4 CONCLUSIONS
A number of general themes emerge from these data. First, they provide
clear support for the hypothesis that foraging environment influences
the genotype-phenotype map for craniofacial skeletal traits (Parsons et
al., 2016; Navon et al., 2021; Zogbaum et al., 2021). More specifically,
our data suggest that pelagic foraging “drives” species- and
environment-specific DE. This may seem counterintuitive as diets that
involve large/hard prey items are generally considered to be the more
mechanically demanding compared to small/soft food (Muschick et al.,
2011; Gunter et al., 2013; Hulsey et al., 2020). However, Navon et al
(2020) showed that in MZ bone matrix was deposited at a fast rate under
pelagic foraging conditions, and speculated that suction feeding imposes
mechanical load on the feeding apparatus as animals repeatedly open and
protrude their jaws. Our data support this assertion, and thus we
consider the foraging treatments utilized here to challenge the feeding
apparatus in two distinct ways (compared to a “standard” flaked food
diet); our benthic treatment was designed to impose high amplitude but
low frequency loading onto the feeding apparatus as animals scrapped
food from rocks, whereas our pelagic treatment translated to higher
frequency but lower amplitude loading as animals repeatedly protruded
their jaws to gather small food items.
Our data also detected evidence for genetic assimilation. In particular,
when considering loci that were DE between species + environments,
patterns in MZ benthic fish resembled those across TRC. Tropheopsspecies, including sp. “red cheek”, are generally found in a
benthic environment (Ribbink et al., 1983), and may have lost a degree
of plasticity as they evolved to specialize on benthic food items. MZ on
the other hand are true generalists in the sense that they routinely
foraging from both the benthic and pelagic zones (Ribbink et al., 1983).
While plasticity has been noted in TRC (Parsons et al., 2014; Navon et
al., 2020), our data suggest that MZ may be more plastic than TRC in
that they mount a more pronounced transcriptional response, at least at
the time point analyzed in this study.
Cell cycle regulation consistently appeared in GO analyses, describing
species differences, as well as plasticity within MZ. This implicates
cell proliferation as an important biological mechanism of species- and
environment-specific bone growth in cichlids. This observation is
notable as our previous work has implicated Hedgehog signaling in the
evolution and plasticity of the cichlid jaw, including the IOP-RA
complex (Hu & Albertson, 2014; Parsons et al., 2016; Navon et al.,
2020). While canonical members of the Hedgehog signaling pathway were
not significantly DE or DA in this dataset (although KIAA0586regulates the signal, Schock et al., 2016), cell proliferation is
well-known to be regulated by this pathway (St. Jacques et al., 1999;
Tiet et al., 2006; Sun & Deng, 2007; Zaman et al., 2019), providing a
potential cellular mechanism through which variation in Hedgehog
signaling leads to differences in bone shape among and within cichlid
species.
Finally, with these large overlapping genome-wide datasets, we were able
to narrow down thousands of DEGs to roughly two dozen that were both DE
and DA. Given that each experiment was conducted at a different time
point, this reduced dataset points to loci whose expression is important
for species divergence over extended periods of time. Among these were
genes that were both sensitive and robust to the environment. Notably,
nearly all of these genes are new to the field of bone biology, and
while some encode known effectors of well studied signaling pathways
(e.g., interleukin/Wnt, Talpid/Hh) and cell behaviors (e.g.,
Casp6/apoptosis, Impdh1b/cell-cycle), others implicate largely novel
mechanisms (e.g., Gnmt/methionine cycle). Thus, this work establishes a
robust foundation for future studies into how genotype and the
environment combine to influence bone formation, remodeling, and
evolution.