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Abstract

The all-female marbled crayfish Procambarus virginalis is a freshwater crayfish which is the only known obligatory parthenogen among the decapod crustaceans. Marbled crayfish are recent descendants of the sexually reproducing slough crayfish Procambarus fallax and have most likely emerged through a recent evolutionary macromutation event in P. fallax. Marbled crayfish reproduce by apomictic parthenogenesis, where oocytes do not undergo meiosis and all offspring are genetically identical clones of the mother. Nevertheless, marbled crayfish show a high degree of phenotypic variation and are a highly invasive species, where (through parthenogenesis) a single animal can establish a whole population. Moreover, they have been distributed via the pet trade and anthropogenic releases, and have formed stable populations in a variety of ecological habitats. Earlier this year, our group performed whole-genome sequencing for 11 marbled crayfish animals from different populations and countries, and found only four non-synonymous single nucleotide variances in coding regions. Since the marbled crayfish’s remarkable adaptability is not due to genetic variability, it is crucial to investigate epigenetic programming in this organism. I present here a comprehensive analysis of DNA methylation in marbled crayfish. Whole- genome bisulfite sequencing data was used to directly compare methylation patterns from multiple replicates in different tissues and from different marbled crayfish and Procambarus fallax animals. These methylation maps were integrated with RNA-seq and ATAC- seq data to comprehensively analyse the interplay between DNA methylation, chromatin accessibility, and gene expression. I found 18% of CpGs in marbled crayfish to be methylated. Repeats showed overall low methylation levels, with the exception of a single class of DNA transposons, which was ubiquitously methylated. DNA methylation was mainly targeted to the coding regions of housekeeping genes in marbled crayfish. In contrast to paradigmatic mammalian methylomes, I only observed very moderate methylation differences between tissues for both gene bodies and promoters. I did, however, identify a set of approximately 700 genes that showed a high variance in their methylation across tissues and animals. Gene body methylation was significantly inversely correlated with gene expression variability. Interestingly, the marbled crayfish shows overall lower methylation levels and higher gene expression variability than its parent species P. fallax. Since plasticity in gene expression can be a beneficial trait for adapting to new environments, this trait might contribute to the marbled crayfish’s adaptive and invasive success. The integrative analysis of DNA methylation, chromatin accessibility, and gene expression revealed that genes with highly methylated gene bodies were located in regions of poorly accessible chromatin and showed stable expression patterns. In contrast, lowly methylated genes were found in more accessible chromatin when stably expressed, and in more condensed chromatin when variably expressed. In this context, gene body methylation might function to stabilise gene expression in regions of limited chromatin accessibility. These findings broaden our knowledge of evolutionary conservation of DNA methylation patterns in invertebrates and provide novel insights on the interplay between gene body methylation, chromatin accessibility, and gene expression.