PONE-D-22-04909Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservationPLOS ONE

Dear Dr. Elizondo-Sancho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Thank you for submitting your manuscript PONE-D-22-04909 “Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation” to PLOS ONE. 

I have now received feedback from three experts in the field. As you can see below, they all found the paper of interest but had a few comments. In particular they found that some details on the analyses are missing which could make the paper more robust and understandable. They provided constructive comments that will help you to improve your paper. 

As a result, I invite you to resubmit a revised version of the paper addressing the comments made by these referees.

With kind regards,

Johann 

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Additional Editor Comments:

Thank you for submitting your manuscript PONE-D-22-04909 “Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation” to PLOS ONE.

I have now received feedback from three experts in the field. As you can see below, they all found the paper of interest but had a few comments. In particular they found that some details on the analyses are missing which could make the paper more robust and understandable. They provided constructive comments that will help you to improve your paper.

As a result, I invite you to resubmit a revised version of the paper addressing the comments made by these referees.

With kind regards,

Johann

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PlosONE review Elizondo-Sancho et al. 2022

In this study, Elizondo-Sancho and collaborators add a significant number of samples to revisit the population structure of the Scalloped Hammerhead shark in the Eastern Tropical Pacific region. By doing so they provide valuable information for the management of this species. Overall, I found the manuscript well written and easy to follow. The genetic techniques used are not cutting-edge but the data is analysed adequately and I believe the conclusions are robust. I only made a few minor comments that I hope will improve the manuscript. Well done to all the authors. Pierre Feutry

L87-91: there are other philopatric shark species, look up genus Glyphis and Bull Shark

L112: are the acronyms ok for the locations in Costa Rica? There is a discrepancy between that sentence (COY, OJO and COS) and figure 1 (COY, OJO, ICO). Cocos Island seems to be missing in the locations from CR

L123-124: along the coast is quite vague and doesn’t really match up with the specificity of the sampling locations.

L149: “Nance et al. (2009 lewini” Typos?

L167 : add a « , » after « among haplotypes”

L191-192: related individuals should only be removed if there is a reason to think there was a bias in the sampling favoring the catch of related individuals, e.g. 2 neonates caught in the same eddy on the same day. On the opposite, if two siblings were caught 3 years apart there is no reason to remove one of them. See paper by Anderson & Waples (2017) for further details

L213-216: need to explain what method was used to choose the “best” K

L220-221: how did you deal with the potential of overfitting? Alpha score? Cross-validation?

L238: delete “relatively low”, this type of comment on the results belongs to the discussion.

L273: What is significant? Less than 0.05 p-value after correction? It’s generally better to provide the confidence interval for the Fst values rather than p-values, could add them in the upper diagonal

L284: delete “duly”

L305-306: move “(Fis)” after “inbreeding coefficients”

L309-311: replace “a greater” and “a lower” by “the greatest” and “the lowest” respectively

L312: replace “less” by “the least” and “greater” by “the most”

L368: most likely a bottleneck is not detected because the population size is not (yet) low enough, is that what you mean by “only a few generations have passed since overfishing started”. Please reword

L368-369: how can you report a “non-detectable bottleneck”? Do you mean that no bottleneck was detected?

L379: the entire mitogenome is one marker, I would reword and say “using more mitochondrial regions”

L380: Feutry et al. 2014 is a better reference for that statement

L404: or more markers, or both. See Foster et al. 2021

L415: not a challenge if you have plenty of markers (generally SNPs)

L430: quite likely there is further structuring, but more markers and/or more individuals are required to demonstrate it. I think the discussion need to emphasize a bit more the high potential for finer scale structure and the need to look into it with appropriate genetic techniques

References

Feutry, P., Kyne, P. M., Pillans, R. D., Chen, X., Naylor, G. J., & Grewe, P. M. (2014). Mitogenomics of the Speartooth Shark challenges ten years of control region sequencing. BMC evolutionary biology, 14(1), 1-9.

Foster, S. D., Feutry, P., Grewe, P., & Davies, C. (2021). Sample size requirements for genetic studies on yellowfin tuna. PloS one, 16(11), e0259113.

Waples, R. S. , & Anderson, E. C. (2017). Purging putative siblings from population genetic data sets: A cautionary view. Molecular Ecology, 26, 1211–1224. 10.1111/mec.14022

Reviewer #2: This research uses mtDNA and microsatellite data to assess the population structure of the Critically Endangered Scalloped Hammerhead Shark in the Eastern Tropical Pacific. These data build upon previous published datasets (which is wonderful!), revealing novel patterns of population structure. Given the highly threatened status of this species and the importance of population genetic data in conservation and management strategies, this is an important paper that need to be published. I am recommending major revisions on the basis that some of the information could be communicated more clearly through rephrasing, consistent use of terms, and some reorganization. I found some statements to be confusing as written, so I suggest rephrasing and being more concise to improve reader comprehension. The use of consistent names or acronyms for sampling sites would also improve clarity surrounding data analysis and results. Some of the information might flow better with some minor reorganization in the introduction and discussion. Specific suggestions regarding these points are below. The manuscript also needs a careful proofread. Comments/examples are provided in the manuscript pdf but these are not comprehensive.

