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The raised criticisms were now satisfactorily answered.
[# PeerJ Staff Note - this decision was reviewed and approved by Jennifer Vonk, a PeerJ Section Editor covering this Section #]
I reviewed the first version of this manuscript, as well as the second version. The authors have now corrected some minor issues, and I am happy with the final version.
All the best,
Barbara van Asch
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I thank the authors for the extensive revision of the first version of the manuscript. Some minor issues remain, however; I hope the authors can successfully satisfy the criticisms raised on the last version.
The revised manuscript is a great improvement over the original version, and I appreciate that the authors put honest effort in recognizing weak aspects and made good use of the reviewers’ suggestions, including mine. I am happy to say that the issues I raised have all been addressed to my satisfaction.
The new version of the network is a massive improvement. Please consider improving the colour legend: remove the underscore in the names of the regions, increase the size of the outline so it does not overlap with the text, the circles and text should not overlap.
Please note that some colours are very difficult to read when placed on a white background. This is the case in Figure 1 (yellow, light grey and light blue). The spacing between the letters in the names of the outgroups should be corrected for a more polished appearance of the figure. Please check this type of issue throughout the manuscript. I understand that the first author is a new graduate starting his career as a researcher, and I congratulate him on the revision of this manuscript. However, I insist that attention to detail is necessary in this profession, and there are still points to be addressed in the writing style and presentation. For example, the phrases “(P. leo, 3 sequences : Accession numbers: KP001498, KP001502 & KP001506) and three non-African leopard subspecies (P. p nimr, P. p fuscia and P. p orientalis : Accession numbers AY035279, EF199743 & KY866876).” have mistakes such us spacing between words and punctuation, lack of full stop in the abbreviated name of the species, and unnecessary use of “&”. Please also note that there are rules for the use semicolon (;) and colon (:). The latter should not be used in e.g. titles of sections.
Congratulations and best wishes for your present and future work.
No issues
No issues
No issues
The revisions are a substantial improvement to the manuscript's readability and validity. The figures were submitted as .eps files, which I do not have the ability to open. Cannot see changes/edits the authors made to figures.
The edits are a welcomed improvement to this section.
I agree with Reviewer 1 and Reviewer 3 that the study's conclusions were a bit far reaching for the analysis conducted by the authors. You've tempered the exaggeration. The revised manuscript is substantially improved.
I cannot view the figures but trust the judgement of Reviewer 1 and Reviewer 3. I will indicate this manuscript as 'accepted' pending R1 and R3 do not suggest further revision. If further revision is requested, please provide figures in a format that is compatible across devices. Thank you.
The manuscript raises serious methodological issues and reveals general carelessness in the presentation of the report. I hope the Authors can successfully address the Reviewers’ criticisms; please be sure to detail your answers when writing your rebuttal letter, as well as marking the changes in the new version.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
LANGUAGE AND STYLE
I strongly recommend English language revision. I used the Abstract to exemplify that there is much room for improvement as it would be too tedious and beyond my role as scientific reviewer to annotate and make suggestions throughout the whole manuscript. Please see below the original text, and some suggestions/corrections in track changes. (“across the continent” appears four times in the Abstract)
“The African leopard (Panthera pardus pardus) is facing a risk of extinction across the continent. Recent studies have explored the genetic diversity and population structure of leopards across the continent to help inform conservation decisions. In Africa, both Both mitochondrial genome and whole genome leopard data sets have been published, and the most notable genetic observation is the presence of two divergent mitochondrial lineages, PAR-I and PAR-II. Both lineages appeared to be distributed widely, with PAR-II more concentrated frequently found in southern Africa. Until now, no study has attempted to date the emergence of either lineage, assess their distribution across the continent, or explore their evolutionary histories in any detail. Therefore, to To investigate these interesting and underappreciated questions, we assembled compiled and analysed the largest and most geographically representative leopard data set of the mitochondrial NADH-5 gene to date. We combined samples (n = 33) collected in an altitudinal transect across the Mpumalanga province of South Africa, where two leopard populations of leopard are known to be in genetic contact, with previously published sequences representing individuals from across the continent (n = 211). We found estimate that the maternal PAR-I and PAR-II lineages diverged approximately 0.7051 (0.44773-0.9632) million years ago (Ma). Through Our spatial and demographic analyses, we show that while PAR-I underwent a mid-Pleistocene population expansion resulting in several closely related haplotypes with little geographic structure across much of its range, PAR-II remained at constant size and may even have declined slightly in the last 0.1 Ma. The higher genetic drift experienced within PAR-II drove a greater degree of structure with little haplotype sharing and unique haplotypes found in central Africa, the Western Cape and, Kwazulu-Natal provinces of South Africa, and the South African Highveld. The phylogeographic structure of PAR-II implies points to its origin in central Africa, but its increasing frequency southward and its exclusive occurrence in south / and south-eastern South Africa suggests that this lineage may have been isolated in South Africa during the mid-Pleistocene. This hypothesis is supported by historical changes in paleoclimate that promoted intense aridification around the Limpopo Basin between 1.03-0.6 Ma, potentially reducing gene flow and promoting genetic drift.”
