Cytogeography of the East Asian Tulips (Amana, Liliaceae)

Round 1

Reviewer 1 Report

The authors of the paper recognized that the genus Amana (Liliaceae), with its limited distribution in the part of research on chromosomes and their cytogeography, remained neglected in relation to related genera. Including all known species and a significant number of locations in the complete genus area, they collected individuals for the given analyzes. However, the setting of the paper, the set hypothesis, as well as the written sector of Materials and Methods are not logically complex. In addition, the research results of this stakeholder group are not clearly presented in the paper. Taking into account all the shortcomings of the work, but also the presented data, the manuscript requires additional editing, so that the scientific contribution of this group of researchers would be properly visible.

The presented Article does have serious flaws, but the presented data should be rewritten in logical order to overcome issue.

 

Comments for author File: Comments.pdf

Author Response

Responses to reviewers

Reviewer 1

• A brief summary

The authors of the paper recognized that the genus Amana (Liliaceae), with its limited distribution in the part of research on chromosomes and their cytogeography, remained neglected in relation to related genera. Including all known species and a significant number of locations in the complete genus area, they collected individuals for the given analyzes. However, the setting of the paper, the set hypothesis, as well as the written sector of Materials and Methods are not logically complex. In addition, the research results of this stakeholder group are not clearly presented in the paper. Taking into account all the shortcomings of the work, but also the presented data, the manuscript requires additional editing, so that the scientific contribution of this group of researchers would be properly visible.

• General concept comments

Article: In relation to the text of the manuscript, the title is clear and unambiguous. It clearly describes the intention of the author.

Review:

Chromosome analysis of seven species of the genus Amana is a significant for understanding of this peculiar genus; however, the attached manuscript shows major inconsistencies and shortcomings in scientific data processing; First of all, the rules of nomenclature are not respected and the paper introduces names for species that have not been validly published, but are analyzed in that regard. This approach is not acceptable for scientific publication, unless a new species are validly published in the paper. It is unaccepted to consolidate one sample (one individual) as one population; as well, for most of the locations (presented as population) it is evident that they are on close range; It is necessary to correct this scientific approach. Another omission, which requires a major, is the introduction of a significant part of unpublished data (of which the author of the manuscript is also the author of this unpublished data), and referring to them firstly in the introduction, and then their analysis and discussion in the Results. This is unacceptable for publication in this format, and it is necessary to either reformulate this segment of the paper in accordance with the scientific rules of publishing the results of the paper, and the nomenclature, or find another acceptable solution. The third objection refers to the writing of the paper: 1) the Introduction states the data that are part of the Results sector; 2) Mterial and methods are incompletely written, 3) the Results include data that are not explained in detail in Material and methods.

The paper deserves to be reorganized and rewritten because all the elements are in the work, but they are not arranged in a logical order.

R: Thank you for your insightful comments!

  For the nomenclature, it is hopeful that two new names (A. nanyueensis and A. tianmuensis) will be validly published in a month. The paper has been sending out for polishing by the journal Plant Diveristy, and the next step is acceptance and online publication. So that we think an acceptable solution is to wait for the publication of that paper. For Amana sp.1 and Amana sp.2, we can simply use these two names.

  For population and location, we do understand the difference between them and we are sorry for the misleading information. Table 1 listed all of our collections, some collections are from the same site/population. So that we reviewed the collection records carefully, all the different collections less than one kilometer apart were considered as the same population. In Tabel 1, we use A, B, C to distinguish different collections from the same population. In this way, the number of populations reduced to 95. Besides, we would like to clarify that we collected multiple individuals for each collection, not a single individual.

  The Introduction does not state the data that are part of the Results section. Two ploidy levels within A. edulis is a finding of previous studies. For Material and methods section, we have rephrased it and added more explanations.

 

• Specific comments

- Unnecessarily long description of the strategy of early spring plants in the introduction, but this is not a significant shortcoming;

R: Thank you for your comments! We described the strategy of early spring plants extensively in the introduction in order to draw out the difference between Amana and other typical spring ephemerals.

 

- When the name of the species are firstly stated, it is recommended to declare the full scientific name of the species

R: Thank you for your correction! We have revised all the scientific names.

 

- Unpublished species names should not be used in the paper, as it is done in the introduction, and later in the results

R: We have found that there may be four more cryptic species in the genus with extensive fieldwork since 2014. A series of observations and experiments have been carried out on Amana sp.3 (named as A. nanyueensis) and Amana sp.4 (named as A. tianmuensis) and the results will be published in a short time. Therefore, we retained the new names of these two species in this paper. At the same time, research on Amana sp.1 and Amana sp.2 is pushing forward but more evidence is needed before publishing them as new species, so that we used Amana sp.1 and Amana sp.2 (but not the new names) in this paper.

  If you think this treatment is unacceptable, we can get rid of Amana sp.1 and Amana sp.2 in the present paper. Is this an acceptable solution?

 

- Line 54-63 - for the Introduction section, too much unpublished data is cited and the later hypothesis is based on them; these data are actually the results of the work, as shown later in the Results

R: Further phylogenomic analyses revealed three clades within Amana and so that we created different maps according to different clades. In this way, the geographical distribution of closely related species can be seen more clearly.

