Table 3. Results of PhyML analyses with test sequence data. Prehistory of Potyviruses Potyvirus divergence; mid-tree2 Potyvirus divergence; outgroup to CAb or PRS3 Species divergence4 0.153 0.074 0.276 0.171 0.149 0.072 0.222 0.172 a b c d e a b c d e Model used GTR+I+G HKY+I+G Sequences1 Outgroup OG+CAb OG+PRS OG+CAb+PRS2CAb OG+CAb+PRS2PRS Mean of rows abcd Mean of rows bcd Mean of rows bcde Outgroup OG+CAb OG+PRS OG+CAb+PRS2CAb OG+CAb+PRS2PRS Mean of rows abcd Mean of rows bcd Mean of rows bcde 1.460 1.666 1.525 1.669 1.669 1.580 1.620 1.330 1.508 1.372 1.550 1.550 1.440 1.477 NA5 1.742 1.447 1.664 1.483 1.584 1.542 1.341 1.592 1.450 1.481 1See Methods and Supporting Information Lists S1 and S2. OG, outgroup; CAb, CAbMV cCP sequences; PRS, PRSV cCP sequences. 2Mid-tree divergence (nucleotide substitutions/site). The mean pairwise difference of cCP sequences linked through the midpoint of the tree. 3Outgroup divergence (nucleotide substitutions/site). The mean pairwise difference between the outgroup cCP sequences and every American CAbMV or PRSV cCP sequence. 4Species divergence (nucleotide substitutions/site). The mean pairwise difference of sequences linked through the basal nodes of the clusters of the American CAbMV (rows b and d) or the PRSV (rows c and e) cCP sequences. 5NA, not applicable. doi:10.1371/journal.pone.0002523.t003 However the PhyML tests also showed that the apparent positions of the basal nodes of the CAbMV and PRSV clusters were clearly influenced by what other sequences were present in the dataset (Table 3). For example the node of the South American CAbMV sequences was at 0.153 ns/s when calculated from the ‘CAbMV plus outgroup’ sequences, but when the PRSV sequences were also added the node of the South American CAbMV sequences was estimated as 0.276 ns/s. This will affect attempts to estimate the age of the radiation for if it is assumed that CAbMV first entered South America 500 YBP, then the age of the potyvirus radiation estimated from the ‘CAbMV plus outgroup’ tree is 5444 YBP, whereas it is only 3023 YBP from the ‘CAbMV plus PRSV plus outgroup’ tree. Therefore for our dating analyses we used, where possible, test sequences from just a single species with the outgroup sequences. Most, but not all, of the results obtained in the BEAST tests (Table 4) were similar to those obtained with PhyML. For example both methods found the age of the potyvirus radiation given by the HKY+I+G model to be less than that given by the GTR+I+G model by a similar amount and, importantly, the BEAST tests showed that the ages of nodes estimated using the ‘uncorrelated lognormal (LN) relaxed clock’ were around 10% older than those obtained using a ‘strict clock’. The two methods used to estimate the position of the potyvirus radiation from the trees gave indistinguishable results. The greatest difference between PhyML and BEAST results was when the radiation was estimated from single representative sequences of each species, rather than when the dataset included sequences also providing intra-species variation. The ML estimate of the radiation was then about 10% less distant, whereas the BEAST estimate was 1.7 to 2.7 times more distant. In summary, the most consistent date estimates obtained by either PhyML or BEAST were those obtained with datasets that contained a mixture of inter-species and intra-species. These estimates were similarly variable whichever method was used; the standard deviations were from 3% to 12% of the means. The HKY+I+G model gave node estimates about 10% less than the GTR+I+G model, and the LN relaxed clock gave estimates about 10% more than a strict clock. The outgroup dataset alone gave inconsistent results. The CAbMV and PRSV sequences used for these tests were representative of those used in the main dating analyses described above, so it is of interest that all the results (Tables 3 and 4) showed the South American CAbMV lineage to be about twice as divergent as the American PRSV lineage, and hence possibly twice as old. Discussion Four independent lines of evidence have given surprisingly similar ML estimates (Table 5) of the date of the initial major radiation of potyviruses. They range from 4532 YBP to 6353 YBP and are based on independent historical isolation and outbreak events over the past 500 years. Table 5 also shows the large effect that the ‘extrapolation ratio’ has on the possible accuracy of the estimates; when the estimate is based on a branch node dated as 75 YBP then an error of one decade in that dating alters the estimated radiation time of the potyviruses by 847 years, whereas when the node is dated as 500 YBP then a 10 year error changes the radiation time by only 111 years. We have most confidence in the results produced by the Australian potyviruses and the American CAbMV and PRSV populations. The mean potyvirus radiation date from these is PLoS ONE | www.plosone.org 7 June 2008 | Volume 3 | Issue 6 | e2523