Variability and Community Composition of Marine Unicellular Eukaryote Assemblages in a Eutrophic Mediterranean Urban Coastal Area with Marked Plankton Blooms and Red Tides
, Natassa Stefanidou 2, Maria Moustaka-Gouni 2, Ulrich Sommer 3 and George Tsipas 1
Round 1
Reviewer 1 Report
It is very hard to give comments back on a manuscript without line numbers, so please excuse the “vague” feedback.
This project collected weekly samples from a single station and spatial sampling from an additional three locations, collected monthly, to address community shifts during red tides and plankton blooms with an attempt to correlate these communities to environmental shifts.
The manuscript is well written and conclusions are well argued for. There is a lot of data in this paper that is clearly laid out, however I have some comments that need to be addressed:
Abstract:
Middle ish in the abstract: Metabarcoding is not the same as metagenomics. Metagenomics is most often referred to when sequencing DNA that has been fragmented and not amplified with a primer. i.e. not using specific assays, but rather random locations on the genome.
Introduction:
End of the first paragraph there is a “with” too much. “produced provide with…” delete the with here.
Materials and methods:
L 2. You should mention immediately which station had samples collected from weekly.
L 20 ish. Were there no replications of the samples?
L 20 ish. The samples should ideally have been kept at -80 instead of -20, how long were the samples stored in the -20 freezer?
Fig 1: This figure is huge, but I am sure those will be made smaller when published.
Section 2.3: How were the PCR products purified?
2.4: When saying normalized do you mean rarefied?
2.4: The authors should consider not rarifying the data and instead follow the recommendations from this paper: Waster not, Want not: Why rarefying microbiome data is inadmissible. Written by Paul McMurdie and Susan Holmes.
This could result in completely different results, which would be worth exploring. The authors should at least check if the results are significantly different. I am imagining this to significantly show different results for fig. 2.
2.5: I am not exactly sure how the network analysis was done. Could you explain this in more detail in the methods? It sounds to me that you did a correlation analysis and not necessarily a co-correlation analysis? Not sure if this is referred to as a network analysis. What I think of when referring to a network analysis I think of something similar to this:
https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/
Results:
3.1: End of paragraph, what are the significant differences based on? Average differences over the year? I am not so sure if it makes sense to compare the stations this way. Also, it would be good to have this as a figure in the supplementary material. Maybe a line plot of the different environmental variables over time with the average?
Fig. 2: The color scheme on this figure is not distinguishable. Change them. There is a strange legend under the legend. I believe this to be the legend of figure 2b, but they should both be written in the same spot. It looks like legend Fig 2b is written in the text (larger font size). Have you thought about adding the Bray-Curtis or some sort of community shift measurement into this figure? Not exactly sure how to do this, but could be an interesting measure to look at that would actually give you a measure for the community at one time point compared to the next. +-
Fig 3: Why are the barplots not all the way up to 100%? Here is also the same problem with the legend as fig 2.
Fig 4: This figure is not legible. The leafs of the tree should ideally be colored or the labels should be colored. Colors could be according to season, fig 5, or other variable that makes sense. IT could also be useful to draw boxes around the leafs that cluster together, to highlight the clusters.
Fig. 6: Why are the figures different sizes? And why are there not 5 figures similarly to fig 1 and 2? It is quite difficult to do a CCA with such few replicates, in some cases you don’t even have three per season. It might be more meaningful to leave this analysis only for WT.
Discussion:
What is MALV and MAST? Explain first use of abbreviations.
I am imagining the discussion to change a bit depending on what the reviewers decide to do with the data. I would be happy to review this again.
Conclusion:
Is the temperature and salinity not a function of a changing water mass, fresh water input, or something similar? I have a feeling that the red tide is not caused by the salinity or temperature but rather something that is a function of that.
