Review Reports - Phytoplankton Biodiversity in Two Tropical, High Mountain Lakes in Central Mexico

Round 1

Reviewer 1 Report

Authors: Estela Cuna, Javier Alcocer, Martha Gaytán, Margarita Caballero

Title: Phytoplankton biodiversity in two tropical, high mountain lakes in Central Mexico

The manuscript addresses an interesting topic concerning phytoplankton diversity in lakes located in volcanic calderas. The authors suggest that based on the analysis of the phytoplankton community of the lakes studied, the impact of climate change could be inferred and the phytoplankton could be used as an indicator of environmental change in the area. The authors analyzed the phytoplankton composition based on samples from March 2000 to March 2001 and provided a list of taxa found in the two lakes of the Nevado de Toluca volcano. However, in my opinion, the results presented are insufficient to prove the hypotheses. The main objection is that the study lasted only 13 months and it was conducted 20 years ago. After 20 years, the situation in the lakes, especially such small ones, could have changed, which has certainly happened.

Besides, the taxonomic identification was based on old, fixed samples, additionally of using an inverted microscope. Not surprisingly, most of the taxa present in the list are identified to the genus level, or the identification is uncertain - "cf." Taxa at the genus rank in most cases do not provide indicative value, since very often within a single genus there are species often with completely different environmental preferences.

I think that to increase the merit of this work it would be good to repeat the study, juxtapose the results with the archival data presented in this manuscript, and perhaps then it would be easier to capture the changes in the phytoplankton of El Sol and La Luna lakes.

Other comments I presented below.

Abstract

line 20&22: Should be Bacillariohyceae, not Bacillariophycea (in Abstract and throughout the text).

lines 24-26: “…we provide for the first time a taxonomic list of the phytoplankton community of Lakes El Sol and La Luna and postulate the reasons for their different biodiversity and composition.” In the Conclusions section, we can find: “HML El Sol and La Luna display high phytoplankton diversity (92 and 63 taxa, respectively) in comparison with other temperate and tropical HML. And lines 242-244: All the taxa found in Lake La Luna were present also in Lake El Sol,…” My question is: Were the diversity and composition different in the lakes studied, as you postulated in the Abstract section if you found all the species from La Luna in El Sol?

Introduction

First of all, I’m not sure what the Authors mean by using the word “biodiversity”. Is this the only list of species included? If so, this should be called species richness. Biodiversity is usually expressed by indices calculated from biomass/frequency of occurrence/and others.

line 68: …y La Luna… Should be “and La Luna” I suppose.

Materials and Methods

This is the part that causes me the most concern about the whole concept of the research problem. The study was conducted 20 years ago during only one calendar year. I think it is very likely that many of the species you showed on the included list don’t occur in these lakes now. It is typical in such small (especially La Luna lake) and astatic lakes. Such lakes are characterized by the huge randomness of phytoplankton composition. Also, statements like “La Luna has higher nitrogen and El Sol higher EC, pH, phosphorus,…” sound a bit awkward here as this information is given on the basis of different papers, within one of them physical-chemical data about these lakes say: “a sample from Nevado de Toluca was obtained in August 1997” (Armienta, M. A., De la Cruz-Reyna, S., & Macıas, J. L. (2000). Chemical characteristics of the crater lakes of Popocatetetl, El Chichon, and Nevado de Toluca volcanoes, Mexico. Journal of Volcanology and Geothermal Research, 97(1-4), 105-125.) So how is it possible to have data about El Sol and La Luna based on “a sample”? In addition, I think we should not say now about climate change if we have data from research done years ago.

Also, identification of species for biological diversity purposes that is the main goal of the manuscript cannot be done on fixed material (Lugol’s solution) and especially using an inverted microscope where there is no possibility to see details of a species which is in a counting chamber. A question to the Authors: how did you determine small algae like Synechocystis minuscula, Chrysococcus minutus, some small greens to the species level using an inverted microscope? Moreover, how did you determine diatoms to the species level using such a microscope?

