Genetic Diversity, Mating System, and Seed Viability Reveal a Trade-Off between Outcrossing and Inbreeding in Pinus yunnanensis var. tenuifolia, an Ecologically Important Conifer Species Growing in a Hot-Dry River Basin Habitat in Southwest China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a very interesting topic: the impact of mating systems on the genetic diversity of seven populations of Pinus yunnanensis var. tenuifolia. These populations inhabit a fragmented range and are adapted to hot and dry climate conditions. The authors examined the genetic variation using nuclear microsatellites in both seeds and sprouts to assess the influence of mating systems (outcrossing vs. inbreeding) on seed quality parameters across 20 families of P. yunnanensis var. tenuifolia within this fragmented range.

The material collected from the specific populations is substantial, the methods applied are appropriate and well-described, and the manuscript is generally well-written. The results and conclusions obtained are highly significant for biologists, conservation geneticists, and foresters.

Detailed comments:

Line 14 – not status; mating type or mating system. In my opinion, the mating system is the best term.

L. 22 - My proposal is: Three seed quality indicators differed significantly between families

L. 60 - It is unclear for me. My proposal is: such as evolution of a distinct variety from the marginal population

L. 75: not stand; population structure

L. 81 – It should be: seed quality traits

L. 82 – please, use the lowercase letter in the word ‘germination’

L. 85-86: It should be: ...and to assess (2) whether the mating type/system has a significant...

L. 90: dot should be placed after ‘et al.’

L. 91: Please, explain ‘…better preserved than…’ what? Gradation of adjectives requires a comparison to something else to show a relative difference.

L. 100 – Please, explain how long were seeds exposed to sunlight?

L. Please, explain - 100 seeds per cone? per mother tree? per population?

L. 112 – It should be: when germination reached its peak

L. 115 – Please, delete ‘further’ because genotyping was not done previously

L. 120-121 - Please explain how the microsatellites were amplified. Were all loci amplified independently, with 12 separate PCRs for each sample, or were panels constructed for multiplex PCRs? If panels were used, how many were there, and how were the specific microsatellites arranged within each panel?

L. 126 - Analytik Jena (?), Germany

L. 151: It should be: mating system

Table 1 - While the sample size is 24? Authors extracted DNA from 24 seeds and 24 sprouts. It should be: 24/24

L. 190 - Please, give a symbol for the values in the first bracket, Φst (Phi) = 0.122/0.102

L. 207-29 - This statement is not clear at the first sight, because as I understand all populations are common, are not they? My proposal: Families from the common populations XQ, BW, BY, CJ, and DT exhibit high levels of coancestry (Figure 1D, 1E), suggesting that gene flow between those populations is relatively limited. Level of coancestry was lower in the WJ and QX populations, possibly indicating gene exchange between them and other localities.

L. 212 - Authors did not mention about IBD test in the Material and Methods chapter.

L. 248 – Explanation is confused because there are many vertical lines in each figure. Please, remove light grey vertical lines to make the figures more clear. In each plot, reader see colours, first of all. My suggestion is: The vertical lines dividing each plot into three coloured parts, from left to right, represent the lower quartile…

L. 251 – It should be: … is not different

L. 251 again – It should be: …lower quartile was different

L. 269 and 287 - All titles of subchapters in the Discussion should start from a capital letter

L. 274 - It should be: common populations.

L. 290, 298, and 366 – genetic diversity can be high/substantial/considerable, but not ‘rich’

L. 293 - It should be: …genetic differentiation between populations cannot be ignored

L. 311 - the space is needed between 'offspring' and bracket

Comments on the Quality of English Language

I am not native speaker but the English language is fine, in my opinion. It only needs some minor improvements.

Author Response

Dear reviewer,

We are very happy with your positive comments on the study. We are also particularly grateful for all your careful and valuable revision suggestions to the manuscript, and we have completed all the changes based on your suggestions to improve the quality of the manuscript. The following is a point-by-point response:

 

Detailed comments:

Line 14 – not status; mating type or mating system. In my opinion, the mating system is the best term.

A: We revised based on your suggestions.

1. 22 - My proposal is: Three seed quality indicators differed significantly between families.

A: We revised based on your suggestions.

