the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 27 Jan 2025
Distribution Patterns and Community Assembly Processes of Eukaryotic Microorganisms in Tibetan Plateau Proglacial Lakes at Different Emergence Stages
Abstract. Proglacial lakes are rapidly expanding due to climate change and glacier retreat. Eukaryotic microorganisms play a crucial role in the biogeochemical cycles of these lakes. However, there is limited understanding of the formation processes of eukaryotic microbial communities and their responses to material cycling in proglacial lakes, which are land reservoirs and new habitats for biological evolution of glacier meltwater, particularly in proglacial lakes at different developmental stages. This study investigates the distribution patterns and community assembly process of eukaryotic microbes in high altitude proglacial lakes, formed during different periods (i.e., 1990s, 2000s and post-2010), located on the central Tibetan Plateau. Using 18S rDNA gene amplification sequencing, in conjunction with neutral community model and a null model, we analyze the spatial dynamics and assembly processes of eukaryotic microbial communities. Our results reveal significant spatial heterogeneity community structure. Characterized by a pronounced geographical distance-decay pattern that intensifies with the age of the proglacial lake, indicating stronger symbiotic relationships and biological nesting. For proglacial lakes formed at different times, ecological shifts account for approximately 80 % of the observed community variations. Water temperature was the primary environmental factors influencing the formation of eukaryotic microbial communities. This study provides valuable data on the distribution patterns and assembly processes of eukaryotic microbial communities in emerging proglacial lakes, enhancing our understanding in the trajectories of eukaryotic microbial communities’ formation in high altitude glacier lakes in the context of climate change, and offering insights into the mechanisms that sustain eukaryotic microbial diversity in extreme environments.
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RC1: 'Comment on egusphere-2024-3688', Anonymous Referee #1, 05 Feb 2025
This is a potentially interesting paper on microbial succession in those lakes that form at the base of retreating glaciers on the Tibetan Plateau. It is interesting, but not surprising that community structure could change over time in these lakes, but the over-selling of the resulst based on three lakes is a substantial weakness.
In general it is over-interpreted and provides speculations as results or even conclusions. It is too long, but does not explain concepts necessary for understanding what or why the authors are performing their analysis, but often over explains the well-known.
The English in places difficult to parse and there is far too much private use of acronyms making it difficult to read.
While the sample size of one lake per age group is not statistically useful, the study, if substantially shortened, would make a useful note.
Given the small sample size, it seems difficult to satisfy the study’s listed aims (Lines 190-193).
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RC2: 'Comment on egusphere-2024-3688', Anonymous Referee #2, 17 Mar 2025
The study focuses on eukaryotic microbial communities in proglacial lakes on the Tibetan Plateau in relation to climate change. It provides novel data about these communities and aims to understand how eukaryotic microbial communities vary among lakes formed at different times and what processes shape their assembly. It is a relevant topic in terms of the emergence of novel proglacial lakes in relation to climate change.
The strength of the article includes the novelty of the sample sites and a large amount of environmental factors measured. The weakness lies in a small number of lakes analysed per “time” factor, especially that the strongest environmental factor affecting the community is temperature, which might be largely affected by local geographic pattern and confound the effects of “time” factor. Also, the conclusion about stochastic vs deterministic processes driving community assembly with time since retreat would require more lakes in each group to be sampled to draw relevant concusions.
The article also seems to be prepared in a rush. There is a chaotic structure, especially in the introduction. Many mistakes can be found, especially in citations.
Below are more specific comments:
The authors put quite a strong emphasis on the depth of the lake in their sampling strategy and subsequent data analysis, however, I find it confusing for the overall article and not really addressed in the study aims nor conclusions. In the data analyses, depth should be one of the factors or the abstract and hypotheses should be rewritten to focus on this aspect more.
I would put emphasis on rewriting and shortening the article for clarity and to avoid speculations based on a small number of sampled lakes per type.
Introduction:
There is a lot of irrelevant information, for example regarding bacterial communities. The structure is confusing, for example focuses on local lakes, then it jumps to global trends of glaciers, then again to global lakes. The authors should rethink the structure and remove redundant fragments, some example can be found below and highlight this issue:
Line 55 – do you mean mountain glaciers?
Line 85 – not sure how marine sediments are relevant
Line 86 – in this paper the chemolithotrophy was mentioned in terms of Archea, the information in the references should be cited properly and should be relevant
Line 126-129 - describe bacteria which is not relevant
Line 150 – what are AM fungi?
Line 154 – is this reference relevant as it mentions birth and death so probably refers to animals?
Line 156 – is subtropical river relevant for the topic?
Methods:
Please add a total number of samples analysed for clarity, were all 3 lakes sampled twice? How were the calculations done for the differences in table S3 – are different layers calculated for all the lakes combined? Same for the periods?
Citations provided in the methods section should be addressed to the original research methods were possible. For example, the primers used are referenced to the author’s previous work whereas they appear to be typical 18S primers known as ‘TAReuk’ and should be references as such. I also advise to provide more details on environmental analyses methods or again point to more broadly used references to facilitate replication of the protocols elsewhere.
There is a lack of consistence in applying statistical methods, especially in the context of analysis of DNA samples vs environmental samples. For DNA analyses the multivariate tests are used, while environmental data are mostly compared pairwise. Multivariate methods such as MANOVA or GLMMs should be used for environmental data.
Line 332 - the spatial heterogeneity of water quality in the same lake is relatively small – I am not sure how to interpret this sentence; does it regard different layers? Which measurements are considered water quality?
Discussion:
No correlation of environmental factors in EL samples – discuss?
I am also concerned about drawing general conclusion like “As the development time of proglacial lakes increases and accelerated glacier retreat, the proportion of stochastic processes first rises and then declines (ML > EL > NL)” based on one lake per development time
Some limitations of the methods used could ideally be discussed, for example the limitations of 18S method in describing the community structure.
Grammar mistakes:
Line 139 – repetition
The bibliography needs to be carefully examined for mistakes, for example line 218 refers to the “J et al, 2003” which clearly is an error of bibliography software. Some citations are provided in capital letter etc.
Line 345 – table S4 does not contain such data
Line 416 – again, Table S5 does not contain such data, probably meant table s6. The article must be reviewed carefully to check the numbering.
