LETTERS Putative greigite magnetofossils from the Pliocene...

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LETTERS

Putative greigite magnetofossils from thePliocene epoch

IULIANA VASILIEV1*, CHRISTINE FRANKE1†, JOHANNES D. MEELDIJK2, MARK J. DEKKERS1,COR G. LANGEREIS1 AND WOUT KRIJGSMAN1

1Paleomagnetic Laboratory ‘Fort Hoofddijk’, Department of Earth Sciences, Utrecht University, Budapestlaan 17, 3584 CD, Utrecht, The Netherlands2Department of Chemistry, Utrecht University, Sorbonnelaan 16, 3545 CA Utrecht, The Netherlands†Current address: Laboratoire des Sciences du Climat et de l’Environnement, CEA-CNRS-UVSQ, Campus du CNRS, Bat. 12, Avenue de la Terrasse, 91198 Gif-sur-YvetteCedex, France*e-mail: [email protected]

Published online: 19 October 2008; doi:10.1038/ngeo335

Magnetotactic bacteria produce chains of magnetite1,2 and/orgreigite3–5 crystals within their cell bodies called magnetosomesthat are permanently magnetized6. They use these magnetsto navigate along geomagnetic field lines to reach theirpreferred habitat7. Greigite magnetosomes have been welldocumented in modern sedimentary environments, but theiridentification in the fossil record remains controversial. Herewe use transmission electron microscopy, electron diffractionpatterns and rockmagnetic analyses to assess the origins ofnanometre-scale greigite crystals found in Pliocene claystonesfrom the Carpathian foredeep of Romania. We find that, likemodern magnetosomal greigite grains, the crystals are singledomain8, with few crystallographic defects and an overall shapeconsistent with an intracellular origin. We suggest these crystalsare magnetosomal in origin, which would place them amongthe oldest greigite magnetofossils identified so far. The crystalsalso carry a primary magnetic signal, which has remained stablesince its acquisition 5.3–2.6 million years ago. We suggest thatgreigite magnetofossils could therefore provide reliable recordsof ancient geomagnetic field variations, and that they could alsobe used as a proxy to assess palaeoenvironmental conditions inlow-oxygen sedimentary environments.

Robust fossil evidence for magnetite magnetofossils dates backto the Cretaceous period9, whereas claims of magnetofossils extendback to the early Proterozoic era10 (∼2 Gyr). Today, these bacteriaare cosmopolitan in distribution and easy to identify in modernenvironments, where they contribute to the biogeochemical cyclingof important elements, including iron, nitrogen, sulphur andcarbon7. These crystals exhibit high chemical purity, specific crystalmorphologies and exceptionally narrow grain-size distribution8,11.

Magnetotactic bacteria achieve directional sensing usingmagnetosomes, which are membrane-bounded chains offerrimagnetic crystals. Two magnetic minerals have beenunequivocally recognized to be produced by magnetotacticbacteria: magnetite1,2 (Fe3O4) and greigite3–5 (Fe3S4).Magnetosomal minerals dominantly form around the oxic–anoxicinterface in aquatic habitats7, reflecting the palaeo-redox conditionsand are of interest for geochemistry and geobiology. Theyare of importance also for palaeomagnetism because, whenpreserved, they can significantly contribute to the primarynatural remanent magnetization (NRM) of sedimentary rocks.

Magnetosomal magnetite is often well preserved and regularlyobserved in geological archives (for example, see refs 9,10). Greigitemagnetosomes have been uncontestedly identified only in recentsoils and lake sediments, although their occurrence has also beenclaimed in Miocene rocks of the Western Carpathian foredeep12.

Authigenic greigite has been known since 1964 (ref. 13) andis preserved in rocks at least as old as the Cretaceous14. However,the reliability of palaeomagnetic data from greigite-bearing rocksis frequently questioned15 because greigite is thermodynamicallymetastable16 and the timing of NRM acquisition by greigite isnot well constrained because of its diagenetic formation15. It wasfurther assumed that greigite would not last long in the geologicalrecord because excess sulphur would cause transformation topyrite16. A recent re-evaluation of greigite’s thermodynamics17,however, suggests that, despite its metastability, greigite maypreserve a primary NRM for geological times14.

Recent greigite-based magnetostratigraphies straightforwardlycorrelate to the geomagnetic polarity timescale18,19 and supportthe formation and preservation of ancient greigite in sedimentaryrocks20. These records come from the Carpathian foredeep ofRomania (Fig. 1), which was part of the Eastern Paratethys(see Supplementary Information, Fig. S1), a large semi-isolatedfresh to brackish-water domain that comprised the present-day Black Sea and Caspian Sea regions18. Large quantities ofdetrital material derived from the uplifted orogen and activevolcanic sources were deposited in shallow-water environmentswith ostracods and molluscs indicative of the euphotic zone21,22.Posfai et al.12 previously conducted a transmission electronmicroscopy (TEM) study of greigite from one sample from Poland’sMiocene Carpathian foredeep and concluded, solely on the basisof analysis of particle size and shape distributions, that thesediment might contain crystals produced by the multicellularmagnetotactic prokaryote.

Thermomagnetic measurements in air, acquiredgyro-remanence during alternating field demagnetization (seeSupplementary Information, Fig. S2) and scanning electronmicroscopy showed that greigite is the key magnetic mineral inthe sedimentary rocks of the Romanian Carpathian foredeep20.Positive reversal tests, a positive fold test and the occurrence ofinclination shallowing (Fig. 1e,f) provided further evidence for anearly acquisition of the NRM20.

