Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.    Need to revise the title in line of findings highlighting significance of ITS markers for Physalis species discrimination.

2.    Revise the introduction highlighting more significance/ economic importance of Physalis species.

3.  Species status of collected samples was first confirmed by taxonomist which was further strengthened by ITS2 barcoding. So, ITS2 barcoding only supplemented the findings of taxonomist. Therefore, instead of using the term species identification, the authors should have used the term species discrimination in the whole manuscript.

4.   Give details of sampling procedure, how sample were collected. Also provide altitude of collection sites and species status of collected sample in S2 table. Author should present S2 table in main manuscript as Table 1 instead of Supplementary file. Author can use DIVA-GIS software to visualize the collected specimens on Map.

5.   Also depict the collected species on Map. Author can present the species richness analysis. For further analysis using DIVA-GIS author can refer any paper on DIVA GIS based analysis for species richness and distribution.

6.   From coordinates data in supplementary files S1, S2 it looks that same species accessions were collected from single coordinates indicating duplication of specimens rather than their different identity. If duplicates of same line/genotypes collected they will tend to group in same groups. In Figure 2 Voucher no. L2, L4, L6, L7, L8, L9, 10 clustered into same groups. These samples were collected from same geographical location Londiani-Sorget (0.1635° S, 35.5931° E). It seems that these are duplicate not different samples since these have been collected from same location.

7.  Author should have used only those accessions of same species which were collected from different geographical locations to avoid any chance of duplication of any accession.

8.    If any morphological or biochemical data collected/generated by author could be included in the manuscript.

9.    Authors need to include the photographs of species from collection site in manuscript.

10. Discussion: Authors did many analysis and estimated many parameters. As different analysis only strengthened the same findings that ITS2 Barcode are ideal for species differentiation and within species diversity as compared to rbcL barcodes in Physalis species. Authors repeated the same sentences many times in discussion. Reduce discussion part within one page only.  

11.   Authors can explain their finding with the help of one Graphic/Figure/flow chart in discussion to summarize the findings of different analysis like BLASTs analysis, Multiple sequence alignments, genetic divergence analysis, nucleotide polymorphism and neutrality test and ABCD.

Author Response

The authors appreciate the time and effort by the editor and reviewers for reviewing this manuscript and for providing insightful comments and suggestions to improve its quality. We have revised the manuscript as per the reviewers’ suggestions and comments. We have incorporated the following reviewer’s specific comments in the revised version of manuscript. The point-by-point responses are provided below.

Comments and Suggestions for Authors

1. Need to revise the title in line of findings highlighting significance of ITS markers for Physalis species discrimination.

Response: Thank for the observation. We have revised the title as suggested.

2. Revise the introduction highlighting more significance/ economic importance of Physalis species.

Response: We have provided additional information on the nutritional and economic importance of Physalis in the introduction section. The significance of phytochemical content of Physalis has also been further described in the introduction.

3. Species status of collected samples was first confirmed by taxonomist which was further strengthened by ITS2 barcoding. So, ITS2 barcoding only supplemented the findings of taxonomist. Therefore, instead of using the term species identification, the authors should have used the term species discrimination in the whole manuscript.

Response: Thank you for the observation. We have used the term “species discrimination” instead of “species identification” throughout the manuscript.  

4. Give details of sampling procedure, how sample were collected. Also provide altitude of collection sites and species status of collected sample in S2 table. Author should present S2 table in main manuscript as Table 1 instead of Supplementary file. Author can use DIVA-GIS software to visualize the collected specimens on Map.

Response: We have provided the details on the sampling procedure, which was purposeful sampling. We have also provided the altitude as well as the assumed species status of the plant samples in Supplementary Table S2. Supplementary Table S2 was not moved to the main text as it is detailed and it will be published supplementary material. We did DIVA-GIS map for the visualization of the different species and not the collected specimens.

5. Also depict the collected species on Map. Author can present the species richness analysis. For further analysis using DIVA-GIS author can refer any paper on DIVA GIS based analysis for species richness and distribution.

Response: The collected species have been depicted on the map. We only managed to show species distribution on the map.

