MSV diversity in African grasses Fig. 1. Complex relationships amongst MSV isolates sampled throughout Africa and the Indian Ocean island of La Re´ union. Whereas tree branches with less than 50 % bootstrap support have been collapsed, those with greater than 70 and 90 % support are labelled with open and filled-in circles respectively. The tree was rooted using Panicum streak virus (isolate Karino; GenBank accession no. L39638) as an outgroup (not shown). Virus names take the form Strainvariant [country–region– laboratory ID–year of isolation]. Variant numbers are equivalent to the subtype designators given in other publications (Owor et al., 2007b; Martin et al., 2001). Wherever representation of older recombination events would have obscured the representation of more recent recombination events, the older events have been displayed as a thinner bar. Hatched regions indicate tracts of sequence either transferred from currently unsampled MSV strains during a relatively recent recombination event, or transferred between ancestral sequences during a more ancient recombination event. Numbers associated with recombination events correspond with those in Supplementary Table S3. Events marked with an asterisk were characterized in Owor et al. (2007b). Positions of genomic features are indicated above the coloured bars: V2, movement protein gene; V1, coat protein gene; C1/C2, replication-associated protein gene; C1, repA gene; LIR, long intergenic region; SIR, short intergenic region. Bar, 0.2 nucleotide substitutions per site. example, Ng-Lag-2007, the only MSV-H isolate we have sampled, has a genome that appears to have been assembled during at least four separate recombination events. Adding to the complexity of interpreting the origins of sequences such as Ng-Lag-2007 is that we cannot know, without better sampling, either how old many of these recombination events are, or the order in which they most likely occurred. For example, lack of clear evidence for where the different pieces of Ng-Lag-2007 have come from indicates that the detected recombination events occurred between either progenitors of the sampled strains (i.e. if they are older events), or divergent, currently unsampled MSV genotypes (i.e. if they are more recent events). Possibly the most interesting amongst the less complex recombinant strains is MSV-A – the strain that causes maize streak disease. Previous analyses of recombination amongst MSV strains (Martin et al., 2001; Padidam et al., 1999) failed to detect that this strain had arisen from an ancient recombination event between MSV-G/MSV-F and MSV-B progenitors. This was because without the newly discovered MSV-G and -F genomes it was not possible to tell that the MSV-A virion sense ORFs were unusually similar to those of the MSV-Bs. Every currently sampled MSV-A genome has an unmistakable trace of this recombination signal (including the most divergent genomes from La Re´union; P57.461029), indicating that the recombination event must have occurred prior to the time of the last common ancestor of all known MSV-As. We must, however, caution that, given the inherent difficulties associated with identifying recombinant sequences in datasets with such high degrees of recomb- ination, it is possible that we have misidentified MSV-A as the recombinant in this sequence exchange. That a recombination event has occurred is very probable, but we cannot be absolutely certain that it is not all of either the MSV-B or MSV-G and MSV-F sequences that are recombinant instead of the MSV-A sequences. It may require either the discovery of non-recombinant close relatives of the MSV-A viruses or fitness studies on laboratory reconstructions of the possible ancestral par- ental and recombinant viruses to prove that it is MSV-A and not the other strains that are recombinant. However, until this information becomes available, the most http://vir.sgmjournals.org parsimonious hypothesis presented by RDP3 is that the MSV-As are recombinant. Partial conservation of recombination patterns amongst geminiviruses Conserved patterns of inter-species geminivirus recomb- ination including recombination hot- and cold-spots have recently been described amongst members of the genus Begomovirus (Lefeuvre et al., 2007a, b; Prasanna & Rai, 2007). Although similarities in intra-strain recombination rates have been demonstrated for MSV-A and variants of the begomovirus species, East African cassava mosaic virus and East African cassava mosaic Kenya virus (Owor et al., 2007b), it is currently unknown whether patterns of inter- species/strain recombination are also conserved between begomoviruses and mastreviruses. We therefore analysed the distribution of breakpoints detected in our African streak virus dataset using the method described by Heath et al. (2006). Despite the relatively low number of unique recombination events analysed (36 in the African streak virus dataset compared with 284 collectively detected in the datasets analysed by Lefeuvre et al., 2007b) there was strong statistical evidence of recombination hot-spots near the coat protein gene (cp)/short intergenic region (SIR) interface and at the virion-strand origin of replication (v- ori), and a recombination cold-spot spanning almost the entire cp (Fig. 2a). Importantly, these hot- and cold-spots are in almost precisely the same locations as those detected previously in begomoviruses (Fig. 2b). Unlike with the begomoviruses, however, in the mastrevirus genomes there is no evidence of a clear recombination hot-spot near the centre of the replication associated protein gene (rep). Also, in the mastrevirus dataset the cp/SIR interface hot-spot is substantially more pronounced than the v-ori hot-spot, whereas the converse is true for the begomoviruses. Despite these differences, this result strongly suggests that similar processes are shaping recombination breakpoint distributions in both genera. Importantly, the observed recombination patterns, including the differences between them, are entirely consistent with recent hypotheses that have invoked a mixture of biochemical and selective forces 2067