748 B. Longdon et al. Sigma virus host switching 0.3 substitutions per site 1* 0.98 (75) 0.51* 1 (71) 0.59 (55) 1 (89) 1(100) 1(100) DMelSV (Hap23) 1(100) DMelSV (AP30) DAnaSV DImmSV DObsSV DTriSV DAffSV MStaSV D. melanogaster D. ananassae D. obscura D. tristis D. affinis D. immigrans M. stabulans Vesiculoviruses Ephemeroviruses Lyssaviruses Figure 1. Bayesian phylogeny of the sigma viruses (left) and their hosts (right). Node labels represent Bayesian posterior sup- ports with maximum-likelihood bootstrap support in brackets. The tree is rooted with the Lyssavirus clade. Non-sigma virus clades are collapsed. Nodes marked with an asterisk had bootstrap support of less than 50%. Drosophila melanogaster and D. ananassae shared a common ancestor approximately 20 million years ago (Ma) but both fall within the D. melanogaster group, which is separated from the obscura group (D. obscura, D. afﬁnis and D. tristis) by approximately 25 Ma. Both of these groups fall within the subgenus Sophophora, while D. immigrans is in the subgenus Drosophila, which separated from Sophophora approximately 40 Ma (dates from [21]). 3. RESULTS We detected novel sigma viruses in four dipteran species, including three species of Drosophila—D. tristis, D. immigrans and D. ananassae—and one member of the Muscidae, Muscina stabulans. We have tentatively named these new viruses as DTriSV, DImmSV, DAnaSV and MStaSV, respectively. This brings the total number of sigma viruses described to seven, and for the ﬁrst time extends their distribution outside the genus Drosophila. We sequenced 1845 – 3006 bp of the RDRP gene from the four viruses (accession-numbers: JF311399 – JF211402). The sequences are highly divergent from one another, with the most closely related pair, DAffSV and DTriSV, having an amino acid sequence identity of 0.73. The other ﬁve genes of sigma viruses have previously been shown to have even greater diver- gence [11]. The new sigma viruses form a clade of dipteran-infecting viruses that also contains the pre- viously described sigma viruses DMelSV, DObsSV and DAffSV (ﬁgure 1). In common with previous phylo- genies [11,22], the sigma virus clade is most closely related to the Ephemerovirus and Vesiculovirus clades (which together form the Dimarhabdoviruses). To test whether the sigma viruses have exclusively cospeciated with their hosts, we compared the host and virus phylogenies. The phylogeny of these host species is extremely well resolved [17,18]. We found the likelihood of the virus tree constrained to follow the topology of the host taxa was signiﬁcantly reduced (SH-test: 2DlnL ¼ 328, p , 0.005). For the Bayesian trees, we also found that the viral phylogeny differed signiﬁcantly from the host topology, with none of the topologies in the posterior sample of trees matching that of the hosts. Therefore, both methods suggest these viruses have switched between host species. Incongruence is owing to two factors: ﬁrst, the pres- ence of DImmSV in a clade of viruses with hosts from a different subgenus of Drosophila to D. immigrans Biol. Lett. (2011) [18]; and also D. obscura is much more closely rela- ted to D. tristis than D. afﬁnis [17], yet a viral clade comprising DTriSV and DAffSV is well-supported. However, although the trees were not congruent, we found that the inferred virus topology was more similar to the host topology than expected by chance. Only 2 per cent of random viral topologies were closer to the host topology than the posterior sample of actual virus topologies were to the host topology [20]. This may imply cospeciation events, but could be owing to other factors such as preferential host switching between clo- sely related species. 4. DISCUSSION We have discovered four new sigma viruses in D. ananas- sae, D. immigrans, D. tristis and M. stabulans. Together with the three existing sigma viruses in other Drosophila, they form a clade of Dipteran-infecting rhabdoviruses. It is probable that these viruses are vertically transmitted as not only are all of the previously known sigma viruses ver- tically transmitted [12], but also vertical-transmission of CO2 sensitivity—the hallmark of sigma virus infection— is known from other Diptera [23,24]. The phylogeny of the viruses reﬂects neither the phylogeny of the hosts, nor the region of the world where they were collected (these viruses were isolated in Europe, Africa and America). Therefore, sigma viruses have switched between host lineages during their evolution. Sigma viruses have highly dynamic interactions with their hosts. In D. melanogaster populations, there has been a recent selective sweep of a gene conferring resistance to DMelSV [25], and this was followed by the sweep of a viral genotype that overcomes host resistance [26]. DObsSV also shows evidence of a recent and rapid sweep [12]. Such rapid changes in host resistance are expected to drive ﬂuctuations in viral prevalence, and may make virus – host associations unstable and short-lived [9]. If so, then the virus will