1. 60 - It is unclear for me. My proposal is: such as evolution of a distinct variety from the marginal population.

A: We apologize for the unclear representation. We have made corrections based on your suggestions.

1. 75: not stand; population structure.

A: We revised based on your suggestions.

1. 81 – It should be: seed quality traits.

A: We revised based on your suggestions.

1. 82 – please, use the lowercase letter in the word ‘germination’.

A: We revised based on your suggestions.

1. 85-86: It should be: ...and to assess (2) whether the mating type/system has a significant...

A: We revised based on your suggestions.

1. 90: dot should be placed after ‘et al.’

A: We revised based on your suggestions.

1. 91: Please, explain ‘…better preserved than…’ what? Gradation of adjectives requires a comparison to something else to show a relative difference.

A: We apologize for the unclear representation, and the sentence has been revised to ”Seven representative wild populations of PYT, which were previously reported in Bai et al. (Bai et al., 2020), were selected for the study”.

1. 100 – Please, explain how long were seeds exposed to sunlight?

A: We have added the description, and that is “The cones were exposed to sunlight from three to seven days”.

1. Please, explain - 100 seeds per cone? per mother tree? per population?

A: That is “per mother tree”, and we corrected.

1. 112 – It should be: when germination reached its peak.

A: We corrected based on your suggestions.

1. 115 – Please, delete ‘further’ because genotyping was not done previously.

A: We corrected based on your suggestions.

1. 120-121 - Please explain how the microsatellites were amplified. Were all loci amplified independently, with 12 separate PCRs for each sample, or were panels constructed for multiplex PCRs? If panels were used, how many were there, and how were the specific microsatellites arranged within each panel?

A: We apologize for the unclear representation, and the revised description is “All microsatellite loci were amplified independently with 12 separate PCRs for each sample”.

1. 126 - Analytik Jena (?), Germany

A: We corrected based on your suggestions.

1. 151: It should be: mating system

A: We corrected based on your suggestions.

Table 1 - While the sample size is 24? Authors extracted DNA from 24 seeds and 24 sprouts. It should be: 24/24

A: We corrected based on your suggestions.

1. 190 - Please, give a symbol for the values in the first bracket, Φst (Phi) = 0.122/0.102

A: We corrected based on your suggestions.

1. 207-29 - This statement is not clear at the first sight, because as I understand all populations are common, are not they? My proposal: Families from the common populations XQ, BW, BY, CJ, and DT exhibit high levels of coancestry (Figure 1D, 1E), suggesting that gene flow between those populations is relatively limited. Level of coancestry was lower in the WJ and QX populations, possibly indicating gene exchange between them and other localities.

A: We corrected based on your suggestions.

1. 212 - Authors did not mention about IBD test in the Material and Methods chapter.

A: We added the description“the Isolation by distance (IBD) among families was tested using mantal test between a matrix of family-level genetic distances and a geographic distances (Jombart, 2022)”in Data analysis section.

1. 248 – Explanation is confused because there are many vertical lines in each figure. Please, remove light grey vertical lines to make the figures more clear. In each plot, reader see colours, first of all. My suggestion is: The vertical lines dividing each plot into three coloured parts, from left to right, represent the lower quartile…

A: We corrected based on your suggestions.

1. 251 – It should be: …is not different.

A: We corrected based on your suggestions.

1. 251 again – It should be: …lower quartile was different.

A: We corrected based on your suggestions.

1. 269 and 287 - All titles of subchapters in the Discussion should start from a capital letter.

A: We corrected based on your suggestions.

1. 274 - It should be: common populations.

A: We corrected based on your suggestions.

1. 290, 298, and 366 – genetic diversity can be high/substantial/considerable, but not ‘rich’.

A: We corrected based on your suggestions.

1. 293 - It should be: …genetic differentiation between populations cannot be ignored.

A: We corrected based on your suggestions.

1. 311 - the space is needed between 'offspring' and bracket.

A: We corrected based on your suggestions.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Article entitled “Genetic diversity …. China” deals with the relationship between mating status and the fitness of seeds in Pinus yunnanensis var. tenuifolia. Using molecular marker technology (SSR markers), authors study the genetic diversity, and using an in-silco method studied mating status of the plant. Authors also established a relationship between mating status and seed quality. My comments are:–

1.      Why there is a big variation in GR and GP, average 40.14% and 12.39%, respectively?

2.      You are not study the effect of any environmental stress/factor? In nature, plant are exposed to lot of stresses which are also an important parameter for the diversity of a plant species.

