Biol. Lett. (2011) 7, 747–750 doi:10.1098/rsbl.2011.0160 Published online 30 March 2011 Evolutionary biology Host-switching by a vertically transmitted rhabdovirus in Drosophila Ben Longdon1,*, Lena Wilfert2, Jewelna Osei-Poku2, Heather Cagney2, Darren J. Obbard1,† and Francis M. Jiggins2,† 1Institute of Evolutionary Biology, and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh EH9 3JT, UK 2Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK *Author for correspondence (b.longdon@ed.ac.uk). †These authors contributed equally to the study. A diverse range of endosymbionts are found within the cells of animals. As these endosym- bionts are normally vertically transmitted, we might expect their evolutionary history to be dominated by host-ﬁdelity and cospeciation with the host. However, studies of bacterial endo- symbionts have shown that while this is true for some mutualists, parasites often move horizon- tally between host lineages over evolutionary timescales. For the ﬁrst time, to our knowledge, we have investigated whether this is also the case for vertically transmitted viruses. Here, we describe four new sigma viruses, a group of vertically transmitted rhabdoviruses previously known in Drosophila. Using sequence data from these new viruses, and the previously described sigma viruses, we show that they have switched between hosts during their evolutionary history. Our results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts. Keywords: rhabdovirus; Drosophila; host-shift; sigma virus; phylogeny; vertically transmitted 1. INTRODUCTION Many animals have intimate associations with protists, bacteria and viruses, which live within the cytoplasm of their cells and are transmitted vertically between generations [1]. Vertical transmission and an inability to survive for long outside of the host mean that endosymbionts might be expected to show extreme host-ﬁdelity and cospeciate with their hosts. Indeed, phylogenies of bacterial endosymbionts show obligate mutualists have remarkably stable associations with their hosts. For example, Buchnera bacteria, which syn- thesize amino acids lacking from the diet of aphids, have been stably vertically transmitted for approxi- mately 150 – 250 Myr [2]. Similar patterns have been found in other mutualists such as Wigglesworthia in tsetse ﬂies [3], Blochmannia in carpenter ants [4] and Blattabacterium in cockroaches and termites [5]. By contrast, parasitic endosymbionts persist for rela- tively short periods in a given host lineage and Electronic supplementary material is available at http://dx.doi.org/ 10.1098/rsbl.2011.0160 or via http://rsbl.royalsocietypublishing.org. Received 11 February 2011 Accepted 7 March 2011 frequently switch host species. For example, there is little or no congruence between the phylogenies of Wol- bachia, [6], Rickettsia [7] and Spiroplasma bacteria [8] and their arthropod hosts. These associations may be unstable as hosts can evolve resistance and drive the parasite to extinction [9]. In contrast to bacterial endosymbionts, little is known about the evolutionary history of vertically trans- mitted viruses. Sigma viruses are vertically transmitted rhabdoviruses previously known from three species of Drosophila—Drosophila melanogaster (DMelSV) [10], Drosophila obscura (DObsSV) and Drosophila afﬁnis (DAffSV) [11]. These viruses are unusual in that they are transmitted vertically through both eggs and sperm [10,12]. Here, we describe four new sigma viruses that each infect a different species of Diptera, and use a phy- logenetic approach to show that sigma viruses have switched between host species during their evolution. 2. MATERIAL AND METHODS (a) Viral discovery and sequencing We collected Drosophila tristis in Derbyshire, UK; Drosophila immi- grans in Marktredwitz, Germany; Drosophila ananassae in Kiliﬁ, Kenya; and Muscina stabulans in Cambridge, UK. Infected ﬂies were detected by exposing them to pure CO2 at 128C for 15 mins. Uninfected ﬂies recover after approximately 30 mins while infected ﬂies remain paralysed [10]. RNA was extracted from paralysed ﬂies, reverse transcribed (see [11]), and ampliﬁed by PCR using multiple degenerate primers targeted to conserved regions of the viral RNA-dependent RNA polymerase gene (RDRP) (electronic supplementary material, table S1). PCR products were sequenced using BigDye reagents (GenePool facility, University of Edinburgh, UK) and once a small region of the RDRP gene had been sequenced, 30 RACE (rapid ampliﬁcation of cDNA ends) was used to obtain further sequence (see [11]). To obtain high-quality sequences, new primers were designed to amplify the fragment sequenced by RACE, and this was re-sequenced in both directions. The host species was conﬁrmed by sequencing mitochondrial COI and/or Cytb genes. Additional species were also collected and tested with the CO2 assay, but we only report those species from which we were able to amplify a sigma virus. (b) Inferring the virus phylogeny The nucleotide sequence of the RDRP genes from sigma viruses and other rhabdoviruses was aligned based on the translated amino acid sequence using CLUSTALW. Alignments were trimmed to contain only a conserved region of the RDRP that could be robustly aligned. Phy- logenies were inferred using maximum-likelihood (ML) (PAUP [13]) and Bayesian (MRBAYES [14]) methods. The ML analysis used a heuristic search with a nearest neighbour interchange algor- ithm and a general time reversible model with a gamma-distributed rate variation and a proportion of invariable sites. This model of sequence evolution was selected by comparing alternative models using Akaike information criterion in MODEL TEST [15]. Node-sup- port was estimated by non-parametric bootstrapping. The Bayesian analysis used the same model of sequence evolution and the Markov chain Monte Carlo was run for 1 million generations, sampled every 100 steps with the ﬁrst 25 per cent of samples being discarded as burn-in. (c) Detecting incongruent tree topologies To detect topological incongruence between host and parasite phylo- genies, we used a Shimodaira–Hasegawa test (SH-test) [16], which compares the likelihood of the viral phylogeny inferred from the data with one constrained to match the host topology [17,18]. We also used a Bayesian approach that identiﬁes the proportion of the pos- terior sample of viral topologies that match the host phylogeny (e.g. [19]). As these approaches compare only topologies (and not branch lengths), they are a conservative test for host switching. Even when topologies are incongruent, some cospeciation or switch- ing between related hosts may make host and virus topologies more similar than expected by chance. To test for topological similarity, we compared the distribution of Robinson-Foulds [20] distance metrics provided by 104 random viral topologies to that derived from the posterior sample of viral topologies. 747 This journal is q 2011 The Royal Society