PONE-D-21-37151Genetic signature of blind reintroductions of Iberian ibex (Capra pyrenaica) in Catalonia, Northeast SpainPLOS ONE

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(T.B. is funded by national funds through CESAM/DBIO under the POSEUR project CP01-MARG-QUERCUS/2018. https://poseur.portugal2020.pt/

E.F. is funded by national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. https://www.fct.pt/

J.F. was supported by a PhD grant from FCT/MCTES (PD/BD/150645/2020), co-financed by European Social Fund POPH-QREN program. https://www.fct.pt/

J.C. was supported by a research contract (CEECIND/01428/2018) from the Fundação para a Ciência e a Tecnologia (FCT). https://www.fct.pt/

FCT/MCTES financed CESAM (UIDP/50017/2020), through national funds. https://www.fct.pt/

“Apoio à Contratação de Recursos Humanos Altamente Qualificados” (NORTE-06-3559-FSE-000045), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement. ForestWISE—Collaborative Laboratory for Integrated Forest & Fire Management, was recognized as a CoLAB by the Foundation for Science and Technology, I.P. (FCT). https://www.forestwise.pt/pt/institution/

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(T.B. is funded by national funds through CESAM/DBIO under the POSEUR project CP01-MARG-QUERCUS/2018. E.F. is funded by national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. J.F. was supported by a PhD grant from FCT/MCTES (PD/BD/150645/2020), co-financed by European Social Fund POPH-QREN program. J.C. was supported by a research contract (CEECIND/01428/2018) from the Fundação para a Ciência e a Tecnologia (FCT). Thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020), through national funds. This work is also a result of the project “Apoio à Contratação de Recursos Humanos Altamente Qualificados” (NORTE-06-3559-FSE-000045), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement. ForestWISE—Collaborative Laboratory for Integrated Forest & Fire Management, was recognized as a CoLAB by the Foundation for Science and Technology, I.P. (FCT). This article/publication is based upon work from COST Action, supported by COST (European Cooperation in Science and Technology). We grateful Josep Vicens Jovani, Xavier Oliver, Maria Josep Vargas, Xavier Sempere, Jordi Xifra and Ignasi de Dalmases, from the Tortosa and Beseit National Game Reserve, the Controlled Game Area of Montserrat, the Game and Fish Section of Girona (DARP) and the Agents Rurals body, for their advice and samples provided.)

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(T.B. is funded by national funds through CESAM/DBIO under the POSEUR project CP01-MARG-QUERCUS/2018. https://poseur.portugal2020.pt/

E.F. is funded by national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. https://www.fct.pt/

J.F. was supported by a PhD grant from FCT/MCTES (PD/BD/150645/2020), co-financed by European Social Fund POPH-QREN program. https://www.fct.pt/

J.C. was supported by a research contract (CEECIND/01428/2018) from the Fundação para a Ciência e a Tecnologia (FCT). https://www.fct.pt/

FCT/MCTES financed CESAM (UIDP/50017/2020), through national funds. https://www.fct.pt/

“Apoio à Contratação de Recursos Humanos Altamente Qualificados” (NORTE-06-3559-FSE-000045), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement. ForestWISE—Collaborative Laboratory for Integrated Forest & Fire Management, was recognized as a CoLAB by the Foundation for Science and Technology, I.P. (FCT). https://www.forestwise.pt/pt/institution/

This article/publication is based upon work from COST Action, supported by COST (European Cooperation in Science and Technology). https://www.cost.eu/

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: 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: Please state clearly in the text:

1. How many individuals were the goat samples used? Whether the 185 muscle samples came from different individuals

2. How are these individuals related?

3. What are the reasons for choosing 14 microsatellite markers?

4. Please refer to the journal requirements to modify the table format.

5. FIG. 2, this labeling method is difficult to identify, so it is suggested to adjust the background color of the satellite map or replace it with a blank background containing latitude and longitude labels.

