Critically Small Contemporaneous Effective Population Sizes Estimated for Stocks of the African Bonytongue in Western Africa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Monitoring the effective population size of African bonytongue （Heterotis niloticus）is of great significance to the resource status of this species in West Africa. The methods and software used in this study to estimate the effective population size are relatively general, and the results have positive effects in the field. However, there are several concerns in the manuscript that call the science of the findings into question. The details are as follows:

1.    The number and distribution of SSR markers in each population should be given in the main text of the manuscript, as well as the markers used in the estimation of effective population size.

2.    The range of Ne estimates for some populations is too large in Table 2. Please give the reasons in the discussion. It should be excluded that the number of SSR markers is insufficient or the distribution of genomic regions is not balanced, which makes the published markers unsuitable for Ne estimation.

3.    The conclusions presented in the manuscript title do not match the results, and necessary changes should be made to make the title and content consistent.

4.    In addition, in my personal opinion, for Ne estimates of multiple populations distributed over such a wide range of water bodies, molecular markers should achieve low levels of genome-wide coverage to improve estimation accuracy.

For the above reasons, I am wary of the results presented in this manuscript and the conclusions drawn therefrom, and more evidence is needed to reassure. Therefore, it is recommended that the editorial department reject the paper and encourage the author to re-submit after adding new and convincing data.

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

Reviewer 1

Monitoring the effective population size of African bonytongue（Heterotis niloticus）is of great significance to the resource status of this species in West Africa. The methods and software used in this study to estimate the effective population size are relatively general, and the results have positive effects in the field. 

However, there are several concerns in the manuscript that call the science of the findings into question. The details are as follows:

1. The number and distribution of SSR markers in each population should be given in the main text of the manuscript, as well as the markers used in the estimation of effective population size.

Our response: We have added a new table (Table 2), which provides the number of SSR markers used in this study. Supplemental Material SM1 has the individual genotypes and the distribution of allele frequencies per population.

2. The range of Ne estimates for some populations is too large in Table 2. Please give the reasons in the discussion. It should be excluded that the number of SSR markers is insufficient or the distribution of genomic regions is not balanced, which makes the published markers unsuitable for Ne estimation.

Our response: We interpret that the reviewer refers to the upper bounds of confidence intervals (CIs), which were infinity in most cases when using NeEstimator, the software program used in our previous submission. The method used by NeEstimator for calculating CIs, which is not a jackknife method, has some limitations, especially for upper bound CI values when using small sample sizes, which result in negative estimations that are reported as infinity. In this version, we implemented three jackknife methods with correction for small sample size to calculate CI, which reduced the number of negative (i.e., infinity) CIs upper bounds. For nine populations, the upper bound was finite and the CI was narrow. Corrections for small sample size were also applied when inferring the Ne point estimates. We have enhanced our explanation of the issue of estimation of negative values (reported as infinite values) with the following text (Discussion, ~line 330): ‘Disequilibrium methods can produce negative estimates, which are reported as infinite. This occurs, because to estimate Ne, the expected contribution of disequilibrium from sources other than drift is subtracted from the total estimated disequilibrium or r². Accordingly, if the sample of individuals and loci is relatively small given the true Ne, mean disequilibrium (r²) can be smaller than the correction for disequilibrium due to sources such as finite sample size, leading to a negative Ne estimate (Waples and Do 2010).’

Because of the problems with NeEstimator when using small sample sizes, in this revised version, we used a different program to estimate Ne (SpEEDNe; M. B. Hamilton, the author of the program is a coauthor in our manuscript’s new version), which computes 16 different Ne estimators, including Waples’ estimators, and also performs simulations to determine which of these Ne estimators are most appropriate for particular datasets. The following text explains the methods/rationale for performing computer simulations to determine the best estimator for our data (from Methods; ~line 162). “Because there are numerous estimators of Ne that employ two-locus disequilibrium (r²), we carried out simulations using SpEED-Ne (Hamilton et al. 2018) to evaluate which estimators exhibited the greatest accuracy and precision with sample sizes of individuals, loci, and numbers of alleles per locus that approximated those of sampled populations. Time-forward simulations were carried out following Hamilton et al. (2018), generating microsatellite genotype data sets with true Ne equal to 10, 25, 50, 100 and 250, each with 250 independent replicates.”

The following text from the Results (~line 215) identifies the two best Ne estimators, which are the ones we present in our results, along with three jackknife methods to calculate CIs, correcting for small sample sizes. “Simulated genotype data (SM3) showed that for true Ne values of 10, 25, and 50, the  UB-AFT estimator, followed by  UB-AFW estimator, provide the most accurate estimates among the 16 compared (i.e., median slightly biased, smaller quartiles ranges, and markedly fewer outliers).” 