3.      Mating status (inbreeding or outcrossing) also depends on the environmental suitability.  Is collecting seeds from a particular location will give a fare idea about mating status.

4.      How genetic diversity was corelated with Seed quality?

5.      Section 3.5 described Relationship between the outcrossing rate and seed quality but there is no description about “Relationship between the inbreeding and seed quality”

6.      Abstract: “This highlights that excessive inbreeding or outbreeding seems to be unfavorable to seed viability”. Is it concluded from the present study? If yes, is data sufficient to support it?

7.      Last two concluding line of Abstract (line 34-37) is not aligning with the final conclusion of the study (Section 5)?

Author Response

Dear reviewer,

We are happy with your careful and valuable revision suggestions to the manuscript, and we have improved the quality of the manuscript. The point by point responses to the comments are below：

Q1.Why there is a big variation in GR and GP, average 40.14% and 12.39%, respectively? 

R: Thank you for your comments. We have discussed the concern in revised manuscript (lines 313-329). The germination rate of seeds in PTY wild populations was 40.14%, which was lower than the germination rate of the P. yunnanensis seeds (GR = 58%), but close to the germination rate of P. kesiya var. langbianensis in the southern region (43%). These results are likely related to the unique habitat of PYT. Compared to P. yunnanensis, PYT and P. kesiya var. langbianensis may have undergone more similar habitat selection effects (Wang et al., 2004). In addition, the GP measured in this study was significantly lower than the GR, indicating that the seed germination period was not concentrated, and the emergence time was asynchronous. We speculated that it may be influenced by long-term habitat selection (stress) while being affected by the mating system (inbreeding or outcrossing level) of the mother tree. The area where PYT is located is a typical seasonal arid and hot region with frequent foehn winds in spring (Feng et al., 2023; Li and Wang, 1981; Xu, 1983). Asynchronous emergence of seedlings may be beneficial for avoiding adverse meteorological conditions and ensuring that some seeds can germinate into seedlings under suitable weather conditions (Huang, 1993). Some studies suggest that high temperatures promote the seed germination of P. yunanensis and P. densata (Huang et al., 2016; Yang et al., 2017; Zhang et al., 2018). It remains to be further explored whether PYT also exhibits similar characteristics.

Q2.You are not study the effect of any environmental stress/factor? In nature, plant are exposed to lot of stresses which are also an important parameter for the diversity of a plant species.

R: Thank you for your question. Actually, we are investigating the seeds germination characteristics of Pinus yunnanensis var. tenuifolia based on temperature and humidity gradient experiments to explore to what extent the special climate of the Nanpan River and Hongshui River basins affects its seed germination.

Q3.Mating status (inbreeding or outcrossing) also depends on the environmental suitability.  Is collecting seeds from a particular location will give a fare idea about mating status.

R: Thank you for your comments, and we agree that harvesting based on environment-particular populations is beneficial to control the effects of environmental conditions on mating systems. However, we wished to assess whether there was a correlation between seed quality and mating systems among mother trees from different populations. Therefore, considering the practicability and workload of the study, we finally chose 960 seeds from 20 mother trees from seven populations as experimental samples.

Q4. How genetic diversity was corelated with seed quality?

R: Thank you for your question. We analyzed the correlation between genetic diversity parameters (Hobs, Hexp, and Fis) and seed quality indicators (TSW, GR, and GP) (Fig. A1). It was found that Hobs only showed a marginal quadratic correlation with TSW, while Hexp showed a significant negative correlation with TSW. However, Hobs and Hexp showed no significant correlation with GR and GP. It is worth noting that Fis showed a significant quadratic correlation with TSW and GR. Although the correlation between Fis and GP does not present a significant level, it shows a similar trend to that between Fis and GR. This is consistent with the results of correlation analysis between mating system parameters and seed quality indicators, indicating that excessive inbreeding and outcrossing are not conducive to seed germination.   