6. It is recommended to start with the introduction or discussion to simplify the article, which should correspond to the conclusion

Reviewer #2: I have evaluated the paper “Genetic signature of blind reintroductions of Iberian ibex (Capra pyrenaica) in Catalonia, Northeast Spain”. In this study, the authors characterize the variability of three Iberian ibex populations from Catalonia by using mitochondrial sequences and 14 microsatellite markers. No signature of introgression with domestic goats is identified and a close affinity between the Tortosa Beseit and Montserrat populations is detected, while the Montgrí population appears to be more differentiated. Conservation and management strategies are also discussed in the light of the results obtained by the authors. This paper can be of interest to readers of PloS One, but a major revision is needed. Please provide a detailed response to each one of the queries outline below indicating in which lines of the new manuscript changes have been made

With regard to the scientific content of the paper

Q1.My main observation is that a representative number of domestic goats must be genotyped for the 14 microsatellites and such data should be included in the Structure plot. Otherwise, it is hard to understand how the authors conclude that microsatellite data corroborate the absence of hybridization with domestic goats (?).An added benefit of genotyping some goats would be to compare their levels of diversity with those of ibexes. A PCA including the 3 ibex populations plus a domestic goat population would be also welcome.

Q2.In the Results section, please divide it in 2 subsections: one for mitochondrial data and another one for microsatellite data. In L315 indicate the FST value even if it is not significant. Sometimes it is difficult to infer whether you are talking about mitochondrial or microsatellite results. This needs to be presented in a much clearer way.

Q3.I have difficulties in understanding the “assignment test”. The basis of such analysis needs to be explained with more detail in the materials and methods section. In L307-8 is explained that the assignment test is based on Structure results and I assume that this refers to microsatellite genotypes, right? Then the clusters should be Tortosa Beseit+Montserrat and Montgrí (K=2), or Tortosa Beseit, Montserrat and Montgrí. So, how an assignment probability can be calculated for domestic goats in Table S3? Based on mitochondrial data? But mitochondrial data are haploid, so no point in making a Structure analysis. The authors are using an admixture model assuming that individual i has inherited some fraction of its genome from ancestors in population k and yielding, as output, the posterior mean estimates of these proportions. Please check whether the term is “posterior probablilities” used in the legend of Fig. 4 is correct in this context (in the non-admixture model it would, but I am not so sure about the admixture model so I advise to verify it). Data presented in Table S3, from where do they come from? What is genetic cluster 1 and 2? Cluster 2 is domestic goats? But not a single domestic goat has been genotyped with microsatellites and subsequently analysed with Structure, right? Each Table and Figure should be meaningful by itself, so please indicate with precision what are you displaying because this part of the text is very confusing. Indicate in a clear way which type of marker and which clusters or popultions are you taking into consideration in each of the analyses. Detailed legends for tables and figures are very helpful to readers. Detailed methodological explanations are also very welcome.

Q4.What is your interpretation about the failure to detect bottlenecks with the Bottleneck software despite the demographic history of these 3 ibex populations? Is the Bottleneck software well suited to detect any type of bottleneck? This should be discussed properly.

Q5.Does it make sense that the Montserrat population is non-inbred (see lines 453-4) and its source population (Tortosa Beseit) is inbred based on FIS measurements despite of having a much larger size? I find it very hard to believe (how a population recently derived from an inbred source population can be non-inbred?). This needs to be discussed. With 46 founders and no additional incoming individuals, it is impossible that the Montserrat population is not inbred. Maybe this is explained by the inherent lack of power of FIS to detect inbreeding when scarce molecular data are available. In any case, an interpretation should be provided to understand this unexpected outcome.

Q6.L135, 370, 371, 387. I am nost sure whether is realistic to hypothesize that Iberian ibexes might be introgressed with Alpine ibexes or vice versa. Their geographic ranges are completely different and they are separated by 1,000 km, so how could the introgression of Iberian ibexes with Alpine ibexes be a threat (L136)? Such hypothesis needs to be justified or discarded as unrealistic.

