the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 08 Apr 2025
Heterogeneity of the horizontal environment drives community assemblages and species coexistence of prokaryotic communities in cold seep sediments
Abstract. Microbes in cold seep sediments play important roles in controlling methane filtration and the global geochemical cycle, but little is known about microbial distribution and community assembly in the horizontal sediment profile. This study conducted a comprehensive investigation of prokaryotic community diversity in sediments from different habitats in a cold seep ecosystem of the South China Sea. Compared to other sites, the prokaryotic community in the methane seep site showed a lower α-diversity. Halobacterota was dominant in methane seep site, while higher abundances of Chloroflexi and Asgardarchaeota were observed in the fauna sites. The assembly process of the bacterial community in the methane seep site was mainly a stochastic process, while the archaeal community was mainly formed by a deterministic process. The prokaryotic community in fauna sites was influenced by both stochastic and deterministic processes. The heterogeneity of the horizontal environment such as the content of CH4, Ba2+, total inorganic carbon, and SO42− influenced prokaryotic community diversity and drove the community assembly processes. Additionally, bacterial species coexisted more closely in methane seep site than in other sites, but archaea did the opposite. Overall, this study revealed how prokaryotes build communities in different cold seep habitats.
- Preprint
(1991 KB) - Metadata XML
-
Supplement
(664 KB) - BibTeX
- EndNote
Status: closed
-
RC1: 'Comment on egusphere-2025-1372', Anonymous Referee #1, 13 Apr 2025
-
AC1: 'Reply on RC1', Jingchun Feng, 31 Jul 2025
Reviewer #1:
This manuscript tries to show that bacterial and archaeal communities at seep sites are assembled based on different degrees of stochastic vs deterministic processes. The low degree of phylogenetic resolution for these communities (mostly phylum, class, and order) and the limited range of biogeochemical parameters (mostly sulfate, methane, DIC, Ca2+) calls into question whether the results are reliable; how these conclusions are reached remains unclear. Throughout the entire manuscript, the writing remains strangely opaque and contorted. Weirdly phrased half-truths and generalities about the microbial ecology of cold seep sites and methane oxidation suggest that these authors are not very familiar with this subject, as also suggested by the inadequate reference list that lacks foundational papers on cold seep ecology and microbial syntrophy by Boetius, Orphan, Knittel, Wegener, or Treude. This reviewer had the impression of reading a manuscript that is trying to reinvent the wheel on cold seep microbial ecology and trying at the same time to infer too much about community assembly, on the basis of inadequate data (routine 16S rRNA gene high-throughput sequencing and some biogeochemistry). Consulting some basic papers on microbial community assembly in marine seeps and sediments would be essential for reconsidering the scientific strategy, and for a fresh start (Ruff et al. 2015 Proc. Natl. Acad. Sci. USA 112:4015-4020; Starnawski et al. 2017, PNAS 114:2940-2945) However, this manuscript cannot be recommended for further consideration.
Response: The authors thanks for the reviewer’s detailed comments and corrections. We will seriously consider these suggestions and make explanations and improvements to the manuscript.
Details:
Suggestion: Line 22: methane filtration? Do you mean methane assimilation or oxidation?
Response: The methanotrophic communities in cold springs can filter out most of the methane and reduce its discharge into seawater, referred to as a microbial filter according to the description of Ruff et al (2019) in The ISME Journal, 1751-7362 (In situ development of a methanotrophic microbiome in deep-sea sediments). In cold seeps, the methane is mainly oxidized by ANMEs into CO2 rather than assimilated. To avoid ambiguity, we will change the methane filtration to methane oxidation:
“… in methane oxidation and the global geochemical cycle…” (on Page 3, Line 21)
Suggestion: Line 35: “coexisted more closely” reads like tight spatial association. You probably mean “coexisted in closer taxonomic associations”
Response: The modifications will be as follows:
“…bacterial network showed higher connectivity and species interactions in methane seep site than in other sites…” (on Page 3, Line 35−36)
Suggestion: Line 44-46: better: “In this type of seepage environment, methane is predominantly oxidized by anaerobic methanotrophic archaea in syntrophic association with sulfate-reducing bacteria (SRB)”
Response: Based on the reviewer’s suggestion and the description of Lin et al (2022), we will made the following modifications:
“In this type of seepage environment, sulfate-driven anaerobic oxidation of methane is the predominant process mediated by methanotrophic archaea and sulfate-reducing bacteria (SRB) (Knittel et al., 2009).” (on Page 4, Line 45−47)
Suggestion: Line 46: Please reference the foundational microbiology papers for anaerobic methane oxidation by Antje Boetius, Katrin Knittel and others. Lin et al. 2022 did not discover anaerobic methane oxidation!
Response: We had read the article of Knittel et al (2009) and updated the reference. The specific literature citations will be as follows:
“In this type of seepage environment, sulfate-driven anaerobic oxidation of methane is the predominant process mediated by methanotrophic archaea and sulfate-reducing bacteria (SRB) (Knittel et al., 2009).” (on Page 4, Line 45−47)
Suggestion: Line 54 ff: better: “Abundant benthic fauna such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate at the seep periphery where methane concentrations are decreasing. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).”
Response: Based on the comments of the reviewer and the research of Feng et al, the revisions will be as follows:
“Abundant benthic faunas such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate there when methane releasing weaken. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).” (on Page 4, Line 54−57)
Suggestion: Line 65: what are “Alvin shrimp”? This should be a different species from Rimicaris exoculata, the common hydrothermal vent shrimp on the Mid-Atlantic Ridge.
Response: According to the research of Li (2015) (Report on two deep-water caridean shrimp species (Crustacea: Decapoda: Caridea: Alvinocarididae, Acanthephyridae) from the northeastern South China Sea), it should be Alvinocaris longirostris Kikuchi & Ohta, 1995 (Alvinocarididae), a species related to the chemosynthetic environment discovered in the South China Sea, which was also observed in the research of Feng (2023b). To avoid misunderstandings, we will made the following corrections:
“…dense populations of mussels, clams, shrimps, and deep-sea crabs…” (on Page 5, Line 64)
Suggestion: Line 74: induce? You probably mean “influence” or “determine”
Response: We had reconsidered the word we used, the revision will be as follows:
“…and interspecific trade-offs determine community composition…” (on Page 5, Line 73)
Suggestion: Line 84ff: “In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).“
Response: The corrections will be as follows:
“In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation in sediments driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).” (on Page 6, Line 88−90)
Suggestion: Line 86 ff: The entire section is awkwardly written and needs to be rephrased for clarity. Try something like this: “At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains a methanotrophic and a methylophaga-related symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).”
Response: Thanks for reviewer’s detailed revisions and polishing. The specific revisions will be as follows:
“At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains methanotrophic and thiotrophic symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts of mussels support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).” (on Page 6, Line 90−99)
Suggestion: Line 96 ff. In this introductory paragraph on microbial studies at cold seeps, the authors begin with a general statement on cold seeps although this paragraph focuses exclusively on work done at the Haima cold seeps. The paragraph should be rephrased to make this focus explicit.
Response: Thank you for reviewer’s suggestions. We have reviewed the relevant research on Haima cold seep and refer the studies of Xu et al (2020), Niu et al (2017) Chen et al (2023) and others. We will rewrite this paragraph as follows:
“Haima cold seep is one of the two active seep sites in the South China Sea at present, and the other one is the F Site cold seep (Feng et al., 2018). At present, Haima cold seep has been regarded as a research hotspot. It was revealed that the ecological and geochemical gradients differed in distinct areas of the Haima cold seep, and the distribution of benthic faunas was associated with methane and sulfides (Xu et al., 2020). Although many studies about microorganisms had been conducted on Haima cold seep, these studies had mainly focused on the distribution and diversity of the microbial communities. It had been reported that there was a preference distribution of specific ANME at different depths in Haima cold seep sediments (Niu et al., 2017). It also revealed an interaction between environment condition and microorganisms, which might played an important role in the carbon and sulfur element cycles (Chen et al., 2023). Moreover, several studies on Haima cold seeps have shown that methane fluids promote microbial aggregation and evolution in sediments (Dong et al., 2023; Niu et al., 2017; Zhong et al., 2023). Environmental heterogeneity in different areas of Haima cold seep system affected the composition of microbial communities, which even was associated to mineral processes here (Liang et al., 2023). However, how horizontal environmental heterogeneity affects community assembly and species coexistence in different areas of Haima cold seep was still unclear.” (on Page 6−7, Line 100−117)
Suggestion: Lines 173-174: please reference the vegan package with a literature or online citation. There are several “vegan” versions, please specify the one that you have actually used.
Response: We have cited the literature of Dixon et al (2003) (VEGAN, a package of R functions for community ecology) on vegan package and marked the vegan version. The specific modifications are as follows:
“…were calculated using the “vegan” package (vers. 2.6−4) in R (Dixon, 2003), including…” (on Page 11, Line 189−190)
Suggestion: Line 232-236: The methane profiles were likely determined with whole sediment samples, but dissolved ions are most likely obtained from porewater, and need to specified as such in manuscript text and figure legends.
