Genetic Diversity and Population Structure of the Spring Orchid Cymbidium goeringii in Korean Distant Islands

Round 1

Reviewer 1 Report

A brief summary

In this paper, Noh et al. used microsatellite markers to examine genetic diversity and the population structure of Cymbidium goeringii, orchid species important especially in horticulture but also in biodiversity preservation. The general topic fits into the concept of island biogeography, and it is not a novelty, but still can be interesting and valuable. I think however, that the authors should firstly work on aims and hypotheses, and re-writing some part of the paper to keep the scientific soundness and to be fully understandable for the readers. The methodology requires additional explanations. Although the scientific interest of the study match the main scope of the journal, the manuscript need much improved to be published.

 

Broad comments

The weakest points of the paper are “Introduction and “Discussion” which may stem from literature deficiency, as the number of papers deal with island biogeography, plants (especially orchids) genetic diversity is enormous, the authors made little use of it. The major part of “Introduction” are species and markers information which rather fits to “Material and methods”, apart from the fact that the general information about various markers (AFLP or RAPD) misses the point here…  One paragraph of the “Introduction” concern the influence of “yellow dust wind” which can be taken as the torn out information based on people beliefs. That needs clarify.

In general I would suggested to shortly introduce what are the main drivers of genetic structure and diversity (so historical processes like re-colonization, current demographic processes – decline in the abundance due to orchid collectors). Then I would go directly to the topic, C. goeringii, what is the problem and why did you study this problem.

There are no stated aims and hypotheses or research question. Authors “examined” and “compered” but these are methods to sort out the problem, verify the hypotheses, which are not there.

Main methodological concern: the genetic diversity based on SSR markers (but not only) is quiet sensitive to the number of individuals genotyped/individuals in populations. There is no information about number of individuals in each sampled population, but the number of samples between populations vary greatly (16-54). In other word, it is very likely to have lower diversity indices calculate for population because you sampled 5 times not 10 times the local gene pool. If you sampled all individuals in populations that’s fine, but if not, maybe you should consider the re-sampling procedure (see for ex. Stachurska-Swakoń et al. 2013 BotJLinnSoc).

Authors applied standard analyses and form of results presentation.       

The manuscript needs an English editing. Some examples are indicated in the specific comments.

 

Specific comments

1. Abstract

Line 18-19 – The population structure …was independent – it should be clarify what those it mean “independent” population structure  

Line 24. … some people believe – that sounds a bit stranger to an academic paper…

1. Introduction

Line 32-37 – characteristic of the genus and species should be mentioned further, and the first part of the Introduction should focus on the main issue that authors should clarify

Line 38-40 - propagate by sexual and vegetative reproduction, I will rather say that “vegetative propagation“ and “sexual reproduction” and “propagate by dust-like seeds”; moreover self-pollination is a type of mating system that greatly affects genetic diversity. If the authors want to mention it, should be more widely explained. Whether species represent the “deceptive” or “rewarding” pollination system is also important in discussing level of genetic diversity 

Line 40 – Male insects… too general, authors should specify this issue, maybe there are some data for other Cymbidium species, for example male bee species

 … pollination mediators… - in literature it state “pollinators”

Line 42 – how these it relate to Cymbidium goeringii?

Line 43-54 – authors should focus on microsatellite information

Line 55-62 – Windsowing orchids seeds could makes the winds the important factor influenced the genetic structure of orchids populations… although there are sparse information about long-distance transport of these seeds, we shouldn’t exclude such possibility. However authors do not rely on any literature data or historical data about winds directions in this area. Presented by authors way of hypothesizing is inappropriate.

Line 63-67 – “examined” and “compared” is a way to respond the research questions which were not be specify.       

 

• Material and Methods and IV. Results - see previous comments

1. Discussion

This issue point to a lack of detail and clarity. I recommended rewriting based on available literature, in conjunction with previously formulated aims and hypotheses.

Line 224-230 – This is a paragraph deals with possible impact of self-pollination on genetic diversity indexes, although there is no published/or not published data about mating and pollination systems of C. goeringii. It should be considered that even in the different populations of one species presented mating and pollination systems may be different. Maybe in the C. goeringii flowers structure something promote selfpollination, as reproductive assurance (RA) but without experiments we cannot be sure what is happening in population.   

Line 234-235 – why the “an open population” exhibit “more genetic stability” – it is not clear, what the authors understands by  “open” and “stability”

Line 231 – 250 – whole paragraph needs to be thoughtful and rewriting, for example:

Authors talk about habitat fragmentation and give the literature about man-made habitats destruction and fragmentation, I do not know if this is the case in studied populations, but they should rather concerned on island populations, separated from main range of the species (how this main range looks like?);

Then, they mentioned about overcollection, which seems to be very important factor influenced the genetic diversity and this conservation aspects should be more strongly emphasized by authors and even placed in the conclusions. The same as the main scopes, concerns the history of colonization, the geographical factors (yellow dust wind, altitudes) that influence/or not (it should be tested) the observed level of genetic variation and genetic distance between populations.

