Model used Sequences1 GTR+I+G strict clock GTR+I+G Outgroup relaxed clock OG+CAb Root age2 22392.7 8404.5 8356.9 7932.3 27167 9357 8527.7 8630.5 Outgroup OG+CAb OG+PRS OG+CAb+PRS-CAb OG+CAb+PRS-CAb mean of rows abcd 11771.6 mean of rows bcd 8231.2 mean of rows bcde OG+PRS OG+CAb+PRS-CAb OG+CAb+PRS-CAb mean of rows abcd 13420.5 mean of rows bcd 8838.4 mean of rows bcde Outgroup OG+CAb OG+PRS OG+CAb+PRS-CAb OG+CAb+PRS-CAb mean of rows abcd 8760.3 mean of rows bcd 7479.2 mean of rows bcde 12603.8 7746.5 7570.4 7120.6 HKY+I+G strict clock HKY+I+G Outgroup relaxed clock OG+CAb Mid tree age3 Species age4 35364 8094.9 8484.4 8609.4 15138.21 8396.3 34967.8 8944.3 8295.7 10368.12 15644 9202.7 14697.5 7013.5 6677.4 6643.04 8757.9 6778 8996.2 7447.1 6276.2 8472.4 7573.2 NA5 653.9 290.2 633.4 292.5 467.5 687.9 700.2 598.3 266.8 563.3 472.9 253.1 459.3 255 360.1 653.6 645.9 474.9 254.9 507.3 a b c d e a b c d e a b c d e a b c d e Table 4. Results of BEAST analyses of test sequence data. OG+PRS OG+CAb+PRS-CAb OG+CAb+PRS-CAb mean of rows abcd 9257.6 mean of rows bcd 8083.1 mean of rows bcde 12780.9 11170.3 8776.3 7736.9 7736.1 1See Methods and Supporting Information: Lists S1 and S2. OG, outgroup, CAb, CAbMV cCP sequences. PRS, PRSV cCP sequences. 2Root age. The mean tree root height as ‘years before present’ (YBP) calculated by BEAST given a prior substitution rate of 1.261024 substitutions/site/year. 3Mid-tree age. The mean divergence (YBP) of all pairs of cCP sequences linked through the mid point of the tree estimated from the two most distant sequences. 4Species age. The mean divergence (YBP) of the American CAbMV (rows b and d) or PRSV (rows c and e) cCP sequences linked through the basal node of each cluster. 5NA, Not applicable. doi:10.1371/journal.pone.0002523.t004 5965+/2394 YBP and the mean evolutionary rate is 1.27361024 ns/s/yr. The large possible extrapolation inaccuracy of the Australian potyvirus evidence is offset by the fact that the data comes from several viruses We have least confidence in the results provided by the PPV epidemic data, as two different strains of the virus with seemingly different evolutionary rates were involved in the epidemic. Also, the time between the start of the outbreak and when it was first recorded is unknown. This ‘eclipse phase’ may have been more Prehistory of Potyviruses than the decade that we assumed, especially as, at the time, plant pathology was in its infancy. Indeed if the calculation is reversed, using the average of the three other radiation estimates, 5965 YBP, then we find that the PPV epidemic probably started about four decades before it was noticed, and this is entirely possible. Thus, to summarize, the ML analyses using a ‘strict clock’ and relying on the data obtained from the Australian and American potyvirus incursions indicate that the evolutionary rate of potyviruses is around 1.27361024 ns/s/yr and their major radiation occurred 5965 YBP. However analyses of the data indicate that a relaxed clock would be more appropriate than a strict clock, and comparisons of representative test cCP sequences have shown that a relaxed clock analysis finds an evolutionary rate 10% less, and hence a date for the potyvirus radiation 10% greater, than a strict clock analysis. Thus our studies indicate that the potyvirus cCP region most probably evolves at a rate of 1.1561024 ns/s/yr and the potyvirus major radiation occurred 6,560 YBP. The evolutionary rate we have estimated for potyviruses is similar to that of 1.161024 ns/s/yr of the mite-borne potyvirid, wheat streak mosaic tritimovirus (WSMV) [13], which was first found in American wheat crops in the 1920s [63] having arrived from Europe shortly before. Other similar published evolutionary rates of plant viruses are those for rice yellow mottle virus, which was estimated from samples collected over 40 years to be 4– 861024 ns/s/yr [14], and of tomato yellow leaf curl begomovirus estimated to be evolving at 4.661024 ns/s/yr from samples collected over 18 years [15]. Although these estimates are for different viruses, and so the differences may be species specific, it is noticeable that the evolutionary rates based on samples collected over a short time span (18 and 40 years) are several-fold greater than those based on longer time scales (100 years or more), as has been noted in studies of the rates of evolution of cellular organisms [64,65]. Not all plant viruses evolve so quickly. The tobamoviruses show evidence of having co-evolved with their hosts, and so it is possible they are more than 100 million years old [11,12] and evolving 10,000 times more slowly than potyviruses. However this range of evolutionary rates is not exceptional as viruses of animals cover the same range, indeed it is possible that a single ‘virus’ may cover that range by changing its ‘life style’; the pol gene of an endogenous lentivirus of European rabbit has been found to have homologues in exogenous (i.e. infectious) lentiviruses [66]. Our results place the potyvirus radiation in the centre of the current geological epoch, the Holocene, during which agriculture and complex human civilization has developed; and even if there had been fortuitously reinforcing combinations of errors in our methods they would have been unlikely to place our estimate of the radiation date outside the Holocene. Is this merely a co- incidence, or was agriculture itself responsible for potyvirus emergence and subsequent speciation? Modern humans first appeared during the Pleistocene around 250,000 years ago, but there is no evidence that animals and plants were domesticated until the last 8,500 to 13,000 years. Agriculture started in at least nine independent regions of the world, each with a different suite of animals and plants [43]. This suggests that the invention of agriculture was driven by a global trigger, rather than by local conditions. The Pleistocene was a period of greatly fluctuating climate. By contrast the Holocene, which started about 11,500 years ago, has been a period of unusually stable and predictable climate [67,68]. Richerson and colleagues [69] have convincingly argued that the Pleistocene climate was too changeable to allow the domestication of animals and plants. However the predictable climate in the Holocene drove the development of agriculture. It allowed human ‘hunter gatherers’ to increase in numbers and PLoS ONE | www.plosone.org 8 June 2008 | Volume 3 | Issue 6 | e2523