Rise to modern levels of ocean oxygenation coincided with the Cambrian radiation of animals

The early diversification of animals (∼630 Ma), and their development into both motile and macroscopic forms (∼575–565 Ma), has been linked to stepwise increases in the oxygenation of Earth's surface environment. However, establishing such a linkage between oxygen and evolution for the later Cambrian ‘explosion' (540–520 Ma) of new, energy-sapping body plans and behaviours has proved more elusive. Here we present new molybdenum isotope data, which demonstrate that the areal extent of oxygenated bottom waters increased in step with the early Cambrian bioradiation of animals and eukaryotic phytoplankton. Modern-like oxygen levels characterized the ocean at ∼521 Ma for the first time in Earth history. This marks the first establishment of a key environmental factor in modern-like ecosystems, where animals benefit from, and also contribute to, the ‘homeostasis' of marine redox conditions.

T he delay between the origin of animals (B800 Ma, as inferred from molecular clocks) 1 and their early diversification in the Ediacaran Period (635-541 Ma) 2 has been suggested to be due to sluggish oxygenation of the Earth surface 3,4 . After the 'Great Oxidation Event' (GOE 5 ) in the early Proterozoic (B2,400-2,100 Ma), atmospheric P O 2 stayed within B0.01 and B10% of the present atmospheric level during the mid-Proterozoic (B2,100-800 Ma (refs 6,7)). It was only after the termination of the Cryogenian glaciations (B635 Ma) that oxygen levels in Earth's surface environment began to increase significantly again 8,9 . Although the lower estimate of Ediacaran atmospheric P O 2 may surpass the minimum oxygen requirement of animals (o0.1% present atmospheric level) 4 , the deeper ocean likely remained predominantly anoxic [10][11][12] . As a consequence, O 2 deficiency 13 , H 2 S toxicity 14 and a scarcity of trace metal micronutrients (such as Mo, Cu and Zn (ref. 15)) may have continued to limit the ecological distribution of eukaryotes.
Eukaryotes, especially animals and planktonic algae, only began to dominate the marine ecosystem during the 'Cambrian explosion' of biological diversity (B520 Ma) 12,16,17 . Many essential aspects of this biotic event, such as increased animal body size 18 , active locomotion, bioturbation 19,20 , carbonate biomineralization 21 , carnivory 13,22 and cropping 17,23,24 , have been linked to a rise in atmospheric oxygen beyond the minimum requirement of animals, and/or widespread ocean oxygenation. However, redox conditions in the early Cambrian oceans, especially the deep ocean, are still controversial. Some studies suggest widespread oxygenation 13,25,26 , while others propose a ferruginous (Fe 2 þ -rich) or even euxinic (H 2 S-rich) deep marine environment 10,27 . Moreover, it has been suggested that oceanic oxygen remained at levels much lower than the modern until the Devonian 28 .
