# Evidence of limited N2 fixation in the Southern Ocean

Shuai Gu, Hugo Berthelot, Yajuan Lin, Weiyi Tang, Julie Robidart, A. Murat

Eren, Hugh Ducklow, Nicolas Cassar

► To cite this version:

Shuai Gu, Hugo Berthelot, Yajuan Lin, Weiyi Tang, Julie Robidart, et al. Evidence of limited N2 fixation in the Southern Ocean. Communications Earth & Environment, 2025, 6 (1), pp.264. 10.1038/s43247-025-02225-0. hal-05037659

HAL Id: hal-05037659

https://hal.univ-brest.fr/hal-05037659v1

Submitted on 21 Apr 2025

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# Evidence of limited N₂ fixation in the Southern Ocean

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Shuai Gu<fnref n="1" />, Hugo Berthelot ⑩<sup>2,12</sup>, Yajuan Lin ⑩<sup>1,2,3</sup>, Weiyi Tang<sup>1,4,5</sup>, Julie Robidart<sup>6</sup>, A. Murat Eren<sup>7,8,9,10</sup>,

Hugh W. Ducklow ⑩<sup>11</sup> & Nicolas Cassar ⑩<sup>1,2</sup> ☒

Biological nitrogen fixation is an important source of new nitrogen, influencing ocean fertility and carbon uptake. While recently documented in Arctic waters, its role in the Southern Ocean remains uncertain. We measured nitrogen fixation along the Western Antarctic Peninsula and at Palmer Station over two austral summer months. Rates from ¹⁵N₂ assay were below conservative detection limits but detectable under less stringent detection thresholds. Continuous acetylene reduction assay provided further support. *nifH* gene sequencing identified Gammaproteobacteria as the dominating identified diazotrophs, while Epsilonproteobacteria contributed disproportionally to *nifH* expression when putative nitrogen fixation was highest. Combined with environmental observations, we hypothesize that vertical water mixing resuspended sediments into the water column and contributed to the limited nitrogen fixation. Given the sporadic and low rates, further research is needed to determine whether nitrogen fixation plays a minor role or represents an overlooked process with biogeochemical significance in the Southern Ocean.

Nitrogen is a fundamental element of life, present in biomolecules such as proteins and nucleic acids. In many regions of the world oceans such as oligotrophic regions, nitrogen availability limits photosynthesis and the biological uptake and sequestration of carbon dioxide (CO₂)<fnref n="1" />. Biological dinitrogen (N₂) fixation to ammonium is a major source of new nitrogen in the ocean² and can support up to half of new production in oligotrophic waters<fnref n="1" />. However, estimates of global N₂ fixation harbor substantial uncertainties, ranging from less than 100 to over 200 Tg N year⁻¹²⁻⁴. By comparison, global ocean denitrification estimates are ~400 Tg N year⁻¹⁵. Other sources of nitrogen, such as riverine input and atmospheric deposition, are not large enough to balance the nitrogen budget in the ocean². The implication is that either the oceanic nitrogen inventory is decreasing (with implications for ocean fertility), or that nitrogen sources (sinks) are under- (over-) estimated. In order to address a potential missing source of nitrogen, researchers are actively looking for other biomes where N₂ fixation may be important. Until recently, N₂ fixation was believed to be conducted predominantly by cyanobacteria such as *Trichodesmium* living in warm tropical and subtropical waters⁶. However, recent studies have not only

expanded the range of habitats over which N₂ fixation may be important, including coastal⁷ and polar oceans⁸, but also the microbial taxa who may be responsible for it⁹⁻¹². In polar waters, most observations of N₂ fixation have been collected in the Arctic with a dominance of diazotrophs more eurythermal than *Trichodesmium* such as UCYN-A and cluster III *nifH* phy lotypes, and with rates ranging from below detection limit to 17.2 nmol N L⁻<fnref n="1" /> d⁻¹⁸,¹³,¹⁴. In contrast, few observations are available in Antarctic waters. Traditional views would dictate that the cold and nitrogen rich waters of the Southern Ocean should not be conducive to N₂ fixation. However, low N₂ fixation rates have been reported in the high-latitude waters of the South Pacific and South Indian Oceans¹⁵,¹⁶. Recently, Shiozaki et al.¹⁷ published evidence of N₂ fixation in eastern Antarctic waters with a particularly high rate (44.4 nmol N L⁻<fnref n="1" /> d⁻<fnref n="1" />) at the ice-edge¹⁷. This high rate has recently been questioned by White et al. as it was calculated from an outlier and the observed UCYN-A abundance was not sufficient to support such a high rate¹⁸. More recently, N₂ fixation was reported during late summers in the coastal waters of Chile Bay at the Western Antarctic Peninsula (WAP)¹⁹.

<footnote n="1">¹Division of Earth and Climate Sciences, Nicholas School of the Environment, Duke University, Durham, NC, USA. ²Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/IFREMER, Institut Universitaire Européen de la Mer (IUEM), Plouzané, France. ³Department of Life Sciences, College of Science, Texas A&M University—Corpus Christi, Corpus Christi, TX, USA. ⁴Department of Geosciences, Princeton University, Princeton, NJ, USA. ⁵College of Marine Science, University of South Florida, St. Petersburg, FL, USA. ⁶Ocean Technology and Engineering Group, National Oceanography Centre Southampton, Southampton, UK. ⁷Helmholtz Institute for Functional Marine Biodiversity, Oldenburg, Germany. ⁸Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany. ⁹Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, Oldenburg, Germany. ¹⁰Max Planck Institute for Marine Microbiology, Bremen, Germany. ¹¹Lamont-Doherty Earth Observatory, Columbia University, Palisades, NY, USA. ¹²Present address: Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany. ✉e-mail: nicolas.cassar@duke.edu</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1** | Map of the sampling region at the Western Antarctic Peninsula. Numbers 1-5 indicate discrete sampling stations from January to February in 2018. The red diamond shows Palmer Station, where time-series observations were conducted from January to March in 2019.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2** | Nitrogen fixation rates measured at Palmer Station. Nitrogen fixation rates (nmol N L⁻¹ d⁻¹) measured by ¹⁵N₂ incubation (blue bars) from January to March and by FARACAS (blue line) from Jan. 27th to 28th in 2019. The error bars show</figcaption></figure>

standard deviation of the triplicate incubations (except for duplicates on Feb. 11th). Rates above the MQR, LOD<sub>Montoya</sub>, or LOD<sub>sd</sub> are depicted with green marks.
<figure><img src="image_6.png" /><figcaption>**Fig. 3** | Times series of environmental parameters from Jan. to Mar. at Palmer Station in 2019. 1-h averaged chlorophyll, water temperature, salinity, wind speed and O₂/Ar ratio are shown by blue lines in each panel. Each red dot represents a 10-min average at the sampling time point for discrete DNA/RNA samples. The shaded</figcaption></figure>

area indicates the time periods when ¹⁵N₂ incubation experiments were performed and the signals were below (gray shaded) or above the MQR or the LOD<sub>Montoya</sub> (green shaded).

