Developmental gene evolution shaping cichlid head morphology

The remarkably species-rich assemblages of cichlid fishes in the three Great East African Lakes, Victoria, Malawi and Tanganyika, represent a natural mutant model system for the study of the factors contributing to the formation of multiple new species within short time periods. This phenomenon is termed adaptive radiation, as the formation of novel species is tightly linked to the specialization towards the available ecological niches. The evolutionary success of cichlid fishes was explained by their key-innovation of two sets of jaws, the oral and the pharyngeal jaws, allowing them to rapidly evolve diverse feeding specializations. The theory of genetic assimilation and the Flexible Stem Hypothesis suggest that (1) a mutation or environmental change triggers the expression of one or more novel, heritable, phenotypic variant(s) within a population; (2) the initially rare variant(s) start to spread (in the case of an environmentally induced change, due to the consistent recurrence of the environmental factor), each creating a differentiated subpopulation expressing one alternative fit variant; and (3) selection on existing genetic variation for the regulation or form of the trait under selection causes it to become genetically fixed in individuals of a subpopulation. Such subpopulations ultimately evolve to new species adapted to alternative ecological niches, at the expense of losing the ancestral phenotypic plasticity. We plan to utilize the great comparative potential of the different evolutionary ages of the radiations in the three Great East African Lakes Victoria, Malawi, and Tanganyika to tackle the validity of the Flexible Stem Hypothesis as a central mechanism of cichlid fish diversification. This concept proposes that the broad range of phenotypic plasticity of ancestral "generalist" species, which allows survival in varied environmental conditions, is progressively reduced to be replaced and expanded by genetically based, specialized phenotypes. Since trophic niche differentiation and the decoupling of oral and pharyngeal jaws in cichlids are considered a key factor facilitating rapid radiation, we will compare two outgroup riverine species with known adaptive jaw phenotypic plasticity to three monophyletic sets of eco-morphologically equivalent model species from three independent lake radiations with increasing evolutionary age. We study the genomic basis of oral and pharyngeal jaw evolution by combining geometric morphometric and genomic analyses. Thereby, we intend to find genetic and gene regulatory correlates to divergent eco-morphologies after they have been shaped during embryogenesis. We assess the scope of phenotypic plasticity for each study species in relation to the divergence time from the riverine generalists by comparing the head-morphology of corresponding wild and captivity-bred individuals via micro-computed tomography as a standardized measure to assess phenotypic plasticity. This allows us to test if the genetic architecture of eco-morphological divergence corresponds to the pattern predicted by the concept of genetic assimilation in accordance with the Flexible Stem Hypothesis. We address this at four levels: non-coding RNAs, the epigenetic regulation via CpG methylation, gene expression, and divergence in coding regions (protein evolution, gene duplications).

The project utilized the great comparative potential of the different evolutionary ages of three independent radiations of cichlid fishes in the East African Lakes Victoria, Malawi, and Tanganyika to tackle the validity of the "Flexible Stem Hypothesis" as a central mechanism of cichlid fish adaptive radiation. This concept proposes that a broad "reaction norm" of phenotypic plasticity in ancestral "generalist" species, which allows survival in varied environmental conditions, is progressively reduced and replaced by genetically based specialized phenotypes, during adaptation to the more stable and predictable lake environment. As oral and pharyngeal jaws are considered a key factor facilitating adaptation and speciation, we concentrated on these structures. To connect the transcriptional activity of shape-relevant gene networks with the morphology of oral and pharyngeal jaws, we compared the three lake radiations and studied gene transcription, alternative splicing and gene methylation patterns in combination with micro-computed tomography. Using 200 whole-transcriptomes from cichlid oral and pharyngeal jaws from the three sister radiations, we tried to identify the genetic and gene regulatory correlates to - convergently evolved - specialized trophic morphologies at two important life stages, after they have been shaped during embryogenesis. Overall, the steady-state jaw transcriptional patterns at two postlarval stages were highly dynamic and species-specific. We found a surprisingly high degree of alternative splicing events compared with gene expression differences among species, stages and trophic types. The distinction between carnivory and herbivory is already present at the onset of independent life. Within the stages we could identify several differentially expressed genes between carnivorous and herbivorous species, connected to Wnt signaling, an important pathway in skeletogenesis and several genes involved in craniofacial morphology. We could identify two genes of interest, that could be contributing to the broader morphologies in herbivory and carnivory (frzb and fgfr2). Our study finds that alternative splicing diverges faster than gene expression during adaptive radiation. In younger radiations, gene expression patterns are conserved across tissues and alternative splicing is radiation-specific. However, at more advanced stages of adaptive radiation, gene expression is radiation-specific and alternative splicing is species-specific. We find that gene expression and alternative splicing diversity is negatively correlated with age of radiation, with younger radiations harbouring greater diversity than older radiations. Interestingly, non-radiating riverine species had the highest transcriptional diversity. Given that the putative ancestor of these radiations was a riverine cichlid, it is possible that an ancestral cache of standing regulatory variation was evolutionarily fine-tuned as ecological opportunities arose. We found little evidence for convergent gene expression underlying convergent trophic adaptations in these parallel cichlid radiations, suggesting low evolutionary constraint and high redundancy in the genotype-phenotype map determining jaw shape. This high redundancy likely facilitated evolutionary tinkering to generate the incredible diversity of cichlid trophic adaptations.