Citation: https://doi.org/10.5194/egusphere-2024-3688-RC2 -
RC3: 'Comment on egusphere-2024-3688', Anonymous Referee #3, 19 Mar 2025
Distribution Patterns and Community Assembly Processes of Eukaryotic Microorganisms in Tibetan Plateau Proglacial Lakes at Different Emergence Stages
Assessment
This paper examining eukaryote assembly processes in proglacial lakes over time is an important concept to examine scientifically. However, this manuscript requires major revisions to be considered for publication. Below are major reasons for this opinion, along with suggestions on how to improve the manuscript in the detailed comments.
- The 3 lakes range in surface area from 3500 m2 (NL) to 6500 m2 (EL) and maximum depths from 2 m (NL) to 6 m (ML). Thus, the lakes differ in size and depth and any differences observed among the lakes may be a result of surface area and size rather than the age of the lakes (formed 1980, formed 1990, formed 2010). Thus, all results comparing microbial communities between lakes and associating them with lake age are questionable.
- The paper does not clearly state for the reader what the sampling unit is and what the sample size is within the statistical analysis. Additionally, there are confusing disrepancies on details related to sampling and these are:
- line 181 indicates that 3 lakes were sampled in 2021 and 2022 and line 228 indicates that 3 lakes were sampled in May 15 and and August 15 in 2022. So was the sampling conducting in 2021 and 2022 or just 2022
- line 229 to 231 describes how during each sampling period (May and August) from 2022 (assuming only sampled 1 year) that sub-samples of water were collected from six locations (surface and bottom at the inlet, center, and outlet of each lake and then the six sub-samples were composited into 1 sample from each lake. Based on this description the sample size for each lake in 2022 is 2 with one sample from May 2022 and 1 sample from August 2022. Thus, for any statistical analysis the authors would only have 2 samples from each lake and a total of six samples from all 3 lakes with only a representative sample for the entire lake.
- With this understanding in mind - reading the results (see Figure 1, 2, 3, and 4) is confusing because the authors compared different aspects of microbial communities between the surface and the bottom of the lakes. How is this possible if all sub-samples were composited into 1 lake sample?
- Additionally, Figure 2 creates more confusion regarding the sample size because it reports a sample size of 66 for NL, 273 for ML, 66 for EL and 273 for surface samples and 274 for bottom samples. How does the sample size increase from 2 for each lake to between 66 and 273 for each lake? Additionally, Figure 3 depicts figures showing plots of distance and bray curtis dissimilarity and there is clearly more than 2 samples within each of the lake types. Figure 3 also shows lots of samples for surface and bottom, which is hard to understand how the authors have multiple samples when all subsamples collected from each lake were combined into one lake sample.
- lines 266 to 314 describe the use of 12 different statistical analyses. This is too many statistical tests for one manuscript, especially since the authors have not described how each statistical test helps them address their two research questions. thus, this section needs to be revised to use fewer statistical tests and clearly describe how each test provides information to help address each research question.
- The results section reports comparisons of environmental variables between the three lakes and reports comparisons of microbial communities between surface and bottom samples and these analyses should not even be presented because they are not part of the stated research questions on lines 187-190.
The two questions of the study were, 1) Do eukaryotic microbial communities vary among proglacial lakes that formed during different time periods? and 2) What is the dominant process – deterministic or stochastic – shaping the assembly of the eukaryotic microbial communities?
Introduction Comments
The introduction could be reduced in length by at least ½ by streamlining content focused on building up to the research questions. Each paragraph should be tightly written and shortened in length. Specific suggestions below describe possible ways to reduce the length of the introduction.
Methods
The sampling by lake category only has one lake per category. Repeat sampling of the n=3 lake sites (sampled 2x each melt season over 2 years or maybe just one year) is not accounted for in the statistical tests used in the manuscript
To improve the paper, the 6 sub samples from the lakes could be used to be equivalent to either 12 samples per lake (x2 sampling events in 2021) or 24 samples per site if sampled over two years. If doing this, however, pseudoreplication needs to be accounted for in the statistical analysis by using Linear mixed effect model which would be appropriate for repeat samples. Again, a revision of the questions needs to be done because we really don’t know if the community differences are from age or size and depth of the lakes.
Other Comments
Throughout the document there are spaces missing before parentheses in conjunction with all citations, with examples in lines 112, 114,118, 122, and 124. Another example of missing spaces is in between parentheses as in line 143 “…theory)(Stegen…).
Below are detailed suggestions by line.
Abstract Comment 1 Title, line 3
The title is misleading as the reader could construe the meaning to refer to the emergence stages of eukaryotic microorganisms. Suggest renaming Emergence stages to “Meltwater Lake Age” or “Meltwater Lake Development Stages”. However, as mentioned earlier, the differences in the lakes and resulting community characteristics could be due to the size of the lakes and depths of the water rather than the developmental age.
Abstract Comment 2 Abstract, line 21
Be more specific than material cycling. Perhaps nutrient cycling and environmental variables (e.g., turbidity, DO%) would clarify.
Abstract Comment 3 Abstract, lines 21-22
You can remove “which are land reservoirs….proglacial lakes” because most readers will know what proglacial lakes are. It would also be helpful to provide a reference describing the length of time it might take for proglacial lakes to mature ecologically (if we know that information). But again, you really can’t test this since the size of the lakes is so different.
Abstract Comment 4 Abstract (line 24 -25)
I suggest you rename and abbreviate the proglacial lakes as 1) Proglacial Lake 1 (L1) – formed in the 1990s; 2) Proglacial Lake 2 (L2) – formed in 2000s; and 3) Proglacial Lake 3 - formed after 2010. You could use these abbreviations throughout the document, which are much clearer than the current abbreviations.
Abstract Comment 5 Abstract, line 26
Remove the comma after sequencing, not necessary
Comment 6 Abstract, line 28
Heterogeneity in community structures (replace what is there)
Abstract Comment 7 Abstract, lines 29-30
The term biological nesting is somewhat unclear to me. Do you mean that communities within the larger microbial communities showed nested symbiotic relationships due to microhabitat factors? (e.g., DO, water depth, turbidity) This analysis does not relate to the stated questions of the paper, and contradicts the methods which state that all sub samples were combined into one representative sample for the lake.