Author Response
Dear Reviewer,
We would like to thank you for your positive disposition towards our work. All the comments and suggestions are considered and respective changes have been made in the revised text, accordingly. All changes are highlighted in red to facilitate reading. Please see the attached word file for a response on your comments and suggestions.
Sincerely on behalf of all the authors,
Dr. Savvas Genitsaris| Academic Associate
International Hellenic University
T +30-2310-807538
Email: [email protected]
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript describes the temporal and spatial changes of protist diversity in a particular urban marine environment (Thessaloniki Bay) over one year using a weekly and/or monthly sampling strategy and high-throughput sequencing (Illumina MiSeq). This study is actually a perfect complement of a previous paper from (globally) the same authors on the same ecosystems, published in Diversity (Genitsaris et al., 2019, doi:10.3390/d11080136), where microscopic analysis of the eukaryotic plankton was performed. In the present study using amplicon sequencing, the authors highlight that the microbial eukaryote community was diverse (including species newly detected in this environment), different between the four studied areas and characterized by seasonal patterns likely due to environmental factors and biotic interactions. The authors identify the salinity and temperature as main environmental factors that explain the variation in protist community structure observed in such coastal ecosystem.
Overall, the manuscript is relatively well written and provides novel information regarding the protist diversity in coastal ecosystems. The extent of the sequencing and sequence processing seems fine, and this could eventually be a substantive dataset that will add to the growing global database of protist sequence information as it relates to species richness and diversity. Also, most of the current studies on the spatial variation of protist diversity have considered relative distant environments and the choice of authors of focusing on four adjacent sampling sites can substantively help to better understand the factors that determine the community structure of such microorganisms.
Having said that, I yet have some remarks/concerns on the proposed work that I believe could help the authors to significantly boosted their manuscript. I indeed think that they have given too much importance in their manuscript on findings that are not completely new for protist ecologists and not enough on novel aspects that they have raised and should reconsider to emphasize more. It is now fully admitted that HTS -and molecular approaches in general- provide deeper insights in the microbial diversity by revealing the presence of species that were not reported before by classical approach. Moreover, the seasonality of the microbial eukaryote communities -and its phytoplanktonic component- in the euphotic layer of aquatic environments is well known and has been previously described both using microscopical and molecular approaches. It is also relatively well known that water temperature and salinity have an impact on the protist community structure.
On the other hand, there is several aspects that the authors have begun to tackle in their paper that could be deeply investigated in my opinion. Indeed, in the introduction, they notably gave a legitimate importance on the functional diversity in the protist community (highlighting that underexplored function were retrieved among this community using HTS tools) and I was surprised that this aspect wasn’t investigated in their study. Indeed, it would have been interesting to also depict the protist community associated to HABs and red tides in the scope of their functional diversity to see if these events promote certain functional groups when they occurred. For instance, answers regarding the eukaryotic parasitism and predation that may potentially control HABs and red tides events could be given (Who are they? Are they “stimulate” or inhibit” by the different phytopanktonic events described here? ...). I believe that the extent and time frequency of samples that the authors have is perfectly for it. In my view, this kind of analysis will consequently boost their study.
My second remark/concern is regarding the network analysis that the authors have done which may be actually considered to highlight the point I raise above regarding the functional diversity of the community. The authors used the MIC-MINE to investigate protist interactions in their network approach and I wonder if this is the most adapted to their samples. Indeed, since the authors have time series data, I thought that a cooccurrence analysis such as eLSA (Xia et al., 2011, doi: 10.186/1752-0509-5-S2-S15) would have been more adapted to their data set. There was a very interesting review by Weiss et al. (2016, [10.1038/ismej.2015.235]) on the suitability of different correlation techniques for network analyses of microbial community data. Can the authors justify why MIC-MINE might be the most adapted to their data set? Furthermore, in their study, the authors did a network analysis after pairwise comparisons of all the samples but it would have been also interesting to use the same approach to explore the relationships among the dominant taxa of eukaryotic plankton (pairwise comparisons of OTUs) as it is usually done with such cooccurrence analysis. Do the authors have performed such analysis? In that case, cooccurrence analysis that take into account time-delay would have been interesting to highlight microbial association such competition or proto-cooperation. Similarly, in the same analysis, relationship between the dominant taxa and environmental variables could be done and will be a good complement to the CCA that shown this relationship at the protist community level.