Response: The methane profiles were measured using sediment samples, and TIC, TOC and other ions were obtained from porewater extracted from sediments. We have made supplements and modifications in the text, and so have the legends of figure.1:
“The porewater was extracted from sediments by Rhizon samplers and kept at 4°C until analysis.” (on Page 9, Line 133−134)
“The Headspace Equilibrium method was used to measure CH4 concentrations in the sediments. Specifically, 5 g of sediment was transferred into a glass vial, and immediately added 5 mL of NaOH (5% w/w). Then the class vial was sealed with butyl rubber stopper and aluminum rolled sheet and shaken for 10 minutes to achieve methane equilibrium between the aqueous and gas phases. The CH4 concentrations of the samples were measured by gas chromatography (Trace 1300, Thermo Fisher, Waltham, MA, USA). Concentrations of TOC, TIC and SO42‒, Cl‒, Ba2+, K+, Ca2+, Mg2+, Fe3+, Cu2+, and Mn2+ in sediments were measured using the porewater extracted from the sediments.” (on Page 9−10, Line 142−150)
“…Specifically, concentrations of CH4 of sediments and concentrations of TIC, TOC, and SO42‒, Cl‒, Ba2+, K+, Ca2+, Mg2+, Fe3+, Cu2+, and Mn2+ of porewater from sediments were measured…” (on Page 36, Line 762−764)
Suggestion: Line 238: “…and the areas with different macrofaunal assemblages…”
Response: The following revisions have been made based on the reviewers' suggestions:
“…the methane seep area (ROV1) and the areas with different macrofaunal assemblages (ROV2, ROV3, and ROV4) …” (on Page 15, Line 258−260)
Suggestion: Line 240: microbially-mediated
Response: The specific modification is as follows:
“…methane drove the microbe-mediated anaerobic oxidation of methane…” (on Page 15, Line 261)
Suggestion: Line 244: “… methane combined with Ca2+ …” does not form authigenic carbonates; DIC and Calcium do.
Response: After reconfirmation, the correction is as following:
“…and dissolved bicarbonate combined with Ca2+ to form authigenic carbonates…” (on Page 15, Line 265−266)
Suggestion: Line 246: “horizontal differences” sounds strange; you want to say that the geochemical gradients differ according to biogeochemical conditions at different Haima cold seep locations.
Response: Based on this suggestion, we replaced the word and made the following specific modifications:
“In short, the geochemical discrepancies were shown according to biogeochemical conditions at different locations of Haima cold seep.” (on Page 15, Line 267−268)
Suggestion: Line 256: better: “… revealed distinct profiles of microbial α-diversity in different sediments … Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites. “
Response: Thanks for reviewer’s detailed suggestions, the specific paragraphs have been revised:
“… revealed distinct profiles of microbial α-diversity in different sediments. Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites.” (on Page 16, Line 277−280)
Suggestion: Line 265: It is not the bacterial community that clusters in different quadrants; the PCoA analyses reveals that clustering patterns for the ROV1-4 communities are concentrated in the third, fourth, first, and first quadrants, respectively. Please rewrite accordingly.
Response: Thanks for reviewer’s detailed suggestions, the specific modification is as follows:
“The PCoA analyses reveals that clustering patterns for the ROV1-4 bacterial and archaeal communities are concentrated in the third, fourth, first, and first quadrants, respectively.” (on Page 16, Line 283−285)
Suggestion: Line 285ff: The taxonomic designations in the text are not genus level, but appear mostly family- and order level. If you want to use order-, family-, or genus-level designations for ANME-1 and ANME-2 bacteria, please consult Chadwick et al. 2022 (PLoS Biol. 2022 Jan 5;20(1):e3001508; doi:10.1371/journal.pbio.3001508) and Laso-Pérez et al 2023 (Nature Microbiol 8, 231–245; doi:10.1038/s41564-022-01297-4) for an updated taxonomy.
Response: Thanks for reviewer’s corrections. After confirming the 16S rRNA sequencing data, we found that "ANME-1b" might be regarded as ANME-1b order and "ANME-2c" was regarded as ANME-2c family. The detailed taxonomic annotations are as follows:
“d__Archaea.p__Halobacterota.c__Methanosarcinia.o__Methanosarciniales.f__ANME.2c.g__ANME.2c; d__Archaea.p__Halobacterota.c__ANME.1.o__ANME.1b.f__ANME.1b.g__ANME.1b.”
Since these clades do not have explicit official names, their commonly used group names are displayed in the sequencing data. To date, ANME has not been isolated in pure culture. ANME-1 were proposed to be placed within their own order Methanophagales (The growing tree of Archaea: new perspectives on their diversity, evolution and ecology). ANME-1b is one of the three subclades of ANME-1, and there is no definite designation for ANME-1b yet. Chadwick et al (2022) proposed that the genus name Candidatus Methanogaster with family Methanogasteraceae for ANME-2c. Although the naming has been modified, ANME-2c is still the commonly used name. Therefore, we name them according to clear classification levels in sequencing data and provided supplementary information. The specific modifications are as follow:
“…ROV1 was dominated by ANME-1b and ANME-2c (genus Candidatus Methanogaster proposed) (Chadwick et al., 2022).” (on Page 17, Line 306−307)
“In addition, ANME-1a mainly inhabited ROV3 and ROV4.” according to order level taxonomy. (on Page 17, Line 308−309)
“Meanwhile, ANME-1b and ANME-2c existed in ROV1, while ANME-1a appeared in ROV4.” according to order, family and order level taxonomy, respectively. (on Page 18, Line 317−318)
Suggestion: Line 261-302: The taxonomic analysis remains at a poorly resolved and physiologically ambiguous level (mostly Phylum); are there any attempts to interpret this diversity in terms of function?
Response: In this section, we have introduced the phylum community composition of bacteria and archaea at each site and provided specific percentages, to illustrate the result that the microbial diversity at each site was different, both at the phylum level and genus level. The functions of these microbes had been discussed in the paragraphs later (on Page 19−20, Line 338−364).
Suggestion: Line 304. Physical distance is biogeographically relevant, but studies of seep habitats have shown a high degree of connectivity (for example, Meyer et al. 2013. Frontiers in Microbiology 4:207; doi:10.3389/fmicb.2013.00207). For sampling sites that are only a few hundred meters or some kilometers apart, distance is irrelevant and only biogeochemical conditions separate microbial communities.
Response: We studied the research of Meyer et al (2013). The main influencing factors of the microbial composition in hydrothermal vent and cold seeps may be different, namely temperature and methane. Methane is an important carbon source in cold seep systems. The methane concentration varies in different areas of a cold seep system, such as strong seepage areas, weak seepage areas, or no seepage areas, resulting in different abundances of methanotrophic microorganisms.
According to the study of Feng et al (2023a) (Tracing the Century-Long Evolution of Microplastics Deposition in a Cold Seep), the intensity of methane seepage varies indifferent cold seep successional periods, and the corresponding indicator faunas are different. As we have observed during sampling process, the Haima cold seep had a distinct ecological gradient, which was consistent with the study of Xu et al (2020) (Spatial distribution of seepages and associated biological communities within Haima cold seep field, South China Sea). The distances between different habitats there were very close. The four sites we had selected showed different methane concentrations and different dominant fauna aggregations, including mussels, clams and sea anemones, which indicate different biogeochemical conditions. To display more clearly, we have added a location map of the sampling sites in the supplementary materials.
Suggestion: Line 315: what exactly is “Biomass energy” ? Also, is the community at these sites bacterivorous and does it use bacterial biomass and energy?
Response: According to Field et al (2008), the term biomass energy can refer to any source of heat energy produced from non-fossil biological materials. (Biomass energy: the scale of the potential resource) Most seepage faunas rely on the organic matter converted from methane by methanotrophic and thiotrophic symbionts (Levin, 2005) (Ecology of cold seep sediments: Interactions of fauna with flow, chemistry and microbes). Here we would like to illustrate that microorganisms convert chemical energy such as methane into bioavailable energy, which supports the survival of faunas there. To avoid misunderstanding, we will modify “biomass energy” to “nutrition”.
Suggestion: Line 319 ff: The entire section is inadequate; large bacteria groups are interpreted in terms of very specific capabilities that are correct only for specialized subgroups. For example, it is not true that the Desulfobacteraceae cooperate with the ANME archaea; very specific lineages of Desulfobacteraceae form syntrophic associations with (some) ANME archaea (Schreiber et al. 2010. Environ Microbiol. 12(8):2327-40. doi: 10.1111/j.1462-2920.2010.02275.x.). Some assertions are wildly speculative, for example Lokiarchaea as methane oxidizers and sulfate reducers (line 333) and Thermoplasmatota as free-ling methanogens (vasty overinterpreted; Line 334).
Response: Thank you for the reviewer’s' corrections. We have revised this section based on the composition of the phylum level community and the LDA results. The analysis of the phylum level or order level depends on the most clearly distinguishable level in the data. The specific modifications are as follows:
“As the results showed, class Gammaproteobacteria of phylum Proteobacteria, were dominant member of ROV1, 3, and 4, which have been found frequently as a dominant bacterial taxon in deep sea sediments (Aoki et al., 2014). Desulfobacterota is a high abundant phylum in bacterial communities, and the SEEP-SRB2 genus is predominant at ROV1. It widely existed in cold seep habitats and involved in mussel symbionts with ANME-2c and mat symbionts with ANME-1 (Kleindienst et al., 2012). The relatively high methane content and biomarkers of ROV1 suggested that ANME-1b and ANME-2c might collaborate with SEEP-SRB2 for AOM. Chloroflexi was also an abundant phylum in bacterial communities and S085 and FW22 belonging to class Dehalococcoidia was the dominant member at ROV2. Studies had shown that some strains are associated with the organic degradation in marine sediments or organic dehalogenation (Löffler et al., 2015; Wasmund et al., 2014). Asgardarchaeota was an abundant phylum in archaeal community of ROV2, and class Lokiarchaeia was the main biomarkers. Lokiarchaeia (Spang et al., 2015) were first discovered in sediments near the Loki’s Castle active vent site. Many members of the Asgardarchaeota phylum including Lokiarchaeia were regarded as anaerobic fermentative heterotrophs involved in the sediment carbon cycle (Busi et al., 2021). Study had found that a representative strain of Lokiarchaeia (Candidatus Prometheoarchaeum syntrophicum MK-D1) from cold seep sediments grew syntrophically with a methanogen and sulfate-reducing bacteria and degraded amino acids and peptides (Imachi et al., 2020). Phylum Thermoplasmatota also dominated in archaeal communities, especially SG8-5 order. SG8-5 were likely to assimilate acetate for heterotrophy (Hu et al., 2021). Though Thermoplasmatota phylum is widely present in various ecosystems, its ecological functions and distribution in marine sediments remain elusive. Studies showed that other orders are host-associated or free-living methanogens (Methanomassiliicoccales) or might be involved in the degradation of organic matter in Marine sediments (Candidatus Yaplasmales) (Borrel et al., 2020; Zheng et al., 2022).” (on Page 19−20, Line 338−364)
Suggestion: Line 338: What is “Microbial reaction intensity”?