Results

The main result from this study is a big table that encompasses a list of species found in El Sol and La Luna lakes. This is a nice concept but we should not draw a conclusion, even in the future, about climate change impact based on phytoplankton composition having 45 taxa out of 92 identified to the genus level, and the next 14 identified as “cf.” or “aff.” which means uncertain identification. I agree with the statement the Authors said that “this information constitutes a baseline necessary for future studies, to evaluate possible changes in phytoplankton species in these lakes that could be related with global and climate changes, enabling them to be used as sentinels of environmental changes in the region.”, but we need more concrete data, f.i. species identified to the species level. There is a lot of species within one genus of which presence means contrary different situations. For example, Pinnularia sp. 1 or 2 from this study, but there are for instance Pinnularia gibba - a very common species typical of alkaline waters, and Pinnularia subcapitata, also very common, which prefers acidic waters and it is an indicator of anthropogenic acidification. They both mean completely different environmental conditions. I’d like to say I don’t mean that all taxa should be identified to species, but in this manuscript taxa of uncertain species affinity consist of the majority.

According to the Materials&Methods part, there was also the information the abundance was counted. It would be good to present such data to characterize the lakes. The readers need some data about the lakes (abundance, phosphorus content, nitrogen content, etc.). I have also a comment concerning dominant species: I think there cannot be 28 dominant species within the assemblage of 92 taxa. It’s almost 30% of all species determined. The issue of dominance is that one species has domination over the rest. There may be also a few co-dominant species that means these species are dominant over the rest. If there are 28 dominant taxa in the entire community, they are not dominant taxa any longer. It would be easier to see the structure of phytoplankton if the data of abundance/numbers had been included.

Check the table and correct italics/non italics like Chromulina sp. or Euastrum cf. oblongum.

In Table 2 there are two different taxa Stenopterobia sp. 1 and Stenopterobia sp. 2 which have the same symbol (Stspp) used in bivariate diagrams of abundance what is misleading to the readers.

Discussion

lines 166-167: there is no data in Table 3 concerning lakes from Italy and Slovakia. Where is “a” or “b” in this table?

lines 200-202: Euglenophytes are not indicators of oligotrophic waters.

line 201: Zignematal species maybe will be a better expression than only Zignematal.

line 208: italics

lines 209-210: Peridinium willei for sure cannot be considered as small size phytoplankton species. It can be up to 60 µm long and up to 70 µm wide.

lines 217-225: Do these data come from this survey in August 1997?

Author Response

Reviewer 1

Comments and Suggestions for Authors

Authors: Estela Cuna, Javier Alcocer, Martha Gaytán, Margarita Caballero

Title: Phytoplankton biodiversity in two tropical, high mountain lakes in Central Mexico

The manuscript addresses an interesting topic concerning phytoplankton diversity in lakes located in volcanic calderas. The authors suggest that based on the analysis of the phytoplankton community of the lakes studied, the impact of climate change could be inferred, and the phytoplankton could be used as an indicator of environmental change in the area. The authors analyzed the phytoplankton composition based on samples from March 2000 to March 2001 and provided a list of taxa found in the two lakes of the Nevado de Toluca volcano. However, in my opinion, the results presented are insufficient to prove the hypotheses. The main objection is that the study lasted only 13 months and it was conducted 20 years ago. After 20 years, the situation in the lakes, especially such small ones, could have changed, which has certainly happened.

The present study aimed to provide the phytoplankton baseline required to be able to compare when other-more recent-studies are undertaken. Without this baseline it is impossible to identify long term changes in the phytoplankton that could be eventually associated to regional or climate change in future works. We changed the text to make it clearer that this is not the objective of the present contribution. We did not pursue to inferred climate changes based on phytoplankton, which would be impossible without a previous reference. The main contribution was to provide the phytoplankton baseline, in addition to contribute to the poorly known tropical high mountain lakes ecology.