6. From coordinates data in supplementary files S1, S2 it looks that same species accessions were collected from single coordinates indicating duplication of specimens rather than their different identity. If duplicates of same line/genotypes collected they will tend to group in same groups. In Figure 2 Voucher no. L2, L4, L6, L7, L8, L9, 10 clustered into same groups. These samples were collected from same geographical location Londiani-Sorget (0.1635° S, 35.5931° E). It seems that these are duplicate not different samples since these have been collected from same location.

Response: The coordinates provided are for the locations of sampling in the Counties. In some places several samples were collected from the same location, because the samples were collected based on availability. In some locations, all the available plants were collected separately.

7. Author should have used only those accessions of same species which were collected from different geographical locations to avoid any chance of duplication of any accession.

Response: From the field all the plants were of the same species based on taxonomist. All the plants collected from all the geographical locations were identified as Physalis peruviana. Therefore, we used all the accessions collected for species discrimination using DNA barcoding.

8. If any morphological or biochemical data collected/generated by author could be included in the manuscript.

Response: We did not collect data on morphological or biochemical data for the Physalis accessions used in this study. This is because morphological characteristics usually results in misidentification due to similarities in the phenotypic characteristics of the different species. For example, Physalis minima and Physalis pubescens are morphologically similar which presents a challenge in their differentiation using their phenotypic characteristics. In addition, morphological and biochemical identification is also affected by environmental/physiological factors, stage of growth and development of plants.

9. Authors need to include the photographs of species from collection site in manuscript.

Response: We have included photos of plants from the collection sites in the manuscript.

10. Discussion:Authors did much analysis and estimated many parameters. As different analysis only strengthened the same findings that ITS2 Barcode are ideal for species differentiation and within species diversity as compared to rbcL barcodes in Physalis species. Authors repeated the same sentences many times in discussion. Reduce discussion part within one page only. 

Response: Thank you for the observation. We have revised the manuscript and reduced the discussion section. 

11. Authors can explain their finding with the help of one Graphic/Figure/flow chart in discussion to summarize the findings of different analysis like BLASTs analysis, Multiple sequence alignments, genetic divergence analysis, nucleotide polymorphism and neutrality test and ABCD.

Response: We have provided a flow chart in the discussion that summarizes the findings of the different analyses used in the study.

Reviewer 2 Report

Comments and Suggestions for Authors

Title: Identification, phylogenetic analysis and molecular diversity of Physalis species based on DNA barcoding

Identification of species through morphological characters/features or through molecular markers are important to identify the different species in particular genus/regions, for efficient utilization for commercial, genetic resource conservation and their effective utilization in breeding programs for genetic improvement. However, morphological identification is affected by environmental/physiological factors, stage of growth and development of plants while Molecular identification is more accurate as it is based on unique nucleotide sequences that are not affected by the morphological characteristics of the species, development stage (growth phase) and environmental/physiological factors.

This study, characterized the physalis species using rbcL and ITS2 barcoding study for the first time Physalis species present in Kenyan. As this study combined the two markers for efficient and reliable identification of Physalis species. The Physalis species are reported to contain rich in water- and fat-soluble vitamins (A, E, K, C and B-complex), minerals (magnesium, potassium, calcium and zinc), fatty acids (such as palmitate and linoleic acid), proteins and sugars.

Overall, this study is novel. For the first time this work collected and characterised using combination of two widely used barcode makers. Thoroughly examined the identified and discriminated into three species namely P. purpurea, P. peruviana and P. cordata. Also identified ITS2 is the most reliable barcode for the Physalis species.  My few suggestions as follows. 

Recommendation: Recommended for publication with minor revision

Abstract: This part may be shortened a bit.

Introduction: It covered the information available on Physalis and available barcodes and their advantages.

Physalis, genus name must be italicized throughout the text.

Font size uniformly followed in the text.

Materials and Methods: Well written

There were 64 Physalis plant samples were collected but only 28 samples were used in this study. Basis for selection of only 28 need to be explained.

2.6 why rbcl sequence is not used for interspecific genetic distance calculation.

Result: Presentation is good and non-redundant.

Table 1. please check alignment length (bp) for ITS2 – 841bp??. As the ITS length ranged between 237-707bp.

3.4. 3.4. Identification of Physalis species based on phylogenetic analysis

In Table 3 & 4. Write the scientific names of Physalis species as P. peruviana, P. cordata, etc.

