Benthic diel oxygen variability and stress as potential drivers for animal diversification in the Neoproterozoic-Palaeozoic

Domain: earth_science
Published: 2025-03-21
Editorial outcome: minor_revision

Abstract
The delay between the origin of animals in the Neoproterozoic and their Cambrian diversification remains perplexing. Animal diversification mirrors an expansion in marine shelf area under a greenhouse climate, though the extent to which these environmental conditions directly influenced physiology and early organismal ecology remains unclear. Here, we use a biogeochemical model to quantify oxygen dynamics at the sunlight sediment-water interface over day-night (diel) cycles at warm and cold conditions. We find that warm temperatures dictated physiologically stressful diel benthic oxic-anoxic shifts over a nutrient-rich shelf. Under these conditions, a population-and-phenotype model further shows that the benefits of efficient cellular oxygen sensing that can offer adaptations to stress outweigh its cost. Since diurnal benthic redox variability would have expanded as continents were flooded in the end-Neoproterozoic and early Palaeozoic, we propose that a combination of physiological stress and ample resources in the benthic environment may have impacted the adaptive radiation of animals tolerant to oxygen fluctuations.

Introduction
Animals diversified dramatically during the first half of the Cambrian Period (540–490 million years ago)1 in the shallow benthic environment2 (Fig. 1A). Multiple studies have investigated how temperature, nutrients and oxygen gradients influenced animal diversity in the Cambrian Period3. However, the specific mechanisms by which these abiotic conditions influenced subsequent changes in animal diversity patterns at the Neoproterozic-Paleozoic boundary remains unresolved, and no single hypothesis fully explains the timing of the event and nor is it likely to4. For example, it is suggested that environmental oxygen concentrations increased above a threshold permissive for a diversity of animal species at some point in time, but we lack understanding of a line of events that would have spurred the diversification of animals after this threshold5. In contrast to how other biotic turnover events are linked to harsh conditions6, the Cambrian explosion is rarely explored as the result of physiological stress. Recent investigations, however, note that recurring marine anoxia and heterogenous water column oxygenation may have accelerated evolutionary innovations at the Neoproterozic-Paleozoic boundary7–9 and over the Phanerozoic Eon6,9. More generally, it is well established that cellular and physiological stress is a driver for change in evolutionary biology10. Rapid evolutionary change in organisms as diverse as Ordovician trilobites and modern fruit flies is associated with environmental fluctuations and resource availability above the needs of survival and maintenance11. For the Cambrian, however, it remains unknown, and we here aim to test, whether diel oxygen fluctuations would have been present to accelerate diversification10.

The benthic Cambrian habitat in which animals primarily diversified2 expanded with large-scale global flooding of continental fragments from Rodinia and Pannotia12,13. In Laurentia, sandy sediment deposits in shallow settings increased in areal extent four-fold during the early Cambrian Period, as demonstrated by macrostratigraphic summaries of rock quantities in North America14 and consistent with strontium isotope data12,15,16 (Fig. 1A). This flooding resulted in a dramatic expansion in sunlit shallow water sediments where benthic microalgae photosynthesis likely regulated the diel oxygen dynamics and seafloor redox conditions, as in modern seas17,18. Given that microalgae contribute substantially to the benthic oxygen budget in the modern coastal ocean19,20 and became ecologically significant in the Cryogenian-Ediacaran21,22, their presence can be presumed to have contributed to pronounced diel benthic oxygen fluctuations in the end-Neoproterozoic and early Palaeozoic. Notably, the microalgae photosynthetic oxygen production and oxygen consumption through heterotrophic respiration at the sediment-water interface is fundamentally controlled by temperature23. Temperature also regulates diffusive transport mechanisms and oxygen solubility in seawater. With increasing temperature, diffusive transport mechanisms increase and oxygen solubility decreases that leads to faster transport rates but less availability of oxygen. During the Cambrian Period, global temperatures were generally defined by a greenhouse climate as indicated by modelling efforts24,25, oxygen isotope palaeothermometry26 and climatically sensitive lithologies27–32 (Fig. 1B, S1). Annual equatorial sea surface temperatures have been estimated as high as 30–38 °C in the Cambrian32. Although reconstructions suggest a variable climate over the post-Marinoan33, both shallow shelf area and global temperature increased between the Cryogenian and the Cambrian periods14,27,34. The higher temperatures would have increased photosynthesis-driven gross oxygen production, the molecular diffusion of oxygen, and the coupled oxygen consumption in benthic microalgae communities. Consequently, oxygen dynamics and variability in oxygen concentrations between day and night likely would have been amplified with higher temperatures, leading to temporally amplified daily redox fluctuations35 and more physiologically stressful conditions for early animals in the expanding shallow benthic habitats.

Physiological stress is a potential driver of the emergence of evolutionary innovations10. In addition to increasing genetic variation (e.g. mutations), environmental stress lets a population probe alternative phenotypic states36. Since beneficial stress-tolerant traits have high heritability under stressful conditions11, increased variance channelled through existing functional systems can both account for similarity and lead to adaptive radiation10. For a scenario where environmental stress leads to adaptive radiation, however, it is suggested that fluctuations are predictable and combined with a surplus of energy to drive organisms’ metabolism (beyond what is needed for survival and maintenance)11. In contrast, constant extreme physiological stress (e.g. saline pools) or low-energy environments (e.g. dark caves) would hinder phenotypic explorations and, therefore, adaptive radiation10,36.

A specific trait that allows extant animals to tolerate environmental fluctuations is cellular oxygen sensing37. Oxygen-sensing mechanisms regulated by Hypoxia-Inducible Factor alpha (HIF-α) allow animals to orchestrate tissue homoeostasis by sensing and responding to redox fluctuations37. Oxygen shortage can be physiologically stressful for multicellular organisms by affecting metabolic homoeostasis38, and extant animal cells coordinate a HIF-α response within minutes by, for example, shifting from aerobic to anaerobic cell metabolism39. This means that oxygen-sensing mechanisms accommodate stress-avoidance traits with high heritability under stressful conditions. Components in the current oxygen sensing mechanism have evolved from prokaryotes (e.g. the Per-ARNT-Sim or PAS domain)40, to choanoflagellates (e.g. the prolyl hydroxylation domain or PHD)41 and within animals37.

Today, all animals with true tissues (excludes Porifera) and a benthic component to their lifecycle (generally excludes Ctenophora) respond transcriptionally to O2 fluctuations via HIF-α39,42 (Fig. 1C). These animals share a joint core to this sensing by an oxygen dependent degradation domain (ODDD) of the bHLH-PAS protein where a proline can be targeted by a PHD protein (if O2 is present) and hinder gene transcription (for details on Molecular dynamics within Hypoxia Inducible Factor (HIF), see supplementary discussion). However, the components of the pathway vary in their presence within animal groups and in terms of how they sense and respond to oxygen fluctuations. For example, an additional C-terminal transactivation domain (CTAD) can enable the protein to bind with transcriptional coactivators of and a factor inhibiting HIF (FIH) can limit the regulation of genes43. Differences in oxygen sensitivity and regulatory roles are also described for the two different prolines (P564 and P402) targeted by PHD, and an N-terminal transactivation domain (NTAD)43. These variations are likely to contribute to fine-tuning of both O2 sensitivity and range of regulated genes44,45, such that rudimentary oxygen sensing mechanisms could have been a functional but evolving system that channelled stress-induced adaptations.

A downside of oxygen sensing functionality via HIF-α is its energy cost for the cell. For example, the pathway involves a continuous production of a protein that is either degraded (in the presence of oxygen) or stabilised to induce a response (at oxygen shortage). Therefore, we here tested trade-offs for oxygen sensing as benefits and costs vary, against the suggestion that fluctuating benthic O2 concentrations led to physiological stress in which improved HIF-α functionality optimised fitness.

In this work, we hypothesise that the combination of physiological stress and the substantial expansion of nutrient-rich sunlit benthic habitats from the late Neoproterozoic to the early Palaeozoic promoted phenotypic probing and, subsequently, the adaptive radiation of animals (Fig. 2A, B). We further hypothesise that stress-induced adaptations could have been channelled through the regulatory system of oxygen-sensing mechanisms. To explore this, we first investigate benthic O2 fluctuations over day- and night cycles (Fig. 2C) at varying temperatures and carbon loads using a 1-dimensional (1D) biogeochemical model (Fig. 2D)46. We then assess trade-offs between the benefits of an efficient oxygen-sensing mechanism and its energetic costs for early animal fitness by modelling ecological (population) and evolutionary (strategy) dynamics of organisms with effective or poor oxygen-sensing mechanisms (eOSM or pOSM) existing in the same environment.

Results
Biogeochemical model: We applied a 1D biogeochemical model to constrain O2 concentration dynamics and predict the concurrent physiological stress imposed by rapidly fluctuating (redox) conditions over a 21-h day and night cycle (early Palaeozoic)47. The partial differential equation model includes oxygen gross production, consumption and diffusive transport equations (Fig. 2C, Supplementary Table 1 for parameter values and supplementary methods for model description). The model results were validated against measured data from modern marine microphytobenthic studies at sunlit shallow-water sandy sediments (Supplementary Fig. 2). As the model is qualitatively generic and reflects key biophysical processes, identical dynamics and shape of profiles would be expected if validated against ocean shelf or even deep-sea sediments48. The modelled dynamics are universal for muddy, mixed, carbonate and sandy sediments and include oxygen scavenging by, e.g. iron and sulphide reduction35. The model was used to derive day and night O2 concentration profiles and to quantify the impact on these of changing temperature and total organic carbon (TOC) availability (see ‘Method’ section for modelled scenarios). Since rapid changes in O2 concentrations can be physiologically stressful to benthic animals when adjustments to their ATP-turnover are less swift49, we also quantified the transition time between day and night conditions.

Biogeochemical model results: These first-order experiments found that in a cold scenario (+5 °C), daily oxygen fluctuations and diel amplitude would have been modest with weak oxic conditions during the day and with hypoxic conditions during night lasting for ~11 h (Fig. 3A, C). In contrast, a warm model scenario (+25 °C) demonstrated profoundly amplified daily fluctuations with abrupt changes from fully oxygenated conditions during the day to true anoxia during the night in less than 0.3 h (Fig. 3B, D). Regardless of changes in TOC (0.1 to 1 relative to Phanerozoic levels3), a hot climate (at +25 °C and +40 °C, compared to a cold at +5 °C) led to O2 concentrations that were high during the day and low during night hours (Fig. 3E), shorter transition times with hypoxic conditions (Fig. 3F) and longer exposure to anoxia at the sediment water interface (Fig. 3G). A visual comparison of the results for 20% PAL versus 50% PAL at varying TOC adjustment factors (Supplementary Tables 3–4) depicts that a delicate balance of sub-modern ppO2, sub-modern TOC and temperature could lead to severe diel redox fluctuations at the sediment-water interface (Supplementary Figs. 4–5). The robustness of the model results with respect to parameter values was tested by running a Monte-Carlo simulation (50.000 times over, see Methods for details).