We have deleted the unpublished data from the Introduction section. Instead, we rephrase the phylogenomic results and explain why we created different maps according to different clades in the Materials and methods section.

 

- Terminology must be improved (eg two kinds of ploidy levels)

R: We have replaced it with “two ploidy levels”.

 

- Not clear meaning of “Vertical dropper”? Botanically incorrect term

R: The materials used in our study are the vertical droppers of Amana (please see the figure below, which is also added in the appendix of manuscript). This term has also come up in previous studies (Auguste, 1972 [1]; Hagiya and Amaki, 1957 [2]). Thus, we retained this term.

[1] Auguste, H. Bulb habit and reproduction in different ploidy forms of Tulipa oculus-solis in Israel. Israel Journal of Botany 1972, 21, 185-196. https://www.researchgate.net/publication/259404440

[2] Hagiya, K.; Amaki, W. Studies on the dropper of tulip bulbs. (I) Classification of tulip droppers and the varietal difference of their formation. 1957, 26, 205-208.

https://doi.org/10.2503/jjshs.26.205

A whole plant of Amana edulis. The red arrow points to the vertical dropper.

 

- The term "population" has been applied in an unacceptable way from a scientific point of view; the authors did not collect entire populations, as given in Table 1 and Materials’, but one individual from each recognized population. However, by reviewing the coordinates, not all locations are at a significant distance to be considered as different populations; Taking into account the derived conclusions, it is necessary to precisely determine the methods and scientific approach in the work, and terminology related to population;

R: Thank you for the insightful comments! Table 1 listed all of our collections, but not each collection represent a different population, because some collections are from the same site/population. So that we reviewed the collection records carefully, all the different collections less than one kilometer apart were considered as the same population. We use A, B, C in Tabel 1 to distinguish different collections from the same population. In this way, the number of populations reduced to 95. Besides, we would like to clarify that we collected multiple individuals for each collection, not a single individual.

 

- line 93 – definition given in Manuscript is not Carnoy solution

R: Thank you for pointing that out! We have revised it.

 

- line 99-100: describe in detail the methods used to create maps (what data, etc.)

R: The latitude, longitude and chromosome number of each collection are used to make the geographical distribution maps of cytotypes through ArcMap 10.2 (Minami et al., 2000 [19]). The Elevation data (30 seconds) is download from WorldClim v2.1 (https://worldclim.org/). We have added this in the manuscript.

 

- Voucher numbers are given in Table 1 but the necessary details are not given in the Material and Methods

R: Voucher specimens were deposited at the Herbarium of Zhejiang University (HZU). We have added this in the manuscript. If any other necessary details are missing, please let us know.

 

- 104-107 - repeated from the introduction (should be here)

R: The introduction briefly describes previous research by other scholars, who only included very few populations of Amana edulis. And our results show a whole picture of the genus that there are two ploidy levels in A. edulis, but other ten species are all diploid except for one A. tianmuensis population from Qingliangfeng Botanical Garden, which has both diploid and triploid. Our results agree with previous studies, but are more detailed and comprehensive.

 

- line 130 is not blank color but black color

R: Thank you for pointing that out! We have corrected it.

 

- line 132 - it is not acceptable to publish unpublished data without explanation in the Material and methods. Rephrase and then publish this data properly.

R: Thank you for your comments! We have rephrased the Material and methods section and added more explanations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Work extremely well conceived. Great sample. Good discussion.

Acceptance after English language revision.

Author Response

Dear reviewer, thank you for reviewing our manuscript and your encouraging comments!

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper is well written, original and interesting, despite relying only on traditional chromosome counts. The interest is however justified by having studied all the species (and many different populations) in the genus Amana. I annotated the manuscript directly, suggesting several corrections, modifications and improvements.

A crucial point is to avoid the use of diploids, tetraploids etc. until the Discussion, since the authors neither reconstructed karyotypes not estimated the genome size.

Also, please pay special attention in integrating the References cited, since a lot of fully pertinent and recent work was omitted.

Comments for author File: Comments.pdf

Author Response

Responses to reviewers

Reviewer 3

• Please also add information about the tribe Tulipeae, to which amana belongs together with Gagea and Tulipa.

https://www.tandfonline.com/doi/abs/10.1080/11263504.2015.1115435.

R: Thank you for your helpful suggestion! We have added a short paragraph about Liliaceae and tribe Tulipeae.

 

(2) Line 76- actually, two of them are seemingly being described so that they shoud be considered as "regular" species.

R: Thank you for your helpful suggestion! We have done it follow your suggestion. A series of observations and experiments have been carried out on Amana sp.3 (named as A. nanyueensis) and Amana sp.4 (named as A. tianmuensis) and the results will be published in a short time. Therefore, we retained the new latin names of these two species in this paper. At the same time, research on Amana sp.1 and Amana sp.2 is pushing forward but more evidence is needed before publishing them as new species, so that we used Amana sp.1 and Amana sp.2 in this paper.

 

(3) You just counted the chromosomes. Ploidy level is just an inference, which can be done (as Discussion) based on available literature. Accordingly, please omit any indication about ploidy levels in results and infer them (by citing appropriate literature) only in Discussion.

