analysis of MSV evolution using the MODELTEST web server (Posada, 2006) Recombination was analysed using the RDP (Martin & Rybicki, 2000), GENECONV (Padidam et al., 1999), BOOTSCAN (Martin et al., 2005a), MAXCHI (Smith, 1992), CHIMAERA (Posada & Crandall, 2001), SISCAN (Gibbs et al., 2000) and 3SEQ (Boni et al., 2007) methods implemented in the RDP3 program (Martin et al., 2005b). Default settings were used throughout and only potential recombination events detected by two or more of the above methods, coupled with phylogenetic evidence of recombination, were considered significant. The severity of Bonferroni correction was minimized by only searching for recombination signals in a single sequence within groups of sequences sharing .99.3 % sequence identity. Using the approach outlined in the RDP3 program manual (http://darwin.uvigo.es/rdp/rdp.html), the approximate breakpoint positions and recombinant sequence(s) inferred for every potential recombination event were manually checked and adjusted where necessary using the phylogenetic and recombination signal analysis features available in RDP3. The distribution of unambiguously detected breakpoint positions of all unique recombination events was analysed for evidence of recombina- tion hot- and cold-spots with RDP3 as described by Heath et al. (2006). Published RDP3 project files describing breakpoint distributions detectable in bipartite and monopartite begomoviruses (Lefeuvre et al., 2007b) were merged in RDP3 and used to produce a composite plot of begomovirus recombination breakpoint distributions. RESULTS AND DISCUSSION Discovery of new MSV strains We cloned and fully sequenced 83 individual MSV genomes sampled primarily from indigenous African grasses presenting with streak symptoms. For preliminary objective classification of these sequences we aligned them with a selection of MSV (n588) and non-MSV (n524) African streak virus genomes and determined pair-wise percentage sequence identities shared between them. All of the new sequences shared greater than 79.1 % identity with previously described MSV isolates (Supplementary Table S2 available in JGV Online) and, based on the current ICTV species demarcation guidelines for the mastreviruses (Stanley et al., 2005), they are all MSV strain isolates. Consistent with previous analyses of African streak virus diversity (Martin et al., 2001; Willment et al., 2002; Shepherd et al., 2008b; Varsani et al., 2008), we further subdivided the sequences into strain groupings. As is clearly indicated by a deep trough between 92 and 94 % identity in a plot of pair-wise MSV sequence identities (Supplementary Fig. S1 available in JGV Online) and, in accordance with Martin et al. (2001), we identified 93 % identity as a ‘natural’ MSV strain demarcation threshold. Using this criterion we classified the MSV isolates into 11 strains (named MSV-A to -K), only five of which (MSV-A to -E) have been described previously (Martin et al., 2001; Schnippenkoetter et al., 2001; Willment et al., 2002). The similarities between the newly determined sequences and previously described MSV isolates allowed us to deduce that they contained all genomic features that have http://vir.sgmjournals.org MSV diversity in African grasses previously been identified as having functional relevance during MSV infections. Evidence of extensive inter-strain MSV recombination As recombination features prominently in geminivirus evolution (Lefeuvre et al., 2007b; Martin et al., 2001; Padidam et al., 1999; Prasanna & Rai, 2007) and can cause phylogeny reconstruction errors (Awadalla, 2003; Penny et al., 2007; Posada & Crandall 2002) we tried to remove, as far as possible, the influence of recombination from the construction of an MSV phylogeny. We therefore analysed the 83 newly sequenced genomes together with all other 112 publicly available African streak virus genomes using a battery of seven recombination analysis methods implemented in the RDP3 program. We found clear evidence of 36 distinct recombination events (detectable by three or more different analysis methods and with good phylogenetic support) spread across 164 of the 195 analysed genomes. Twenty-seven of these events were detected in 157 of the 172 analysed MSV sequences (Fig. 1; Supplementary Table S3 and Supplementary RDP3 project file). Due to such a high proportion of the analysed MSV sequences being detectably recombinant (91.3 %), it proved more difficult to produce a recombination-free MSV phylogeny than we had anticipated. The primary problem was that there was no fraction of the sequence alignment longer than 519 nt (alignment positions corres- ponding to nt sites 311 and 760 in MSV-Ns, taking position 1 as the first A residue 39 of the virion strand origin of replication) that was unbroken by detectable recombination breakpoints in any of the sequences. As this 519 nt region is quite conserved amongst the MSV isolates, phylogenetic analyses focusing on it lacked sufficient power to resolve relationships amongst individuals within par- ticular strains (see Supplementary Fig. S2 for this recombination-free phylogeny). To provide a general description of the relationships between all of the sequences we therefore opted to simply construct a maximum-likelihood tree using the full genome sequences, essentially ignoring recombination, and present it together with a breakdown of the sequence mosaics that must be considered when interpreting its topology (Fig. 1). Despite recombination undermining our confidence in the accuracy of this MSV phylogeny, there is good bootstrap support (.70 %) for all of our tentative MSV strain classifications. It should, however, be pointed out that the recombination analysis indicated that only four of the 11 strains (MSV-B, MSV-E, MSV-G and MSV-I) are pre- dominantly represented by sequences that are not the products of inter-strain recombination events involving exchanges of more than 30 % of their genomes. Some of the recombinant strains, such as MSV-H and -F, appear to have quite complex mosaic structures. For 2065