Abstract Comment 8, line 31
Perhaps instead of ecological shifts, which I think of as large shifts in ecological function due to an overarching factor such as climate change, can you explain what you mean by ecological shifts? Please provide details such as the physicochemical properties of proglacial lakes that develop over time. Also, are the community variations you refer to eukaryotic variations? If so, specify here.
Abstract Comment 9, line 32
Factors should be singular as factor
Abstract Comment 10, Line 34
Suggest replacing “emerging” with “proglacial lakes of different meltwater developmental age”, or similar to redefine. 30-year-old lakes are not in the emergence stage.
Abstract Comment 11 line 35
Shouldn’t this be “community assembly processes” rather than formation?
Comment 12 Introduction lines 47-72
Compress this content into 2-3 sentences. You give a lot of statistics around lake changes, glacier changes, and regional changes. Since one of the major comments in the review is to reduce the length of the introduction, this is a long paragraph that could be reduced in length. Focus on the few important facts that directly relate to your research questions.
Comment 13 Introduction line 69
How is the expansion of proglacial lakes altering regional hydrological dynamics? Give a concrete example in the sentence. What is a potential impact on ecosystems due to the expansion of the proglacial lakes?
Comment 14 Introduction lines 74 – 75
Doing a quick literature search, I found quite a few alpine lake and glacier forefront lake papers focusing on eukaryotes with a couple below. Perhaps there are more papers related to prokaryotes which is something you can contrast with less emphasis being done on eukaryotes and in particular on eukaryotic community assemblage. https://academic.oup.com/femsec/article/91/4/fiv010/596383
https://www.sciencedirect.com/science/article/abs/pii/S1574954121001564
Bacteria
https://link.springer.com/article/10.1007/s00300-020-02770-8
https://link.springer.com/article/10.1007/s00300-023-03137-5
Comment 15 Introduction line 83
Do you have any citation that describes what chemical processes eukaryotes rely on due to turbidity of proglacial lakes? Also, at the end of line 83, replace was with “is”.
Comment 16 Introduction lines 85
Not all sediments beneath ice sheets are marine sediments. Suggest removing marine in front of sediments. Again, in the same line the space between “sheet” and “(Hood et al….” is missing.
Comment 17 Introduction line 87-90
Define mixotrophic symbiosis for the reading audience. Planktonic eukaryotic organisms in oligotrophic environments are able to obtain nutrients mixotrophically through photosynthesis and heterotrophy. Mixotrophic symbiosis is when a protozoa or algae uses both photosynthesis and organic nutrients from symbiont organisms.
Comment 18 Introduction line 92
The first part can be simplified “The eukaryotic community structure and diversity can undergo…” Also, what types of changes can they undergo over time? Can you reference particular changes that have been observed? It would improve this to give specific examples.
Comment 19 Introduction lines 99-109
This paragraph feels like a repeat of the previous paragraph. I suggest combining the two paragraphs into one succinct paragraph. Look at the first few lines of this paragraph and the previous paragraph.
Comment 20 Introduction lines 114-115
What does the continuous formation glacial lakes mean? (you need an of before lakes). Do you mean that due to the continuous recession of glaciers due to climate change that new lakes are constantly being formed?
Comment 21 Introduction line 116-118
This sentence is a repeat of similar information from the previous paragraph.
Comment 22 Line 119
What do you mean by stronger growth? Do you have a reference to any studies supporting that eukaryotic microorganisms have more resilience in extreme conditions? I think I understand what you mean but I think prokaryotic microorganisms are well-known for their extremophile adaptations and you might need to clarify how eukaryotes specifically have better growth (and clarify what you mean by stronger growth).
Comment 23, Line 124
Are there any studies that have found a relationship with turbidity and eukaryotes in proglacial waters?
Second comment – the Peter & Sommaruga study examined four lakes, not three.
Comment 24, Line 126
Do any of the three lakes you examined have hydrological connectivity with the glacier? Have any of the three lakes lost connectivity with the glacier?
Comment 25, Line 128
Which physico-chemical (spelled wrong in manuscript) did the Freimann study find has relevance to the bacteria groups and which bacteria taxa?
Comment 26, lines 135-136
You do not need (parentheses around the phrase (such as ….salinity)
Comment 27, Line 139
“In this context” is repeated twice. Remove one.
Comment 28, Lines142-143
Suggest adding 1) before “deterministic” and 2) before “stochastic”
Comment 29, Line 144
Instead of ecosystems or biological types tell us a couple of types of ecosystems that the paper refers to. It is unclear what “biological types” means in this context.
Comment 30 line 150
Instead of ecosystem would “studied habitat” be more precise in describing the area of study for the microbial communities?
Comment 31, Line 150
What is AM fungal communities? Please spell out all acronyms the first time you introduce.
Comment 32 , Line 158
You don’t have to put the m a.s.l. in parentheses. “at high-altitude regions between 5000-5500 m a.s.l.”
Comment 33, line 161
Better reflect than what? You need to tell us what the high-altitude lakes are more natural than (e.g., lowland (<3000 m a.s.l.) alpine lakes or similar example.
Comment 34, lines 163-164
It looks like you started to make a list in your sentence and created a cutoff. Either use an and or continue your sentence to complete it.
Comment 35 , lines 164-165
Again, this sentence has a grammatical structure issue. You need a verb after “melting and retreat”…
Comment 36, lines 166-168
This sentence needs work. Tighten and use a different word from construct.
Comment 37 lines 170-184
This whole section should be moved to the methods section. Here you don’t need to talk about 18S sequencing technology, this is for the methods section. Moreover, 18S sequencing in itself cannot tell us how microorganisms adapt to specific environmental conditions, this information needs to be combined with environmental data collected in the field and from weather station data. Suggest removing the content from this section and discussing how you combined the 18S and environmental data and other genomic data in the methods.
The research question paragraph should focus on the objective of understanding community assembly processes of lakes with different developmental times and then go right into your questions. The rest of lines 170-184 should go in the methods.
Comment 38 Methods Section
This is a general comment reflected in overall comment to add spaces before parentheses. Lines 197 and 198 both have missing spaces.
Comment 39 Line 199
KQGRG should be in parentheses and the rest in the sentence. Are the GPS coordinates correct?
Comment 40, Line 203
There is a space between -6.4 and degree symbol, remove the space here and make sure any other temperature readings are presented in the same format.
Comment 41, line 208
These lakes were found to have formed – reword to These lakes formed in the 1980s, 1990s, and 2010s. Cut down the extra wording.
