Figure 4.

Two-dimensional surface area of cave erythrocytes is significantly larger than surface erythrocytes. Erythrocytes from each population were stained with Wright–Giemsa and visualized under light microscopy (a–d, scale bar 10 µm). The total (a–d, yellow ring) and nuclear (a–d, dark blue ring) two-dimensional area was calculated for each erythrocyte in ImageJ²⁹ (v2.0.0). The total surface area was significantly higher in all cave populations compared to surface (p < 0.00003) but did not differ from each other (e). Although the total two-dimensional area was higher in cavefish, there were no differences in the two-dimensional area of the nuclei (f), suggesting the differences in total two-dimensional area are due to differences in the cytoplasm, where hemoglobin is found. Two outliers were detected (the highest Chica value and the lowest Pachón value). Removal of these outliers did not change statistical significance (Table S6).