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Lost world of complex life and the late rise of the eukaryotic crown

Nature volume 618, pages 767–773 (2023)Cite this article

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Abstract

Eukaryotic life appears to have flourished surprisingly late in the history of our planet. This view is based on the low diversity of diagnostic eukaryotic fossils in marine sediments of mid-Proterozoic age (around 1,600 to 800 million years ago) and an absence of steranes, the molecular fossils of eukaryotic membrane sterols1,2. This scarcity of eukaryotic remains is difficult to reconcile with molecular clocks that suggest that the last eukaryotic common ancestor (LECA) had already emerged between around 1,200 and more than 1,800 million years ago. LECA, in turn, must have been preceded by stem-group eukaryotic forms by several hundred million years3. Here we report the discovery of abundant protosteroids in sedimentary rocks of mid-Proterozoic age. These primordial compounds had previously remained unnoticed because their structures represent early intermediates of the modern sterol biosynthetic pathway, as predicted by Konrad Bloch4. The protosteroids reveal an ecologically prominent ‘protosterol biota’ that was widespread and abundant in aquatic environments from at least 1,640 to around 800 million years ago and that probably comprised ancient protosterol-producing bacteria and deep-branching stem-group eukaryotes. Modern eukaryotes started to appear in the Tonian period (1,000 to 720 million years ago), fuelled by the proliferation of red algae (rhodophytes) by around 800 million years ago. This ‘Tonian transformation’ emerges as one of the most profound ecological turning points in the Earth’s history.

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Fig. 1: Geological time chart comparing the molecular fossil, microfossil and phylogenetic records of early eukaryote evolution.
Fig. 2: Juxtaposition of biological and geological hopanoids and steroids, and the evolution of the sterol biosynthetic pathway through time.
Fig. 3: The succession of fossil steroids and bacterial hopanoids through time.

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Data availability

All processed data generated during this study are included in this published article and its supplementary information files. Raw data are available from the corresponding author on reasonable request.

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Acknowledgements

J.J.B. acknowledges funding support from Australian Research Council grants DP160100607, DP170100556 and DP200100004, and P.A. and P.S. acknowledge funding support from the Centre National de la Recherche Scientifique and the Université de Strasbourg. B.J.N. acknowledges doctoral and postdoctoral fellowships by the Australian National University, CSIRO Office of the Chief Executive, and the Central Research and Development Fund of the University of Bremen. A.J.M.J. publishes with the permission of the Executive Director, Northern Territory Geological Survey. The authors thank D. Edwards and the organic geochemistry team at Geoscience Australia (GA) for oil samples from the National Collection; and the Geological Survey of Western Australia (GSWA), the Northern Territory Geological Survey (NTGS), M. Brasier, N. Butterfield, J. Chen, T. Gallagher, E. Grosjean, K. Kirsimae, A. Lapland, M. Moczydłowska, S. Porter, N. Sheldon, E. Sperling and S. Zhang for rock specimens and extracts.

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Author notes

  1. These authors contributed equally: Jochen J. Brocks, Benjamin J. Nettersheim

Authors and Affiliations

  1. Research School of Earth Sciences, The Australian National University, Canberra, Australian Capital Territory, Australia

    Jochen J. Brocks, Benjamin J. Nettersheim, Amber J. M. Jarrett, Nur Güneli, Tharika Liyanage, Lennart M. van Maldegem & Janet M. Hope

  2. MARUM–Center for Marine Environmental Sciences and Faculty of Geosciences, University of Bremen, Bremen, Germany

    Benjamin J. Nettersheim

  3. Université de Strasbourg, CNRS, Institut de Chimie de Strasbourg UMR 7177, Strasbourg, France

    Pierre Adam & Philippe Schaeffer

  4. Northern Territory Geological Survey, Darwin, Northern Territory, Australia

    Amber J. M. Jarrett

  5. GFZ German Research Center for Geosciences, Potsdam, Germany

    Christian Hallmann

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Contributions

J.J.B. and B.J.N. interpreted the data and wrote the paper with contributions from C.H. and all co-authors. B.J.N., A.J.M.J., N.G., T.L. L.M.v.M, J.M.H. and J.J.B. conducted the biomarker analyses. B.J.N. and J.M.H. conducted pyrolysis and ring-opening experiments. P.A. and P.S. synthesized standards, conducted RuO4 oxidation experiments and assisted with compound identification. J.J.B. conceived the project and compiled data and figures.

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Correspondence to Jochen J. Brocks or Benjamin J. Nettersheim.

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Extended data figures and tables

Extended Data Fig. 1 Diagenetic and pyrolysis products of ur- and protosterols.