Here we use sedimentary molybdenum (Mo) isotope compositions to trace the evolution of global ocean redox state over this critical period for animal evolution. Global marine redox conditions can be inferred from the sedimentary Mo record because of its redox-sensitive deposition and isotope fractionation mechanisms 9,[29][30][31][32] . In the modern oxic open oceans, Mo is present as the conservative oxyanion molybdate MoO 2 À 4 À Á at relatively high concentrations (its salinity-normalized concentration is B107 nmol kg À 1 ) 33 . The modern open-ocean seawater (OSW) Mo reservoir is enriched in heavy isotopes (modern d 98/95 Mo OSW ¼ þ 2.34%, relative to NIST-SRM-3134 (ref. 34)) relative to the dominant input from rivers (d 98/95 Mo Rivers ¼ þ 0.7% (ref. 35)). This arises because a major sink for Mo in the modern oceans is the slow adsorption on particulate manganese oxides under widespread oxic conditions, and because this process is accompanied by a À 3% isotopic fractionation D Sediment-OSW , that is, d 98/95 Mo Sediment À d 98/95 Mo OSW ¼ À 3% (refs 29,36)). When bottom-water dissolved oxygen is low or absent, H 2 S may be present in pore waters of organic-rich reducing sediments (on some continental margins) or in the water column (for example, Black Sea and regions of intense upwelling). Under these conditions, Mo deposition can be accelerated by one to two orders of magnitude 37,38 , leading to smaller isotopic fractionations D Sediment-OSW ¼ 0 to À 0.7% (ref. 39)) due to more quantitative sequestration from the dissolved phase in these settings. An expansion of such sulphidic conditions will therefore cause the d 98/95 Mo value of seawater to decrease towards the riverine input value. Seawater d 98/95 Mo will most likely be recorded in sediments deposited under euxinic conditions because of quantitative removal of aqueous Mo. However, importantly, measured d 98/95 Mo values of sediments provide a minimum constraint on contemporaneous seawater isotopic composition because all known sedimentary Mo sinks record d 98/95 Morseawater 28 . We determined Mo concentrations and isotope compositions (see Methods) of well-preserved organic-rich marine sediments of Cryogenian to early Cambrian age. We also assess local water column redox conditions using iron speciation (see Methods) in the same sediments. We find that the tempo of ocean oxygenation, as reconstructed by the Mo isotope and concentration profiles, was in step with the early Cambrian bioradiation.

Results
Geologic setting and samples. Late Neoproterozoic to Cambrian successions are well developed in South China. They are fossiliferous, with several unique biotas in various environments (Supplementary Note 1) and provide one of the best candidates for constraining the co-evolution of marine oxygenation and life 9,14,40 . We collected black shales and organic-rich cherts from shallow-to-deep water successions on the south-eastern margin of the Yangtze platform ( Supplementary Figs 1 and 2). The ages of our samples span the mid-Cryogenian to the early Cambrian (B660-520 Ma). Ediacaran and early Cambrian successions in the Yangtze Gorges area represent shallow-water facies, while deeper water facies occur in northwestern Hunan and southern Anhui provinces (see Supplementary Note 1).
Mo isotopes. Our Mo concentration and isotopic data, together with published data from the late Archaean to early Cambrian, are compiled in Supplementary Data 1 and shown in Fig. 1 and Supplementary Fig. 2. After several moderate peaks ( þ 1.5% to þ 1.7%) without a distinct increase in Mo abundance in the late Archaean (B2,500-2,750 Ma) 41

Discussion
Our data provide an estimate for the lower limit of coeval seawater Mo isotopic composition and document the fact that d 98/95 Mo OSW rose to a level higher than ever before during the early Cambrian, peaking at modern levels (B þ 2.3%) at B520 Ma. The rarity of black shales deposited under fully euxinic conditions makes a continuous record of the precise d 98/95 Mo of seawater difficult to obtain. However, where sulphidic black shale data (or phosphatic data, which have been suggested also to record seawater values 25 ) are available, their d 98/95 Mo data are lower than those at 520 Ma-that is, þ 1. Mo data from the mid-Cryogenian to the early Cambrian. In the timescale, three major glaciations are marked as snowflakes, S1 to S4 denote the first four Cambrian stages. The colour of the data points denotes local redox: sulphidic (Fe Py /Fe HR 40.7, red), ferruginous (Fe Py /Fe HR o0.7, orange), anoxic (blue, when Fe speciation data are not available, Fe/Al40.5, trace metal enrichments and other sedimentary characteristics are used to discriminate anoxic conditions) and unknown (grey, no above mentioned data are available, also included are typical carbonates and phosphates, to which Fe-S-C systematics redox