Alternative DNA Markers to Detect Guam-Specific CRB-G (Clade I) Oryctes rhinoceros (Coleoptera: Scarabaeidae) Indicate That the Beetle Did Not Disperse from Guam to the Solomon Islands or Palau

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article [diversity-3046504] entitled (Alternative DNA markers to detect Guam-specific CRB-G (clade I) Oryctes rhinoceros (Coleoptera: Scarabaeidae) indicate that the beetle did not disperse from Guam to the Solomon Islands or Palau) needs major revision for publication.

Here, some of comments to the authors

Comments

1-      All citation in the text should be numerical NOT as [authors, year]

2-      There is difficulty to mention line numbers, thus, it should be added in the MS

3-      add family and order of all scientific names of insects as Spodoptera frugiperda and Helicoverpa

4-      What is the meaning of vs. in [2014 vs. Arnemann et al. 2018]. Do you mean [and]?

5-      Table 1: write only [Table 1. Details of Oryctes rhinoceros (CRB) samples used in this study] as a title, and remove the remaining in the text.

6-      The same comment for Table 2 and add explanations as footnotes

7-      Figure 1: abbreviate the legend and explain the other information in the text

8-      At the beginning of page 12: remove this citation to discussion section.

9-      Add conclusion to indicate the important results and recommendations

10-   References should be as MDPI style

Author Response

R1

The article [diversity-3046504] entitled (Alternative DNA markers to detect Guam-specific CRB-G (clade I) Oryctes rhinoceros (Coleoptera: Scarabaeidae) indicate that the beetle did not disperse from Guam to the Solomon Islands or Palau) needs major revision for publication.

Here, some of comments to the authors

Comments

1- All citation in the text should be numerical NOT as [authors, year]

[Authors] Fixed

2- There is difficulty to mention line numbers, thus, it should be added in the MS

[Authors] noted

3- add family and order of all scientific names of insects as Spodoptera frugiperda and Helicoverpa

[Authors] Fixed

4- What is the meaning of vs. in [2014 vs. Arnemann et al. 2018]. Do you mean [and]?

[Authors] we have changed it to ‘versus’

5- Table 1: write only [Table 1. Details of Oryctes rhinoceros (CRB) samples used in this study] as a title, and remove the remaining in the text.

[Authors] We have updated Table 1 details, but we feel that certain details were needed to enable readers to understand the table without needing to refer back to the main text, including e.g., gene names and basic explanation of ‘GenBank Accession number’ as sample codes.

6- The same comment for Table 2 and add explanations as footnotes

[Authors] Updated and simplified. As we have made significant changes to the revised manuscript, the explanations were no longer needed and have been removed.

7- Figure 1: abbreviate the legend and explain the other information in the text

[Authors] Fixed

8- At the beginning of page 12: remove this citation to discussion section.

[Authors] We have not understood what the reviewer has requested here, so we have not taken any action on this point.

9- Add conclusion to indicate the important results and recommendations

[Authors] Fixed

10- References should be as MDPI style

[Authors] Fixed

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Tay et al. present an alternative mtDNA loci for the identification of CRB from Guam. They discovered these loci through the sequencing of mitogenomes.  They use phylogenetic analysis to show that the standard COI locus lacks resolution while the new loci ATP6 and COIII resolve the Guam CRBs as monophyletic. These new diagnostic markers provide valuable tools for the study of RCB. In addition, the authors conclude that Guam is not the source of CRB found in other localities.  All analyses appear to be executed correctly. However, additional analyses would provide more detailed insight to the spread of RCB to different locations and thus, the conclusions may need revision. 

 

Major suggestions to improve this manuscript.

 

In the introduction the authors present the use of mitogenomes as a great advantage over using just one locus.  But yet, they do not use the mitogenomes they generated for this study to reconstruct a phylogeny.  A phylogeny reconstructed with whole mitogenomes could result in a different topology then presented in Fig. 1a,b and thus refute their conclusion that that Guam is not the source of CRB found in other localities

 

Furthermore, using the phylogeny based on APT6 and COIII (Fig. 1b) to conclude that Guam is not the source of CRB found in other localities is not appropriate.  These loci were chosen because they include diagnostic SNPs (like synapomorphies) thus biasing the phylogenetic analyses for a monophyletic group of Guam individuals.  The entire mitogenomes should be used to reconstruct the phylogeny so to reduce author bias in locus choice.

