Vanilla pompona Schiede (Vanilloideae-Orchidaceae): Morphological Variation of the Labellum in the Mexican Localities of Veracruz, Puebla, Jalisco and Oaxaca

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions and corrections highlighted.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Please see the answer in the point-by-point responses in the section below.

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Please see the answer in the point-by-point responses in the section below

Are the results clearly presented?

Can be improved

Please see the answer in the point-by-point responses in the section below

Are the conclusions supported by the results?

Can be improved

Please see the answer in the point-by-point responses in the section below

 3. Point-by-point response to Comments and Suggestions for Authors

 Comments 1: Introduction: it seems to me that the writing of the manuscript is heterogeneous. Whereas the methods, Results, and Discussion are interesting and readable, the Introduction is confusing. In lines 31-35, some redundant information can be seen (of course V. pompona belongs to V. pompona!). Please, rewrite the Introduction to avoid unnecessary and repetitive information.

Response 1: Thank you for pointing this out, we apologize for the confusion in the wording. We consider deleting this paragraph: [In the revised manuscript this change can be found - page number: 1, paragraph 1 of the introduction, and lines 33-35]

V. pompona pertains to the family Orchidaceae, subfamily Vanilloideae, tribe Vanilleae, genus Vanilla, species V. Pompona [5,6].

However, we believe it is important to mention that there are three subspecies of V. pompona: [In the revised manuscript this change can be found - page number: 1, paragraph 1 of the introduction, and lines 35-37]

 Three subspecies have been reported for V. pompona: subsp. pompona native to México, subsp. grandiflora reported in Peru and Brazil and subs. pittieri reported in Costa Rica [4-7].  

Comments 2: Again, in the Methods, some contradictory info is given. It is said that there is not much information about the species, but it is also said that genetic erosion has taken place (lines 75-76). To detect genetic erosion it is necessary to have a really good idea of the population genetics and structure. Please, check these affirmations.

Response 2: We agree with this comment, because it has not yet been reported in any research that V. pompona populations present genetic erosion. Therefore, we consider that it would be appropriate to delete the following paragraph: [In the revised manuscript this change can be found - page number: 2, paragraph 4 of the introduction, and lines 74-77].

It is not known whether clonal reproduction, disappearance of the habitat where the species is distributed, such as deciduous tropical forests, gallery forests, and tropical pine and oak forests [9], and problems of genetic erosion [29] have caused low genetic variation in V. pompona. 

 Comments 3: In lines 395-402, it is suggested that the observed variation could be partly caused by pollinators. It is cited an example dealing with a very different and distantly related species (V. palmarum) that is pollinated by hummingbirds. As the authors mention, all evidence points to bee pollination in V. pompona, especially by large Euglossine bees. The fact is that there is -so far- no evidence that different populations of V. pompona are pollinated by different bees and justifying the observed variation in pollinators seems unsubstantiated and weak. In fact, the authors themselves bring no evidence regarding this. Please, do not contribute to the literature that uses pollinators as a "catch-all" trick. The study of orchid pollination is a serious discipline, not a place for unsubstantiated speculation.

Response 3: Thank you for your comment, we also consider that the example about Vanilla palmarum was not appropriate. Therefore, we decided to delete this information:  [In the revised manuscript this change can be found - page number: 14, paragraph 3 of the discussion, and lines 398-401].

An example of pressure from pollinators is reported for Vanilla palmarum Salzm. ex Lindl., whose flowers have no aroma, and the labellum underwent a modification to guide the beak of the hummingbird Amazilia fimbriata Gmelin to the nectar chamber [49].  

In addition, for a better understanding, we restructured the paragraph and described the bee species that have been reported to pollinate V. pompona populations in countries such as Brazil, Costa Rica and Peru. [In the revised manuscript this change can be found - page number: 14, paragraph 3 of the discussion, and lines 402-405].

Variation into the six labellum morphotypes of V. pompona identified in this study may also have been in part caused by pressure from pollinators or by genetic drift, which has been reported for other orchids [48]. V. pompona has a reward strategy for attracting pollinators offering both nectar and fragrance, for example, in some populations in Brazil the pollinators of this species are Eulaema bombiformis Packard, E. cingulate Fabricius and E. meriana Olivier [50]. While for Costa Rica and Peru, it has been reported that males of the species E. cingulata Fabricius are the specific pollinators of this plant [51]. 

Comment 4. : In the summary of the evaluation format, the reviewer pointed out that the conclusions could be improved. 

