The Genetic Differences and Structure of Selected Important Populations of the Endangered Taxus baccata in the Czech Republic
Round 1
Reviewer 1 Report
This study focused on the genetic conservation of T. baccata in the Czech Republic, with molecular tools of 7 SSR loci. This is a straightforward study without too deep a connotation. There are apparent deficiencies regardless of the sampling, the particularity of the subject matter, the analysis of population genetics, and the depth of conservation application. My opinion is as follows.
Major comments:
Mayol et al. (2015) had already explored the whole population genetic structure of T. baccata around Europe, using a fantastic 4992 samples from 238 populations. I have noticed that Mayol et al. (2015) also sampled some populations from Czech, but their sampling seemd to be different from this study. This may imply that the sampling is not comprehensive in this study. Also, the purpose of this research-the preservation of "rare and endangered" yew does not seem to be as important as the authors mentioned because it is not really so rare. Besides, suppose the authors really want to consider the issue of conservation genetics, it will be better to analyze together with other published data. What I mean is to get their data and analyze them together to understand whether the genetic structure of the yew population in Czech is really unique among the entire European populations, and to which group its structure in the entire European population belongs, rather than just comparing the Ho, He, and Fst of this study with other studies. When the sampling strategy is different, such a comparison is of little significance, and it has nothing to do with conservation.
Regarding data analysis, I think this is just a simple population genetic analysis, not an analysis aimed at conservation genetic research. Whether the genetic diversity is increasing or decreasing, how were the genetic responses when facing climate change or anthropogenic disturbance, whether these so-called extant populations are in a crisis of genetic decline, and whether the pressure of fragmentation mentioned in the INTRODUCTION is really affecting the genetic structure, and whether the genetic variation and genetic structure really reflect dioecy and zoochory, were not analyzed or discussed.
The DISCUSSION part is even more disappointing. Many discourses can be put forward without the analysis or evidence provided in this study. The content of the discussion is not targeted but more like an introduction statement. The comparison with other studies, as mentioned above, is less meaningful and cannot support the conclusions. In short, the level of this study is far below the level of Forests.
Minor comments:
Abstract: Please remove the number before each sentence
L35-37: I do not understand the causality between the low adaptability to fragmentation and the characteristic of dioecy and zoochory. Please explain it.
L38: RGD happens when the population size is not infinite. It was not caused by fragmentation.
L74: About the sampling, I wonder the adequality of the sampling. In the INTRODUCTION, the authors mentioned that this species was rare and endangered in the Czech Republic. Thus the sampling population was only 3+1. However, whether these four populations are the only distribution of the yew was not well explained. Some other populations unsampled, namely the ghost populations, may interbreed with the studied populations, i.e., gene flow. I believe such a scenario could have happened because Taxus baccata is widely distributed in Europe. In this situation, the impact of ghost populations cannot be ignored, especially when conservation genetics is the focus issue.
L102: Frankly speaking, only 7 SSR loci seemed too few to represent the genome-wide diversity pattern in such a huge genome-size species. I guess that the authors used the same markers as Mayol et al. (2015), Litkowiec et al. (2018), or/and Maroso et al. (2021), probably for convenience for the comparison (sorry that I did not carefully check the loci). However, I have not seen too detailed comparisons in the discussion, except for the estimates of He and Ho.
Fig. 2: There is no biological and statistical meaning for the PCoA for only four populations at the population level.
L220 and Fig. 3: I understand that the GenAlex calculates the "within-individual" variance in AMOVA. However, it is actually meaningless. Can you explain the biological meaning of the 56% genetic variation within individuals?
L317: Introgression or genetic contamination usually interprets the genetic admixture between species instead of between populations. I can understand what the authors want to express here, but the expression of the sentence should be more precise.
Author Response
Comments and Suggestions for Authors
This study focused on the genetic conservation of T. baccata in the Czech Republic, with molecular tools of 7 SSR loci. This is a straightforward study without too deep a connotation. There are apparent deficiencies regardless of the sampling, the particularity of the subject matter, the analysis of population genetics, and the depth of conservation application. My opinion is as follows.
Major comments:
- Mayol et al. (2015) had already explored the whole population genetic structure of T. baccata around Europe, using a fantastic 4992 samples from 238 populations. I have noticed that Mayol et al. (2015) also sampled some populations from Czech, but their sampling seemd to be different from this study. This may imply that the sampling is not comprehensive in this study. Also, the purpose of this research-the preservation of "rare and endangered" yew does not seem to be as important as the authors mentioned because it is not really so rare. Besides, suppose the authors really want to consider the issue of conservation genetics, it will be better to analyze together with other published data. What I mean is to get their data and analyze them together to understand whether the genetic structure of the yew population in Czech is really unique among the entire European populations, and to which group its structure in the entire European population belongs, rather than just comparing the Ho, He, and Fst of this study with other studies. When the sampling strategy is different, such a comparison is of little significance, and it has nothing to do with conservation.
