Round 1

Reviewer 1 Report

This research paper reports the genetic characterization of several representative natural populations of the relict and medicinal subshrub Capparis spinosa based on a SNP dataset generated by dd-RAD genome sequencing.

The paper is original and well written with results clearly presented and properly discussed. I am confident it may be of great interest for the readers working on plant genetic diversity and biodiversity conservation. In order make this paper even more attractive for the scientific community, I suggest to improve the graphical resolution and overall quality of the figures (some details are actually unreadable).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Review of Article

Population genetic structure and biodiversity conservation of a relict and medicinal subshrub Capparis spinosa in arid Central Asia

This is a well-analyzed and decently written manuscript about an important native plant to the region of interest, which is noted to be understudied. Overall, the manuscript is readable, but there are phrases that do not make sense throughout, where the authors’ points can be guessed at, but not clear. This is commonly associated with overuse or compounded adverbs ending in “-ly” that are not understandable in the general English (UK/US) lexicon. Therefore, I suggest a native English speaker service or peer review to check these phrases so that I need not correct them all.

I am most interested in the ecological and biogeographical inferences that can be made from the generated data. The authors utilized RAD-Seq, a common molecular approach for non-model organisms. Issues in plants include a number of assumptions, much like its predecessor, AFLPs. I think the authors did a fairly thorough job with analyses and treated the data appropriately, with the glaring omission of an AMOVA and pairwise Fst over pairwise Nei’s GD in the SI. I believe these two analyses, the AMOVA as a table and a pairwise Fst figure in the manuscript itself will strengthen the paper.

Overall, I find this paper relevant, an important contribution to geologically-based biogeographical patterns of genetic relatedness, or lack thereof. IBD ought to be discussed briefly in the introduction to be consistent with the discussion, as it appears that IBD and metapopulation dynamics are the main forces resulting in these extant analyses and populations of C. spinosa. Further, it is important for the authors to familiarize themselves with these concepts for inclusion in the paper, showing a well-founded knowledge base in these areas and demonstrate the relevancy of these concepts concisely in the writing and flow of the total paper.

I am recommending major revisions to allow the authors ample time to make a stronger case for conservation of this relict plant species in their study area(s) based on biogeographical and metapopulation dynamics in natural resources management and conservation, which I read as the major “take-home” message. It seems that the authors become entangled with verbose language in their effort to present this important study.

Minor comments:

The abstract does not mention genetic diversity from the outset. Should be included in the list of pop gen analyses.

Introduction seems to draw from metapopulation theory (see Hanski) and source-sink dynamics, where isolated populations possess higher intrinsic extinction rates (where GD is low) as compared to source populations (larger populations with spatially closer populations for admixture, where GD would be expected to be high, and intrinsic extinction rates would be comparatively lower). This would be good to include in the paper to strengthen the conservation & management of natural resources and biodiversity protection using a biogeographical framework.

Introduction sentence on line 35 with citation #4 re: albatrosses, see review by Sakai et al 2000. May help provide a more general framework and more appropriate citation.

Line 37 for citations 5 & 6, see Zomlefer et al. 2018 in Systematic Botany. JL Hamrick’s contribution in leading plant pop gen remains a great legacy from Stebbins’ work.

Lines 37-40 deal with mechanisms of biogeography, including both vicariance and dispersal mechanisms. This may be better phrased so that the foundation for the paper is stronger of C. spinsoa.

Line 115: “A certain amount of previous studies have hitherto been carried out on the genetic

variation and diversity of C. spinosa.” Where are the citations for this statement? Maybe consider a semicolon is related to the next statement.

Line 122-125: “However, the residual (change to relict) wild populations remaining in arid Central Asia(add ,) dominated by a temperate continental deserts climate(add comma) (have been shown a) lack of concern(hard stop period). Valuable information on (the) genetic structure and diversity conservation of C. spinosa in this region have(change to are) relatively scarcely (delete –ly) been involved to date (delete this phrase and change to and are of significant biogeographical and conservation interest to [add whom]).”

