Unveiling unique microbial nitrogen cycling and nitrification driver in coastal Antarctica

Domain: earth_science
Published: 2024-04-12
Editorial outcome: major_revision

Abstract
Largely removed from anthropogenic delivery of nitrogen (N), Antarctica has notably low levels of nitrogen. Though our understanding of biological sources of ammonia have been elucidated, the microbial drivers of nitrate (NO −) 3 cycling in coastal Antarctica remain poorly understood. Here, we explore microbial N cycling in coastal Antarctica, unraveling the biological origin of NO − via oxygen isotopes in soil and lake sediment, and through the reconstruction of 1968 metagenome-assembled genomes from 29 microbial phyla. Our analysis reveals the metabolic potential for microbial N fixation, nitrification, and denitrification, but not for anaerobic ammonium oxidation, signifying a unique microbial N-cycling dynamic. We identify the predominance of complete ammonia oxidizing (comammox) Nitrospira, capable of performing the entire nitrification process. Their adaptive strategies to the Antarctic environment likely include synthesis of trehalose for cold stress, high substrate affinity for resource utilization, and alternate metabolic pathways for nutrient-scarce conditions. We confirm the significant role of comammox Nitrospira in the autotrophic, nitrification process via 13C-DNA-based stable isotope probing. This research highlights the crucial contribution of nitrification to the N budget in coastal Antarctica, identifying comammox Nitrospira clade B as a nitrification driver.

Introduction
Antarctica, a region largely insulated from anthropogenic nitrogen (N) deposition, exhibits notably low N concentrations1. These diminished N levels play an integral role in sustaining ecological equilibrium in the continent’s barren, ice-free terrains2,3. Consequently, elucidating the intricacies of microbial N-cycling pathways in Antarctic ecosystems—including diazotrophy (N fixation), nitrification, anaerobic ammonium oxidation (anammox), and denitrification—is paramount4. Diazotrophic Cyanobacteria are the primary producers of biologically accessible ammonium (NH +) in these environments5. Alongside NH +, 4 4 nitrate (NO −) constitutes a significant inorganic N pool in polar 3 biomes6,7. The synthesis of NO − in Antarctic ecosystems can be 3 attributed to both abiotic atmospheric deposition, particularly within mineral-rich Antarctic soils8, and biotic nitrification processes. Still, the discrete contributions of these sources to the coastal Antarctic NO − 3 reservoir remain inadequately characterized, necessitating further investigation.

Nitrification plays a pivotal role in the global biogeochemical N cycle9 and contributes significantly to the emissions of nitrous oxide (N O)10, a potent greenhouse gas. The process of nitrification encom- 2 passes the oxidation of ammonia (NH ) to NO − via the intermediate 3 3 nitrite (NO −). Traditionally, these two metabolic steps have been 2 attributed to distinct microbial guilds: ammonia-oxidizing bacteria (AOB)11 and archaea (AOA)12, responsible for converting NH to NO −, 3 2 and nitrite-oxidizing bacteria (NOB)13, which facilitate the subsequent oxidation of NO − to NO −. Beyond these canonical nitrifiers, the 2 3 recently identified complete ammonia oxidizers (comammox) within the genus Nitrospira, specifically lineage II, perform full nitrification on its own14,15. These organisms are increasingly recognized for their sig- 2 nificant role in nitrification across diverse ecosystems, including both engineered systems16 and natural environments17. The contribution of comammox bacteria to nitrification and subsequent NO − production 3 in Antarctic ecosystems, however, remains an enigma.

Phylogenetically, comammox Nitrospira cluster into clades A and B14, distinguished by their respective ammonia monooxygenase genes (amoA). Physiological assessments of clade A comammox Nitrospira have revealed an exceptionally high NH affinity, suggestive of an oli- 3 gotrophic niche adaptation18,19. In stark contrast, clade B comammox Nitrospira, despite their widespread distribution in various habitats, 2 including those characterized by low temperatures such as the Tibetan Plateau20,21 and Arctic permafrost22, have yet to be cultured for physiological scrutiny.

This study aims to unravel the complexities of the microbial N cycle in the ice-free regions of East Antarctica, identifying the primary sources of NO − and examining the potential roles of micro- 3 organisms in the nitrification process. Our research is strategically centered on the Larsemann Hills (LH)23, a vast ice-free rocky land- scape in East Antarctica, dotted with over a hundred oligotrophic lakes shrouded in ice. Through a comprehensive approach, we dis- cover that (i) the origin of NO − is primarily the biological nitrifica- 3 tion process; (ii) the microbial N cycle is distinct, encompassing most microbial N-cycling processes except for the anammox path- way; and (iii) the comammox Nitrospira clade B serves as an abun- dant and active driver of nitrification, possessing various survival strategies against the cold and oligotrophic conditions of the coastal Antarctic environment.

Results
Isotopic signatures indicate the biological origin of nitrate
Depending on how NO − is produced, the composition of its oxygen 3 isotopes differs, allowing differentiation between abiotic and biotic sources24. For biologically produced NO −, one oxygen atom (O) is 3 expected from atmospheric oxygen (O) and two from the surround- 2 ing water (H O)24. Biologically produced NO − would have a δ18O of 2 3 ~0.6‰, since δ18O of atmospheric O is 23.9‰25 and the measured δ18O 2 of Larsemann Hills (LH) lake water is –12.7±1.5‰ (Supplementary Table 1). In addition, while biologically produced NO − has a Δ17O of 3 0‰, which is identical to that of O and H O, atmospheric NO − is 2 2 3 usually characterized by high oxygen isotopic ratios26,27 and can reach Δ17O≥35‰ in the atmosphere in Antarctica26. Atmospheric NO − in 3 Antarctica is mainly produced by the reaction of nitrogen oxides (NO ), ozone (O), and hydroxyl radicals (OH·)27.

x 3
The mean δ18O of NO − in sediments from LH lake is 4.6‰. The 3 annual mean δ18O of NO − in snow and the atmosphere at the coastal 3 Zhongshan station, situated in LH, is approximately 92‰28. From these, the contributions of atmospheric deposition and biological production to sedimentary NO − pool can be quantified by isotope 3 mass balancing, indicating that the nitrification process constitutes 95% of the NO − in sediments. In addition, limited data of sediment 3 NO − Δ17O produced a mean of ~1.3‰, which strengthens the observed 3 >90% contribution of nitrification considering the very high Δ17O of atmospheric NO − at Zhongshan station (~32‰)28. Similarly, the 3 isotope mass balance of δ18O and Δ17O suggested ~96% of soil NO − in 3 the two study areas is from nitrification. Thus, we demonstrated here that sediment and soil NO − predominantly originated from biological 3 production in and nearby the investigated lakes.

Unique microbial nitrogen cycle in coastal Antarctica
The primary production of NO − is largely attributed to the microbial 3 nitrification process, rather than atmospheric precipitation. Conse- quently, the origin of NH +, the substrate for nitrification, can be traced 4 back to biological N fixation, as well as mineralization. The qPCR- based quantification of functional N-cycling genes (Fig. 1) revealed a comparable amount of N fixation (nifH, encoding nitrogenase) to nitrification genes (amoA and nxrB, encoding ammonia mono- oxygenase subunit A and nitrite oxidoreductase subunit B, respec- tively), with quantities ranging from 102 to 104 copies ng−1 DNA. The relatively low quantity of the gene encoding hydroxylamine dehy- drogenase (hao) (<10 copies ng−1 DNA) can be attributed to the low coverage of the applied primers (targeting AOB rather than comam- mox Nitrospira, Supplementary Table 2) and the absence of the bac- terial hao gene in the genomes of AOA29, which are of notable abundance in the studied samples.

