The molecular record of Cryogenian sponges – a response to Antcliffe (2013)

Molecular fossils or lipid biomarkers, which are preserved in ancient sedimentary rocks and petroleum that have undergone a mild thermal history, have been studied for over five decades (e.g. Eglinton et al. 1964, 1966; Blumer and Snyder 1965) and can yield valuable and unique insights concerning past ocean redox structure, plankton successions and biospheric evolution (e.g. Brocks and Pearson 2005; Peters et al. 2005; Knoll et al. 2007; Briggs and Summons 2014, for recent reviews). Currently, the earliest compelling hydrocarbon biomarker record for Metazoa comes from a ~100 million year record reported by Love et al. (2009) of distinctive Neoproterozoic–Cambrian steroids known informally as 24-isopropylcholestanes (24-ipc), which are derived from sterols produced in abundance by demosponges (McCaffrey et al. 1994). The most extensively documented record for these compounds extends to interglacial sedimentary rocks of the Cryogenian period (713–635 Ma) in a thermally immature succession of Neoproterozoic – early Cambrian marine sedimentary rocks from the Huqf Supergroup of the South Oman Salt Basin (SOSB). The concentration of biomarkers varies in different sedimentary rocks due to lithological and thermal maturity controls, so as well as reporting absolute abundances of 24-ipc steranes detected in each rock sample, Love et al. (2009) normalized their data and documented changes in the ratio of 24-ipc to the structurally similar (see Fig. 1), but analytically resolvable, sterane isomer 24-n-propylcholestane (24-npc), which originates principally from the sterols of marine pelagophyte algae (Giner et al. 2009). 24-npc is typically the dominant C30 sterane detected in marine Phanerozoic oils and rocks from all lithologies (Moldowan 1984; Moldowan et al. 1985, 1990), and there is no evidence that 24-npc steranes can be significantly converted into 24-ipc steranes, or vice versa, either during low temperature diagenesis or higher temperature burial maturation (e.g. Moldowan et al. 1990; Summons et al. 1992; McCaffrey et al. 1994; Zhang et al. 2000). Demosponges, particularly those from the order Halichondrida (Love et al. 2013), are the only living taxon known to produce their major sterols with a 24-ipc carbon skeleton. 24-ipc-related compounds can constitute >99% of the total sterols in membrane lipids in the demosponge Pseudaxynissa (Hofheinz and Oesterhelt 1979), and 24-ipc is among the major sterols in other genera such as Topsentia, Collocalypta, Halichondria and Epipolasis. Love et al. (2009) studied 64 source rock samples encompassing carbonates, siltstones and shales, of appropriate thermal maturity for biomarker preservation (verified independently by Rock-Eval pyrolysis screening). This set of samples provided stratigraphic coverage of all formations of the Neoproterozoic–Cambrian Huqf Supergroup available in drill core. In a related study, Grosjean et al. (2009) verified the anomalously high relative abundances of 24-ipc in a large suite of SOSB rocks as well as in petroleum samples produced from various SOSB formations, while Kelly et al. (2011) found similar patterns of 24-ipc steranes in Neoproterozoic oils from east Siberia, including some oils potentially sourced from putative Cryogenian source rocks. Anomalously high abundances of 24-ipc steranes are also characteristic features of certain Neoproterozoic – early Cambrian oils and rock extracts from India (Peters et al. 2005), Pakistan (Grantham et al. 1987) and Australia (McCaffrey et al. 1994). These rocks and oils possess unusual biomarker characteristics which are also prominent in SOSB oils and rocks (e.g. C29 sterane dominance in the C27–C30 range, high 24-ipc/npc ratio, presence of an unusual mid-chain methylalkane series) and which are only commonly detected together in Neoproterozoic – early Cambrian samples as noted previously (Peters et al. 1995, 2005; Love et al. 2009; Kelly et al. 2011). The SOSB biomarker record indicated the presence of demosponges in the Cryogenian period, significantly before the appearance of obvious spicules in the fossil record but at a time consistent with predictions of molecular clocks (Sperling et al. 2010), including clocks that predated our biomarker study (Berney and Pawlowski 2006). More recently, we have detected elevated 24-ipc/24-npc ratios in Permian strata from China which contain abundant demosponge siliceous spicules (Love et al., 2013), and this is part of an ongoing