Finally, I would recommend the authors to clearly said in the introduction that this study follows a previous work from the same authors already published where the different periods of blooms were identified and analysis of the environmental variables were done. In my view, this is not clearly stated in the introduction and may lead to confusion. Thus, the authors could focus this manuscript on the novel data and analysis they made and may shorten as much as they can any data or analysis that have been previously done in the above-mentioned paper (for instance, paragraphs in line with the environmental variables, see comments below). On the other hand, I think that the different bloom and non-bloom periods that occurred in their sampling area are not enough identified in this study. I would recommend the authors to clearly show these periods in the different figures/tables for helping any readers that haven’t actually red their previous work.
Lastly, I would have really appreciated that the authors provide a document containing line numbers in order to facilitate the review process. This make difficult the referee’s works, notably for pointing out specific part of the text that should be revised by the authors. I strongly recommend the authors to provide such document in the future review process steps. Below, I will then cite the page number and the beginning and end of sentences/paragraphs to which my specific comments are referring to.
-Abstract-
P1 - “...the known and unexplored diversity...”: The combined use of the two adjectives “known” and “unexplored” is for me contradictory. In other words, if a diversity/environment is truly unexplored, nothing is by definition known from that system. Eventually, the use of “underexplored diversity” could be a more adapted adjective and it might be what the authors wanted to say here. If not, I yet don’t think it is not necessary to describe how is the studied diversity and the authors could simply say instead that they have studied the taxonomic diversity.
P1 - “In almost all samplings, ...”: Replace “In almost all samplings, ...” by either “in almost all sampling waters/stations/points, ...” or “in almost all samples, ...”.
P1 - “The metagenomic analysis ...”: Apart if I missed it, I don’t think that the authors present any metagenomic sequencing data sets in that manuscript but rather amplicon-based sequencing datasets. Please clarify and correct. Furthermore, in that sentence, I would suggest to say that the analysis “has detected” rather than “detected” the known unicellular groups of the Bay.
P1 - “Furthermore, OTUs closely related to known harmful species ...”: Can the authors clarify what they meant by “low numbers”? Do they mean “in low abundance” (number of reads) or “in low richness” (number of OTUs)? In both cases, I don’t think that the conclusion made by the authors is then accurate but rather contradictory. Indeed, it suggests that the OTUs related to harmful taxa are either in low abundance and/or are represented by a few numbers of species, suggesting then that most of the eukaryotic biodiversity is not due to the presence of such species of importance for human health by most likely by other eukaryotic taxa with potentially no negative impacts on the ecosystem. Consequently, the part of the eukaryotic diversity due to species with potentially negative impacts on human health is relatively low and less important that we could primarily suggests based on the type of samples analyzed (i.e., red tides and phytoplanktonic blooms). If so, this constitutes an interesting point that the authors may emphasized in the abstract.
- 1. Introduction-
P1 - “During the past decade...”: “Insight” could be in plural as the authors listed several aspects where new insights were obtained on the unicellular eukaryotes. Furthermore, I would suggest to delete “unicellular eukaryotic” in “into the unicellular eukaryotic diversity” since the authors already specify at the end of the same sentence to what the different aspects listed are referring to.
P1 - “Furthermore, previously undiscovered or underestimated trophic strategies ...”: Can the authors clarify and cite as example what are these previously underestimated trophic strategies that are now central to marine ecosystem functioning? Overall, I thought this sentence unclear and I don’t really understand what the authors want to point out here, especially the end of this sentence with the use of the subordinating conjunction “while” that suggest the study of the environmental effects and biotic relationships are not linked with the “new trophic strategies” which I believe is not true. Can you rephrase and clarify your thought here?
