Review Reports

Round 1

Reviewer 1 Report

This study contributes with new taxonomic information of parabathynellids from a poorly studied and difficult to Access environments, subterranean waters from South Korea. I consider this study highly valuable as it contributes to understand distribution patterns based on morphological and molecular analyses. This paper further describes two new species based on morphological attributes and infer about the phylogenetic position of the group base don 18S RDNA. This study, however, has some pitfalls and format problems that should be addressed prior to be accepted for publication.

The manuscript needs to be improved in the following sections:

Abstract: The background, main objective of the research and methodology are missing. It is recommended to include missing sections and a broader discussion.

Keywords: It is recommended not to include the name of the new species as keywords and make a better selection of other keywords to gain visibility.

Introduction: Information on the importance of parabatynellids, global distribution, diversity and evolutionary relationships as subterranean crustaceans are missing. Please include the biological importance of the group. Please include a brief description of what is the type of environment from where the new species come from. In Figure 1A, please remove uninformative lines (right and bottom). Figure 1B, does not present any coordinate reference or illustrative reference (names of country or sea) to clearly identify what region is referring to. Please include such information.  The figure caption does it is not specified the name of region shown.

Methodology: The tables are unordered; it is first presented table 3 and then table 2. Please correct them to be sequential numbered.  The information presented at the end of the methodology section should be included in the results section. Table 3 and figure 13 are results, please include in the respective section. In the description of the study area, it is mentioned the Namhangang river, but not shown in Figure 1 A. Please modify the figure 1 and add the missing information. Table 1 is incomplete since it lacks the sequence numbers from GenBank. If this information is still in progress, please specify it in the manuscript. In table 3, names of species are confused and repetitive. Please use a better organization. After figure 1, it is shown the figure 13, this is not a correct and logical order. Please use a logical figure numerical order.

Results: In table 2, please reword as follows “… among Nipponbathynella spp.” by “… among species of Nipponbathynella”. The organization of the table is confusing. On first line the names are not appropriate organized. First column describing morphological attributes are highly confused, please use a better organization. The symbology is not described, please describe what “?” and bold word means. The order of the tables in the manuscript is not correct, table 2 must be placed before table 3.

Discussion: It is recommended to more profoundly discuss the ecological importance of parabatynellid group, particularly that of new species. The species distribution must also be better discussed. Please described what is the significance of p-value for phylogenetic analysis.

In the following text “The morphological affinity between the genera Nipponbathynella, Arisubathynella and Parabatynella has been reported in previous studies [6, 11, 12]”. Please discuss what does previous studies described.

In this text  “Our analysis based on molecular data shows the validity of the five genera that were previously classified as one 'morphogenus' based mainly on morphological characteristics (see Molecular analysis). These results provide important data for the genetic pattern of the family Parabathynellidae, not only in the Korean peninsula but also in other regions”, please specifies the location from molecular data comes from and discusses what does “other regions” mean.

References: The references cited are mostly old and relatively few for the entire research. It is recommended to make broader documental research including more recent papers.  

The english lenguage must be improved.

Author Response

Response to Reviewer 1 Comments

 

Point 1: [Abstract] The background, main objective of the research and methodology are missing. It is recommended to include missing sections and a broader discussion.

Response 1: We added the missing sections pointed out by reviewer.

 

Point 2: [Keywords] It is recommended not to include the name of the new species as keywords and make a better selection of other keywords to gain visibility.

Response 2: We deleted the name of the new species in keywords and added ‘Korean peninsula’, ‘Syncarida’ as keywords.

 

Point 3: [Introduction] Information on the importance of parabathynellids, global distribution, diversity and evolutionary relationships as subterranean crustaceans are missing. Please include the biological importance of the group. Please include a brief description of what is the type of environment from where the new species come from. In Figure 1A, please remove uninformative lines (right and bottom). Figure 1B, does not present any coordinate reference or illustrative reference (names of country or sea) to clearly identify what region is referring to. Please include such information. The figure caption does it is not specified the name of region shown.

Response 3: We added the details about parabathynellid’s ecology, distribution, evolutionary aspect in the first paragraph; We removed the uninformative lines in the Figure 1A and added the names of countries in the Figure 1B.

 

Point 4: [Methodology] The tables are unordered; it is first presented table 3 and then table 2. Please correct them to be sequential numbered. The information presented at the end of the methodology section should be included in the results section. Table 3 and figure 13 are results, please include in the respective section. In the description of the study area, it is mentioned the Namhangang river, but not shown in Figure 1 A. Please modify the figure 1 and add the missing information. Table 1 is incomplete since it lacks the sequence numbers from GenBank. If this information is still in progress, please specify it in the manuscript. In table 3, names of species are confused and repetitive. Please use a better organization. After figure 1, it is shown the figure 13, this is not a correct and logical order. Please use a logical figure numerical order.

Response 4: We corrected the numbers of the tables (Table 2 and 3) in all text.; the sentence ‘the type materials of the new species examined in this study have been deposited in the collection at the National Institute of Biological Resources, Korea (NIBR) (Table 1).’ is presented also in “type materials” in the result section.; we changed the Hangang River to Namhangang River, which is a part of the Hangang River in South Korea, in Figure 1A.; We filled the Table 1 with voucher numbers and GenBank numbers.; We merged the cells in the species column and assigned them to two columns in Table 1; We shifted the Figure 13 after Figure 12.

 

Point 5: [Results] In table 2, please reword as follows “… among Nipponbathynella spp.” by “… among species of Nipponbathynella”. The organization of the table is confusing. On first line the names are not appropriate organized. First column describing morphological attributes are highly confused, please use a better organization. The symbology is not described, please describe what “?” and bold word means. The order of the tables in the manuscript is not correct, table 2 must be placed before table 3.

Response 5: We used horizontal lines to divide the table into sections to increase visibility. We have added details about the table to the legend.

 

Point 6: [Discussion] It is recommended to more profoundly discuss the ecological importance of parabathynellid group, particularly that of new species. The species distribution must also be better discussed. Please described what is the significance of p-value for phylogenetic analysis.

Response 5: High branch support indicates greater confidence in the evolutionary relationship represented by that branch. Higher p-values suggest stronger support for the branch. This is so general that I don't think it's necessary to include it in the discussion.

 

Point 7: In the following text “The morphological affinity between the genera Nipponbathynella, Arisubathynella and Parabathynella has been reported in previous studies [6, 11, 12]”. Please discuss what does previous studies described.

Response 6: We discussed the morphological affinities between Korean Arisubathynella and European Iberobathynella and Parabathynella in detail in our most recent paper by the same authors (Ji & Min, 2023), which we cite here. Additionally, the morphological closeness of Nipponbathynella and Arisubathynella was mentioned in the Introduction.

 

Point 8: In this text “Our analysis based on molecular data shows the validity of the five genera that were previously classified as one 'morphogenus' based mainly on morphological characteristics (see Molecular analysis). These results provide important data for the genetic pattern of the family Parabathynellidae, not only in the Korean peninsula but also in other regions”, please specifies the location from molecular data comes from and discusses what does “other regions” mean.

Response 7: This means that it can be an example of a genetic standard that can distinguish the genus of parabathynellid species occurring not only in Korea but also in 'other regions' using the 18S gene sequence. So, we changed “the genetic standard of the family Parabahtynellidae” to “the genetic standard for genus delimitation of the family Parabathynellidae”.

 

Point 8: [References] The references cited are mostly old and relatively few for the entire research. It is recommended to make broader documental research including more recent papers.

Response 8: We added relevant, more recent papers to the manuscript, and these papers were added to the Reference list.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Figure 6 on page 10 - Try to get rid of the black background around thoracopod VIII

Figure 12 on page 17 - Draw thoracopod VIII. Don’t only provide an SEM picture.

Use 18S rRNA all over the manuscript, and not 18S rDNA or simply 18S.

COI and 18S rRNA accession numbers will be provided after the article will be accepted.

Congratulations on the nice work!

Author Response

Response to Reviewer 2 Comments

 

Point 1: Figure 6 on page 10 - Try to get rid of the black background around thoracopod VIII.

Response 1: We changed the black area to white.

 

Point 2: Figure 12 on page 17 - Draw thoracopod VIII. Don’t only provide an SEM picture.

Response 2: We provided the drawing of male thoracopod VIII. The SEM picture and newly drawed picture are in Figure 13.

 

Point 3: Use 18S rRNA all over the manuscript, and not 18S rDNA or simply 18S.

Response 3: We changed 18S or 18S rDNA to 18S rRNA gene.

 

Point 4: COI and 18S rRNA accession numbers will be provided after the article will be accepted.

Response 4: we provided GenBank numbers with voucher numbers according to instruction of Reviewers.

Author Response File: Author Response.pdf

Reviewer 3 Report

The legende of Figure 8 is completely corrupted. The authors obviously had cancelled one panel (B) from the figure plate and then forgot to relabel the panels and to adjust the legend. Nonetheless, the panels are correctly referred to in the main text.

Author Response

Response to Reviewer 3 Comments

 

Point 1: The legende of Figure 8 is completely corrupted. The authors obviously had cancelled one panel (B) from the figure plate and then forgot to relabel the panels and to adjust the legend. Nonetheless, the panels are correctly referred to in the main text.