Response 4. For a better understanding based on the results, we restructured the conclusions. [In the revised manuscript this change can be found - page number: 15, paragraph 1 of conclusions, and lines 426-436].

Six morphotypes of the labellum of V. pompona were obtained from localities in the states of Veracruz, Puebla, Oaxaca and Jalisco, Mexico. Each morphotype was distinguished mainly by size and shape, which shows that there is infraspecific variation in the germplasm of V. pompona. The variation that exists in the labels of this species was possibly associated with pollinator pressure and the presence of geographic barriers, which play a very important role in the types of environments in which specimens of V. pompona develop. This plant is wild; therefore, the environment plays a very important role in the variation related to phenological factors, since, for the locality of Cazuelas, Papantla Veracruz, plants with different flowering dates during the year were found. Knowledge of the infraspecific variation of the species is important for the conservation and preservation of the species in Mexico.

Response to Comments on the Quality of English Language

Point 1: The reviewer only mentions that a moderate revision of the English language is required.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted in red in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

 Please see the answer and revisions in the point-by-point responses in the section below.

Are all the cited references relevant to the research?

Must be improved

Please see the answer and revisions in the point-by-point responses in the section below

Is the research design appropriate?

Must be improved

Please see the answer in the point-by-point responses in the section below

Are the methods adequately described?

Must be improved

Please see the answer in the point-by-point responses in the section below

Are the results clearly presented?

Can be improved

Please see the answer and revisions in the point-by-point responses in the section below

Are the conclusions supported by the results?

Can be improved

Please see the answer and revisions in the point-by-point responses in the section below

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The reviewer pointed out in the manuscript to include in the title (Vanilloideae-Orchidaceae).

Response 1: We agree with this comment. We restructured the title to read as follows:

 Vanilla pompona Schiede (Vanilloideae-Orchidaceae): Morphological variation of the labellum in localities of Veracruz, Puebla, Jalisco and Oaxaca, Mexico. [In the revised manuscript this change can be found – page number: 1, in the title, and line: 2]

Comments 2: The ms. of Dr. Viveros-Antonio and colleagues provide a population-level morphometric analysis of the widespread Vanilla pompona. It is an important approach to understanding morphological variation within species, but there are several points to be readdressed prior to acceptance of this manuscript.

The abstract needs some improvement

Response 2: We appreciate the reviewer's comments in the abstract. We made the corrections indicated in the manuscript. We also add the word "flower" in the key words, as suggested by the reviewer.  Changes in the abstract were marked in red. [In the revised manuscript this change can be found – page number: 1, in the abstract, and lines: 16,18, 19, 20,21,22, 27, 30]

Abstract: Vanilla pompona is the third most important commercial species of the genus Vanilla, but the its morphometric variation of its flowers is little known. In orchids, infraspecific variation is mostly expressed in the labellum. This study had the objective of analyzing the floral variation of V. pompona collections from localities in Veracruz, Puebla, Jalisco and Oaxaca, in Mexico. During the flowering period, we obtained 55 collections of V. pompona flowers, and the labellum of each flower was dissected. We used ImageJ to measure and portray 54 lines and 7 angles in these labella.  and in the dissected labellum of each flower 54 lines and 7 angles were measured and portrayed with the ImageJ program. With the data obtained, we performed an analysis of variance, a principal components analysis, and a cluster analysis. The results showed significant differences among the collections and localities in the means of the lines of the basal, middle, and apical regions of the labellum. Also, six morphotypes were obtained, and we found that variation was associated with factors such as the environment, geographic barriers, and pollinator pressure. Moreover, we observed variation in flowering periods dates in one locality of Veracruz. We concluded that there is infraspecific variation within and between the collection localities of V pompona of the Atlantic region (Puebla and Veracruz) and the Pacific region (Jalisco and Oaxaca), Mexico.

Keywords: orchids; morphometry; flower vanilla; infraspecific variation.

Comments 3: Please, include information on the distribution of V. pompona in the introduction.

Response 3: Thank you for your comment. We added information about the distribution of V. pompona. The following paragraph was added and citations and references were also updated in the manuscript. [In the revised manuscript this change can be found – page number: 1, paragraph: 1, and lines: 36-40]

 The presence of V. pompona has been reported in the Guadeloupe Islands, Martinica and Dominica [1], in some regions of Guyana and Suriname [7], Costa Rica [8], Honduras and Panamá [4], Perú [9] in the state of Maranhão, Brazil [10]. In Mexico, this plant is distributed in Oaxaca, Nayarit, Puebla, Michoacán, Guerrero, Veracruz and Jalisco [11].

