LETTERS

Evidence of giant sulphur bacteria inNeoproterozoic phosphoritesJake V. Bailey1, Samantha B. Joye3, Karen M. Kalanetra3, Beverly E. Flood2 & Frank A. Corsetti1

In situ phosphatization1 and reductive cell division2 have recentlybeen discovered within the vacuolate sulphur-oxidizing bacteria.Here we show that certain Neoproterozoic Doushantuo Formation(about 600 million years BP) microfossils, including structurespreviously interpreted as the oldest known metazoan eggs andembryos3–10, can be interpreted as giant vacuolate sulphur bac-teria. Sulphur bacteria of the genus Thiomargarita have sizesand morphologies similar to those of many Doushantuo micro-fossils, including symmetrical cell clusters that result from mul-tiple stages of reductive division in three planes. We also proposethat Doushantuo phosphorite precipitation was mediated by thesebacteria, as shown in modern Thiomargarita-associated phospho-genic sites, thus providing the taphonomic conditions that pre-served other fossils known from the Doushantuo Formation.

Fossil cyanobacteria11, spherical organic-walled microfossils knownas acritarchs12, algae13, and putative microscopic metazoans4,7,8,14 havebeen described from the Neoproterozoic (about 599 6 4.2 Myr BP

15)Doushantuo Formation in South China, but the unit is perhaps mostnoted for producing the world’s oldest animal eggs (Megasphaera) andanimal embryos (Parapandorina)6,9,10. Initially regarded as algae16,solitary spheroids were reinterpreted as animal eggs because of theirexternal envelope and large size, and multiple bodies enclosed byenvelopes were reinterpreted as embryos on the basis of their sizeand apparent evidence for reductive division by non-rigid cells9,10,17.These putative eggs and embryos, along with other Doushantuomicrofossils commonly regarded as metazoans4–8,14, currently providethe earliest direct evidence of animal life.

However, several questions remain about the origins and preser-vational context (taphonomy) of this unprecedented accumulationof cellular microfossils. Conspicuously absent are intermediatestages linking very abundant single bodies interpreted as eggs(Megasphaera) and less common low-cell-number clusters inter-preted as early blastula stages (Parapandorina), with the very rare,controversial structures interpreted as gastrulae, larvae and adultforms3,7–9,14. Furthermore, recent X-ray tomography investigationsreport no evidence of epithelialization by means of gastrulation inputative Doushantuo embryos, a developmental process that is ubi-quitous in modern metazoan embryos18. Taphonomic studies advoc-ate that a bias towards the preservation of early developmental stagescould result from the resistance conferred by the embryonic envel-ope19,20. However, many specimens lack all or part of the outer envel-ope, a condition that if present before mineralization would result indegradation within hours20. Preservation of blastomeres within anintact fertilization envelope has been achieved in laboratory experi-ments, but experimental conditions required large concentrations(100 mM) of 2b-mercaptoethanol as a hypothetical simulant ofextremely sulphidic conditions20. Even if conditions were capableof continuously preventing autolysis and microbial degradation of

eggs and embryos, evidence for a specific molecular mechanism ofphosphatization in these cells is lacking21. Here we suggest thatMegasphaera inornata and Parapandorina represent solitary andreductively dividing giant vacuolate sulphur bacteria. This explana-tion is consistent with the morphology, taphonomic robustnessand palaeogeochemical conditions required to explain manyDoushantuo globular microfossils, while providing a biogeochemicalmechanism for phosphatization, which probably facilitated the pre-servation of other Doushantuo fossils.

In modern organic-rich marine sediments, sulphur-oxidizingbacteria, such as Beggiatoa, Thioploca and Thiomargarita oxidizehydrogen sulphide generated by bacterial sulphate reduction1,2,22–24.Thiomargarita, the focus of our investigation, is currently the largestknown bacterium, with average cell diameters between 100 and400 mm (some cells grow as large as 750 mm)22,24. Thiomargarita cellsare generally spherical and appear hollow, with the central vacuoleoccupying most of the cell volume2,22,24 (Figs 1a–e and 2a, b).Thiomargarita stores nitrate, presumably used as the oxidant forsulphide oxidation, at concentrations thousands of times that ofseawater. A thin (0.5–2.0 mm thick) outer layer of cytoplasm sur-rounding the vacuole contains abundant spherical inclusions1,22,24.Thiomargarita specimens from the Gulf of Mexico occur primarilyas solitary cells or clusters of cells surrounded by a mucus-filledsheath (Fig. 2a, b)2. Dense botryoidal clusters of smaller sulphur-globule-containing cells (about 20mm) are also observed22. WhereasThiomargarita cells from Namibia commonly divide in one plane, andmore rarely in two planes22, recently described Thiomargarita cellsfrom the Gulf of Mexico undergo division along three planes, resultingin clusters of two, four and eight cells2 (Fig. 1b–e, and SupplementaryFigs 1–5). No statistically significant difference was observed in thebiovolumes of cell clusters, regardless of cell number, an observationconsistent with reductive cellular division2. Clusters of three cellsoccasionally resulted from an incomplete second stage of reductivedivision (Fig. 1c)2.