Population and phenotype model: After finding that daily benthic redox fluctuations on an expanding Cambrian shelf would have presented animals with severe daily O2 fluctuations, we explored the ecological (population) and evolutionary (strategy) dynamics of species with effective and poor oxygen-sensing mechanisms (eOSM or pOSM), respectively, existing in these environments. To do this, we used a mathematical modelling framework named G functions50,51 that simultaneously captures ecological (population) and evolutionary (oxygen-sensing strategy) dynamics over time. In other words, the same set of equations can qualitatively capture the interplay between changes in oxygen levels, ecological dynamics and species competition, and adaptation via oxygen sensing mechanisms. The capacity to effectively sense and respond to changing oxygen levels (rate of phenotypic switching) presumably incurs a cost, but it would be an adaptive advantage for an organism to swiftly change from aerobic to anaerobic cell metabolism in this environment. For example, for anaerobic metabolism to produce ATP that matches that of aerobic metabolism (per glucose molecule), eighteen times more glucose is required. An increased metabolic rate is feasible, and the organism can remain highly functional, if also rate-limiting enzymes keep up. Expression of phosphofructokinase, the rate limiting and irreversible step in this pathway, is rapidly upregulated by HIF-α49. Organisms with eOSM would have the benefit of responding quickly to changing oxygen conditions, but at some cost. The costs of rapid cellular phenotypic plasticity could include cells refining oxygen sensing or maintaining the intra-cellular machinery to rapidly upregulate anaerobic metabolism (or their rate-limiting enzymes). Our model extends the classic Lotka-Volterra competition equations and imposes a cost for efficient oxygen-sensing. Namely, the population dynamics are given by Eq. (1):

dx_i/dt = x_i G(v,u,x) | v=u_i (1)

where the per capita growth rates of a species are given by Eq. (2):

G(v,u,x) = r/K(v) * (K(v) - sum_{j=1}^n x_j) - ds (2)

and the carrying capacity is a function of how well the focal individual’s strategy matches the environment, Eq. (3):

K(v) = K_m exp(-(v-γ)^2 / 2σ_k^2) (3)

In these equations, v represents a species’ cellular metabolism phenotype (strategy), u=(u1, u2) and x=(x1, x2) are the strategies and population sizes of each species in the population, respectively. The evolutionary dynamics are then derived by Fisher’s fundamental theorem52 as Eq. (4):

du_i/dt = s_i dG/dv | v=u_i (4)

As a simplified L-V competition model, we assume that competition among species is independent of their strategies. All individuals have the same adverse impacts on each other and compete equally in a density-dependent fashion. This leads to growth in a logistic manner, wherein the carrying capacity depends on how well adapted the species is to its (continually changing) environment. Finally, we included a cost of oxygen sensing through the last term in the G-function. We assume this cost scales linearly with the capacity for oxygen sensing. The eOSM and pOSM species are differentiated solely by the eOSM species having a higher capacity for oxygen sensing, s, than the pOSM species. Thus, the eOSM species can switch its cellular metabolism more rapidly than the pOSM species. For further details on model derivation, please consult the ‘Methods’ section.

To capture the ecological (population) and phenotype (strategy) dynamics of each species (eOSM and pOSM), we performed three simulations, in which we let the optimal phenotypic strategy (γ) vary in a sinusoidal fashion with added stochastic noise. We simulated this over 500 time-steps (pseudotime; i.e. an arbitrary time unit) to capture the dynamics before the continental flooding, as in the early Palaeozoic. For simplicity and visualisation purposes, the flooding that led to the expanded shallow shelf area was implemented as a switch. To simulate the flooding, we increased the periodic O2 fluctuations (driven by microalgal production and consumption), stochastic O2 fluctuations (driven by nutrient cycling), or both.

Population and phenotype model results: The results of the simulations can be seen in Fig. 4A–C. When oxygen fluctuated stochastically, the benefit of tracking environmental oxygen levels with an efficient oxygen sensing was greatly outweighed by its cost and pOSM species dominated (Fig. 4A). When oxygen fluctuated periodically (as if daily), eOSM species dominated the population within ~500 (pseudo) time-steps after the switch, suggesting the benefit of perceiving and responding to redox fluctuations in a spatiotemporal manner (Fig. 4B). In the case of both periodic and stochastic fluctuations, eOSM species were greatly favoured, which drove the pOSM species to extinction within 250 (pseudo) time-steps (Fig. 4C). Although a qualitative estimate in pseudo-time, the biological event rate exceeds that of fluctuations, and both are instant in comparison to atmospheric changes of oxygen concentrations. The trends observed in the plots of eco-evolutionary dynamics generally hold for a wider range of parameter values (for details on Model of population dynamics and speed of phenotypic plasticity, see Supplementary discussion and Supplementary Figs. 7–8). In all cases, the pOSM and eOSM species attempt to track the environment, but the eOSM species, with their improved oxygen-sensing capability, displayed greater changes in strategy. Note that the cost of the oxygen sensing mechanism has a large influence on the outcome of the competition experiments (Supplementary Fig. 8). The competition experiments demonstrated that eOSM species are favoured across the range of costs of the mechanism tested in environments with stochastic and periodic fluctuations, and favoured for most tested costs (d<0.09) in environments with periodic fluctuations. In contrast, pOSM species are favoured for most tested costs (d>0.01) in environments with only stochastic fluctuations. The shape and magnitude of the cost function was chosen arbitrarily for the purposes of this qualitative modelling study, but for a given environment, there will be a threshold value for d, below which eOSM species will outcompete pOSM species and above which pOSM species will outcompete eOSM species. Furthermore, it is possible that extreme costs can overshadow the main trends observed in this in silico study.

The population and phenotype model is agnostic to which organisms would represent pOSM or eOSM species. To relate the phenotype to the Cambrian Period (Fig. 4D, E) and the early evolution of animals, we mapped differences in the composition of HIF-driven oxygen sensing across the animal clade based on sequences in ref. 53 and ref. 43 (Fig. 4F, supplementary discussion and Supplementary Table 4, for details). While HIFs that today compose the OSM of modern animals contain components (e.g. genes, domains, or proteins) that can be traced back to the origins of yeast and even to prokaryotes37, its building blocks expand and differ in functions within and between early branching animals. For example, oxygen sensing in animals with e.g. only P564 would be simpler than in animals e.g. with P564 and P402 or with P564 and CTAD. Components to the OSM like the P402, CTAD, NTAD and Factor Inhibiting HIF (FIH) regulate a wide scope of target genes at a wider range of conditions, some of them with clear importance for development37. From what we know of the importance of these components in Bilateria54–56, the different configurations amongst animals43,53 reflect varying efficiency and costs to sense and respond to redox fluctuations.

Methods
Biogeochemical model: Model parameters were tuned for sandy sediments and to mimic cold (5 °C) and warm (25 °C, and 40 °C) shelf conditions with 20–50% of present atmospheric levels (%PAL) of O2 and 0.1–1 times the Phanerozoic load of organic carbon. While the term hypoxia was originally used to describe internal stress on an animal, it has also come to describe the external ocean medium94. The specific definitions of anoxic to oxic conditions used here are: anoxic <0.02 µM, severely hypoxic between 0.02 and 22 µM, hypoxic between 22 and 65 µM and oxic >65 µM95.

To test whether the model results were robust with respect to parameter values, we ran a Monte-Carlo simulation of the model 50,000 times over a large range of ppO2, TOC and temperature responses for production and respiration rates (Q10 Prod and Q10 Resp), accommodating for most possible seafloor conditions (Supplementary Table 1). These simulations supported the generality of the model by demonstrating that a short transition time (<1 h) from daytime oxic to nighttime anoxia/severe hypoxic conditions in the diffusive boundary layer (DBL) was met in 67% of the cases and that the model output is primarily sensitive to TOC adjustment factors and ppO2 (various scenarios shown in Supplementary Fig. S6). A compilation of TOC contents in more than 13,650 shale samples from the end-Neoproterozoic and Phanerozoic eons indicates that TOC in the Cryogenian were lower than in the Cambrian Period and the Phanerozoic overall3. Quantitative differences arise if parameters are set for muddy, mixed, or carbonate sediments, but the same fluctuating patterns and qualitative trends remain alike48.

Population and phenotype model: The G function framework we use allows us to track both how the population sizes of the species change over time (ecology) as well as how their strategies evolve in response to changing environments (evolution). We modelled the population dynamics of two species, one with high phenotypic plasticity for cellular metabolism in response to dissolved oxygen conditions and one with low (i=1, 2), Eq. (1):

dx_i/dt = x_i G(v,u,x) | v=u_i (1)

where the fitness-generating function, G(v,u,x), describes the expected per capita growth of a species as influenced by its cellular metabolism phenotype (strategy), v, the strategies of each species in the population, u=(u1, u2), and the population sizes of each species, x=(x1, x2). In this case, the relevant strategy describes an abstract trait that allows cells of the multicellular organism to switch along a continuum between anaerobic and aerobic metabolism. Thus, this equation captures the change in the population size of a given species as a product of its current population size and its per capita growth rate. We let the rate of phenotypic switching of cells of the organism depend on (a) the slope of the fitness gradient and (b) how fast the species can change phenotype in response to the fitness gradient. The slope of the fitness gradient is given by dG/dv. How fast the species can change in response to this fitness gradient depends on the oxygen sensing mechanism; the more developed this is, the more quickly a species can switch. These components together allow for the derivation of the equation for phenotype dynamics52, Eq. 2:

du_i/dt = s_i dG/dv | v=u_i (5)

where s_i is a measure of the capacity for cellular oxygen sensing that allow the organisms to closely track and respond to their environment. To define our fitness-generating function, G, we used simplified Lotka-Volterra (L-V) competition equations, Eq. 3:

G(v,u,x) = r/K(v) * (K(v) - sum_{j=1}^n x_j) - ds (6)

We let the carrying capacity be a function of the focal individual’s strategy. We assume that there is some optimal strategy value γ. The closer the individual’s strategy is to γ, the higher its carrying capacity, Eq. 4:

K(v) = K_m exp(-(v-γ)^2 / 2σ_k^2) (7)