The other reason I recommend major revision is that some data analyses are not presented, and this dataset may reveal additional novel and important information about the species with some further analysis. The data for HWE are not presented, so the p-values are unknown, as is the scale for these analyses. Was HWE significant calculated for all individuals pooled or for each sample site? This is important because of the assumptions of HWE (e.g. pooling two populations or not-pooling a single population could cause violations in the HWE assumptions). P values should also be reported for all statistics throughout. For example, Table 2 has ‘significant values in bold’, but the threshold used is not specified to give this more meaning. The authors state a correction was applied, so presumably it is not 0.05. Bottleneck tests are also mentioned, but no methods or results presented. Additional statistics that should be considered are: 1) exact tests, 2) DEST for mtDNA, 3) a hierarchical STRUCTURE analysis, 4) genetic diversity indices for the identified populations (rather than sampling sites), and 4) set up the AMOVA as a hypothesis. See specific comments below regarding some of these.

Line 64-65: All populations experience genetic drift, but the effects are more pronounced in small populations. Suggest rephrasing to make this clear.

Line 98: Suggest rephrasing from “sorting out the genetic diversity…”. For example, could change to “it is important to assess population structure and genetic diversity between potential nursery areas in this region”.

Line 100-103: Remove, as this is methods.

Line 112: Add sample sizes for each site.

Line 123: Here is says samples were collect from juveniles, but the introduction said YOY. Which is it? Similarly, what life stages were sampled in the other studies where the data is used here? This needs to be stated, and potential caveats addressed in the discussion since life stage sampled is needed to interpret the data more fully.

Line 153: The entire mtDNA control region?

Line 136: Remove “species-specific”. These primers may have been designed for S. lewini, but that does not mean they are species-specific. That would involve cross testing in other species to make sure they do not cross-amplify DNA from other species.

Lines 146, 159: More details are needed for how PCR products were cleaned and sequenced, as well as for fragment analysis, e.g. what size standard was used?

Line 156: Suggest including cycle numbers in Table S2 since they were not the same across all loci.

Line 165: Add sample sizes for these locations.

Line 173: Suggest exact tests here too.

Line 191-192: I understand why the authors removes FS from the analyses, but does this then impact population structure and genetic diversity statistics in the other direction? Since relatedness/sibship approaches can be used to elucidate population structure and natal philopatry, it might be worth exploring the data without this removal of FS and/or going further with relatedness analyses. See next comment as well.

Line 202-204: Were these comparisons also made between individuals at different sites? Suggest doing so to compare to the within-nursery data. It would also be interesting to look at sibship between different nursery areas as well as within nursery areas.

Line 210: Why not calculate DEST for mtDNA too?

Line 213: Suggest a hierarchical STRUCTURE if any of the identified populations (GUA, COS, PAN) have >1 sampling site. This is not really clear to me, as per the below comment for line 282. Also suggest looking at delta K

Table 1: Suggest calculating genetic diversity indices for each of the identified populations; this will give an estimate of diversity at scales relevant to management.

Line 250-252: Suggest rephrasing these sentences as they are confusing. I suspect the authors are trying to say that there were two common haplotypes across all sampling sites, but they were found at different frequencies in Mexico compared to Central America and Colombia.

Line 262-265: This statement is long and confusing. An AMOVA should be set up to test a specific hypothesis.

Line 282: I’m finding the acronyms and verbiage surrounding locations somewhat confusing. Here, GUA, COS, and PAN are mentioned. GUA and PAN are labelled on the map, but COS is not. I’m guessing that COS includes samples from >1 site, but the same may also be true for PAN based on the map. Later, there is reference to regions (e.g., 372) but it is difficult to follow given the inconsistencies. Suggest explaining sampling sites/ countries (pooled or not), regions (countries pooled?), etc. early on and then using the same language throughout the manuscript.

Line 285: “population-specific”- what was this defined by?

Line 291: Were all FS pairs from the same sampling site? This could be an interesting discussion point.

Line 308, 311, etc: Suggest reporting actual P-value. Was a correction applied to these statistical tests? If so, what was the new threshold?

Line 316-319: These statements seem to be contradictory. Were they all non-significant?

Line 321: Why were the samples from the Cocos Islands not included in analyses? For example, FST, DEST, Structure, etc.? The sample size wasn’t huge, but still worth including in the structure plot at a minimum.

Line 339-340: The statement “The average withing sampling site….” is confusing; suggest rephrasing.