Response: We want to express that the aggregation of the bacteria such as Chloroflexi and Asgardarchaeota at some sediment layers may strengthen the corresponding reactions here, like organic matter degradation or anaerobic fermentation. To avoid misunderstanding, we will modify “microbial reaction intensity” to “microbial activity”.
Suggestion: Line339: If microbial communities consist predominantly of some major phyla (such as Halobacterota and Proteobacteria), this is not sufficient reason to call them “similar”. Phylum-level resolution is not adequate for such claims.
Response: Thanks for reviewer’s correction, the specific modification is as follows:
“Accordingly, the environmental similarity and the same quadrant distribution of the samples of ROV3 and ROV4 correspond to the fact that both sites were dominated by Proteobacteria and Halobacterota.” (on Page 20, Line 369−371)
Suggestion: Lines 345ff: these sections discussing the geochemical parameters of seep sites do not offer anything new. Methane, sulfate, Calcium DIC all are “relevant”, and of course it is known that sulfate is used as terminal electron acceptor of methane oxidation. The entire section reads like a laborious attempt to reinvent the wheel.
Response: We must admit that the content of these sections is some basic knowledge about cold seeps, as many studies have shown. However, we must clarify that we do not intend to use these geochemical data to illustrate new discoveries, but rather to demonstrate the differences in the impact of geochemical parameters on prokaryotes and discuss the possible intrinsic connections between the environment and microbial communities.
Suggestion: Line 381ff: These sections discussing microbial community assembly seem to be based on poorly-resolved taxonomic analyses, mostly phylum, class- and order-level. Is this sufficient to distinguish between deterministic vs stochastic processes in community assembly? Different percentages of homogenizing dispersal, undominated, heterogeneous and homogeneous selection, dispersal limitation and homogeneous selection are assigned to bacterial and archaeal communities, while the reader wonders how these statements (with 0.1% accuracy!) are possible.
The writing continues to be vague, for example in statements such as “The interaction of methane-related substances was an important factor that promotes microbial aggregation ” Is this a new way of saying that sulfate-dependent methane oxidation is performed by syntrophic consortia?
Response: The NTI and βNTI values in this section were both calculated based on OTU data to obtain the results of community assembly. It seems that there is no clear restriction on at which level random processes and deterministic processes should be discussed. Stegen et al (2012) (Stochastic and deterministic assembly processes in subsurface microbial communities) had analyzed based on the phylogenetic distance of OTUs; Måren et al (2018) (Changing contributions of stochastic and deterministic processes in community assembly over a successional gradient) discussed them at species level. Here, based on the previous chapter's explanation of the diversity of community microbial composition and the environmental influencing factors of diversity, we use the null model to infer the dominant ecological factors (homogeneous selection, heterogeneous selection, undominated, homogenizing dispersal, and dispersal limitation) affecting these communities.
For the accuracy of 0.1%, the explanation is that we had calculated the βMNTD between different layers of microorganisms at each site (a total of 21 groups) and obtained the βNTI values. According to the discrimination method in materials and methods, the dominant ecological factors corresponding to each group were matched respectively. Then we calculated the proportion of groups with the same ecological factors among all groups at each site. For example, there were 18 groups showed Homogeneous selections and 3 groups showed Homogenizing dispersal in archaeal data of ROV1, and it was calculated that the proportion of Homogeneous selection is 85.7%. The methods had been mentioned in the research of Stegen et al (2013) (Quantifying community assembly processes and identifying features that impose them) and Tripathi et al (2018) (Soil pH mediates the balance between stochastic and deterministic assembly of bacteria). We will further supplement in the materials and methods to make it clear to the readers.
The description of the sentence “The interaction of methane-related substances was an important factor that promotes microbial aggregation” was to once again emphasize the conclusion of the previous paragraph, indicating that methane and related ions had a significant impact on the selective aggregation and community composition of prokaryotic microorganisms in the Haima cold seeps. Here, the microorganisms do not merely refer to methanotrophic and thiotrophic symbionts, but also refer to the other members of the microbial community at each site, especially those with low abundance. The abundance of AOM-related microorganisms was indeed related to the intensity of methane seepage. The response mechanisms of rare microorganisms to different cold seep habitats and whether they have important ecological functions still require more research to further explore.
Suggestion: Line 450 ff: The discussion text moves to "species coexistence" while the archaean and bacterial groups that are discussed here are all named on the phylum level. Note that the figure shows "phylum-level microbial co-occurence (Fig 6). There is a big difference between phylum and species.
Response: Thanks for reviewer’s correction. The co-occurrence network figures were visualized based on OTU data, with each node representing an OTU. Therefore, these figures could illustrate the coexistence of archaea and bacterial species at four sites. To clarify the classification of each OTU, we colored the OTUs of the same phylum with the same color. We admit that the explanation of the figure. 6 was not clear enough. We must correct that "Phylum-level" refers to the fact that the network figures were colored based on Phylum level to identify of the connections between the phylum to which different OTUs belong.
The detailed modification is as follows:
“Fig. 6. co-occurrence patterns of operational taxonomic units (OTUs) at sites ROV1, ROV2, ROV3, and ROV4 in networks and links of topological parameters with sites; co-occurrence networks of (a) bacterial and (b) archaea communities at different sites. Different colors represent different phyla, lines represent connections between microbes, each node represents an OTU, and node sizes represent the proportion of OTUs of the phylum; network topological parameters (average degree, average clustering coefficient, average path length and modularity) of co-occurrence of (c) bacterial and (d) archaea communities at different sites.” (on Page 40, Line 807−814)
Citation: https://doi.org/10.5194/egusphere-2025-1372-AC1
-
AC1: 'Reply on RC1', Jingchun Feng, 31 Jul 2025
-
RC2: 'Comment on egusphere-2025-1372', Hongchen Jiang, 16 Jul 2025
This manuscript investigates the horizontal heterogeneity of prokaryotic communities in cold-seep sediments of the South China Sea and explores the driving mechanisms of community assembly and species coexistence. By integrating environmental parameters, microbial metabarcoding, and statistical modeling, the study reveals that methane concentration and other geochemical factors significantly influence community diversity and assembly processes. Key findings include: (1) stochastic processes dominate bacterial community assembly in methane seep sites (ROV1), while deterministic processes primarily shape archaeal communities; (2) bacterial and archaeal co-occurrence patterns exhibit contrasting niche differentiation across habitats; and (3) CH₄, Ba²⁺, and total inorganic carbon emerge as critical drivers of microbial distribution. This research advances our understanding of microbial biogeography in cold-seep ecosystems and highlights the pivotal role of environmental heterogeneity in shaping prokaryotic community structure. The findings hold significant implications for elucidating methane-cycling mechanisms and predicting ecosystem responses to environmental change.
Some suggestions and comments:
1) Lines 71–95: The comparative analysis of neutrality theory and niche theory is rather brief and does not clearly answer core questions such as whether random processes dominate the characteristics of high-productivity environments.
It is recommended to cite the latest theoretical models (e.g., Stegen et al., 2012), quantify the contribution rate of deterministic processes, and discuss their potential connection with cold seep fluid flux. Stegen, J. C. et al. (2012). Estimating and mapping ecological processes influencing microbial community assembly. Frontiers in Microbiology, 3, 370.
2) Lines 120-123: Only four sampling points (ROV1-ROV4) are set, and the actual distance between sampling points and geological background differences are not specified, which may limit the representativeness of the results.
It is recommended to supplement the sampling point distribution map and geological feature description, and increase the basis for supplementary sampling.
Citation: https://doi.org/10.5194/egusphere-2025-1372-RC2 -
AC2: 'Reply on RC2', Jingchun Feng, 31 Jul 2025
Reviewer #2:
This manuscript investigates the horizontal heterogeneity of prokaryotic communities in cold-seep sediments of the South China Sea and explores the driving mechanisms of community assembly and species coexistence. By integrating environmental parameters, microbial metabarcoding, and statistical modeling, the study reveals that methane concentration and other geochemical factors significantly influence community diversity and assembly processes. Key findings include: (1) stochastic processes dominate bacterial community assembly in methane seep sites (ROV1), while deterministic processes primarily shape archaeal communities; (2) bacterial and archaeal co-occurrence patterns exhibit contrasting niche differentiation across habitats; and (3) CH₄, Ba²⁺, and total inorganic carbon emerge as critical drivers of microbial distribution. This research advances our understanding of microbial biogeography in cold-seep ecosystems and highlights the pivotal role of environmental heterogeneity in shaping prokaryotic community structure. The findings hold significant implications for elucidating methane-cycling mechanisms and predicting ecosystem responses to environmental change.