In this study, we provide evidence of potential but limited N₂ fixation rates in Antarctic waters. We focus our study on the Western Antarctic Peninsula (Fig. 1), a region which has experienced warming, sea ice retreat, and changes in phytoplankton community structure over the last decades²⁰⁻²². We conducted a survey of N₂ fixation along the WAP shelf from January to February in 2018, and time-series observations at Palmer Station during the growing season from January to March in 2019. The rates measured by ¹⁵N₂ incubation method were below the most stringent detection limit at all offshore stations in 2018 and from February to March at Palmer Station in 2019. However, some measured N₂ fixation rates were above less stringent detection limits suggesting potential N₂ fixation. This was corroborated by estimates with our high-frequency instrument FARACAS, a flow-through method that measures N₂ fixation continuously based on acetylene reduction²³. While Shiozaki et al.¹⁷ found a dominance of UCYN-A in *nifH* gene expression¹⁷, our *nifH* gene amplicon sequencing results showed that Proteobacteria were likely the dominant diazotroph in our study region. Combined with the analysis of environmental properties, we hypothesize that the diazotrophs were associated with the sediments. In our study, the potential N₂ fixation fluxes were sporadic and low. Additional work is needed to determine if N₂ fixation in Antarctic waters is an interesting biological process with little biogeochemical implications, or if our limited observations encountered an important new biome of N₂ fixation.

# Results and Discussion

## Measuring low N₂ fixation rates

Due to relatively low fluxes compared to the available pool, measuring N₂ fixation in the ocean using ¹⁵N₂ assays has long been a methodological challenge and a matter of discussion, with methodological bias toward potential over-estimation²⁴ or under-estimation²⁵⁻²⁷. More recently, Gradoille et al.²⁸ pointed out the need to qualify systematically the detection limits when using ¹⁵N₂ assays in particular when reporting low rates to avoid interpreting false positives²⁸. They proposed two ways to qualify the detection threshold. The first one is to propagate all the experimental uncertainties associated with each parameter used for the determination of the rate, defined as to the minimal quantifiable rate (MQR)²⁹. The second one is to define a minimum ¹⁵N enrichment between the start and end of the incubations of 4‰ (0.00146 atom%) according to Montoya et al.³⁰, referred here as the limit of detection (LOD<sub>Montoya</sub>). In addition, we defined an alternative limit of detection (LOD<sub>sd</sub>) using three times the standard deviation of the initial ¹⁵N abundance in our samples as a threshold. In our experiments, while the isotopic composition of the particulate organic N (PON) was measured in triplicate after the incubations, the initial value was measured in monoplicate only. In order to define the LOD<sub>sd</sub>, we modeled the standard deviation of the initial ¹⁵N abundance in PON using the observed trend over time at Palmer station (see the “Method” section for more details).

N₂ fixation was detected at seven, one, and none of the time points out of 18 at Palmer Station depending on whether we use the MQR, LOD<sub>Montoya</sub>,
<figure><img src="image_7.png" /><figcaption>**Fig. 4** | Time series of diazotroph community composition at class/phylum level, identified from *nifH* sequencing. The left and right panels show DNA and RNA, respectively. Only samples with more than 1000 reads are included. The black and</figcaption></figure>

white squares indicate samples for which N₂ fixation rates were above or below detection thresholds, respectively.

and LOD<sub>sd</sub> as thresholds, respectively (Fig. 2). Interestingly, the only sample above the LOD<sub>Montoya</sub> is not above the MQR. This discrepancy is due to the fact that only the <sup>15</sup>N enrichment difference between the beginning and the end of the incubation in the particulate matter is used to calculate LOD while all the parameters used in the calculation of the N<sub>2</sub> fixation rate are included to calculate MQR. Nevertheless, since the N<sub>2</sub> fixation rates presented here were always close to the different detection limits used (Supplementary Data 1 and 2), we interpret them as potential N<sub>2</sub> fixation rates.

## Time series of nitrogen fixation measurements and other environmental factors

<sup>15</sup>N<sub>2</sub> fixation rates were above the MQR between Jan. 24th and Feb. 22nd (with the exception of Feb. 17th) with rates ranging 0.21–6.59 nmol L<sup>-1</sup> d<sup>-1</sup> when detected and then remained below the MQR for the rest of the studied period. N<sub>2</sub> fixation rates were only above the LOD<sub>Montoya</sub> on Mar. 5th at a rate of 1.53 nmol L<sup>-1</sup> d<sup>-1</sup>. These measured N<sub>2</sub> fixation rates were comparable to most of the maximum N<sub>2</sub> fixation rates reported by Shiozaki et al.<sup>17</sup>, except for one of their extremely high value (44.4 nmol N L<sup>-1</sup> d<sup>-1</sup>) at Station E near the sea ice edge<sup>17</sup>, although our study was conducted during austral summer when there was no sea ice formation in the northern WAP. This rate was also comparable to the rates in surface waters under light conditions between 2013 and 2017 reported by Alcamán-Arias et al.<sup>19</sup> at Chile Bay, but considerably lower than those in 2018 and 2019<sup>19</sup>. On a more global scale, between Jan. 24th and Feb 14th N<sub>2</sub> fixation rates were higher than the 7th decile of the compiled rates reported in Shao et al.<sup>31</sup>. However, when normalized to the high biomass observed in the study region (N<sub>2</sub> fixation rate/particulate organic N), our rates (<0.001 d<sup>-1</sup>) are much lower than what is measured in subtropical waters (typically ranging 0.01–0.1 d<sup>-1</sup>)<sup>31</sup>, suggesting a limited impact of N<sub>2</sub> fixation in this Antarctic ecosystem. To capture temporal patterns of N<sub>2</sub> fixation at higher resolutions than <sup>15</sup>N<sub>2</sub> incubations, measurements by FARACAS were carried out, which also showed detectable N<sub>2</sub> fixation from Jan. 27th to 28th (Fig. 2), ranging from 1.2 to 5.8

