Pollen Morphological Peculiarities of Selected Mimosoideae Taxa of Hainan Island and Their Taxonomic Relevance

Round 1

Reviewer 1 Report

The article is an interesting contribution to the knowledge of the pollen morphology of the local flora of Hainan island. However, it requires many clarifications and changes. In the introduction, the authors present only traditional taxonomic approach of the Fabaceae family, including Mimosaceae [lines 30-42]. They omit the modern taxonomic position of the mimosae (APG system), according to which it belongs to Mimosoid clade. This has consequences, for example, in the smaller number of genera and species currently assigned to mimosae (i.e. Mimosoideae – less than in the formal Mimosaceae). In the section Introduction the authors write „Mimosaceae is the subfamily of Fabaceae, sometimes treated as a separated family (Shinwari et al. 2014)”[lines 30-31]. However, cited Shinwari et al. (2014) refer to the subfamily Mimosoidae, and not to the family Mimosaceae.

The authors write: „Light and scanning electron microscopy plays a crucial role in the determination of the ultra-structure of the plant materials” [lines 53-54]. Commonly an ultra-structure (or rather ultrastructure) means biological fine structure (i.e. as of a cell, including pollen grain) not visible through an ordinary microscope. Ultrastructure can be viewed with SEM microscopy, however TEM microscopy is a standard technique for viewing ultrastructure. So a term ultrastructure mainly refers to the deeper layers of a given biological object (as pollen grain – its wall structure), than the pecularities of its surface (exine micromorphology).

All species listed in the table 1, currently analyzed, according to the APG system belonged to the Mimosoid clade and Caesalpinioideae subfamily (which belongs to the Fabaceae family). I want to emphasize once again the necessity to revise systematic affiliation of investigated taxa, or at least to clearly define according to whom the systematic system is adopted.

In the subsection Light Microscopy [lines 106-107] the authors refer to Punt at al. (2007). This article is not cited in the section References.

In the subsection referring to quantitative analysis [lines 118-121] authors do not specify the number of pollen grains analyzed for individual species. So what is the point of using a statistical program (SPSS software) without specifying the basic assumptions?

The authors conclude that „The LM and SEM micrographs were illustrated in (Figure 1-4) and were summarized in (Table 1)[lines 124-125]. However, table 1 only consists the geographical characteristics of species. On the other hand, I recommended to support a table with synthetic list of grain characteristics of the investigated species (– comparison).

            The figures (1-4) presented in the reviewed article require changes. First of all, they do not contain illustrative material for all investigated species, both in LM and SEM. The arragement of pollen species in the figures is surprising. For example, SEM photos of the Mimosa pudica pollen grains are scattered over two figures (fig. 1 and 2; why?). The micrographs 2 h-i are probably incorrectly described. The SEM photos are not always of good quality, they lack a clear scale of magnification. Figure captions contain only species names. There is no information on morphological details worth paying attention to.

Generally, in the section Results and Discussion, the authors do not always clearly distinguish their results from the data, resulting from the literature. So it seems that a better solution would be to separate the results and discussion sections.

When describing the exine sculpture of particular species (almost all), the authors usually mention several types of it (e.g. Mimosa diplotricha „exine sculpturing was predominantly areolate, verrucate, scabrate and sparsely gemmate” [lines 146-147]). At the same time, there is no detailed information – as it is which structure actually dominates, whether the types mentioned occur together or on different pollen grains, etc.

The sentence [lines 146-147]: „In comparison with previous studies, the exine sculpturing is characterized by tubercules that were inconsistent with our results except for tetrads type of pollen (Caulton, 2019) should be corrected. It is difficult to understand who really observed tubercules. In given reference (Caulthon, 2019) two authors should be cited: Agashe and Coulthon (2019). Furthermore, they (i.e. Agashe and Coulthon) mention about tubercles, not tubercules.

The authors investigated two pollen samples of Mimosa diplotricha (subsection , 3.2. Mimosa diplotricha [lines 151-169]) originated from different sites. However, in table 1 only one localization is given. The average reader is rather unable to locate „Beihualing tropical rain forest”. It is important because the authors found the variability of pollen grains between the two Mimosa diplotricha populations, explaining it by the influence of climatic and geographic factors.