Comment 42 , Lines 211-212
Suggest renaming the oldest lake as Lake 1 (L1), 1990s lake as Lake 2 (L2) and newest as Lake 3 (L3). This will help readership understand the order into which they were formed since this is the emphasis of the paper.
Comment 43 , Lines 207-224
This information could all be presented in a table which would be much easier to remember and summarize than everything written out. In addition, you could incorporate additional environmental data collected, numbers of samples combined and nutrient data.
Comment 44 , Line 225
You mention studying the lakes at different depths. However, this is not a part of your research questions, and the way you combined the microbial data does not allow for examination of the eukaryotic communities at different depths. Remove all references to different depths as this is out of the scope of the current manuscript.
Comment 45, Lines 229-230
Since you combine the samples, you only have one sample per lake. Remove all discussion of representing communities at different depths. Leave how you collected the samples at different depths in the methods, but since you combined the samples, you can not examine questions related to depths, nor do you propose to do this.
Comment 46, Line 132
Remove the plb acronym for the lake bottom throughout the manuscript. This is not necessary.
Comment 47, Line 232
What does PVDF stand for? Spell out the first time you introduce an acronym.
Comment 48, Lines 236 - 238
Either remove the reference to Hanna Instruments or put the entire company information. I suggest removing it since water quality meters are fairly standard. For the other instrument references, you need to provide complete company and location information if you are keeping this information.
Comment 49, Line 248
Were should be is.
Comment 50 , Line 252
Remove space after 2 and before % and repeat throughout document.
Comment 51 , Line 299
Specify that the E=environmental and S=spatial
Comment 52, Line 300
Cite R and version after last sentence
Comment 53. Line 301-302
All packages need citations and versions
Comment 54 Line 315
Add s to Result
Comment 56 Lines 306-310
Suggest to add a table with definitions for each of the features of a network and node-level features. These could be in the supplemental document.
Comment 57 Line 315
Add “s” to Result
Comment 58 Line 317
Correct misspelling of measurements
Comment 59 Line 319-320
Explain wavelet singular spectrum transformation. Also, spell out WT, EC, and sal. It is not necessary to abbreviate water quality parameters.
Comment 60 Line 321
What is the formula for the water quality index?
The statistical results are not valid as the correct test which accounts for pseudoreplication was not used. Which posthoc test did you use to tell the difference between the groups? Again, the results are not valid because pseudoreplication was not taken into account with inflated degrees of freedom and potentially incorrect conclusions based on the test used.
Comment 61 Line 324-325
Again, I do not understand why you are comparing the habitat parameters between the different sections of the water column, as there were no questions related to comparing the substrate and column variables. And the results are not valid as there is pseudoreplication.
Extremely significant differences? Take away the extremely significant.
Comment 62 Lines 339-342
Again, renaming the lakes as L1, L2, and L3 will make more sense when reading the results.
The statistical results here are again questionable due to the tests used that did not take into account pseudoreplication. Because pls and plb differences do not address any of the research questions in the paper, these should be removed. The microbial indicators of different layers within the proglacial lakes did not address any questions, so remove these.
Comment 63 Figure 1
None of these statistical comparisons had enough independent replicates to have valid results. A minimum of three independent samples is needed (e.g., three years of samples from each of the three lakes during the same month). It is not clear which samples were used, but if two temporal samples were taken during the melt season (e.g., June and September) for each of the three lakes, then season should be a factor considered because season could influence the results. Therefore, as understood, there are two samples for each lake being compared, which is not a valid statistical test.
Remove pls and plb as they are not relevant to any of the research questions of the paper.
Rename lakes as L1, L2, and L3 and put the exact date range of lake formation rather than early, middle, and late.
Comment 64 Figure 2
N=66 and 273. What are these samples? Based on the sampling methods, there should only be like 2-4 repeat samples for each lake (with correction for the one year/two year question for sampling earlier in comments). What are the higher numbers of 66 and 273 representing?
Comment 65 Figure 3
Remove results for pls and plb as they were not in the original research questions.
Comment 66 Lines 393-405
It again seems like the Mantel test does not account for the spatial autocorrelation in the data. Inflated type 1 error rates and unreliable statistical estimates may be the result for the lake community comparisons.
Although interesting, the results for pls and plb are not related to any research questions in the introduction.
Comment 67 Figure 4
Which samples are represented in a) analysis? If plb and pls are within the analysis, it is suggested to remove those samples. For b) what are the different taxonomic levels in the VPA analysis? Do you mean you examined eukaryotes at the OTU, Order, Family, Class levels? Be specific here.
Figure 4, lines 411-412. Rename lakes and remove lake surface and lake bottom, or incorporate these samples into the L1, L2, and L3 samples without distinguishing them as separate analyses.
Comment 68 Line 416 and Table S5
What do you mean by different taxonomic levels? Are you referring to different taxonomic levels of the eukaryotes? If so, where is this information displayed? Also, Table S5 doesn’t seem to have network nodes and edges. Is the Table misnumbered?
Comment 69 , line 446
Do you mean composition analysis?
Comment 70, line 447
What is meant by “the same kind” in this sentence? And can you also explain why they exhibit competitive or antagonistic interactions?
Comment 71, lines 468-482
This paragraph mixes both results and discussion. Remove all results from the discussion and place them in the results section for your null and neutral model analyses.
Comment 72, lines 524-531
The glacier-lake connectedness should be a part of the introduction and methods to describe how the three lakes of different developmental ages were connected or disconnected from the glacier water source. This should be written at the beginning of the methods instead of towards the end of the discussion.
Comment 73, line 532
should be but was, not were.
Comment 74, Figure 8
Figure 8 – Is this necessary to include in the paper
Comment 75, Lines 560-561 – again make sure to update to L1, L2, and L3 for more clarity throughout the document.
Comment 76, Line 580 – Conclusion – It is not necessary to begin the conclusion restating your methods in the first sentence.
Comment 77, Line 582 – you can remove “at different levels” as it is redundant with developmental stages.
Comment 77, Line 583 – with only three lakes in this study, it is suggested not to make a broad statement about microbial diversity increasing by elevation – it could be due to the developmental stage, water temperature, or other factor.