nmol N L<sup>-1</sup> d<sup>-1</sup>. These rates were comparable to <sup>15</sup>N<sub>2</sub> incubation measurements. The stir bar in FARACAS incubator was found to be stuck before 9 pm on Jan. 28th and could have caused overestimation of N<sub>2</sub> fixation rates between 4 am and 9 pm because of ethylene buildup in the incubator. The discrepancy between the N<sub>2</sub> fixation rates measured by two different methods could also be caused by uncertainties in the conversion ratio between acetylene reduction and N<sub>2</sub> fixation and release of newly fixed N into dissolved phase not captured by the <sup>15</sup>N<sub>2</sub> incubation. The increasing N<sub>2</sub> fixation trends at night as observed after 9 pm on 28th is consistent with the higher rates under dark conditions at the WAP<sup>19</sup>, potentially contributed by the heterotrophic diazotrophs (as described in the next section).

The days before Feb. 2nd and 14th, when N<sub>2</sub> fixation rates were at their highest, were characterized by a drastic change in environmental conditions. Strong winds on Jan 22nd (>15 m/s) caused considerable water mixing with a substantial drop in biological oxygen saturation (as estimated with O<sub>2</sub>/Ar) at the ocean surface from around +10% to below -10% (Fig. 3). The water temperature did not change substantially, but salinity increased from 33.4 to 33.8, the highest value observed over the study period (Fig. 3) and was consistent with upwelling of bottom waters or Upper Circumpolar Deep Water (salinity 34.62–34.68)<sup>32</sup>. Vertical profiles of water density at station B 1 km from Palmer Station also showed elevated values in the surface layer during this time (Supplementary Fig. 1), indicating the enhanced vertical water mixing associated with the strong winds. Nutrients (N and P, Supplementary Fig. 1) also increased during this water mixing event, and triggered a diatom bloom in early February, as shown by the increase in chlorophyll, O<sub>2</sub>/Ar ratios (Fig. 3), and particulate organic N (Supplementary Data 1). The color of the filtered biomass, when N<sub>2</sub> fixation was measurable, was also visually distinct from other samples (Supplementary Fig. 2), indicating potentially different plankton communities or particle sources. Based on these environmental conditions, and our *nifH* analyses showing likely sediment-associated diazotrophs (see “Discussion” below), we
<figure><img src="image_8.png" /><figcaption>**Fig. 5** | Maximum likelihood phylogenetic analysis of potential diazotroph communities based on *nifH*. The analysis includes a total of 206 *nifH* sequences, color coded by source: blue and purple for the top 15 *nifH* amplicon sequencing variants (ASVs) in DNA and RNA samples in this study, respectively, with overlap in *nifH* ASV 3; red labels denote top ASVs identified in eastern Antarctica waters by</figcaption></figure>

Shiozaki et al.¹⁷; pink labels for sequences identified from Chile Bay by Alcamán-Arias et al.¹⁹. Additional *nifH* sequences from other environmental samples are represented in various colors. Bootstrap values were determined from 1000 iterations (blue circles).

hypothesize that the strong winds resuspended sediments in the shallow water column (bottom depth within 10 m).

The importance of sedimentary N₂ fixation is increasingly evident³³,³⁴, especially in coastal oceans³⁵⁻³⁸. Mixing could inoculate the water column with sedimentary diazotrophs³⁹ and/or alleviate water column diazotrophic iron limitation through resuspension⁴⁰,⁴¹. Limited organic matter availability (with lower chlorophyll and negative O₂/Ar ratios), or higher N/P ratios (Supplementary Fig. 1) may explain the undetectable N₂ fixation on other windy days later during the growing season. Further studies are needed to ascertain the factors controlling diazotrophy in the region.

To expand the spatial coverage of N₂ fixation observations along the WAP, we conducted ¹⁵N₂ incubations at 5 different stations along the WAP during the 2018 PAL-LTER cruise from Jan 22nd to Feb 3rd (Fig. 1). All N₂ fixation rates were below the detection limits (LOD<sub>montoya</sub>, LOD<sub>sd</sub>, and MQR) except for station 150.00 where the measured N₂ fixation rate of 1.9 nmol L⁻¹ d⁻¹ which was associated with a high standard deviation (±8.3 nmol L⁻¹ d⁻¹) exceeded the LOD<sub>montoya</sub> of only 0.3 nmol L⁻¹ d⁻¹. As opposed to the time-series sampling site at Palmer Station, where bottom depth is 10–20 m, these 5 regional cruise stations were farther offshore, with the water depth ranging from 250 to 460 m. The absence of detectable N₂ fixation rates in surface waters at these offshore deeper stations also aligns with our hypothesis that sedimentary diazotrophs are the main contributors to measurable N₂ fixation rates in the study region.

## Diazotroph community composition in Antarctica

DNA and RNA were extracted from a total of 55 filters collected from Jan. 13th to Mar. 24th in 2019. *nifH* genes were amplified from all 55 DNA samples. The sequences show the presence of putative diazotrophs in the WAP coastal waters (Fig. 4). A maximum likelihood phylogenetic tree was built for the top 15 *nifH* ASVs among DNA and RNA samples from this study (named as “*nifH* ASV 1”, etc.) together with the top 15 ASVs from eastern Antarctica (named as “ASV001” to “ASV015” in Shiozaki et al.)¹⁷, 18 OTUs from Chile Bay¹⁹ and other representative *nifH* sequences from environmental samples or diazotroph isolates (Fig. 5).