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IncEI = 71.8 IncEI = 67.1

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Figure 1 Greigite-based magnetostratigraphy of the Badislava valley (central plots). Circles and triangles represent high-temperature (HT) and low-temperature (LT)components, respectively. Shaded bands indicate intervals of delayed low-temperature acquisition. a, Thermomagnetic run for greigite-bearing samples.b–d, Demagnetization diagrams illustrating the transition from reversed (d) to normal polarity (b) via a sample (c) recording two antipodal directions. e,f, Histograms ofinclinations on using the elongation/inclination (E/I) correction method29. Coloured lines refer to expected (IncGAD, yellow), original (Incorg, blue), unflattened (IncEI, green) andmost frequent bootstrapped (red, with 95% error bounds: dashed red lines) mean inclinations.

An earlier scanning electron microscopy study on theRomanian rocks20 revealed octahedral greigite crystals of aninorganic origin that range 400–1,000 nm in size. The number ofgreigite particles, however, was remarkably small when comparedwith the high initial intensity of the samples (10–65 mA m−1).Consequently, the presence of an extra magnetic carrier in a smaller(magnetofossil) grain-size range was suspected. To identify theprecise nature of greigite formation in these rocks, we used acombination of mineral magnetic methods and TEM. In addition,we evaluated our results using the six criteria for magnetofossil

identification of Kopp and Kirschvink23: (1) the contextual andpalaeomagnetic evidence for a primary origin, (2) the presenceof a significant single-domain magnetic phase, (3) size and shapedistributions characteristic of magnetosome crystals, (4) evidencefor chains of crystals, (5) evidence for chemical purity and(6) high-resolution TEM (HRTEM) evidence for crystallographicperfection. Our earlier palaeomagnetic and palaeoenvironmentalresearch of the Carpathian foredeep fulfilled the first criterion. Theother five criteria require an extensive TEM study combined withrockmagnetic experiments. For TEM imaging, we used magnetic

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Figure 2 Fossil elongated prismatic greigite magnetosomes. a, TEM micrograph of a prismatic, slightly elongated {100}+{111} greigite crystal, typical ofmagnetosomes, very close to the idealized crystal habit (inset). b, HRTEM detail of the greigite crystal. The inset shows well-ordered lattice fringes from the area in thedashed square. c, EDS from the crystal with distinct Fe and S peaks; O, Al and Si are from the background signal of the clay flake; Cu peaks originate from the TEM grid.d, Selected-area electron diffraction pattern with the same orientation as b. b and d were recorded with 41◦ -tilt difference from a.

concentrates obtained from approximately 20 g of powdered rocksamples (see Supplementary Information).

TEM analyses on these extracts identified numerousnanoparticles (Figs 2a, 3a,b and Supplementary Information,Figs S3,S4) that seemed to be chemically pure and have fewcrystallographic defects, passing two criteria (chemical andcrystallographic perfection)23. The crystals span a grain-sizerange of 20–75 nm, implying that they are magnetically singledomain, although smaller, probably superparamagnetic grains werealso distinguished. Many of the particles are irregular in shape,but exhibit strong diffraction (Fig. 3a), indicating that they arehighly crystalline. Other particles show elongated (Fig. 2a andSupplementary Information, Fig. S3) and hexagonal (Fig. 3a)outlines when viewed in projection, which is characteristic ofelongated prismatic and truncated cuboctahedral grains and pointsto cubic crystal symmetry. Energy-dispersive X-ray spectroscopy(EDS) analysis shows that most of particles consist of iron andsulphur (Figs 2c and 3c). HRTEM (Fig. 2b) and single-crystalselected-area electron diffraction patterns (Fig. 2d) indicate thatthe measured d-spacing corresponds to those of greigite. The size

distribution of 20–75 nm, the elongated prismatic or cuboctahedralcrystal morphologies and the elemental composition all indicatethat these greigite crystals have a magnetosomal origin. The roughlyprismatic (Fig. 2a and Supplementary Information, Fig. S3)and cuboctahedral (Fig. 3a,b) magnetosomes are magneticallysingle domain (see Supplementary Information, Fig. S4) andwould have been responsible for the magnetotactic reactionof the living organism4. Isothermal remanent magnetizationcomponent analysis revealed a very small dispersion parameterof approximately 0.10–0.15 log units20, which is also indicative of amagnetosomal origin of the magnetic crystals12. The single-domaingreigite with the grain-size distribution and shape typical ofmagnetofossils, having truncated-edge crystal morphology, passestwo more magnetofossil identification criteria23.

The last and most difficult to fulfil criterion in rock records isthe presence of magnetosomal chains, because both diagenesisand magnetic extraction techniques can contribute to chaindisruption24. We identified a few single-domain magnetitemagnetosomes (see Supplementary Information, Fig. S5),although their contribution to the NRM is minimal because




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Figure 3 Fossil cuboctahedral greigite magnetosomes. a, TEM micrograph ofgreigite crystals; some are close to a zone-axis orientation producing strongdiffraction contrasts. b, Magnification shows the cuboctahedron {100}+{111}crystal morphology found in both magnetite and greigite magnetosomes; manyparticles have well-defined edges. The inset represents the idealized crystal habit.c, EDS from crystals in b; see also caption to Fig. 2. The intensity of S and Fe peaksis lower than that of the Cu peaks because of the very small crystals and/orpositioning of the material near the copper bars of the grid, catching morestray radiation.

the samples are demagnetized below 400 ◦C. We conclude thatour greigite magnetofossils pass five out of six criteria formagnetosome identification23.

