A social niche breadth score reveals niche range strategies of generalists and specialists

Generalists can survive in many environments, whereas specialists are restricted to a single environment. Although a classical concept in ecology, niche breadth has remained challenging to quantify for microorganisms because it depends on an objective definition of the environment. Here, by defining the environment of a microorganism as the community it resides in, we integrated information from over 22,000 environmental sequencing samples to derive a quantitative measure of the niche, which we call social niche breadth. At the level of genera, we explored niche range strategies throughout the prokaryotic tree of life. We found that social generalists include opportunists that stochastically dominate local communities, whereas social specialists are stable but low in abundance. Social generalists have a more diverse and open pan-genome than social specialists, but we found no global correlation between social niche breadth and genome size. Instead, we observed two distinct evolutionary strategies, whereby specialists have relatively small genomes in habitats with low local diversity, but relatively large genomes in habitats with high local diversity. Together, our analysis shines data-driven light on microbial niche range strategies.


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Reviewer #1 (Remarks to the Author):
In this manuscript Bastiaan von Meijenfeldt et al. present the results of their work on using a large dataset of environmental sequencing data to explore niche range strategies in different microbial taxa. The approach they use is to compare the communities that each microbial taxa resides in and based on that define their niche breadth. This is an interesting approach and could potentially highlight the physicochemical dimensions of their niche or whether they have promiscuous or specific interactions with other members of the community. Having a score for social niche breadth sounds exciting but having a rigorous and consistent way to interpret this score is equally important. While this score could be a mean for relative comparisons, there are some inconsistencies in the provided discussions that need clarification. In the results section there are remarks as low/high local diversity, low/high social niche breadth, low/high diversity specialists but the thresholds for these delineations are not clear to the reader. It is essential to clarify these in the text. I see the meadian value provided in Figure 2d but still don't know how the SNB values provided in the text should be interpreted. For example in this section of the results SNB values are very close and difficult to interpret without prior knowledge of ecological strategies of these taxa. " Taxa with relatively low SNB for their ranks include known specialists such as Christensenella (SNB = 0.24), but also the taxa Pelagibacteraceae (SNB = 0.25) and Prochlorococcus (SNB = 0.22), which hold some of the most abundant organisms on Earth. These taxa, known for their highly streamlined genomes, are found in aquatic samples with a uniform microbial composition (Supp. fig. 5b) and thus have a low SNB. The genus Roseobacter (SNB = 0.30), whose members are considered marine generalists with large genomes and a versatile metabolism, is found in more diverse samples (Supp. fig. 5b) and has an SNB close to the median of all genera." For example, different representatives of family Pelagibacteraceae dominate marine and freshwater ecosystems (SAR11 vs LD12 respectively) and the overall community of marine and freshwater ecosystems are rather different. One would argue that at the family level Pelagibacteraceae should be rather generalist than specialist. The title of this section "Generalists compete for dominance, specialists are stable at low abundance" does not seem to corroborate with the dominance of what you identify as specialists in the paragraph before (i.e., Pelagibacteraceae and Prochlorococcus) I highly recommend authors to add a brief description of the type of data they obtain from MGnify regarding the community composition and what are the main steps in these analyses and how it differs for metagenomes, amplicon datasets and metatranscriptomes. How these different analysis pipelines used for each as authors call "experiment type" will affect their comparative analysis. The alpha diversity obtained by metagenome and amplicon data for the same sample is not the same, many low abundance taxa might not be detected in the metagenome or multiple ribosomal operons could affect the abundance calculations based on amplicon analysis, etc… will such differences impact their analysis? In the analyses provided in this manuscript each piece of data is collected from a different source and as I understand genomes are not reconstructed from the same datasets used for community comparisons how could this affect the interpretations of the pangenome, genome size, etc.
Methods: I have some comments regarding the datasets used in this study.
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-In experiment type section it is confusing as to what authors mean by category "unknown"? this could be very confusing and could potentially affect the results in ways that are difficult to discuss or conclude.
-Also, I would not say that abundance calculations based on metagenomes and amplicon is comparable to those calculated from metatrnscriptomes. In the metatrsncriptome datasets, activity of each taxa is affecting the final representation of reads affiliated to this taxa whereas in the other two methods abundance is not affected by the activity per se. I would suggest removing metatranscriptomes from this study.
