Two Routes to Land: Genomic Underpinnings of Parallel Aerial Egg Deposition in Aquatic Old-World Pila and New-World Pomacea (Ampullariidae)

The evolution of aerial oviposition in Old-World Pila and New-World Pomacea apple snails—diverged since the Gondwanan breakup—offers a powerful model for probing genomic adaptations underpinning key evolutionary innovations. We generate a chromosomal-level genome for Pila celebensis and a scaffold-level genome for Pila pesmei, revealing a genus-specific doubling in genome size driven by transposable element expansions. Analyses of macrosynteny and topologically associating domains (TAD) identified lineage-specific chromosomal rearrangements associated with positive selection in gene blocks enriched for environmental sensing, metabolism, and stress response. Breakpoints in aerial egg layers preferentially are localized within TADs, suggesting convergent rewiring of gene regulation. Gene family evolution revealed parallel expansions in cellulases, β-D-xylosidases, and immune genes, alongside convergent positive selection in aquaporins critical for aerial osmoregulation. Perivitelline fluid (PVF) proteomics uncovered the central role of PVF1, likely acquired via ancient horizontal gene transfer (HGT) from viruses in the Ampullariidae ancestor in the Jurassic. Subsequent duplications enabled lineage-specific adaptation; PVF1 in aerial eggs shows parallel increases in hydrophobicity and aromatic residues (notably phenylalanine), enhancing desiccation resistance. Collectively, these convergent genomic mechanisms—structural rearrangement, gene family dynamics, and HGT-driven innovation—underpin the independent evolution of aerial oviposition in Pila and Pomacea, providing a multi-layered blueprint for understanding key ecological transitions.