 

In addition, once a phylogeny based on mitogenomes is reconstructed the authors should estimate the likelihood of the geographic origins for the internal nodes of the phylogeny using Statistical Dispersal Extinction Cladogenesis with software such as BioGeoBEARS.  This analysis will provide the authors and readers a detailed likelihood of Guam as the source of other RCB introductions.   

 

Minor issues:   

 

Page 2: “focus on this mitocnondrial haplotype” correct to, “focus on this mitochondrial haplotype”

 

Page 9: “It is likely that future detailed genetic assessments of CRB will provide the resolving power required to further elucidate CRB invasion histories” This sentence is in a different font as the rest of the article. 

 

Comments on the Quality of English Language

No major issues detected. 

Author Response

R2

Comments and Suggestions for Authors

Tay et al. present an alternative mtDNA loci for the identification of CRB from Guam. They discovered these loci through the sequencing of mitogenomes. They use phylogenetic analysis to show that the standard COI locus lacks resolution while the new loci ATP6 and COIII resolve the Guam CRBs as monophyletic. These new diagnostic markers provide valuable tools for the study of RCB. In addition, the authors conclude that Guam is not the source of CRB found in other localities. All analyses appear to be executed correctly. However, additional analyses would provide more detailed insight to the spread of RCB to different locations and thus, the conclusions may need revision.

[Authors] We thank R2 for the review and input. Whilst the reviewer is correct that we wished to demonstrate Guam as not the source of other “CRB-G” found in other locations, we wished to highlight that this study was not to “provide more detailed insight to the spread RCB [sic] to different location”. To do so would require significantly more samples and especially of many more native CRB populations.

 

Major suggestions to improve this manuscript.

In the introduction the authors present the use of mitogenomes as a great advantage over using just one locus. But yet, they do not use the mitogenomes they generated for this study to reconstruct a phylogeny. A phylogeny reconstructed with whole mitogenomes could result in a different topology then presented in Fig. 1a,b and thus refute their conclusion that that Guam is not the source of CRB found in other localities

[Authors] The mitogenome phylogeny based on 13 PCGs was constructed and the result was almost identical to the two-genes phylogeny that we presented in Fig. 1b. To support this, we have now included it as a supplemental figure. We further elaborated this in our responses below.

Note that Fig. 1a was the phylogeny that would have been obtained by continual usage of the partial mtCOI gene which is the current and widely accepted approach. It is not the approach that we are advocating in this study.

 

Furthermore, using the phylogeny based on APT6 and COIII (Fig. 1b) to conclude that Guam is not the source of CRB found in other localities is not appropriate. These loci were chosen because they include diagnostic SNPs (like synapomorphies) thus biasing the phylogenetic analyses for a monophyletic group of Guam individuals. The entire mitogenomes should be used to reconstruct the phylogeny so to reduce author bias in locus choice.

[Authors] We respectfully disagreed with the reviewer that our conclusion “that Guam is not the source of CRB found in other localities is not appropriate.” for the fact we clearly stated in L172 – 174 (of the original submitted manuscript version) that “The assembled and annotated mitogenome from the Guam 04-Or5 specimen (collected in 2014) provided evidence that all Guam individuals examined here shared the same mitogenome (Table 1).” To further clarified this, we have now edited the sentence by adding the word “fully” in L187 such that it reads “The fully assembled and annotated mitogenome from the Guam 04-Or5 specimen (collected in 2014) provided evidence that all Guam individuals examined here shared the same mitogenome (Table 1) that was uniquely identified only on Guam” (now L187-189).

Synapomorphies are relevant largely for between species comparison/phylogenetic analysis, where the concern of shared derived characters could influence on placement of basal and derived phylogenetic positions between species. For understanding of within species genetic relationship especially based on non-recombination genetic marker systems such as the maternally inherited mitochondrial genomes, synapomorphies (i.e., shared characters such as SNPs) are derived from their shared (i.e., inherited) maternal lineages, and the unique SNPs identified in the true ‘CRB-G’ were not shared with other populations (i.e., not shared with other ancestral ‘common’ characters/SNPs with other populations).

The unique SNPs in ATP6 and COIII in Guam CRB distinguished this maternal lineage from other CRB maternal lineages, and was based on genome survey of full mitochondrial genome dataset, and this helped to avoid misinterpretation/biases for monophyly of Guam from potential synapomorphic trait-associated complications of ancestral relationship with other CRB populations surveyed.