While there are some similarities in diazotrophic communities between different Antarctic waters (e.g., clustering of *nifH* ASV 1 and ASV013, *nifH* ASV 14 and Ued OTU03, ASV004 and Ued OTU12), most of the top *nifH* ASVs from our study are distinct from those in eastern Antarctic or Chile Bay waters (Fig. 5). The top sequences from our study are also different from Arctic samples based on the phylogenetic tree (Fig. 5). For example, UCYN-A was described as a major contributor to N₂ fixation in eastern Antarctica⁷ and the Arctic waters¹⁴,⁴². In contrast, no *nifH* ASVs from our study sites at the WAP are close to UCYN-A (ASV001) on the phylogenetic tree (Fig. 5). The absence of UCYN-A was also reported in the Chile Bay waters¹⁹, indicating distinct diazotroph communities between eastern and western Antarctic waters. In our study, Proteobacteria were the most well-represented diazotrophic phylum, with Gammaproteobacteria as
the dominant class (Fig. 4). 22 out of the 55 RNA samples showed posi-
tive *nifH* RT-PCR amplifications. Shown on Fig. 4 are the 13 RNA samples
with more than 1000 reads per library after removal of the non-*nifH* gene
sequences. Epsilonproteobacteria and Bacteroidia dominated the RNA
samples. The discrepancy between DNA and RNA *nifH* compositions
indicates that some rare diazotrophs may contribute disproportionally to N₂
fixation rates.

Potential for sedimentary diazotrophy

Some Epsilonproteobacteria can fix N₂ and have been shown to be poten-
tially important in high-latitude coastal sediments⁴³. We hypothesize that
Epsilonproteobacteria were likely key diazotrophs at the WAP. When
measurable N₂ fixation rates and substantial *nifH* RNA reads were detected
(Fig. 4), the most abundant RNA reads were identified as Epsilonproteo-
bacteria, with *nifH* ASV 35 being the most abundant ASV on Jan. 28 and
Feb. 2 and *nifH* ASV 108 and *nifH* ASV 152 accounting for >70% of the
reads on Feb. 23. Strains under genus *Arcobacter* were among the top blast
hits for both *nifH* ASV 108 and *nifH* ASV 152. *Arcobacter* has been reported
in diverse habitats including estuarine sediments¹⁴,⁴⁵. The top blast hit was
*Candidatus Sulfurimonas* under genus *Sulfurimonas* (score = 468,
e-value = 2e−127) for *nifH* ASV 35. Genus *Sulfurimonas* is under the order
Campylobacterales, which has been reported in high-latitude ocean
sediments⁴⁶,⁴⁷. Similarly, species of genus *Sulfurimonas* can be dominant in
pelagic and sedimentary redoxclines⁴⁸, again agreed with the hypothesis that
*nifH* ASV 35 resulted from resuspended sediments. Furthermore, *nifH* ASV
35, *nifH* ASV 108 and *nifH* ASV 152, which were the dominantly expressed
RNA ASVs during measurable N₂ fixation in our study, clustered together
with sequences isolated from sedimentary samples (Fig. 5). *nifH* ASV 1 and
*nifH* ASV 20 cluster with ASV013 on the phylogenetic tree, together with
four other sedimentary samples, further supporting a sedimentary origin.
Notably, Chile Bay diazotrophs also clustered together with sedimentary
samples but differed from the top *nifH* ASVs in our RNA samples on the
phylogenetic tree (Fig. 5).

A bloom of the genus *Arcobacter* and *Sulfurimonas* was reported after
oxygenation of sediments collected from the coastal Baltic Sea<sup>43</sup>, a similar
eutrophic high latitude coastal environment to the WAP, which was tra-
ditionally believed unfavorable for N<sub>2</sub> fixation. A recent study also showed
that N<sub>2</sub> fixation occurred under simulated sediment resuspensions with
coastal Baltic Sea sediments, dominated by sulfur-reducing bacteria<sup>49</sup>.
Reported to be prominent in diazotrophic communities from temperate
estuary sediments<sup>39</sup>, sulfur-reducing Deltaproteobacteria were also present
at our study site. Sediment resuspension at the WAP likely led to a re-
oxygenation similar to one observed in the Baltic Sea study<sup>43</sup>. The authors
hypothesized that re-oxygenation may have led to favorable microaerophilic
conditions for these genera, and high heterogeneity in sediments created
micro niches which permitted anaerobic N<sub>2</sub> fixation<sup>43</sup>. N<sub>2</sub> fixation may also
have been stimulated by subdued inhibition by ammonium when resus-
pended and diluted into the water column<sup>50</sup>.

Considering that a large portion of the *nifH* sequences were not
identified based on the currently available reference database (Fig. 4), an
alternative to the sediment source is that we may have encountered new
heterotrophic N₂ fixers. Recent studies have shown that heterotrophic N₂
fixers may be more important in the ocean than previously thought⁹⁻¹². Our
study demonstrates that they may also be important in Antarctic waters. For
example, many of our *nifH* ASVs also showed substantial levels of sequence
identity (i.e., over 85%) to *nifH* genes found in genomes of prevalent het-
erotrophic bacterial diazotrophs recently reconstructed from surface ocean
metagenomes¹⁰.

In our study, characterization of the community was also performed on
days when N₂ fixation was below the detection limit. On some of these days,
the *nifH* gene was successfully amplified (RT-PCR) from RNA samples
(Fig. 4) (e.g., Mar. 24th). This could result from low absolute abundance of
active diazotrophs or below detection limit N₂ fixation, or a change in
diazotroph community between the start of the incubations and molecular
sampling. For example, an RNA sample collected in the afternoon of Jan.

27th, right before the second ¹⁵N₂ incubation, displayed notably different
active taxa based on *nifH* gene expression, dominated by *nifH* ASV 8
under class Bacteroidia (93.7%). The top blast hit of *nifH* ASV 8 is an
uncultured bacterium from the OMZ water column (score = 348,
e-value = 2e-91). This substantial change in *nifH* gene expression within
10 h could be caused by diel variability or changes in water masses, perhaps
associated with tidal activity.

