title: Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.
abstract: 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 (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. 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.
authors.affiliation: Sierra Nevada Research Institute, University of California Merced, 5200 N. Lake Road, Merced, CA, 95343, USA.|Climate and Ecosystem Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Moyes
authors.affiliation: Sierra Nevada Research Institute, University of California Merced, 5200 N. Lake Road, Merced, CA, 95343, USA.|Climate and Ecosystem Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Lara M
authors.initials: LM
authors.lastname: Kueppers
authors.affiliation: Physical and Life Sciences Directorate, Lawrence Livermore National Lab, 7000 East Avenue, Livermore, CA, 94550, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pett-Ridge
authors.affiliation: Life and Environmental Sciences, School of Natural Sciences, University of California Merced, 5200 N. Lake Road, Merced, CA, 95343, USA.
authors.forename: Dana L
authors.initials: DL
authors.lastname: Carper
authors.affiliation: Molecular Biophysics and Integrated Bioimaging Division, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Nick
authors.initials: N
authors.lastname: Vandehey
authors.affiliation: Molecular Biophysics and Integrated Bioimaging Division, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: James
authors.initials: J
authors.lastname: O'Neil
authors.affiliation: Sierra Nevada Research Institute, University of California Merced, 5200 N. Lake Road, Merced, CA, 95343, USA.|Life and Environmental Sciences, School of Natural Sciences, University of California Merced, 5200 N. Lake Road, Merced, CA, 95343, USA.
authors.forename: A Carolin
authors.initials: AC
authors.lastname: Frank
mesh_terms: D000114:Acetylene / Q000378:metabolism; D001419:Bacteria / Q000378:metabolism; D017753:Ecosystem; D060026:Endophytes / Q000378:metabolism*; D005030:Ethylenes / Q000378:metabolism; D016013:Likelihood Functions; D009586:Nitrogen Fixation; D009587:Nitrogen Isotopes; D009591:Nitrogenase / Q000378:metabolism; D010802:Phylogeny; D028223:Pinus / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D012987:Soil / Q000737:chemistry
chemical_list: D005030:Ethylenes; D009587:Nitrogen Isotopes; D012987:Soil; C036216:ethylene; D009591:Nitrogenase; D000114:Acetylene
keywords: 13N radioisotope; Pinus flexilis; acetic acid bacteria; acetylene reduction; conifer; limber pine; nitrogen fixation; subalpine