Some suggestions and comments:
Suggestion: 1) Lines 71–95: The comparative analysis of neutrality theory and niche theory is rather brief and does not clearly answer core questions such as whether random processes dominate the characteristics of high-productivity environments.
It is recommended to cite the latest theoretical models (e.g., Stegen et al., 2012), quantify the contribution rate of deterministic processes, and discuss their potential connection with cold seep fluid flux. Stegen, J. C. et al. (2012). Estimating and mapping ecological processes influencing microbial community assembly. Frontiers in Microbiology, 3, 370.
Response: We have cited the research of Stegen et al (2011, 2015) to supplement the content, and point out that methane from cold seeps is an important selective force influencing microbial communities. The specific modification is as follows:
“Even within a system, the relative impacts of these ecological processes in different communities vary greatly (Stegen et al., 2015). In most communities, both stochastic and deterministic factors work together. For instance, increased environmental filtering and dispersal limitation lead to a decrease species richness (Stegen et al., 2011). Additionally, when interspecific competition is lower than intraspecific competition and leads to niche differentiation, species begin to coexist (Gravel et al., 2011). As a representative abiotic factor in cold seep environments, methane can be an important selective force for biological community.” (on Page 5−6, Line 77−85)
Suggestion: 2) Lines 120-123: Only four sampling points (ROV1-ROV4) are set, and the actual distance between sampling points and geological background differences are not specified, which may limit the representativeness of the results.
It is recommended to supplement the sampling point distribution map and geological feature description, and increase the basis for supplementary sampling.
Response: Thanks for the reviewer’s suggestions. We will supplement the sampling site location map and add the feature descriptions of the sampling sites. The specific modifications and location map are as follows:
“The four sampling sites were ROV1, ROV2, ROV3 and ROV4 respectively. Of the four sites, the ROV1 site was identified as a methane seep area with strong methane seepage. The ROV2, ROV3, and ROV4 sites were defined areas with faunas and methane seepage were weak, which were respectively colonized by abundant mussels, clams and sea anemones. The distance between the four sites was 4.6−12.2 km.” (on Page 9, Line 134−139)
Citation: https://doi.org/10.5194/egusphere-2025-1372-AC2
-
AC2: 'Reply on RC2', Jingchun Feng, 31 Jul 2025
Status: closed
-
RC1: 'Comment on egusphere-2025-1372', Anonymous Referee #1, 13 Apr 2025
This manuscript tries to show that bacterial and archaeal communities at seep sites are assembled based on different degrees of stochastic vs deterministic processes. The low degree of phylogenetic resolution for these communities (mostly phylum, class, and order) and the limited range of biogeochemical parameters (mostly sulfate, methane, DIC, Ca2+) calls into question whether the results are reliable; how these conclusions are reached remains unclear. Throughout the entire manuscript, the writing remains strangely opaque and contorted. Weirdly phrased half-truths and generalities about the microbial ecology of cold seep sites and methane oxidation suggest that these authors are not very familiar with this subject, as also suggested by the inadequate reference list that lacks foundational papers on cold seep ecology and microbial syntrophy by Boetius, Orphan, Knittel, Wegener, or Treude. This reviewer had the impression of reading a manuscript that is trying to reinvent the wheel on cold seep microbial ecology and trying at the same time to infer too much about community assembly, on the basis of inadequate data (routine 16S rRNA gene high-throughput sequencing and some biogeochemistry). Consulting some basic papers on microbial community assembly in marine seeps and sediments would be essential for reconsidering the scientific strategy, and for a fresh start (Ruff et al. 2015 Proc. Natl. Acad. Sci. USA 112:4015-4020; Starnawski et al. 2017, PNAS 114:2940-2945) However, this manuscript cannot be recommended for further consideration.
Details:
Line 22: methane filtration? Do you mean methane assimilation or oxidation?
Line 35: “coexisted more closely” reads like tight spatial association. You probably mean “coexisted in closer taxonomic associations”
Line 44-46: better: “In this type of seepage environment, methane is predominantly oxidized by anaerobic methanotrophic archaea in syntrophic association with sulfate-reducing bacteria (SRB)”
Line 46: Please reference the foundational microbiology papers for anaerobic methane oxidation by Antje Boetius, Katrin Knittel and others. Lin et al. 2022 did not discover anaerobic methane oxidation!
Line 54 ff: better: “Abundant benthic fauna such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate at the seep periphery where methane concentrations are decreasing. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).”
Line 65: what are “Alvin shrimp”? This should be a different species from Rimicaris exoculata, the common hydrothermal vent shrimp on the Mid-Atlantic Ridge.
Line 74: induce? You probably mean “influence” or “determine”
Line 84ff: “In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).“
Line 86 ff: The entire section is awkwardly written and needs to be rephrased for clarity. Try something like this: “At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains a methanotrophic and a methylophaga-related symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).”
Line 96 ff. In this introductory paragraph on microbial studies at cold seeps, the authors begin with a general statement on cold seeps although this paragraph focuses exclusively on work done at the Haima cold seeps. The paragraph should be rephrased to make this focus explicit.
Lines 173-174: please reference the vegan package with a literature or online citation. There are several “vegan” versions, please specify the one that you have actually used.
Line 232-236: The methane profiles were likely determined with whole sediment samples, but dissolved ions are most likely obtained from porewater, and need to specified as such in manuscript text and figure legends.
Line 238: “…and the areas with different macrofaunal assemblages…”
Line 240: microbially-mediated
Line 244: “… methane combined with Ca2+ …” does not form authigenic carbonates; DIC and Calcium do.
Line 246: “horizontal differences” sounds strange; you want to say that the geochemical gradients differ according to biogeochemical conditions at different Haima cold seep locations.
Line 256: better: “… revealed distinct profiles of microbial α-diversity in different sediments … Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites. “
Line 265: It is not the bacterial community that clusters in different quadrants; the PCoA analyses reveals that clustering patterns for the ROV1-4 communities are concentrated in the third, fourth, first, and first quadrants, respectively. Please rewrite accordingly.
Line 285ff: The taxonomic designations in the text are not genus level, but appear mostly family- and order level. If you want to use order-, family-, or genus-level designations for ANME-1 and ANME-2 bacteria, please consult Chadwick et al. 2022 (PLoS Biol. 2022 Jan 5;20(1):e3001508; doi:10.1371/journal.pbio.3001508) and Laso-Pérez et al 2023 (Nature Microbiol 8, 231–245; doi:10.1038/s41564-022-01297-4) for an updated taxonomy.
Line 261-302: The taxonomic analysis remains at a poorly resolved and physiologically ambiguous level (mostly Phylum); are there any attempts to interpret this diversity in terms of function?
Line 304. Physical distance is biogeographically relevant, but studies of seep habitats have shown a high degree of connectivity (for example, Meyer et al. 2013. Frontiers in Microbiology 4:207; doi:10.3389/fmic.2013.00207). For sampling sites that are only a few hundred meters or some kilometers apart, distance is irrelevant and only biogeochemical conditions separate microbial communities.
Line 315: what exactly is “Biomass energy” ? Also, is the community at these sites bacterivorous and does it use bacterial biomass and energy?
Line 319 ff: The entire section is inadequate; large bacteria groups are interpreted in terms of very specific capabilities that are correct only for specialized subgroups. For example, it is not true that the Desulfobacteraceae cooperate with the ANME archaea; very specific lineages of Desulfobacteraceae form syntrophic associations with (some) ANME archaea (Schreiber et al. 2010. Environ Microbiol. 12(8):2327-40. doi: 10.1111/j.1462-2920.2010.02275.x.). Some assertions are wildly speculative, for example Lokiarchaea as methane oxidizers and sulfate reducers (line 333) and Thermoplasmatota as free-ling methanogens (vasty overinterpreted; Line 334).
Line 338: What is “Microbial reaction intensity”?
Line339: If microbial communities consist predominantly of some major phyla (such as Halobacterota and Proteobacteria), this is not sufficient reason to call them “similar”. Phylum-level resolution is not adequate for such claims.
Lines 345ff: these sections discussing the geochemical parameters of seep sites do not offer anything new. Methane, sulfate, Calcium DIC all are “relevant”, and of course it is known that sulfate is used as terminal electron acceptor of methane oxidation. The entire section reads like a laborious attempt to reinvent the wheel.
Line 381ff: These sections discussing microbial community assembly seem to be based on poorly-resolved taxonomic analyses, mostly phylum, class- and order-level. Is this sufficient to distinguish between deterministic vs stochastic processes in community assembly? Different percentages of homogenizing dispersal, undominated, heterogeneous and homogeneous selection, dispersal limitation and homogeneous selection are assigned to bacterial and archaeal communities, while the reader wonders how these statements (with 0.1% accuracy!) are possible.
The writing continues to be vague, for example in statements such as “The interaction of methane-related substances was an important factor that promotes microbial aggregation ” Is this a new way of saying that sulfate-dependent methane oxidation is performed by syntrophic consortia?
Line 450 ff: The discussion text moves to "species coexistence" while the archaean and bacterial groups that are discussed here are all named on the phylum level. Note that the figure shows "phylum-level microbial co-occurence (Fig 6). There is a big difference between phylum and species.
In sum, this manuscript attempts a bioinformatics-based analysis of community assembly patterns where basic underpinnings and assumptions on how to measure community structure [esp. on taxonomic scale and resolution] are never clarified. The fairly limited spectrum of biogeochemical parameters is not discussed in its relevance to microbial physiology. Finally, the physiology and ecology of syntrophic methane oxidation at seeps is treated as an essentially unknown research field; however, plentiful good publications from over 25 years of research exist that should be studied before attempting a new start.