The aim of this study was to demonstrate the presence of unique SNPs that would more accurately differentiate Guam CRB (i.e., CRB-G) from other non-Guam (and therefore misidentified) ‘CRB-G’, and to support this, we therefore chose to present a phylogeny that was constructed based on the two gene regions of the diagnostic SNPs. Identification of the unique SNPs was based on whole mitochondrial genome data and the respective phylogeny (with same topological patterns) now also presented in Fig. S2.

We also constructed the phylogeny based on the 13 mitochondrial genome protein coding genes, excluding tRNAs, rRNA, intergenic regions (due to presence of gaps), as well as the control region (due to length differences and difficulties of sequencing/assembly) to confirm that the topology of the phylogeny remained almost identical as the topology of the phylogeny reported in Fig. 1b in the manuscript. Specifically, based on the 13 mitochondrial genome PCGs, Guam, Philippines, Indonesia and Malaysia clustered as their own clades, and introduced Palauan individuals clustered with both Philippines and Indonesia. The only difference being the placement of the Solomon Islands CRB individual in a separate branch by itself. Philippines remained the clade closes to Gaum overall, while one individual (Phil-01) shown to cluster strongly with Guam. We have provided the mitogenome phylogeny as a supplemental figure.

 

In addition, once a phylogeny based on mitogenomes is reconstructed the authors should estimate the likelihood of the geographic origins for the internal nodes of the phylogeny using Statistical Dispersal Extinction Cladogenesis with software such as BioGeoBEARS. This analysis will provide the authors and readers a detailed likelihood of Guam as the source of other RCB introductions.   

[Authors] The suggestion by the reviewer to further analyse the phylogeny using BioGeoBEARS is noted. We would, however, like to point out that this study was not aimed to identify the origin/s of the invasive populations, but rather to demonstrate that in the current published literature where the CRB-G has been linked with widespread rapid dispersal to other Pacific Island nations largely reflected limitations of the partial mtCOI marker, and hence, in incorrectly identified CRB-G on other non-Guam Pacific Island nations. To identify the native origins of various introduced CRB populations would require surveying of many more native populations and significantly increasing the sampling size per population. The current examples of native and introduced CRB populations included less native than introduced populations, i.e., only three native range populations (i.e., Philippines, Indonesia, Malaysia) and four introduced populations (i.e., Taiwan, Solomon Islands, Guam, Palau) and is therefore inappropriate to be used to infer/estimate native range (i.e., more native range populations and greater number of individuals per native population would be needed to ensure confidence in identifying the potential origin of a particular invasive population).

 

Minor issues:   

Page 2: “focus on this mitocnondrial haplotype” correct to, “focus on this mitochondrial haplotype”

[Authors] Fixed

Page 9: “It is likely that future detailed genetic assessments of CRB will provide the resolving power required to further elucidate CRB invasion histories” This sentence is in a different font as the rest of the article. 

[Authors] Fixed

Reviewer 3 Report

Comments and Suggestions for Authors

Invasions of alien organisms are a global environmental problem with historical background and uncertain prospects associated with changes in biodiversity and obvious economic damage. The latter is determined by the strategy of invaders, who are pests of agriculture and forestry, carriers of new pathogens for aquaculture, poisonous to farm animals and humans, as well as allergens.

To control and limit invasion processes, it is necessary to establish the routes of introduction, including donor populations, including the analysis of natural factors limiting numbers, for example, specific pathogens and predators.

Currently, molecular biology methods have established donor populations, including for insects, for example, the Colorado potato beetle (Schoville et al., 2018). confirms a one-time introduction to Europe after a major genetic bottleneck.

The species Oryctes rhinoceros damages palm trees, including coconut and oil palm trees.

The natural range is from tropical Asia, from India and the Maldives, to the Philippines and southern China.

Accidentally introduced to the tropical islands of the Pacific Ocean - Samoa (1909) from Ceylon, where it penetrated and settled on the nearest islands from 1912 to 1963), later penetrated to Guam (2007), Hawaii (2013), Solomon Islands (2015) , Vanuatu and New Caledonia (2019).

Over the years, many species of predators and parasitoids have been identified, such as the Oryctes rhinoceros nudivirus (OrNV) virus, and many species have been introduced to the invaded Pacific islands (doi: 10.1093/aesa/saaa057), with little control or regulation available. except for labor-intensive manual collection. Accurate identification of the donor population, where there was a long-term co-evolution of the invader species and consumer organisms: predators, parasites and other pathogens, will allow us to identify promising agents for population regulation.

