Exploring the influence of atmospheric CO2 and O2 levels on the utility of nitrogen isotopes as proxy for biological N2 fixation.

Biological N2 fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ15N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ15N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric pO2 was lower and pCO2 was higher. Here, we explore δ15N signatures under three atmospheres with (i) elevated CO2 and no O2 (Archean), (ii) present-day CO2, and O2 and (iii) future elevated CO2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ15N and the associated fractionation factor epsilon (ε = δ15Nbiomass - δ15NN2) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ε ranges between 3.70‰ and -4.96‰ with a mean ε value of -1.38 ± 0.95‰, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set revealed correlations of isotopic fractionation of BNF with CO2 concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of ε to species type, C/N ratios and toxin production in cyanobacteria, albeit it within a small range (-1.44 ± 0.89‰). We therefore conclude that δ15N is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ε) observed in the toxin-producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation.IMPORTANCENitrogen is an essential element of life on Earth; however, despite its abundance, it is not biologically accessible. Biological nitrogen fixation is an essential process whereby microbes fix N2 into biologically usable NH3. During this process, the enzyme nitrogenase preferentially uses light 14N, resulting in 15N depleted biomass. This signature can be traced back in time in sediments on Earth, and possibly other planets. In this paper, we explore the influence of pO2 and pCO2 on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO2, light, and toxin-producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin-producing Nodularia and Nostoc species that offer insight into why some organisms produce these N-rich toxic secondary metabolites.

Dating Ammonia-Oxidizing Bacteria with Abundant Eukaryotic Fossils.

Evolution of a complete nitrogen (N) cycle relies on the onset of ammonia oxidation, which aerobically converts ammonia to nitrogen oxides. However, accurate estimation of the antiquity of ammonia-oxidizing bacteria (AOB) remains challenging because AOB-specific fossils are absent and bacterial fossils amenable to calibrate molecular clocks are rare. Leveraging the ancient endosymbiosis of mitochondria and plastid, as well as using state-of-the-art Bayesian sequential dating approach, we obtained a timeline of AOB evolution calibrated largely by eukaryotic fossils. We show that the first AOB evolved in marine Gammaproteobacteria (Gamma-AOB) and emerged between 2.1 and 1.9 billion years ago (Ga), thus postdating the Great Oxidation Event (GOE; 2.4 to 2.32 Ga). To reconcile the sedimentary N isotopic signatures of ammonia oxidation occurring near the GOE, we propose that ammonia oxidation likely occurred at the common ancestor of Gamma-AOB and Gammaproteobacterial methanotrophs, or the actinobacterial/verrucomicrobial methanotrophs which are known to have ammonia oxidation activities. It is also likely that nitrite was transported from the terrestrial habitats where ammonia oxidation by archaea took place. Further, we show that the Gamma-AOB predated the anaerobic ammonia-oxidizing (anammox) bacteria, implying that the emergence of anammox was constrained by the availability of dedicated ammonia oxidizers which produce nitrite to fuel anammox. Our work supports a new hypothesis that N redox cycle involving nitrogen oxides evolved rather late in the ocean.

Co-evolution of early Earth environments and microbial life.

Two records of Earth history capture the evolution of life and its co-evolving ecosystems with interpretable fidelity: the geobiological and geochemical traces preserved in rocks and the evolutionary histories captured within genomes. The earliest vestiges of life are recognized mostly in isotopic fingerprints of specific microbial metabolisms, whereas fossils and organic biomarkers become important later. Molecular biology provides lineages that can be overlayed on geologic and geochemical records of evolving life. All these data lie within a framework of biospheric evolution that is primarily characterized by the transition from an oxygen-poor to an oxygen-rich world. In this Review, we explore the history of microbial life on Earth and the degree to which it shaped, and was shaped by, fundamental transitions in the chemical properties of the oceans, continents and atmosphere. We examine the diversity and evolution of early metabolic processes, their couplings with biogeochemical cycles and their links to the oxygenation of the early biosphere. We discuss the distinction between the beginnings of metabolisms and their subsequent proliferation and their capacity to shape surface environments on a planetary scale. The evolution of microbial life and its ecological impacts directly mirror the Earth's chemical and physical evolution through cause-and-effect relationships.

Timing the evolution of phosphorus-cycling enzymes through geological time using phylogenomics.

