Characterization of the Pollen Trapped by Populus L. Seeds during the Dispersion Season

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a palynological paper named as “Characterization of the pollen trapped by Populus seeds during the dispersion season”. However, there are several questions should be resolved before the paper is published.

Here are my comments:

1.      How to understand this finding: “A tendency of higher 22 pollen concentrations found in the afternoon samples, both airborne and on the seeds, was observed.”? The trend does not seem obvious in Figure 4.

2.      Names of pollen genus in the figures should be expressed as italic.

3.      How about the significant influence between wind speed and airborne pollen concentration? Please explain the reason for this relation.

4.     Line 107, why not collecting the seeds from ground?

5.      If possible, more sampling areas should be added to improve the accuracy of data.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Thank you very much for your revision and pertinent suggestions. We hope we have responded to the comments in the best way possible. Changes in the manuscript are in yellow colour.

The authors present a palynological paper named as “Characterisation of the pollen trapped by Populus seeds during the dispersion season”. However, there are several questions should be resolved before the paper is published.

Here are my comments:

1. How to understand this finding: “A tendency of higher 22 pollen concentrations found in the afternoon samples, both airborne and on the seeds, was observed.”? The trend does not seem obvious in Figure 4.

Response: The authors intention was to highlight a tendency that there were more days with higher pollen concentrations during the afternoon compared with the morning (4 vs 2 in seeds and 4 vs 3 in air). That is why we mentioned it as a tendency and not a trend. It was likely to happen that higher concentrations were found in the afternoon hours. The sentence was corrected to better correspond to what is observed in Figure 4: “Also, a tendency towards more days with higher pollen concentration in the afternoon compared with the morning one was observed on airborne and tuft-like seeds.”

2. Names of pollen genus in the figures should be expressed as italic.

Response: Indeed in figure 5 the pollen genus are not expressed in italics because it is a column graphic made in excel and the legend of the x-axis is programmed as a bulk legend and we cannot change each name independently.

3. How about the significant influence between wind speed and airborne pollen concentration? Please explain the reason for this relation.

Response: The explanation for the negative significant correlation between wind speed and airborne pollen was added to the text as followed: “ This negative relationship can indicate that the airborne pollen sampled can originate mostly from the vicinity area (Pérez-Badia et al., 2011; Markey et al., 2022).” Both references were added to the text.

4. Line 107, why not collecting the seeds from ground?

Response: The author thought about this because it would be much easier to collect the samples and have a lot more mass. However, seeds in the ground could bias our analysis. Seeds settling in the soil, difficultly resuspending, can be exposed to the environment for many days. They will probably attach pollen and other debris already deposited in the soil (for example, particles that are heavier and would bias the number of pollen/mg). So, we limited our sampling to what was in the air.

5. If possible, more sampling areas should be added to improve the accuracy of data.

Response: We agree with the reviewer. However, the logistics of this study protocol make its concretization difficult. Also, adding more sampling areas implies more sampling years, which cannot be done presently.    

Reviewer 2 Report

Comments and Suggestions for Authors

A wonderful piece of work. It is an interesting topic and the study was carried out scientifically sound.

Some minor corrections include:

Please also consider the forensic aspect of your work. Palynology is important also to forensics and there are a couple of studies on the pollen content of dust that would strengthen also your findings. It is not unusual that your Populus hair seed sample does content a different composition than the aerobiologic samples. Please refer to this aspect as well as to the work already known in that field.

p. 2 line 70: add "or both"; it is most probable that pollen collected in the seed hairs derives from the air and the soil.

References: I could not find reference 21 in the text. Please check if you really used it anywhere and adopt the order if necessary.

Figure 3: The colors are quite similar. It would be great to find scales, that could be also discriminated if it was only black and white, but even if not so, please choose a broader range of colors than now. It will be easier for the reader.

Congratulations to your work, it was interesting to read.

Author Response

Thank you very much for your revision that allowed us to improve the manuscripts. We hope we have responded to the comments in the best way possible. Changes in the manuscript are in yellow colour.