Fig. A1 Relationship between genetic diversity parameters (Hobs, Hexp, and Fis) and seed quality indicators (TSW, GR, and GP)

Q5.  Section 3.5 described Relationship between the outcrossing rate and seed quality but there is no description about “Relationship between the inbreeding and seed quality”

R: Thank you for your comments. Inbreeding consists mainly of selfing (1-t_m) and biparental inbreeding (t_m-t_s), and the relationship between biparental inbreeding and seed quality has been demonstrated in Fig. 5 and analyzed in the text (lines 287-293). Since selfing rate (1-t_m) is inversely proportional to multilocus outcrossing rate (t_m), the relationship between multilocus outcrossing rate and seed quality indirectly reflects the relationship between selfing and seed quality; Since most of the families detected based on seeds and sprouts had very low selfing rates, resulting in a severely skewed distribution of t_m values, it was difficult to well detect the relationship between selfing rate and seed quality. This may be due to the fact that most of the selfed offspring were mainly aborted early in seed development (Discussion section lines 414-418), rendering most of them unavailable for DNA amplication and genotyping. Therefore, the main genetic effect of biparental inbreeding on seed quality was detected in this study.

Q6.  Abstract: “This highlights that excessive inbreeding or outbreeding seems to be unfavorable to seed viability”. Is it concluded from the present study? If yes, is data sufficient to support it? 

R: Thank you for your comments. It is generally believed that mating systems mixed with inbreeding and outcrossing are beneficial for many plants to adapt to the environment, over inbreeding or outbreeding likely to be detrimental to population maintaining and expanding,  but there has been little empirical support (Goodwillie et al., 2005; Hargreaves and Eckert, 2014; Peterson and Kay, 2015). Our study based on 12 microsatellite loci analyzing mating system data from 20 open-pollinating families (24 seeds and 24 sprouts per family, totally 960 individuals) from seven populations partly supports the above hypothesis. Based on the analysis of changes in mating system parameters and genetic diversity parameters before and after seed germination, as well as regression analysis of mating system parameters and seed quality indicators, consistent results were obtained. This, to some extent, reflects the robustness of the results of this study. The sample size in the present study is comparable to that of some previous literatures focus on mating system (López-Villalobos and Eckert, 2018; Zaruma et al., 2024; Zhang et al., 2019). It should be noted that our study was based on seed germination test conducted indoors condition, which concluded that excessive outcrossing or inbreeding were not favorable to PYT seed germination, whereas seed germination under wild conditions is affected by many environmental conditions. It has been reported that Pinus yunanensis seeds have better germination ability after stimulation by high temperature (Huang et al., 2016; Yang et al., 2017; Zhang et al., 2018). Whether the seed germination of PYT under wild conditions is stimulated or inhibited by special environmental factors needs further exploration (as a limitation for this study, we have added this statement in the conclusions and limitations section).

Q7. Last two concluding line of Abstract (line 34-37) is not aligning with the final conclusion of the study (Section 5)?

R: Thank you for your comments. For conciseness, our inferences are provided directly at the end of the abstract. As a further explanation, we have included the extrapolation at the end of the conclusion.

References

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Feng, S.-S., Huang, C.-H., Tang, M.-Y., Jiang, W.-X., Bai, T.-D., 2023. Geographical variation of needles phenotypic and anatomic traits between populations of Pinus yunnanensis var. tenuifolia and its environmental interpretation. Chinese Journal of Plant Ecology 47, 1116–1130. https://doi.org/10.17521/cjpe.2023.0041

Goodwillie, C., Kalisz, S., Eckert, C.G., 2005. The Evolutionary Enigma of Mixed Mating Systems in Plants: Occurrence, Theoretical Explanations, and Empirical Evidence. Annual Review of Ecology, Evolution, and Systematics 36, 47–79. https://doi.org/10.1146/annurev.ecolsys.36.091704.175539

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Zaruma, D.U.G., Sebbenn, A.M., Rossini, B.C., Pardo, A.C.F., Silvestre, M.A.M., Cambuim, J., da Silva, A.M., Pupin, S., Rodrigues, C.J., Marino, C.L., de Moraes, M.L.T., 2024. Mating system and inbreeding depression in Hymenaea stigonocarpa. Tree Genetics & Genomes 20, 1–12. https://doi.org/10.1007/s11295-024-01639-9

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Response to my comments is satisfactory.