Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study builds meaningfully on previous works and provides relevant contribution to studying speciation, a topic of wide interest and importance in evolutionary biology. The MS is clearly written and provides novel and convincing evidence of sympatry between the W and S species in Listvyanka, which is a particularly interesting and relevant finding. However, some issues regarding sample origin and experimental design should be considered before publication, as outlined below.

1. Since the W individuals were collected from Listvyanka (the site where both W and S species coexist) and some of them were not genotyped, there is a risk they had been incorrectly assigned to the W species (as postulated by the authors in lines 485-486). For this reason, I recommend that all experiments involving non-genotyped animals from this site be excluded. If the parental species in such crosses cannot be confirmed, the reliability of these results is weakened.
2. After excluding non-genotyped individuals, if the number of experiments involving the W species becomes too small to draw robust conclusions, the authors should consider removing the analyses of W species from the MS. The experiments involving the E and S species already provide substantial and interesting findings.
3. Lines 472-474 (“In the case of the only juvenile in the WxS cross we suspect contamination from another tank, as at the moment there were no ovigerous females present in this cross.”): If contamination is indeed the most plausible explanation, the authors should clarify how they ensured the integrity and reliability of the remaining experiments. Specifically, what measures were taken to prevent cross-tank contamination elsewhere in the study?
4. Was the presence of natural hybrids or backcross individuals in Listvyanka investigated? Based on the legend of Figure 9, I assume that the authors genotyped the animals from Listvyanka only for the COI gene. If this assumption is correct, I would like to ask why the authors did not sequence the 18S gene fragment. Including this nuclear marker could help determine whether the W and S species are merely in sympatry in Listvyanka, or if they are in true secondary contact.

Author Response

Comment 1: The study builds meaningfully on previous works and provides relevant contribution to studying speciation, a topic of wide interest and importance in evolutionary biology. The MS is clearly written and provides novel and convincing evidence of sympatry between the W and S species in Listvyanka, which is a particularly interesting and relevant finding. However, some issues regarding sample origin and experimental design should be considered before publication, as outlined below.

Response 1: We are very grateful to the Reviewer for positive assessment of our work and valuable recommendations! Please find point-by-point responses to the questions and concerns.

 

Comment 2: 1st Since the W individuals were collected from Listvyanka (the site where both W and S species coexist) and some of them were not genotyped, there is a risk they had been incorrectly assigned to the W species (as postulated by the authors in lines 485-486). For this reason, I recommend that all experiments involving non-genotyped animals from this site be excluded. If the parental species in such crosses cannot be confirmed, the reliability of these results is weakened.

and Comment 3: 2nd After excluding non-genotyped individuals, if the number of experiments involving the W species becomes too small to draw robust conclusions, the authors should consider removing the analyses of W species from the MS. The experiments involving the E and S species already provide substantial and interesting findings.

Response 2: Thank you for the suggestion! Yes, we understand and share the concern and also considered this option prior to submitting the manuscript. However, the logic of the manuscript does not work like this, because the decision to genotype a large sample came from the results of the experiment where we observed unexpected juveniles. In our previous works with smaller samples, we only observed W animals in Listvayanka and had no reason to think otherwise. If we remove all experiments related to animals from Listvyanka, it is borderline to cherry-picking, as we only retain the data we can easily explain. Moreover, in this case, there is no logical reason to include genotyping from this spot to the manuscript, but we consider these data as important for multiple reasons. First, it is an example of a natural secondary contact of previously divided species. Second, it provides new light for the interpretation of the results we obtained earlier in a similar experiment (https://doi.org/10.3390/ijms231810858). Third, Listvyanka is one of the most easily accessible places on Baikal and thus a popular spot for scientific research for many groups. Thus, the suggested restructuring leaves out an important piece of information, which in turn is not large enough to be published separately.

So, after many discussions and considerations between co-authors, we decided to choose the path of maximal honesty and describe the results as is.

