Review Reports - Morphological and Mitochondrial Evidence Supporting New Records of Leatherleaf Slugs (Gastropoda: Veronicellidae) in Mexico

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has great merit as it addresses species of a poorly studied family of terrestrial mollusks, which also exhibit significant morphological differences compared to other land snails. Additionally, many species within this family are of medical importance, acting as intermediate hosts for nematodes that cause disease, and of economic relevance, with several species recognized as agricultural pests. However, there are important points that should be addressed by the authors before the manuscript can be considered for publication.

Below are comments and suggestions:

When discussing the distribution of D. occidentalis, I suggest being more specific, as this species is not found in tropical and subtropical regions “around the world.” Its records are limited to the Americas and, in the case of South America, are mostly in northern regions. Please clarify and specify these distribution records.
There is no doubt about the presence of D. occidentalis in the country, as the authors provide both morphological and molecular data that support its occurrence.
Regarding the record of Simrothula prismatica, however, I recommend that the authors provide more conclusive evidence. The photos of the penis and the female part of the reproductive system are not clear, and no molecular data are presented. In the image provided for this species, the penis appears to be uncoiled (note that it is a turgid organ and may display various degrees of coiling). Additionally, the base may still be partially covered by the penial sheath.
In the image of the penial gland of D. occidentalis, please position the gland with its papilla facing upward (as was done for S. prismatica).
The photos of the female reproductive structures (panel D of Figures 2 and 3) are not satisfactory. The structures should be more clearly separated in this region, especially highlighting the copulatory bursa. After dissociation, I suggest producing a drawing (vector illustration) based on the photo.
The photo of the penis of S. prismatica is not clear. The penis must be uncoiled before photographing. Micro-pins can be used to properly position the structure. Its shape is currently not visible. It also appears that the penial sheath was not fully removed from the specimen in Figure 3, and what is indicated as the vas deferens might actually be part of the retractor muscle. Please verify this.
The authors need to deposit their newly generated sequences in GenBank. I only found sequences previously submitted by other authors listed in the manuscript.
The phylogenetic tree must include the sequences obtained from what was identified as S. prismatica, even if there are no matching sequences available in GenBank.

Author Response

Answers to Reviewer 1 of the review on Manuscript ID: taxonomy-3765458 titled “Morphological and Mitochondrial Evidence Supporting New Records of Leatherleaf Slugs (Gastropoda: Veronicellidae) in Mexico”
1. Summary
We appreciate the comments and corrections made to our manuscript. We considered all the observations and implemented the suggested modifications to improve the quality of the document. Please find below the detailed responses and the corresponding revisions/corrections.
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	Done
Are all figures and tables clear and well-presented?	Must be improved	Done
Is the research design appropriate?	Yes
Are the methods adequately described?	Yes
Are the results clearly presented?	Must be improved	Done
Are the conclusions supported by the results?	Must be improved	Done
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: When discussing the distribution of D. occidentalis, I suggest being more specific, as this species is not found in tropical and subtropical regions “around the world.” Its records are limited to the Americas and, in the case of South America, are mostly in northern regions. Please clarify and specify these distribution records.
R: In lines 63-65, we have specified the distribution of the species.
Comments 2: Regarding the record of Simrothula prismatica, however, I recommend that the authors provide more conclusive evidence. The photos of the penis and the female part of the reproductive system are not clear, and no molecular data are presented. In the image provided for this species, the penis appears to be uncoiled (note that it is a turgid organ and may display various degrees of coiling). Additionally, the base may still be partially covered by the penial sheath.
R: Photographs of these structures have been added, and molecular information is presented. Comments 3: *In the image of the penial gland of D. occidentalis, please position the gland with its papilla facing upward (as was done for S. prismatica).* R: Done Comments 4: The photos of the female reproductive structures (panel D of Figures 2 and 3) are not satisfactory. The structures should be more clearly separated in this region, especially highlighting the copulatory bursa. After dissociation, I suggest producing a drawing (vector illustration) based on the photo. R: A better separation of the structures of the female reproductive system was achieved, and the photographs are presented. We hope this will be sufficient to demonstrate the differences between the specimens presented here. Comments 5: The photo of the penis of S. prismatica is not clear. The penis must be uncoiled before photographing. Micro-pins can be used to properly position the structure. Its shape is currently not visible. It also appears that the penial sheath was not fully removed from the specimen in Figure 3, and what is indicated as the vas deferens might actually be part of the retractor muscle. Please verify this R: Done Comments 6: The authors need to deposit their newly generated sequences in GenBank. I only found sequences previously submitted by other authors listed in the manuscript. R. Done *Comments 7: The phylogenetic tree must include the sequences obtained from what was identified as S. prismatica, even if there are no matching sequences available in GenBank.* R: Done
4. Response to Comments on the Quality of English Language
Point 1:
R: We have performed an extensive revision of the English Language. We expect that the current manuscript fulfills the quality issue.
5. Additional clarifications
[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe two new veronicellid slug species from Mexico, based on morphological data and DNA sequences.