Comment 78 Lines 585-857 – again, with only three lakes in the study, it is difficult to make a general statement about stochastic assembly processes. More lakes should be examined using more robust statistical comparison techniques before suggesting a relationship with stochastic processes.
Comment 79, Lines 587 and 588 – The structure of the sentence is incorrect. There is a verb missing. The second sentence can be modified to “Proglacial lakes that are still connected with glaciers are more affected by glacial meltwater inputs”
Comment 80, Line 590 – Sentence starting with “As proglacial….should be present tense. Also what do they become separated from? Is it the feeder glacier or another proglacial lake?
Comment 81, Line 591 – Put the sentence “Consequently,… into present tense. …are influence by …..
Comment 82, Line 592 – the sentence describing that lakes are influenced by multiple factors is vague. In line 593 do you mean eukaryotic communities?
Citation: https://doi.org/10.5194/egusphere-2024-3688-RC3
Status: closed
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RC1: 'Comment on egusphere-2024-3688', Anonymous Referee #1, 05 Feb 2025
This is a potentially interesting paper on microbial succession in those lakes that form at the base of retreating glaciers on the Tibetan Plateau. It is interesting, but not surprising that community structure could change over time in these lakes, but the over-selling of the resulst based on three lakes is a substantial weakness.
In general it is over-interpreted and provides speculations as results or even conclusions. It is too long, but does not explain concepts necessary for understanding what or why the authors are performing their analysis, but often over explains the well-known.
The English in places difficult to parse and there is far too much private use of acronyms making it difficult to read.
While the sample size of one lake per age group is not statistically useful, the study, if substantially shortened, would make a useful note.
Given the small sample size, it seems difficult to satisfy the study’s listed aims (Lines 190-193).
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RC2: 'Comment on egusphere-2024-3688', Anonymous Referee #2, 17 Mar 2025
The study focuses on eukaryotic microbial communities in proglacial lakes on the Tibetan Plateau in relation to climate change. It provides novel data about these communities and aims to understand how eukaryotic microbial communities vary among lakes formed at different times and what processes shape their assembly. It is a relevant topic in terms of the emergence of novel proglacial lakes in relation to climate change.
The strength of the article includes the novelty of the sample sites and a large amount of environmental factors measured. The weakness lies in a small number of lakes analysed per “time” factor, especially that the strongest environmental factor affecting the community is temperature, which might be largely affected by local geographic pattern and confound the effects of “time” factor. Also, the conclusion about stochastic vs deterministic processes driving community assembly with time since retreat would require more lakes in each group to be sampled to draw relevant concusions.
The article also seems to be prepared in a rush. There is a chaotic structure, especially in the introduction. Many mistakes can be found, especially in citations.
Below are more specific comments:
The authors put quite a strong emphasis on the depth of the lake in their sampling strategy and subsequent data analysis, however, I find it confusing for the overall article and not really addressed in the study aims nor conclusions. In the data analyses, depth should be one of the factors or the abstract and hypotheses should be rewritten to focus on this aspect more.
I would put emphasis on rewriting and shortening the article for clarity and to avoid speculations based on a small number of sampled lakes per type.
Introduction:
There is a lot of irrelevant information, for example regarding bacterial communities. The structure is confusing, for example focuses on local lakes, then it jumps to global trends of glaciers, then again to global lakes. The authors should rethink the structure and remove redundant fragments, some example can be found below and highlight this issue:
Line 55 – do you mean mountain glaciers?
Line 85 – not sure how marine sediments are relevant
Line 86 – in this paper the chemolithotrophy was mentioned in terms of Archea, the information in the references should be cited properly and should be relevant
Line 126-129 - describe bacteria which is not relevant
Line 150 – what are AM fungi?
Line 154 – is this reference relevant as it mentions birth and death so probably refers to animals?
Line 156 – is subtropical river relevant for the topic?
Methods:
Please add a total number of samples analysed for clarity, were all 3 lakes sampled twice? How were the calculations done for the differences in table S3 – are different layers calculated for all the lakes combined? Same for the periods?
Citations provided in the methods section should be addressed to the original research methods were possible. For example, the primers used are referenced to the author’s previous work whereas they appear to be typical 18S primers known as ‘TAReuk’ and should be references as such. I also advise to provide more details on environmental analyses methods or again point to more broadly used references to facilitate replication of the protocols elsewhere.
There is a lack of consistence in applying statistical methods, especially in the context of analysis of DNA samples vs environmental samples. For DNA analyses the multivariate tests are used, while environmental data are mostly compared pairwise. Multivariate methods such as MANOVA or GLMMs should be used for environmental data.
Line 332 - the spatial heterogeneity of water quality in the same lake is relatively small – I am not sure how to interpret this sentence; does it regard different layers? Which measurements are considered water quality?
Discussion:
No correlation of environmental factors in EL samples – discuss?
I am also concerned about drawing general conclusion like “As the development time of proglacial lakes increases and accelerated glacier retreat, the proportion of stochastic processes first rises and then declines (ML > EL > NL)” based on one lake per development time
Some limitations of the methods used could ideally be discussed, for example the limitations of 18S method in describing the community structure.
Grammar mistakes:
Line 139 – repetition
The bibliography needs to be carefully examined for mistakes, for example line 218 refers to the “J et al, 2003” which clearly is an error of bibliography software. Some citations are provided in capital letter etc.
Line 345 – table S4 does not contain such data
Line 416 – again, Table S5 does not contain such data, probably meant table s6. The article must be reviewed carefully to check the numbering.
Citation: https://doi.org/10.5194/egusphere-2024-3688-RC2 -
RC3: 'Comment on egusphere-2024-3688', Anonymous Referee #3, 19 Mar 2025
Distribution Patterns and Community Assembly Processes of Eukaryotic Microorganisms in Tibetan Plateau Proglacial Lakes at Different Emergence Stages
Assessment
This paper examining eukaryote assembly processes in proglacial lakes over time is an important concept to examine scientifically. However, this manuscript requires major revisions to be considered for publication. Below are major reasons for this opinion, along with suggestions on how to improve the manuscript in the detailed comments.
- The 3 lakes range in surface area from 3500 m2 (NL) to 6500 m2 (EL) and maximum depths from 2 m (NL) to 6 m (ML). Thus, the lakes differ in size and depth and any differences observed among the lakes may be a result of surface area and size rather than the age of the lakes (formed 1980, formed 1990, formed 2010). Thus, all results comparing microbial communities between lakes and associating them with lake age are questionable.