-What is the threshold for differentiating between saline and non-saline. Does this refer to marine salinity or hypersaline? Clumping a large range of salinity together would be misleading.
I understand all the limitations caused by unknown ranks and taxa in meta-analysis. It would be great if authors discuss how different approaches they have tested for dealing with the unknown taxa would affect their results and conclusions. This could be a section in the supplementary results and discussions.
Since in the submitted file there was no line number, I copy here the part of text I am referring to in each comment. Minor comments: -All figures are packed with datapoints but presented in very small sizes both for the main figures and the supplementary figures. I highly recommend authors to take advantage of the available space and make their plots larger and less compact.
-I find the first sentence of the abstract disconnected from the rest of the abstract text -implying a low turnover rate and stable community composition of highly diverse habitats Maybe this could be concluded if these datasets are timeseries of the same sampling point otherwise how the comparison of alpha and beta diversity could signal low turnover rate? -Most annotated biomes have low beta diversity reflecting consistent microbial composition. It could be reformulated to mention that these biomes have low beta diversity indexes in comparison with each other inside one biome -We used the observation that microbiomes are biosensors Please add references -Niche breadth across the tree of life and at different taxonomic ranks Prokaryotic tree of life -The distribution of SNB differs per taxonomic rank, where high-ranking taxa tend to be generalists and low-ranking taxa specialists I agree with the explanation authors provide after this sentence, but I still feel resistance towards how this sentence in formulated. Clumping these high taxonomic ranks specifically for ubiquitous phyla such as proteobacteria with high diversity of ecological strategies in their representatives could be very misleading. I recommend some reformulation in this paragraph. -Indeed, the four best-studied phyla, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria… I don't think the higher representation of these taxa in solely due to them being more studied. Most environment are dominated by representatives of these taxa in metagenomic studies as well.
-Candidate phyla have a low SNB compared to established phyla What do you mean by established? Maybe validly described? Although I don't think having a candidate status has an actual effect on the SNB. Maybe it is a side effect of most these candidate phyla being Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. among rare taxa? I think here you mean candidate phyla radiation rather than candidate phyla in general? -Phyla with relatively specialised genera include… Please provide stats on how prevalent specialized genera are in these phyla compared to the phyla with more generalized genera -In conclusion, species in specialist genera are genomically more similar than species in generalist genera What do you mean here, genome size? Pangenome size? Genomic content and functions? -in the section about coding density and streamlining please add the name of relevant taxa. Are these specialists animal associated groups with streamlined genomes or free living groups with streamlined genomes such as SAR11? There are know differences in the streamlining route for these groups. -as they require specific partners in their local communities I am curious as your data could provide any hint towards whether these groups have specific hosts or rather a diverse range of partners in different environments? -In cases where identical genomes had different taxonomic taxa What do you mean by taxonomic taxa? Taxonomic affiliation maybe? -A function was considered present in a species if at least 50% of its genomes contained it. This sounds odd to me. Do you mean as a part of the core or in general? This threshold needs more explanation to make the logic clearer.
-carried out via the false discover rate (FDR) as suggested in discovery Reviewer #2 (Remarks to the Author):

Comments to the Author
The article entitled "On specialists and generalists: niche range strategies across the tree of life" reports an analysis of environmental sequencing datasets publicly available to assess for each possible taxon the degree of variation of its surrounding community. To do so, the authors introduce a new metrics named SNB for Social Niche Breadth reflecting the community similarity between samples in which a specific microbial taxon occurs, with this score being low for organisms always detected in compositionally similar samples (called here social specialists) and this score being the highest for organisms that are found in sample with distinct community structure (social generalist). The SNB scores are then analysed in regards to biomes, metabolism, genomic features and compared to previous work on generalist and specialist features and (a little bit) to the ecological theory. The implementation of this new metric is exciting and opportune in the meta'omic era, and could be more easily adopted by the research community with some improvements of the manuscript, that are developed in the following, and that could also include a conceptual schema of SNB. The objectives of this work could also be more clearly stated, to clarify if the aim is here introducing a new metric and comparing it to the ones currently is use, or if it is to extract new features for understanding generalist/specialist (evolutionary strategy, …). The postulates related to the creation of SNB score could also be formalised. The article is well concise and globally easy to read, with the notable exception of the partitioning of Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
the main text and supplementary text that has to be revised in my opinion. The English is fluid and no typos were noted.