For most (9 of 12) populations, we obtained narrower and finite CIs (shown in Table 3; formerly Table 2). For comparison, we retained the NeEstimator (Waples) values in the table.   

3. The conclusions presented in the manuscript title do not match the results, and necessary changes should be made to make the title and content consistent.

Our response: This is probably again referring to the infinity upper CI values. With the new CI (and point estimates) calculations, we consider that the title and the results are consistent.

4. In addition, in my personal opinion, for Ne estimates of multiple populations distributed over such a wide range of water bodies, molecular markers should achieve low levels of genome-wide coverage to improve estimation accuracy.

Our response: As mentioned above, the computer simulations show that the estimators we selected should be fairly precise for our data. These arguments are more thoroughly developed in our revised Discussion (~line 268): “The computer simulations we conducted indicated that Ne estimates obtained using r²_D with AFT and the small sample size bias correction (UB) are expected to be fairly precise for the genotypic data used, especially when Ne  50, which encompasses the Ne estimates we obtained for nine populations. This is consistent with expectations on the performance of the disequilibrium N_e estimation approach, with which precise estimates for relatively small (Ne < 200) isolated populations can be obtained, and small populations are not likely to be mistaken for large ones (Waples and Do 2010). Waples (Guest Box 10 in Allendorf et al. 2013) indicates that if Ne is relatively small (< 100), reasonably precise estimates can be obtained using samples of 25–50 individuals with 5–10 moderately variable loci; however, considerably more data are needed to achieve comparable precision if the population is relatively large (Ne ∼ 500–1000 or higher). Gilbert and Whitlock (2015) used simulations to compare the performance of seven Ne estimation methods under different scenarios of migration with three different Ne values (50, 500 and 5000), and found that the disequilibrium method (implemented in NeEstimator v2.0 in their study) outperformed the other methods in conditions of isolation for Ne = 50. This method also performed well in scenarios of low migration and small Ne. Accordingly, because the Heterotis populations studied herein exhibit genetic differentiation consistent with isolation (Hurtado et al., 2013; Oladimeji et al., 2022), the small Ne estimates we obtained are likely reflective of the true Ne.”

For the above reasons, I am wary of the results presented in this manuscript and the conclusions drawn therefrom, and more evidence is needed to reassure. Therefore, it is recommended that the editorial department reject the paper and encourage the author to re-submit after adding new and convincing data.

Our response: As mentioned above, for this resubmission we used a new program, SpEEDNe, that estimates Ne using 16 different estimators, and performs simulations to compare them. The author of SpEEDNe is one of the coauthors. We conducted computer simulations to determine the best estimator (among the 16) for the range of Ne values obtained, under the sample sizes and loci number we used. We implemented three jackknife methods with correction for small sample size to calculate CI. The CIs of most populations (9 out of twelve) were narrow and finite. We are confident that the populations for which we report small Ne, indeed have a small Ne. While more data are always desirable, we consider that our revised approach demonstrates that the data we used are adequate for the inferences we make. 

Reviewer 2 Report

Comments and Suggestions for Authors

Review of Critically Small Contemporaneous Effective Population Sizes for Stocks of the African Bonytongue in West Africa by Hurtado et al.

 

General Comments

            This is a very well written, interesting study with potentially important conservation implications. My main concern is whether or not these “populations” should be considered separately as done here. The authors need to demonstrate that these are distinct populations of potential concern and that the genotypic differences presented in cited sources that their results are based on do not represent sampling error among those sources.

Specific Comments

·       Introduction, line 92. Are there total population estimates for the populations considered in this study? How isolated are they from each other? Are there estimates of gene flow between the populations? What is the areal extent of the distribution of each population? These questions are aimed at whether or not study populations should be considered genetically distinct (as assumed here) or pockets of genetic variation within a larger connected population.

·       Introduction, Table 1. In caption, delete “of live weight.”

·       Introduction, Table 1. Why are percentages in parentheses?

·       Introduction, Table 1. Eliminate “(9^th globally) in last column. It is not consistent with the column title and is already presented in the text.

·       Introduction, Table 1. For the row titles, replace “inland” with “all inland fisheries.”

·       Introduction, lines 94-104. Information on genetic differentiation is presented in two different ways in these lines. Only for southern Nigeria are F_ST values referenced. It would be useful to state the relative genetic differentiation and F_ST values for all. In this context, what does “lower but significant F_ST values” mean? Lower than other sites in your study? Statistically significant?

·       Methods, line 111-112. When were the genotypic data in these citations collected? How contemporary were the different data sets?

·       Methods, lines 113-116. These lines repeat the descriptions in lines 94-106. A simple reference to the early text would suffice.

·       Methods, lines 117-119. Why was this not done for the other three countries?

