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Lombardo et al. argue that, if our hypothesis is correct, ADEs should be continuous rather than patchy. However, alluvium deposition can be a patchy process and the distribution of large and small ADE patches can be predicted regionally based on fluvial geomorphology. For example, 89% of all known ADEs have been predictively mapped using elevation, distance to bluff, and geological provenance as the key predictors (with a false negative rate of 6.5% and a false positive rate of 4.7%)10. Predicted areas include small and large ADE patches, up to several square kilometres in size, and indicate that ADEs cover ~154,000 km2 mostly in central and western Amazonia. This may seem to be a very large area ( >3% of the Amazon basin) but it is only a fraction of the projections found in some of the most cited anthropogenic theory literature11. Assuming the same excess fertility observed at our site, the creation of those ADEs would have required a prohibitive amount of biomass burning, in areas 800–1680 times larger (Fig. 1), which is inconsistent with the centralised small-scale deposition proposed by Lombardo et al. In this regional scenario, it remains unclear how many Amazons would have been needed to build the already-mapped ADEs.Lombardo et al. centre their opinion on settlements in other parts of the Amazon basin, under different socioecological and geomorphological contexts, and where the data we have developed are not available for comparison. Their narrative conflates the Brazilian lowland with other regions, such as the Llanos de Moxos and other systems in the Bolivian-Peruvian foreland basins, where older archeological sites occur. Their comments about the mineral composition of ADEs appear to contradict recent discoveries (made by some of their co-authors)12 which show that some oxides found at our ADE site bear “no relationship to anthropogenic activity” because “their sources are attributed to the weathering of micas, feldspars, mafic minerals (pyroxene), and sodic plagioclase” that are not found locally. To explain the inconsistency between those findings and the current theory of ADE formation, Macedo et al. argue that “sediment depositions in floodplain soils” that “are not related to human occupation” should be considered. That suggestion is consistent with our data which indicate deposition of exogenous materials to the site prior to the invention of agriculture in central Amazonia.Our study area is on a Tertiary terrace, and we acknowledge in our paper that it lies above the modern 100-year flood height for Manaus. However, significant Pleistocene and Holocene tectonic activity and river aggradation/degradation demonstrably affected the flood height over time. A complex neotectonic history has affected terrace elevations, nutrient deposition, and remobilisation, as well as flood heights and aggradation, resulting in higher base levels that were many metres above flood waters today in past millennia13,14,15. In addition, rivers transported and dispersed sediments from the Andes to the lowland, which were re-mobilised, and re-deposited in patchy patterns, from floodplains several times between 20 and 5 thousand years ago16,17,18. Such mineral inputs by past avulsion events may have occurred earlier in the Quaternary and remain as a relict soil where it has not subsequently eroded19. The older weathered sediments on the upper terraces lining the river look nothing like recent alluvium and the distribution of elements and their assemblages at our site are consistent with alluvial deposits in other sites. This process is explained in studies cited by Lombardo et al. (e.g., Pupim et al.), which note several periods of river aggradation, that support our hypothesis.As explained in our original paper, our data do not preclude a more recent human effect on the local landscape. The wisdom of indigenous populations, manifested in the application of waste materials to agricultural sites (since at least the late Holocene), may have further enriched ADEs or countered their natural degradation. Recent studies12, 16, 17, which post-date the studies that Lombardo et al. cite to argue against a geogenic influence, reveal a dynamic neotectonic history and support our hypothesis. Thus, the extent to which other ADE sites originated from depositional processes should be investigated based on evidence that goes beyond those presented by Lombardo et al. More

