*4.1. Specimen Collection*

Euthanized male and female *E. Coqui* were obtained from Atlanta Botanical Garden (Atlanta, GA, USA). The females were tested as a species-specific control as females do not tend to inflate their gular tissue. The number of biological replicates was limited because these frogs were taken from live populations, and excessive sampling of frogs causes ecological instability. In total, four male ECs and two female ECs were collected for all the experiments. Although the age of the frogs may influence the mechanics of the tissue, *E. Coqui* was obtained from a population, and an exact age could not be determined. The estimated age was between 1 and 5 years. In total, four euthanized male *X. Laevis* and four male *X. Muelleri* were obtained from Xenopus Express Inc. These species were used as negative controls as they do not display any external tissue inflation during calls. All specimens were euthanized on-site before either fixation or freezing and were transported overnight. Samples used for tensile testing were received frozen in ice and immediately dissected for sample collection. Samples were then stored at −20 ◦C until testing was performed. Fixed samples were used for histology and microscopy, while frozen samples were used for tensile tests and biochemical assays. The skin tissue of the desired regions was dissected in the laboratory with surgical scissors and scalpels. The specimen's gular and leg skin tissue were of particular interest; areas examined can be seen in Figure 1b. Rat bladders were dissected in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee (RA17M330, 7/23/2018).

## *4.2. Uniaxial Tensile Test*

We determined the mechanical properties of the unfixed and hydrated skin tissues by conducting uniaxial tensile tests using the Instron Tensile testing equipment (MTS Criterion ™ 40). Samples (*n* = 3) per group (male *EC:* three biological repeats; female *EC:* two biological repeats; male *XL:* three biological repeats; male *XM:* three biological repeats) for both gular skin and leg were cut into small rectangular pieces and measured for their thickness, width, and length. Cut samples ranged from 1 mm to 3 mm wide and 4 mm long. Average sample thickness (0.1–0.4 mm) was measured using an electronic caliper, which varied due to di fferences in species and the site of extraction (gular vs. leg). Generally, *EC* samples were thinner than those of *XL*/*XM*. The sample dimensions were limited to the size of the frogs, as EC is extremely small (~1.5 inches for male). Reference state dimensions were established after mounting the tissue onto the apparatus and tared before testing began. Preconditioning was not performed prior to tensile loading. We also assumed that the tissues would behave isotropically under tensile loading. We acknowledge that the biaxial test would be more predictive of tissue expansion; however, because of the small size of *EC*, we opted for comparative uniaxial analysis. Each side of the cut samples was then glued in between two pieces of thick paper to increase the surface area that is clamped onto the instrument, similar to a study conducted by Dahms et al. [42]. The strain was calculated using the distance between the clamps. The samples were extended with a strain rate of 0.5 mm/min, using a 5N load cell. Engineering stress, membrane tension, strain, and secant modulus (at 20% strain) were calculated and visualized with GraphPad Prism 8.0. Secant moduli were calculated using the stress and strain values before and after 20% strain.