We assume that deviations of strategy values from γ decrease carrying capacity in a Gaussian fashion. We allow γ to change over time as a result of fluctuating oxygen levels in the environment. A particular dissolved oxygen level corresponds monotonically to a particular value for γ. In the model, the ecological impact of a species’ phenotype to oxygen levels comes through its carrying capacity. As a simplified L-V competition model, we assume that competition among species is independent of their strategies. All individuals have the same adverse impacts on each other and compete equally in a density-dependent fashion. This leads to growth in a logistic manner, wherein the carrying capacity depends on how well adapted the species is to its (continually changing) environment. Finally, we included a cost of oxygen sensing through the last term in the G-function. We assume this cost scales linearly with the capacity for oxygen sensing. The eOSM and pOSM species are differentiated solely by the eOSM species having a higher capacity for oxygen sensing, s, than the pOSM species. Thus, the eOSM species can switch its cellular metabolism more rapidly than the pOSM species.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_3": "I am honestly a little disappointed to be reviewing this manuscript for the fourth time and finding the authors disregard my suggestions yet again. I really am just trying to help the authors anchor an interesting modelling approach in a representative and up-to-date reading of the Ediacaran-Cambrian literature. I have outlined many suggestions about how the Ediacaran biota relate to eumetazoan clades in previous reviews, so I won’t repeat them here. I suggest a reframe of the discussion to avoid making poorly supported overstatements about a driver of the Cambrian explosion and instead describe an interesting mechanism that may have impacted animal diversification at some point between the Cryogenian and Cambrian.\nAs per my previous reviews, I remain critical of the authors’ overly confident descriptions of Cambrian vs Cryogenian climate and evolutionary trajectories. All that I think is really required here is a broader acknowledgment and appreciation that these mechanisms may have been important at some point between the Cryogenian and Cambrian but that we are not currently able to reconcile exactly when. I think that uncertainty is fine in the context of an interesting modelling study and don’t really understand why the authors continue to try to overinterpret their results in an outdated evolutionary and climatic framework here.\nI agree with the similar comments of reviewer 1 and really struggle to understand why we have gone through all of these rounds of review when the authors simply could have taken our suggestions on board. I can’t help but feel that it is a waste of everyone’s time to be debating this in rebuttal letters when the authors could have incorporated our referenced suggestions with ease. New analyses may be reasonably out of scope, but a discussion reframe would have been straightforward for the authors to incorporate at any juncture.\nIn its current form, I cannot recommend this paper for publication in Nature Communications. As emphasised repeatedly in my multiple reviews, fixing these fundamental issues really wouldn’t be that hard but (unfortunately) the authors don’t seem willing to do so.\nLine 37 – I don’t see the relevance of the reference to cancer here and strongly suggest removing.\nLine 61 – interpretation of Sr isotope records is contentious. They certainly don’t “demonstrate” this. I suggest you qualify the statement or remove.\nLine 86 – “coupled to” – correlated with? A potential driver of?\nLine 130 – specifically in the Cambrian? Why not anchor your hypotheses on this broad distinction between the Cryogenian and early Paleozoic. The more specific your time references here, the less compelling your narrative, in my opinion.\nLine 326 – “we conjecture that oxygen sensing mechanisms evolved within early animals at the Cambrian flooding event” – I don’t understand how you can invoke oxygen sensing evolution in early animals at ~520Ma (critical objection being the use of ‘early’). Yet again, I feel like the discussion here is completely missing an informed perspective on the divergence times of early animals. Surely this is way too late for these higher level divergences to be associated with innovation in OSMs? If you disagree then please justify.\nFigure 4 – as I have said in every review of this paper, I firmly feel that the evolutionary timelines invoked for different metazoan lineages here is very out of date. For example, when we look at the Bilateria illustration in Fig 4F, what about the Ediacaran bilaterians? Dickinsonia, Spriggina and bilaterian trace makers are all fully disregarded here so far as I can tell. Similar stories can be applied to your Cnidaria and Porifera extents.\nLines 482 onwards – refer to above and previous comments on how out of date I feel this discussion of different lineages is. I strongly recommend that this would have to be updated to represent current understanding of the history of these lineages in order for this manuscript to be published in this form. I have provided detailed arguments on this in previous reviews and really don’t understand why the authors are so resistant to doing this. If they fundamentally disagree with the literature that I cite then it would be useful to justify this in the text and put forward an argument that is neutral to these reconstructions. Currently this out of date discussion just weakens a perfectly good modelling study in my opinion, and will only frustrate readers in the Neoproterozoic community. Having spent the time to outline these arguments to the authors multiple times between my four reviews of this manuscript, I honestly am quite frustrated that they have not taken the small amount of time it would take to incorporate this more up to date discussion into their narrative.\nLine 511 – “For the first time, a cascade of physical, functional, and ecological factors aligns directly with the Cambrian Period.” I would argue this is patently untrue. Why overreach like this? There is a cool underexplored mechanism here that you have the opportunity to tell the reader about. Why frustrate the reader by making claims like this that are clearly dismissed by a quite surficial understanding of the literature?\nFig 5 caption – see notes above.", "reviewer_4": "The authors have done a reasonable job of addressing the substantive points from my previous reviews. I would be happy to see this manuscript published following a few minor revisions which I still think are necessary and which I hope the authors find helpful.\nThere are some line-by-line comments below, but first I would strongly encourage the authors to fully revise the text to separate their general, first order, modelling results from their interpretation of how this would play out in the Neoproterozoic-Phanerozoic transition. In particular, this is very generally applicable, first order modelling. At various places, the text could be clarified to show that:\na. The first order modelling of oxygen concentration cycles was done for a range of T, TOC, and [O2] conditions which could be interpreted broadly in a geographical and temporal way (e.g. for various places on present-day Earth or times in the geological past). Keeping this distinction clear throughout would help emphasise the broader applicability of the concepts modelled here and would widen the appeal of the study.\nb. The first order modelling of pOSM and eOSM species could also be shown as of much broader relevance than it is currently presented as. In particular, the authors acknowledge that there are different differences in OSM efficiency between different animal clades (bilaterians, cnidarians, placozoans, poriferans, ctenophores). The focus in the results section on presenting this work as predominantly about the differences between bilaterians and other animals hides the wider implications which could be seen more broadly in modern and other palaeo-ecological settings. To keep to the early animal example, such an approach could be applied to the redox oscillations (on a different time scale, but same principle) of the later Ediacaran world, rather than just the Cambrian.\nFinally, I do hope to see this published soon and I think the authors are very nearly there with it.\nLine-by-line comments:\nLine 9: Proterozoic-Phanerozoic, or Neoproterozoic-Palaeozoic, would be preferable to Precambrian-Cambrian.\nLines 27 to 28: ditto Line 9.\nFig. 1: Please define the dashed box label “ROI”.\nLine 51 (Fig. 1 caption): rephrase to make one sentence about the blue-red transition; it doesn’t currently flow. E.g. “+25 °C marking a transition from icehouse to greenhouse climate states”. Otherwise, the caption is much improved, thanks.\nLines 72 to 74: climatically sensitive lithologies have also contributed substantially to this picture (e.g. Boucot, A.J., Xu, C., Scotese, C.R. and Morley, R.J. 2013. Phanerozoic Paleoclimate: An Atlas of Lithologic Indicators of Climate, 1st ed. SEPM Concepts in Sedimentology and Paleontology11).\nLines 74 to 76: something has gone wrong here, I think. Please check for grammar and punctuation.\nLine 109: “ctenophores” or “Ctenophora”.\nLine 117: this paragraph is good but, as someone who is not a biologist or biochemist, I only follow the implications of it through having already reviewed the paper. At Line 117 you could be a bit more explicit that each of the additions to the HIFa pathway increased its efficacy and so represent step-wise acquisitions of O2 sensitivity across the animal tree.\nFig. 2: I reiterate that the maps in A and B should have the same colour scales – they are representing different temperatures of water mass, but still water mass. At present, this looks more like topography than flooded continental area and mis-represents that the continents were not completely flooded in the Cambrian. I still think the maps add confusion as this is not a latitude, or even geography, based study, but more about flooded continental shelf. The onshore-offshore profiles show this better. This is particularly pertinent as the next section talks immediately about a 1D model, no horizontal (map) resolution needed.\nLine 168: “or anoxia” is superfluous. Just need “O2 concentration.” As levels oxygenation are defined below (lines 172 to 174).\nLines 168 to 169: I don’t follow the grammar of “Since quick O2 changes require benthic animals to conform their ATP-turnover”. Should this be “conform to their ATP-turnover rate”? or something else?\nLines 176 to 178: I thought from their last response that the authors were refraining from calling the scenarios “Cryogenian” and “Cambrian” and instead sticking to “cold/warm/hot” or “icehouse/greenhouse” and then interpreting these later as reflecting Cryogenian and Cambrian. Presenting it in this way would emphasise that this is a first-order simulation experiment, not an actualistic one, particularly as some Cryogenian intervals were quite possibly greenhouse or super-greenhouse climate states. This would also emphasise the broader applicability of the work: this is just looking at pO2 variation under different T, TOC, [O2] conditions – these apply in the modern world (e.g. by ocean basin, by latitude) and in numerous other deep-time contexts. The generality of this is a strength that could be much better shown.\nFig. 3: I again urge the authors to make this figure easier to follow and assess. It should be split up or made bigger, or something. I also suggest again plotting the blue and yellow bands in Fig 3E either on the same scale or on separate plots: their numeric range is two orders of magnitude different and it is still not obvious without the caption which coloured envelope relates to which y-axis scale, particularly as the same blue and yellow are used for different oxygen concentrations in figs 3A & 3B.\nLine 319: “d >= 0.01” includes “d >= 0.09”. I think a “, respectively” is needed to clarify that these values relate to stochastic and periodic fluctuations, respectively?