The authors comprehensively characterized geochemistry and microbial communities of two seep sites in Monterey Bay, and observed very little DNA and RNA belonging to anaerobic methanotrophic archaea (ANME), particularly at Clam Field. They further supported this with gene quantification, microscopy, and sediment incubations under varying methane partial pressures, and posited that ANME may be outcompeted by other taxa in the presence of more complex hydrocarbons. They concluded that such a surprising absence of ANME has the potential to revise estimates of methane flux into the hydrosphere.   

This manuscript is well-written, and the data are presented and interpreted in a logical order. I particularly appreciate how the authors contextualized their results as they presented them. I recommend accepting this manuscript once a few (mostly minor) details are clarified in the text. Hopefully my comments below are useful.

Methods

Lines 187-188: What was the minimum 16S sequencing depth of any sample? Were any samples discarded due to low read depth?

Line 192: Please specify if the published mcrA sequences were manually compiled, or cite a relevant source.

Results

Line 295-296: Re “while the relative abundance of Bacteroidota decreased with sediment depth (Fig. 4a)” Just from looking at Fig. 4a, Bacteroidota look somewhat consistent with depth here- please justify with statistics or remove.

Was no cDNA recovered from three of the samples at Clam Field seep-edge (Fig. 4a)?

Line 336 “Seep-SRB1, the group containing many known obligate ANME symbionts” Please provide a citation or two.

Lines 356-358: If I’m understanding correctly, the two highly abundant rows corresponding to ANME-2c in Extrovert Cliff both represent one ASV that cannot be resolved between the two reference sequences (and so its relative abundance is split between the two of them). I think it would minimize confusion if this were described more explicitly (particularly in the figure legend).

Line 431: Please briefly justify the comparison to US Atlantic Margin seeps in particular: were they simply the most convenient to compare because similar data were collected by the same research group?

Discussion

Lines 478-483: I see some similar phrasing between this and the third paragraph of the introduction of the Semler et al 2022 AEM study. This feels borderline unnecessarily picky to point out, given the same first author, but I mention it just in case the editor or publisher disagrees with me.  

I understand that the lack of methane-dependent sulfate reduction and inability to enrich ANME in the clam field incubations would be considered a “negative” result, but why not comment on the Desulfobacterota that increased over time (Fig. S5?) Could these be the hydrocarbon degraders implicated in 553-555, and/or are they particularly good at thriving in sulfidic conditions?

The manuscript by Semler & Dekas describes the biochemistry and microbial ecology of two cold seep sites located in the Monterey Bay area. The authors report low abundances of ANME archaea quantified by 16S rRNA gene and transcript sequencing, together with ddPCR and mcrA sequencing, complemented by microscopic identification of typical ANME-SRB aggregates, low rates of potential activity and lack of response to the presence of methane during prolonged incubations. These microbial ecology observations are complemented by geochemical measurements of relevant compounds (methane, sulfide, sulfate) and isotopic measurements of methane.

The manuscript is well written and tackles an interesting question. The data seem to be carefully collected and are described and analyzed with scientific rigor. Conclusions follow environmental observations or experimental outcomes and hypotheses are presented as such in case they could not be confirmed by experimentation/observation. My comments are all minor. I congratulate the authors for this very interesting piece of work

I have two main comments on the reviewed manuscript. While the DAPI staining nicely shows aggregates of cells at Extrovert Cliff, their taxonomic identity is unclear. The authors are aware of this (L399) and describe them as putative ANME aggregates (Fig S2 caption) and claim that an ANME-typical morphology was detected (L508). Would it be possible to explain this in the discussion? Is this morphology really typical? Is there any previous experience with such an indirect classification? This would help the reader to see the validity of the experimental strategy.

My second comment relates to the extended incubation. I wonder why only the Clam Field site was chosen for incubation, even though AOM for the other site was more likely but still low compared to other cold seep sites. I suggest stating the reason in one sentence. Direct observation/quantification of sulphate-dependent AOM at Extrovert Cliff would allow comparison with other sites. 

Minor comments: 

L19 - I feel that the last sentence of an abstract needs polishing.         

L56 – please consider to include the new clade names for (doi: 10.1371/journal.pbio.3001508.)

L58 and elsewhere – you use ‘symbiont’ for the ANME-SRB aggregates while I find ‘syntrophs’ much more fitting, as it describes the type of metabolic interaction

L183 - Was it possible to overlap majority of pair-end reads after trimming? What was the length of overlap in DADA2 pipeline? 

L188 - 53K mcrA reads per sample are not in line with 39K reads reported in the Results (L352), please check. 

L195 - Please specify the substitution model which was used to construct the reference tree. 

L393 - Converting mcrA copy number per well to cells per g assumes mcrA gene to be a single copy gene which is not true in 100% cases, cell numbers can be overestimated. 

L289 and elsewhere – please use 16S rRNA gene amplification rather than 16S rRNA amplification

L306 -as inferred by the presence of…

L429 - Methods section is missing the description of combining ASV matrices from the current and previous studies, please add such description.  

L605 - ENA accession number is still private, please either release the dataset or share a reviewer's link.