Review Reports

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

-

Is the research design appropriate?

Yes

-

Are the methods adequately described?

Yes

-

Are the results clearly presented?

Yes

-

Are the conclusions supported by the results?

Yes

-

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: The territory of the authors' own research mostly coincided with that indicated in the literature. However, 123 species previously mentioned were not discovered by the authors (a total of 153 species were discovered, 84 of which were new. Consequently, only 69 species that were mentioned earlier were found by the authors. 192 - 69 = 123). How can we explain the loss of such a large number of species? Perhaps there were errors in the definition? Have environmental changes affected the distribution of certain species? Maybe these species that were mentioned earlier no longer live on the territory of Bolivia?

Response 1: We acknowledge the reviewer’s observation regarding the discrepancy in species counts between our study and the existing literature. There are several factors that may explain this difference:

Geographical focus: Our sampling points are primarily located in the Altiplano and Andean regions of Bolivia. In contrast, much of the existing literature on Bolivian rotifers focuses on the Amazonian region, which is known for its higher biodiversity. Consequently, the absence of sampling points in the Amazonian zone in ULRA collection likely contributed to the lower number of species recorded, as this region is richer in species diversity compared to the Andean region.

Sampling effort and methodology: Some of the referenced literature encompasses long-term studies that include samples from both rainy and dry seasons. These extended and repeated sampling efforts increase the likelihood of capturing a broader range of species. In comparison, our study represents a snapshot of the local biodiversity, which may not fully reflect the temporal variability in species presence.

Environmental and temporal changes: It is also plausible that environmental changes have affected the distribution and presence of certain species. ULRA collection samples have been taken a long many years, so we may have shifts in species composition over time, potentially explaining the absence of previously recorded species in our current study.

Taxonomic Revisions and Identification: We have taken considerable care to verify our species identifications against the latest taxonomic literature. It is possible that some of the species not found in our study have undergone taxonomic revisions or synonymizations, which might account for discrepancies in species lists.

We believe that the differences in species records are attributable to the factors outlined above rather than errors in species identification, and we added some lines to the manuscript to clarify this observation (lines 184 to 197):

“In our study, only 69 species that were mentioned earlier by other authors were found in the ULRA samples collection. The discrepancy between the number of rotifer species identified in our study and those reported in previous literature can be attributed to sever-al factors. Our research primarily focused on sampling sites in the Altiplano and Andean regions, whereas much of the existing literature emphasizes the Amazonian region of Bo-livia, known for its higher biodiversity. The absence of sampling in the Amazonian region likely contributed to the lower number of species recorded in our study. Additionally, differences in sampling effort and methodology may have played a role; some of the earlier studies involved long-term monitoring across different seasons, increasing the likelihood of detecting a wider range of species. Environmental changes over time may have also influenced species distributions, potentially leading to the absence of some species previously recorded. Lastly, taxonomic revisions and careful verification of species identifications against the latest literature may have resulted in some species being synonymized or reclassified, contributing to the observed discrepancies in species counts.”

Comment 2: Tables and figures in the article clearly illustrate information presented. However, there seems to be an error in Table 1. In the family Epiphanidae, the column "own observations" indicates "0", which should probably be "5".

Response 2: Agree. We have, accordingly, modified the table.

Comments 3: Page 5, line 4 from the bottom: "467" is not a reference to a literary source, but rather the number of Rotifera species found in Brazil. It is likely that this number should be deduced from the square brackets.

Response 3: Agree. Thanks for your observation. It was changed.

Additional clarifications

Reviewer 3 have suggested a reference that increased the number of literature species in 3, so please note the changes in results, table 1 and supplementary material.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We have made some changes to improve it.

Is the research design appropriate?

Can be improved

As we worked with samples from diverse projects along the time, it is impossible for us to change the research design.

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: In the table, Bdelloidea and some families are incorrectly arranged

Response 1: Agree. We have, accordingly, modified the table.

