Adaptive variation in craniofacial structure contributes to resource specialization and speciation, but the genetic loci that underlie craniofacial adaptation remain unknown. Here we show that alleles of the hedgehog pathway receptor Patched1 ( Ptch1 ) gene are responsible for adaptive variation in the shape of the lower jaw both within and among genera of Lake Malawi cichlid fish. The evolutionarily derived allele of Ptch1 reduces the length of the retroarticular (RA) process of the lower jaw, a change predicted to increase speed of jaw rotation for improved suction-feeding. The alternate allele is associated with a longer RA and a more robustly mineralized jaw, typical of species that use a biting mode of feeding. Genera with the most divergent feeding morphologies are nearly fixed for different Ptch1 alleles, whereas species with intermediate morphologies still segregate variation at Ptch1 . Thus, the same alleles that help to define macroevolutionary divergence among genera also contribute to microevolutionary fine-tuning of adaptive traits within some species. Variability of craniofacial morphology mediated by Ptch1 polymorphism has likely contributed to niche partitioning and ecological speciation of these fishes.

Adaptive variation in the craniofacial skeleton is a key component of resource specialization and habitat divergence in vertebrates, but the proximate genetic mechanisms that underlie complex patterns of craniofacial variation are largely unknown. Here we demonstrate that the Hedgehog (Hh) signaling pathway mediates widespread variation across a complex functional system that affects the kinematics of lower jaw depression—the opercular four-bar linkage apparatus—among Lake Malawi cichlids. By using a combined quantitative trait locus mapping and population genetics approach, we show that allelic variation in the Hh receptor, ptch1, affects the development of distinct bony elements in the head that represent two of three movable links in this functional system. The evolutionarily derived allele is found in species that feed from the water column, and is associated with shifts in anatomy that translate to a four-bar system capable of faster jaw rotation. Alternatively, the ancestral allele is found in species that feed on attached algae, and is associated with the development of a four-bar system that predicts slower jaw movement. Experimental manipulation of the Hh pathway during cichlid development recapitulates functionally salient natural variation in craniofacial geometry. In all, these results significantly extend our understanding of the mechanisms that fine-tune the craniofacial skeletal complex during adaptation to new foraging niches.

Thyroid hormone (TH) directs the growth and maintenance of tissues throughout the body during development and into adulthood, and plays a particularly important role in proper ossification and homeostasis of the skeleton. To better understand the roles of TH in the skeletogenesis of a vertebrate model, and to define areas of the skeleton that are particularly sensitive to developmental TH, we examined the effects of hypo‐ and hyperthyroidism on skeletal development in zebrafish. Performing a bone‐by‐bone anatomical assessment on the entire skeleton of adult fish, we found that TH is required for proper ossification, growth, morphogenesis, and fusion of numerous bones. We showed that the pectoral girdle, dermatocranium, Weberian apparatus, and dentary are particularly sensitive to TH, and that TH affects development of skeletal element regardless of bone type and developmental origin. Indeed, the hormone does not universally promote ossification: we found that developmental TH prevents ectopic ossification in multiple thin bones and within connective tissue of the jaw. In all, we found that TH regulates proper morphogenesis and ossification in the majority of zebrafish bones, and that the requirement for the hormone extends across bone types and developmental profiles. Anat Rec, 302:1754–1769, 2019. © 2019 American Association for Anatomy

Understanding the developmental processes that underlie the production of adaptive variation (i.e. the ‘arrival of the fittest’) is a major goal of evolutionary biology. While most evo-devo studies focus on the genetic underpinnings of adaptive phenotypic variation, factors beyond changes in nucleotide sequence can also play a major role in shaping developmental outcomes. Here, we document a vigorous but enigmatic gaping behaviour during the early development of Lake Malawi cichlid larvae. The onset of the behaviour precedes the formation of bone, and we predicted that it might influence craniofacial shape by affecting the mechanical environment in which bone develops. Consistent with this, we found that both natural variation and experimental manipulation of this behaviour induced differential skeletal development that foreshadows adaptive variation in adult trophic morphology. In fact, the magnitude of difference in skeletal morphology induced by these simple shifts in behaviour was similar to those predicted to be caused by genetic factors. Finally, we demonstrate that this mechanical-load-induced shift in skeletal development is associated with differences inptch1expression, a gene previously implicated in mediating between-species differences in skeletal shape. Our results underscore the complexity of development, and the importance of epigenetic (sensuWaddington) mechanisms in determining adaptive phenotypic variation.

Phenotypic novelties are an important but poorly understood category of morphological diversity. They can provide insights into the origins of phenotypic variation, but we know relatively little about their genetic origins. Cichlid fishes display remarkable diversity in craniofacial anatomy, including several novelties. One aspect of this variation is a conspicuous, exaggerated snout that has evolved in a single Malawi cichlid lineage and is associated with foraging specialization and increased ecological success. We examined the developmental and genetic origins for this phenotype and found that the snout is composed of two hypertrophied tissues: the intermaxillary ligament (IML), which connects the right and left sides of the upper jaw, and the overlying loose connective tissue. The IML is present in all cichlids, but in its exaggerated form it interdigitates with the more superficial connective tissue and anchors to the epithelium, forming a unique ligament–epithelial complex. We examined the Transforming growth factor β (Tgfβ) → Scleraxis (Scx) candidate pathway and confirmed a role for these factors in snout development. We demonstrate further that experimental up-regulation of Tgfβ is sufficient to produce an expansion of scx expression and concomitant changes in snout morphology. Genetic and genomic mapping show that core members of canonical Tgfβ signaling segregate with quantitative trait loci (QTL) for snout variation. These data also implicate a candidate for ligament development, adam12, which we confirm using the zebrafish model. Collectively, these data provide insights into ligament morphogenesis, as well as how an ecologically relevant novelty can arise at the molecular level. Significance Biologists have long been captivated by novel traits because they provide insights into both the origin of and constraints on morphological variation. The iconic adaptive radiations of cichlid fishes have led to incredible diversity of form, including some species with an exaggerated snout. This novelty is mechanically integrated with the upper jaw, appears to be under directional selection, and is found in one of the most ecologically successful cichlid lineages. We used protein manipulation, gene expression, and genetic mapping to implicate the Tgfβ pathway in the development of this unusual trait. Given the functions of Tgfβ signaling in tissue proliferation, migration, invasion, and organ fibrosis, this represents an example of the cooption of existing pathways in the evolution of novelty.