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The biotic induction of OAE-2The rapid proliferation of select microbial communities at 427.54 mcd likely represents a pre-OAE biotic perturbation (pre-OAE BP) presaging the protracted period of widespread marine deoxygenation during OAE-2, and progressive deoxygenation predating the +CIE7 (Fig. 4). At the beginning of the pre-OAE BP (427.54 mcd), abruptly elevated tetrapyrroles and crenarchaeol concentrations signify an abrupt increase in primary production by photoautotrophs and chemoautotrophs residing above the chemocline. Increased volumes of precipitating biogenic snow concordantly consumed oxygen, expanding the preexisting OMZ as anaerobic bacteria thrived based on accelerated obGDGTs synthesis. Euxinia did not penetrate the photic zone at the outset of the productivity bloom as isorenieratane was not detected and heightened rates of microbial sulfate reduction were seemingly transient, inferred from the DAGEs profile, and limited to pre-OAE BP initiation. The lack of a well-stratified water column, evinced by absent to low concentrations of halophilic archaeal lipids (i.e., extended archaeols), relatively low rates of microbial sulfate reduction, and a dense oxygenic microbial plate likely precluded the development of PZE initially.Establishing a definitive causal mechanism for the pre-OAE BP is difficult, but the concomitance of LIP activity with the productivity spike is intriguing. Application of a linear sedimentation rate from OAE-2 to the pre-OAE BP interval following previous works6,7 approximated the pre-OAE BP occurring 220 ± 4 kyr before OAE-2, lasting for ~100 kyr (427.54–426.88 mcd; see Estimating the duration of the pre-OAE BP in Supplementary Information for rationale and calculation). Significantly, this was roughly coincident with the onset of LIP activity (~200–300 kyr before OAE-2) inferred from marine osmium isotope stratigraphy27. Similarities in the modern planktonic community response, such as elevated productivity and compositional changes, between the 2018 Kilauea eruption28 and the pre-OAE BP reinforce inference of a potential magmatic trigger for this event (see Evidence for LIP trigger of the pre-OAE biotic perturbation in Supplementary Information for additional details).A constant, yet overall lower, nutrient and trace metal inventory6 (Fig. S4) combined with a redox-driven shift in fixed N species (from NO3− to NH4+)15, potentially leading to a fixed N shortage29 via intensified denitrification and annamox reactions30, were probable culprits in the failure to sustain prolific rates of primary production beyond 100 kyr at the Demerara Rise. The gradual decline in biomass production, indicated by decreasing tetrapyrrole and crenarchaeol profiles (Fig. 4), was accompanied by a notable shift in deep water communities. Sulfate-reducing bacteria exerted increasing predominance over methanogenic archaea, a trend coeval with the primary productivity spike and extending well into the OAE (Fig. 3). A collapse of autotrophic communities to pre-perturbation levels was concordant with the progressive shoaling of H2S-laden waters. Continued vertical migration of the chemocline intruded the photic zone, producing PZE that enabled anoxygenic photosynthesis by Chlorobiaceae (Fig. 4). Unlike the overall oscillatory character of PZE throughout the studied section, this protracted phase of PZE was sustained until the onset of OAE-2 (426.43–426.00 mcd, Figs. 3 and 4) and is approximately contemporaneous with a thallium (Tl) isotope excursion7 (426.40–426.30 mcd).The positive Tl isotope excursion represents the progressive expansion of bottom water anoxia predating OAE-2 by 43 ± 11 kyr6,7. However, evidence for a causal mechanism of pre-OAE deoxygenation remains indeterminate. Our comprehensive biomarker inventory provides an interpreted sequence of events culminating in the regional to global expansion of anoxia predating OAE-2. A protracted phase of enhanced primary productivity began ~220 ± 4 kyr prior to OAE-2, increasing localized production and export of organic carbon at Demerara Rise. Similar productivity spikes likely occurred in settings of comparable paleogeographic configuration (e.g., equatorial, continental margins/shelves), seeding the oceans with fixed carbon. Continued scavenging of marine oxygen via organic carbon remineralization resulted in OMZ expansion locally, and likely initiated oxygen drawdown in much of the proto-North Atlantic Ocean. Stratigraphic records of sulfur isotopes of pyrite (δ34Spyrite) from the proto-North Atlantic and Tethys Oceans11 validate the areal extrapolation of our interpretations. A gradual decline in δ34Spyrite values at Demerara Rise begins at 427.50 mcd, nearly identical to the onset of the pre-OAE BP (427.54 mcd, Fig. 4). Correlation of δ34Spyrite in a global transect (Western Interior Seaway, proto-North Atlantic, Tethys) revealed consistent behavior in δ34Spyrite prior to the +CIE, indicating increasingly expansive marine deoxygenation on a global scale11. Over ~100 kyr, increased regional biomass production induced pervasive marine anoxia, inhibiting Mn-oxide formation, producing the observed positive Tl isotope excursion, and ultimately, the globally observed +CIE reflecting enhanced organic carbon burial signaling the onset of OAE-2. Thus, the local biotic signal recorded at ODP Site 1258 underlines the crucial role the Demerara Rise, and similar undocumented settings, served in initiating deoxygenation of the global ocean.Microbial ecological dynamics during and after OAE-2Changes in microbial community compositions during