R: In introduction, some previous studies have shown that the basic chromosome number of Amana, Tulipa and Erythronium is x = 12 (Peruzzi, 2009 [1]) and there are two ploidy levels in A. edulis, diploid (2n = 2x = 24 in the northernmost populations, such as Liaoning and Henan provinces, China) and tetraploid (2n = 4x = 48 in the southern-most population, such as Zhejiang Province, China) (Deng et al., 2016 [2]). Based on these literatures, we think it is such a simple inference and probably self-evident. So that we couldn’t resist the temptation to use it right away in the results section.

[1] Peruzzi, L.; Leitch, I. J.; Caparelli, K.F. Chromosome diversity and evolution in Liliaceae. Annals of Botany 2009, 103, 459–475.

doi:10.1093/aob/mcn230, available online at www.aob.oxfordjournals.org

[2] Deng, A.H.; Li, K.; Chen, Y.; Liu, C.Y.; Guo, Q.S.; Zhu, Z.B.; Miao, Y.Y. Karyotype analysis of different populations of Tulipa edulis. Chinese Herbal Medicine 2016, 39, 493–498. doi:10.13863/j.issn1001-4454.2016.03.008

 

(4) you just counted the chromosomes. Ploidy level is just an inference, which ca be done (as Discussion) based on available literature. Accordingly, please omit any indication about ploidy levels in results and infer them (by citing appropriate literature) only in Discussion

R: Thank you for your insightful comments! We have added the research progress and results in our Discussion.

 

(5) I suggest to reverse the order of information provided: first country, then province, then county, then exact locality

R: Thank you for your helpful suggestion! We have modified Table 1 following your suggestion.

 

(6) Move to Discussion. No references in Results!

R: You are right, we have modified it following your suggestion.

 

(7) First of all, you should infer, in the light of available literature, that your cytotypes could be reliably identified as diploid, triploid, and tetraploid levels, respectively.

R: Please see answer to comment 3. For the same reason, we did not infer the ploidy levels here.

 

(8) Line 169- More in general, this is throughly discussed also in the recent work by Carta et al. (2020). Incidentally, in the supplemental material of the same paper, also the ancestral chromosome number for Liliaceae is inferred, i.e. n = 7.

https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.16668

Line 171-173 There is a study focussed on Liliaceae!! https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.13769

Line 185-186 See also: https://compcytogen.pensoft.net/article/1774/

R: Thank you for your insightful comments! We have added the research progress and results in our Discussion.

 

(9) That's a pity that some karyotype reconstruction / karyotype asymmetry estimation was not possible, I guess due to the average quality of metaphasic plates...

R: Thank you for your comments! Actually, we did karyotype reconstruction / karyotype asymmetry estimation for some populations. However, the quality of metaphasic plates is not good enough. Besides, the morphology of chromosome varies greatly between different populations of the same species, we couldn’t find a meaningful pattern, so that we decided not to show those results.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I found that the resubmitted Mansucript is improved and all answers that were apointed in the proces of revision are accepted or explained in detail

 

Only data, that are not harmonized through is the number of the samples (see the comments in the attached file)

Comments for author File: Comments.pdf

Author Response

Responses to reviewers

Reviewer 1

I found that the resubmitted Mansucript is improved and all answers that were apointed in the proces of revision are accepted or explained in detail

 

Only data, that are not harmonized through is the number of the samples (see the comments in the attached file)

 

• Line 45 “differ from other typical spring ephemerals”

compare to which species/genus or plants groups is this statement relevant?

Or, it is known that ephemeral spring species are not found commonly in subtropical regions.

R: Thank you for your helpful comments!

  We rephrased it as “However, the genus Amana spreads to subtropical regions (such as Zhejiang, southern Anhui and northern Jiangxi, China), differ from classic spring ephemerals such as tulips, trout lilies, crocuses, daffodils, and hyacinths.”

 

• Line 61, 62 “Wang et al., in press”

This reference

R: This paper has just been accepted by the journal “Plant Diversity” on March 1st, and it will be published online soon, hopefully in March. So we rephrased it as “Wang et al., 2022”.

 

• Line 76 “95”

In the Abstract, 90 populations were stated

R: Thank you for your insightful comments!

  95 is the total number, among which 5 are from previous studies. By the way, we delete one population because it might represent another new species, during our recent field work, we found several more populations of it. Thus we rephrase it as “In this study, we included 94 populations (89 from present study and five from previous studies) representing all 11 species ……”

 We also drew this out in the caption of Fig. 2, as “The five populations centered with a white dot are based on previous studies (Deng, 2016 [19]; Sato, 1943 [59]; Noguchi and Kowano, 1974 [60]).”

 

• Line 89 “A total of 90 populations”

I will repeat at this place, that researchers didn't collect populations, rather a samples (consisting of 1 individual) from 90 populations;

Written in present state - some can conclude that large number of individuals were collected

R: Dear reviewer, we do collected multiple individuals for each collection, normally around 10, not just a single individual. Thus we think our statement is appropriate.

Author Response File: Author Response.docx