1 INTRODUCTION
A major ongoing challenge in biological research is to understand the
origin and maintenance of biodiversity, with broad implications in
conservation, ecology and evolutionary biology. Traditionally, these
endeavors have involved characterizing the forces and mechanisms
operating above the organismal level (e.g., selection, environmental
change [Schluter 2000; Callaghan et al., 2004; Burns et al., 2009;
Siepielski et al., 2014]) or within the organism (e.g., genetic and
developmental mechanisms [Kawajiri et al., 2014; Hohenlohe, 2014;
Margres et al., 2015]). Understanding the intersection of extrinsic
and intrinsic forces (Mccairns & Bernatchez, 2012; van Heerwaarden &
Sgrò, 2017; Laitinen & Nikoloski, 2019; Levis et al., 2020) holds
significant potential to advance the field.
African cichlids are a hyperdiverse group of fishes that have long been
used as an evolutionary model (Kocher, 2004; Seehausen, 2006), and have
been especially useful in revealing both the genetic and environmental
factors that contribute to biodiversity (McKaye et al., 1984; Sturmbauer
& Meyer, 1992; Genner et al., 2004; Ding et al., 2015; Malinsky et al.,
2018). In Africa, approximately 2000 cichlid species have arisen over
the past ~5 million years, which is unparalleled
compared to the speciation rates of other vertebrates (Seehausen, 2006).
Moreover, cichlid diversity is pronounced across several phenotypic
axes, including coloration (Seehausen et al., 1999; Maan et al., 2006;
Salzburger, 2009), activity levels (Lloyd et al., 2021), as well as
reproductive and foraging behaviors (Balshine-Earn & Earn, 1998; Genner
et al., 1999; Lopez-Fernandez et al., 2014). Variation in feeding
architecture, which relates to the foraging niche exploited by each
species/population, is another critical axis of cichlid diversity (e.g.,
Cooper et al, 2010). Cichlid craniofacial variation is largely
continuous, but there are also examples of extreme or discontinuous
variants (reviewed by Powder and Albertson, 2016). In general terms,
cichlids partition their foraging niche along a benthic-pelagic
ecomorphological axis, with concomitant shifts in foraging anatomy
(Young et al 2009; Cooper et al. 2010; Conith and Albertson, 2021). For
instance, species inclined toward a benthic mode of feeding tend to have
steeply descending facial profiles, small eyes positioned toward the top
of their heads, and short, robust oral jaws with closely-spaced,
multicuspid teeth optimal for biting and scraping (e.g., Figure 1A,B).
On the opposite end of this spectrum, pelagic feeders tend to possess
longer, streamlined heads, large eyes, and long, up-turned oral jaws
with large, widely spaced teeth optimal for suction/ram feeding (e.g.,
Figure 1C,D) (Albertson et al., 2003; Cooper et al., 2010).
Significant efforts over the past 20 years have focused on
characterizing the genetic basis of cichlid craniofacial variation
(e.g., Albertson et al., 2005; Roberts et al., 2011; Powder et al.,
2014; Hu & Albertson, 2017; Singh et al., 2017; DeLorenzo et al.,
2022). In addition, cichlids have long been a model of phenotypic
plasticity (Meyer 1987; Wimberger 1991; Huysseune 1995;
Machado-Schiaffino et al., 2014; Schneider et al., 2014; Meuthen et al.,
2018; Navon et al., 2020), which is defined as the ability of a single
genotype to produce a range of phenotypes in response to environmental
inputs. Plasticity is critical for organismal survival in an era of
rapid environmental change (Willis et al., 2008; Sih et al., 2011;
Gugger et al., 2015; Karasz et al., 2022; Morgan et al., 2022). It can
also influence the direction and/or speed of future evolutionary change
by exposing new phenotypic and genetic variants to natural selection
(Ledon-Rettig et al., 2010 ProcB; McGuigan et al., 2011 evol; Landy et
al., 2020 PNAS; Campbell et al., 2021). In spite of its importance
across a range of biological disciplines, there are many outstanding
questions about plasticity, including its genetic basis and evolutionary
potential (Gibert 2017). Plasticity is well documented in cichlids
across a range of morphological traits including full body,
craniofacial, oral jaw and pharyngeal jaw shapes (Huysseune, 1995;
Muschick et al., 2011; Gunter et al., 2013; Parsons et al., 2014; Navon
et al., 2020). A notable theme that has come from these data is that
closely related species can differ in either their magnitude or pattern
of plasticity in response to the same stimulus (Parsons et al., 2014;
Navon et al., 2020), suggesting that plasticity itself is an evolvable
trait. If true, then plasticity must also have an explicit genetic basis
(Kuttner et al., 2014; Lafuente et al., 2018; Diouf et al., 2020);
however, understanding plasticity at this level has proven challenging
(Gibert 2017).
Previous efforts in our lab have sought to describe the genetic basis of
plasticity, and have described roles for Wnt (Parsons et al., 2014) and
Hh (Hu & Albertson 2017; Navon et al., 2020) signaling, respectively.
In addition, QTL analyses in cichlids have demonstrated the critical
importance of the environment in determining the genotype-phenotype
(G-P) relationship. Specifically, the genetic basis of variation in
multiple hard and soft tissue traits was shown to depend, almost
entirely, on the foraging environment in which the animals were reared
(Parsons et al., 2016; Zogbaum 2021). Such genetic mapping studies led
to the discovery of crocc2 as an environmentally-dependent
regulator of jaw shape (Gilbert & Tetrault et al., 2021). Ciliary
rootlet coiled-coil 2 (crocc2 ) encodes a protein that is a major
structural component of the primary cilium’s rootlet (Yang et al.,
2002). Primary cilia are important mechanosensors that help cells sense
and respond to environmental stimuli, but roles of the rootlet in
mechanosensing are less clear (Styczynska-Soczka & Jarman, 2015).
Notably, this gene was only implicated in regulating cichlid jaw shape
in the mechanically demanding benthic/biting environment (Parsons et al
2016; Gilbert & Tetrault et al., 2021), and functional analyses in
zebrafish showed that mutations in crocc2 led to degeneration of
cilia, decreased mechanosensing abilities, dysmorphic bone shapes, and
mis-regulation of gene networks in bone tissue (Gilbert & Tetrault et
al., 2021). Together, this incipient literature has implicated a small
handful of genes that contribute to mechanosensitive signal transduction
pathways (e.g., Hh) and structural components of the cell (e.g.,
rootlets) in the evolution and plasticity of cichlid bone shape. Here we
seek to advance this research program by taking a genome-wide approach.
In particular, to address the question of genetic and epigenetic control
of plasticity in the cichlid feeding apparatus, we utilize two
complementary methods of assessing transcriptional output: RNA-seq to
analyze gene expression, and ATAC-seq to assess chromatin accessibility.
We focused on an important functional complex - i.e., the
interopercle-retroarticular (IOP-RA) complex - which (1) is part of the
opercle 4-bar linkage chain, (2) helps to drive lower jaw depression,
(3) is comprised of hard and soft tissues, (4) varies among Malawi
cichlids in a manner that predicts foraging mode/habitat, and (5) has
been shown to be plastic in previous research (Figure 1; Hu and
Albertson 2014; 2017; Navon et al 2021). Our goals are to identify genes
that are both differentially expressed (DE) and differentially
accessible (DA) between species and environments.