Benthic archaea as potential sources of tetraether membrane lipids in sediments across an oxygen minimum zone

Benthic archaea comprise a significant part of the total prokaryotic biomass in marine sediments. Recent genomic surveys suggest they are largely involved in anaerobic processing of organic matter, but the distribution and abundance of these archaeal groups are still largely unknown. Archaeal membrane lipids composed of isoprenoid diethers or tetraethers (glycerol dibiphytanyl glycerol tetraether, GDGT) are often used as archaeal biomarkers. Here, we compare the archaeal diversity and intact polar lipid (IPL) composition in both surface (0–0.5 cm) and subsurface (10–12 cm) sediments recovered within, just below, and well below the oxygen minimum zone (OMZ) of the Arabian Sea. Archaeal 16S rRNA gene amplicon sequencing revealed a predominance of Thaumarchaeota (Marine Group I, MG-I) in oxygenated sediments. Quantification of archaeal 16S rRNA and ammonia monoxygenase (amoA) of Thaumarchaeota genes and their transcripts indicated the presence of an active in situ benthic population, which coincided with a high relative abundance of hexose phosphohexose crenarchaeol, a specific biomarker for living Thaumarchaeota. On the other hand, anoxic surface sediments within the OMZ and all subsurface sediments were dominated by archaea belonging to the Miscellaneous Crenarchaeota Group (MCG), the Thermoplasmatales and archaea of the DPANN (superphylum grouping Micrarchaeota, Diapherotrites, Aenigmarchaeota, Nanohaloarchaeota, Parvarchaeota, Nanoarchaeota, Pacearchaeota and Woesearchaeota). Members of the MCG were diverse, with a dominance of subgroup MCG-12 in anoxic surface sediments. This coincided with a high relative abundance of IPL GDGT-0 with an unknown polar head group. Subsurface anoxic sediments were characterized by higher relative abundance of GDGT-0, -2 and -3 with dihexose IPL types, GDGT-0 with a cyclopentanetetraol molecule and hexose, as well as the presence of specific MCG subgroups, suggesting that these groups could be the biological sources of these archaeal lipids.

1. Although the authors go to great length rebutting the comments of both reviewers, for most instances their reasoning is not reflected in the revised manuscript (in particular regarding the detailed technical explanations in the second report of reviewer 2). Although the manuscript would be acceptable for publication after minor revisions, I am taken aback by the authors' reluctance to make meaningful changes to the text after three revisions. The review process should provide an opportunity for the authors to make their reasoning more accessible to the readers instead of trying to just brush off criticism. We want to understand your research and help make it more impactful. It is sad that the authors did not grasp this opportunity.
We are sorry that the reviewer feels that we missed the chance to improve our manuscript during the past revisions. We really appreciate the detailed comments by both reviewers during this lengthy process, but we also feel that we also addressed these comments with great detail and modified our manuscript accordingly. The reviewer also recommended to re-analyze the samples, which as discussed before is not feasible due to the decay of the compounds in the already extracted samples. However, in our last reply to the reviewer's comments we included new data comparing the different analytical methods by using other freshly extracted samples. This addition contributes to the clarity of the manuscript and addresses the reviewer's concerns. Also, we have added many caveats and statements to our manuscript regarding our analytical approach, such as the lack of quantitation and the underestimation of the MH-GDGTs. Furthermore, we have addressed all line by line comments and made modifications as recommended. We believe this demonstrates our willingness to consider the reviewer's comments. However, it is clear we have differing scientific views on the topic of this paper and we retain the right to express our views. ). For details on the QPCR conditions, efficiency and R 2 of the QPCR assays see Table S2.  replicates.