The process by which the authors identified the two newly recorded species should be made more transparent. The morphological characteristics of the genera Simrothula and Diplosolenodes should be specified, as should the criteria for distinguishing the species Simrothula prismatica (Simroth, 1914) and Diplosolenodes occidentalis (Guilding, 1824) from congeneric species.

Simrothula prismatica must be included in the molecular phylogenetic analysis, even though no DNA sequences of this species were previously available. However, it is important that it is considered in the analysis for future comparisons of the Mexican specimen with other specimens.

If additional veronicellid slug species from Mexico are to be identified, all species known from Mexico must be considered, not only the two newly recorded species. Accordingly, please add the five veronicellid slug species already known from Mexico to Table 2.

Minor issues:

Line 181. Onchidella in italics.

Lines 199–202: Make this sentence separate and reword it (it is not clearly understandable).

Lines 244–247: A review by Barratt et al. (2016, Parasitology, 143: 1087–1118) showed that the host spectrum of Angiostrongylus cantonensis is broad. Therefore, the presence of different veronicellid slug species is probably not significant. Nevertheless, the spread of veronicellid slug species increases the risk.

Figs 2–3. The individual figures are too small, despite there being enough space. Use the full width of the page for the figures and enlarge the individual figures.

Fig. 4. The labelling of the tree is hardly readable. Use the full width of the page for the figure and increase the font size of the labels.

Author Response

Answers to Reviewer 2 of the review on Manuscript ID: taxonomy-3765458 titled “Morphological and Mitochondrial Evidence Supporting New Records of Leatherleaf Slugs (Gastropoda: Veronicellidae) in Mexico”
1. Summary
We appreciate the comments and corrections made to our manuscript. We considered all the observations and modifications suggested to improve the quality of the document. Please find below the detailed responses and the corresponding revisions/corrections.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Are all figures and tables clear and well-presented?	Must be improved	Done
Is the research design appropriate?	Yes
Are the methods adequately described?	Yes
Are the results clearly presented?	Must be improved	Done
Are the conclusions supported by the results?	Yes
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: The process by which the authors identified the two newly recorded species should be made more transparent. The morphological characteristics of the genera Simrothula and Diplosolenodes should be specified, as should the criteria for distinguishing the species Simrothula prismatica (Simroth, 1914) and Diplosolenodes occidentalis (Guilding, 1824) from congeneric species.
R: Photographs of diagnostic structures for both species were added, and an effort was made to ensure that the information presented in Table 2 clarified the differences between species.
Comments 2: *Simrothula prismatica* must be included in the molecular phylogenetic analysis, even though no DNA sequences of this species were previously available. However, it is important that it is considered in the analysis for future comparisons of the Mexican specimen with other specimens.
R: S. prismatica was included in the phylogenetic analysis and the sequences obtained in this study were submitted to the GenBank. Comments 3: If additional veronicellid slug species from Mexico are to be identified, all species known from Mexico must be considered, not only the two newly recorded species. Accordingly, please add the five veronicellid slug species already known from Mexico to Table 2. R: Done

4. Response to Comments on the Quality of English Language
Point 1:

R: We have performed an extensive revision of the English Language. We expect that the current manuscript fulfills the quality issue.

