Sampling and physicochemical characteristics at Mariana Trench
The depth transect at the Challenger Deep of Mariana Trench was sampled on two cruises at 0, 2,000, 4,000, 8,000, 9,600, 10,400 and 10,500 m depths. Ammonia concentration was uniform across the transect and ranged from 17.5 to 26.7 nM (Additional file 1: Table S1). Likewise, nitrite concentration was low and constant over the depth, never exceeding 0.11 µM (Additional file 1: Table S1). There was an increase in nitrate concentration with increasing depth, i.e., nitrate ranged between 34-39 µM at >2,000 m while in the surface its concentration was 0.01-0.32 µM. There was a slight decrease in pH from the surface (8.24) to the bottom (7.8) of the trench. Salinity remained constant throughout the different sampling depths. Temperature generally decreased with the depth of seawater and ranged from 29ºC at the surface to approximately 1 ºC at the bottom of the trench. There was a marked increase in silicate concentration over depth and the concentration ranged between 0.42 and 159 µM.
Diversity and distribution of archaea along the depth transect
A total of 190 Gbp raw metagenomic data was retrieved at various depths (0, 2,000, 4,000, 8,000, 9,600, 10,400 and 10,500 m) from two cruises in the Challenger Deep. Binning and assembly of these data resulted in hundreds of bins including four thaumarchaeal MAGs (MTA1, MTA4, MTA5 and MTA6 [short for Mariana Trench Archaea]) representing four distinct deep-sea clades of AOA (Table 1). Phylogenetic analyses were conducted based on 16S rRNA, amoA genes (found in metagenomes) and 60 concatenated ribosomal proteins in order to investigate the evolutionary relationships between these deep-sea Thaumarchaeota (Fig. 1a and Additional file 1: Figure S1). Relative abundances of different thaumarchaeotal clades were also examined through metagenomic amoA genes to determine the differences in their distribution patterns in various samples along the vertical transect (Fig. 1b). Furthermore, sequencing of the environmental 16S rRNA genes was conducted using two different primer sets to elucidate the distribution of these ammonia oxidizers (Fig. 1c).
Primers targeting both Archaea and Bacteria were used in 16S rRNA gene sequencing. However, results of the two primer sets showed apparent differences, likely indicating a PCR bias, e.g. relative abundance of Thaumarchaeota estimated by the 341F/802R primers was three times greater than that by the 515F/806R primers at 8,000 m (Fig. 1c). Nevertheless, similar patterns were shown in the vertical distribution of Thaumarchaeota estimated from the metagenomic amoA genes and 16S rRNA gene amplicons with both primer sets (Fig. 1b and 1c). For example, both 16S rRNA gene primers retrieved almost no thaumarchaeotal sequences in 0 m samples, which was consistent with previous results  (Depth 0 m metagenomics analysis where very few sequences were present, Fig. 1b). Furthermore, both methods predicted the highest relative abundance of AOA in 2,000 m samples, ranging from 5.9% to 14.9% of total prokaryotes. Previous estimation based on different methods (such as DAPI nucleic acid staining or primers targeting 16S rRNA genes) indicated that 20-75% of total sequences belonged to Thaumarchaeota at a similar depth [8, 28]. Between the two primers sets, the 341F/802R set is more likely to reflect the real distribution pattern of AOA, because the results from this primer sets are more consistent both with previously published studies and with our metagenomic dataset. Although our results gained using two methods (16S rRNA and amoA genes) predicted the abundance to be lower than in previous studies, considering the existence of 16S rRNA PCR bias and the highly conservative estimation method of metagenomic amoA (explained in Material and Methods), the inconsistency between these results is moderately small and within an acceptable range.