165
In this study, we analyzed both IPLs and DNA/RNA extracts from sediments previously collected along the Arabian

166
Sea Murray Ridge within the OMZ (885 mbsl), just below the lower interface (1306 mbsl), and well below the OMZ

Archaeal IPL-GDGTs in the surface and subsurface sediments
A range of IPL-GDGTs (GDGT-0 to 4 and crenarchaeol) with the IPL-types monohexose (MH), dihexose (DH) and hexose-phosphohexose (HPH) was detected in surface and subsurface sediments across the Arabian Sea OMZ (Table  with one dihexose moiety; Table 2) were detected and identified based on their mass spectral characteristics (Fig. S2).

224
Below the OMZ, in partly and fully oxygenated surface sediments at 1306, 2470 and 3003 mbsl (Table 1)

247
As the Thaumarchaeota MGI was dominant in oxygenated sediments at 1306, 2470 and 3003 mbsl (Fig. 1b), we further analyzed the diversity of this group by performing a more detailed phylogeny of the recovered 16S rRNA gene reads as 'Ca. Nitrosoarchaeum koreensis MY1' or environmental 16S rRNA gene sequences from marine sediments (Fig.3).

259
The diversity of Thaumarchaeota MG1 was further assessed by amplification, cloning and sequencing of the archaeal

286
In this study, we assessed the changes in benthic archaeal diversity and abundance in sediments of the Arabian Sea 298

299
We detected large differences in archaeal diversity between the surface sediment deposited within the OMZ and those  (Table 5).
For example, OTU-2 becomes progressively more abundant with increasing water depth, suggesting that this OTU is favored at the higher oxygen concentrations found in the surface sediment at 3003 mbsl. OTU-4 was closely affiliated with 'Ca. Nitrosopelagicus brevis', a pelagic MG-I member, which indicates that this DNA is most likely derived from the overlying water column (Table 5), and thus should be considered to represent fossil DNA.

326
There is a discrepancy between the 16S rRNA gene copy numbers and the amoA gene copy numbers within the 327 sediments (Fig. 5). AmoA gene copies were consistently lower than the 16S rRNA gene copies, even within sediments

334
Members of the DPANN Woesearchaeota were only present in the surface sediment at 885 mbsl but not in the subsurface anoxic sediments at 885 and 1306 mbsl, suggesting that their presence here is not solely dependent on the 336 absence of oxygen but possibly also on the OM composition and availability in surface and subsurface sediments.

337
Alternatively, the DPANN Woesearchaeota 16S rRNA gene signal could also originate from the water column and 338 deposited in the surface sediment at 885 mbsl as fossil DNA as observed for the case of Thaumarchaeota as mentioned 339 above.

341
The archaeal diversity in the subsurface sediment (1012 cm) from both 885 and 1306 mbsl (i.e. dominated by MCG,

342
MBG-B, -D and -E) is similar to that observed in the surface sediment at 885 mbsl. This supports that oxygen 343 availability is an important factor for determining the diversification of archaeal groups (Fig. 1b). MCG, one of the 344 dominant archaeal groups in these sediments, showed substantial differences in the distribution of its subgroups (Table  4). All subsurface sediments had a high intra-group diversity of MCG. This is in contrast with the surface sediment at  crenarchaeol was predominantly present with DH as the predominant IPL-type (Table 2). This is considered to be a fossil signal of Thaumarchaeota deposited from the water column due to a higher preservation potential of glycolipid fossil since evidence for active Thaumarchaeota is lacking.

406
The low relative abundance of GDGT-0 IPLs in the surface sediment at 885 mbsl (Table 2) is remarkable. Only MH-

407
GDGT-0 was detected in low relative abundance (0.3 %), whereas any other of the IPL-types with GDGT-0 as CL that 408 were screened for in our study (Table S2; Fig. 1b

725
Extracted OTUs from the Arabian Sea sediments assigned as MCG were inserted in the tree. The number of detected 726 reads per OTU per samples are indicated. Per MCG subgroup the relative abundance is given as detected at the different stations and sediments depths, this is also noted in Table 4. Scale bar represents a 2% sequence dissimilarity.  Table 4. Scale bar represents a 2% sequence dissimilarity.

743
(OPD) in the sediment, and TOC content and pore water composition of the surface (0-0.5 cm) sediment a