OAE-2 were apparent, signified by a shift in the normalized total biomarker pool (Fig. 3) and variations in the absolute concentrations of individual biomarkers (Fig. 4). In general, OAE-2 was defined by an expansion and diversification of intermediate and deep water communities (426.00–423.07 mcd), followed by a period of instability leading to the termination of the OAE (423.07–422.00 mcd). Photo- and chemoautotrophs residing above the chemocline were adversely affected, evinced by relatively low, invariant tetrapyrrole and crenarchaeol profiles (Fig. 4). Based on these observations, we divided OAE-2 into two periods defined by contrasting paleoenvironmental conditions modulating the microbial inhabitants of Demerara Rise.The first period of OAE-2 (426.00–423.07 mcd, Fig. 4) was marked by the intrusion of a euxinic OMZ into the photic zone. Elevated, yet fluctuating isorenieratane concentrations suggest relatively persistent PZE of varying vertical extent, in agreement with previous investigations using biomarkers and nitrogen isotopes at nearby sites12,13,31. During this interval, microbial sulfate reduction was likely active as DAGEs continually increased, aligning with estimates of expanded seafloor euxinia32. The co-occurrence of abundant extended archaeols and isorenieratane intimates the role that density stratification served in maintaining the protracted PZE of OAE-2, substantiating concurrent findings based on neodymium33 and oxygen isotopes34. Vertical nutrient advection via upwelling35 led to preferential exposure to expanding intermediate water communities tolerant to sulfidic conditions in the OMZ. Scavenging of a potentially limited fixed N inventory30, depleted in NO3− and predominated by NH4+[ 15,29, and inhibition of efficient nutrient transfer by pronounced density stratification likely induced severe N deficiency in surface water communities, explaining the relatively muted productivity of oxygenic photoautotrophs (i.e., tetrapyrroles) and chemoautotrophs (i.e., crenarchaeol) observed (Fig. 4). The concentration and predominant utilization of fixed N in the OMZ led to the proliferation and diversification of intermediate and deep water microbial taxa, while a shoaling chemocline led to increased nutrient (i.e., fixed N) competition between photoautotrophs and retreating Thaumarchaeota as highlighted by our biomarker inventory and the nitrogen isotopic record31. These findings challenge previous interpretations of highly productive, predominantly eukaryotic primary producers reliant on the upwelling of isotopically depleted NH4+ during OAE-215. Instead, the decline of C30-17-nor-DPEP (Fig. S5; Supplementary Data 3), a source-specific tetrapyrrole diagenetically derived from algal chlorophyll-c36, and reconstructed water column conditions during OAE-2 indirectly support a rise in cyanobacteria, diazotrophs able to fix N2, in oxygenated, nutrient-depleted shallow waters. Increased cyanobacterial contribution is further supported by C and N stable isotopes16,37, as well as the prominence of potentially phylum-specific biomarkers across OAE-2 (e.g., 2-methylhopanoids6,14).Fig. 5: Contrasting biogeochemical conditions between the pre-OAE BP and OAE-2.a, b Microbial ecology and water column conditions during the pre-OAE BP, reflecting high primary production of organic carbon (a) and OAE-2, characterized by relatively lower organic carbon production, but substantially enhanced biomass preservation (b). c, d Averaged fractional abundances of individual biomarkers throughout the pre-OAE BP (c) and OAE-2 (d). Biomarker source organisms are abbreviated as follows: phytoplankton (P), ammonia oxidizing archaea (AOA), sulfur oxidizing bacteria (SOB), unknown anaerobic bacteria (UAB), sulfate reducing bacteria (SRB), halophilic archaea (HA), methanogenic archaea (MA).Full size imageA reversal from the formerly outlined conditions typified the second period of OAE-2 (423.07–421.99 mcd, Fig. 4). Destabilization of the stratified water column and reduced production of H2S led to deepening and contraction of the euxinic OMZ. The observed decline in halophilic archaea, coincident with an overall decline in Chlorobiaceae populations, is roughly coeval with positive neodymium isotopic excursions observed across the proto-North Atlantic33 attributed to the enhanced latitudinal commingling of proto-North Atlantic water masses38. Although detrimental to sustained PZE, the persistence of a well-developed anaerobic bacterial community (i.e., obGDGTs) suggests the lasting presence of a non-euxinic OMZ despite improved bottom water circulation. A premature recovery of the chemoautotrophic Thaumarchaeota, inhabiting the base of the photic zone, relative to the shallower dwelling obligately oxygenic phototrophs (Fig. 3) likely reflects reduced toxicity associated with retreating euxinic waters, lessened resource competition with [primarily] Chlorobiaceae, and a competitive advantage tied to preferential exposure to upwelled nutrients and tolerance to low O2 conditions.The termination