Overall, the introduction would benefit if it opened with the description of the study site and the focal organism (starting at line 61 to 125), then move into the mechanisms of genetic diversity (GD throughout), population genetic structure, IBD & metapops, and finally, expectations and assumptions made by the scientists.

Methods:

All analyses seem appropriate, including outgroups for a rooted phylogenetic analysis; however, what I find most glaring, is the missing hierarchical AMOVA for total genetic variation partitioning, within and across groups. I recommend ARLEQUIN by Excoffier. I am confused how pairwise Fst can be derived without a hierarchical AMOVA. Can this omission be justified? How can pop gen be done without an AMOVA to validate PCoA results?

Results:

First sentence of section 3.2 does not make readable sense. I get the authors’ point, but I think it should be reworded in a clearer sentence structure.

In text, parenthetical citations of figures and SI, the main paper figures/tables should precede the SI figures/tables for readability.

Map should include source, datum, N arrow, etc. The basics of a GIS created map. An ecoregional map may serve better than a satellite image.

Table 1 can be shrunk down and these data be placed in the SI. The map is good and covers what is needed. Color coding the table groups to the map may be helpful.

Figure 2 after the asterisk, is unclear what this means.

Figure 3: why a circular phylogenetic tree? Bootstrap values are nearly invisible @ 150%. Consider a tree with just the groups, followed by a tree in the SI that includes bootstraphs with all 263 individuals. Take a whole page as necessary. In this case, the results cannot be utilized if printed so small.

Figure 4, the sum of variation in the PCoAs for the 2 main axes are low for all (total and within group). Each axis includes the amount of variation explained by PCoA, but are small and require zooming in to 150% to see. Recommend adding the sums of variation explained in each (a, b, c) into the text associated with Fig. 4 caption.

Recommend pairwise Fst over Nei’s genetic distances in the SI. IBD should be introduced in the introduction as a possible outcome of analyses.

Is there a way to make Table 2 more readable? As in, some of the less informative indices into the SI?

Figure 5 should follow pairwise Fst figure. Figure 5 should also include the y=mx+b result of the regression on the figure and the slope in the caption (r = 0.499, p < 0.001) (Figure 5). Since currently Figure 5 follows a very large table, it is separated from that portion of the text and is difficult for the reader. This is an important result and the authors, I’m sure, wish for it to be clear.

Figure 6 really ought to be Figure 1, and be associated with the introduction/methods and not seeming like an afterthought at the end of the results.

Even more minor comments

Abstract

Line 12: “our purpose is to evaluate” change verb to past tense for consistency “…was to..”

Line 14-15: “from dd-RAD sequencing, principal components analysis, maximum likelihood phylogenetic trees and ADMIXTURE clustering,” add Oxford comma in listing analyses between “trees” & “and”

Line 15: “the significant genetic structure and differentiation were explored.” Suggest to modify to “…population structure and differentiation were explored.”

Suggestion re: Keywords—try not to repeat search criteria in your keywords that are already in the title. This provides additional search criteria to make the paper more discoverable.

Introduction

Line 37: “but ARE also…” (add the verb)

Line 41: w/c suggest “nonnegligible” to “contributory”

Human activity, encroachment into natural areas (for recreation, agriculture, etc.) more than influences biodiversity. Consider the Anthropocene and the spatial and temporal scale of discussion in the introduction and discussion, the areas where the authors can make their case more impactful.

Line 134: “Restriction (add hyphen) site”. This can really be defined in the methods. Most molecular ecologists are familiar with RAD-Seq.

Methods: Ensure sequence data is deposited appropriately prior to or concurrent with publication. DRYAD is a general standard.

Principal Components Analysis is now abbreviated as PCoA as compared to Principle Coordinates Analysis (PCA). Pls change throughout.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The conservation of useful plant genetic resources is becoming a major challenge in the face of major climate changes. On the other hand, natural resources are also strongly influenced by anthropogenic factors. This shows the need not only to assess but also to preserve plant species diversity. Thus, the main goal of these studies to investigate the genetic diversity of Capparis spinosa populations is relevant in both scientific and practical terms. The selected region is in Central Asia, and this type of research is being conducted for the first time. However, the presentation of studies of C. spinosa populations has some limitations.

