Functional stomach loss preceding body inflation evolution in Tetraodontiformes: Evidence of gene loss and rapid evolution.

Tetraodontiformes species exhibit several evolutionary innovations, notably the convergent evolution of two traits in some clades: defensive body inflation and the loss of acid-peptic digestion. It has been widely believed that the evolution of body inflation drove the loss of gastric digestion, as ingesting seawater during inflation would hinder acidification and ultimately lead to the loss of gastric digestion. Leveraging accumulating genome resources, we investigated the presence, synteny, gene expression, and rapid evolution of nine genes associated with acid-peptic digestion across seven species from four Tetraodontiformes families, representing two branches that underwent convergent evolution. In the first evolutionary branch, all agastric species-including the species lacking inflation capability (Mola mola)-exhibit loss or pseudogenization of eight gastric-function-related genes. Among these, only pga1 is retained, though it lacks digestive functionality. In another branch, the greenfin horse-faced filefish (Thamnaconus septentrionalis), which possesses a functional stomach, maintains solely pga2 as its single pepsinogen gene, with pga1 and pgc having been lost, while slc26a9 and vsig1 show evidence of rapid evolution. These findings suggest that the convergent loss of gastric digestion likely occurred before the evolution of body inflation in both branches, supporting an "unfunctional-stomach-first" evolutionary scenario.