Phenotypic covariation among suites of traits may constrain or promote diversification both within and between species, yet few studies have empirically investigated this relationship. In this study we investigate whether phenotypic covariation of craniofacial traits is associated with diversification in an adaptive radiation of pupfishes found only on San Salvador Island, Bahamas (SSI). The full radiation of pupfishes includes generalist, durophagous, and lepidophagous pupfish species. We compare phenotypic variation and covariation (i.e., P matrix) between 1) allopatric populations of generalist pupfish from neighboring islands and estuaries in the Caribbean, 2) SSI pupfish populations not containing the full radiation of fishes, and 3) SSI pupfish populations containing the full radiation in sympatry. Additionally, we interrogate the patterns observed in the P-matrices of two independent F2 hybrid crosses derived from the two most morphologically distinct members of the radiation to make inferences about the underlying mechanisms contributing to the variation in craniofacial traits in SSI pupfishes. We found that the P matrix of SSI generalist populations not found in sympatry with specialists exhibited higher levels of mean trait correlation, constraints, and integration with simultaneously lower levels of flexibility compared to generalist populations on other Caribbean islands and sympatric populations of all three species found on SSI. We also document that while many craniofacial traits appear to be produced via additive genetic effects, variation in key traits such as head depth, maxilla length, and lower jaw length may be produced via non-additive genetic mechanisms. Ultimately, this study suggests that differences in phenotypic covariation significantly contribute to producing and maintaining organismal diversity.

Adaptive radiations offer an excellent opportunity to understand the eco-evolutionary dynamics of gut microbiota and host niche specialization. In a laboratory common garden, we compared the gut microbiota of two novel trophic specialists, a scale-eater and a molluscivore, to a set of four outgroup generalist populations from which this adaptive radiation originated. We predicted an adaptive and highly divergent microbiome composition in the specialists matching their rapid rates of craniofacial diversification in the past 10 kya. We measured gut lengths and sequenced 16S rRNA amplicons of gut microbiomes from lab-reared fish fed the same high protein diet for one month. In contrast to our predictions, gut microbiota largely reflected 5 Mya phylogenetic divergence times among generalist populations in support of phylosymbiosis. However, we did find significant enrichment of Burkholderiaceae bacteria in both lab-reared scale-eater populations. These bacteria sometimes digest collagen, the major component of fish scales, supporting an adaptive shift. We also found some enrichment of Rhodobacteraceae and Planctomycetacia in lab-reared molluscivore populations, but these bacteria target cellulose. Minor shifts in gut microbiota appear adaptive for scale-eating in this radiation, whereas overall microbiome composition was phylogenetically conserved. This contrasts with predictions of adaptive radiation theory and observations of rapid diversification in all other trophic traits in these hosts, including craniofacial morphology, foraging behavior, aggression, and gene expression, suggesting that microbiome divergence proceeds as a nonadaptive radiation.