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Dear Dr. Seabra and colleagues:
Thanks for revising your manuscript based on the concerns raised by the reviewer. I now believe that your manuscript is suitable for publication. Congratulations! I look forward to seeing this work in print, and I anticipate it being an important resource for groups studying spittlebug systematics. Thanks again for choosing PeerJ to publish such important work.
Best,
-joe
[# PeerJ Staff Note - this decision was reviewed and approved by Nigel Andrew, a PeerJ Section Editor covering this Section #]
Dear Dr. Seabra and colleagues:
Thanks for revising your manuscript. The reviewers are mostly satisfied with your revision (as am I). Great! However, there are a few issues still to entertain. Please address these ASAP so we may move towards acceptance of your work.
Please note that Reviewer 1 kindly provided a marked-up version of your manuscript.
Best,
-joe
The language has been improved in the areas that were pointed out by myself and other reviewers of the previous version. In some newly-added sections, I have suggested a few additional corrections to minor grammatical errors, which I have marked with comments on the attached annotated PDF.
A few specific comments for continuity:
-Both "colonization" and "colonisation" are used in the manuscript. Either is acceptable, but I would stick with one or the other.
-Similarly, both "color" and "colour" are used in the manuscript.
-Both "Structure" and "STRUCTURE" are used in relationship to the model-based population structure analysis performed. I would stick with one or the other.
I also appreciate the authors switching to color-blind friendly colors in their figures, which I believe will improve accessibility for all readers.
I have no additional comments, as the added details provided in response to my and other reviewer's suggestions have addressed my previous concerns.
I have no additional comments.
I am satisfied by and appreciate the edits made by the authors. I would suggest one additional, very minor round of close reading to tighten up the flow, but am otherwise looking forward to seeing the final manuscript published.
No comment
No comment
No comment
The authors of "Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data" have adequately responded to all of the feedback from my initial review and have made all necessary revisions. At this point, I feel that the manuscript is prepared for publication in PeerJ.
Dear Dr. Seabra and colleagues:
Thanks for submitting your manuscript to PeerJ. I have now received three independent reviews of your work, and as you will see, the reviewers raised some concerns about the research. Despite this, these reviewers are optimistic about your work and the potential impact it will have on research studying microbial community structure in aquifers associated with arsenic. Thus, I encourage you to revise your manuscript, accordingly, taking into account all of the concerns raised by the three reviewers.
Please aim to improve the overall clarity of the manuscript, especially by broadening the background and hypotheses presented in the Introduction, adding more descriptions in the Material sand Methods, describing the biology of P. spumarius beyond your current text, and improving the accessibility of the figures.
Fortunately, there is a lot of additional criticism here for you to consider (mostly minor). If you choose to address these concerns, I encourage you to revise your work and resubmit. However, I strongly encourage you to take into account all of the concerns raised by the reviewers. While the concerns of the reviewers are relatively minor, this is a major revision to ensure that these original reviewers have a chance to evaluate your responses to their concerns.
Please note that Reviewer 1 kindly provided a marked-up version of your manuscript.
I look forward to seeing your revision, and thanks again for submitting your work to PeerJ.
Good luck with your revision,
-joe
The language is understandable and professional throughout, but there are occasional grammatical errors or oddities and a number of long sentences. The long sentences in particular can make the point of a section a little hard to grasp. I have commented on some of these items in the annotated PDF, but a close reading for maximum clarity would be beneficial prior to full acceptance.
The figures are illustrative of the major findings of the work and have text that is of a readable size. I will note however that the color scheme used is not color-blind friendly, and that the resolution of the images in my copy of the PDF were not very high, and so some items were a bit pixelated.
I checked and was able to access the raw data and scripts associated with analyses in this study.
The methods as outlined are generally sufficient to allow replication, barring a handful of concerns/suggestions.
First, it is not clear how the insect specimens were collected, nor how they were stored between the collection date and use in the various experiments. I think it would be appropriate to add in these details (~L185).
Second, why does the sampling strategy for P. tesselatus from Morocco (7 individuals from multiple sites) differ so much from the sampling strategy employed for P. spumaris (L192)? Is it a protected or harder to locate species? I don't think this impacted the results of the 9 population work (the differentiation in the RAD data is strong), and the authors do exclude that population from another set of analyses and discuss how this difference in sampling may have impacted summary statistics, but I do think a justification of the sampling difference would be good to include.