investigation. Antcliffe (2013; see also Antcliffe et al. 2014) questioned the significance of the 24-ipc record presented by Love et al. (2009) for dating the origin of sponges, considering that it was more likely a product of marine (pelagophyte) algae, and argued that sponges appeared much later as part of a more rapid Cambrian explosion. Such an inference, however, is based on a misinterpretation of the lipid biomarker record. The significant gap between the earliest record of 24-ipc and the first uncontested sponge body fossils remains – Antcliffe's attempt to fix it by negating the biomarker evidence is fallacious. Antcliffe (2013, p. 917) argued that ‘several different pelagophyte (Class Pelagophyceae part of the Stramenopiles within the Chromaveolata [sic]) algae are also capable of producing these exact compounds, and may similarly have done so in deep time’. But the C30 sterols produced by pelagophytes have a 24-npc skeleton and any sterols with a 24-ipc skeleton that they generate are in concentrations two orders of magnitude lower than that of 24-npc (Giner et al. 2009; see Table 1), as addressed in our original paper. These proportions are reflected in the steranes to which the sterols transform during diagenesis. We are not aware of any published study which suggests that 24-ipc sterols in pelagophyte cultures are anything more than minor trace sterols under any particular growth conditions (see Table 1). 24-ipc sterols are found in trace quantities with respect to the 24-npc compounds, which are the major sterols, and usually the 24-ipc sterols are below detection limits in any case. Love et al. (2009, supplementary information, section 2) stated that: ‘It is an aberration of an enzymatic process intended to produce 24-n-propylidenecholesterol (and related structures) and does not constitute a biosynthetic capacity to make abundant 24-isopropylcholesterol given optimal growth conditions’. Antcliffe (2013, p. 917) asserted that ‘modern marine algae are also reported to produce structural isomers that are compositionally identical to the sponge marker; they do this in copious quantities’. This is incorrect. Structural isomers are, by definition, different chemical compounds and 24-ipc and 24-npc steranes are distinct compounds (see Fig. 1) that can be readily resolved using standard capillary gas chromatography. No algae, other than the pelagophytes in trace or undetectable amounts with the caveats as discussed above, are known to produce 24-ipc sterols that are compositionally identical to the sponge marker (Volkman et al. 1998; Volkman 2003, 2005). He also claimed (2013, p. 917) that ‘24-isopropylcholestane can be produced by diagenetic alteration of compounds produced in large quantities by algae’, but this is inconsistent with the generally low Phanerozoic baseline values of 24-ipc/npc sterane ratios (typically 0.2–0.3) for marine rock extracts and oils (see section 10 of Love et al. 2009) and as illustrated in the figure showing temporal abundance patterns of 24-ipc in the Brocks and Butterfield (2009) commentary article. Any hypothetical reaction mechanism that might potentially convert 24-npc to 24-ipc steranes during diagenesis or subsurface burial maturation must then be minor from this well-established Phanerozoic baseline ratio constraint and so secondary transformation cannot account for the consistently high 24-ipc/24-npc ratios found for Huqf Supergroup rocks. As we stated in Love et al. (2009), ‘The ratio of 24-ipc/24-npc (for all 4 regular geoisomers) was measured for extracts and pyrolysates and found to be anomalously high (0.52–16.1, with an average value of 1.51, Tables S1–S2) in all Neoproterozoic-early Cambrian SOSB samples in comparison with Phanerozoic and mid-Proterozoic oils and bitumens reported previously (typically <0.3S1)’. The original Table S3 is reproduced here as Table 1, which shows that the extremely low ratio values of 24-ipc/24-npc (0.00–0.01) reported for pelagophyte algal cultures are at least two orders of magnitude lower than the typical ratios found in South Oman rock extracts and pyrolysates (average = 1.51). Antcliffe (2013) also referred to a single cell genomics study (Siegl et al. 2011) which claimed that poribacterial symbionts of marine sponges could possibly synthesize 24-ipc sterols. But a subsequent study by the same workers showed this to have yielded a false result (Kamke et al. 2013). The Aplysina aerophoba sponge specimen used by Siegl et al. (2011) does not make significant amounts of 24-ipc among its major