Our Carpathian samples thus comprise two distinctly differenttypes of greigite, generated by two different formation mechanisms(Fig. 1a): (1) small (20–75 nm) slightly prismatic elongated andcuboctahedral crystals of magnetosomal origin and (2) largeroctahedral grains (400–1,000 nm) of authigenic origin. Close topolarity reversals, the thermal demagnetization diagrams showthe presence of two different (even antipodal) NRM componentsin a single specimen (Fig. 1b–d). Unexpectedly, the hysteresisloop and the first-order reversal curves (see SupplementaryInformation, Fig. S6) indicate only a unimodal coercive forcedistribution, which is unusual for a specimen recording twodifferent directions. We therefore fitted the isothermal remanentmagnetization acquisition curve25, with two magnetic componentshaving approximately the same mean acquisition field (B1/2) buta significantly different dispersion parameter (see SupplementaryInformation, Fig. S6). This translates into two different grain-sizedistributions, having the same mean coercivity. Magnetosomes areknown for their narrow coercivity switching field distribution (lowdispersion parameter)12 and therefore we tie the narrow dispersionparameter in our samples to the greigite magnetosomes. The widerdistribution would thus be related to the authigenic phase ofgreigite. We acknowledge that authigenic greigite has a narrowgrain-size distribution26 (when compared with other magneticminerals), but magnetosomal greigite has even lower dispersionparameter values than the authigenic greigite phases. First-orderreversal curve analysis27 of thermally treated samples shows thatgreigite survives up to 350 ◦C and is replaced at ∼360 ◦C, by a non-magnetic phase13. This behaviour is consistent with the thermaldemagnetization spectra of our samples, indicating that greigitewas indeed the NRM carrier.

Authigenic or early diagenetic greigite formed later than themagnetosomes, deeper in the sediment and therefore acquireda later magnetic field. The delayed NRM acquisition is easilyobservable and directionally traceable from the demagnetizationdiagrams, especially in the intervals that straddle polarityreversals (Fig. 1). The low-temperature component must beattributed to diagenetic greigite because it records the delayedcomponent (Fig. 1c and Supplementary Information, Fig. S7). Thehigh-temperature component represents greigite magnetosomes,which formed close to the sediment–water interface7 or in thewater column28 at the time of the deposition and thus record themagnetic field without significant delay. Generally, larger grainsresist thermal demagnetization and, in the case of greigite, thermalalteration longer. Here, the ∼10 times smaller magnetosomeswith their high chemical purity and few crystallographic defectsseem to persist to the highest temperatures. The larger, diageneticpseudo-single-domain greigite particles would have a less stablemagnetization, explaining partially why the larger fraction isless resistant to thermal demagnetization than the smaller one.The origin of the two components is furthermore confirmed bytheir different inclination values distinguishable in the thermaldemagnetization diagrams. We obtain mean inclinations of52.0◦ for the high-temperature (Fig. 1e) and of 60.4◦ for thelow-temperature component (Fig. 1f). This implies that inclinationshallowing has significantly affected the high-temperaturecomponent (Fig. 1e), caused by dewatering and compaction of thetop sediment layer. In contrast, no significant inclination error isobserved for the low-temperature component (Fig. 1f), because theauthigenic greigite forms later in the sediment, when compactionhad largely come to an end. Applying the inclination errorcorrection method using the field model TK03.GAD (ref. 29) onboth data sets corrects the high-temperature and low-temperatureinclinations to 65.5◦ and 64.9◦, respectively. Both corrected valuesare remarkably similar and indistinguishable from the expectedinclination at the site latitude (IGAD = 63.6◦).

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Palaeomagnetism is widely used in earth sciences for platetectonic reconstructions, for dating and correlation of marineand continental sequences and for studying geomagnetic fieldbehaviour. Here, we show that magnetosomal greigite can survivegeological times and that a primary NRM component canbe extracted, noticeably enhancing the value of greigite forpalaeomagnetic studies, including records of rapid geomagneticvariations. We emphasize the importance of small demagnetizationsteps in the 300–360 ◦C temperature range, because magnetosomalgreigite survives heating up to 350–360 ◦C, whereas authigenicgreigite is removed at 290–300 ◦C.

In some conditions, greigite magnetosomes may have agreater preservation potential than magnetite magnetosomesbecause the latter ultimately dissolve under anoxic conditionswhereas the former persist—having been formed under suchconditions. Greigite magnetofossils might be expected to be moreabundant in higher productivity, more sulphidic sediments, butthese environments are still insufficiently studied23. The greigite-producing bacteria prefer reduced conditions and are probablyanaerobic sulphate reducers. The high preservation capacityof greigite magnetosomes may help to detect environmentalvariations, expressed by biogeochemical changes in sedimentarybasins. The magnetofossil record may serve as tracers of localchanges in oxygen level and provide an underexploited archive ofthe long-term evolution of marine redox stratification importantin characterizing anoxic/euxinic sedimentary environments such asthe Oceanic Anoxic Events.

