Overlap in identity and expression direction of DE gene sets
within lineages
To determine if transcripts exhibiting expression plasticity in response
to rearing with predators were more likely to also exhibit evolutionary
divergence between high- and low-predation populations, we evaluated
overlap in transcripts differentially expressed between populations and
between rearing conditions as well as their directions of expression
change. To test the hypothesis that initial plastic responses following
colonization of low-predation environments might drive subsequent
divergence, we specifically compared pattern of ancestral plasticity
(i.e. high-predation fish reared with and without predators) to genetic
divergence (high- versus low-predation fish reared in the novel
predator-free environment) within each drainage. We used post-hoc tests
of simple effects to identify DE genes in these comparisons and estimate
log-fold gene expression changes. We then used log-fold expression
changes to determine whether gene expression differences were in the
same/concordant direction (i.e. upregulated in high-predation
populations and in response to rearing with predator cues, or
down-regulated in high-predation populations and in response to rearing
with predator cues) or opposite/non-concordant direction (upregulated in
high-predation populations but down-regulated in response to rearing
with predators, or vice versa).
Within each drainage, we compared the overlap in genes with both
statistically significant genetic and plastic expression changes. We
compared the association between directions of plasticity and divergence
(concordant vs. non-concordant) in (1) those genes with both significant
ancestral plasticity and significant population differences, and (2) all
transcripts with significant population differences in expression,
including those without significant expression plasticity. The second
comparison addressed the possibility that even subthreshold expression
plasticity in response to rearing environment could influence patterns
of expression divergence, especially given the high false negative rate
in our conservative DE analyses. Because these comparisons rely on two
comparisons to a single group (high predation fish reared without
exposure to predator cues), the estimates of plasticity and divergence
are dependent. We adapted a parametric bootstrap method (Efron &
Tibshirani, 1993) similar to the approach implemented by (W. C. Ho &
Zhang, 2019) but accounting for conditional probabilities. We tested
whether the conditional probabilities of gene expression divergence
differed for genes that exhibited ancestral plasticity and those that
did not. We further tested whether conditional probabilities for
upregulated or downregulated genes in the low predation population
compared to the high predation population differed depending on the
direction of plasticity. We calculated parametric bootstrap confidence
intervals for these differences in conditional probabilities as detailed
in the Supplemental Methods, with confidence intervals that do not
include zero indicating association between divergence and plasticity.