Periodic Reporting for period 1 - SpecIAnt (Speciation in ants: Unraveling the genetic, epigenetic and molecular basis of speciation using wood ants) Berichtszeitraum: 2018-09-01 bis 2020-08-31 Zusammenfassung vom Kontext und den Gesamtzielen des Projekts This project will contribute to answering some of the outstanding questions in speciation with three aims. First, I will confirm candidate speciation genes that I have discovered. Second, I will investigate the molecular basis of reproductive isolation using protein modeling and network analysis, testing the idea that speciation genes do not evolve independent of each other, but instead are part of same functional networks. Third, I will combine genomic data on three levels to study the relative roles of genes, gene expression and epigenetics in speciation. To achieve these goals I will use ant species of the Formica rufa s.str. and capitalize on my previous studies on hybridization in this group. In addition to the above aims I will develop an outreach programme on evolution and through collaboration with the Finnish Environment Institute help to elucidate the role of hybrids for forest ecosystems. Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse WP1: Validation and functional annotation of genes and gene networks being candidates for reproductive isolationSpeciation underlies the generation of novel biodiversity. Yet, there is much to learn about how natural selection shapes genomes during speciation. Selection is assumed to act against gene flow at barrier loci, promoting reproductive isolation. However, evidence for gene flow and selection is often indirect and we know very little about the temporal stability of barrier loci. We first identified candidate barrier loci and then tested for barrier stability in a sample collected ten years later and use survival analysis to provide a direct measure of natural selection acting on barrier loci. We find multiple candidate barrier loci most of which are predicted to fall into a single protein interaction network. Surprisingly, a proportion of them are not stable after ten years, natural selection apparently switching to favoring introgression in the later sample. Barrier instability and natural selection for introgressed alleles could be due to environment-dependent selection, emphasizing the need to consider temporal variation in natural selection and the stability of barrier loci in future speciation work. These results have been submitted to a journal (Kulmuni et al. 2019.WP2: Gene expression and methylation patterns underlying reproductive isolationDuring speciation the genomic architecture of diverging species may diverge to the extent that genes or genomic regions from different species become incompatible if brought together in hybrids. These genomic regions then restrict gene flow between species, and thus contribute to reproductive isolation. I propose that a similar divergence can evolve in gene expression and methylation, and that these also contribute to reproductive isolation. The aim of this WP was to discover gene expression and methylation patterns associated to hybrid male breakdown, contrasting expression in males who will survive to those that will die. However, surprisingly, WP1 revealed that natural selection acted opposite to what we had previously shown (Kulmuni & Pamilo, 2010, PNAS) and the individuals who were expected to experience negative selection were actually favored on our sampling year. Due to this finding we revised our study questions regarding gene expression and decided not to proceed to methylation studies, but instead try to understand why there seems to be yearly fluctuations in natural selection acting on hybrids (followed up in WP3).We contrasted individuals with more- or fewer-introgressed alleles, to investigate the number of differentially expressed genes and their functions associated with introgression. These results are currently being written up as a manuscript (Beresford, Morandin, Butlin, Kulmuni, manuscript).WP 3: Identify distribution of hybrids and parental species in Finland and correlate it with environmental variablesThe aim of this WP was to map the distribution of hybrids throughout Finland and understand which environmental variables could be affecting the fitness and survival of hybrid ants. The Finland-wide survey is still on-going.The intriguing result of potential fluctuating selection from WP1, led to further investigations of which environmental variables could be affecting the fitness and survival of hybrid ants. We hypothesised that since the two parental species are distributed in different latitutudes, F. aquilonia being northern and F. polyctena being southern, they could be adapted into different temperatures. Following from that we investigated the potential role of temperature in shaping the genetic variability in a hybrid population between Formica polyctena and F. aquilonia wood ants using long term population genetic data spanning 14 years. We find that the frequencies of both parental-like alleles in the hybrid population co-vary with temperature over the years in males but not in females. Results suggest hybridization can lead to sex-specific outcomes that are dependent on ecological factors and that genetic variability resulting from hybridization could help hybrids cope with varying temperature. These results are currently under review in Evolution (Martin-Roy & Kulmuni 2019.WP 4: Develop an outreach programme, suitable for high school students, to demonstrate the importance of evolution for our daily livesDuring 2017 and 2018 we developed and executed an outreach event about evolution and visited one high school, one daycare and one primary school.Altogether three manuscripts have been submitted during the MCSA funding period. I have presented the results obtained in the MCSA in one International conference (Gordon Conference for Speciation, California) and as an invited speaker at a international workshop (SMBE workshop in speciation genomics). After receiving the MCSA funding I have been able to expand my research group from one PhD student into three PhD students, 1 post doc and 2 MSc's. I have also received three other major grants with a total value of > 1M €, which have enabled me to expand the study questions being addressed in my group. Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts) This project set out to combine population level data on genomics, transcriptomics, epigenetics and fitness to understand the role of epigenetics in speciation and how natural selection acts on specific genomic regions during speciation and hybridization. In the end I combined genomics and transcriptomics and developed a SNP panel to measure fitness in the natural population. These tools allowed me to go beyond state-of-the-art by studying evolution in action in nature. I was also able to cement a new model system (hybridizing wood ants) in speciation and hybridization research. Due to its unique aspects, like haplodiploidy and possibility to collect long-term genetic data on the same populations, it will allow me to go beyond current state-of-the-art in speciation. The MCSA project laid the foundations for my future research where I will study outcomes of hybridization through time and space.Results from the WP3 of the MCSA project have implications for adaptation under climate change as they suggest genetic variation acquired through hybridization could help populations cope with fluctuating temperature (Martin-Roy & Kulmuni 2019). Understanding how wood ants, a keystone species in boreal forests, copes with fluctuating temperature has implications for healthy forest ecosystems.