- The paper does not clearly state for the reader what the sampling unit is and what the sample size is within the statistical analysis. Additionally, there are confusing disrepancies on details related to sampling and these are:
- line 181 indicates that 3 lakes were sampled in 2021 and 2022 and line 228 indicates that 3 lakes were sampled in May 15 and and August 15 in 2022. So was the sampling conducting in 2021 and 2022 or just 2022
- line 229 to 231 describes how during each sampling period (May and August) from 2022 (assuming only sampled 1 year) that sub-samples of water were collected from six locations (surface and bottom at the inlet, center, and outlet of each lake and then the six sub-samples were composited into 1 sample from each lake. Based on this description the sample size for each lake in 2022 is 2 with one sample from May 2022 and 1 sample from August 2022. Thus, for any statistical analysis the authors would only have 2 samples from each lake and a total of six samples from all 3 lakes with only a representative sample for the entire lake.
- With this understanding in mind - reading the results (see Figure 1, 2, 3, and 4) is confusing because the authors compared different aspects of microbial communities between the surface and the bottom of the lakes. How is this possible if all sub-samples were composited into 1 lake sample?
- Additionally, Figure 2 creates more confusion regarding the sample size because it reports a sample size of 66 for NL, 273 for ML, 66 for EL and 273 for surface samples and 274 for bottom samples. How does the sample size increase from 2 for each lake to between 66 and 273 for each lake? Additionally, Figure 3 depicts figures showing plots of distance and bray curtis dissimilarity and there is clearly more than 2 samples within each of the lake types. Figure 3 also shows lots of samples for surface and bottom, which is hard to understand how the authors have multiple samples when all subsamples collected from each lake were combined into one lake sample.
- lines 266 to 314 describe the use of 12 different statistical analyses. This is too many statistical tests for one manuscript, especially since the authors have not described how each statistical test helps them address their two research questions. thus, this section needs to be revised to use fewer statistical tests and clearly describe how each test provides information to help address each research question.
- The results section reports comparisons of environmental variables between the three lakes and reports comparisons of microbial communities between surface and bottom samples and these analyses should not even be presented because they are not part of the stated research questions on lines 187-190.
The two questions of the study were, 1) Do eukaryotic microbial communities vary among proglacial lakes that formed during different time periods? and 2) What is the dominant process – deterministic or stochastic – shaping the assembly of the eukaryotic microbial communities?
Introduction Comments
The introduction could be reduced in length by at least ½ by streamlining content focused on building up to the research questions. Each paragraph should be tightly written and shortened in length. Specific suggestions below describe possible ways to reduce the length of the introduction.
Methods
The sampling by lake category only has one lake per category. Repeat sampling of the n=3 lake sites (sampled 2x each melt season over 2 years or maybe just one year) is not accounted for in the statistical tests used in the manuscript
To improve the paper, the 6 sub samples from the lakes could be used to be equivalent to either 12 samples per lake (x2 sampling events in 2021) or 24 samples per site if sampled over two years. If doing this, however, pseudoreplication needs to be accounted for in the statistical analysis by using Linear mixed effect model which would be appropriate for repeat samples. Again, a revision of the questions needs to be done because we really don’t know if the community differences are from age or size and depth of the lakes.
Other Comments
Throughout the document there are spaces missing before parentheses in conjunction with all citations, with examples in lines 112, 114,118, 122, and 124. Another example of missing spaces is in between parentheses as in line 143 “…theory)(Stegen…).
Below are detailed suggestions by line.
Abstract Comment 1 Title, line 3
The title is misleading as the reader could construe the meaning to refer to the emergence stages of eukaryotic microorganisms. Suggest renaming Emergence stages to “Meltwater Lake Age” or “Meltwater Lake Development Stages”. However, as mentioned earlier, the differences in the lakes and resulting community characteristics could be due to the size of the lakes and depths of the water rather than the developmental age.
Abstract Comment 2 Abstract, line 21
Be more specific than material cycling. Perhaps nutrient cycling and environmental variables (e.g., turbidity, DO%) would clarify.
Abstract Comment 3 Abstract, lines 21-22
You can remove “which are land reservoirs….proglacial lakes” because most readers will know what proglacial lakes are. It would also be helpful to provide a reference describing the length of time it might take for proglacial lakes to mature ecologically (if we know that information). But again, you really can’t test this since the size of the lakes is so different.
Abstract Comment 4 Abstract (line 24 -25)
I suggest you rename and abbreviate the proglacial lakes as 1) Proglacial Lake 1 (L1) – formed in the 1990s; 2) Proglacial Lake 2 (L2) – formed in 2000s; and 3) Proglacial Lake 3 - formed after 2010. You could use these abbreviations throughout the document, which are much clearer than the current abbreviations.
Abstract Comment 5 Abstract, line 26
Remove the comma after sequencing, not necessary
Comment 6 Abstract, line 28
Heterogeneity in community structures (replace what is there)
Abstract Comment 7 Abstract, lines 29-30
The term biological nesting is somewhat unclear to me. Do you mean that communities within the larger microbial communities showed nested symbiotic relationships due to microhabitat factors? (e.g., DO, water depth, turbidity) This analysis does not relate to the stated questions of the paper, and contradicts the methods which state that all sub samples were combined into one representative sample for the lake.
Abstract Comment 8, line 31
Perhaps instead of ecological shifts, which I think of as large shifts in ecological function due to an overarching factor such as climate change, can you explain what you mean by ecological shifts? Please provide details such as the physicochemical properties of proglacial lakes that develop over time. Also, are the community variations you refer to eukaryotic variations? If so, specify here.
Abstract Comment 9, line 32
Factors should be singular as factor
Abstract Comment 10, Line 34
Suggest replacing “emerging” with “proglacial lakes of different meltwater developmental age”, or similar to redefine. 30-year-old lakes are not in the emergence stage.
Abstract Comment 11 line 35
Shouldn’t this be “community assembly processes” rather than formation?
Comment 12 Introduction lines 47-72
Compress this content into 2-3 sentences. You give a lot of statistics around lake changes, glacier changes, and regional changes. Since one of the major comments in the review is to reduce the length of the introduction, this is a long paragraph that could be reduced in length. Focus on the few important facts that directly relate to your research questions.