The thaumarchaeal community exhibited a pronounced change over the depth transect in both methods (16S rRNA and amoA genes), in agreement with previous studies [24, 28]. As expected, the thaumarchaeal community of shallower depths (2,000 to 4,000 m) were dominated (95.92 %) by the gamma AOA with only a small proportion (1.56 %) of the thaumarchaeal community at 2,000 m being beta AOA, which have been previously reported in shallower waters [24, 28]. The beta AOA have been reported to exist predominantly in lower epipelagic and upper mesopelagic zone (depth 50 ~ 500 m) [14, 24, 28]. These AOA were detected in our deep sea samples at low relative abundances (0.91 %) suggesting that they might not be native to these depths. Again, in agreement with earlier reports, the abundance of alpha AOA was considerably higher at the greatest depths and accounted for approximately 70% of all archaea at 8,000 m depth. The gamma AOA were also relatively abundant (39.09 %) in these >6,000m samples. Unexpectedly, our study also retrieved sequences most likely related to the thermophilic AOA clade, which includes the genus Candidatus Nitrosocaldus typically found in hot springs [32-34] (amoA of MTA5 was clustered with Ca. Nitrosocaldus in Additional file 1: Figure S1b, Fig. 1a). The sequences related to Ca. Nitrosocaldus were predominantly found in the 2,000 m samples, which is surprising given that the temperature at 2,000 m in Mariana Trench is ~ 2.3 °C (Additional file 1: Table S1). This is, to our knowledge, the first time, that sequences related to Ca. Nitrosocaldus have been reported in either a saline environment or an ecosystem with a high hydrostatic pressure.
Microorganisms in water samples can be divided into free-living (0.2 ~ 3 µm) and particle-associated (>3 µm) fractions by membrane filter sizes. Microorganisms abundant in free-living fraction are usually considered to be planktonic, while those found in particle-associated fraction might attach to particulate organic matter. According to the relative abundance estimates from samples below 200 m, Thaumarchaeota were consistently less abundant in the particle-associated samples than in the free-living samples, suggesting that most Thaumarchaeota along the water column are planktonic. However, the gamma AOA in 10,400 and 10,500 m are equally abundant in the particle-associated samples and in the free-living samples, indicating that several members of the gamma AOA clade might have undiscovered interactions with particulate organic matter.
Four thaumarchaeotal MAGs (MTA1, MTA4, MTA5 and MTA6) were retrieved from our samples. In addition to these MAGs, other thaumarchaeal fragments (short contigs or scaffolds) binned with other Bacteria or Archaea were also detected, resulting in a highly “contaminated” bin (a bin merging sequences from different strains or species). MAG MTA1 harbors a near-complete genome sequence belonging to alpha AOA, which predominate the hadal thaumarchaeotal community. MAG MTA4, recovered from binning of 2,000 m water samples, is a member of the gamma AOA. Most previous studies of deep-sea thaumarchaeotal SAGs have mainly focused on this clade [20, 21], which are also present in all of our deep-sea samples (especially abundant in 2,000 and 4,000 m samples). Binning of samples from other depths did not result in higher quality assemblies of gamma AOA genomes, thus only MTA4 was analyzed to examine the potential functions of this clade. However, due to the low completeness and quality of MAG MTA4, previously published high-quality SAGs of the same clade were used for the subsequent comparative genomics analyses. MAG MTA6 is nearly identical to Ca. Nitrosopelagicus brevis CN25  with ANI ≈ 98% and affiliated with the beta AOA clade.
Intriguingly, our study retrieved a MAG (MTA5) representing the thermophilic thaumarchaeotal clade, which contains the archaeal genus Ca. Nitrosocaldus [32-34]. This was very surprising given that organisms belonging to this clade have been previously reported exclusively in fresh water hot springs. The phylogenetic placement of MAG MTA5 corresponds to the amoA gene and ribosomal proteins, suggesting that this is not a chimeric genome of multiple lineages nor a result of assembly or binning errors (Fig. 1a and Additional file 1: Figure S1). All AMO subunits were present in the MAG MTA5, indicating that this organism is a putative ammonia oxidizer. However, the sequencing coverage was low (×10) and further studies will be required to investigate the presence, metabolism and ecological function of this clade of AOA in the deep sea.
Although the gamma AOA were more abundant than the alpha AOA in shallower samples (2,000 and 4,000 m in Fig. 1b and 1c), it was difficult to recover high-quality genomic bins belonging to the gamma AOA from these samples (only one gamma AOA MAG (MTA4) was recovered with a low completeness of 24.84%). The greater species diversity within the gamma AOA might explain this result and accordingly, both ANI and tetranucleotide frequency correlation coefficient values (TETRA)  indicate that the alpha AOA may consist of a single phylotype, whereas the gamma AOA have multiple phylotypes (Additional file 1: Figure S2). A recent study also suggested that the genomes of the alpha AOA might experience less gene flow due to presence of genes encoding a thrombospondin-like extracellular structure . This structure contains five Ca2+-binding domains and may regulate the cellular structure for adhesion, thus leading to the smaller divergence of the alpha AOA . Furthermore, the phylogenetic distances of other genes (such as the amoA and the ribosomal protein genes) among the gamma AOA were greater than those of the alpha AOA. It is interesting to note that another highly redundant merged bin with > 400% contamination was generated in our binning process. This bin contained fragments of the gamma AOA and multiple amoA genes (Table 1). It is likely that multiple strains or species of gamma AOA were too similar to be distinguished and thus were placed into this bin. This would also explain why no high quality gamma AOA MAG was recovered in our study even if gamma AOA were abundant in the samples. The contaminated metagenomic bin was omitted from subsequent analyses due to its poor quality.
