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Nitrogen isotopes reveal independent origins of N2-fixing symbiosis in extant cycad lineages

Nature Ecology & Evolution volume 8, pages 57–69 (2024)Cite this article

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Abstract

Cycads are ancient seed plants (gymnosperms) that emerged by the early Permian. Although they were common understory flora and food for dinosaurs in the Mesozoic, their abundance declined markedly in the Cenozoic. Extant cycads persist in restricted populations in tropical and subtropical habitats and, with their conserved morphology, are often called ‘living fossils.’ All surviving taxa receive nitrogen from symbiotic N2-fixing cyanobacteria living in modified roots, suggesting an ancestral origin of this symbiosis. However, such an ancient acquisition is discordant with the abundance of cycads in Mesozoic fossil assemblages, as modern N2-fixing symbioses typically occur only in nutrient-poor habitats where advantageous for survival. Here, we use foliar nitrogen isotope ratios—a proxy for N2 fixation in modern plants—to probe the antiquity of the cycad–cyanobacterial symbiosis. We find that fossilized cycad leaves from two Cenozoic representatives of extant genera have nitrogen isotopic compositions consistent with microbial N2 fixation. In contrast, all extinct cycad genera have nitrogen isotope ratios that are indistinguishable from co-existing non-cycad plants and generally inconsistent with microbial N2 fixation, pointing to nitrogen assimilation from soils and not through symbiosis. This pattern indicates that, rather than being ancestral within cycads, N2-fixing symbiosis arose independently in the lineages leading to living cycads during or after the Jurassic. The preferential survival of these lineages may therefore reflect the effects of competition with angiosperms and Cenozoic climatic change.

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Fig. 1: Nitrogen isotope fractionation in the terrestrial nitrogen cycle.
Fig. 2: Nitrogen isotopic analyses of select fossil samples.
Fig. 3: Foliar nitrogen isotope data from cycad (blue) and non-cycad (red) fossils across the last ∼250 Myr.
Fig. 4: Evolutionary ecology of cycads.

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Data availability

Fossil occurrence data were downloaded from the Paleobiology Database (https://paleobiodb.org/) by means of the paleobioDB package in R (v.0.7.0). Non-N2-fixing plant nitrogen isotope data were downloaded from the TRY plant trait database (https://www.try-db.org/). Source data are provided with this paper.

Code availability

All code generated in this study is available in Supplementary Codes 1, 2, 3 and 4 as well as at https://github.com/m-kipp/cycad-evolution.

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Acknowledgements

We dedicate this paper to N. Nagalingum, whose pioneering research laid the foundations for this work by showing evidence of dramatic changes in recent cycad evolution and who also generously helped to locate suitable fossil cycad specimens for nitrogen isotope analysis in an early stage of this study. We also thank numerous individuals and institutions who assisted with providing access to or information about fossil collections, including P. Wilson Deibel, K. Anderson, D. Hopkins, R. Eng, M. Rivin, R. Serbet, B. Atkinson, P. Mayer, J. Watson, M. Pole, C. Liu, J. Wang, G. Shi, S. Manchester, H. Wang, A. Hendy, C. Jaramillo, P. Hayes, L. Stevens, J. Todd and T. Güner. A. Schauer is thanked for tireless technical assistance. M.A.K. acknowledges support from an NSF Graduate Research Fellowship and an Agouron Institute Postdoctoral Fellowship in Geobiology. Funding for isotopic analyses was provided by the University of Washington Royalty Research Fund and NASA Exobiology grant NNX16AI37G to R.B., as well as by a Paleontological Society student grant to M.A.K.

Author information

Authors and Affiliations

  1. Department of Earth & Space Sciences, University of Washington, Seattle, WA, USA

    Michael A. Kipp & Roger Buick

  2. Virtual Planetary Laboratory, NASA Astrobiology Institute, Seattle, WA, USA

    Michael A. Kipp, Eva E. Stüeken & Roger Buick

  3. Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA

    Michael A. Kipp

  4. Division of Earth and Climate Sciences, Nicholas School of the Environment, Duke University, Durham, NC, USA

    Michael A. Kipp

  5. School of Earth & Environmental Sciences, University of St. Andrews, St. Andrews, UK

    Eva E. Stüeken

  6. Department of Biology, University of Washington, Seattle, WA, USA

    Caroline A. E. Strömberg & William H. Brightly

  7. Burke Museum of Natural History and Culture, Seattle, WA, USA

    Caroline A. E. Strömberg

  8. Department of Knowledge, Royal BC Museum, Victoria, British Columbia, Canada

    Victoria M. Arbour

  9. Botanical Department, Hungarian Natural History Museum, Budapest, Hungary

    Boglárka Erdei

  10. School of Biological Sciences and the Environment Institute, University of Adelaide, Adelaide, South Australia, Australia