Line 124-131, data must be included in the table 3.

Figure 3. X- & y- axis values are not visible.

Discussion: Appropriate discussion with right conclusion.

Author Response

The authors appreciate the time and effort by the editor and reviewers for reviewing this manuscript and for providing insightful comments and suggestions to improve its quality. We have revised the manuscript as per the reviewers’ suggestions and comments. We have incorporated the following reviewer’s specific comments in the revised version of manuscript. The point-by-point responses are provided below.

Comments and Suggestions for Authors

Title: Identification, phylogenetic analysis and molecular diversity of Physalis species based on DNA barcoding

Identification of species through morphological characters/features or through molecular markers are important to identify the different species in particular genus/regions, for efficient utilization for commercial, genetic resource conservation and their effective utilization in breeding programs for genetic improvement. However, morphological identification is affected by environmental/physiological factors, stage of growth and development of plants while Molecular identification is more accurate as it is based on unique nucleotide sequences that are not affected by the morphological characteristics of the species, development stage (growth phase) and environmental/physiological factors.

This study, characterized the physalis species using rbcL and ITS2 barcoding study for the first time Physalis species present in Kenyan. As this study combined the two markers for efficient and reliable identification of Physalis species. The Physalis species are reported to contain rich in water- and fat-soluble vitamins (A, E, K, C and B-complex), minerals (magnesium, potassium, calcium and zinc), fatty acids (such as palmitate and linoleic acid), proteins and sugars.

Overall, this study is novel. For the first time this work collected and characterised using combination of two widely used barcode makers. Thoroughly examined the identified and discriminated into three species namely P. purpurea, P. peruviana and P. cordata. Also identified ITS2 is the most reliable barcode for the Physalis species.  My few suggestions as follows. 

Recommendation: Recommended for publication with minor revision

Abstract: This part may be shortened a bit.

Response: We have shortened the abstract as suggested.

Introduction: It covered the information available on Physalis and available barcodes and their advantages.

Physalis, genus name must be italicized throughout the text.

Response: We have italicized all the Physalis names within the text.

Font size uniformly followed in the text.

Response: We have used Font sizes based on the journal guidelines.

Materials and Methods: Well written

There were 64 Physalis plant samples were collected but only 28 samples were used in this study. Basis for selection of only 28 need to be explained.

Response: The 28 samples used were those that were successfully amplified and sequenced for both barcode markers. This has also been explained in the manuscript.

2.6 Why rbcl sequence is not used for interspecific genetic distance calculation.

Response: Sequences based on rbcL gene were not used for the interspecific genetic distance calculation because they were not able to discriminate Physalis. Interspecific genetic distance study requires the efficient discrimination of species as it compares the genetic distance between species. ITS2 is the only primer that was able to discriminate species. This explains why rbcL sequences were not subjected to interspecific genetic distance calculation.

Result: Presentation is good and non-redundant.

Table 1. Please check alignment length (bp) for ITS2 – 841bp??. As the ITS length ranged between 237-707bp.

The length of the alignment is correct at 841bp. Though the range of sequence length for the ITS2 gene sequences was between 237-707 bp, the alignment length given as an overall value for the combined gene sequences was 841bp and this has been presented in Supplementary Figure S1 for verification.

3.4. 3.4. Identification of Physalis species based on phylogenetic analysis

In Table 3 & 4. Write the scientific names of Physalis species as P. peruviana, P. cordata, etc.

Response: Scientific names of P. peruviana, P. cordata and P. purpurea have been corrected in Tables 3, 4 and 5.

Figure 3. X- & y- axis values are not visible.

Response: We have improved X and Y axis values visibility and included labels of DNA barcode gaps identified.

Discussion: Appropriate discussion with right conclusion.

Reviewer 3 Report

Comments and Suggestions for Authors

A manuscript entitled,” Identification, phylogenetic analysis and molecular diversity of Physalis species based on DNA barcoding “was reviewed for Crops.

DNA barcoding is a powerful technology for biodiversity classification and identification. For plants, MatK, RbcL, TrnH, and ITS are mainly used for DNA sequence barcoding.

