Simulated heatwave alters intertidal estuary greenhouse gas fluxes

Domain: earth_science
Published: 2025-11-25
Editorial outcome: major_revision

Abstract
Intertidal estuarine habitats are inundated by seawater and uncovered with every tidal cycle, with potential exposure to both marine and atmospheric heatwaves. Little is known about the role of intertidal soft sediment ecosystems in the carbon cycle and how increasing extreme temperature events may affect carbon flux dynamics. Here we conducted a multi-day experiment simulating a low tide atmospheric heatwave at two estuary intertidal flats (sandy/muddy) to test the responses of macrobenthic biodiversity and fluxes of methane (CH4) and carbon dioxide (CO2). Results show heatwave simulation increases CO2 uptake at the sandy site and causes a switch from efflux (source) to influx (sink) of CO2 at the muddy site. Raw CH4 fluxes are unchanged by the temperature treatment but effect sizes relative to controls are greatest in muddy sediments. We provide evidence for cumulative effects of heatwave duration on macrobenthic biodiversity and greenhouse gas fluxes and show that increasing muddiness (often associated with degradation) and increasing duration of heatwave events may change the carbon source/sink status of estuaries.

Introduction
Increasing anthropogenic greenhouse gas (GHG) emissions are changing our planet’s climate and causing losses of biodiversity, ecosystem function, and ecosystem services worldwide1–3. Climate change is altering temperature regimes, and increasing the frequency, intensity and duration of atmospheric and marine heatwaves4–7. Intertidal habitats in estuaries and along the coast are particularly vulnerable because they submerge and emerge (being alternately exposed to seawater and air) during every tidal cycle8. Situated at the land-sea margin, they will also bear the impacts of increased storm events that deliver increased sediments, nutrients and pollution from land and larger waves and tidal forces from the sea.

Temperature modulates all physiological processes and thus governs the functioning and biogeochemistry of ecosystems, including the carbon cycle9. In general, metabolic rates of organisms increase with temperature, but this is constrained by thermodynamics and the thermal sensitivities of particular species10. Climate change is increasing mean temperatures, but extreme events and increased temperature variation are likely to drive ecosystem change as organisms reach their thermal tolerance limits11,12. Short-term extreme events such as heatwaves are strong drivers of ecosystem dynamics and resilience13–15 and are known to have long-term irreversible consequences for coastal populations, communities and their ecosystem functions [e.g. refs. 16,17]. Cumulative effects of prolonged heat exposures are expected to differ from effects of single short-term heat exposures18. Consequently, there have been many calls for studies that provide a deeper understanding of these impacts [e.g. ref. 4]. Intertidal organisms may be particularly vulnerable to extreme temperature events because they are already living close to their thermal maxima19.

Most research on GHG fluxes in intertidal coastal ecosystems has focussed on vegetated habitats (i.e., mangrove, saltmarsh, and seagrass for their ‘blue carbon’ potential20,21). In contrast, relatively little is known about the role of ‘unvegetated’ (microphytobenthos dominated) intertidal flats in the carbon cycle22, and how they will be influenced by higher frequency and intensity of extreme temperature events. Interest in the effects of changes in GHG emissions in intertidal habitats with broad areal coverage is driven by the potential for reinforcing feedbacks. If GHG effluxes (CO2, CH4) increase with increasing temperatures, this could create a feedback that contributes to accelerated climate warming23.

Intertidal flats may be a significant reservoir for carbon22, but their GHG emissions may offset their rates of carbon uptake24,25. The contributions of estuaries in global GHG flux calculations are often limited to water-air fluxes, i.e., excluding sediment-air fluxes in the intertidal zone26–28. For many estuaries, the contributions of intertidal sediments as carbon sinks remain uncertain, hampering efforts to constrain carbon budgets in estuaries with large proportions of intertidal area29. A further complication is that intertidal environments can vary greatly in sediment and habitat types present, tidal ranges, exposure times, and freshwater inputs that are likely to influence GHG fluxes.

Photosynthesis by microphythobenthos is a carbon sink (i.e., CO2 fixation). However, it also generates oxygen as a byproduct, which increases rates of organic matter remineralisation and CO2 release30 relativeto other oxidants. Organic matter remineralisation continues until available oxidants are consumed or all oxidisable organic carbon is utilised. Methane is primarily produced (methanogenesis) in anoxic sediments and is the final step in organic matter degradation producing CH4 and CO2 30,31. Elevated temperature can be detrimental to microphytobenthic biomass and productivity, and can cause changes in community composition32. Photosynthetic capacity of microphytobenthos can increase with temperature33 but may be inhibited at temperatures greater than 25°C34 or 35°C33. Temperature influences CH4 production through direct effects on methanogens, and via indirect effects on carbon mineralisation and substrate availability (because mineralisation increases with temperature, which results in more carbon substrate available and therefore faster depletion of other electron acceptors)35.

There is a lack of data and understanding of the drivers of CH4 and CO2 fluxes in estuarine systems, especially spatial and temporal variability and dynamics36,37. GHG fluxes on intertidal flats are known to have diurnal patterns, and fluctuations in water level have a significant effect on CO2 and CH4 fluxes38–40. Fluxes of GHGs are influenced by temperature, generally because warming increases microbial activity38,40, therefore heatwave simulation is expected to alter CO2 and CH4 fluxes through direct and indirect effects on the microbial community, including the microphytobenthos. The influence of temperature on microbial communities is likely to have a large influence on the biogeochemical response to heating because thermal tolerance and adaptation will vary among species41, which will differentially impact different biogeochemical pathways. For example, net CH4 flux is the balance of activity of CH4-producing methanogens and CH4-consuming methanotrophs, but temperature may affect these processes differently30. Temperature increases have been shown to accelerate organic matter breakdown and increase GHG production42.

Changing sedimentary environments in estuaries, especially increasing bed sediment muddiness (silt + clay content), is the product of intensified anthropogenic land use, which can lead to changes in habitats and species composition, reduced ecosystem functioning, and loss of ecosystem services43–45. Consequently, sediment mud content is used as a proxy for estuary degradation46. Increasing sediment mud content can also reduce ecosystem interaction network complexity, which is an important indicator of ecosystem resilience47–49. Sediment characteristics, especially mud content, are known to be a key regulator of carbon fluxes in tidal flats50. Estuaries, at the interface of freshwater and marine ecosystems and subject to increasing sediment and organic inputs resulting from human activities, may be at risk of rapidly changing GHG emission status (switching from sinks to sources of CO2 51). Methane emissions increase as aquatic ecosystems become more impacted by humans (especially through increases in organic inputs), and they are generally higher in freshwater ecosystems than marine ecosystems31,52. In this study, we aim to evaluate the influence of low tide temperature extremes and subsequent impacts on GHG fluxes in unvegetated estuary intertidal flats with different sedimentary environments.

To investigate the influence of a low-tide atmospheric heatwave event on sediment-air fluxes of CO2 and CH4 we conducted a week-long field experiment using Open Top Chambers (OTCs) to increase low-tide sediment temperatures of two contrasting intertidal flats, one sandy and one muddy. We hypothesised that heatwave simulation would 1) increase the uptake of CO2 by stimulating photosynthetic activity of microphytobenthos (we expect surfaces to be warmed, and photosynthesis therefore enhanced, to a greater extent than within-sediment microbial respiration), and 2) increase the emission of CH4 by stimulating methanogenesis. We also hypothesised that 3) longer exposure to low tide heatwave simulation (i.e. cumulative days) would result in a greater effect on fluxes (i.e. effect size; the difference between control and treatment plot values). We chose heatwave treatment durations based on the definitions of atmospheric and marine heatwaves; events with at least three53 and five days54, respectively. The 5-day treatment duration was used to satisfy the temporal conditions of heatwave definitions, and the longer duration treatment (seven days) provided an extension of this, allowing us to begin to test for cumulative effects of slightly longer heatwave duration. We expected that 4) heatwave simulation would negatively affect the benthic macrofaunal communities through loss of sensitive species. Finally, we postulated that 5) treatment effects (and hypotheses 1–4) would be site-dependent where the muddy site would show less resilience to heatwave simulation than the sandy site.

Results
The OTCs used to experimentally alter temperatures within the sediment surface significantly increased both the mean and maximum temperatures during incubation periods (Supplementary Table 1, Figs. S1–S3). During incubations at the sandy site, sediment temperatures in control plots were on average 24.9°C and increased maximally to 30.3°C, and in treatment plots on average 26.3°C with 36.1°C maximum. During incubations at the muddy site, sediment temperatures in controls were on average 25.3°C and increased maximally to 30.5°C, and in treatment plots, sediment temperatures were on average 27.3°C with maximums up to 36.3°C. Mean sediment temperatures were greater at the muddy site (26.6±0.15°C) than the sandy site (25.8±0.13°C) but there was no difference in the maximum sediment temperature reached at each site. The strength of the treatment effect on mean and maximum temperature depended on Site, as indicated by a significant Site×Treatment interaction (Supplementary Table 1). The weather conditions varied throughout the experiment week, creating variability in the daily mean and maximum sediment temperatures measured in control and treatment plots (Figs. S1–S3, Supplementary Table 1). Similarly, the magnitude of the treatment effect on sediment temperatures varied throughout the week. Since the incubation days were offset between sites (i.e. Day 1 at the muddy site was a day later than the sandy site), the sandy site experienced six consecutive hot days followed by a warm day, whereas the muddy site experienced five consecutive hot days and two warm days (Figs. S1–S3).

Sediment properties were significantly different between sites, with higher sediment mud (Pseudo-F=281, p=0.0001) and organic matter content (Pseudo-F=21.7, p=0.0001) at the muddy site than at the sandy site (Fig. 1c, d, Supplementary Table 2). Seven days of daytime low tide heatwave simulation resulted in a reduction of sediment organic matter content at both sites (Pseudo-F=27.5, p=0.0001), but there was no evidence for this effect after only five consecutive days (Fig. 1c, Supplementary Table 2). Heatwave treatment also resulted in decreased biomass of Chla (Pseudo-F=7.12, p=0.002), with greater effect magnitudes at the sandy site (Pseudo-F=15.8, p=0.0002, Supplementary Table 2, Fig. 1a).

