SIMS analyses of the oldest known assemblage of microfossils document their taxon-correlated carbon isotope compositions J. William Schopfa,b,c,d,1, Kouki Kitajimad, Michael J. Spicuzzad, Anatoliy B. Kudryavtsevb, and John W. Valleyd aDepartment of Earth, Planetary, and Space Sciences, University of California, Los Angeles, CA 90095; bCenter for the Study of Evolution and the Origin of Life, University of California, Los Angeles, CA 90095; cMolecular Biology Institute, University of California, Los Angeles, CA 90095; and dWisconsin Astrobiology Research Consortium, Department of Geoscience, University of Wisconsin, Madison, WI 53706 Contributed by J. William Schopf, November 17, 2017 (sent for review October 16, 2017; reviewed by David J. Bottjer, Andrew Czaja, and Yanan Shen) Analyses by secondary ion mass spectroscopy (SIMS) of 11 specimens and physiological characteristics of these exceedingly ancient fossil of five taxa of prokaryotic filamentous kerogenous cellular micro- microbes remain to be established. Initially formally described as fossils permineralized in a petrographic thin section of the ∼3,465 Ma “prokaryotes Incertae Sedis”—nonnucleated microorganisms of Apex chert of northwestern Western Australia, prepared from the uncertain and undefined systematic relations (ref. 2, p. 643)—the same rock sample from which this earliest known assemblage of present study suggests a solution to this unresolved problem. cellular fossils was described more than two decades ago, show their We here present results of in situ analyses of 11 specimens of δ13C compositions to vary systematically taxon to taxon from −31‰ five taxa of permineralized microscopic fossils embedded in to −39‰. These morphospecies-correlated carbon isotope composi- Apex chert petrographic thin section 4 of 6/15/82-1H prepared tions confirm the biogenicity of the Apex fossils and validate their from the same rock sample at the same time as the six holotype- morphology-based taxonomic assignments. Perhaps most signifi- and paratype-containing sections previously archived at the 13 cantly, the δ C values of each of the five taxa are lower than those NHM (sections 4 of 6/15/82-1B through 6/15/82-1G; refs. 1 and − ‰ of bulk samples of Apex kerogen ( 27 ), those of SIMS-measured 2). The indigenousness and syngenicity of the permineralized − ‰ fossil-associated dispersed particulate kerogen ( 27.6 ), and those fossils to their encompassing chert matrix is shown by optical − ± ‰ typical of modern prokaryotic phototrophs ( 25 10 ). The SIMS microscopy supported by Raman spectroscopy which also es- EARTH, ATMOSPHERIC, δ13 AND PLANETARY SCIENCES data for the two highest C Apex taxa are consistent with those of tablishes their kerogenous composition. The biogenicity and δ13 extant phototrophic bacteria; those for a somewhat lower C taxon, taxonomic relations of the analyzed fossils are documented by with nonbacterial methane-producing Archaea; and those for the two their demonstrably cellular cylindrical filamentous morphology; lowest δ13C taxa, with methane-metabolizing γ-proteobacteria. Al- the taxon-defining size ranges of their medial cells and, where though the existence of both methanogens and methanotrophs has preserved, the morphology of their terminal cells; and their been inferred from bulk analyses of the carbon isotopic compositions morphometric comparison with previously reported specimens of pre-2,500 Ma kerogens, these in situ SIMS analyses of individual EVOLUTION from the same rock. Analyses of each of the 11 specimens by microfossils present data interpretable as evidencing the cellular pres- ervation of such microorganisms and are consistent with the near- secondary ion mass spectroscopy (SIMS) document the carbon basal position of the Archaea in rRNA phylogenies. isotope compositions of the five taxa studied. The taxon-correlated SIMS carbon isotope data reported here Apex chert | Archaea | Archean | methanogens | methanotrophs reaffirm the carbonaceous, kerogenous (rather than mineralic) Significance idely regarded as among the oldest known evidence of life, Wthe morphologically diverse cellular carbonaceous (ker- ogenous) microscopic fossils of the ∼3,465 Ma Apex chert, sys- Although the existence of the Archaea (one of three all- tematically described more than two decades ago (1, 2), have encompassing domains of life) in the Archean Eon (4,000 to been a focus of controversy. Perhaps spurred by a reluctance to 2,500 million years ago) has been inferred from carbon isotopes affirm the veracity of “claims for life in the earliest 2.0–2.5 billion in bulk samples of ancient rocks, their cellular fossils have been years of Earth’s history” (3), some workers have suggested the unknown. We here present carbon