Many of the single-celled globular Doushantuo Formation micro-fossils exhibit sizes, morphologies and cellular division geometriesconsistent with those of Thiomargarita. The Doushantuo microfossilMegasphaera inornata, for example, is a large unornamented spher-oidal microfossil (about 500 mm in diameter that may or may not besurrounded by a thin (about 10 mm) phosphatic envelope9 (Fig. 1a9),similar to that of large single cells of Thiomargarita (Fig. 2a, b). In theabsence of additional morphological differentiation, such sphericalbodies convey limited information, highlighting a perpetual issue inthe investigation of morphologically conservative fossil structures.

A more striking comparison can be made between certainThiomargarita clusters and the Doushantuo microfossil known asParapandorina9,10. Typically, Parapandorina contains even-numbered(2n) internal bodies (2, 4, 8, 16, 32) and shows little or no apparent

1Department of Earth Sciences and 2Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA. 3Department of Marine Sciences,University of Georgia, Athens, Georgia 30602, USA.

Vol 445 | 11 January 2007 | doi:10.1038/nature05457

198Nature ©2007 Publishing Group

change in diameter, regardless of the number of internal bodies pre-sent, suggesting that the internal bodies resulted from reductive cel-lular division9,10 (Fig. 1b9–e9). The observation of reductive divisionled to their interpretation as animal embryos, as this process was atthat time unknown outside the Metazoa in cells of comparable size.The recent discovery of reductive division in Thiomargarita nowwarrants a re-evaluation of the Doushantuo structures. BothThiomargarita and Parapandorina consist of several polyhedral to

spherical bodies surrounded by a thin envelope, show reductive celldivision, and are of similar size (Fig. 1a–e, a9–e9, and SupplementaryFigs 1–6). Thiomargarita and Parapandorina cells both seem to bedistorted by adjacent cells, suggesting a non-rigid cell wall, a factpreviously used to discount the latter’s interpretation as an alga17

(see Supplementary Information for a detailed discussion of cell clus-ter geometry). Thiomargarita cells also include abundant inclusionsand globules that can contain sulphur, polyphosphate or glycogen(labelled ‘i’ in Fig. 2a)22 and can form larger aggregates (Sup-plementary Fig. 6). Subcellular structures in some Parapandorinaspecimens18 may have resulted from the diagenetic alteration of suchinclusions in ancient Thiomargarita.

Three reductive divisions (eight-cell stage) are the maximumnumber currently observed in Thiomargarita, although such divisioncould continue to produce clusters with more cells. Reductive divi-sion in Thiomargarita and other bacteria is thought to be a survivalresponse to starvation2. We postulate that Thiomargarita cells, ifentombed within precipitating hydroxyapatite and cut off from theirvital metabolic substrates, sulphide and nitrate, could continue todivide, perhaps resulting in a morphology similar to that of the rareParapandorina specimens that contain more than eight cells. Of the56 specimens examined in ref. 18, four-cell Parapandorina specimenswere the most abundant (28%), with 74% of specimens containingeight or fewer cells. Specimens with more than 16 cells were lessabundant (18%). In the 207 specimens examined in ref. 25,Parapandorina specimens with four internal bodies were also themost abundant (48%), and those containing more than 16 cells wereabsent. Parapandorina is much less common than Megasphaera inDoushantuo phosphorites, which is consistent with the interpreta-tion of Megasphaera inornata as abundant solitary cells that dividereductively as a stress response, producing rare Parapandorina.