This is a misunderstanding and we have made changes in the methods section to make it clear that taxonomic identification was made using both, fresh and fixed samples which were observed under a light microscope at different magnifications (400 to 1000 x) and in the case of diatoms using cleaned material processed with hydrogen peroxide and when needed samples were also observed with a scanning electron microscope to confirm identification. The counts were performed using sedimentation chambers under the inverted microscope, which is a widely accepted method for phytoplankton quantification. Also, it is important to mention that identification of samples was done in the laboratory only a few days after sample collection, quantification had to be finished before the next set of samples arrived in the laboratory. So, all the samples were identified and counted within the next month after sampling.

We agree with the referee that it would be quite interesting to undertake such a study. Our research group is certainly planning such a study in the future and compare the findings after 20 years. In this way, it would be possible to capture the changes in the phytoplankton of El Sol and La Luna lakes and to identify if climate change and/or other forcing agents were responsible of such changes. However, this is work that will be undertaken in the future and will involve one or two years of monthly sampling. Such results cannot be included in the present research which objective is to provide the necessary baseline for later comparisons either by our research group or maybe other research groups.

Other comments I presented below.

Abstract

line 20&22: Should be Bacillariohyceae, not Bacillariophycea (in Abstract and throughout the text).

Done

Yes of course, diversity between the lakes was different! Species richness was higher in Lake El Sol compared to la Lake La Luna, with 29 taxa found in El Sol that are not present in La Luna, even though all the taxa present in La Luna were found in El Sol. This is a very clear nested pattern of species richness reduction in the smaller lake that also had more extreme environmental conditions (e.g., more acidic pH). We have modified the discussion so that this point is clearer.

Introduction

Biodiversity refers to the variety of live forms found in a certain place on earth, and it can be measured by different metrics. For example, species richness (the number of species present), other metrics include diversity indexes where species presence is weighted by its abundance, such as the Shannon-Wiener index. So, there is no mistake in using the term biodiversity when referring to the species richness of a certain region. Besides, since we involved not only the phytoplankton species richness but also dominance, and in the new updated version of the manuscript we also included abundance and diversity indexes, we think the term is appropriate.

line 68: …y La Luna… Should be “and La Luna” I suppose.

Done

Materials and Methods

This is a misconception of the reviewer, all samples were identified and quantified a few days after collection (as explained previously) during 2000 and 2001. We are aware that today it is quite likely the phytoplankton assemblage may have changed. However, the objective of the manuscript was to provide a phytoplankton baseline, in addition to contribute to the poorly known tropical high mountain lakes ecology.

Also, statements like “La Luna has higher nitrogen and El Sol higher EC, pH, phosphorus,…” sound a bit awkward here as this information is given on the basis of different papers, within one of them physical-chemical data about these lakes say: “a sample from Nevado de Toluca was obtained in August 1997” (Armienta, M. A., De la Cruz-Reyna, S., & Macıas, J. L. (2000). Chemical characteristics of the crater lakes of Popocatetetl, El Chichon, and Nevado de Toluca volcanoes, Mexico. Journal of Volcanology and Geothermal Research, 97(1-4), 105-125.) So how is it possible to have data about El Sol and La Luna based on “a sample”?

The physical and chemical data were measured in situ or in water samples that were collected at the same time as the phytoplankton samples, every month between March 2000 to March 2001. The physical and chemical data are now presented in Table 1. Only basic ionic composition was determined based on a single sampling date, but ionic composition is relatively constant in these lakes (see for example Ibarra et al. 2020), so there is no need to repeat this analysis monthly.

In addition, I think we should not say now about climate change if we have data from research done years ago.

We are not dealing with climate change, we have changed the text to make this clear. Nevertheless, we provide the phytoplankton baseline required to further studies looking for evidence of climate change. New studies would be able to compare the current findings with our study to find out if there have been changes (e.g., composition) in the phytoplankton assemblages, and if climate change is to be blame.

Phytoplankton species were identified in fresh (unfixed) samples through optical microscopy (Carl Zeiss with phase contrast) under 400 – 1000 x only a few days after sample collection. Diatoms were identified using cleaned material processed with hydrogen peroxide and when needed samples were also observed with a scanning electron microscope to confirm identification. Once identified, cell counts were done on fixed samples by the standard Utermhöl method with a Carl 94 Zeiss D inverted microscope using 50- and 100-ml sedimentation chambers. We have now provided a detailed methodology on how phytoplankton were analyzed to avoid confusion.