Received 28 April 2008; accepted 26 September 2008; published 19 October 2008.

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203, 1355–1356 (1979).3. Farina, M., Esquivel, D. M. S. & Lins de Barros, H. G. P. Magnetic iron-sulphur crystals from a

magnetotactic microorganism. Nature 343, 256–258 (1990).4. Mann, S., Sparks, N. H. C., Frankel, R. B., Bazylinski, D. A. & Jannasch, H. W. Biomineralization of

ferrimagnetic greigite (Fe3S4) and iron pyrite (FeS2) in a magnetotactic bacterium. Nature 343,258–261 (1990).

5. Bazylinski, D. A., Heywood, B. R., Mann, S. & Frankel, R. B. Fe3O4 and Fe3S4 in a bacterium. Nature366, 218–218 (1993).

6. Johnsen, S. & Lohmann, K. J. The physics and neurobiology of magnetoreception. Nature Rev. 6,703–712 (2005).

7. Bazylinski, D. A. & Frankel, R. B. Magnetosome formation in prokaryotes. Nature Rev. 2,217–230 (2004).

8. Diaz Ricci, J. C. & Kirschvink, J. L. Magnetic domain state and coercivity predictions for biogenicgreigite (Fe3S4): A comparison of theory with magnetosome observations. Geophys. J. Res. 97,17309–17315 (1992).

9. Hounslow, M. W. & Maher, B. A. Quantitative extraction and analysis of the carriers of magnetizationin sediments. Geophys. J. Int. 124, 56–74 (1996).

10. Chang, S.-B. R. & Kirschvink, J. L. Magnetofossils, the magnetization of the sediments, and theevolution of magnetite biomineralization. Annu. Rev. Earth Planet. Sci. 17, 169–195 (1989).

11. Moskowitz, B. M. Biomineralization of magnetic minerals. Rev. Geophys. 33, 123–128 (1995).12. Posfai, M. et al. Crystal-size distributions and possible biogenic origin of Fe sulfides. Eur. J. Mineral.

13, 691–703 (2001).13. Skinner, B. J., Erd, R. C. & Grimaldi, F. S. Greigite, the thio-spinel of iron; a new mineral.

Am. Mineral. 49, 543–555 (1964).14. Reynolds, R. L. et al. Magnetization and geochemistry of greigite bearing Cretaceous strata, North

Slope Basin, Alaska. Am. J. Sci. 294, 485–528 (1994).15. Roberts, A. P. & Weaver, R. Multiple mechanisms of remagnetization involving sedimentary greigite

(Fe3S4). Earth Planet. Sci. Lett. 231, 263–277 (2005).16. Berner, R. A. Thermodynamic stability of sedimentary iron sulfides. Am. J. Sci. 265, 773–785 (1967).17. Rickard, D. & Luther, G. W. Chemistry of iron sulfides. Chem. Rev. 107, 514–562 (2007).18. Vasiliev, I. et al. Towards an astrochronological framework for the eastern Paratethys Mio-Pliocene

sedimentary sequences of the Focsani basin (Romania). Earth Planet. Sci. Lett. 227, 231–247 (2004).19. Vasiliev, I., Krijgsman, W., Stoica, M. & Langereis, C. G. Mio-Pliocene magnetostratigraphy in the

southern Carpathian foredeep and Mediterranean-Paratethys correlation. Terra Nova 17,374–387 (2005).

20. Vasiliev, I. et al. Early diagenetic greigite as a recorder of the palaeomagnetic signal inMiocene–Pliocene sedimentary rocks of the Carpathian foredeep (Romania). Geophys. J. Int. 171,613–629 (2007).

21. Panaiotu, C. E., Vasiliev, I., Panaiotu, C. G., Krijgsman, W. & Langereis, C. G. Provenance analysis as akey to orogenic exhumation: A case study from the East Carpathians (Romania). Terra Nova 19,120–126 (2007).

22. Stoica, M., Lazar, I., Vasiliev, I. & Krijgsman, W. Mollusc assemblages of the Pontian and Daciandeposits in the Topolog-Arges area (southern Carpathian foredeep - Romania). Geobios 40,391–405 (2007).

23. Kopp, R. E. & Kirschvink, J. L. The identification and biogeochemical interpretation of fossilmagnetotactic bacteria. Earth Sci. Rev. 86, 42–61 (2007).

24. Kobayashi, A. et al. Experimental observation of magnetosome chain collapse in magnetotacticbacteria: Sedimentological, paleomagnetic, and evolutionary implications. Earth Planet. Sci. Lett.245, 538–550 (2006).

25. Kruiver, P. P., Dekkers, M. J. & Heslop, D. Quantification of the magnetic coercivity components bythe analysis of acquisition curves of isothermal remanent magnetisation. Earth Planet. Sci. Lett. 189,269–276 (2001).

26. Rowan, C. J. & Roberts, A. P. Magnetite dissolution, diachronous greigite formation, and secondarymagnetizations from pyrite oxidation: Unravelling complex magnetizations in Neogene marinesediments from New Zealand. Earth Planet. Sci. Lett. 241, 119–137 (2006).

27. Pike, C. R., Roberts, A. P. & Verosub, K. L. Characterising interactions in fine magnetic particlesystem using first order reversal curves. J. Appl. Phys. 85, 6660–6667 (1999).