Major comments 1 -While 'Social niche breadth (SNB)' is often stated in the text, 'Niche breadth' is also used throughout the manuscript, with sometimes an ambiguity between the "metabolic" niche breadth (the historical definition of niche breadth, i.e. similarly to Hutchinson niche; also sometimes called "general niche breadth" in the manuscript) and the SNB introduced in this work. Similarly, a carful revision of the text where, 'generalist' and 'specialist' are rename 'social generalist' or 'social specialist' when opportune (similarly to 'habitat generalist' or 'habitat specialist' in other publications to differentiate with the historical names of generalist/specialist that refers to observations of the metabolic flexibility) would clarify the manuscript. (e.g. "for the vast majority of taxa, the SNB score is lower than expected based on random permutations (Fig. 2a-c), indicating that all microbes are *[SOCIAL]* specialists to some extent because they occur in a non-random subset of all samples."; "Whereas local communities are typically dominated by generalists, the relative abundance of generalists is more variable across samples than that of specialists, whose abundance is relatively stable.": Are you here mentioning *social* generalists and specialists accordingly to some of your results, or is it a general statement, that would then need a reference?). Similarly, 'diversity', 'alpha-diversity' and sometimes 'richness' are used. The definition of 'diversity' is so much dependent of the authors (and the readers!) that I would advice to uniquely use one of the latters.
2 -Some concepts or justifications are not well enough developed (or referenced), and data interpretations are not always in the main text. Mainly, expressing clearly the rationales and hypothesis behind the new concept of SNB would be necessary to fully exploit the provided discussion and comparison to previous study (e.g. relationship between niche breadth and genome size, …) a-Information on SNB score are all at the end of the manuscript in the methods section. Although it is the right place to present the formular, a bit of background (for example stating that SNB is a dissimilarity score based of Spearman correlation) would be useful at the beginning of the results, where the score is introduced. Indeed, most of the recent publications treats social interactions using networks and metrics based on graph theory, and it would be useful making clear very early on that this is not the case here. b-How is SNB score filling a gap in ecological studies? How does is compare to trophic niche breadth or habitat niche breadth, etc, that are named in the introduction? How does the SNB score compare or differ from co-occurrence networks or any other microbial interactions network used in a massive number recent studies? Why is SNB read out more accurate / appropriate to target ecological question such as deterministic vs neutral assembly process mentioned in the introduction? Etc. Please, describe better the advantages of SNB in the context of the state of the art. c-The traditional definition of ecological niche breadth includes the fundamental and the realised niche. This latter is the n-dimensional space that a taxon actually inhabits in the presence of interspecific competition. Similarly, are you scoring the fundamental social niche breadth? I have the sensation that you are hypothesizing that the sum of all realized social interactions found in your 22 thousand of sample results in the fundamental social niche? Have you checked if the number of samples allows to 'saturate' the list of social partners for at least most of the taxon?
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. d-What are the postulates in the background of SNB? Probably at least that genetic information of the SSU summarize the metabolic capacity of an organism and that metabolic capacity determines the biogeographical distribution of the taxon. Also, a taxon is considered actively inhabiting in a biome sample if DNA is in sufficient quantity to produce at least 1 read per 10 000. Etc e-Strong statement such as "Also, it remains difficult to quantify the degree of similarity between categorical biomes." would deserve a justification or a good reference. f-Data interpretations better read in the text than in the figure captions.
