Background and Aims The reduction of plant productivity by low atmospheric CO 2 partial pressure (pCO 2 ) during the last glacial period is proposed as a limiting factor for the establishment of agriculture. Supporting this hypothesis, previous work has shown that glacial pCO 2 limits biomass in the wild progenitors of C 3 and C 4 founder crops, in part due to the direct effects of glacial pCO 2 on photosynthesis. Here, we investigate the indirect role of pCO 2 mediated via water status, hypothesizing that faster soil water depletion at glacial (18 Pa) compared to postglacial (27 Pa) pCO 2 , due to greater stomatal conductance, feeds back to limit photosynthesis during drying cycles.Methods We grew four wild progenitors of C 3 and C 4 crops at glacial and post-glacial pCO 2 and investigated physiological changes in gas exchange, canopy transpiration, soil water content and water potential between regular watering events. Growth parameters including leaf area were measured.Key Results Initial transpiration rates were higher at glacial pCO 2 due to greater stomatal conductance. However, stomatal conductance declined more rapidly over the soil drying cycle in glacial pCO 2 and was associated with decreased intercellular pCO 2 and lower photosynthesis. Soil water content was similar between pCO 2 levels as larger leaf areas at post-glacial pCO 2 offset the slower depletion of water. Instead the feedback could be linked to reduced plant water status. Particularly in the C 4 plants, soil-leaf water potential gradients were greater at 18 Pa compared with 27 Pa pCO 2 , suggesting an increased ratio of leaf evaporative demand to supply.Conclusions Reduced plant water status appeared to cause a negative feedback on stomatal aperture in plants at glacial pCO 2 , thereby reducing photosynthesis. The effects were stronger in C 4 species, providing a mechanism for reduced biomass at 18 Pa. These results have added significance when set against the drier climate of the glacial period.