28. Simmons, S. L., Sievert, S. M., Frankel, R. B., Bazylinski, D. A. & Edwards, K. J. Spatiotemporaldistribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond.Appl. Env. Microbiol. 70, 6230–6239 (2004).

29. Tauxe, L. Inclination flattening and the geocentric axial dipole hypothesis. Earth Planet. Sci. Lett. 233,247–261 (2005).

Supplementary Information accompanies the paper at www.nature.com/naturegeoscience.

AcknowledgementsThis work was financially supported by the Netherlands Research Centre for Integrated Solid EarthSciences (ISES) and the Netherlands Geosciences Foundation (ALW) with support from theNetherlands Organization for Scientific Research (NWO). J. Kirschvink is thanked for providing thenew code for the single-domain stability field of greigite.

Author contributionsI.V. initiated the project, undertook the analyses and provided the interpretation. C.F. and J.D.M.assisted and advised on TEM microscopy. C.G.L. assisted with the NRM analyses and the TK03.GADcorrection. M.J.D. and W.K. advised and assisted throughout.

Author informationReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions.Correspondence and requests for materials should be addressed to I.V.


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1

Supplementary Information to accompany

Putative greigite magnetofossils from the Pliocene epoch

Iuliana Vasiliev, Christine Franke, Johannes D. Meeldijk, Mark J. Dekkers, Cor G.

Langereis, Wout Krijgsman

Guide to Supplementary Information

Supplementary Methods

Supplementary Palaeomagnetic and Rockmagetic Properties

Methods

p. 2

Supplementary Microscopy Methods p. 3

Geological setting and sedimentary environment p. 4

Supplementary Figures and Legends

Supplementary Figure 1. Palaeogeography of the study area. p. 5

Supplementary Figure 2. Gyroremanent magnetisation in

greigite.

p. 6

Supplementary Figure 3. Greigite prismatic magnetofossil. p. 7

Supplementary Figure 4. Magnetofossil greigite plotted on the

SD stability field of magnetite.

p. 8

Supplementary Figure 5. Magnetite magnetofossils. p. 9

Supplementary Figure 6. Palaeomagnetic and rock magnetic

measurements performed on sample RR 122.

p. 10

Supplementary Figure 7. Characteristic thermal

demagnetization behaviour.

p. 11

Supplementary References cited in the supplementary information



2

Supplementary Methods: Palaeomagnetism and Rock Magnetism

To assess the magnetic mineralogy, we performed thermomagnetic runs in air with a

modified horizontal translation type Curie balance1 with a sensitivity of

approximately 5×10−9

Am2. Runs during different heating (solid lines) and cooling

(dashed lines) cycles were performed at a rate of 10 °C min-1

. Approximately 70 mg

of powdered sample was put into a quartz glass sample holder held in place by quartz

wool. Total magnetization is plotted in a series of runs to increasingly higher

temperatures (100, 200, 300, 350, 450, 500 and 700 °C, respectively). Each six

seconds a data point was recorded, equivalent to one degree Celsius. The applied field

was cycled between 150 and 300 mT. An alternating gradient magnetometer

(Princeton Measurements Corporation, MicroMag Model 2900 with 2T magnet, noise

level 2×10−9

Am2) was used to successively measure hysteresis loops, FORC

diagrams, IRM acquisition (after having used the demagnetization option of the

MicroMag) and backfield demagnetization curves, all at room temperature. The

saturation magnetization (Ms), the saturation remanent magnetization (Mrs) and

coercive force (Bc) were determined from the measured hysteresis curves, after

applying the correction for the paramagnetic contribution on a mass-specific basis. To

further assess the magnetic domain state, the effects of magnetic interactions and the

magnetic mineralogy, FORC diagrams were measured2. IRM acquisition curves,

acquired with the MicroMag System, were decomposed into coercivity components

using the fitting program of Kruiver3, limited to symmetric distributions in the log-

space. Palaeomagnetic results have been previously documented for the studied

sedimentary rocks4-7

and extensive rock magnetic investigations were presented

elsewhere7.



3

Supplementary Methods: Electron Microscopy

For TEM analyses, magnetic extracts were obtained using the extraction procedure

described by Dekkers8. The extraction was performed on 20 grams of grinded rock

samples. The resulting powders were well dispersed by ultrasonic agitation for forty

minutes in demineralised argon-purged water. Sodium polyphosphate

[Na4P2O7·10H2O] was used as a peptising agent to keep the clay mineral particles

dispersed. The obtained suspension was put on the extracting column for three hours.

The prolonged ultrasonic agitation was necessary because the rock samples were

strongly lithified clays. The resulting extracts were washed with demineralised water

in three steps using a ‘magnetic finger’9 to purify the grains from remaining clay

flakes. To further remove adhering clay mineral coatings we used the cleaning

procedure described by Franke et al.,10

. After a second agitation in the ultrasonic bath

for another two minutes to disperse the magnetic concentrate, carbon coated TEM

copper grids were dipped into the extract and subsequently dried in air. For efficiency

we used a magnetic finger attached to the exterior of the vial that attracted the

particles mostly on one side of the copper grid. We took care not to attract too much

of the coarser particles. For all TEM analyses, a FEI Tecnai 20 FEG transmission

electron microscope was used at an acceleration voltage of 200 kV in bright field

mode, at high resolution, or for recording electron diffraction patterns. Elemental

compositions were identified by using energy dispersive spectroscopy.



