Figure 3. Radial neighbor-joining tree of the cCP genes of 71 isolates of plum pox virus (red branches) and 47 outgroup potyviruses and ryegrass mosaic rymovirus (black branches). The number of isolates of each strain cluster is in parentheses. doi:10.1371/journal.pone.0002523.g003 therefore that the diversity of the PPV-M population has been acquired over the past 90 years plus an ‘eclipse’ period of 10 years during which the significance of the disease was not recognized or recorded, then the evolutionary rate of PPV-M will have been 1.461024 ns/s/yr. The PPV cCP sequences differ from those of the outgroup potyviruses by a mean of 1.269+/20.0696 ns/s, so that if the expected differences are linearly related to elapsed time, then the potyvirus radiation possibly occurred around 4532 years before present (YBP). Australian potyviruses. Agriculture was not established in Australia before the continent was colonized by Europeans in the Table 1. Potyviruses found both in Australia and elsewhere. Prehistory of Potyviruses late 18th century [38]. Thirty eight potyvirus species have been isolated in Australia and had their cCP sequences determined [39](List S5). Of those thirty eight viruses, twenty are cosmopolitan (Table 1) in that they are also found elsewhere in the world, whereas eighteen (Table 2) have only been found in Australia. Most of the cosmopolitan viruses are well studied crop pathogens that cause significant damage wherever they occur. Most of the Australian populations of the cosmopolitan viruses are much less diverse than their corresponding world populations, none are more diverse and some have only recently appeared in Australia. They are most probably immigrants carried to Australia during the past two centuries. By contrast the eighteen endemic potyviruses are mostly found in native plants or weeds, only two infect crop species, and most of their populations are much more diverse than those of the immigrant viruses indicating that they have been in Australia longer. There is no information about when individual immigrant viruses entered Australia, so it is not possible to compare the population diversity of each virus with its date of entry and thereby directly obtain measures of their evolutionary rates. However, if our conclusion that they have all entered during the past two centuries, is correct, then the average diversity of the populations can be assumed to have accumulated while they have been in Australia. Phylogenies calculated from the cCP sequences of the immigrant viruses indicated that seven probably entered Australia on only one occasion. The populations of these viruses had a mean basal divergence of 0.0176 ns/s (Table 1), and on average differed from the nearest overseas cCP by only twice as much, 0.0327 ns/s. The cCP sequences of the Australian immigrant viruses differed from those of the outgroup potyviruses by a mean of 1.4904 ns/s. The diversity and number of plants imported to Australia has greatly increased since agriculture was established two centuries ago, especially since the Second World War when the number of vessels, tonnage of goods and number of human migrants has increased exponentially [40] so it is likely that the mean time of potyvirus entry is less than 100 years. If we assume it is 75 years then the average evolutionary rate of the potyvirus populations is Name Apium virus Y Celery mosaic virus Johnson grass mosaic virus Papaya ringspot virus Sugarcane mosaic virus Sweet potato feathery mottle virus - strain C Sweet potato virus Y Mean 3 4 25 6 12 5 5 8.6 1Divergences calculated from ML trees (GTR+I+G) of the cCP sequences as the mean pairwise nucleotide substitutions/site of sequences linked through the basal node. 2Outgroup potyviruses of the same lineage omitted. 3NA, not applicable; a partial cCP sequence of a New Zealand isolate has been reported, its sequence is 97% identical to the Australian sequences. 4The atypical cCP sequence of PRSV from Lagenaria siceraria in Pakistan (AB127935) was excluded; divergence when that sequence is included is given in parentheses. Note: cCP sequences have also been reported for Australian isolates of bean common mosaic, bean yellow mosaic, clover yellow vein, Ornithogalum mosaic, pea seed- borne mosaic, peanut mottle, Pleione virus Y, sweet potato feathery mottle-strain RC, turnip mosaic and watermelon mosaic viruses, but their phylogenies indicate that different lineages have entered Australia, and hence on more than one occasion. Partial cCP sequences also available for Euphorbia ringspot virus (1 sequence), potato virus Y (3 sequences) and zucchini yellow mosaic virus (4 sequences) with nearest overseas sequences with 98%, 97–99% and 93–100% identities respectively in BLAST searches. doi:10.1371/journal.pone.0002523.t001 PLoS ONE | www.plosone.org 4 June 2008 | Volume 3 | Issue 6 | e2523 Number of Australian sequences Divergence1 of Australian sequences Divergence from closest overseas sequence Divergence of world population Divergence from outgroup potyviruses2 0.027 0.003 0.034 0.015 0.034 0.009 0.003 0.018 NA3 0.020 0.133 0.025 0.026 0.028 0.012 0.041 - 0.020 0.133 0.316 0.047 0.189 0.146 0.1864(0.378) 1.334(1.329) 1.467 1.458 1.645 1.414 1.595 1.520 1.490