The Genetic Characterization of the Canarian Endemic Palm (Phoenix canariensis) by Simple Sequence Repeats and Chloroplast Markers: A Tool for the Molecular Traceability of Phoenix Hybridization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the effort of the authors to serve this research which has a significant advancement in Genetic characterization of the Canarian endemic (Phoenix canariensis) by SSR and chloroplast markers. The current manuscript shows are promise, there are some major revisions that must be addressed before acceptance

·       This research entitled “Genetic characterization of the Canarian endemic Palm 2 (Phoenix canariensis) by SSR and chloroplast markers: a tool to molecular traceability of Phoenix hybridization” holds significant academic value. The topic addressed considerable scientific depth.

·       In the abstract; One of the main conservation problems affecting the endemic palm from The Canary lands (Phoenix canariensis) is the anthropogenic hybridization with other Phoenix species, especially Phoenix dactylifera which has been introduced extensively throughout its geographical range. Rephrase this sentences line number 26,27 and have some words have start with capital word in the mid of sentences improved the whole abstract.

·       Our goal here were: i) to detect a molecular tracer that could reliably distinguish 31 between Phoenix canariensis and P. dactylifera in the Canary archipelago and ii) to characterize the 32 presence and extent of genetic hybridization events between the two species. Its need in the abstract, write clear the objectives in the last paragraph of introduction.

·       Can you briefly explain why Phoenix canariensis is particularly important to the Canary Islands?

·       Keywords, writing words in the same format.

·       Rephrase this sentence and make scientifically sense of this sentences” Frequent interspecific hybridizations 50 among species and the scarcity of suitable taxonomic morphological and anatomical char- 51 acters to distinguish different Phoenix species have motivated the search for useful DNA 52 specific identification” But the first paragraph of introduction writes clearer.

·       Can you provide more detail on how the extent of hybridization was quantified using these genetic markers?

·       Figure 1. a) PCA a and b figure increase the resolution 300 dpi or above.

·       Section 3.1. Genetic characterization of Phoenix canariensis and P. dactylifera and their hybrids, check previous article and compared your result, write and explain clearly every result. improve the sentences and write scientifically

·       In the discussion part rephrase the sentences, Of the alleles detected, more than half were specific to any of the two species, as suggested 316 by the allelic loadings of the PCA (Figure 1b). Overall, our data supported previous epectations in Phoenix genus about the ability of the SSR markers to discriminate Phoenix species.

·       What are the specific genetic markers or molecular tracers proposed in this study, and how do they compare in sensitivity and accuracy to other existing markers for detecting hybridization and introgression in Phoenix species?

·       Elaborating on this point can provide readers with a clearer understanding of the advancements and potential benefits your proposed tracers offer, as well as their practical application in conservation and hybrid identification efforts.

·       The conclusion section should be improved write the significance of the study.

·       The manuscript with improved grammar and clarity.

·       Please cite and follow these papers, how to write the abstract, introduction, etc.

 

1.      Umar Zeb, Azizullah Azizullah, Xiukang Wang, Sajid Fiaz, Hanif Khan. (2021). Comparative genome sequence and phylogenetic analysis of chloroplast for evolutionary relationship among Pinus species. Saudi Journal of Biological Sciences. https://doi.org/10.1371/ journal.

2.      Zeb, U., Dong, W. L., Zhang, T. T., Wang, R. N., Shahzad, K., Ma, X. F., & Li, Z. H. (2020). Comparative plastid genomics of Pinus species: Insights into sequence variations and phylogenetic relationships. Journal of Systematics and Evolution, 58(2), 118-132.      

3.      Zeb, U., Aziz, T., Azizullah, A., Zan, X. Y., Khan, A. A., Bacha, S. A. S., & Cui, F. J. (2024). Complete mitochondrial genomes of edible mushrooms: features, evolution, and phylogeny. Physiologia Plantarum, 176(3), e14363.

4.      Zeb, U., Wang, R., Dong, P., Wang, N., Zhang, T., Lin, C., & Wang, X. (2019). Characterization of the complete chloroplast genome sequence of Pinus pumila (Pinaceae). Mitochondrial DNA Part B, 4(1), 290-291.

 

Comments on the Quality of English Language

Improve english

Author Response

In the abstract; One of the main conservation problems affecting the endemic palm from The Canary lands (Phoenix canariensis) is the anthropogenic hybridization with other Phoenix species, especially Phoenix dactylifera which has been introduced extensively throughout its geographical range. Rephrase this sentences line number 26,27 and have some words have start with capital word in the mid of sentences improved the whole abstract.

We have changed.

The objectives were also modified in the last paragraph of introduction.

We also have explained the relevance of P. canariensis in the archipelago. It is the plant symbol of the Canary Islands and the extension in all island is very relevant.

Respect to the question:  Can you provide more detail on how the extent of hybridization was quantified using these genetic markers?