a′′a

c′c

γ

β

α

e e′

d′d

b′b

Figure 1 | Comparisons of light micrograph images of translucentunmineralized modern Thiomargarita cells (left column) with scanningelectron microscopy images of opaque mineralized Doushantuomicrofossils (right column). a, Solitary Thiomargarita cell from the Gulf ofMexico (after ref. 2). b, Two-cell cluster of Thiomargarita. c, Three-cellThiomargarita cluster, thought to result from the incomplete division of atwo-cell cluster. Greek letters identify each of the three cells. d, TetragonalThiomargarita tetrad resulting from reductive division in two planes.e, Offset between opposing cells in rhomboidal Thiomargarita tetradsresembles offset in some Doushantuo tetrads9 and cross-furrows in four-cellblastulas. Arrows indicate thin sheath surrounding cell cluster.a9, Megasphaera inornata, from the Doushantuo Formation9. b9, Twoappressed hemispherical bodies enclosed by an external envelope (after ref.9). c9, Thiomargarita triplets occasionally result from incomplete division,which results in two cells with a combined volume roughly equal to the thirdundivided cell9. This Doushantuo globular triplet shows similar relativevolumes. After ref. 5. d9, Parapandorina tetrad resulting from division in twoplanes. Modified after ref. 5. e9, Doushantuo rhomboidal tetrad modifiedafter ref. 9. Scale bars, 150mm (b9); 100mm (a–e, a9, c9–e9).

i

i

a b

Figure 2 | Phase contrast images of Thiomargarita cellular structure. a, Amulti-layered ultrastructure (white arrows) surrounding cytoplasm thathosts abundant spherical inclusions (labelled ‘i’ with black arrows) and (b) amucus-filled sheath (white arrows) that hosts abundant microbial filaments.Scale bars, 100mm (a); 60 mm (b).

NATURE | Vol 445 | 11 January 2007 LETTERS

199Nature ©2007 Publishing Group

Phosphorite deposition is relatively uncommon and poorly under-stood throughout the geological record. Recently, sulphide-oxidizingbacteria have been shown to mediate phosphorite deposition inmodern environments1. Thiomargarita cell accumulations corre-late with increased pore water phosphate and accumulations ofphosphorus-containing minerals, which amounted to more than50 g kg21 of dry sediment1. Laboratory experiments suggest thatmetabolically driven phosphate release by Thiomargarita controlsphosphate mineral precipitation, thus providing a microbiallymediated mechanism of phosphorite formation1, as was previouslyproposed for related sulphide-oxidizing bacteria13. Unlike animaleggs and embryos, the ambient internal and external chemical envir-onments mediated by these microorganisms are conducive tophosphatization, providing a biogeochemical explanation for theunusually high phosphate concentrations responsible for the preser-vation of Doushantuo microfossils.

The unusual abundance of globular microfossils in theDoushantuo Formation has long been considered problematic forthe animal embryo interpretation26; mass spawning and concen-tration by sedimentary processes have been proposed as possiblesolutions9,27. More parsimoniously, Thiomargarita cells of similarsizes (about 500 mm) can be found in great abundances (up to107 cells m22) and would not require unusual circumstances toexplain large accumulations of their fossils24 (see Supplemen-tary Information). In addition, stages of reductive division inThiomargarita are separated by months to years2, allowing a longerwindow for the observed preferential preservation of two-cell, four-cell and eight-cell clusters, unlike invertebrate embryos that developfrom the fertilized egg, through the blastula stages to a gastrula in amatter of a few hours28. The extremely long intervals between reduct-ive division in Thiomargarita might also explain why cell clusters withmore than eight cells have not yet been observed.

Figure 3a shows Thiomargarita cells from Namibia covered byfilamentous bacteria interpreted to be symbiotic sulphate-reducingbacteria22. Thiomargarita cells from the Gulf of Mexico also com-monly occur with filamentous and spheroidal bacteria. Doushantuoglobular microfossils, but not acritarchs and algae, are commonlyassociated with phosphatized filaments and spheroids previouslyinterpreted to be fossilized infesting bacteria21 (Fig. 3b).Taphonomic studies indicate that infesting bacteria rapidly destroyembryonic animal cells20, making the preferential co-preservation ofembryos and the offending degradative bacteria unlikely, althoughnot unprecedented21. Conversely, the preservation of bacteria withtheir natural host Thiomargarita cells requires no such fortuitousmineralization of transient putrefying remains.

The geochemical conditions during the deposition of theDoushantuo Formation, but not necessarily the palaeoenvironmentitself, may have been analogous to modern upwelling zones whereThiomargarita was discovered24. Multiple lines of palaeontological

and lithological evidence point to enriched nutrient concentrationsin Doushantuo depositional environments12. Additionally, indica-tors of sulphidic conditions, which would have been a requirementfor Thiomargarita, are abundant in the Doushantuo Formation.Sulphur and oxygen isotope values of sulphate from DoushantuoFormation phosphorites provide evidence of probable bacterial sul-phate reduction, perhaps coupled to sulphide oxidation29. Abundantpyrite in phosphorites and in microfossils21 is also suggestive of sul-phidic conditions. In an environment with nutrient-rich, productivesurface waters and euxinic bottom waters, the remains of planktonicalgae, and perhaps metazoans, from the overlying water column,along with allochthonous benthic organic debris, would occurtogether with abundant sulphide-oxidizing bacteria as in the moderncounterparts, resulting in fossil assemblages rich in phosphaticspheroidal and globular forms, such as those observed in theDoushantuo Formation.