4

Geological setting and sedimentary environment

The samples have been taken from riverbeds where the rock surfaces were freshly

cleaned by the stream. The sedimentary sequence consists of an alternation of blue to

grey sandstones, siltstones and clays. Our sections start stratigraphically in deposits of

late Sarmatian age and end at Romanian (Eastern Paratethys nomenclature11

). In the

Supplementary Figure 1 we show the uppermost Miocene and Pliocene, greigite-

bearing, part of the section. The older (upper Miocene part) has magnetite as a

magnetic carrier. The appearance of greigite in Chron C3r (between 6.0 and 5.5 Ma)

is most likely related to regional tectonic and/or climatic events that reshaped the

basin configuration and consequently changed drastically the palaeoenvironmental

conditions. However, the presence of benthic ostracods12,13

and molluscs13,14

throughout the entire upper Miocene-Pliocene time interval demonstrates that the

lowermost water column remained sufficiently oxygenated for these organisms to

live. Anoxic conditions favouring greigite formation could therefore only have been

present within the sediments, related to degradation of organic matter during rapid

burial. Greigite formation in the Romanian Carpathian foredeep palaeoenvironment

occurred in a very high sedimentation rate setting (60-150 cm/kyr4), a situation

similar to south-western Taiwan15-18

and eastern New Zealand19,20

. High

sedimentation rates accompanied by rapid burial of organic matter will lead to a

completely anoxic diagenetic environment close to the sediment/water interface21,22

.

In such settings, the authigenic greigite in the Eastern Carpathian foredeep likely has

formed through bacterial mediation during early diagenesis, up to kyrs after

deposition.



Rimnicu Sarat Valley

(45.55ºN and 26.88ºE)

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

4600

4800

5000

5200

5400

5600

5800

6000

6200

6400

6600

6800

7000

7200

Dacia

n

Meot.

Po

ntian

Ro

ma

nia

n

180 360

Declination

-90 0 90

Inclination

Black Sea

Ukraine

Turkey

Romania

Hungary

Bulgaria

Germany

Italy

C3r

T

N

C

C2A

rC

2A

n.1

n

0ºE 20ºE 40ºE 60ºE

25ºE

30ºE

35ºE

40ºE

45ºE

50ºE

55ºE

0 400

km

800

10ºW 10ºE 30ºE 50ºE

Late Miocene/early Pliocene of the Paratethys

Dan u b eDan u b e

Vol g

a

Vol g

a

Black Sea

Casp

ian

Se

a

Aral

Atla

ntic

Oce

an

Mediterran

ean Sea

France

Poland

Russia

Ukraine

Turkey

550

600

650

700

750

800

850

900

950

1000

1050

1100

Str

ati

gra

ph

ic l

eve

l (m

)

180 360

Declination

-90 0 90

Inclination

Po

ntian

Dacia

n

C3r

T

N

Badislava Valley

(45.15ºN and 24.53ºE)

a

b

Panel b

Supplementary Figure. 1

Palaeogeography of the study area. (a) Schematic

palaeogeographic map of the late Miocene/early

Pliocene, showing the Paratethys area and the present-

day land contribution; (b) map of the study areas

modified after Vasiliev et al., 20077 with the indication

the geographic position of the sections in front of the

Romanian Carpathians. The polarity zones with

palaeomagnetic data for the studied river sections:

Badislava and Rîmnicu Sarat. Black (white) denotes

normal (reversed) polarity. The indicatives for the most

important subchrons are displayed (C = Cochiti, S =

Sidufjall, N = Nunivak, T = Thvera). The names of the

substages are according to the Eastern Paratethys

nomenclature11. Mean latitudes and longitudes (in

decimal degrees) are given in the header of the

magnetostratigraphy columns.



BD110.1BN

up/W

Inti = 11 mAm-1

100 mT 9080 0 mT

a

100 mT807060

BD114.1B

N

up/W

Inti = 3 mAm-1

b

Supplementary Figure 2.

Gyroremanent magnetisation in greigite. (a) and (b) are tilt corrected Zijderveld

diagrams of typical alternating field (AF) demagnetisation behaviour; open (closed)

circles are projections on the vertical (horizontal) plane. Initial NRM intensities (Inti) are

given. The values represent milliTesla (mT). AF demagnetisation was performed by an

in-house developed robot, which let the samples pass-through a 2G Enterprises SQUID

magnetometer (noise level 10-12 Am2). After 45 mT AF demagnetisation, the samples

were increasingly acquiring a gyroremanent magnetisation much larger than the initial

NRM. (a) BD 110.1B is the sister sample of BD 110.1A (Fig. 1c); note the remarkable

difference between thermal and AF demagnetisation. (b) BD 114.1B is the sister sample

of BD 114.1A which has identical thermal demagnetisation behaviour as sample BD

115.1A (Fig. 1b).



Panel c

20 nm

Panel d

Panel b

10 nm10 nm

(111) 5.7A

5 nm

68o

53o

2

3

1 {200} {111}

{111}

a b

c d


Greigite prismatic magnetofossil. (a) TEM micrograph of elongated prismatic

greigite crystal with well-defined truncated edges. There is non-uniform contrast

indicating changing thickness as expected from the outline of the crystal. (b) High

magnification of the area indicated in panel a. Three sets of lattice fringes: set 1

corresponds to the {200} plane, sets 2 and 3 correspond to the {111} plane. Set 2

forms angles of 68 º and 53 º with sets 3 and 1, respectively. (c) High

magnification of the lattice image of the area indicated in panel a. The bands

between the lines indicate a d-spacing of 5.7 Å, corresponding to (111) greigite

reflection. (d) High magnification of the lattice image from a truncated edge of the

crystal.