Reviewer 2

A wonderful piece of work. It is an interesting topic and the study was carried out scientifically sound. Some minor corrections include:

Please also consider the forensic aspect of your work. Palynology is important also to forensics and there are a couple of studies on the pollen content of dust that would strengthen also your findings. It is not unusual that your Populus hair seed sample does content a different composition than the aerobiologic samples. Please refer to this aspect as well as to the work already known in that field.

Response: The reviewer is very accurate, palynology also contributes to forensics. Indeed, there are some interesting studies quantifying pollen, including using eDNA. In the case of our study, we focussed only on samples collected from air, not from the ground, where dust can also be found. So, if the reviewer does not mind, we opted not to discuss this aspect because, in our opinion, it is a marginal subject to the core discussion of our findings.

1. 2 line 70: add "or both"; it is most probable that pollen collected in the seed hairs derives from the air and the soil.

Response: Change was done accordingly.

References: I could not find reference 21 in the text. Please check if you really used it anywhere and adopt the order if necessary.

Response: Thank you for this correction, indeed reference 21 was named as 22 in the text (page 3 line 116). It was corrected

Figure 3: The colors are quite similar. It would be great to find scales, that could be also discriminated if it was only black and white, but even if not so, please choose a broader range of colors than now. It will be easier for the reader.

Response: Thank you for your suggestion. We change the range colors.

Reviewer 3 Report

Comments and Suggestions for Authors

General comments for the authors:

This work deals with the presence of pollen in hairy seeds of the genus Populus L. collected during several days in an urban environment during seed dispersion period. The methodology used was already partially proposed by different aerobiological societies and articles and seems adequate and reproducible for the purpose of the study. The results are clearly and correctly presented, although they need to be revised from time to time due to possible divergences between data shown in the text and in the tables. Perhaps it would have been advisable to increase the number of sampling years to make the statistical analysis and the relationship between atmospheric pollen content and poplar seed hairs more consistent. Finally, the discussion is well structured and referenced, although the authors could consider reflecting on other aspects related to the influence of meteorological factors or differences between pollen types present in seeds and not in the atmosphere.

The research presented is in the scope of aerobiology journal and therefore publishable.  

Specific comments for the authors:

a) Title

Line 2.  According to botanical nomenclature, the generic name Populus, the first time it is mentioned in a text, must bear the abbreviated name of the author who described it. In this case: Populus L.

b) Authors of the manuscript

Line 4. The surname of the second author should begin with a capital letter: Castro.

1. Introduction section

Lines 47-48. Based on botanical nomenclature, perhaps the following proposed wording would be more appropriate: “Trees of the genus Populus L., popularly known as poplars, are common trees present in urban green spaces, particularly the species P. alba L., P. nigra L., P. tremula L., P. x canadensis Moench. and P. x canescens (Aiton) Sm.”

Furthermore, it is proposed to add this sentence to indicate that the presence of poplars in cities is not only restricted to urban environments: “Some of these species are typical of riparian formations associated with watercourses that run through many urban environments.” At this point, you could add the following reference or any other reference you think suitable: https://www.sciencedirect.com/science/article/pii/S0048969721049561

Line 56. Here you can incorporate the reference [31] of the manuscript or the reference Ribeiro et al. (2009): https://www.sciencedirect.com/science/article/pii/S0013935108002557

2. Material and Methods

Lines 100-101. The specific epithets "alba" and "nigra" should be italicised. In addition, scientific names of species named for the first time in the text: Populus deltoides W. Bartram ex Marshall and Populus nigra var. italica Münchh.

Lines 106-122. A few questions or reflections regarding seed collection and seed treatment.

I believe that the seeds were not collected from the same poplar species. It might be interesting for future studies to consider whether there are differences between seeds from different species due to differences in the pilosity of the seeds.

Acetolysis could destroy several pollen grains and fungal spores. Have you considered using another method that avoids the use of the acetolytic method? This would make the identification analogous to the aerobiological methodology and would also allow discriminating the presence of fungal spores.

Line 143. The authors could consider incorporating a reference to aerobiological methodology. (i.e.  Galán, C., et al. "Management and Quality Manual." Spanish Aerobiology Network (REA). Servicio Publicaciones Universidad de Córdoba, Córdoba, Spain (2007).