Comments 4: Not necessary. It should be in the title

Response 4: We agree with this comment, so the taxonomic description of V. pompona was removed. [In the revised manuscript this change can be found – page number: 1, paragraph: 1, and lines: 40-42]

 V. pompona pertains to the family Orchidaceae, subfamily Vanilloideae, tribe Vanilleae, genus Vanilla, species V. Pompona [5,6].

We include "Vanilloideae-Orchidaceae" in the title as was suggested by the reviewer. Please see answer in comment 1.

However, we consider that it is important to mention the subspecies that have been described for V. pompona, since they have a different distribution in different countries. Therefore, we have written the following paragraph. [This change can be found - page number: 1, paragraph 1 of the introduction, and lines 35-36].

Three subspecies have been reported for V. pompona: subsp. pompona native to Mexico, subsp. grandiflora reported in Peru and Brazil and subsp. pittieri reported in Costa Rica [4-7].

 Comments 5: Although it is in IUCN website I cant understand how they accept it. Vanilla pompona is a widespred species and would never fit in one of the IUCN critearia.

Response 5: We appreciate the comment. We take into account the IUCN's description of this specie. The following paragraph shows the IUCN's justification for the reasons they considered for including V. pompona on the list of endangered species.

The IUCN reports for V. pompona that there are only a few isolated wild subpopulations, therefore, the population is considered to be highly fragmented. The habitat quality and quantity are being continuously reduced by land use change, especially for agriculture and cattle grazing. These impacts have led to a continuing decline in the number of mature individuals and a population decline that warrants its listing as Endangered (Herrera-Cabrera et al., 2020).

Reference

Herrera-Cabrera, B.E.; Hernández, M.; Vega, M.; Wegier, A. Vanilla pompona (amended version of 2017 assessment). The IUCN Red List of Threatened Species 2020: 2020. e.T105878897A173977322. https://dx.doi.org/10.2305/IUCN.UK.2020-2.RLTS.T105878897A173977322.en. Accessed January 10, 2023.

Comments 6: superfluous

Response 6: We agree with this comment and suggestion to eliminate this paragraph on labellum characteristics, because it is not relevant information for the manuscript. [In the revised manuscript this change can be found - page number: 2, paragraph 3 of the introduction, and lines 58, 60, 61, 62].

 The V. pompona flower is made up of sepals, petals, labellum, penicillate callus, column, stigma, and one ovary [4]. In orchids, the labellum is a modified petal [13], which owes its existence and aspect to the presence of specific genes involved in the evolutionary floral development of this plant family [14,15].

Comments 7: Include it after the next sentence

Response 7: We agree with the suggestion to move the sentence on the function of the labellum. We made the change in the manuscript and it is described as follows:  [In the revised manuscript this change can be found - page number: 2, paragraph 3 of the introduction, and lines 65, 66].

The labellum, as a reproductive structure of orchids, is less susceptible to the environment [15], and for this reason, it is used to identify species within this family [4,17] and to analyze infraspecific variation [18,19]. It functions as a platform for insects to land on and to attract pollinators [20].

Comments 8: It is too short for a paragraph

Response 8:  We also agree that the paragraph on genetic variation is very short. So, we considered joining this paragraph with the next one, because the information is related. [In the revised manuscript this change can be found - page number: 2, paragraph 4 of the introduction, and lines 76- 79].

Comments 9: ??? morphological variation in orchid flowers?

Response 9:  We agree with the comment. We modify the sentence and the change is marked in red. [In the revised manuscript this change can be found - page number: 3, paragraph 1 of the introduction, and line 99].

In the study of morphological variation of orchid flowers, it is important to use an integrated approach, with classic taxonomy [21].

Comments 10: Please include other recent and relevant references on morphometric studies using Orchidaceae not only local studies.

Response 10. Thanks for the comment. We add information from recent references on morphometric studies in orchids. For a better understanding, the paragraph was rewritten and new references were added. [In the revised manuscript this change can be found - page number: 3, paragraph 1 of the introduction, and lines 101-106].