In the subsection 3.3. Leucaena leucocephala [lines 170-183] authors explained the differences between their results and Aftab and Perveen (2006) due to the different geographical regions - different altitudes and different environmental factor. However, they did not include different pollen preparation (lack of acetolysis in current investigation, and application of this method by Aftab and Perveen), which may have effect on pollen morphology. It is worth noting that Leucaena leucocephalis and Entada phaseoloides (other subsection [lines 226-237]) are two only species with pollen shed in monad and there in no explanation of this phenomena in current article, from the systematic point of view of the studied group of plants.

            In relation to Albizia lebback [lines 197-198] the authors cited: „Besides, they also mentioned the allergy-causing pollen” (i.e. Caulton, 2019). I do not understand the relationship of this fact with current research.

Authors stated: „The tricolporate, subprolate, size 30 x 35μm and faintly reticulate exine pattern of the Entada spiralis pollen have been found in the previous study” [lines 234-235]. However, they do not explain about what study.

The section conclusion also requries redrafting. The sentence: ‘The subfamily Mimosaceae taxa collected from different geographical regions of the Hainan Island were analyzed by using both light and scanning electron microscopy” [lines 248-249] can not be consider as conclusion. The sentence: ”This study explored and fills the palynological gap of the studied taxa which was not previously investigated”. [lines 249-250] is very general. Authors should provide specific information, which species have not been described in the palynological literature so far and which features of the pollen grains are of real importance in the taxonomy of the taxa studied.

In section reference, authors should pay attention to the repetition of citations of articles (see Aftab, R., &Perveen, A. (2006);  Khan, S. U., Zafar, M., Ullah, R., Shahat, A. A., Ahmad, M., Sultana, S., & Malik, K. (2021); Perveen, A., &Qaiser, M. 1998)

Summing up, the article requires many changes and should not be published in its current form.

Author Response

Response to reviewer 1.

The article is an interesting contribution to the knowledge of the pollen morphology of the local flora of Hainan island. However, it requires many clarifications and changes. In the introduction, the authors present only traditional taxonomic approach of the Fabaceae family, including Mimosaceae [lines 30-42]. They omit the modern taxonomic position of the mimosae (APG system), according to which it belongs to Mimosoid clade. This has consequences, for example, in the smaller number of genera and species currently assigned to mimosae (i.e. Mimosoideae – less than in the formal Mimosaceae). In the section Introduction the authors write „Mimosaceae is the subfamily of Fabaceae, sometimes treated as a separated family (Shinwari et al. 2014)”[lines 30-31]. However, cited Shinwari et al. (2014) refer to the subfamily Mimosoidae, and not to the family Mimosaceae.

Ans: Thank you very much for your useful and detail comments. Along with the traditional system of classification, now we provided detail about the modern taxonomic position as well. For example; The Fabaceae is one of the diverse families found in most ecosystems of the earth planet. It has been divided into three subfamilies; Papilionoideae, Caesalpinioideae and Mimosoideae (Lewis, et al., 2005). In recent Fabaceae classification, the Legume Phylogeny Working group (LPWG 2017) recognized six subfamilies and the traditional subfamily, Mimosoideae is presented as a clade nested in the Caesalpinioideae with new discrimination.  In the present study, we followed the Lewis et al., (2005) classification. Furthermore, the recent classification which only proposes changes at the subfamily level and has not yet solved the taxonomic problem of the tribes in the Mimosoid clad does not affect the interpretation of our results.

The authors write: „Light and scanning electron microscopy plays a crucial role in the determination of the ultra-structure of the plant materials” [lines 53-54]. Commonly an ultra-structure (or rather ultrastructure) means biological fine structure (i.e. as of a cell, including pollen grain) not visible through an ordinary microscope. Ultrastructure can be viewed with SEM microscopy, however, TEM microscopy is a standard technique for viewing ultrastructure. So a term ultrastructure mainly refers to the deeper layers of a given biological object (as pollen grain – its wall structure), than the pecularities of its surface (exine micromorphology).

Ans: Thank you very much for your detail comments about the ultrastructure. We corrected these mistakes in the manuscript.

All species listed in the table 1, currently analyzed, according to the APG system belonged to the Mimosoid clade and Caesalpinioideae subfamily (which belongs to the Fabaceae family). I want to emphasize once again the necessity to revise the systematic affiliation of investigated taxa, or at least to clearly define according to whom the systematic system is adopted.

