Barcoding Mullets (Mugilidae): Genetic Characterization of Exploited Species in Southern Peninsular India

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Dear authors,

I believe that the manuscript with the new changes is excellent, removing any doubts that was raised in the older version. I would just change some small details like:

Remove author and year from title

Change in keyword "genbank" for something more useful like Indian Ocean, Asia...

Author Response

No answer need

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The manuscript has been revised according to my opinion, it can be considered to accept.

Author Response

no answer needed

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

This manuscript aimed to fill the knowledge gap and data deficiency regarding the phylogenetic diversity of mullets occurring in India. It confirmed 21 species of mullets under 8 genera. The overall analysis and writeup have been improved than the previous version of the manuscript. However, there are some concerns which I feel must be addressed.

The major concern is the sampling design itself. The sampling design neither carries the aims of the research nor does it support the title of the manuscript. When you haven't sampled fish with certain systematic sampling from the wild habitats, you may not claim that you have filled the existing knowledge gap and data deficiency. You have collected fish from the fish markets and in such conditions, fishermen might have captured the fish of their interest only and many species might have been left. With the present title, the readers of the paper will be expecting the sampling from the water bodies, not from the market.

Additionally, I wonder how you georeferenced the geographic origin of those samples for the preparation of maps. How accurate and specific localities for the samples were obtained for those fish which were collected from the market?

One option to overcome these shortcomings could be to mention clearly in the title itself about the sampling effort. You have claimed that the study represents the 'Indian subcontinent'- however, with this sampling effort, I doubt it does. The second concern in the title is- 'reveals undescribed diversity'; is it true? Which new species/groups that were not previously recorded in the subcontinent have been described in this study? I could not figure it out. 

These issues are to be clarified in the paper. 

Comments for author File: Comments.pdf

Author Response

Que: Remove author and year from title

Que: The major concern is the sampling design itself. The sampling design neither carries the aims of the research nor does it support the title of the manuscript. When you haven't sampled fish with certain systematic sampling from the wild habitats, you may not claim that you have filled the existing knowledge gap and data deficiency. You have collected fish from the fish markets and in such conditions, fishermen might have captured the fish of their interest only and many species might have been left. With the present title, the readers of the paper will be expecting the sampling from the water bodies, not from the market.

Ans: In response to the reviewer's concern about the sampling design, we have revised the title of the manuscript to "Barcoding mullets (Mugilidae): genetic characterization of exploited species in southern peninsular India." This modification is made to better align the title with the actual sampling approach employed. The revised title, not only aptly reflects the implemented sampling methodology but also underscores the genetic characterisation of commercially valuable species presented in the manuscript.

Que: I wonder how you georeferenced the geographic origin of those samples for the preparation of maps. How accurate and specific localities for the samples were obtained for those fish which were collected from the market?

Ans: To georeference the geographic origin of the samples and prepare maps, specific methods were employed based on the fishing gear used for mullet harvesting. Samples obtained directly from cast net and Chinese dipnet operations were collected on-site, providing accurate and identifiable locations. In the case of stake nets (are fixed nets) allowed for precise location determination. For mullets captured through gillnets, information on capture locations was gathered from fishers. The georeferencing of these locations was achieved through passive means, incorporating fishing-related data. This involved utilizing details such as distance and direction from landing centers, as provided by fishers or vendors. It is worth noting that the method of identifying probable fishing locations using distance and direction from landing centers is an established practice in Indian scenario, even in marine waters (Padua et al., 2021 and Varghese et al., 2023).

Que: One option to overcome these shortcomings could be to mention clearly in the title itself about the sampling effort. You have claimed that the study represents the 'Indian subcontinent'- however, with this sampling effort, I doubt it does. The second concern in the title is- 'reveals undescribed diversity'; is it true? Which new species/groups that were not previously recorded in the subcontinent have been described in this study? I could not figure it out. 

Ans: The previous title of the manuscript included the term 'Indian subcontinent' as a result of incorporating newly generated COI sequences, alongside sequences from mugilid specimens in GenBank originating from the broader Indian region. While our primary sampling focused on the southern part of India, our intent was to conduct a thorough phylogenetic analysis. However, we recognize that the term 'Indian subcontinent' may not precisely represent the geographical extent of our sampling efforts. We value the reviewer's suggestion to refine the title to accurately convey the study's scope and sampling design.