    Robert S. Hill & Miriam Slodownik

  11. Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA

    Kirk R. Johnson

  12. Department of Palaeontology, National Museum, Prague, Czech Republic

    Jiří Kvaček

  13. Department of Botany, School of Natural Sciences, Trinity College Dublin, Dublin, Ireland

    Jennifer C. McElwain

  14. National Geographic Society, Washington, DC, USA

    Ian M. Miller

  15. Research Division, Swedish Museum of Natural History, Stockholm, Sweden

    Vivi Vajda

  16. Department of Geology, Lund University, Lund, Sweden

    Vivi Vajda

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Contributions

M.A.K., E.E.S., C.A.E.S. and R.B. designed the study. C.A.E.S., V.M.A., B.E., R.S.H., K.R.J., J.K., J.C.M., I.M.M., M.S. and V.V. provided fossil specimens. M.A.K. and E.E.S. conducted the isotopic measurements. M.A.K., E.E.S., C.A.E.S. and R.B. analysed the data. W.H.B. conducted the ASR, with input from M.A.K., E.E.S., C.A.E.S. and R.B. M.A.K. wrote the manuscript, with input from all authors.

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Correspondence to Michael A. Kipp.

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Extended data

Extended Data Fig. 1 δ15N vs. C/N for all studied units.

(A) Soma flora, (B) Most Formation, (C) Gatuncillo Formation, (D) Chuckanut Formation, (E) Macquarie Harbor Formation, (F) Castle Rock flora, Denver Formation, (G) Comox Formation, Nanaimo Group, (H) Yorkshire flora, Cloughton Formation, (I) Primulaelv Formation, Kap Stewart Group, (J) Lunz flora, Lunz Formation, (K) Thale flora, Lower Keuper (L) Fremouw Formation. Cycad data shown as blue circles; non-cycad data as red crosses. Grey bands denote range of C/N ratios observed in modern cycads23; dashed lines denote range of δ15N values observed in modern cycads23. Fossil cycad foliage predominantly falls within the range of C/N ratios observed in modern plants. Fossil cycad foliage also overwhelmingly overlaps with the C/N ratios of other analysed plants, with one stark exception (Thale flora, panel K). In that case, the lack of δ15N vs. C/N correlation within either cycads or non-cycads suggests that diagenetic processes (which would impart a δ15N vs. C/N correlation) did not appreciably alter the isotopic composition of either group, or create a postdepositional isotopic offset between the two groups. Overall, the C/N data suggest that postdepositional alteration is unlikely to have imparted or obscured the isotopic trends observed across units.

Source data

Extended Data Fig. 2 TN content of fossil over matrix.

(A) Soma flora, (B) Most Formation, (C) Gatuncillo Formation, (D) Chuckanut Formation, (E) Macquarie Habor Formation, (F) Castle Rock flora, Denver Formation, (G) Comox Formation, Nanaimo Group, (H) Yorkshire flora, Cloughton Formation, (I) Primulaelv Formation, Kap Stewart Group, (J) Lunz flora, Lunz Formation, (K) Thale flora, Lower Keuper, (L) Fremouw Formation. Fremouw Formation samples were permineralized and thus did not allow a separate characterization of carbonaceous compression fossil versus matrix. Cycad data shown as blue circles; non-cycad data as red crosses. Dashed lines denote range of δ15N values observed in modern cycads23. Isotopic trends within and between units are not correlated with the N concentration of recovered foliage. Recovered fossil material has on average an order of magnitude more nitrogen than the background matrix, indicating that the isotopic signatures derive from the foliage and not soil organic matter.

Source data

Extended Data Fig. 3 Relative genus richness of Cycadales and Angiospermae.

Lines separately denote genus richness estimated via bootstrap resampled range through genus richness, TRiPS estimated genus richness and Chao1 estimated genus richness. Calculations are described in Methods.

Supplementary information

Reporting Summary

Supplementary Code 1

Analysis of geochemical data.

Supplementary Code 2

Ancestral state reconstruction.

Supplementary Code 3

Analysis of Paleobiology Database.

Supplementary Code 4

Functions required for TRiPS analysis.

Source data

Source Data Extended Data Fig. 1

Elemental and isotopic data.

Source Data Extended Data Fig. 2

Affinity of cycad taxa in Paleobiology Database.

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Kipp, M.A., Stüeken, E.E., Strömberg, C.A.E. et al. Nitrogen isotopes reveal independent origins of N2-fixing symbiosis in extant cycad lineages. Nat Ecol Evol 8, 57–69 (2024). https://doi.org/10.1038/s41559-023-02251-1

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