With the results indicating that resuspended sediments may have
contributed to measurable N₂ fixation in coastal Antarctic waters, our study
challenges the traditional view that polar, coastal, and nutrient-rich oceans
are inhospitable to diazotrophs and adds to recent studies expanding their
potential niches⁷,¹⁷,¹⁹,⁵¹⁻⁵⁵. However, our observations showed N₂ fixation to
be low (or absent when using more stringent thresholds) and sporadic. The
major diazotroph communities were distinct from other studies in Antarctic
waters¹⁷,¹⁹, showing the Southern Ocean to be a potentially diverse N₂ fixer
habitat. This time-series dataset also complements our understanding of
temporal variabilities of N₂ fixation in Antarctica waters. It remains to be
determined how widespread diazotrophy is in Antarctic waters and how it is
influenced by sediment resuspension. Furthermore, given that vertical
mixing is common in this region, sediment resuspension may also influence
elemental cycling within the water column⁵⁶. In light of the disproportionate
influence of climate change in polar regions (e.g., warming, stratification,
and community changes), further studies are needed to examine the
importance of N₂ fixation in this newly discovered biome.

Methods

High-resolution continuous measurement of N₂ fixation rates
(FARACAS)

High frequency measurements of aquatic N₂ fixation were conducted at Palmer Station, Antarctica, from January to March in 2019 using the FARACAS system²³. Due to technical issues, the FARACAS data were only usable at the start of the deployment during January 27–28th. Unfiltered seawater was continuously pumped into the laboratory from 6 m depth in Arthur Harbor, west of Palmer Station. C₂H₂ gas was prepared in Tedlar bag by reacting high-purity calcium carbide (CaC₂, Alfa Aesar) with ultrapure water, and then dissolved in 0.2 µm filtered seawater to a saturation of 70% (v/vv%). The 70% C₂H₂ tracer water was then mixed with unfiltered seawater at a ratio of 1:6, reaching a final saturation of 10%. This water mixture was pumped into a modified 9 L glass incubator, where flow-through incubation was performed. Ambient temperature was maintained by a water-jacket outside of incubator and light was simulated using an LED-wrap based on the solar altitude. The incubator was thoroughly acid washed every two days. C₂H₄ production rates were measured during the incubation using a Picarro CRDS (cavity ring-down laser absorption spectroscopy) C₂H₄ analyzer (model G1106, Santa Clara, CA), and converted to N₂ fixation rates using a factor of 4:1.

¹⁵N₂ incubation

Concurrently, discrete ¹⁵N₂ addition incubations were conducted from late
January to March 2019 at Palmer Station, following the protocols outlined in
Mohr et al.²⁵. Briefly, ¹⁵N₂ enriched water was made by dissolving 10 mL ¹⁵N₂
tracer (Cambridge Isotope Laboratories, 98%+) into 1 L 0.2 µm filtered
seawater after degassing under vacuum for more than 1 h. Surface seawater
was collected into 4 L transparent polycarbonate bottles directly from the
shore adjacent to the inlet of unfiltered water source for the FARACAS system
at Palmer Station or into 2.5 L transparent polycarbonate bottles from Niskin
bottles during the 2018 LTER cruise. In case of unfavorable weather condi-
tions at Palmer Station, water was collected from the tap in the Palmer Station
pump house. ¹⁵N₂ enriched water was injected to the bottom of each triplicate
bottles (5% v:v) at Palmer Station or directly injected as ¹⁵N₂ during the 2018
LTER cruise, after which the triplicate bottles were incubated for 24 h in an
outdoor transparent tank, with continuously circulating surface seawater
controlling the temperature. Dissolved ¹⁵N₂ samples were collected in gas
tight Exetainer vials (Labco) from initial ¹⁵N₂ enriched water as well as from
each bottle after incubation. The samples were poisoned with HgCl₂ and
stored until analysis using Membrane Inlet Mass Spectrometer (Bay Instruments). Particulate matter was recovered by filtering the water through pre-combusted 25 mm GF/F filters (Whatman) at the end of each incubation. Non-incubated particulate matter was also sampled at each time point (in monoplicate) to reflect the natural ¹⁵N/¹⁴N ratio in the environment. Filters were stored at -80 °C and dried before N isotope analysis using an elemental analyzer coupled with a mass spectrometer. N₂ fixation rates were calculated according to Montoya et al.³⁰. At Palmer Station, the natural (i.e., non-incubated) ¹⁵N abundance of particulate matter measured at each time point in monoplicate showed a scattered pattern but a linear increasing trend over the time course of the survey (Sieve Bootstrap Based Test, "notrend_test" function in "funtimes" R package, *p*-value < 0.0001). We assume that this trend reflects an ecological reality and the scattered nature of the samples is mostly due to analytical uncertainty. In order to account for this, we modeled the linear increasing trend (r = 0.75, *p*-value = 0.0003, *n* = 18, Supplementary Fig. 3) and used the fitted values to define the initial ¹⁵N abundance in particulate matter for each time point. During the 2018 LTER cruise, no pattern in the ¹⁵N abundance of non-incubated was observed and the average of all values was used for all stations. To determine the LOD<sub>sd</sub>, the standard deviation of the initial ¹⁵N abundance in the PON was calculated using the rolling standard deviation method with a width of 3, to account for the trend, leading to a value of 0.00063 atom%. LOD<sub>Montoya</sub> and MQR were calculated according to Gradoville et al.²⁸.

## DNA and RNA sample collection

DNA and RNA samples were collected twice a day around mid-day and mid-night from January 13th to March 24th in 2019. In order to avoid potential contamination within the pipes between Palmer Station pump house and the wet lab, unfiltered seawater was collected into acid-washed LDPE Cubitainers (Thermo Scientific) from the tap in the pump house. A peristaltic pump was used to filter seawater onto 0.22 µm polyethersulfone membrane filters (Millipore) or 0.22 µm Sterivex filters (Millipore). The filtration was limited to no more than 30 min to minimize RNA degradation, achieving a filtration volume ranging between 600–4000 mL. The filters were flash frozen immediately using liquid nitrogen and then stored at -80 °C until further analysis.