3 -Data analysis: a-The paragraph "Taxa with relatively low SNB for their ranks include known specialists such as Christensenella […] also have a high SNB, highlighting the diversity of their anaerobic habitats." is comparing SNB score-based assessment of niche breadth with more traditional metabolically-base niche breadth assessment. This could make a lot more sense if the hypothesis behind SNB and how it compared to previously introduced assessment of lifestyle would be discussed first (see also comment 2b and 2c). Is this paragraph, for the authors, a kind a validation that their SNB score is accurate to determine niche breadth? b-"… indicating that genomic signatures of SNB are generalizable across habitat and phylogeny": This hold true only if you demonstrate first that SNB scores have a genomic signature. c-"…at the genus rank we found that specialists are younger than generalists ( Fig. 3b)": Can you explain how you conclude that from this figure? I was unable to track this information. d-"The correspondence between community heterogeneity and genomic heterogeneity confirms …": I don't know where to find that in the data. What are the metrics used to assess "community heterogeneity" (SNB?) and "genomic heterogeneity" (heterogeneity of the independent genomic features?). Which statistical test was used? e-The final result section "Generalist pan genomes reflect fluctuating habitats, low and high diversity specialists differ in metabolic adaptations", social niche breadth is compared to metabolic niche breadth. This is a very important step to know if SNB is a metrics that allow to characterize the specificity of community assembly or of SNB can also be used as a proxy for metabolic niche breadth. However, no clear conclusion are made. The related figure 5 is also difficult to examine: would it be possible to have the data in a bar chart, with bar length being the 'fraction of genome' and the orientation of the bar indicating if enriched in social specialist (negative) or social generalist (positive)? f-Maybe more of curiosity questions: have you seen a difference in average SNB at the domain level between archea and bacteria, as archea are supposably a very specialized domain? It seems that you have sample with no bacteria ( "Samples from similar annotated biomes cluster together based on…"; "Samples are separated by host-association and salinity"; "Most samples from the plants biome are derived" from seagrasses and macro algae from kelp forests, which is why they cluster near marine samples"; "Taxa richness differs per annotated biome and taxonomic rank. The low number of annotated species is a consequence of a Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. relatively unexplored biosphere". "Annotated biomes with high alpha diversity have low beta diversity." b-In figure 2b, and related text, an upper taxonomical level is always more generalist than its daughter taxon. This is very much expected as mentioned in the text. However, it leads to a representation where it is almost impossible to compare SNB scores across a taxonomical level due to the color gradient constrain (phyla are all orange, species are all blue). Once the fact that "highranking taxa tend to be generalists and low-ranking taxa specialists", wouldn't it be useful to somehow re-scale the color to use the full color gradient to each level to be able to visualize if, towards the leaves, some branches of the tree are more made of social generalist, social specialist or a mix of both? c-As mentioned in comment 3e, the figure 5 is difficult to read. Another representation of the data might be envisaged.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. biomarkers? 4-"organisms that occur in diverse sample": Does 'diverse' mean here with a high biodiversity/richness or in a high number of sample or even compositionally different?
Results 5-"…, implying a low turnover rate…": Do you mean a turn over in the species composition (invasion, extinction, …) or at the cell level (generational turn over)? 6-"Most annotated biomes have low beta diversity reflecting consistent microbial composition": I am a bit uncomfortable with this statement, as 'consistent' is a bit too strong. It can also be that number of taxon of the accessory community is small relative to the number of taxon making the core community, but this can represent a large number of taxon in absolute number. 7-"We used the observation that microbiomes are biosensors …": observation from who? Depending, please give a reference. 8-"Only recently discovered, candidate phyla generally require specific growth conditions and are thus difficult to cultivate, consistent with their low SNB." The link between being difficult to cultivate and low SNB is unclear to me as the authors as using sequencing data from environmental sample, and as such, that did not require cultivation steps. 9-"have relatively generalised genera": please rephrased. 10-"At the generalist end of the spectrum are ubiquitous taxa": Do you mean cosmopolitan? Is there a reference for that? 11-"…while specialists are adapted to become dominant within their habitats.": Please, add 'in stable conditions' or something similar. 12-"Whereas local communities are typically dominated by generalists": what is a local community in the context of your work? 13-"Specialists have a low but constant abundance near carrying capacity (K-selected), and some (but not all) generalists are opportunistic taxa that reach high relative abundance when circumstance permits (r-selected)": can you please add a reference, maybe specific to prokaryotes? 14-At first instance "genomic features" is used in the text, please add a few words to explain what is meant, and how many independent features are considered. 15-"…but at the genus rank we found that specialists are younger than generalists ( Fig. 3b)". Figure  3b is displaying the data at the family level, so at the taxonical rank just above. So is it at the family of genus rank that social specialists are found younger than social generalists? 16-"because genome size estimates are often based on cultivated microbes that differ markedly from environmentally derived genomes, previous suggestions of a positive correlation between genome size and niche range are likely biased": please rephrase this sentence, as every work has its own inherent bias, including the present work. 17-"…for example, the genus Polyangium with a mean genome size of 12.7Mb." Can you add the SNB score too, so it reads "for example the genus Polyangium with a mean genome size of 12.7Mb and a SNB of XX".
