A glimpse into the dark – the bacterial and archaeal diversity of tropical anchialine cave sediments

  1. Kenneth Meland
  2. Thomas M. Iliffe
  3. Stefan Thiele

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Abstract

Using ion torrent sequencing of the V4 region of the 16S rRNA gene, we investigated the sediment from four anchialine caves on Eleuthera (Bahamas). Anchialine caves are secluded, landlocked marine ecosystems, with high degrees of endemism. In the cave sediments, the bacterial and archaeal communities differ from marine communities and between caves, while often resembling those from low oxygen or anaerobic marine sediments. The waters of all caves investigated here were mostly saline and oxygenated, with Windermere Abyss differing from the other caves. This was mirrored by the bacterial and archaeal communities, with members of the wb1-A12 clade, Nitrosopumilaceae , members of the NB1-j phylum, and Actinomarinales being dominant in Preacher’s Blue Hole, Valentine’s Cave, and Bung Hole while Desulfatiglans , Bathyarchaeia , and members of the GIF3 and Sva0485 clades were dominant in Windermere Abyss. All communities showed taxa found in oxygenated and anoxic sediments, thus indicating a variety of chemoautotrophic lifestyles, including methane-, nitrogen- and sulphur cycling. The communities were different from other caves and included understudied or otherwise rare taxa, partially from deep sea sediments, implying that the isolation might be an evolutionary factor, and indicating that anchialine caves could be a window into the deep sea.

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  1. Version published to 10.1099/acmi.0.000622.v3 on Access Microbiology
  2. Access Microbiology

    Thank you for making the changes needed and addressing the reviewers comments thoroughly- it made me job nice and easy. Congratulations on a very nice short report on these understudied and difficult to access sites.

  3. Version published to 10.1099/acmi.0.000622.v2 on Access Microbiology
  4. Access Microbiology

    Both reviewers agree that your work is scientifically sound and of interest and importance to the field. Reviewer 2 has flagged several points which need some modification or clarification. Please can you address these.