Citation: https://doi.org/10.5194/egusphere-2025-1372-RC1 -
AC1: 'Reply on RC1', Jingchun Feng, 31 Jul 2025
Reviewer #1:
This manuscript tries to show that bacterial and archaeal communities at seep sites are assembled based on different degrees of stochastic vs deterministic processes. The low degree of phylogenetic resolution for these communities (mostly phylum, class, and order) and the limited range of biogeochemical parameters (mostly sulfate, methane, DIC, Ca2+) calls into question whether the results are reliable; how these conclusions are reached remains unclear. Throughout the entire manuscript, the writing remains strangely opaque and contorted. Weirdly phrased half-truths and generalities about the microbial ecology of cold seep sites and methane oxidation suggest that these authors are not very familiar with this subject, as also suggested by the inadequate reference list that lacks foundational papers on cold seep ecology and microbial syntrophy by Boetius, Orphan, Knittel, Wegener, or Treude. This reviewer had the impression of reading a manuscript that is trying to reinvent the wheel on cold seep microbial ecology and trying at the same time to infer too much about community assembly, on the basis of inadequate data (routine 16S rRNA gene high-throughput sequencing and some biogeochemistry). Consulting some basic papers on microbial community assembly in marine seeps and sediments would be essential for reconsidering the scientific strategy, and for a fresh start (Ruff et al. 2015 Proc. Natl. Acad. Sci. USA 112:4015-4020; Starnawski et al. 2017, PNAS 114:2940-2945) However, this manuscript cannot be recommended for further consideration.
Response: The authors thanks for the reviewer’s detailed comments and corrections. We will seriously consider these suggestions and make explanations and improvements to the manuscript.
Details:
Suggestion: Line 22: methane filtration? Do you mean methane assimilation or oxidation?
Response: The methanotrophic communities in cold springs can filter out most of the methane and reduce its discharge into seawater, referred to as a microbial filter according to the description of Ruff et al (2019) in The ISME Journal, 1751-7362 (In situ development of a methanotrophic microbiome in deep-sea sediments). In cold seeps, the methane is mainly oxidized by ANMEs into CO2 rather than assimilated. To avoid ambiguity, we will change the methane filtration to methane oxidation:
“… in methane oxidation and the global geochemical cycle…” (on Page 3, Line 21)
Suggestion: Line 35: “coexisted more closely” reads like tight spatial association. You probably mean “coexisted in closer taxonomic associations”
Response: The modifications will be as follows:
“…bacterial network showed higher connectivity and species interactions in methane seep site than in other sites…” (on Page 3, Line 35−36)
Suggestion: Line 44-46: better: “In this type of seepage environment, methane is predominantly oxidized by anaerobic methanotrophic archaea in syntrophic association with sulfate-reducing bacteria (SRB)”
Response: Based on the reviewer’s suggestion and the description of Lin et al (2022), we will made the following modifications:
“In this type of seepage environment, sulfate-driven anaerobic oxidation of methane is the predominant process mediated by methanotrophic archaea and sulfate-reducing bacteria (SRB) (Knittel et al., 2009).” (on Page 4, Line 45−47)
Suggestion: Line 46: Please reference the foundational microbiology papers for anaerobic methane oxidation by Antje Boetius, Katrin Knittel and others. Lin et al. 2022 did not discover anaerobic methane oxidation!
Response: We had read the article of Knittel et al (2009) and updated the reference. The specific literature citations will be as follows:
“In this type of seepage environment, sulfate-driven anaerobic oxidation of methane is the predominant process mediated by methanotrophic archaea and sulfate-reducing bacteria (SRB) (Knittel et al., 2009).” (on Page 4, Line 45−47)
Suggestion: Line 54 ff: better: “Abundant benthic fauna such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate at the seep periphery where methane concentrations are decreasing. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).”
Response: Based on the comments of the reviewer and the research of Feng et al, the revisions will be as follows:
“Abundant benthic faunas such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate there when methane releasing weaken. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).” (on Page 4, Line 54−57)
Suggestion: Line 65: what are “Alvin shrimp”? This should be a different species from Rimicaris exoculata, the common hydrothermal vent shrimp on the Mid-Atlantic Ridge.
Response: According to the research of Li (2015) (Report on two deep-water caridean shrimp species (Crustacea: Decapoda: Caridea: Alvinocarididae, Acanthephyridae) from the northeastern South China Sea), it should be Alvinocaris longirostris Kikuchi & Ohta, 1995 (Alvinocarididae), a species related to the chemosynthetic environment discovered in the South China Sea, which was also observed in the research of Feng (2023b). To avoid misunderstandings, we will made the following corrections:
“…dense populations of mussels, clams, shrimps, and deep-sea crabs…” (on Page 5, Line 64)
Suggestion: Line 74: induce? You probably mean “influence” or “determine”
Response: We had reconsidered the word we used, the revision will be as follows:
“…and interspecific trade-offs determine community composition…” (on Page 5, Line 73)
Suggestion: Line 84ff: “In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).“
Response: The corrections will be as follows:
“In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation in sediments driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).” (on Page 6, Line 88−90)
Suggestion: Line 86 ff: The entire section is awkwardly written and needs to be rephrased for clarity. Try something like this: “At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains a methanotrophic and a methylophaga-related symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).”
Response: Thanks for reviewer’s detailed revisions and polishing. The specific revisions will be as follows:
“At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains methanotrophic and thiotrophic symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts of mussels support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).” (on Page 6, Line 90−99)
Suggestion: Line 96 ff. In this introductory paragraph on microbial studies at cold seeps, the authors begin with a general statement on cold seeps although this paragraph focuses exclusively on work done at the Haima cold seeps. The paragraph should be rephrased to make this focus explicit.
Response: Thank you for reviewer’s suggestions. We have reviewed the relevant research on Haima cold seep and refer the studies of Xu et al (2020), Niu et al (2017) Chen et al (2023) and others. We will rewrite this paragraph as follows:
“Haima cold seep is one of the two active seep sites in the South China Sea at present, and the other one is the F Site cold seep (Feng et al., 2018). At present, Haima cold seep has been regarded as a research hotspot. It was revealed that the ecological and geochemical gradients differed in distinct areas of the Haima cold seep, and the distribution of benthic faunas was associated with methane and sulfides (Xu et al., 2020). Although many studies about microorganisms had been conducted on Haima cold seep, these studies had mainly focused on the distribution and diversity of the microbial communities. It had been reported that there was a preference distribution of specific ANME at different depths in Haima cold seep sediments (Niu et al., 2017). It also revealed an interaction between environment condition and microorganisms, which might played an important role in the carbon and sulfur element cycles (Chen et al., 2023). Moreover, several studies on Haima cold seeps have shown that methane fluids promote microbial aggregation and evolution in sediments (Dong et al., 2023; Niu et al., 2017; Zhong et al., 2023). Environmental heterogeneity in different areas of Haima cold seep system affected the composition of microbial communities, which even was associated to mineral processes here (Liang et al., 2023). However, how horizontal environmental heterogeneity affects community assembly and species coexistence in different areas of Haima cold seep was still unclear.” (on Page 6−7, Line 100−117)
Suggestion: Lines 173-174: please reference the vegan package with a literature or online citation. There are several “vegan” versions, please specify the one that you have actually used.
Response: We have cited the literature of Dixon et al (2003) (VEGAN, a package of R functions for community ecology) on vegan package and marked the vegan version. The specific modifications are as follows:
“…were calculated using the “vegan” package (vers. 2.6−4) in R (Dixon, 2003), including…” (on Page 11, Line 189−190)
Suggestion: Line 232-236: The methane profiles were likely determined with whole sediment samples, but dissolved ions are most likely obtained from porewater, and need to specified as such in manuscript text and figure legends.
Response: The methane profiles were measured using sediment samples, and TIC, TOC and other ions were obtained from porewater extracted from sediments. We have made supplements and modifications in the text, and so have the legends of figure.1:
“The porewater was extracted from sediments by Rhizon samplers and kept at 4°C until analysis.” (on Page 9, Line 133−134)
“The Headspace Equilibrium method was used to measure CH4 concentrations in the sediments. Specifically, 5 g of sediment was transferred into a glass vial, and immediately added 5 mL of NaOH (5% w/w). Then the class vial was sealed with butyl rubber stopper and aluminum rolled sheet and shaken for 10 minutes to achieve methane equilibrium between the aqueous and gas phases. The CH4 concentrations of the samples were measured by gas chromatography (Trace 1300, Thermo Fisher, Waltham, MA, USA). Concentrations of TOC, TIC and SO42‒, Cl‒, Ba2+, K+, Ca2+, Mg2+, Fe3+, Cu2+, and Mn2+ in sediments were measured using the porewater extracted from the sediments.” (on Page 9−10, Line 142−150)
“…Specifically, concentrations of CH4 of sediments and concentrations of TIC, TOC, and SO42‒, Cl‒, Ba2+, K+, Ca2+, Mg2+, Fe3+, Cu2+, and Mn2+ of porewater from sediments were measured…” (on Page 36, Line 762−764)
Suggestion: Line 238: “…and the areas with different macrofaunal assemblages…”
Response: The following revisions have been made based on the reviewers' suggestions:
“…the methane seep area (ROV1) and the areas with different macrofaunal assemblages (ROV2, ROV3, and ROV4) …” (on Page 15, Line 258−260)
Suggestion: Line 240: microbially-mediated
Response: The specific modification is as follows:
“…methane drove the microbe-mediated anaerobic oxidation of methane…” (on Page 15, Line 261)
Suggestion: Line 244: “… methane combined with Ca2+ …” does not form authigenic carbonates; DIC and Calcium do.