Methods 18-"Taxa that were represented by less than 5 reads were removed before dissimilarity calculations": 5 reads in the total dataset or per sample? 19-"For some of the ecological dissimilarity": which ones? 20-"pairwise comparisons can be based on few taxa": Do you mean that it is the case "pairwise Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
comparisons was based on a reduced number taxa", or that even with a reduced number of taxa the analysis is robust or meaningful "pairwise comparisons can be computed albeit based on few taxa"? Displays 21-"Both colour-coding and size of markers represents the Shannon entropy": Why depicting the same metrics twice? 22-"Blue lines indicate samples with low alpha diversity and yellow lines indicate samples with high alpha diversity": Would it be possible to add a color scale as the alpha-diversity is here represented by a color gradient. 23-"Annotated biomes are arranged according to alpha diversity": is it the cumulative alpha-diversity of all samples of the biome (Boolean 'or') or the average alpha diversity?
Reviewer #3 (Remarks to the Author): The authors present a very interesting and original study that uses the distribution of taxa with respect to overall microbiome composition in diverse natural habitats on a large scale as a proxy for their ecological niche breadth. They take advantage of the huge amount of existing data to bring some standard approaches (e.g. the study of co-occurrence of different species/taxa) to an entirely new scale, both data-wise and conceptually. This results, I believe, in an important empirical contribution. It uncovers many new patterns, some of them expected (larger pan-genome in generalists than in specialists) and other, largely unexpected and surprising given common assumptions in the field, such as the dominance of generalists in terms of abundance. Overall, the methodology is state of the art, and its robustness is shown using appropriate controls, random permutation tests, and comparison to alternative technical solutions.
The study is largely descriptive, and I tend to favor more hypothesis-driven analyses. But I do believe that in this case, the novelty of the observed patterns has an important value by itself, and studying the principles and mechanisms that produce them would require substantial work that is probably beyond the scope of this study. For instance, the authors discuss the differences in genomic content and size between specialists and generalists in low vs high diversity communities, as well as functional enrichment patterns, to a somewhat coarse-grained extent, which is probably a limitation imposed by the large scale of the study.
In summary, I enjoyed reading the paper. I think it is an important contribution to the field and opens many new questions and opportunities for future work. If any criticism, I found some figures very data-dense, which somewhat hinders their straightforward visual interpretation (e.g. fig 4d or Fig 5). Especially in a study of this scale, I like figures that summarize the main, big-picture findings in a more graphically interpretable way (with more detailed figures and of course the data itself available as a supplement to those that want to delve into the details). But this is a matter of personal choice, and of course, it is up to the authors how they present their data.

Djordje Bajic
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reviewer #4 (Remarks to the Author):
The manuscript by von Meijenfeldt and colleagues proposes a community diversity-based metric for identifying ecological generalists and specialists. The main idea is that species living in very similar communities are likely to be specialises while those living in communities with diverse compositions are generalists. The underlying assumption is that the community diversity is an indicator of the environmental selection pressures. Once this definition is accepted, the authors use it to investigate distinguishing features of generalists compared to those of the specialists. This revealed differences in abundance profiles (generalists "stochastically" dominating while specialists being at low but constant abundance) as well as in their pangenome structures.
Overall, we find the topic highly interesting and of potential broad interest. The conclusions, however, require much stronger support.
