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. 2012;7(8):e42433.
doi: 10.1371/journal.pone.0042433. Epub 2012 Aug 3.

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination

Chris R Smith  1 , Navdeep S Mutti, W Cameron Jasper, Agni Naidu, Christopher D Smith, Jürgen Gadau
Affiliations

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination

Chris R Smith et al. PLoS One. 2012.

Abstract

Background: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events.

Methodology/principal findings: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP). Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD) is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids) were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination.

Conclusions/significance: These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic hybridization appear to have altered genomic methylation levels suggesting a possible link between changes in overall DNA methylation and the origin and regulation of genetic caste determination in P. barbatus.

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Conflict of interest statement

Competing Interests: The research that is presented in this article was performed while N. S. Mutti was at the Arizona State University, Tempe, AZ. N. S. Mutti is currently employed at DuPont, therefore Dr. Mutti's current contact information was updated to reflect change of employment. DuPont played no role in designing, conducting or funding the research presented in the article, therefore it won't alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. In the dependent lineage (genetic caste determining) Pogonomyrmex, queens obligately mate within and between lineages (the lineage pair J1/J2 are pictured here) in order to produce a functional colony.
Hybrid matings produce workers (horned symbols), while within lineage, “pure”, matings produce reproductive females (future queens). Diagnostic microsatellite markers can be used to assess the parentage of individuals at any point during development, even prior to their physical differentiation.
Figure 2
Figure 2. Methylation profile for three genes.
The red rectangle represents the portion of this gene that was amplified by the nested primers. Below the full gene figure is a zoom view of the amplified gene area. Below the amplified area are squares representing the methylation profile of the area. Open squares represent an unmethylated CpG. Black squares represent a methylated CpG. Red squares represent a methylated CpA, Blue squares represent a methlyated CpC, Green squares represent a Methylated CpT. “W” stands for “worker”. “Q” stands for “queen”. Below each methylation profile is an “N>1” sequence which displays sites found to be methylated in more than one sample. The number of squares represents the number of times the site was found. A “Q” or a “W” was placed under the site denoting if the multiple-sample site was relegated to that caste.
Figure 3
Figure 3. Global trends of CpG methylation.
a) The proportion of methylated loci increases in queens in adulthood, but is constant for workers over development. Only adult workers and virgin queens differed, with virgin queens having significantly more methylated DNA (P<0.05). In b) variation in the proportion of methylated loci between workers of different hybrid origin. Labels indicate the maternal lineage; all workers are hybrids between the J1 and J2 lineages. The direction of hybridization affected the degree of methylation, with workers from J1 mothers and J2 fathers being 17% more methylated (P<0.05) than those from the reciprocal cross. In c) a comparison between four lineages: P. barbatus and P. rugosus have normal (environmental) caste determination and ancestrally hybridized to give rise to the J lineages (which have genetic caste determination). The J lineages are significantly less methylated (P<0.05) than their parental species. Together, b) and c) show that two successive rounds of hybridization both changed the degree of genome methylation present in workers. All error bars are 95% C.I. Numbers inside the bars indicate sample sizes, and the number of colonies from which individuals were sampled (in parentheses).
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Publication types

Grants and funding

This study was funded primarily by a Post-doctoral Interdisciplinary Research in the Life Sciences (PIRLS) grant from Arizona State University to C. R. Smith and N. S. Mutti. Additional funding was provided by a grant from the National Science Foundation (IOS-0920732) to J. Gadau and C. R. Smith. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.