22Phenotypic plasticity allows organisms to change their phenotype in response to shifts in 23 the environment. While a central topic in current discussions of evolutionary potential, a 24 comprehensive understanding of the genetic underpinnings of plasticity is lacking in 25 systems undergoing adaptive diversification. Here we investigate the genetic basis of 26 phenotypic plasticity in a textbook adaptive radiation, Lake Malawi cichlid fishes. 27Specifically, we crossed two divergent species to generate an F 3 hybrid mapping 28 population. At early juvenile stages, hybrid families were split and reared in alternate 29 foraging environments that mimicked benthic/scraping or limnetic/sucking modes of 30 feeding. These alternate treatments produced variation in morphology that was broadly 31 2 similar to the major axis of divergence among Malawi cichlids, providing support for the 32 flexible stem theory of adaptive radiation. Next we found that the genetic architecture of 33 several morphological traits was highly sensitive to the environment. In particular, of 22 34 significant quantitative trait loci (QTL), only one was shared between environments. In 35 addition, we identified QTL acting across environments with alternate alleles being 36 differentially sensitive to the environment. Thus, our data suggest that while plasticity is 37 largely determined by loci specific to a given environment, it may also be influenced by 38 loci operating across environments. Finally, our mapping data provide evidence for the 39 evolution of plasticity via genetic assimilation at an important regulatory locus, ptch1. In 40 all, our data address longstanding discussions about the genetic basis and evolution of 41 plasticity. They also underscore the importance of the environment in affecting 42 developmental outcomes, genetic architectures, morphological diversity, and 43 evolutionary potential.

Phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes under different environmental conditions, is critical for the origins and maintenance of biodiversity; however, the genetic mechanisms underlying plasticity as well as how variation in those mechanisms can drive evolutionary change remain poorly understood. Here, we examine the cichlid feeding apparatus, an icon of both prodigious evolutionary divergence and adaptive phenotypic plasticity. We first provide a tissue-level mechanism for plasticity in craniofacial shape by measuring rates of bone deposition within functionally salient elements of the feeding apparatus in fishes forced to employ alternate foraging modes. We show that levels and patterns of phenotypic plasticity are distinct among closely related cichlid species, underscoring the evolutionary potential of this trait. Next, we demonstrate that hedgehog (Hh) signaling, which has been implicated in the evolutionary divergence of cichlid feeding architecture, is associated with environmentally induced rates of bone deposition. Finally, to demonstrate that Hh levels are the cause of the plastic response and not simply the consequence of producing more bone, we use transgenic zebrafish in which Hh levels could be experimentally manipulated under different foraging conditions. Notably, we find that the ability to modulate bone deposition rates in different environments is dampened when Hh levels are reduced, whereas the sensitivity of bone deposition to different mechanical demands increases with elevated Hh levels. These data advance a mechanistic understanding of phenotypic plasticity in the teleost feeding apparatus and in doing so contribute key insights into the origins of adaptive morphological radiations.

Adaptive radiations are often characterized by the rapid evolution of traits associated with divergent feeding modes. For example, the evolutionary history of African cichlids is marked by repeated and coordinated shifts in skull, trophic, fin and body shape. Here, we seek to explore the molecular basis for fin shape variation in Lake Malawi cichlids. We first described variation within an F mapping population derived by crossing two cichlid species with divergent morphologies including fin shape. We then used this population to genetically map loci that influence variation in this trait. We found that the genotype-phenotype map for fin shape is largely distinct from other morphological characters including body and craniofacial shape. These data suggest that key aspects of fin, body and jaw shape are genetically modular and that the coordinated evolution of these traits in cichlids is more likely due to common selective pressures than to pleiotropy or linkage. We next combined genetic mapping data with population-level genome scans to identify wnt7aa and col1a1 as candidate genes underlying variation in the number of pectoral fin ray elements. Gene expression patterns across species with different fin morphologies and small molecule manipulation of the Wnt pathway during fin development further support the hypothesis that variation at these loci underlies divergence in fin shape between cichlid species. In all, our data provide additional insights into the genetic and molecular mechanisms associated with morphological divergence in this important adaptive radiation.

Cichlid fishes exhibit rapid, extensive, and replicative adaptive radiation in feeding morphology. Plasticity of the cichlid jaw has also been well documented, and this combination of iterative evolution and developmental plasticity has led to the proposition that the cichlid feeding apparatus represents a morphological “flexible stem”. Under this scenario, the fixation of environmentally sensitive genetic variation drives evolutionary divergence along a phenotypic axis established by the initial plastic response. Thus, if plasticity is predictable then so too should be the evolutionary response. We set out to explore these ideas at the molecular level by identifying genes that underlie both the evolution and plasticity of the cichlid jaw. As a first step, we fine-mapped an environment-specific QTL for lower jaw shape in cichlids, and identified a non-synonymous mutation in the ciliary rootlet coiled-coil 2 (crocc2), which encodes a major structural component of the primary cilium. Given that primary cilia play key roles in skeletal mechanosensing, we reasoned that this gene may confer its effects by regulating the sensitivity of bone to respond to mechanical input. Using both cichlids and zebrafish, we confirmed this prediction through a series of experiments targeting multiple levels of biological organization. Taken together, our results implicate crocc2 as a novel mediator of bone formation, plasticity and evolution.

Physical principles and laws determine the set of possible organismal phenotypes. Constraints arising from development, the environment, and evolutionary history then yield workable, integrated phenotypes. We propose a theoretical and practical framework that considers the role of changing environments. This 'ecomechanical approach' integrates functional organismal traits with the ecological variables. This approach informs our ability to predict species shifts in survival and distribution and provides critical insights into phenotypic diversity. We outline how to use the ecomechanical paradigm using drag-induced bending in trees as an example. Our approach can be incorporated into existing research and help build interdisciplinary bridges. Finally, we identify key factors needed for mass data collection, analysis, and the dissemination of models relevant to this framework.Using the ecomechanical approach to understand the rules of life All forms of life must comply with physical laws, resulting in a series of 'universal' or 'hard' constraints (see Glossary) [1,2]. Although these constraints limit the possible phenotypes, 'local' or 'soft' constraints emerge as a consequence of ecological, developmental, and evolutionary processes that determine which phenotypes are adaptive. Thus, any realized phenotype is the result of: (i) physical principles and processes; (ii) the context in which the organism performs the manifold tasks required for growth, survival, and reproduction (i.e., organismenvironment interactions); and (iii) its evolutionary history [1,3]. HighlightsAll organisms must comply with physical laws, which place rigid or hard constraints on survival and reproduction. Ecomechanics is the expression of that interplay, and assumes a central role when considering organismal development, ecology, and evolution.