\nLine 332: “cnidarians” (lowercase “c”) or “Cnidaria”.\nLines 326 to 342: here is an opportunity to illustrate the impact of stepwise acquisition of increasingly ‘e’OSMs, with each clade being slightly more ‘e’ than those earlier branching clades. I think I made a similar point before, but here the key is not the ‘bilaterians are best’ but that each clade made some slight improvements in their OSM. The text of this paragraph could again be strengthened by saving until the end of the paragraph the specific case of early animal evolution through the Neoproterozoic-Phanerozoic transition and keeping the initial interpretation as hypothetical discussion of organisms with less (‘p’) or more (‘e’) efficient oxygen sensing mechanisms.\nFig. 4: This is a lot better, and I really like the addition of panel F. I do again ask about the colour variation in the diel bar – are we to read anything into the yellow-grey-green transitions and their placement?\nFig. S1: this is really hard to interpret, particularly for anyone who has difficulty with their colour vision. I would urge the authors again to simplify this and use colour palettes that are robust to common colour vision deficiencies."}, "author_response": "Point by Point Response to Reviewers’ comments\nReviewer #3 (Remarks to the Author):\nI am honestly a little disappointed to be reviewing this manuscript for the fourth time\nand finding the authors disregard my suggestions yet again. I really am just trying to help\nthe authors anchor an interesting modelling approach in a representative and up-to-\ndate reading of the Ediacaran-Cambrian literature. I have outlined many suggestions\nabout how the Ediacaran biota relate to eumetazoan clades in previous reviews, so I\nwon’t repeat them here. I suggest a reframe of the discussion to avoid making poorly\nsupported overstatements about a driver of the Cambrian explosion and instead\ndescribe an interesting mechanism that may have impacted animal diversification at\nsome point between the Cryogenian and Cambrian.\nWe appreciate the very direct review that made us see the Reviewer’s points in a new\nand clearer light. We also appreciate the reviewer comments that have been indeed\nhelpful as we now have fully absorbed them. In response, we have now put more effort\ninto mediating, merging, and balancing the work in the direction stressed by the\nreviewer, downscaled the (over)statements and updated the evolutionary framework\nand added new literature citations. Indeed, by absorbing more current literature from\nthe Neoproterozoic community, it seems now clearer that several of our statements\nwere too generalized, out of date, and without enough nuance. We hope the new text\nreflects a more educated and humble tone.\nWe have taken the changes as follows (plus smaller edits in additional instances):\n• Changed in title, abstract (row14, 28, 37, 45 etc) and throughout to capture a\npossible mechanism in a wider time interval, e.g., end-Neoproterozoic and early\nPaleozoic or Neoproterozoic-Paleozoic boundary.\n• Edited to ‘Ediacaran and early Paleozoic’ instead of Cambrian (row 500).\n• Added ‘Ediacaran and Cambrian periods’ (row 501).\n• We edited to clarify that cnidaria are represented in the Ediacaran (row 545).\n• Changed to “energy changed with the Neoproterozoic to early Palaeozoic\nflooding events” (row 570).\n• Removed that ‘physical, functional, and ecological factors align’ (row 573).\n• Changed to “Previous explorations into the animal diversification in the\nNeoproterozoic-early Palaeozoic” (row 601).\n• Changed to “Our results suggest that severe daily redox variations in an\nexpanding benthic shallow shelf created niches in the end-Neoproterozoic and\nearly Palaeozoic that could have promoted adaptive radiation (row 614).\nAs per my previous reviews, I remain critical of the authors’ overly confident\ndescriptions of Cambrian vs Cryogenian climate and evolutionary trajectories. All that I\nthink is really required here is a broader acknowledgment and appreciation that these\nmechanisms may have been important at some point between the Cryogenian and\nCambrian but that we are not currently able to reconcile exactly when. I think that\nuncertainty is fine in the context of an interesting modelling study and don’t really\nunderstand why the authors continue to try to overinterpret their results in an outdated\nevolutionary and climatic framework here.\nYes, we agree (now). With a humbled perspective on the ‘long fuses’ to animal\ndiversification from within the Neoproterozoic, we agree with the reviewer that a more\nmodest claim that the proposed mechanisms may have been important and with\nimpact, therein are both fair and more scientifically sustainable. We have fully re-drawn\nthe phylogenetic tree (although simplified) and its divergence estimate in Figure 1 and\nspecifically in panel 4F. The phylogeny of animal lineages is based on (Philippe,\nBrinkmann et al. 2011), with approximate divergence between lineages based on\n(Telford, Budd et al. 2015) and (Dunn, Liu et al. 2021). Although their estimates di^er, we\nplace the total group Metazoa at 574-600 Ma or mid-Ediacaran, while Cnidarian is\nplaced at 574 Ma from (Dunn, Liu et al. 2021).\nThis comment indeed made us realize and now emphasize that components of the HIF\nsystem also evolved within animal groups, such as the Protostomia . The implications of\nfully absorbing this input are (as the reviewer likely saw long before us), that pOSM vs\neOSMs could have been found both within and between animal groups. If OSM evolved\nwithin, e.g., Cnidarian, the range of adaptations would likely have di^ered within\nEdiacaran cnidarians as well as between, e.g., non-Ediacaran and Ediacaran\ncnidarians. Therefore, we now map and further discuss extant di^erences of HIF\ncomponents between and within animal groups. We have made recovered the\nsequences used in Mills et al., 2018 and Graham and Presell 2017 to compare. Of\ncourse, the annotations have changed some so a few components – such as P564 in\nNematostella and Trichoplax – are indeed less certain today (see di^erences in the new\nTable S4). However, an increase more complex combinations across the Metazoa\nappears with particular high diversity in the species tested from within Protostomia (see\nnew phylogeny in Figure 4F). We think this comment significantly forwarded the\ndiscussion on how these many components to the OSM may have been adaptations to\nchanges in the shelf environment. We have also changed the text, such as on row 354:\n“This could separate Placozoa (pOSM) from Cnidaria (eOSM), or pOSM from\neOSM species within cnidarians or bilaterians in both the Ediacaran (Dunn, Liu et\nal. 2021) or Cambrian (Fig. 4F).”\nHumbled by the experience of not fully knowing the Neoproterozoic literature and\nphylogenetic studies of the Ediacaran biota, we decided not to venture out into\ncomparisons of between e.g., Charnia or Nematostella or Dickinsonia, Kimberella, or\nNematoda. However, based on how we know that the di^erent components to the OSM\nof humans (e.g., CTAD, NTAD, FIH, P402, P564) lead to di^erent sensing and responding\nto O2 fluctuations, we can presume that animals with slightly different compositions\n(whether Charnia or Nematostella) had slightly different OSMs. Or, similarly, that\nbilaterians like Dickinsonia, Kimberella, or Nematoda may have had as di^erent OSM as\nwe see between extant protostomes. But again, in the current manuscript, we will avoid\nopening another discussion on the details of Ediacaran organisms other than paying\nthem respect as Metazoa.\nI agree with the similar comments of reviewer 1 and really struggle to understand why\nwe have gone through all of these rounds of review when the authors simply could have\ntaken our suggestions on board. I can’t help but feel that it is a waste of everyone’s time\nto be debating this in rebuttal letters when the authors could have incorporated our\nreferenced suggestions with ease. New analyses may be reasonably out of scope, but a\ndiscussion reframe would have been straightforward for the authors to incorporate at\nany juncture.\nAgain, we acknowledge this point and believe that we have finally taken these very\nvaluable suggestions onboard in this current version, include those described above\nand in the thorough rewrite of the manus. We apologies for rounds of review and\nappreciate the reviewer’s patience.\nIn its current form, I cannot recommend this paper for publication in Nature\nCommunications. As emphasized repeatedly in my multiple reviews, fixing these\nfundamental issues really wouldn’t be that hard but (unfortunately) the authors don’t\nseem willing to do so.\nYes, we are willing to and have now had the bandwidth to improve the manuscript.\nThanks for the frank wake-up comment.\nLine 37 – I don’t see the relevance of the reference to cancer here and strongly suggest\nremoving.\nYes, we can see this is distracting. Removed.\nLine 61 – interpretation of Sr isotope records is contentious. They certainly don’t\n“demonstrate” this. I suggest you qualify the statement or remove.\nThanks, and we agree. We have edited to let the estimate acquired via Macrostrat\n‘demonstrate’ the increase, as? this would only be consistent with the Sr record.\nLine 86 – “coupled to” – correlated with? A potential driver of?\nThanks yes. Now changed to ‘a potential driver of’.\nLine 130 – specifically in the Cambrian? Why not anchor your hypotheses on this broad\ndistinction between the Cryogenian and early Paleozoic. The more specific your time\nreferences here, the less compelling your narrative, in my opinion.\nYes, finally we see this point and are able to ‘kill’ the darling or narrow focus on the\nCambrian. Changed to ‘from the late Neoproterozoic to the early Paleozoic’ throughout.\nLine 326 – “we conjecture that oxygen sensing mechanisms evolved within early\nanimals at the Cambrian flooding event” – I don’t understand how you can invoke\noxygen sensing evolution in early animals at ~520Ma (critical objection being the use of\n‘early’). Yet again, I feel like the discussion here is completely missing an informed\nperspective on the divergence times of early animals. Surely this is way too late for\nthese higher level divergences to be associated with innovation in OSMs? If you\ndisagree then please justify.\nYes, the reviewer is entirely correct that this is too late to fully innovate OSM. What we\nmean to say is that adaptations within the OSM may have been selected for. As\nmentioned above, we have redrawn the representations of animal phylogeny and\ndivergence times (Figure 1 and 4F), which have forced us to re-evaluate a previously\nnaïve view on the changes of OSM. To the extent that components of OSMs are studied\noutside of humans, it is clear that the function and differences are much more\ncomplicated than just having HIF-1a or not. It is also more complicated that some\nanimal groups have specific OSM components within their group. The fine detail to how\nOSM components differ within animals groups, but also increases its complexity from\nsponges to vertebrates is now visible in Figure 4F. With this is mind, it is also clear that\nthe many components to OSMs (e.g., CTAD, P564, NTAD, FIH) in extant animals each fill\nfundamental roles in the sensitivity and response to chemical variations (in internal and\nexternal environments). We have therefore rewritten the entire paragraph from row 353\n(and at instances in the text above):\n“The popula+on and phenotype model is agnos+c to what organisms\nwould represent p The popula+on and phenotype model is agnos+c to\nwhich organisms would represent pOSM or eOSM species, which could be,\nfor example, Porifera vs Cnidaria, Cnidaria vs Bilateria, or invertebrates vs\nvertebrates within Bilateria. Here, we suggest that adapta+ons of oxygen\nsensing mechanisms can have been selected for within animals at events\nthat led to specifically periodically fluctua+ng redox condi+ons (Fig. 4D-\nE). While HIFs that today compose the OSM of modern animals contain\ncomponents (e.g., genes, domains, or proteins) that can be traced back to\nthe origins of yeast and even to prokaryotes(Hammarlund, Flashman et\nal. 2020 and references therein), its building blocks expand within and\nbetween early branching animals, including poriferans, cnidarian, or\nmolluscs. Oxygen sensing in animals with e.g., only P564 would be simpler\nthan in animals e.g., with P564 and P402 or with P564 and CTAD. This\ncould separate Placozoa (pOSM) from Cnidaria (eOSM), or species with\npOSM from eOSM within cnidarians or bilaterians; whether living in the\nEdiacaran(Dunn, Liu et al. 2021) or Cambrian (Fig. 4F). Components to the\nOSM like the P402, CTAD, NTAD and Factor Inhibi+ng HIF (FIH) regulate a\nwide scope of target genes at a wide range of condi+ons, some of them\nwith clear importance for development(Hammarlund, Flashman et al.