30 Polyomaviruses (PyVs) are small, double-stranded DNA tumor viruses carried by diverse 31 vertebrates. PyVs have previously been considered highly host restricted in mammalian hosts, 32 with host-switching events thought rare or nonexistent. Prior investigations have revealed 33 short-range host-switching events of PyVs in two different African bat species within the 34 horseshoe bat genus Rhinolophus. Herein, we have conducted a systematic investigation of PyVs 35 in 1,083 archived bat samples collected from five provinces across China, and identified 192 36 PyVs from 186 bats from 15 host species within 6 families (Rhinolophidae, Vespertilionidae, 37 Hipposideridae, Emballonuridae, Miniopteridae and Pteropodidae) representing 28 38 newly-described PyVs, indicative of extensive genetic diversity of bat PyVs. Surprisingly, two 39 PyVs were identified in multiple bat species from different families, and another PyV clustered 40 phylogenetically with PyVs carried by bats from a different host family, indicative of three 41 inter-family PyV host-switching events. The time to most recent common ancestor (tMRCA) of 42 the three events was estimated at 0.02-11.6 million years ago (MYA), which is inconsistent with 43 the estimated tMRCA of their respective bat hosts (36.3-66.7 MYA), and is most parsimoniously 44 explained by host-switching events. PyVs identified from geographically separated Chinese 45 horseshoe bat species in the present study showed close genetic identities, and clustered with 46 each other and with PyVs from African horseshoe bats, allowing assessment of the effects of 47 positive selection in VP1 within the horseshoe bat family Rhinolophidae. Correlation analysis 48 indicated that co-evolution with their hosts contributed much more to evolutionary divergence of 49 PyV than geographic distance. In conclusion, our findings provide the first evidence of . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/627158 doi: bioRxiv preprint 50 inter-family host-switching events of PyV in mammals and challenge the prevailing evolutionary 51 paradigm for strict host restriction of mammalian PyVs. 52 53 Author summary 54 Since the discovery of murine polyomavirus in the 1950s, polyomaviruses (PyVs) have been 55 considered both genetically stable and highly host-restricted in their mammalian hosts. In this 56 study, we have identified multiple cases of host-switching events of PyVs by large scale 57 surveillance in diverse bat species collected in China. These host-switching events occurred 58 between bat families living in the same colony, indicating that a large population with frequent 59 contacts between different bat species may represent an ecological niche facilitating PyV 60 host-switching. The cases studied involved members of bats from several families, including 61 horseshoe bats, which were previously found to harbor a number of highly virulent viruses to 62 both humans and...