It should be noted that different haplotypes may have different resistance to pathogen infection. The CRB-G haplotype noted in Guam appears to be resistant to Oryctes rhinoceros nudivirus (OrNV) (Marshall et al., 2017, https://doi.org/10.1016/j.jip.2017.07.006) , later found a high incidence of OrNV infection in all three haplotypes (CRB-G, CRB-PNG and CRB-S) in the Pacific region (Etebari et al., 2021).

In this regard, the study is relevant, including for assessing resistance to pathogens.

 

Notes:

1. For a better understanding of the material, it is advisable to supplement the article with a map, taking into account the authors’ conclusion that Oryctes rhinoceros from the island of Guam was not a source for secondary invasion.

2. In the “Introduction” section, it is generally desirable to indicate the objects on which the research was conducted, references (Hebert et al., 2004; Rugman-Jones et al., 2013; Tay et al., 2016; De Barro et al., 2011 ; Behere et al., 2007; Tay et al., 2017a). I believe that attention should be paid to the analysis of study data specifically using nuclear and mitochondrial markers, supplementing this section, for example, with data (Reil et al., 2016; Etebari et al., 2021). The latter work notes that “mitochondrial lineages are not an accurate reflection of the dispersal history.”

Thus, I believe that the authors need to provide justification and a more detailed analysis of the chosen research methods and markers. There is information in the “Discussion” section, but it is not structured enough and speaks about research prospects, which is more suitable for the “Conclusions” section. For example, “Given that the WGS/multigene approaches can provide more comprehensive evidence than single gene analyzes (e.g. partial mtCOI), and we have found exactly this result with this analysis, we suggest that a detailed study using these more detailed genetic assessments is needed to further improve current understanding of CRB invasion biology."

2. Link to unpublished data “The CRB is a hitchhiker pest (Hoffmann et al. in press) and is continuing to disperse to new locations, being recently reported in the Marshall Islands (The Marshall Islands Journal 2023) and multiple Hawaiian islands (HDOA 2024)” does not allow reviewing this paragraph.

In general, it is necessary to improve the design and formatting of the text, to characterize the identified haplotypes using different markers of Oryctes rhinoceros (Linnaeus, 1758).

Author Response

R3

Comments and Suggestions for Authors

Invasions of alien organisms are a global environmental problem with historical background and uncertain prospects associated with changes in biodiversity and obvious economic damage. The latter is determined by the strategy of invaders, who are pests of agriculture and forestry, carriers of new pathogens for aquaculture, poisonous to farm animals and humans, as well as allergens.

To control and limit invasion processes, it is necessary to establish the routes of introduction, including donor populations, including the analysis of natural factors limiting numbers, for example, specific pathogens and predators.

Currently, molecular biology methods have established donor populations, including for insects, for example, the Colorado potato beetle (Schoville et al., 2018). confirms a one-time introduction to Europe after a major genetic bottleneck.

The species Oryctes rhinoceros damages palm trees, including coconut and oil palm trees.

The natural range is from tropical Asia, from India and the Maldives, to the Philippines and southern China.

[Authors] We have not included Maldives as the natural range because we are unsure where this authority was from. Multiple authorities including Level (1969) have identified the natural range of the CRB as from India to Philippines and southern China, the Maldives was not mentioned, although it was mentioned to be present also in the Maldives in Bedford (1980) citing Catley (1969), and subsequently cited by studies as part of the CRB’s native range. We reconfirmed that Catley (1969, PANS 15, 18-30) did not mention the Maldives as part of the CRB’s native range, nor was the Maldives included in the map as a native range (Cately 1969, Fig. 1), and we therefore have chosen to omit the Maldives as part of the CRB’s native range. Further, OrNV was required to be introduced to the Maldives to manage the CRB, indicating that this was likely an introduced population that established without the co-evolved OrNV pathogen.

Accidentally introduced to the tropical islands of the Pacific Ocean - Samoa (1909) from Ceylon, where it penetrated and settled on the nearest islands from 1912 to 1963), later penetrated to Guam (2007), Hawaii (2013), Solomon Islands (2015) , Vanuatu and New Caledonia (2019).