proxies cannot easily be applied). The dashed lines mark the average d 98/95 Mo value of modern seawater ( þ 2.34%) and the riverine input  14 analysed the iron speciation systematics of samples obtained from a shore-to-basin transect and proposed that sulphidic zones were sandwiched between ferruginous waters on continental margins through the Ediacaran Period. Owing to geochemical similarities with Mo, U concentrations in sediments can also reflect the redox state of the global ocean 26 , although the onset of U enrichment in sediments requires less reducing conditions than that of Mo 51 . Hence, U concentrations in sediments deposited under both sulphidic and anoxic non-sulphidic conditions can indicate the size of the ocean uranium reservoir, which is proportional to the level of ocean oxygenation. Available data show that U concentrations in anoxic sediments are generally higher for the early Cambrian than for the Ediacaran, and also peaked at B520 Ma (see Supplementary Fig. 3 28)). We also note that in the modern oceans there are large areas of moderately oxic seafloor (mOx, B14% (ref. 38)), where the sulphidic zone is deep in the sediment column and the bottom-water O 2 concentration is higher than or comparable to the weakly oxic condition. In these settings Mn oxide-related Mo is quantitatively remobilized from shallow sediments and released back to seawater 37,38,54 . Such areas are important but not relevant to the mass balance model since they are neither a sink nor a source for Mo. Our modelling results (Fig. 2) suggest that Mo removal under oxygenated bottom waters (including both strongly and weakly oxic conditions) must account for more than 94% of the total Mo sink when d 98/95 Mo OSW reaches modern-like levels ( þ 2.3%), but can be as low as 33% when d 98/95 Mo OSW is þ 1.6%, which is the highest observed value for the Proterozoic. The modern-like d 98/95 Mo OSW value also requires that the strongly oxic sink accounts for more than 42% of the total oxic sink, while a d 98/95 Mo OSW value of þ 1.6% would allow weakly oxic sink to account for up to 91% of the total oxic sink.
The areal proportions of the three redox conditions are further explored through incorporating Mo accumulation rates (see Methods). There is a wide range in estimated Mo accumulation rates for modern euxinic settings (F' Eux ), from 12,000 mg m À 2 yr À 1 (ref. 32) to 4,800 mg m À 2 yr À 1 (ref. 38), and we here choose the lower rate to avoid overestimating both the difference between euxinic and oxic sinks, and the oxic area fraction. To maintain not only mass but also isotope balance of the modern oceanic Mo cycle, a F' sOx value of 40 mg m À 2 yr À 1 , which is higher than previous estimate of 27.5 mg m À 2 yr À 1 (ref. 38), is required. Our modelling results (Fig. 2)   event was broadly contemporaneous with the Cambrian bioradiation (Fig. 1). The early Cambrian bioradiation is characterized by the emergence of nearly all major bilaterian body plans 1 . This developmental/morphological complexity is a long-term consequence of eukaryotic multicellularity, which had originated by the late Mesoproterozoic (B1,200 Ma) and which diversified during the Ediacaran 12,17,55 . However, the Ediacaratype biota (B575-541 Ma) was dominated by soft-bodied sessile epibenthic osmotrophs, and there is no unambiguous evidence for motile bilaterians 56,57 , except towards the very end of the Ediacaran 58 . From the terminal Ediacaran to the earliest Cambrian (B580-529 Ma), shallow bioturbation 20 and phosphatic or aragonitic small shelly fossils characterize the initial phase of the 'Cambrian explosion' 59 . During Cambrian Stage 2 (B529-521 Ma), diverse deeper burrows and complex trace fossils record active bioturbation of sediments and mark the so-called 'substrate/agronomic revolution' 20 , while small shelly fossils further diversified and calcitic taxa began to appear 59,60 . Nearly all bilaterian body plans had appeared by early Cambrian Stage 3 (ref. 1), the major phase of the 'Cambrian explosion'.
The apparently abrupt appearance of large, motile and diverse animal forms, following a prolonged and obscure history of phylogenetic evolution, has frequently been explained by changes in the physical environment, especially redox conditions 1,13 , although this conclusion is not without controversy 4 . Our data clearly indicate a spatial waning of anoxia (including euxinic and non-sulphidic conditions) in the early Cambrian ocean, which may have been a pre-condition for the transition to a modern marine ecosystem supporting diversified animals. If anoxia were widespread, even though early animals may have inhabited patchy oxic environments, intrusion/upwelling of anoxic water would have stifled their success 14,61 . Diminished euxinia may also have relieved the Proterozoic trace metal micronutrient crisis 15 .
