PONE-D-24-18040Phylogeographic pattern of chestnut pest Niphades castanea Chao (Coleoptera: Curculionidae) in China based on mtDNA and ITS markersPLOS ONE

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Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The paper studied genetic variance of the chestnut pest Niphades castanea in China. The authors genotyped 170 bettles from several locations.

The paper suffers from lack of scope. The aim formulated by authors was to analyse genetic diversity of the N. castanea " hoping to supply evidence for developing scientific strategies for prevention and control", yet neither results nor discussion addresses this aim.

In my opinion the paper should be considerably shortened. There are many paragraphs that add nothing new to the paper. For instance, second paragraph of the Introduction can be removed, as it states generally known facts about the genetic variance in general. Similarly, the first paragraph of the Discussion is very general and does not add any specific information relevant to the scope of the study.

I am also concerned about the finding of the neutrality tests. In particular, the Tajima's D is sensitive to demographic processes and with any "external" information about processes in the population it is not possible to state whether negative D resulted from purifying selection or population expansion. Any interpretation of the results should be treated with caution. The authors may refer to Pentinsaari et al. 2016 DOI: 10.1038/srep35275

Writing needs some improvement. E.g. in the first paragraph of the Introduction, words as "important" or "particular" are unnecessarily repeated.

Most of the results (e.g. section 3.1, 3.2 and most of the section 3.3) could be summarized in one or two sentences. Authors unnecessary repeat information from tables, which btw should be placed as Supplementary. They also provide many unimportant details such as AC/GC ratio. The only part of the results that really provides information relevant to the aim of the study are haplotype networks and section 3.5. Thus, the results should be revised and considerably shortened.

On the other hand, the core results are not adequately presented. I could not find AMOVA table nor the description. The Fig 6 is hard to read, I suggest providing a classic Fst matrix instead.

I'm not convinced that the selection of the markers was right for the study. In line the authors wrote "ITS1 is a very conserved

gene, which was not suitable to distinguish N. castanea populations at the species levels in this study" which questions its use in the paper.

An important question which is not covered in the discussion is the human impact on the population structure studied in the paper. Neither Introduction nor the Discussion mention that. Yet, I imagine that in the case of a cultivated species, human activities may be a main driver of the population structure. How likely it is that the pests were also transferred between locations by humans e.g. with a harvest? Is the origin of the host trees known? I would expect that at least some of the populations were planted by humans.

Section 2.1. A map of the locations would be helpful. The table itself can go to the Supplementary materials.

Section 2.4. Were sequences from N. castanea from other locations (outside China) included? Why was Dorysthenes paradoxu selected as an outgroup?

Section 3.1 The first paragraph of the Results is in fact description of the study site and should be moved to the Methods.

Section 3.2 is obsolete. Is does not provide any useful information given the goal of the paper.

Table 3. Why were selection tests calculated?

Table 4-6. Should be moved to the Supplementary materials, as the result is better pictured in haplotype networks

Figure 4B and 5B. Why there are some haplotypes not connected to the network?

Figure 6 is unnecessarily complicated. Instead, I suggest presenting classic Fst matrix between studied sites.

66 I can't see what makes N. castanea an "excellent model" for evolutionary studies. There is no arguments supporting this statement.

96 It is not quality of a genome, as the authors didn't study genomes. A gel can visualise a quality of the DNA isolates.

132 What were the selection tests calculated? All markers (ITS and mtDNA) are expected to be selectively neutral

180 This sentence should be rephrased: "amplification products of ITS1 gene WERE FOUND in 143 N. castanea individuals.", not "existed"

181 Relatively low variability of COI (mtDNA) is typical for this gene and mitochondrial markers in general, did the authors expected otherwise?

190 There is no sense in comparing ITS and COI/COII, as one is a nuclear gene, and the others are mtDNA. Simply because of that they are expected to differ in the levels of polymorphisms.