\n2020). […]”\nFigure 4 – as I have said in every review of this paper, I firmly feel that the evolutionary\ntimelines invoked for different metazoan lineages here is very out of date. For example,\nwhen we look at the Bilateria illustration in Fig 4F, what about the Ediacaran\nbilaterians? Dickinsonia, Spriggina and bilaterian trace makers are all fully disregarded\nhere so far as I can tell. Similar stories can be applied to your Cnidaria and Porifera\nextents.\nYes. We now agree with this point and see how others have painstakingly mapped\nanimal evolution in the deeper Neoproterozoic. Indeed, the acknowledgement of work\nthat places total-group Eumetaoza into the mid-Cryogenian (Philippe, Derelle et al.\n2009, Telford, Budd et al. 2015, Dunn, Liu et al. 2021) forces us to view the di^erences in\noxygen sensing mechanisms, not only between Cnidaria and Bilateria, but also between\nProtostomia and Deuterostomia. A more careful mapping of the diversity of OSM also\nwithin Bilateria (using e.g., Mills and Canfield 2014, Graham and Presnell 2017, Song,\nModjewski et al. 2022) not only makes us realize that the previous representation in\npanels E-F were too general and simplistic. Instead, the comment and reading certainly\nmakes it so clear that the argument and mechanism we wish to put forward are those of\nselection, adaptation and diversification. See also Table S4 for comparison of what\nthese analyses gave in 2017, 2018, and today.\nIn response:\n• We have re-made panel E and F. In E, we’ve only depicted the conditions that we\nargue contribute to the mechanism at the PC/C boundary (dashed). We aim for\nthis dashed box to fill emphasize that we by no means make claims to accurately\ntime the Neoproterozoic roots of animal evolution.\n• We depict the simplified phylogeny of animal evolution in Panel F. This phylogeny\nstretches into the Neoproterozoic (of panel D and in Figure 1) but without an\nadjacent exact time line. The nodes are built on Telford et al., 2015 and with\nnodes placed in general terms from the Dunn et al., 2021 paper. We feel the\nupdated Panel F serves two purposes: to make a more current representation of\nthe deep roots of metazoan lineages plus that the diversity of OSMs are much\nmore than just having HIF-a or not.\nLines 482 onwards – refer to above and previous comments on how out of date I feel\nthis discussion of different lineages is. I strongly recommend that this would have to be\nupdated to represent current understanding of the history of these lineages in order for\nthis manuscript to be published in this form. I have provided detailed arguments on this\nin previous reviews and really don’t understand why the authors are so resistant to\ndoing this. If they fundamentally disagree with the literature that I cite then it would be\nuseful to justify this in the text and put forward an argument that is neutral to these\nreconstructions. Currently this out of date discussion just weakens a perfectly good\nmodelling study in my opinion, and will only frustrate readers in the Neoproterozoic\ncommunity. Having spent the time to outline these arguments to the authors multiple\ntimes between my four reviews of this manuscript, I honestly am quite frustrated that\nthey have not taken the small amount of time it would take to incorporate this more up\nto date discussion into their narrative.\nAgain, we appreciate the patience of the reviewer to prod us to pay attention to the\nadvancements in the Neoproterozoic literature. After reading this literature better, we\nfully agree and have gone over the manuscript with a new lens at hand. Apart from\nchanges to” late Neoprotererozic-early Paleozoic” throughout (see above), we also add\nnotes to the redrawn phylogeny and expansion of pOSM and eOSM species in both the\nEdiacaran and Paleozoic:\n• Oxygen sensing in animals with e.g., only P564 would be simpler than in animals\ne.g., with P564 and P402 or with P564 and CTAD. This could separate Placozoa\n(pOSM) from Cnidaria (eOSM), or species with pOSM from eOSM within\ncnidarians or bilaterians; whether living in the Ediacaran (Dunn, Liu et al. 2021) or\nCambrian (Fig. 4F).\n• The divergence of animal clades (one of several possible time-calibrated trees ,\nbased on (Philippe, Brinkmann et al. 2011), with approximate divergence\nbetween lineages based on (Telford, Budd et al. 2015) and (Dunn, Liu et al.\n2021)). Boxes represent early branching clades and Bilateria that today perform\ncellular oxygen sensing mechanisms with the Hypoxia-Inducible Factor (HIF)\nsystem (filled orange) or not (grey), based on (Graham and Presnell 2017, Mills,\nFrancis et al. 2018). The positions of Ctenophora and Placozoa are based on\n(Philippe, Brinkmann et al. 2011).\nLine 511 – “time, a cascade of physical, functional, and ecological factors aligns\ndirectly with the Cambrian Period.” I would argue this is patently untrue. Why overreach\nlike this? There is a cool underexplored mechanism here that you have the opportunity\nto tell the reader about. Why frustrate the reader by making claims like this that are\nclearly dismissed by a quite surficial understanding of the literature?\nThanks yes, we see this now. This is now changed to “The underexplored mechanism for\nthe selection of a cellular machinery to handle redox fluctuations can be inferred within\nthe framework of physical, functional, and ecological changes in the end-\nNeoproterozoic and early Palaeozoic.”\nFig 5 caption – see notes above.\nAlso here, the text and labels in the figure are updated. Thanks for these comments.\nReviewer #4 (Remarks to the Author):\nThe authors have done a reasonable job of addressing the substantive points from my\nprevious reviews. I would be happy to see this manuscript published following a few\nminor revisions which I still think are necessary and which I hope the authors find\nhelpful.\nWe are grateful to the reviewer´s comments here and previously. We have addressed\nthese comments in full, as explained below.\nThere are some line-by-line comments below, but first I would strongly encourage the\nauthors to fully revise the text to separate their general, first order, modelling results\nfrom their interpretation of how this would play out in the Neoproterozoic-Phanerozoic\ntransition. In particular, this is very generally applicable, first order modelling. At\nvarious places, the text could be clarified to show that:\na. The first order modelling of oxygen concentration cycles was done for a range of T,\nTOC, and [O2] conditions which could be interpreted broadly in a geographical and\ntemporal way (e.g. for various places on present-day Earth or times in the geological\npast). Keeping this distinction clear throughout would help emphasize the broader\napplicability of the concepts modelled here and would widen the appeal of the study.\nWe agree and have made an e^ort to separate results from interpretations as well as\nmaking them time-agnostic. For example, in the section on the biogeochemical model\nand results, we made the following changes:\n• Row 166: Removed ‘Cambrian’ for a later reference to ‘(early Paleozoic)’.\n• All references to Cryogenian/Cambrian climate are replaced with cold or warm.\n• Rephrased the text to be^er describe the basic func+onality of the biogeochemical\nmodel used to quan+fy sediment O2 dynamics (l. 165-186).\n• Rephrased the text to make the immediate modelling results more clear in a non-\ninterpreted way based on the basic biophysical responses (l. 188-238), e.g. (l.\n188) “These first-order experiments, we found that in an icehouse scenario, daily\noxygen fluctuations and diel amplitude would have been modest with weak oxic\nconditions during the day and with hypoxic conditions during night lasting for ~11\nh (Fig. 3A, 3C). In the historically later greenhouse scenario, daily fluctuations\nwere profoundly amplified and would have demonstrated abrupt changes from\nfully oxygenated conditions during daytime to true anoxia during nighttime in less\nthan 0.3 h (Fig. 3B, 3D).”\n• Edited to row 412: “By applying two lines of first-order reconstructions”.\nb. The first order modelling of pOSM and eOSM species could also be shown as of\nmuch broader relevance than it is currently presented as. In particular, the authors\nacknowledge that there are different differences in OSM efficiency between different\nanimal clades (bilaterians, cnidarians, placozoans, poriferans, ctenophores). The focus\nin the results section on presenting this work as predominantly about the differences\nbetween bilaterians and other animals hides the wider implications which could be\nseen more broadly in modern and other paleo-ecological settings. To keep to the early\nanimal example, such an approach could be applied to the redox oscillations (on a\ndifferent time scale, but same principle) of the later Ediacaran world, rather than just\nthe Cambrian.\nWe fully agree and the comments from both the reviewers have encouraged us to revise\nthat previously simplistic view. Indeed, the figure panel 4F is re-drawn to emphasize how\nmuch the components of OSM di^er between, but also within, animal groups. The text is\nalso changed to capture this. For example, at row 343, we summarize the results with\nthe following changes to the text:\n“The population and phenotype model is agnostic to which organisms would\nrepresent pOSM or eOSM species, which could be, for example, Porifera vs\nCnidaria, Cnidaria vs Bilateria, or invertebrates vs vertebrates within Bilateria.\nHere, we suggest that adaptations of oxygen sensing mechanisms can have been\nselected for within animals at events that led to specifically periodically\nfluctuating redox conditions (Fig. 4D-E). While HIFs that today compose the OSM\nof modern animals contain components (e.g., genes, domains, or proteins) that\ncan be traced back to the origins of yeast and even to prokaryotes41 and references therein,\nits building blocks expand within and between early branching animals, including\nporiferans, cnidarian, or molluscs. Oxygen sensing in animals with e.g., only P564\nwould be simpler than in animals e.g., with P564 and P402 or with P564 and CTAD.\nThis could separate Placozoa (pOSM) from Cnidaria (eOSM), or species with\npOSM from eOSM within cnidarians or bilaterians; whether living in the\nEdiacaran17 or Cambrian (Fig. 4F). Components to the OSM like the P402, CTAD,\nNTAD and Factor Inhibiting HIF (FIH) regulate a wide scope of target genes at a\nwide range of conditions, some of them with clear importance for development41.”\nFinally, I do hope to see this published soon and I think the authors are very nearly there\nwith it.\nMuch appreciated, thanks so much for all the input.\nLine-by-line comments:\nLine 9: Proterozoic-Phanerozoic, or Neoproterozoic-Paleozoic, would be preferable to\nPrecambrian-Cambrian.\nWe agree and have changed to Neoproterozoic-Paleozoic here, plus edited ‘PC/C’ also\nonwards in the manuscript.\nLines 27 to 28: ditto Line 9.\nThanks, yes. Edited here and throughout the manuscript.\nFig. 1: Please define the dashed box label “ROI”.\nDefined.\nLine 51 (Fig. 1 caption): rephrase to make one sentence about the blue-red transition; it\ndoesn’t currently flow. E.g. “+25 °C marking a transition from icehouse to greenhouse\nclimate states”. Otherwise, the caption is much improved, thanks.\nEdited.