·       Methods, lines 120. It is stated here that contemporary Ne was estimated for all populations. Based on text that follows, you did this for all study populations in the three countries evaluated, but based on its placement, the statement could be misinterpreted as applying to just Cameroon populations.

·       Results, line 160-161. Provide in parentheses the names of the other southern locations you are referring to.

·       Results, lines 169-170. This statement should be moved to a point earlier in the ms when infinite values are first discussed.

·       Results, Table 2. Why are the confidence intervals calculated for raw values only and not for life span-adjusted values?

·       Results, Table 2. The presentation of infinite values is a bit confusing and misleading since it implies an infinitely large population size. “Not determined” would be more appropriate with a footnote saying why. You say this is due to sampling error, but if the raw value can’t be computed (e.g., Nkam and Nyong rivers), how is a lower confidence limit calculated? A bit more explanation may be in order.

·       Discussion, lines 178 and 182. Here and elsewhere, the terms “Ne estimates” and “Ne estimations” are used interchangeably. Choose one.

·       Discussion, line 209. Briefly describe STRUCTURE analysis.

·       Discussion, line 312. Add at end of sentence “because this designation applies to the species as a whole.”

Author Response

General Comments

            This is a very well written, interesting study with potentially important conservation implications. My main concern is whether or not these “populations” should be considered separately as done here. The authors need to demonstrate that these are distinct populations of potential concern and that the genotypic differences presented in cited sources that their results are based on do not represent sampling error among those sources.

Our response: Regarding the concerns about whether the populations are genetically distinct, in our previous articles (Hurtado et al. 2013 and Oladimeji et al. 2022), we demonstrated that the populations from Benin and Nigeria were genetically distinct, and discussed their conservation concerns. These studies were led by Hurtado, and included most coauthors, and we are using their datasets for this study. Thus, we do not consider we need to further demonstrate the population genetic differences in this study, as they were subject of thorough analyses in these previous papers. In the case of the Cameroon populations (Wikondi et al. 2023), the authors of that study showed genetic differences, but did not thoroughly interpret them. This is why we are including population genetic analyses of their populations, which clearly show that their five populations are genetically differentiated (Supplemental Materials SM2).

Specific Comments

• Introduction, line 92. Are there total population estimates for the populations considered in this study? How isolated are they from each other? Are there estimates of gene flow between the populations? What is the areal extent of the distribution of each population? These questions are aimed at whether or not study populations should be considered genetically distinct (as assumed here) or pockets of genetic variation within a larger connected population.

Our response: As mentioned above, the previous studies in Benin and Nigeria demonstrated the different populations considered in this study are genetically differentiated. For Cameroon, we are providing analysis of population genetic differentiation.

• Introduction, Table 1. In caption, delete “of live weight.”

      Our response: We revised the caption for Table 1 to: ‘Inland capture (live weight) of all fishes and Heterotis niloticus in Africa, Nigeria and Benin, and for all fishes in Cameroon.’

• Introduction, Table 1. Why are percentages in parentheses?

      Our response: Parentheses have been removed.

• Introduction, Table 1. Eliminate “(9^thglobally) in last column. It is not consistent with the column title and is already presented in the text.

Our response: We have made the suggested change.

• Introduction, Table 1. For the row titles, replace “inland” with “all inland fisheries.”

Our response: We eliminated “inland” in the row titles as the title specifies ‘Inland capture’.

• Introduction, lines 94-104. Information on genetic differentiation is presented in two different ways in these lines. Only for southern Nigeria are F_STvalues referenced. It would be useful to state the relative genetic differentiation and F_ST values for all. In this context, what does “lower but significant F_ST values” mean? Lower than other sites in your study? Statistically significant?

Our response: This text was removed. We refer to the previous studies that identified these populations as genetically differentiated, where readers can obtain more details on the degree of differentiation and statistical significance.

• Methods, line 111-112. When were the genotypic data in these citations collected? How contemporary were the different data sets?

Our response: Collection years are shown in Table 2.

• Methods, lines 113-116. These lines repeat the descriptions in lines 94-106. A simple reference to the early text would suffice.

Our response: The text has been substantially modified; there shouldn’t be as much redundancy in the revised version.

• Methods, lines 117-119. Why was this not done for the other three countries?

Our response: As explained above, we present analyses of population genetic differentiation for Cameroon only, because the authors did not thoroughly interpret their results. For Benin and Nigeria, we presented those analyses in our previous papers.

• Methods, lines 120. It is stated here that contemporary Ne was estimated for all populations. Based on text that follows, you did this for all study populations in the three countries evaluated, but based on its placement, the statement could be misinterpreted as applying to just Cameroon populations.

Our response: The text has been modified to increase clarity.