The title should be corrected. The term ‘Biodiversity’ indicate the different kinds of life, i.e., the diversity of animals, plants, fungi, and even microorganisms. In my opinion the term ‘diversity’ is more acceptable.  

Keywords. The keywords almost completely repeat the words in the title. I would suggest changing at least part of it to make it easier and faster for the reader to find the publication.

 Abstract is adequate and contains short information on methods and results of this study.

Introduction. This section is very extensive. As many as 47 references were discussed. This would be more appropriate for a review article. In the article presenting the investigations, the most important aim is to discuss the problem and this is done in p.1 lines 30-60 and p. 2-3, lines 90-113 and 139-149. Detailed descriptions of the climate given in p. 2, lines 61-89 should be avoided or substantially shortened. I consider it is necessary to justify the innovativeness of the research or methods employed. I missed that in this section.

Materials and methods. Data processing and methods used are presented. I have a question about collecting samples for research. How could the authors justify that 5 individuals are enough to assess the genetic diversity of a population?  On the other hand, is 100 meters a sufficient distance to separate shrubby plant populations? The number and collection of samples essentially determine the correctness of the experiment.

Results. The obtained data are presented in 2 tables, 6 figures and supplements. In my opinion, Table 1 and Figures 1 and 6. should be moved to the Materials and methos section. In Figures 2, 3, 4 and Table 2, it is necessary to provide an explanation of what the abbreviations EP, ST, NT, WT, ET and WH mean. Visual information in the manuscript, i.e., figures, tables should provide clear information to the reader without the body text. I would suggest to the authors to avoid, as far as possible, the repetitive information in the text that is given in the table. (p. 9, lines 286-292 and etc.). Therefore, I would suggest that authors should carefully review the tables and figures and description of results.

Discussion. The discussion is the place in the manuscript where authors should discuss their research findings in the context of other studies. This section is overloaded with various information on results of this study (p. 15, lines 475-500) without discussion. So this section should base the innovation of the manuscript and must be corrected also.

Conclusions. The conclusions briefly discuss the object and research methods, but I missed the generalization and the significance of the research. In this section, the authors should to emphasize the importance for the future use of C. spinosa genetic resources.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors did a great job on these major revisions and this is a much stronger draft of this manuscript. Brava! Thank you for the opportunity to review this paper and its significance on relict plant species located in rare or endangered communities. Very minor comments appended below.

As requested, here is the direct link to Sakai et al., 2000, which may be beneficial in this and future studies: The Population Biology of Invasive Species (annualreviews.org)

Line 113: "peculiarly" to "particularly"

Line 114-115: whereupon (change to whereas) artificial (change to anthropogenic or external) interference undoubtedly further weakens their (add) fitness: decreases in survivorship and reproduction, and increases extinction risks

Line 125: delete "in the long term."

Line 142: if this is the first occurrence of these abbreviations, pls. spell out. ADMIXTURE clustering, PCoA and ML phyloge

Line 148, add comma after "accordingly"

Line 201: remove "firstly"

Figure 4 colors to be applied in Figure 5 would be helpful for consistency and results interpretation for readers.

In the SI, there appears to be a bobble/irregularity around K=10, in the Bayesian probability in the program ADMIXTURE. A suggestion for future work is to evaluate beyond K=10, and go as high up as the number of populations and not regions. There may be more, finer-scale population structure than this at a geographically large scale. 

Line 305: ...variation among the six geographical(add)ly isolated groups...

Line 367: facilitating closer reduced genetic distances between these populations

Line 402 (indicate the meters are elevation inside the parentheses)

Line 415: ...leading to the discontinuous gene...

Line 420: ...difficult to maintain their gene...

Line 434: ...hotspots of genetic diversity...

Line 440: ...particularly important to monitoring..., minimize, restore

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors did a great job and revised the manuscript by making changes to all chapters.

Author Response

Response to Reviewer 3 Comments

Point 1: The authors did a great job and revised the manuscript by making changes to all chapters.

Response 1: We thank Reviewer 3 for the comments, which are important for improving our manuscript.