 

Comment 4: 3rd Lines 472-474 (“In the case of the only juvenile in the WxS cross we suspect contamination from another tank, as at the moment there were no ovigerous females present in this cross.”): If contamination is indeed the most plausible explanation, the authors should clarify how they ensured the integrity and reliability of the remaining experiments. Specifically, what measures were taken to prevent cross-tank contamination elsewhere in the study?

Response 4: Thank you for your question. Yes, in this case, we consider contamination the most plausible explanation, as there were no females with eggs in marsupia remaining in this particular tank at the moment we found this juvenile. All crosses were in experimental tanks were placed next to each other, and we always exchanged water in them at the same day in order to ensure most similar environmental conditions. Unfortunately, a nine-month-long experiment with ~900 approximately 1-mm long juveniles analyzed could have led to some errors. We ensure the reliability of the results by genotyping all adults and juveniles the origin of which was doubtful, and also by the fact that we made conclusions only from multiple consecutive and corroborating sources of evidence (presence of amplexuses, then females with eggs, then juveniles, juveniles were genotyped).

 

Comment 5: 4th Was the presence of natural hybrids or backcross individuals in Listvyanka investigated? Based on the legend of Figure 9, I assume that the authors genotyped the animals from Listvyanka only for the COI gene. If this assumption is correct, I would like to ask why the authors did not sequence the 18S gene fragment. Including this nuclear marker could help determine whether the W and S species are merely in sympatry in Listvyanka, or if they are in true secondary contact.

Response 5: Thank you for this interesting question! Actually, we genotyped 18S for animals surviving after hybrid crosses and for all S individuals found in Listvyanka to rule out methodological issues before drawing important conclusions, but only a random selection of ~20 animals of the W species from Listvyanka was genotyped for both markers. The 18S genotyping system we developed was not ideal due to a very few differences between the sequences. It has a specific primer matching the E. verrucosus W sequence and a control primer matching both sequences. It allows for the distinction between the two species, but a hybrid individual would also provide a W-like signal in this test. However, while making the revision, we finished the analysis of the 100 animals from Listvyanka now, and all of them belonged to the same species according to the COI and 18S marker sequences (see updated Figure 9 and Table S6). It allows us to confidently conclude that the S species animals we see in Listvyanka are not hybrid. This information is now added to the manuscript (lines 527-259 in the file with tracked changes).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive experimental study on reproductive isolation among three cryptic species of the Eulimnogammarus verrucosus complex in Lake Baikal. The authors provide robust evidence for both prezygotic and postzygotic barriers, supported by genetic, behavioral, and developmental data. The study is well-designed, methodologically sound, and addresses an important topic in evolutionary biology—speciation and reproductive isolation in cryptic species. The findings are novel and contribute significantly to our understanding of how reproductive barriers operate in natural systems. The manuscript is generally well-written and well-supported by data. I recommend acceptance after minor revisions.

1. Introduction: The introduction is thorough but could be more concise. Consider condensing the literature review on amphipod reproductive studies to focus more directly on the gaps this study aims to fill.
2. Methods: The description of the mate choice trials and crossing experiments is clear, but the sample sizes for some trials (e.g., only 31 mate choice trials) are modest. A brief justification for the sample size or a note on statistical power would be helpful. Clarify whether the “no-choice” and “with-choice” terminologies are standardized in the field, as they are used throughout without definition.
3. Results: The genotyping results from the SxW cross showing only S juveniles are well-explained, but the possibility of early mortality of W individuals could be discussed more explicitly. Figure 5 and 6 are informative but could benefit from clearer labeling (e.g., which combinations are control vs. experimental).
4. Discussion: The discussion of the “inhibition of one sex” in E-species crosses is intriguing but speculative. Consider tempering the conclusions or proposing specific follow-up experiments. The link between genetic distance and reproductive isolation is well-supported, but a more explicit comparison with other amphipod systems (e.g., Gammarus) would strengthen the broader implications.

1.  

Comments on the Quality of English Language

The manuscript is generally well-written, but there are occasional grammatical inconsistencies and awkward phrasings (e.g., “eggs falling out from the brood pouches” → “eggs released from the brood pouches”). A thorough proofreading is recommended.

