Evolution of DNA Methylation across Insects

Abstract

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects.

Keywords: DNA methylation; molecular evolution.; phylogenetic comparisons; social behavior; whole genome bisulfite sequencing.

Fig. 1.

WGBS reveals extensive variation of DNA methylation in insects. (A) Genomic levels of DNA methylation in insects ranges from zero (all Diptera examined) to ∼14% (Blattella asahinai). Higher ranges are observed for coding regions; zero (all Diptera examined) to ∼42% (Blattella germanica). Overall, levels are highest in Blattodea, and do not always associate with social species. (B) A species tree constructed from nuclear and mitochondrial loci, which was used in Phylogenetic Generalized Least Squares (PGLS) analysis. Results from this analysis revealed that there is no correlation between social behavior and DNA methylation (table 1). Values at nodes are posterior probabilities <0.95; all blank nodes have ≥0.95 posterior probability.

Fig. 2.

Patterns of DNA methylation and social behavior across the insect tree of life. Relationships of 123 insect species, and outgroups Catajapyx aquilonaris (Dipluran) and Daphnia pulex (Crustacea) investigated with DNA methyltransferases, sociality, division of labor and DNA methylation scored as a binary (presence/absence) trait. The tree was constructed from 58 nuclear protein coding loci, and was used in Pagel’s test for evolutionary dependence. All nodes except one had posterior probability of 1.00, which is indicated on the phylogeny. (B) A chronogram of insect order relationships with sociality, division of labor and DNA methylation scored as binary (presence/absence) trait. The chronogram was modified from Misof et al. 2014. For (A) and (B), traits are represented as shaded boxes above each species or order. Half-filled boxes indicate the trait is variable within the corresponding order.

Fig. 3.

Evolution of DNMT1, 2 and 3 across Insecta and other Arthropoda. Relationships of DNMT1, 2, and 3 in insects, Diplura and Ixodida (Arachnida). DNMT2 can be found in all insect orders investigated, while DNMT1 and 3 are more order-poor. The insect order for each sequence is provided in square brackets following the GenBank or genome annotation accession number. The tree was rooted to DNMT2.

Fig. 4.

Divergent nonneutral evolution of DNMT1a and DNMT1b in Apoidea. (A) Hypothesized relationships among DNMT1 in Hymenoptera suggests a duplication event shared by the superfamilies Apoidea (bees) and family Formicidae (ants) gave rise to what is referred to as DNMT1a and DNMTb (supplementary fig. S3, Supplementary Material online). DNMT1b appears to have been lost from Formicidae (ants), whereas both DNMT1a and DNMT1b were retained in Apoidea (families Apidae, Halictidae, and Megachilidae). Divergent selection between DNMT1a and DNMT1b in Apoidea suggests the former is under relaxed purifying selection and the latter is under purifying selection. Bombyx mori (Lepidoptera) was used to root the tree, and was excluded from PAML analyses. (B) Several sites in DNMT1b were identified as under positive selection (yellow circles), with one site (T432) in the CXXC zinc finger domain, and three (V575, A581, S919) in the BAH domain. The crystal structure of Apis mellifera DNMT1b was predicted from Mus musculus DNMT1 using the program HHpred with default settings (Hildebrand et al. 2009). Details including red spheres, dark and light blue colouring specify Zn²⁺ions, CXXC domain and s-adenosyl-l-homocysteine, the secondary product from the DNA methylation reaction it performs, according to Mus musculus DNMT1, respectively. Bootstrap support values are characterized as shaded circles. dN/dS (ω) values are for the most preferred branch model.