Seed Germination Requirements of the Threatened Local Greek Endemic Campanula pangea Hartvig Facilitating Species-Specific Conservation Efforts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to read this interesting article. Indeed, the issues of reproduction of rare plants are currently not well studied, which limits conservation in culture. The authors have chosen a somewhat non-standard approach to the study of the germination process of seeds of one of the bluebell species. The authors have prepared a fairly detailed literature review, methods and results are described in detail. The reliability of the data presented is beyond doubt. However, when reading the article, it seems that the authors fail to convey important issues to the reader in order to understand the relevance and significance of the article. So, it is not entirely clear what the relevance of the study is if the control samples give high germination without treatment. What can be the reason for the problems of germination of seeds of this species (poorly formed embryo or density of the seed peel) Are fresh seeds mentioned, what does this mean? As you know, bluebell does not belong to those plants that reproduce with green seeds, like some buttercups or peonies. Fresh means, in this case, ripe not earlier than the previous growing season? Or have some other criteria of freshness been used? Speaking about the effect of temperature on germination, the authors mention that bluebells living in higher latitudes germinate at higher temperatures later in germination. This question remains unclear to the reader, because spring comes later in the northern regions and the temperature rises gradually. It would be possible to bring greater clarity to this issue when using such a concept as the sum of effective temperatures, if, of course, it is possible to do this on the basis of available sources. I think that the relevance of this work is much higher than the authors indicate, since the fact that this species germinates worse at higher temperatures indicates high risks for the conservation of this species in the face of climate change. The authors need to work on the presentation of the material in the article so that their thoughts become more understandable to the reader. The article can be published after a little revision.

Author Response

Reply to the first Reviewer

Thank you for the opportunity to read this interesting article. Indeed, the issues of reproduction of rare plants are currently not well studied, which limits conservation in culture. The authors have chosen a somewhat non-standard approach to the study of the germination process of seeds of one of the bluebell species. The authors have prepared a fairly detailed literature review, methods and results are described in detail. The reliability of the data presented is beyond doubt.

Authors’ response: We would like to thank the reviewer for his/her kind comments.

 

However, when reading the article, it seems that the authors fail to convey important issues to the reader in order to understand the relevance and significance of the article. So, it is not entirely clear what the relevance of the study is if the control samples give high germination without treatment. What can be the reason for the problems of germination of seeds of this species (poorly formed embryo or density of the seed peel).

Authors’ response: We would like to thank the reviewer for this comment. Indeed, the control seeds showed high germination percentages in a narrow temperature range of 15-20°C. With the treatment of seeds with GA₃, the possibility of extending this temperature range was investigated. According to the results, the seed germination percentage at 10°C was significantly lower in control (35.00%) than that in GA₃ pretreated seeds (78.33%). From a practical view, the results of GA₃ treatment provide evidence for broadening the temperature range and successful germination of C. pangea seeds from 10 to 20°C, thus facilitating in vivo mass reproduction over a wider seasonal spectrum as an effort to enhance its conservation and sustainable exploitation.

Regarding the problems of germination of seeds of this species, it has been reported that seeds with non-deep physiological dormancy may germinate over a narrow range of temperatures. In the case of C. pangea, the results of germination test indicate that seeds exhibit non-deep physiological dormancy, however, further investigation is required to establish the existence of morphological dormancy (underdeveloped embryo) (see track changes in the revised manuscript).

 

Are fresh seeds mentioned, what does this mean? As you know, bluebell does not belong to those plants that reproduce with green seeds, like some buttercups or peonies. Fresh means, in this case, ripe not earlier than the previous growing season? Or have some other criteria of freshness been used?

Authors’ response: We would like to thank the reviewer for this comment helping to eliminate an misinterpretation. The relevant text has been modified into 'freshly collected mature seeds' which refers to mature seeds used in a germination experiment right after their collection from the wild (see track changes). In the present study, Campanula pangea stored seeds were used in the germination experiment.

 

Speaking about the effect of temperature on germination, the authors mention that bluebells living in higher latitudes germinate at higher temperatures later in germination. This question remains unclear to the reader, because spring comes later in the northern regions and the temperature rises gradually. It would be possible to bring greater clarity to this issue when using such a concept as the sum of effective temperatures, if, of course, it is possible to do this on the basis of available sources.

