Highlighting the Importance of Correct Sex Identification in Chondrichthyan Genomic Studies, Using the White Shark as an Example

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Through genomic analyses of white shark samples, this manuscript has elucidated the potential discrepancies between phenotypic sex determinations and genetic data, thereby underscoring the critical importance of accurate sex identification in chondrichthyan genomic research. The manuscript presents a comprehensive case study that scrutinizes errors in sex identification within existing genomic databases and advocates for researchers to furnish detailed and precise information—including sex, sampling locations, and life histories—when publishing genomes of sharks, rays, or any other organisms. This practice not only enhances the precision of scientific investigations but also bolsters the utility and interpretability of genomic data in future research. However,  the manuscript coulb be accepted after some modification as following: 

Abstract
1. The abstract is well-written and clear.

Introduction

1.some relevant citations should be added when discribing how high-throughput sequencing facilitates the construction of reference genomes, reveals insights into developmental processes, the basis of genetic diseases, and the diversity of life.

2. a more thorough overview of the current understanding and research gaps in elasmobranch sex determination mechanisms should be provided.

3. In lines 69–75, the authors suggest that this may reflect “dynamism in sex determination.” Are there any literature that supports such sexual dynamism in white sharks or other elasmobranchs? If it is the individual’s sex case, what implications for future research on sex determination mechanisms?

4. Are there any challenges in sex identification specific to white sharks or other elasmobranchs? What biological factors might contribute to discrepancies between genotypic and phenotypic sex in these species? 

5. In lines 75–78, the authors emphasize the importance of accurate sex annotation. Provide some specific examples of applications where precise sex information is critical?

Materials and Methods

1. The Materials and Methods section is well-written. However, I am curious about the rationale for selecting these four specific ocean regions.

Result and Discussion

1. Provide some additional genetic or sequence-level data to further validate the pseudoautosomal characteristics of the 2 Mb region or discuss potential methods for future validation in the discussion section.

2. In lines 183–185, the authors noted that accurate assignment of sex is crucial for studying sex determination mechanisms. Could the authors elaborate on the potential misleading effects that incorrect sex identification may introduce?

4. In lines 193–194,  which mention some conflicts between visual and genetic sex identification, but do not discuss the specific causes of these discrepancies in depth. For example, may some external environmental factors, developmental stages, or individual differences contribute to inconsistencies in sex identification?

Reference

1. More references need to be cited.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript. We appreciate your insightful comments and suggestions. Please find the detailed point-by-point responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

 

Introduction

Comments 1: some relevant citations should be added when discribing how high-throughput sequencing facilitates the construction of reference genomes, reveals insights into developmental processes, the basis of genetic diseases, and the diversity of life.

Response 1: Thank you for your suggestion. We have cited sources we believe to be relevant to these statements. These include examples of developmental genes revealed by examining reference genomes, the application of genomic sequences to identify variations and their connection to diseases, studies of genomic evolution through comparative genomics, and the application of genomes to conservation biology. All examples above were made possible by high-throughput reference genome assemblies.

Comments 2: a more thorough overview of the current understanding and research gaps in elasmobranch sex determination mechanisms should be provided.

Response 2: Thank you for your suggestion. We have included descriptions that summarize the current state of understanding regarding the elasmobranch sex determination mechanisms in lines 72-75.

Comments 3: In lines 69–75, the authors suggest that this may reflect “dynamism in sex determination.” Are there any literature that supports such sexual dynamism in white sharks or other elasmobranchs? If it is the individual’s sex case, what implications for future research on sex determination mechanisms?

Response 3: Thank you for the comment. To our knowledge, the evidence of sexual dynamism among the elasmobranchs came from the reports of intersexual (or hermaphroditic) individuals, although not among the white sharks. While the presence of hermaphroditic individuals had been suggested to be the cause of environmental contamination [1], cases of skewed sex ratio or sex reversal such as those of common frogs [2] or chickens [3] have not been reported in elasmobranch species. Our speculation that intersexuality or sex reversal could be the potential mechanism is based on the relatively abundant records of intersexual elasmobranchs, indicating plasticity in the development of sexual traits. If future studies can verify these speculations, it could modify our current understanding of the elasmobranch sex determination mechanism from a mammalian-like genetic sex determination system to a more dynamic view where environmental factors could potentially affect the phenotypic sex.

Comments 4: Are there any challenges in sex identification specific to white sharks or other elasmobranchs? What biological factors might contribute to discrepancies between genotypic and phenotypic sex in these species?

Response 4: To our knowledge, there are no substantial records of discrepancies or difficulties regarding the sex identification of white sharks until the reports of Delva-Devloo et al. [5]. Sexes of sharks and rays are generally reported without much ambiguity due to the presence of claspers in males even prior to sexual maturity [4]. Sex-associated genetic markers in elasmobranchs, as far as we can tell, did not exist before Delva-Devloo et al. [5]. Therefore, the conflict between genotypic or genetic sex and phenotypic sex is a and has not been widely assessed.

