Diverse winter communities and biogeochemical cycling potential in the under-ice microbial plankton of a subarctic river-to-sea continuum

ABSTRACT Winter conditions greatly alter the limnological properties of lotic ecosystems and the availability of nutrients, carbon, and energy resources for microbial processes. However, the composition and metabolic capabilities of winter microbial communities are still largely uncharacterized. Here, we sampled the winter under-ice microbiome of the Great Whale River (Nunavik, Canada) and its discharge plume into Hudson Bay. We used a combination of 16S and 18S rRNA gene amplicon analysis and metagenomic sequencing to evaluate the size-fractionated composition and functional potential of the microbial plankton. These under-ice communities were diverse in taxonomic composition and metabolically versatile in terms of energy and carbon acquisition, including the capacity to carry out phototrophic processes and degrade aromatic organic matter. Limnological properties, community composition, and metabolic potential differed between shallow and deeper sites in the river, and between fresh and brackish water in the vertical profile of the plume. Community composition also varied by size fraction, with a greater richness of prokaryotes in the larger size fraction (>3 µm) and of microbial eukaryotes in the smaller size fraction (0.22–3 µm). The freshwater communities included cosmopolitan bacterial genera that were previously detected in the summer, indicating their persistence over time in a wide range of physico-chemical conditions. These observations imply that the microbial communities of subarctic rivers and their associated discharge plumes retain a broad taxonomic and functional diversity throughout the year and that microbial processing of complex terrestrial materials persists beneath the ice during the long winter season. IMPORTANCE Microbiomes vary over multiple timescales, with short- and long-term changes in the physico-chemical environment. However, there is a scarcity of data and understanding about the structure and functioning of aquatic ecosystems during winter relative to summer. This is especially the case for seasonally ice-covered rivers, limiting our understanding of these ecosystems that are common throughout the boreal, subpolar, and polar regions. Here, we examined the winter under-ice microbiome of a Canadian subarctic river and its entry to the sea to characterize the taxonomic and functional features of the microbial community. We found substantial diversity in both composition and functional capabilities, including the capacity to degrade complex terrestrial compounds, despite the constraints imposed by a prolonged seasonal ice-cover and near-freezing water temperatures. This study indicates the ecological complexity and importance of winter microbiomes in ice-covered rivers and the coastal marine environment that they discharge into.

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Reviewer #1 (Comments for the Author): The authors analyzed microbial communicites and metagenomic composition of winter samples from the Great Whale River and its plume into Hudson bay.The study provides insights into the winter microbiome of ice-covered subarctic rivers and associated coastal marine watersOverall, the study is well-designed and the manuscript is written nicely.I only have several minor questions.
2. what happened to the >3um fraction of the "R.3" sample, as it is missing in Figure 3 and 4 3.The authors found prokaryote community compositiondiffered primarily by size fraction and then by sampling group, but opposite for eukaryotes.They need to discuss this findings in more detail.4. Important comparisons should be marked if they are significant.
Reviewer #2 (Comments for the Author): The authors investigated diversity and community structure of bacteria and eukaryotes using metabarcoding approaches in two size fractions of plankton along an ice-covered estuarine gradient of a subarctic river.Meanwhile, they also examined geochemical cycling-relevant genes to infer functional potential using metagenomics.They found the bacterial community structure was different between <3 and > 3 micron size fractions, but not for eukaryotes of these two sizes.They concluded that the microbial communities of subarctic rivers and their associated discharge plumes retain a broad taxonomic and functional diversity throughout the year.This work contributes by documenting the microbial diversity and spatial changes in a very special environment and season.Although the manuscript is well written, there are flaws that well-known nutrients like dissolved inorganic nitrogen species, dissolved silicate, and phosphate data are lacking.Apart from that, several aspects can be improved during revisions.
There are discussions on environmental factors influencing community composition of bacteria and microeukaryotes (lines 448-479).However, this is largely descriptive, without any statistic support.Multivariate analysis such as RDA or CCA can be performed.In fact, correlations between environmental variables and alpha diversity should be performed to specify the points in discussion as well.
Lines 246, 352, 372, 375, 376 and so on: Statistical significance should be supplied for these comparisons."The prevalence and abundance of genes associated with the nitrogen cycle indicate that nitrogen fixation was likely absent during the winter, whereas nitrification and denitrification were likely operating." This does not make sense to me.The abundance of nif genes in the metagenomes was relative, not absolute quantity.Also bearing in mind what you are looking into is the genetic potential, instead of the expressed genes (activities).

