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The demographic history of Rhinolophus hipposideros (lesser horseshoe bat) was reconstructed across its European, North African and Middle-Eastern distribution prior to, during and following the most recent glaciations by generating and analysing a multimarker data set. This data set consisted of an X-linked nuclear intron (Bgn; 543 bp), mitochondrial DNA (cytb-tRNA-control region; 1630 bp) and eight variable microsatellite loci for up to 373 individuals from 86 localities. Using this data set of diverse markers, it was possible to determine the species' demography at three temporal stages. Nuclear intron data revealed early colonization into Europe from the east, which pre-dates the Quaternary glaciations. The mtDNA data supported multiple glacial refugia across the Mediterranean, the largest of which were found in the Ibero-Maghreb region and an eastern location (Anatolia/Middle East)-that were used by R. hipposideros during the most recent glacial cycles. Finally, microsatellites provided the most recent information on these species' movements since the Last Glacial Maximum and suggested that lineages that had diverged into glacial refugia, such as in the Ibero-Maghreb region, have remained isolated. These findings should be used to inform future conservation management strategies for R. hipposideros and show the power of using a multimarker data set for phylogeographic studies.

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Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.