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.
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RC1: 'Comment on egusphere-2025-1372', Anonymous Referee #1, 13 Apr 2025
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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
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