Our data also indicate unprecedentedly widespread oxygenation in the early Cambrian ocean. The expansion of strongly and moderately oxic seafloor from o80% during the Precambrian to 497% during the early Cambrian may have provided a significant increase in the size of habitable space for animals. Animals favour continental marginal areas that have abundant nutrients but are also prone to anoxia because of high primary productivity and the consequent high O 2 demand. When 497% of the seafloor was well oxygenated, there must have been vast space (460% of the continental margins) for animals to flourish, given that modern continental margins occupy B7% of the world's seafloor. This expansion of oxygenated seafloor took place against the backdrop of the rising sea level in the early Cambrian 62 , creating numerous new oxygenated ocean margin settings and liberating animals that possibly suffered from fluctuating redox conditions on the continental shelf during Ediacaran time 14,61 . Moreover, importantly, the expansion of oxic seafloor to a modern extent may imply near-modern average marine oxygen concentrations. Although animals, especially those of sponge-grade and meiofauna, could survive at modest oxygen levels 4,63 , the large size and ecological dominance of the characteristic Cambrian fauna could not have developed without abundant oxygen. Metabolically expensive behaviours, such as active locomotion, bioturbation and muscular carnivory, require a high oxygen consumption rate for efficient aerobic respiration 13,20 . Moreover, oxygenation would have favoured aerobic over anaerobic respiration in the deep ocean, resulting in a decrease in alkalinity and thus suppressing carbonate deposition on/in the seafloor 21 . This, consequently, would have increased surface ocean carbonate supersaturation, which may have reduced the physiological cost of carbonate skeleton construction and facilitated the evolutionary arms race 13 .
Animal ecosystem engineers may also have contributed to ocean oxygenation 12,17 . The early originating suspension-feeding sponges had low O 2 requirements 63 , and may have helped consume the large dissolved organic carbon reservoir that acted as a major redox buffer in the Proterozoic ocean 12 . Planktonic animals and algae 64 diversified nearly simultaneously in the early Cambrian, and likely enhanced the efficiency of the biological pump 12,17,65 , lowering oxygen demand in the water column. Once benthic motile animals became widespread, they deepened O 2 penetration depth through bioturbation, which helped P-retention and in turn limited primary production and stabilized marine oxygenation 12 .
In sum, a pervasively well-oxygenated ocean would have played a critical role in the 'Cambrian explosion', during which newly evolved animals and ecosystems affected both carbon and nutrient cycling, first facilitating and then stabilizing more widespread oxygenation in the world's oceans 12,17 . Through a combination of high-resolution palaeoenvironmental and palaeobiological studies, we are reaching a more comprehensive understanding of the complex, dynamic interactions and feedbacks that helped to bring modern-like ecosystems into being.

Methods
Samples. Fresh rock samples were powdered using an agate mill. Trace metal and Mo isotope analyses were carried out with an Element II ICP-MS and a Neptune MC-ICP-MS, respectively, at the University of Bristol. Samples were digested by standard HF-HNO 3 -HCl methods at 180°C for 472 h.
Mo isotope composition. Mass spectrometry and mass bias correction of Mo isotope data by the double spike technique were carried out as previously described 35 . Mo isotope data are reported using the d notation, relative to the Mo standard NIST-SRM-3134 (with its d 98/95 Mo value of þ 0.25% (refs 34,66)) where The internal precision of Mo isotopic measurements ranges between 0.02% and 0.05% (2SE of 30 integrations) for all our samples. The external reproducibility of a series of replicates (entire procedure from digestion of powder onwards) of a black shale sample is 0.02% (2SD, six replicates).