Main points: 1. The central pillar of the study is the metric termed 'social niche breadth' (SNB). Yet, the manuscript is quite shallow on the justification of the used formula and its potential pitfalls. A rank correlation based metric needs to be used with much caution when several members of the community are very low abundance (a typical case in many habitats including soil). In such cases, the technical artifacts (DNA extraction, amplification, data processing etc.) mean that the estimated abundances are only order of magnitude estimates with ranks having no real meaning. How sensitive are the results to use if this metric? Using a combination of different dissimilarity metrics will be much more robust. 2. Another potential confounder is the niche alteration by microbes -many microbes secrete small molecules like amino acids that can alter the niches. The implications of such metabolic interactions on the analysis should be discussed as it can underestimate the true habitat diversity. 3. The manuscript seems to question the "classic" ecological theory that specialists have fitness advantage at the expense of versatility. Yet, this should be more prominently discussed. What explanations the data/analysis offers against the classical view? Both sides of the arguments should be clearly laid out and potential pitfalls discussed. An orthogonal analysis of longitudinal datasets will be important in this context. 4. The choice of including just a few meta-transcriptomics datasets is puzzling, it does not add anything to the study but rather adds potential confounder. Further, it is unclear how 16S vs shotgun metagenomics estimates are pooled together. I recommend analysing the two separately to check the robustness of the conclusions. 5. The manuscript is poor on clearly reporting the effect sizes, which are modest in many cases and thus potentially sensitive to the choices of the metrics and any biases in the data. 6. Repetition of the main analyses in a subset of habitats would be very insightful in testing the robustness/generalisation of the conclusions drawn. 7. Given the pan-genome level differences between generalists and specialists, it becomes imperative to discuss whether the "true functional taxonomic unit" should be the sub-species/strain rather than species or higher. 8. The enrichment analyses in the last figure are way to descriptive without any insights. I suggest Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. moving the long lists to supplementary while presenting a noteworthy summary in the main figure/text. 9. Introduction would benefit from explicit mentioning of classic ecological theory re. niche occupation & competitive exclusion (terms of these theories are referred to in the Results section, such as rstrategists and K-strategists, but would be good to briefly address in introduction). A more substantial explanation of what falls under the term 'niche breadth' and the literature usage of this will also be important to include. 10. "To test this hypothesis, we compared SNB of microbes to their predicted maximal growth rates": the growth rate prediction is a tricky business. Why not use growth rates calculated from metagenomic samples with tools like coptr, iRep etc.? 11. Terms like "low diversity specialists" are unclear and warrant clear definitions. 12. Figure captions are sometimes unclear (e.g. Fig.2 panel e: what is the key for the letters?). Sometimes citations to the supplementary are spelled out completely or shortened. Consistency might be good. 13. Regarding the genomes in generalist genera being more variable in size, what is the hypothesis? What is the literature suggesting? What functions are coded on these variable regions? 14. Please consider making a github repository to share the code and the results 15. "Lachnospiraceae (SNB = 0.50) and its genera (Supp. table 3), obligate anaerobes that were previously regarded as specialists 23 also have a high SNB" :Why Lachnospiraceae have a high SNB, where the diversity of their habitats come from? In general these concepts of "high diversity habitats" and "low diversity habitats" are not completely clear. ********************END********************

13
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Decision Letter, first revision:
22nd December 2022 Dear Dr. Dutilh, Thank you for submitting your revised manuscript "On specialists and generalists: niche range strategies throughout the tree of life" (NATECOLEVOL-220717078A). It has now been seen again by the original reviewers 2-4 and their comments are below. The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Ecology & Evolution, pending minor revisions to satisfy the reviewers' final requests and to comply with our editorial and formatting guidelines.
