Horizontal gene transfer (HGT) is an important factor in bacterial evolution that can act across species boundaries. Yet, we know little about rate and genomic targets of cross-lineage gene transfer and about its effects on the recipient organism's physiology and fitness. Here, we address these questions in a parallel evolution experiment with two Bacillus subtilis lineages of 7% sequence divergence. We observe rapid evolution of hybrid organisms: gene transfer swaps ∼12% of the core genome in just 200 generations, and 60% of core genes are replaced in at least one population. By genomics, transcriptomics, fitness assays, and statistical modeling, we show that transfer generates adaptive evolution and functional alterations in hybrids. Specifically, our experiments reveal a strong, repeatable fitness increase of evolved populations in the stationary growth phase. By genomic analysis of the transfer statistics across replicate populations, we infer that selection on HGT has a broad genetic basis: 40% of the observed transfers are adaptive. At the level of functional gene networks, we find signatures of negative, positive, and epistatic selection, consistent with hybrid incompatibilities and adaptive evolution of network functions. Our results suggest that gene transfer navigates a complex cross-lineage fitness landscape, bridging epistatic barriers along multiple high-fitness paths.

In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off.

Bacterial transformation, a common mechanism of horizontal gene transfer, can speed up adaptive evolution. How its costs and benefits depend on the growth environment is poorly understood. Here, we characterize the distributions of fitness effects (DFE) of transformation in different conditions and test whether they predict in which condition transformation is beneficial. To determine the DFEs, we generate hybrid libraries between the recipient Bacillus subtilis and different donor species and measure the selection coefficient of each hybrid strain. In complex medium, the donor Bacillus vallismortis confers larger fitness effects than the more closely related donor Bacillus spizizenii. For both donors, the DFEs show strong effect beneficial transfers, indicating potential for fast adaptive evolution. While some transfers of B. vallismortis DNA show pleiotropic effects, various transfers are beneficial only under a single growth condition, indicating that the recipient can benefit from a variety of donor genes to adapt to varying growth conditions. We scrutinize the predictive value of the DFEs by laboratory evolution under different growth conditions and show that the DFEs correctly predict the condition at which transformation confers a benefit. We conclude that transformation has a strong potential for speeding up adaptation to varying environments by profiting from a gene pool shared between closely related species.

9Horizontal gene transfer is an important factor in bacterial evolution that can act across 10 species boundaries. Yet, we know little about rate and genomic targets of cross-lineage 11 gene transfer, and about its effects on the recipient organism's physiology and fitness. 12Here, we address these questions in a parallel evolution experiment with two Bacillus 13 subtilis lineages of 7% sequence divergence. We observe rapid evolution of hybrid 14 organisms: gene transfer swaps ~12% of the core genome in just 200 generations, and 15 60% of core genes are replaced in at least one population. By genomics, transcriptomics, 16fitness assays, and statistical modeling, we show that transfer generates adaptive evolution 17 and functional alterations in hybrids. Specifically, our experiments reveal a strong, 18repeatable fitness increase of evolved populations in the stationary growth phase. By 19 genomic analysis of the transfer statistics across replicate populations, we infer that 20 selection on HGT has a broad genetic basis: 40% of the observed transfers are adaptive. 21At the level of functional gene networks, we find signatures of negative and positive 22 selection, consistent with hybrid incompatibilities and adaptive evolution of network 23 functions. Our results suggest that gene transfer navigates a complex cross-lineage fitness 24 landscape, bridging epistatic barriers along multiple high-fitness paths. 25 26 Significance statement 27In a parallel evolution experiment, we probe lateral gene transfer between two Bacillus subtilis 28 lineages close to the species boundary. We show that laboratory evolution by horizontal gene 29 transfer can rapidly generate hybrid organisms with broad genomic and functional alterations. 30By combining genomics, transcriptomics, fitness assays and statistical modeling, we map the 31 selective effects underlying gene transfer. We show that transfer takes place under genome-32 wide positive and negative selection, generating a net fitness increase in hybrids. The 33 evolutionary dynamics efficiently navigates this fitness landscape, finding viable paths with 34 increasing fraction of transferred genes. 35 36

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