Interactive comment on “ Microbial assemblages on a cold-water coral mound at the SE Rockall Bank ( NE Atlantic ) : interactions with hydrography and topography ” by J . D . L . van Bleijswijk

Abstract. This study characterizes the microbial community composition over Haas Mound, one of the most prominent cold-water coral mounds of the Logachev Mound province (Rockall Bank, NE Atlantic). We outline patterns of distribution vertically – from the seafloor to the water column – and laterally – across the mound – and couple these to mound topography and hydrography. Samples of water, sediment and Lophelia pertusa were collected in 2012 and 2013 from locations that were chosen based on high definition video surveys. Temperature and current measurements were obtained at two sites at the summit and foot of Haas Mound to study near-bed hydrodynamic conditions. Overlaying water was collected from depths of 400 m as well as 5 and 10 m above the bottom using a CTD/Rosette system. Near-bottom water, sediment and L. pertusa mucus and skeleton samples were obtained with a box corer. Of all these biotopes, Roche GS-FLX amplicon sequencing targeting both Bacteria and Archaea was carried out, augmenting our understanding of deep sea microbial consortia. The pattern of similarities between samples, visualized by multi-dimensional scaling (MDS), indicates a strong link between the distribution of microbes and the specific biotopes. The microbial operational taxonomic unit (OTU) diversity was the highest in near-bottom water, which was sampled in the coral framework. For the first time, Thaumarchaeota marine group I (MGI) were found in L. pertusa mucus; Endozoicomonas was detected in skeleton, mucus and near-bottom water, whereas Mycoplasma was only detected in skeleton and near-bottom water, however not in mucus. Analysis of similarities (ANOSIM) indicates that overlaying water is well-mixed at 400 m depth but less so at 5 and 10 m above the bottom, where the composition of microbial communities differed significantly between summit, slope and off-mound. At all locations, the near-bottom water differed significantly from water at 5 m above the bottom, illustrating that the near-bottom water in between the coral framework represents a separate microbial habitat. Furthermore, the observed spatial heterogeneity in microbial communities is discussed in relation to environmental conditions.

been done here, then the data would contain pseudoreplicated units.Performing multiple PCRs on a single sample should be a step when preparing for 454 sequencing.At the very least, conducting replicate PCRs provides evidence that DNA template is actually present and will amplify using the chosen primers.However, treating pseudoreplicated units as replicate units-as appears to have been done here-will violate key statistical assumption of independence of samples.I would suggest the authors conduct their analysis on only one replicate per sample, which would appear to reduce their N from 126 to 42.
Response: We pooled the methodological replicates for all biotopes, resulting in 40 samples.
Is the description of experiments and calculations sufficiently complete and precise to allow their reproduction by fellow scientists (traceability of results)?I do not think it would be possible for others to reproduce this work given the manuscript's present format.For example, there appears to be either a miscommunication or misunderstanding about the next generation sequencing (NGS) platform used.The author's cite a "Roche 454 GS-FLX Titanium sequencer."This instrument should be referred to as the "Roche GS-FLX Sequencer using Titanium Chemistry."Regardless, the authors describe sending 7 pooled samples to Macrogen for sequencing using the above NGS platform on "1/8 lane each."To the best of my knowledge, the GS-FLX instrument uses a picotitre plate.DNA capture beads containing sequence template-DNA amplified via emulsion PCR-are flowed over the plate and captured in nano-sized wells.Sequencing of the DNA template library, therefore, occurs within individual wells.There are millions of wells per plate allowing for multiplexing different tagged samples on a single plate.Illumina platforms, such as the HiSeq, use lanes.It would be helpful if the authors would rectify this apparent conflict.
Response: We agree that there were some sloppy and incorrect descriptions in the former version of the manuscript.We revised the methods section.

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Do the authors give proper credit to related work and clearly indicate their own new/original contribution?Archaea have previously been reported in association with L. pertusa by Emblem et al. (2012).It may behoove the authors to conduct a more thorough literature review before making claims of first-discovery.However, it could be that the authors are âAËŸTinË G factâAËŸT the first to report Archaea in association with L. pertusa growing on a carbonate Ë G mound in the Logachev Mound Province.
Response: We now give deserved credit to Emblem et al. and changed our text.To our knowledge and this of another anonymous reviewer, it was the first time that Archaea were found in mucus of L. pertusa.
Is the overall presentation well-structured and clear?No. Please see comments and suggested edits in the supplemental pdf provided.Most of these suggestions are copyedits and can easily be included if accepted.Doing so may strengthen the overall presentation and clarity of this manuscript.However, there are other potential issues that may require the authors to re-analyze the entire data set (i.e., pseudoreplication caused by the inclusion of triplicate PCRs in the sample set).
Response: we re-analysed the data and revised the figures and manuscript (see pdf supplement).
Is the language fluent and precise?Fluency has been demonstrated but there are numerous grammatical errors and a recurring theme of imprecision.The English language is inherently ambiguous.Sadly, this means great attention must be paid to word selection and grammar to ensure statements of objectives, methods, and conclusions cannot be misinterpreted or misunderstood.Though the authors demonstrate good command of the English language, it is recommended they revisit the entire text to ensure the appropriate use of punctuation, grammar, verb tense, and paragraph cohesion.Some suggestions have been provided in the supplement pdf.
Response: We revised the entire text and have accepted most suggestions provided in the supplement.
Are mathematical formulae, symbols, abbreviations, and units correctly defined and used?There are numerous instances where units of measurement appear to be missing.These should be included when and wherever appropriate.Additional issues exist whereby the authors do not introduce abbreviations in the body of the text despite their use in Tables and Figures (e.g., near-bottom water = w_bc in Table 1; water column above the mound = w_CTD in Table 3).There are also some general inconsistencies throughout the text with regard to the use of abbreviations.For example, in the Abstract the term "5 + 10 m above bottom (ab)" is used.Later in the text this is written as "5 and 10 m ab."It is recommended that the "+" be replaced with "and" throughout the text and that the abbreviation "ab" not be used in the abstract.Generally, acronyms should not be used in the abstract unless the term is to be used frequently.
Response: We deleted acronyms from the abstract and used more consistent descriptions for sample categories.
Should any parts of the paper (text, formulae, figures, tables) be clarified, reduced, combined, or eliminated?Figures 6 and 7 are very difficult to interpret due to their present size and quantity of information.It would be helpful to readers if these charts would be enlarged so each one occupies a single page.
Response: Figures 6 and 7 are enlarged and revised to make them well readable.
Detailed responses to comments from the supplement file P1510: Is it appropriate to propose a hypothesis in the abstract?Further, this does not seem to be one of the overall conclusions derived from your present work.You might consider removing this statement from the Abstract and relocating it to your Discussion/Conclusions.