Clonal Diversity and Fine-Scale Genetic Structure of a Keystone Species: Ilex aquifolium
Round 1
Reviewer 1 Report
This is an interesting study examining human activity and clonal diversity and population structure of Ilex aquifolium.
However, I believe that the analysis methods remain questionable and require additions.
For example, this study was analyzed using SSRs created in other Ilex species.
As the authors themselves point out, there are concerns about the effects of low polymorphisms and null alleles. Although the possibility of somatic mutations (overestimation of the number of genets) is discussed in the paper, I feel that the underestimation due to low polymorphisms is not explained enough. There have been cases where clones have been identified as the same genet due to a small number of polymorphisms.
The method should include a disclaimer that the analysis is being carried out in consideration of this issue.
The definitions of the following words are also ambiguous and confusing. They should be unified through fertility.
The figures and tables also need to be corrected. Details are as follows.
L53-54
From reading the discussion and other information, it seems that not only the climate but also past land use may be a factor. Are there any reports based on this?
Results.
3.1. genetic diversity
It is unclear how clones are defined in the calculation.
Are all DNA samples (stems) included in the calculation or only genets?
It should be described in methods.
3.2. Clonal assignment
L275
The total samples are 171(125+46) isn’t it? The number of samples seems to differ from the method.
Why only 94 samples in A and B, not 262?
L382-398
Isn't the high He because the calculation includes clonal repeats? If the impact on sexual reproduction is considered, it should also be calculated for MLGs only.
Fig1.
The extent of Zones 1 and 2 is not clear in this fig. Please show details.
Do the circles in Fig. 1 show the location of “all” trees >20 cm? This figure is confusing, especially in zone 1.
What size trees were covered by the pie chart measurements?
The pie charts (A, B) on the map are very difficult to read. They should be shown in a separate diagram.
Dotted lines are confusing (small stream, contour, site range).
Fig2.
Please show the study area as a circle.
The No in the legend is misleading and should be excluded from the legend. A textual explanation is sufficient.
Fig5.
Please give axis titles.
Fig6.
Please give axis titles.
In fig. 6, the result for the South was shown first. But the order is different in other results (1st North and then South).
Fig. 6 is confusing.
Table 1
The number of samples is not shown in this table. Please add such information.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This study by Vega et al. Evaluated the genetic structure of English holly grown in in a sub-Mediterranean mixed forest of Central Spain. It is well written article and provided significant data for the readers of the journal. The number of loci utilized is low and thus reducing its reliability of conclusion. However, the number of samples used are large enough. It is a good piece of work.
Author Response
Dear reviewer, thanks for your kind review, we appreciate it.
Although we agree that the number of loci is low and polymorphism is not very high, we concluded that diversity levels are high enough to assess the big picture of genetic status of the species in the area and that, together with the large sample size, we have a robust image of the clonal conformation and reproduction patterns in this species.
Reviewer 3 Report
In this study, Clara Vega et al. reported the fine-scale genetic structure analysis of Ilex aquifolium, by asing the important question if recent forest management have impact on intraspecific genetic diversity patterns. Seven microsatellite loci were identified and used to assess the contribution of clonal and sexual reproduction. Authors showed that management activities are not negatively affecting the genetic diversity. This study is well designed and performed in a high standard. Data are organized in a logic manner and presented in good quality.
Author Response
Dear reviewer, thanks for your kind review, we really appreciate it.