Important comparisons should be marked if they are significant.
Response: The limnological variables comparisons are descriptive given the low number of replicates per sampling group (three), which limits the statistical power of analyses such as ANOVA, as does the presence of missing values (see . In response to this review comment we have rephrased part of the text to clarify that when we indicate greater abundance of a gene in a sampling group, we are referring to the results of the differential abundance analysis of KOs that were involved in pathways, reactions and modules presented in Figure 5 and that were found to be significantly differentially abundant (adjusted p-values ≤ 0.01; tested with package DESeq2 that can handle low numbers of replicates) along the river between the shallow and the deeper sites (RSh vs. R), and in the vertical plume profile between surface water and brackish water at 4 m depth (PS vs. P4M).S2 and S3) presenting the DESeq2 results for these KOs (log2fold change and adjusted p-values).
d) To make the text easier to read, we opted not to include the p-value after every comparison as they were all significant.However, we have added the following sentence for clarification in the first paragraph of this result section: "In the following section, when genes are indicated to be differentially abundant in one of the sampling groups, we refer to these identified genes".(Lines 312-313).

Reviewer 2:
The authors investigated diversity and community structure of bacteria and eukaryotes using metabarcoding approaches in two size fractions of plankton along an ice-covered estuarine gradient of a subarctic river.Meanwhile, they also examined geochemical cycling-relevant genes to infer functional potential using metagenomics.They found the bacterial community structure was different between <3 and > 3 micron size fractions, but not for eukaryotes of these two sizes.

Response:
We thank the reviewer for this very good summary, but would like to clarify one point.We also found differences in microbial eukaryote communities, both in terms of community structure, as indicated by a significant PERMANOVA result (Lines 270-271 in the revised manuscript), and in terms of richness, as indicated by the Wilcoxon signed-rank test for paired samples result (Lines 291-292).
RSh vs R; panel a) and between plume surface and brackish water (PS vs P4M; panel b).The genes shown are restricted to those implicated in the reactions outlined in Figure 5 for nitrogen, carbon and sulfur metabolism and photosynthesis/pigments." c) We added two supplementary tables (Supplementary Table S2 and S3) presenting the DESeq2 results for these KOs (log2fold change and adjusted p-values).
d) To make the text easier to read, we opted not to include the p-value after every comparison (Lines 352 and so on) as they were all significant.However, we have added the following sentence for clarification in the first paragraph of this result section: "In the following section, when genes are indicated to be differentially abundant in one of the sampling groups, we refer to these identified genes".(Lines 312-313).Response: Figure 5 provides an overview of the pathways/reactions discussed in the results.Standard deviations were added to illustrate the variability of the sums within each sampling group.Due to the small sample size (3) per group, which resulted in low statistical power, we refrained from conducting statistical analysis to compare the sum of normalized gene abundances (reads/recA reads) between sampling groups.This is for this same reason we did not perform ANOVA on our limnological variables.Instead, we chose to assess the differential abundance of each KO involved in the pathways/reactions presented in Figure 5, as this analysis is suitable for low numbers of replicates.The following sentence was added to the legend of Figure 5: "Significantly differentially abundant genes (adjusted p-values ≤ 0.01) between shallow and deeper river sites and between plume surface and brackish water are presented in Figures 6a and 6b." "The prevalence and abundance of genes associated with the nitrogen cycle indicate that nitrogen fixation was likely absent during the winter, whereas nitrification and denitrification were likely operating." This does not make sense to me.The abundance of nif genes in the metagenomes was relative, not absolute quantity.Also bearing in mind what you are looking into is the genetic potential, instead of the expressed genes (activities).
Response: We agree that this was overextending.The sentence was changed to "The prevalence of genes associated with nitrogen fixation was lower than for nitrification and denitrification."(Lines 493-494).
March 5, 2024 1st Revision -Editorial Decision Re: Spectrum04160-23R1 (Diverse winter communities and biogeochemical cycling potential in the under-ice microbial plankton of a subarctic river-to-sea continuum) Dear Dr. Marie-Amelie Blais: Thank you for your efforts in revision by following the reviewers' comments.Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Figure 5 :
Figure 5: Error bars have been annotated, but significance was lacking for all these comparisons.
a) We added the threshold p-value (≤ 0.01) used to determine KOs for which the z-score was calculated, at Line 247.b) We changed the legend of Figure 6a and 6b, it now reads: "Figure 1. a-b) Z-score (SD from the row mean, calculated from normalized gene abundance reads/recA reads) of significantly differentially abundant genes (adjusted p-values ≤ 0.01) between shallow and deeper river sites (RSh vs R; panel a) and between plume surface and brackish water (PS vs P4M; panel b).The genes shown are restricted to those implicated in the reactions outlined in Figure 5 for nitrogen, carbon and sulfur metabolism and photosynthesis/pigment." c) We added two supplementary tables (Supplementary Table

Figure 5 :
Figure 5: Error bars have been annotated, but significance was lacking for all these comparisons.