\nLines 72 to 74: climatically sensitive lithologies have also contributed substantially to\nthis picture (e.g. Boucot, A.J., Xu, C., Scotese, C.R. and Morley, R.J. 2013. Phanerozoic\nPaleoclimate: An Atlas of Lithologic Indicators of Climate, 1st ed. SEPM Concepts in\nSedimentology and Paleontology11).\nThanks, yes. Now added.\nLines 74 to 76: something has gone wrong here, I think. Please check for grammar and\npunctuation.\nIndeed, yes. The sentence is now changed.\nLine 109: “ctenophores” or “Ctenophora”.\nEdited.\nLine 117: this paragraph is good but, as someone who is not a biologist or biochemist, I\nonly follow the implications of it through having already reviewed the paper. At Line 117,\nyou could be a bit more explicit that each of the additions to the HIFa pathway\nincreased its efficacy and so represent step-wise acquisitions of O2 sensitivity across\nthe animal tree.\nThanks, we can see this now. We have edited the last part of the paragraph as follows:\n“These animals share a joint core to this sensing by an oxygen dependent\ndegradation domain (ODDD) of the bHLH-PAS protein where a proline can be\ntargeted by a PHD protein (if O is present) and hinder gene transcription (see\n2\nalso Supplementary Information). However, the components of the pathway vary\nin their presence within animal groups and in terms of how they sense and\nrespond to oxygen fluctuations. For example, an additional C-terminal\ntransactivation domain (CTAD) can enable the protein to bind with transcriptional\ncoactivators of and a factor inhibiting HIF (FIH) can limit the regulation of\ngenes.21 Di^erences in oxygen sensitivity and regulatory roles are also described\nfor the two di^erent prolines (P564 and P402) targeted by PHD, and an N-\nterminal transactivation domain (NTAD).21”\nFig. 2: I reiterate that the maps in A and B should have the same colour scales – they are\nrepresenting different temperatures of water mass, but still water mass. At present, this\nlooks more like topography than flooded continental area and mis-represents that the\ncontinents were not completely flooded in the Cambrian. I still think the maps add\nconfusion as this is not a latitude, or even geography, based study, but more about\nflooded continental shelf. The onshore-offshore profiles show this better. This is\nparticularly pertinent as the next section talks immediately about a 1D model, no\nhorizontal (map) resolution needed.\nWe concur and have removed the upper visualization of the flooded continents.\nLine 168: “or anoxia” is superfluous. Just need “O2 concentration.” As levels\noxygenation are defined below (lines 172 to 174).\nThanks, yes. This is edited now.\nLines 168 to 169: I don’t follow the grammar of “Since quick O2 changes require benthic\nanimals to conform their ATP-turnover”. Should this be “conform to their ATP-turnover\nrate”? or something else?\nThanks, and we see the confusion. Indeed, we meant to say (and have now changed the\nsentence to): “Since quick O changes can be physiologically stressful to benthic\n2\nanimals where adjustments to their ATP-turnover are less quick, we also measure the\ntransition time between day- and nighttime conditions.”\nLines 176 to 178: I thought from their last response that the authors were refraining\nfrom calling the scenarios “Cryogenian” and “Cambrian” and instead sticking to\n“cold/warm/hot” or “icehouse/greenhouse” and then interpreting these later as\nreflecting Cryogenian and Cambrian. Presenting it in this way would emphasize that this\nis a first-order simulation experiment, not an actualistic one, particularly as some\nCryogenian intervals were quite possibly greenhouse or super-greenhouse climate\nstates. This would also emphasise the broader applicability of the work: this is just\nlooking at pO2 variation under different T, TOC, [O2] conditions – these apply in the\nmodern world (e.g. by ocean basin, by latitude) and in numerous other deep-time\ncontexts. The generality of this is a strength that could be much better shown.\nYes, we completely agree (now) and have edited throughout the manuscript to avoid\nthese premature associations. Here in this part, all Cryogenian or Cambrian are\nstricken.\nFig. 3: I again urge the authors to make this figure easier to follow and assess. It should\nbe split up or made bigger, or something. I also suggest again plotting the blue and\nyellow bands in Fig 3E either on the same scale or on separate plots: their numeric\nrange is two orders of magnitude different and it is still not obvious without the caption\nwhich coloured envelope relates to which y-axis scale, particularly as the same blue\nand yellow are used for different oxygen concentrations in figs 3A & 3B.\nWe see the reviewer’s point and we have therefore:\n• Changed the color coding to be the same as in the A-B modeling results.\n• Moved each field closer to its y-axis to clarify.\n• Emphasized in the caption that the data is primarily represented by the symbols\n(not the fields).\n• We have increased font size of labels to 16 pts.\nLine 319: “d >= 0.01” includes “d >= 0.09”. I think a “, respectively” is needed to clarify\nthat these values relate to stochastic and periodic fluctuations, respectively?\nThanks for the sharp eye that in fact made us see a mistake in the interpretation of the\ncompetition experiment results (previously also noted by the reviewer as confusing).\nTherefore, we have now revisited the results and both changed and expanded on this\ndiscussion. As seen in Fig. S8, it is clear that eOSM can hold the cost of a more e^icient\noxygen sensing mechanism in specifically environments with periodic and stochastic\nfluctuations. In environments with only period fluctuations, eOSM can carry a fairly high\ncost (until 0.08). In contrast, in environments with only stochastic fluctuations, the cost\nof an e^icient oxygen sensing mechanism is soon too high and would favor pOSM. We\nare very grateful for the poke to revisit this experiment, since it emphasizes the potential\nrole for period fluctuations to have another evolutionary force than the stochastic\nfluctuations that we have discussed more in the field (e.g., e^ects of upwelling etc).\nChanges in short:\n• Row 329-335 is rewritten.\n• In the caption of the modeling results, we emphasize that a total of 33\ncompetition simulations were run, across three environmental conditions and\neleven di^erent costs of the oxygen sensing mechanism (d) and that – for each of\nthese simulations – the competition experiment is run until one of the species\ngoes extinct while the other remains extant.\n• We have updated the Figure S6, so that periodic fluctuations is denoted with an X\ninstead, and is therefore visible also when below the circle.\n• We have also added the code for these competition experiments in SI.\nLine 332: “cnidarians” (lowercase “c”) or “Cnidaria”.\nThanks, this is now edited together with the sentence.\nLines 326 to 342: here is an opportunity to illustrate the impact of stepwise acquisition\nof increasingly ‘e’OSMs, with each clade being slightly more ‘e’ than those earlier\nbranching clades. I think I made a similar point before, but here the key is not the\n‘bilaterians are best’ but that each clade made some slight improvements in their OSM.\nThe text of this paragraph could again be strengthened by saving until the end of the\nparagraph the specific case of early animal evolution through the Neoproterozoic-\nPhanerozoic transition and keeping the initial interpretation as hypothetical discussion\nof organisms with less (‘p’) or more (‘e’) efficient oxygen sensing mechanisms.\nWe are grateful for this view and comment. We have now added to the paragraph after\nmodeling results:\n“The population and phenotype model is agnostic to what organisms would\nrepresent pOSM or eOSM species. The pOSM vs eOSM could be, for example,\nPorifera vs Cnidaria, Cnidaria vs Bilateria, invertebrate bilaterian vs vertebrates,\nor any combination within animal groups.”\nWe also edited that “adaptations of rudimentary oxygen-sensing capacities gave certain\nanimals a competitive advantage”. We have also updated Figure 4E with a more detailed\nschematic of the di^erences within OSM of animals.\nFig. 4: This is a lot better, and I really like the addition of panel F. I do again ask about\nthe colour variation in the diel bar – are we to read anything into the yellow-grey-green\ntransitions and their placement?\nWe are grateful to the reviewer pointing this out again; a point that we know has\nremained important, but hard to visualize. The reviewer’s point made us rotate the\ndirection of the color variation 90 degrees. The distinct color, aimed to visualize that\ndaily benthic O fluctuations were severe, is now stretching over the entire time frame\n2\nthat we define as our region of interest (ROI). A contrasting color bar for modest daily\nbenthic oxygen fluctuations (imagine less distinct color differences) would likely bring\nthis point home more clearly, especially if placed in at the end of the Cryogenian.\nHowever, we hesitate to expand indications for both temperatures or metabolic energy\noutside of our ROI since it adds uncertainty. We think the least uncertain assumptions,\nthat global temperatures were lower at the end of the Cryogenian, is clear enough from\nFigure 1. We hope that you agree.\nFig. S1: this is really hard to interpret, particularly for anyone who has difficulty with\ntheir colour vision. I would urge the authors again to simplify this and use colour\npalettes that are robust to common colour vision deficiencies.\nYes. We have now changed the color of the Mills et al., record to just dark gray (removed\nour coloring). Also, we added direct links to both manuscripts (Mills et al., and also\nBergmann et al.,) so that comparison is easier for the reader."}, {"round": 2, "reviewer_comments": {"reviewer_3": "Firstly, apologies to the authors for the delay in submitting my review at the end of a busy year. I am genuinely really pleased to see the changes that the authors have made in response to my comments and am glad that some directness seems to have paid off in this case. I think the increased generality and nuance here will (despite making broader claims) lead to a much more well received manuscript when the community have the opportunity to read it. Now that the authors have addressed so many of my key (hopefully constructive!) criticisms, I am happy to recommend this manuscript for publication in Nature Communications without any further rounds of review.", "reviewer_4": "Firstly, the text is much clearer this time round, and I was pleased to read an improved version. Secondly, I don’t have a problem with the fundamental science of this work (though I remain perplexed about why the results of the 40 C simulations are not really included), and I think it does have the broad relevance suitable for Nature Communications. Thirdly, however, I do still feel that there are improvements needed regarding how the work is contextualised and presented, but I think most of these are more personal preference than strictly necessary.\nTo reiterate a point from my previous reviews, this is a first-order one dimensional modelling study that has very broad applicability to evolutionary biology and ecology, and that is a major advantage of this work. I think this broad relevance still gets lost in (quite a lot of) unnecessary introduction/discussion about the Neoproterozoic/Cryogenian and Palaeozoic/Cambrian.\nA point that emphasises this is that my immediate thought on reaching line 94 (“Physiological stress is a potential driver of the emergence of evolutionary innovations…”) was “here is the start of the introduction”. The majority of the essential information for the introduction is in lines 94 to 151, with then one or possibly two paragraphs of Neoproterozoic-Phanerozoic transition context at line 140. Setting the work up in this way would help readers understand that this has broader applicability and that an interesting application of this work is to the Proterozoic-Phanerozoic transition. A similar point applies to the current framing of the discussion which could be reorganised to lead with and emphasise the general results of the study and then discuss what this would mean in the case of the Proterozoic-Phanerozoic transition.