Collection of intestinal contents from wild fish
Field collection of Astyanax mexicanus for this study was conducted under permit no. SGPA/DGVS/03634/19 granted by the Secretaría de Medio Ambiente y Recursos Naturales to Ernesto Maldonado. Total intestinal contents were collected from wild Pachón cavefish and Río Choy surface fish in July 2019 using the following protocol: Captured fish were placed in their environmental water and euthanized by immersion in MS-222 on the day of capture. The gut was removed by making a cut posterior to the stomach and directly at the urogenital pore. The gut was transferred to sterile Petri dish and the contents were washed out by inserting a sterile syringe into one end and expelling 200 µL sterile PBS. The contents and PBS were transferred to 300 µL RNA later. The samples were stored at room temperature for four days, shipped on ice, and stored at -20°C until DNA extraction was carried out. The PBS and RNA later used to collect samples were included in the DNA extraction and sequencing pipeline and did not produce read counts high enough to analyze. Thirteen out of twenty wild samples that were collected had enough read counts to analyze: five Pachón (3 males, 1 female, 1 undifferentiated) and six Río Choy (3 males, 3 females). The weight of fish used for sequencing is shown in Figure S1A .