Ans: Thank you very much for your useful comments. And we incorporated the suggested changes in the manuscript as well. The Fabaceae has been divided into three subfamilies; Papilionoideae, Caesalpinioideae and Mimosoideae (Lewis, et al., 2005). While in the recent Fabaceae classification, the Legume Phylogeny Working group (LPWG 2017) recognized six subfamilies and the traditional subfamily, Mimosoideae is presented as a clade nested in the Caesalpinioideae with new discrimination. Hence, In the present study, we followed the Lewis et al., (2005) classification. Furthermore, the recent classification which only proposes changes at the subfamily level and has not yet solved the taxonomic problem of the tribes in the Mimosoid clad, does not affect the interpretation of our results.

In the subsection Light Microscopy [lines 106-107] the authors refer to Punt at al. (2007). This article is not cited in the section References.

Ans: Thank you for highlighting the missing citation and we provided it.

In the subsection referring to quantitative analysis [lines 118-121] authors do not specify the number of pollen grains analyzed for individual species. So what is the point of using a statistical program (SPSS software) without specifying the basic assumptions?

Ans: Thank you for your useful comment and now we specified the total number of pollen grains for each analyzed sample.

The authors conclude that „The LM and SEM micrographs were illustrated in (Figure 1-4) and were summarized in (Table 1)[lines 124-125]. However, table 1 only consists the geographical characteristics of species. On the other hand, I recommended to support a table with synthetic list of grain characteristics of the investigated species (– comparison).

Ans: We are thankful to your useful suggestion and we improved the table 1 with pollen morphological traits.

The figures (1-4) presented in the reviewed article require changes. First of all, they do not contain illustrative material for all investigated species, both in LM and SEM. The arragement of pollen species in the figures is surprising. For example, SEM photos of the Mimosa pudica pollen grains are scattered over two figures (fig. 1 and 2; why?). The micrographs 2 h-i are probably incorrectly described. The SEM photos are not always of good quality, they lack a clear scale of magnification. Figure captions contain only species names. There is no information on morphological details worth paying attention to.

Ans: Thank you very much for your useful comments and suggestions and we incorporated all these changes and highlighted the text red. We added figure 4 the missing material. The pollen of Mimosa pudica are scattered in fig 1and fig 2 because we take many SEM micrographs in different views and showed various features , i.e. General view of the pollen, close view of the exine sculpturing, polar view, equatorial view, oblique equatorial view, polar area etc. Besides we modified the figures captions as per your comments. 

Figure 1. Acacia confusa (a-c), a) general view of the pollen, b) close view of the exine sculpturig Entada phaseoloides (d-g), d) oblique polar view, e) equatorial view, f) showing sunken colpus, g) showing zono-colpus. Mimosa pudica  wanning (h,i), h-i) showing oblique equatorial view. Scale bar: a= 10 µm, b= 4 µm, c=10 µm,  d=10 µm,  e= 10 µm , f-g= 10 µm , h-i= 3 µm.

Figure 2. SEM micrographs showing the pollen grains of Mimosa pudica (a-c Wanning), a) close view of exine sculpturing, b) showing polar area, c) equatorial view. Mimosa diplotricha (d-g Beihualing),  d) equatorial view, e) polar view, f) close view of the exine, g) prolate shape pollen. Mimosa diplotricha (h-i Haikou), h) polar view, i) close view of the exine sculpturing. Scale bar: a) Scale bar: a= 1 µm, b-c= 4 µm, c=10 µm,  d-e=10 µm, f= 3 µm, g-h= 10 µm , i= 3 µm.

Figure 3. Showing the SEM micrographs of Albizia lebback pollen grain (a-c), a) showing quadrangular shape pollen in polar view outline, b) showing close view of individual grain of the polyad, c) inaperturate pollen. Leucaena leucocephala (d-f), d) polar view, e) close view of the colpus surface membrane, f) showing apocolpium region. The scale for a) 10 µm, b) 5 µm , c) 10 µm. d) 30 µm, e-f) 5 µm.

