Gut microbiome diversity and divergence among taxa
We sequenced a total of 11,152,147 reads across all samples (Table S2). We identified 5,174 bacterial taxa in 48 samples. Similar to other ray-finned fishes (Youngblut et al. 2019), proteobacteria is the predominate microbial taxon (Figure S3). We did not find any significant differences among species in Chao1 or Shannon diversity indices (Kruskal-Wallace [pairwise], P > 0.05; Figure 1). San Salvador Island pupfishes clustered together relative to the three outgroup generalist species, indicating strong host phylogenetic signal associated with overall microbiome diversity (Figure 2; Figure S4). Water and tissue controls were scattered throughout the NMDS plots but were clearly distinct from Cyprinodon microbiome samples with the exception of one tissue control that clustered near the outgroup species, possibly due to contamination during dissections (Figure 2).
Multiple regression analyses of the effects of dietary specialization (generalist, scale-eater, or molluscivore) and the fixed effect of population origin (two different lakes on San Salvador Island, Lake Cunningham, North Carolina, and El Potosí) on NMDS axes 1 and 2 confirmed that population origin and scale-eating had a significant effect on microbiome divergence along both axes (NMDS1: scale-eatersP = 0.001; NMDS2: scale-eaters P = 0.018).