Ancient CO₂ levels favor nitrogen fixing plants over a broader range of soil N compared to present

Small inreases in CO₂ stimulate nitrogen fixation and plant growth. Increasing soil N can inhibit nitrogen fixation. However, no studies to date have tested how nitrogen fixing plants perform under ancient CO₂ levels (100 MYA), when nitrogen fixing plants evolved, with different levels of N additions. The aim of this study was to assess if ancient CO₂, compared to present, favors nitrogen fixers over a range of soil nitrogen concentrations. Nitrogen fixers (Alnus incana ssp. rugosa, Alnus viridis ssp. crispa, and Alnus rubra) and their close non-nitrogen fixing relatives (Betula pumila, Betula papyrifera, Betula glandulosa) were grown at ancient (1600 ppm) or present (400 ppm) CO₂ over a range of soil N levels, equivalent to 0, 10, 50, and 200 kg N ha^-1 year^-1. The growth of non-N fixing plants increased more than N fixing plants in response to the increasing N levels. When grown at an ancient CO₂ level, the N level at which non-nitrogen fixing plant biomass exceeded nitrogen fixing plant biomass was twice as high (61 kg N ha^-1 year^-1) as the N level when plants were grown at the ambient CO₂ level. Specific nodule activity was also reduced with an increasing level of soil N. Our results show there was a greater advantage in being a nitrogen fixer under ancient levels of CO₂ compared with the present CO₂ level.