5. Additional clarifications

As for minor issues, the corresponding changes were made in the hope that they would be resolved satisfactorily.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. Introduction, line 30 e 31

The Veronicellidae have a predominantly tropical and subtropical distribution, mainly in the Americas, but they are also present in parts Africa and Asia.

Suggestion: “Veronicellids are predominantly distributed across tropical and subtropical regions, occurring naturally in the Americas, Africa, and Asia.”

Veronicellids are very diverse in Africa and Asia as well, in their tropical areas.

2. Introduction, p. 3, line 89-94

Comment: To include the report of D. occidentalis in Brazil as well.

3. Table 1

Laevicaulis sp.

Belocaulus sp.

Comment: “sp.” “should not be italicized”

4. Figure 2 is duplicated

Comment: I understand it is duplicated because it shows both the previous version and the modified one. However, in the modified version, it seems that the penis is not positioned properly. I have the impression that the part located near the letter B should be positioned upwards. Please check this. Note that the base of the penis base is the most enlarged portion.

The same happens with the Figure 3. But, I understood that is because it includes the previous version and the modificated.

Also, unfortunately, the female portion is not well visualized and does not appear to be completely dissociated. A drawing might help the authors illustrate more clearly what they intend to show.

5. Results

177-179: The bursa duct (db) is thicker than in Diplosolenodes occidentalis and does not have the bifurcation.

Comment: It is not clear what the authors mean when they say that the bursa does not have a bifurcation. I was wondering if you are talking about the “canalis junctor” or “ducto de ligação” that all veronicellids have. Please revise this statement.

6. The sequences obtained for the individuals from the Tapachula municipality exhibited a 97% similarity with the Diplosolenodes occidentalis GenBank sequence KM489366.1, while those corresponding to the individuals from Huehuetán demonstrated a 98.36% similarity with this same sequence.

Comment: This sequence from Tapachula is what you considered S. prismatica?

I believe they are the same species (=D. occidentalis), based on your morphological and molecular data. It is important to keep in mind that this species shows variation in external coloration.

I suggest re-examining the penial gland of the specimen you refer to as S. prismatica to check for the presence of one or two short tubules, similar to those you observed in D. occidentalis I believe such tubules may be present.
Additionally, I recommend reviewing other internal morphological characteristics that you considered to differ from D. occidentalis (as the bursa copulatrix and the retractor muscle of the penis).
Finally, I also suggest re-evaluating your sequence from Tapachula to verify whether it requires any editing, as its similarity may actually exceed 97%. In any case, they seem to correspond to different haplotypes of the same species.

Also, S. prismatica is considered a synonym of S. fuhrmanni. Please see Gomes et al. (2006) for more details, also to analyse the morphology of this species.

7. Table. 2. Note that D. occidentalis has light, very short lines regularly distributed across its entire notum, in addition to the spots. This feature is very characteristic and distinguishes this species from S. plebeia, for example, which shows only spots and occasionally some brown lines.

8. You stated that:

The sequences obtained from the individuals from the Tapachula municipality exhibited 97% similarity with the Diplosolenodes occidentalis GenBank sequence KM489366.1, while those from Huehuetán showed 98.36% similarity with this same sequence.

Comment:
At the same time, you state: “Conversely, given the lack of available sequences for the genus Simrothula, it was not possible to compare our molecular results with those of related taxa.”

Considering this statement, I understood that the specimens from Tapachula are what you refer to as S. prismatica. So, why did you state that no sequences were available? Also, the sequences of “S. prismatica” are in figure 4.

I believe they represent different haplotypes of the same species. Please, check this.

9. Page 12. You stated: “Additionally, the identification of Simrothula prismatica in the United States is reported, although without current evidence of established breeding populations.”

Comment: Simrothula prismatica in the United States? I did not understand this statement.