In addition to N fixation and nitrification processes, we also observed the significant metabolic potential of denitrifiers, as indi- cated by the high abundances (>103 copies ng−1 DNA) of genes encoding the sequential reduction of NO − (nar, nap), NO − (nir), nitric 3 2 oxide (NO, nor), and N O (nos) (Fig. 1). The relatively higher abun- 2 dances (>104 copies ng−1 DNA) of nirS and nirK genes (Fig. 1) can likely be attributed to their presence not only in denitrifiers but also in nitrifiers. Interestingly, we found substantial quantities (102 to 104 copies ng−1 DNA) of the functional gene for dissimilatory nitrate reduction to ammonium (DNRA, nrfA) and assimilatory nitrite reduc- tion (ANR, nasA) (Fig. 1). The potential for DNRA and ANR is wide- spread among phylogenetically diverse microorganisms and these processes ensure the retention of bioavailable inorganic N in an ecosystem.

Strikingly, the hzo gene (encoding hydrazine oxidoreductase), a biomarker for the anammox process, was not detected in any of the tested sediment and soil samples. Anammox bacteria catalyze the anaerobic oxidation of NH + using NO − as an electron acceptor, pro- 4 2 ducing N as the final product. These bacteria were first identified in 2 wastewater treatment systems30 and were subsequently discovered in various environments, including marine31, coastal32, terrestrial33, and engineering systems34. The absence of anammox functional markers in coastal Antarctica suggests unique microbial N-cycling properties in this remote region.

Diverse microbiomes and the microbial N-cycling processes
From an extensive dataset exceeding 450 gigabases of sequencing data, we managed to reconstruct a dereplicated collection of 724 high- quality and 1244 medium-quality35 metagenome-assembled genomes (MAGs) (Supplementary Data 1). The recovered genomes encompass 29 distinct phyla (Supplementary Data 1 and 2), marking, to the best of our knowledge, the most comprehensive inventory of Antarctica coastal soil and sediment genomes to date. On average, we obtained around 60 MAGs per sample site studied (Fig. 2a). The most abundant phyla included Actinobacteriota, Pseudomonadota, Bacteroidota, Chloroflexota, Verrucomicrobiota, Acidobacteriota, Patescibacteria, Planctomycetota, and Gemmatimonadota (Fig. 2a and Supplementary Data 1), aligning with findings from other Antarctic surveys5,36,37. Cya- nobacteria also featured among the top ten most abundant MAGs (Supplementary Data 1), a deviation from the Mackay Glacier Region, where they were largely absent in most soil samples37. We identified only five archaeal MAGs within the Thermoproteota phylum, all of which were further classified as ammonia-oxidizing archaea (AOA) within Group I.1b.

To comprehend the metabolic strategies that sustain the abun- dant bacterial life in these extremely nutrient-poor sediments and soils, we examined the distribution and affiliation of 52 marker genes conserved across different energy conservation and carbon acquisi- tion pathways37 in the MAGs we retrieved. As predicted, genes for aerobic respiration were encoded by nearly all community members (Fig. 2b and Supplementary Fig. 1). Consistent with observations from the Mackay Glacier Region37, a significant number of the MAGs appear to fix carbon via the Calvin-Benson-Bassham (CBB) cycle (212 MAGs) or the 3-hydroxypropionate cycle (345 MAGs) (Fig. 2b). These mechan- isms facilitate the generation of biomass through means that are either separate from or complementary to photoautotrophy, a role pre- dominantly carried out by Cyanobacteria (Supplementary Fig. 1).

Genomic analysis revealed that the most abundant and widespread community members encoded trace gas oxidation genes. In a pattern similar to previous discoveries37,38, carbon monoxide (CO) dehy- drogenases (CoxL) were exclusive to Actinobacteriota and Chloro- flexota (Fig. 2b). Interestingly, uptake [NiFe] hydrogenases were encoded by MAGs from Acidobacteriota, Chloroflexota, and Verruco- microbiota (Fig. 2b), which aligns with observations from temperate soil where Acidobacteriota are known to be active atmospheric H 2 consumers39, albeit with slight differences.

In the realm of N metabolism, a comprehensive array of functional genes was identified within the MAGs we analyzed (Fig. 2b). N fixation 2 appears to be predominantly carried out by taxa within Acidobacter- iota, Cyanobacteria, Desulfobacterota, Myxococcota, and Verrucomi- crobiota (Fig. 2b). Consistent with the quantitative analyses, the hzs gene, indicative of the anammox process, was not present in the retrieved MAGs. While a total of 96 Planctomycetota MAGs were obtained, subsequent verification through taxonomic classification confirmed that none of these MAGs belong to the anammox family Brocadiaceae (Fig. 2b and Supplementary Data 2). The absence of anammox process maybe attributed to the optimal temperature range of 12–17°C, which is characteristic of anammox process found in similarly cold Arctic fjord sediments40, suggesting a limited tolerance to low temperatures. This assertion is further reinforced by the absence of anammox bacteria in the McMurdo Dry Valleys (an Ant- arctic desert)41 and in Arctic regions42 as well. The pervasive distribu- tion of denitrification genes across nearly all phyla underscores the robust and widespread microbial denitrification activity in Antarctica, corroborating previous research that highlights the ubiquity of cold- adapted denitrifiers across diverse Antarctic ecosystems43. Regarding the nitrification process, the marker genes-amoA (associated with ammonia oxidation) and nxrB (linked to nitrite oxidation) were found exclusively in the Thermoproteota and Nitrospirota phyla (Fig. 2b). Although Antarctic soil nitrification was previously reported back in 199744, attributed to AOB genera Nitrosospira and Nitrosomonas, as well as AOA within group I.1b45–48, no investigation has yet been con- ducted into the presence and function of the recently discovered comammox bacteria within the Nitrospira genus14,15. Here, we present data showing the prevalence of the comammox Nitrospira amoA gene in Antarctic lake sediments and soils, strongly suggesting the activity of comammox Nitrospira in this region.

Abundant nitrification drivers in coastal Antarctica
The quantitative evaluation of nitrification-related functional genes, including amoA and nxrB (Supplementary Fig. 2), established that AOA and comammox bacteria were the predominant nitrifiers in the ana- lyzed soils and sediments (Supplementary Fig. 3). Our metagenomic analysis corroborates these findings, having identified only AOA and Nitrospira MAGs among the various nitrifying groups (Fig. 2b), sug- gesting they outnumber AOB. Furthermore, a more detailed analysis of the abundance and community structure of all identified nitrifying groups was conducted using amplicon amoA and nxrB sequencing.

Subsequent phylogenetic assessment revealed an unexpected dom- inance of clade B comammox Nitrospira in this environment (Supple- mentary Table 5 and Supplementary Fig. 8). This was surprising given the successful extraction of diversity information from AOA (Supple- mentary Fig. 6) and AOB (Supplementary Fig. 7) in the samples investigated (Supplementary Results and Discussion).

Among the dereplicated MAGs, five were classified as Group I.1b- AOA within the genera Nitrosocosmicus and Nitrospharea of the Ther- moproteota phylum. This categorization stemmed from GTDB taxo- nomic classification, phylogenomic assessments, and average nucleotide identity (ANI) analyses (Supplementary Data 2 and Supple- mentary Fig. 4). These findings are in agreement with the data obtained from amoA amplicon sequencing, as discussed in the Supplementary Results and Discussion section. Together with their previous detection in Arctic soils49,50, this suggests a capacity of Nitrosocosmicus-like AOA to thrive in cold and nutrient-deficient conditions.

Of the seven Nitrospirota MAGs analyzed, four were verified as comammox Nitrospira, and the remaining three belonged to the exclusively nitrite-oxidizing lineages II and IV of Nitrospira. This clas- sification was substantiated by phylogenomic and average nucleotide identity (ANI) analyses (Fig. 3a, b, Supplementary Table 4, and Sup- plementary Fig. 5), and these results concur with the phylogenetic assessment based on the amoA and nxrB gene of Nitrospira (Supple- mentary Figs. 8, 9). The notably high abundance of these Nitrospira MAGs in the soil and sediment metagenomes studied (reaching up to 0.173%, Supplementary Table 3) further emphasize their significant roles in nitrification within these ecosystems.