We explain in the text that the degree of hybridisation is detected through the STRUCTURE and NEWHYBRID software (Figure 2) as well as by counting the number of exclusive alleles of the Canarian Nuclear Genotype (CNG) (Table 2).

We have improved the quality of figure 1.

The discussion was also slightly modified.

We also have improved the conclusion.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Genetic characterization of the Canarian endemic palm (Phoenix canariensis) by SSR and chloroplast markers: a tool for molecular traceability of Phoenix hybridization" is interesting from the standpoint of conservation biology and the genetic impact of alien species on native species. In their study, the authors utilized nuclear SSR and chloroplast minisatellite markers to investigate genetic hybridization events between the Canarian endemic species Phoenix canariensis and its continental relative, P. dactylifera. They successfully developed molecular markers capable of identifying pure Phoenix canariensis genotypes. A total of 19 SSR loci and one cpDNA locus were analyzed in a large number of plants from Gran Canaria, Tunisia, and the Iberian Peninsula. The paper is technically sound and is well-presented. I didn't notice any major flaws in the work. However, in the introduction, I think there was a lack of information about palm species that grow in the Canary Islands, which could potentially cross with Phoenix canariensis. I'm also wondering whether hybrids are only possible between Phoenix canariensis and P. dactylifera in Gran Canarian.

Line 115. “…we used nuclear SSR and chloroplast minisatellite markers to characterize genetic hybridization events between the Canarian endemism Phoenix canariensis and its continental relative P. dactylifera .”  In my opinion, it would be clearer and more accurate to say "...we utilized nuclear SSR and chloroplast minisatellite markers to characterize genetic hybridization events between the Canarian endemic species and its continental relative...”

Line 123. “…for conservation the of Phoenix ….”

Lines 256-258. There are 7 loci in question, but only 6 are specified.

In the beginning of the Discussion section, the article states that the SSR genetic diversity in this study is comparable to other analyses that used similar markers in a wider geographical range of Phoenix. However, the manuscript does not provide the usual genetic diversity parameters (Fis, HO, HE, Na). Therefore, it would be helpful to present some parameters of the PC, HB, and PD groups in the results and then compare them with the findings of reference [8]. Otherwise, the sentence should be removed or rewritten.

Author Response

We modified lines 115 and 123 as the referee suggests.

With respect to: Lines 256-258. There are 7 loci in question, but only 6 are specified.

One of the loci (P15) shows a duplication only in P. canariensis, that is why they are labelled as P15A and P15B. It is clearer in the text now.

In the beginning of the Discussion section, the article states that the SSR genetic diversity in this study is comparable to other analyses that used similar markers in a wider geographical range of Phoenix. However, the manuscript does not provide the usual genetic diversity parameters (F_IS, HO, HE, Na). Therefore, it would be helpful to present some parameters of the PC, HB, and PD groups in the results and then compare them with the findings of reference [8]. Otherwise, the sentence should be removed or rewritten. We consider that is not necessary, it is not informative for the proposal of the paper. However, we could add a table as supplementary material.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Genetic characterization of the Canarian endemic Palm (Phoenix canariensis) by SSR and chloroplast markers: a tool to molecular traceability of Phoenix hybridization” by Isabel Saro and collaborators aims to contribute to mitigate conservation problems related to the anthropogenic-driven hybridization of the endemic palm from The Canary Islands (Phoenix canariensis) with the introduced Phoenix dactylifera. The main objective of the study was to develop molecular diagnostic markers of pure taxa and to evaluate the degree of hybridization. The paper is a short, mainly methodological manuscript but has the virtue to confirm with an extensive database (N=433) that a combination of nuclear and chloroplast markers can be valuable tools to identify P. canariensis. The paper is well written and the applied markers and analytical tools are sound as well the interpretation of results.

Please found below comments that need to be addressed.

I found the morphological characterization of hybrids a main concern. In particular, Figure S1 should be part of the main text. A detailed description of morphological traits of hybrids should be provided and included in Table 1.

A description of each pure species’ habitats and those of the hybrid individuals need to be included. Are the hybrids found in sympatry with pure taxa?

In Figure 1a samples corresponding to putative hybrid morpho-geographic types HBGC and HBTF should be identified by a distinct symbol.

The authors clearly state in the introduction that the studied species present conservation relevance given that identification of pure and hybrid populations is a very subjective task currently. Later in Table 1 authors provide a list of morphological traits that were used to assign studied pure Phoenix species’ samples and they express that hybrid individuals were recognized as having intermediate characteristics between pure taxa. However, when looking at those traits, it is not clear what characteristics hybrid individuals will show in terms of for example presence of offshoots if pure species have either occasional presence or absence of them. Therefore, more details are needed on how hybrids are identified in terms of their morphology. This is important because results presented in Table 3 clearly show that whereas most pure taxa match distinct and mostly diagnostic genotypes, hybrids do not. Authors recognize in the discussion that “ a significant fraction of the hybrids detected in this study were mistaken for pure specimens of P. canariensis”. A thorough and quantitative evaluation of hybrid individuals in terms of their morphology should be performed for example using multivariate discriminant analysis to confirm their hybrid / introgressed / pure status. If not possible, these issues should be further addressed in the discussion.