StableSD

MetastableSD

Multidomain

Cuboidal

ElongatePrismatic

ElongateIrregular

Superparamagnetic

Length

(nm

)

Shape Factor (Width/Length)

300

100

30

0 0.2 0.4 0.6 0.8 10.1 0.3 0.5 0.7 0.9

a

200 nm

b

c

0.00

Fre

quency

Particle lengh (nm)12 16 20 24 28 32 36 40

0.05

0.10

0.15

0.20

0.25


Magnetofossil greigite plotted on the SD

stability field of magnetite. (a) The diagram is

redrawn after Kopp and Kirschvink, 200723 and is

a function of shape factor (width/length ratio) and

length. For details see23. Shaded regions mark

size and shape of crystals from magnetotactic

bacteria. The shape fields in the diagram are based

upon the specific crystal morphologies produced by

living magnetite-producing magnetotactic bacteria,

not greigite-producing ones. The elongated

prismatic greigite crystals in our samples fit on the

cuboidal area of Kopp and Kirschvink23. We still

interpret them as prismatic because they are

different from the cuboctahedral crystals in our

samples. In addition, the cuboctahedral crystals are

smaller than all existing data so far. The

importance of the magnetic interaction at these

critical sizes has been shown to be very

important24. (b) TEM micrograph of a cluster of

cuboctahedral greigite crystals; (c) the size

distributions of the greigite crystals from panel b.

These measurements are subject to bias because,

at this size, even with the excellent resolution, it is

difficult to obtain the true size values of the crystals.

Therefore, we chose to count the particle three

times, then stacked these measurements (in total

443) and run the statistics. The mean size value of

the particles is 24.61 nm with 3.71 as standard

deviation. The histogram shows a clearly skewed

(to the left) distribution, which indicates a

magnetosomal origin of these crystals25,26.



a

100 nm

0 2 4 6 8 10

0

100

200

300

400

500

Fe

SiFeO

Cu

1 3 5 7 9

Energy [keV]

Counts

(s-1

)

Fe Cu

C

b


Fossil magnetite magnetosomes. (a) TEM micrograph of prismatic magnetite

crystals. In the upper left part of the image they are aligned in a (disrupted) chain

and in the lower right part they are clustered in no preferred order. (b) Energy-

dispersive X-ray spectrum from the crystals of the chain in the image showing

the distinct peaks for Fe and O of iron oxide. Al and Si peaks are caused by the

background signal of the clay mineral flake; the C and Cu peaks originate from

the carbon coated copper TEM grid.



SF = 3SF = 3

20 40 60 80 100 120

Bc (mT)

-30

-10

10

30

40

50

-50

-40

-20

0

20

Bu (

mT

)

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 00.1 -0.1

0

4

8

12

16

20

15

30

45

0

1 10 100 1,000

1 10 100 1000

IRM

(10-3

X A

m2kg-1

)IR

M (

10

-3 X

Am

2kg-1

)

LinearAquisitionPlot

GradientAquisitionPlot

Applied field (mT)

B1/2 = 74.1 mTDP = 0.11

magnetosomes

B1/2 = 74.1 mTDP = 0.36authigenic

0 100 200 300 400

0.0

0.2

0.4

0.6

0.8

1.0

Norm

aliz

ed inte

nsity

Temperature (oC)

240

210

180 oC

N

up/W

380

270

Inti = 16 mAm-1

0 100 200 300 400 500 600 700

0.006

0.014

0.022

0.030

0.026

0.018

0.010

To

tal m

ag

ne

tisa

tio

n (

Am

2kg

-1)

Temperature (oC)

Bc = 28.8 mT

Bcr = 62.4 mT

Mr/Ms = 0.30

0

8

-8

4

-4

0-300 300

B (mT)

M (

10-3

x A

m2kg

-1)

a

e

c

b

f

d


Palaeomagnetic and rock magnetic measurements performed on sample RR 122. (a) Zijderveld diagram (after tilt correction)

with the direction of the low (blue arrow) and high (red arrow) temperature component. Correspondingly coloured numbers are the

temperature steps taken to calculate the directions of the two temperature components. See also caption to figure 1. (b)

Thermomagnetic runs during different heating (red solid lines) and cooling (blue dashed lines) show an irreversible decrease in

magnetization caused by the greigite decomposition reaction chain, between 200 and 400 ºC. A slight increase in the total

magnetization is visible at ~500 ºC, indicating the formation of new magnetic minerals because of oxidation of an iron sulphide.

This temperature is higher than the ones known for alteration of iron sulphides. (c) Normalized decay curve represented as

absolute (blue diamonds) and difference vector sum (red circles) values. There are two major inflections in the decay curves

arising from the combined effect of greigite alteration and magnetic unblocking. These extrapolated lines (to ~290 ºC and ~350

ºC) indicate two magnetic carriers, represented by blue and red areas. The hatched area indicates a possibly viscous (e.g.

laboratory induced) magnetisation removed up to 100 ºC. (d) Hysteresis loop shown between ±300 mT. (e) FORC diagram,

indicating (highly interacting) SD particles. (f) IRM acquisition curve, decomposed into coercivity components using a log-normal

fitting program3. Open blue squares are measured data points. The components are marked with green and purple lines on the

linear acquisition plot (LAP) and equivalently coloured shading on the gradient acquisition plot (GAP). Both components have the

same coercivity (B1/2), but different dispersion parameters (DP) indicating different grain size distribution. For additional details,

see also the supplementary palaeomagnetic and rock magnetic methods section.



