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Abstract

Genes diverge in form and function in multiple ways over time; they can be conserved, acquire new roles, or eventually be lost. However, the way genes diverge at the functional level is little understood, particularly in plastic systems. We investigated this process using two distantly related nematode species, Allodiplogaster sudhausi and Pristionchus pacificus. Both these nematodes display environmentally-influenced developmental plasticity of mouth-form feeding structures. This phenotype can be manipulated by growth on particular diets, making them ideal traits to investigate functional divergence of developmental plasticity genes between organisms. Using CRISPR-engineered mutations in A. sudhausi mouth-form genes, we demonstrate examples of the various ways ancestral genes regulate developmental plasticity and how these roles can progressively diverge. We examined four ancestral genes, revealing distinct differences in their conservation and functional divergence in regulating the mouth phenotype in both species. Specifically, certain genes retain the same characteristics, while others have acquired a new function. Additionally, two ancestral genes retain their functions as switch genes, which completely prevent a phenotype, and the other two display quantitative effects, with knockouts in these genes displaying intermediate phenotypes. Remarkably, despite the evolutionary distance, all genes examined were involved in mouth-form regulation. Finally, multiple gene knock-out mutants were engineered, with key sulfatase-encoding genes acting downstream of all others, suggesting they play a major role in mouth-form plasticity. Together, this study represents the first mutant-based functional analysis of the evolution of developmental plasticity between two highly diverged species, offering new insights into the genetic mechanisms underlying phenotypic evolution.