Authors’ response: The optimum temperature for seed germination of lowland species in the Mediterranean is relatively low (≤ 15°C). This is an ecological adaptation of the species, as it ensures that germination takes place in late autumn when rainfall is sufficient, thus increasing the length of the growing season before the onset of the summer drought. However, high mountain species in the Mediterranean region that are exposed to different environmental conditions have different seed germination behaviour than lowland species. It has been observed that the seeds of many mountainous Mediterranean species can easily germinate without treatment, reaching their optimum point at relatively high temperatures. Compared to low altitudes, the growing season at high altitudes occurs later in the year, as winter environmental conditions are harsher, and summer conditions are milder. Therefore, to ensure successful germination and establishment, plants of higher altitudes tend to germinate at higher temperatures (see the modified fourth paragraph in the Discussion section of the revised manuscript).

To accommodate the reviewer’s comment the following information has been added, in the revised version of the manuscript (see track changes):

“In the Mediterranean-type ecosystem characterized by high seasonality (hot dry summers and cold wet winters), the optimum temperature for seed germination of lowland species is relatively low (≤ 15°C) [33, 49, 50]. This represent an ecological adaptation of several species, as it ensures that germination takes place in late autumn when rainfall is sufficient, thus increasing the length of the growing season before the onset of the summer drought. However, Mediterranean high mountain species are exposed to different environmental conditions and therefore have different seed germination behaviour than lowland species.” (see track changes in the revised manuscript).

 

I think that the relevance of this work is much higher than the authors indicate, since the fact that this species germinates worse at higher temperatures indicates high risks for the conservation of this species in the face of climate change. The authors need to work on the presentation of the material in the article so that their thoughts become more understandable to the reader. The article can be published after a little revision.

Authors’ response: We would like to thank the reviewer for this comment. According to the results of the germination test, the germination percentage decreased significantly (33%) at high temperature. Rising temperatures due to climate change will probably result in a decrease of the populations of species. The aim of this study was to investigate the germination behaviour and develop a species-specific germination protocol that will contribute to conservation and sustainable exploitation. Undoubtedly, in a future research line, the response of C. pangea seed germination under different scenarios of temperature rise due to climate change could be investigated. To accommodate the reviewer’s comment, we have added the following text in the revised version of the manuscript (see track changes in the second paragraph of the discussion): “The significant reduction in germination percentage at the higher (25°C) examined temperature (33%) raises some concerns about the future adaptability of this species to climate change impact, and therefore, it should be further investigated.”.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The proposed article aims at the elaboration of the optimal strategy for ex situ conservation of Campanula pangea seeds, a local endemic species of North East Greece. The topic is relevant. C. pangea is a recently described species of sect. Involucratae which occurs exclusively on Mt Pangeo, Eastern Macedonia (single-mountain endemic). To define the conditions favorable for germinations, the Authors investigated the germinating ability of seeds under various temperatures and gibberellic acid (GA₃) pre-treatment. The optimal temperature range for C. pangea seeds was shown to constitute 5-20°C and seeds pre-treatment in GA₃ solution (1000 mg L-1 GA₃) to increase gemination ability at 10°C. The experiments have demonstrated that the C. pangea seeds are characterized by the non-deep physiological dormancy. The results of the study will help to establish a specialized species-specific protocol for ex situ propagation and conservation.

 

The title of the article corresponds to the content.

The content of the proposed article is briefly described and contextualized with respect to previous and present theoretical background and empirical research on the problem of ex situ conservation of threatened plant species.

The methods are clearly stated and described in details. The adequate statistics methods are applied for treatment of the results.

The manuscript is written in good language. The data are well presented, illustrated and comprehensively discussed.

 

Comments.

 

1. The article reports on the development of ecological (bioclimatic) profile which is based exclusively on the climatic characteristics of the sites of the species natural habitats. However, it is known (Krigas et al., 2012) that an ecological profile of a plant species implies a characterization of its relationship with the environment, including information about its ecological needs, adaptations, and place in the community. It includes the species attitude to light, water, temperature, soil type, and its role in plant communities. The only climatic characteristics (temperatures, precipitations) are apparently insufficient be called ecological (bioclimatic as indicated on Figure 3E) profile.

 

2. Please specify the choice of 1000 mg L^-1 GA₃ concentration for the experiments. Why only that concentration was verified?

 

3. Please check content of Table 1. There is inconsistence with the text. The text on the Lines 199-201: “The two-way ANOVA analysis revealed that the effects of incubation temperature (p 199 = 0.001) and GA₃ (p = 0.003) on Campanula pangea seed germination, as well as the effects 200 of their interaction (p = 0.000) were statistically significant (Table 1).” In the Table: p = 0.000 for temperature, p= 0.003for GA₃, and p=0.001 for interaction of both factors.