Comment 13 Introduction line 69
How is the expansion of proglacial lakes altering regional hydrological dynamics? Give a concrete example in the sentence. What is a potential impact on ecosystems due to the expansion of the proglacial lakes?
Comment 14 Introduction lines 74 – 75
Doing a quick literature search, I found quite a few alpine lake and glacier forefront lake papers focusing on eukaryotes with a couple below. Perhaps there are more papers related to prokaryotes which is something you can contrast with less emphasis being done on eukaryotes and in particular on eukaryotic community assemblage. https://academic.oup.com/femsec/article/91/4/fiv010/596383
https://www.sciencedirect.com/science/article/abs/pii/S1574954121001564
Bacteria
https://link.springer.com/article/10.1007/s00300-020-02770-8
https://link.springer.com/article/10.1007/s00300-023-03137-5
Comment 15 Introduction line 83
Do you have any citation that describes what chemical processes eukaryotes rely on due to turbidity of proglacial lakes? Also, at the end of line 83, replace was with “is”.
Comment 16 Introduction lines 85
Not all sediments beneath ice sheets are marine sediments. Suggest removing marine in front of sediments. Again, in the same line the space between “sheet” and “(Hood et al….” is missing.
Comment 17 Introduction line 87-90
Define mixotrophic symbiosis for the reading audience. Planktonic eukaryotic organisms in oligotrophic environments are able to obtain nutrients mixotrophically through photosynthesis and heterotrophy. Mixotrophic symbiosis is when a protozoa or algae uses both photosynthesis and organic nutrients from symbiont organisms.
Comment 18 Introduction line 92
The first part can be simplified “The eukaryotic community structure and diversity can undergo…” Also, what types of changes can they undergo over time? Can you reference particular changes that have been observed? It would improve this to give specific examples.
Comment 19 Introduction lines 99-109
This paragraph feels like a repeat of the previous paragraph. I suggest combining the two paragraphs into one succinct paragraph. Look at the first few lines of this paragraph and the previous paragraph.
Comment 20 Introduction lines 114-115
What does the continuous formation glacial lakes mean? (you need an of before lakes). Do you mean that due to the continuous recession of glaciers due to climate change that new lakes are constantly being formed?
Comment 21 Introduction line 116-118
This sentence is a repeat of similar information from the previous paragraph.
Comment 22 Line 119
What do you mean by stronger growth? Do you have a reference to any studies supporting that eukaryotic microorganisms have more resilience in extreme conditions? I think I understand what you mean but I think prokaryotic microorganisms are well-known for their extremophile adaptations and you might need to clarify how eukaryotes specifically have better growth (and clarify what you mean by stronger growth).
Comment 23, Line 124
Are there any studies that have found a relationship with turbidity and eukaryotes in proglacial waters?
Second comment – the Peter & Sommaruga study examined four lakes, not three.
Comment 24, Line 126
Do any of the three lakes you examined have hydrological connectivity with the glacier? Have any of the three lakes lost connectivity with the glacier?
Comment 25, Line 128
Which physico-chemical (spelled wrong in manuscript) did the Freimann study find has relevance to the bacteria groups and which bacteria taxa?
Comment 26, lines 135-136
You do not need (parentheses around the phrase (such as ….salinity)
Comment 27, Line 139
“In this context” is repeated twice. Remove one.
Comment 28, Lines142-143
Suggest adding 1) before “deterministic” and 2) before “stochastic”
Comment 29, Line 144
Instead of ecosystems or biological types tell us a couple of types of ecosystems that the paper refers to. It is unclear what “biological types” means in this context.
Comment 30 line 150
Instead of ecosystem would “studied habitat” be more precise in describing the area of study for the microbial communities?
Comment 31, Line 150
What is AM fungal communities? Please spell out all acronyms the first time you introduce.
Comment 32 , Line 158
You don’t have to put the m a.s.l. in parentheses. “at high-altitude regions between 5000-5500 m a.s.l.”
Comment 33, line 161
Better reflect than what? You need to tell us what the high-altitude lakes are more natural than (e.g., lowland (<3000 m a.s.l.) alpine lakes or similar example.
Comment 34, lines 163-164
It looks like you started to make a list in your sentence and created a cutoff. Either use an and or continue your sentence to complete it.
Comment 35 , lines 164-165
Again, this sentence has a grammatical structure issue. You need a verb after “melting and retreat”…
Comment 36, lines 166-168
This sentence needs work. Tighten and use a different word from construct.
Comment 37 lines 170-184
This whole section should be moved to the methods section. Here you don’t need to talk about 18S sequencing technology, this is for the methods section. Moreover, 18S sequencing in itself cannot tell us how microorganisms adapt to specific environmental conditions, this information needs to be combined with environmental data collected in the field and from weather station data. Suggest removing the content from this section and discussing how you combined the 18S and environmental data and other genomic data in the methods.
The research question paragraph should focus on the objective of understanding community assembly processes of lakes with different developmental times and then go right into your questions. The rest of lines 170-184 should go in the methods.
Comment 38 Methods Section
This is a general comment reflected in overall comment to add spaces before parentheses. Lines 197 and 198 both have missing spaces.
Comment 39 Line 199
KQGRG should be in parentheses and the rest in the sentence. Are the GPS coordinates correct?
Comment 40, Line 203
There is a space between -6.4 and degree symbol, remove the space here and make sure any other temperature readings are presented in the same format.
Comment 41, line 208
These lakes were found to have formed – reword to These lakes formed in the 1980s, 1990s, and 2010s. Cut down the extra wording.
Comment 42 , Lines 211-212
Suggest renaming the oldest lake as Lake 1 (L1), 1990s lake as Lake 2 (L2) and newest as Lake 3 (L3). This will help readership understand the order into which they were formed since this is the emphasis of the paper.
Comment 43 , Lines 207-224
This information could all be presented in a table which would be much easier to remember and summarize than everything written out. In addition, you could incorporate additional environmental data collected, numbers of samples combined and nutrient data.
Comment 44 , Line 225
You mention studying the lakes at different depths. However, this is not a part of your research questions, and the way you combined the microbial data does not allow for examination of the eukaryotic communities at different depths. Remove all references to different depths as this is out of the scope of the current manuscript.
