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In a subarctic climate, the seasonal shifts in temperature, precipitation, and plant cover drive the temporal changes in the microbial communities in the topsoil, forcing soil microbes to adapt or decline. Many organisms, such as mound-building ants, survive the cold winter owing to the favorable microclimate in their nest mounds. We have previously shown that the microbial communities in the nest of the ant Formica exsecta are significantly different from those in the surrounding bulk soil. In the current study, we identified taxa, which were consistently present in the nests over a study period of three years. Some taxa were also significantly enriched in the nest samples compared with spatially corresponding reference soils. We show that the bacterial communities in ant nests are temporally stable across years, whereas the fungal communities show greater variation. It seems that the activities of the ants contribute to unique biochemical processes in the secluded nest environment, and create opportunities for symbiotic interactions between the ants and the microbes. Over time, the microbial communities may come to diverge, due to drift and selection, especially given the long lifespan (up to 30 years) of the ant colonies., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)