Two high-quality comammox MAGs, Nitrospira sp. La1-X1 and Nitrospira sp. La3-X1, have genome sizes of 4.26 Mb and 3.78 Mb, respectively (Supplementary Table 3). The amoA gene sequences from La1-X1 and La3-X1 align closely with the dominant amoA OTUs and with previously published sequences from clade B comammox (Fig. 4c and Supplementary Fig. 8). Consistent with the amoA-based phylogeny, phylogenomic analysis also demonstrates a distinct clustering of these comammox MAGs within clade B (Fig. 3a). For La1-X1, the ANI was highest with MAG Nitrospira sp. palsa 1310, which was identified from Arctic permafrost soil22. The ANI between these genomes is 96.67% (Fig. 3b), surpassing the species threshold of 95%51, suggesting that these genomes represent different strains of the same Nitrospira species, potentially adapted to cold environments. In contrast, La3-X1 is grouped within a clade B subset that includes MAGs from drinking water treatment systems and glacier surface soil52, but it did not share high ANI values with any other clade B genomes (≤85%, Fig. 3b). This suggests that La3-X1 represents a unique so far undetected group within clade B.

Metabolic potential and surviving strategy
The clade B comammox MAGs Nitrospira sp. La1-X1 and Nitrospira La3- X1 harbor the complete genetic machinery for NH and NO − oxidation, 3 2 the respiratory chain, and the reduced tricarboxylic acid (rTCA) cycle, which is the conserved CO fixation pathway in Nitrospira (Fig. 3c, d). 2 These core metabolic features are highly conserved in comammox Nitrospira, as reported in previous studies53,54. Similar to other Nitrospira genomes52, La1-X1 and La3-X1 do not encode nitric oxide reductase (NOR), which is crucial for enzymatic NO production55. They do carry 2 genes for urea transport and hydrolysis by the urease, indicating the use of urea as an alternative ammonia source, as shown for other comam- mox Nitrospira14,15,56. In addition, like some Nitrospira that have the confirmed ability to use hydrogen and formate as alternative energy sources57,58, the comammox MAGs La1-X1 and La3-X1 contain genes for formate and hydrogen oxidation. The formate dehydrogenases of comammox are similar to their nitrite-oxidizing counterparts. In con- trast, comammox bacteria possess a 3b-type [NiFe] hydrogenase, which is rarely identified in canonical Nitrospira and its physiological role remains unclear54,59. While the capacity for formate oxidation is widely distributed in clade B, the 3b-type [NiFe] hydrogenase has been mainly identified in clade A comammox species54 (Fig. 3d). However, the pre- sence of this hydrogenase type in a few clade B genomes52,54 including La3-X1 challenges the clade specificity of this feature.

In addition to the canonical FF H+-driven ATPase, La1-X1 and La3- 1 0 X1 encode a potentially Na+-pumping FF ATPase, previously detected 1 0 in the haloalkalitolerant nitrite-oxidizing Ca. Nitrospira alkalitolerans60, marine nitrite-oxidizing Ca. Nitronereus thalassa61 and clade A comammox Ca. Nitrospira kreftii19. Similar to these genomes, La3-X1 also possesses a Na+-translocating NADH:ubiquinone oxidor- eductase (NQR), a feature missing in La1-X1 (Fig. 3c). These Na+- pumping enzyme complexes might represent an adaption of La3-X1 to saline or haloalkine conditions similar to other Nitrospira.

The disaccharide trehalose is one of several solutes known to protect bacteria against cold stress, and may also protect against other harmful environmental conditions, such as osmotic stress62. La1-X1 possesses a trehalose-6-phosphate synthase (OtsA) and the corre- sponding phosphatase (OtsB) to potentially produce the non-reducing disaccharide in two steps from UDP-glucose, as shown for E. coli63. In contrast, La3-X1 encodes several other trehalose synthesis enzymes in one gene cluster, indicating that this species might convert NDP- glucose (trehalose synthase) and maltose (trehalose synthase/amy- lase) to trehalose. However, all these trehalose synthesis pathways have also been identified in Nitrospira from mesophilic environments and might thus not be a distinguishing feature for cold adaptation. In addition to trehalose synthesis, La1-X1 and La3-X1 possess other fea- tures for coping with different stresses. Whereas a gene cluster encoding a nitrile hydratase has been detected in La1-X1 and La3-X1, the genome lacks an amidase to degrade the produced amides further to the corresponding carboxylic acid and ammonia, which could serve as an energy source. An example of a potential amidase is a putative formamidase identified in a clade B MAG obtained from the Rifle aquifer52. Other detoxification mechanisms include catalases and Fe or Mn superoxide dismutases (SOD) for reactive oxygen defense identi- fied in both clade B genomes, and a periplasmic Cu-Zn SOD, which is present in La3-X1 and many other clade B genomes, but absent in La1- X1 (Fig. 3c, d).

An adaptation to oligotrophic conditions (Supplementary Table 1) might be the methionine salvage pathway (MSP) of La1-X1 (Fig. 3c). This pathway recycles the sulfur-containing intermediate 5’-methyl- thioadenosine back to methionine, thus allowing the use of reduced sulfur compounds under sulfur limitation. However, similar to the high-quality genome of the nitrite-oxidizing Nitrospira lenta57 and other clade B genomes, the mtnE gene encoding the last step in this pathway is missing in La1-X1. Whether other enzymes might complete the MSP in these Nitrospira species remains to be determined.

Nitrification and N O production activity
2
We have demonstrated that comammox Nitrospira potentially serves as crucial drivers of nitrification in coastal East Antarctica, and further investigated their survival strategies from a genomic per- spective. Additionally, we confirmed the nitrification activity of comammox Nitrospira using DNA-SIP on two lake sediment samples (LA1 and LA2) and one soil sample (LS4). DNA-SIP incubations were conducted at 4°C (for LA1) and 10°C (for LA1, LA2, and LS4), and the production of NO − served as an indicator of nitrification activity 3 (Fig. 4a and Supplementary Fig. 11). During the incubations, the comammox Nitrospira were actively growing, as evidenced by the peak shifts of their DNA in the 13C-treatment (Fig. 4b and Supple- mentary Fig. 4). Subsequent sequencing of the amoA genes from the labeled DNA revealed clade B amoA OTUs, including those of comammox MAGs La1-X1 and La3-X1, in both the 4°C and 10°C incubations (Fig. 4c). Despite the temperature used in the DNA-SIP incubations not being identical to the in situ condition, our data strongly suggest an active role for clade B comammox Nitrospira in nitrification.

The addition of NH + resulted in an increase of NO − production, 4 3 however, did not stimulate higher N O production compared to 2 incubations without NH + addition (Fig. 4a), indicating the low N O 4 2 production potential of comammox Nitrospira55. The addition of chlorate, a potential comammox-specific inhibitor64, slightly reduced the production of NO − and N O (Fig. 4a). However, since chlorate can 3 2 also influence other nitrite-oxidizing Nitrospira species as well as denitrifiers, the observed reductions in NO − and N O levels cannot be 3 2 solely attributed to changes in comammox activity. Notably, prior research has shown the resilience of clade B comammox Nitrospira to freeze-thaw cycles, which underscore the endurance of this particular nitrifier group in environments with low and even subfreezing temperatures65. Raising the temperature (10°C) stimulated the activity of AOA (in LA2) and AOB (in LA2 and LS4) (Supplementary Fig. 4, Supplementary Results and Discussion), indicating that these nitrifiers are more competitive under elevated temperature conditions, an important finding to predict ecosystem changes in response to global warming.

Implications and outlook
In this study, we elucidated the unique microbial N cycle in pristine and oligotrophic coastal Antarctic soil and lake sediments, identifying the microbial nitrification process as the primary pathway for NO − pro- 3 duction. Our metagenomic and quantitative functional gene-targeted analyses both revealed clade B comammox Nitrospira to be a key nitrifier in these environments. This finding not only expands our understanding of microbial diversity but also underscores the pivotal role of specific microbial groups in biogeochemical cycling in extreme environments. Our results also revealed fascinating patterns of niche differentiation between clade A and B comammox Nitrospira and canonical nitrifiers. Clade B species might have superior affinity for their substrate ammonia, and are potentially best adapted to survive and thrive in cold and oligotrophic environments. This niche differ- entiation could have significant implications for N-cycling in cold environments and it will be important to determine how the nitrifier and overall microbial communities will react to global warming.

