Discussion
Using a common garden experiment we show that differences in gut
microbial diversity across Cyprinodon pupfish species largely
reflect phylogenetic distance among generalist populations in support of
phylosymbiosis (Bordenstein and Theis 2015), rather than novel trophic
specializations as predicted by adaptive radiation theory. Our study is
highly consistent with Ren et al. (2016) which also found limited
microbiome divergence and minimal associations with ecomorph in an
adaptive radiation of Puerto Rican Anolis lizards, even within
wild lizards. Gut microbiome diversity has also been found to associate
more strongly with geography than phylogeny (Godoy-Vitorino et al.,
2012) or a combination of geography, diet, and host phylogeny
(Antonopoulou et al., 2019). These emerging studies of microbiome
divergence within adaptive radiations of hosts provide an important
counterpoint to the classic expectation of rapid phenotypic
diversification and speciation during adaptive radiation (Schluter 2000;
Stroud and Losos 2016; Martin and Richards 2019; Gillespie et al. 2020).
A major caveat is that we did not examine the microbiota of
wild-collected animals feeding on their diverse natural resources of
macroalgae, scales, and snails. Scales form up to 50% of the diet in
scale-eaters (Martin and Wainwright 2013) and wild gut microbiome
samples surely would have revealed more substantial differences in
microbiome diversity and composition among generalist and specialist
species on San Salvador Island. However, our goal with this common
garden study using lab-reared animals fed an identical generalist-type
diet for one month was to uncover any genetically based microbiome
differences in these taxa by eliminating environmental effects as much
as possible. Pupfishes exhibit no parental care and deposit external
eggs on the substrate so vertical transmission also appears highly
unlikely (but see Satoh et al. 2019 for a potential example of vertical
transmission in a scale-eating cichlid). Furthermore, by including two
lab-reared colonies of each generalist and specialist species on San
Salvador from genetically differentiated and ecologically divergent lake
populations (Martin et al. 2016; Richards and Martin 2017), we aimed to
connect significant differences in microbiome composition observed in
our specialist species to their specialized diets, rather than their
lake environment or genetic background. This provides strong evidence of
genetic divergence in the host associated with trophic specialization.
These results are all the more surprising because trophic specialists
show very little genetic differentiation from generalists
(Fst = 0.1 – 0.3; Martin and Feinstein 2014;
Richards et al. 2020). Indeed, there are only a few thousand nearly
fixed or fixed SNPs (Fst > 0.95)
between scale-eaters and molluscivores out of over 10 million
segregating SNPs and as few as 157 fixed SNPs and 87 deletions in
scale-eaters (McGirr and Martin 2020). However, this minimal set of
genetic differences may be driving differences in gut microbiome
composition. Intriguingly, the only fixed coding indel uncovered so far
in this system is a fixed deletion in all scale-eater populations of the
fifth exon of the gene gpa33 (McGirr and Martin 2020). This gene is
expressed exclusively in the intestinal epithelium and mice knockouts
display a range of inflammatory intestinal pathologies in mice (Williams
et al. 2015), suggesting it may play a role in shifting the gut
microbiota of scale-eaters that we observed in this study. Overall,
metabolic processes were the single most enriched category among all
differentially expressed genes between these trophic specialists at the
8 dpf larval stage, accounting for 20% of differential expression
(McGirr and Martin 2018).