Functional divergence of conserved developmental plasticity genes between two distantly related nematodes

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 divergence in regulating mouth phenotype in both species. Loss of certain genes results in similar developmental disruptions in both species, while for others they are distinct. Additionally, two ancestral genes retain their functions as switch genes, which completely prevent a phenotype, and the other two display quantitative effects, with knock-outs in these genes displaying intermediate phenotypes. Remarkably, despite the evolutionary distance, all genes examined were involved in mouth-form regulation. Finally, key sulfatase-encoding genes act downstream of the other genes, 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.