of OAE-2 was marked by the temporary re-establishment of microbial community compositions mirroring those observed prior to the pre-OAE BP (Figs. 3 and 4). Contraction of the OMZ led to a deep chemocline, with PZE restricted to the basal photic zone as the production of reduced sulfide species diminished. The Thaumarchaeota continued the recovery initiated towards the latter half of OAE-2, accompanied by the rebounding oxygenic photoautotrophs. However, the recovery of shallow autotrophic communities was halted by an episode of PZE (421.19–421.04 mcd) based on abrupt increases in isorenieratane concentrations (Fig. 4). Temporary development of pronounced density stratification likely facilitated the accumulation of H2S in the lower to intermediate photic zone, producing the short-lived PZE episode. Interestingly, covariant responses observed in additional biomarker profiles (e.g., obGDGTs) to PZE during OAE-2 were not evident across this post-OAE interval, possibly due to the transient nature of PZE at this time. For example, the initial increase in isorenieratane concentrations at the onset of OAE-2 was not immediately accompanied by shifts in other biomarker classes (e.g., obGDGTs; Fig. 4), suggesting frequent recurrences of PZE may be required to illicit a major microbial ecological response as observed later during the OAE. Still, this brief episode of post-OAE PZE (421.19–421.04 mcd) coincides with a positive organic carbon isotope excursion9 (Fig. S5), trace metal drawdown6 (Fig. S4), and minor positive Tl isotope excursion7 at the Demerara Rise. Prior study7 tentatively attributed this interval to enhanced carbon burial during a post-OAE deoxygenation event of smaller magnitude, with subsequent work revealing continued pyrite burial post-OAE 211. Our biomarker inventory revealed some environmental consistencies (e.g., PZE) between this interval and OAE-2, but the overall biotic response to this post-OAE geochemical perturbation was relatively subdued and requires additional sampling and investigation to properly constrain.Broader implicationsThe recognition of the pre-OAE BP and evolving water column conditions at Demerara Rise highlights additional complexities of a dynamic ocean relevant to interpretations of OAE-2 and the +CIE. Enhanced, sustained, and widespread carbon burial is required to produce the +CIE used to define OAE-28,10. Still, the principal forcing, productivity or preservation, remains enigmatic as evidence for the former mounts12,39.Based on the tetrapyrrole profiles (Fig. 4) primary production was greatest during the pre-OAE BP and relatively muted throughout OAE-2 at Demerara Rise, assuming minimal alteration to the genetic tetrapyrrole stratigraphic signal. Biomass preservation was presumedly enhanced during OAE-2 through sulfurization11, as the OMZ transitioned from anoxic to euxinic and penetrated the photic zone, yet low tetrapyrrole concentrations persist. Previous work noted a similar discrepancy between preservation potential and porphyrin abundance, postulating a paucity of trace metals to chelate with the free-base porphyrins induced poor preservation as desulfurization did not reveal additional porphyrin content16. However, both the pre-OAE BP and OAE-2 were characterized by relatively depleted trace metal inventories6 (Fig. S4), yet exhibit contrasting tetrapyrrole profiles, suggesting relative changes in primary production were the predominate control on the stratigraphic distribution of tetrapyrroles across the studied interval at the Demerara Rise. The strong covariance between tetrapyrrole and crenarchaeol concentrations reinforces the interpretation tetrapyrroles faithfully reflect primary production (Fig. S6). Crenarchaeol, a biosynthetic product of chemoautotrophic archaea (Thaumarchaeota) comprising up to 20% of all archaea and bacteria in the modern ocean40, is structurally distinct from the tetrapyrroles making it likely that diagenetic alteration of the two biomarkers is not consistent in rate or form. Thus, the positive correlation between key proxies for major contributors to primary production, the photoautotrophs and chemoautotrophs, minimizes concern for the integrity of the biotic signal at Demerara Rise (see Tetrapyrroles as a record of primary production in Supplementary Information for additional details).These findings provide direct evidence for a causal mechanism resulting in both the Tl isotope excursion and +CIE as previously described. It is highly probable the pre-OAE BP was not exclusive to the Demerara Rise based on the immense and presently unconstrained organic carbon burial required to produce the +CIE10. Further characterization of comparable localities to Demerara Rise may reveal similar high productivity events, as primed, highly productive settings likely capitalized on exogenous nutrient delivery via efficient upwelling to the photic zone prior to stratification during OAE-2. Hence, OAE-2 and the +CIE were not coincident with heightened surface water productivity relative to the pre-OAE BP at