Diet Metabarcoding Reveals Extensive Dietary Overlap between Two Benthic Stream Fishes (Zingel asper and Cottus gobio) and Provides Insights into Their Coexistence

Round 1

Reviewer 1 Report

The authors used metabarcoding approach and field survey to study the dietary overlap of Zingel asper and Cottus gobio collected from three sites respectively in Aug., Sep., and Nov.. The molecular evidence and macroinvertebrate sampling provide very good basis for the discussion of prey preferences, and the analyses are adequate.  However, this MS suffers from some concerns and should be published after major revision.

1. The conclusion of seasonal variations of prey items is problematic because all site are samples once and the results contain no temporal information. If the authors wish to make any seasonal comparisons, they should sample at least twice in different seasons. If not, the experimental design include two variables, sites and season. How could you conclude any variation is a temporal consequence without the consideration of locality? It is better to narrow down the conclusion to report the food contents of these two protected species.
2. Metabarcoding is the first word of the title. However, this word appears for the first time in the M&M section. There is very much room for a better review of this methods and related studies in the very short INTRODUCTION.
3. Primer sequences were not provided or properly cited.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The approach described in the manuscript is promising for the development of fish diet studies.

Broad comments

Introduction: Given the contribution of metabarcoding to the work, it is necessary to include information about the relevance of this method.

Materials and Methods: The information about the studied species is theoretical and would not be in Materials and methods. What determines the choice of seasons? The difference between the selections was only 10 weeks.

Results: The results description is almost clear. However, several aspects of Figure and tables must be ameliorated. See specific comments.

Discussion: Authors correctly discussed the results from the perspective of previous studies and the purpose of the study.

Specific comments

Table 1 – Check dates and seasons, September in Summer?

Figure 1, which is referenced in Materials and Methods, is better divided into 2: map and results separately.  

Table 2 and 3 – You can not specify the season.

Author Response

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Author Response File: Author Response.pdf