\nSome specific points (using the line numbers in the merged pdf 508067_2_merged_1731277702.pdf):\nLines 6-7: “… the mechanisms linking these environmental conditions to physiology and then early organismal ecology are unclear.” Unless making a very general point that organisms’ (animals? see next comment) physiology provides the mechanistic link between environment and ecology, in the context of this study I understand this point to imply an strong environmental control on early animal diversification, which is an unproven assumption; if it’s my mistaken assumption then apologies for that. I would suggest rephrasing this sentence to be more circumspect about whether there was an environmental control. This is something the authors do actually go on to show a mechanism for.\nLines 6-7: “… the mechanisms linking these environmental conditions to physiology and then early organismal ecology are unclear.” Do the authors here mean “organismal” or “animal”, in the context of their study?\nLine 47: “temporal extent of potentially global glaciations of the Cryogenian”? The geographic extent of Cryogenian glacial conditions is not a settled topic.\nLine 47: “(blue vertical fields)”? To distinguish from the blue colour in the temperature profile.\nLine 51: “surface air temperature”?\nLine 64: suggest deleting “alone”; its current inclusion suggest that there was a greater than four-fold increase in shallow settings globally, which may or may not be true but can’t be inferred from one continent alone. It would be okay to keep if the authors referred to an area rather than a relative proportion.\nLine 67: suggest also citing Peters & Gaines (currently ref 23) as they also drew the link with Sr isotopes, and consider also mentioning Nd isotopes which may tell a similar story.\nLine 81-2: (a) if including oxygen isotope evidence, I would suggest including reference to Wotte et al. (2019) as well; (b) for modelling, I would suggest referencing Nardin et al. (2011) who modelled late Cambrian climate (reference on line 79 is to the whole Cambrian Period) and at least one of Scotese et al. (2021) or Valdes et al., (2020) who modelled Phanerozoic-scale temperatures including the Cambrian; (c) I would suggest also citing Wong Hearing et al. (2021) (already ref 37) here because reference is made to both climate modelling and climatically sensitive lithologies.\nLine 113: please spell out “PAS” at first use.\nLine 118: what different points are citations 43 and 46 referencing here? (i.e. why are they split around “(Fig. 1C)”?)\nFig 2: much better. Please make sure all abbreviations are spelled out in the caption.\nLines 150-1: stick with one or the other of “and”/”or” for “effective and poor”/(“eOSM or pOSM”).\nLine 156: “interface”\nLine 161: Following the points above about this being a general study, not need to reference Cambrian daylength in the caption.\nLine 182: purely personal preference, but I’d suggest “day” and “night” rather than “daytime” and “nighttime” here and elsewhere.\nLine 192-5: I appreciate the removal of “Cryogenian” and “Cambrian”, but I would also avoid using “icehouse” and “greenhouse” here. I would suggest sticking to cold/cool/warm/hot as this could also reflect latitudinally different temperatures. The very generally applicable modelling work could be used to infer something about e.g. different locations in the modern ocean, like the difference between a polar setting with a steep bathymetric gradient and tropical setting with a wide shallow sea.\nLine 199: consider a different descriptor (e.g. “hot” rather than “warm”) for the 40 C runs. There is a lot of difference between a 25 C ocean and a 40 C ocean, and one might expect major mortality at 40 C.\nFig 3: I again ask the authors why they have not included the 40 C simulations as z-t-O2 space plots?\nFig 3E: I make one last attempt to ask the authors to clarify Fig 3E. As I said before, the y-axis scales are very different, the dashed versus solid outlines are almost impossible to differentiate, and although it’s slightly more intuitive with one colour abutting each axis, it is still not explicit which axis applies to which symbol set or colour.\nLine 235-240: these sentences are purely discussion, not results, and are not needed here.\nLine 273-4: do the authors mean “growth of an individual” or “increase in population size”? Not entirely clear at present.\nLine 354-377: from “which could be, for example, …” is pure discussion material. I would suggest moving all of it to the Discussion.\nLine 419: I think “+/-“ symbol in this context is “approximately” rather than “positive 5 or negative 5 degrees C”? Suggest replacing with “~” or “about”.\nLine 465: all organisms alive now will surely “use refined mechanisms”; surely here the meaning is that\nLine 496-7: first clause needs referencing.\nLine 511: I don’t quite follow the grammar here – how does higher productivity offer large areas?\nLine 526: grammar of the inserted text doesn’t quite work.\nLine 533: the climate of the Ediacaran Period is still rather uncertain. Depending on which lines of evidence you follow, there may have been one long (Wang et al., 2023a, 2023b) or multiple shorter (Linnemann et al., 2022; Niu et al., 2024) glaciations in the late Ediacaran. What the climate of the early Ediacaran was like is particularly uncertain.\nLine 580-4: This is a great point and is worthy of a bit more here.\nFig 5: something odd with the colouring of the mid-Neoproterozoic panel – blue for sky and for water?\nFig. S1: this is still requite hard to follow, though removing the red has helped. Again, I would suggest simplifying this, or making it a multi-panel plot."}, "author_response": "Point by Point Response to Reviewers’ comments\nReviewer #3 (Remarks to the Author):\nFirstly, apologies to the authors for the delay in submitting my review at the end of a busy year. I am genuinely really pleased to see the changes that the authors have made in response to my comments and am glad that some directness seems to have paid off in this case. I think the increased generality and nuance here will (despite making broader claims) lead to a much more well received manuscript when the community have the opportunity to read it. Now that the authors have addressed so many of my key (hopefully constructive!) criticisms, I am happy to recommend this manuscript for publication in Nature Communications without any further rounds of review.\nWe are very grateful to the reviewers very constructive comments and continuous work to help us improve the manuscript. We are also happy that the reviewer considers the changes as sufficient and the current version suitable for publication. We agree it may be well received by the community.\nReviewer #4 (Remarks to the Author):\nFirstly, the text is much clearer this time round, and I was pleased to read an improved version. Secondly, I don’t have a problem with the fundamental science of this work (though I remain perplexed about why the results of the 40 C simulations are not really included), and I think it does have the broad relevance suitable for Nature Communications. Thirdly, however, I do still feel that there are improvements needed regarding how the work is contextualised and presented, but I think most of these are more personal preference than strictly necessary. To reiterate a point from my previous reviews, this is a first-order one dimensional modelling study that has very broad applicability to evolutionary biology and ecology, and that is a major advantage of this work. I think this broad relevance still gets lost in (quite a lot of) unnecessary introduction/discussion about the Neoproterozoic/Cryogenian and Palaeozoic/Cambrian. A point that emphasises this is that my immediate thought on reaching line 94 (“Physiological stress is a potential driver of the emergence of evolutionary innovations…”) was “here is the start of the introduction”. The majority of the essential information for the introduction is in lines 94 to 151, with then one or possibly two paragraphs of Neoproterozoic-Phanerozoic transition context at line 140. Setting the work up in this way would help readers understand that this has broader applicability and that an interesting application of this work is to the Proterozoic-Phanerozoic transition. A similar point applies to the current framing of the discussion which could be reorganised to lead with and emphasise the general results of the study and then discuss what this would mean in the case of the Proterozoic-Phanerozoic transition.\nWe are grateful for the effort and very constructive comments that the reviewer provides. Regarding the +40C scenario, we reply in full below. In essence, based on the step between +5C and the +25C step also represents the presumed increase in TOC loading, we evaluate that a second step to +40C does not add much information or power to visualize the suggested change in temperature-driven redox fluctuations, see figure for comparison below. (The probability of all factors aligning to the suggested change in temperature-driven redox fluctuations is also evaluated in the Monte-Carlo simulation). In the now revised manuscript, we agree that the reviewers suggestions to e.g., remove and restructure text improved the structure. We describe these changes in detail below.\nSome specific points (using the line numbers in the merged pdf 508067_2_merged_1731277702.pdf):\nLines 6-7: “… the mechanisms linking these environmental conditions to physiology and then early organismal ecology are unclear.” Unless making a very general point that organisms’ (animals? see next comment) physiology provides the mechanistic link between environment and ecology, in the context of this study I understand this point to imply an strong environmental control on early animal diversification, which is an unproven assumption; if it’s my mistaken assumption then apologies for that. I would suggest rephrasing this sentence to be more circumspect about whether there was an environmental control. This is something the authors do actually go on to show a mechanism for.\nThanks yes, we can see that straw man now. We have rephrased this sentence to\n‘Animal diversification mirrors an expansion in marine shelf area under a greenhouse climate, though the extent to which these environmental conditions directly influenced physiology and early organismal ecology remains unclear.’.\nLines 6-7: “… the mechanisms linking these environmental conditions to physiology and then early organismal ecology are unclear.” Do the authors here mean “organismal” or “animal”, in the context of their study?\nGood point. With ‘organismal’, we mean to also include how also e.g., benthic microalgae were influenced by the expansion and, therefore, interacted with the ecology of animals and other organisms. Although the double meaning with organismal may not be entirely clear here in the abstract, we leave the sentence as is to possibly include also the other organisms.\nLine 47: “temporal extent of potentially global glaciations of the Cryogenian”? The geographic extent of Cryogenian glacial conditions is not a settled topic.\nYes, changed.\nLine 47: “(blue vertical fields)”? To distinguish from the blue colour in the temperature profile.\nYes, changed.\nLine 51: “surface air temperature”?\nYes, changed.\nLine 64: suggest deleting “alone”; its current inclusion suggest that there was a greater than four-fold increase in shallow settings globally, which may or may not be true but can’t be inferred from one continent alone. It would be okay to keep if the authors referred to an area rather than a relative proportion.\nAgreed, changed.\nLine 67: suggest also citing Peters & Gaines (currently ref 23) as they also drew the link with Sr isotopes, and consider also mentioning Nd isotopes which may tell a similar story.\nYes and thanks for the mention on Nd isotopes. Both Peters & Gaines 2012 and Wei et al., 2019 are added.\nLine 81-2:\n(a) if including oxygen isotope evidence, I would suggest including reference to Wotte et al. (2019) as well;\nAgreed and added.