Results

The main result from this study is a big table that encompasses a list of species found in El Sol and La Luna lakes. This is a nice concept, but we should not draw a conclusion, even in the future, about climate change impact based on phytoplankton composition having 45 taxa out of 92 identified to the genus level, and the next 14 identified as “cf.” or “aff.” which means uncertain identification. I agree with the statement the Authors said that “this information constitutes a baseline necessary for future studies, to evaluate possible changes in phytoplankton species in these lakes that could be related with global and climate changes, enabling them to be used as sentinels of environmental changes in the region.”, but we need more concrete data, f.i. species identified to the species level. There is a lot of species within one genus of which presence means contrary different situations. For example, Pinnularia sp. 1 or 2 from this study, but there are for instance Pinnularia gibba - a very common species typical of alkaline waters, and Pinnularia subcapitata, also very common, which prefers acidic waters and it is an indicator of anthropogenic acidification. They both mean completely different environmental conditions. I’d like to say I don’t mean that all taxa should be identified to species, but in this manuscript taxa of uncertain species affinity consist of the majority.

The reason that many taxa were not identified to species level was that they could not be clearly assigned to described species considering most taxonomic keys deal with temperate environments and very little is known from tropical inland waters. Moreover, new species are currently being recorded in these lakes, such is the case of Temnogametum iztacalense. But new taxa descriptions is a long process that will be gradually done but it is out of the scope of this work.

According to the Materials&Methods part, there was also the information the abundance was counted. It would be good to present such data to characterize the lakes.

The data were included as Supplementary Information as also requested by other referees.

The readers need some data about the lakes (abundance, phosphorus content, nitrogen content, etc.).

We have now included a new table (Table 1) with data on the physical and chemical data of the lakes as requested.

I have also a comment concerning dominant species: I think there cannot be 28 dominant species within the assemblage of 92 taxa. It’s almost 30% of all species determined. The issue of dominance is that one species has domination over the rest. There may be also a few co-dominant species that means these species are dominant over the rest. If there are 28 dominant taxa in the entire community, they are not dominant taxa any longer.

As mentioned in the statistical analysis section (Materials and Methods), dominance was assigned according to the Olmstead & Tukey diagram based on abundance versus frequency of occurrence. The diagram classifies the taxa in four categories: representative / dominant (high abundance and frequency), abundant/temporal (high abundance, low frequency), constant/frequent (low abundance, high frequency), and rare (low abundance, low frequency).

We agree with the reviewer that in this case the term dominant might not be the best choice and we changed it in the text for “representative” taxa when appropriate. In the revised manuscript we restricted the use of dominant species to those that have abundances of 50% or higher.

It would be easier to see the structure of phytoplankton if the data of abundance/numbers had been included.

The abundance data are now provided as Supplementary Information as also requested by other referees.

Check the table and correct italics/non italics like Chromulina sp. or Euastrum cf. oblongum.

Errors in tables 1 and 2 were corrected

In Table 2 there are two different taxa Stenopterobia sp. 1 and Stenopterobia sp. 2 which have the same symbol (Stspp) used in bivariate diagrams of abundance what is misleading to the readers.

The diagram was corrected to clarify which is species 1 and which is species 2.

Discussion

lines 166-167: there is no data in Table 3 concerning lakes from Italy and Slovakia. Where is “a” or “b” in this table?

We replaced the Table. The data concerning lakes from Italy and Slovakia are now clearly visible.

lines 200-202: Euglenophytes are not indicators of oligotrophic waters.

We agree that they are not indicative of oligotrophic conditions, but they are indicative of mesotrophic conditions, so we indicate that together with Zygnematales they indicated oligo-mesotrophic conditions.

line 201: Zignematal species maybe will be a better expression than only Zignematal.