Dear reviewer, we thank for your evaluation. Regarding to the sampling and to the rarity of T. baccata in the Czech Republic, we revised the INTRODUCTION. Mayol et al. (2015) have sampled 4 populations from the Czech Republic, in different sites, than we used, however, their chosen sample size was lower (13,30,36, and 26 individuals in each population), compared to our number of samples. We agree that analysis of our data together with already published data would be more informative. However, we can not compare our data together with other published data, because we do not have source data (microsatellite allele sizes of analyzed trees) from their Genetic Analyser. Moreover, each Genetic Analyzer instrument has a unique laser, CCD detector, and optical alignment, which causes signal differences between different systems. From this reason, we think that comparing the Ho, He or Fst is more accurate. The aim of our study was to determine the level of genetic diversity and the genetic structure of yew populations not through the whole Czech Republic, but just in Lužické Mountains and in populations close to this territory, to avoid eventual inappropriate mixing of sub-populations, where the genetic data have not been published yet. Yew sampling in the Czech Republic is a bit complicated because it is always subject to the permission of the locally competent nature conservation authorities, as it is a specially protected plant species and occurs mostly in specially protected areas, sometimes also in hard-to-reach places.
- Regarding data analysis, I think this is just a simple population genetic analysis, not an analysis aimed at conservation genetic research. Whether the genetic diversity is increasing or decreasing, how were the genetic responses when facing climate change or anthropogenic disturbance, whether these so-called extant populations are in a crisis of genetic decline, and whether the pressure of fragmentation mentioned in the INTRODUCTION is really affecting the genetic structure, and whether the genetic variation and genetic structure really reflect dioecy and zoochory, were not analyzed or discussed.
We thank for your evaluation. In our study, the most important issue was to evaluate the seed orchard in Lužické Mountains and avoid eventual inappropriate mixing of sub-populations. Unfortunately, we do not have data from the Czech Republic, describing disturbances, which you mentioned. However, we tried to specify some of your suggestions in the INTRODUCTION (the copy is below), to improve the text.
“The decline and reduction of populations was mainly caused by a combination of targeted logging, used methods of forestry management (sudden changes in light conditions) and pastoralism (eradication due to horse poisoning). At present, almost all seedlings from natural regeneration are decimated by high numbers of hunting populations of even-toed ungulates, so the stability of populations of long lived tree species can often be only apparent.”
- The DISCUSSION part is even more disappointing. Many discourses can be put forward without the analysis or evidence provided in this study. The content of the discussion is not targeted but more like an introduction statement. The comparison with other studies, as mentioned above, is less meaningful and cannot support the conclusions. In short, the level of this study is far below the level of Forests.
We thank for your evaluation. We modified the discussion and added more comparisons with some other studies. We hope that our additions and corrections have improved our text.
Minor comments:
- Abstract: Please remove the number before each sentence
We removed all numbers in the abstract.
- L35-37: I do not understand the causality between the low adaptability to fragmentation and the characteristic of dioecy and zoochory. Please explain it.
L38: RGD happens when the population size is not infinite. It was not caused by fragmentation.
We thank for your evaluation. We modified and revised these sentences.
- L74: About the sampling, I wonder the adequality of the sampling. In the INTRODUCTION, the authors mentioned that this species was rare and endangered in the Czech Republic. Thus the sampling population was only 3+1. However, whether these four populations are the only distribution of the yew was not well explained. Some other populations unsampled, namely the ghost populations, may interbreed with the studied populations, i.e., gene flow. I believe such a scenario could have happened because Taxus baccata is widely distributed in Europe. In this situation, the impact of ghost populations cannot be ignored, especially when conservation genetics is the focus issue.
We thank for your evaluation. We added the INTRODUCTION with detailed information about sampling and hope this will improve our text.
- L102: Frankly speaking, only 7 SSR loci seemed too few to represent the genome-wide diversity pattern in such a huge genome-size species. I guess that the authors used the same markers as Mayol et al. (2015), Litkowiec et al. (2018), or/and Maroso et al. (2021), probably for convenience for the comparison (sorry that I did not carefully check the loci). However, I have not seen too detailed comparisons in the discussion, except for the estimates of He and Ho.