isotope analyses of 11 mi- ∼ Apex fossils to be chert-embedded mineralic pseudofossils crobial fossils from the 3,465-million-year-old Western Aus- composed of “abiotic graphite” (4, 5), barium carbonate (6, 7), tralian Apex chert from which we infer that two of the five or hematite in secondary veinlets (8). Other studies implied that species studied were primitive photosynthesizers, one was an the fossils are nonindigenous clay mineral needle-like crystallites Archaeal methane producer, and two others were methane “ ” consumers. This discovery of Archaea in the Archean is consis- (9) or suggested them to be composed of vermiculate-like “ ” minerals produced via a “nonbiological formation model” in- tent with the rRNA tree of life, confirms the earlier disputed volving the hydration and exfoliation of mica flakes followed by biogenicity of the Apex fossils, and suggests that methane- cycling methanogen−methanotroph communities were a sig- their surficial adsorption of later-introduced hydrocarbons (10). ’ Principal deficiencies of these suggestions are that carbonaceous nificant component of Earth s early biosphere. (kerogenous) cellular microbe-like assemblages of nonbiologic Author contributions: J.W.S., K.K., M.J.S., and J.W.V. designed research; J.W.S., K.K., pseudofossils are evidently unknown in the geological record; M.J.S., A.B.K., and J.W.V. performed research; J.W.S., K.K., M.J.S., and J.W.V. analyzed abiologically produced kerogenous particulate organic matter is data; and J.W.S. and J.W.V. wrote the paper. similarly unreported from the geological record; and virtually none Reviewers: D.J.B., University of Southern California; A.C., University of Cincinnati; and of these studies is reported to have been based on examination of Y.S., University of Science and Technology of China. the scores of demonstrably kerogenous (4, 11, 12) morphometrically The authors declare no conflict of interest. diverse well-characterized (1, 2) originally described Apex specimens Published under the PNAS license. archived at London’s Natural History Museum (NHM) (2). 1To whom correspondence should be addressed. Email: [email protected]. Although the earlier disputed biogenicity of the Apex fossils This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. seems largely to have been laid to rest (12), the biological affinities 1073/pnas.1718063115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1718063115 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 composition of the exceedingly ancient Apex fossils; reinforce Biological Affinities of the Apex Fossils. SIMS δ13C data for the 11 the widely assumed (but difficult to firmly establish) validity of Apex fossils analyzed (Figs. 1–3andTable1)provideameansto the use of cellular and organismal morphology for the assign- assess the physiology and biological affinities of the five taxa studied. ment of ancient microbes to biologically meaningful taxonomic The δ13C values of the individual fossils reported here range from categories; and provide insight into the physiology and biological −29.8‰ to −44.1‰ (Table 1), the data showing that each of the affinities of the five Apex taxa examined. five morphometrically defined taxa has a characteristic carbon iso- tope composition that varies taxon to taxon from the highest in δ13C Results and Discussion (an unnamed unicell, −30.9‰,andPrimaevifilum minutum, Geologic Setting. Geologically initially mapped as a shallow ma- −31.6‰)tothelowest(Archaeocillatoriopsis disciformis, −39.2‰, rine facies (13, 14), the fossiliferous locality (3) of the ∼3,465 Ma and P. amoenum, −39.4‰). The SIMS-determined δ13Cvaluesof Apex chert (15) has more recently been reinterpreted to be a the fossils thus differ from those of bulk samples of Apex kerogen brecciated and altered hydrothermal vein deposit (16). The (−27‰; ref. 25) and those of SIMS measurements acquired away 13 11 specimens of five taxa of permineralized microscopic fossils from the fossils to determine the δ C values of particulate kerogen analyzed here are embedded in Apex chert petrographic thin dispersed in the chert matrix (∼−27.6‰; Table S1 and Dataset S2). 13 section 4 of 6/15/82-1H prepared from a rock sample collected As discussed below, the microfossil δ C values are similar to from outcrop in 1982 (cf. refs. 1 and 2). those of extant prokaryotic phototrophs, methanogenic Archaeans, Although a hydrothermal environment has been suggested to and γ-proteobacterial methanotrophs. be unlikely for preservation of delicate fossil microbes (4, 5, 9), Direct evidence of the biological affinities of the Apex fossils is biota-prohibiting hydrothermal temperatures for the genesis of limited to their (i) cellular and organismal morphologies and (ii) the Apex chert have