Phytoplankton Diversity of a Natural Karst Lake Combining Morphological and Molecular Approaches

Round 1

Reviewer 1 Report

This study combines eDNA metabarcoding and traditional morphological methods together to explore the phytoplankton biodiversity of is a natural karst lake and to compare the disadvantages of each method. Overall, this is a very interesting work, especially for subsequent environmental monitoring. I have some minor issues listed below.

1. 93 lines： The map does not have scale, latitude and longitude and it is suggested to add  in Figure 1.

2. 116-124 lines： The phytoplankton sample preservation based on morphological method is described in the paper, but the phytoplankton collection method is not described specifically.

3. 245-248lines： The morphological method found cyanobacterial phylum with high biomass, which belongs to prokaryotes, but this paper only did eDNA test for eukaryotes, why not for prokaryotes?

Author Response

We'd like to thank you for your helpful comments and suggestions, which have helped greatly improve the manuscript. We've also addressed all your concerns and summarize the changes you've made. Our responses to the comments are listed below. The line numbers we mention in the responses refer to the manuscript version with the track changes.

Reviewer #1:

This study combines eDNA metabarcoding and traditional morphological methods together to explore the phytoplankton biodiversity of is a natural karst lake and to compare the disadvantages of each method. Overall, this is a very interesting work, especially for subsequent environmental monitoring. I have some minor issues listed below.

1. 93 lines： The map does not have scale, latitude and longitude and it is suggested to add  in Figure 1.

Response 1. Thank you very much for your suggestion. We have added the map with scale, latitude and longitude in Figure 1. We also added map of Croatia and picture of the Lake.

2. 116-124 lines： The phytoplankton sample preservation based on morphological method is described in the paper, but the phytoplankton collection method is not described specifically.

Response 2. The phytoplankton collection method is described in lines 105 - 109. The biological and chemical water samples were collected using the vertical sampler (Hydro-Bios Apparatebau Gmbh, Germany). For the morphological analysis of phytoplankton, discrete samples were taken at 5 m depth intervals (from the surface to the bottom) together with the composite samples taken from the euphotic zone on all stations.

3. 245-248lines： The morphological method found cyanobacterial phylum with high biomass, which belongs to prokaryotes, but this paper only did eDNA test for eukaryotes, why not for prokaryotes?

Response 3. Thank you very much for this comment. We agree that eDNA metabarcoding should also be performed for prokaryotes. Unfortunately, eDNA metabarcoding for eukaryotes and prokaryotes uses two different gene markers, thus providing two different datasets, which cannot be used as one collective set. The differences between these two datasets preclude a joint analysis in one set, unlike in the case of the morphological approach. This part of eDNA metabarcoding for phytoplankton communities is still controversial and not fully clarified. A recent study by Hanžek et al. (2021) on phytoplankton communities in natural karstic lakes (including Lake Visovac) in Croatia acknowledged that, when comparing the two approaches, the descriptive species play the most significant role in establishing the dominant functional groups and assessing the ecological status. As eukaryotic algae were the main descriptive species in the study by Hanžek et al. (2021), as well as in this study, we also used only the eukaryotic gene marker. Hopefully, it will be possible to include prokaryotes and eukaryotes within the same eDNA metabarcoding procedure in the near future.

Reference:

Hanžek, N.; Gligora Udovič, M.; Kajan, K.; Borics, G.; Várbíró, G.; Stoeck, T.; Žutinić, P.; Orlić, S.; Stanković, I. Assessing Ecological Status in Karstic Lakes through the Integration of Phytoplankton Functional Groups, Morphological Approach and Environmental DNA Metabarcoding. Ecological Indicators 2021, 131, 108166, doi:10.1016/j.ecolind.2021.108166.

Author Response File: Author Response.docx

Reviewer 2 Report

The current manuscript entitled “Phytoplankton diversity of a natural karst lake combining morphological and molecular approaches” by Šimunović et al. investigated the phytoplankton community of a natural karstic lake by combining and comparing the morphological and molecular approach to check the applicability of eDNA metabarcoding as a biomonitoring tool. After a careful reading, I found this manuscript interesting and suitable for publication in the water MDPI journal. However, authors are requested to make certain changes in the current version. I suggest minor revision and my specific comments are:

1.      This study was done in the Krka River springs at the base of Dinara Mountain near the city of Knin in Croatia, however, the authors did not mention the location and country name in the title and abstract which makes it confusing. Please correct accordingly.

2.      The abstract lacks the major numerical findings. Please revised accordingly.

3.      Remove bold from line 20.

4.      Avoid using personal words in the manuscript (e.g., we, us, our, etc.).

5.      The authors made several statements in the manuscript (in both the introduction and discussion sections) but they did not provide supporting references. Please correct this problem carefully.