Figure 4: Showing the SEM micrographs of the pollen. Acacia mangium (a) showing octad polyads. Mimosa bimucronata (b) showing bitetrad polyads. Albizia julibrissin (c) showing 12-grains polyads.

Figure 5. Light micrographs of pollen; Calliandra haematocephala (a-b), a) showing equatorial view, b) polar view. Entada phaseoloides (c) polar view. Mimosa diplotricha (d) showing tetrad-type pollen. Mimosa pudica (e), showing triyad type pollen. Scale bar 50 µm.

Generally, in the section Results and Discussion, the authors do not always clearly distinguish their results from the data, resulting from the literature. So it seems that a better solution would be to separate the results and discussion sections.

Ans: Thank you for your useful suggestions. We revised the discussion section and clearly discuss our results with previously published literature. We avoid the confusing sentences and now it is easy to read and understandable for the readers.

When describing the exine sculpture of particular species (almost all), the authors usually mention several types of it (e.g. Mimosa diplotricha „exine sculpturing was predominantly areolate, verrucate, scabrate and sparsely gemmate” [lines 146-147]). At the same time, there is no detailed information – as it is which structure actually dominates, whether the types mentioned occur together or on different pollen grains, etc.

Ans: Thank you very much for your useful comments. We avoid this confusion and make it clear easy to read and understandable throughout the results.

The sentence [lines 146-147]: „In comparison with previous studies, the exine sculpturing is characterized by tubercules that were inconsistent with our results except for tetrads type of pollen (Caulton, 2019) should be corrected. It is difficult to understand who really observed tubercules. In given reference (Caulthon, 2019) two authors should be cited: Agashe and Coulthon (2019). Furthermore, they (i.e. Agashe and Coulthon) mention about tubercles, not tubercules.

Ans: We make these changes in the manuscript and highlighted the text red. We also corrected the citation and cited it in the references section.

Agashe, S. N., & Caulton, E. (2019). Pollen and spores: applications with special emphasis on aerobiology and allergy. CRC Press.

The authors investigated two pollen samples of Mimosa diplotricha (subsection , 3.2. Mimosa diplotricha [lines 151-169]) originated from different sites. However, in table 1 only one localization is given. The average reader is rather unable to locate „Beihualing tropical rain forest”. It is important because the authors found the variability of pollen grains between the two Mimosa diplotricha populations, explaining it by the influence of climatic and geographic factors.

Ans: We added Mimosa diplotricha  in the table along with its detail.

In the subsection 3.3. Leucaena leucocephala [lines 170-183] authors explained the differences between their results and Aftab and Perveen (2006) due to the different geographical regions - different altitudes and different environmental factor. However, they did not include different pollen preparation (lack of acetolysis in current investigation, and application of this method by Aftab and Perveen), which may have effect on pollen morphology. It is worth noting that Leucaena leucocephalis and Entada phaseoloides (other subsection [lines 226-237]) are two only species with pollen shed in monad and there in no explanation of this phenomena in current article, from the systematic point of view of the studied group of plants.

Ans: Thank you very much for useful comments and we incorporated in the manuscript as well, that variation in acetolysis techniques may also affect the pollen morphology. Besides, Leucaena leucocephalis and Entada phaseoloides are two only species with pollen shed in monad which is found different from rest of the species. So such traits are found useful at generic as well at species level. For example the Acacia and Albizzia have a polyad type pollen while Leucaena and Entada shed in monad.

In relation to Albizia lebback [lines 197-198] the authors cited: „Besides, they also mentioned the allergy-causing pollen” (i.e. Caulton, 2019). I do not understand the relationship of this fact with current research.

Ans: Thank you for your useful suggestions and we deleted the irrelevant sentence.

Authors stated: „The tricolporate, subprolate, size 30 x 35μm and faintly reticulate exine pattern of the Entada spiralis pollen have been found in the previous study” [lines 234-235]. However, they do not explain about what study.

Ans: We correct this mistake in the ms and provided detial about the mentioned statement. Furthermore, Rao & Lee (1970), documented the pollen flora of Singapore and Malaya and observed the tri-colporate, subprolate, size 30 x 35µm and faintly reticulate exine pattern of the Entada spiralis pollen.