 

Minor editorial comments

Table 2 abbreviation for P. dactylifera occidental and oriental should be different instead of PD for both

Author Response

I found the morphological characterization of hybrids a main concern. In particular, Figure S1 should be part of the main text. A detailed description of morphological traits of hybrids should be provided and included in Table 1.

We fully agree with you. Figure S1 is very illustrative to distinguish between P. canariensis and P. dactylifera. However, Figure S1 has not been included as the main part of the text due to lack of space.

We want to clarify that we have detected in the populations and localities analysed that there is an extremely wide range of variation in the morphological characters of the hybrids. This is one of the reasons why we are looking for a reliable molecular marker. The variation is enormous, very high, and we are now trying to find out which morphological characters could be used in more detail to allow us to separate morphologically the different hybrids (P. canariensis x P. dactylifera) we are finding. According to our preliminary results that we are working on, it affects many more variables than the ones indicated in table 1, but we are still working on that study. For example, it seems to affect the size of the leaves, the size and number of leaflets, the size of the petiole and pseudo-petioles, the size and thickness of spikes... and there are even hybrids that appear with offshoot, but others, being hybrids, do not have it. It is therefore very difficult to include a single morphological character that can be assigned to hybrids. 

We would like to give as an example our recent typing of the hybrid Phoenix x arehuquensis; P. canariensis x P. reclinata (Obon et al. 2024; South African Journal of Botany 168 (2024)124-129). 

A description of each pure species’ habitats and those of the hybrid individuals need to be included. Are the hybrids found in sympatry with pure taxa?

The situation of P. canariensis, P. dactylifera and their hybrids in the archipelago is very variable. We do not have palm groves of P. dactylifera, but individuals dispersed. The problem are the hybrids. Fortunately, there are still natural populations on several islands, where we found pure specimens of P. canariensis, in which there do not seem to be any hybrid or date palm specimens in the vicinity. Since the aim of our work was to analyse the degree of hybridisation and genetic introgression in hybrid specimens, sampling was directed at certain populations and localities where hybrids grow sympatrically and sub-spontaneously with canary specimens. We indicate this circumstance in material and methods.

In Figure 1a samples corresponding to putative hybrid morpho-geographic types HBGC and HBTF should be identified by a distinct symbol.

They are included in different colours. We understand that this is not a relevant aspect to include and that it could clutter the figure. Nor have we put different symbols to Pd242 or Pd254. We believe that this figure is more illustrative and simpler as it is.

The authors clearly state in the introduction that the studied species present conservation relevance given that identification of pure and hybrid populations is a very subjective task currently. Later in Table 1 authors provide a list of morphological traits that were used to assign studied pure Phoenix species’ samples and they express that hybrid individuals were recognized as having intermediate characteristics between pure taxa. However, when looking at those traits, it is not clear what characteristics hybrid individuals will show in terms of for example presence of offshoots if pure species have either occasional presence or absence of them. Therefore, more details are needed on how hybrids are identified in terms of their morphology. This is important because results presented in Table 3 clearly show that whereas most pure taxa match distinct and mostly diagnostic genotypes, hybrids do not. Authors recognize in the discussion that “ a significant fraction of the hybrids detected in this study were mistaken for pure specimens of P. canariensis”. A thorough and quantitative evaluation of hybrid individuals in terms of their morphology should be performed for example using multivariate discriminant analysis to confirm their hybrid / introgressed / pure status. If not possible, these issues should be further addressed in the discussion.

As mentioned above, the differentiation and distinction of hybrids from a morphological point of view is sometimes subjective and very difficult. There are numerous types of hybrids with a very wide range of morphological variation. On the one hand, being a dioecious species, it is quite possible (we do not know) that depending on whether the female is a canary or a date palm, the hybrid may be morphologically different. For example, there is some sexual dimorphism in specimens of P. canariensis (not yet published). Furthermore, we do not know (it has not been studied) which main variables mark and host the hybrids, as we have found an enormous variation. In addition to this, there is the physiological situation of the specimen. If we are in a period of drought, the specimen is morphologically different and can also confuse the type of individual. Furthermore, we have found (described in figure 2) that there is a wide range of hybrids depending on the level of crossbreeding. It is possible that in many cases we are dealing with 3rd or 4th generation hybrids, i.e. they are neither F1 nor F2. In fact, we have detected a group of specimens, which, having morphological characters of P. canariensis, harbour chlorotype 242 (mentioned in results and discussion). Therefore, these hybrids are possibly ancestral and are the consequence of different crosses with canary specimens, diluting their morphological-hybrid character and becoming more similar to canary specimens. In conclusion, the variation of hybrids is considerably wide and we cannot delimit them into a single type.

Minor editorial comments

Table 2 abbreviation for P. dactylifera occidental and oriental should be different instead of PD for both

Modified