Line 353 and elsewhere: Population declines can lead to a loss of genetic diversity, but that does not mean that: 1) population declines always cause declines in genetic diversity, or 2) that all populations with low diversity have undergone recent declines. This section seems to attribute the observed levels of genetic diversity to recent population declines, but this is not actually known. Elasmobranchs have some of the slowest rates of mutation among vertebrates, so genetic diversity accumulates slowly and can be low even in the absence of population declines. Suggest developing this section to be more comprehensive of genetic diversity in elasmobranchs, perhaps bringing in phylogeography (e.g. how recently might these populations been founded?)

Line 356: Levels of genetic diversity were not calculated for the central-southern ETP overall- they were calculated by sampling sites from what I can tell. Suggest analyzing genetic diversity for the identified populations to back up this statement. It also makes more sense from a management perspective to analyze data for each identified population.

Line 358-359: Take care with verbiage. Genetically distinct populations do not mean they resulted from independent evolutionary history. All populations of this species in the ETP likely have a common evolutionary history. This is evidenced by the presence of two common haplotypes shared across populations.

Line 367: It is stated that a bottleneck was not detected, but no data are presented to support this. Suggest either including bottleneck tests (with discussion on caveats of the various statistical approaches) or deleting the statement about the detection of bottleneck tests.

Line 373: The sentence on this line is confusing, suggest rephrasing.

Line 376: The term “sub-population’ has a specific meaning for the IUCN species assessments, which is cited as the source of this definition. The IUCN definition of ‘sub-population’ is not the same as used in population genetics. I suggest the authors read this definition more carefully and rework this point. The data presented in this paper does not support further splitting the EP sub-population of this species, as per the IUCN definition. Within this region, the identification of distinct population units is important to inform management, so suggest focusing on that.

Line 388-391; 397; 400-401; 436: The phrasing on these lines need some work as it is difficult to understand/follow. For example, line 388 mentions oceanographically dynamic regions and then uses the phrase ‘mixing zone’. Is this referring to a physical mixing zone or ‘mixing’ meaning gene flow? Line 400 mentions “high sampling effort” but not what locations fit this category. Etc.

Line 407: What age classes were sampled in these other studies?

Line 415: The discussion on relatedness could be built upon more. For example, what were the challenge for assessing sibship in this study? Could take some of the analysis further as well to support more discussion, as mentioned in previous comments.

Lines 423-429: This ought to be discussed in the population structure section. Philopatry is the logical explanation for the observed population structure, so integrate there. I also suggest either doing additional analyses on relatedness to develop this section more fully, or use the relatedness statistics to support the population structure findings.

Figure 1: Add sample sizes to caption or figure

Figure 2: It is difficult to see the ticks or count them.

Figure 5: What is the difference between the gray and black arrows? Specify in the caption.

Reviewer #3: In their article, Elizondo-Sancho describe the scalloped hammerhead’s population genetic structure and connectivity across nursery grounds from the Eastern Tropical Pacific, using a combination of mtDNA sequences and microsatellite markers. They employ commonly used population genetic analyses, and report stronger patterns of genetic structure than previously reported, suggestive of natal philopatry. They go on discussing the implications of these findings in terms of management of local populations.

Generally, the study is well conducted and I have no major issue with the analyses. Some important details (e.g. regarding STRUCTURE analyses) are missing, and I think some of the interpretations may be unwarranted. But these are fairly minor issues that I think can be very easily addressed by the authors with a minor revision. below I provide some detailed comments:

• Lines 50-51: I am not sure what the value of reporting Ho and allelic richness at microsatellite markers in the abstract is. These are very highly dependent on marker type (bialellic, tri-alellic, how where the markers selected).

• Line 87: i would rephrase as "allele frequency differences through time", since divergence usually refers to accumulation of mutations, while here the authors are talking of the effects of drift.

• Lines 214_216 and in general STRUCTURE analyses:

The authors do not explain how they chose the value for K they report in the results. What method was used to choose K (Evanno’s method? Other)? How do STRUCTURE plots look for different values of K? IS there any way to assess the admixture proportions? For SNPs data it's common to use evalAdmix (http://www.popgen.dk/software/index.php/EvalAdmix ), to evaluate pairwise correlation of residuals matrix between individuals. I am not sure whether there is an equivalent approach for microsatellite data.

Also, the admixture proportions reported in the figure are a bit difficult to reconcile with both the general population structure (Fst) and relative migration rates inferred: how is it that PAN and GUA show the lowest relative migration rates but the highest levels of admixture?

I am also not sure why the Authors have not reported structure analyses and DAPC of the entire dataset (including the samples from previous studies).

• DivMigrate analyses: what measure of genetic differentiation was used to estimate relative migration patterns ? Also please give more details on the method (including citation of the method implemented in divMigrate: Sunqvist et al 2016, Ecol Evol https://doi.org/10.1002/ece3.2096 ). How are relative migration rates scaled? i.e. is the highest migration rate given as 1?

