Ga2019,10,465 10of14 mCG divergence betw moters and gene bodies was not correlated with ex ession divergence of the corresponding orthologs (Figure S8D-J and Figure S9D-J:Mann-Whitney-Wilcoxon test,p-values> 0.05).This suggests that both MPs and MGBs have little contribution to the overall gene expression divergence between the two mushroom species at all three developmental stages,although we cannot rule out the possibility that the interspecific expression differences of a small subset of genes are regulated by differential MPs and/or MGBs 4.Discussion toliensis and P.,as two widely culti ated edibl mu me Afte asp d f 3.845lMa transitions in the two mushroom species.We found that overall DNA methylation levels and atterns(CG methylome)are highly conserved between the two species and largely stable across the major developmental stages,while overall gene expression profiles(transcriptomes)manifest development-dependent interspecihic divergence with fruit body showing the largest difterence.Thus sudy provi important epigenome and transcriptome information in relation to developmen such asp in TEe hylum These ties of DNA methylation are fully d in o ervation is that both the content and composition of T between the two mushrooms,Pt and Pe,albeit their-18 MYA divergence.In line with conservation of TEs,Pt and Pe showed similar methylation landscapes in both TEs and genic regions.This may suggest the high efficiency of DNA methylation as a genome defense system results also accord vith previous studie demonstrating the high conservation of global DNA methylation patterns in 18,26 tha atic major develop ental transitions in both ears to play limited developmental roles in the fungus kingdom20 We found that compared with TEs,expressed protein-coding genes contain very low levels of mCG in general.Nevertheless,still hundreds of genes were identified to contain methylated promoters in both mushrooms.For these genes,a significant negative correlation exists between mCGs and gene prenvels,suggesting that tor a subset of genes,DNA methylaton likely plays regulatory r expre in Ple adjacen to TEs tended to be siler 1 .We idor that it that DNA methylation detected in g ic regions of the two mushrooms s cies may have heen the ov-products derived from adiacent tes rather than being modified via direct targeting.However our current data cannot directly test this scenario,and therefore,we cannot rule out alternative possibilities such as some unique properties of the TE-adjacent genes(relative to TE-remote genes)for direct n s do suggest that,when oc ringitplays some role n ulating comple proce ggreg d afte of cellula eration.stipe elongating and capGenes 2019, 10, 465 10 of 14 mCG divergence between promoters and gene bodies was not correlated with expression divergence of the corresponding orthologs (Figure S8D–J and Figure S9D–J; Mann-Whitney-Wilcoxon test, p-values > 0.05). This suggests that both MPs and MGBs have little contribution to the overall gene expression divergence between the two mushroom species at all three developmental stages, although we cannot rule out the possibility that the interspecific expression differences of a small subset of genes are regulated by differential MPs and/or MGBs. 4. Discussion Pleurotus toliensis and P. eryngii, as two widely cultivated edible mushrooms, have been commercialized as nutritional, medicinal and animal feed products [42–44]. After ~18 MYA (Million Years Ago) divergence, the two species differ in aspects of habitat preference, growth condition, wood-decaying enzymes and fruit body phenotypes [3,8,45]. Major goals of this work were to establish the methylome landscapes and global gene expression profiles during major developmental transitions in the two mushroom species. We found that overall DNA methylation levels and patterns (CG methylome) are highly conserved between the two species and largely stable across the major developmental stages, while overall gene expression profiles (transcriptomes) manifest development-dependent interspecific divergence with fruit body showing the largest difference. Thus, our study provides important epigenome and transcriptome information in relation to development and divergence of the two mushroom species. DNA methylation shows conserved properties, such as predominant confining to CG contexts, preferential localization in TEs, and largely depletion in genic regions, across the entire fungal phylum [18]. These properties of DNA methylation are fully recapitulated in our results. A remarkable observation is that both the content and composition of TEs are very similar between the two mushrooms, Pt and Pe, albeit their ~18 MYA divergence. In line with conservation of TEs, Pt and Pe showed similar methylation landscapes in both TEs and genic regions. This may suggest the high efficiency of DNA methylation as a genome defense system in Pleurotus [19]. Our results also accord with previous studies demonstrating the high conservation of global DNA methylation patterns in both ascomycetes and basidiomycete [17,18,26]. Our results show that mCG levels are static across major developmental transitions in both mushrooms, and which applies to both TEs and protein-coding genes. This lends further support to the previously proposed notion that, in contrast to animals and plants [14,21], DNA methylation appears to play limited developmental roles in the fungus kingdom [20]. We found that compared with TEs, expressed protein-coding genes contain very low levels of mCG in general. Nevertheless, still hundreds of genes were identified to contain methylated promoters in both mushrooms. For these genes, a significant negative correlation exists between mCGs and gene expression levels, suggesting that for a subset of genes, DNA methylation likely plays regulatory role in their expression in Pleurotus. Previous studies indicate that genes adjacent to TEs tended to be transcriptionally silenced by DNA methylation [8,19,20]. In line with this observation, we also found that mCG levels of methylated promoters reduce gradually as distances to their nearest TE increase. We consider that it is possible that DNA methylation detected in genic regions of the two mushrooms species may have been the by-products derived from adjacent TEs rather than being modified via direct targeting. However, our current data cannot directly test this scenario, and therefore, we cannot rule out alternative possibilities such as some unique properties of the TE-adjacent genes (relative to TE-remote genes) for direct targeting by the methylation machinery. Irrespective of origin of the low level genic methylation in Pleurotus, our results do suggest that, when occurring, it plays some a role in regulating gene expression. Fruit body formation involves complex developmental processes in basidiomycete fungi. Transition from vegetative mycelium to primordium requires the aggregation of cells into compact hyphal knots [46]. Then, fruit body is formed after a battery of cellular events including differentiation of primitive hymenium, karyogamy generation, stipe elongating and cap expanding [47]. Conceivably