In the study of morphological variation in orchid flowers, it is important to use an integrated approach, with classical taxonomy [21], also with morphometric analysis of the labellum as has been used in different orchid species, for example: to differentiate between species of the genus Scaphyglottis [37], also to differentiate between these three species; Dryadella edwallii Luer, D. wuerstlei Luer, and D. zebrina Luer [38]. In the evaluation of morphological variation of Pseudolaelia vellozicola Hoehne populations [39] and populations of Pseudorchis straminea Fernald and P. albida Á.Löve and D.Löve [40]. In addition to studies on morphological variation in species of the Vanilla genus [22-25].

Comments 11: again? wasn't the second objective supposed to be related to biotic/abiotic factors that might influence the variation?

Response 11 Thanks for the comment. Certainly the wording is confusing, so part of the paragraph was modified to make the objectives of the study clearer and more precise. [In the revised manuscript this change can be found - page number: 3, paragraph 1 of the introduction, and lines 108-115].

In orchids, biotic and abiotic factors, such as temperature and light, have been reported to influence the flowering period [41,42]. This study it will contribute knowledge on the labellum variation related to V. pompona flowering dates. In V. pompona the morphological variation of flowers on the Mexican Atlantic slope and the Pacific Coast is unknown. For this reason, the study aimed to determine morphological variation of the V. pompona labellum in collections from localities of Veracruz, Puebla, Oaxaca, and Jalisco. We believe that this knowledge is useful for conservation, management, and use of this species.

Comments 12:  ??

Response 12. Thank you for the comment regarding the translation of the first subtitle of Materials and Methods. The translation correction has already been done. [In the revised manuscript this change can be found - page number: 5, paragraph 1 of the materials and methods, and line 149].

Plant material

 Comments 13:  why not CVA? it is the most important analysis. Please check recent publications on morphometrics.

Response 13.  Thank you for your comment. We reviewed recent publications on morphometric analysis, as suggested by the reviewer. We would like to explain the reasons why we used principal component analysis (PCA) in our study:

In orchid flower morphometry studies, the most commonly used multivariate analyses are Canonical Variables Analysis (CVA) and Principal Component Analysis (PCA) (De Luna et al., 2020). CVAs are used to explore the separation of a priori defined groups (Lattin et al., 2003; Manly, 1986). A priori groups should consist of 12 to more than 30 repetitions (De Luna and Gómez-Velasco, 2008; Ramírez-Sánchez et al., 2016). Recent studies have been reported on the morphometric analysis of orchids where CVA is used to differentiate between species of the genus Epidendrum (Pessoa et al., 2021), delimitation of the species Trichocentrum cepula, T. caatingaense, T. sprucei (Camelo-Júnior et al., 2022) and to distinguish species of the genus Scaphyglottis (Araújo et al., 2023).

Principal component analysis (PCA) is used to reduce dimensions and explore the total structure of the database according to Wickens (1995) and Lattin et al. (2003). The principal component analysis is performed based on the correlation matrix between the selected variables (Sneath and Sokal, 1973).

In the study of the variation of the labellum of V. pompona, no a priori defined groups were formed, because the necessary characters to form them do not exist, which was one of the reasons why the CVA was not used. In our study we analyzed the infraspecific variation of the labellum of V. pompona, not the variation between species, so it was considered appropriate to use PCA. To verify the PCA, a cluster analysis was performed.

Also in our specimens we have three to five replicates per collection, and to use the CVA the groups must be formed from 12 to more than 30 replicates (De Luna and Gómez-Velasco, 2008; Ramírez-Sánchez et al., 2016). It was not possible to have more than 5 replicates per collection due to the distance between the collection localities and because flowers are ephemeral (only last one day) (Soto-Arenas, 2009. Therefore, we decided to apply the PCA.

Principal Component Analysis was adequate to identify which of the 61 characters evaluated (54 traits and 7 angles) were important in defining groups. PCA has also been used in other orchids, for example, in the floral and vegetative morphometry of five species of Pleurothallis (Orchidaceae) (Borba et al., 2002). As well as in the evaluation of floral morphological variation within the species Laelia anceps subsp. dawsonii f. chilapensis (Salazar-Rojas et al., 2010) and in the taxonomic update of Dryadella (Orchidaceae: Pleurothallidinae) based on morphometric analyses of three species from the Brazilian Atlantic rainforest (Imig et al., 2022).