## DNA and RNA extraction, nested PCR and *nifH* gene sequencing

DNA and RNA were extracted from 0.22 µm membrane or 0.22 µm Sterivex filters using Allprep DNA/RNA mini kit (Qiagen) following the manufacture's instruction with an additional 2-min bead-beating step at 30 Hz using 0.2 g of 0.1 mm Zr beads. DNA and RNA were eluted using 50 uL EB buffer or RNase-free water respectively. RNA samples were further cleaned using RNase-Free DNase set (Qiagen) and the RNA Clean & Concentrator kit (Zymo) to remove DNA, and finally eluted in 15 µL RNase free water. DNA and RNA concentrations were measured on a Qubit fluorometer using Qubit DNA/RNA HS Assay Kit. In order to synthesize cDNA from RNA samples for *nifH* gene amplification, reverse transcription was conducted using QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer's guidelines. *nifH* genes were then amplified using a nested PCR protocol (nifH3 and nifH4 primers during the 1st round, nifH1 and nifH2 primers with Illumina adapter during the 2nd round) as described by Turk et al.⁵⁷. The thermal cyle of nested PCR was 95 °C for 5 min followed by 35 cycle of 1 min at 95 °C, 55 °C, 72 °C, respectively, and then 5 min at 72 °C. Finally, customized dual indexed barcodes (6 bp) with heterogenous spacers⁵⁸ were added through a ligation process using KAPA HiFi ReadyMix⁵⁹. The thermal cycle of ligation PCR was 95 °C for 3 min followed by 8 cycle of 30 s at 95 °C, 55 °C, 72 °C, respectively, and then 5 min at 72 °C. Samples were pooled in equal molar concentrations before sending for Illumina sequencing at Duke Center for Genomic and Computational Biology in one MiSeq 300PE run.

## Sequence data processing

Raw sequencing data was demultiplexed using QIIME 1 (version 1.9.1). The reads were then merged, and barcodes, primers and adapters were trimmed

using BBDuk (version 1.1.2). ASVs were inferred using DADA2 pipeline after quality filtering and dereplication⁶⁰. Chimeras were then removed, and taxonomy was assigned using the function "assignTaxonomy" with reference *nifH* ARB database⁶¹. All ASVs were blasted against Refseq database using blastx in DIAMOND⁶² and the ASVs that do not encode nitrogenase were filtered out as non-*nifH* sequences. Some of the featured ASVs were further blasted on National Center for Biotechnology Information (NCBI) database for more information. In total, 25403 ASVs were identified from 55 DNA samples and 22 RNA samples. The 55 DNA samples had an average of 101846 reads and the 13 RNA samples (>1000 reads) had an average of 41147 reads. All *nifH* gene sequencing data has been deposited in NCBI.

Phylogenetic analyses were conducted in MEGA7 (version 7.0.26)⁶³. 206 sequences were first aligned using ClustalW and then constructed the phylogenetic tree by Maximum Likelihood method based on the Tamura-Nei model⁶⁴. Bootstrap values were calculated based on 1000 times of replications.

## Environmental parameters

Temperature, salinity, chlorophyll, and wind speed data were downloaded from the Antarctic Meteorological Research Center (AMRC) FTP site (ftp://amrc.ssec.wisc.edu/pub/palmer/) and averaged over a 10-min time span at the sampling time or an 1-hour time span during the time series. Temperature was measured at the Palmer Station pump house using a Seabird SBE 38 thermometer. Conductivity was measured using Seabird SBE45 thermosalinograph in the Palmer Station aquarium lab. On the days when these temperature and conductivity data were not available, data were interpolated based on the Palmer Station tide gauge sensor data and salinity was calculated⁶⁵ in order to obtain a wider time coverage. Chlorophyll was measured using a WetLab fluorometer (FLRT 3759) in the aquarium lab. Wind speed was measured at the weather station in the backyard of Palmer Station. O₂/Ar ratios were measured in the wet lab using an equilibrator inlet mass spectrometer (EIMS)⁶⁶ and averaged to the same 10-min or 1-h resolution as other environmental parameters.

## Data availability

Source data used to create all the figures are available on Figshare (https://doi.org/10.6084/m9.fishshare.28386431.v2). The sequence datasets generated for this study have been deposited at DDBJ/ENA/GenBank under the accession KIWL00000000. The version described in this paper is the first version, KIWL01000000.

## Code availability

No custom code or mathematical algorithm used to be deemed central to the conclusions.

Received: 17 March 2024; Accepted: 19 March 2025;

Published online: 05 April 2025

## References

1. Karl, D. et al. The role of nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean. *Nature* **388**, 533 (1997).

2. Gruber, N. The marine nitrogen cycle: overview and challenges. *Nitrogen Mar. Environ.* **2**, 1–50 (2008).

3. Luo, Y.-W., Lima, I. D., Karl, D. M., Deutsch, C. A. & Doney, S. C. Data-based assessment of environmental controls on global marine nitrogen fixation. *Biogeosciences* **11**, 691–708 (2014).

4. Tang, W., Li, Z., & Cassar, N. Machine learning estimates of global marine nitrogen fixation. *J. Geophys. Res. Biogeosciences* **124**, 717–730 (2019).

5. Codispoti, L. A. An oceanic fixed nitrogen sink exceeding 400 Tg N a⁻¹ vs the concept of homeostasis in the fixed-nitrogen inventory. *Biogeosciences* **4**, 233–253 (2007).

6. Capone, D. G. et al. Nitrogen fixation by Trichodesmium spp.: an important source of new nitrogen to the tropical and subtropical North Atlantic Ocean. *Glob. Biogeochem. Cycles* **19**, 2 (2005).
7. Tang, W. et al. Revisiting the distribution of oceanic N 2 fixation and estimating diazotrophic contribution to marine production. *Nat. Commun.* **10**, 831 (2019).

8. Sipler, R. E. et al. Preliminary estimates of the contribution of Arctic nitrogen fixation to the global nitrogen budget. *Limnol. Oceanogr. Lett.* **2**, 159–166 (2017).

9. Delmont, T. O. et al. Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes. *Nat. Microbiol.* **3**, 804–813 (2018).

10. Delmont, T. O. et al. Heterotrophic bacterial diazotrophs are more abundant than their cyanobacterial counterparts in metagenomes covering most of the sunlit ocean. *ISME J.* **16**, 927–936 (2022).