An important concern is that this method assumes migration-drift equilibrium, I doubt this is a reasonable assumptions when it comes to long-lived marine animals with large Ne whose habitat has been affected by glaciations. See for example Maisano-Delser et al paper in Heredity on black-tip reef sharks and Walsh et al. paper in Heredity on grey reef sharks. So these results need to be interpreted with caution (as the authors of the package diveRsity themselves say).

• The authors mention low levels of genetic diversity (referring to pi and haplotype diversity). Low with respect to what? Other populations of the same species, other coastal sharks, or other marine fish? a pi of 0.0016 does not seem very low, but again this depends on what the reference is. What does seem interesting is the high degree of geographical heterogeneity in these estimates.

• Regarding migration rates, the authors mention that “Analysis of the extent and direction of gene flow showed no significant movement between coastal sampling sites.”. I am not sure how this conclusion was reached. There is no real analyses of the extent of gene flow, as measures of geneflow are "relative" (no absolute values ). Also the analyses assume migration-drift equilibrium and an island model, so the authors must be careful in interpreting the results.

• The authors mention that the low diversity of mtDNA is consistent with overexploitation. (Lines 352-353). No evidence is presented that the low levels of genetic diversity of this species are linked in any way to recent population declines. Given the generation time of scalloped hammerheads i find this hypothesis extremely unlikely.

None of the analyses the author presented allow any inference of recent changes in Ne, and to my knowledge such analyses would require extensive two-locus statistics (LD) obtained for a great portion of the genome, along with good linkage maps (for example, using the method developed by Santiago: https://doi.org/10.1093/molbev/msaa169) . Also please note that most studies on genetic diversity of sharks concluded that patterns of genetic diversity were almost certainly unrelated to recent population declines but rather reflect the species history of colonization/range expansion/isolation. See work on grey nurse sharks (Stow et al 2006 Biology Letters and subsequent paper in Molecular Ecology about grey nurse sharks https://doi.org/10.1098/rsbl.2006.0441 https://doi.org/10.1111/j.1365-294X.2009.04377.x , and recent work on blacktip reef shark by Stefano Mona and Maisano-Delser https://doi.org/10.1038/s41437-018-0164- , as well as work on grey reef sharks just published in heredity https://doi.org/10.1038/s41437-022-00514-4 ).

If the authors want to test this hypothesis they could try to use the R package “migraine” to detect possible bottlenecks, but they should also be aware that these estimates could be biased by complex demographic histories (e.g. https://doi.org/10.1038/s41437-018-0164-0 ).

• Lines 368-369: I am not sure what is meant by "non-detectable bottleneck effect". It could very well be that overharvesting may have reduced census size while having negligibly effects on effective population size. A non-detectable effect is not an effect at all?

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Reviewer #1: Yes: Pierre Feutry

Reviewer #2: No

Reviewer #3: No

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PONE-D-22-04909R1Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservationPLOS ONE

Dear Dr. Elizondo-Sancho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Dear Mariana Elizondo-Sancho,

I have now received the comments of one of the original reviewer who only highlighted a minor point to address.

Please address it in a revised version and I will be happy to accept your work for publication in Plos One.

Kind regards

Johann

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Kind regards,

Johann Mourier, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Mariana Elizondo-Sancho,

I have now received the comments of one of the original reviewer who only highlighted a minor point to address.

Please address it in a revised version and I will be happy to accept your work for publication in Plos One.

Kind regards

Johann

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Reviewers' comments:

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Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: The authors addressed most of my concerns.

A further comment to be addressed in a minor revision (no need to send this back to me for review)

In the discussion about genetic diversity, the authors mention the slow mutation rate for mtDNA in sharks. Note that genetic diversity is a product of Ne and mutation rate, i.e. nucleotide diversity is determined by the mutation scaled population size (or the population scaled mutation rate, if you prefer). So that at equilibrium pi= = 4Ne (where is the mutation rate). So the authors are correct that the mutation rate alone does not explain low nucleotide diversity, but this does not suggest at all that nucleotide diversity could reflect overexploitation. It most likely (given generation time and time for pi to reach equilibrium) reflects historical demographic events. So the point that diversity is likely shaped by long term Ne or other demographic events should be made, in my opinion. Indeed, theta (and pi at equilibrium) are indeed a measure of population size given a mutation rate.

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Reviewer #3: No

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Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation

PONE-D-22-04909R2

Dear Dr. Elizondo-Sancho,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Johann Mourier, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your effort in revising your manuscript.

I am now happy to recommend your work to be published in Plos One. I hope you enjoyed the reviewing process to help you improve your manuscript.

Kind regards

Johann

Reviewers' comments: