#Discussion

Research on DNA methylation to date has revealed that its extent and function varies considerably among organisms. While several studies have compared DNA methylation patterns between distantly related taxa (\cite{9032274}, \cite{20395474}, \cite{22328716}), our analysis focused on a single taxonomic group, the reef-building scleractinian corals. Within this group, we took a comparative approach of six species to provide a comprehensive evaluation of germline methylation patterns. In three of these species, representing three independent studies, we found that genes expressed differentially in response to environmental stressors exhibited significantly lower levels of methylation. This work adds to a small but growing body of evidence supporting an association between hypomethylation and gene expression plasticity (\cite{22232607}, \cite{25511458}).

As in most other invertebrate taxa surveyed (\cite{20799955}, \cite{22328716}), we observed patterns of CpG O/E that were indicative of bimodal distributions in all of the coral transcriptomes. All distributions were dominated by a relatively high CpG O/E fraction, suggesting that the majority of genes in reef corals experience relatively low levels of methylation. A similar pattern was observed in a genome-wide analysis of the sea anemone *Nematostella vectensis* (\cite{20395474}, \cite{22328716}), and in an analysis of exons in *A. millepora* \cite{25511458}. In contrast, CpG O/E profiles in other invertebrates, such as the oyster *Crassostrea gigas* and the sea squirt *Ciona intestinalis*, suggest larger low-CpG O/E fractions (\cite{20799955}, \cite{22328716}). This is consistent with higher genome-wide levels of CpG methylation in *C. intestinalis* and *C. gigas* than in *N. vectensis* (21.6%, 15%, and 9.4%, respectively; \cite{20395474}, \cite{24987376}). Levels of DNA methylation broadly reflect evolutionary relationships (\cite{9032274}, \cite{20395474}), so it could be speculated that coral methylation is similar to that of *N. vectensis*. However, phylogenies derived from methylation patterns may differ considerably from those derived from protein sequences, suggesting lineage-specific changes in methylation \cite{22328716}. Lineage-specific changes in methylation could reflect differences in life history strategies \cite{22328716}. 

Across reef coral species, ranking of biological processes according to mean CpG O/E was largely consistent, corroborating a trend reported in other studies of invertebrate gene body methylation (\cite{20799955}, \cite{22328716}, \cite{25511458}). With little variation between coral species, the biological processes enriched in high CpG O/E values (predicted to have low CpG DNA methylation) reflect genes involved in inducible functions, while processes associated with the low CpG O/E values reflect genes for essential housekeeping functions. Housekeeping genes are ubiquitously expressed across tissue types, and tend to be evolutionarily conserved. Indeed, higher levels of germline DNA methylation are associated with gene orthology among invertebrate taxa (\cite{22328716}, \cite{21693438}, \cite{17420183}), suggesting a protective effect that contrasts with the tendency for methylated cytosines to experience higher mutation rates than nonmethylated nucleotides. However, it may be that DNA methylation has an overall protective influence despite the mutagenic effect on CpGs, or that CpG mutations have largely run their course over time in heavily methylated genes, with fewer methylated CpGs left for substitutions \cite{21693438}. One hypothesis for the role of gene body methylation is that it may facilitate consistent expression of ubiquitously expressed core genes needed for essential biological functions (\cite{7732579}, \cite{22232607}, \cite{24397979}). While there have been some reports of negative associations between gene body methylation and gene expression in invertebrates (\cite{17420183}, Riviere et al. 2013), positive or bell-shaped relationships have been reported in several taxa, including corals (\cite{20395474}, \cite{24282674}, \cite{25511458}, \cite{22577155}). In *A. millepora*, Dixon et al. \cite{25511458} reported that the most highly expressed genes in a reciprocal transplant experiment were those exhibiting higher levels of gene body methylation. These genes were enriched for housekeeping functions. A similar phenomenon of increased expression among hypermethylated genes was reported for *C. gigas*, in addition to an inverse relationship between DNA methylation and variation in expression between tissues \cite{24282674}. High transcript abundances among highly methylated genes may reflect their widespread expression across cell and tissue types (\cite{24282674}, \cite{25511458}). 

In contrast to hypermethylation and consistent expression, our finding of relatively low methylation among differentially expressed genes in response to thermal stress and ocean acidification in the three acroporid corals supports the hypothesis that hypomethylation is associated with transcriptional plasticity \cite{22232607}. Sparse methylation of gene bodies, and the tendency for genes involved in inducible functions to have lower methylation levels, is thought to leave these genes open to a greater variety of transcriptional opportunities \cite{22232607}. Potential sources of transcriptional variation could include access to alternative start sites, increased sequence mutations, exon skipping, and transient methylation \cite{22232607}. Such transcriptional variation could contribute to phenotypic plasticity, and it might be particularly beneficial among species that experience variable environments that require constant tuning of gene expression, such as corals and other sessile organisms. Dixon et al. \cite{25511458} found support for this theory in *A. millepora*, showing that genes differentially expressed in response to transplantation to new environments were significantly enriched in the low methylation (high CpG O/E) component. 

In our analysis, lower methylation among differentially expressed genes was at least partially attributable to increased representation of genes involved in inducible functions relative to those with housekeeping functions. This was especially true in *A. hyacinthus*. While unsurprising, this highlights an important caveat to our analysis; low methylation among differentially expressed genes could simply reflect the fact that environmental stressors elicit higher expression of inducible genes relative to housekeeping genes, and that these genes simply happen to exhibit different methylation patterns. Furthermore, it remains unclear whether gene body DNA methylation actively regulates genes, as opposed to the alternative hypothesis that it is simply a byproduct of an open chromatin state \cite{22577155}. For example, gene body methylation may reflect exposure of DNA to DNA methyltransferases (DNMTs) as a consequence of unpacked chromatin during transcription \cite{22577155}. Further studies will be needed to evaluate causative relationships between DNA methylation and transcriptional activity. Additionally, despite the utility of CpG O/E to infer germline methylation patterns, experimental work on transient methylation in somatic cells and how it might influence transcription is needed. However, experimental studies on social insects have already provided compelling evidence for links between differential DNA methylation, transcription, and phenotypic plasticity (\cite{18339900}, \cite{19556545}). Studies of DNA methylation in plants have gone even further, having already incorporated epigenetics into ecological research \cite{18021243}. 

There is a great deal of interest in the adaptive potential of corals in the face of continued environmental change. Perhaps the most intriguing aspect of DNA methylation is that its effects may extend beyond an individual organism’s lifetime and be transferred to successive generations. Although the epigenomes of some organisms, such as mammals, are extensively reprogrammed during meiosis and embryogenesis, in some cases DNA methylation can be passed on to offspring \cite{24719220}. This is especially true in plants (\cite{21515434}, \cite{24679529}), but there is also evidence for inheritance of DNA methylation in oysters \cite{Olson_2014}. Transgenerational epigenetic inheritance is a potentially transformative biological concept, but its extent and significance is only just beginning to be understood, and remains largely unstudied in the vast majority of organisms (\cite{23416892}, \cite{24679529}). 

Our results illustrate patterns of gene body DNA methylation that are similar across coral species and largely consistent with what has been found in other invertebrates. This work serves as a basis with which to further characterize and compare DNA methylation in corals and related taxa. Within the context of environmental challenges faced by corals, our analysis found broad support for an inverse relationship between gene body methylation and differential gene expression. This suggests that the potential role of DNA methylation in the response of corals to environmental change warrants further investigation.