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The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation 1-3 . Although the divergence observed at some host-resistance 4-6 and parasiteinfectivity 7-9 genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

SummaryThe wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss . Both polar and non-polar mutations in the sixth gene of the wss operon ( wssF ) or adjacent downstream genes ( wssGHIJ ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa , which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.

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