Molecular bases of morphological diversity in cichlid fishes

Abstract

Key innovations are characters promoting species richness in the monophyletic group sharing it, relative to a sister taxon not exhibiting it. Key innovations can even induce adaptive radiations for example by increasing the available new niche space to the lineage, facilitating reproductive isolation or reducing extinction rates. In chapter I the specialized morphology of the tiger tail seahorse, Hippocampus comes, exhibiting numerous characteristics and key innovations, is investigated using a full genome sequencing and de novo assembly approach. Derived traits in seahorses (and some allies) include a toothless tubular mouth, bony plates covering their whole body, male pregnancy within a brooding pouch and the loss of caudal and pelvic fins. Using comparative genomics, increased amino-acid and nucleotide evolution rates were identified in the seahorse genome compared with other teleost fish. Expansion of an astacin metalloprotease gene family was identified that is highly expressed at different stages of the seahorse male pregnancy in the brood pouch. Furthermore, seahorses lost enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes that might explain the lack of mineralized teeth. In addition, tbx4, a major regulatory of hind limb development in tetrapods, could not be found in the seahorse genome. Knock-out of tbx4 in zebrafish led to a loss of pelvic fins – a phenotype resembling that of the seahorse. Key innovations may lead to species proliferation and, in the most extreme cases, adaptive radiations, i.e. the extremely rapid emergence of new species corresponding to ecologically distinct niches from a single common ancestral population. Some of the largest and most rapid adaptive radiations are found in East African haplochromine cichlids, radiating in the three Great Rift lakes, Lake Victoria, Lake Malawi and Lake Tanganyika. Cichlid fishes possess a second set of functional jaws, pharyngeal jaws, that presumably facilitated their trophic diversification, which is why these jaws are considered a key innovation in cichlids. In some species, pharyngeal jaws were found to respond plastically to varying diets, raising the question about the evolutionary consequences of phenotypic plasticity in this key ecological trait. Chapter II reviews the increasing evidence that phenotypic plasticity can facilitate population divergence by promoting phenotypic diversification and, eventually, genetic divergence. Phenotypic plasticity is the ability of organisms with a given genotype to develop different phenotypes according to environmental stimuli, resulting in individuals that are better adapted to local conditions. The chapter illustrates how a plastic ‘ancestral’ lineage, after colonizing a new habitat, phenotypically diversifies and how these diverse phenotypes can then be genetically fixed via a process called ‘genetic assimilation’ (a ‘flexible-stem’ scenario). Additionally, molecular mechanisms are reviewed that illustrate how genetic fixation of a formerly plastic phenotype may work, solely by random mutations and without the need for a ‘cost of plasticity’. It is described how genetic assimilation contributes to cryptic genetic variation, but also how it can lead to non-adaptive responses. Predictions about expected phenotypic, genetic and transcriptional patterns induced during a flexible-stem radiation are formulated and illustrated. Furthermore, it is noted that the degree of inducible adaptive and non-adaptive plasticity is expected to vary across lineages at different stages of genetic assimilation. Analyses of these patterns can inform on the state of genetic assimilation in candidate lineages. It is reasoned that, depending on the environment, phenotypic plasticity can promote lineage diversification and divergence, and increase the rate of evolution. The chapter also exemplifies proposed patterns and conclusions using the cichlids as a model system. It is concluded that available evidence supports a flexible stem scenario for at least some cichlid radiations. In spite of their ecological importance, the developmental regulatory networks underlying plastic phenotypes often remain uncharacterized. In chapter III the regulatory basis of phenotypic plasticity in the lower pharyngeal jaw of the cichlid Astatoreochromis alluaudi is investigated, a model species in the study of adaptive plasticity. By raising juvenile A. alluaudi on either soft or hard diets for between one to eight months, the temporal regulation of previously identified candidate genes could be monitored during the plastic response. Morphological divergence of phenotypes of the two diet groups could be observed between three to five months of treatment, which are preceded by a consistent change in candidate gene expression patterns. It is concluded that investigated genes are likely contributing to the plastic response and pharyngeal jaw bone remodeling in this cichlid. Candidate genes were found to be strikingly co-expressed according to functional categories and transcription factor binding site analysis was performed to examine the prospective regulatory basis of this co-regulation. Based on these results a candidate gene regulatory network putatively underlying lower pharyngeal jaw plasticity is proposed, including evidence for a modular organization but also cross-talk among these modules, which presumably facilitates the plastic remodeling of this highly integrated morphological structure. Chapter IV investigates whether adaptive diversity in pharyngeal jaw phenotypes found in one of the most extensive adaptive radiations of cichlids, the modern haplochromines, is likely to have originated in a flexible stem. Juveniles of five cichlid species from within the modern haplochromines, representing ‘basal’ non-radiating generalist and ‘derived’ radiating specialists, were fed on either a soft or a hard diet to induce a plastic response in the lower pharyngeal jaws. The measured morphological adaptive plastic response was determined to be most pronounced in the most basal generalist, while the more specialized species had considerably lower levels of plasticity. This suggests that plasticity was reduced during trophic specialization in this radiation via genetic assimilation. In contrast, non-adaptive plastic responses were identified to be more pronounced in specialized species, coinciding with predictions made in chapter II of this thesis. Two candidate genes that potentially have undergone genetic assimilation are identified. It is concluded that in this cichlids’ radiation the degree of adaptive phenotypic plasticity was reduced by genetic assimilation during trophic specialization to suit progressively the more narrow ecological niches of each species. Besides the wide diversity of trophic characteristics, cichlids are famously known for their outstanding diversity in body colorations across species, but also across development within species and between sexes. The evolution of the latter was suggested to be driven by Fisher’s run-away selection, which could be evident by sexual dimorphism not only in body coloration, but also the visual system. In chapter V, sexually monomorphic and dimorphic cichlid species, both from the Afrotropics as well as the Neotropics, are investigated for being sexually dimorphic in visual systems, however, no evidence was found supporting this. Nonetheless, rod opsin expression was highly variable across all species, while interspecies variations in cone opsin expression were limited to Afrotropic species. By predicting candidate cichlids’ effective retina sensitivities and their body colorations in their corresponding habitats, evidence is found indicating that both abiotic factors (such as the available ambient light spectrum and its brightness) as well as biotic factors (here: conspecific body colorations) effecting opsin expression.