Response: After reconfirmation, the correction is as following:
“…and dissolved bicarbonate combined with Ca2+ to form authigenic carbonates…” (on Page 15, Line 265−266)
Suggestion: Line 246: “horizontal differences” sounds strange; you want to say that the geochemical gradients differ according to biogeochemical conditions at different Haima cold seep locations.
Response: Based on this suggestion, we replaced the word and made the following specific modifications:
“In short, the geochemical discrepancies were shown according to biogeochemical conditions at different locations of Haima cold seep.” (on Page 15, Line 267−268)
Suggestion: Line 256: better: “… revealed distinct profiles of microbial α-diversity in different sediments … Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites. “
Response: Thanks for reviewer’s detailed suggestions, the specific paragraphs have been revised:
“… revealed distinct profiles of microbial α-diversity in different sediments. Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites.” (on Page 16, Line 277−280)
Suggestion: Line 265: It is not the bacterial community that clusters in different quadrants; the PCoA analyses reveals that clustering patterns for the ROV1-4 communities are concentrated in the third, fourth, first, and first quadrants, respectively. Please rewrite accordingly.
Response: Thanks for reviewer’s detailed suggestions, the specific modification is as follows:
“The PCoA analyses reveals that clustering patterns for the ROV1-4 bacterial and archaeal communities are concentrated in the third, fourth, first, and first quadrants, respectively.” (on Page 16, Line 283−285)
Suggestion: Line 285ff: The taxonomic designations in the text are not genus level, but appear mostly family- and order level. If you want to use order-, family-, or genus-level designations for ANME-1 and ANME-2 bacteria, please consult Chadwick et al. 2022 (PLoS Biol. 2022 Jan 5;20(1):e3001508; doi:10.1371/journal.pbio.3001508) and Laso-Pérez et al 2023 (Nature Microbiol 8, 231–245; doi:10.1038/s41564-022-01297-4) for an updated taxonomy.
Response: Thanks for reviewer’s corrections. After confirming the 16S rRNA sequencing data, we found that "ANME-1b" might be regarded as ANME-1b order and "ANME-2c" was regarded as ANME-2c family. The detailed taxonomic annotations are as follows:
“d__Archaea.p__Halobacterota.c__Methanosarcinia.o__Methanosarciniales.f__ANME.2c.g__ANME.2c; d__Archaea.p__Halobacterota.c__ANME.1.o__ANME.1b.f__ANME.1b.g__ANME.1b.”
Since these clades do not have explicit official names, their commonly used group names are displayed in the sequencing data. To date, ANME has not been isolated in pure culture. ANME-1 were proposed to be placed within their own order Methanophagales (The growing tree of Archaea: new perspectives on their diversity, evolution and ecology). ANME-1b is one of the three subclades of ANME-1, and there is no definite designation for ANME-1b yet. Chadwick et al (2022) proposed that the genus name Candidatus Methanogaster with family Methanogasteraceae for ANME-2c. Although the naming has been modified, ANME-2c is still the commonly used name. Therefore, we name them according to clear classification levels in sequencing data and provided supplementary information. The specific modifications are as follow:
“…ROV1 was dominated by ANME-1b and ANME-2c (genus Candidatus Methanogaster proposed) (Chadwick et al., 2022).” (on Page 17, Line 306−307)
“In addition, ANME-1a mainly inhabited ROV3 and ROV4.” according to order level taxonomy. (on Page 17, Line 308−309)
“Meanwhile, ANME-1b and ANME-2c existed in ROV1, while ANME-1a appeared in ROV4.” according to order, family and order level taxonomy, respectively. (on Page 18, Line 317−318)
Suggestion: Line 261-302: The taxonomic analysis remains at a poorly resolved and physiologically ambiguous level (mostly Phylum); are there any attempts to interpret this diversity in terms of function?
Response: In this section, we have introduced the phylum community composition of bacteria and archaea at each site and provided specific percentages, to illustrate the result that the microbial diversity at each site was different, both at the phylum level and genus level. The functions of these microbes had been discussed in the paragraphs later (on Page 19−20, Line 338−364).
Suggestion: Line 304. Physical distance is biogeographically relevant, but studies of seep habitats have shown a high degree of connectivity (for example, Meyer et al. 2013. Frontiers in Microbiology 4:207; doi:10.3389/fmicb.2013.00207). For sampling sites that are only a few hundred meters or some kilometers apart, distance is irrelevant and only biogeochemical conditions separate microbial communities.
Response: We studied the research of Meyer et al (2013). The main influencing factors of the microbial composition in hydrothermal vent and cold seeps may be different, namely temperature and methane. Methane is an important carbon source in cold seep systems. The methane concentration varies in different areas of a cold seep system, such as strong seepage areas, weak seepage areas, or no seepage areas, resulting in different abundances of methanotrophic microorganisms.
According to the study of Feng et al (2023a) (Tracing the Century-Long Evolution of Microplastics Deposition in a Cold Seep), the intensity of methane seepage varies indifferent cold seep successional periods, and the corresponding indicator faunas are different. As we have observed during sampling process, the Haima cold seep had a distinct ecological gradient, which was consistent with the study of Xu et al (2020) (Spatial distribution of seepages and associated biological communities within Haima cold seep field, South China Sea). The distances between different habitats there were very close. The four sites we had selected showed different methane concentrations and different dominant fauna aggregations, including mussels, clams and sea anemones, which indicate different biogeochemical conditions. To display more clearly, we have added a location map of the sampling sites in the supplementary materials.
Suggestion: Line 315: what exactly is “Biomass energy” ? Also, is the community at these sites bacterivorous and does it use bacterial biomass and energy?
Response: According to Field et al (2008), the term biomass energy can refer to any source of heat energy produced from non-fossil biological materials. (Biomass energy: the scale of the potential resource) Most seepage faunas rely on the organic matter converted from methane by methanotrophic and thiotrophic symbionts (Levin, 2005) (Ecology of cold seep sediments: Interactions of fauna with flow, chemistry and microbes). Here we would like to illustrate that microorganisms convert chemical energy such as methane into bioavailable energy, which supports the survival of faunas there. To avoid misunderstanding, we will modify “biomass energy” to “nutrition”.
Suggestion: Line 319 ff: The entire section is inadequate; large bacteria groups are interpreted in terms of very specific capabilities that are correct only for specialized subgroups. For example, it is not true that the Desulfobacteraceae cooperate with the ANME archaea; very specific lineages of Desulfobacteraceae form syntrophic associations with (some) ANME archaea (Schreiber et al. 2010. Environ Microbiol. 12(8):2327-40. doi: 10.1111/j.1462-2920.2010.02275.x.). Some assertions are wildly speculative, for example Lokiarchaea as methane oxidizers and sulfate reducers (line 333) and Thermoplasmatota as free-ling methanogens (vasty overinterpreted; Line 334).
Response: Thank you for the reviewer’s' corrections. We have revised this section based on the composition of the phylum level community and the LDA results. The analysis of the phylum level or order level depends on the most clearly distinguishable level in the data. The specific modifications are as follows:
“As the results showed, class Gammaproteobacteria of phylum Proteobacteria, were dominant member of ROV1, 3, and 4, which have been found frequently as a dominant bacterial taxon in deep sea sediments (Aoki et al., 2014). Desulfobacterota is a high abundant phylum in bacterial communities, and the SEEP-SRB2 genus is predominant at ROV1. It widely existed in cold seep habitats and involved in mussel symbionts with ANME-2c and mat symbionts with ANME-1 (Kleindienst et al., 2012). The relatively high methane content and biomarkers of ROV1 suggested that ANME-1b and ANME-2c might collaborate with SEEP-SRB2 for AOM. Chloroflexi was also an abundant phylum in bacterial communities and S085 and FW22 belonging to class Dehalococcoidia was the dominant member at ROV2. Studies had shown that some strains are associated with the organic degradation in marine sediments or organic dehalogenation (Löffler et al., 2015; Wasmund et al., 2014). Asgardarchaeota was an abundant phylum in archaeal community of ROV2, and class Lokiarchaeia was the main biomarkers. Lokiarchaeia (Spang et al., 2015) were first discovered in sediments near the Loki’s Castle active vent site. Many members of the Asgardarchaeota phylum including Lokiarchaeia were regarded as anaerobic fermentative heterotrophs involved in the sediment carbon cycle (Busi et al., 2021). Study had found that a representative strain of Lokiarchaeia (Candidatus Prometheoarchaeum syntrophicum MK-D1) from cold seep sediments grew syntrophically with a methanogen and sulfate-reducing bacteria and degraded amino acids and peptides (Imachi et al., 2020). Phylum Thermoplasmatota also dominated in archaeal communities, especially SG8-5 order. SG8-5 were likely to assimilate acetate for heterotrophy (Hu et al., 2021). Though Thermoplasmatota phylum is widely present in various ecosystems, its ecological functions and distribution in marine sediments remain elusive. Studies showed that other orders are host-associated or free-living methanogens (Methanomassiliicoccales) or might be involved in the degradation of organic matter in Marine sediments (Candidatus Yaplasmales) (Borrel et al., 2020; Zheng et al., 2022).” (on Page 19−20, Line 338−364)
Suggestion: Line 338: What is “Microbial reaction intensity”?