sterols, and SMT (sterol methyltransferase) genes are not specific for 24-ipc sterol synthesis in any case. Subsequent genomics investigation of the same poribacterial symbionts have shown that these, like all known bacteria, do not have the genetic capacity to make 24-alkylkated sterols (Kamke et al. 2013). Antcliffe (2013, p. 917) claimed that ‘it is also possible that contamination by petroleum derived lubricating oil used when coring while extracting these compounds from subsurface layers, has affected the data’. The only lubricating oils with sufficiently high 24-ipc/24-npc ratios to influence the signal in soluble rock extracts come from similarly old Neoproterozoic–Cambrian sequences (McCaffrey et al. 1994; Peters et al. 1995; Kelly et al. 2011), and these were not used. Organic geochemists, in any case, are acutely aware of the dangers of contamination, and we reported experiments that demonstrate unequivocal generation of 24-ipc from the immobile kerogen component of SOSB sediments using catalytic hydropyrolysis (Love et al. 1995, 2009), performed in parallel with the more conventional solvent-extractable biomarker analyses. Having a robust kerogen-bound 24-ipc record from SOSB strata was an important self-consistency check for verifying that these steranes were syngenetic with the host rocks and that these compounds had not migrated in from other, possibly younger, strata. Kerogen is a high molecular weight and insoluble organic polymer formed during early diagenesis (in the water column and/or shallow subsurface) and which cannot migrate once incorporated within the host sediment. Ancient sedimentary kerogen usually contains an abundant bound biomarker pool linked by strong covalent bonds, and this bound pool remains well preserved and quantitatively important well into the later stages of oil-window thermal maturity for ancient rocks (Murray et al. 1998; Lockhart et al. 2008). We can recognize the distinctive isomeric patterns that characterize kerogen-bound steranes and hopanes in pyrolysates released by covalent bond cleavage (Love et al. 1995), as we described in section 1 of Love et al. (2009). Thus, our kerogen-bound 24-ipc record rigorously ground-truthed the temporal range of the abundant Cryogenian–Cambrian 24-ipc steranes that we reported for SOSB strata in Love et al. (2009). While Antcliffe's attempt to discredit the biomarker evidence is based on misunderstandings, his observation that all the Neoproterozoic organisms that might have produced 24-ipc ‘are unavailable for analysis by the modern organic chemist and cannot be eliminated from the list of possible producers’ is, of course, self-evident. However, future studies may reveal an evolutionary phylogeny for key enzymes in 24-isopropylcholesterol biosynthesis (Volkman 2005) and, in the meantime, sponges remain the only credible producer given the sterane ratios and absolute abundances in Neoproterozoic–Cambrian oils and rocks from South Oman, eastern Siberia, India, Pakistan and Australia. As regards the age of the molecular fossil occurrences, Antcliffe is only partially correct that ‘although the compounds are widely reported as c. 751 Ma, they are younger than 645 Ma’. Note that Love et al. (2009) did not claim an age of c. 751 Ma for the occurrence of 24-ipc. They identified high 24-ipc/24-npc ratios in samples from interglacial strata from the SOSB, that is, older than the Marinoan-aged cap carbonates found there but younger than the underlying glacial of Sturtian age. From our abstract, ‘Biomarker analysis has yet to reveal any convincing evidence for ancient sponges pre-dating the first globally extensive Neoproterozoic glacial episode (the Sturtian, ~713 Myr ago in Oman)’. As was pointed out previously (Love et al. 2009, section 11), 24-ipc steranes are either below detection limits or present only in trace amounts from pre-Sturtian-age Neoproterozoic rocks and oils, including rock bitumens from the c. 742 Ma Walcott Member of the Chuar Group (Summons et al. 1988), the c. 700–800 Ma Visingsö Group and the c. 850 Ma Bitter Springs Formation (McCaffrey et al. 1994) and from the c. 1.1 Ga Nonesuch Formation (Pratt et al. 1991). 