Chemical structures in purple and cyan are biogenic precursors. Structures in black are fossil lipids detected in mid-Proterozoic sedimentary rocks and generated in pyrolysis experiments of the respective biolipids. Green arrows signify biosynthetic reactions, black dashed arrows point to products of diagenesis (and laboratory pyrolysis). Note that the dashed arrows do not imply direct product-precursor relationships as diagenetic reactions commonly involve numerous intermediates and complex reaction networks. All structures in black, apart from X, were found in mid-Proterozoic bitumens. TAS = triaromatic steroids, MAL = monoaromatic lanosteroids, DAL = diaromatic lanosteroids. ‘x,4,24-Me3TAS’ signifies a TAS methylated at C-4, C-24 and at an unknown position x.

Extended Data Fig. 2 Mass spectra and elution behaviour of cyclosterane and its cleavage products in a severely biodegraded migrabitumen (12Z083, drill core MY4, 103.3 m).

a, M/z 412 to 300 partial ion chromatograms of cyclosterane and successive side-chain cleavage products. ‘x’ indicate absence or low concentration of pseudohomologs indicative of side-chain branching positions. R is the pentacyclic core of cyclosterane, ‘*’ denotes a chiral centre. b, Total ion current (TIC) of the saturated hydrocarbon fraction of the biodegraded oil, highlighting the biodegradation resistance of the two cyclosterane isomers k1 and k2. ‘~’ marks truncated signal of internal standard. c, Mass spectra of the cyclosterane side-chain cleavage products. See text for explanation of red and blue dashed lines. d, Juxtaposition of the mass spectra of cyclosterane isomer k2 (upper panel) and cycloartane from the NIST 95 library (lower panel) (the mass spectrum of k1 is not shown as it is nearly identical to k2). e, Suggested major MS fragmentation of hypothetical cyclosterane structure I and cycloartane.

Extended Data Fig. 3 Mass chromatograms and spectra of protosteranes.

a, M/z 259 partial mass chromatograms of a co-injection experiment of Eocene bitumen Y2 containing 8β(H),9α(H)- lanostane l4 (see text) and 1,640 Ma Barney Creek Fm (B03178). b, MRM 414 → 259 showing the elution positions of lanostane isomers l1 to l4, and the results of pyrolysis experiments on cycloartenol, lanosterol and euphenol (3β,13α,14β,17α-lanost-8-en-3-ol). c, Mass spectra of signal m3 of euphenol pyrolysis, l4 in BCF sample B03178 and lanostane in the Y2 standard. d, e, MRM chromatograms highlighting the relative elution positions of stigmastanes, lanostane, cyclosteranes and C27 hopanes in (d) the 1,440 Ma Tieling Fm (sample 17B101) and (e) an Ordovician oil from Australia (GA#299). TTP1 and 2 are Tetracyclic Terpane isomers66. Note that 8β(H),9α(H)-lanostane l4 co-elutes with TTP2, but that its presence can be recognized by an elevated TTP2 peak or a shoulder trailing the peak. The chromatograms are identified by MRM precursor → product transitions and relative signal heights in % relative to the highest signal.

Extended Data Fig. 4 Mass chromatograms and spectra of monoaromatic lanosteroids (MAL).

a, M/z 379 mass chromatogram identifying the 20S and 20R isomers of C29 MAL in sample B03163 from the 1,640 Ma Barney Creek Fm (black) and coinjection experiment with an authentic MAL standard on a DB-5MS capillary column (red). b, Authentic C29 MAL standard. c, C28 MAL of sample 14B211 from the 725 Ma Kanpa Fm showing an immature isomer distribution (S << R). d, C28 MAL generated through pyrolysis of lanosterol (sample BEX20150624). e, C28 MAL of sample B03163 from the 1,640 Ma Barney Creek Fm showing a mature isomer distribution (S ≈ R). seco-hop = monoaromatic 8,14-secohopanoids (see Extended Data Fig. 8a). f, Mass spectra of signals labelled in (a) to (e). a1 to a4 are chromatographic signal identifiers for geological bitumens, and A1 to A4 identifiers for corresponding authentic standards and pyrolysis products. Ancient bitumen chromatograms are in black, standard chromatograms in blue.

Extended Data Fig. 5 Mass chromatograms of diaromatic lanosteroids (DAL).

a, d, g, M/z 404, 375 and 361 mass chromatograms identifying isomers of C28, C29 and C30 DAL generated through pyrolysis of cycloartenol. b, e, h, C28, C29 and C30 DAL of sample 14B211 from the 725 Ma Kanpa Fm showing an immature isomer distribution (20S << 20R). c, f, i, C28, C29 and C30 DAL of sample B03162c from the 1,640 Ma Barney Creek Fm with a mature isomer distribution (20S ≈ 20R). b1 to b10 are chromatographic signal identifiers for geological bitumens, and B1 to B10 identifiers for pyrolysis products of authentic standards in blue.