The section conclusion also requries redrafting. The sentence: ‘The subfamily Mimosaceae taxa collected from different geographical regions of the Hainan Island were analyzed by using both light and scanning electron microscopy” [lines 248-249] can not be consider as conclusion. The sentence: ”This study explored and fills the palynological gap of the studied taxa which was not previously investigated”. [lines 249-250] is very general. Authors should provide specific information, which species have not been described in the palynological literature so far and which features of the pollen grains are of real importance in the taxonomy of the taxa studied.

Ans: Thank you very much for your useful comments on this section. We revised the conclusion accordingly

In section reference, authors should pay attention to the repetition of citations of articles (see Aftab, R., &Perveen, A. (2006);  Khan, S. U., Zafar, M., Ullah, R., Shahat, A. A., Ahmad, M., Sultana, S., & Malik, K. (2021); Perveen, A., &Qaiser, M. 1998)

Ans: We carefully check the references and arrange it accordingly.

Summing up, the article requires many changes and should not be published in its current form.

Ans: Thank you very much for your precious time and useful comments and suggestions on our manuscript. We respond point by point to all your suggested queries and highlighted the text red in the manuscript where we make changes. We improved the quality of the manuscript as per your useful comments.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

The manuscript needs a major revision:

- Carefully, define how many and which species are actually studied;

- Improve the writing of results and discussion.

- Explore the discussion with more previous studies.

- Improve image quality, especially in LM (avoid excessive use of photoshop);

-Improve legends of images, as they are not very informative and contain a lot of mistakes.

- The article did not follow the journal's guidelines. See instructions for authors.

All considerations, suggestions and/or corrections are highlighted in the attached PDF file.

Comments for author File: Comments.pdf

Author Response

Response to reviewer 2.

The manuscript needs a major revision:

- Carefully, define how many and which species are actually studied;

Ans: Thank you very much for your comment. Now we carefully mentioned the studied species in table 1. Please see table 1 in the manuscript.

- Improve the writing of results and discussion.

Ans: Thank you for your useful comments and we revised the results and discussion section carefully

- Explore the discussion with more previous studies.

Ans: We revised the discussion section and discuss our results with previously published literature.

- Improve image quality, especially in LM (avoid excessive use of photoshop);

Ans: Thank you very much for your useful suggestions and we added figure 4. Besides, we add a scale bar in the caption of each figure.

-Improve legends of images, as they are not very informative and contain a lot of mistakes.

Ans: We carefully revised all the captions and add detail about the observed traits.

- The article did not follow the journal's guidelines. See instructions for authors.

Ans: Thank you very much for your suggestions, we carefully check and arrange the manuscript as per the guidelines of the journal.

All considerations, suggestions and/or corrections are highlighted in the attached PDF file.

Ans: Thank you very much for your precious time, your valuable comments and suggestions. We respond point by point to all the suggested queries and highlighted the text red where we make any changes.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear authors,

The manuscript has been revised again and at this moment, I would like to highlight your efforts to improve the discussion with more previous studies. However, I noticed that just part of my previous considerations was revised, please, review the old and new considerations in the attached PDF file. I emphasize that the manuscript still does not follow the guidelines of the journal. See instructions for authors.

All considerations, suggestions and/or corrections are highlighted in the attached PDF file.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

Thank you very much for sending us the decision and constructive reviews on our ms. We incorporated all the suggested queries in the manuscript and highlighted the text in blue color where we make any changes. We are also thankful to the reviewer for their useful comments on our ms. If you need any assistance please free to contact us.

Response to reviewers

Abstract

Line 12,13,14, 15, 17, 18 21, 25; All the suggested queries have been incorporated in the manuscript.

Keywords: Line 30: Make changes as per comments

Introduction

Line 115, 121,122: Modified as per comments

Materials and Methods

We make changes in table 1 and also added the missing species Albizia lebbeck.

We also removed the suggested column as your comments.

Line 137: Modified

Results and Discussion

Line 155-156: We corrected the mistake

Line: 159, 164,171, 177, We corrected the arrangement of the figures and reduce their size.

We arranged all the species in alphabetical order.

Line 231,235: We carefully check and corrected the mistake.

Line 271: We added Albizia lebbeck in the table as well.

Conclusion: line 384: We corrected the mistake.

References

We arranged all the references as per the guidelines of the journal.

Author Response File: Author Response.docx