In this study only samples with Mo-enrichment factor (Mo EF , which is defined as the ratio of ( Iron speciation. We further filtered our samples to include only those deposited under anoxic conditions. We accessed the local redox conditions by iron speciation. Iron in carbonate (Fe Carb ), oxides (Fe Ox ) and magnetite (Fe Mag ) was sequentially extracted at the Newcastle University using the method described in ref. 67. Iron in pyrite (Fe Py ) is calculated from pyrite sulphur measured by the chromium reduction method, given a stoichiometry for pyrite of FeS 2 . Total organic carbon (TOC) contents were measured at the University College London using a Leco C/S analyser after acidification with 6 N HCl.
In general, under an anoxic (O 2 -free, with or without H 2 S) water column, highly reactive Fe (Fe HR ) usually constitutes more than 38% of total Fe (Fe T ) because of syngenetic formation of either iron sulphides in sulphidic waters or nonsulphidized minerals (Fe carbonate, Fe III oxides or magnetite) in ferruginous waters. A Fe Py /Fe HR ratio of 0.7 (ref. 11) is used to discriminate between ferruginous (o0.7) and sulphidic (40.7) anoxia in the water column.
Mass balance model of Mo cycle. The parameters used in the model are listed in the Supplementary Table 1. We divide the Mo sinks into a strongly oxic sink (F sOx , bottom-water dissolved oxygen 410 mM, where O 2 penetrates 41 cm below the sediment-water interface), a weakly oxic sink (F wOx , bottom-water dissolved oxygen o10 mM, with a shallow sulphidic zone in the reducing sediments) and a euxinic sink (F Eux , H 2 S is present in bottom water) 37 greater than 11 mM, an average fractionation of À 0.5% (D Eux À OSW ) is used for the euxinic sink 28 . The Mo isotope composition of open ocean seawater (d 98/95 Mo OSW ) is determined by the fractions of the fluxes to sediments in the three redox settings (f Eux , f wOx and f sOx ) with the assumption of steady-state, and setting the input to the ocean at the modern value of the riverine flux: Isotope mass balance is described by: where each d Output equals (d 98/95 Mo OSW þ D Output ). We define: and solve the above three equations to get the d 98/95 Mo OSW as a function of k sOx and f Eux , and then plot the contours of d 98/95 Mo OSW in Fig. 2. The Mo output rates (F' Output , g m À 2 yr À 1 ) in various redox settings are assumed to be controlled by first-order kinetics with respect to the coeval Mo reservoir in the open ocean (R), that is, each F' Output ¼ F' Output0 Â R/R 0 (subscript 0 denotes the modern value). Replacing each f Output ( ¼ F Output /F Rivers ) in the isotope mass balance equation (3) with: together with the mass balance equation (2), we can get the d 98/95 Mo OSW as a function of areal fractions of the three redox settings f A Eux , f A wOx and f A sOx . In our sensitivity analyses (see Supplementary Fig. 4) we first investigated the impact of setting both D Eux À OSW and d Rivers to zero. This results in even smaller areas of euxinia and greater predominance of oxic bottom waters for modern-like d 98/95 Mo OSW , including a major contribution of strongly oxic conditions.
In modern weakly oxic settings, although the uppermost few centimetres of sediments show heterogeneous D Sediment-OSW ranging from À 1.2% to À 0.2%, the D Sediment-OSW of deep sediments converge on a common value of À 0.7%, which more likely represents the true average D wOx-OSW over long timescales 39 . However, we further investigated the impact of uncertainty in D wOx-OSW . A smaller fractionation, such as D wOx-OSW of À 0.2%, results in a similar situation to the previous sensitivity analyses, that is, even greater contributions are required from oxic sinks for modern-like d 98/95 Mo OSW . Although D wOx-OSW of À 1.2% would allow smaller contributions from oxic sinks, modern-like d 98/95 Mo OSW still requires/reflects a predominance of oxic sinks.