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Reviewer #2 (Remarks to the Author): The article, now entitled "On specialists and generalists: niche range strategies across throughout the tree of life" has been diligently revised. New analyses, displays and methods are reported. At this stage, I only have a few additional minor comments: Minor comments 1-Abstract line 13-14 "specialists are restricted to a single environment". If it is about the 'traditional' definition of specialist, I would rather argue that they are restricted to narrow *conditions*, albeit these conditions can be found in diverse environments as long as micro-niches in these biomes display these restricted conditions. Otherwise, it should be "habitat specialists are restricted to a single environment". 2-Line 36-37 "Niche breadth definitions that assess the full n-dimensional niche space have been based on occurrence in microbiomes". This sentence remains unclear to me. 3-Line 141: "small phyla": does it mean low diversity (LD) phyla? 4-Line 157: "Taxa with high and low SNB are dispersed throughout the prokaryotic tree of life (Fig. Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 2d),": fig 2d is representing z-score and not directly SNB. 5-Line 194 and 262 *social* generalist? 6-Line.268-269: "It was previously suggested that generalist species are evolutionary younger than specialist species 27". I think here, it is habitat specialist and habitat generalist 7- Figure 1 (i) panel c: can the sample be ordered to have a Gaussian shape? Or why is the curve irregular? (ii) Panel f "Annotated biomes with high mean alpha diversity have low beta diversity." Albeit true for the biome with extreme high alpha diversity and extreme high beta diversity, there is also biomes with both low alpha and beta diversity. The response is generally satisfactory. 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If you have any further questions, please feel free to contact me.
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Reviewer #1: Remarks to the Author: I appreciate author's thorough revisions of the manuscript and supplementary information to improve reproducibility.
After reading the authors response to my previous comment regarding the "candidatus" taxa I specifically find this justification not too accurate. Many candidatus taxa are mainly lacking the culture to get a valid description and could be among highly abundant and ubiquitous lineages as well. I would recommend authors to remove this section (Line 145-156) as it is not among the main findings of their study and relies highly on the correct designation of candidatus status that might not be reliably annotated and updated in different databases.
Reading the revised version of the manuscript, I suggest some minor edits and changes: Line 26-social niche Line 60-quantify social niche range I like the expanded explanation in lines 49 -61. Line141-144-this sentence reads a bit confusing. Please rephrase to explain the impact of taxon rank jump on SNB clearer. Line 370-the evolutionary history of different types based on SNB is not specifically the focus of this study and I would recommend authors to rephrase this sentence "Generalist genera are older than specialist genera"

Supplemental data
Line 39-fix the typo Reviewer #2: Remarks to the Author: The article, now entitled "On specialists and generalists: niche range strategies across throughout the tree of life" has been diligently revised. New analyses, displays and methods are reported. At this stage, I only have a few additional minor comments: Minor comments 1-Abstract line 13-14 "specialists are restricted to a single environment". If it is about the 'traditional' definition of specialist, I would rather argue that they are restricted to narrow *conditions*, albeit these conditions can be found in diverse environments as long as micro-niches in these biomes display these restricted conditions. Otherwise, it should be "habitat specialists are restricted to a single environment".
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. study and relies highly on the correct designation of candidatus status that might not be reliably annotated and updated in different databases.
We agree that the definition of 'candidatus' taxa is that they are not yet validly described, e.g. because they lack a culture isolate. While in theory they could be among highly abundant and ubiquitous lineages, we do observe that they have a relatively low SNB and would like to mention this. We would like to keep the discussion of 'candidatus' phyla in, but we have toned it down into: "Many phyla with the "candidatus" status have a low SNB compared to validly described phyla (Supp. Fig. 17). The connection between the candidatus status and low SNB may reflect a discovery bias of these phyla where widespread lineages (generalists) tend to be discovered and described sooner than rare ones (specialists). Specialised organisms in candidate phyla may require specific growth conditions, as reflected in their relatively consistent social communities and low SNB. Reading the revised version of the manuscript, I suggest some minor edits and changes: Line 26-social niche We have rewritten this sentence to "Together, our analysis shines data-driven light on microbial niche range strategies." We agree that we analyse 'social' niche range but we believe that for the final sentence of the abstract it's important to mention that this new approach shines new light on niche range strategies 'in general' and we thus did not add 'social' here.
Line 60-quantify social niche range We have added 'social'.
I like the expanded explanation in lines 49 -61.

Thank you!
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Line141-144-this sentence reads a bit confusing. Please rephrase to explain the impact of taxon rank jump on SNB clearer.