Regarding the phrase "'reveals undescribed diversity" in the previous title, it was used in light of our phylogenetic analysis, which identified distinct genotypes, potentially representing new species (denoted in the manuscript and the Phylogenetic tree as Ellochelon sp., Osteomugil sp. D, and Planiliza macrolpis cf. H) or the resurrection of previously synonymized species (Mugil cephalotus and Mugil (Osteomugil) amarulus). However, we acknowledge that assigning new names or resurrecting species requires more detailed morphological investigations. As a result, the term "reveals undescribed diversity" has been omitted from the present title to accurately reflect the cautious interpretation of our findings.

Que: Line 105 inwards, this paragraph contains the information about the data analysis and should come after the DNA extraction, PCR and sequencing.

Ans: Rearranged the paragraph accordingly.

Que: No need to use four maps, you can use different symbols (not only circles) and colours to indicate the localities in a single bigger and clearer map. Scientific name in the legend are to be italicized.

Ans: In response to the reviewers' suggestion to use a single map with different symbols and colors, we initially attempted this approach. However, due to the presence of certain locations harboring a higher number of species, even by employing various symbols and colors also led to potential overlap issues, resulting in reduced distinguishability. To address this challenge and enhance clarity, we have categorized the eleven species into four distinct groups, ensuring minimal overlap among locations within each group. This adjustment aims to provide a clearer representation on the map. Furthermore, we have implemented the suggestion to italicize scientific names in the legend, aligning with proper formatting. Additionally, we have unitalicized the prefix "cf" in the scientific names as presented in Figure 2 and Figure 4.

Editing done in the manuscript is highlighted in the revised version.

 

Round 2

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Thank you for considering the comments and improving the manuscript. 

All the best!

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript makes a great contribution to the identification of Mugilidae in India, contributing greatly to elucidate identification problems that have been present in other articles. The manuscript was very well done and I believe that only minor details need to be corrected before publication. The author and year for each genus and species is mandatory the first time it is used in the text (there is only one correct case in the manuscript). Furthermore the phylogeny with the wrong IDs was extremely confusing. I recommend indicating in the tree who was previously misidentified (with a *) in other articles and keeping Table 2 with the complete information.

Comments for author File: Comments.pdf

Author Response

• The author and year for each genus and species were added the first time they were used in the manuscript.
• We have limited the scope of the current manuscript to a discussion of Mugilidae species diversity in India. Although we recognize the concern regarding the potential misidentification of sequences in the database, we acknowledge that this should be addressed within a broader context through a more comprehensive analysis.

Reviewer 2 Report

Comments and Suggestions for Authors

1. The information of COI sequences should be described in the results.

2. The figure resolution of the phylogenetic tree is low.

3. I suggest that the authors should provide the clear photos of species.

4. Genetic distance analysis is recommended.

 

5. Many sequences on NCBI are inaccurate. 

 

Author Response

• Detailed information about the newly generated sequences is presented in Table 1 and Figure 1 (location map). Information on the complete dataset, including Species ID, Location, GenBank Accession Number, and BOLD ID, is provided in Supplementary Table 1. Comparative sequences, which encompass topotypic sequences and other sequences relevant to the context of the manuscript, are highlighted within their respective sections in the Results and Discussion.
• High-resolution figures have been added to the manuscript.
• Added a Figure 4. presents the phylogenetic tree based on the newly generated sequences, including a photo of each species.
• Figure 3 has been added, featuring a matrix diagram that represents pairwise genetic distances among 21 species groups of Indian mullets.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript carried out a phylogenetic analysis of Mullets based on mitochondrial Cytochrome Oxidase Subunit 1 gene fragment. It has employed freshly generated 78 sequences and the sequences retrieved from GenBank. The primary concern is the size of the marker used in the study does not match the claims made by the authors. A short mitochondrial DNA fragment, which is one of the conserved regions of the mtDNA has been used in the study.

Another primary concern is use of tables and figures in the manuscript needs extensive improvement. Table 1 could go into the supplement and authors could provide sampling localities of 78 sequences generated during this study in a map. It would be more meaningful. 