P2 - “It is evident that the plethora…”: Delete “with”.
P2 - “Thus, they have received the attention of marine microbiologists using HTS tools”: The authors can actually emphasize here that there is an increase in the attention of marine microbiologists thanks to the development of HTS tools.
P2 - “These studies have revealed high and novel diversity...”: The authors can emphasize here a bit more that these studies are scarce.
P2 - “Here we use HTS to investigate...”: What do the authors meant by “frequent-resolution”? Please clarify.
Overall, in that paragraph, I thought it missed 1-2 sentences that explain about the harmful algal blooms/red tides that justify the study of their importance in coastal areas. In other words, the authors can say few words about them (who they are? What are the consequences for human health and ecosystem functioning) in order to justify the in-depth study of the unicellular eukaryote community in this study.
P2 - “The main questions were...”: Please replace “,” by “?” in “(i) Do the assemblages include previously undetected taxa in the area, (ii) How do these taxa contribute to the phenomena of blooms, red tides and mucilage aggregates?”
- 2. Materials and Methods -
P2 - “The sampling procedure...”: In that sentence, the authors may clarify somehow that the monthly samples that were taken from the three other adjacent sites were done also, at the same time, in the WT for clarity (which justify why there is in total 48 common samples).
P2 - “In order to better illustrate ...”: The authors should clearly differentiate “samples” and “sampling dates” throughout the manuscript. Indeed, in the monthly sampling of the 4 sites, there is only 12 sampling dates for a total of 48 samples. Please check and correct throughout the text.
P3 -Table 1- legend caption: Replace “Sampling Sites” by “Sampling sites”.
P3 - “During all samplings...”: The term “During all samplings” sounds incorrect. I would suggest to replace it by “For all samples”.
P4 - Figure 1: Can the authors add a scale to the figure in order to evaluate the distance between the different sampling sites?
P4 - “These primers have been found to successfully amplify...”: The authors specify here that this set of primers amplify all major high-level eukaryotic taxonomic groups. Does this assumption results of an in silico analysis done by the authors (and if so, how did they do it?) or from previous analysis of these primers from the literature? Please clarify.
P4 - “The PCR amplification step and the sequencing...”: Please replace by “The PCR amplification and sequencing steps ...”.
P4 - “Nitrate/nitrite (NO3−/NO2−), …”: Writing “Nitrate/Nitrite (NO3−/NO2−)” suggests that the authors have calculated the ratio of the two nitrogen sources while, as I understand, they actually have considered the sum of both components to get an estimate of the concentration of nitrogen oxides in their samples. Please rephrase to avoid any confusion. Furthermore, here and elsewhere, I would recommend to write “Nitrate + Nitrite” instead of ‘Nitrate & Nitrite” for clarity. The authors could also define an abbreviation for the nitrogen oxides as “NOx (Nitrate + Nitrite [NO3−+ NO2−])”.
P5 - “The resulting dataset was normalized …”: Can the authors specify how they proceed for the normalization of the dataset (using which program)?
P5 - “We chose this course of action as...”: I don’t believe that this explanation of the normalization approach is needed since it is nowadays a common approach in comparative analysis of microbial diversity.
P5 - “All data analyses described in ...”: No need to specify that the data analyses were described in methods and results. The authors can directly say that “All data analysis was performed on...”.
P5 - “For the network analysis ...”: The authors used the MIC-MINE to investigate protist interactions in their network approach. There was a very interesting review by Weiss et al. (2016, [10.1038/ismej.2015.235]) on the suitability of different correlation techniques for network analyses of microbial community data. Could the authors please comment on if they followed the recommendations in this article? Especially, since the authors have time series data, I thought that an LSA analysis would have been more adapted to their data set. Did the authors have consider this temporal component in their analysis?