• Results, line 160-161. Provide in parentheses the names of the other southern locations you are referring to.

Our response: The text has been modified to increase clarity.

• Results, lines 169-170. This statement should be moved to a point earlier in the ms when infinite values are first discussed.

Our response: This was removed. In the Discussion, we explain why infinity is reported in Linkage Disequilibrium methods.

• Results, Table 2. Why are the confidence intervals calculated for raw values only and not for life span-adjusted values?

Our response: This was removed.

• Results, Table 2. The presentation of infinite values is a bit confusing and misleading since it implies an infinitely large population size. “Not determined” would be more appropriate with a footnote saying why. You say this is due to sampling error, but if the raw value can’t be computed (e.g., Nkam and Nyong rivers), how is a lower confidence limit calculated? A bit more explanation may be in order.

Our response: A thorough explanation is provided in the Discussion.

• Discussion, lines 178 and 182. Here and elsewhere, the terms “Ne estimates” and “Ne estimations” are used interchangeably. Choose one.

Our response: We use “estimates” throughout the text.

• Discussion, line 209. Briefly describe STRUCTURE analysis.

Our response: A brief description of the STRUCTURE results of Wikondi et al. (2023) is provided (~line 213).

• Discussion, line 312. Add at end of sentence “because this designation applies to the species as a whole.”

Our response: added “; a designation that applies to the species as a whole” ~line 468

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled “Critically small contemporaneous effective population sizes for 2 stocks of the African bonytongue in West Africa” aims to estimate the effective population size in Heterotis niloticus populations in Nigeria, Benin, and Cameroon, assessing genetic indicators of inbreeding depression and the risk of losing evolutionary potential. I believe that this manuscript constitutes a noteworthy advancement in the conservation of the species, providing essential support for the sustainable management of its populations amidst various anthropogenic and environmental pressures.

In providing constructive feedback, I would appreciate more comprehensive detailed information regarding the samples employed in this study. While I understand that the data are sourced from published papers, I am seeking specifics in additional information such as the data type, number of microsatellites, number of fish samples. Are genetic data collected over multiple generations to estimate changes in Ne over time? This approach can provide insights into historical trends in population size. The quality and quantity of genetic data, including markers and sample sizes, play a crucial role in the precision of Ne estimates. Could you provide a summary detail for each population, and clarify whether all available data were incorporated into the analysis? This additional information will not only enhance the transparency of the study and a more comprehensive understanding of the methodology.

Also, please pay careful attention to the reference format of the journal when citing sources in your manuscript.

Author Response

Reviewer 3

The manuscript titled “Critically small contemporaneous effective population sizes for 2 stocks of the African bonytongue in West Africa” aims to estimate the effective population size in Heterotis niloticus populations in Nigeria, Benin, and Cameroon, assessing genetic indicators of inbreeding depression and the risk of losing evolutionary potential. I believe that this manuscript constitutes a noteworthy advancement in the conservation of the species, providing essential support for the sustainable management of its populations amidst various anthropogenic and environmental pressures.

In providing constructive feedback, I would appreciate more comprehensive detailed information regarding the samples employed in this study. While I understand that the data are sourced from published papers, I am seeking specifics in additional information such as the data type, number of microsatellites, number of fish samples. Are genetic data collected over multiple generations to estimate changes in Ne over time? This approach can provide insights into historical trends in population size. The quality and quantity of genetic data, including markers and sample sizes, play a crucial role in the precision of Ne estimates. Could you provide a summary detail for each population, and clarify whether all available data were incorporated into the analysis? This additional information will not only enhance the transparency of the study and a more comprehensive understanding of the methodology.

Our response: We agree that adding this information enhances the paper. This was also suggested by the other reviewers. We have included this information in Table 2 and Supplemental Material SM1.

Also, please pay careful attention to the reference format of the journal when citing sources in your manuscript.

Our response: According to the guidelines of the journal, there is not a format for the references, However, we are willing to work with the editorial office on this if necessary, once the manuscript is accepted.

• Your references may be in any style, provided that you use the consistent formatting throughout. It is essential to include author(s) name(s), journal or book title, article or chapter title (where required), year of publication, volume and issue (where appropriate) and pagination. DOI numbers (Digital Object Identifier) are not mandatory but highly encouraged. The bibliography software package EndNote, Zotero, Mendeley, Reference Manager are recommended.
• When your manuscript reaches the revision stage, you will be requested to format the manuscript according to the journal guidelines.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In this updated edition, I observe the author's thorough responses to the queries I raised in the previous round of review comments, enhancements in analytical methodologies, and systematic revisions to the manuscript's maintext. In my view, the scholarly quality of this manuscript has been significantly enhanced and it now meets publication standards.