Author Response

Comment 1: This manuscript presents a comprehensive experimental study on reproductive isolation among three cryptic species of the Eulimnogammarus verrucosus complex in Lake Baikal. The authors provide robust evidence for both prezygotic and postzygotic barriers, supported by genetic, behavioral, and developmental data. The study is well-designed, methodologically sound, and addresses an important topic in evolutionary biology—speciation and reproductive isolation in cryptic species. The findings are novel and contribute significantly to our understanding of how reproductive barriers operate in natural systems. The manuscript is generally well-written and well-supported by data. I recommend acceptance after minor revisions.

Response 1: We are very grateful to the Reviewer for the time invested in helping us improve our work and for the positive assessment! Please find below responses to particular comments.

 

Comment 2: 1st Introduction: The introduction is thorough but could be more concise. Consider condensing the literature review on amphipod reproductive studies to focus more directly on the gaps this study aims to fill.

Response 2: This part of the Introduction is much shorter now, and we added a sentence to explain one of the most important gaps we wanted to fill (lines 136-138 in the file with tracked changes). The added sentence reads “Unfortunately, most of these studies did not follow the development of embryos up to hatching and thus could not assess postzygotic reproductive barriers”.

 

Comment 3: 2nd Methods: The description of the mate choice trials and crossing experiments is clear, but the sample sizes for some trials (e.g., only 31 mate choice trials) are modest. A brief justification for the sample size or a note on statistical power would be helpful. Clarify whether the “no-choice” and “with-choice” terminologies are standardized in the field, as they are used throughout without definition.

Response 3: 

1. We understand that larger sample sizes in mate choice trials could have allowed us to make more robust conclusions, but we were limited by the number of amplexuses that could have been sampled and transported within one day, and we were not equally lucky at all three sampling places. However, we only draw conclusions about non-random mating in general and do not interpret them in the sense of any correlation between genetic similarity and amplexus formation. Indeed, a statistical analysis can be used to justify our conclusions. A chi-squared test for 22 conspecific : 1 hetetrospecific amplexuses formed in this experiment confirms non-random mating with p < 0.0001. This is in clear contrast to the results we obtained with geographically separated populations of a related morphological species Eulimnogammarus cyaneus, for which we recorded 5 amplexuses with animals from the same population and 5 amplexuses with animals from different populations (https://doi.org/10.1002/jez.2891). We have added the statistical information to the manuscript (line 403 in the file with tracked changes) and are grateful for this suggestion.

2. Thank you for this remark! After re-analysis of the literature, we came to the conclusion that the most widely used combination is “choice experiment” vs. “no-choice experiment” (for example, https://doi.org/10.1016/j.jembe.2004.04.010 and https://doi.org/0.1007/s00265-013-1564-z for mating experiments or https://doi.org/10.1016/S0022-0981(97)00162-7 and https://doi.org/10.7717/peerj.5929 for food-related behavioral trials). Thus, we corrected “with-choice experiments/trials” to “choice experiments/trials” (lines 125 and 587 in the file with tracked changes).

 

Comment 4: 3rd Results: The genotyping results from the SxW cross showing only S juveniles are well-explained, but the possibility of early mortality of W individuals could be discussed more explicitly. Figure 5 and 6 are informative but could benefit from clearer labeling (e.g., which combinations are control vs. experimental).

Response 4: 

1. The ratio of sexes in the end of this experiment was 1:1 (Figure 5), and they were genotyped to confirm the expected species of origin (Table S6).

2. We added labels “control combinations” and “experimental combinations” to figures 5 & 6 (as well as 7 and 8, which have the same panels) and hope they are now as easy to read as possible. Thank you for this suggestion!

 

Comment 5:

4th Discussion: The discussion of the “inhibition of one sex” in E-species crosses is intriguing but speculative. Consider tempering the conclusions or proposing specific follow-up experiments. The link between genetic distance and reproductive isolation is well-supported, but a more explicit comparison with other amphipod systems (e.g., Gammarus) would strengthen the broader implications.