Immediate and cumulative effects of heatwave on carbon fluxes
Sediments at the sandy site consistently showed uptake of CO2. Rates of CO2 uptake increased in the treatment plots over time (Fig. 2a–c, Supplementary Table 3), an effect that did not occur in controls plots (Supplementary Table 4). CO2 flux was not immediately (i.e. after two consecutive days of simulated heatwave) affected at the sandy site, but after five and seven consecutive days of simulated heatwave there was significantly more CO2 uptake by sediments in treatment plots relative to controls.

For the first 5 days of the experiment, sediments at the muddy site were emitting CO2 under natural conditions (Fig. 2d, e). The temperature treatment on average resulted in a switch to net CO2 uptake by the sediments as measured on Days 2 and 5. However, on Day 7 which was cooler than previous days (Figs. S1, S2), control plots were taking up CO2 and experimental warming resulted in higher rates of CO2 uptake (Fig. 2f, Supplementary Table 3). Similar to the sandy site, control plot CO2 fluxes at the muddy site did not differ significantly by day (Supplementary Table 4).

The magnitude of difference (i.e. the treatment effect size) between control and treatment plot CO2 fluxes was significantly greater on Day 5 and 7 than it was on Day 2 at both sites (Fig. 3a, b). Repeated daytime low tide heatwave simulation resulted in a greater amount of CO2 uptake compared to controls at both sites, although the effect was less at the muddy site (Fig. 3a, b, Supplementary Table 3).

Throughout the experiment at both sites, all plots were emitting CH4 (Fig. 4). Under ambient conditions (control plots) fluxes varied significantly by day and were greater at the muddy site compared with the sandy site (Supplementary Table 4). Heat treatment did not have a significant effect on CH4 fluxes at either site at any time during the experiment (Fig. 4, Supplementary Table 3).

Although there was no absolute effect of warming on CH4 fluxes, treatment effects varied with site and heatwave duration (Fig. 3c, d). At the sandy site, the treatment effect on CH4 emissions was negative throughout the experiment and did not change significantly with increasing days of heatwave simulation (Fig. 3c). At the muddy site, there were significant differences in the treatment effect size with increasing heatwave duration (Fig. 3d). Two days of low tide heatwave treatment resulted in greater CH4 emissions than control sediments, but after 5- and 7-days CH4 emissions were less than control plots (Fig. 3c, d).

Relationship between maximum sediment temperature and fluxes
For all plots combined, maximum sediment temperature did not affect CO2 flux, but site, date, and incubation day all had significant main effects (Supplementary Table 5, Fig. 5a). For CH4 flux, there were significant main effects of maximum sediment temperature, site, and incubation day (Fig. 5b, Supplementary Table 5). There was a significant interaction effect of maximum temperature and site on CO2 flux but not CH4 flux (Supplementary Table 5).

Influence of cumulative heating on macrofaunal community structure
The macrofaunal community structure was distinct between the sites, the sandy site had a greater number of taxa per core than the muddy site and a similar number of individuals (Supplementary Table 6, Fig. 1e, f, Fig. 6). The sandy site was characterised by moderate densities of cockles (Austrovenus stutchburyi, ~7 individuals core−1) with other dominant taxa including Halopyrgus pupoides gastropods, and Ceratonereis spp polychaetes. The muddy site was characterised by the presence of burrowing crabs (Hemiplax hirtipes, ~1–2 individuals core−1) and other dominant taxa included Paracorophium excavatum (amphipod), and Arthritica sp. 5 (bivalve). Experimental warming significantly altered the community composition with the effect being dependent on site, and a greater effect on the community composition at the muddy site compared with the sandy site (Fig. 1e, f, Fig. 6, Supplementary Table 6). At the sandy site, community composition on Day 7 was more similar to controls than that on Day 5. The direction of change in community structure for the 5- and 7-day treatments was similar at the muddy site, however at the sandy site, there was obvious divergence between the two treatment durations (Fig. 6). The simulated low tide heatwave did not affect the number of species, but the number of individuals present reduced in both the 5-day and 7-day duration treatments (Supplementary Table 6). Key bioturbating species were negatively impacted by the simulated heatwave; Paracorophium excavatum accounted for the greatest amount of dissimilarity between control and heatwave treatments at both the sandy and muddy sites (Supplementary Tables 7, 8).

Methods
The low tide heatwave experiment was carried out in Waihī Estuary, Bay of Plenty, in Northern New Zealand from 16 to 23 February 2024 (Fig. 7). Waihī Estuary is a barrier enclosed estuary with a large proportion of intertidal area (57%), a catchment composed primarily of low-lying agricultural land use85. It has a high level of freshwater input through channelised agricultural drains, delivering high nutrient loads (488 kg nitrogen and 46 kg phosphorus per year on average) and fine sediment inputs (13.8 tonnes per year)86. The circulation in some parts of the estuary is restricted and some areas in the upper reaches can be described as hypoxic and degraded with high incidence of nuisance macroalgae. Deposition of terrestrially derived fine sediments (‘mud’) and poor flushing in the upper parts of the estuary has resulted in a gradient of sediment type (muddy to sandy).

Experimental design
Two experimental sites with contrasting sediment types (sandy, muddy) ~200 m apart were established in the mid-reaches of Waihī Estuary (Fig. 7a–c). This area contained expanses of relatively sandy and relatively muddy sediment but did not exhibit any other signs of eutrophication. The duration of low tide emergence was approximately 5 h. At each site, a total of 30 plots were established with 10 replicates each for control, short duration (5 days of heatwave simulation), and long duration (7 days of daytime low tide heatwave simulation) treatments. Wooden stakes with temperature loggers (EnvLoggers, Electric Blue CRL) positioned 1 cm below the sediment surface were installed in each plot 2 weeks prior to commencement of the experiment. Each logger recorded the temperature every minute for the entire duration of the experiment. Replicates of each treatment type were arranged in a randomised block design in a 3 column × 10 row array (OTCs 5 m apart) at each site to prevent imbalances in sample allocation with respect to measured or unmeasured environmental heterogeneity across the sites.

Low tide heatwave simulation treatment
To elevate sediment temperature during tidal emersion, Open Top Chambers (OTCs) were placed on treatment plots for ~4 h during daytime low tides every day for five or seven consecutive days (short and long duration treatments, respectively) at each site. OTCs were conical (base dia. 0.8 m, top dia. 0.3 m), constructed of 1.5 mm thick polycarbonate sheeting and were deployed with the bottom edge flush with the sediment. OTCs provided passive warming of the treatment plot sediments but prevented convective cooling by the wind. Control and treatment plots solar radiation levels were similar (Fig. S4), and the conical open top design minimised increases in relative humidity. Temperature differences between control and treatment plots (i.e., the warming effect of the OTCs) were dependent on daily weather conditions. The greatest amount of warming (relative to controls) occurred on sunny days with a breeze, and the least occurred on cloudy, still days. Following low-tide incubations, OTCs were removed so there was no interference with high-tide (or night time low-tide) hydrodynamics, faunal behaviour, immigration or emigration of organisms. To enable time to conduct the sampling required at each site, the establishment of warming treatments was offset by a day between sites; sandy site OTC incubations were from 16/2/24 to 22/2/24 and muddy site OTC incubations were from 17/2/24 to 23/2/24.

Gas flux
CO2 and CH4 gas fluxes were measured in control plots every day at both sites and in OTC treatment plots on days 2, 5, and 7 at each site. Gas fluxes in control plots were measured first so that treatment plots could be measured following at least three hours of OTC incubation. Daily sampling of controls at both sites enabled us to account for weather and avoid confounding site and date in subsequent statistical analyses.

A Li-7810 portable trace gas analyser (LiCor, Nebraska, USA) and static chamber with opaque lid were used to measure fluxes of CO2 and CH4 between the sediment and the atmosphere (i.e. across the sediment-air interface). A circular stainless-steel chamber (40 cm dia., 22.9 L vol.) with a removable lid enclosed a 0.11 m2 area of sediment (Fig. S5). To reduce the influence of sediment disturbance on flux measurements, chamber bases were placed in the plots ~10 min prior to incubations by gently pushing them to a depth of 2 cm. We used a line drawn 2 cm above the lower edge of the chamber base to ensure constant depth and chamber volume between plots. We alternated three chamber bases to reduce the time between flux measurements at each plot to <1 min. Bases contained a trough filled with water to create a seal with the lid, which was connected via tubing to the gas analyser. The concentration of CO2 and CH4 was recorded every second for 4 min (measurement rate 1 Hz, flow rate 300 ml min−1). The Li-7810 gas analyser uses Optical Feedback-Cavity Enhanced Absorption Spectroscopy (OF-CEAS), which utilises the response in the infra-red of the gas being measured. Precision for CH4 at 1 Hz is 0.1 nmol mol−1 while for CO2 it is 0.05 μmol mol−1. The instrument was calibrated before the field campaign using four tanks with CH4 and CO2 amount fractions determined by the World Meteorological Organisation Central Calibration Laboratory; the CH4 is reported on the WMO X2004A scale and the CO2 on the WMO X2007 scale. The accuracy, determined through repeated determinations of a known gas for three-minute averaging, is 0.1 nmol mol−1 for CH4 and 0.1 μmol mol−1 for CO2.

Flux values were calculated using the goFlux package87 in R software v4.3.288. Flux peaks were manually identified using incubation start times and a 30 s deadband period. The goFlux function was used to calculate both a linear (LM) and non-linear (HM) flux estimate, and the best.flux function was used to automatically select the best flux estimate based on criteria scores for model fit (AICc, MAE, g. factor, MDF). In most cases, the HM model provided the best flux estimate. CO2 fluxes were expressed in mmol m−2 h−1 and CH4 fluxes in nmol m−2 h−1.