Author Response

For research article

Response to Reviewer X 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/Can be improved/Must be improved/Not applicable
Are all the cited references relevant to the research?	Yes/Can be improved/Must be improved/Not applicable
Is the research design appropriate?	Yes/Can be improved/Must be improved/Not applicable
Are the methods adequately described?	Yes/Can be improved/Must be improved/Not applicable
Are the results clearly presented?	Yes/Can be improved/Must be improved/Not applicable
Are the conclusions supported by the results?	Yes/Can be improved/Must be improved/Not applicable
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Introduction, line 30 e 31 The Veronicellidae have a predominantly tropical and subtropical distribution, mainly in the Americas, but they are also present in parts Africa and Asia. Suggestion: “Veronicellids are predominantly distributed across tropical and subtropical regions, occurring naturally in the Americas, Africa, and Asia.” Veronicellids are very diverse in Africa and Asia as well, in their tropical areas.
Response 1: We agree with this comment; the statement was corrected.
Comments 2: Introduction, p. 3, line 89-94 Comment: To include the report of D. occidentalis in Brazil as well
Response 2: Agree. We have modified this point. Line 70 Brazil was added. Comments 3: Table 1 Laevicaulis sp. Belocaulus sp. Comment: “sp.” “should not be italicized” Response 3: Done. Comments 4: Figure 2 is duplicated Comment: I understand it is duplicated because it shows both the previous version and the modified one. However, in the modified version, it seems that the penis is not positioned properly. I have the impression that the part located near the letter B should be positioned upwards. Please check this. Note that the base of the penis base is the most enlarged portion. The same happens with the Figure 3. But, I understood that is because it includes the previous version and the modificated. Also, unfortunately, the female portion is not well visualized and does not appear to be completely dissociated. A drawing might help the authors illustrate more clearly what they intend to show Response 4: We have modified the composition and added the diagrams of the female reproductive in both species. Comments 5: Results 177-179: The bursa duct (db) is thicker than in Diplosolenodes occidentalis and does not have the bifurcation. Comment: It is not clear what the authors mean when they say that the bursa does not have a bifurcation. I was wondering if you are talking about the “canalis junctor” or “ducto de ligação” that all veronicellids have. Please revise this statement. Response 5: We have corrected the statement. Comments 6: The sequences obtained for the individuals from the Tapachula municipality exhibited a 97% similarity with the Diplosolenodes occidentalis GenBank sequence KM489366.1, while those corresponding to the individuals from Huehuetán demonstrated a 98.36% similarity with this same sequence. Comment: This sequence from Tapachula is what you considered S. prismatica? I believe they are the same species (=D. occidentalis), based on your morphological and molecular data. It is important to keep in mind that this species shows variation in external coloration. I suggest re-examining the penial gland of the specimen you refer to as S. prismatica to check for the presence of one or two short tubules, similar to those you observed in D. occidentalis I believe such tubules may be present. Additionally, I recommend reviewing other internal morphological characteristics that you considered to differ from D. occidentalis (as the bursa copulatrix and the retractor muscle of the penis). Finally, I also suggest re-evaluating your sequence from Tapachula to verify whether it requires any editing, as its similarity may actually exceed 97%. In any case, they seem to correspond to different haplotypes of the same species. Also, S. prismatica is considered a synonym of S. fuhrmanni. Please see Gomes et al. (2006) for more details, also to analyse the morphology of this species. Response 6: An analysis of genetic distances was performed (Table 1 in appendix) among genera of the family Veronicellidae using sequences available in the GenBank, and in the case of the genus Simrothula, those obtained in this study, the genetic divergence between Simrothula and Diplosolenodes is 1% (0.01). In some mollusc groups, valid species with divergences of less than 1% have been documented, provided that other characters (such as morphological and anatomical characters in this case) support their differentiation as Dayrat (2010) mentioned. Based on the support values obtained in the phylogenetic tree and species delimitation analyses, we considered the validity of Diplosolenodes occidentalis and Simrothula prismatica is supported. Although the genetic distance between them is low, the percentage of divergence can vary considerably between lineages (Puillandre et al., 2012; Kekkonen & Hebert, 2014). We also conducted species delimitation analyses (SDP, bPTP and GMYC) detecting two independent lineages (see appendix). We also re-examined the specimens and found clear differences among them, such as the presence of a constriction at the base of the bursa in Simrothula, forming a bud-like structure, which is not present in Diplosolenodes. The tissue that characterizes the bursa of Diplosolenodes could not be identified. However, the short tubules in the penial gland are absent in Simrothula. Based on these differences, we consider the specimens to belong to Simrothula. It would be necessary to collect additional specimens to completely solve any doubts. We, the authors, await the reviewer’s judgment, as it is important for us that there be no uncertainty. Should this evidence deemed non-sufficient, we remain open to your kind comments. According to MolluscaBase (2025), Simrothula prismatica is currently recognized as a valid species. However, previous studies (e.g., Gomes et al., 2006) have considered it a synonym of S. fuhrmanni based on morphological comparisons. In this study, we follow the nomenclature of MolluscaBase, referring to our specimens as S. prismatica. Comments 7: Table. 2. Note that D. occidentalis has light, very short lines regularly distributed across its entire notum, in addition to the spots. This feature is very characteristic and distinguishes this species from S. plebeia, for example, which shows only spots and occasionally some brown lines Response 7: We added this feature to D. occidentalis. Comments 8: You stated that: The sequences obtained from the individuals from the Tapachula municipality exhibited 97% similarity with the Diplosolenodes occidentalis GenBank sequence KM489366.1, while those from Huehuetán showed 98.36% similarity with this same sequence. Comment: At the same time, you state: “Conversely, given the lack of available sequences for the genus Simrothula, it was not possible to compare our molecular results with those of related taxa.” Considering this statement, I understood that the specimens from Tapachula are what you refer to as S. prismatica. So, why did you state that no sequences were available? Also, the sequences of “S. prismatica” are in figure 4. I believe they represent different haplotypes of the same species. Please, check this. Response 8: There may be some confusion since the sequences in Fig. 4 are those obtained in this study. The GenBank accession numbers of the sequences were added in the paragraph, as there are indeed no sequences available in GenBank. Comments 9: Page 12. You stated: “Additionally, the identification of Simrothula prismatica in the United States is reported, although without current evidence of established breeding populations.” Comment: Simrothula prismatica in the United States? I did not understand this statement. Response 9: In the work “New records of leatherleaf slugs (Gastropoda: Veronicellidae) for Nuevo León, Mexico, with an updated checklist of the species found in North America”, De Luna et al. (2004) report that the species has been intercepted in the United States, although without establishing reproductive populations. We modified the statement.
4. Response to Comments on the Quality of English Language
Point 1:
Response 1: (in red)
5. Additional clarifications
[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