the Demerara Rise. Rather, antecedent increases in primary production locally facilitated the initiation of the OAE as a mechanism to consume marine oxygen and subsequently enhance organic carbon preservation globally. This highlights how OAE-2, and perhaps other OAEs in the geologic record, were not instantaneously induced but rather a gradual transition stemming from sustained forcing(s). In addition, the occurrence of the pre-OAE BP well before the established onset of OAE-2 reveals how fluctuations in primary production can be linked to marine deoxygenation but may not necessarily be concurrent. As shown here, OAE-2 at the Demerara Rise was preceded by elevated primary production that progressively attenuated towards event onset. While the hallmark features of an OAE are well-established, further identification and refinement of trends preceding widespread anoxia in the past will improve our understanding of how marine deoxygenation develops, as well as our ability to assess planetary health today.A shift from a productivity- to preservation-dominant system during OAE-2 at Demerara Rise, and possibly similar paleogeographic settings experiencing the pre-OAE BP, facilitated substantial organic carbon burial producing the +CIE. Distinct shifts in water column chemistry and structure from the pre-OAE BP to OAE-2 imparted considerable changes on microbial life, which altered the primary driver governing biomass sequestration (Fig. 5). Yet, both intervals reveal relatively comparable carbonate-corrected total organic carbon values6 (Fig. S5), signifying enhanced preservation as a critical component of organic carbon burial during OAE-2 at Demerara Rise. Consequently, this work suggests that sustained increases in primary production prior to OAE-2 initiated and regulated pre-OAE deoxygenation, resulting in a progressive shift to preservation as the primary control on organic carbon accumulation in sediments. Expanding euxinia and attendant changes to biogeochemical cycling adversely affected primary producers while simultaneously enhancing organic matter preservation via sulfurization11. Flourishment of Thaumarchaeota in oligotrophic settings in the modern open ocean41, and lack thereof during OAE-2 based on diminished crenarchaeol concentrations, underscores the scarcity of bioessential elements (e.g., fixed N) caused by microbial utilization of electron acceptors further down the redox ladder due to intensified marine anoxia, ultimately limiting primary production. The switch from a productivity to preservation model, reconstructed using biomarkers (Fig. 5) and initially suggested based on drawdown of the trace metal inventory6, was also concomitant with relative warming4. Simulated projections of the marine microbial response to continued global warming in the future revealed similar biotic trends (e.g., decreased primary productivity) to warming-induced oceanographic changes42 (e.g., intensified stratification) observed during OAE-2. Thus, an abundance of proxy- and model-based results paired with conceptual evidence suggest relatively low production and enhanced preservation of organic carbon throughout OAE-2 at the equatorial Demerara Rise.The pre-OAE BP may foreshadow greater regional trends observed during OAE-2. Equatorial upwelling centers, like Demerara Rise, are spatially restricted and represent regions of already high primary production before OAE-2. Climatic shifts concurrent with OAE-2 may have produced favorable conditions for elevated primary productivity in regions unable to capitalize on or exposed to allochthonous nutrient delivery prior to the +CIE. While the pre-OAE BP offers a causal mechanism for the Tl isotope excursion and +CIE initiation, areal expansion of organic carbon preservation and production is necessary to sustain enhanced organic carbon burial for the duration of the +CIE.Continued development of preexisting proxies is critical to extract and clarify current understandings of major climatic events in Earth history. Although reliant on excellent preservation of the microbial signal, the analytical and interpretative approach used here enables simultaneous examination of a wide array of biomarkers, producing a more holistic reconstruction of oceanographic changes inferred from microbial ecological variations spanning the surface to the sediment. This is timely, as investigations of the sedimentary archives become increasingly valuable analogs to understand the response of modern oceans to natural and anthropogenic forcings. Similarities between the pre-OAE BP and modern, climate-driven marine deoxygenation are concerning, while particular attention to preexisting highly productive settings may hold the key to forecasting the geologically rapid transition to a global OAE. Even though natural processes are currently beyond our control, stifling anthropogenic catalysts of climate change may decelerate the unfortunate, progressive suitability of OAEs as climate analogs in the future. More

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