 References

Araújo, A.M.D; Pessoa, E; Giacomin, L. Is the labellum informative to distinguish species of Scaphyglottis (Orchidaceae)? Insights from geometric morphometrics. Acta Bot. Brasil. 2023, 37, 1-12. doi: 10.1590/1677-941X-ABB-2023-0017

Borba, E.L; Shepherd, G.J; BERG, C.V.D; Semir, J. Floral and vegetative morphometrics of five Pleurothallis (Orchidaceae) species: correlation with taxonomy, phylogeny, genetic variability and pollination systems. Ann. Bot. 2002, 90(2). 219-230. doi: 10.1093/aob/mcf168

Camelo-Júnior, A.E; Ferreira, A.W; Andrade, I.M; Mayo, S.J; Nollet, F; Silva, J.L; Pessoa, E. M. Species delimitation in the Trichocentrum cepula (Oncidiinae, Orchidaceae) complex: a multidisciplinary approach. System. Biodivers. 2022, 20(1), 1-18. https://doi.org/10.1080/14772000.2022.2099478

De Luna, E; Gómez-Velasco G. 2008. Morphometrics and the identification of Braunia andrieuxii and B. secunda (Hedwigiaceae: Bryopsida). Syst. Bot. 2008, 33(2), 219-228. doi: 10.1600/036364408784571608

De Luna, E. Integrando análisis morfométricos y filogenéticos: de la sistemática fenética a la mor­fometría filogenética. Acta Bot. Mex., 2020, 127, 1-50. DOI: 10.21829/abm127.2020.1640

Imig, D.C; Righetto-Mauad, S.A.V; da Silva‐Pereira, V; Toscano de Brito, A.L.V; Smidt, E.D.C. Taxonomic update of Dryadella (Orchidaceae: Pleurothallidinae) based on morphometric analyses of three species of the Brazilian Atlantic rainforest. Feddes Repert. 2022,134(1), 66-79. doi: 10.1002/fedr.202200038

Lattin, J; Carroll J.D; Green P.E. Analyzing Multivariate Data. Thomson Brooks and Cole. Pacific Grove, USA. 2003; 556 pp.

Manly, B.F.J. Multivariate Statistical Methods: A Primer. Chapman and Hall. London, UK. 1986; 159 pp.

Pessoa, E.M; Cordeiro, J.M; Felix, L.P; Almeida, E.M; Costa, L; Nepomuceno, Á; Van den Berg, C. Too many species: morphometrics, molecular phylogenetics and genome structure of a Brazilian species complex in Epidendrum (Laeliinae; Orchidaceae) reveal fewer species than previously thought. Bot. J. Linn. Soc. 2021, 195(2), 161-188. https://doi.org/10.1093/botlinnean/boaa071

Ramírez-Sánchez, M.M; De Luna E; Cramer C. Geometric and traditional morphometrics for the assessment of char­acter state identity: multivariate statistical analyses of char­acter variation in the water mite genus Arrenurus (Acari, Hydrachnidia, Arrenuridae). Zool. J. Linn. Soc. 2016, 177(4): 720-749. doi: 10.1111/ zoj.12384

Salazar-Rojas, V.M.; Herrera-Cabrera, B.E.; Soto-Arenas, M.Á.; Castillo-González, F. Morphological variation in Laelia anceps subsp. dawsonii f. chilapensis Soto-Arenas Orchidaceae in traditional home gardens of Chilapa, Guerrero, Mexico. Genet. Resour. Crop. Evol. 2010, 57(4), 543-552. doi: 10.1007/s10722-009-9492-5

Sneath, P.H.A.; Sokal, R.R. Numerical taxonomy. The principles and practices of numerical classification. San Francisco: W. H. Freeman and Co. 1973.

Soto-Arenas, M.A. Recopilación y Análisis de la Información Existente sobre las Especies Mexicanas del Género Vanilla. Reporte Intermedio. CONACYT. México. 2009; 76 pp.

Wickens, T. D. The Geometry of Multivariate Statistics. Law­rence Erlbaum Associates, Inc. Hillsdale, USA. 1995; 165 pp.

Comments 14: In the summary of the evaluation format, the reviewer pointed out that the conclusions could be improved. 

Response 14. For a better understanding based on the results, we restructured the conclusions. [In the revised manuscript this change can be found - page number: 15, paragraph 1 of conclusions, and lines 446-456].