I could not figure out in the manuscript about what was the length (in bp) of the alignment used for the phylogenetic analysis. 

Results: Why is it necessary to construct a phylogenetic tree using all 239 sequences? Take some representative sequences from each group (could be unique haplotypes from each group) and use those sequences to construct a tree. If you want, move this long tree to supplement. Make the images easier for readers to understand. How can readers grasp information from a figure that is itself 4 pages long? AND how 239? Including the outgroup?

Line 240 onwards and Table 2: Is it logical to say that sequences in GenBank are misidentified? Somewhere in the manuscript you have mentioned about possibilities of hybridization. In light of hybridization, how do you justify the claim of misidentification? Your sampling itself is not from the in situ habitats of the Mullets. So, your sampling efforts are not comprehensive. In such case, you might have missed many species. 

Most importantly, mitochondrial barcoding gene (a fragment of Cytochrome Oxidase Subunit 1) is highly conserved. Multiple species can have same identical sequences of barcoding segments. Therefore, you need to be cautious while making this claim of misidentification. 

Table 3 and Table 4. Are they the primary concern of your manuscript? If yes, it should have come in the results, not in the discussion. Authors need to follow the basic practice of writing scientific literature. 

I have made some other comments in the annotated PDF. 

All the best!

 

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minor English improvment needed. 

Author Response

• Over the past two decades, the use of the mitochondrial cytochrome oxidase subunit I (COI) gene in fish species identification has significantly advanced scientific research. Hebert et al. 2003 proposed that a single gene sequence, specifically COI, could effectively differentiate the majority of animal species, solidifying its standing as a global bioidentification system. In 2005, the incorporation of the COI marker into the worldwide program iBOL marked a pivotal moment, emphasizing its global recognition and the extensive documentation in numerous publications. Moreover, COI has played a pivotal role in resolving taxonomic ambiguities, particularly in cases where observable and definitive morphological differences were lacking, as exemplified by studies conducted by Bickford et al. in 2007 and Hyde et al. in 2014. The robustness of COI for species delimitation gained further support when a study by Durand et al. in 2017, employing three mitochondrial markers for Mugilidae phylogeny, yielded results consistent with species delimitation based solely on the COI marker, affirming its reliability in species identification. The practical utility of COI is underscored by the work of Polyakova et al. (2013) and Naz et al. (2023), who effectively demonstrated its capacity to recognize and unambiguously identify fish species.
• Table 1 in the previous version of the manuscript has been relocated to the Supplementary Materials in the present form. Instead, we have introduced a new table that encompasses the details of the 78 newly generated sequences, and we have also included a study map.
• The lengths of the sequences in base pairs (bp) are provided in Table 1, and the length of the outgroup used in the analysis is also mentioned in the manuscript.
• The comprehensive tree with 239 sequences in the previous version of manuscript has been relocated to the Supplementary Material in the present form. We have created two additional phylogenetic trees: first one using representative sequences, comprising a total of 76 sequences, including an out-group, from each clade as delineated in the comprehensive phylogenetic tree, and the second one utilizing 78 newly generated sequences along with an out-group for this analysis.
• We have limited the scope of the current manuscript to a discussion of Mugilidae species diversity in India. Although we recognize the concern regarding the potential misidentification of sequences in the database, we acknowledge that this should be addressed within a broader context through a more comprehensive analysis.
• While it's acknowledged that interspecific hybridization cannot be ruled out when employing the COI gene , it is important to note that there have been no previous reports of natural interspecific hybridization within the Mugilidae family. Furthermore, even in cases where hybridization may be possible, it is unlikely to explain the frequency of instances where discrepancies arise in species identification between morphological characteristics and mitochondrial DNA.
• The inclusion of Table 3 and Table 4 in the previous version of the manuscript served to enhance the overall strength and comprehensiveness of our study. Table 3, which focuses on the discrepancies observed in the morphological characters used in prior studies of Indian mullets, emphasizes the significance of our current research, which employs a molecular approach. Additionally, Table 4 addresses the reliability of topotypic sequences utilized in our analysis. We have relocated both of these tables, containing additional information, to the Supplementary Materials section.