Sediment and macrofaunal sampling
At the end of the experimental treatment period (Day 5 for 5-day plots, and Day 7 for control and 7-day plots) cores were collected for analysis of sediment grain size, organic matter content, and microphytobenthic pigments (core size: dia. 2.6 cm, depth 2 cm) and benthic macrofaunal community composition (core size: 13 cm dia. 15 cm depth). Macrofaunal cores were sieved over 500 µm mesh, preserved in 70% isopropyl alcohol and stained with Rose Bengal. All individuals were counted and identified to the lowest possible taxonomic resolution (usually species). Sediment cores were frozen and stored at −20°C. Wet sieving (after digestion in 6% hydrogen peroxide for 48 h) was used to measure the cumulative percentage mass of sediment size fractions89. Sediment organic matter content was quantified as loss on ignition (400°C for 5.5 h) after drying to a constant weight (60°C)90. The microphytobenthic biomass of the sediments (i.e., chlorophyll a content) was quantified by extracting chlorophyll a from freeze-dried sediments by boiling in 95% ethanol, then analysing the extract on a spectrophotometer (UV1800; Shimadzu, Kyoto, Japan)91. All sediment and biological samples were collected under the Fisheries New Zealand Special Permit 842-4 to Earth Sciences New Zealand (formerly the National Institute of Water and Atmospheric Research).

Data analysis
Sediment temperature data (from 1 cm below the sediment surface) were trimmed, such that we only analysed data from periods when the OTCs were positioned atop treatment plots at each site. Tests for differences in mean and maximum temperature between sites and treatments was carried out using Permutational Analysis of Variance (PERMANOVA) on normalised data, with Euclidean distance matrices and 9999 permutations92. Pair-wise post-hoc tests (9999 permutations) were conducted where main tests showed significant differences. Tests for differences in sediment properties (Chla, phaeophytin, mud content, organic content) were similarly performed.

To analyse the effect of heatwave treatment on fluxes, treatment plot flux data were combined (i.e., 5-day and 7-day plots) and separate PERMANOVAs were conducted for CH4 and CO2 fluxes for each site for Days 2, 5 and 7. Treatment plot data were combined because at day two and five all OTC plots were not statistically different. To understand the treatment effect on fluxes, the magnitude of difference between control (average for each site (i) on each day (k)) and heat treatment plots (i.e. effect size) was calculated using Eq. (1). To test if the immediate effect of warming was different to the cumulative effect of multiple days of heating, separate PERMANOVAs for each site were conducted comparing differences in flux magnitude between Days 2, 5 and 7.

Flux magnitude difference Day i, Site k = Treatment flux Day i, Site k − Average control flux Day i, Site k (1)

To understand the role of sediment temperature on CO2 and CH4 fluxes, PERMANOVAs were conducted with maximum sediment temperature as a continuous covariable, and site, date, and OTC incubation Day as fixed factors. Maximum sediment temperature was used as we expected this to be a stronger driver of change than mean sediment temperature. Including the date allowed us to account for any differences between days that may have influenced the analysis (i.e., since the OTC incubations and measurements commenced one day later at Site 2 than Site 1). This analysis also allowed investigation of interaction between temperature and site.

To understand how experimental warming influenced the relationship between temperature and gas fluxes, we similarly performed PERMANOVAs with maximum sediment temperature as a continuous covariable with control and treatment data from both sites.

