*, which have more spread out crimp structures. Additionally, the tissue sample of the genus *Xenopus* has a nearly linear collagen structure. Interestingly, *XL* and *XM* have distinct regions where the collagen is perpendicularly aligned. These areas seemingly discretize the tissue into collagen segments.

**Figure 3.** Tissue morphology by histology of gular tissue dissected from various species of frogs. Scale bar 100 μm. Muscle bundles (black arrows), mucous glands (red arrows), collagen structure (green arrows), perpendicularly aligned collagen (yellow arrows), urothelium (orange), lamina propria (purple), and detrusor muscle (grey).

Histological cross-sections of leg tissue show similar trends with the collagen structures between the frog species. *EC* tissue has a more pronounced crimp structure compared to *XL* and *XM*. Once again, the muscle bundles and mucous glands constitute a more substantial portion of the tissue in *Xenopus* samples. Collagen in the male *EC* leg has a less compact crimp structure than that found in the gular tissue. Similar to the gular tissue of the *Xenopus* samples, the collagen structure is more linear and has minimal crimping. Leg tissues do not exhibit the same inflatable function as the gular tissue; therefore, it does not require the crimped collagen structure. Lastly, sectioning of fresh rat bladder showed an abundance of muscle bundles with intermittent collagen fibers, dissimilar to the frog tissues. Collagen ultrastructures found in gular tissues were not visually identified in the rat bladder seen in Figures 3 and 4.

**Figure 4.** Hematoxylin and eosin (H&E) staining of leg tissue dissected from various species of frogs. Scale bar 100 μm. Muscle bundles (black arrows), mucous glands (red arrows), collagen structure (green arrows), perpendicularly aligned collagen (yellow arrows), urothelium (orange), lamina propria (purple), and detrusor muscle (grey).