Phosphorus plays a crucial role in controlling biological productivity, but geological estimates of phosphate concentrations in the Precambrian ocean, during life's origin and early evolution, vary over several orders of magnitude. While reduced phosphorus species may have served as alternative substrates to phosphate, their bioavailability on the early Earth remains unknown. Here, we reconstruct the phylogenomic record of life on Earth and find that phosphate transporting genes (pnas) evolved in the Paleoarchean (ca. 3.6-3.2 Ga) and are consistent with phosphate concentrations above modern levels ( > 3 µM). The first gene optimized for low phosphate levels (pstS; <1 µM) appeared around the same time or in the Mesoarchean depending on the reconstruction method. Most enzymatic pathways for metabolising reduced phosphorus emerged and expanded across the tree of life later. This includes phosphonate-catabolising CP-lyases, phosphite-oxidising pathways and hypophosphite-oxidising pathways. CP-lyases are particularly abundant in dissolved phosphate concentrations below 0.1 µM. Our results thus indicate at least local regions of declining phosphate levels through the Archean, possibly linked to phosphate-scavenging Fe(III), which may have limited productivity. However, reduced phosphorus species did not become widely used until after the Paleoproterozoic Great Oxidation Event (2.3 Ga), possibly linked to expansion of the biosphere at that time.

Evaluating the multiple sulfur isotope signature of Eoarchean rocks from the Isua Supracrustal Belt (Southwest-Greenland) by MC-ICP-MS: Volcanic nutrient sources for early life.

On the anoxic Archean Earth, prior to the onset of oxidative weathering, electron acceptors were relatively scarce, perhaps limiting microbial productivity. An important metabolite may have been sulfate produced during the photolysis of volcanogenic SO2 gas. Multiple sulfur isotope data can be used to track this sulfur source, and indeed this record indicates SO2 photolysis dating back to at least 3.7 Ga, that is, as far back as proposed evidence of life on Earth. However, measurements of multiple sulfur isotopes in some key strata from that time can be challenging due to low sulfur concentrations. Some studies have overcome this challenge with NanoSIMS or optimized gas-source mass spectrometry techniques, but those instruments are not readily accessible. Here, we applied an aqua regia leaching protocol to extract small amounts of sulfur from whole rocks for analyses of multiple sulfur isotopes by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). Measurements of standards and replicates demonstrate good precision and accuracy. We applied this technique to meta-sedimentary rocks with putative biosignatures from the Eoarchean Isua Supracrustal Belt (ISB, >3.7 Ga) and found positive ∆33S (1.40-1.80‰) in four meta-turbidites and negative ∆33S (-0.80‰ and -0.66‰) in two meta-carbonates. Two meta-basalts do not display significant mass-independent fractionation (MIF, -0.01‰ and 0.16‰). In situ Re-Os dating on a molybdenite vein hosted in the meta-turbidites identifies an early ca. 3.7 Ga hydrothermal phase, and in situ Rb-Sr dating of micas in the meta-carbonates suggests metamorphism affected the rocks at ca. 2.2 and 1.7 Ga. We discuss alteration mechanisms and conclude that there is most likely a primary MIF-bearing phase in these meta-sediments. Our new method is therefore a useful addition to the geochemical toolbox, and it confirms that organisms at that time, if present, may indeed have been fed by volcanic nutrients.

Nitrogen isotopes reveal independent origins of N2-fixing symbiosis in extant cycad lineages.

Cycads are ancient seed plants (gymnosperms) that emerged by the early Permian. Although they were common understory flora and food for dinosaurs in the Mesozoic, their abundance declined markedly in the Cenozoic. Extant cycads persist in restricted populations in tropical and subtropical habitats and, with their conserved morphology, are often called 'living fossils.' All surviving taxa receive nitrogen from symbiotic N2-fixing cyanobacteria living in modified roots, suggesting an ancestral origin of this symbiosis. However, such an ancient acquisition is discordant with the abundance of cycads in Mesozoic fossil assemblages, as modern N2-fixing symbioses typically occur only in nutrient-poor habitats where advantageous for survival. Here, we use foliar nitrogen isotope ratios-a proxy for N2 fixation in modern plants-to probe the antiquity of the cycad-cyanobacterial symbiosis. We find that fossilized cycad leaves from two Cenozoic representatives of extant genera have nitrogen isotopic compositions consistent with microbial N2 fixation. In contrast, all extinct cycad genera have nitrogen isotope ratios that are indistinguishable from co-existing non-cycad plants and generally inconsistent with microbial N2 fixation, pointing to nitrogen assimilation from soils and not through symbiosis. This pattern indicates that, rather than being ancestral within cycads, N2-fixing symbiosis arose independently in the lineages leading to living cycads during or after the Jurassic. The preferential survival of these lineages may therefore reflect the effects of competition with angiosperms and Cenozoic climatic change.