Comment 45, Lines 229-230
Since you combine the samples, you only have one sample per lake. Remove all discussion of representing communities at different depths. Leave how you collected the samples at different depths in the methods, but since you combined the samples, you can not examine questions related to depths, nor do you propose to do this.
Comment 46, Line 132
Remove the plb acronym for the lake bottom throughout the manuscript. This is not necessary.
Comment 47, Line 232
What does PVDF stand for? Spell out the first time you introduce an acronym.
Comment 48, Lines 236 - 238
Either remove the reference to Hanna Instruments or put the entire company information. I suggest removing it since water quality meters are fairly standard. For the other instrument references, you need to provide complete company and location information if you are keeping this information.
Comment 49, Line 248
Were should be is.
Comment 50 , Line 252
Remove space after 2 and before % and repeat throughout document.
Comment 51 , Line 299
Specify that the E=environmental and S=spatial
Comment 52, Line 300
Cite R and version after last sentence
Comment 53. Line 301-302
All packages need citations and versions
Comment 54 Line 315
Add s to Result
Comment 56 Lines 306-310
Suggest to add a table with definitions for each of the features of a network and node-level features. These could be in the supplemental document.
Comment 57 Line 315
Add “s” to Result
Comment 58 Line 317
Correct misspelling of measurements
Comment 59 Line 319-320
Explain wavelet singular spectrum transformation. Also, spell out WT, EC, and sal. It is not necessary to abbreviate water quality parameters.
Comment 60 Line 321
What is the formula for the water quality index?
The statistical results are not valid as the correct test which accounts for pseudoreplication was not used. Which posthoc test did you use to tell the difference between the groups? Again, the results are not valid because pseudoreplication was not taken into account with inflated degrees of freedom and potentially incorrect conclusions based on the test used.
Comment 61 Line 324-325
Again, I do not understand why you are comparing the habitat parameters between the different sections of the water column, as there were no questions related to comparing the substrate and column variables. And the results are not valid as there is pseudoreplication.
Extremely significant differences? Take away the extremely significant.
Comment 62 Lines 339-342
Again, renaming the lakes as L1, L2, and L3 will make more sense when reading the results.
The statistical results here are again questionable due to the tests used that did not take into account pseudoreplication. Because pls and plb differences do not address any of the research questions in the paper, these should be removed. The microbial indicators of different layers within the proglacial lakes did not address any questions, so remove these.
Comment 63 Figure 1
None of these statistical comparisons had enough independent replicates to have valid results. A minimum of three independent samples is needed (e.g., three years of samples from each of the three lakes during the same month). It is not clear which samples were used, but if two temporal samples were taken during the melt season (e.g., June and September) for each of the three lakes, then season should be a factor considered because season could influence the results. Therefore, as understood, there are two samples for each lake being compared, which is not a valid statistical test.
Remove pls and plb as they are not relevant to any of the research questions of the paper.
Rename lakes as L1, L2, and L3 and put the exact date range of lake formation rather than early, middle, and late.
Comment 64 Figure 2
N=66 and 273. What are these samples? Based on the sampling methods, there should only be like 2-4 repeat samples for each lake (with correction for the one year/two year question for sampling earlier in comments). What are the higher numbers of 66 and 273 representing?
Comment 65 Figure 3
Remove results for pls and plb as they were not in the original research questions.
Comment 66 Lines 393-405
It again seems like the Mantel test does not account for the spatial autocorrelation in the data. Inflated type 1 error rates and unreliable statistical estimates may be the result for the lake community comparisons.
Although interesting, the results for pls and plb are not related to any research questions in the introduction.
Comment 67 Figure 4
Which samples are represented in a) analysis? If plb and pls are within the analysis, it is suggested to remove those samples. For b) what are the different taxonomic levels in the VPA analysis? Do you mean you examined eukaryotes at the OTU, Order, Family, Class levels? Be specific here.
Figure 4, lines 411-412. Rename lakes and remove lake surface and lake bottom, or incorporate these samples into the L1, L2, and L3 samples without distinguishing them as separate analyses.
Comment 68 Line 416 and Table S5
What do you mean by different taxonomic levels? Are you referring to different taxonomic levels of the eukaryotes? If so, where is this information displayed? Also, Table S5 doesn’t seem to have network nodes and edges. Is the Table misnumbered?
Comment 69 , line 446
Do you mean composition analysis?
Comment 70, line 447
What is meant by “the same kind” in this sentence? And can you also explain why they exhibit competitive or antagonistic interactions?
Comment 71, lines 468-482
This paragraph mixes both results and discussion. Remove all results from the discussion and place them in the results section for your null and neutral model analyses.
Comment 72, lines 524-531
The glacier-lake connectedness should be a part of the introduction and methods to describe how the three lakes of different developmental ages were connected or disconnected from the glacier water source. This should be written at the beginning of the methods instead of towards the end of the discussion.
Comment 73, line 532
should be but was, not were.
Comment 74, Figure 8
Figure 8 – Is this necessary to include in the paper
Comment 75, Lines 560-561 – again make sure to update to L1, L2, and L3 for more clarity throughout the document.
Comment 76, Line 580 – Conclusion – It is not necessary to begin the conclusion restating your methods in the first sentence.
Comment 77, Line 582 – you can remove “at different levels” as it is redundant with developmental stages.
Comment 77, Line 583 – with only three lakes in this study, it is suggested not to make a broad statement about microbial diversity increasing by elevation – it could be due to the developmental stage, water temperature, or other factor.
Comment 78 Lines 585-857 – again, with only three lakes in the study, it is difficult to make a general statement about stochastic assembly processes. More lakes should be examined using more robust statistical comparison techniques before suggesting a relationship with stochastic processes.
Comment 79, Lines 587 and 588 – The structure of the sentence is incorrect. There is a verb missing. The second sentence can be modified to “Proglacial lakes that are still connected with glaciers are more affected by glacial meltwater inputs”
Comment 80, Line 590 – Sentence starting with “As proglacial….should be present tense. Also what do they become separated from? Is it the feeder glacier or another proglacial lake?
Comment 81, Line 591 – Put the sentence “Consequently,… into present tense. …are influence by …..
Comment 82, Line 592 – the sentence describing that lakes are influenced by multiple factors is vague. In line 593 do you mean eukaryotic communities?
Citation: https://doi.org/10.5194/egusphere-2024-3688-RC3
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