We do not suggest that the Doushantuo microbiota is composedentirely of sulphur bacterial remains, because there are many struc-tures in the Doushantuo that clearly do not resemble sulphur bac-teria. Furthermore, the hypothesis posed here does not invalidate thepossibility that some Doushantuo globular microfossils are indeedanimal eggs and embryos, because the two hypotheses are not mutu-ally exclusive. Instead, our reassessment provides a morphologicallyplausible alternative interpretation of the most abundant globularmicrofossils (Megasphaera inornata and Parapandorina) as giantvacuolate sulphur bacteria, and offers a mechanism of preserva-tion by means of microbially mediated phosphate release byThiomargarita, as noted in modern habitats. Given the possibilitythat Megasphaeara inornata and Parapandorina represent giant bac-teria, and the controversy surrounding other putative metazoans inthe Doushantuo Formation3,30, the interpretation of Doushantuoglobular microfossils as metazoan embryos deserves further scrutiny.

METHODSFor Thiomargarita sample collection, sediment samples from a variety of hydro-

carbon seeps in the Gulf of Mexico were collected during a research expedition

on the R/V Seward Johnson in July 2002. Push cores were collected with the

Johnson Sea-Link II submersible2. Sediment samples were preserved in 2.5%

glutaraldehyde at 4 uC until microscopic analysis. Microscopic images and mea-

surements of cell diameters were made with phase-contrast microscopy, with the

exception of Fig. 1e, which was stained with fluorescein isothiocyanate (FITC)

and imaged with a Leica TCS-SP Laser Scanning Confocal Microscope fitted

with an argon ion laser (488 nm).

Received 26 July; accepted 20 November 2006.Published online 20 December 2006.

1. Schulz, H. N. & Schulz, H. D. Large sulfur bacteria and the formation ofphosphorite. Science 307, 416–418 (2005).

2. Kalanetra, K. M., Joye, S. B., Sunseri, N. R. & Nelson, D. C. Novel vacuolate sulfurbacteria from the Gulf of Mexico reproduce by reductive division in threedimensions. Environ. Microbiol. 7, 1451–1460 (2005).

3. Xiao, S., Yuan, X. & Knoll, A. H. Eumetazoan fossils in terminal Proterozoicphosphorites? Proc. Natl Acad. Sci. USA 97, 13684–13689 (2000).

4. Li, C., Chen, J. & Hua, T. E. Precambrian sponges with cellular structures. Science279, 879–882 (1998).

5. Chen, J.-Y. The Dawn of Animal World (Jiangsu Science & Technology, Nanjing,2004).

6. Chen, J.-Y. et al. Phosphatized polar lobe-forming embryos from the Precambrianof southwest China. Science 312, 1644–1646 (2006).

7. Chen, J.-Y. et al. Precambrian animal life: probable developmental and adultcnidarian forms from southwest China. Dev. Biol. 248, 182–196 (2002).

8. Chen, J.-Y. et al. Precambrian animal diversity: New evidence from high resolutionphosphatized embryos. Proc. Natl Acad. Sci. USA 97, 4457–4462 (2000).

9. Xiao, S. & Knoll, A. H. Phosphatized animal embryos from the NeoproterozoicDoushantuo Formation at Weng’an, Guizhou, South China. J. Paleontol. 74,767–788 (2000).

10. Xiao, S., Zhang, Y. & Knoll, A. H. Three-dimensional preservation of algae andanimal embryos in a Neoproterozoic phosphorite. Nature 391, 553–558 (1998).

11. Awramik, S. M. et al. Prokaryotic and eukaryotic microfossils from a Proterozoic/Phanerozoic transition in China. Nature 315, 655–658 (1985).

12. Zhou, C., Brasier, M. D. & Xue, Y. Three-dimensional phosphatic preservation ofgiant acritarchs from the Terminal Proterozoic Doushantuo Formation in Ghizhouand Hubei Provinces, South China. Palaeontology 44, 1157–1178 (2001).

a b

Figure 3 | Filaments associated with Thiomargarita cells and Doushantuomicrofossils. a, Thiomargarita samples from Namibia (modified after ref.22) and the Gulf of Mexico are often covered with filamentous bacteria thatare probably sulphate-reducing symbionts. b, Doushantuo globularmicrofossils, such as this Parapandorina specimen, are commonly coveredwith filaments thought to be mineralized embryo-infesting bacteria (afterref. 9). Scale bars, 50 mm (a); 100mm (b).