On-line chloride removal from ion chromatography for trace-level analyses of phosphite and other anions by coupled ion chromatography-inductively coupled plasma mass spectrometry.

RATIONALE: Ion chromatography (IC) combined with inductively coupled plasma mass spectrometry (ICPMS) is an ideal tool for measuring low concentrations of anionic species such as phosphite; however, the high concentration of chloride and other anions in natural solutions may negatively impact chromatographic separation and data quality. METHOD: We developed an on-line mechanism of removing chloride from a sample within an ion chromatograph, using an additional valve and a separation column that transfers chloride to waste while phosphite and most other anions are retained. We installed this system in a coupled IC/ICPMS system (ICS6000 and Element 2 in medium-resolution mode) and determined linearity and detection limits. In addition, we measured phosphorus species by NMR for comparison as an alternative method for phosphite determination. RESULTS: Chloride was fully removed from the samples while phosphite was retained and could be analysed by IC/ICPMS. Concentrations could be measured down to 0.003 µmol/L and possibly less with good linearity over the explored range (up to 1.615 µmol/L; r2  = 0.999). In contrast, the detection limit by NMR was 6.46 µmol/L. CONCLUSIONS: The on-line removal mechanism works well for simplifying sample matrices. It removes the need for costly pre-analytical sample treatment with OnGuard columns. We confirm that IC/ICPMS is the most powerful technique for quantifying phosphite in natural solutions. The new chloride-removal method may also be applicable to analyses of other anions.

Are Large Sulfur Isotope Variations Biosignatures in an Ancient, Impact-Induced Hydrothermal Mars Analog?

Discrepancies have emerged concerning the application of sulfur stable isotope ratios as a biosignature in impact crater paleolakes. The first in situ δ34S data from Mars at Gale crater display a ∼75‰ range that has been attributed to an abiotic mechanism. Yet biogeochemical studies of ancient environments on Earth generally interpret δ34S fractionations >21‰ as indicative of a biological origin, and studies of δ34S at analog impact crater lakes on Earth have followed the same approach. We performed analyses (including δ34S, total organic carbon wt%, and scanning electron microscope imaging) on multiple lithologies from the Nördlinger Ries impact crater, focusing on hydrothermally altered impact breccias and associated sedimentary lake-fill sequences to determine whether the δ34S properties define a biosignature. The differences in δ34S between the host lithologies may have resulted from thermochemical sulfate reduction, microbial sulfate reduction, hydrothermal equilibrium fractionation, or any combination thereof. Despite abundant samples and instrumental precision currently exclusive to Earth-bound analyses, assertions of biogenicity from δ34S variations >21‰ at the Miocene Ries impact crater are tenuous. This discourages the use of δ34S as a biosignature in similar environments without independent checks that include the full geologic, biogeochemical, and textural context, as well as a comprehensive acknowledgment of alternative hypotheses.

Exploring the Effects of Residence Time on the Utility of Stable Isotopes and S/C Ratios as Proxies for Ocean Connectivity.

Various geochemical proxies have been developed to determine if ancient sedimentary strata were deposited in marine or nonmarine environments. A critical parameter for proxy reliability is the residence time of aqueous species in seawater, which is rarely considered for proxies relying on stable isotopes and elemental abundance ratios. Differences in residence time may affect our ability to track geologically short-lived alternations between marine and nonmarine conditions. To test this effect for sulfur and nitrogen isotopes and sulfur/carbon ratios, we investigated a stratigraphic section in the Miocene Oberpullendorf Basin in Austria. Here, previous work revealed typical seawater-like rare earth element and yttrium (REY) systematics transitioning to nonmarine-like systematics. This shift was interpreted as a brief transition from an open marine depositional setting to a restricted embayment with a reduced level of exchange with the open ocean and possibly freshwater influence. Our isotopic results show no discernible response in carbonate-associated sulfate sulfur isotopes and carbon/sulfur abundance ratios during the interval of marine restriction inferred from the REY data, but nitrogen isotopes show a decrease by several permil. This observation is consistent with the much longer residence time of sulfate in seawater compared with REY and nitrate. Hence, this case study illustrates that the residence time is a key factor for the utility of seawater proxies. In some cases, it may make geochemical parameters more sensitive to marine water influx than paleontological observations, as in the Oberpullendorf Basin. Particular care is warranted in deep time, when marine residence times likely differ markedly from the modern.

