Final Report Summary - GENTS (Genome Evolution in attine aNTS) The originally research included two major tasks: 1. Construction of chromosome mapping for A. echinatior and 2. Identification of imprinting genes in ant brains using transcriptome and epigenomic data. The first program has been combined with also devising a chromosome may for the inquiline social parasite A. insinuator. The second project had to be abandoned in its original version owing to lack of sufficiently replicated samples, but has been replaced by another citting edge genomic project on A. echinatior. My project has been handicapped by unexpected difficulties in rearing sufficient samples for obtaining the necessary biomass to successfully apply the (epi)genomic sequencing activities that were planned. As time went by and project 2 seemed increasingly less feasible, we decided to increase the level of ambition in project 1, so it came to include two sister species that split only a few million years ago, but that have significant differences in life-histories (one having evolved to be a social parasite of the other) so that the likelihood of chromosome re-arrangement seems relatively high. However, the social parasite species is rare and reproduces slowly in the lab. Nonetheless, the 100 full brother males for both species have been obtained, their DNA isolated, and they will be sent to BGI Shenzhen for sequencing.The second project hinged on sufficient availability of so-called “selfish patrilines”. That is lineages within a colony of A. echinatior that have the same father and that coexist with lineages having a different father (all colony members have the same mother queen). Such patrilines are called “selfish” if they produce more than their fair share of new (dispersing) virgin queens and fewer workers than the average patriline in a colony. We had hoped to have screened colonies available from a previous study documenting these selfish patrilines, but only one of these survived (haplessly the only one of that previous batch without selfish patrilines) and a massive new genotyping effort that we undertook with new lab colonies did not produce patrilines with sufficiently deviating caste ratios to make this project worth the investments in time and money.I therefore shifted my attention to the epigenetic regulation of caste differentiation and focused on the RNA-editing, an underappreciated epigenetic mechanisms in regulating gene function, to clarify its role in brain gene expression regulation across the three female castes of A. echinatior. We performed large-scale DNA and RNA sequencing with replicate samples from three colonies to identify genomic sites where RNA differed from DNA. We found that RNA-editing was pervasive across many ant genes expressed in the brains of small workers, large workers and gynes (virgin queens), with over ten thousand RNA-editing evens existing in over 800 genes. These RNA-editing genes appeared to be functionally enriched for neurotransmission, circadian rhythm, temperature response, RNA splicing, and carboxylic acid biosynthesis. About 10% of the RNA-editing sites were highly conserved between A. echinatior and other ant species whose genomes have been sequenced and might thus have been functionally important in early ant evolution. Another finding from this work was the varied editing levels of the same sites between castes, which suggested that RNA editing might be a general mechanism that shapes caste behavior in ants after the genetic code has been transcribed. This is the first time that RNA-editing regulation has been explored in a eusocial insect species. The paper has been accepted by Nature Communications and will be published online soon.During the second year of the MC grant, we also initiated a new project to assess mutation load and selection across the different stages of ant development. Ant males are haploid and produce enormous quantities of sperm in a matter of weeks, so that the millions of sperm stored in a queen’s spermatheca after mating should contain large numbers of sperm with slightly deleterious mutations, which may cause deleterious effect on individual fitness of workers (colony growth) and new virgin queens founding offspring colonies after dispersal, when such damaged sperm is actually used to fertilize eggs. We thus expect that mutational damage will gradually decrease going from eggs, to young larvae, to older larvae and to adults and that nursing workers will be particularly keen to raise only the least burdened offspring into new queens. Queens, however, need to produce millions of eggs during their long lives, so here mutations may accumulate when ovary tissues mutate somatically. This is fundamentally different from normal species where males are diploid, such as humans, where females are born with all the eggs they will ever have. We have completed a demanding sampling program to obtain all material for genome resequencing that will allow us to quantify the origin and regulation of mutational load in Acromyrmex echinatior. The samples for this project will be sent to BGI for sequencing 15 October 2014. In the past two years, I have been co-leading the genomic project for the dampwood termite (Zootermopsis nevadensis) together with Prof. Jürgen Liebig from Arizona State University. In this work, we compared the termite genome with eusocial Hymenoptera and identified several genomic transition significantly associated with its profound differences in mating biology relative to the Hymenoptera. This work was published in Nature Communications on 20th May 2014. At the same time, I also completed another termite genome sequencing project my host group (Associate Professor Michael Poulsen and Professor Jacobus Boomsma) on the fungus-growing termite (Macrotermes natalensis). We obtained the draft genome of the termite, its Termitomyces fungal symbiont, and several caste-specific termite gut metagenomes allowing us to study the shift in composition of the gut microbiota and the complementary roles of these bacteria in this complex symbiosis that also involves fungus gardens. The manuscript is under review now in PNAS.