Cytochrome Oxidase Subunit II: Potential Marker for the Identification of Forensically Significant Species of Coleoptera—A Preliminary Study
, Anup Kumar Kesavan 3, Nadiyah M. Alabdallah 4, Mohammed A. Alshehri 5, Samy Sayed 6, Mohammad Javed Ansari 7,* and Madhu Bala 1,*
Round 1
Reviewer 1 Report
The presented MS is a simple but still interesting study on the barcoding of in forensically important beetles. The main objective of the study was to provide the reference data base for the 11 taxa. I would say that the goal was obtained by the authors s but the MS needs some revision. Here are some issues that authors should address:
- The justification for using COXII over the most commonly used COXI is week. This could be better explained in the Introduction. Moreover, there is no information about COXI or other markers availability for the studied taxa. This would be important for readers to know if other options for barcoding these taxa are available. Moreover, if the COXI sequences can be obtained for these species from public data bases, the authors could conduct similar analysis on this popular barcode to make better and direct comparison with their COXII marker.
- The M&M section is missing some important information that I marked directly within the PDF file.
- The most important issue in this MS is that the authors did not pay much attention to accessing intraspecific variability within the studied species. Although there is no direct and easily accessible information on the total number of specimens and DNA sequences analyzed in the study, the sample size seems to be extremely low.
- I also cannot find a strong justification for the phylogenetic analysis presented in the study. The goal was to create a reference data base with DNA sequences. The DNA sequences should be useful for fast species discrimination. I think that the only goal of phylogenetic analysis here would be to check if the general topology of the tree is in line with the current systematics of beetles and not more. Other elaborations on the phylogenetic affinities are not relevant here due to the super-small phylogenetic sample sizes which cannot give confident results. The authors did not mention anything about the DNA barcoding rules, e.g., barcoding gap and did not attempted to check this in their data set.
Comments for author File: 
Comments.pdf
Author Response
Response of reviewer 1 comments
- The justification for using COXII over the most commonly used COXI is week. This could be better explained in the Introduction. Moreover, there is no information about COXI or other markers availability for the studied taxa. This would be important for readers to know if other options for barcoding these taxa are available. Moreover, if the COXI sequences can be obtained for these species from public data bases, the authors could conduct similar analysis on this popular barcode to make better and direct comparison with their COXII marker.
Response: Strong justification of using COXII over COXI is added in introduction section. For few studied taxa COXI sequences were obtained and submitted to GenBank but data was unpublished. Apart from COXI and COXII we obtained sequences for 12s gene also and submitted to GenBank, but data is again unpublished.
- The M&M section is missing some important information that I marked directly within the PDF file.
Response: All the relevant missing information added in M&M section.
- The most important issue in this MS is that the authors did not pay much attention to accessing intraspecific variability within the studied species. Although there is no direct and easily accessible information on the total number of specimens and DNA sequences analysed in the study, the sample size seems to be extremely low.
Response: In divergence and nucleotide composition section we add additional information regarding intraspecific and interspecific variability. Information regarding total number of specimens and DNA sequences analysed added in M&M section. Sample size is low for some species. Multiple sequences were submitted for few species at different time interval that’s why non identical accession numbers were obtained for species.
- I also cannot find a strong justification for the phylogenetic analysis presented in the study. The goal was to create a reference data base with DNA sequences. The DNA sequences should be useful for fast species discrimination. I think that the only goal of phylogenetic analysis here would be to check if the general topology of the tree is in line with the current systematics of beetles and not more. Other elaborations on the phylogenetic affinities are not relevant here due to the super-small phylogenetic sample sizes which cannot give confident results. The authors did not mention anything about the DNA barcoding rules, e.g., barcoding gap and did not attempt to check this in their data set.
Response: We agree that phylogenetic trees represent the general topology where the species belonging to one family are clustered together in a group. These trees also help to find if there was any incongruence between morphological and molecular data due to uncertainty in morphological identification. Although it was only possible for those whose reference sequences were found in GenBank. Purpose of the present study was to create the reference data indeed as the reviewers implies as due to low sample size it was not possible to resolve the phylogenetic relationships. Nevertheless, these trees help to distinguish the phylogeny and species delineation. We will add information regarding barcoding rules and barcoding gaps We will add information regarding barcoding rules and barcoding gaps. Regarding barcoding gaps, since our data related to present study is not much sizeable and haven’t get enough data from GenBank so, it’s difficult to infer any conclusions related to barcoding gaps.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The text in figures 3 and 4 is not of publishable quality and the font is blurry with poorly defined margins and difficult to read. This quality issue should be resolved prior to publication.
Author Response
Response of Reviewer 2 comments
The text in figures 3 and 4 is not of publishable quality and the font is blurry with poorly defined margins and difficult to read. This quality issue should be resolved prior to publication.
Response: Corrections are incorporated in text.
Reviewer 3 Report
I have marked one or two typological details that could be attended to to finally polish this manuscript. The novel results are noted and the authors congratulated on this forward leap in forensic entomology for India.
Comments for author File: Comments.pdf
Author Response
Response of reviewer 3 comments
have marked one or two typological details that could be attended to finally publish this manuscript. The novel results are noted, and the authors congratulated on this forward leap in forensic entomology for India.
Response: All the typological errors mentioned by reviewer corrected and incorporated in manuscript.
Reviewer 4 Report
I support my opinion from the first review. The research, although methodologically correct, adds very little (basically nothing) to the knowledge on the identification of forensically important beetles. Showing that COII sequences differ between eleven beetle species is not enough to get published in an internationally recognized journal.
Author Response
Response of reviewer 4 comments
I support my opinion from the first review. The research, although methodologically correct, adds very little to the knowledge on the identification of forensically important beetles. Showing that COII sequences differ between eleven beetle species is not enough to get published in an internationally recognized journal.
Response: Since it is a preliminary study from this part of world off course having forensic significance. During forensic analysis accurate identification of every forensically relevant species is crucial. Sometimes even a single species in sufficient to solve cases of homicides, suicides, or unnatural deaths. Molecular studies supplemented in accurate identification of these species if sometimes morphologically it’s difficult to identify them. First attempt is being made to prepare small molecular database of these forensically relevant beetle species from India which aids in accurate identification. Secondly some contributions were made towards GenBank data through this study.
Round 2
Reviewer 1 Report
I have no further corrections or suggestions to the revised version of the MS.
Author Response
Response to reviewer 1 comment
Comment-I have no further corrections or suggestions to the revised version of the MS.
Response: Reviewer have no further correction or suggestions to the revised manuscript.
Reviewer 4 Report
The manuscript lacks novelty that warrants publication. The fact that the COII gene can be used as a molecular marker to identify species is not new.
Author Response
Response to reviewer 4 comment
Comment: The manuscript lacks novelty that warrants publication. The fact that the COII gene can be used as a molecular marker to identify species is not new.
Response: The purpose of the study was to create a database specifically for forensically important beetles as the molecular data was found insufficient for the species included in this study from India. So, an attempt was made to create a database for forensic fauna using COII gene and on finding the positive results we found that it can be used a molecular marker for forensically important species also.
During literature survey, there are various other genes such as COI, 16s rRNA etc. which are used as molecular markers repeatedly and a good number of studies were based upon them, but the data based upon COII was scarce especially in India. Once again, the focus was to create database using it for the Indian species.
We agree that COII gene is not new when it comes to molecular identification of species, but the data based upon this gene is new for the maximum species included in the present study from India. Submission of the sequences of these species will contribute to implementation of a national database, increasing the amount of information available for some forensically relevant Coleoptera species from India.