\n(b) for modelling, I would suggest referencing Nardin et al. (2011) who modelled late Cambrian climate (reference on line 79 is to the whole Cambrian Period) and at least one of Scotese et al. (2021) or Valdes et al., (2020) who modelled Phanerozoic-scale temperatures including the Cambrian;\nThanks for all three of these, where the Valdes et al was new to us and much appreciated. Nardin and Valdes are added.\n(c) I would suggest also citing Wong Hearing et al. (2021) (already ref 37) here because reference is made to both climate modelling and climatically sensitive lithologies.\nAgreed and added.\nLine 113: please spell out “PAS” at first use.\nChanged.\nLine 118: what different points are citations 43 and 46 referencing here? (i.e. why are they split around “(Fig. 1C)”?)\nGood point. The Semenza reference (44) refers to a first description of the role of HIF-1a for responding to variations in oxygen concentrations (although fairly unspecific) and the Loenarz reference (47) to a first mapping of HIF-1a across the animal clade. We have now moved them together. Changed.\nFig 2: much better. Please make sure all abbreviations are spelled out in the caption.\nYes, we have added explanations to the model parameters: Model parameters are atmospheric oxygen (ppO ), temperature (T), salinity (S), production Q10 (Q10p),\n2\nrespiration Q10 (Q10r), and TOC adjustment factor (c ).\noc\nLines 150-1: stick with one or the other of “and”/”or” for “effective and poor”/(“eOSM or pOSM”).\nCorrected now, to ‘or’.\nLine 156: “interface”\nEdited.\nLine 161: Following the points above about this being a general study, not need to reference Cambrian daylength in the caption.\nPoint taken and both the description and reference are deleted (albeit moved to its explanation in SI).\nLine 182: purely personal preference, but I’d suggest “day” and “night” rather than “daytime” and “nighttime” here and elsewhere.\nChanged throughout, and we agree the text got crisper.\nLine 192-5: I appreciate the removal of “Cryogenian” and “Cambrian”, but I would also avoid using “icehouse” and “greenhouse” here. I would suggest sticking to cold/cool/warm/hot as this could also reflect latitudinally different temperatures. The very generally applicable modelling work could be used to infer something about e.g. different locations in the modern ocean, like the difference between a polar setting with a steep bathymetric gradient and tropical setting with a wide shallow sea.\nFair enough. After the change, I can see the sentences may have a longer lifespan.\nLine 199: consider a different descriptor (e.g. “hot” rather than “warm”) for the 40 C runs. There is a lot of difference between a 25 C ocean and a 40 C ocean, and one might expect major mortality at 40 C.\nYes, changed.\nFig 3: I again ask the authors why they have not included the 40 C simulations as z-t-O2 space plots?\nCertainly, and we discussed it internally over the review process. First, we have felt that the hot scenario is pushing the limits on expected, realistic surface ocean temperatures and may open unwanted discussion on whether the ocean could actually have surface water of +40C (and whether the clumped isotope data is reasonable etc). Although the reviewer has a fair point that this is a modeling exercise, we may also want to depict a change in fluctuations that can be imposed on an imagined sediment-water interface. Also, we evaluate that the major shift in temporal redox shifts occurs especially between +5 and +25 degrees, where we also replicate the presumed increase in TOC. If we use the higher TOC load for +40C, the model results are compared below. Although we do not think that this comparison adds information to the main manuscript, we share the model and look forward to the model´s future use ; including further exploration of other temperature-driven changes.\nFig 3E: I make one last attempt to ask the authors to clarify Fig 3E. As I said before, the y-axis scales are very different, the dashed versus solid outlines are almost impossible to differentiate, and although it’s slightly more intuitive with one colour abutting each axis, it is still not explicit which axis applies to which symbol set or colour.\nYes, we see the risk for an over-filled panel. Since our point is to actually contrast these temperature different patterns, we want to avoid splitting them up into separate panels. However, we have now emphasized the symbols in different color, increased the thickness of the outline, and rephrased the caption.\nLine 235-240: these sentences are purely discussion, not results, and are not needed here.\nRemoved.\nLine 273-4: do the authors mean “growth of an individual” or “increase in population size”? Not entirely clear at present.\nWe agree it was not clear. We mean increase in the population size. Thank you for inviting us to clarify this and we have done so in the manuscript. The per capita growth rate captures the rate at which the population of the ith species grows per individual. So we are talking about an increase in population size, normalized by the total population size. We added an explanation that we at that specific instance refer to a species:\n“where the fitness-generating function, 𝐺(𝑣,𝒖,𝒙), describes the expected per capita growth of a species as influenced by its cellular metabolism phenotype (strategy), v, the strategies of each species in the population, u = (u , u ), and the population sizes of\n1 2\neach species, x = (x , x ).”.\n1 2\nLine 354-377: from “which could be, for example, …” is pure discussion material. I would suggest moving all of it to the Discussion.\nWe thank the reviewer for this comment and do indeed agree that it was, from start, mostly discussion material. However, the last round of revision resulted in an expanded map and analysis of oxygen sensing components across animals that is not published before. To emphasize this new result, we have edited the section. We have also moved the discussion material to the discussion. What remains here is as follows:\n“The population and phenotype model is agnostic to which organisms would represent pOSM or eOSM species. To relate the phenotype model, the flooding event and early animal diversification in the Cambrian Period (Fig. 4D-E) to the early evolution of animals, we mapped diDerences in the composition of HIF-driven oxygen sensing across the animal clade based on sequences in 18 and 19 (Fig. 4F, SI text and Table S4 for details). While HIFs that today compose the OSM of modern animals contain components (e.g., genes, domains, or proteins) that can be traced back to the origins of yeast and even to prokaryotes42 and references therein, its building blocks expand and diDer in functions within and between early branching animals.. For example, oxygen sensing in animals with e.g., only P564 would be simpler than in animals e.g., with P564 and P402 or with P564 and CTAD. Components to the OSM like the P402, CTAD, NTAD and Factor Inhibiting HIF (FIH) regulate a wide scope of target genes at a wide range of conditions, some of them with clear importance for development42. From what we know of the importance of these components in Bilateria59-61, the diDerent configurations amongst animals18,19 reflect varying eDiciency and costs to sense and respond to redox fluctuations.”\nLine 419: I think “+/-“ symbol in this context is “approximately” rather than “positive 5 or negative 5 degrees C”? Suggest replacing with “~” or “about”.\nWe agree. The tracked addition of a ‘+’ in the previous version made it also look as if the ‘+’ sign was underscored. So it was never a “+/-“ sign and remains to be only “+5C”.\nLine 465: all organisms alive now will surely “use refined mechanisms”; surely here the meaning is that\nThanks yes. This is edited for clarity to: “Eukaryotic cells (like those of animals) transport this insoluble but bio-essential ferric iron across its membrane with mechanism like the chelator ferric reductase68.”.\nLine 496-7: first clause needs referencing.\nYes, thanks. References Maxwell et al, Iwai et al., and Lando et al., are added.\nLine 511: I don’t quite follow the grammar here – how does higher productivity offer large areas?\nWe agree this was an unclear sentence. We rephrased to clarify, as follows: “The higher productivity offers extensive habitats and biotic feedbacks where species themselves create environmental heterogeneity that, in turn, promotes the evolution and coexistence of even more species”.\nLine 526: grammar of the inserted text doesn’t quite work.\nWe agree, now edited.\nLine 533: the climate of the Ediacaran Period is still rather uncertain. Depending on which lines of evidence you follow, there may have been one long (Wang et al., 2023a, 2023b) or multiple shorter (Linnemann et al., 2022; Niu et al., 2024) glaciations in the late Ediacaran. What the climate of the early Ediacaran was like is particularly uncertain.\nWe thank the reviewer for pointing this out and have clarified that the timing or pattern of warming of the Ediacaran remains uncertain. Indeed, staggered events of cooling or warming may have further implications for selection of cellular mechanisms to cope with stress. The first part of the sentence now reads: “With the warm climate and enlarged continental shelves of the Ediacaran (as a shift from possibly one79,80 or several 81,82 extensive glaciations) and Cambrian periods, […]”.\nLine 580-4: This is a great point and is worthy of a bit more here.\nWe agree and have clarified and expanded the argument.\nFig 5: something odd with the colouring of the mid-Neoproterozoic panel – blue for sky and for water?\nIn fact, yes. We intend for the color coding to represent cold (blue) or warm (pink) in both air and water at the icehouse versus greenhouse climate. We have clarified that in the caption.\nFig. S1: this is still quite hard to follow, though removing the red has helped. Again, I would suggest simplifying this, or making it a multi-panel plot.\nWe agree and have split these this SI Figure 1 up into two panels, a and b.\nReferences cited:\nLinnemann, U., Hofmann, M., Gärtner, A., Gärtner, J., Zieger, J., Krause, R., Haenel, R., Mende, K., Ovtcharova, M., Schaltegger, U., Vickers-Rich, P., 2022. An Upper Ediacaran Glacial Period in Cadomia: the Granville tillite (Armorican Massif) – sedimentology, geochronology and provenance. Geol. Mag. 159, 999–1013. https://doi.org/10.1017/S0016756821001011\nNardin, E., Goddéris, Y., Donnadieu, Y., Hir, G.L., Blakey, R.C., Pucéat, E., Aretz, M., 2011. Modeling the early Paleozoic long-term climatic trend. Geol. Soc. Am. Bull. 123, 1181–1192. https://doi.org/10.1130/B30364.1\nNiu, Y., Shi, G.R., Zhang, Q., Jones, B.G., Wang, X., Zhao, G., 2024. Ediacaran Cordilleran-type mountain ice sheets and their erosion effects. Earth-Sci. Rev. 249, 104671. https://doi.org/10.1016/j.earscirev.2023.104671\nScotese, C.R., Song, H., Mills, B.J.W., van der Meer, D.G., 2021. Phanerozoic paleotemperatures: The earth’s changing climate during the last 540 million years. Earth-Sci. Rev. 215, 103503. https://doi.org/10.1016/j.earscirev.2021.103503\nValdes, P.J., Scotese, C.R., Lunt, D.J., 2020. Deep Ocean Temperatures through Time. Clim. Past Discuss. 1–37. https://doi.org/10.5194/cp-2020-83\nWang, R., Shen, B., Lang, X., Wen, B., Mitchell, R.N., Ma, H., Yin, Z., Peng, Y., Liu, Y., Zhou, C., 2023a. A Great late Ediacaran ice age. Natl. Sci. Rev. nwad117. https://doi.org/10.1093/nsr/nwad117\nWang, R., Yin, Z., Shen, B., 2023b. A late Ediacaran ice age: The key node in the Earth system evolution. Earth-Sci. Rev. 247, 104610. https://doi.org/10.1016/j.earscirev.2023.104610\nWong Hearing, T.W., Pohl, A., Williams, M., Donnadieu, Y., Harvey, T.H.P., Scotese, C.R., Sepulchre, P., Franc, A., Vandenbroucke, T.R.A., 2021. Quantitative comparison of geological data and model simulations constrains early Cambrian geography and climate. Nat. Commun. 12, 3868. https://doi.org/10.1038/s41467-021-24141-5\nWotte, T., Skovsted, C.B., Whitehouse, M.J., Kouchinsky, A., 2019. Isotopic evidence for temperate oceans during the Cambrian Explosion. Sci. Rep. 9, 6330. https://doi.org/10.1038/s41598-019-42719-4"}]

Decision letter