In addition, in this paragraph the authors defined two classes of OTUs based on their read abundance. First, the most abundant OTUs based on a threshold of the overall abundance of 0.1% of the total reads (all sites combined I suppose) and studied in the network analysis. Second, the dominant OTUs defined as OTUs with relative number of reads > 1 % of the total number of reads in each site and considered in a CCA. As I suppose, the different thresholds for defining these two classes are not chosen randomly and are based on the literature, am I right? If so, I would recommend the author to specify it and maybe used terms defined in the literature for defining the two classes to avoid confusion. For instance, Logares et al., 2015 (doi:10.1016/j.resmic.2015.09.009) have define “instantly abundant” and “regionally abundant”, the OTUs are respectively abundant in one or all location. Also, I wonder why the authors doesn’t have consider both the dominant OTUs in all sites (regional abundant OTUs with > 0.1% of the total reads) and the dominant OTUs in each site (instantly abundant OTUs with > 1% of reads in each site) in the CCA to see what may explain regionally and locally the diversity variation observed. It would have been interesting to have such analysis.
-Results-
P6 - “3.1. Environmental Variables”: As I understand in this paragraph, and as specified in the material and methods, these measurements and comparisons between environmental variables were previously made and were published recently in a paper from the same authors. In this manuscript, the interests of such data are for the determination of putative environmental factors that may explained the variation in the unicellular eukaryotic diversity in a CCA analysis (see P12 - “3.4. Environmental Effects on Unicellular Eukaryotic Communities”). Since these data and analysis of the environmental variables were previously shown in a previous paper, I don’t believe that the authors have to present them again in this manuscript as results per se. I would recommend to move this section “3.1” of the results in the Materials and Methods and the section relative to the measurements of the environmental parameters. There, the authors could describe briefly and refer to the previous published articles for having more details on the environmental variables.
P6 - “Nutrients, i.e. SiO4, PO4, NO3, …”: The authors already define the signification of the abbreviation “POP” in the Material and Methods, there is no need to repeat it here.
P6 - “Especially on the 22 March ... »: Add a comma between 2017 and “at”.
P6 - “The ratio of observed to expected ...”: Since the authors present this ratio “observed/expected richness”, it would be interesting to actually have the value of such ratio that appears in the two supplementary tables (Tables S1 and S2). If so, the authors should refer to these tables when they present the ratio value in the text.
P6 - “Indeed, OTU_2 …”: These results are not shown in any items (Table or Figure). First, the authors could actually refer to the Table 2 for the taxonomic assignment of the OTU_2 to Noctiluca scintillans. Second, the fact that OTU_2 represent almost all the reads of one date in WT constitutes an interesting result but, apart if I missed it, isn’t been shown in any figure. The authors could eventually “use” the Figure S7 for it, since this heatmap highlights already the dates where OTUs of interest representing more than 10% of the sample’s total number of reads are shown. Indeed, instead of only one color, they could use a range of color from 10 to 100% to highlight when this OTU dominate the dataset (as it is the case for OTU_2, here).
P7 - “These spikes of …”: Here, the authors introduce information regarding the different blooms, red tides ... that appears nowhere in the text (including table/figure). Since the specificity of the studied ecosystem is the presence of such algal blooms, it would have been interesting to know for each of them, the dates when these events occurred to understand the different observed patterns. This could be in the form of a short paragraph and/or shown in (existing) figures/tables.
P7 - “Even so, Dinoflagellata …”: Please refer to the table and/or figure that present the results presented in that sentence.
P7 - “During the warm months of summer, ...”: Same remark. Moreover, I am not sure this is totally true. As the text is, it seems that the diatoms are only present in warm months and dinoflagellates the rest of the year but, apart if I am mistaken the colors, I saw higher abundance of Bacillariophyta in other samples than the one from this season. For instance, in the last week of May 2017, mid-November and in February 2018 (last sample). These two samples seem actually the ones where the diatoms are dominated the community. Furthermore, dinoflagellates are also well represented in summer (particulary in July).