We thank for your evaluation. We tried to add some additional data to the discussion, according to the SSR loci comparison. However, in most of the papers, characteristics of using loci, excepted Ho and He, are very often missing.
- 2: There is no biological and statistical meaning for the PCoA for only four populations at the population level.
We thank for your evaluation. The PCoA analysis was chosen just to visualize similarities and dissimilarities of our data. However, we decided to remove the Figure 3 and the content related with the PCoA analysis.
- L220 and Fig. 3: I understand that the GenAlex calculates the "within-individual" variance in AMOVA. However, it is actually meaningless. Can you explain the biological meaning of the 56% genetic variation within individuals?
We agree that including “within ind” level into GeneAlEx calculation might be meaningless. The variation is unique to the individual, but is unpredictable, given the phenotype. Thus, we modified the AMOVA interpretation.
- L317: Introgression or genetic contamination usually interprets the genetic admixture between species instead of between populations. I can understand what the authors want to express here, but the expression of the sentence should be more precise.
We thank for your evaluation. We modified the part with “introgression”.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Taxus baccata is an important medicinal tree, well known for its Taxol content and its utilization in Cancer treatments. Due to its lower growth rate and overcollection, T. baccata became an endangered species in across all of Europe. The current study concentrated on the SSR-based genetic diversity of four Taxus baccata population naturally grown in limited area of the Czech Republic. The number of SSR markers utilized in the study could be considered as low, but the results indicated their high discrimnation capacity among and in the Taxus populations. The methodolgy, results and disscussions parts of the artcile was written very well with a fluent english. The results obtained in the study could be utilized to establish new Taxus orchards and determined genetic divertsity is important for insitu and exsitu conservation programs. Therefore, the article is quite novel and attractive for resercehrs working on Taxus and related tree species.
Author Response
Dear reviewer, we thank for your evaluation.
Reviewer 3 Report
The authors investigated four endangered yew populations in Czech. The populations are L (seed orchard collected from 18 Luzicke localities), J (Jilovske), and B (Brezinske) in northern Bohemia and M (Moravsky Karst) in Moravia. I wonder whether any other natural populations exist in Czech and whether the four populations are representative samples from the distributional range in Czech. It is necessary to explain the basic information of the distributional records in Czech at first. Mayol et al (2015) illustrated a good map of the species distributional range. Show the range of Luzicke Mountains where 18 sources were collected.
This species has been intensively studied (Dubreuil 2010; Chybicki 2011; Mayol 2015; Litkowiec 2018; Gargiulo 2019). Although only 7 loci were examined, the findings are useful for conservation in Czech. In spite of dioecy (complete outcrossing), Fis > 0.1 suggests mating with relatives and structuring within populations, as shown in previous studies.
In Spain (Dubreuil 2010) where genetic diversity is low at the periphery of species distributional range (Mayol 2015), strong inbreeding and structuring were observed. In Poland (Litkowiec 2018) and UK (Gargiulo 2019) where genetic diversity is relatively high (Mayol 2015), geographical genetic structuring is relatively weak. Because Czech is also at the center of species distributional range, the observed low genetic differentiation is reasonable.
It is necessary to incorporate the present findings into sufficient knowledge across Europe.
The most important issue of this study is to evaluate the seed orchard in Luzicke. However, the authors did not state the strategy of conservation using the seed orchard clearly. It is necessary to show a guideline such as Litkowiec (2018) in Poland.
Because the manuscript is immature, extensive revision is necessary.
Author Response
Dear reviewer, we thank for your evaluation. We have revised our text extensively, we tried to incorporate all present findings into discussion and specify our conservation strategy. We hope this will improve our text.
Round 2
Reviewer 1 Report
This is the second time that I have evaluated the manuscript. I have reviewed the manuscript in light of the comments that were previously raised by myself. I questioned the insufficient sampling and inadequate analytical strategy on the conservation issue in the previous comments. The authors have provided some modifications to the introduction and minor corrections regarding the analyses in this version. However, the problem of sampling was still not resolved. Although the authors rebutted that they only focused on the Czech populations instead of all European populations, I still cannot accept this explanation. This is because it is still irrational to assess the population genetic structure without considering genotypes outside an artificial divided administrative area (such as a country). In addition, only the concept of conservation was put forward in this article, without substantive suggestions or actions. However, these suggestions do not seem to require such a laborious study. The advice of the conservation strategy provided in the last sentence of this article in the revised version also lacked sufficient supports of scientific evidence. Thus, I am sorry that no positive evaluation was given for this revised article.
Reviewer 3 Report
The findings are useful to conserve Czech populations of this endangered species.
