Evidence that host identity drives differences in A. mexicanus microbiome composition
We next tested whether the identity of the host population impacts microbiome composition independent of habitat by comparing the intestinal microbiome of six-month-old fish from the Río Choy River, Pachón Cave, Tinaja Cave, and Molino Cave that have been bred in the laboratory for generations, were surface sterilized as embryos, and were raised on the same recirculating water system and diet. In this controlled setting, we found that Tinaja and Molino cavefish had significantly lower microbial species richness compared to surface fish suggesting that host genetics alone can impact alpha diversity, the number and distribution of microbial species in the intestine (Figure 3A : Shannon Index, Figure S2B : Inverse Simpson Index).
In addition to impacting microbial species richness, we found that morphotype identity had a significant impact on microbiome composition (Table 1 , Beta Diversity Permanova, p=0.002). To visualize the differences in microbiome composition we summarized ASV abundances into Bray-Curtis dissimilarities and performed principle coordination analysis (PCoA, Figure 3B ). In the PCoA plot comparing laboratory raised fish, the centroid of Tinaja cavefish samples is furthest from surface fish samples and the ellipses estimating covariance overlap except for Pachón and Tinaja cavefish samples (Figure 3B ). PCoA plots of unweighted unifrac and Jaccard distances differently show that the Pachón centroid is ordinated furthest from surface fish and that ellipses estimating covariances overlap in all samples (Figure S3 ). To determine which laboratory-reared morphotypes harbored significantly different microbiomes, we performed a pairwise post-hoc test (Table 2 ). We found that the microbiome of surface fish was significantly different from Pachón and Tinaja cavefish (p.adj = 0.025 and 0.020) but not Molino cavefish (p.adj = 0.075). These results suggest that Tinaja and Pachón cavefish have acquired genetic changes that result in establishment of a different microbiome from surface fish despite being raised in the same habitat. We also found that samples from Pachón and Tinaja cavefish were significantly different from each other suggesting distinct cavefish populations have genetic differences that impact microbiome composition (p.adj = 0.006). Since the fish were all raised under identical conditions, these results suggest that morphotype identity alone accounts for differences in microbiome composition in A. mexicanus .