In addition, we have successfully obtained in total of 1968 MAGs, significantly expanding the existing microbial genomic data for Ant- arctica. These MAGs provide invaluable insights into the microbial diversity and their metabolic capabilities in the extreme and unique environment of coastal Antarctica. The availability of these MAGs will undoubtedly facilitate further research into the intricate relationships between microbial communities, biogeochemical cycles, and climate change in polar regions and beyond.

This study underscores the importance of understanding the unique microbial N cycle in coastal Antarctica. We have uncovered clade B comammox Nitrospira to be a nitrification driver in low- temperature environments, and investigated their survival strategy and potential impact on climate change through the production of greenhouse gas N O55,66. It is crucial to further investigate how 2 comammox Nitrospira evolve and survive in Antarctic ecosystems, meeting one of the six priorities for Antarctic science67. Recognizing the climatological significance of N-cycling and biogeochemical pro- cesses, future research should continue to monitor these microbes, as they could hold the key to understanding the broader implications of microbial activity on our planet’s climate.

Methods
Sample collection and treatment
Samples were collected in Larsemann Hills (LH), the second-largest ice-free land in East Antarctica with an area of ~50 km2. LH has a cold and dry continental climate, with an annual mean temperature of ~ −10 oC and the temperatures occasionally above 0 oC in summer68 lead to this region typically free of snow cover in summer. As a result of seasonal snow cover and the glacier melting, a number of land- locked lakes are developed in this region. Surface sediment (the upper ~5 cm) and surface water samples were collected from six lakes (LA1-LA6) in LHs in February 2020 (Fig. 1a). The surface sediment samples were collected using a stainless-steel spade on the shore of lakes, with typical water depths of ~150–200 cm. Approximately 1 L of surface water near the lake shore was also sampled with clean poly- ethylene (PE) bottles, a portion of which was used to measure tem- perature, pH, salinity, and conductivity utilizing a multi-probe water quality meter (YSI Professional Plus series; Supplementary Table 1). The remainder of the water samples was filtered using 0.22-μm polytetrafluoroethene (PTFE) filters for chemical analyses. Addi- tionally, a surface soil sample (the top ~5 cm) was collected near each lake, ~100 m from the lake shore, using a clean stainless spatula. At each sampling site, larger gravels were removed firstly, and five soil sub-samples (four corners and the center of a square) were collected at a distance of 5–10 m and then mixed to obtain a representative sample (LS1-LS6). For a comprehensive understanding of N-cycling processes in the ice-free areas in coastal Antarctica, surface soil samples (SVL1–SVL10) were also collected in February 2022 at Inex- pressible Island, South Victoria Land where the climate is comparable to that of LH, following the same sampling protocols. All of the sediment and soil samples were stored in sealed PE bags, and all samples were transported to the laboratory at temperatures of ~ −20°C for subsequent analysis.

Physiochemical analysis
In the laboratory, ~50 g of the sediment and soil samples were freeze- dried in 50-mL clean centrifuge tubes (ALPHA 1-4/LD, Martin Christ Inc.). After drying, the samples were homogenized using an agate mortar and pestle, and subsequently passed through a 1 mm sieve for further chemical analysis. For determining total organic carbon (TOC) content, approximately 5 g of samples were digested with 10% HCl (v/v) to remove carbonate. Then, TOC was measured with an automatic element analyser (Elementar, VARIO EL III), with acetanilide used as the external standard. The detection limit (DL) of the TOC was estimated to be ~0.005%. All samples were measured in triplicate, yielding a relative standard deviation (1σ) of <10% for each sample. For chemical ion analysis, ~5 g of freeze-dried samples were placed in sterile 50-mL centrifuge tubes and suspended in 25 mL Milli-Q water (18.2 MΩ). The solution was then ultrasonicated for 40 min. The supernatant was first centrifuged for 15 min at 3000 × g, and then filtered through 0.22-μm PTFE filters for nutrient determination. Nutrient concentrations (NH +, 4 NO −, and PO 3−) in the sediment, soil extracts, and lake water samples 3 4 were determined using an Aquion RFIC ion chromatograph (IC, Thermo Scientific, USA), equipped with the analytical columns CS12A (2×250 mm), AS11-HC (2×250 mm), methanesulfonic acid (MSA), and potassium hydroxide (KOH) as eluents for cations and anions, respectively. In addition, the concentrations of SiO 2- were determined 3 using an automated QuAAtro™ nutrient analyser (Seal Analytical Ltd., UK). During sample analysis, replicate determinations (n=5) were performed, and 1σ for all species was <5%.

Isotopic analysis of nitrate
The isotopic composition of NO − was determined using the bacterial 3 denitrifier method at the Environmental Stable Isotope Laboratory of East China Normal University (ECNU-ESIL). Briefly, the denitrifying bacterium Pseudomonas aureofaciens, which lacks the N O reductase 2 enzyme, quantitatively transforms NO − into gaseous N O69,70. The δ15N 3 2 and δ18O of the generated N O were measured in duplicates using 2 isotope-ratio mass spectrometry (IRMS, Thermo Scientific Delta V). The Δ17O of NO − was separately analyzed through the thermal 3 decomposition of N O into N and O 71, followed by measurements at 2 2 2 m/z 32, 33, and 34 on the IRMS. The pooled standard deviation (1σ ) p was employed to ascertain the measurement precision of the overall denitrifier method72,73. The 1σ of all duplicate samples executed in at p least two different batches was 0.6‰ for δ15N (n=10), 0.3‰ for δ18O (n=10), and 0.8‰ for Δ17O (n=8). However, due to the limited amounts of NO − in the samples, only three sediment and five surface 3 soil samples were analyzed for Δ17O of NO −.
3
In addition, lake water stable isotopes (δ18O and δ2H) were ana- lyzed using laser absorption spectrometry (TIWA-45EP, Los Gatos Research, Inc.). To ensure quality control, replicate analyses (n=5) were performed, yielding relative standard deviations of 0.05‰ and 0.2‰ for δ18O and δ2H, respectively.

DNA extraction, quantification, and sequencing analysis
Total DNA was extracted from 0.5 g sediment/soil samples using the FastDNA SPIN kit (MP Biomedicals, Santa Ana, CA) according to the manufacturer’s protocols. The final DNA quality and quantity were determined using Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, China). A detailed description of the quantitative PCR (qPCR) analysis for functional N-cycling genes is provided in the Sup- plementary Methods section. It also elaborates on the procedures for PCR and high-throughput amplicon sequencing, as well as the sub- sequent phylogenetic analysis of nitrification genes.

Metagenomic sequencing
The total DNA from the original sediment and soil samples was sequenced on the Illumina HiSeq X ten platform using a 150-bp paired- end library at Beijing Novogene Biotech Co., Ltd. (Beijing, China). The NEXTFLEX Rapid DNA-Seq Library Prep Kit 2.0 (Bioo Scientific, Austin, TX, USA) was used for DNA library preparation with an insert size of ~300 bp according to the manufacturer’s recommendations. DNA was sheared using a Covaris S220 Focused Ultrasonicator to create 150 bp fragments. Subsequently, raw metagenomic sequencing data in FastQ format was generated using the RTA (Real-Time Analysis) v3.4.4 and bcl2fastq (btq) v2.16. Each individual sample yielded a range of 20–50 gigabases of sequencing data.