Six morphotypes of the labellum of V. pompona were obtained from localities in the states of Veracruz, Puebla, Oaxaca and Jalisco, Mexico. Each morphotype was distinguished mainly by size and shape, which shows that there is infraspecific variation in the germplasm of V. pompona. The variation that exists in the labels of this species was possibly associated with pollinator pressure and the presence of geographic barriers, which play a very important role in the types of environments in which specimens of V. pompona develop. This plant is wild; therefore, the environment plays a very important role in the variation related to phenological factors, since, for the locality of Cazuelas, Papantla Veracruz, plants with different flowering dates during the year were found. Knowledge of the infraspecific variation of the species is important for the conservation and preservation of the species in Mexico.

Response to Comments on the Quality of English Language

Point 1: The reviewer only mentions that a moderate revision of the English language is required.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions and corrections highlighted.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Are all the cited references relevant to the research?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Please see the answer in the point-by-point responses in the section below

Are the conclusions supported by the results?

Can be improved

 3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: This version presents some improvements if compared with the first one. Yet, I still believe that one important weakness is appealing to "pollinator pressures" to justify labellum variability. Would bees react differently among morphotypes? No evidence of this is presented and, in my opinion, invoking pollinator pressures as a main cause without evidence is speculation. Thus, I strongly suggest that the authors lower the tone and explicitly say that such pollinator pressures must be proven. Yes, authors have detected morphotypes and shown that these morphotypes are phylogenetically distributed and aren't aleatory. Okay, but the proposed pollinator pressures have to be demonstrated too. As I said in my previous review, pollination studies are serious science and cannot be treated as a "catch-all", convenient explanation without supporting evidence. Please, mention pollinator pressures as a POSSIBLE/TO BE TESTED possibility. All the best wishes.

Response 1: An apology for the way we worded the paragraph on pollinator pressure on labellum variation. We have restructured the paragraph, taking into account your appropriate recommendation on this aspect. We hope that in this version we have done it in the right way. [In the revised manuscript this change can be found - page number: 14, paragraph 3 of the discussion, and lines 392-400].

 Vanilla pompona has a reward strategy to attract pollinators by offering them both nectar and fragrance [48]. Different studies have documented the presence of specific pollinators for this species, as in some populations in Brazil, where Eulaema bombiformis Packard, E. cingulate Fabricius and E. meriana Olivier have been described [48]. While in Costa Rica and Peru, males of the species E. cingulata Fabricius have been reported as the specific pollinators of this plant [49]. The variation in the six V. pompona labellum morphotypes identified in this study could be associated in part by pollinator pressure, as has already been reported for other orchids [50]. However, pollinator pressure in V. pompona is only one possibility, which needs to be tested.

Response to Comments on the Quality of English Language

Point 1:

Minor editing of English language is required.

I think the text needs moderate English improvement

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted in red in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Must be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Must be improved

Please see the answer in the point-by-point responses in the section below

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

 3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The new version is better, but the authors should include the reasons they used only PCA in the new version of the ms.  

Response 1: We appreciate the recommendation. In this new version we have included the reasons why principal component analysis was used in our study. [In the revised manuscript this change can be found – page number: 6, paragraph: 3, and lines: 175-195]

 In orchid flower morphometry studies, the most commonly used multivariate analyses are Principal Component Analysis (PCA) and Canonical Variables Analysis (CVA) [44]. Canonical Variables Analysis are used to explore the separation of a priori defined groups [45,46]. These groups should consist of 12 to more than 30 replicates [47,48]). Recently, studies have been carried out in orchids based on morphometric analysis of flowers where CVA is used to differentiate between species of the genus Epidendrum [49], to delimit the species Trichocentrum cepula, T. caatingaense, T. sprucei [50] and to distinguish species of the genus Scaphyglottis [51]. While principal component analysis (PCA) is used to reduce dimensions and explore the overall structure of the database according to Wickens [52] and Lattin et al. [45], and is performed based on the correlation matrix between the selected variables [53].

In the study of the variation of the labellum of V. pompona, no a priori defined groups were formed, because the necessary characters to form them do not exist, which was one of the reasons why the CVA was not used. Another reason is that in our specimens we have three to five replicates per collection, and we could not have more due to the distance between the collection localities and because the flowers are ephemeral (they only last one day) [13]. In addition, in this study we analyzed the infraspecific variation of the labellum of V. pompona, not the variation between species, so we considered it appropriate with the 61 traits (lines and angles) defined previously for V. pompona [23], a multivariate principal components analysis (PCA) and a cluster analysis with a mean distance between clusters were performed [53]. SAS statistical software version 9.0 (2002) was used [54].

Response to Comments on the Quality of English Language

Point 1: Minor editing of English language is required.

The ms. needs to be checked by a native speaker