Warming and altered precipitation independently and interactively suppress alpine soil microbial growth in a decadal-long experiment

Reviewer #1 (Public Review):

Warming and precipitation regime change significantly influences both above-ground and below-ground processes across Earth's ecosystems. Soil microbial communities, which underpin the biogeochemical processes that often shape ecosystem function, are no exception to this, and although research shows they can adapt to this warming, population dynamics and ecophysiological responses to these disturbances are not currently known. The Qinghai-Tibet Plateau, the Third Pole of the Earth, is considered among the most sensitive ecosystems to climate change. The manuscript described an integrated, trait-based understanding of these dynamics with the qSIP data. The experimental design and methods appear to be of sufficient quality. The data and analyses are of great value to the larger microbial ecological community and may help advance our understanding of how microbial systems will respond to global change. There are very few studies in which the growth rates of bacterial populations from multifactorial manipulation experiments on the Qinghai-Tibet Plateau have been investigated via qSIP, and the large quantity of data that comprises the study described in this manuscript, will substantially advance our knowledge of bacterial responses to warming and precipitation manipulations.

Reviewer #2 (Public Review):

Summary: The authors aimed to describe the effect of different temperature and precipitation regimes on microbial growth responses in an alpine grassland ecosystem using quantitative 18O stable isotope probing. It was found that all climate manipulations had negative effects on microbial growth, and that single-factor manipulations exerted larger negative effects as compared to combined-factor manipulations. The degree of antagonism between factors was analyzed in detail, as well as the differential effect of these divergent antagonistic responses on microbial taxa that incorporated the isotope. Finally, a hypothetical functional profiling was performed based on taxonomic affiliations. This work gives additional evidence that altered warming and precipitation regimes negatively impact microbial growth.

Strengths: A long term experiment with a thorough experimental design in apparently field conditions is a plus for this work, making the results potentially generalisable to the alpine grassland ecosystem. Also, the implementation of a qSIP approach to determine microbial growth ensures that only active members of the community are assessed. Finally, particular attention was given to the interaction between factors and a robust approach was implemented to quantify the weight of the combined-factor manipulations on microbial growth.

Weaknesses: The methodology does not mention whether the samples taken for the incubations were rhizosphere soil, bulk soil or a mix between both type of soils. If the samples were taken from rhizosphere soil, I wonder how the plants were affected by the infrared heaters and if the resulting shadow (also in the controls with dummy heaters) had an effect on the plants and the root exudates of the parcels as compared to plants outside the blocks? If the samples were bulk soil, are the results generalisable for a grassland ecosystem? In my opinion, it is needed to add more info on the origin of the soil samples and how these were taken.

The qSIP calculations reported in the methodology for this work are rather superficial and the reader must be experienced in this technique to understand how the incorporators were identified and their growth quantified. For instance, the GC content of taxa was calculated for reads clustered in OTUs, and it is not discussed in the text the validity of such approach working at genus level.

The selection of V4-V5 region over V3-V4 region to quantify the number of copies of the 16S rRNA gene should be substantiated in the text. Classic works determined one decade ago that primer pairs that amplify V3-V4 are most suitable to assess soil bacterial communities. Hungate et al. (2015), worked with the V3-V4 region when establishing the qSIP method. Maybe the number of unassigned OTUs is related with the selection of this region.

Report of preprocessing and processing of the sequences does not comply state of the art standards. More info on how the sequences were handled is needed, taking into account that a significant part of the manuscript relies on taxonomic classification of such sequences. Also, an OTU approach for an almost species-dependent analysis (GC contents) should be replaced or complemented with an ASV or subOTUs approach, using denoisers such as DADA2 or deblur. Usage of functional prediction tools underestimates gene frequencies, including those related with biogeochemical significance for soil-carbon and nitrogen cycling.

Reviewer #3 (Public Review):

Summary: In this paper, Ruan et al. studied the long-term impact of warming and altered precipitations on the composition and growth of the soil microbial community. The researchers adopted an experimental approach to assess the impact of climate change on microbial diversity and functionality. This study was carried out within a controlled environment, wherein two primary factors were assessed: temperature (in two distinct levels) and humidity (across three different levels). These factors were manipulated in a full factorial design, resulting in a total of six treatments. This experimental setup was maintained for ten years. To analyze the active microbial community, the researchers employed a technique involving the incorporation of radiolabeled water into biomolecules (particularly DNA) through quantitative stable isotope probing. This allowed for the tracking of the active fraction of microbes, accomplished via isopycnic centrifugation, followed by Illumina sequencing of the denser fraction. This study was followed by a series of statistical analysis to identify the impact of these two variables on the whole community and specific taxonomic groups. The full factorial design arrangement enabled the researchers to discern both individual contributions as well as potential interactions among the variables

Strengths: This work presents a timely study that assesses in a controlled fashion the potential impact of global warming and altered precipitations on microbial populations. The experimental setup, experimental approach and data analysis seem to be overall solid. I consider the paper of high interest for the whole community as it provides a baseline to the assessment of global warming on microbial diversity.

Weaknesses: While taxonomic information is interesting, it would have been highly valuable to include transcriptomics data as well. This would allow us to understand what active pathways become enriched under warming and altered precipitations. Non-metabolic OTUs hold significance as well. The authors could have potentially described these non-incorporators and derived hypotheses from the gathered information. The work would have benefited from using more biological replicates of each treatment.