Line 145. In the aerobiological sampling for the morning and afternoon periods, perhaps the data would be more comparable if 6 hours in the morning period were also considered

Line 148. Why did not use Spearman's correlation coefficient? Airborne pollen levels do not follow a normal distribution. For this reason, Pearson's correlation coefficient could also be used but the data should be log-transformed.

3. Results

Line 159. After reviewing the results presented in table 1, I counted 18 pollen types in the aerobiological samples, not 19 as in the text.

Line 160. The authors indicate that all pollen types observed in the atmosphere were found in the seed hairs. However, according to table 1, Juglans pollen was identified in aerobiological samples during the 2018 sampling but not in seeds. Please check the data.

4. Discussion

Line 226. “Populus” must be written in italics.

Line 244. In this way, the authors could consider including rainy days for future studies. This could determine the influence of precipitation on how seed hairs trap pollen grains. I make this comment because I am unaware of the existence of studies or references related to these processes and I was surprised that the authors have discarded rainy days to carry out this type of study even though they coincide with the dispersion period of poplar seeds.

Author Response

Thank you very much for your assertive review. We hope we have responded to the comments in the best way possible. Changes in the manuscript are in yellow colour.

Reviewer 3

General comments for the authors: This work deals with the presence of pollen in hairy seeds of the genus Populus L. collected during several days in an urban environment during seed dispersion period. The methodology used was already partially proposed by different aerobiological societies and articles and seems adequate and reproducible for the purpose of the study. The results are clearly and correctly presented, although they need to be revised from time to time due to possible divergences between data shown in the text and in the tables. Perhaps it would have been advisable to increase the number of sampling years to make the statistical analysis and the relationship between atmospheric pollen content and poplar seed hairs more consistent. Finally, the discussion is well structured and referenced, although the authors could consider reflecting on other aspects related to the influence of meteorological factors or differences between pollen types present in seeds and not in the atmosphere.

The research presented is in the scope of aerobiology journal and therefore publishable.

 Specific comments for the authors:

Line 2.  According to botanical nomenclature, the generic name Populus, the first time it is mentioned in a text, must bear the abbreviated name of the author who described it. In this case: Populus L.

Response: It was corrected

Authors of the manuscript. Line 4. The surname of the second author should begin with a capital letter: Castro.

Response: It was corrected

Lines 47-48. Based on botanical nomenclature, perhaps the following proposed wording would be more appropriate: “Trees of the genus Populus L., popularly known as poplars, are common trees present in urban green spaces, particularly the species P. alba L., P. nigra L., P. tremula L., P. x canadensis Moench. and P. x canescens (Aiton) Sm.”

Response: Thank you for the suggestion. It was corrected accordingly.

Furthermore, it is proposed to add this sentence to indicate that the presence of poplars in cities is not only restricted to urban environments: “Some of these species are typical of riparian formations associated with watercourses that run through many urban environments.” At this point, you could add the following reference or any other reference you think suitable: https://www.sciencedirect.com/science/article/pii/S0048969721049561

Response: Thank you very much for the suggestion. It was added the sentence.

Line 56. Here you can incorporate the reference [31] of the manuscript or the reference Ribeiro et al. (2009): https://www.sciencedirect.com/science/article/pii/S0013935108002557

Response: The reference [31] was added.

Lines 100-101. The specific epithets "alba" and "nigra" should be italicized. In addition, scientific names of species named for the first time in the text: Populus deltoides W. Bartram ex Marshall and Populus nigra var. italica Münchh.

Response: Thank you for the correction. It was changed accordingly.

Lines 106-122. A few questions or reflections regarding seed collection and seed treatment. I believe that the seeds were not collected from the same poplar species. It might be interesting for future studies to consider whether there are differences between seeds from different species due to differences in the pilosity of the seeds.

Response: That is a very interesting suggestion. Thank you very much. Indeed, there could be some differences in the ability to “catch” pollen depending on the features of the seed's pilosity.

