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Dear Authors,
I am pleased to accept this revised manuscript for publication in PeerJ - Congratulations! If you could please remove 'Lesperance et al. 2021' reference from the manuscript, following reviewer#2 recommendation noted below. This can be done at the proof stage.
I also take the opportunity to thank the reviewers for their valuable contribution in improving this work.
With warm regards,
Xavier
Final Reviewer#2 comment:
With regards to the changes on what is now line 151, I recommend the authors remove the reference to Lesperance et al., 2021. My intention with the suggestion was for the authors to update methods of LOD determination to more recent protocols that are more specific to the eDNA field, but the Bustin et al, 2009 methods are acceptable here and it would be better to exclude the new reference than to refer to it in this context. No other changes are necessary in my view.
[# PeerJ Staff Note - this decision was reviewed and approved by Robert Toonen, a PeerJ Section Editor covering this Section #]
I am satisfied with the revisions.
With regards to the changes on what is now line 151, I recommend the authors remove the reference to Lesperance et al., 2021. My intention with the suggestion was for the authors to update methods of LOD determination to more recent protocols that are more specific to the eDNA field, but the Bustin et al, 2009 methods are acceptable here and it would be better to exclude the new reference than to refer to it in this context. No other changes are necessary in my view.
No comment
no comment
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Dear Authors,
My apologies for the considerable delay in returning this assessment, but one original reviewer became unresponsive despite multiple reminders, and I've had to find another reviewer at a later stage.
I have now received two independent reviews of your study. While both reviewers clearly recognised the quality/novelty of your work, they have collectively raised a number of minor issues that will need to be addressed in your revised manuscript.
Overall, the reviewers have provided you with excellent suggestions on how to improve the manuscript, and I be looking forward to receiving your revised manuscript along with a point-by-point response to their comments.
With warm regards,
Xavier
Clear language throughout the manuscript.
A more thorough review of reptile eDNA studies would be ideal and also the use of eDNA to broaden verified species distribution records (see “General Comments” below).
Manuscript structure and accompanying figures/tables are good. I recommend slight adjustments to the locator map in Figure 1 (see “General Comments” below) for a broader international audience.
The experimental design is sound. The authors designed a sensitive, probe-based qPCR assay for common musk turtle, with a limit of detection of 10 copies per PCR reaction. eDNA occurrence data across Southern Ontario, Canada was used in conjunction with previously verified occurrence records from the Natural Heritage Information Centre (NHIC) to create Species Distribution Models incorporating climate, elevation and landscape variables.
Methods are described with sufficient information and there is thorough supplementary information for scientists looking to replicate or conduct similar research.
The overall research is original.
The main findings are valid. The musk turtle qPCR primers worked well in both the laboratory and field, with no qPCR detections resulting from non-template controls nor DNA extracts from co-occurring turtles. The species-distribution models incorporating eDNA data predicted greater musk turtle presence towards the north of the study region compared to the NHIC-only model.
Line 35: Maybe change to “abundance and/or presence/absence data”. Is not “presence-only data” still presence-absence data?
Line 41: I wouldn’t say hindering... Conservation and management efforts are not being applied broadly because of an underestimation of the species range.
Line 50: eDNA is certainly used for the non-invasive detection of species. Might be worth looking into whether eDNA (on its own) has been used to re-calibrate species distribution ranges (such as those on IUCN red list) or whether eDNA data needs to be combined with other data sources to provide greater certainty. Its one thing to publish an eDNA paper, another to change verified species distribution ranges – which is what is largely used to guide government conservation and management efforts.
Line 70-74. Re-word; it is a very long sentence.
Figure 1: It is hard to tell as a non-North American where exactly the sampling area is. Please include a zoomed out locator map of Canada/USA with the sampling area highlighted. The current locator map which includes some of the US and Canadian states/provinces is still hard to distinguish.
Line 147: Sub-sub?
Line 236: There are quite a few aquatic reptile eDNA studies now. Please see this latest review (below) to expand your discussion, particularly in relation to reptile-shedding issues and whether it may or may not be relevant to turtle eDNA detectability. A review of applications of environmental DNA for reptile conservation and management - Nordstrom - 2022 - Ecology and Evolution - Wiley Online Library.
The manuscript is well written and follows a good logical flow. The targeted species is ecologically important and refining both aquatic reptile detection methods as well as distribution modelling are valuable areas of eDNA research.
Line 139: Much work has been done on LOD determination in eDNA studies since 2009. I suggest referring to more recent studies (e.g., Lesperance et al., 2021) in order to base your LOD in more eDNA-specific statistics.
Figure 3: The figure title is rather confusing and contains errors. Please clarify what is meant by “B > A or vice-versa”. Readers should not have to refer to supplemental information to understand a figure – I suggest briefly explaining what is meant by B > A or considering another way to visualize this. It is also confusing to have capital letters A and B signifying both the sub-figures and components of your statistical analysis being visualized in the figure.
Sub-figures C and D are described as “subfigure B-D”.
Quadrants ii and iii of sub-figures C and D seem to be switched in the order described.
The fonts on sub-figures C and D are extremely small. I suggest resizing, or if necessary moving some components of this figure to supplemental to make room for larger print.
Table 1: Please define abbreviations in the title or table headings.
While I find your modelling protocol well considered and described, there are several other approaches being taken to tackle this challenge (some specific examples are referred to in Burian et al., 2021, and there are other more recent examples). I would like to see some of these mentioned and compared with in order to justify the use of your workflow.
Line 17: Here and in several other places you refer to an eDNA “protocol” that was developed. Does this refer to the eDNA assay qPCR analysis, or does it include the sampling efforts, interpretation and modelling stages? While any robust sampling effort must be informed by species life history, consider emphasizing what aspects of the workflow were developed to specifically target musk turtle eDNA and what these decisions were based on. If “protocol” merely refers to the assay and qPCR conditions, please clarify this.
Line 113: Phenol-chloroform DNA isolations have been shown to be less replicable than kit-based isolation methods in some studies (e.g., Deiner et al, 2015). Please provide justification for using these methods or note the possible impacts on your study in the discussion.
Line 123: How were species determined to be relevant to alignment? Were no other species considered in the assay design? What about species whose ranges may also be larger than currently known and therefore possibly co-occurring? Mention any resources used to determine species relevance to assay design.
Line 130: How many positive replicates out of 3 were determined to represent a positive or negative result? What was the chosen threshold for replicate number and positive/negative call based on? Please provide more specifics.
Line 134: Is a blank control a no-template control? How many replicates of blank control were performed per plate? Were positive controls used?
Line 135: Which co-occurring turtles were tested? How was DNA sample quality determined? Were neat specimen DNA isolates used to test assay or were they diluted to represent ecologically relevant concentrations? Were permits required for tissue collection?
I am satisfied with the quality of work and validity of findings in this manuscript.
Line 14: Suggest changing “quantify” to more appropriate word such as “determine” or “establish”.
Line 24: Unclear what “additional” eDNA evidence is versus the eDNA detections mentioned in the previous sentence.
Line 30: Again "quantify" seems inappropriate here as you are determining boundaries.
Lines 43-48: Unnecessary to list all conventional methods, especially for other taxa.
Line 93: this sentence belongs in the intro or discussion.
Line 106: “slowing-moving” should be “slow-moving”
Line 113: add space after “buffer”.
Line 119: Extra space between “method” and “in”.
Line 186: Rephrase or change “resulted” to resulting
Line 213: extra space after “presences”.
Line 216: Change additional to “addition”.
Lines 216-218: Confusing sentence, consider revising.
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