For review article

Response to Reviewer X 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. [This is only a recommended summary. Please feel free to adjust to it. We do suggest maintaining a neutral tone and thanking the reviewers for their contribution although the comments may be negative or off-target. If you disagree with the reviewer's comments, please include any concerns you may have in the letter to the Academic Editor.]
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Is the work a significant contribution to the field?		[Please give your response if necessary. Or you can also give your corresponding response in the point-by-point response letter. The same as below]
Is the work well organized and comprehensively described?
Is the work scientifically sound and not misleading?
Are there appropriate and adequate references to related and previous work?
Is the English used correct and readable?
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: [Paste the full reviewer comment here.]
Response 1: [Type your response here and mark your revisions in red] Thank you for pointing this out. I/We agree with this comment. Therefore, I/we have….[Explain what change you have made. Mention exactly where in the revised manuscript this change can be found – page number, paragraph, and line.] “[updated text in the manuscript if necessary]”
Comments 2: [Paste the full reviewer comment here.]
Response 2: Agree. I/We have, accordingly, done/revised/changed/modified…..to emphasize this point. Discuss the changes made, providing the necessary explanation/clarification. Mention exactly where in the revised manuscript this change can be found – page number, paragraph, and line.] “[updated text in the manuscript if necessary]”
4. Response to Comments on the Quality of English Language
Point 1:
Response 1: (in red)
5. Additional clarifications
[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]