Technical comment on "Reexamination of 2.5-Ga 'whiff' of oxygen interval points to anoxic ocean before GOE".

Many lines of inorganic geochemical evidence suggest transient "whiffs" of environmental oxygenation before the Great Oxidation Event (GOE). Slotznick et al. assert that analyses of paleoredox proxies in the Mount McRae Shale, Western Australia, were misinterpreted and hence that environmental O2 levels were persistently negligible before the GOE. We find these arguments logically flawed and factually incomplete.

Mid-latitudinal habitable environment for marine eukaryotes during the waning stage of the Marinoan snowball glaciation.

During the Marinoan Ice Age (ca. 654-635 Ma), one of the 'Snowball Earth' events in the Cryogenian Period, continental icesheets reached the tropical oceans. Oceanic refugia must have existed for aerobic marine eukaryotes to survive this event, as evidenced by benthic phototrophic macroalgae of the Songluo Biota preserved in black shales interbedded with glacial diamictites of the late Cryogenian Nantuo Formation in South China. However, the environmental conditions that allowed these organisms to thrive are poorly known. Here, we report carbon-nitrogen-iron geochemical data from the fossiliferous black shales and adjacent diamictites of the Nantuo Formation. Iron-speciation data document dysoxic-anoxic conditions in bottom waters, whereas nitrogen isotopes record aerobic nitrogen cycling perhaps in surface waters. These findings indicate that habitable open-ocean conditions were more extensive than previously thought, extending into mid-latitude coastal oceans and providing refugia for eukaryotic organisms during the waning stage of the Marinoan Ice Age.

Hydrothermal Regeneration of Ammonium as a Basin-Scale Driver of Primary Productivity.

Hydrothermal vents are important targets in the search for life on other planets due to their potential to generate key catalytic surfaces and organic compounds for biogenesis. Less well studied, however, is the role of hydrothermal circulation in maintaining a biosphere beyond its origin. In this study, we explored this question with analyses of organic carbon, nitrogen abundances, and isotopic ratios from the Paleoproterozoic Zaonega Formation (2.0 Ga), NW Russia, which is composed of interbedded sedimentary and mafic igneous rocks. Previous studies have documented mobilization of hydrocarbons, likely associated with magmatic intrusions into unconsolidated sediments. The igneous bodies are extensively hydrothermally altered. Our data reveal strong nitrogen enrichments of up to 0.6 wt % in these altered igneous rocks, suggesting that the hydrothermal fluids carried ammonium concentrations in the millimolar range, which is consistent with some modern hydrothermal vents. Furthermore, large isotopic offsets of ∼10‰ between organic-bound and silicate-bound nitrogen are most parsimoniously explained by partial biological uptake of ammonium from the vent fluid. Our results, therefore, show that hydrothermal activity in ancient marine basins could provide a locally high flux of recycled nitrogen. Hydrothermal nutrient recycling may thus be an important mechanism for maintaining a large biosphere on anoxic worlds.

Phylogenomic Evidence for the Origin of Obligate Anaerobic Anammox Bacteria Around the Great Oxidation Event.

The anaerobic ammonium oxidation (anammox) bacteria can transform ammonium and nitrite to dinitrogen gas, and this obligate anaerobic process accounts for up to half of the global nitrogen loss in surface environments. Yet its origin and evolution, which may give important insights into the biogeochemistry of early Earth, remain enigmatic. Here, we performed a comprehensive phylogenomic and molecular clock analysis of anammox bacteria within the phylum Planctomycetes. After accommodating the uncertainties and factors influencing time estimates, which include implementing both a traditional cyanobacteria-based and a recently developed mitochondria-based molecular dating approach, we estimated a consistent origin of anammox bacteria at early Proterozoic and most likely around the so-called Great Oxidation Event (GOE; 2.32-2.5 Ga) which fundamentally changed global biogeochemical cycles. We further showed that during the origin of anammox bacteria, genes involved in oxidative stress adaptation, bioenergetics, and anammox granules formation were recruited, which might have contributed to their survival on an increasingly oxic Earth. Our findings suggest the rising levels of atmospheric oxygen, which made nitrite increasingly available, was a potential driving force for the emergence of anammox bacteria. This is one of the first studies that link the GOE to the evolution of obligate anaerobic bacteria.

Diagenetic nutrient supplies to the Proterozoic biosphere archived in divergent nitrogen isotopic ratios between kerogen and silicate minerals.