Several factors may contribute to the discrepancy between genotypic and phenotypic sex identification. Other than the likelihood of misidentification during visual inspection due to the poor condition of the sourced individual or the specimen being a juvenile, intersexuality and sex reversal can also be potential causes. Although not yet assessed in elasmobranchs, intersexual individuals may exhibit an external sexual phenotype with a contradicting genotype. In environmental-induced sex reversals such as those of amphibians [6] and teleosts [7], genotypic and phenotypic sex discrepancies are expected.

Comments 5: In lines 75–78, the authors emphasize the importance of accurate sex annotation. Provide some specific examples of applications where precise sex information is critical?

Response 5: Thank you for your comment. Accurate sex annotation for samples allows studies that inform sex-specific migratory or demographic history, which can be applied to inform effective conservation status and practices to the species of interest [8–10]. Accurate sex annotations for the genomic sequences are also essential for the detection of sex-specific sequences or variations, which are critical for the studies of sex determination [11–14] and molecular marker design [5,12]. This has been incorporated in lines 40 – 42 and 58- 65.

 

Materials and Methods

Comments 1: The Materials and Methods section is well-written. However, I am curious about the rationale for selecting these four specific ocean regions.

Response 1: Thank you for your comment. We added context to the Materials and Methods. The samples were selected from these ocean basins to reduce the possibility of skewed genetic variation caused by population structure. The white shark is known to be widely distributed and with some degree of phylogeographic patterns [16,17]. As the reference white shark genome and the individual of Marra’s assembly were from two different populations [17], we have chosen these samples to ensure our standards for males and females at least include individuals from these populations to minimize potential biases.

 

Result and Discussion

Comments 1: Provide some additional genetic or sequence-level data to further validate the pseudoautosomal characteristics of the 2 Mb region or discuss potential methods for future validation in the discussion section.

Response 1: Thank you for the comment. We believe that the evidence we have collected so far does not allow us to unambiguously validate this region as the pseudoautosomal region. Our assertion of this region as a “putative PAR” is based on its location on a male heterogametic chromosome and the similar read coverage between males and females. Several methods can be applied to support this claim, such as the female-to-male read coverage ratio as applied by Yamaguchi et al. [13] and Wu et al. [14], although this method applies the same rationale. The other method that could potentially support the identity of this region as a PAR is through a thorough variant calling. In a heterogametic sex chromosome (i.e., Y) that is differentiated enough from its homolog (i.e., X), the heterozygosity should be 0 except for the PAR. However, our preliminary test based on genetic variation does not demonstrate such a clear distinction between regions with and without heterozygous sites, which could potentially be caused by several confounding factors such as repeating elements that are inappropriately mapped to the reference genome. Therefore, we consider this region to be regarded as a putative PAR until further evidence emerges. To further validate the presence of a PAR, one could conduct cytological investigations to observe the formation of chiasmata between the X and the Y chromosome or fluorescence in situ hybridization.

Comments 2: In lines 183–185, the authors noted that accurate assignment of sex is crucial for studying sex determination mechanisms. Could the authors elaborate on the potential misleading effects that incorrect sex identification may introduce?

Response 2: As described in our response to Introduction comments 5, the development of sex-specific molecular markers or the identification of sex-specific regions both required the accurate sex identification of the source materials. Misidentification could potentially confuse the analyses involved in discovering sex-specific markers and diminish the sequence differences one would otherwise observe when comparing the between sex Fst elevated heterozygous variants in males (e.g. [18]), or the difference in genomic coverage between XX females and XY males as in this study.

Comments 3: In lines 193–194,  which mention some conflicts between visual and genetic sex identification, but do not discuss the specific causes of these discrepancies in depth. For example, may some external environmental factors, developmental stages, or individual differences contribute to inconsistencies in sex identification?

Response 3: Thank you for your comment. As we do not have direct access to the specimen of interest, and the sex determination systems of the elasmobranchs are not yet well understood, we could only speculate on the causes of such discrepancies. As mentioned in our response to Introduction comment 4, reports of conflicting sex identification among elasmobranchs are recent. While all your suggestions are highly reasonable and could contribute to our observations, more studies are required to provide a comprehensive explanation. Therefore, we described the speculations made by [5] in lines 207-208 and emphasized how our discovery and suggestions provide a pathway for future investigations.

 

Reference

Comments 1: More references need to be cited.

Response 1: Thank you for the suggestion. We have made the modifications based on your suggestions and cited relevant references.

 

References cited in responses

1. Yano, K.; Tanaka, S. Hermaphroditism in the Lantern Shark Etmopterus Unicolor (Squalidae, Chondrichthyes). Jpn. J. Ichthyol. 1989, 36, 338–345, doi:10.11369/jji1950.36.338.