11. Farnelid, H. et al. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria. *PloS ONE* **6**, e19223 (2011).

12. Bombar, D., Paerl, R. W. & Riemann, L. Marine non-cyanobacterial diazotrophs: moving beyond molecular detection. *Trends Microbiol.* **24**, 916–927 (2016).

13. Blais, M. et al. Nitrogen fixation and identification of potential diazotrophs in the Canadian Arctic. *Glob. Biogeochem. Cycles* **26**, 3 (2012).

14. Shiozaki, T. et al. Diazotroph community structure and the role of nitrogen fixation in the nitrogen cycle in the Chukchi Sea (western Arctic Ocean). *Limnol. Oceanogr.* **63**, 2191–2205 (2018).

15. Hörstmann, C. et al. Hydrographic fronts shape productivity, nitrogen fixation, and microbial community composition in the southern Indian Ocean and the Southern Ocean. *Biogeosciences* **18**, 3733–3749 (2021).

16. Raes, E. J. et al. N2 Fixation and new insights into nitrification from the ice-edge to the equator in the South Pacific Ocean. *Front. Mar. Sci.* **7**, 389 (2020).

17. Shiozaki, T. et al. Biological nitrogen fixation detected under Antarctic sea ice. *Nat. Geosci.* **13**, 729–732 (2020).

18. White, A. E., Granger, J. & Turk-Kubo, K. Questioning high nitrogen fixation rate measurements in the Southern Ocean. *Nat. Geosci.* **15**, 29–30 (2022).

19. Alcamán-Arias, M. E. et al. Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula. *Microorganisms* **10**, 1140 (2022).

20. Lin, Y. et al. Decline in plankton diversity and carbon flux with reduced sea ice extent along the Western Antarctic Peninsula. *Nat. Commun.* **12**, 1–9 (2021).

21. Stammerjohn, S., Massom, R., Rind D. & Martinson D. Regions of rapid sea ice change: an inter-hemispheric seasonal comparison. *Geophys. Res. Lett.* **39**, 6 (2012).

22. Montes-Hugo, M. et al. Recent changes in phytoplankton communities associated with rapid regional climate change along the western Antarctic Peninsula. *Science* **323**, 1470–1473 (2009).

23. Cassar, N., Tang, W. Y., Gabathuler, H. & Huang, K. Method for high frequency underway N-2 fixation measurements: flow-through incubation acetylene reduction assays by cavity ring down laser absorption spectroscopy (FARACAS). *Anal. Chem.* **90**, 2839–2851 (2018).

24. Dabundo, R. et al. The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements. *PloS ONE* **9**, e110335 (2014).

25. Mohr, W., Wallace, G. & LaRoche, D. W. J. Methodological underestimation of oceanic nitrogen fixation rates. *PloS ONE* **5**, e12583 (2010).

26. Großkopf, T. et al. Doubling of marine dinitrogen-fixation rates based on direct measurements. *Nature* **488**, 361 (2012).

27. Wannicke, N. et al. New Perspectives on Nitrogen Fixation Measurements Using 15N2 Gas. *Front. Mar. Sci.* **5**, 120 (2018).

28. Gradoville, M. R. et al. Diversity and activity of nitrogen-fixing communities across ocean basins. *Limnol. Oceanogr.* **62**, 1895–1909 (2017).

29. White, A. E. et al. A critical review of the 15N2 tracer method to measure diazotrophic production in pelagic ecosystems. *Limnol. Oceanogr. Methods* **18**, 129–147 (2020).

30. Montoya, J. P., Voss, M., Kahler, P. & Capone, D. G. A simple, high-precision, high-sensitivity tracer assay for N (inf2) fixation. *Appl. Environ. Microbiol.* **62**, 986–993 (1996).

31. Shao, Z. et al. Global oceanic diazotroph database version 2 and elevated estimate of global N2 fixation. *Earth Syst. Sci. Data* **15**, 3673–3709 (2023).

32. Moffat, C., Owens, B. & Beardsley, R. On the characteristics of circumpolar deep water intrusions to the west Antarctic Peninsula continental shelf. *J.Geophys. Res. Oceans* **114**, C5 (2009).

33. Newell, S. E., McCarthy, M. J., Gardner, W. S. & Fulweiler, R. W. Sediment nitrogen fixation: a call for re-evaluating coastal N budgets. *Estuaries Coasts* **39**, 1626–1638 (2016).

34. Fulweiler, R., Nixon, S., Buckley, B. & Granger, S. Reversal of the net dinitrogen gas flux in coastal marine sediments. *Nature* **448**, 180 (2007).

35. Gardner, W. S. et al. Nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA) support nitrogen dynamics in Texas estuaries. *Limnol. Oceanogr.* **51**, 558–568 (2006).

36. McCarthy, M. J., Newell, S. E., Carini, S. A. & Gardner, W. S. Denitrification dominates sediment nitrogen removal and is enhanced by bottom-water hypoxia in the Northern Gulf of Mexico. *Estuaries Coasts* **38**, 2279–2294 (2015).

37. Wang, R. et al. Nitrogen fixation in surface sediments of the East China Sea: occurrence and environmental implications. *Mar. Pollut. Bull.* **137**, 542–548 (2018).

38. Yao, X. et al. Nitrogen fixation occurring in sediments: contribution to the nitrogen budget of lake Taihu, China. *J. Geophys. Res. Biogeosciences* **123**, 2661–2674 (2018).

39. Hallström, S. et al. Pelagic N2 fixation dominated by sediment diazotrophic communities in a shallow temperate estuary. *Limnol. Oceanogr.* **67**, 364–378 (2021).

40. Burdige, D. J. & Christensen, J. P. Iron biogeochemistry in sediments on the western continental shelf of the Antarctic Peninsula. *Geochim. et Cosmochim. Acta* **326**, 288–312 (2022).

41. Sherrell, R. M., Annett, A. L., Fitzsimmons, J. N., Roccanova, V. J. & Meredith, M. P. A 'shallow bathtub ring' of local sedimentary iron input maintains the Palmer Deep biological hotspot on the West Antarctic Peninsula shelf. *Philos. Trans. R. Soc. A Math. Phys. Eng. Sci.* **376**, 20170171 (2018).