Response: We want to express that the aggregation of the bacteria such as Chloroflexi and Asgardarchaeota at some sediment layers may strengthen the corresponding reactions here, like organic matter degradation or anaerobic fermentation. To avoid misunderstanding, we will modify “microbial reaction intensity” to “microbial activity”.
Suggestion: Line339: If microbial communities consist predominantly of some major phyla (such as Halobacterota and Proteobacteria), this is not sufficient reason to call them “similar”. Phylum-level resolution is not adequate for such claims.
Response: Thanks for reviewer’s correction, the specific modification is as follows:
“Accordingly, the environmental similarity and the same quadrant distribution of the samples of ROV3 and ROV4 correspond to the fact that both sites were dominated by Proteobacteria and Halobacterota.” (on Page 20, Line 369−371)
Suggestion: Lines 345ff: these sections discussing the geochemical parameters of seep sites do not offer anything new. Methane, sulfate, Calcium DIC all are “relevant”, and of course it is known that sulfate is used as terminal electron acceptor of methane oxidation. The entire section reads like a laborious attempt to reinvent the wheel.
Response: We must admit that the content of these sections is some basic knowledge about cold seeps, as many studies have shown. However, we must clarify that we do not intend to use these geochemical data to illustrate new discoveries, but rather to demonstrate the differences in the impact of geochemical parameters on prokaryotes and discuss the possible intrinsic connections between the environment and microbial communities.
Suggestion: Line 381ff: These sections discussing microbial community assembly seem to be based on poorly-resolved taxonomic analyses, mostly phylum, class- and order-level. Is this sufficient to distinguish between deterministic vs stochastic processes in community assembly? Different percentages of homogenizing dispersal, undominated, heterogeneous and homogeneous selection, dispersal limitation and homogeneous selection are assigned to bacterial and archaeal communities, while the reader wonders how these statements (with 0.1% accuracy!) are possible.
The writing continues to be vague, for example in statements such as “The interaction of methane-related substances was an important factor that promotes microbial aggregation ” Is this a new way of saying that sulfate-dependent methane oxidation is performed by syntrophic consortia?
Response: The NTI and βNTI values in this section were both calculated based on OTU data to obtain the results of community assembly. It seems that there is no clear restriction on at which level random processes and deterministic processes should be discussed. Stegen et al (2012) (Stochastic and deterministic assembly processes in subsurface microbial communities) had analyzed based on the phylogenetic distance of OTUs; Måren et al (2018) (Changing contributions of stochastic and deterministic processes in community assembly over a successional gradient) discussed them at species level. Here, based on the previous chapter's explanation of the diversity of community microbial composition and the environmental influencing factors of diversity, we use the null model to infer the dominant ecological factors (homogeneous selection, heterogeneous selection, undominated, homogenizing dispersal, and dispersal limitation) affecting these communities.
For the accuracy of 0.1%, the explanation is that we had calculated the βMNTD between different layers of microorganisms at each site (a total of 21 groups) and obtained the βNTI values. According to the discrimination method in materials and methods, the dominant ecological factors corresponding to each group were matched respectively. Then we calculated the proportion of groups with the same ecological factors among all groups at each site. For example, there were 18 groups showed Homogeneous selections and 3 groups showed Homogenizing dispersal in archaeal data of ROV1, and it was calculated that the proportion of Homogeneous selection is 85.7%. The methods had been mentioned in the research of Stegen et al (2013) (Quantifying community assembly processes and identifying features that impose them) and Tripathi et al (2018) (Soil pH mediates the balance between stochastic and deterministic assembly of bacteria). We will further supplement in the materials and methods to make it clear to the readers.
The description of the sentence “The interaction of methane-related substances was an important factor that promotes microbial aggregation” was to once again emphasize the conclusion of the previous paragraph, indicating that methane and related ions had a significant impact on the selective aggregation and community composition of prokaryotic microorganisms in the Haima cold seeps. Here, the microorganisms do not merely refer to methanotrophic and thiotrophic symbionts, but also refer to the other members of the microbial community at each site, especially those with low abundance. The abundance of AOM-related microorganisms was indeed related to the intensity of methane seepage. The response mechanisms of rare microorganisms to different cold seep habitats and whether they have important ecological functions still require more research to further explore.
Suggestion: Line 450 ff: The discussion text moves to "species coexistence" while the archaean and bacterial groups that are discussed here are all named on the phylum level. Note that the figure shows "phylum-level microbial co-occurence (Fig 6). There is a big difference between phylum and species.
Response: Thanks for reviewer’s correction. The co-occurrence network figures were visualized based on OTU data, with each node representing an OTU. Therefore, these figures could illustrate the coexistence of archaea and bacterial species at four sites. To clarify the classification of each OTU, we colored the OTUs of the same phylum with the same color. We admit that the explanation of the figure. 6 was not clear enough. We must correct that "Phylum-level" refers to the fact that the network figures were colored based on Phylum level to identify of the connections between the phylum to which different OTUs belong.
The detailed modification is as follows:
“Fig. 6. co-occurrence patterns of operational taxonomic units (OTUs) at sites ROV1, ROV2, ROV3, and ROV4 in networks and links of topological parameters with sites; co-occurrence networks of (a) bacterial and (b) archaea communities at different sites. Different colors represent different phyla, lines represent connections between microbes, each node represents an OTU, and node sizes represent the proportion of OTUs of the phylum; network topological parameters (average degree, average clustering coefficient, average path length and modularity) of co-occurrence of (c) bacterial and (d) archaea communities at different sites.” (on Page 40, Line 807−814)
Citation: https://doi.org/10.5194/egusphere-2025-1372-AC1
-
AC1: 'Reply on RC1', Jingchun Feng, 31 Jul 2025
-
RC2: 'Comment on egusphere-2025-1372', Hongchen Jiang, 16 Jul 2025
This manuscript investigates the horizontal heterogeneity of prokaryotic communities in cold-seep sediments of the South China Sea and explores the driving mechanisms of community assembly and species coexistence. By integrating environmental parameters, microbial metabarcoding, and statistical modeling, the study reveals that methane concentration and other geochemical factors significantly influence community diversity and assembly processes. Key findings include: (1) stochastic processes dominate bacterial community assembly in methane seep sites (ROV1), while deterministic processes primarily shape archaeal communities; (2) bacterial and archaeal co-occurrence patterns exhibit contrasting niche differentiation across habitats; and (3) CH₄, Ba²⁺, and total inorganic carbon emerge as critical drivers of microbial distribution. This research advances our understanding of microbial biogeography in cold-seep ecosystems and highlights the pivotal role of environmental heterogeneity in shaping prokaryotic community structure. The findings hold significant implications for elucidating methane-cycling mechanisms and predicting ecosystem responses to environmental change.
Some suggestions and comments:
1) Lines 71–95: The comparative analysis of neutrality theory and niche theory is rather brief and does not clearly answer core questions such as whether random processes dominate the characteristics of high-productivity environments.
It is recommended to cite the latest theoretical models (e.g., Stegen et al., 2012), quantify the contribution rate of deterministic processes, and discuss their potential connection with cold seep fluid flux. Stegen, J. C. et al. (2012). Estimating and mapping ecological processes influencing microbial community assembly. Frontiers in Microbiology, 3, 370.
2) Lines 120-123: Only four sampling points (ROV1-ROV4) are set, and the actual distance between sampling points and geological background differences are not specified, which may limit the representativeness of the results.
It is recommended to supplement the sampling point distribution map and geological feature description, and increase the basis for supplementary sampling.
Citation: https://doi.org/10.5194/egusphere-2025-1372-RC2 -
AC2: 'Reply on RC2', Jingchun Feng, 31 Jul 2025
Reviewer #2:
This manuscript investigates the horizontal heterogeneity of prokaryotic communities in cold-seep sediments of the South China Sea and explores the driving mechanisms of community assembly and species coexistence. By integrating environmental parameters, microbial metabarcoding, and statistical modeling, the study reveals that methane concentration and other geochemical factors significantly influence community diversity and assembly processes. Key findings include: (1) stochastic processes dominate bacterial community assembly in methane seep sites (ROV1), while deterministic processes primarily shape archaeal communities; (2) bacterial and archaeal co-occurrence patterns exhibit contrasting niche differentiation across habitats; and (3) CH₄, Ba²⁺, and total inorganic carbon emerge as critical drivers of microbial distribution. This research advances our understanding of microbial biogeography in cold-seep ecosystems and highlights the pivotal role of environmental heterogeneity in shaping prokaryotic community structure. The findings hold significant implications for elucidating methane-cycling mechanisms and predicting ecosystem responses to environmental change.
Some suggestions and comments:
Suggestion: 1) Lines 71–95: The comparative analysis of neutrality theory and niche theory is rather brief and does not clearly answer core questions such as whether random processes dominate the characteristics of high-productivity environments.
It is recommended to cite the latest theoretical models (e.g., Stegen et al., 2012), quantify the contribution rate of deterministic processes, and discuss their potential connection with cold seep fluid flux. Stegen, J. C. et al. (2012). Estimating and mapping ecological processes influencing microbial community assembly. Frontiers in Microbiology, 3, 370.
Response: We have cited the research of Stegen et al (2011, 2015) to supplement the content, and point out that methane from cold seeps is an important selective force influencing microbial communities. The specific modification is as follows:
“Even within a system, the relative impacts of these ecological processes in different communities vary greatly (Stegen et al., 2015). In most communities, both stochastic and deterministic factors work together. For instance, increased environmental filtering and dispersal limitation lead to a decrease species richness (Stegen et al., 2011). Additionally, when interspecific competition is lower than intraspecific competition and leads to niche differentiation, species begin to coexist (Gravel et al., 2011). As a representative abiotic factor in cold seep environments, methane can be an important selective force for biological community.” (on Page 5−6, Line 77−85)
Suggestion: 2) Lines 120-123: Only four sampling points (ROV1-ROV4) are set, and the actual distance between sampling points and geological background differences are not specified, which may limit the representativeness of the results.