Are the sampling locations selected for each country representative of the variation expected (especially for areas where the spittlebug is introduced)?
What type of PE sequencing was used? 2x100? (L248)
Next (L306), although I think 10 runs of STRUCTURE are probably sufficient to get a sense of the general pattern, I would have liked to see more replicate runs performed (perhaps 50-100). The authors could consider using the program ADMIXTURE (http://dalexander.github.io/admixture/), which uses the same statistical model but performs faster, if computational resources were a concern limiting replicate run number.
Related (L308), it is not explicitly stated if the deltaK method is used for selecting the best K (although the citations within the harvester program utilized by the authors suggest it was).
Although I think the authors treat their results with appropriate caution in the discussion and when drawing ultimate conclusions of their findings, I was struck by two results in particular.
First, the relatively small number of SNPs is surprising given A) the large number of reads recovered per individual, and B) the fairly permissive (p = 0.5) missing data filter allowed in calling SNPs from STACKS. Although the authors do note that the genome size is large, given that the average coverage is ~100x for the recovered loci and the proportion of heterozygous sites observed, it seems like there should be more SNPs. As their results were the same after removal of ~10% of their dataset (see second point), I don't necessarily think their results were strongly impacted in this case, but will note that population structuring methods can be influenced by relatively slight changes in allele frequency in smaller datasets.
Secondly, I am also a little surprised by how many Fst outliers were detected in this dataset (almost 10% of markers), although this may be a reflection of the wide range of locations and limited sampling sites that the organisms were sampled from, as the authors note.
I think these results build nicely on previous work in the system by providing a genomic-scale glimpse at differentiation, as well as by integrating some phenotypic data from the male genitalia. This work also contributes some explanations for both previously noted and newly discovered findings, which seem plausible when considered in the light of the insect's natural history information. As noted in several locations by the authors in the discussion, this work also provides a nice starting point for future work that can clarify the genetic landscape, degree of gene flow between populations of P. spumaris, and this species interactions with P. tesselatus.
I have attached an annotated PDF with suggestions for improving the clarity of the manuscript (see Basic Reporting section) as well as on some methodological questions (see experimental design).
The manuscript reports relevant findings about Philaenus population structure. The experimental questions and the way the authors pursued them are thoroughly described.
The experimental design is solid
The results are well reported and clearly discussed
The article provides relevant insights about Philaenus genera.
I suggest minor changes that might improve the draft:
1) I consider the difference between spumarius and tesselatus at nuclear genome level, while no differentiation is possible by just mitochondrial analysis, as the main finding of this paper. Try to better stress in the astract the importance of developing alternative and integrative approaches for the taxonomy of a group of species, instead of centering the discussion of the reflection of your findings on Xylella fastidiosa control.
2) I would remove any reference to Xylella fastidiosa transmission, since it is not clear how your findings could contribute to the development of a control strategy either directly or indirectly
In the manuscript “Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data”, the authors use a combination of morphological, mtDNA, and RAD-sequencing methodologies to explore the evolutionary relationship between Philaenus spumarius and P. tesselatus. These species of spittlebugs are found throughout Europe, as well as in Northern Africa and the USA and they are of particular concern because they are a vector for a bacterial plant pathogen, X. fastidiosa. Previous work has identified P. tesselatus and P. spumarius as two distinct species using morphological data, but this is not reflected using mtDNA. The authors use collections from 9 sites in Europe, Africa, and North America to first, determine if genomic data can clarify the status of P. tesselatus and next, to look for evidence of selection on P. spumarius populations. The RAD-seq data supports previous claims that P. spumarius and P. tesselatus are two distinct species, despite a lack of evidence using mtDNA haplotypes. Further, they find evidence for positive selection on P. spumarius populations that is correlated with a handful of environmental variables as well as a signal of isolation by distance among the populations. They also find evidence to support the origin populations of several colonization events in the USA and AZO sites. The authors conclude with a discussion of the limitations of their work and the possibility that Wolbachia has had an impact on the mitochondrial DNA in P. spumarius, explaining the lack of differentiation in mtDNA compared to both morphological and RAD-seq data.