Assembly and binning of metagenomes
Raw reads were processed using fastp v0.19.774 for adapter trimming, quality filtering, and per-read quality trimming. The fourteen quality- controlled metagenomes were individually assembled and co- assembled using MEGAHIT v1.1.375 with default parameters (k-mers: 21, 29, 39, 59, 79, 99, 119, 141). Each of the assembly was initially binned using the binning module (–metabat2 –maxbin2 –concoct; –metabat2 for co-assembly) in the metaWRAP pipeline76 v1.3.2, and were con- solidated using DAS Tool v1.1.277 with default parameters. After a dereplication check using dRep v3.0.078 (-comp 50 -con 10) and com- pleteness and contamination evaluation using CheckM v1.1.379, 1968 MAGs were obtained, and the taxonomy of each MAG was assigned using GTDB-Tk v1.5.080 with the Genome Taxonomy Database (Release 06-RS202). In the process of annotating metabolic functions, the genomes that were extracted were examined using DIAMOND81 v0.9.14 against 52 tailor-made protein databases, which consisted of marker genes representing common metabolic traits for energy conservation as well as C and N fixation37. To confirm the existence of crucial metabolic genes in the MAGs, maximum-likelihood phylogenetic trees were generated to confirm their phylogenetic affiliation The relative abundances (in percentage) for all dereplicated MAGs were deter- mined by aligning each sample’s clean paired-end reads to the MAGs utilizing CoverM v0.6.1 (https://github.com/wwood/CoverM) in genome mode, applying the default configurations. Moreover, the trim- med reads were incorporated into CLC Genomics Workbench version 20.0 (CLC Bio, Qiagen, Germany), and the de novo assembly algorithm of CLC was employed to search for amoA and nxrB sequences. Contigs that contained either amoA or nxrB genes were selected for phyloge- netic analysis, in conjunction with amplicon sequences.

Phylogenomic analysis and genome annotation
Genomes and MAGs classified by the GTDB-Tk database R20282 as Nitrospiracea, with estimated genome completeness ≥70% and contamination ≤10%, were downloaded from NCBI. Dereplication was performed using the drep v2.4.278 dereplicate workflow with cut- offs for estimated genome completion ≥70% and contamination ≤10%, but otherwise default settings. In addition to these 95 Nitrospi- racea genomes, seven Nitrospira lineage IV and two clade B gen- omes were included in the phylogenetic analysis using the UBCG pipeline for the extraction and concatenated alignment of 91 single- copy core genes83. In addition, two Leptospirillum genomes (GCF_000284315.1, GCF_000299235.1) were included as outgroup. A maximum-likelihood phylogenetic tree was constructed using IQ- TREE v1.6.1284 with 1000 ultrafast bootstrap replications and the GTR+F+I+G4 model identified by the implemented Modelfinder85. ANI analysis of all clade B genomes was performed using the OrthoANI86. Gene calling and automatic genome annotation were performed using the MicroScope platform87, and annotations of selected features were manually checked and refined. For analyzing the distribution patterns of selected key features, 17 comammox clade B genomes were annotated using prokka v.1.14.688 with the “--gcode 11” and “--metagenome” options to obtain all protein sequences for generating a BLAST database. The distribution of selected key proteins within clade B was analyzed by conducting a BLASTp search against this database with default settings, except for an e-value cutoff of 1e-6. Only hits with an identity ≥35% (pident) and a query coverage ≥80% (qcovs) were reported as present. Phyloge- nomic and ANI analyses were carried out on the retrieved AOA MAGs, using representative genomes (which include Group I.1a and Group I.1b-AOA) and Group I.1b Nitrosocosmicus AOA genomes as references, respectively.