Done

line 208: italics

Done

lines 209-210: Peridinium willei for sure cannot be considered as small size phytoplankton species. It can be up to 60 µm long and up to 70 µm wide.

We agree with the incorrect use of the small size, although our specimens are in the smallest size ranging 20 µm. It has been corrected in the text.

lines 217-225: Do these data come from this survey in August 1997?

No, nutrient and chlorophyll concentration were measured at the same time as the phytoplankton (March 2000 to March 2001).

Author Response File: Author Response.docx

Reviewer 2 Report

Cuna et al. investigated phytoplankton communities in two neighboring alpine lakes in Central Mexico. The presented data provide new insight into phytoplankton diversity in high mountain lakes especially in tropical areas. The manuscript is well written and is of general interest.

Comments:

1) Phytoplankton data were collected more than twenty years ago. Some words about why analysis took so long should be given. Also, it should also be discussed if phytoplankton composition is still the same in these alpine lakes after more than two decades.

2) It is written in Materials and Methods that samples for phytoplankton were collected along full year. It is also indicated that samples were three different depths were collected. Thus, some spatiotemporal data are available. Presenting such patterns, possibly in the Supplement, would be useful.

3) Some micrographs, at least about the newly found morphospecies should be presented.

4) Nutrient levels and other physicochemical data should be presented either in the Materials and Methods or in the Results section, rather than in the Discussion.

5) The two rightmost columns of Table 3 are not visible in the downloaded manuscript.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Comments:

All samples were identified and quantified a few days after collection (as explained previously) during 2000 and 2001. We are aware that today it is quite likely the phytoplankton assemblage may have changed. We made changes in the text to make it clear that the objective of the manuscript is to provide a phytoplankton baseline for future work by us or by others, in addition to contribute to the poorly known tropical high mountain lakes ecology.

To give an explanation to the reader we modified the following paragraphs:

“Moreover, the present study analyzes comparatively the phytoplankton biodiversity of these two unique tropical HML and provides insights on the differences found. This information constitutes a necessary baseline for future studies, which could evaluate possible changes in phytoplankton species in these lakes.”

“All analyses were completed at the time, but preparation of the material for publication was not possible until now. Hence, these valuable historical data are an important baseline for further studies”

We obtained only quantitative samples from three water depths. Since the lakes’ shallowness and continuous mixing, the three water depths turned out to be similar and then treated as replicates. Since the aim of the study is the phytoplankton biodiversity, the phytoplankton dynamics is statistically summarized and included in the results.

3) Some micrographs, at least about the newly found morphospecies should be presented.

Unfortunately, we do not have “good quality” photographs to be included given that the pixel resolution of the photographs taken 20 years ago is not acceptable by modern standards.

4) Nutrient levels and other physicochemical data should be presented either in the Materials and Methods or in the Results section, rather than in the Discussion.

Nutrients and other environmental variables are now provided as Table 1 in the results section.

5) The two rightmost columns of Table 3 are not visible in the downloaded manuscript.

The table is now complete, all columns are visible.

Author Response File: Author Response.docx

Reviewer 3 Report

I have completed the revision of the ms by Cuna et al. “Phytoplankton biodiversity in two tropical, high mountain lakes in Central Mexico”. In this ms the authors documented the phytoplankton species inhabiting these two extreme, tropical ecosystems, and they compared these aquatic environments with the phytoplankton diversity from other Mexican inland waters. The topic is very interesting, and authors collected very valuable data. However, I have several comments regarding the paper, and I feel that it will need significant editing before being ready for publication. I outline several of my concerns below and have included an annotated PDF with several more comments. I hope these are useful for ms improvement.

After reading the manuscript, I found that the authors mix a lot the results and discussion sections, I suggest that the authors restructure the ms. In my opinion, the manuscript would be greatly improved if the authors delved into the fish introduction in Lake El Sol. In this sense, I think the authors could get more out of their results. Finally, I consider it relevant to include the lake area as a possible factor that structures the diversity of phytoplankton, since Lake El sol is much larger than Lake La Luna.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

Comments and Suggestions for Authors

In the new version, work has been done on restructuring the results and discussion sections and we have taken up the aspect of the introduction of fish as well as the size of the lakes to get more out of our results.

