were manipulated using BIOEDIT [17]. The initial collation and alignment of sequences was done using CLUSTALX [18] and its neighbor-joining tree (NJ; [19]) option. The resulting trees were viewed using TREEVIEW [20]. Synonymous and non-synony- mous differences between sequences were estimated using the DnDscan method [21] and presented and graphed using EXCEL. Some distance estimates were presented as histograms using SBHistogram (http://www.sb-software.com/sbhisto/) Only the ‘coherently evolving’ region of the potyvirus coat protein genes (cCP; defined in Results) were used for phylogenetic comparisons; the cCP region of potato virus Y genome (Genbank Reference Sequence NC_001616 [22]) encodes the sequence - DVNAG- at its N-terminus and the C-terminus of the VP at the other. A representative set of 47 different cCP sequences (List S1) was used both as a template for all alignments and as an outgroup together, when required, with that of ryegrass mosaic rymovirus (RGMV) as an outlier to the outgroup. These sequences were aligned via their encoded amino acid sequences using the Transalign program (kindly supplied by Georg Weiller) and CLUSTALX [18] with default parameters, and gave an alignment with 720 nucleotides and gaps (240 codons); the alignment of the cCPs of these ‘outgroup potyviruses’ will be provided on request. The aligned outgroup sequences were checked for incongruent relationships, which might have resulted from recombination, using the RDP package version 3.22 [23] with default settings and a Bonferroni corrected P-value cut off of 0.05, and they were also checked by the PHYLPRO program (Weiller 1998). The phylogenetic relationships of sequences were inferred and compared mostly using the maximum likelihood method PhyML (ML)[24]. Limited tests were also done using the program BEAST [25] to assess whether a Bayesian method gave trees that were of different size or topology from those obtained by ML methods. We also checked whether both of the above methods and also the maximum parsimony method PAUP (version 4.0b10) with the tree branch reconnection [26] and neighbor-joining (NJ) method [19] gave trees with similar topologies. Default settings were used for all analyses except where specified. To compare the evolutionary rates obtained using different evolutionary models and methods we assembled four small representative sets of aligned cCP sequences. The basis of each set was the 47 outgroup potyvirus cCPs, which provided for each set the same unresolved root (see Results). One set was just the 47 species sequences, to another was added the 29 cCP sequences (List S2) of cowpea aphid-borne mosaic virus (CAbMV), to a third was added 30 representative cCP sequences (List S2) of papaya ringspot virus (PRSV), and the fourth had all three, outgroup plus CAbMV plus PRSV sequences; the four test sets thus contained a total of 47, 76, 77 and 106 sequences, but all had the same root. For each of the four test sequence sets MODELTEST 3.7 [27] favoured the general time-reversible model [28] with gamma- distributed rate variation [29] and a proportion of invariable sites (GTR+I+G) and the Hasegawa, Kishina, Yano model (HKY+I+G) [30] using both the Akaike information criterion (AIC) and likelihood ratio test [31]. Likelihood scores using a single clock for all branches (strict clock) or branch-specific clocks (relaxed clock) models were obtained in PAUP and compared using the clock likelihood ratio test (http://www.molecularevolution. org/cdc/resources/lrt.php; http://www.stat.tamu.edu/,west/ applets/chisqdemo.html). Trees were obtained using BEAST with either ‘strict’ or ‘relaxed’ clocks so that the effect of this difference on dating could be assessed; for the ‘relaxed’ clock the rates for different branches were distributed according to a lognormal distribution. Each BEAST analysis involved at least five million Monte Carlo Markov Chain (MCMV) generations with samples Prehistory of Potyviruses taken every 1000, and ESS values assessed by Tracer 1.4 of at least 50; duplicate analyses gave results that differed on average by less than 2% of the mean. Divergence times were estimated from the full sets of cCP sequences aligned with the outgroup cCPs by comparing pairwise distances inferred as ML estimates by PhyML and date estimates by BEAST using the GTR+I+G model for both. The Newick format trees obtained from PhyML or BEAST were converted to distance matrices using PATRISTIC [32]. The position of a node (single or polytomous) was then calculated as the mean pairwise patristic distance of all pairs of sequences connected through that node; this ‘basal divergence’ is double the mean length of the individual branches, which, combined with an estimate of the date of the node, provide an estimate of their ‘evolutionary rate’. Results The sequences There are more than 4,000 potyvirus sequences in the Genbank database. Most are from the 3-terminal region of the genome, which encodes the coat protein gene. The 59terminal region of the coat protein gene is frequently repetitive, whereas that encoding the core and C-terminus of the coat protein gene is not, and seems to have evolved in a coherent hierarchical way by point mutations and by occasional homologous recombination [33], so we call this region the ‘‘coherently-evolving coat protein’’ (cCP) region and used it for our phylogenetic studies. Trees with closely similar topologies were obtained from all regions of the potyvirid genome, except its variable 59-terminal region and the 59-terminal region of the coat protein gene, using maximum likelihood, parsimony or distance methods, and this topology did not change when the genome was divided by windows of fixed length rather than gene by gene [34]. Phylogenies calculated from the cCP region of 227 sequences representing all genera of the potyvirids place all potyviruses in a compact cluster with a surprisingly uniform radial branch length (Fig. 1 and List S3). The potyvirus cluster was consistently connected in all taxonomies by a short branch to the rymovirus cluster, and the rymoviruses were connected by much longer branches to the other potyvirid genera (Fig. 1). There are several ways in which the distinctive topology of the potyvirus cluster may have arisen. Most likely it originated as a major speciation event, a star-burst, from which all lineages have diverged at similar rates with several of those lineages also forming small sub-lineages. Alternatively, if potyvirus genes have a limited capacity for change, then the star-burst may reflect mutational saturation. However when the pairwise differences of the 227 potyvirid cCP sequences were separated into non-synonymous (NS) and synonymous (S) differences and were plotted against the total number of differences (Fig. 2), S differences can be seen to have saturated and not increase further after total differences exceeded about 0.20–0.30 uncorrected differences/site (ud/s), whereas NS differences between the cCPs of potyvirus species had not saturated. The large number of points around 0.36 ud/s represent pairwise comparisons between potyvirus species, where- as comparisons between the cCPs of species of different potyvirid genera ranged from 0.5–0.7 ud/s. This indicates that, unless the mutability of potyvirus cCP genes is quite different from that of the cCPs of other potyvirids, then the potyvirus topology is not the consequence of mutational saturation. Finally there is also the possibility that the potyvirus topology is the result of extensive recombination between sequences. However a check of the 47 outgroup potyvirus cCPs using the RDP3 package of programs found only five cCPs with tentative evidence of recombination, PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2523