DNA-stable isotope probing (SIP) incubation and analysis
Lakes sediments (LA1 and LA2) and the soil sample (LS4) were selected for DNA-SIP microcosm incubation experiments (Supplementary Fig. 10) to mimic a low-temperature oligotrophic environment. (Note that these three sampling locations are distributed almost uniformly throughout LH.) Microcosms were constructed in 120 mL serum bot- tles containing 10 g sediments or soil at 60% of maximum water- holding capacity and were incubated for 56 days at 10°C in the dark. For each sample, two different treatments were established in tripli- cate microcosms. The 13CO microcosms were amended with 5% (v/v) 2 13CO (99 atom%; Sigma-Aldrich Co., St. Louis, MO, USA) plus 2 a approximately 5 µg 15N-NH Cl-N g−1 dry weight soil/sediment (d.w.s.), 4 while the 12CO control treatments received 5% (v/v) 12CO plus ~5 µg 2 2 14N-NH Cl-N g−1 d.w.s. The water content was restored weekly over an 4 8-week incubation period by opening the bottles. Additional labeled or unlabeled NH Cl was supplied (approximately once every 4 2–3 weeks) to maintain ~5 µg NH Cl-N g−1 d.w.s. during the incubations. 4 The sediment/soil samples were destructively sampled after 56 days of incubation and transferred immediately to –80°C for subsequent molecular analysis. The remaining ~2 g of sediments were used for end-point quantification of NH +, NO −, and NO − concentrations. The 4 2 3 LA1 sediment sample was additionally chosen for incubation at 4°C, adhering to the same procedure previously outlined for a period of 84 days (12 weeks). In addition to the ammonium substrate, 50 μM chlorate, a specific comammox inhibitor64, was introduced to study the activity of comammox Nitrospira. Moreover, the production of N O64 was actively monitored during these 4°C DNA-SIP incubations. 2 The fractionation of DNA post-DNA-SIP incubations, along with the subsequent quantification analysis of functional nitrification groups, is described in the Supplementary Methods.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "As an enthusiast of the microbial ecology of extreme environments, I consider this study quite excifing, refreshing and robust. I could be really picky about fime-series measurements in different seasons, but I do understand the human, resource and weather limitafions to develop these kinds of studies in such a harsh environment as Antarcfica. So, I celebrate how the authors combined diverse methods to build a solid study to start filling the gap about the nitrificafion process and funcfional N-cycling genes in this unique environment. Besides the solid combinafion of culture dependent and independent work, I find the microcosm rate measurements at representafive temperatures of the ecosystem a strong point of the study. The metagenomic analysis and the recovery of MAGs is robust and provides a great resource to the scienfificcommunity. The high point of the genomic analysis is the idenfificafion and predominance of commamox Nitrospira capable of performing the enfire nitrificafion process. I found no methodological weaknesses in this manuscript and it is wriften clearly and concisely. I do have some minor comments that are not affecfing the outcome of my decision, but I'd thank the authors for sending some feedback about them:\nResults:\n- Some sentences sound too general across the manuscript and can be more clear with the addifion of some specific informafion in brackets. i.e.L. 209/210: ...suggesfing their relafively high abundances (Supplementary Table 3). This could be improved with \"...suggesfing their relafively high abundances (>X%) (Supplementary Table 3)\".This is an example but I've noficed the same paftern in different lines.\n- Based on the high number of MAGs that the authors recovered, I'd love to see the esfimafions about how much of the microbial community is represented by this set of genomes/MAGs. SingleM appraise could help to get some esfimafions about this.\nMethods:\n- Not clear to me how the 52 metabolic marker genes database was built or if they used another similar database already reported. Include reference if necessary.\nFigures:\n-Fig. 1.a: Add distance bar for Larsemann/South Victoria maps.\nSuppl. MyM\n- There is a typo at the end of page 2. \"in Larsemann Hills aera\". Please correct it.\n- OTUs vs ASVs (QIIME1 versus QIIME2): I've noficed that for the amoA amplicon sequencing, the authors used OTUs by clustering the sequences at 95% similarity cutoff. If they were presented only 16S data instead ofamoA genes, I'd strongly recommend using QIIME2 and an ASVs approach instead OTUs/QIIME1 due to this approach being a liftle outdated and ASVs offer a higher resolufion of the microbial community. So, my quesfion out of curiosity is: why did the authors decide to use a clustering approach to define OTUs instead of using ASVs?", "reviewer_2": "In this manuscript, the authors delve into the microbial N cycling processes specific to the coastal regions of Antarcfica, with a parficular emphasis on East Antarcfica. The work is a meficulously conducted analysis that paints a detailed picture of the N cycle in the region under invesfigafion. The study is intriguing and represents significant work, encompassing physicochemical and isotopic analyses, quanfificafion of funcfional genes, metagenomic and comparafive genomic analyses, and 13C-DNA-based SIP experiments. The authors have observed unique N cycling processes and idenfified Commamox bacteria—previously not found—as the key players in nitrificafion within Antarcfica's oligotrophic and ice-free areas. This discovery is poised to capfivate those researching biogeochemical N cycling and polar science.\nFrom a methodological standpoint, the authors have employed a comprehensive array of tools, including isotopic analysis of nitrate, extensive quanfificafion of funcfional N-cycling marker genes, deep metagenomic sequencing, and detailed comparafive genomic analysis. Obtaining a mulfitude of MAGs, especially some high-quality Commamox genomes, is not trivial. Furthermore, implemenfing the 13C-DNA-based SIP method at two different temperatures (4 and 10 ºC) on selected sediment and soil samples validated the role of detected Commamox Nitrospira in nitrificafion. The authors have ufilized virtually available methods to substanfiate their findings.\nThe manuscript is well-structured and wriften, making for an engaging read. Despite the complexity and breadth of content related to microbial N cycling quanfificafion, the authors have presented their findings in a clear, structured, and succinct manner. The logical progression of the narrafive effecfively draws the reader's aftenfion to the novel nitrificafion driver, clade B Commamox Nitrospira. Since their discovery, this is a significant stride, marking the first in-depth invesfigafion of complete nitrifiers in the Antarcfic environment. The findings that Commamox bacteria, parficularly clade B, are highly abundant and acfive in the area studied are surprising. They will inspire further research into these microbial communifies' ecophysiological roles and evolufionary trajectories in cold and oligotrophic environments.\nOverall, this work meets the journal's requirements and is of high value to the reader. And I have some comments and suggesfions before being accepted.\nMajor concerns:\n(1) The sampling fime is precise. However, we don’t know when the incubafion experiments were conducted, which was related to nitrogen transformafion and microbial acfivifies.\n(2) The relafive abundance of microbial N cycle genes is defined by copies of ng DNA in Figure 1bcd. Some works indicated that ‘Measuring the total count and concentrafion of microbes or genes (absolute abundance) provides a richer source of informafion than relafive abundance and can correct some conclusions drawn from relafive abundance data (hftps://doi.org/10.1038/s41587-023-01754-3)’. Thus, it would be good to have absolute quanfitafive data if possible.\n(3) De-noising algorithm analysis (ASVs), rather than OTUs, is more accurate and recommended.\n(4) Why were only three soil (LS4) and sedimental (LA1 and LA2) samples selected for the SIP experiment? What were the selecfion criteria?\n(5) The ammonium and nitrate contents were low (Supplementary Table 1). During the SIP incubafion, approximately 5 μg g-1 of 15N labeled NH4+ was added as the substrate. About 40 μg g-1 of nitrate and ~ 20 ppm of N2O were detected after the 56-day incubafion (Figure 4a). Do you have the isotopic abundance values of nitrate? This is important to illustrate the nitrogen transformafion processes.\nMinor concerns:\n(1) The abstract could be more succinct. Some abbreviafions can be omifted.\n(2) Did the authors find any excifing properfies in the AOA genomes? Especially in the nutrient-poor environment of this study.\n(3) Line 48: Consider referring to it as the ‘ice-free Antarcfic region,’ ‘Coastal East Antarcfica,’ or ‘Coastal Antarcfica’.\n(4) Lines 63-69: lack references.\n(5) Line 244: ‘La3-bin1’ should be corrected to ‘La3-X1’. Right?\n(6) Line 287: Please correct the spacing issue in ‘Fe or Mn’.\n(7) Line 437: Number ‘seven’ should be replaced with ‘fourteen’. Right?\n(8) Line 458: Please confirm the term ‘all available’ as databases are regularly updated.\n(9) Lines 485-486: Could you clarify what ‘in situ flooding condifions’ mean?\n(10) Line 504: It should read ‘OTU sequences’.\n(11) Fig. 3a: Please double-check the phylogenomic tree; ‘palsa 1315’ appears twice.\n(12) Fig. 3d: AHL is missing from the boftom part of the figure.\n(13) Figure 4a: What is ‘CK’ treatment? No descripfion is found in Secfion ‘Material and Methods’ either.\n(14) Supplementary Fig. 6 and Supplementary Fig. 7: Please confirm the accuracy of the colored shadows.\n(15) Supplementary Fig. 9: The stray ‘25’ is in the middle of the figure; please remove it."}, "author_response": "We are grateful to the reviewers for their careful reading of the manuscript, construcve cricisms, posive comments and encouraging remarks, all of which have significantly enhanced the quality of our resubmited paper. Below, we have addressed each reviewer's comments (noted in black) with our responses (in blue):\nReviewers’ Comments\nReviewer #1 (Remarks to the Author):\nAs an enthusiast of the microbial ecology of extreme environments, I consider this study quite excing, refreshing and robust. I could be really picky about me-series measurements in different seasons, but I do understand the human, resource and weather limitaons to develop these kinds of studies in such a harsh environment as Antarcca. So, I celebrate how the authors combined diverse methods to build a solid study to start filling the gap about the nitrificaon process and funconal N-cycling genes in this unique environment. Besides the solid combinaon of culture dependent and independent work, I find the microcosm rate measurements at representave temperatures of the ecosystem a strong point of the study. The metagenomic analysis and the recovery of MAGs is robust and provides a great resource to the scienfic community. The high point of the genomic analysis is the idenficaon and predominance of commamox Nitrospira capable of performing the enre nitrificaon process. I found no methodological weaknesses in this manuscript and it is writen clearly and concisely. I do have some minor comments that are not affecng the outcome of my decision, but I'd thank the authors for sending some feedback about them:\nResponse: We thank the reviewer for these very posive comments on the general significance of our manuscript.