Evidence for Multiple Independent Expansions of Fox Gene Families Within Flatworms.

Expansion and losses of gene families are important drivers of molecular evolution. A recent survey of Fox genes in flatworms revealed that this superfamily of multifunctional transcription factors, present in all animals, underwent extensive losses and expansions during platyhelminth evolution. In this paper, I analyzed Fox gene complement in four additional species of platyhelminths, that represent early-branching lineages in the flatworm phylogeny: catenulids (Stenostomum brevipharyngium and Stenostomum leucops) and macrostomorphs (Macrostomum hystrix and Macrostomum cliftonense). Phylogenetic analysis of Fox genes from this expanded set of species provided evidence for multiple independent expansions of Fox gene families within flatworms. Notably, FoxG, a panbilaterian brain-patterning gene, appears to be the least susceptible to duplication, while FoxJ1, a conserved ciliogenesis factor, has undergone extensive expansion in various flatworm lineages. Analysis of the single-cell atlas of S. brevipharyngium, combined with RNA in situ hybridization, elucidated the tissue-specific expression of the selected Fox genes: FoxG is expressed in the brain, three of the Fox genes (FoxN2/3-2, FoxO4 and FoxP1) are expressed in the pharyngeal cells of likely glandular function, while one of the FoxQD paralogs is specifically expressed in the protonephridium. Overall, the evolution of Fox genes in flatworms appears to be characterized by an early contraction of the gene complement, followed by lineage-specific expansions that have enabled the co-option of newly evolved paralogs into novel physiological and developmental functions.