The microendemic origins of scale-eating in Cyprinodon pupfishes provides unexpected insights into rapid speciation, new vertebrate gene function, & the regulation of human cancer genes

Authors: Christopher Martin

Year: 2023

Abstract

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology that also provides an opportunity to uncover new genes and regulatory networks with potential clinical relevance. Highly conserved genetic regulatory pathways shared across diverse vertebrate species have been shaped by adaptive evolution to produce a tremendous diversity of phenotypes. This diversity can be harnessed to access the genetic underpinnings of human clinical and natural variation that is absent from model systems. Emerging non-model systems are known as ‘evolutionary mutant’ models and have proven to be a powerful complement to research on model systems. Fundamental investigation of the genetic basis of adaptive phenotypes can lead to better diagnosis, prevention, and treatment of human diseases. Here I review a decade of my lab’s work investigating the rapid evolutionary transition from a generalist algae-eating pupfish (Cyprinodon variegatus) to scale-eating (C. desquamator) and molluscivore (C. brontotheroides) specialists endemic to the desert-like subtropical hypersaline lakes of San Salvador Island in the Bahamas. We show that colonizing these niches occurred in stages, beginning with selection on standing genetic variation for feeding behavior, then aided by adaptive introgression from diverse sources, and ending with selection on de novo mutations in key craniofacial genes. We discovered that only 157 single-nucleotide polymorphisms (SNPs) and 87 deletions are fixed between these two specialists despite extensive phenotypic divergence in their craniofacial morphology, metabolism, and behavior. In many cases, only a single highly divergent SNP or structural variant is found in the regulatory, intronic, or (rarely) coding region of genes associated with these phenotypic axes. This provides a key advantage for identifying causal adaptive variants due to minimal genetic differentiation among highly divergent trophic ecotypes replicated across lake populations. For example, using the pupfish evolutionary mutant system, we recently demonstrated a new role for galr2 in vertebrate craniofacial development. We confirmed the loss of a putative Sry transcription factor binding site in the upstream region of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among species using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel function for Galr2 in craniofacial development and jaw elongation by exposing embryos to drugs that inhibit Galr2 activity. Galr2-inhibition reduced Meckel’s cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We also identified additional candidate adaptive variants in pupfish for human genes that also play a key role in cancer, including gpa33, prlh, and twist1. We are now using CRISPR-Cas9 genome editing to confirm a causal role for these variants. By combining candidate gene and variant discovery with functional genetics, we aim to demonstrate the flexibility and power of non-model systems to gain new insights into the developmental genetics of human diseases. Our findings also illustrate the growing utility of linking adaptive variation in non-model systems, such as desert fishes, with novel vertebrate gene functions.