Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N₂-fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis-AAB association is consistent across years, we re-sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N₂, we incubated twigs with ¹³N₂-enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using ¹³N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N₂ fixation in P. flexilis foliage. N₂ diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6 μg N m^-2 d^-1 to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stable N₂-fixing strategy for long-lived conifers. This novel source of biological N₂ fixation has fundamental implications for understanding forest N budgets.