 

4. I suggest to correct graph 6 in Table 1: toe replace “Sign.” by “p”.

Author Response

Reply to the second Reviewer

The proposed article aims at the elaboration of the optimal strategy for ex situ conservation of Campanula pangea seeds, a local endemic species of North East Greece. The topic is relevant. C. pangea is a recently described species of sect. Involucratae which occurs exclusively on Mt Pangeo, Eastern Macedonia (single-mountain endemic). To define the conditions favorable for germinations, the Authors investigated the germinating ability of seeds under various temperatures and gibberellic acid (GA₃) pre-treatment. The optimal temperature range for C. pangea seeds was shown to constitute 5-20°C and seeds pre-treatment in GA₃ solution (1000 mg L-1 GA₃) to increase gemination ability at 10°C. The experiments have demonstrated that the C. pangea seeds are characterized by the non-deep physiological dormancy. The results of the study will help to establish a specialized species-specific protocol for ex situ propagation and conservation.

The title of the article corresponds to the content.

The content of the proposed article is briefly described and contextualized with respect to previous and present theoretical background and empirical research on the problem of ex situ conservation of threatened plant species.

The methods are clearly stated and described in details. The adequate statistics methods are applied for treatment of the results.

The manuscript is written in good language. The data are well presented, illustrated and comprehensively discussed

Comments

1. The article reports on the development of ecological (bioclimatic) profile which is based exclusively on the climatic characteristics of the sites of the species natural habitats. However, it is known (Krigas et al., 2012) that an ecological profile of a plant species implies a characterization of its relationship with the environment, including information about its ecological needs, adaptations, and place in the community. It includes the species attitude to light, water, temperature, soil type, and its role in plant communities. The only climatic characteristics (temperatures, precipitations) are apparently insufficient be called ecological (bioclimatic as indicated on Figure 3E) profile.

 Authors’ response: To accommodate the reviewer’s comment, we have eliminated the term ‘ecological profile’ throughout the revised version of the manuscript; instead, we have used the term ‘bioclimatic profile’ in line with the reviewer’s suggestion (see scattered track changes).

 

1. Please specify the choice of 1000 mg L^-1 GA₃ concentration for the experiments. Why only that concentration was verified?

Authors’ response: In a recently published study on Campanula cretica for which we had an adequate number of wild-sourced available seeds, seeds treated with GA₃ (500 and 1000 mg L^-1) at high temperatures (20 and 25°C) showed higher germination percentages than control seeds and those cold-stratified for 25 days. No significant difference was observed between the germination percentages of seeds treated with 500 and 1000 mg L^-1 of GA₃.

(Panagiotidou T.-N., Anestis I., Pipinis E., Kostas S., Tsoktouridis G., Hatzilazarou S., Krigas N. (2025). GIS Bioclimatic profile and seed germination of the endangered and protected Cretan endemic plant Campanula cretica (A. DC.) D. Dietr. for conservation and sustainable utilization. Agriculture, 15, 1161)

 In the present study, first of all, we had a limited number of available wild-sourced seeds, therefore it was not possible to apply a second concentration of GA₃ solution (for example 500 mg L^-1), and we had to choose either 500 or 1000 mg L^-1. Based on own unpublished data in another two Campanula species (C. pelviformis and C. lyrata) with enough seeds for experimentation, the seed treatment with 1000 mg L^-1 of GA₃ was chosen as it significantly improved the seed germination at 25°C compared to seed treatment with 500 mg L^-1 of GA₃.

To address this comment, we have added the following sentence in section 2.2 of the revised version of the manuscript (see track changes): “Informed by results of previous studies on seeds of Campanula species (C. cretica [38], C. pelviformis and C. lyrata (unpublished data)) and due to the limited number of wild-sourced available seeds, the treatment of seeds with 1000 mg L^-1 of GA₃ was chosen for experimentation”.

  

1. Please check content of Table 1. There is inconsistence with the text. The text on the Lines 199-201: “The two-way ANOVA analysis revealed that the effects of incubation temperature (p199 = 0.001) and GA₃ (p = 0.003) on Campanula pangea seed germination, as well as the effects 200 of their interaction (p= 0.000) were statistically significant (Table 1).” In the Table: p = 0.000 for temperature, p= 0.003 for GA₃, and p=0.001 for interaction of both factors.

Authors’ response: We thank the reviewer for the comment. The sentence has been corrected (see track changes in the revised manuscript)

 

1. I suggest to correct graph 6 in Table 1: toe replace “Sign.” by “p”.

Authors’ response: We thank the reviewer for the comment. The label of column 6 in Table 1 has been corrected. The “Sig.” has been replace by “p value”. (see track changes in the revised manuscript).

 

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigated the germination characteristics of Campanula pangea seeds native to Greece and obtained results that were appropriate for the purpose.

