PONE-D-22-34041Niche overlap across landscape variability between two large herbivores using dietary eDNA metabarcoding in the Białowieża Primeval forest (Poland)PLOS ONE

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1. Feces were only sampled in 1 month (June 2019) thus it seems the title and hypotheses could be edited to focus more on what you were actually able to test given the limitations of your sampling schedule and eDNA of samples collected.

Recommended title: Niche overlap across landscape variability during summer between two large herbivores using dietary eDNA metabarcoding

2. It is not clear why the models are presented in the Results instead of the Methods?

3. Please remove V and replace with "among-individual diet variation." It does not seem like it is necessary to reference this by V each time it is presented and I am also not sure why it is defined twice (Methods and Results).

4. As mentioned by both reviewers, predation risk needs more detail and justification. It appears that predation risk is based solely on the herbiscapes but the reader should not have to read that to determine predation risk. Instead of a half sentence (Line 147), more details on how "Predation risk was inferred based on landscape use..." is needed. Otherwise, the validity of your assessment needs to be determined based on reviewing Bubnicki et al. 2019.

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript aims at assessing diet composition and niche overlap within and between red deer and European bison, and correlating such information with habitat quality and predator density, to support management actions. The approach is sound and mostly well explained, I just miss a comparison of the results with information on plant availability in the area and the inclusion of the species movement abilities while discussing the results.

General comments

• I honestly feel that using this approach to estimate predator abundance is an over ambitious goal, since other disturbances and the overall landscape of fear could influence this result. Talking of perception of predator density from the prey species is acceptable, but it should be discussed mentioning the possibility that other factors could also contribute to such perception.

• I missed a parallel with the observed diet composition and plants available in the areas considered. eDNA cannot be used to infer proportions in the diet, however, detecting abundant or scarce species in the diet would provide valuable information in this context.

• The species movement should be considered when discussing the results, as scats are not deposited where consumption occurred.

Specific comments

L36, L109, Fig.1: red deer instead of Red deer.

L65-66: this aspect could be better highlighted, since diet composition influences the overall body condition of the species, but also its impact on the vegetation.

L82: probably here the authors meant “study areas”.

L117: Probably better to replace “It” with “The area” or something alike.

L121: without forestry nor ungulate management.

L122: Considering the previous sentence mentions the whole Polish part of the forest, the BNP and the state forest, I’d suggest to replace “It” with a more explicit subject here.

L149: It seems like “both” refers to predation risk + habitat quality here, but I think the authors likely meant both species, please clarify.

L154-156: Where steel beads used in this process? Usually, nitrogen is added when working with plant material, so I was wondering if the authors used the steel beads as recommended instead.

L162-168: the lab procedure is sound and sufficiently explained, I would just suggest to add a reference to the supplementary information and specify the information on the Qubit Kit.

L171-172: Please specify the filtering criteria and add a reference to the supplementary information.

L174: the filtering in this step is clearly explained and sound.

L176-177: this sentence might be a bit challenging for readers with no previous experience with the method, I’d suggest making it a bit more beginner friendly.

L205: Please break down these values per species as well.

Fig.1: is not colour blind friendly. Additionally, please use a larger font for the x-axis.

L212-216: please briefly mention the differences among areas as well.

Supplementary Fig. 3: Please add test values.

L237-238: very good point!

L279: “in summer” is not really necessary here.

L283-284: This is consistent with NMDS results.

L285-288: as mentioned elsewhere, this statement is not fully supported by your results, so it should be better addressed here. Or you could move lines 301-304 here.

L304: could also be playing a role.

Reviewer #2: The authors conduct an interesting study of bison and red deer diets in the Bialowieza ecosystem. They find interesting associations between diet and ecosystem variables and they use appropriate metabarcoding methods. I believe the authors should consider their terminology in some places and consider the value of using both V and overlap. I also believe this is a good study and can be appropriate for publication in PLoS ONE after revisions.

Comments

Abstract: I do not believe that intraspecific dietary dissimilarity is an adequate indicator of predation risk; I think this section of the Abstract should be dramatically tempered. Without a comprehensive survey of possible contributing factors to red deer dietary dispersion (and wolf risk being the only strong predictor of it), it would be disingenuous to recommend that ecosystem managers solely need to measure red deer diets to quantify predation risk.