To explore the influence of enhanced warming on the macrofaunal community structure, a Bray-Curtis similarity matrix was created with square-root transformed data. We then used a bootstrapped metric Multi-Dimensional Scaling (MDS) ordination (50 bootstraps per group, minimum rho of 0.99) and a two-way PERMANOVA (9999 permutations) with Site (1, 2) and Treatment (Control, 5 day, 7 day) as fixed factors. Post-hoc pair wise tests were performed where significant interactions occurred (9999 permutations). A Similarity Percentage Breakdown (SIMPER) analysis was performed to analyse the species driving differences between treatments (i.e., which species increased or decreased the most due to heatwave simulation). All PERMANOVAs were performed as first described unless otherwise noted. PERMANOVAs, MDS and SIMPER analyses were carried out using Primer v7 software with the PERMANOVA addon93. All other data visualisations were conducted using ‘ggplot2’ from the ‘ggpubr’ package94 with R software version 4.5.188.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study aimed to investigate the impacts of simulated heatwave events on GHG emissions, macrobenthic communities, and sediment characteristics in tidal flats. Despite increasing concerns about climate extremes, the effects of rising extreme temperature events on the carbon cycle and biodiversity of tidal flats remain poorly understood. To address this gap, the researchers developed a smart OTC system that passively warmed sediment in treatment plots while minimizing convective cooling by wind—allowing for practical field deployment during low tide. The study’s findings are noteworthy: heatwave simulation enhanced CO₂ uptake at sandy sites and induced a shift from CO₂ sources to sinks in muddy sediments. CH₄ fluxes showed stronger responses in muddy sediments, and cumulative heatwave duration had significant effects on both macrobenthos and GHG emissions. These results suggest that prolonged extreme temperature events could substantially alter the carbon dynamics of estuaries. However, several concerns and issues should be addressed before the manuscript is deemed suitable for publication.\nSpecific comments:\n1. The manuscript reports that the muddy site exhibited lower resilience to heatwave simulation compared to the sandy site. However, the rationale behind this observation is not clearly articulated. The authors are encouraged to elaborate on the underlying mechanisms that may explain this differential response.\n2. The manuscript includes both short (5-day) and long (7-day) heatwave simulation treatments. The rationale for selecting these specific durations should be clarified. Why were 5 and 7 days chosen as representative of short- and long-duration heatwaves? Do these durations reflect ecologically relevant or observed extremes in the study region? A more detailed background information of heatwave events and justification of the experimental design would improve the clarity and robustness of the study.\n3. Figure 6. The results show that at the muddy site, community structures after both 5-day and 7-day heatwave simulations shifted in the same direction. In contrast, at the sandy site, the community responses diverged between the two durations. This contrasting pattern raises interesting ecological questions but currently lacks sufficient explanation in the manuscript. A deeper discussion of these site-specific dynamics would enhance the interpretation of the findings and their ecological implications.\n4. The manuscript uses sediment muddiness as a proxy for the degradation status of estuarine tidal flats. The rationale behind this statement should be more clearly explained and supported with more relevant literature.\n5. The results toward the end of the experiment are intriguing, particularly the observed differences in CH₄ flux effect sizes between Days 5 and 7 at the sandy site. The authors suggest that cooler temperatures during the final two days may have enabled some recovery, potentially confounding the interpretation of the effects of longer heatwave duration. This raises an important concern regarding the consistency of experimental conditions and the attribution of observed effects specifically to heatwave duration. Would it be possible to assess the robustness of the findings by excluding the data from the final two days of the experiment? This would help clarify whether the observed patterns are truly due to cumulative heatwave exposure or influenced by post-heatwave recovery.\n6. The study simulated heatwave conditions primarily during daytime low tides, yet in actual extreme warming events, elevated nighttime air and sediment temperatures are also expected. These elevated nighttime temperatures could exacerbate physiological and biogeochemical stress, potentially compounding the effects observed in this study. Given this discrepancy, the authors should discuss how this limitation may influence the interpretation of their results.\n7. The sites in the estuary received a high level of freshwater input via channelised agricultural drains, which likely contribute substantial nutrient loads. Additionally, the high incidence of nuisance macroalgae suggests eutrophic conditions. Could these factors be biasing the GHG emission measurements? Also, the absence of clear control sites makes it difficult to disentangle natural variability from anthropogenic impacts.\n8. The study presents a multi-day experimental simulation of a low tide atmospheric heatwave at two tidal flats to examine biodiversity responses. However, the term 'biodiversity' may be too broad in this context, as the study focuses specifically on the benthic macrofaunal community. For clarity and precision, it would be more accurate to refer to this explicitly throughout the manuscript.\n9. The term 'effect size' is not clearly defined in the manuscript. Is it referring to the difference in values between treatment and control groups? Providing a clear definition and explanation of how effect size was calculated would improve clarity for readers.\n10. Table A1 lacks an indication for Sites 1 and 2. In addition, given the significant interactions observed between site, treatment, and day, it’s unclear whether the reported mean temperatures can reliably indicate a treatment effect. Similar concerns apply to other tables.\n11. How long is the low tide period during which the experiment was conducted? I’m curious about the feasibility of carrying out this manipulation in the field—specifically, whether the duration of low tide provided sufficient time to complete incubation for 3 hours and measurements across all 30 plots. Additional detail on timing and logistics would help assess the practicality and consistency of the experimental setup.\n12. Several relevant studies have not been cited for comparison. For instance, research on tidal flats as CO₂ sinks—closely related to factors such as chlorophyll a concentrations, mud content, and sediment characteristics—should be considered (e.g., Global Change Biology, DOI: 10.1111/gcb.15107; Frontiers in Marine Science, DOI: 10.3389/fmars.2021.670180).", "reviewer_2": "The authors conducted a short-term warming experiment using open-top chambers in two intertidal flats with distinct macrofaunal communities and sediment characteristics. They placed OTC on the sediment for approx. 4 hours during one low tide event each day for 5 or 7 days, raising the soil by 1-2 °C on average over those 4 hours. They measured CH4 and CO2 fluxes 3 times during the experiment and sampled the sediment at the end. Although CO2¬ flux was not correlated with sediment temperature and CH4 flux was, there was no different in net CH4 flux between control and warmed plots while CO2 uptake increased with warming.\nMy main concern with this manuscript is that I’m not convinced the authors can extrapolate heatwave effects on GHG fluxes from a 5-7 day experiment that was not replicated over time and in which warming was only applied for a max of 4 hours per day. While there is potential for it to contribute to our understanding of how brief heatwaves will affect GHG dynamics in intertidal flats, the paper needs substantial revision in order to be beneficial to the scientific community, particularly in the analysis, data presentation, and discussion of warming and flux data.\nThe introduction and discussion can be substantially refined to focus more on what the authors’ data actually show, whereas currently the discussion especially feels more like explanations of what they expected to find. For example, the authors discuss many reasons why CH4 emissions vary (e.g. lines 238-255), but do not link these potential mechanisms back to their experiment to hypothesize what might have driven the patterns they observed.\nLastly, given that temperature was measured at 2 depths (1 cm and 5 cm) every minute for the entire week, this would be a stronger paper if it included an hourly time series of sediment temperature by treatment (ideally including both depths), rather than just summaries of what occurred at the soil surface during the 4-hour incubation. The authors acknowledge that a natural heatwave would have continuous warming (line 296), but without seeing the continuous temperature data over the week a reader can’t determine how to interpret the results from this experiment in the context of comparing to a natural heatwave.\nLine 188 – Maximum sediment temperature doesn’t seem to be a reasonable correlate without more understanding of how it relates to the time when the fluxes were measured. Presumably the relationships would be stronger using sediment temperature during the flux measurement period, as max temperature could have occurred in the following hour.\nLine 191 - Why would the muddy site have lower CH4 emissions in the OTC compared to the controls, but also a significant positive correlation with soil temperature? Does this indicate that it’s day to day fluctuation in temperature that’s more important than short-term heatwaves?\nLine 264-265 – “…processes or mechanisms that control gas release from the sediments” sounds like the authors are discussing methods of gas transport (e.g. diffusion or ebullition), but the rest of the paragraph is discussing controls on CH4 production.\nLine 320-321 – Were CH4 emissions actually significantly higher in OTC on Day 2?\nLine 323-324 – This is an interesting concept, but not something highlighted in the results so it’s hard to see if there actually were stronger effects of warming on CH4 and CO2 emissions from the muddy site compared to the sandy site.\nLine 420 - Why was the sediment temperature data trimmed and only the shallow depth used?\nLine 414 – When were sediment/macrofaunal cores collected for the control plots?\nLine 425 – Why were the two heatwave treatments combined for statistical analysis?\nFig 1 – Which treatments are significantly different from others?\nFig 2,3,4 – Which treatments are included in each box plot? Do the OTC boxes from Day 2 and Day 5 include fluxes from both 5- and 7-day warming treatments and then Day 7 OTC is only 7-day?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to reviewers Nature Communications manuscript NCOMMS-25-29176A\nTitle: Simulated heatwave alters intertidal estuary greenhouse gas fluxes:\nduration and degradation state determine response\nWe would like to thank the reviewers for their supportive and helpful\ncomments that have helped us improve our manuscript. We provide a point-\nby-point response below (our responses in blue italic text) along with a tracked\nchanges version of the manuscript. For specific changes, we provide line\nreference numbers for the tracked changes document.\nWe would like to note that we found a data error for the CH treatment effect\n4\nsizes used to generate Figure 3. The figure has been fixed, and a small change\nmade in the reported results. This error affected the effect size plot and\ninterpretation only, and there are no longer significant differences in the CH\n4\nflux treatment effect magnitude between days at the Sandy site. This does not\nsignificantly alter study outcomes or interpretation but strengthens the\nconclusions that heatwave treatment effects were greater at the muddy site.\nAll other data have been re-checked and confirmed as correct.\nReviewer #1\n1. The manuscript reports that the muddy site exhibited lower resilience to\nheatwave simulation compared to the sandy site. However, the rationale\nbehind this observation is not clearly articulated. The authors are\nencouraged to elaborate on the underlying mechanisms that may explain\nthis differential response.\nWe have added content to the introduction that clarifies the use of\nsediment mud content as a proxy for degradation in estuaries and\ndescribes the relationship between increasing sedimentation in estuaries\nand reducing resilience:\n(L93-100) “Changing sedimentary environments in estuaries, especially\nincreasing bed sediment muddiness (silt + clay content), is the product of\nintensified anthropogenic land use, which can lead to changes in habitats and\nspecies composition, reduced ecosystem functioning, and loss of ecosystem\nservices [43-45]. Consequently, sediment mud content is used as a proxy for\nestuary degradation [46]. Increasing sediment mud content can also reduce\necosystem interaction network complexity, which is an important indicator of\necosystem resilience [47-49]. Sediment characteristics, especially mud content,\nare known to be a key regulator of carbon fluxes in tidal flats [50]”\nThe following references have been added:\n1\n45. Thrush, S.F., et al., Habitat change in estuaries: Predicting broad-scale\nresponses of intertidal macrofauna to sediment mud content. Marine Ecology\nProgress Series, 2003. 263: p. 101-112.\n46. Thrush, S.F., et al., Muddy waters: Elevating sediment input to coastal and\nestuarine habitats. Frontiers in Ecology and the Environment, 2004. 2(6): p.\n299306.\n47. Thrush, S.F., J.E. Hewitt, and A.M. Lohrer, Interaction networks in coastal soft-\nsediments highlight the potential for change in ecological resilience. Ecological\nApplications, 2012. 22(4): p. 1213-1223.\n48. Douglas, E.J., et al., Changing intra- and interspecific interactions across\nsedimentary and environmental stress gradients. Ecosphere, 2023. 14(1): p.\ne4373.\n49. Douglas, E.J., A.M. Lohrer, and C.A. Pilditch, Biodiversity breakpoints along\nstress gradients in estuaries and associated shifts in ecosystem interactions.\nScientific Reports, 2019. 9(1): p. 17567.\nWe also amended a sentence in the final paragraph of the discussion that now\nreads:\n(L408-410) “This has implications for ecosystem resilience, since degraded\necosystems typically have lower species and trait diversity, and reduced\ninteraction network complexity, making them more vulnerable to disturbance\nevents such as heatwaves [81-84].”\n2. The manuscript includes both short (5-day) and long (7-day) heatwave simulation\ntreatments. The rationale for selecting these specific durations should be clarified.