Is similar or different... ? Please rewrite your hypothesis.

Done

Add a summary table with average values of the main physical and chemical variables of both lakes.

Done

Did you only get quantitative samples? did not obtain formaldehyde fixed qualitative samples?

We obtained only quantitative samples from three water depths. Since the lakes’ shallowness and continuous mixing, the three water depths turned out to be similar and then treated as replicates.

please add the depth of the midpoint sample

The lakes are astatic, then we added the average depth of both lakes as reference.

add concentration, 1%?

Done

Please indicate in the figure which are Sol and Luna lakes.

Done

Since table 1 is long and occupies more than one page, it is recommended to place the column names on all pages.

Done

I recommend relocating this paragraph to the discussion, since they are not results obtained in this work.

Done

Table 3 and the related results (line 168-169) should be in the Results section.

We think this comparison table with published information is not result but discussion.

In your hypothesis you mentioned: "that the lakes Sol and Luna present likely higher phytoplanktonic diversity compared to their temperate equivalents."

In my opinion you should include this part of the hypothesis in the Discussion section, and also mention which factors could drive the difference in diversity of the phytoplankton among HML lakes and the temperate ones.

We added the following paragraph. The trend of increasing species diversity from the poles towards the tropics or the LDG (latitudinal diversity gradient) has been long postulated in terrestrial and marine ecosystems (e.g., Hillebrand 2004). However, the mechanisms behind this trend are still far from being understood, representing one of the significant contemporary biogeography challenges (e.g., Willig et al. 2003). The reduced climatic variation existing in areas close to the Equator favors tropical species to develop small thermal tolerance ranges and a restricted dispersion. These features, especially in mountainous environments (e.g., HML), generate rapid isolation between populations and develop new species (Polato et al. 2018). Nevertheless, there is no evidence connecting ecological processes gradients within communities at a local scale directly to the geographic gradient in biodiversity (Usinowicz et al. 2017).

But in line 83-84 you mentioned that Luna lake presented higher nitrogen, particularly nitrate concentrations than El Sol [8,9]. Please clarify this point.

We replaced “nutrient concentration” by “trophic status” to avoid confusion. Despite the higher nitrate concentration in Lake La Luna, the very low SRP availability (P = limiting nutrient of the primary productivity) lead to ultraoligotrophic condition as indicated by the very low chlorophyll-a concentration. On the contrary, with lower nitrates concentration but higher SRP availability, Lake El Sol becomes more productive (oligotrophic), displaying higher chlorophyll-a concentration.

There is much literature regarding the effects of introducing rainbow trout in naturally fishless environments. Below, I suggest some works that you can cite.

We added the following paragraph. Fish introduction is associated to biodiversity loss in shallow lakes (Izaguirre et al. 2018). However, phytoplankton biodiversity in fish-stocked Lake El Sol was higher than in the fishless Lake La Luna. Rainbow trout, in addition to promoting turbidity, also change the food webs, generate trophic cascade effects, and change the plankton functional groups with Cyanobacteria as a dominant group in fish-stocked lakes (e.g., Izaguirre and Saad 2014, Lancelotti et al 2016, Saad et al. 2018). However, Cyanobacteria were dominant in fishless Lake La Luna, while Chlorophytes in fish-stocked Lake El Sol, the opposite to expected.

I recommend that the authors look at the results again and pay special attention to the species of cyanobacteria that are present in Lake El Sol (but are not present in Luna lake), it could be due to the stocking of trout (?) (See the suggested bibliography).

None of the Cyanobacteria found in Lake El Sol (e.g., Anabaena cf. lapponica, Anabaena sp., Limnoraphis birgei, Merismopedia sp., Synechocystis minuscula, Pseudoanabaena sp., Chrysococcus minutus, Dinobryon cf. Sociale, Mallomonas cf. Acaroides, Mallomonas sp. 2) are abundant (<0.01%). Chlorophytes are the dominant group in Lake El Sol, differing from the expected Cyanobacteria in fish-stocked lakes.