Comment 2: The authors said that the sampling effort is enough for the Altiplano, but it has to be supported by a statistical analysis so the statement could be rejected or not. Additionally, the total number of water bodies found in the country and the proportion sampled is not mentioned. I suggest some estimators like jack 2, chao 2, bootstrap.

Response 2: Thank you for your suggestion, we agree. We have conducted a Chao2 analysis and modified the text in lines 133-143.

“The map of all sampling points (Figure 1) shows that two of Bolivia's three principal basins (the Amazon and La Plata) are severely under-sampled, indicating that there are potentially more rotifer species in this basin that have yet to be documented. On the other hand, the species accumulation curve for the Altiplano basin started to level off, although no clear plateau was observed (Figure 2). To evaluate the adequacy of our sampling effort, for the Altiplano basin, we conducted a Chao2 analysis that provides an estimate of the total species richness, including both the observed species and those that are likely present but not detected in the samples. In our study, the Chao2 value (estimated total number of rotifer species in the Altiplano) was 116, while the observed number of species in this region was 106. This suggests that our sampling effort has captured a substantial and representative portion of the rotifer diversity of Altiplano basin.”

Comments 3: The authors suggest molecular and metabarcoding analyses. If it has been little studied in the country, it will generate a lot of data that is difficult to find in databases. I suggest mentioning this and also discussing the need for taxonomy in the country, its ecological and evolutionary importance as well as social importance in recreational areas. What differentiates it from other countries or, without considering that, highlights the impact of knowledge in this area of research in the country because of the evolutionary process and environmental differences that could be important for other researchers in the world. If the ecological importance of the environment and the variations in which organisms evolve are highlighted, it could emphasize the need to continue studies and call for a larger non-local audience.

Response 3: Thanks for your observation. We have clarified the idea in lines 215-236:

“The taxonomy of rotifers in South America is complex and constantly evolving. Numerous species in the region have not been thoroughly described, and debates persist regarding the taxonomic status of several common species. Recent advancements in molecular techniques, such as DNA barcoding and metabarcoding, have provided valuable in-sights into these taxonomic ambiguities, facilitating more precise species identification and diversity assessments [37]. Although the application of molecular and metabarcoding analyses in Bolivia has been limited, the potential for generating extensive datasets that are currently underrepresented in global databases is significant. This situation highlights the pressing need for taxonomic research within the country, which holds profound eco-logical, evolutionary, and societal implications. Enhanced taxonomic efforts are essential not only for understanding ecological roles of rotifer species and informing conservation strategies but also for the effective ecological management of natural and recreational areas.

The diverse ecological landscapes of Bolivia, including high-altitude Andean lakes and the rich Amazon basin, offer exceptional opportunities to study not only rotifers diversity but also rotifers adaptation and evolution under varied environmental pressures. To achieve a comprehensive understanding of rotifer ecology and distribution in Bolivia, further extensive sampling, particularly in the under-sampled Amazon and La Plata basins, is imperative. The integration of contemporary taxonomic and molecular methodologies is expected to uncover numerous additional species. Establishing standardized sampling protocols and developing a national database for rotifer records would greatly facilitate future research efforts.”

Comment 4: It mentions that "many freshwater ecosystems in the region are threatened by pollution" How many? From Bolivia or other countries? it's unclear the message

Response 4. Thank you for pointing out the need for clarification.

We were talking about South America region, as is it mentioned in the previous sentence: “The conservation status of rotifers in South America is largely unknown. Many freshwater ecosystems in the region are threatened by pollution,…..”.

We have updated the manuscript in lines 248 to 253 to specify and cite a relevant review that document the extent of pollution in South America:

“South America hosts some of the world's most varied water resources, featuring the largest river (the Amazon), the longest mountain range (the Andes), and the driest region (the Atacama Desert) on the planet. But the conservation status of rotifers in South America is largely unknown. Many freshwater ecosystems in this region are increasingly threatened by various forms of pollution, habitat destruction, and climate change, between other pressures [37], …..”

We believe this will clarify the message and provide a more accurate representation of the environmental challenges faced by emphasize the environmental pressures faced by aquatic habitats in Bolivia and the broader region of South America.