\nResults:\n- Some sentences sound too general across the manuscript and can be more clear with the addion of some specific informaon in brackets. i.e. L. 209/210: ...suggesng their relavely high abundances (Supplementary Table 3). This could be improved with \"...suggesng their relavely high abundances (>X%) (Supplementary Table 3)\". This is an example but I've noced the same patern in different lines.\nResponse: We thank the reviewer for this point. We have revised the whole manuscript accordingly (lines 120, 125-130 and 165).\n- Based on the high number of MAGs that the authors recovered, I'd love to see the esmaons about how much of the microbial community is represented by this set of genomes/MAGs. SingleM appraise could help to get some esmaons about this.\nResponse: We appreciate the reviewer's comment. We used CoverM (version 0.6.1) to esmate the coverage of the MAGs in the samples invesgated. The total relave abundances ranged from 29.6% to 54.5%, with an average of 41.6% (see Supplementary Data 2).\nMethods:\n- Not clear to me how the 52 metabolic marker genes database was built or if they used another similar database already reported. Include reference if necessary.\nResponse: We thank the reviewer for this point. The metabolic marker genes database is based on a previous publicaon. The related reference has been added accordingly (lines 161 and 446).\nFigures:\n-Fig. 1.a: Add distance bar for Larsemann/South Victoria maps.\nResponse: We thank the reviewer for this point. Distance bars were added to the maps. Addionally, we revised Fig. 1 in accordance with the reviewers' comments.\nSuppl. MyM\n- There is a typo at the end of page 2. \"in Larsemann Hills aera\". Please correct it.\nResponse: We thank the reviewer for this point. It has been corrected.\n- OTUs vs ASVs (QIIME1 versus QIIME2): I've noced that for the amoA amplicon sequencing, the authors used OTUs by clustering the sequences at 95% similarity cutoff. If they were presented only 16S data instead of amoA genes, I'd strongly recommend using QIIME2 and an ASVs approach instead OTUs/QIIME1 due to this approach being a litle outdated and ASVs offer a higher resoluon of the microbial community. So, my queson out of curiosity is: why did the authors decide to use a clustering approach to define OTUs instead of using ASVs?\nResponse: We appreciate the reviewer's point. We indeed used the QIIME2 plaorm, not QIIME1, for the analysis. We have updated the related informaon in the Supplementary Methods. The QIIME2 codes used in this study are also provided in Appendixes in the Supplementary Informaon. We agree with the reviewer that for a 16S rRNA gene dataset, ASV analysis is the most commonly used and recommended method. For funconal marker genes, such as nxrB, clustering based on 95% similarity is recommended (Pester et al., 2014). This is because mulple non-idencal copies of the marker gene within one genome can lead to an overesmaon of diversity. Therefore, we used a more stringent approach and clustered the ASV sequences obtained via QIIME2 into OTUs using a 95% similarity threshold.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors delve into the microbial N cycling processes specific to the coastal regions of Antarcca, with a parcular emphasis on East Antarcca. The work is a meculously conducted analysis that paints a detailed picture of the N cycle in the region under invesgaon. The study is intriguing and represents significant work, encompassing physicochemical and isotopic analyses, quanficaon of funconal genes, metagenomic and comparave genomic analyses, and 13C-DNA-based SIP experiments. The authors have observed unique N cycling processes and idenfied Commamox bacteria—previously not found—as the key players in nitrificaon within Antarcca's oligotrophic and ice-free areas. This discovery is poised to capvate those researching biogeochemical N cycling and polar science.\nFrom a methodological standpoint, the authors have employed a comprehensive array of tools, including isotopic analysis of nitrate, extensive quanficaon of funconal N-cycling marker genes, deep metagenomic sequencing, and detailed comparave genomic analysis. Obtaining a multude of MAGs, especially some high-quality Commamox genomes, is not trivial. Furthermore, implemenng the 13C-DNA-based SIP method at two different temperatures (4 and 10 ºC) on selected sediment and soil samples validated the role of detected Commamox Nitrospira in nitrificaon. The authors have ulized virtually available methods to substanate their findings.\nThe manuscript is well-structured and writen, making for an engaging read. Despite the complexity and breadth of content related to microbial N cycling quanficaon, the authors have presented their findings in a clear, structured, and succinct manner. The logical progression of the narrave effecvely draws the reader's atenon to the novel nitrificaon driver, clade B Commamox Nitrospira. Since their discovery, this is a significant stride, marking the first in-depth invesgaon of complete nitrifiers in the Antarcc environment. The findings that Commamox bacteria, parcularly clade B, are highly abundant and acve in the area studied are surprising. They will inspire further research into these microbial communies' ecophysiological roles and evoluonary trajectories in cold and oligotrophic environments.\nOverall, this work meets the journal's requirements and is of high value to the reader. And I have some comments and suggesons before being accepted.\nResponse: We thank the reviewer for these very posive comments on the general significance of our work and on the complementary nature of the methodology used to reveal novel insights into the N-cycling processes in the Antarcc ecosystem.\nMajor concerns:\n(1) The sampling me is precise. However, we don’t know when the incubaon experiments were conducted, which was related to nitrogen transformaon and microbial acvies.\nResponse: We appreciate the reviewer's comment. The soil and lake sediment samples were immediately stored at -20 °C aer collecon and were maintained at this temperature during transportaon. The samples arrived at the low temperature laboratory (-20oC) in East China Normal University in May 2020. The 10 °C -SIP incubaon was performed in June 2020. The 4°C SIP incubaon was conducted following the 10 °C SIP incubaon.\nWe agree with the reviewer that the incubaon me may affect nitrogen transformaon and microbial acvies. However, considering the long-term low temperature in the Antarcc region, storing the samples at -20°C may have litle effect on the acvity of cryogenic microorganisms. Addionally, the successful labeling of funconal nitrifiers and the observed nitrate producon acvity further support the recovery of related microbes.\n(2) The relave abundance of microbial N cycle genes is defined by copies of ng DNA in Figure 1bcd. Some works indicated that ‘Measuring the total count and concentraon of microbes or genes (absolute abundance) provides a richer source of informaon than relave abundance and can correct some conclusions drawn from relave abundance data (htps://doi.org/10.1038/s41587-023-01754-3)’. Thus, it would be good to have absolute quantave data if possible.\nResponse: We appreciate the reviewer's comment. We agree with the reviewer and have provided the quantave source data in both copies. g-1 and copies. ng-1 DNA formats in Supplementary Data 1 and revised Fig. 1 in accordance with the reviewers' comments. The reason we used copies. ng-1 DNA data in Fig.1, rather than copies. g-1, is due to the high heterogeneity of the soil/sediment samples (mixing with sands or even rocks). These samples have very low organic mater/biomass and are resistant to extracon of high-quanty/quality DNA, making copies. ng-1 DNA data more meaningful for comparisons. This is also why we did not perform a metagenomic analysis on all soil samples (2 out of 10 sequenced) from South Victoria Land, due to the limitaons posed by the extracon of high-quanty/quality DNA.\n(3) De-noising algorithm analysis (ASVs), rather than OTUs, is more accurate and recommended.\nResponse: We appreciate the reviewer's comment, which was also raised by Reviewer 1 (see comment and response above). All our amplicon data were analyzed through QIIME2 ASVs, with representave sequences clustered based on >95% similarity being named OTUs. While ASV analysis is very common for the 16S rRNA gene, there was a concern about the potenal for overesmang funconal marker gene diversity due to non-idencal gene copies within the same genome. As a result, we adopted a more stringent approach and clustered the funconal ASVs into OTUs.\n(4) Why were only three soil (LS4) and sedimental (LA1 and LA2) samples selected for the SIP experiment? What were the selecon criteria?\nResponse: We appreciate the reviewer's point. In fact, the SIP experiment is not a high-throughput method and it's not realisc to apply it to all samples. Furthermore, there is no significant difference among the samples in terms of the abundances of funconal n-cycling genes (Fig. 1), as well as the metagenomic output (Fig. 2). Therefore, it's not necessary to conduct SIP incubaons with all samples. The reasons we chose LA1, LA2, and LS4 for further SIP analysis are: 1) these samples represent different types (soil and sediment) and 2) they are distributed almost uniformly throughout the study area LH; that is, LA1 is next to the Antarcc ice sheet; LA2 is in the center of LH; and LS4 is adjacent to the coastal line (Fig.1). This point was included in the revised manuscript (lines 485-486).\n(5) The ammonium and nitrate contents were low (Supplementary Table 1). During the SIP incubaon, approximately 5 μg g-1 of 15N labeled NH4+ was added as the substrate. About 40 μg g-1 of nitrate and ~ 20 ppm of N2O were detected aer the 56-day incubaon (Figure 4a). Do you have the isotopic abundance values of nitrate? This is important to illustrate the nitrogen transformaon processes.