Extended Data Fig. 6 Mass spectra of DAL given in Extended Data Fig. 5.

b1 to b10 are chromatographic signal identifiers for geological bitumens, and B1 to B10 identifiers for pyrolysis products of cycloartenol. Vertical labels ‘13 Aug 18 35’ are unique identifiers for individual GC-MS experiments. Signals marked ‘x’ are from coeluting compounds.

Extended Data Fig. 7 Mass chromatograms of triaromatic steroids (TAS).

a, M/z 259 mass chromatogram of TAS with two methylations in the ring system (Me2TAS) in sample 13N14 from the 1,100 Ma El Mreïti Group, and b, in the pyrolysate of an enrichment mixture of cycloartenol (CA) and 24-methylene cycloartenol (MCA). c, M/z 245 chromatogram of A-ring methylated TAS (MeTAS) of the pyrolyzed CA and MCA mixture, d, of sample 10J093 of the ~750 Ma Chuar Group, e the El Mreïti Gr (as above), and f, the Phanerozoic-based AGSOSTD oil reference standard. TAS dinosteroids in grey. g, M/z 231 mass chromatogram of TAS without methylations in the ring system of the Chuar Gr, h, the El Mreïti Gr and i, the AGSOSTD (samples as above). j, M/z 253 chromatogram of monoaromatic steroids (MAS) of sample 14B212 of the ~725 Ma Kanpa Fm and k, the AGSOSTD. Colour coding as in panel (e). MAS compound identification and roman numeral nomenclature follows Figure 13.107 and 13.108 in ref. 67.

Extended Data Fig. 8 Mass chromatograms of aromatic hopanoids in the 1,640 Ma Barney Creek Fm (12B117), and thermal maturity evaluation of the sample set.

Mass chromatograms of a, m/z 365 identifying regular aromatic 8,14-secohopanoids, b, m/z 351 regular 28-nor-8,14-secohopanoids, c, m/z 414 8,14-secohopanoids with a fluorene moiety, d, m/z 416 and e, m/z 402 8,14-secohopanoids with an acenaphthene moiety, and f, m/z 191 benzohopanoids. g,h, Evaluation of possible bias in the aromatic steroid and hopanoid record caused by thermal maturity based on cross plots of thermal maturity indicator Rc(MPR) against (g) sample age, and (h) the relative abundance of aromatic hopanoids and steroids (H/(H+S)). Rc(MPR) = computed vitrinite reflectance (Rc) based on the Methyl Phenanthrene Ratio (MPR, see Supplementary Table 1).

Extended Data Fig. 9 Confirmation of the elution position of S and R 4,24-dimethyl triaromatic cholesteroids in SIR m/z 245 traces on DB-5MS (left) and VZ-200 (right) gas chromatographic columns.

a, c, The AGSOSTD standard, b, e, the ~1,300 Ma Velkerri Formation, Roper Group (sample 230692, drill core Altree-2, 410.55 m). Note that the 20S-isomer of the 4,24-dimethyl triaromatic cholesteroid co-elutes with the 20R-isomer of 4-methyl triaromatic cholesteroid on the DB-5MS capillary column, but is resolved on VZ-200. d, A-ring methylated 24-methly triaromatic cholesteroid standard formed by pyrolysis of ergosterol (Supplementary Methods). 4,24-dimethyl triaromatic cholesteroids were also generated through pyrolysis of the C31 ursterol 24-methylene cycloartenol (Extended Data Fig. 7c).

Extended Data Table 1 Information on Cryogenian to Palaeoproterozoic geological samples
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Supplementary information

Supplementary methods and discussion

Reporting Summary

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Supplementary Table 1

Aromatic biomarker data. Relative abundance data of all aromatic steroids, protosteroids and hopanoids used to assemble Figs. 1a and 2b, including a key to the colours and reference to individual compounds identified in Extended Data Figs. 1–9. Also included is methylphenanthrene-based thermal maturity data.

Supplementary Table 2

Saturated biomarker data. Relative abundance data of all saturated steranes, protosteranes and hopanes used to assemble Fig. 2a, including a key to the colours, definition of ratios, and references to data taken from the literature.

Supplementary Table 3

Geological Samples. Characterization of all samples analysed in this study from the Palaeoproterozoic to the Cenozoic, including sample names, ages, basins, regions, groups, formations, drill core and outcrop names, sample depths and a brief description of lithology.

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Brocks, J.J., Nettersheim, B.J., Adam, P. et al. Lost world of complex life and the late rise of the eukaryotic crown. Nature 618, 767–773 (2023). https://doi.org/10.1038/s41586-023-06170-w

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