P8 - Figure 2 - legend caption: Please find a generic title common to the two panels and then specify the difference between panel a and b in a second sentence
P9 - Figure 3 - legend caption: Same remark than before, I recommend to have a generic title that explain the whole figure first following with sentence that specify the difference between the two panels.
Furthermore, in the panel a, I found it difficult to distinguish and differentiate the different colors in each bar of the barplot. Is it possible to make the bars a little bit wider to help the reader to see the difference between samples?
P10 - “During the common samplings...”: For clarity, replace “During the common samplings in the 4 sites, the total number of OTUs corresponding to 12 samplings for each site, was 1153, 1195, 1362, and 1071 for WT, AR, MH, and HB, respectively (Supplementary Table S2).” By ““During the monthly sampling, common to the 4 sites (n= 12 per site), the total number of OTUs was 1153, 1195, 1362, and 1071 for WT, AR, MH, and HB, respectively (Supplementary Table S2).”
P10 - “Overall, 560 OTUs …”: Refer to the corresponding figure that highlight that results (i.e., Figure S4).
P10 - “Dinoflagellata in all cases comprised...”: Please refer to the corresponding figure that show such results.
P10- “Furthermore, > 40 % ...”: It also seems that diatoms are also very important in WT both in November 2017 and February 2018, don’t they?
P10 - “It is noteworthy ...”: In the manuscript, the authors wrote either “α -diversity” or alpha-diversity”. I would recommend to choose one of the two terms and keep it throughout the text.
P11 - Figure 4 and P12 - Figure 5: These are potentially very interesting results. I have however several questions/concerns about these approaches.
First, I yet wonder if the authors did the same type of analysis on the temporal survey in WT (weekly sampling). It would indeed have been interesting to see if the same clustering could be observed in both analysis with a shorter timescale of sampling. Did the authors have tested such analysis for the temporal survey in WT?.
Second, since the authors have time series data, I thought that a cooccurrence analysis such as eLSA (Xia et al., 2011, doi: 10.186/1752-0509-5-S2-S15) would have been more adapted to their data set. Indeed, with such analysis time-delayed co-occurrence/associations between OTUs can be observed. Did the authors have consider this temporal component in their analysis?
P12 - “Concerning the relationship between all samples…”: The authors said that the environmental variables were included in the CCA analysis according to their p-values. Do they mean that only environmental parameters with a significative p values were added to the plot? this is unclear. Furthermore, can they specify in the material and methods what test did they do to determine this p-value and what are these p-values? Was it a Monte Carlo permutation tests (n=999)? If so, it would have been interesting to performed a forward selection to only identify the set of parameters that best/significantly explain the observed clustering of samples (for instance with a p < 0.05).
P12 - Figure S6: I have the same remark than above. Please clarify if the environmental variables in the different CCA plots are the ones that likely explain a significant part (p < 0.05) of the changes in abundance of the dominant OTUs.
P14 - Figure S7- legend caption: Please correct “OUT” by “OTU”. Moreover, the term “of the per sample number of reads” is a bit unclear here and in the main text. Do the author mean “of the sample’s total number of reads”? Please clarify.
P16- “Fungi have been previously found...”: If the authors want to discuss about the recurrent presence of fungal sequences and of the other “new players” they cited in the sampling area in line with the senescence of blooms, they should first highlight them in the Results section. For now, this is not shown enough and the link between these “new players” and the different blooms are not clear at all. This may constitute an interesting finding.
P16 - “The metagenomic characterization ...”: Since the authors only target one specific gene (18S rDNA gene) using an amplicon-based approach, the term “metagenomic” is not adapted here.
P17 - “Furthermore, during these phenomena ...”: As the authors highlight the difference between bloom and non-blooms periods in the rate of newly introduced OTUs in the system, it is important to clearly show when these periods occurred. For instance, in Figure 2 that concerns the point raised by the authors here, it is unclear for the readers that haven’t read the previous paper written by the same authors that identified the different bloom periods in the different sites, where and when these blooms occurred. It should be clearly identified in this figure.
