Deciphering the evolutionary mechanisms shaping the regulatory circuitry underlying the novel brooding organ in syngnathids (pipefishes & seahorses) using cutting-edge single-cell multiomics

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The diversity of life reflects the multifaceted adaptations organisms evolved to adjust to an everchanging environment. New phenotypic traits can evolve due to gradual change to already existing traits (making them “homolog”), or they can arise as entirely new characteristics without any phenotypic precursor. Such “de novo” traits may still show homology on the level of gene regulation though (“deep homology”): novel traits may coopt regulatory circuitry of other preexisting traits, which can accelerate the evolution of novelty and complexity. Syngnathids (pipefish and seahorse) show such a novel trait: male pregnancy via specialized brooding organs. Across species, brooding organs may be simple (in the pipefish Nerophis ophidion the brooding organ merely serves to attach the eggs to the father’s belly, where they remain exposed to seawater), complex (in the seahorse Hippocampus erectus the brooding organ on the fish’s tail forms a pouch-like structure that holds the eggs in a regulated micro-environment shielded from seawater), or of intermediate complexity (in the pipefishes Syngnathus typhle and Doryrhamphus excisus). This project aims to investigate the molecular and evolutionary mechanisms underlying brooding organs to further our understanding of how evolutionary novel structures can arise. For this purpose, tissue samples of the four aforementioned syngnathids will be collected across brooding organ development. In three sub-projects (i.) the organs’ morphological developments will be comparatively described via histology and immunohistology, (ii.) the brooding organs’ transcriptome-wide gene expression patterns will be comparatively analyses across developmental stages, and (iii.) robust regulatory gene networks will be generated via single-cell multiomics, integrating RNA-seq and ATAC-seq from the same individual cells, of selected individuals. Subsequently, morphological characteristics will be associated to gene expression characteristics, cell types will be identified per developmental stage and species, trait-specific gene regulatory networks will be identified, and upstream transcription regulators orchestrating these gene regulatory networks determined. By comparative analyses of networks among brooding organ stages and between species, unique genes/regulatory elements and regulatory interactions can be identified. These analyses will reveal, for instance, if convergent brooding organ phenotypes evolved via parallel regulatory evolution and if the development of more complex phenotypes indeed recapitulates its evolution on a regulatory level, as suggested by morphological observations. Thus, this study will provide valuable insights into the developmental biology, molecular basis and evolutionary mechanisms underlying the syngnathids’ novel brooding organs and discuss more broadly the evolutionary mechanism underlying the evolution of novel traits, ranging from de novo evolution to deep homology.