42. Cheung, S. et al. High biomass turnover rates of endosymbiotic nitrogen-fixing cyanobacteria in the western Bering Sea. *Limnol. Oceanogr. Lett.* **7**, 501–509 (2022).

43. Broman, E., Sachpazidou, V., Pinhassi, J. & Dopson, M. Oxygenation of hypoxic coastal Baltic Sea sediments impacts on chemistry, microbial community composition, and metabolism. *Front. Microbiol.* **8**, 2453 (2017).

44. Collado, L. & Figueras, M. J. Taxonomy, epidemiology, and clinical relevance of the genus Arcobacter. *Clin. Microbiol. Rev.* **24**, 174–192 (2011).

45. Sasi Jyothsna, T., Rahul, K., Ramaprasad, E., Sasikala, C. & Ramana, C. V. Arcobacter anaerophilus sp. nov., isolated from an estuarine sediment and emended description of the genus Arcobacter. *Int. J. Syst. Evolut. Microbiol.* **63**, 4619–4625 (2013).

46. Rasigraf, O., Schmitt, J., Jetten, M. S. & Lüke, C. Metagenomic potential for and diversity of N-cycle driving microorganisms in the Bothnian Sea sediment. *MicrobiologyOpen* **6**, e00475 (2017).

47. Jorgensen, S. L. et al. Correlating microbial community profiles with geochemical data in highly stratified sediments from the Arctic Mid-Ocean Ridge. *Proc. Natl. Acad. Sci. USA* **109**, E2846–E2855 (2012).

48. Henkel, J. V. et al. Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov., thiotrophic manganese oxide reducing chemolithoautotrophs of the class Campylobacteria
isolated from the pelagic redoxclines of the Black Sea and the Baltic Sea. *Syst. Appl. Microbiol.* **44**, 126155 (2021).

49. Liesirova, T. et al. Nitrogen-fixing sulfate reducing bacteria in shallow coastal sediments under simulated resuspension. *Estuar. Coast. Shelf Sci.* **280**, 108165 (2022).

50. Darnajoux, R., Reji, L., Zhang, X. R., Luxem, K. E. & Zhang, X. Ammonium sensitivity of biological nitrogen fixation by anaerobic diazotrophs in cultures and benthic marine sediments. *J. Geophys. Res. Biogeosciences* **127**, e2021JG006596 (2022).

51. Conley, D. J. et al. Controlling eutrophication: nitrogen and phosphorus. *Science* **323**, 5917 (2009).

52. Zehr, J. P. & Paerl, H. W. Molecular ecological aspects of nitrogen fixation in the marine environment. In *Microbial Ecology of the Oceans* (ed. Kirchman, D. L.) Ch. 13 481–525 (Wiley, 2008).

53. Voss, M., Croot, P., Lochte, K., Mills, M. & Peeken, I. Patterns of nitrogen fixation along 10 N in the tropical Atlantic. *Geophys. Res. Lett.* **31**, 23 (2004).

54. Sohm, J. A. et al. Nitrogen fixation in the South Atlantic Gyre and the Benguela upwelling system. *Geophys. Res. Lett.* **38**, 16 (2011).

55. Mulholland, M. R. et al. High rates of N2 fixation in temperate, western North Atlantic coastal waters expand the realm of marine diazotrophy. *Glob. Biogeochem. Cycles* **33**, 826–840 (2019).

56. Pedersen, J. N., Bombar, D., Paerl, R. W. & Riemann, L. Diazotrophs and N2-fixation associated with particles in coastal estuarine waters. *Front. Microbiol.* **9**, 2759 (2018).

57. Turk, K. A. et al. Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic. *ISME J.* **5**, 1201 (2011).

58. Fadrosh, D. W. et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. *Microbiome* **2**, 1–7 (2014).

59. Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. *Appl. Environ. Microbiol.* **79**, 5112–5120 (2013).

60. Callahan, B. J. et al. DADA2: high-resolution sample inference from Illumina amplicon data. *Nat. Methods* **13**, 581–583 (2016).

61. Moynihan, M. A. nifHdada2 GitHub repository. Zenodo. https://doi.org/10.5281/zenodo.3958370 (2020).

62. Buchfink, B., Reuter, K. & Drost, H. -G. Sensitive protein alignments at tree-of-life scale using DIAMOND. *Nat. Methods* **18**, 366–368 (2021).

63. Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. *Mol. Biol. Evol.* **33**, 1870–1874 (2016).

64. Tamura, K. & Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. *Mol. Biol. Evol.* **10**, 512–526 (1993).

65. Hill, K., Dauphinee, T. & Woods, D. The extension of the practical salinity scale 1978 to low salinities. *IEEE J. Ocean. Eng.* **11**, 109–112 (1986).

66. Cassar, N. et al. Continuous high-frequency dissolved O2/Ar measurements by equilibrator inlet mass spectrometry. *Anal. Chem.* **81**, 1855–1864 (2009).

## Acknowledgements

We thank the scientists, scientific support personnel, the crew on R/V Laurence M. Gould, and the PAL-LTER team for their assistance in sample

collection. We are grateful to Hans Gabathuler for instrument support. This work is supported by NSF OPP-1643534 to N.C. and NSF OPP-1440435 to H.W.D.

## Author contributions

N.C. designed the study. S.G. performed measurements and collected samples at Palmer Station, with contributions from W.T. and H.W.D. H.B. conducted measurements at offshore stations. S.G. and H.B. analyzed nitrogen fixation data. S.G. performed molecular sequencing with contributions from J.R. and Y.L. S.G. analyzed environmental data. S.G., Y.L., and A.M.E. conducted sequencing data analyses. S.G. and N.C. wrote the manuscript with contributions from all co-authors.

## Competing interests

The authors declare no competing interests.

## Additional information

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s43247-025-02225-0.

**Correspondence** and requests for materials should be addressed to Nicolas Cassar.

**Peer review information** *Communications Earth & Environment* thanks Douglas Capone and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Clare Davis and Alice Drinkwater. A peer review file is available.

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