P17- “In addition to microscopic data...”: The importance of MALVs sequences is not clearly shown in the manuscript and the results section. Please add few words about these parasitic groups to support the statement made here. The putative negative impacts of parasitic OTUs and could have been better investigated through a network analysis at the OTU level to identify the pairs of OTUs involving such organisms in the studied system (see comments above).
P17 - “In the present study, MIC pairwise comparisons of dominant OTUs ...”: Apart if I missed it, the authors present the MIC pairwise comparisons of all samples by considering only the dominant OTUs but is not a direct pairwise comparisons of the dominant OTUs that indeed would highlight putative biotic associations. Do the authors did such analysis? This would be interesting to have it in the manuscript, especially if no indications of significant correlations are observed between OTUs are observed but rather associations with abiotic factors. This would constitute a very interesting finding.
P18 - “Even though microscopy data ...”: Same remark than above about the use of the term “metagenomic” in this study.
Author Response
Dear Reviewer,
We would like to thank you for your positive disposition towards our work. All the comments and suggestions are considered and respective changes have been made in the revised text, accordingly. All changes are highlighted in red to facilitate reading. Please see tha attached word for a point-by-point response to your comments and suggestions,
Sincerely, on behalf of all the authors
Dr. Savvas Genitsaris| Academic Associate
International Hellenic University
T +30-2310-807538
Email: [email protected]
Author Response File: Author Response.docx
Reviewer 3 Report
This is a very intensive examination of the eukaryotic microbial assemblages of Thessaloniki Bay, a very highly anthropogenically impacted inner embayment of the Aegean Sea, in Greece. The work exhaustively examines the variability and diversity of the eukaryotic microbiome of the Bay for over a full year's period from 2017 to 2018. The work ties in tightly to a previous publication, listed as item #33 in the Literature Cited, which took a more conventional microscopical analysis of the region over the same time period. A second in a pair of top quality papers that describe the impact of such disturbance on microbe populations. Carefully done, close attention to all of the required details. Recommend publication. Only very minor adjustments needed for the English.
Not 'true' metagenomic sequencing because all of the samples were amplified prior to MiSeq analyses, but nevertheless acceptable because of the primer types, which were appropriately chosen.
Only one question, which is relatively minor but could have an impact on the final outcome:
Towards the bottom of page 5 of 23, the authors discarded any streptophytes from the data set, but this could include some unicellular flagellates. I am not sure why the authors would have done this.
Otherwise, the work is very well described, will be of interest to the readership, and provides new insight into microbial activities in eutrophic environments.
Author Response
Dear Reviewer,
We are thankful for your positive disposition towards our manuscript. Your comments were considered and the necessary changes were made accordingly in the revised manuscript. These are highlighted in red to facilitate reading. Please see the attached word, for a point-by-point response to your comments.
Sincerely, on behalf of all the authors,
Dr. Savvas Genitsaris| Academic Associate
International Hellenic University
T +30-2310-807538
Email: [email protected]
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors have done a thorough work with the revised version and addressed and/or defended many of the issues that I have raised. Their manuscript has greatly gained in clarity and now is much more effective in highlighting the diversity and seasonality of the protistan community associated to phytoplanktonic blooms. Overall, their manuscript scientifically sounds for me and I am overall happy with the new version of the manuscript. I believe this manuscript is close to be the final version. Yet, I have some last minor comments to address in order to, I hope, improve some aspects of the study that I list points by points below:
L22-25: I acknowledge the authors for clarifying the meaning of the term “low numbers” here, which refer to OTUs in low abundance. Consequently, As I understand, this means that they have retrieved sequences affiliated to known species involved in harmful blooms within both OTUs in high (dominant) and low abundance (rare?), am I right? If so, I don’t believe that the conclusions made by the authors are the most suitable.