Over the years, many species of predators and parasitoids have been identified, such as the Oryctes rhinoceros nudivirus (OrNV) virus, and many species have been introduced to the invaded Pacific islands (doi: 10.1093/aesa/saaa057), with little control or regulation available. except for labor-intensive manual collection. Accurate identification of the donor population, where there was a long-term co-evolution of the invader species and consumer organisms: predators, parasites and other pathogens, will allow us to identify promising agents for population regulation.

It should be noted that different haplotypes may have different resistance to pathogen infection. The CRB-G haplotype noted in Guam appears to be resistant to Oryctes rhinoceros nudivirus (OrNV) (Marshall et al., 2017, https://doi.org/10.1016/j.jip.2017.07.006) , later found a high incidence of OrNV infection in all three haplotypes (CRB-G, CRB-PNG and CRB-S) in the Pacific region (Etebari et al., 2021).

In this regard, the study is relevant, including for assessing resistance to pathogens.

Notes:

1. For a better understanding of the material, it is advisable to supplement the article with a map, taking into account the authors’ conclusion that Oryctes rhinoceros from the island of Guam was not a source for secondary invasion.

[Authors] We have now provided a map showing where our analysed samples originated from.

 

2. In the “Introduction” section, it is generally desirable to indicate the objects on which the research was conducted, references (Hebert et al., 2004; Rugman-Jones et al., 2013; Tay et al., 2016; De Barro et al., 2011 ; Behere et al., 2007; Tay et al., 2017a). I believe that attention should be paid to the analysis of study data specifically using nuclear and mitochondrial markers, supplementing this section, for example, with data (Reil et al., 2016; Etebari et al., 2021). The latter work notes that “mitochondrial lineages are not an accurate reflection of the dispersal history.”

Thus, I believe that the authors need to provide justification and a more detailed analysis of the chosen research methods and markers. There is information in the “Discussion” section, but it is not structured enough and speaks about research prospects, which is more suitable for the “Conclusions” section. For example, “Given that the WGS/multigene approaches can provide more comprehensive evidence than single gene analyzes (e.g. partial mtCOI), and we have found exactly this result with this analysis, we suggest that a detailed study using these more detailed genetic assessments is needed to further improve current understanding of CRB invasion biology."

[Authors] We thank the reviewer for the suggestion. We have now provided a new paragraph (L76-L86) to link and justify why reassessment of the mtCOI marker is needed

 

2. Link to unpublished data “The CRB is a hitchhiker pest (Hoffmann et al. in press) and is continuing to disperse to new locations, being recently reported in the Marshall Islands (The Marshall Islands Journal 2023) and multiple Hawaiian islands (HDOA 2024)” does not allow reviewing this paragraph.

[Authors] All of these citations are publicly accessible, and the details are provided in the References.

In general, it is necessary to improve the design and formatting of the text, to characterize the identified haplotypes using different markers of Oryctes rhinoceros (Linnaeus, 1758).

[Authors]: We thank R3 for this suggestion. The text has now been substantially changed, with a new full mitogenome protein coding gene phylogeny provided (Fig. S2) to support the identified haplotypes differentiating Guam CRB-G (clade I) from other CRB.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors did all my comments, thus, i suggest to be accepted as it is

Author Response

no comments from R1 

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript is an improvement. 

Author Response

no comments from R2

Reviewer 3 Report

Comments and Suggestions for Authors

The reviewer thanks you for your responses to comments. The authors made significant changes to the manuscript. Responses to comments have been received.

Comment: 

1. In the text, you must include a link numbered 61 “61. Cheng, C. T.; Jeng, M. L.; Tsai, J.F.; Li, C. L.; Wu, L.W. Two mitochondrial genomes of Taiwanese rhinoceros beetles, Oryctes rhinoceros and 781 Eophileurus chinensis (Coleoptera: Scarabaeidae). Mitochondrial DNA B Resour 2021, 6, 2260-2262, doi:10.1080/23802359.2021.1948364.”

Author Response

Comment 1: In the text, you must include a link numbered 61 “61. Cheng, C. T.; Jeng, M. L.; Tsai, J.F.; Li, C. L.; Wu, L.W. Two mitochondrial genomes of Taiwanese rhinoceros beetles, Oryctes rhinoceros and 781 Eophileurus chinensis (Coleoptera: Scarabaeidae). Mitochondrial DNA B Resour 2021, 6, 2260-2262, doi:10.1080/23802359.2021.1948364.

Reply: The reference #61 was already included in the main text (in Table 1 section). We have re-formatted the references and ref. #61 is now #46.