6.      The map of the study area should include geocoordinate information. Also, put a full map of Croatia in the top corner to clearly indicate where the location exists.

7.      References for standard sampling and analytical procedures are missing.

8.      Correct the ionic valences, they should be superscripted and not subscripted.

9.      Is it okay to use eDNA in the manuscript while the authors analyzed SSU rRNA samples? If I am not wrong, it should be corrected in the whole manuscript.

10.   Table 1: Define all abbreviations under the table footer.

11.   The results and discussion sections are written nicely.

12.   Line 632-637: Put this content in a new heading namely Conclusion.

Author Response

We'd like to thank you for your helpful comments and suggestions, which have helped greatly improve the manuscript. We've also addressed all your concerns and summarize the changes you've made. Our responses to the comments are listed below. The line numbers we mention in the responses refer to the manuscript version with the track changes

Reviewer #2:

The current manuscript entitled “Phytoplankton diversity of a natural karst lake combining morphological and molecular approaches” by Šimunović et al. investigated the phytoplankton community of a natural karstic lake by combining and comparing the morphological and molecular approach to check the applicability of eDNA metabarcoding as a biomonitoring tool. After a careful reading, I found this manuscript interesting and suitable for publication in the water MDPI journal. However, authors are requested to make certain changes in the current version. I suggest minor revision and my specific comments are:

1. This study was done in the Krka River springs at the base of Dinara Mountain near the city of Knin in Croatia, however, the authors did not mention the location and country name in the title and abstract which makes it confusing. Please correct accordingly.

Response 1. Thank you for your observation. Respectfully, we consider ommitting the spatial details which render the manuscript a somewhat local character, such as location and country name in the title and abstract, would be beneficial in terms of increasing the overall visibility and international renown of the Journal. Of course, if there are reasonable objections to our notion, we will gladly add the required details.

2. The abstract lacks the major numerical findings. Please revised accordingly.

Response 2. Thank you for this suggestion. We have added a sentence with some of the most important numerical findings in the abstract (Lines 17-19).

LINE 17: "A total of 51 phytoplankton taxa were found using morphological approach, whilst molecular approach discovered 97 ASVs that corresponded to algal community.”

3. Remove bold from line 20.

Response 3. As a rule, the Reynolds functional groups (FGs) or coda are officially written in bold to differentiate from other parts of text. In continuation, we note several relevant articles which follow this rule, by the creator of this grouping, as well as some of his colleagues and esteemed scientific names:

Reynolds, C.S.; Huszar, V.; Kruk, C.; Naselli-Flores, L.; Melo, S. Towards a Functional Classification of the Freshwater Phytoplankton. Journal of Plankton Research 2002, 24, 417–428, doi:10.1093/plankt/24.5.417.

Padisák, J.; Crossetti, L.O.; Naselli-Flores, L. Use and Misuse in the Application of the Phytoplankton Functional Classification: A Critical Review with Updates. Hydrobiologia 2009, 621, 1–19, doi:10.1007/s10750-008-9645-0.

Padisák, J., Borics, G., Grigorszky, I. et al. Use of Phytoplankton Assemblages for Monitoring Ecological Status of Lakes within the Water Framework Directive: The Assemblage Index. Hydrobiologia 553, 1–14 (2006). https://doi.org/10.1007/s10750-005-1393-9.

Kruk, Carla & Devercelli, Melina & Huszar, Vera & Hernández Atilano, Esnedy & Beamud, Sara & Diaz, Mónica & Silva, Lúcia & Marley, Angel. (2017). Classification of Reynolds phytoplankton functional groups using individual traits and machine learning techniques. Freshwater Biology. 62. 10.1111/fwb.12968.

Kozak, A.; Budzyńska, A.; Dondajewska-Pielka, R.; Kowalczewska-Madura, K.; Gołdyn, R. Functional Groups of Phytoplankton and Their Relationship with Environmental Factors in the Restored Uzarzewskie Lake. Water 2020, 12, 313. https://doi.org/10.3390/w12020313

4. Avoid using personal words in the manuscript (e.g., we, us, our, etc.).

Response 4. Thank you for your suggestion. We have made substitutions of all personal words throughout the manuscript (with the option „track changes“).

5. The authors made several statements in the manuscript (in both the introduction and discussion sections) but they did not provide supporting references. Please correct this problem carefully.

Response 5. The newly added references are provided in the manuscript („with the option track changes“). The following references were added in the introduction:

1. Myers, N.; Mittermeier, R.A.; Mittermeier, C.G.; da Fonseca, G.A.B.; Kent, J. Biodiversity Hotspots for Conservation Priorities. Nature 2000, 403, 853–858, doi:10.1038/35002501.
2. Gaedke, U. Trophic Dynamics in Aquatic Ecosystems. In Encyclopedia of Inland Waters; Elsevier, 2009; pp. 499–504 ISBN 978-0-12-370626-3.