We agree and have rewritten to: "There are many phyla that have low SNB scores and contain few classes, orders, and families compared to the dominant ones described above. These low-scoring phyla contain relatively few described classes, orders, and families so we observe that the distribution of SNB scores is more skewed towards social specialism at the phylum rank (median SNB = 0.36) than at those lower ranks (median SNB = 0.38-0.43, see Fig. 2d)." Line 370-the evolutionary history of different types based on SNB is not specifically the focus of this study and I would recommend authors to rephrase this sentence "Generalist genera are older than specialist genera" We have rewritten to remove the focus on the evolutionary process but rather on the observation: "Generalist genera are older than specialist genera, and have large and open pan genomes with which they have adapted to different habitats."

Supplemental data
Line 39-fix the typo Thanks, we have fixed the typo.
Reviewer #2: Remarks to the Author: The article, now entitled "On specialists and generalists: niche range strategies across throughout the tree of life" has been diligently revised. New analyses, displays and methods are reported. At this stage, I only have a few additional minor comments: Minor comments 1-Abstract line 13-14 "specialists are restricted to a single environment". If it is about the 'traditional' Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. definition of specialist, I would rather argue that they are restricted to narrow *conditions*, albeit these conditions can be found in diverse environments as long as micro-niches in these biomes display these restricted conditions. Otherwise, it should be "habitat specialists are restricted to a single environment".
We agree and have rewritten the next sentence to clarify: "Although a classical concept in ecology, niche breadth has remained challenging to quantify for microbes because it depends on an objective definition of the environmental conditions." 2-Line 36-37 "Niche breadth definitions that assess the full n-dimensional niche space have been based on occurrence in microbiomes". This sentence remains unclear to me.

We have rewritten to:
"The full n-dimensional niche space of organisms {htj} has previously been assessed based on their occurrence in different environments." 3-Line 141: "small phyla": does it mean low diversity (LD) phyla?
We have rewritten this section to: "There are many phyla that have low SNB scores and contain few classes, orders, and families compared to the dominant ones described above. These low-scoring phyla contain relatively few described classes, orders, and families so we observe that the distribution of SNB scores is more skewed towards social specialism at the phylum rank (median SNB = 0.36) than at those lower ranks (median SNB = 0.38-0.43, see Fig. 2d)." 4-Line 157: "Taxa with high and low SNB are dispersed throughout the prokaryotic tree of life (Fig. 2d),": fig 2d is representing z-score and not directly SNB.

Yes we agree, and have rewritten to:
"Taxa with high and low Z-scores are dispersed throughout the prokaryotic tree of life (Fig. 2d)." Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
6-Line.268-269: "It was previously suggested that generalist species are evolutionary younger than specialist species 27". I think here, it is habitat specialist and habitat generalist Yes you are right, we have rewritten to: It was previously suggested that habitat generalist species are evolutionary younger than habitat specialist species {10.1038/s41467-017-01265-1}. Figure 1 (i) panel c: can the sample be ordered to have a Gaussian shape? Or why is the curve irregular? (ii) Panel f "Annotated biomes with high mean alpha diversity have low beta diversity." Albeit true for the biome with extreme high alpha diversity and extreme high beta diversity, there is also biomes with both low alpha and beta diversity.

7-
(i) The distribution is cut off at 50,000 reads, because this is one of our selection criteria, which is why the shape is asymmetrical. We have added this cut-off in the figure itself, and have changed the caption to make this more clear: "Total number of taxonomically annotated reads per samples (n = 22,518 samples). Boxplot shows interquartile range and median. No samples with less than 50,000 reads were selected." (ii) We very much agree with your observation about the alpha and beta diversity and have added in the caption of the figure: "Annotated biomes with high mean alpha diversity have low beta diversity, whereas both low and high beta diversity is found among annotated biomes with low mean alpha diversity."

Supplementary
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1 Line 39: "social niche 39 breadt" should read breadth Thanks, we have fixed the typo.
Reviewer #3: Remarks to the Author: On my side, everything adressed, good to go.

Thanks for this!
Reviewer #4: Remarks to the Author: The response is generally satisfactory. My only remaining concern is that "available upon request" for data/code is not a good scientific practice.
We agree and will add all the code (including the code for making the figures) to a Zenodo DOI.

Final Decision Letter:
27th February 2023 Dear Dr Dutilh, We are pleased to inform you that your Article entitled "A social niche breadth score reveals niche range strategies of generalists and specialists", has now been accepted for publication in Nature Ecology & Evolution.
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