Response 5:

1. Thank you for this suggestion! Yes, we totally agree that this is just an observation and a hypothesis, and additional experiments are needed to test it. So, we changed “inhibition” (which is indeed a hypothesis) to “preferential mortality” (which we indeed saw) in order to more closely follow the data (line 617 in the file with tracked changes). We also added a sentence to the end of this paragraph:

“However, additional experiments are required to test this hypothesis, such as direct behavioral trials recording interaction between males of different species.” (lines 623-624).

2. We added the most direct comparison possible of our results with those obtained in the G. pulex / G. fossarum with directly comparing K2P distances with those within the Eulimnogammarus verrucosus complex (Discussion, lines 580-586). This is indeed a great suggestion, thank you!

 

 

Comment 6:

Comments on the Quality of English Language

The manuscript is generally well-written, but there are occasional grammatical inconsistencies and awkward phrasings (e.g., “eggs falling out from the brood pouches” → “eggs released from the brood pouches”). A thorough proofreading is recommended.

Response 6:

Thank you for your suggestion! We had the paper proofread.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript is well prepared. In what follows, unclear or incorrect sentences are listed; I hope the authors reconsider them carefully:

Line 2: Incompatibility → Incompatibilities.

22 (and others): interspecies → interspecific.

54: possible not → not possible.

101: judge about →　assess.

162: (Gerstfelt, 1858) → express by numerals of literatures [XX].

Fig. 2, B: 6^th fig from the left: ♂S　→　♂E.

240-241: from their size: not make sense.

Fig 3: explain the mark of asterisks.

252: Typical embryonic developmental stages in [42] (S7 etc.) are to be described with morphological or embryological terms, e.g., morula, blastula, gastrula stages, since the photos are not clear.

Author Response

Comment 1: This manuscript is well prepared. In what follows, unclear or incorrect sentences are listed; I hope the authors reconsider them carefully:

Response 1: We would like to thank the reviewer for work and for their kind words. We appreciate and share the concerns raised by the Reviewer and updated the text accordingly; we also had it proofread. Please find more particular responses below.

 

Comment 2: Line 2: Incompatibility → Incompatibilities.

Response 2: Corrected, thank you!

 

Comment 3: 22 (and others): interspecies → interspecific.

Response 3: Corrected, thank you!

 

Comment 4: 54: possible not → not possible.

Response 4: Corrected, thank you!

 

Comment 5: 101: judge about →　assess.

Response 5: Corrected as suggested, thank you!

 

Comment 6: 162: (Gerstfelt, 1858) → express by numerals of literatures [XX].

Response 6: References are rearranged, thank you!

 

Comment 7: Fig. 2, B: 6^th fig from the left: ♂S　→　♂E.

Reply 7: Indeed, there was a mistake in the figure. It is now corrected, and we are very grateful to the Reviewer for noticing it.

 

Comment 8: 240-241: from their size: not make sense.

Reply 8: Indeed, there was an unfortunate typo, it should have been “from the side”. It is now corrected (line 261 in the file with tracked changes). Thank you!

 

Comment 9: Fig 3: explain the mark of asterisks.

Reply 9: These asterisks denote the p-value as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (Wilcoxon–Mann–Whitney test with Holm’s correction for multiple comparisons). This information has been added to the figure 3 caption. We are grateful to the Reviewer for this suggestion.

 

Comment 10: 252: Typical embryonic developmental stages in [42] (S7 etc.) are to be described with morphological or embryological terms, e.g., morula, blastula, gastrula stages, since the photos are not clear.

Reply 10: Thank you for this suggestion! The eggs of this species are black up to very advanced stages of development, and thus we had to use the number of nuclei as a proxy, exactly as done in the manuscript [42] we reference. This classification does not provide a clear correspondence between the transition between the number of cells and morula => blastula stages, so we did not include this information. However, we added the term “blastula” when we refer to the critical S7 stage (lines 472-473 in the file with tracked changes), and we also explain that this developmental stall occurs prior to gastrulation in the Discussion section (line 646-652).