TP 12

C3n.4n

~4.99 Ma

Thvera

0.0

0.2

0.4

0.6

0.8

1.0

0 100 200 300 400

Norm

aliz

ed inte

nsity

Temperature (oC)

N

up/W

285-360

20oC

Inti = 4 mAm-1

TP 22

C3r.2r

~4.70 Ma

0.0

0.2

0.4

0.6

0.8

1.0

0 100 200 300 400

Norm

aliz

ed inte

nsity

N

up/W

210

360Inti = 8 mAm-1

20oC

150

RR 097

C2An.1n

~2.80 Ma

Gauss

0.0

0.2

0.4

0.6

0.8

1.0

Norm

aliz

ed inte

nsity

0 100 200 300 400

N

up/W

400

240

340

Inti = 2 mAm-1

20oC

180

240

RM 034

C3An.1n

~6.05 Ma

0.0

0.2

0.4

0.6

0.8

1.0

0 100 200 300 400

Norm

aliz

ed inte

nsity

N

up/W

390oC

20oC

150

Inti = 2 mAm-1


Characteristic thermal demagnetization behaviour. On the left hand side of

each panel, Zijderveld diagrams are plotted after tilt correction. Blue and red

arrows as in caption to Supplementary Fig. 1. On the right hand side are the

corresponding normalized decay curves. We show examples from two valleys that

generated greigite-based magnetostratigraphy (RR and RM from Rîimnicu Sarat

and TP from Topolog sections). (a) Sample with different normal HT and LT

components showing a decay curve suggesting two main magnetic carriers for the

respective components; (b) sample showing dual polarity recorded within the same

sample at the transition from normal to reverse. The delayed LT component was

acquired during the later reversed field. From Topolog valley: (c) sample TP 22 was

magnetized in a reversed magnetic field and has again two distinguishable

components; (d) sample TP 12 shows dual polarity recorded within the same

sample at the reversal from normal to reversed.

d

a

b

c



12

Supplementary References

1. Mullender, T. A. T., van Velzen, A. J. & Dekkers, M. J. Continuous drift

correction and separate identification of ferromagnetic and paramagnetic

contribution in thermomagnetic runs. Geophys. J. Int. 114, 663-672 (1993).

2. Roberts, A. P., Pike, C. R. & Verosub, K. L. First-order reversal curve

diagrams: A new tool for characterising the magnetic properties of natural

samples. J. Geophys. Res. 102, 28461-28475 (2000).

3. Kruiver, P. P., Dekkers, M. J. & Heslop, D. Quantification of the magnetic

coercivity components by the analysis of aquisition curves of isothermal

remanent magnetisation. Earth Planet. Sci. Lett. 189, 269-276 (2001).

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Paratethys Mio-Pliocene sedimentary sequences of the Focsani basin

(Romania). Earth Planet. Sci. Lett. 227, 231-247 (2004).

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magnetostratigraphy in the southern Carpathian foredeep and Mediterranean-

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6. Dupont-Nivet, G., Vasiliev, I., Langereis, C. G., Krijgsman, W. & Panaiotu,

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constrained by paleomagnetism. Earth Planet. Sci. Lett. 236, 234-387 (2005).

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(Romania). Geophys. J. Int. 171, 613-629 (2007).

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1988).



13

9. von Dobeneck, T., Petersen, N. & Vali, H. Baktrielle Magnetofossilen -

Palaomagnetische spuren einer ungewohlichen Bakteriengruppe.

Geowissenschaften in unserer Zeit 5, 27-35 (1987).

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separation to concentrate magnetic particles. Geophys. J. Int. 170, 1053-1066

(2007).

11. Rögl, F. & Daxner-Hock, G. Late Miocene Paratethys Correlation. The

Evolution of the Western Eurasian Neogene Mammal Faunas, 487 (1996).

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(Editura Academiei Romane, Bucharest, 1995).

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Pontian and Dacian deposits from the Topolog-Arges area (southern

Carpathian foredeep - Romania). Geobios 40, 391-405 (2007).

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14

18. Kao, S.-J., Horng, C.-S., Roberts, A. P. & Liu, K.-K. Carbon-sulfur-iron

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bacterial sulphate reduction: the G model tested. Limnol. Oceanogr. 29 (1984).

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anoxic marine sediments. Geochim. Cosmochim. Acta 51, 645-659 (1987).

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interpretation of fossil magnetotactic bacteria. Earth Sci. Rev. 86, 42-61

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24. Muxworthy, A. R. & Williams, W. Critical single-domain/multidomain grain

sizes in noninteracting and interacting elongated magnetite particles:

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sulfides. Eur. J. Mineral. 13, 691-703 (2001).



15

26. Kruiver, P. P. & Passier, H. F. Coercivity analysis of aquisition curves of

magnetic phases in sapropel S1 related to variation in redox conditions,

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LETTERS Putative greigite magnetofossils from the Pliocene...

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