\nResponse: We appreciate the reviewer's comment. For the 10 °C -SIP, the incubaon period was 8 weeks, during which the NH + substrate was added weekly in approximately 5 ppm increments, resulng in a final cumulave product of around 40 μg g-1 nitrate. For the 4°C -SIP, the incubaon period was 12 weeks, and the NH + substrate was added every 2-3 weeks. This also resulted in accumulaon of nitrate. Given the extremely low inial quanes of NH + and NO - in the original samples (as shown in Supplementary Table 1), all accumulated NO - can be considered as a product derived from the NH + substrate.\nWe agree with the reviewer that the rao between labeled and unlabeled nitrate is an intriguing aspect to consider when understanding the significance of internal N sources versus added NH + during dissimilatory processes. However, the primary aim of the experiment was to label metabolically acve cells that ulized the provided labeled N and C for assimilatory processes, incorporang these elements into their DNA. The reason we applied 15N labeled NH +, in addion to 13C-CO during the SIP incubaon, was to enhance the incorporaon of heavy C/N into acve nitrifiers. These methods are commonly employed in similar SIP incubaon setups (Sun et al., 2022; Wang et al., 2019).\nMinor concerns:\n(1) The abstract could be more succinct. Some abbreviaons can be omited.\nResponse: We thank the reviewer for this comment. We have removed the abbreviaons and shortened the abstract within 150 words, as required by Nature Communications.\n(2) Did the authors find any excing properes in the AOA genomes? Especially in the nutrient-poor environment of this study.\nResponse: We appreciate the reviewer's point. AOA has previously been reported in coastal Antarcc soils (Orz et al., 2020) and thus is not the focus of this study. Nevertheless, we screened the annotaons of the retrieved AOA genomes and did not find uniquely significant features so far. As more Antarcc-originated AOA genomes or isolates become available, we will be able to conduct further genomic or even physiological invesgaons regarding their survival strategies and metabolic potenals.\nLine 48: Consider referring to it as the ‘ice-free Antarcc region,’ ‘Coastal East Antarcca,’ or ‘Coastal Antarcca’.\nResponse: We thank the reviewer for spong this. We have made revisions accordingly.\n(3) Lines 63-69: lack references.\nResponse: We thank the reviewer for this point. Related references have been cited (lines 56-61).\n(4) Line 244: ‘La3-bin1’ should be corrected to ‘La3-X1’. Right?\nResponse: We thank the reviewer for this correcon. Changes have been made accordingly (lines 230-241).\n(5) Line 287: Please correct the spacing issue in ‘Fe or Mn’.\nResponse: We thank the reviewer for this correcon. Change has been made accordingly (line 283).\n(6) Line 437: Number ‘seven’ should be replaced with ‘fourteen’. Right?\nResponse: We thank the reviewer for this correcon. We have changed the number accordingly (line 435).\n(7) Line 458: Please confirm the term ‘all available’ as databases are regularly updated.\nResponse: We thank the reviewer for this point. Changes have been made accordingly (line 457).\nLines 485-486: Could you clarify what ‘in situ flooding condions’ mean?\nResponse: We thank the reviewer for this point. Changes have been made accordingly (lines 484-485).\n(8) Line 504: It should read ‘OTU sequences.\nResponse: We thank the reviewer for this point. Changes have been made as suggested (lines 506-507).\n(9) Fig. 3a: Please double-check the phylogenomic tree; ‘palsa 1315’ appears twice.\nResponse: We thank the reviewer for this correcon. The correcon has been made (Fig.3). In addion, we double-checked all our figures to ensure there are no similar mistakes. The informaon of reference genomes, including those for comammox Nitrospira, AOA and NOB Nitrospira, is listed in Supplementary Table 4. Moreover, the source data for comammox and AOA ANI analysis can be found in Supplementary Data 5 and Supplementary Data 6, respecvely.\n(10) Fig. 3d: AHL is missing from the botom part of the figure.\nResponse: We thank the reviewer for this point. We have added the related informaon of AHL accordingly (Fig.3).\n(11) Figure 4a: What is ‘CK’ treatment? No descripfion is found in Secon ‘Material and Methods’ either.\nResponse: We appreciate the reviewer's suggeson. We have accordingly added the related informaon for 'CK' in the legend of Fig. 1.\n(12) Supplementary Fig. 6 and Supplementary Fig. 7: Please confirm the accuracy of the colored shadows.\nResponse: We thank the reviewer for this point. Changes have been made accordingly.\n(13) Supplementary Fig. 9: The stray ‘25’ is in the middle of the figure; please remove it.\nResponse: We thank the reviewer for this point. Changes have been made accordingly.\nReferences:\nOrtiz M, Bosch J, Coclet C, Johnson J, Lebre P, Salawu-Rotimi A, et al. Microbial nitrogen cycling in Antarctic soils. Microorganisms 2020; 8.\nPester M, Maixner F, Berry D, Rattei T, Koch H, Lucker S, et al. NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira. Environ. Microbiol. 2014; 16: 3055-71.\nSun X, Zhao J, Zhou X, Bei Q, Xia W, Zhao B, et al. Salt tolerance-based niche differentiation of soil ammonia oxidizers. ISME J. 2022; 16: 412-422.\nWang B, Qin W, Ren Y, Zhou X, Jung MY, Han P, et al. Expansion of Thaumarchaeota habitat range is correlated with horizontal transfer of ATPase operons. ISME J. 2019; 13: 3067-3079."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you to the authors for addressing all my comments and making the changes accordingly.\nNo major comments or changes required from my end.\nJust a small clarificafion about CoverM (approach used by the authors) and SingleM appraise (approach suggested by me in the previous revision). Please, keep this in mind for your future work.\nBoth approaches complement each other, but they indicate/measure different things. While CoverM is giving you the relafive abundances of the MAGs in the community, SingleM is esfimafing the total number of taxa in the community based on binned and unbinned sequences and indicafing how many of that total are represented by the recovered MAGs.\ni.e. CoverM output\nMAG_1 5%\nMAG_2 3%\nMAG_3 2%\nSubtotal: 10% of mapped reads\ni.e. SingleM output:\n10 different species detected by SingleM appraise and present in your community, but only 3 species represented in your MAGs in the above example. 3 out of 10 (30% of the total species recovered in the MAGs).\nI hope this clarificafion helps. Take a look at this link for your future work.\nhftps://bio-protocol.org/exchange/minidetail?type=30&id=9291876", "reviewer_2": "I am glad that the authors have revised the manuscript according to the comments, and this manuscript is much improved in terms of data presentafion and descripfion. I consider that this manuscript has fulfilled the requirements for publicafion in Nature Communicafion.\nSome minor suggesfions:\n(1) The annual mean air temperature is ~-10 ºC in the sampling sites. I agree that storing the samples at -20 ºC may have liftle effect on the microbial acfivifies. What’s the in-situ temperature of soil and sediment in this region? This data will befter indicate the low impact of sample preservafion temperature on microbial acfivifies.\n(2) Compared to the in situ condifion, the incubafion condifion of 13C-DNA-SIP was more specific. Thus, it is recommended that the limitafions of the SIP results in this manuscript should be stated."}, "author_response": "We thank the reviewers for their encouraging remarks and further comments. The manuscript has been revised accordingly. Below, we have addressed each reviewer's comments (noted in black) with our responses (in blue):\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThank you to the authors for addressing all my comments and making the changes accordingly.\nNo major comments or changes required from my end.\nResponse: We are delighted to receive this news and wish to express our gratude to the reviewer for the insighul comments on our manuscript.\nJust a small clarificaon about CoverM (approach used by the authors) and SingleM appraise (approach suggested by me in the previous revision). Please, keep this in mind for your future work.\nBoth approaches complement each other, but they indicate/measure different things. While CoverM is giving you the relave abundances of the MAGs in the community, SingleM is esmang the total number of taxa in the community based on binned and unbinned sequences and indicang how many of that total are represented by the recovered MAGs.\ni.e. CoverM output\nMAG_1 5%\nMAG_2 3%\nMAG_3 2%\nSubtotal: 10% of mapped reads\ni.e. SingleM output:\n10 different species detected by SingleM appraise and present in your community, but only 3 species represented in your MAGs in the above example. 3 out of 10 (30% of the total species recovered in the MAGs).\nI hope this clarificaon helps. Take a look at this link for your future work.\nhtps://bio-protocol.org/exchange/minidetail?type=30&id=9291876\nResponse: We appreciate the reviewer's insighul comments on the disnct algorithms and outputs between CoverM and SingleM. Indeed, we plan to incorporate SingleM evaluaons in our future work.\nReviewer #2 (Remarks to the Author):\nI am glad that the authors have revised the manuscript according to the comments, and this manuscript is much improved in terms of data presentaon and descripon. I consider that this manuscript has fulfilled the requirements for publicaon in Nature Communicaon.\nResponse: We are delighted to receive this news, and we express our gratude for the reviewer's construcve feedback and correcons.\nSome minor suggesons:\n(1) The annual mean air temperature is ~-10 ºC in the sampling sites. I agree that storing the samples at -20 ºC may have litle effect on the microbial acvies. What’s the in-situ temperature of soil and sediment in this region? This data will beter indicate the low impact of sample preservaon temperature on microbial acvies.\nResponse: We appreciate the reviewer for raising this point. The in-situ temperatures of soil and sediment in this region while we were collecng the samples were also low (around 0 °C), even in summer. This also suggests that the preservaon of samples at low temperatures has a minimal impact on microbial acvies.\n(2) Compared to the in situ condion, the incubaon condion of 13C-DNA-SIP was more specific. Thus, it is recommended that the limitaons of the SIP results in this manuscript should be stated.\nResponse: We appreciate the reviewer for this comment. The limitaons of the SIP results in this manuscript have already been stated in the manuscript (Lines 309-310)."}]

Decision letter