I recommend not to include again numbers, here you must highlight the more important findings...

Done

Reviewer 4 Report

The dominant species were identified, did you also monitor the quantity of species and calculate the biodiversity index?
All the taxa found in Lake La Luna were present also in Lake El Sol, but the “Dominant taxa were 28 species in El Sol and 22 species in La Luna, of which 12 are common to both lakes”, why the dominant taxa were different?
Study area, how far away are the two lakes?

Author Response

Reviewer 4

Comments and Suggestions for Authors

The dominant species were identified, did you also monitor the quantity of species and calculate the biodiversity index?

Species were quantified and the diversity index calculated; these data are now included in the text.

All the taxa found in Lake La Luna were present also in Lake El Sol, but the “Dominant taxa were 28 species in El Sol and 22 species in La Luna, of which 12 are common to both lakes”, why the dominant taxa were different?

In the introduction, third paragraph, it says: Despite their closeness and similar geologic context, they differ in physical and chemical characteristics [9] and in their biological communities known so far (e.g., zooplankton and macrobenthos, 10,11, 12,13]. The morphometric, physical, and chemical differences are at least partially responsible of the differences. Also, as mentioned in the discussion the introduction of rainbow trout in the 1950´s should also impacted the aquatic communities. We consider your suggestion for the discussion.

Study area, how far away are the two lakes?

They are 600 m apart, this is mentioned in the introduction, third paragraph: “These lakes are about 600 m away from each other, inside the crater of the Nevado de Toluca volcano”.

Round 2

Reviewer 3 Report

After reading the revised version of the ms by Cuna et al. “Phytoplankton biodiversity in two tropical, high mountain lakes in Central Mexico”, I greatly appreciate the changes incorporated into the ms, but I still found several flaws. Although the authors re-wrote the hypothesis, the introduction lacks theoretical information to frame the hypothesis. I also found that many interpretations and conclusions presented in the Discussion are not derived from the results presented and need to be re-written. These are not supported by the analyzes carried out in this work. I consider that the ms needs substantial revision before its publication.

I provide below more specific comments in each section, I hope these are useful for ms improvement.

Introduction

I believe that the manuscript would benefit from increasing the paragraphs on theoretical framework related to the hypothesis (e.g., lake size, trophic status, limnological characteristics), and reducing the emphasis on Lake El Sol and La Luna information, those lines could be in the section of the study area.

Line 66-68: The authors suggest that “This information constitutes a necessary baseline for future studies, which could evaluate possible changes in phytoplankton species in these lakes.” However, this study was carried out 20 years ago and surely the conditions have changed, if possible I recommend going once again to take samples and evaluate in what state these lakes are today.

M&M

Line 101: be consistent and use parentheses.

Results

Line 183-184: “La Luna the phytoplankton density averaged 155 607 ± 144 026 mm3 m-3”. Is biomass, not density.

Discussion

The authors stated in the Introduction that they compared HML tropical lakes with other temperate HML aquatic environments, however this issue is only addressed in the Discussion. On this regard, in my opinion they should put Table 5 in Results section.

276: “without” in lowercase

Line 328-329: The authors mention “Three drivers match our findings on their effects on the phytoplankton biodiversity of Lakes El Sol and La Luna: surface area, pH, and UVR (Table 5)”. However, they did not perform any statistical analysis to reach that conclusion. I suggest you perform at least some multivariate analysis between the physical and chemical variables and phytoplankton abundance or another similar analysis.

Two tables “5” are mentioned: one related to "Phytoplankton taxonomic richness" and the last table about the "drivers", I think the latter should be numbered as table 6.

Line 343- 344: Again, the authors mention that “Finally, the low pH combined with the high incident UVR could be the most important drivers in reducing the phytoplankton diversity in Lake La Luna…”. But there is no analysis to support this claim.

Supplementary material

A parenthesis is missing in the caption of the supplementary material. Authors must define "NF", which appears in the table. Also, since table is long and occupies more than one page, I recommend to place the column names on all pages.

