*2.1. Mechanical Properties*

The uniaxial tensile tests illustrate marked differences between the frogs of genus *Xenopus* and those of *Eleutherodactylidae*, in addition to location-dependent properties. Figure 2 shows representative stress vs. strain and membrane tension characteristics of the species' gular skin tissue and leg skin tissue, with rat bladder as a comparison. Mean and standard deviation (SD) values are summarized in Table 1. It is evident in both Figure 2A,B that *Xenopus* tissues were stiffer relative to those of *Eleutherodactylidae,* as observed by the steep rise in the stress vs. strain curve before ultimate tensile strength (UTS) was reached. As shown in Table 1, there is a general trend of leg tissue being stiffer at 20% elongation, indicated by a higher secant modulus.

**Figure 2.** Representative uniaxial stress–strain curves of (**A**) gular skin tissue and (**B**) leg skin of different anuran species and their comparison to the rat bladder.

Furthermore, male *EC* tissues had lower secant modulus values than all samples tested, highlighting their uniqueness. Interestingly, gular skin tissue from *XM* displayed similar elongation at failure when compared to male *EC*, which were, on average, 350% and 398%, respectively. However, *XM* required much higher stress to elongate the same amount as *EC*, shown in Figure 2A. *XL* behaved as expected, with a very steep slope (indicative of a high secant modulus) followed by quick failure at 104% and 108% for gular and leg tissue, respectively, in stark contrast to samples from *EC* (gular: 398%, leg: 348%). Gular tissue of the male *EC* exhibited an average ultimate tensile strength (UTS) of 1263 ± 134 kPa, half that of its female counterpart (2142 ± 1789 kPa) and a quarter that of the *XL* (4461 ± 2215 kPa) and *XM* (4156 ± 1973 kPa). Comparisons between male and female *EC* show that male gular tissue exhibited higher elongation at break, of ~400% and ~340%, respectively. Figure 2B illustrates the stress vs. strain behavior of leg tissue, displaying similar trends as gular tissue. *Xenopus* samples had higher secant modulus values than *Eleutherodactylidae* in addition to a lower elongation at failure. The most striking observation is the clear difference between the final elongations between the tissue types. In all cases, the gular tissue of a single specimen had a greater elongation than the leg tissue.

Further comparisons with male rat bladder illustrated that there is a closer biomechanical resemblance of male *EC* gular tissue to the urinary bladder. The rat bladder had an average elongation of ~410%, similar to that of male and female *EC* tissues. Additionally, it had a mean peak stress of ~3000 kPa, highlighting its ability to retain urine and prevent failure.

We determined the secant modulus for assessing the stiffness of the sample. From Table 1, it is evident that leg tissues were generally stiffer than the same species' gular tissue, indicated by a higher mean value of the secant modulus. Male EC exhibited slightly higher mean secant modulus values of 373 kPa and 318 kPa for gular and leg tissue, respectively. As expected, the male EC had lower secant modulus values compared to the other species, and XL leg tissue exhibited the highest mean secant modulus of 4863 kPa. In general, leg tissues had higher secant modulus values than gular tissues. However, these trends did not show any statistical significance when compared to the rat bladder (Table 1).