\nWhy were 5 and 7 days chosen as representative of short- and long-duration\nheatwaves? Do these durations reflect ecologically relevant or observed extremes in\nthe study region? A more detailed background information of heatwave events and\njustification of the experimental design would improve the clarity and robustness of\nthe study.\nThe heatwave simulation treatment durations were selected based on the\ndefinitions of both marine and atmospheric heatwaves. Atmospheric heatwaves\nhave recently been defined as at least three consecutive days [1] and marine\nheat waves are defined as at least 5 consecutive days [2], therefore we used 5\ndays as the minimum duration to be considered a heatwave. The 7-day\nsimulation was as long as possible given logistical and budgetary constraints,\nthough the extra 2 days of heat treatment allowed us to begin to test for\ncumulative effects of slightly longer heatwave duration. We have added this\njustification to the final paragraph of the introduction that describes the\nhypotheses:\n2\n(L116-120) “We chose heatwave treatment durations based on the definitions\nof atmospheric and marine heatwaves; events with at least three [53] and five\ndays [54], respectively. The 5-day treatment duration was used to satisfy the\ntemporal conditions of heat wave definitions, and the longer duration treatment\n(seven days) allowed us to begin to test for cumulative effects of slightly longer\nheatwave duration.”\n3. Figure 6. The results show that at the muddy site, community structures after\nboth 5-day and 7-day heatwave simulations shifted in the same direction. In\ncontrast, at the sandy site, the community responses diverged between the two\ndurations. This contrasting pattern raises interesting ecological questions but\ncurrently lacks sufficient explanation in the manuscript. A deeper discussion of\nthese site-specific dynamics would enhance the interpretation of the findings and\ntheir ecological implications.\nWe have added a sentence to the results:\n(L238-240) “The direction of change in community structure for the 5- and 7-day\ntreatments was similar at the muddy site, however at the sandy site, there was\nobvious divergence between the two treatment durations (Figure 6).”\nWe have also added this detail to the discussion which now reads:\n(L326-335) “There were contrasting patterns in the community response to the\ntwo heatwave treatment durations. At the muddy site the community structure\nshifted in the same direction indicating cumulative effects of increasing heatwave\nduration. In contrast, at the sandy site there was divergence between the\ncommunity composition of the 5- and 7-day treatments, and the 7-day heat\ntreatment plots were more similar to the control plots than the 5-day treatment.\nThis could be due to the cooler days experienced at the end of the experiment\n(Days 6 and 7) which may have allowed some recovery and may be reflected in\nthe CH flux effect size difference between Days 5 and 7 at the sandy site. At\n4\nboth sites there were reductions in the abundance of bioturbating amphipods,\nParacorophium excavatum, in the treatment plots.”\n4. The manuscript uses sediment muddiness as a proxy for the degradation\nstatus of estuarine tidal flats. The rationale behind this statement should be\nmore clearly explained and supported with more relevant literature.\nWe have added content to the introduction that clarifies the use of\nsediment mud content as a proxy for degradation in estuaries and\ndescribes the relationship between increasing sedimentation in estuaries\nwith reducing resilience.\n5. The results toward the end of the experiment are intriguing, particularly the\nobserved differences in CH flux effect sizes between Days 5 and 7 at the sandy\n4\nsite. The authors suggest that cooler temperatures during the final two days may\nhave enabled some recovery, potentially confounding the interpretation of the\neffects of longer heatwave duration. This raises an important concern regarding the\nconsistency of experimental conditions and the attribution of observed effects\nspecifically to heatwave duration. Would it be possible to assess the robustness of\nthe findings by excluding the data from the final two days of the experiment?\nThis would help clarify whether the observed patterns are truly due to cumulative\nheatwave exposure or influenced by post-heatwave recovery.\nWe would like to note that we found a data error for the CH treatment effect sizes\n4\nat the sandy site used to generate Figure 3 (previously Figure 4). The figure has\nbeen fixed, and a small change made to the reported results so that there are no\nlonger significant differences in the CH flux treatment effect magnitude between\n4\ndays at the Sandy site. This error affected the effect size plot and interpretation\nonly; all other data has been re-checked and confirmed as correct. This does not\nsignificantly alter study outcomes or interpretation but strengthens the conclusions\nthat heatwave treatment effects were greater at the muddy site.\nAlthough there were cooler days towards the end of the experiment, sediment\ntemperatures were still very warm (>23°C during incubation times on 22-23\nFebruary) and OTC treatments continued to elevate mean and maximum sediment\ntemperatures above control values. So although the OTC temperatures in the final\ndays were not as warm as on days 1-5, they had not returned to normal levels so\ncannot be truly considered post heatwave. In our future work we plan to\ninvestigate post heatwave recovery. We have altered the text in the results section\nso that we use the word ‘warm’ instead of ‘cool’ to better describe the\ntemperatures on the 22 and 23 February.\n(L139-142) “Since the incubation days were offset between sites (i.e. Day 1 at the\nmuddy site was a day later than the sandy site), the sandy site experienced six\nconsecutive hot days followed by a warm day, whereas the muddy site\nexperienced five consecutive hot days and two warm days (Figure A2, A3, A4).”\nOur day 2 measurements (in addition to the day 5 and day 7 measurements)\nare suggestive of a position relationship between heatwave duration on flux\neffect sizes. The effect sizes measured represent an integration of both the\ninstantaneous effect of temperature on flux and the historical effects on the\nsediments that will influence biogeochemistry (i.e. sublethal effects on\n4\nmacrofauna). Unfortunately, in the current study we are unable to tease\napart these effects.\nWe agree with the reviewer that omitting the final two days of experimental data\nwould provide a ‘cleaner’ result and potentially clarify the story, however we\nprefer to still present these. Although day 7 results are more difficult to explain,\nthey provide interesting insights into the dynamic response of fluxes to heat\nstress and temporal weather conditions.\n6. The study simulated heatwave conditions primarily during daytime low tides, yet in\nactual extreme warming events, elevated nighttime air and sediment temperatures are\nalso expected. These elevated nighttime temperatures could exacerbate physiological\nand biogeochemical stress, potentially compounding the effects observed in this study.\nGiven this discrepancy, the authors should discuss how this limitation may influence the\ninterpretation of their results.\nWe thank the reviewer for this comment. This is indeed something that is likely\nto magnify effects seen in our study. We have added text to the discussion to\nemphasise this point. Furthermore, we have added a sentence that mentions the\npossible compounding effects of the future cooccurrence of marine heat waves\nand atmospheric heatwaves. This section now reads:\n(L362-369) “Nighttime low tide air and sediment temperatures were not elevated\nin our study but it is possible that they would be in a real-world heatwave event,\nwhich in turn could exacerbate physiological and biogeochemical stress and\ntherefore compound the effects seen in our study. Coupling of marine and\natmospheric heatwaves is likely [e.g. 72] and may reduce the metabolic reprieve\nprovided by high tides [73]. Coincidentally, during our experiment there was a\nmoderate marine heatwave on the east coast of New Zealand [74] which may\nhave contributed to ecosystem stress and the cumulative effects we measured.”\n7. The sites in the estuary received a high level of freshwater input via channelised\nagricultural drains, which likely contribute substantial nutrient loads. Additionally, the\nhigh incidence of nuisance macroalgae suggests eutrophic conditions. Could these\nfactors be biasing the GHG emission measurements? Also, the absence of clear control\nsites makes it difficult to disentangle natural variability from anthropogenic impacts.\nWe chose to incorporate control plots within each site rather than trying to\nfind suitable control sites further afield.\nWe chose two sites that were relatively close together, roughly the same distance\naway from the closest freshwater input point, and similar in terms of elevation\n(period of inundation). The overt difference was sediment type (muddiness level).\nnuisance macroalgae, anoxic sediment) and we are confident that these factors did\nnot bias our GHG measurements. We have amended the descriptions in the\nMethods section to clarify this.\n(L436-438) “The circulation in some parts of the estuary is restricted and some\nareas in the upper reaches can be described as hypoxic and degraded with high\nincidence of nuisance macroalgae.”\n(L447-449) “This area contained expanses of relatively sandy and relatively muddy\nsediment but did not exhibit any other signs of eutrophication.”\n8. The study presents a multi-day experimental simulation of a low tide atmospheric\nheatwave at two tidal flats to examine biodiversity responses. However, the term\n'biodiversity' may be too broad in this context, as the study focuses specifically on the\nbenthic macrofaunal community. For clarity and precision, it would be more accurate to\nrefer to this explicitly throughout the manuscript.\nAs suggested, we have changed the terminology. In the appropriate places in\nthe manuscript, we have changed ‘biodiversity’ to ‘macrobenthic biodiversity’.\n9. The term 'effect size' is not clearly defined in the manuscript. Is it referring to the\ndifference in values between treatment and control groups? Providing a clear definition\nand explanation of how effect size was calculated would improve clarity for readers.\nEffect size refers to the difference in flux magnitude between the control and\ntreatment plots within a site. We have clarified this definition in the Methods\nsection. (L528)\nWe have also added an explanation of effect size when it is first introduced in\nthe hypothesis section of the Introduction:\n(L113-115) “We also hypothesised that 3) longer exposure to low tide heatwave\nsimulation (i.e. cumulative days) would result in a greater effect on fluxes (i.e.\neffect size; the difference between control and treatment plot values).”\n10. Table A1 lacks an indication for Sites 1 and 2. In addition, given the significant\ninteractions observed between site, treatment, and day, it’s unclear whether the\nreported mean temperatures can reliably indicate a treatment effect. Similar concerns\napply to other tables.\nWe have changed the Site 1 & 2 labels in Table A1 to ‘sandy’ and ‘muddy’. Although\nthe reviewer is correct that higher-order interactions were detected, the strength of\nheating in our treatments was driven by real-world weather conditions (ambient air\n6\ntemperatures and wind speeds during daytime low tides), which varied naturally\nduring the study. We also explained why heating effects may differ in sandy versus\nmuddy sediments (i.e., site), e.g., due to differences in sediment permeability and\nlow tide water content (L398-401). Therefore, interactions of day and site with\ntreatment with were not unexpected. Nevertheless, our data clearly show that\nsediments inside open top chamber treatments were consistently warmer than\ncontrols (see maximum and mean sediment temperature data in Figures A2 and A3,\nrespectively), and we present data from the different treatments by date and site in\nthese figures so that readers wishing to see how the interactions manifest are able to\ndo so.\n11. How long is the low tide period during which the experiment was conducted? I’m\ncurious about the feasibility of carrying out this manipulation in the field—specifically,\nwhether the duration of low tide provided sufficient time to complete incubation for 3\nhours and measurements across all 30 plots. Additional detail on timing and logistics\nwould help assess the practicality and consistency of the experimental setup.\nThis experiment was indeed logistically challenging and required precise planning to\nachieve the measurements within the low tide emergence period. We have added the\nfollowing details to the methods section:\n(L448-449) “The duration of low tide emergence was approximately 5 hours.”\n(L476-477) “Gas fluxes in control plots were measured first so that treatment\nplots could be measured following at least three hours of OTC incubation.”\n(L486-487) “We alternated three chamber bases to reduce the time between flux\nmeasurements at each plot to <1 minute.”\n12. Several relevant studies have not been cited for comparison. For instance, research\non tidal flats as CO sinks—closely related to factors such as chlorophyll a\n2\nconcentrations, mud content, and sediment characteristics—should be considered\n(e.g., Global Change Biology, DOI: 10.1111/gcb.15107; Frontiers in Marine Science,\nDOI: 10.3389/fmars.2021.670180).\nWe thank the reviewer for bringing these studies to our attention; we have now\nincorporated them into our manuscript. Please note that the endnote reference\nchanges do not show up as tracked changes in the manuscript.\n(L98-99) “Sediment characteristics, especially mud content, are known to be key\nregulators of carbon fluxes in tidal flats [50].”\n(L270-271) “...studies have shown intertidal flats to be net CO sinks with uptake\n2\nrelated to Chl a concentrations [50, 55].”\n7\n(L271-273) “Positive relationships between microphytobenthic productivity and\ntemperature have been shown elsewhere with maximum values\noccurring around 30°C [56].”\nReviewer #2\nThe authors conducted a short-term warming experiment using open-top chambers in\ntwo intertidal flats with distinct macrofaunal communities and sediment\ncharacteristics. They placed OTC on the sediment for approx. 4 hours during one low\ntide event each day for 5 or 7 days, raising the soil by 1-2 °C on average over those 4\nhours. They measured CH and CO fluxes 3 times during the experiment and sampled\n4 2\nthe sediment at the end. Although CO flux was not correlated with sediment\n2\ntemperature and CH flux was, there was no different in net CH flux between control\n4 4\nand warmed plots while CO uptake increased with warming.