Acetolysis could destroy several pollen grains and fungal spores. Have you considered using another method that avoids the use of the acetolytic method? This would make the identification analogous to the aerobiological methodology and would also allow discriminating the presence of fungal spores.

Response: We could just destroy the seeds and proceed without acetolysis, just staining and identification. It was not done in this case because the acetolysis could allow us to eliminate other less resistant organic material present in the seeds. We can incorporate the suggestion in future studies. We also did not look for fungal spores, as we are not experts on their identification, so their destruction was not a concern. Our experience with acetolysis has shown us that it could be problematic in paleosamples and mainly for pollen types not adapted for anemophilous pollination. In our opinion, if the acetolysis process is rigorously done, complying with the exact proportions of the acetolysing mixture and reaction times, the destruction of anemophilous pollen types is not an issue.

Line 143. The authors could consider incorporating a reference to aerobiological methodology. (i.e.  Galán, C., et al. "Management and Quality Manual." Spanish Aerobiology Network (REA). Servicio Publicaciones Universidad de Córdoba, Córdoba, Spain (2007).

Response: The reference was added.

Line 145. In the aerobiological sampling for the morning and afternoon periods, perhaps the data would be more comparable if 6 hours in the morning period were also considered

Response: Thank you for your comment. We have chosen to initiate at 7 a.m. (totalizing five hours in the morning period) because it would be more comparable with the seed dispersal period, which starts at the first sunlight hours. In May, at 6 a.m., it is still night.

Line 148. Why did not use Spearman's correlation coefficient? Airborne pollen levels do not follow a normal distribution. For this reason, Pearson's correlation coefficient could also be used but the data should be log-transformed.

Response: Not all our data followed a normal distribution, even with log-transformation and other transformation methods. This is why we choose Spearman’s correlation.

Line 159. After reviewing the results presented in table 1, I counted 18 pollen types in the aerobiological samples, not 19 as in the text.

Response: Thank you for your correction. Change was done. “A total of 26 different pollen taxa were identified on the tuft-like seeds, while only 18 were observed on the airborne pollen samples, ….”

Line 160. The authors indicate that all pollen types observed in the atmosphere were found in the seed hairs. However, according to table 1, Juglans pollen was identified in aerobiological samples during the 2018 sampling but not in seeds. Please check the data.

Response: Thank you for your correction. Change was done. “…, but apart from Juglans pollen type, all the others were concordant with the ones observed on the tuft-like seed samples. (Table 1)”.

Line 226. “Populus” must be written in italics.

Response: Correction done.

Line 244. In this way, the authors could consider including rainy days for future studies. This could determine the influence of precipitation on how seed hairs trap pollen grains. I make this comment because I am unaware of the existence of studies or references related to these processes and I was surprised that the authors have discarded rainy days to carry out this type of study even though they coincide with the dispersion period of poplar seeds.

Response: We opted not to use rainy days mainly because of the washing effect of rain. During these days, two scenarios happened. If there was persistent rain, no seeds were dispersed or transported through the air (that was the case in 2017 where we could only flawlessly execute the sampling protocol in two days). When only sparse showers occurred, seed dispersion either did not happen or when it did, it occurred at distinct hours of the ones established in our protocol. For example, in 2018, we had seed dispersal occurring only in the late afternoon, and therefore we could not compare the morning and afternoon periods.

Reviewer 4 Report

Comments and Suggestions for Authors

This study aims to identify and quantify the pollen trapped by the Poplar tuft-like seeds and compare it with the amount and species-variation of airborne pollen concentrations. The samples were collected for a selected days of the season 2017 and 2018 in Porto, Portugal. The manuscript is clear and well structured. The methods and results are clearly presented. The results were discussed in the context of the previous research. I recommend the paper for publication.

Author Response

Reviewer 4

This study aims to identify and quantify the pollen trapped by the Poplar tuft-like seeds and compare it with the amount and species-variation of airborne pollen concentrations. The samples were collected for a selected days of the season 2017 and 2018 in Porto, Portugal. The manuscript is clear and well structured. The methods and results are clearly presented. The results were discussed in the context of the previous research. I recommend the paper for publication.

Response: Thank you very much for your comments.