2. Matsuba, C.; Miura, I.; Merilä, J. Disentangling Genetic vs. Environmental Causes of Sex Determination in the Common Frog, Rana Temporaria. BMC Genet. 2008, 9, 3, doi:10.1186/1471-2156-9-3.
3. Thompson-Davis, D.C. The Effect of Temperature on Sex Determination in the Domestic Chicken (Gallus Domesticus). Ph.D., The University of Manchester (United Kingdom): England, 1996.
4. Pratt, H.L. CHAPTER 13 - Reproduction in the Male White Shark. In Great White Sharks; Klimley, A.P., Ainley, D.G., Eds.; Academic Press: San Diego, 1996; pp. 131–138 ISBN 978-0-12-415031-7.
5. Devloo-Delva, F.; Gosselin, T.; Butcher, P.A.; Grewe, P.M.; Huveneers, C.; Thomson, R.B.; Werry, J.M.; Feutry, P. An R-Based Tool for Identifying Sex-Linked Markers from Restriction Site-Associated DNA Sequencing with Applications to Elasmobranch Conservation. Conserv. Genet. Resour. 2024, 16, 11–16, doi:10.1007/s12686-023-01331-5.
6. Wallace, H.; Badawy, G.M.I.; Wallace, B.M.N. Amphibian Sex Determination and Sex Reversal. Cell. Mol. Life Sci. CMLS 1999, 55, 901–909, doi:10.1007/s000180050343.
7. Valdivieso, A.; Wilson, C.A.; Amores, A.; da Silva Rodrigues, M.; Nóbrega, R.H.; Ribas, L.; Postlethwait, J.H.; Piferrer, F. Environmentally-Induced Sex Reversal in Fish with Chromosomal vs. Polygenic Sex Determination. Environ. Res. 2022, 213, 113549, doi:10.1016/j.envres.2022.113549.
8. Tsai, W.-P.; Sun, C.-L.; Punt, A.E.; Liu, K.-M. Demographic Analysis of the Shortfin Mako Shark, Isurus Oxyrinchus, in the Northwest Pacific Using a Two-Sex Stage-Based Matrix Model. ICES J. Mar. Sci. 2014, 71, 1604–1618, doi:10.1093/icesjms/fsu056.
9. Wilson Sayres, M.A. Genetic Diversity on the Sex Chromosomes. Genome Biol. Evol. 2018, 10, 1064–1078, doi:10.1093/gbe/evy039.
10. Phillips, N.M.; Devloo-Delva, F.; McCall, C.; Daly-Engel, T.S. Reviewing the Genetic Evidence for Sex-Biased Dispersal in Elasmobranchs. Rev. Fish Biol. Fish. 2021, 31, 821–841, doi:10.1007/s11160-021-09673-9.
11. Pinto, B.J.; Keating, S.E.; Nielsen, S.V.; Scantlebury, D.P.; Daza, J.D.; Gamble, T. Chromosome-Level Genome Assembly Reveals Dynamic Sex Chromosomes in Neotropical Leaf-Litter Geckos (Sphaerodactylidae: Sphaerodactylus ). J. Hered. 2022, 113, 272–287, doi:10.1093/jhered/esac016.
12. Gamble, T.; Castoe, T.A.; Nielsen, S.V.; Banks, J.L.; Card, D.C.; Schield, D.R.; Schuett, G.W.; Booth, W. The Discovery of XY Sex Chromosomes in a Boa and Python. Curr. Biol. 2017, 27, 2148-2153.e4, doi:10.1016/j.cub.2017.06.010.
13. Yamaguchi, K.; Uno, Y.; Kadota, M.; Nishimura, O.; Nozu, R.; Murakumo, K.; Matsumoto, R.; Sato, K.; Kuraku, S. Elasmobranch Genome Sequencing Reveals Evolutionary Trends of Vertebrate Karyotype Organization. Genome Res. 2023, 33, 1527–1540, doi:10.1101/gr.276840.122.
14. Wu, J.; Liu, F.; Jiao, J.; Luo, H.; Fan, S.; Liu, J.; Wang, H.; Cui, N.; Zhao, N.; Qu, Q.; et al. Comparative Genomics Illuminates Karyotype and Sex Chromosome Evolution of Sharks. Cell Genomics 2024, 0, doi:10.1016/j.xgen.2024.100607.
15. Gamble, T.; Coryell, J.; Ezaz, T.; Lynch, J.; Scantlebury, D.P.; Zarkower, D. Restriction Site-Associated DNA Sequencing (RAD-Seq) Reveals an Extraordinary Number of Transitions among Gecko Sex-Determining Systems. Mol. Biol. Evol. 2015, 32, 1296–1309, doi:10.1093/molbev/msv023.
16. Andreotti, S.; Von Der Heyden, S.; Henriques, R.; Rutzen, M.; Meÿer, M.; Oosthuizen, H.; Matthee, C.A. New Insights into the Evolutionary History of White Sharks, Carcharodon Carcharias. J. Biogeogr. 2016, 43, 328–339, doi:10.1111/jbi.12641.
17. Wagner, I.; Smolina, I.; Koop, M.E.L.; Bal, T.; Lizano, A.M.; Choo, L.Q.; Hofreiter, M.; Gennari, E.; Sabata, E. de; Shivji, M.S.; et al. Genome Analysis Reveals Three Distinct Lineages of the Cosmopolitan White Shark. Curr. Biol. 2024, 34, 3582-3590.e4, doi:10.1016/j.cub.2024.06.076.
18. Gammerdinger, W.J.; Conte, M.A.; Acquah, E.A.; Roberts, R.B.; Kocher, T.D. Structure and Decay of a Proto-Y Region in Tilapia, Oreochromis Niloticus. BMC Genomics 2014, 15, 975, doi:10.1186/1471-2164-15-975.

