Figure 5. Radial neighbor-joining tree of the cCP genes of 256 isolates of papaya ringspot virus (coloured branches), two of zucchini yellow fleck virus (ZYFV), one of Moroccan watermel- on mosaic virus, with 46 outgroup potyviruses and ryegrass mosaic rymovirus (RGMV) (black branches) as an outgroup. The PRSV isolates came from the Indian subcontinent (green branches), or from East Asia (blue branches) or from the Americas (red branches). The number of isolates of each species is in parentheses. doi:10.1371/journal.pone.0002523.g005 carried by slaves from Africa 500 YBP then the evolutionary rate of CAbMV is 1.39761024 ns/s/yr, and the potyvirus radiation was, at most, 5565 YBP. The other virus that was probably involved in the great plant diaspora is papaya ringspot virus (PRSV). This virus causes a very damaging disease of papaya and is only distinguished from a potyvirus of cucurbits, originally called watermelon mosaic virus 1 [53], by its ability to infect papaya. The two viruses are now known as the papaya and watermelon strains of PRSV, namely PRSV-P and PSRV-W, but gene sequence evidence shows that individual PRSV-P populations have arisen independently from local PRSV-W populations in regions where papaya has been introduced as a crop [54,55]. The cCP sequences of over 300 isolates of PRSV have been reported and form a complex phylogenetic tree (Fig. 5 and List S7). The most diverse are those from India and Sri Lanka, and, excluding one atypical Pakistan isolate from the calabash gourd (AB127935) that should perhaps be considered a distinct species, the world population of PRSV has a basal node divergence of 0.186+/20.019. Two major PRSV radiations have arisen from different branches of the Indian population. The earliest includes all the isolates in east and south east Asia (China, Indonesia, Malaysia, Philippines, Taiwan, Thailand and Vietnam). The other includes all the isolates from the Americas with small separate sub-clusters of isolates from Hawaii, Taiwan and Australia, and has a basal node divergence of 0.075+/20.012 ns/s. The mean difference between the American lineage PRSV cCPs and the outgroup potyviruses is 1.494+/ 20.148 ns/s. Thus, if the evolutionary rates of PRSV and CAbMV are similar then the basal node divergences of their American populations, 0.075 and 0.1397, suggest that PRSV was Prehistory of Potyviruses carried to the Americas only 300YBP. This difference in the basal node divergences of the two populations was confirmed in all the tests done with different evolutionary models described below (Tables 3 and 4). There is, however, no published experimental evidence that PRSV is seed-borne, but the fact that its phylogeny indicates that it has migrated, at different times, from India via East Asia to several Pacific islands and, separately, from India to the Americas and from there, recently, to two Pacific islands and Australia is only plausibly explained by seed transmission. Published results of tests found no seed transmission of PRSV in papaya and Cucurbita spp. [56], but these are both natives of the Americas, and are probably recent hosts, whereas the phylogeny of PRSV indicates that its long term hosts are Old World cucurbits, and it is therefore much more likely to have been carried in seeds of plants, such as Citrullus lanatus, Cucumis spp., Lagenaria siceraria, Luffa spp., Momordica charantia and Trichosanthes cucumerina [52]. The earliest trade between India and the Americas was established at least 50 years later than that between Africa and South America. Goa was annexed in 1510 by the Portugese and became the base for repairing and provisioning their Asian fleet, which traded spices and silks to Europe from outposts such as Macau. It is possible that PRSV may have been carried in infected seed directly from Goa to Bahia (now called Salvador) in the late 16th century, however very few boats made that trip [57], and it is more likely to have been carried to South America via Europe probably in the early 18th century. At that time the Europeans, especially the British and the French, as part of their imperial ambitions, collected and distributed plants through the worldwide networks of botanic gardens they had established [58–60]; ‘‘Kew Gardens under (Joseph) Banks direction assumed the task of acting as the British Empire’s botanical clearing house’’ [61], and its 1768 list of plants [62] includes 21 cucurbit species including all but one of those named above. So, although PRSV might have been transported from India to South America as early as 450 YBP, it is more likely to have been carried there only 300 years ago. If the more recent date is correct then the evolutionary rate of PRSV is 1.2561024 ns/s/yr and the major radiation of the potviruses 5976 YBP. Dating by other models and methods Quantitative comparisons were also made of trees calculated from four representative sets of aligned cCP sequences. These were comprised of the outgroup potyvirus cCPs aligned with either 29 CAbMV or 30 PRSV cCPs, or both (see Methods). Trees were obtained using PhyML [24] and BEAST [25]. Comparisons of strict and relaxed clock models by clock likelihood ratio tests of the sequences favoured the relaxed clock (branch-specific rates) model, and the BEAST analyses of the four representative sets confirmed this as, with the relaxed clock, all sets of sequences gave only positive ‘coefficient of variation’ values; mean 0.245 and mean lower and upper 95% highest probability density values of 0.145 and 0.335. The PhyML tests (Table 3) showed that estimates of the age of the potyvirus radiation obtained using the HKY+I+G model were, on average, about 10% less than those obtained with the GTR+I+G model. The only other consistent difference between the two models was that the root of the tree was apparently about 10% less distant when it was estimated from the outgroup sequences alone (i.e. when all sequences were from different species), than when one or two of the species was represented by several sequences. There was no consistent difference in the apparent age of the root of the tree when that position was calculated as the midpoint of all the sequences in the tree rather than the mean pairwise difference between the outgroup sequences and the species represented by several sequences (i.e. CAbMV or PRSV). PLoS ONE | www.plosone.org 6 June 2008 | Volume 3 | Issue 6 | e2523