Non-genetic inheritance of induced defense morphologies across multiple unexposed generations of <i>Daphnia lumholtzi</i>

Environmental variation can induce phenotypic changes through adaptation of populations and acclimation of individuals. While genetic adaptation creates persistent change within a population via allele frequency shifts, reversible plastic phenotypes can be inherited through non-genetic mechanisms, independent of DNA sequence change. Most transgenerational plasticity studies in animals examine generations where embryonic or germline exposure to environmental cues cannot be definitively excluded. Empirical evidence for persistence definitively beyond this critical threshold in animals remains scarce, particularly for morphological defenses induced by vertebrate predators. We measured phenotypic effects of vertebrate predator exposure in the clonally reproducing water flea, Daphnia lumholtzi, isolating environmental effects from genetic variation. We exposed the G0 generation to fish conditioned media, then measured morphological defenses in definitively unexposed G3, G4, and G5 generations, characterizing the temporal dynamics of non-genetic inheritance. Predator-induced morphologies persisted through G4 before receding in G5, demonstrating that non-genetic effects extend well beyond the embryonically exposed G1 and germ cell exposed G2 generations. This provides rare empirical evidence for transgenerational plasticity lasting through G4, without confounding genetic variation. We also examined ontogenetic patterns of somatic and defensive trait development, revealing trait-specific temporal dynamics in transgenerational effect expression and decay. These results highlight how current and ancestral environments interact to determine phenotypic variation across generations and underscore the ecological significance of non-genetic inheritance in natural populations, particularly for understanding population responses to environmental change and predator reintroduction.