Reviewer 2 Report

Comments and Suggestions for Authors

I read the paper by Lee et al. with real interest because the topic is extremely current and interesting. It is a very well written manuscript, well structured, clear and complete. The methodologies are clearly described. The results are well presented and are enriched by coherent and explanatory figures. The discussions support the results. The references are also adequate. The authors could only add a paragraph in the introduction dedicated to the experimental model (the white shark) emphasizing how these results are important also because they were obtained for a charismatic species and of high conservation interest.

Author Response

Dear Reviewer,

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 the re-submitted files.

Response: Thank you very much for your comment. We really appreciate your compliments and interest. However, we believe our current data do not provide substantial evidence to support significant insights into the conservation status or management of white sharks. Although the results demonstrated in this study can potentially have conservation implications where the estimation of population sex ratio or sex-specific behaviors are involved (e.g., [1,2]), the present study reports a single case of sex identification discrepancy which may not be sufficient to alter the current understanding of white shark conservation. We, therefore, suggest vigilant monitoring and thorough documentation of future sampling efforts, which may highlight the conservation implication of our discovery when evidence arises.

References cited in the responses

1. Bruce, B.; Bradford, R. Segregation or Aggregation? Sex-Specific Patterns in the Seasonal Occurrence of White Sharks Carcharodon Carcharias at the Neptune Islands, South Australia. J. Fish Biol. 2015, 87, 1355–1370, doi:10.1111/jfb.12827.
2. Kanive, P.E.; Rotella, J.J.; Jorgensen, S.J.; Chapple, T.K.; Hines, J.E.; Anderson, S.D.; Block, B.A. Size-Specific Apparent Survival Rate Estimates of White Sharks Using Mark–Recapture Models. Can. J. Fish. Aquat. Sci. 2019, 76, 2027–2034, doi:10.1139/cjfas-2018-0142.

Reviewer 3 Report

Comments and Suggestions for Authors

 found this paper well organized and well written. However I cannot adequately review it as I realize after accepting the review request that I have no expertise in the genetic method used thus cannot adequately critique their study design and interpretation. I also feel that they expand shark conservation implications in the conclusion as Im not sure how their recommendation will affect shark management. Unfortunately I recommend you find another reviewer that can adequately judge this paper. My apologies. 

Author Response

Dear Reviewer,

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 the re-submitted files.

Response: Thank you very much for your comment. Thank you very much for your comment. We appreciate your interest and suggestion in the management of white sharks. We believe our current data do not provide substantial evidence to support significant insights into the conservation status or management of white sharks. Although the results demonstrated in this study can potentially have conservation implications where the estimation of population sex ratio or sex-specific behaviors are involved (e.g., [1,2]), the present study reports a single case of sex identification discrepancy which may not be sufficient to alter the current understanding of white shark conservation. We, therefore, suggest vigilant monitoring and thorough documentation of future sampling efforts, which may highlight the conservation implication of our discovery when evidence arises.

References cited in the responses

1. Bruce, B.; Bradford, R. Segregation or Aggregation? Sex-Specific Patterns in the Seasonal Occurrence of White Sharks Carcharodon Carcharias at the Neptune Islands, South Australia. J. Fish Biol. 2015, 87, 1355–1370, doi:10.1111/jfb.12827.
2. Kanive, P.E.; Rotella, J.J.; Jorgensen, S.J.; Chapple, T.K.; Hines, J.E.; Anderson, S.D.; Block, B.A. Size-Specific Apparent Survival Rate Estimates of White Sharks Using Mark–Recapture Models. Can. J. Fish. Aquat. Sci. 2019, 76, 2027–2034, doi:10.1139/cjfas-2018-0142.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved much, it could be accepted.