Author Response

Referee 3 – 2^nd review

We have included the theoretical frame for the hypothesis as requested.

I also found that many interpretations and conclusions presented in the Discussion are not derived from the results presented and need to be re-written. These are not supported by the analyzes carried out in this work. I consider that the ms needs substantial revision before its publication.

We have included statistical analysis (MANOVA) showing the variables (environmental and biological) that are statistically different in both lakes.

The discussion is supported in the reported effects of the different drivers on the phytoplankton biodiversity. Lake La Luna characteristics (smaller, more acidic, more transparent, ultraoligotrophic, with no macrophytes and fishless) resulted in a lower phytoplankton species richness, density, and biomass than in Lake El Sol (larger, less acidic, less transparent, oligotrophic, with some macrophytes inhabiting the littoral zone, and with rainbow trout).

We analyzed the most important drivers that could be explaining the difference in the phytoplankton species richness between both lakes. However, out of the six most important drivers known to affect the phytoplankton biodiversity, 50% (3 out of six) of the cases match the expected response (higher or lower species richness). We provide possible explanations for this.

I provide below more specific comments in each section, I hope these are useful for ms improvement.

Introduction

As mentioned before, we have included the theoretical frame for the hypothesis as requested and provide the corresponding literature support.

Line 66-68: The authors suggest that “This information constitutes a necessary baseline for future studies, which could evaluate possible changes in phytoplankton species in these lakes.” However, this study was carried out 20 years ago and surely the conditions have changed, if possible, I recommend going once again to take samples and evaluate in what state these lakes are today.

To be able to respond to the comment “However, this study was carried out 20 years ago and surely the conditions have changed” it is necessary to know which were the characteristics of these lakes 20 years ago. With no baseline, there is not possible to know if the ecosystems have changed and if so, in which direction and the magnitude of the change. This is exactly the main contribution of this study. Unfortunately, under COVID-19 conditions access to National Parks in Mexico is prohibited.

The comparison between today and 20 years ago characteristics constitutes another -future- investigation since it would require: 1) a new one-year study, and 2) to count with a reference baseline, which is the aim of the present manuscript. We are very interested in undertaking a new survey on both lakes and then we would be able to make the comparison. However if these data are published now, they might be used not only by our research group but by all the scientific community in Mexico and elsewhere.

M&M

Line 101: be consistent and use parentheses.

Done

Results

Line 183-184: “La Luna the phytoplankton density averaged 155 607 ± 144 026 mm3 m-3”. Is biomass, not density.

Done

Discussion

Since Table 5 includes mostly published data from other lakes, and this information is provided as a comparison, we consider is more pertinent to discussion than to results sections. The percentage contribution of the dominant phytoplankton groups, and the species richness of Lakes El Sol and La Luna are already provided in the results.

276: “without” in lowercase

Done

We included a new statistical analysis (MANOVA) showing that the variables are statistically different between both lakes. Since the manuscript focus on phytoplankton biodiversity, we look for identifying the drivers explaining the difference of biodiversity between both lakes. Once identified the variables that differ in both lakes, we discussed the published positive or negative effects of these different drivers on the phytoplankton diversity of Lakes El Sol and La Luna.

Two tables “5” are mentioned: one related to "Phytoplankton taxonomic richness" and the last table about the "drivers", I think the latter should be numbered as table 6.

Done

Statistical analyzes showed the variables in which the lakes differ and therefore the probable cause of the difference in biodiversity. Subsequently, we analyze/discuss, based on the bibliography, the reported effects of the aforementioned variables on the biodiversity of phytoplankton. In this way, the variables that we consider are responsible for the differences in biodiversity in the lakes are proposed. However, we mentioned in the manuscript it must be taken into account that the differences in some variables, such as trophic status, in the literature are evidenced in contrasting situations such as oligotrophic versus eutrophic, while in Lakes La Luna and El Sol the difference is subtle between ultra-oligo and oligotrophic, respectively.

Supplementary material

Done

Author Response File: Author Response.pdf