 

We have also added the following references in the discussion section:

Wilken, S.; Huisman, J.; Naus-Wiezer, S.; Van Donk, E. Mixotrophic Organisms Become More Heterotrophic with Rising Temperature. Ecology Letters 2013, 16, 225–233, doi:10.1111/ele.12033.

Reynolds, C.S.; Huszar, V.; Kruk, C.; Naselli-Flores, L.; Melo, S. Towards a Functional Classification of the Freshwater Phytop-lankton. Journal of Plankton Research 2002, 24, 417–428, doi:10.1093/plankt/24.5.417.

6. The map of the study area should include geocoordinate information. Also, put a full map of Croatia in the top corner to clearly indicate where the location exists.

Response 6. Thank you very much for your suggestion. We have added the map with scale, latitude and longitude in Figure 1. Also, we have added the full map of Croatia with the location and picture of the Lake.

7. References for standard sampling and analytical procedures are missing.

Response 7. We have added the missing standard sin the Materials and methods section, line 128-130.

LINE 128: „All sampling and analytical procedures were done according to following standards: HRN EN ISO 5667-3:2012, HRN EN 15204:2008, HRN EN 16695:2015 [27–29].“

References were also added in the References list:

27. HRN EN ISO 5667-3:2012 Water Quality — Sampling — Part 3: Preservation and Handling of Water Samples.
28. HRN EN 15204:2008 Water Quality -- Guidance Standard on the Enumeration of Phytoplankton Using Inverted Microscopy (Utermöhl Technique).
29. HRN EN 16695:2015 Water Quality -- Guidance on the Estimation of Phytoplankton Biovolume.
30. Correct the ionic valences, they should be superscripted and not subscripted.

Response 8. We have thoroughly checked the whole manuscript for mistakes regarding the ionic valences and, as far as we are aware all ionic valences are written correctly.

9. Is it okay to use eDNA in the manuscript while the authors analyzed SSU rRNA samples? If I am not wrong, it should be corrected in the whole manuscript.

Response 9. Thank you for this comment and we agree that the choice of PCR primers is a critical step for species identification in eDNA samples. However, environmental DNA (eDNA) is a mixture of genomic material derived from living organisms and their remains, present in different types of environmental samples, without prior isolation of the target organisms we want to detect (in our case, from water). Primers are designed that can be specific to a particular taxonomic group depending on the type of gene being amplified. There are two main types of markers in eDNA metabarcoding: protein-coding genes (e.g. COI, rbcL) and ribosomal genes (e. g., 18S, 16S, 12S, ITS, 23S; Cao et al., 2022; Pawlowski et al., 2020; Shaw et al., 2019; Stoeck et al., 2018). And in our choice, we used ribosomal genes marker of 18S for eDNA metabarcoding.

References:

Cao, Y.; Lei, Y.; Fang, J.K.H.; Li, T. Molecular diversity of foraminiferal eDNA in sediments and their correlations with environmental factors from the Yellow Sea; Ecological Indicators 2022, 142 109294. https://doi.org/10.1016/j.ecolind.2022.109294

Pawlowski, J.; Apothéloz-Perret-Gentil, L.; Mächler, E.; Altermatt, F. Environmental DNA Applications in Biomonitoring and Bioassessment of Aquatic Ecosystems; Federal Office for the Environment, Bern. Environmental Studies. no. 2010: 71 pp., 2020; ISBN DOI: 10.5167/uzh-187800

Shaw, J.L.A.; Weyrich, L.S.; Hallegraeff, G.; Cooper, A. Retrospective eDNA assessment of potentially harmful algae historical ship ballast tank and marine port sediments. Molecular Ecology 2019, 28: 2476-2485. https://doi.org/10.1111/mec.15055

Stoeck, T.; Pan, H.; Dully, V.; Forster, D.; Jung, T. Towards an eDNA metabarcode-based performance indicator for full-scale municipal wastewater treatment plants. Water Research 2018, 144: 322-331. https://doi.org/10.1016/j.watres.2018.07.051

 

10. Table 1: Define all abbreviations under the table footer.

Response 10. Thank you for your suggestion. All abbreviations were moved to Table 1 footer.

11. The results and discussion sections are written nicely.

Response 11. We kindly thank you for this observation.

12. Line 632-637: Put this content in a new heading namely Conclusion.

Response 12. Thank you for your suggestion. The content in line 632-637 is now placed in a new heading Conclusion.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript is well written and interesting. The methods are correct and reliable. The choice of markers is also appropriate and the study well planned. In the future, it would be good to extend the research to other markers as well. In view of the above, I recommend accepting the article in its current form.

Author Response

We are thankful for your observation.