LETTERS NATURE | Vol 445 | 11 January 2007

200Nature ©2007 Publishing Group

13. Zhang, Y., Yin, L., Xiao, S. & Knoll, A. H. Permineralized fossils from the TerminalProterozoic Doushantuo Formation, South China. Paleontol. Soc. Mem. 50, 1–52(1998).

14. Chen, J.-Y. et al. Small bilaterian fossils from 40 to 55 million years before theCambrian. Science 305, 218–222 (2004).

15. Barfod, G. H. et al. New Lu-Hf and Pb-Pb age constraints on the earliest animalfossils. Earth Planet. Sci. Lett. 201, 203–212 (2002).

16. Xue, Y., Tang, T., Yu, C. & Zhou, C. Large spheroidal chlorophyta fossils from theDoushantuo Formation phosphoric sequence (late Sinian), central Guizhou,South China. Acta Palaeontol. Sin. 34, 688–706 (1995).

17. Xiao, S. Mitotic topologies and mechanics of Neoproterozoic algae and animalembryos. Paleobiology 28, 244–250 (2002).

18. Hagadorn, J. W. et al. Cellular and subcellular structure of Neoproterozoic animalembryos. Science 314, 291–294 (2006).

19. Martin, D., Briggs, D. E. G. & Parkes, R. J. Decay and mineralization of invertebrateeggs. Palaios 20, 562–572 (2005).

20. Raff, E. et al. Experimental taphonomy shows the feasibility of fossil embryos. Proc.Natl Acad. Sci. USA 103, 5846–5851 (2006).

21. Xiao, S. & Knoll, A. H. Fossil preservation in the Neoproterozoic Doushantuophosphorite Lagerstatte, South China. Lethaia 32, 219–240 (1999).

22. Schulz, H. N. in The Prokaryotes: An Evolving Electronic Resource for theMicrobiological Community (eds Dworkin, M. et al. ) Æhttp://141.150.157.117:8080/prokPUB/chaprender/jsp/showchap.jsp?chapnum5513æ(2006).

23. Jørgensen, B. B. & Nelson, D. C. in Sulfur Biogeochemistry—Past and Present (edsAmend, J. P., Edwards, K. J. & Lyons, T.W.) 63–81 (Geol. Soc. Am. Spec. Pap. 379,Boulder, Colorado, 2004).

24. Schulz, H. N. et al. Dense populations of a giant sulfur bacterium in Namibian shelfsediments. Science 284, 493–495 (1999).

25. Dornbos, S. Q. et al. Precambrian animal life: Taphonomy of phosphatizedmetazoan embryos from southwest China. Lethaia 38, 101–109 (2005).

26. Xue, Y. S., Tang, T. F. & Yu, C. L. ‘Animal embryos,’ a misinterpretation ofNeoproterozoic microfossils. Acta Micropaleontol. Sin. 16, 1–4 (1999).

27. Xiao, S. & Knoll, A. H. Embryos or algae? A reply. Acta Micropaleontol. Sin. 16,313–323 (1999).

28. Costello, D. P. & Henley, C. Methods for Obtaining and Handling Marine Eggs andEmbryos (Marine Biological Laboratory, Woods Hole, Massachusetts, 1971).

29. Goldberg, T., Poulten, S. W. & Strauss, H. Sulphur and oxygen isotope signaturesof late Neoproterozoic to early Cambrian sulphate, Yangtze Platform, China:Diagenetic constraints and seawater evolution. Precambr. Res. 137, 223–241(2005).

30. Bengston, S. & Budd, G. Comment on ‘Small bilaterian fossils from 40 to 55 millionyears before the Cambrian’. Science 306, 1291a (2004).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank D. Bottjer, D. Caron, A. Jones, R. Schaffner,S. Douglas, A. Thompson and D. Nelson for discussions, advice and/or assistance.This project was funded by the US National Science Foundation Graduate ResearchFellowship, Earth Sciences, and Life in Extreme Environments programs, as well asthe NASA Exobiology Program. Submersible operations in the Gulf of Mexico weresupported by the US Department of Energy and the National Oceanic andAtmospheric Administration’s National Undersea Research Program.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to J.V.B.(jvbailey@usc.edu).

NATURE | Vol 445 | 11 January 2007 LETTERS

201Nature ©2007 Publishing Group