Comment 5: Some general doubts about the knowledge presented in the manuscript, Are there long-term study data? What environmental factors are known to influence the distribution of rotifers? What is the quality of the sites where rotifers inhabit? Can they be used in quality indices? there are other important contributions of rotifer research in applied science?

Response 5. Thank you for your comments and questions.

Regarding the query about long-term study data, we would like to clarify that our samples were collected from various studies conducted over different years. These studies provide snapshots of rotifer communities at specific times and locations rather than continuous long-term datasets. Consequently, the temporal and spatial variability inherent in these data means they are not directly comparable across years or sites.

Environmental factors are known to influence the distribution of rotifers. However, the physicochemical variables associated with our samples are not yet consolidated into a comprehensive database. We recognize the importance of this information and are actively working to compile and analyze these variables, which will form the basis of a future manuscript.

In terms of water quality, our sampling locations range from pristine environments to more impacted areas. While rotifers can serve as bioindicators due to their sensitivity to changes in water quality, the current dataset does not include sufficient environmental data to use them reliably in water quality indices at this time.

Additional clarifications

Reviewer 3 have suggested a reference that increased the number of literature species in 2, so please note the changes in results, table 1 and supplementary material.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We have made some changes to improve it.

Is the research design appropriate?

Yes

-

Are the methods adequately described?

Can be improved

We have made some changes to improve it.

Are the results clearly presented?

Yes

-

Are the conclusions supported by the results?

Yes

-

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: Authors should include more updated literature dealing with the identification keys for rotifers, like: de Paggi, S. B. J., Wallace, R., Fontaneto, D., & Marinone, M. C. (2020). Phylum rotifera. In Thorp and Covich's freshwater invertebrates (pp. 145-200). Academic Press, and re-evaluate the rotifer records they have for Bolivia, because it is highly possible that some species records (including new records) can belong to species described/reported after the 2000 decade. In my consideration, identifying species based on literature for more than 20 years, is not updated and may cause species misidentification.

Response 1: We appreciate the reviewer’s suggestion to include more updated literature, specifically the reference to de Paggi et al. (2020). In response to this suggestion, we have thoroughly consulted the recommended literature.

In our review of the taxonomic keys and species distributions provided in de Paggi et al. (2020), we didn’t identify any specific records that were not previously unaccounted for in our database, but we found the original reference; Segers et al., 1994, cited in de Paggi et al. (2020). Previously we had spent unsuccessful efforts to obtain this publication (we only had access to the abstract), but fortunately we were able to find it in the last few days, with which we found 3 specific records that were not previously unaccounted for in our database. We have made the necessary corrections in the manuscript, Table 1, and the supplementary material.

To ensure the accuracy of our species identifications, we reassessed our specimens by comparing their photographs (when available) with the most recent taxonomic resources. This allowed us to confirm the correspondence with current taxonomic classifications. Two specialist coauthors have also updated and curated our list of species names accordingly.

However, we face limitations in reviewing all species identifications due to the large volume of our dataset, which includes more than 400 samples from over 200 locations. Given these constraints, it is currently impractical to re-evaluate every identification within the scope of this study. We have prioritized the most critical updates and corrections based on the available resources and will continue to refine our database for future studies.

Comment 2: It would be great if authors can include in S2 table the dates rate species where collected in the table title (i. e. “ S2. List of species of Rotifers observed from 2005 to date in samples of the Unit of Limnology and Aquatic Resources from Bolivia territory.

Response 2: Agree. We have, accordingly, modified the table.

Comments 3: Moreover, it is confusing to follow two different tables, one for the recorded species and other for the obtained in the current study, I recommend the authors to provide a single supplementary table that includes in taxonomical order the rotifer species checklist. For the cases that a species have been recorded in this current document, authors may include the word “this study” in the reference section of the table

Response 3: Agree. Thanks for your observation. Both tables were merged.

Additional clarifications

We have found the statement “You will find other specific comments on the attached revised manuscript.” But the attached document didn’t correspond to our manuscript. We will be happy to answer any other concern that the reviewer may have.