24-ipc steranes are absent from the abundant and diverse lipid biomarker assemblages reported most recently for thermally immature Middle Proterozoic rocks from Australia, China and Mauritania spanning 1.1–1.6 Ga in age (e.g. Brocks et al. 2005; Blumenberg et al. 2012; Flannery and George 2014, Luo et al. 2015). From the Gubrah Formation in Oman, the age of an ash bed within the Sturtian event was dated at 713 Ma (Bowring et al. 2007), consistent with the most recent age constraints for diamictites associated with the Sturtian glaciation at c. 716 Ma (Macdonald et al. 2010). Although its termination has been proposed as late as 640 ± 4.7 Ma (Kendall et al. 2009), this Re–Os age may be correlative with the c. 635 Ma Marinoan age and a new temporal constraint from a post-Sturtian Re–Os date of 662.4 ± 3.9 Ma for the Rapitan Group in northwest Canada (Rooney et al. 2014) suggests an interlude between the Sturtian and Marinoan glaciations. A maximum age for the top of the Marinoan diamictite in South Oman, just below the cap carbonate from the Lahan-1 core, of <645 Myr was reported from U–Pb detrital zircon geochronology (Bowring et al. 2007). This is compatible with an apparently synchronous age for the Marinoan cap carbonate worldwide from dated ash beds layers at c. 635 Ma based on dates obtained from deposits in the Ghaub Formation in Namibia (635.5 ± 0.6 Ma), as well as the Nantuo Formation (636.3 ± 4.9 Ma) and the Doushantuo Formation (635.2 ± 0.2 Ma) in South China (Hoffmann et al. 2004; Condon et al. 2005; Zhang et al. 2008). Our oldest sample from SOSB for which we reported abundant free and kerogen-bound 24-ipc steranes lay stratigraphically below the Ediacaran Nafun Group, as well as below a cap carbonate and diamictite also interpreted as Marinoan in age. Love et al. (2009, supplementary information, section 5) stated ‘The Ghadir Manquil Fm. sediment from GM-1 core was a lime mudstone found approximately 150 m below the base of a diamictite that we interpret as Marinoan age because of the stratigraphic position directly beneath both the Nafun Gp sedimentary package and the underlying (Marinoan) cap carbonate’. Thus, the oldest samples with high 24-ipc steranes ratios and abundances most likely occur in strata dated between c. 635 and c. 713 Ma. We agree that a 635 Ma date is the most appropriate for any molecular clocks using a sponge biomarker as a calibration point for the minimum divergence between demosponges and hexactinellids. However, a 713 Ma calibration is normally set as a soft-bound maximum divergence age constraint on crown-group demosponges based on the absence of abundant 24-ipc in pre-Sturtian-age rocks (e.g. younger clock estimates are allowed; Sperling et al. 2010; Erwin et al. 2011), and we note that removing the sponge biomarker calibration point for constraining crown-group demosponges actually resulted in older and not younger estimates for the origin of animals (Sperling et al. 2010). In summary, sterols with a rare isopropyl substituent at C-24 are biosynthesized by demosponges, primarily of the order Halichondrida (Giner and Djerassi 1990; Giner 1993; Love et al. 2013), but are found in all four major clades of demosponges. In contrast, sterols with an n-propyl substituent at C-24 are biosynthesized by pelagophyte algae as major lipids (Giner et al. 2009), but with only trace or undetectable amounts of 24-ipc sterols present (see Table 1). These sterol structures, and those of the fossil steranes that are their diagenetic counterparts, are distinct and cannot be interconverted to any significant degree during diagenesis and thermal maturation in sediments. It is regrettable that no organic geochemist was asked to review the Antcliffe (2013) paper as it contains fundamental errors and false assumptions concerning organic geochemical methods and principles. While much work remains in interpreting the demosponge biomarker record, including the genomic and functional context of these molecules in the source organisms and continued interrogation of the pre-Sturtian ancient biomarker record, the speculative points raised by Antcliffe (2013) regarding a possible origin from marine algae and contamination by drilling fluids are weakly grounded. Such issues were already addressed in the supplementary information accompanying the Love et al. (2009) paper and no new data was presented by Antcliffe (2013) to support his claims. Accordingly, the most parsimonious explanation for the extraordinary Neoproterozoic occurrence of high absolute and relative abundances of 24-ipc steranes, originating between 635 and 713 Ma, is that they represent chemical fossils of demosponges or their ancestors. Both authors’ research is funded by the NASA Astrobiology Institute. GDL also acknowledges support from the NSF Earth-Life Transitions program (NSF-EAR 1338299).

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