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.