\nextrapolate heatwave effects on GHG fluxes from a 5-7 day experiment that was not\nreplicated over time and in which warming was only applied for a max of 4 hours per\nday. While there is potential for it to contribute to our understanding of how brief\nheatwaves will affect GHG dynamics in intertidal flats, the paper needs substantial\nrevision in order to be beneficial to the scientific community, particularly in the analysis,\ndata presentation, and discussion of warming and flux data.\nThe experiment duration was chosen based on the temporal components of the\ndefinitions of both atmospheric (3 days) and marine heat waves (5 days). Please\nsee response to Reviewer 1 Comment 2.\nThree- to five hours of low tide emergence is typical of mid-tide level intertidal\nflats in most New Zealand estuaries, and many other estuaries globally. Thus,\nwe consider that four hours of warming is a realistic simulation of an\natmospheric heatwave during the day in an intertidal estuarine ecosystem.\nOur experiment was conducted during a week when low tides were during the\nmiddle part of the day (highest incident sunlight irradiance). Additionally, this\nwas a period of spring tides (i.e. the lowest tides of the month), which\nmaximised tidal flat exposure time. Due to the shifts in tidal times by ~48\nminutes per day, coincidence of spring low tides with the hottest 4 hours of\nthe day for a whole week, would be a rare occurrence and could not occur for\nmore than 7 consecutive days.\nThe introduction and discussion can be substantially refined to focus more on what the\nauthors’ data actually show, whereas currently the discussion especially feels more like\nexplanations of what they expected to find. For example, the authors discuss many\n8\nreasons why CH emissions vary (e.g. lines 238-255), but do not link these\n4\npotential mechanisms back to their experiment to hypothesize what might have\ndriven the patterns they observed.\nThe introduction has been revised, and changes have been made so that it\nbetter frames the study particularly with regards to defining heatwaves and\nthe effects of sediment mud content in estuaries.\nThe discussion has been significantly revised. We have added text and\nreferences to clarify and strengthen statements throughout. We have\nalso removed two paragraphs as suggested by another reviewer.\nWe have altered text in the paragraph mentioned above to strengthen links\nwith our results. (L289-290)\nLastly, given that temperature was measured at 2 depths (1 cm and 5 cm) every\nminute for the entire week, this would be a stronger paper if it included an hourly time\nseries of sediment temperature by treatment (ideally including both depths), rather\nthan just summaries of what occurred at the soil surface during the 4-hour incubation.\nThe authors acknowledge that a natural heatwave would have continuous warming (line\n296), but without seeing the continuous temperature data over the week a reader can’t\ndetermine how to interpret the results from this experiment in the context of comparing\nto a natural heatwave.\nThe temperatures were measured by loggers situated 1 cm above and 5 cm\nbelow the sediment surface (L451-452). For brevity, data from the loggers\npositioned above the sediment surface (i.e., in the air) were not presented\nin the study. We have therefore removed mention of these loggers from the\nmethods section to avoid confusion.\nWe have provided an additional figure (Figure A4) in the supplementary material\nthat presents the temperature time series for each treatment at each site. From\nthis figure you can see that average sediment temperatures diverge from control\nplots during the OTC treatment and return to ambient temperatures following\ntidal immersion. It is also clear that tidal fluctuations are the main controller of\nsediment temperatures in both control and treatment plots. Solar radiation is an\nimportant driver of sediment temperature in the intertidal, and this is evident on\n22 & 23 February when there was significant cloud cover relative to the clear\ndays earlier in the experiment (Figure A4).\nLine 188 – Maximum sediment temperature doesn’t seem to be a reasonable correlate\nwithout more understanding of how it relates to the time when the fluxes were\n9\nmeasured. Presumably the relationships would be stronger using sediment\ntemperature during the flux measurement period, as max temperature could have\noccurred in the following hour.\nWhile we explored using mean sediment temperatures in our analyses (which\nshowed similar results), we chose to use the maximum sediment temperature\nas it represents an extreme condition that is expected to be a greater driver of\necological change.\nMaximum temperatures usually occurred towards the end of the incubation\nperiods, however there were not significant temperature increases in the final\nhour of incubation (Figure A4).\nLine 191 - Why would the muddy site have lower CH emissions in the OTC compared to\n4\nthe controls, but also a significant positive correlation with soil temperature? Does this\nindicate that it’s day to day fluctuation in temperature that’s more important than short-\nterm heatwaves?\nWe thank the reviewer for this comment. We are unsure of the answer, but it\nshould be noted that the positive correlation with sediment temperatures is\nbased on the maximum temperatures recorded each day whereas the control-\nOTC treatment comparisons integrate the temperature regime over a number of\nhours each day and cumulatively for five or seven days.\nLine 264-265 – “...processes or mechanisms that control gas release from the\nsediments” sounds like the authors are discussing methods of gas transport (e.g.\ndiffusion or ebullition), but the rest of the paragraph is discussing controls on CH\n4\nproduction.\nThis paragraph has been removed as suggested by another reviewer.\nLine 320-321 – Were CH emissions actually significantly higher in OTC on Day 2?\neffect. Please note that we encountered a data error for the CH treatment effect\n4\nsizes used to generate Figure 3. Figure 3 has been amended, and minor text\nalterations have been made to the results section. We have also altered the\nsentence in the discussion so that it specifies effects at the muddy site:\n(L390-395) “This may explain the change in the magnitude of difference of\nCH fluxes between controls and treatments (i.e. effect size) observed over\n4\ntime at the muddy site: after two days of simulated low tide heatwave, the\ntreatment effect on CH emissions was positive, whereas after five or seven\n4\ndays, the treatment effect was negative.”\nresults so it’s hard to see if there actually were stronger effects of warming on CH\n4\nand CO2 emissions from the muddy site compared to the sandy site.\nWe thank the reviewer for the comment. The text being referred to is in the\nDiscussion section and is mainly for context (based on literature). However,\nwe admit that our analyses did not specify an interactive effect and\ntherefore we have altered this sentence which now reads:\n(L396-398) “This research demonstrates differing effects of atmospheric\nheatwaves (and increasing temperature maxima) in different sedimentary\nenvironments which has implications for estuaries experiencing increasing\nsediment mud content.”\nLine 420 - Why was the sediment temperature data trimmed and only the shallow\ndepth used?\nSediment temperature was only measured at one depth within the sediment\ncolumn. The other logger recorded air temperatures above the surface of the\nsediment (L452). The two data streams were generally highly in sync and\nmirrored each other as temperatures varied over time, but also therefore\nprovided duplicate or redundant information. Previous research has similarly\nshown that above-sediment temperatures reflect within-sediment temperatures\n[3]. We used the sediment temperatures (rather than air or water temperatures)\nbecause they were less variable and more relevant to the locations of the\norganisms and materials that influence CO and CH fluxes (i.e., sediment-\n2 4\nassociated microbes, microphytes, organic matter, macrofauna. Data was\ntrimmed to only include values from the OTC incubation period and therefore\nclearly demonstrate the treatment effect on sediment temperature. Across the\nentire record temperature shows high variability driven by tidal fluctuations\n(Figure A4) as well as the frequent sampling interval (1 min) and high number of\nobservations (n = 10080 over 7 days).\nLine 414 – When were sediment/macrofaunal cores collected for the control plots?\nSampling of control plot sediment and macrofaunal cores was conducted at the\nend of the experiment on day 7. This is stated in the Methods (L505-506).\nLine 425 – Why were the two heatwave treatments combined for statistical analysis?\nWe chose to combine the data from all OTC plots for the analysis because at day\n2 and 5 all OTC plots were effectively treated the same. We have added\nthis detail to the Methods section:\n11\n(L526-527) “Treatment plot data were combined because at day two and five\nall OTC plots were not statistically different.”\nFig 1 – Which treatments are significantly different from others?\nSignificant differences between treatments in sediment characteristics are\nreported in Table A2. We have chosen not to add significance notation to this plot\nas it makes it crowded and difficult to read; however, this detail is provided in the\ntext (see L143-150).\nFig 2,3,4 – Which treatments are included in each box plot? Do the OTC boxes from Day 2 and\nDay 5 include fluxes from both 5- and 7-day warming treatments and then Day 7 OTC is only 7-\nday?\nCorrect, OTC boxes include all data from both treatments for Day 2 & 5, and only\nfrom Day 7 treatments for Day 7. We have added this text to the legend of\nFigures 2 and 4:\n“OTC boxes include data from both 5- and 7-day treatment plots for days 2 and\n5 (n = 20), and 7-day treatment plots only for day 7 (n = 10).”\nAnd Figure 3:\n“Day 2 and Day 5 boxes include data from both 5- and 7-day treatment plots (n =\n20), and Day 7 boxes include data from 7-day treatment plots only (n = 10).”\nReviewer 3\nThis study presents an impressive and timely in-situ warming experiment that\ninvestigates how atmospheric heatwaves influence greenhouse gas (GHG) fluxes in an\nintertidal estuary. By examining the interplay between heatwave duration, benthic\nmacrofaunal communities, and sedimentary environments, the authors reveal context-\ndependent effects on CO and CH fluxes. One key result is that warming altered the\n2 4\nCO2 dynamics depending on habitat characteristics, with larger uptake in sandy\nsediments and turning the muddy site to a source.\nThis work addresses a vital and topical issue, as climate change is driving more frequent\nand intense heatwaves that may alter carbon cycling and feedbacks in productive but\nvulnerable coastal ecosystems. The experimental design — combining field realism with\nmulti-factor analysis — adds strength to the study.\nWhile the key results are highly valuable and the setup was quite remarkable, the\nmanuscript would benefit from additional clarifications and detail. Given that the study\nis framed within the context of heatwaves, it would be helpful to more clearly explain\n12\nhow the experimental warming during low tide fits within this framework. Clearly\nstating the context can be important particularly for intertidal systems that naturally\nexperience strong temperature fluctuations.\nA suggested by the reviewer, we have added more details and clarified the\nexperimental warming design in the context of heatwaves and intertidal habitats.\nPlease see our response to reviewer 1 comment 2. Briefly, we used five days\nbecause it is the minimum duration to be considered a marine heatwave [2].\nBased on logistical and budgetary constraints, an extra 2 days of heat treatment\n(the 7-day simulation) allowed us to begin to test for the cumulative effects of\nslightly longer heatwave duration. We have added this justification to the final\nparagraph of the Introduction that describes the hypotheses: (L116-120). Also in\nthe Introduction section, we provided reasoning on the importance of assessing\nheatwaves effects on intertidal habitats.\nAlso, referring to “heating days” may be somewhat misleading, as the treatment\neffectively involved a short-term heat pulse – an important distinction that could\nmake the observed effects even more pronounced. Potentially, the authors might\nwant to include and/or discuss, that during the time of the experiment an actual\nmoderate marine heatwave occurred on the east coast of New Zealand. Relevant\nmetrics from this event, available onw marineheatwaves.org, provided here:\nWe have altered text throughout so that we refer to ‘daytime low tide heatwave\nexposure’ (or similar) instead of ‘heatwave days’ or ‘heating days’.\nThank you for drawing our attention to the marine heatwave tracker. We have\nadded a sentence to the discussion that mentions the marine heatwave and its\nimplications for our results:\n(L367-369) “Coincidentally, during our experiment there was a moderate marine\nheatwave on the east coast of New Zealand [74] which may also have\ncontributed to ecosystem stress and the cumulative effects we measured.”\nAdditionally, I would also recommend removing the results section of “Treatment\neffect size increased with consecutive heatwave days” and integrate the results into\nthe previous section of “Immediate and cumulative effects of heatwave on carbon\nflux”. This would help streamlining the results and avoid redundancy.\nThank you for this comment, we have acted on these recommendations.\nWe have also reordered the figures and figure citations accordingly.\nLanguage and format editing should also be revised throughout the manuscript.\nWe have carefully reviewed the manuscript for language and formatting errors.\nMore specific comments below\nversion.\nBest Regards,\nNorman Göbeler\nIntroduction:\nLine 69: I would suggest rephrasing to “Methane is primarily produced...”\nWe have made the suggested change.\nLine 110: Is there a specific reason why H4 is excluded from H5? Additionally, the\nconclusion that sandy sites might be more resilient to warming could benefit from\nstronger justification or supporting evidence. Clarifying the rationale behind this\nhypothesis would improve the argument.\nThe exclusion of H4 from H5 was a mistake which we have fixed.\nWe have added content to the introduction that clarifies the use of\nsediment mud content as a proxy for degradation in estuaries and\ndescribes the relationship between increasing sedimentation in estuaries\nwith reducing resilience:\n(L93-98) “Changing sedimentary environments (especially increasing mud\n(silt/clay) content) in estuaries are the product of intensified anthropogenic land\nuse, which can lead to changes in habitats and species composition, reduced\necosystem functioning, and loss of ecosystem services [43-45]. Consequently,\nsediment mud content is often used as a proxy for estuary degradation [46].\nIncreasing sediment mud content can also reduce ecosystem interaction\nnetwork complexity an important indicator of ecosystem resilience [47-49].”