  5. Access Microbiology

    Comments to Author

    This very interesting paper gave a glimpse into the microbial sediment communities of anchialine caves. Generally, cave sediments are often overlooked, despite anchialine caves being found worldwide; thus, microbial diversity within cave sediments is an important study area. However, there is a key weakness of this study, which is, as the authors pointed out, the low number of samples available for analysis. Despite this weakness, I believe this is a significant work that adds to our understanding of anchialine cave diversity; therefore, I support its publication in Access Microbiology. You can find my minor comments below. Title "Bacterial and archaeal diversity" - You mention both bacteria and archaea in the title but you don't differentiate between the two in the text. You could separate the two diversities and explain their taxa separately to better reflect your title. Lines 33-34 "implying that the isolation might be an evolutionary factor" - This reads like the microbial evolution caused them to be isolated instead of what actually happens which is that isolation can cause microbial evolution. Needs rephrasing. Line 46 "high degrees of endemism" - needs expanding Line 46-48 - This sentence is true for all ecosystems not only anchialine caves, please rephrase. Line 52-53 - The studies on the cave water column showed communities that differed from those found in marine sediments. Is there a reason why this is surprising? Was it expected they would be similar? Line 53 - The water column communities weren't like marine sediments but were similar to low oxygen/anaerobic communities in marine environments - so were they similar to marine waters? Coastal sites? Deep-sea environments? This needs to be more specific. Line 59-61 - Here you're introducing the aim/hypothesis of the study, you need to be more specific. It's confusing because you go from the water communities (lines 49-54) to sediments (lines 55-59) and back to the water column in line 60. Needs reshuffling. Line 70 - "Falcon tubes". Falcon is the company, just say 50 mL (conical) centrifuge tubes. Then you can provide the company name and the country in brackets. Line 72 - Out of curiosity, why were the water profiles measured diagonally? Why not take the measurements from the middle of the cave or directly above the sediment sampling site? Line 74 - Does the "single samples" mean only one sediment tube per location/depth? Lines 80-81 - "Samples were taken on the slope and at the bottom of the cave" I'm assuming the depths for these are at 25m and 30m respectively as in your figure - It would be clearer if depth is mentioned here. Lines 92-93 - If I understand correctly, one tube of sediment was collected per sampling point - was the sediment within the tube then homogenised? Then DNA was extracted once per sample and amplified three times? Lines 95-96 - What reagents did you use for the amplification? This information is required for replication purposes. Lines 106 -107 - "Silva SSU Ref NR v138" This is normally clustered at 99%. Was this the case for the one you used? Please add more details. Lines 108-109 - removal of samples with less than 10000 sequences seems extreme. Could you please explain why this was chosen? Or do you mean less than 10 sequences? Line 109 - ML tree. You mention it but it's not present anyway. Can you add it to supplement or justify why it's not included? Line 126 - and everywhere else were "rel. abundance" is mentioned. Please write it in full or don't write it at all. In this instance you could add in the sentence in line 142 that the relative abundance was calculated and then remove it from the parenthesis. Line 133 - "found at both depth" should be "depths" Line 133 and below - Why does the relative abundance not have standard error anymore? Did you not sequence all your technical replicates? Line 193 - Please change 7.0 to "7.0%" Lines 148-149 - "similar physicochemical parameters" Valentine's hole has a lower salinity (30 instead of 34 PSU) and Bung Hole has considerably lower dissolved oxygen than the other two and is actually very close to the Windermere Abyss concentrations. Can you please expand on how they are similar in the results section? Line 153 - "difference in the water column indicate". This should be "could indicate" Line 158 - You use Proteobacteria to say the communities "hint towards oxygenated conditions". Proteobacteria are too varied a phylum to hint at anything. Similarly Planctomycetota have members who anaerobically oxidise ammonia etc. I think this sentence needs reconsidering/rephrasing. Line 162 - "Chloroflexi". So in Windermere Abyss Chloroflexi suggest low oxygen or anoxic conditions but in Valentine's Cave, Bung Hole and Preacher's Blue they suggest oxygenated conditions? Like my previous comment, I think this needs reconsidering/rephrasing. Line 170 - "Actinomarinales". If they're normally found in the water columns, what could they be doing in the sediments? Please expand. Line 183 - "managed to cross the freshwater-salinity barrier". What does this mean? That they can survive in both low and high salinities? This needs to be rephrased. Line 190 - "dihydrogen-sulfite". Just write "hydrogen sulfide" as mentioned in your methods section on line 89. Also did you measure this yourself since you don't reference it on lines 89-90? Lines 192-193 - "the Eleuthera caves resemble to some extent the microbiomes of Blackwood Sinkhole". You only make this comparison in the text when discussing Windermere Abyss and you've also said the microbial communities in Windermere Abyss are different to the other three sampling sites. Please expand here. Which classes were found to be similar? Lines 194-196 - "most striking difference". You mention that the absence of Campylobacteria is striking but you give no suggestions why this could be happening. Was that the only taxon that did not exist between the caves? Also, does not having Campylobacteria really indicate that the cave ecosystems "differ significantly"? This section needs expanding and/or rephrasing. Lines 201-203 - This sentence needs rephrasing because it implies that discovering taxa usually found in deep-sea sediments causes changes in the cave microbiomes and their evolution. You discovering their existence doesn't cause any changes. Line 205 - "tropic to polar bacteria" The tropic vs polar comparison hasn't been mentioned before this point. This needs to be mentioned further in the text if you want to add it in your conclusions. Lines 207-208 - "differ significantly from other cave ecosystems". How do they differ significantly from other cave ecosystems? This needs expanding in the discussion because in lines 175-177 you mention Rokubacteriales and how they're found in karst cave systems Lines 210-211 - "historic ocean" and "window into the past". How do your data provide a window into the past? This feels contradictory to line 203 where you mention anchialine caves could reflect deep-sea communities. This needs rephrasing. Am I correct in assuming you mean that the communities in your caves could show what the deep-sea communities were like before they evolved to thrive in those environments? Is that what you mean by "window in the past"? Line 366 - Figure 1 legend "different samples on Phylum level". This should be "at Phylum level" Figure 1A - Y-axis label change "Rel. abundance" to "Relative abundance" and all writing would benefit from being larger. Also in x-axis it says "Valentin's Cave" instead of "Valentine's Cave" Figure 1B - Have you run any stats on these to check they are actually (not) different between caves or only what CAP showed? Is anything differentially abundant?

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    The anchialine caves are one of the most difficult habitats to study. Selecting samples from such caves for a microbiological comparative approach is of high importance for knowledge in general. The results are correctly analyzed and the discussion is scientifically sound and all the aspects linked to microorganisms from related habitats is discussed. I propose the publication of this manuscript as it is.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Version published to 10.1099/acmi.0.000622.v1 on Access Microbiology