L73: there is a parenthesis missing

L89-98: please add a description of what the herbiscapes are in Bialowieza so the reader has an understanding of Bubnicki et al's results without needing to look up their paper; adding this will help center the reader on your study system and understand what factors go into herbiscapes. Please also add a citation to the Bialowieza-specific study

Throughout: I am also a bit uncomfortable with using niche overlap to describe intraspecific diet similarity. I realize that the author's use Pianka's overlap to quantify this so it technically is niche overlap, but I wonder if it might be easier to term the intraspecific values something like "dietary similarity" and reserve the term "niche" for the interspecific comparisons.

Methods: The section "Herbiscapes" needs to come before the section "Sample collection" because on L135 the authors state "per area per species" yet the reader has not been told that there are five areas yet

DNA metabarcoding methods: What was the told number of reads per library? It seems quite strict to remove sequences with <100 reads when many studies either remove only singletons or those with <10 reads

Methods: why use V and overlap for within species comparisons? Don't they show the same thing?

Fig.2: please add some color to the NMDS; it will make it much easier to distinguish the species

L227: "Food quality" should be "Habitat quality", right?

L227: The model structure is not completely clear to me; are the categorical predictors nested within species? Is this like having the categorical predictor as a fixed effect and per-species random intercepts?

L238-239: there is only two types of species interactions here (potential competition/facilitation between deer and bison; herbivory between the two species and their food plants). I can guess that the authors are referring to bison intraspecific overlap, deer intraspecific overlap, and deer-bison overlap. If I'm right, please rewrite this part to state that clearly, currently it is not clear.

Modelling methods: I'd like some more justification about the modelling approach for niche overlap. Why include "species interaction" as a predictor rather than just having three separate models (one for each species' intraspecific comparisons and one for interspecific overlap)? This will make it easier to interpret model output. Given that Area is just a proxy for predation risk and habitat quality, why not test those directly from the start (with Area as a random intercept)? It doesn't seem like there is much need for the Overlap ~ Area model

L283: there's not really a relationship between NMDS dispersion and dietary diversity at the population level; the NMDS is showing dietary differentiation among deer samples not higher diversity in the population diet; of course those two things may be correlated, but deer could still have a highly diverse diet and be completely clumped in the NMDS (e.g. all deer eat the same very diverse diet)

L286-287: I would intuitively agree with the statement that bison are more grazers than deer, but your data do not support this. Graminoids are a tiny fraction of both species' diets and deer eat more graminoids than bison (Supp Fig 1). Given that your samples were collected in summer, when bison should eat the most amount of graminoids according to the literature, this makes your results all the more important, and provides an important counterpoint to the idea that bison are not good browsers.

L296: Fig. 3 does not show dietary diversity. I suggest rephrasing as dietary dispersion

L299: Given than bison in Bialowieza are not grazers (based on your results), does it make sense to attribute this effect to grazing

L304: do bison forage in forest gaps in Bialowieza?

L296-308: alternative explanations for lower intraspecific dietary differentiation in bison. Deer have narrower muzzles enabling them to be more selective when foraging and potentially creating between-individual variation when low-biomass species are eaten; bison have larger digestive systems allowing for more dietary mixing over time and potentially more similarity between individuals; deer are more head-up foragers than bison creating more potential for vertical niche partitioning between deer individuals

L316-317: wouldn't greater overlap be related to behavioral similarities rather than differences?

L320: could mention the role of resource abundance and palatability in determining niche overlap. If there are just a few common plants, it's likely there will be high overlap. If there are a few plants of high quality, both species should eat them

L390-394: I suggest removing these sentences; I do not think you can make this claim without a comprehensive assessment of red deer diet dissimilarity across sites and predictor variables.

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Niche overlap across landscape variability during summer between two large herbivores using dietary eDNA metabarcoding in the Białowieża Primeval forest (Poland)

PONE-D-22-34041R1

Dear Dr. Dear Dr. Mas-Carrió,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

I appreciate the authors revisions and the detailed explanation for each point raised by myself and both reviewers.

Reviewers' comments: