Phenotypic plasticity is an important feature of biological systems that is likely to play a major role in the future adaptation of organisms to the ongoing global changes. It may allow an organism to produce alternative phenotypes in responses to environmental cues. Modifications in the phenotype can be reversible but are sometimes enduring and can even span over generations. The notion of phenotypic plasticity was conceptualized in the early 20 th century by Richard Woltereck. He introduced the idea that the combined relations of a phenotypic character and all environmental gradients that influence on it can be defined as "norm of reaction". Norms of reaction are specific to species and to lineages within species, and they are heritable. He postulated that reaction norms can progressively be shifted over generations depending on the environmental conditions. One of his biological models was the water-flee daphnia. Woltereck proposed that enduring phenotypic modifications and gene mutations could have similar adaptive effects, and he postulated that their molecular bases would be different. Mutations occurred in genes, while enduring modifications were based on something he called the Matrix. He suggested that this matrix (i) was associated with the chromosomes, (ii) that it was heritable, (iii) it changed during development of the organisms, and (iv) that changes of the matrix could be simple chemical substitutions of an unknown, but probably polymeric molecule. We reasoned that the chromatin has all postulated features of this matrix and revisited Woltereck's classical experiments with daphnia. We developed a robust and rapid ATAC-seq technique that allows for analyzing chromatin of individual daphnia and show here (i) that this technique can be used with minimal expertise in molecular biology, and (ii) we used it to identify open chromatin structure in daphnia exposed to different environmental cues. Our result indicates that chromatin structure changes consistently in daphnia upon this exposure confirming Woltereck's classical postulate.

Background: This study therefore describes the classical experimental system postulated by Richard Woltereck 100 years ago: the adaptive morphological phenotypic plasticity of daphnia . Phenotypic plasticity is an important feature of biological systems that is likely to play a major role in the adaptation of organisms exposed to an environmental stimulus and it is increasingly related to epigenetic mechanisms. Several studies have started to identify the epigenetic basis of phenotypic plasticity of daphnia including non-coding RNAs, covalent modifications at the histone tails and DNA methylation however no study has yet investigated those effect on the genome-wide chromatin structure. The aim of this work was to study for the first time the overall genome-wide chromatin structure of Daphnia pulex in the context of the iconic complex defense response to predation. We developed a robust and rapid ATAC-seq technique that allows for analyzing chromatin of individual daphnia and show here (i) that this technique can be used with minimal expertise in molecular biology, and (ii) we used it to identify open chromatin structure in daphnia exposed to different environmental cues.Results: Our results encouraged the expected induction of anti-predatory morphological changes in the stress treatment was significantly higher than that of daphnia from the control treatment. The developed ATAC-seq technique can be used to characterize chromatin structures of individuals even those that are small and thus with few biological material, making it possible to determine epigenetic polymorphisms relatively easily and at reasonable cost in full populations. In addition, we deliver evidence that chromatin structure changes upon stimuli from the environment.Conclusion: We report here an extremely fast and straightforward method to map the chromatin status of individuals using small amounts of input biological material. W e show here that changes in the environment, such as predator presence the chromatin structure is profoundly reorganised confirming Woltereck ’s classical postulate.