GEOLOGY, July 2008 543 Geology, July 2008; v. 36; no. 7; p. 543–546; doi: 10.1130/G24690A.1; 2 figures; 1 table. © 2008 The Geological Society of America. For permission to copy, contact Copyright Permissions, GSA, or editing@geosociety.org. ABSTRACT Late Pennsylvanian seep limestones (ca. 300 Ma) enclosed in the Ganigobis shales in southern Namibia formed by microbial activity. The process that induced carbonate precipitation was the anaerobic oxidation of methane. The presence of 13 C-depleted pentamethylico- sane (PMI) (–113‰) and a mixture of crocetane and phytane (–112‰) in concert with similarly 13 C-depleted pseudohomologous series of regular isoprenoids reveals that methanotrophic archaea oxidized methane anaerobically at the ancient seep site. Biphytane and a C 39 pseudohomologue are other archaeal molecular fossils with δ 13 C val- ues of –99‰ and –97‰, respectively. The former presence of sulfate- reducing bacteria as the syntrophic partners of methanotrophic archaea in the anaerobic oxidation of methane is indicated by isotopi- cally depleted iso- and anteiso-alkanes. These compounds most prob- ably derive from non-isoprenoidal monoethers and diethers, synthates of sulfate-reducing bacteria. These findings show that anaerobic oxi- dation of methane is at least 300 m.y. old, extending the record of this process for ~140 m.y. As the molecular fossils of archaea and bac- teria are preserved in a product of their own metabolic activity (i.e., methane-derived carbonates with δ 13 C values as low as –51‰), the syngenicity of molecular fossils and enclosing deposits is unambigu- ous. This reveals that microbially formed rocks can represent excel- lent archives for studying past biogeochemical processes. Keywords: anaerobic oxidation, methane, archaea, sulfate-reducing bac- teria, seeps, isoprenoids, limestones. INTRODUCTION The anaerobic oxidation of methane is the key metabolism at modern marine methane seeps and induces carbonate formation. It is mediated by a consortium of methane-oxidizing archaea and sulfate-reducing bacteria (e.g., Hinrichs et al., 1999; Boetius et al., 2000; Orphan et al., 2001). A number of studies of the molecular inventory of seep deposits revealed new insights into anaerobic oxidation of methane and resulted in the descrip- tion of numerous lipid biomarkers with low δ 13 C values that typify this process and the organisms mediating it (Elvert et al., 1999; Hinrichs et al., 1999; Thiel et al., 1999; Pancost et al., 2000). Ancient seep limestones have been identified by their macrofossil inventory, petrographical features, low δ 13 C carbonate values, and lipid biomarkers (for reviews see Peckmann and Thiel, 2004; Campbell, 2006). Limestones consisting of a suite of authigenic carbonate phases typify modern and ancient seeps. Precipita- tion of these carbonates results from an increase in alkalinity caused by anaerobic oxidation of methane (Peckmann and Thiel, 2004; Campbell, 2006). Although δ 13 C carbonate values lower than -30‰ reveal that these car- bonates formed as a consequence of the oxidation of methane, they provide no direct insight into either the biogeochemical process or the affiliation of the microorganisms involved. To date, lipid biomarkers are the only tool to extract this sort of information from ancient seep limestones. The use of lipid biomarkers in studying ancient seep limestones is limited by biodegradation, thermal maturity, and dilution by secondary migration (Goedert et al., 2003; Birgel et al., 2006a). Biodegradation does not seem to be a serious issue for biomarker preservation in seep limestones, as molecules are well protected from consumption due to early engulfment in authigenic carbonates. Secondary migration is also not a significant factor, because extrinsic compounds can easily be recognized by their different carbon isotopic composition. Moreover, separation of compounds closely bonded to carbonate minerals (intra- crystalline) from secondary compounds (extracrystalline) by a succes- sive extraction-dissolution-extraction procedure leads to fractions of lipids that are mostly endemic to the ancient seep sites (Thiel et al., 1999). Thermal maturity, however, significantly restricts the applicabil- ity of lipid biomarkers. To date, only a very limited number of Paleozoic seep limestones have been recognized based on faunal, petrographical, and isotopical evidence (Himmler et al., 2008; Table 1). Earlier attempts to constrain biogeochemical processes at Paleozoic seeps (Hollard Mound; Peckmann et al., 1999; Dzieduszyckia deposit; Peckmann et al., 2007; Iberg deposit; Peckmann et al., 2001) failed because of the high maturity of the seep limestones studied. In this study we provide robust biomarker and isotope evidence that methane was oxidized in the same manner in the Paleozoic as it is at mod- ern marine seeps today. We show that anaerobic oxidation of methane was mediated by methanotrophic archaea and sulfate-reducing bacteria since at least the Late Pennsylvanian. This extends the record of a major biogeo- chemical process for ~140 m.y. SAMPLES AND METHODS Limestone samples were collected from the Ganigobis shale of the Late Pennsylvanian glaciomarine Dwyka Group of Namibia (Fig. 1). Petrographical and paleontological features as well as stable isotope data of the carbonates were presented in Himmler et al. (2008). The prepa- ration, cleaning, and decalcification procedure of the limestone sample discussed here was performed as described in Birgel et al. (2006a). Extraction was carried out with a microwave extraction system (CEM Corporation MARS X) at 80 °C and 600 W with CH 2 Cl 2 :MeOH (3:1). The separation of the resulting extracts was done by column chromatog- raphy into four fractions of increasing polarity. In the following, we dis- cuss only the hydrocarbon fraction. Hydrocarbons were measured using a gas chromatography–mass spectrometry system (Thermo Electron Cor- poration Trace MS) equipped with a 30 m RTX-5 MS fused silica column (0.32 mm i.d., 0.25 μm film thickness). The carrier gas was He. The gas chromatography (GC) temperature program used was as follows: 60 °C, 1 min isothermal; from 60 to 150 °C at 10 °C min –1 ; from 150 to 320 °C at 4° min –1 ; 22 min isothermal. Identification of the measured compounds was based on GC retention times and mass spectra in comparison with published data. Compound-specific carbon isotope analyses were carried out with a Hewlett Packard 5890 gas chromatograph via a Thermo Elec- tron GC-combustion-interface to a Finnigan MAT 252 MS. GC conditions were identical to those described above. Carbon isotopes are given as δ values (δ 13 C in ‰) relative to the Vienna Peedee belemnite standard. Sev- eral pulses of CO 2 gas of known δ 13 C value at the beginning and the end of the runs were used for calibration. Instrument precision was checked using a mixture of n-alkanes with known isotopic composition. *E-mail: peckmann@uni-bremen.de. A new constraint on the antiquity of anaerobic oxidation of methane: Late Pennsylvanian seep limestones from southern Namibia Daniel Birgel MARUM–Center for Marine Environmental Sciences, Universität Bremen, 28334 Bremen, Germany Tobias Himmler André Freiwald Geozentrum Nordbayern, FG Paläoumwelt, Universität Erlangen, 91054 Erlangen, Germany Jörn Peckmann* MARUM–Center for Marine Environmental Sciences, Universität Bremen, 28334 Bremen, Germany