Nitrogen isotopes and abundances in sedimentary rocks have become an important tool for reconstructing biogeochemical cycles in ancient ecosystems. There are two archives of nitrogen in the rock record, namely kerogen-bound amines and silicate-bound ammonium, and it is well documented that the isotopic ratios of these two archives can be offset from one another. This offset has been observed to increase with metamorphic grade, suggesting that it may be related to the bonding environment in differing nitrogen host phases and associated equilibrium isotope fractionation. However, theoretical bounds for this effect have not been established, and it remains possible that some isotopic offsets predate metamorphism. In support of this hypothesis, we report an unexpectedly large isotopic offset of 4-5‰ in siltstones of very low metamorphic grade from the late Mesoproterozoic Diabaig Formation in NW Scotland (1.0 Ga). Carbon to nitrogen ratios of bulk rocks are 2-3 times lower than in other Mesoproterozoic sections. The rocks also contain early-formed phosphate concretions and display wrinkled surfaces on bedding planes, indicative of fossilised microbial mats. Collectively, these data are most parsimoniously interpreted as evidence of diagenetic ammonium release from microbial mats into porewaters, followed by partial oxidation to nitrite or nitrate at the sediment-water interface. This process would render residual ammonium in clays isotopically heavy, while the resulting nitrite or nitrate would be relatively lighter and captured in new biomass, leading to the observed isotopic divergence. The same diagenetic degradation pathway likely also liberated phosphate that was trapped within concretions. Diagenetic release of nutrients is known to occur in modern settings, and our data suggest that nitrogen isotopes may be a way to track this local sedimentary nutrient source in past environments. Lastly, we speculate that diagenetic nutrient recycling within Proterozoic microbial mats may have created a favourable niche for eukaryotic organisms in shallow waters.

Contrasting nutrient availability between marine and brackish waters in the late Mesoproterozoic: Evidence from the Paranoá Group, Brazil.

Understanding the delayed rise of eukaryotic life on Earth is one of the most fundamental questions about biological evolution. Numerous studies have presented evidence for oxygen and nutrient limitations in seawater during the Mesoproterozoic era, indicating that open marine settings may not have been able to sustain a eukaryotic biosphere with complex, multicellular organisms. However, many of these data sets represent restricted marine basins, which may bias our view of habitability. Furthermore, it remains untested whether rivers could have supplied significant nutrient fluxes to coastal habitats. To better characterize the sources of the major nutrients nitrogen and phosphorus, we turned to the late Mesoproterozoic Paranoá Group in Brazil (~1.1 Ga), which was deposited on a passive margin of the São Francisco craton. We present carbon, nitrogen and sulphur isotope data from an open shelf setting (Fazenda Funil) and from a brackish-water environment with significant riverine input (São Gabriel). Our results show that waters were well-oxygenated and nitrate was bioavailable in the open ocean setting at Fazenda Funil; the redoxcline appears to have been deeper and further offshore compared to restricted marine basins elsewhere in the Mesoproterozoic. In contrast, the brackish site at São Gabriel received only limited input of marine nitrate and sulphate. Nevertheless, previous reports of acritarchs reveal that this brackish-water setting was habitable to eukaryotic life. Paired with previously published cadmium isotope data, which can be used as a proxy for phosphorus cycling, our results suggest that complex organisms were perhaps not strictly dependent on marine nutrient supplies. Riverine influxes of P and possibly other nutrients likely rendered coastal waters perhaps equally habitable to the Mesoproterozoic open ocean. This conclusion supports the notion that eukaryotic organisms may have thrived in brackish or perhaps even freshwater environments.

Mercury abundance and isotopic composition indicate subaerial volcanism prior to the end-Archean "whiff" of oxygen.

Earth's early atmosphere witnessed multiple transient episodes of oxygenation before the Great Oxidation Event 2.4 billion years ago (Ga) [e.g., A. D. Anbar et al., Science 317, 1903-1906 (2007); M. C. Koehler, R. Buick, M. E. Barley, Precambrian Res. 320, 281-290 (2019)], but the triggers for these short-lived events are so far unknown. Here, we use mercury (Hg) abundance and stable isotope composition to investigate atmospheric evolution and its driving mechanisms across the well-studied "whiff" of O2 recorded in the ∼2.5-Ga Mt. McRae Shale from the Pilbara Craton in Western Australia [A. D. Anbar et al., Science 317, 1903-1906 (2007)]. Our data from the oxygenated interval show strong Hg enrichment paired with slightly negative ∆199Hg and near-zero ∆200Hg, suggestive of increased oxidative weathering. In contrast, slightly older beds, which were evidently deposited under an anoxic atmosphere in ferruginous waters [C. T. Reinhard, R. Raiswell, C. Scott, A. D. Anbar, T. W. Lyons, Science 326, 713-716 (2009)], show Hg enrichment coupled with positive ∆199Hg and slightly negative ∆200Hg values. This pattern is consistent with photochemical reactions associated with subaerial volcanism under intense UV radiation. Our results therefore suggest that the whiff of O2 was preceded by subaerial volcanism. The transient interval of O2 accumulation may thus have been triggered by diminished volcanic O2 sinks, followed by enhanced nutrient supply to the ocean from weathering of volcanic rocks causing increased biological productivity.