Author Response

A brief summary

In this paper, Noh et al. used microsatellite markers to examine genetic diversity and the population structure of Cymbidium goeringii, orchid species important especially in horticulture but also in biodiversity preservation. The general topic fits into the concept of island biogeography, and it is not a novelty, but still can be interesting and valuable. I think however, that the authors should firstly work on aims and hypotheses, and re-writing some part of the paper to keep the scientific soundness and to be fully understandable for the readers. The methodology requires additional explanations. Although the scientific interest of the study match the main scope of the journal, the manuscript need much improved to be published.

Broad comments

The weakest points of the paper are “Introduction and “Discussion” which may stem from literature deficiency, as the number of papers deal with island biogeography, plants (especially orchids) genetic diversity is enormous, the authors made little use of it.

The major part of “Introduction” are species and markers information which rather fits to “Material and methods”, apart from the fact that the general information about various markers (AFLP or RAPD) misses the point here… 

[Answer] Thank you very much for your comments. We revised this sentence in the opinion of the reviewer: Among the several genetic markers, plant molecular markers called simple sequence repeats (SSR) have been most frequently applied in phylogenetic analysis and classification of Cymbidium species [7-12].

 

One paragraph of the “Introduction” concern the influence of “yellow dust wind” which can be taken as the torn out information based on people beliefs. That needs clarify.

In general I would suggested to shortly introduce what are the main drivers of genetic structure and diversity (so historical processes like re-colonization, current demographic processes – decline in the abundance due to orchid collectors). Then I would go directly to the topic, C. goeringii, what is the problem and why did you study this problem.

There are no stated aims and hypotheses or research question. Authors “examined” and “compered” but these are methods to sort out the problem, verify the hypotheses, which are not there.

Main methodological concern: the genetic diversity based on SSR markers (but not only) is quiet sensitive to the number of individuals genotyped/individuals in populations. There is no information about number of individuals in each sampled population, but the number of samples between populations vary greatly (16-54). In other word, it is very likely to have lower diversity indices calculate for population because you sampled 5 times not 10 times the local gene pool. If you sampled all individuals in populations that’s fine, but if not, maybe you should consider the re-sampling procedure (see for ex. Stachurska-Swakoń et al. 2013 BotJLinnSoc).

Authors applied standard analyses and form of results presentation.       

The manuscript needs an English editing. Some examples are indicated in the specific comments.

[Answer] Thank you very much for your comments. We sampled all individuals in the populations of the sites. We also checked English by an English editing service center.

Specific comments

1. Abstract

Line 18-19 – The population structure …was independent – it should be clarify what those it mean “independent” population structure  

[Answer] Thank you very much for your comments. We revised this sentence: The population structure in the mainland was genetically separated with the populations in the islands.

 

Line 24. … some people believe – that sounds a bit stranger to an academic paper…

[Answer] Thank you very much for your comments. We deleted this sentence: These results implied that the populations of spring orchids distributed in Yeonggwang-gun and Heuksan Island seemed to not be influenced by the yellow dust winds.

 

1. Introduction

Line 32-37 – characteristic of the genus and species should be mentioned further, and the first part of the Introduction should focus on the main issue that authors should clarify

[Answer] Thank you very much for your comments. We revised the corresponding paragraph by strengthening their ecological characteristics.

 

Line 38-40 - propagate by sexual and vegetative reproduction, I will rather say that “vegetative propagation“ and “sexual reproduction” and “propagate by dust-like seeds”; moreover self-pollination is a type of mating system that greatly affects genetic diversity. If the authors want to mention it, should be more widely explained. Whether species represent the “deceptive” or “rewarding” pollination system is also important in discussing level of genetic diversity 

 [Answer] Thank you very much for your comments. We revised the corresponding paragraph as your comment.

 

Line 40 – Male insects… too general, authors should specify this issue, maybe there are some data for other Cymbidium species, for example male bee species

 … pollination mediators… - in literature it state “pollinators”

 [Answer] Thank you very much for your comments. We have revised the "pollinators" and described this in detail: Male insects belonging to the Hymenoptera, Diptera, and Coleoptera have been reported as pollinators mediators in some orchid species [5], but no specific mediators have been reported to date in C. goeringii, which is seemed to have no sexually deceptive and rewarding pollination system.

 

Line 42 – how these it relate to Cymbidium goeringii?

 [Answer] Thank you very much for your comments. We deleted this sentence.

