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Bedrock-soil geochemistry dominates free-living nitrogen fixation in soils but not in litter via interspecific microbial associations

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Abstract

Free-living nitrogen fixation is an important pathway for nitrogen inputs into soil and litter ecosystems. Here we compared free-living nitrogen fixation in forest soils and litter layers between limestone and clastic rock. We found that free-living nitrogen fixation activity is largest in litter followed by limestone soils and lowest in clastic rock soils. Compared to clastic rock soils, limestone soils had higher pH, exchangeable calcium, and greater abundances of diazotrophs and phoD-harboring bacteria, along with increased microbial network complexity and stability. Network analysis identified phoD-harboring bacteria as having strong associations with diazotrophs and arbuscular mycorrhizal fungi, indicating their key role in phosphorus supply for soil free-living nitrogen fixation. In contrast, litter free-living nitrogen fixation activity remained consistent across lithologies due to similar microbial populations. These results suggest that higher soil pH and calcium in limestone promote free-living nitrogen fixation activity by fostering beneficial microbial associations, an effect absent in litter.

Data availability

The data that support the findings of this study are available via figshare at https://figshare.com/s/29d6c417beec414399dc. We deposited all the raw sequencing data reported in this study in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation, under project number PRJNA1169648. The accession IDs range from SRR30898894 to SRR30899051.

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Acknowledgements

We thank the team of Prof. Dejun Li for providing the basic data on litter. This study was supported by the State Key Program of National Natural Science Foundation of China (42430512); the Joint Funds of the National Natural Science Foundation of China (U23A20155); the National Natural Science Foundation of China (42377041; 42361144886); the National Key Research and Development Program (2022YFF1300705); Natural Science Foundation of Hunan Province (2023JJ20044; 2022JJ40535); the Science and Technology Innovation Program of Hunan Province (2023RC3202); the Guangxi Natural Science Foundation Program (2025GXNSFDA069041; 2022GXNSFAA035621; AB22080097); the Youth Innovation Promotion Association of the Chinese Academy of Sciences Program to Dan Xiao (2023383), Chinese Academy of Sciences International Partnership Program—Future Partnership Network (2025) to Dan Xiao, and CAS Scholarship to Dan Xiao.

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Authors and Affiliations

  1. Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China

    Dan Xiao, Wei Zhang, Peilei Hu, Mingming Sun, Jie Zhao, Hongsong Chen & Kelin Wang

  2. Huanjiang Agriculture Ecosystem Observation and Research Station of Guangxi, Guangxi Key Laboratory of Karst Ecological Processes and Services, Huanjiang Observation and Research Station for Karst Ecosystems, Chinese Academy of Sciences, Huanjiang, China

    Dan Xiao, Wei Zhang, Peilei Hu, Mingming Sun, Jie Zhao, Hongsong Chen & Kelin Wang

  3. Department of Environmental Microbiology, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland

    David R. Johnson

  4. Institute of Ecology and Evolution, University of Bern, Bern, Switzerland

    David R. Johnson

  5. Centre for Planetary Health and Food Security, Griffith University, Brisbane, QLD, Australia

    Zhihong Xu

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  1. Dan Xiao
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  3. Wei Zhang
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Contributions

All authors contributed intellectual input and assistance to this study and manuscript. D.X., W.Z., J.Z., H.C., and K.W. designed the research and developed the original framework. D.X., P.H., and M.S. collected the samples and performed the experiment and data analysis. W.Z., Z.X., and K.W. contributed ideas to the manuscript. D.X., D.R.J., W.Z., and K.W. contributed to the writing of the manuscript. The authors read and approved the final manuscript.

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Wei Zhang or Kelin Wang.

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Communications Earth and Environment thanks Dionisios Gasparatos and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Xiao, D., Johnson, D.R., Zhang, W. et al. Bedrock-soil geochemistry dominates free-living nitrogen fixation in soils but not in litter via interspecific microbial associations.
Commun Earth Environ (2026). https://doi.org/10.1038/s43247-026-03456-5

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