\nMethods:\nLine 357: It seems that a cross-reference to Figure 7 may be more appropriate here than to\nFigure 1. Please confirm and adjust accordingly.\nThank you for identifying this mistake which is now fixed.\nLine 387: Please provide the data that support the similar levels of solar radiation in the\ntreatment and control plots.\nWe have added reference to a new plot in the supplementary materials (L\n465, Figure A5) that displays the light data.\nLine 404: Was the volume measured when placed in the field? How was the placement of 2\ncm into the sediment ensured? Was this accounted for when calculating the fluxes?\ndrawn guide line around the side of the chamber to ensure a constant depth and\nvolume between plots. We have added this detail to the Methods section:\n(L485-486) “We used a line drawn 2cm above the lower edge of the chamber\nbase to ensure constant depth and chamber volume between plots.”\nLine 405: Is Figure 7e the correct reference here?\nWe have removed this reference.\nLine 410: The flux calculation method needs to be expanded. For example, clarify whether\na linear model or the HM model was used, and if both were considered, which one provided\na better fit.\nWe have expanded the flux calculation methodology as suggested, and now\ninclude details of model fit and selection:\n(L499-502) “The goFlux function was used to calculate both a linear (LM) and\nnon-linear (HM) flux estimate, and the best.flux function was used to\nautomatically select the best flux estimate based on criteria scores for model fit\n(AICc, MAE, g.factor, MDF). In most cases, the HM model provided the best flux\nestimate.”\nLine 416: While referencing standard protocols is appreciated, please briefly\nsummarize key aspects of the macrofaunal sampling, such as sieve mesh size and\nsample preservation.\nWe have added this detail to the Methods:\n(L508-509) “Macrofaunal cores were sieved over 500 µm mesh, preserved in 70%\nisopropyl alcohol and stained with Rose Bengal. Sediment cores were frozen and\nstored at -20°C.”\nLines 414-418: As this experiment was conducted in parallel to Lam-Gordillo et al., 2025,\ncould you please explain how you dealt with the collection of the 10 bivalves per plot?\nHow were they accounted for? Did this study utilize the same 2.6 cm in diameter cores as in\nLam-Gordillo et al., 2025?\nMacrofauna and sediment cores were collected before the 10 bivalves used for the\nLam-Gordillo et al. 2025 study (there was plenty of space and bivalves in the\nremaining un-cored plot area).\nYes, these were the same sediment cores used in Lam-Gordillo et al. 2025. We\nhave corrected the diameter reported in the Methods (L507).\nPERMANOVA analyses throughout. What distance measure was used? How many\npermutations were performed? Were the fluxes normalized prior to analysis?\nWe have added more detail about our analyses throughout the Data\nAnalysis section of the Methods.\nLine 435: Specify why you used the maximum temperature.\nWe chose to use the maximum sediment temperature as we expected this to be\na stronger driver of change than the mean. We have added this detail\nto the Methods:\n(L537-539) “Maximum sediment temperature was used as we expected this\nto be a stronger driver of change than mean sediment temperature.”\nLine 444: Was nMDS or MDS applied? Figure 6 states MDS. Please provide more details.\nA metric MDS was applied as noted in the legend to Figure 6. We have corrected a\nmistake in the Methods which had referred to nMDS and have also provided more\ndetail in the data analysis section of the Methods. (L540-542)\nResults:\nLine 134 & Table A2: While the organic content is statistically different between the\nsites, it would be helpful to clarify whether an approximately 0.2% higher organic\nmatter content at the muddy site is ecologically meaningful enough to support its\ncharacterization as a “degraded environment.” Additional context or justification\nwould strengthen this point.\nA 0.2% difference in organic content is likely not ecologically significant on\nits own. However, we characterised the muddy site as more degraded based\non the higher proportion of fine sediments (mud content- silt/clay fraction).\nPlease see responses to earlier comments regarding mud content as a proxy\nfor degradation.\nSection following Line 171: This section tends to repeat results already presented\nearlier. I would recommend treating the effect size analyses as complementary to the\nflux data, and integrating them directly into the relevant sections to streamline the\nnarrative and avoid redundancy.\nAs suggested, we have integrated the treatment effect sizes results into\nthe previous section.\nFigure 6: Please clarify if this is MDS or nMDS.\nThis is a metric MDS plot, please see response to earlier comment.\nLine 220-222: This simplifies the complexity of heatwave definitions and involved\nparameters and impacts. Please revise.\nWe have added the following text to revise this statement:\n(L252-259) “A heatwave can be broadly described as “a period of consecutive\ndays where conditions are excessively hotter than normal”, however there are\nmany different atmospheric heatwave definitions and indices used which are\nusually specific to the region and impact of interest (e.g. human health,\nagriculture) [53]. Alongside metrics of frequency, intensity and extent, persistence\nis a key component that is used to define heatwaves; with increasing duration\nassociated with increasing impacts [6].”\nLine 223-227: Please provide supporting references that explicitly link sediment\nmuddiness to environmental degradation.\nAppropriate references [46-49] have been added (L262). Please see response to\nearlier comments (especially Reviewer 1 Comment 1) about links between estuary\ndegradation and sediment mud content. Please note that Endnote reference\nchanges do not appear as tracked changes in the document.\n46. Thrush, S.F., et al., Muddy waters: Elevating sediment input to coastal and\nestuarine habitats. Frontiers in Ecology and the Environment, 2004. 2(6): p. 299-\n306.\n47. Thrush, S.F., J.E. Hewitt, and A.M. Lohrer, Interaction networks in coastal\nsoft-sediments highlight the potential for change in ecological resilience. Ecological\nApplications, 2012. 22(4): p. 1213-1223.\n48. Douglas, E.J., et al., Changing intra- and interspecific interactions across\nsedimentary and environmental stress gradients. Ecosphere, 2023. 14(1): p.\ne4373.\n49. Douglas, E.J., A.M. Lohrer, and C.A. Pilditch, Biodiversity breakpoints\nalong stress gradients in estuaries and associated shifts in ecosystem\ninteractions. Scientific Reports, 2019. 9(1): p. 17567.\nLine 232-233: Unclear what “negative effect” here refers to, please rephrase and clarify. To\nclarify we have replaced “have a negative effect on” with “reduce” (L270)\nLine 234-237: This statement is currently vague and would benefit from further\ndevelopment and clearer support. In particular, considering that both\nmicrophytobenthos (MPB) biomass and macrofaunal abundance declined, could the\nobserved increase in CO uptake be due to reduced community respiration?\n2\n(L273-277) “However, in our study, microphytobenthic biomass declined in\nresponse to experimental heating inside OTCs while photosynthesis appeared\nto have been positively influenced by temperature increases. The effects of\ntemperature on microphytobenthos are species specific, with photoinhibition\nand recovery of photoinhibition having different, species-specific temperature\nthresholds [57].”\nYes, reduced community respiration could indeed have contributed to the\nincrease in net CO uptake. We have clarified the statement earlier in the\n2\nparagraph that mentions this (L268). We provide further discussion of the\ninfluence of macrofaunal respiration later in the Discussion (L336-348).\nLine 248-249: Additional references are needed to support this statement. Moreover, how\nis this conclusion supported when methane fluxes were not directly influenced by the\nwarming treatment in this study?\nAlthough methane fluxes were not directly increased by OTC treatment, the\ncorrelation analysis of methane flux and maximum sediment temperature shows a\npositive slope. We have altered the sentence so that it better links to this result, and\nwe have added appropriate references:\n(L289-292) “The positive correlation between maximum sediment temperature and\nCH emission in our study suggests that with increasing frequency of warm summer\n4\nevents and increasing temperature maxima in intertidal estuary ecosystems, CH\n4\nemissions may begin to trend upwards [59, 62].”\n59. Yvon-Durocher, G., A. P. Allen, D. Bastviken, R. Conrad, C. Gudasz, A. St-\nPierre, N. Thanh-Duc, and P. A. del Giorgio. 2014. Methane fluxes show\nconsistent temperature dependence across microbial to ecosystem scales.\nNature 507:488-491.\n62. Rosentreter, J. A., D. T. Maher, D. V. Erler, R. H. Murray, and B. D. Eyre.\n2018. Methane emissions partially offset “blue carbon” burial in mangroves.\nScience Advances 4:eaao4985.\nParagraph 256-265: This paragraph, in its current form, does not contribute\nsubstantially to the discussion. I would recommend revising to either better integrate it\nwith key findings or consider omitting it.\nWe agree that this paragraph does not provide significant benefit to the paper,\nand we have therefore chosen to omit it.\nParagraph 266-287: The decline in macrofaunal abundance deserves further\nexplanation. Were there any signs of mortality observed? Given that the plots were\n18\nmostly open and horizontal movement of organisms was possible, could escape\nbehaviour be a plausible explanation? If so, what implications might this have for\ninterpreting responses during real-world atmospheric heatwaves?\nNo direct signs of macrofaunal mortality were observed (for example, gaping\nbivalves present on the sediment surface).\nDuring OTC incubations, macrofauna would have been unable to escape by\nexiting across the sediment surface because the chambers formed a seal at\nthe sediment surface. Sub-surface movement of fauna would have been\npossible, although expected to be minimal (given the internal diameter of the\nchambers relative to the sizes of the organisms and their movement rates at\nlow tide). Migration of fauna out of plots outside of the OTC incubation times\n(which was ~19 hours of each 24 h day) would have been completely\nuninhibited. We have added more detail to this paragraph addressing the\ndeclines in macrofaunal abundance:\n(L311-320) “The significant declines in macrofaunal abundance in treatment plots\ncould be indicative of mortality, or behavioural responses that resulted in\nemigration from the treatment plots, but neither of these were observed directly.\nOTCs formed a seal at the sediment surface preventing surface emigration\nduring incubation times, however subsurface emigration (i.e. burrowing and\nlateral movement) may have been possible for some mobile species. Faunal\nmovement from treatment plots may also have occurred outside of the OTC\nincubation periods. While we cannot discern whether the decline of macrofaunal\nabundance was from mortality or emigration (or both), we theorise that the\nsimulated conditions were unsuitable for some of the macrofauna, and that real-\nworld heatwaves would have similar community effects.”\nA future study analysing macrofaunal functional traits responses to heatwave\nsimulation is planned, and we expect this to answer some of these questions\naround interpreting responses to real world heatwaves. We have added the\nfollowing sentence to acknowledge this:\n(L320-323) “Future studies of macrofaunal functional trait responses to\nheatwaves will provide insights into the vulnerabilities and implications of\nfuture heat wave events for animals with, for example, different living habit\n(e.g. attached, burrower, free living, etc.) or mobility (e.g. limited, mobile,\nsessile) traits [64].”\nFurthermore, please consider integrating findings from the parallel study more thoroughly;\nhow was metabolic stress expressed? Were any indicators of anaerobic metabolism (e.g.,\naccumulation of reduced metabolites) observed that might point to suppressed aerobic\nactivity?\ndiscussion with results from the parallel study:\n(L341-348) “Bivalve metabolism typically relies on oxygen generating a steady\nsupply of energy to support various physiological processes [69]. Elevated\ntemperatures can reduce concentrations of dissolved oxygen in sedimentary pore\nwater and accelerate metabolic rates in bivalves, which leads to increased energy\ndemands and may cause a shift from aerobic to anaerobic respiration pathways\n[68]. Indeed, the metabolic responses (i.e. metabolites and metabolic pathways)\nof this key bivalve to warming during this experiment were related to effects on\nenergy metabolism, accumulation of stress-related metabolites, disruption of\namino acid metabolism and impacts on lipid metabolism [68].”\nLine 285-286: Reference 58 states: “Cockles covered by shallow water pools moved\nup when exposed to thermal stress, while burrowing deeper into the sediment in the\nabsence of water pools.” Please clarify how this is relevant here. Please also\nconsider your references 54 here.\nZhou et al. 2022 (previously ref 54) has been added here. We think that Zhou et\nal. 2023 (previously ref 58) is relevant here since cockles showed deeper\nburrowing in response to thermal stress in the absence of water pools on the\nsediment, and in our study, there were not water pools covering the sediment.\nThis study also showed increased migratory behaviour, and we have added that\ndetail to the sentence [L351-353].\nParagraph 288-298: This paragraph is particularly valuable—thank you for this\nthoughtful integration of tidal dynamics and their ecological implications in the\ncontext of this study. It adds essential nuance to the interpretation.\nThank you for this supportive comment.\nLine 299-304: I recommend either removing this section or expanding on it with\nclearer relevance to the study’s findings and broader implications.\nWe thank the reviewer for this comment. In the interests of brevity and\nacknowledging that our study did not directly assess the potential for\nlegacy effects, we have removed the paragraph.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have read through the manuscript and the response to reviews' comments. I found that the response to my comments are fine. I do not have further comments.", "reviewer_2": "The authors have adequately addressed my concerns in the revised manuscript.", "reviewer_3": "The revision is excellent—thank you for your thorough work!\nOne minor suggestion: While I appreciate the detailed descriptions of the PERMANOVA analyses, you might consider stating the general approach once (e.g., \"PERMANOVAs were conducted as described unless otherwise noted\") to avoid repetition and improve flow.\nCheers,\nNorman", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter
[Editorial Note: See attachment]

