Non-genetic inheritance of induced defense morphologies across multiple unexposed generations of Daphnia lumholtzi

Teaser TextCan the ghosts of environments past shape organismal phenotypes of future generations? In an experiment with clonal Daphnia lumholtzi, we reveal that predator-induced changes in body shape in one generation can persist across several unexposed generations, even when the predator signal is long gone. Exposing only the first generation to predator cues, we tracked morphological shifts through five generations of genetically identical individuals. We found that these induced plastic phenotypes persisted to the F4 generation before fading in F5. This discovery supports the importance of non-genetic inheritance and underscores the powerful interplay between ancestral and current environments in shaping organismal form and function. 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. However, most transgenerational plasticity studies examine generations where direct embryonic or germline exposure to environmental cues cannot be excluded. Empirical evidence for persistence definitively beyond the critical threshold for distinguishing true transgenerational plasticity from in utero exposure remains scarce, particularly for vertebrate predator-induced defenses. 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 F0 generation to fish conditioned media, then measured morphological defenses in definitively unexposed F3, F4, and F5 generations characterizing the temporal dynamics of non-genetic inheritance. Predator-induced morphologies persisted through F4 before receding in F5, demonstrating that non-genetic effects extend well beyond the embryonically exposed F1 and germ cell exposed F2 generations. This provides rare empirical evidence for transgenerational plasticity lasting through F4, 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 underscores the ecological significance of non-genetic inheritance in natural populations, particularly for understanding population responses to environmental change and predator reintroduction. Characterizing molecular mechanisms underlying transgenerational phenotypic plasticity remains critical for predicting persistence and ecological consequences of non-genetic inheritance.