In general, I feel that this manuscript is an excellent example of the ways that genetic, genomic, and morphological data can be used together to generate convincing results about the evolutionary history of organisms. The authors utilize a widespread dataset with an appropriate number of specimens included at most sites. They analyzed their datasets with caution that is reflected in their methodology and further discussed in the discussion section. This work also contributes to the current understanding of drivers of evolution and speciation and offers new considerations as to the impacts of Wolbachia on mtDNA analyses. For these reasons, I feel that this manuscript would be of general interest to the readers of PeerJ. However, I have several recommendations that need to be addressed to improve the clarity of the manuscript, particularly to provide more explanation in the methods, broaden some of the ideas presented in the introduction, better explain the biology of P. spumarius, and improve the accessibility of the figures.
In the introduction, the authors could expand their explanation of the importance of understanding the evolutionary history of P. spumarius for models of risk assessment, as described in Lines 108-112. Addressing this more specifically would be helpful to understand why Xylella fastidiosa is relevant to the main topics of the manuscript when the methods/results are focused on the biology of P. spumarius.
All Figures: The combination of red and green is not an accessible combination of colors for people that are colorblind. Consider using alternative colors to benefit these readers, especially in the Structure plots that lack an alternative method to visualize the differences between groups.
Throughout the manuscript, some sentences could be restructured to improve clarity and ease of reading. For example, Lines 350-351 says “We did not exclude any variable at the start of the study based on their correlations, but we tested for correlation the ones that were significantly associated.” I think the second part of this sentence could be revised to make it easier for readers to understand when the authors tested for correlation. I have included a few more similar examples in the comments below.
Experimental design adequately tests the research questions and successfully pairs morphological and genetics data together.
The bioinformatic and statistical methods included an appropriate amount of caution throughout and I appreciated the authors' discussion of possible caveats as part of the discussion section.
Lines 185-193: It is unclear from the methods section how insects were collected. If P. spumarius is polyphagous, were insects collected off of a single host, multiple host plant species, or by using an alternative method? These collection methods could have an impact on the authors findings, as heterozygosity and population structure could also be influenced by host use or attraction to a pheromone bait or by variation in collection methods across sites. Further, has work been done to assess host preferences or host-associated differentiation within P. spumarius or is the species truly indiscriminate among the host plants listed? In general, a broader discussion of the biology of P. spumarius would be helpful – including mention of generation time and life cycle.
Lines 203-210: The description of aedeagus measurements does not include an explanation about how the measurement data was collected – manually or using a computer program for morphometric analysis? It would also be helpful to describe any data preprocessing that occurred before the PCAs described in the next paragraph.
All underlying data has been provided and is accessible on NCBI if not included in the supplemental materials. See general comments below for a couple links that failed to work in the methods section.
Discussion includes some speculation about the impact of Wolbachia on the mtDNA findings. The authors make it clear that this includes some speculation and I feel the commentary is an interesting addition to the end of the manuscript.
Minor Concerns
Line 124: Capitalize “new” in “New Hampshire”
Line 221: Revise “including RNase A treatment step” to say “including an RNase A treatment step”
Line 246: The link included in this line requires a university login and was inaccessible to me. Please consider revising this to a more publicly available source.
Line 250: The link in this line is out of date and no longer maintained. Please consider updating it to their new website.
Line 261: Change “ran” to “run”
Line 318: Change “correlation” to “correlations”
Line 386: Should the word “variables” be changed to “variable”? I’m not entirely sure what this sentence is trying to say. Please consider revising for clarity.
Lines 486-487: Consider revising this sentence for clarity – “However, there were a few individuals that were genetically more similar to others more distant geographically,…”
Lines 587-589: What is known about the phylogenetic relationship and/or divergence timing between P. spumarius, P. tesselatus, and other species found in the region? P. spumarius and P. tesselatus appear to be closely related, but I am curious about the remaining species in the region that the authors describe in the introduction as additional contributors to gene flow.
Line 632: Consider removing “however” from this sentence for clarity.
Line 762: Some references are in a smaller font than the rest of the manuscript.
Figure 2 Legend: Italicize Philaenus in the first line.
Figure 3: Is it possible to move the vector labels to make them easier to read without the overlapping data points?
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