A preliminary study into the use of tree-ring and foliar geochemistry as bio-indicators for vehicular NOx pollution in Malta.

Emissions from traffic over the past few decades have become a significant source of air pollution. Among the pollutants emitted are nitrogen oxides (NOx), exposure to which can be detrimental to public health. Recent studies have shown that nitrogen (N) stable isotope ratios in tree-rings and foliage express a fingerprint of their major N source, making them appropriate for bio-monitoring purposes. In this study, we have applied this proxy to Aleppo pines (Pinus halepensis) at three distances from one of the busiest roads in Malta, a country known to suffer from intense traffic pollution. Our results showed that N and organic carbon (C) stable isotope ratios in tree-rings do not vary over the period 1980-2018 at any of the investigated sites; however, statistically significant spatial trends were apparent in both tree-rings and foliage. The roadside and transitional sites exhibited more positive δ15N and more negative δ13C values compared to those at a rural control site. This is likely due to the incorporation of 15N-enriched NOx and 13C-depleted CO2 from traffic pollution. Sampled top-soil also exhibited the δ15N trend. Our results constitute the first known application of dendrogeochemistry to atmospheric pollution monitoring in Malta.

Using modern low-oxygen marine ecosystems to understand the nitrogen cycle of the Paleo- and Mesoproterozoic oceans.

During the productive Paleoproterozoic (2.4-1.8 Ga) and less productive Mesoproterozoic (1.8-1.0 Ga), the ocean was suboxic to anoxic and multicellular organisms had not yet evolved. Here, we link geologic information about the Proterozoic ocean to microbial processes in modern low-oxygen systems. High iron concentrations and rates of Fe cycling in the Proterozoic are the largest differences from modern oxygen-deficient zones. In anoxic waters, which composed most of the Paleoproterozoic and ~40% of the Mesoproterozoic ocean, nitrogen cycling dominated. Rates of N2 production by denitrification and anammox were likely linked to sinking organic matter fluxes and in situ primary productivity under anoxic conditions. Additionally autotrophic denitrifiers could have used reduced iron or methane. 50% of the Mesoproterozoic ocean may have been suboxic, promoting nitrification and metal oxidation in the suboxic water and N2 O and N2 production by partial and complete denitrification in anoxic zones in organic aggregates. Sulfidic conditions may have composed ~10% of the Mesoproterozoic ocean focused along continental margins. Due to low nitrate concentrations in offshore regions, anammox bacteria likely dominated N2 production immediately above sulfidic zones, but in coastal regions, higher nitrate concentrations probably promoted complete S-oxidizing autotrophic denitrification at the sulfide interface.

A new constraint on the antiquity of ancient haloalkaliphilic green algae that flourished in a ca. 300 Ma Paleozoic lake.

It is established that green algae and land plants progressively colonized freshwater and terrestrial habitats throughout the Paleozoic Era, but little is known about the ecology of Paleozoic saline lakes. Here, we report lipid biomarker and petrographic evidence for the occurrence of a green alga as a major primary producer in a late Paleozoic alkaline lake (Fengcheng Formation; 309-292 Ma). A persistently saline and alkaline lacustrine setting is supported by mineralogical and lipid biomarker evidence alongside extremely enriched δ15 Nbulk values (+16 to +24‰) for the lake depocenter. The prominence of C28 and C29 steroids, co-occurring with abundant carotene-derived accessory pigment markers in these ancient rocks, is suggestive of prolific primary production and elevated source inputs from haloalkaliphilic green algae. The high C28 /C29 -sterane ratios (0.78-1.29) are significantly higher than the typical marine value reported for late Paleozoic rocks (<0.5) and thus are associated with certain groups of chlorophytes. Adaptation to such extreme lacustrine environments, aided by enhanced biosynthesis of certain cell membrane lipids, likely played an important role in the evolution and physiological development of ancient green algae.