The authors chose RbcL (plastid origin) and ITS2(nuclear origin) as barcodes for identifying Kenya Physalis species from seven counties in Kenya. Since it might be difficult to discriminate Physalis species by only observing plant morphology, results in this manuscript might be helpful for the conservation and utilization of Kenya Physalis species.

 Questions and Comments:

 Abstract:

Too long a description. Please condense it so that the readers can quickly grasp its content before they give up understanding a long abstract.

M&M:

2.2 RNAse-----RNase.

2.4 highest similarity -----How did you choose “most similar” sequences? Bit scores or E-value in Blastn table or percent identity? If multiple factors are considered, please describe how they lined up.

Results:

2.4 and 3.4------In fig.2, it seems that the phylogenetic tree was constructed by a combination of calculated genetic distances of ITS2 and rbcL. I am not sure how they treated two species whose tree position is different with either ITS2-based or rbcL-based phylogenetic difference.

Please describe in the Method section clearly.

Discussion

The discriminating power in ITS2 is higher than that of the rbcL barcode.

This might not be surprising, because the rbcL barcode from a protein-coding region should take a selection pressure, while ITS2 is an interspace(intron) between two rRNA and should be relatively tolerant in natural mutation.  

Moreover, in another paper, “Newmaster et al. compared ~10,300 rbcL sequences (with each more than 1,000 bp) collected from GenBank by using a distance method, and found that rbcL did not recognize all plant species but distinguished plants within the same genus.” (Kang, (2017). DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Scientific Reports, 7(1), 1-9.

Please discuss.

Comments on the Quality of English Language

good.

Author Response

The authors appreciate the time and effort by the editor and reviewers for reviewing this manuscript and for providing insightful comments and suggestions to improve its quality. We have revised the manuscript as per the reviewers’ suggestions and comments. We have incorporated the following reviewer’s specific comments in the revised version of manuscript. The point-by-point responses are provided below.

Comments and Suggestions for Authors

A manuscript entitled,” Identification, phylogenetic analysis and molecular diversity of Physalis species based on DNA barcoding “was reviewed for Crops.

DNA barcoding is a powerful technology for biodiversity classification and identification. For plants, MatK, RbcL, TrnH, and ITS are mainly used for DNA sequence barcoding.

The authors chose RbcL (plastid origin) and ITS2(nuclear origin) as barcodes for identifying Kenya Physalis species from seven counties in Kenya. Since it might be difficult to discriminate Physalis species by only observing plant morphology, results in this manuscript might be helpful for the conservation and utilization of Kenya Physalis species.

 Questions and Comments:

 Abstract:

Too long a description. Please condense it so that the readers can quickly grasp its content before they give up understanding a long abstract.

Response: The abstract has been shortened as suggested.

M&M:

2.2 RNAse-----RNase.

Response: The abbreviation of RNase has been corrected

2.4 highest similarity -----How did you choose “most similar” sequences? Bit scores or E-value in Blastn table or percent identity? If multiple factors are considered, please describe how they lined up.

Response: Sequence similarity based on BLASTn analysis was based on E-value and percentage identity. GenBank sequences with the highest percentage identity and an E-value of 0.0 or a close equivalent if none with a E-value of 0.0 were chosen as most similar sequences to the experimental sequences. This information has been added to the manuscript in section 2.4.

Results:

2.4 and 3.4------In fig.2, it seems that the phylogenetic tree was constructed by a combination of calculated genetic distances of ITS2 and rbcL. I am not sure how they treated two species whose tree position is different with either ITS2-based or rbcL-based phylogenetic difference.

Please describe in the Method section clearly.

Response: The phylogenetic tree was not constructed based on a distance method but rather through Bayesian inference (BI). This information has been included in the methodology section.

Discussion

The discriminating power in ITS2 is higher than that of the rbcL barcode.

This might not be surprising, because the rbcL barcode from a protein-coding region should take a selection pressure, while ITS2 is an interspace(intron) between two rRNA and should be relatively tolerant in natural mutation.  

Moreover, in another paper, “Newmaster et al. compared ~10,300 rbcL sequences (with each more than 1,000 bp) collected from GenBank by using a distance method, and found that rbcL did not recognize all plant species but distinguished plants within the same genus.” (Kang, (2017). DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Scientific Reports, 7(1), 1-9.

Please discuss.

Response: The information has been added to the discussion section and elaborated.