Intestinal microbiome of A. mexicanus cavefish and surface fish in their natural habitats
We first analyzed the microbiome of A. mexicanus surface fish and cavefish morphotypes by comparing fish that were captured in the Rio Choy River and Pachón Cave, respectively. The Pachón Cave is perpetually dark, and previous studies analyzing gut contents of field caught fish indicate that adults are opportunistic generalists with a seasonally variable diet of mostly detritus and bat guano . In contrast, surface fish have a normal day/night cycle and consumes insects, crustaceans, and annelids . In addition to consuming distinct diets, the fish reside in water with dissimilar chemical qualities; water in the Pachón cave at the time of collection was lower in temperature, dissolved oxygen, conductance, copper, and phosphate (Table S1 ). Despite these distinct habitats, we did not observe significant differences in the richness or composition of the intestinal microbiome between fish collected at each site (Shannon Index one-way ANOVA with Tukey’s post hoc test p.adj = 0.84, Beta diversity Permanova with pairwise post hoc test p.adj = 0.098). However, when we examined the proportional abundance of taxa, we found that the fish were dominated by only one or two phyla and the dominant phyla differed between morphotypes (Figure 2 ). Firmicutes were the most abundant phylum in surface fish while Proteobacteria and Bacteroidota were the most abundant phyla in Pachón cavefish (Figure 2A,B , average proportional abundance (APA) of Firmicutes = 0.15 Pachón, 0.64 surface, Proteobacteria = 0.62 Pachón, 0.03 surface, Bacteroidota = 0.12 Pachón, 0.09 surface). We also observed differences in pathogenic classifications; one of the Pachón cavefish samples (p11) had mostly Spirochaetota of the genusBrevinema which was not found in wild surface fish samples and is likely a pathogen . Unlike Pachón cavefish samples, Surface fish samples were dominated by the genus Clostridium (APA = 0.008 Pachón, 0.52 surface). In addition, two surface fish samples (s3, s4) had a high proportion of the genus Cetobacterium which was found at very low abundance in only one Pachón cavefish (APA = 0.07 Pachón, 0.20 surface). The data suggests that in their natural habitat, A. mexicanussurface fish and cavefish gut microbiomes are dominated by only a few microbes and that the most abundant phyla differ between populations.