I think this is a research result suitable for publication in this journal.

However, there are some areas that need to be partially reviewed and revised.

1. Figure 1: It would be better to indicate the date the photo was taken in the figure.
2. 2. Line 157: four (4) --> The numbers in parentheses do not need to be indicated. Other parts also need to be revised.
3. 3. In this study, GA was treated at a concentration of 1000 ppm. Then, why was the treatment based on that concentration? An explanation based on evidence is needed.
4. 4. This experiment used fluorescent lights. Then, what was the light intensity?
5. 5. Figure 2: Figure 2 presents the photo of the seed well. However, it would be better to revise it so that the surface of the seed can be confirmed by adjusting the brightness and contrast.
6. 6. Figure 2: Figure (D) is said to germination. However, judging from the photo, it seems that the cotyledons protruded first. It would be better to explain in the title of the figure.
7. 7. Line 199~201: It seems that the p value is incorrectly written. For example, in Table 1, the effect of temperature is written as '0.000' and the interaction effect is written as '0.001'.
8. 8. Table 2: It would be better to add a horizontal line between incubation temp and temperature.
9. 9. Line 223: It seems that Figure 4 --> should be modified to Figure 3.
10. 10. Line 229: Figure 3 --> Figure 4 ??
11. 11. Line 232: Figure 3 --> Figure 4 ??
12. 12. Line 259~260: The germination rate was high at 20 degrees. However, here, it is expressed that the temperature expanded from 10 to 20 degrees. Therefore, it would be better to modify the expression a little.
13. 13. Line 264~266: The author said that since the seeds germinated from the 4th day, morphological dormancy was excluded. However, if the seeds have immature embryos and germinate within 30 days, this is usually considered morphological dormancy. Therefore, it is a misinterpretation to exclude morphological dormancy because germination occurred early from the 4th day. Therefore, the dormancy type of the seeds needs to be reviewed and revised overall.
14. 14. Line 276~293: I agree with the effect of temperature on germination. However, in the case of plants with small seeds, light requirements may still remain even after low-temperature treatment or GA treatment. In other words, even if the temperature is suitable for germination, there are cases where germination does not occur because light conditions are not provided. Therefore, I think the interpretation of this part should be supplemented a little.

Author Response

Reply to the third Reviewer

 This study investigated the germination characteristics of Campanula pangea seeds native to Greece and obtained results that were appropriate for the purpose.

I think this is a research result suitable for publication in this journal.

However, there are some areas that need to be partially reviewed and revised.

1. Figure 1: It would be better to indicate the date the photo was taken in the figure.

Authors’ response: As suggested by the reviewer, we have indicated in the legend of Figure 1 the date of the original photos.

 

1. Line 157: four (4) --> The numbers in parentheses do not need to be indicated. Other parts also need to be revised.

Authors’ response: We thank the reviewer for the comment. The number in parentheses has been deleted (see track changes in the revised manuscript)

 

1. In this study, GA was treated at a concentration of 1000 ppm. Then, why was the treatment based on that concentration? An explanation based on evidence is needed.

Authors’ response: In a recently published study on Campanula cretica for which we had an adequate number of wild-sourced available seeds, seeds treated with GA₃ (500 and 1000 mg L^-1) at high temperatures (20 and 25°C) showed higher germination percentages than control seeds and those cold-stratified for 25 days. No significant difference was observed between the germination percentages of seeds treated with 500 and 1000 mg L^-1 of GA₃.

(Panagiotidou T.-N., Anestis I., Pipinis E., Kostas S., Tsoktouridis G., Hatzilazarou S., Krigas N. (2025). GIS Bioclimatic profile and seed germination of the endangered and protected Cretan endemic plant Campanula cretica (A. DC.) D. Dietr. for conservation and sustainable utilization. Agriculture, 15, 1161)

 

In the present study, first of all, we had a limited number of available wild-sourced seeds, therefore it was not possible to apply a second concentration of GA₃ solution (for example 500 mg L^-1), and we had to choose either 500 or 1000 mg L^-1. Based on own unpublished data in another two Campanula species (C. pelviformis and C. lyrata) with enough seeds for experimentation, the seed treatment with 1000 mg L^-1 of GA₃ was chosen as it significantly improved the seed germination at 25°C compared to seed treatment with 500 mg L^-1 of GA₃.

To address this comment, we have added the following sentence in section 2.2 of the revised version of the manuscript (see track changes): “Informed by results of previous studies on seeds of Campanula species (C. cretica [40], C. pelviformis and C. lyrata (unpublished data) and due to the limited number of wild-sourced available seeds, the treatment of seeds with 1000 mg L^-1 of GA₃ was chosen for experimentation”.

 

1. This experiment used fluorescent lights. Then, what was the light intensity?

Authors’ response: We thank the reviewer for the comment. To address this comment, we have added the following information in the revised manuscript “and a light intensity of 82 μmol m^-2 s^-1” (see track changes).

  

1. Figure 2: Figure 2 presents the photo of the seed well. However, it would be better to revise it so that the surface of the seed can be confirmed by adjusting the brightness and contrast.

Authors’ response: The quality of photo has been improved in the revised manuscript as suggested by the reviewer.

 

1. Figure 2: Figure (D) is said to germination. However, judging from the photo, it seems that the cotyledons protruded first. It would be better to explain in the title of the figure.

Authors’ response: We thank the reviewer for the comment. To avoid any confusion to readers regarding the criterion for determining seed germination, the photo D of Figure 2 has been replaced in the revised version of the manuscript with a new one.

 

1. Line 199~201: It seems that the p value is incorrectly written. For example, in Table 1, the effect of temperature is written as '0.000' and the interaction effect is written as '0.001'.

Authors’ response: We thank the reviewer for the comment. The sentence has been corrected (see track changes in the revised manuscript).

 

1. Table 2: It would be better to add a horizontal line between incubation temp and temperature.

Authors’ response: We thank the reviewer for the comment. Table 2 has been corrected (see track changes in the revised manuscript).

 

1. Line 223: It seems that Figure 4 --> should be modified to Figure 3.

Authors’ response: We thank the reviewer for the comment. The number of figure has been corrected (see track changes in the revised manuscript).

 

1. Line 229: Figure 3 --> Figure 4 ??

Authors’ response: We thank the reviewer for the comment. The number of figure has been corrected (see track changes in the revised manuscript).

 

1. Line 232: Figure 3 --> Figure 4 ??

Authors’ response: We thank the reviewer for the comment. The number of figure has been corrected (see track changes in the revised manuscript).

 

1. Line 259~260: The germination rate was high at 20 degrees. However, here, it is expressed that the temperature expanded from 10 to 20 degrees. Therefore, it would be better to modify the expression a little.

Authors’ response: According to the germination test results, the control seeds showed the highest germination percentages in a narrow temperature range of 15-20°C. The treatment of seeds with GA₃ resulted in wider temperature range and successful germination of C. pangea seeds from 10 to 20°C.

To accommodate the reviewer’s comment and to be more accurate, this sentence has been rephrased as: “The temperature range for successful germination after treatment of seeds with 1000 mg L^-1 of GA₃ extended from 10 to 20°C, though germination remained low at 25°C.” (see track changes in the revised manuscript).

 

1. Line 264~266: The author said that since the seeds germinated from the 4th day, morphological dormancy was excluded. However, if the seeds have immature embryos and germinate within 30 days, this is usually considered morphological dormancy. Therefore, it is a misinterpretation to exclude morphological dormancy because germination occurred early from the 4th day. Therefore, the dormancy type of the seeds needs to be reviewed and revised overall.

Authors’ response: We agree with the comment of the reviewer. It is probable that the embryo of C. pangea seeds is underdeveloped, indicating the presence of morphological dormancy. However, this was not investigated because it was not possible to examine the size of the embryo due to the small size of the seeds. To accommodate the reviewer’s suggestion,  we have rephrased this sentence as: “According to the above results, the seeds of C. pangea may have non-deep physiological dormancy; however, the possibility of morphological dormancy (underdeveloped embryo) cannot be excluded since the size of the embryo was not examined.” (see track changes in the revised manuscript).

Accordingly, the dormancy type of C. pangea seeds has been revised throughout the new version of the manuscript (see track changes in the revised manuscript).

 

1. Line 276~293: I agree with the effect of temperature on germination. However, in the case of plants with small seeds, light requirements may still remain even after low-temperature treatment or GA treatment. In other words, even if the temperature is suitable for germination, there are cases where germination does not occur because light conditions are not provided. Therefore, I think the interpretation of this part should be supplemented a little.

Authors’ response: We agree with the comment made by the reviewer. Apart from temperature, the light affects seeds germination, especially in plants with small seeds. However, in the present study due to limited availability of seeds the effect of light on seed germination was not investigated.

In response to the reviewer’s comment, we have added in the revised manuscript: “Apart from temperature, another factor that affects seed germination is light, especially in plants with small seeds. Undoubtedly, it would be useful to conduct further experiments to draw conclusions about the effect of light on seed germination.” (see track changes in the revised manuscript).