It has been hypothesised that the effects of pollutants on phenotypes can be passed to subsequent generations through epigenetic inheritance, affecting populations long after the removal of a pollutant. But there is still little evidence that pollutants can induce persistent epigenetic effects in animals. Here we show that low doses of commonly used pollutants induce genome-wide differences in cytosine methylation in the freshwater crustacean Daphnia pulex. Uniclonal populations were either continually exposed to pollutants or switched to clean water, and methylation was compared to control populations that did not experience pollutant exposure. While some direct changes to methylation were only present in the continually exposed populations, others were present in both treatments, suggesting that these modifications had persisted for 8 months (> 15 generations). We also identified modifications which were only present in the populations that had switched to clean water, indicating a long-term legacy of pollutant exposure distinct from the persistent effects. Differentially methylated CpGs tended to be highly methylated and were more likely to be in promoters and exons than elsewhere in the genome. Modifications that had been observed in both continually and switched treatments had the highest methylation, and were generally hypomethylated, while modifications found just in the switched treatment tended to be hypermethylated. In a second experiment we confirmed that sub-lethal doses of the same pollutants generate effects on life-histories for at least three generations following the removal of the pollutant. Our results demonstrate that even low doses of pollutants can induce stable transgenerational epigenetic effects that are stably transmitted over many generations. Persistent effects are likely to influence phenotypic development, which could contribute to the rapid adaptation, or extinction, of populations confronted by anthropogenic stressors.
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