194 I don't think describing AC to GC ratio provides any useful information.

216 Please, don't use "pops" instead of "populations".

348-353 Markers used in the study have well described properties. I see no sense in reporting this as something "find" in the paper. This is was should be known and expected from the very beginning.

354 There is no data supporting this claim. The paper did not provide any data to test whether the changes were "historical" (what I understand as "happened during human presence in the area") or happened before.

Reviewer #2: Mao et al. present an analysis of population genetic structure for Niphades castanea a pest of chestnut. They use mitochondrial COI and COII and nuclear ITS1 region to estimate the relationships among 15 populations. They found population genetic structure based on the mitochondrial genes but little for the ITS1 locus. These are typical patterns found with other beetle population genetic studies. The methods are appropriate and the results are explained in detail. However, the discussion is shallow.

A major issue with the analyses is that the author examined the mitochondrial genes separately. Why? The genes should only differ in the rates of nucleotide substitution but not overall evolution- these genes are inherited as a single unit in the mitochondrial genome. Describing the differences in rates of nucleotide substitution is OK but haplotypes, geneflow, and phylogeny should have been analyzed as one unit including the COI and COII genes. This manuscript would be greatly improved by including analyses that combine these two genes.

Another major issue is the discussion which basically repeats the results and gives some reference to the neutrality of genetic loci. However, the authors do not discuss these results in comparison to other beetle phylogeographic studies in or out of China. This type of discussion would help readers understand if the patterns observed for Niphades castanea are typical or deviant. Adding this type of discussion would greatly improve the manuscript.

Specific issues:

1. There are typographical errors throughout the manuscript. Please review again. Be sure all scientific names are italic. Also, italics are used throughout the manuscript for non-Latin words which I assume is for emphasis. I would advise not to do this but it is a decision for the editor.

2. Maximum parsimony and maximum likelihood are confusedly used in some instances:

Line 146: “…performed till no shorter equally parsimonious trees were obtained.” Like the authors state in the above sentences, IQ-Tree produces maximum likelihood trees thus the tree search would end with a maximum likelihood tree (or set of trees), not most parsimonious trees.

Lines 375-376: “The analysis of COI and COII sequences revealed consistent results between

Maximum Parsimony (MP) and Neighbor-Joining (NJ) analyses.” The use of maximum parsimony was not detailed in the Materials and Methods.

3. Lines 349 -352: “Nuclear marker ITS1 displayed more variability, diversity, GC content, average evolutionary divergence over sequence pairs, and lower indel variability than plastid markers COI and COII. However, ITS1 is a very conserved gene…”. If ITS1 is more variable then how is it more conserved?

4. Table 4- “Information on the COI haplotype of COII shared within different pops” This title does not make sense. Perhaps delete table with given the combine analysis of COI and COII will make this table unnecessary?

Reviewer #3: My major concern lies in the phylogenetic analyses in this study.

The focal tree should rely on the model-based ML analyses, but the author seemed to stick to the out-of-date analyses based on parsimony and NJ tree.

Line 15-16， fruit insect pest of chestnuts (Castanea spp.), Needing rewording.

Lines 36, 379: Castanea and N. castanea in italics

Line 81, ‘N.’ should in full generic name here.

Iine 143, by IQ-TREE (Yu et al. 2020) with maximum likelihood (ML) analysis. Please add details about the parameters, model selection, and model used in the phylogenetic analyses. You cannot only say ‘evaluated by IQ-TREE’; the readers need to know the details.

Line 375, Maximum Parsimony (MP) and Neighbor-Joining (NJ) analyses were compared, but how about the ML method using IQTREE for these gene markers? You should also discuss the differences and why.

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Reviewer #1: Yes: Agnieszka Kloch

Reviewer #2: No

Reviewer #3: No

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Unexpectedly complex distribution pattern of chestnutpest Niphades castanea Chao (Coleoptera: Curculionidae) based on mtDNA and ITS markers

PONE-D-24-18040R1

Dear Dr. YunLi Xiao,

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