Local adaptation of plants along environmental gradients provides strong evidence for clinal evolution mediated by natural selection. Plants have developed diverse strategies to mitigate stress, for example, drought escape is a phenological strategy to avoid drought stress, while polyploidy was proposed as a genomic adaptation to stress. Polyploidy as an adaptation to aridity (an environmental parameter integrating temperature and precipitation) was previously documented in annual Brachypodium spp. (Poaceae) in the Western Mediterranean. Here, we examined whether polyploidy or phenology are associated with aridity in annual Brachypodium spp. along the aridity gradient in the Eastern Mediterranean. Using flow cytometry, we determined ploidy levels of plants from natural populations along the Israeli gradient, spanning ∼424 km from mesic Mediterranean to extreme desert climates. In a common garden we recorded time of seedling emergence, flowering and senescence. We tested whether the proportion of allotetraploids in the populations and phenological traits were associated with aridity. Contrary to a previous study in the Western Mediterranean, we found no effect of aridity on the proportion of allotetraploids and diploids within populations. Interestingly, phenology was associated with aridity: time of emergence was later, while flowering and senescence were earlier in desert plants. Our results indicate that in the Eastern Mediterranean, adaptation of Brachypodium to aridity is mediated mainly by phenology, rather than ploidy level. Therefore, we suggest that genome duplication is not the main driver of adaptation to environmental stress; rather, phenological change as a drought escape mechanism may be the major adaptation.

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6Word count: 7 Running short title: Adaptation to stress in Brachypodium along aridity gradient. 8 9 2 Summary 1 0• Plants have diverse strategies to cope with stress, including early flowering to "escape" 1 1 abiotic stress and late flowering to mitigate biotic stress. Plants are usually exposed to 1 2 multiple stresses simultaneously, but little is known about the impact of multiple co-1 3 occurring stresses on plant evolution. 1 4 • We tested for adaptation to both aridity and interspecific competition of the model plant 1 5Brachypodium spp., collected along the aridity gradient in Israel. We recorded flowering 1 6 time and estimated fitness in a controlled watering experiment, with treatments mimicking 1 7Mediterranean and arid precipitation, and in two common gardens located in the extremes of 1 8 the gradient (i.e., desert and mesic Mediterranean). At the latter we also manipulated 1 9interspecific competition to examine the combined effect of competition and aridity. 2 0 • Plants from arid environments always flowered earlier, but we found no selection on 2 1 flowering time in the watering experiment. In the common gardens, however, the direction 2 2 of selection on flowering time differed between sites and competition treatments.2 3 • We conclude that interactions between aridity and competition drive local adaptation of 2 4 Brachypodium in the Eastern Mediterranean basin. Variation in flowering time is an 2 5important adaptive mechanism to aridity and multiple selection agents can have interactive 2 6 effects on the evolution of this trait. 2 7 2 8