Comparison of the intestinal microbiome between wild and laboratory-reared A. mexicanus
To understand how similar the microbiome of A. mexicanus in their wild habitat is to those raised in the laboratory, we compared the microbiomes of field collected surface fish and Pachón cavefish to their laboratory-reared counterparts that had been bred in the laboratory for generations and were raised on the same diet and in the same water. The laboratory habitat is different from both the cave and river habitats. In the laboratory, the fish experience lower temperature and pH, higher conductance and dissolved oxygen, an invariable light cycle, more crowded growth conditions, and a consistent high-nutrient diet (Table S1 ). We found that laboratory-raised fish had greater richness of microbial species in the intestinal microbiome compared to fish in the wild habitat (Figure 3A , Shannon Index,Figure S2 , total observed species, phylogenetic index); alpha-diversity was significantly different in samples from surface fish and Pachón cavefish collected in the field compared to surface fish and Pachón cavefish raised in the lab (Shannon Diversity Tukey’s post-hoc test, wild surface vs lab surface FDR p.adj = 0.001, wild Pachón vs lab Pachón FDR p.adj = 0.008). Since fish are colonized with microbes after hatching, our results suggest the laboratory supports a richer microbial diversity than either natural environment.
To obtain a graphical representation of the differences in microbiome composition between the wild and the laboratory-reared fish we summarized ASV abundances into Bray-Curtis dissimilarities and performed principle coordination analysis (PCoA, Figure 3B ). In the PCoA plot, wild surface fish and wild Pachón cavefish form two partially overlapping clusters that are separate from laboratory-raised fish. We found that habitat (wild versus lab) significantly impacted microbiome composition (Table 1, adonis R function, or Permanova, p =0.002,); beta-diversity was significantly different in samples from surface fish and Pachón cavefish collected in the wild compared to surface fish and Pachón cavefish raised in the lab (Table 2,pairwise post-hoc test, wild surface vs lab surface FDR p.adj = 0.005, wild Pachón vs lab Pachón FDR p.adj = 0.006). In summary, the intestinal microbiome of A. mexicanus in the laboratory has increased richness and altered composition compared to A. mexicanus in their natural habitat. These results have implications for researchers investigating the evolution of cavefish traits that may be influenced by microbiome composition.