Review of Douglas et al. Simulated heatwave alters intertidal estuary greenhouse gas
fluxes: duration and degradation state determine response
Nature Communications, May 2025
This study presents an impressive and timely in-situ warming experiment that investigates how
atmospheric heatwaves influence greenhouse gas (GHG) fluxes in an intertidal estuary. By
examining the interplay between heatwave duration, benthic macrofaunal communities, and
sedimentary environments, the authors reveal context-dependent effects on CO₂ and CH₄
fluxes. One key result is that warming altered the CO2 dynamics depending on habitat
characteristics, with larger uptake in sandy sediments and turning the muddy site to a source.
This work addresses a vital and topical issue, as climate change is driving more frequent and
intense heatwaves that may alter carbon cycling and feedbacks in productive but vulnerable
coastal ecosystems. The experimental design — combining field realism with multi-factor
analysis — adds strength to the study.
While the key results are highly valuable and the setup was quite remarkable, the manuscript
would benefit from additional clarifications and detail. Given that the study is framed within the
context of heatwaves, it would be helpful to more clearly explain how the experimental warming
during low tide fits within this framework. Clearly stating the context can be important
particularly for intertidal systems that naturally experience strong temperature fluctuations.
Also, referring to “heating days” may be somewhat misleading, as the treatment effectively
involved a short-term heat pulse – an important distinction that could make the observed
effects even more pronounced. Potentially, the authors might want to include and/or discuss,
that during the time of the experiment an actual moderate marine heatwave occurred on the
east coast of New Zealand. Relevant metrics from this event, available on
marineheatwaves.org, provided here:
Additionally, I would also recommend removing the results section of “Treatment effect size
increased with consecutive heatwave days” and integrate the results into the previous section
of “Immediate and cumulative effects of heatwave on carbon flux”. This would help
streamlining the results and avoid redundancy.
Language and format editing should also be revised throughout the manuscript.
More specific comments below
Thank you for this very interesting manuscript and I am looking forward to the final version.
Best Regards,
Norman Göbeler
Introduction:
Line 69: I would suggest rephrasing to “Methane is primarily produced…”
Line 110: Is there a specific reason why H4 is excluded from H5? Additionally, the conclusion
that sandy sites might be more resilient to warming could benefit from stronger justification or
supporting evidence. Clarifying the rationale behind this hypothesis would improve the
argument.
Methods:
Line 357: It seems that a cross-reference to Figure 7 may be more appropriate here than to
Figure 1. Please confirm and adjust accordingly.
Line 387: Please provide the data that support the similar levels of solar radiation in the
treatment and control plots.
Line 404: Was the volume measured when placed in the field? How was the placement of 2 cm
into the sediment ensured? Was this accounted for when calculating the fluxes?
Line 405: Is Figure 7e the correct reference here?
Line 410: The flux calculation method needs to be expanded. For example, clarify whether a
linear model or the HM model was used, and if both were considered, which one provided a
better fit.
Line 416: While referencing standard protocols is appreciated, please briefly summarize key
aspects of the macrofaunal sampling, such as sieve mesh size and sample preservation.
Lines 414-418: As this experiment was conducted in parallel to Lam-Gordillo et al., 2025, could
you please explain how you dealt with the collection of the 10 bivalves per plot? How were they
accounted for? Did this study utilize the same 2.6 cm in diameter cores as in Lam-Gordillo et
al., 2025?
Line 426 & other PERMANOVA applications: Please expand on the details of the PERMANOVA
analyses throughout. What distance measure was used? How many permutations were
performed? Were the fluxes normalized prior to analysis?
Line 435: Specify why you used the maximum temperature.
Line 444: Was nMDS or MDS applied? Figure 6 states MDS. Please provide more details.
Results:
Line 134 & Table A2: While the organic content is statistically different between the sites, it
would be helpful to clarify whether an approximately 0.2% higher organic matter content at the
muddy site is ecologically meaningful enough to support its characterization as a “degraded
environment.” Additional context or justification would strengthen this point.
Section following Line 171: This section tends to repeat results already presented earlier. I
would recommend treating the effect size analyses as complementary to the flux data, and
integrating them directly into the relevant sections to streamline the narrative and avoid
redundancy.
Figure 6: Please clarify if this is MDS or nMDS.
Discussion:
Line 220-222: This simplifies the complexity of heatwave definitions and involved parameters
and impacts. Please revise.
Line 223-227: Please provide supporting references that explicitly link sediment muddiness to
environmental degradation.
Line 232-233: Unclear what “negative effect” here refers to, please rephrase and clarify.
Line 234-237: This statement is currently vague and would benefit from further development
and clearer support. In particular, considering that both microphytobenthos (MPB) biomass
and macrofaunal abundance declined, could the observed increase in CO₂ uptake be due to
reduced community respiration?
Line 248-249: Additional references are needed to support this statement. Moreover, how is
this conclusion supported when methane fluxes were not directly influenced by the
warming treatment in this study?
Paragraph 256-265: This paragraph, in its current form, does not contribute substantially to the
discussion. I would recommend revising to either better integrate it with key findings or
consider omitting it.
Paragraph 266-287: The decline in macrofaunal abundance deserves further explanation. Were
there any signs of mortality observed? Given that the plots were mostly open and horizontal
movement of organisms was possible, could escape behavior be a plausible explanation? If so,
what implications might this have for interpreting responses during real-world atmospheric
heatwaves? Furthermore, please consider integrating findings from the parallel study more
thoroughly; how was metabolic stress expressed? Were any indicators of anaerobic
metabolism (e.g., accumulation of reduced metabolites) observed that might point to
suppressed aerobic activity?
Line 285-286: Reference 58 states: “Cockles covered by shallow water pools moved up when
exposed to thermal stress, while burrowing deeper into the sediment in the absence of water
pools.” Please clarify how this is relevant here. Please also consider your references 54 here.
Paragraph 288-298: This paragraph is particularly valuable—thank you for this thoughtful
integration of tidal dynamics and their ecological implications in the context of this study. It
adds essential nuance to the interpretation.
Line 299-304: I recommend either removing this section or expanding on it with clearer
relevance to the study’s findings and broader implications.