Q7.L193, sequencing protocol should be explained

Q8.L325, why a high HWE departure is expected? The numbers of microsatellites in HWD are quite low when populations are considered individually, and I am not sure if it is correct to merge three different populations into a single one and then state that 8/14 microsatellites were in disequilibrium. In other words, could population stratification contribute to this HW departure?

Q9.L404,is it correct to say that TBNGR population has a high diversity? If compared to domestic goats, sure that it does not. This population in 1966 had around 400-500 individuals.

Q10. Both microsatellite genotypes and mitochondrial sequences should be made available. Please include an availability statement in the manuscript.

With regard to formal issues:

Q11. There are many typos that should have been corrected before all authors approved the final submission of the paper.

In many places populations are named in (at least) 2 different manners

e.g Montserrat (correct) and Monserrat (incorrect, please ammend)

Cataluña and Catalonia (use Catalonia since it is an English paper)

Montgrí (correct) and Montgri (incorrect)

Tortosa y Beceit (Admixture plot, incorrect), Tortosa-Beseit (correct), Beseit (L451, incorrect), Tortosa i Beseit (correct), Tortosa and Beseit (correct). Although 3 terms are correct, this population needs to be named with just one single denomination, not multiple different names. Please pick one of these denominations and use it consistently throughout the paper.

The format of the references is also completely inconsistent, as if anybody had revised it. Even if a free-format is allowed, this does not imply than the references can be written in multiple inconsistent formats. Some examples: L572, 3-37.3. (??), plus minus sign in L587 and many other places, L584, a DOI is provided, but only for this ref. , L596,, lacks volume number and pages, L663, 1994 November, L664 PMID etc etc. Please use a free but coherent format to cite papers, and make sure that you are citing them correctly.

Other formal issues:

L138, Iberian peninsula

L174, none of the animals were hunted

L182-3, from the 185 muscle samples, please indicate the number of samples for each population (between parentheses).

L187, 2.5 microliters of 10x buffer

L188, please recheck whether dNTP concentrations is 10 micromolar. It seems quite low to me. MgCl2, the 2 should be subindexed

L199, electrophoresed, not sequenced (if you are talking about microsatellites)

L209, I would say diagnosis

L216, electropherograms

L217, visual inspection (not manual)

L247, indicate how many individuals from each population were successfully sequenced

L292, do you mean 96 ibexes with data for 14 microsatellites?

L308, STRUTUCTURE

L324, sensible means “of good judgement”. Replace by sensitive

L369, gene flow

L370, Alpine

L402, nuclear what?

L423,424, FST without subindexing ST

L451, matrilineal

L497, genetic diversity (not traits)

L508, not sure vein territories is correct, maybe neighboring?

The resolution of the network Figure is too low and the names of the populations are unreadable (please write them with a larger font).

In Table 2, what is the meaning of IAM = xxx and SMM = dxxx? Besides, abbreviations could be defined in a footnote rather than in the legend of the table.

Figure 2, needless to write gene pool 1, 2 and 3. Do not superimpose K values onto the Figure

Instead of Supporting materials I would say Supporting or Supplementary Tables. I see a list of references below Table S2, but I am not sure whether refs corresponding to Table S1 are mentioned anywhere (?).

Table S3, decimals separated with points, not commas

This list of errors/typos is not exhaustive, so I advise all authors to take a thorough look at the revised paper.

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Reviewer #1: No

Reviewer #2: No

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Genetic signature of blind reintroductions of Iberian ibex (Capra pyrenaica) in Catalonia, Northeast Spain

PONE-D-21-37151R1

Dear Dr. Barros,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Yes

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

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. 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 #2: The authors have addressed my queries correctly. I have a few amendments that the authors might take into consideration:

L123, that have

L149, at stake

L260, Fig. 3?

L377, MonTserrat

L441-4, I would rewrite this sentence (this population appears 4 times and a populaion carried out by few individuals sounds awkward)

L520, might occur

L542, Agents Rurals corp

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #2: No