Microbial Ecology

, Volume 71, Issue 3, pp 604–615 | Cite as

The Diversity and Co-occurrence Patterns of N2-Fixing Communities in a CO2-Enriched Grassland Ecosystem

  • Qichao Tu
  • Xishu Zhou
  • Zhili He
  • Kai Xue
  • Liyou Wu
  • Peter Reich
  • Sarah Hobbie
  • Jizhong Zhou
Environmental Microbiology


Diazotrophs are the major organismal group responsible for atmospheric nitrogen (N2) fixation in natural ecosystems. The extensive diversity and structure of N2-fixing communities in grassland ecosystems and their responses to increasing atmospheric CO2 remain to be further explored. Through pyrosequencing of nifH gene amplicons and extraction of nifH genes from shotgun metagenomes, coupled with co-occurrence ecological network analysis approaches, we comprehensively analyzed the diazotrophic community in a grassland ecosystem exposed to elevated CO2 (eCO2) for 12 years. Long-term eCO2 increased the abundance of nifH genes but did not change the overall nifH diversity and diazotrophic community structure. Taxonomic and phylogenetic analysis of amplified nifH sequences suggested a high diversity of nifH genes in the soil ecosystem, the majority belonging to nifH clusters I and II. Co-occurrence ecological network analysis identified different co-occurrence patterns for different groups of diazotrophs, such as Azospirillum/Actinobacteria, Mesorhizobium/Conexibacter, and Bradyrhizobium/Acidobacteria. This indicated a potential attraction of non-N2-fixers by diazotrophs in the soil ecosystem. Interestingly, more complex co-occurrence patterns were found for free-living diazotrophs than commonly known symbiotic diazotrophs, which is consistent with the physical isolation nature of symbiotic diazotrophs from the environment by root nodules. The study provides novel insights into our understanding of the microbial ecology of soil diazotrophs in natural ecosystems.


nifH Soil diazotrophs Community structure Co-occurrence patterns Elevated CO2 



We thank James W. Voordeckers for editing this paper. This work is supported by the U.S. Department of Agriculture (project 2007-35319-18305) through the NSF-USDA Microbial Observatories Program, by the Department of Energy under contract DE-SC0004601 through Genomics: GTL Foundational Science, Office of Biological and Environmental Research, and by the National Science Foundation under grants DEB-0716587 and DEB-0620652 as well as grants DEB-0322057, DEB-0080382 (the Cedar Creek Long Term Ecological Research project), DEB-0218039, DEB-0219104, DEB-0217631, and DEB-0716587 (BioComplexity, LTER and LTREB projects), the DOE Program for Ecosystem Research, and the Minnesota Environment and Natural Resources Trust Fund.

Conflict of Interest


Supplementary material

248_2015_659_MOESM1_ESM.docx (86 kb)
Fig. S1 Ordination analysis of the nifH community structure using unweighted PCoA (A) and NMDS analysis based on Bray-Curtis distance matrix (B). A trend of separation could be found in both analyses. (DOCX 86 kb)
248_2015_659_MOESM2_ESM.docx (318 kb)
Fig. S2 Response ratio analysis of significantly changed nifH OTUs. Relative abundance and genus assignment for these OTUs were also included. Error bars plotted at the right side of the dashed line indicate significantly increased relative abundance at eCO2, while error bars plotted at the left side indicate significantly decreased relative abundance at aCO2. (DOCX 317 kb)
248_2015_659_MOESM3_ESM.docx (227 kb)
Fig. S3 All nifH-centered modules identified in this study. Only modules with >5 nodes were included. Diamond nodes represent nifH OTUs. Circular nodes represent 16S OTUs. (DOCX 226 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Qichao Tu
    • 1
    • 2
  • Xishu Zhou
    • 2
    • 3
  • Zhili He
    • 3
  • Kai Xue
    • 3
  • Liyou Wu
    • 3
  • Peter Reich
    • 4
    • 5
  • Sarah Hobbie
    • 4
  • Jizhong Zhou
    • 2
    • 6
    • 7
  1. 1.Department of Marine Sciences, Ocean CollegeZhejiang UniversityHangzhouChina
  2. 2.Institute for Environmental Genomics and Department of Microbiology and Plant BiologyThe University of OklahomaNormanUSA
  3. 3.School of Minerals Processing and BioengineeringCentral South UniversityChangshaChina
  4. 4.Department of Forest ResourcesUniversity of MinnesotaSt. PaulUSA
  5. 5.Hawkesbury Institute for the EnvironmentUniversity of Western SydneyRichmondAustralia
  6. 6.Earth Science DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  7. 7.State Key Joint Laboratory of Environment Simulation and Pollution Control, School of EnvironmentTsinghua UniversityBeijingChina

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