Archaeal MTA1 MAG from the hadal zone
MAG MTA1 is one of the first high-quality draft thaumarchaeotal genomes from the hadal zone which meets the recently proposed quality standards for MAGs and SAGs (completeness > 90%, contamination < 5%, containing all three rRNA genes and enough tRNA genes) . The MTA1 MAG is 100% complete and belongs to the alpha AOA, the most abundant free-living archaeal clade at 8,000m depth in the Mariana Trench. Given the vast abundance of these archaea in the hadal zone and the major gaps in our knowledge of their lifestyle and environmental adaptation, we focused subsequent analyses on this MAG. MAG MTA1 was therefore used to predict adaptations and metabolism of archaea in the hadal zone and key predictions were validated by examining the transcriptional activity of genes in the predicted pathways.
The estimated size of a closed circular genome of MTA1 is ~1.3 Mb, which is among the smallest thaumarchaeotal genomes reported, and is similar to that of Ca. N. brevis CN25 (1.23 Mb), Ca. Nitrosomarinus catalina SPOT01 (1.36 Mb), and several near complete SAGs of the gamma AOA. All of these deep-sea AOA genomes are streamlined compared to other thaumarchaeotal strains (other complete marine Thaumarchaeota are >1.6 Mb; Table 1).
To get a better overview of the MTA1 MAG, genes were annotated with Archaeal Clusters of Orthologous Genes database (arCOG) , and a comparison of arCOG categories was conducted with several other Thaumarchaeota, including representatives of epipelagic Nitrosopumilus and Ca. Nitrosopelagicus strains and of the gamma AOA clade (Additional file 1: Figure S3). MTA1 MAG has fewer genes associated with cell wall, membrane and envelope biogenesis (category M) than either Ca. N. brevis CN25 or Ca. N. catalina SPOT01. Other categories with relatively high gene number reductions are categories R and S, which both represent genes with unknown functions.
While gamma AOA are the dominant clade in the “ordinary” deep sea, the alpha AOA emerge and dominate the archaeal community in most samples from the greatest depths (>8,000 m; at least in Mariana Trench). A comparison between the gamma AOA and the alpha AOA was performed to examine their unique genes based on arCOG categories (Additional file 1: Figure S4). In most categories the gamma AOA possessed more unique genes than the alpha AOA, especially in the categories M and R (M: cell wall, membrane and envelope biogenesis; R: general function predicted only), indicating their larger genomic inventories.
Central metabolism of alpha AOA in the hadal zone
The potential metabolic pathways of MAG MTA1 were examined (Fig. 2). Unsurprisingly, the overall predicted metabolic map of MTA1 is similar to that of other previously described representatives of the genus Nitrosopumilus, such as the type strain of this genus, Nitrosopumilus maritimus SCM1  (Additional file 1: Table S2). For the core pathways that enable Thaumarchaeota to grow chemolithoautotrophically, ammonia oxidation and carbon fixation by the modified 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycles are considered essential. Like many other marine Thaumarchaeota, MAG MTA1 contains a set of genes involved in the utilization of urea. Various Nitrosopumilus strains can grow on urea as their sole energy source [11, 39, 40] and urea is a common molecule in the sea water. The genetic potential of MAG MTA1 predicts that ammonia is oxidized in the periplasm by AMO, and electrons produced in this step are transferred by blue copper-containing proteins to a quinone reductase and then to the main electron transfer chain. Carbon fixation is carried out by the modified 3-HP/4-HB pathway, which has two major parts: one contains two carboxylation reactions (consuming two bicarbonate molecules) transforming acetyl-CoA via 3-hydroxypropionate to succinyl-CoA, and the other transforms succinyl-CoA to 4-hydroxybutyrate and then back to two acetyl-CoA via multiple enzymes including 4-hydroxybutyryl-CoA dehydratase (hcd), a key enzyme in this pathway. This pathway is thought to be the most energy-efficient one in carbon fixation under aerobic conditions, and perfectly suits the lifestyles of archaea under low energy supplies . Other ubiquitous pathways of marine AOA, such as the incomplete tricarboxylic acid cycle and non-oxidative pentose phosphate pathway, are also conserved in MTA1.