 

Line 43-54 – authors should focus on microsatellite information

 [Answer] Thank you very much for your comments. We have added more details: Microsatellite markers are frequently used as popular genetic markers in forensic biology and population genetics because the cost of development is inexpensive and the functional diversity of the population can be usefully analyzed through the representation of transcribed gene [6].

We revised some sentence: Among the several genetic markers, plant molecular markers called simple sequence repeats (SSR) have been most frequently applied in phylogenetic analysis and classification of Cymbidium species [7-12].

We have detailed the microsatellite information of Cymbidium goeringii.: Microsatellites can provide information about genetic diversity, inbreeding probability, genetic differentiation, and population structure of C. goeringii [10,12,13]. Hyun et al. (2012) developed 21 polymorphic microsatellites from SSR-enriched genomic libraries of C. goeringii and applied them in the analysis of four populations (East Korea, West Korea, China, and Japan) [9]. Lee et al. (2020) determined the sequences of 13 microsatellites for molecular authentication of ten commercially high-priced cultivars of Korean C. goeringii with characteristic flower phenotypes [12].

Line 55-62 – Windsowing orchids seeds could makes the winds the important factor influenced the genetic structure of orchids populations… although there are sparse information about long-distance transport of these seeds, we shouldn’t exclude such possibility. However authors do not rely on any literature data or historical data about winds directions in this area. Presented by authors way of hypothesizing is inappropriate.

[Answer] Thank you very much for your comments. As your comment, following sentence was added: It was proposed that yellow dusts contain pollutants of heavy metals, viruses, bacteria and fungi (Kang and Kim, 2014). In addition, several other points were edited.

Line 63-67 – “examined” and “compared” is a way to respond the research questions which were not be specify.       

[Answer] Thank you very much for your comments. As your comments, the last paragraph of Introduction was considerably revised: Few studies have been performed on the population genetic diversity of C. goeringii in small islands far from the mainland. In the present study, we investigated the genetic diversity and population structure of C. goeringii populations collected from small islands far from the mainland by the analyses of 11 microsatellite loci. In particular, in order to estimate the effect of yellow dust on the C. goeringii populations in Korea, Ulleung-Island and Heuksan-Island, respectively located in the east and west sides of Korea, were selected as the survey sites. We also compared the genetic structure and origin of C. goeringii distributed in Korea, China, and Japan.

Material and Methods and IV. Results - see previous comments

Discussion

This issue point to a lack of detail and clarity. I recommended rewriting based on available literature, in conjunction with previously formulated aims and hypotheses.

Line 224-230 – This is a paragraph deals with possible impact of self-pollination on genetic diversity indexes, although there is no published/or not published data about mating and pollination systems of C. goeringii. It should be considered that even in the different populations of one species presented mating and pollination systems may be different. Maybe in the C. goeringii flowers structure something promote selfpollination, as reproductive assurance (RA) but without experiments we cannot be sure what is happening in population.   

 [Answer] Thank you very much for your comments. As your advice, we alleviated the overall degree of suggestion.

Line 234-235 – why the “an open population” exhibit “more genetic stability” – it is not clear, what the authors understands by “open” and “stability”

 [Answer] Thank you very much for your comments. As your advice, following sentences were deleted fom the text: This suggested that the population of YG is an open population, which is why it exhibits more genetic stability than that do the other two populations. Meanwhile, the two populations of UL and HS are relatively closed populations with limited migratory flow.

Line 231 – 250 – whole paragraph needs to be thoughtful and rewriting, for example:

Authors talk about habitat fragmentation and give the literature about man-made habitats destruction and fragmentation, I do not know if this is the case in studied populations, but they should rather concerned on island populations, separated from main range of the species (how this main range looks like?); Then, they mentioned about overcollection, which seems to be very important factor influenced the genetic diversity and this conservation aspects should be more strongly emphasized by authors and even placed in the conclusions. The same as the main scopes, concerns the history of colonization, the geographical factors (yellow dust wind, altitudes) that influence/or not (it should be tested) the observed level of genetic variation and genetic distance between populations.

 [Answer] Thank you very much for your comments. As your advice, the last paragraph of the “Discussion” was completely rewritten as followed: As the first population genetic study of spring orchids distributed in the small distant islands, our study suggests that the genetic diversities of small isolated populations are just slightly lower than that of the inland population and are genetically separated from inland population. In addition, this study concluded that the contribution of yellow dust to the spring orchid distribution in the west side of Korea was absent or very weak. This study will be useful for understanding the genetic diversity and population structure of isolated populations as well as for the conservation of C. goeringii.

Reviewer 2 Report

In their study, Woo Noh et al. analyzed the genetic diversity and population structure of Cympidium goeringii in Korean Distant Islands. The study is well structured and well written. My opinion is that only minor additions need to be made to this initial version of the manuscript. Specifically, I would like to see some general information that should be stated in the introduction section.