Please rewrite the final part of the Abstract so that the conclusions of your study and their impact on conservation and management of leopards are clearer. I cannot agree that the authors “highlight several interesting aspects of leopard biology” – there is no mention of biological aspects in this study except for an apparent association between size and mitochondrial lineage. Please also note that the statement “our interpretations require further testing with a denser sample of nuclear genomes” implies that you used nuclear genomes in your analyses, which was not the case.
“Interestingly, we ascertained that the two leopard populations previously sampled in Mpumalanga were are in fact PAR-I and PAR-II, and that the two ? are in secondary contact in north-eastern South Africa. Although our interpretations require further testing with a denser sample of nuclear genomes, we highlight several interesting aspects of leopard biology and provide a first, although tentative, genetic explanation for the smaller observed size of leopards at the southernmost end of their range in Africa. If our interpretation holds true, it will have significant consequences for the conservation management of the leopard in South Africa.”
EXPERIMENTAL DESIGN
I detected issues in the experimental design.
Median-joining network: why were outgroups included in the MJ network? Networks are, by definition, rootless trees useful for displaying relationships between closely related haplotypes. The inclusion of outgroups does not make sense and creates unnecessary reticulations. In fact, if PAR-I and PAR-II are as diverged as the Bayesian tree suggests, the two lineages should be shown in two separate networks to show the relationships among the haplotypes within each lineage. The colour scheme can be simplified if PAR-I and PAR-II are shown separately because you can use the same colour for each country/region in both networks, and the figure would be more reader-friendly.
Phylogenetic tree: nodal supports inferior to 0.5 is extremely low. In fact, according to many authors, posterior probabilities inferior to 0.85 are negligible. This means that the reader has no way of assessing the nodal support of the phylogeny. This issue must be addressed, and low nodal support must be acknowledged and taken into account by the authors in their assessment of the phylogeny. Another issue: why do you present the consensus tree and not the best tree? This aspect may drastically influence the phylogeny. The colour scheme is misleading for the reader: please highlight the two clusters of haplotypes that form PAR-I and PAR-II e.g. using a line, and use the same colour for each country/region.
Dating: the authors must acknowledge and discuss the fact that their time estimates are based in a single, mitochondrial gene. This aspect is not negligible and deserves appropriate treatment within this study.
I detected many issues in the description of the methods, some of them linked to inadequate use of terminology specific to population genetics / phylogeography. For example, “maternal” appears in many instances where “matrilineage” or “matrilinear” should be written. Please note that “maternal” and “matrilinear” are very different concepts. Please see pdf for some highlights.
At this point, the validity of the findings is compromised due to experimental issues (MJ network, nodal support of phylogenetic tree, consensus vs best tree, lack of clarity due to confusing colour schemes in both the tree and the MJ network. I give suggestions in the experimental section.
The study was conducted somewhat appropriately and addresses a relevant knowledge gap. However, I see numerous analytical issues that must be addressed (which I will highlight later). The general presentation has much room for improvement - it seems to stem directly from a postgraduate thesis and requires the attention of a senior author with experience in reporting population and phylogenetic analyses. I’ll use the section starting in line 266 as an example: the title should be “Mitochondrial phylogeographic structure” instead of “Spatial genetic structure”; the text includes statements that do not belong in the M&M; redundant explanation of how pie charts were plotted on a map, including the colours – this can be simply given on the figure itself.
I believe that the manuscript will greatly benefit from a detailed revision by senior researchers who are familiar with the standards of international journals such as PeerJ, and the standards of scientific communications in the field of population genetics. I highlighted some issues in the pdf but not extensively, as it would be extremely tedious due to the apparent lack of attention to detail from the authors. It is my opinion that this manuscript was not ready for submission, and should have been polished with regards to presentation, language and style in addition to methodological issues that I highlight.
Please also note that natural specimens do not have nationalities: e.g., “South African leopards” is incorrect (as well as “Mpumalanga samples” and similar wordings). Please also note that “sample” and “specimen” are not interchangeable terms and should be used correctly and in context.
I wish the authors all the best for their revision.
