Temperature sensitivity of the interspecific interaction strength of coastal marine fish communities

Reviewer #1 (Public Review):

The authors have studied the effect of temperature on the interspecific interaction strength of coastal marine fish communities, using eDNA samples. Their introduction describes the state of the art concerning the dynamics of interspecific interactions in ecological communities. This introduction is well written and highly information dense, summarizing all that the reader needs to know to further understand their study setup and execution.

The authors hypothesize that water temperature changes could have an effect on the interspecific interaction strength between marine fishes, and they studied this with a two year long, bi-weekly eDNA sampling campaign at 11 study sites in Japan with different temperature gradients. These 550 water samples were analysed for fish biodiversity through eDNA-metabarcoding using MiFish primers. By using the most abundant fish species as an internal spike in and quantifying the copy numbers from this species by qPCR, the authors were able estimate DNA copy numbers for the total dataset. From the 50 most frequently detected fish species in these samples they showed that temperature affected the interspecific interaction strength between some species. Their work provides a highly relevant approach to perform species-interaction strength analysis based on eDNA biodiversity assessments, and as such provides a research framework to study marine community dynamics by eDNA, which is highly relevant in the study of ecosystem dynamics. The models and analytical methods used are clearly described and made available, enabling application of these methods by anyone interested in applying it to their own site and species group of interest.

Strengths: The authors have a study setup that is suitable to measure the effects of temperature of the eDNA diversity, and have taken a large number of samples and all appropriate controls to be able to accurately measure and describe these dynamics. The applied internal spike in to enable relative eDNA copy number quantification is convincing.

Weaknesses: The authors aim to study the relationship between species interaction strength and ecosystem complexity, and how temperature will influence this. However, there is only limited ecological context discussed explaining their results, and a link with climate change scenario's is also limited. A further discussion of this would have strengthened the manuscript.

The authors were able to find a correlation between water temperature and interaction strengths observed. However, since water temperature is dependent on many environmental variables that are either directly or indirectly influencing ecosystem dynamics, it is hard to prove a direct correlation between the observed changes in community dynamics and the temperature alone.

Reviewer #2 (Public Review):

In this work Ushio et al. combine environmental DNA metabarcoding with novel statistical approaches to demonstrate how fish communities respond to changing sea temperatures over a seasonal cycle. These findings are important due to the need for new techniques that can better measure community stability under climate change. The eDNA metabarcoding dataset of 550 water samples over two years is, I feel, of sufficient scale to provide power to detect fine-scale ecological interactions, the experiments are well controlled, and the statistical analysis is thorough.

The major strengths of the manuscript are: (1) the magnitude of the dataset, which provides densely replicated sampling that can overcome some of the noise associated with eDNA metabarcoding data and scale up the number of data points to make unique inferences; (2) the novel method of transforming the metabarcode reads using endogenous qPCR "spike-in" data from a common reference species to obtain estimates of DNA concentration across other species; and (3) the statistical analysis of time-series and network data and translating it into interaction strengths between species provides a cross-disciplinary dimension to the work.

I feel like this kind of study showcases the power of eDNA metabarcoding to answer some really interesting questions that were previously unobtainable due to the complexities and cost of such an exercise. Notwithstanding the problems associated with PCR primer bias and PCR stochasticity, the qPCR "spike-in" method is easy to implement and will likely become a standardised technique in the field. Further studies will examine and improve on it.

Overall I found the manuscript to be clear and easy to follow for the most part. I did not identify any serious weaknesses or concerns with the study, although I am not able to comment on the more complex statistical procedures such as the "unified information-theoretic causality" method devised by the authors. The section on limitations of the study is important and acknowledges some issues with interpretation that need to be explained. The methods, while brief in parts, are clear. The code used to generate the results has been made available via a GitHub repository. The figures are clear and attractive.