Synteny between MTA1 and the type strain of Thaumarchaeota
An alignment between the MTA1 genome and the type strain Nitrosopumilus maritimus SCM1 was performed to assess the genome arrangement and the conservation of synteny (Fig. 3). Although the MTA1 genome is not closed, the gene organisation within the contigs is robust due to the high sequencing depth (×97). The genome organisation of MTA1 is largely similar to that of SCM1 and the order of MTA1 contigs could be inferred from the SCM1 genome (Fig. 3). There are three large insertions on the MTA1 genome as well as multiple minor genomic rearrangements compared to the SCM1 genome (Fig. 3). Interestingly, several unique genes are located near the insertion sites, including the glycine cleavage system on contig 2. In addition, multiple unique genes were located near the gaps between the contigs, e.g. the set of atypical A-type ATPase genes.
Unusual bioenergetics of archaea in the hadal zone
The MTA1 MAG contains two sets of A-type ATP synthase genes, which was considered unusual among published marine AOA genomes until very recently (Fig. 4). The first four steps of the electron transfer chain are conserved between MTA1 and other marine Thaumarchaeota, but the complex V, the archaeal-type ATP synthase, is atypical for most marine AOA. The atypical ATP synthase of MTA1 falls within the same phylogenetic cluster as sequences for the gamma AOA, the terrestrial acidophilic AOA Ca. Nitrosotalea , neutrophilic Ca. Nitrosocosmicus [43-45] and several acidophilic or hyperthermophilic archaea in other phyla. In contrast, the typical ATP synthase set in MTA1 is conserved in most other Thaumarchaeota and Crenarchaeota (Fig. 4c). During the review of this current manuscript, Wang and colleagues published a study demonstrating that the distinct, atypical ATP synthase found in the deep sea AOA, and in AOA genera Ca. Nitrosotalea and Ca. Nitrosocosmicus, is a key adaptation to low pH and, most likely, also to elevated pressures .
Wang and colleagues confirmed that the transcriptional activity of the atypical ATP synthase is elevated at low pH and that the heterologous expression of this operon confers to E.coli the ability to grow faster at low pH. This strongly suggests that this operon is a V-type ATPase involved in pumping out protons and maintaining pH homeostasis . Interestingly, the related euryarchaeal ATPase / ATP synthase sequences (Fig. 4c) couple the gradient of Na+ to ATP synthesis instead of proton pumping  and the subunit c of ATPase / ATP synthase contains the ion binding motifs which determine the preference for H+ or Na+. Analyses of the subunit c sequences of the MTA1 imply that the two distinct ATPase / ATP synthase sets are coupled to Na+ or H+, respectively (Fig. 4b) . A combination of sodium and proton motive force is present in many marine bacteria, e.g. Vibrio species found in the deep sea  and the Marine Group II Euryarchaeota, which are ubiquitous in the marine environment, have putative Na+-coupling ATP synthases . However, there is no direct experimental evidence for the coupling of ATP synthesis to either H+ or Na+ gradients in Thaumarchaeota and the findings by Wang and colleagues favour the explanation that this protein is involved in proton extrusion.
Previous phylogenetic analysis suggested these ATP synthases are spread among archaea and bacteria through horizontal gene transfer (HGT) . The gene synteny surrounding the typical ATP synthase of MTA1 is conserved in other Thaumarchaeota (Fig. 4a), and the phylogeny of the subunit A of this ATP synthase is congruent with that of the 16S rRNA and ribosomal proteins genes. In contrast, the downstream and upstream genes of the atypical ATPase / ATP synthase set in MTA1 are different from other Thaumarchaeota (Fig. 4a). Furthermore, linear regression results of tetranucleotide frequency divergencies indicate that the atypical ATPase / ATP synthase was likely acquired through a horizontal gene transfer (Additional file 1: Figure S5). If these ATPases / ATP synthases were horizontally acquired, it is most likely that they originated from the gamma AOA. The topology of the phylogenetic tree (Fig. 4c) implies that the ATPases / ATP synthases of all the Thaumarchaeota were transferred horizontally from Euryarchaeota. This is in agreement with the conclusions by Wang and colleagues who suggested that the ATPase operon has been horizontally transferred between TACK and DPANN superphyla and Euryarchaeota .
Intriguingly, genes putatively associated with Na+ bioenergetics are relatively common in the MTA1 MAG. In addition to ubiquitous transporters, such as Na+/Ca+ antiporters, NhaP-type Na+(K+)/H+ antiporters and Na+-dependent bicarbonate transporters, present in other epipelagic Nitrosopumilus genomes, a subset of unique transporters was found only in the alpha AOA and gamma AOA (Additional file 1: Table S3). For example, a putative transporter similar to the NhaD-type Na+/H+ antiporter was present in MAG MTA1 and closely related SAGs of the same AOA clade . In addition, a unique putative Na+/solute symporter gene (Na+/glucose symporter superfamily, similar to the PutP-type Na+/proline symporter) was present in MTA1. These genes are all predicted to require a Na+ gradient or other monovalent cations across the membrane, although these predictions are pending experimental validation in Thaumarchaeota. Likewise, functionally similar Na+/H+ antiporter and Na+/solute symporter genes are present in the genomes of the genus Candidatus Nitrosotalea . However, the identities between these genes in Ca. Nitrosotalea and MTA1 genes are too low (only approximately 20%) for them to be considered homologues.
Adaptation of archaea to the extreme pressure in the hadal zone
For organisms living in the hadal zone, one of the major challenges is to adapt to the extremely high hydrostatic pressure. Under high hydrostatic pressure, proteins from organisms accustomed to ambient atmospheric pressures undergo denaturation . Osmoprotectants, also called osmolytes or compatible solutes, are produced as one of the major mechanisms to adapt to extreme pressures . Some representatives of the genus Nitrosopumilus have the genetic potential to synthesize the osmolyte ectoine [22, 38]. Mannosylglycerate has also been reported as an osmolyte in the hot spring AOA Nitrososphaera gargensis . In contrast to some of the previously published AOA genomes, no genes involved in biosynthesis of these osmoprotectants could be detected in the MTA1 MAG.
The MTA1 MAG harbours an extra genomic island associated with inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS), which may be involved in adaptation to high hydrostatic pressure. These genes participate in the biosynthesis of di-myo-inositol phosphate (DIP), which is a key osmoprotectant previously found in many hyperthermophilic archaea and bacteria [55, 56]. Coding sequences for these two enzymes have merged into a single open reading frame in the MTA1 MAG and an additional inositol-1-monophosphatase (IMPA) gene copy is located in the vicinity of the merged gene. The IMPA gene is usually present as a single copy in other previously sequenced archaeal genomes and is normally responsible for the hydrolysis of myo-inositol monophosphate to generate phosphate and myo-inositol, a usual osmoprotectant and a precursor of DIP. These two genes, in addition to two other genes annotated as encoding a TATA-box binding protein and an AsnC family transcriptional regulator, respectively, formed a small genomic island in MAG MTA1 and a previously published SAG which belongs to the same AOA clade (Additional file 1: Figure S6). Production of myo-inositol has been previously postulated as a key adaptation mechanism of archaea to the deep sea [24, 29] but there is no prior evidence that these genes are transcribed and required for the survival under high pressure. To validate this prediction, the DIPPS/IPCT transcripts were quantified by RT-qPCR in this study and were shown to be relatively abundant (up to ~3,000 copies per liter) in our cold sea water samples at 4,000 m to 10,500 m depths. Indeed, these transcripts were most abundant in 8,000 m deep samples where the abundance of alpha AOA was also the highest (temperature ~1.96 ºC, Fig. 5). This provides novel evidence that (i) these archaeal populations are active in the hadal zone and (ii) the production of the osmolyte myo-inositol may be required for the survival under high hydrostatic pressure. The unexpected finding of these DIPPS/IPCT homologues in both thermophiles and the MTA1 MAG implies that microbes adapt to different harsh environmental factors through similar mechanisms.
The MTA1 MAG has a glycine cleavage system along with the genes involving in lipoylation, which could also play a role in osmoregulation . Glycine cleavage system and lipoate-related genes are present in several gamma AOA SAGs, indicating that the accumulation or utilization of glycine might be ubiquitous in deep-sea archaeal clades (Additional file 1: Table S4). The glycine cleavage system was also recently reported in alpha, gamma and delta AOA lineages in the Mariana and Ogasawara Trenches . Apart from osmoprotectants, chaperones may help proteins fold properly and maintain their functions under high hydrostatic pressure . In most marine Thaumarchaeota, there are only two gene copies of thermosomes (group II chaperonins) [59, 60]. MAG MTA1 has an additional thermosome encoding gene located near the unique Na+/solute symporter and urease genes (Additional file 1: Figure S7b). The extra thermosome gene is phylogenetically distinct (Additional file 1: Figure S7a), suggesting a distinct function compared to the typical thermosomes and potential unique advantages in protein folding and proper functioning under high hydrostatic pressure.
Autotrophy vs heterotrophy in deep-sea archaea
Over the years there has been a continuous debate as to whether the lifestyle of marine archaea is primarily autotrophic, mixotrophic or heterotrophic [4, 61, 62]. There is evidence that some marine archaea can take up and utilize organic compounds [61-63], while ammonia-oxidizing archaea in the marine environment are typically considered autotrophs able to fix their own inorganic carbon. Trench environments are particularly interesting in this respect as these habitats are considered less oligotrophic than the upper layers of the ocean and their primary production is thought to be driven by the sinking organic nutrients . To gain a better understanding of the preferred lifestyles of deep-sea archaea and their capacity for mixotrophy and the uptake of organic compounds, we compared the amino acid and inorganic ion transporter genes between alpha AOA and gamma AOA clades (Additional file 1: Table S5). Interestingly, the genomes from the alpha AOA clade contained a greater number (57% more) of transporters for the uptake of organic compounds than those belonging to gamma AOA clade. The presence of these additional transporter genes in the alpha AOA would be parsimonious with a less oligotrophic lifestyle and the suggestion that primary production in the deepest seas is driven by sinking organic carbon. This would also be an attractive explanation for the different distribution patterns of the alpha AOA and the gamma AOA between the hadal zone and upper layers. However, it is not clear how this would fit together with the presence of the 3-HP/4-HB pathway for autotrophic carbon fixation in the alpha AOA.
Evidence of autotrophy in MTA1
Considering the presence of both the inorganic carbon fixation pathway and the large complement of predicted transporters for organic compounds in the MTA1 MAG, we further investigated whether the lifestyle of archaea in the hadal zone is autotrophic. To address this question we monitored the abundance and transcription of key autotrophy marker genes, amoA and hcd, from alpha AOA by q-PCR on DNA and cDNA (Fig. 6). The amoA gene encodes for the a subunit of ammonia monooxygenase, whilst hcd encodes the key enzyme of the archaeal carbon fixation 3-HP/4-HB pathway and both are required for autotrophic growth in AOA. Consistent with the metagenomics data (Fig. 1b), the amoA and hcd gene transcripts of alpha AOA were most abundant in samples at 8,000 m. Furthermore, the abundance of amoA and hcd gene transcripts mirrored their gene abundance levels, i.e. most of these genes were in samples at 4,000 to 10,500 m and were absent in samples shallower than 2,000 m. Given such high amoA and hcd gene transcript levels in the hadal zone (Fig. 6), it is most likely that MTA1 AOA and, moreover, the alpha AOA, are important autotrophic ammonia oxidizers in these aphotic waters. Thaumarchaeota have been previously demonstrated to drive dark carbon fixation at 3,000 m depth in the Mediterranean Sea , but to our knowledge this is the first report documenting the transcription of the key genes in the thaumarchaeal carbon fixation pathway at >10,000 m depth and in the trench environment. It is also worth noting that previously characterized marine AOA have an extremely high affinity for NH4+ and the ammonium concentration remained constantly above the reported Km throughout the depth transect in our dataset (Additional file 1: Table S1) . AOA in the hadal zone are therefore unlikely to be limited for ammonium. Collectively, this suggests that Thaumarchaeota in the hadal zone grow autotrophically and may play important, understudied roles in nitrogen and carbon cycling in the deep ocean. In addition, these deep-sea archaea have the genetic potential for uptake of many organic compounds, suggesting that under certain conditions they may be able to metabolize organic carbon.