It is recommended to supplement the sampling point distribution map and geological feature description, and increase the basis for supplementary sampling.
Response: Thanks for the reviewer’s suggestions. We will supplement the sampling site location map and add the feature descriptions of the sampling sites. The specific modifications and location map are as follows:
“The four sampling sites were ROV1, ROV2, ROV3 and ROV4 respectively. Of the four sites, the ROV1 site was identified as a methane seep area with strong methane seepage. The ROV2, ROV3, and ROV4 sites were defined areas with faunas and methane seepage were weak, which were respectively colonized by abundant mussels, clams and sea anemones. The distance between the four sites was 4.6−12.2 km.” (on Page 9, Line 134−139)
Citation: https://doi.org/10.5194/egusphere-2025-1372-AC2
-
AC2: 'Reply on RC2', Jingchun Feng, 31 Jul 2025
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 275 | 63 | 16 | 354 | 22 | 12 | 27 |
- HTML: 275
- PDF: 63
- XML: 16
- Total: 354
- Supplement: 22
- BibTeX: 12
- EndNote: 27
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
This manuscript tries to show that bacterial and archaeal communities at seep sites are assembled based on different degrees of stochastic vs deterministic processes. The low degree of phylogenetic resolution for these communities (mostly phylum, class, and order) and the limited range of biogeochemical parameters (mostly sulfate, methane, DIC, Ca2+) calls into question whether the results are reliable; how these conclusions are reached remains unclear. Throughout the entire manuscript, the writing remains strangely opaque and contorted. Weirdly phrased half-truths and generalities about the microbial ecology of cold seep sites and methane oxidation suggest that these authors are not very familiar with this subject, as also suggested by the inadequate reference list that lacks foundational papers on cold seep ecology and microbial syntrophy by Boetius, Orphan, Knittel, Wegener, or Treude. This reviewer had the impression of reading a manuscript that is trying to reinvent the wheel on cold seep microbial ecology and trying at the same time to infer too much about community assembly, on the basis of inadequate data (routine 16S rRNA gene high-throughput sequencing and some biogeochemistry). Consulting some basic papers on microbial community assembly in marine seeps and sediments would be essential for reconsidering the scientific strategy, and for a fresh start (Ruff et al. 2015 Proc. Natl. Acad. Sci. USA 112:4015-4020; Starnawski et al. 2017, PNAS 114:2940-2945) However, this manuscript cannot be recommended for further consideration.
Details:
Line 22: methane filtration? Do you mean methane assimilation or oxidation?
Line 35: “coexisted more closely” reads like tight spatial association. You probably mean “coexisted in closer taxonomic associations”
Line 44-46: better: “In this type of seepage environment, methane is predominantly oxidized by anaerobic methanotrophic archaea in syntrophic association with sulfate-reducing bacteria (SRB)”
Line 46: Please reference the foundational microbiology papers for anaerobic methane oxidation by Antje Boetius, Katrin Knittel and others. Lin et al. 2022 did not discover anaerobic methane oxidation!
Line 54 ff: better: “Abundant benthic fauna such as deep-sea white clams and sea anemones appear at seep sites after initial microbial colonization, and mussels gradually dominate at the seep periphery where methane concentrations are decreasing. Carbonate rocks develop at older, late-stage seeps (Feng et al., 2023a).”
Line 65: what are “Alvin shrimp”? This should be a different species from Rimicaris exoculata, the common hydrothermal vent shrimp on the Mid-Atlantic Ridge.
Line 74: induce? You probably mean “influence” or “determine”
Line 84ff: “In the early stage of a cold seep, methane seepage predominates, and favors anaerobic methane oxidation driven by anaerobic methanotrophic archaea (ANME) and SRB (Cui et al., 2019).“
Line 86 ff: The entire section is awkwardly written and needs to be rephrased for clarity. Try something like this: “At later stages of seep development, bivalves with diverse chemosynthetic bacterial symbionts appear in seep habitats. For example, the deep-sea mussel Idas sp. contains a methanotrophic and a methylophaga-related symbiont, which collectively supported chemosynthesis (Duperron et al., 2008). Previous studies have shown that methanotrophic and thiotrophic symbionts support the carbon needs of their host already during early growth stages (Duperron et al., 2011). The species composition and abundance of symbionts may vary greatly within a host species at different sites, in consequence of different biogeochemical conditions (Duperron et al., 2007).”
Line 96 ff. In this introductory paragraph on microbial studies at cold seeps, the authors begin with a general statement on cold seeps although this paragraph focuses exclusively on work done at the Haima cold seeps. The paragraph should be rephrased to make this focus explicit.
Lines 173-174: please reference the vegan package with a literature or online citation. There are several “vegan” versions, please specify the one that you have actually used.
Line 232-236: The methane profiles were likely determined with whole sediment samples, but dissolved ions are most likely obtained from porewater, and need to specified as such in manuscript text and figure legends.
Line 238: “…and the areas with different macrofaunal assemblages…”
Line 240: microbially-mediated
Line 244: “… methane combined with Ca2+ …” does not form authigenic carbonates; DIC and Calcium do.
Line 246: “horizontal differences” sounds strange; you want to say that the geochemical gradients differ according to biogeochemical conditions at different Haima cold seep locations.
Line 256: better: “… revealed distinct profiles of microbial α-diversity in different sediments … Bacterial α-diversity peaked at the ROV2 site and had its lowest value at ROV1; archaeal α-diversity was the lowest at ROV1, and differed significantly from the other three sites. “
Line 265: It is not the bacterial community that clusters in different quadrants; the PCoA analyses reveals that clustering patterns for the ROV1-4 communities are concentrated in the third, fourth, first, and first quadrants, respectively. Please rewrite accordingly.
Line 285ff: The taxonomic designations in the text are not genus level, but appear mostly family- and order level. If you want to use order-, family-, or genus-level designations for ANME-1 and ANME-2 bacteria, please consult Chadwick et al. 2022 (PLoS Biol. 2022 Jan 5;20(1):e3001508; doi:10.1371/journal.pbio.3001508) and Laso-Pérez et al 2023 (Nature Microbiol 8, 231–245; doi:10.1038/s41564-022-01297-4) for an updated taxonomy.
Line 261-302: The taxonomic analysis remains at a poorly resolved and physiologically ambiguous level (mostly Phylum); are there any attempts to interpret this diversity in terms of function?
Line 304. Physical distance is biogeographically relevant, but studies of seep habitats have shown a high degree of connectivity (for example, Meyer et al. 2013. Frontiers in Microbiology 4:207; doi:10.3389/fmic.2013.00207). For sampling sites that are only a few hundred meters or some kilometers apart, distance is irrelevant and only biogeochemical conditions separate microbial communities.
Line 315: what exactly is “Biomass energy” ? Also, is the community at these sites bacterivorous and does it use bacterial biomass and energy?
Line 319 ff: The entire section is inadequate; large bacteria groups are interpreted in terms of very specific capabilities that are correct only for specialized subgroups. For example, it is not true that the Desulfobacteraceae cooperate with the ANME archaea; very specific lineages of Desulfobacteraceae form syntrophic associations with (some) ANME archaea (Schreiber et al. 2010. Environ Microbiol. 12(8):2327-40. doi: 10.1111/j.1462-2920.2010.02275.x.). Some assertions are wildly speculative, for example Lokiarchaea as methane oxidizers and sulfate reducers (line 333) and Thermoplasmatota as free-ling methanogens (vasty overinterpreted; Line 334).
Line 338: What is “Microbial reaction intensity”?
Line339: If microbial communities consist predominantly of some major phyla (such as Halobacterota and Proteobacteria), this is not sufficient reason to call them “similar”. Phylum-level resolution is not adequate for such claims.
Lines 345ff: these sections discussing the geochemical parameters of seep sites do not offer anything new. Methane, sulfate, Calcium DIC all are “relevant”, and of course it is known that sulfate is used as terminal electron acceptor of methane oxidation. The entire section reads like a laborious attempt to reinvent the wheel.
Line 381ff: These sections discussing microbial community assembly seem to be based on poorly-resolved taxonomic analyses, mostly phylum, class- and order-level. Is this sufficient to distinguish between deterministic vs stochastic processes in community assembly? Different percentages of homogenizing dispersal, undominated, heterogeneous and homogeneous selection, dispersal limitation and homogeneous selection are assigned to bacterial and archaeal communities, while the reader wonders how these statements (with 0.1% accuracy!) are possible.
The writing continues to be vague, for example in statements such as “The interaction of methane-related substances was an important factor that promotes microbial aggregation ” Is this a new way of saying that sulfate-dependent methane oxidation is performed by syntrophic consortia?
Line 450 ff: The discussion text moves to "species coexistence" while the archaean and bacterial groups that are discussed here are all named on the phylum level. Note that the figure shows "phylum-level microbial co-occurence (Fig 6). There is a big difference between phylum and species.
In sum, this manuscript attempts a bioinformatics-based analysis of community assembly patterns where basic underpinnings and assumptions on how to measure community structure [esp. on taxonomic scale and resolution] are never clarified. The fairly limited spectrum of biogeochemical parameters is not discussed in its relevance to microbial physiology. Finally, the physiology and ecology of syntrophic methane oxidation at seeps is treated as an essentially unknown research field; however, plentiful good publications from over 25 years of research exist that should be studied before attempting a new start.