The authors use clear language through the introduction and discussion. Methods and Results are almost unavoidably full of jargon with molecular analysis papers. Suggestions to improve readership would be simple one sentence explanations of tests and why they are chosen. Many folks in the field will know what these tests are or where to find the information to look it up but do your reader a favor and make that information readily accessible. Think about reach, you want a non-molecular focused inidividual to be able to pick up your manuscript, understand it, and apply the knowledge, and hopefully cite you! Incorporate rationale for the tests you use, it will aid in scientific literacy of your manuscript, which should always be a primary goal.
Literature-cited adequately addresses the background of previously conducted molecular studies of African leopards. Manuscript hypotheses and support is self-contained.
I found several of your figures difficult to read. They should stand on their own and convey relatively quickly the 'big ideas' you wish the reader to comprehend. Specifically, in Figure 2: the coloring used on the haplotype network makes it nearly impossible to distinguish which samples belong to which haplotypes in the network. Consider a system that uses labels and attempts to color-coordinate by geography to orient the reader.
Figure 4, you reference this figure four times in your manuscript drawing the reader to population declines/contraction in the last 20 Ka, 100 Ka and expansion around 200 Ka but this isn't as clear with the figure alone. I would suggest placing markers/arrows, or some other designation to highlight areas you wish to draw to the reader's attention. Make comprehension of your data/figures paramount.
No Comment.
This was an interesting manuscript and it was nice to see additional novel sampling for leopards. mtDNA studies are increasingly falling out of favor with the accessibility of more comprehensive and robust sampling/analyses practices. I caution the authors in overextending their findings using <500 bp of mtDNA.
For example, see lines 355-357 of the manuscript. Please exercise extreme caution in making such a statement on the basis of <500 bp of mtDNA. You’re seeing only half the story (maternal inheritance) and looking at a VERY small section of the mitochondrial genome. Statements of panmixia need to be vetted against much longer sequences across numerous loci in both mitochondrial and critically, nuclear genomes. Previous studies that have concluded panmixia in African felids have been overturned. See Bertola et al. 2015 (lions) and Charruau et al. 2011 (cheetahs). Something to consider
Premature statements will strike readers as hyperbolic and can negatively impact the conclusions drawn from the manuscript. Overall, the manuscript is well-written and the conclusions supported by your data but please exercise caution in 'putting the cart before the horse' with statements like this.
The only issues I have are 1) the use of an individual loci to draw conclusions of panmixia (see above) and 2) the first sentence of the abstract (Lines 35-36), which is stated bluntly but not supported elsewhere in the manuscript.
Line 35-36: "The African leopard (Panthera pardus pardus) is facing a risk of extinction across the continent." This is a bold statement that is not supported 1) in your manuscript, or 2) elsewhere in the scientific literature. Populations are declining across most of their range within Africa, though they are still found in North, West, Central, East, and Southern Africa. Species facing extinction, on a continental scale, would be extirpated throughout much of this range. I found this statement hyperbolic and unsupported by the text in the body of the manuscript. Either justify this statement, modify it to more accurately reflect regional populations (yes, facing extinction in North Africa w/ 97% range contraction), or remove it as to not mislead the reader.
The paper is well written with a lot of detail. In my opinion it can be made shorter with to the point writing. It reads like an MSc thesis. The rest is all fine for publication.
I made some comments in the text that needs to be addressed for clarity.
The paper is interesting but in a way fail to meet one of the criteria set out - to help making management decisions based on the science presented. In the current form, all the nuclear evidence is speculative (or drawn from work already published) and this leaves the recommendation based on a mtDNA fragment only. Female gene-flow in leopard is most likely limited and this is not addressed in the paper. Reduced female gene-flow and male dominated gene-flow needs to be considered when measuring the impact of the current work. In my opinion the current experimental design and marker used does not contribute much towards the conservation of leopard. I indicated some problems with sample sizes using the Fat analyses and also the absence of posterior probabilities on the trees. The usage of Fu's FS and disregarding selection is controversial to indicate expansion. Mismatch distributions are better suited.
The main contribution of the work (new knowledge) is the dating of the divergences among two close mtDNA lineages. The meaning of this is not fully explored and linking this to acidification is not very convincing. A more thorough exploration of structure in other cats (for example Lions) may also beneficial. There is a distant lack of comparative patterns found in other species in Africa. The contact zone in the eastern parts of Southern Africa is not unique among mammals and some ideas can be obtained from comparative phylogeography.
The impact and novelty of the work is very limited as outlined above. The finings seem valid and can be replicated based on the methods described.
Please see comments in the text provided
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