Figure 1. Unrooted phylogenetic tree showing the relation- ships of the cCP gene sequences of 227 potyvirids including 183 potyviruses. The ‘other potyvirids’ include species of Maclur- avirus (maclura mosaic, Narcissus latent and cardamom mosaic viruses), Bymovirus (barley mild mosaic, barley yellow mosaic, oat mosaic, wheat spindle streak mosaic and wheat yellow mosaic viruses), Tritimovirus (brome streak mosaic, oat necrotic mottle and wheat streak mosaic viruses), Ipomovirus (cucumber vein yellowing and sweet potato mild mottle viruses) and Rymovirus (ryegrass mosaic, Agropyron mosaic and Hordeum mosaic viruses). BVY is blackberry virus Y; an unassigned potyvirid species. The tree was calculated from the cCP regions using the PhyML program with the HKY+I+G model. doi:10.1371/journal.pone.0002523.g001 and that was given by only with a single method, Siscan, out of the seven in RDP3 and by PHYLPRO, and only one cCP, that of onion yellow dwarf virus, gave tentative evidence of recombination by two methods. So we conclude that the topology of the potyviruses is most probably a star-burst resulting from a major radiation event. Dating the Potyvirus Major Radiation Only in the last decade have significant numbers of potyvirus gene sequences been determined, and there is no reason to believe that comparisons of non-contemporaneous sequences obtained over such a short period of time would allow the ‘time signal’ to be distinguished with any accuracy from the ‘population variation signal’, as even the simplest calculation using uncorrected distances shows that it would involve a several hundreds-fold extrapolation. Therefore we searched for historical isolation and outbreak events that correlated with the phylogenetic relationships of potyvirus cCP sequences, and could provide longer time scales from which to estimate the date of the radiation. Four independent lines of evidence were found: Prehistory of Potyviruses Figure 2. Graph of the number of pairwise synonymous (blue) and non-synonymous (red) differences between the cCP genes of the 227 potyvirids in Fig. 1 plotted against total number of differences. cCP genes of different outgroup potyviruses mostly have total differences of around 0.36 uncorrected differences/site, corre- sponding to 64% identity or 1.5 nucleotide substitutions/site. doi:10.1371/journal.pone.0002523.g002 Plum pox virus. Long historical records exist for most Prunus species (stonefruits). Chinese literature records cultivation of the peach, Prunus persica, over the past 3000 years. Pliny (23–79AD) mentioned twelve distinct varieties of plum, some of which were taken to Great Britain by the Romans, where they were recorded in William Langland’s Piers Ploughman (1362) [35]. Significantly however, there is no known record of the very obvious and damaging symptoms of plum pox virus (PPV) until around 1915 in Bulgaria. Plum pox, ‘‘Sharka’’ in Slavic, then spread north and east across Europe in a well-documented epidemic infecting most cultivated and wild Prunus species [36], and it has recently spread to the Americas; it was first found in Chile in 1992 and in North America in 1999 [37]. Comparisons of the 67 cCP genes (List S4) of PPV isolates collected from throughout the area of this outbreak (Fig. 3) placed most of the isolates into two major clusters. These two clusters had been distinguished by serological tests before gene sequencing became routine, and they are called D (Dideron) and M (Marcus) strains of PPV. The D strain cCP sequences came from isolates collected throughout Europe, Eurasia and the Americas, whereas the M strain cCPs came only from Europe, and there are also four cCP sequences from three other distinct strains. The mean difference of all pairs of sequences linked through the basal node of the M strain cluster was 0.028+/20.004 nucleotide substitutions/site (ns/s), but only 0.009+/20.002 ns/s for the D strain. It might seem most likely that the epidemic of the last 90 years was of the more widely dispersed D strain. However the M strain causes much more severe symptoms and ‘‘is considered to be the epidemic form of the virus’’ [36], whereas the D strain may have spread further, but more recently, merely because infected plants show few or ephemeral symptoms and so plants chosen for propagation and dispersal may have been infected with this strain until more sensitive detection methods stopped its inadvertent spread in horticultural stock. Plum pox disease first appeared in Bulgaria 90 years ago, but the epidemic in stonefruits probably started several years before. If we assume PLoS ONE | www.plosone.org 3 June 2008 | Volume 3 | Issue 6 | e2523