As I see (but I might be wrong), the fact that the authors have retrieved both dominant and low abundant OTUs were related to known harmful species together suggest the (relatively high?) presence of taxa with potential negative impacts on human health. However, the novel finding of the authors is within these low abundance OTUs that are affiliated to HABs. Indeed, I wonder if the authors wanted instead to point it out here that the part of microbial diversity with potential impact on human health are actually higher/richer than previously suggested/detected with classical approaches (microscopy, Sanger sequencing, ...) as these low abundant OTUs might be undetected with these approaches (below the detection limit of these methods). If so, as these two mentioned sentences are, this is not clear and the authors might rephrase them to clarify their points.
L180-181: Replace by “per Logares et al.” by “by Logares et al.”.
L194: Delete "the" in “...on the 22 March 2017...”.
L416-422: In the revised version, the MIC-MINE analysis was removed by the authors. They can still mention it in discussion since this one doesn’t show any indications of significant correlations between dominant OTUs, and between OTUs and environmental factors (which is a result per se) but they have to be clear on the fact that this analysis in NOT in the present study. Please rephrase this part to specify why this analysis was done and the fact that it is not presented here.
Author Response
The authors have done a thorough work with the revised version and addressed and/or defended many of the issues that I have raised. Their manuscript has greatly gained in clarity and now is much more effective in highlighting the diversity and seasonality of the protistan community associated to phytoplanktonic blooms. Overall, their manuscript scientifically sounds for me and I am overall happy with the new version of the manuscript. I believe this manuscript is close to be the final version. Yet, I have some last minor comments to address in order to, I hope, improve some aspects of the study that I list points by points below:
We would like to thank again the reviewer for his/her helpful suggestions and comments that significantly contributed to the improvement of our original manuscript. All new corrections based on these comments and suggestions are highlighted in red to facilitate reading. Previous corrections that are accepted are now incorporated in the new manuscript.
L22-25: I acknowledge the authors for clarifying the meaning of the term “low numbers” here, which refer to OTUs in low abundance. Consequently, As I understand, this means that they have retrieved sequences affiliated to known species involved in harmful blooms within both OTUs in high (dominant) and low abundance (rare?), am I right? If so, I don’t believe that the conclusions made by the authors are the most suitable. As I see (but I might be wrong), the fact that the authors have retrieved both dominant and low abundant OTUs were related to known harmful species together suggest the (relatively high?) presence of taxa with potential negative impacts on human health. However, the novel finding of the authors is within these low abundance OTUs that are affiliated to HABs. Indeed, I wonder if the authors wanted instead to point it out here that the part of microbial diversity with potential impact on human health are actually higher/richer than previously suggested/detected with classical approaches (microscopy, Sanger sequencing, ...) as these low abundant OTUs might be undetected with these approaches (below the detection limit of these methods). If so, as these two mentioned sentences are, this is not clear and the authors might rephrase them to clarify their points.
We are sorry for the confusion. Here we meant that additionally to the dominant OTUs responsible for the blooms and red tides, few other rare OTUs were detected within and around blooms, that belonged to known harmful taxa, however not easily detectable by classical microscopy. We have added a relevant sentence to clarify.
L180-181: Replace by “per Logares et al.” by “by Logares et al.”.
Done.
L194: Delete "the" in “...on the 22 March 2017...”.
Done.
L416-422: In the revised version, the MIC-MINE analysis was removed by the authors. They can still mention it in discussion since this one doesn’t show any indications of significant correlations between dominant OTUs, and between OTUs and environmental factors (which is a result per se) but they have to be clear on the fact that this analysis in NOT in the present study. Please rephrase this part to specify why this analysis was done and the fact that it is not presented here.
Thank you for the suggestion. We have now added a sentence indicating that we performed such analysis to identify potential significant correlations among OTUs and between OTUs and environmental variables and have added that these data are not shown.