Response 1: We corrected the legend of Figure 8.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thanks for providing a corrected version for this manuscript, the document is much better than the previous one. The arguments and figures/tables are well and logically presented and therefore the results may be more sound for scientific community. I however found some minor editing problems that could be easily corrected. Please see the attached document. An important issue that I found is the facts that in spite COI sequences are presented, the analysis of them are weak and are not presented in an integrative way with the morphological identification. Usually, the integration of both disciplines are the basement of the so call integrative taxonomy. Please provide at least an NJ identification tree. Furthermore, the authors provide evidence of two genes, COI and 16S but again they are analyzed separately. I strongly recommend concatenate the genes and use a technique of species delimitation such as generalized mixed Yule coalescent-model or automatic barcode gap discovery. 

The phylogeny presented is based on one gene only, and the inferences of the resulting tree are relatively deep in an evolutionary framework. I do suggest include additional evidence of other markers, why COI is not used for phylogeny? In case the addion of other markers is not possible, then the authors must be craefully with their evolutive inferences and may focus on genetic diversity.

Comments for author File: Comments.pdf

I am not native english speaker, but I found minor problems with english lenguage

Author Response

Point 1: Please describe immediately after collection, picking? please describe.

Response 1: We added “and picked using 200 µl pipet under the stereomicroscope (SZX12, Olympus, Japan).”.

 

Point 2: it is unclear this sentence, two loci of each one? please clarify.

Response 2: We added a comma (,) after “two loci of CO1 and 18S rRNA” to clarify the meaning.

 

Point 3: Why you obtained consensus sequences? The objective of the study is testing genetic divergences and potential intraspecific divergences. consensus sequences make you lose information and increase uncertainity on results.

Response 3: We did not obtain a consensus sequence from two different species. To clarify the meaning, we changed the sentence to “We combined forward and reverse sequences of raw data of CO1 and 18S rRNA genes using Geneious v. 8.1.9 for the final sequences.”.

 

Point 4-1 please describe how many sequences come from your sequencing procedure.

Point 4-2: Please describe from where the other sequences come from.

Response 4: We changed “The final alignments of the 18S data set using Geneious v.8.1.9 (Biomatters, Auckland, New Zealand) consisted of 1,790 bp for 45 sequences.” to “The final alignments of the 18S data set using Geneious v.8.1.9 (Biomatters, Auckland, New Zealand) consisted of 1,790 bp for 45 sequences including sequences of two new Nipponbathynella species. The remaining 43 sequences were taken from NCBI (https://www.ncbi.nlm.nih.gov/gene).”

 

Point 5: Please describe this abbrevation.

Response 5: We added the description “(Adenine/Guanine/Cytosine/Thymine)” for “Ns”.

 

Point 6: Please clarify why you provide pairwise distance at specific level using 18s RNA, what is the significance?. Why is not more appropriate do in it with COI? COI is species specific and will provide you additional information to infer genetic divergences at specific level.

Response 6: CO1 sequences are useful for distinguishing genetic differences between different species within the same genus (species level discrimination). The CO1 distance between our two new species is more than 20%, very clear that they are distinct species (see Molecular analysis). Table 2 assesses whether the five genera in Korea are also molecularly valid by providing 18S pairwise distances for the five genera in Korea that were previously described only morphologically in Korea.

 

Point 7: Why reproductive structures are not described. Usually, these structures are determinant for species identification.

Response 7: In the order Bathynellacea, thoracopod VIII of male and female serves as the reproductive organ. The thoracopod VIIIs were described in Figure 6, 11, and 12.

 

Point 8: IN all taxonomic figures, please add reference to know where the described structures are, as in figure 6B. for example, where are the nipple-like protrusions, where are ctenidia.

Response 8: We added the reference for each appendage. Since the names like “nipple-like protrusion” are not the general name of Bathynellacea, using these names as references to pictures may be misleading. So, we denote on the figure with arrows and added that to the descriptions.

 

Point 9: The authors must analize more in deep, the COI results of his study. At the moment, the evidence presented using this gene is weak. Please consider using statistical analysis of species delimitation such as generalized mixed yule-coalescent model and or utomatic barcode gap discovery (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html)

Response 9: Our two new species are morphologically distinct from other species clearly within the genus Nipponbathynella. Moreover, the CO1 distances we obtained between the two new species also provide molecular evidence that they are distinct species. Unfortunately, as mentioned in the discussion, we lack CO1 sequences within the Nipponbathynella genus to which the two new species can be compared. Consequently, the morphological evidence and the analyzed CO1 (20.7-20.8%) and 18S (0.7%) distances are sufficient to indicate that the two species are distinct and the new species.

Author Response File: Author Response.pdf