 

 

Introduction

A general paragraph about the orchid family and why this family is significant from a conservation point of view would be a good addition.

L.51: C. goeringii in italics

I would also like to see some information about the general distribution of C. goeringii.

Author Response

In their study, Woo Noh et al. analyzed the genetic diversity and population structure of Cympidium goeringii in Korean Distant Islands. The study is well structured and well written. My opinion is that only minor additions need to be made to this initial version of the manuscript. Specifically, I would like to see some general information that should be stated in the introduction section.

Introduction

A general paragraph about the orchid family and why this family is significant from a conservation point of view would be a good addition.

L.51: C. goeringii in italics

I would also like to see some information about the general distribution of C. goeringii.

[Answer] Thank you very much for your comments. We corrected the manuscript by your advices. Particularly, more information for C.goeringii distribution was provided in the first paragraph of Introduction.

Round 2

Reviewer 1 Report

The general comment about revised version of the manuscript  

The authors Noh et al. submitted the revised manuscript that concern genetic diversity and the population structure of mainland and island populations of Cymbidium goeringii from China and Korea.

In the first-round of the review authors were encourage to improve some parts of the manuscript, particularly aims, introduction and discussion, which authors agreed. In my opinion authors improved indicated parts, however they still needs some amendments.  

There is still some misunderstanding parts, as examples:

-in introduction: The authors written about pollination and mating system in C. goeringii, but I have impression that they not exactly understand this issue, thus the conclusions are incorrect.

  -in introduction: The impact of the yellow dust wind on orchid genetic structure is one of the main aim of this paper, in revised paper this thread was expanded, but still some important information are missing, for example level and time of C. goeringii  fruiting – quiet important in terms of seed dispersal and observed genetic structure.

- in responses to the one of the comment, authors specify that all individuals from study populations of C. goeringii were collected, but this important information is not in the text, moreover all the investigated populations are extremely small (information in Tab. 1 sample size), which certainly affects inbreeding level, it should be emphasized.

-discussion: authors conclude about self-pollination based on mixotrophism, that based on literature, in may opinion unfounded conclusion, especially that pollination system in different populations of the same species may differ.    

 

Author Response

Response to the Comments by reviewers

The general comment about revised version of the manuscript

The authors Noh et al. submitted the revised manuscript that concern genetic diversity and the population structure of mainland and island populations of Cymbidium goeringii from China and Korea.

In the first-round of the review authors were encourage to improve some parts of the manuscript, particularly aims, introduction and discussion, which authors agreed. In my opinion authors improved indicated parts, however they still needs some amendments.

There is still some misunderstanding parts, as examples:

-in introduction: The authors written about pollination and mating system in C. goeringii, but I have impression that they not exactly understand this issue, thus the conclusions are incorrect.

[Answer] Thank you very much for your comments. We deleted following sentence because it didn't seem to fit the purpose of this study: Male insects belonging to the Hymenoptera, Diptera, and Coleoptera have been reported as pollinators mediators in some orchid species [5], but no specific mediators have been reported to date in C. goeringii, which is seemed to have no sexually deceptive and rewarding pollination system.

Then, we integrated remain sentences into a paragraph.

-in introduction: The impact of the yellow dust wind on orchid genetic structure is one of the main aim of this paper, in revised paper this thread was expanded, but still some important information are missing, for example level and time of C. goeringii fruiting – quiet important in terms of seed dispersal and observed genetic structure.

[Answer] Thank you very much for your comments. As the comment, following sentence was added into the corresponding section: Very tiny C. goeringii seeds with about 1.2-1.8 mm long could easily be seen blown out from the bursted pods by wind in spring.

- in responses to the one of the comment, authors specify that all individuals from study populations of C. goeringii were collected, but this important information is not in the text, moreover all the investigated populations are extremely small (information in Tab. 1 sample size), which certainly affects inbreeding level, it should be emphasized.

[Answer] Thank you very much for your comments. We added following sentences with a reference (Pruett and Winker, 2008): In general, sample size can affect the levels of alleles such as Ho, He, and allele per locus [24], suggesting that inbreeding coefficient and genetic diversity can be affected, but this pattern was not seen in in our results (Table 1). In our study, the the sample sizes for all populations were mostly a sample size [24] known to be stable (20 or more) than the generally described small sample sizes (5-10).

-discussion: authors conclude about self-pollination based on mixotrophism, that based on literature, in may opinion unfounded conclusion, especially that pollination system in different populations of the same species may differ.

[Answer] Thank you very much for your comments. We thought that this was an unfounded conclusion, so we deleted the introduction and discussion sections.