The genus Microtus is one of the most speciose mammalian genera and at present, it consists of at least 90 species distributed across Palearctic and Nearctic. It represents an example of one of the most rapid radiations among mammals. Recognition of factors driving such radiations, whether it was an ecological or climatic change, is focal for understanding the mechanism of species formation and divergence. However, in spite of numerous studies, phylogenetic relationships within Microtus are not fully resolved, and the divergence times of the main lineages are uncertain and based only on imprecise paleontological calibrations. These relationships should be resolved with higher confidence using genome-wide data while direct molecular clock calibration may be provided by the DNA sequences from paleontological specimens. The main goal of the presented research project is to reconstruct the genome-based phylogeny of Microtus voles and to provide direct calibration of this phylogeny using genome sequences of Middle and Late Pleistocene vole specimens. To accomplish this, we will generate medium coverage genomes from modern individuals of 15-20 selected species from various Microtus subgenera. Moreover, we will obtain vole specimens from two key paleontological sites – Denisova cave (Altai, Russia) and Geißenklösterle cave (Germany). The chronology of both sites is well recognized, and they contain deposits ranging in age from ca. 250 to 40 ka and 90 to 30 ka, respectively. In addition, specimens from other sites like Obłazowa 2 (40-30 ka; Poland) and Emine Bair Khosar (120-10 ka; Crimea, Ukraine) will be included to increase the geographic and temporal scope of the analyzed specimens. Sequencing of ancient genomes from tiny rodent molars is highly challenging. DNA in palaeontological remains is present in the minute amounts, is highly fragmented and modified. DNA extracts are also highly contaminated with microbial DNA. However, preliminary experiments conducted by the PI showed that DNA in rodent molars is exceptionally well-preserved and enables genome-wide sequencing even for very old specimens. The state-of-art DNA extraction and library preparation methods combined with high-throughput sequencing will be used to generate low to middle coverage genomes from at least 15 paleontological specimens with the best-preserved DNA. We will use molecular dating approach based on mitochondrial phylogenies to validate the stratigraphic dating of specimens which are out of range of radiocarbon dating. This will result in a dataset comprising modern and ancient genomes of multiple Microtus species. It will be used to infer and calibrate the phylogeny of the Microtus genus. The amount and nature of the genome-wide data gives a promise of highly supported reconstruction while the age of the sequenced palaeogenomes (up to 200 ka) of the reliability of the calibration. Many of the vole species live in sympatry, genomic data will allow testing whether admixture occurred between particular species and to determine the timing and direction of the admixture events. Although, often sympatric, vole species frequently differ in their habitat preferences or occupy distinct ecological niches. Selection scans of the genomes of present-day individuals may potentially identify lineage-specific adaptations and genes under selection in particular species associated with different habitats. The presence of ancient genomes may allow identifying when the potentially adaptive variants first appeared. Previous studies of equine, woolly mammoth and cave bear palaeogenomes substantially influenced the understanding of the evolution of these taxa, thus we believe this project will provide a valuable contribution to the reconstruction of the evolutionary history of Microtus voles and to understanding speciation and species formation processes.