Summary: RDP3 is a new version of the RDP program for characterizing recombination events in DNA-sequence alignments. Among other novelties, this version includes four new recombination analysis methods (3SEQ, VISRD, PHYLRO and LDHAT), new tests for recombination hot-spots, a range of matrix methods for visualizing over-all patterns of recombination within datasets and recombination-aware ancestral sequence reconstruction. Complementary to a high degree of analysis flow automation, RDP3 also has a highly interactive and detailed graphical user interface that enables more focused hands-on cross-checking of results with a wide variety of newly implemented phylogenetic tree construction and matrix-based recombination signal visualization methods. The new RDP3 can accommodate large datasets and is capable of analyzing alignments ranging in size from 1000×10 kilobase sequences to 20×2 megabase sequences within 48 h on a desktop PC. Availability: RDP3 is available for free from its web site http://darwin.uvigo.es/rdp/rdp.html Contact:darrenpatrickmartin@gmail.com Supplementary information: The RDP3 program manual contains detailed descriptions of the various methods it implements and a step-by-step guide describing how best to use these.

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian phylogeographic inference and recombination analyses to available TYLCV sequences (including those of 15 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV's diversification and movements throughout the world. In agreement with historical accounts, our results suggest that the first TYLCVs most probably arose somewhere in the Middle East between the 1930s and 1950s (with 95% highest probability density intervals 1905–1972) and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and -IL strains. Despite the global distribution of TYLCV we found no convincing evidence anywhere other than the Middle East and the Western Mediterranean of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the center of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, the evidence indicates that the region is epidemiologically isolated, which suggests that novel TYLCV variants found there are probably not direct global threats. We instead identify the Mediterranean basin as the main launch-pad of global TYLCV movements. Tomato yellow leaf curl virus (TYLCV) poses a serious threat to tomato production throughout the temperate regions of the world. Our analysis, using a suite of bioinformatic tools applied to all publically available TYLCV genome sequences, suggests that the virus probably arose somewhere in the Middle East between the 1930s and 1950s and that its global spread only began in the 1980s after the emergence of two strains – TYLCV-Mld and -IL. In agreement with others, we also find that the highly invasive TYLCV-IL strain has jumped at least twice to the Americas – once from the Mediterranean basin in the early 1990s and once from Asia in the early 2000s. Although our results corroborate historical accounts of TYLCV-like symptoms in tomato crops in the Jordan Valley in the late 1920s, they indicate that the region around Iran is both the current center of TYLCV diversity and is the site where the most intensive ongoing TYLCV evolution is taking place. However, our analysis indicates that this region is epidemiologically isolated suggesting that novel TYLCV variants found there are probably not direct global threats. Moreover, we identify the Mediterranean basin as the main launch-pad of global TYLCV movements.

The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.

Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool.