Symbioses shape feeding niches and diversification across insects

For over 300 million years, insects have relied on symbiotic microbes for nutrition and defence. However, it is unclear whether specific ecological conditions have repeatedly favoured the evolution of symbioses, and how this has influenced insect diversification. Here, using data on 1,850 microbe–insect symbioses across 402 insect families, we found that symbionts have allowed insects to specialize on a range of nutrient-imbalanced diets, including phloem, blood and wood. Across diets, the only limiting nutrient consistently associated with the evolution of obligate symbiosis was B vitamins. The shift to new diets, facilitated by symbionts, had mixed consequences for insect diversification. In some cases, such as herbivory, it resulted in spectacular species proliferation. In other niches, such as strict blood feeding, diversification has been severely constrained. Symbioses therefore appear to solve widespread nutrient deficiencies for insects, but the consequences for insect diversification depend on the feeding niche that is invaded.

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Reviewers' comments:
Reviewer #1 (Remarks to the Author): In this communication by Cornwallis and colleagues, the authors apply an elegant phylogenetic approach to test various hypotheses that, over the years, have come to define the field of insect symbiosis. Chiefly, why do obligate symbioses evolve? And how do they influence diversification in insects? The manuscript is extremely well-written, the approach is novel, and the results appear to be exciting and of interest to a broad audience. Nonetheless, I do have a few comments and questions.
First, the definition of 'obligate symbiosis' is unusual. Obligate dependence is typically and primarily defined experimentally, where symbiont loss corresponds to a net decrease in survivorship, development, and fitness. Here, obligate dependence was determined by the presence of morphological structures exclusively associated with symbiosis (e.g., bacteriocytes) (Line 64; and 533). But where information on symbiont localization is lacking, data on the impact of symbiont loss was then used to determine the obligate status of a mutualism. This is problematic since a number of insect-microbe symbioses feature bacteriomes, or symbiotic organs, but the benefit to the insect host is either untested, or only context-dependent, most evidently in weevils and beetles relying on tyrosine supplementation for cuticle hardening. Applying a widely accepted definition of obligate symbiosis is thus critical for redefining the initial working dataset, for which much of the downstream analyses, and exciting findings, depends on.
The authors did an exceptional job curating the dataset at the center of this study. This was not a trivial effort, and I am certain that the field will benefit immensely from this work. Nonetheless, a few statements suggest a greater familiarity is still necessary with the literature and the range of obligate symbioses insects engage in. This is most evident in the claim that only four insect families harbor defensive symbionts ( One of the most exciting findings reported in this study, and for which there are many, is the connection between B vitamin deficiency in an insect's diet and its propensity to engage in an obligate symbiotic partnership. Given a wave of recent studies, the authors find ample experimental evidence to support their claim. This was supplemented with an elegant analysis to delineate between the functional convergence of these symbionts from the reliance on a restricted set of symbiotic partners. The implication here is that hosts evolved dependence on a broad range of microbes that in turn associate with insects feeding on low vitamin diets. Missing, however, is the metabolic conservation of the biosynthetic pathways involved. Specifically, are B vitamin pathways, especially B5 and B9, enriched in the reduced (and not-so-reduced) genomes of bacterial insect symbionts? And given the central role B vitamin deficiency appears to play in the evolution of obligate symbioses, are B vitamin biosynthesis pathways more conserved than those that encode other host-beneficial factors (e.g., amino acid)? Given the publicly available genomic resources for many of the cited study systems, such 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/. an analysis would complement the rigorous phylogenetic approaches at the center of this study.
Reviewer #2 (Remarks to the Author): I have read and reviewed the manuscript "Symbiont-driven niche expansion shapes the adaptive radiation of insects", which is a very interesting and well written manuscript. The authors explore the roles that obligate symbionts had for niche evolution and diversification of insects. Obligate symbionts seem to be key to insects that have diets with low levels of vitamin B, allowing them to expand their niche preferences. However, diversification following feeding niche evolution depended on the niche invaded. While symbionts might have helped herbivorous insects to diversify, the opposite happened to blood-feeding insects.
I have enjoyed reading the manuscript and commend the authors for synthesising so many analyses into a fairly condensed manuscript (except for the methods section). My comments are in the interest of making the main text clearer for the readers. Some of my doubts got answered when reading the methods, but I kept them here because the authors might find it worth to try and clarify these sentences in the main text.
-So many Lepidoptera tips in the tree! Seems like there is a taxonomic bias. How does that affect the analyses? -Which insect life stage is considered? -If families can have both states (obligate/non obligate), then how did you classify them into either one of the states? -"This pattern of food utilisation explains the current distribution of obligate symbiosis remarkably well, where over 90% of insect species feeding on blood, phloem, xylem and wood have obligate symbionts" -what about the other way around? The percentage of species with symbionts that feed on blood, phloem, xylem or wood? -Please include color legend in both panels of fig 4.
-Lines 273-274 -"lineages with higher rates of obligate symbiosis were also more specious", but obligatory symbiosis is binary in the figure.
-287-289 -Isn't this the main dataset? Maybe it should say "excluding"? -Lines 510-512. Please explain why vertically transmitted symbionts were not included in coevolution analyses.
-Lines 568-569 -"if species fed in more than one of the following niches". Do you mean single species feeding in more than one niche, right? It could be clearer, so that there's no conflict with the next sentence.
-Line 614 -I suggest "diversification can be modelled using three…" because when I first read this, I thought all three approaches had been used in this study.
-Line 727 -Which 2 binary traits? Low or high B vitamin levels and presence/absence of obligate symbionts? I understand section 3 has more general explanations whereas section 4 presents the details, but it would be much easier to follow if those sections where connected by referring to each other in the text.
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-Line 757 -remove 'of' before 'node'. -Methods section 4.3.1 -Is there a reason for comparing the focal node to both descendant nodes instead of comparing the focal node to it's parent node, which would mean that only events along one branch are considered? I find the classification based on at least one descendant being obligate/nonobligate unnecessarily more complicated than simply comparing nodes at the beginning and end of a branch.
-Supplementary tables are hard to follow because there's not much explanation. The connection to the text is there, but there's a lot of information in the tables that is hard to figure out what it means. -Couldn't find the code repository at osf.io.
Reviewer #3 (Remarks to the Author): In this paper, the authors address the question whether the engaging in a symbiotic relationship is linked to the tendency of insect taxa to diversify. To address this issue, the authors perform a metaanalysis, in which they analyze more than 1,800 different symbiont-host interaction across different insect families. Using the existence of morphological structures that house the insects' symbionts or diagnostic experiments as a proxy for the interaction being obligate, the authors test whether i) insects with obligate symbionts are more likely to diversify taxonomically and ii) if this is linked to the insects' diet. The latter question has been analyzed by correlating the abundance of the nutrient in the focal food source to the presence of obligate symbionts (Fig. 2). In addition, the most likely ancestral composition of food sources in terms of symbiont-provided nutrients (i.e. vitamins, amino acids) has been calculated to correlate the change to the derived state with the association with obligate symbionts (Fig. 3).
This paper addresses an important question and is the first one to systematically verify whether obligate symbiosis is correlated with an increased rate of diversification. Previous studies have tested this idea using a much smaller set of host-symbiont interactions (e.g.: doi.org/10.1098/rspb.2012.2820). Thus, the main advance of this paper is that a large dataset has been analyzed using the same statistical approach, thus making the results directly comparable.
My main concerns are that the data is presented in a way that is difficult to understand. Moreover, the paper does not present novel findings that was not known before. Finally, I think that besides species richness also the rates of speciation of symbiont-associated and symbiont-free clades should be compared.
Below, I will elaborate on these points in more detail.
Main points: (1) The data is consistently presented in a way that is extremely difficult to understand. This applies in particular to the figures that I find hard to grasp since neither the main text nor the figure legend provides sufficient information to understand it. Thus, it remains frequently unclear what exactly has been done and what is shown. While this information is presented in detail in the materials and methods, I think that also the main text (and the figures in particular) should be sufficiently clear to understand the main points without having to refer to the methods section.

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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/. -What does the difference between "non to Ob" versus "Ob to Ob" show? How are these two cases linked with each other. I do not see this to be a meaningful comparison.
-Wouldn't it be possible to show the difference in the estimated ancestral and currently used level of vitamin (y-axis) depending on whether an obligate interaction evolved during this time or not (xaxis)? I think this might help to clarify this graph.
-It would be helpful to mention how many cases were considered in each of the different groups.
-Also: Did the authors correct for multiple testing in this graph? d) Figure 4: -In panel A please explain what the two different colours mean. Also, the number of cases below should be mentioned in the same order as the data is shown. -Panel B: Please explain the colour code. Also: why is "NonObligate" written in capital letters and in one word without hyphen?
(2) I find the main conclusions not very surprising. It was known beforehand that obligate symbioses were particularly common in insects that feed on plant sap, xylem, and blood, because essential nutrients are low in these food sources. In these cases, insects can only use these food sources when they have symbionts to complement them with the missing essential metabolites. Thus, it is a circular argument to then conclude that these taxa are more species-rich. This is self-evident, because these insects can only use these food sources when they have the corresponding symbionts. Sister clades, from which these lineages derive, should obviously be less diverse.
(3) The key argument is based on data of species richness of insect clades that are or are not associated with obligate bacterial symbionts (Fig. 4). However, I think that also the rate of speciation should be considered, which could be correlated to the presence of obligate symbionts. If the pattern also holds in this case, this would strengthen the argument even more. (5) Verification analysis: The author say they repeated the analyses to examine the robustness of the 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/. results. However, the results of these analysis are not shown. To allow the reader to really judge how much the presented results depend on the set of examples studied, the results of these control analyses should be presented graphically and using statistics (in the supplementary information).
(6) The discussion is way too short and should link the main findings to the existing literature.
(7) I think the work that has been done does not warrant to talk about niche expansion, since it remains unclear how the size of the original niche was and whether there was just a shift (i.e. use of a different niche) or a niche expansion (i.e. access that a new niche in addition to the previous niche). Thus I recommend rewording the title.
Minor comment: -Page 6 line 106: I think the original papers rather than a review should be cited in this context, to give credit to the authors making this discovery.
Reviewer #4 (Remarks to the Author): In this manuscript, the authors are reporting on the results of a meta-analysis on the correlation between the occurrence of obligate symbionts in insects and the feeding ecology as well as species richness of their host families. Based on an extensive literature survey, they compile an impressive set of data on the presence or absence of obligate symbionts across 400 insect families, as well as the nutritional composition of the insects' food sources based on available data for the general food types. They report on 16 independent origins of obligate symbioses in insects and identify several B vitamins as the only nutritional components whose deficiencies significantly associate with the evolution of symbiosis. Reconstructing ancestral feeding niches and their nutritional composition, the authors test whether symbioses evolved before or after switches to B vitamin-deficient diets and find evidence for the former. Finally, they assess the association between feeding niche, symbiosis, and species richness across the different insect families, finding evidence for a significant impact of feeding niche on species richness, and for symbiosis being associated with increased species richness in herbivorous families.
I have reviewed this paper previously for another journal, and the authors have made important changes and additions to the analyses and the manuscript in response to the reviewers' comments. However, there are in my view still a few major drawbacks in the analyses that cast some doubt on the validity of the main conclusions that the authors present, and there are several overstatements in the manuscript, especially pertaining to the causal link between dietary B vitamin content and the evolution of symbiosis. Nevertheless, this is an impressive meta-analysis that provides an interesting overview of evolutionary patterns in insect symbioses and their possible links to dietary transitions of the hosts.
Major comments: 1. While I understand the rationale for collapsing taxa on the family level, this entails a couple 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/.
shortcomings for the analyses, which are not or only partly considered in the paper and in my view could have a severe impact on the results: a. Several insect families contain symbiotic and non-symbiotic taxa (e.g. Lygaeidae, Formicidae, Melyridae, Chrysomelidae, etc), with sometimes multiple evolutionary transitions from non-symbiotic to symbiotic. Considering the families as ancestrally symbiotic pushes the transition to an earlier node than is actually true. This causes problems when trying to infer the evolutionary order of events (diet shift and origin of symbiosis). b. It can impact the analysis of the impact of symbiosis on diversification (see below). 2. One of the most interesting conclusions of the study is that dietary specialization followed the acquisition of symbionts, rather than the other way around. Even though I intuitively tend to agree that this makes sense, I am not convinced that the data presented here provide compelling evidence for this scenario. The conclusion is based on the data presented in Figure 3, indicating that differences in vitamin B5 and B9 levels were lowest on branches from symbiotic to symbiotic nodes. When looking at the phylogeny presented in Figure 1, however, it appears that the vast majority of the branches connecting sym-sym nodes remain within the same feeding niche. If I understand correctly, than the nutrient composition of the diets of non-extant taxa were estimated by reconstructing this taxon's most likely feeding niche (l. 714-723 and 780-786) and then using the average nutritional values for this feeding niche as a proxy. If this is the case (and the methods are not clear in this regard), then all branches connecting taxa with the same feeding niche should get a difference of 0 in all nutritional values. Looking at Fig. 1 quickly shows that the vast majority of sym-to-sym node connections remain within the same feeding niche, so the plot for this category should show a lot of zeroes, while all values are below zero in Fig. 3. I am not sure how to explain this. Maybe I misunderstand the reconstruction of ancestral diets? The only other way that I can see how the authors may have inferred ancestral diets is by extrapolating the actual nutrient content levels of extant species' feeding niches to vitamin contents of presumed ancestral diets, essentially averaging out when shifts between feeding niches occurred. If vitamin levels of ancestral diets were inferred from the extrapolated nutrient contents of diets of extant species, though, I do not think it is a valid approach, as changes in nutrient levels during transitions within feeding niches cannot be reliably reconstructed this way. 3. Estimating the impact of symbiosis on diversification: a. The sister taxa approach that the authors are using is commendable. However, I see two problems with this analysis: i. Given that the origin of symbioses is collapsed on the family level, the analysis may not capture the necessary comparisons of symbiotic versus non-symbiotic sister taxa. ii. I don't understand which taxa actually were compared. The authors describe 16 origins and 8 losses of symbiosis, but they indicate that 123 sister comparisons were done (l. 960). For this analysis, only the 24 transitions between symbiotic and non-symbiotic status seem to be relevant, so I do not understand where this large number of sister taxa comparison comes from. Again, it would be very helpful to have a clear list of origins and losses of symbiotic relationships presented, as well as the sister taxa comparisons that were used for this analysis. As it stands, it is impossible to evaluate the soundness of the approach. b. The other two approaches are problematic, since it is not clear how the non-symbiotic families included in the phylogeny were selected, so it remains unclear how much of a sampling bias there is. For example, the most diverse order, the beetles, are only represented by 11 families in total, out of which 9 are considered to be symbiotic, even though there are more than 170 beetle families, most of which are non-symbiotic (and some really large non-symbiotic families are missing, e.g. 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/. Staphylinidae, Tenebrionidae, as well as the phytophagous Buprestidae). Furthermore, the data presented in Figure 4a cannot be correct. There are an estimated 1 million described insect species to date (10^6), but some individual families in the figure are indicated to have 10^9 species.
l. 1: The title seems somewhat overstated, since the manuscript does not demonstrate niche expansions (rather: shifts), nor does it establish a causal link between symbiosis and adaptive radiations. l. 80: It is important to somewhere explicitly list the 16 reconstructed evolutionary origins of symbioses and the eight losses, as they are difficult to impossible to extract from Fig. 1 and Table S5. l. 159: Here and elsewhere, the authors put too much weight on the B vitamins as the nutritional components driving the evolution of symbiosis, while it has been shown that other factors can be the ones driving this (e.g. eAAs, digestive enzymes). In particular in ll. 307-308: This is correlational, so causality cannot be inferred! Stating that, based on this analysis of inferred ancestral diets, B vitamins are the only nutrients found to significantly correlate with the presence of symbionts is fine, but claiming that this was the driving force behind the evolution of symbiosis is not. l. 610-611: This seems like a strange approach. Given that there are missing values for some dietary components in some diets, this can have a strong impact on the mean, especially if a number of micronutrients is missing, pushing the mean towards much lower values. I understand the rationale for avoiding the problem of compositional data (although I am not sure whether using this standardization by dividing by the mean instead of the sum actually solves this, since the results will still depend on the other dietary components), but the approach the authors are using created a substantial problem when comparing nutrient composition across diets.

Reviewer #5 (Remarks to the Author):
This manuscript seeks to study the macroevolution of symbiosis in insects. The paper is well written; however, I felt a little dazzled by results and p-mcmc values. So much so that I became slightly confused about which ones are the most important and which are linked to which hypothesis. I think in any revision I would recommend the author go through the manuscript with the reader in mind a really link the results to the specific hypotheses and expectations.
My expertise is in phylogenetic comparative methods so I'm reading the manuscript from that perspective. I think the analyses on the whole look well done. The tree itself is very difficult and I think that needs to be made more clear in any revision. The dating and topological uncertainty of the tree is not really considered. Whilst I appreciate it would be far outside the scope of this study to produce a better foundation tree -I think this need to be discussed more throughout.
I have a few important concerns that I think the authors should consider: 1. Number of species in a family. The number of species estimated to be in each family is a critical estimate to many of the analyses in this study. However, I think it will be quite likely that the number of species estimated in each family will be highly correlated with research effort, which will in turn is likely to be associated with occurrences of symbiosis and other covariates important here. So given 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/. this, I think the author need to control for research effort (associated with each family) in their analyses.
2. Stochastic mapping and Discrete. I am slightly confused about why the authors need to use both stochastic mapping and Discrete. I would suggest they use the best model (dependent/independent) and then use that to determine the ancestral stats of the nodes of interest. This part of the methods was very unclear -why do you need Stochastic mapping at all here?
3. The significance and difference of rates in the Discrete mode. The authors say they use the posterior distribution of rates to determine whether the rates are significantly different to each other. However, there is a reversible-jump procedure available in BayesTraits that is specifically designed to reduce dimensionality and determine if there are differences among rates -I think the authors should use that.

Author Rebuttal to Initial comments
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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/.
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/.
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/.
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/.
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/.
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/.
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/.
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/.
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/.
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/.
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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/.
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/.
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/.
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/.
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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/.
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/.
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/.
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/.

Decision Letter, first revision:
12th December 2022 *Please ensure you delete the link to your author homepage in this e-mail if you wish to forward it to your co-authors.

Dear Charlie,
Your revise manuscript entitled "Symbioses shape feeding niches and diversification across insects" has now been seen by the same five reviewers, whose comments are attached. You will see that the first four reviewers are largely satisfied with the revisions but our phylogenetics expert still has major concerns which will need to be addressed before we can offer publication in Nature Ecology & Evolution. We will therefore need to see your responses to the criticisms raised and to some editorial concerns, along with a revised manuscript, before we can reach a final decision regarding publication.
We therefore invite you to revise your manuscript taking into account all reviewer and editor comments. Please highlight all changes in the manuscript text file in Microsoft Word format.
We are committed to providing a fair and constructive peer-review process. Do not hesitate to contact us if there are specific requests from the reviewers that you believe are technically impossible or unlikely to yield a meaningful outcome.
When revising your manuscript: * Include a "Response to reviewers" document detailing, point-by-point, how you addressed each reviewer comment. If no action was taken to address a point, you must provide a compelling argument. This response will be sent back to the reviewers along with the revised manuscript. * If you have not done so already please begin to revise your manuscript so that it conforms to our Article format instructions at http://www.nature.com/natecolevol/info/final-submission. Refer also to any guidelines provided in this letter.
* Include a revised version of any required reporting checklist. It will be available to referees (and, potentially, statisticians) to aid in their evaluation if the manuscript goes back for peer review. A revised checklist is essential for re-review of the paper.
Please use the link below to submit your revised manuscript and related files:

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<strong>Note:</strong> This URL links to your confidential home page and associated information about manuscripts you may have submitted, or that you are reviewing for us. If you wish to forward 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/. this email to co-authors, please delete the link to your homepage.
We hope to receive your revised manuscript within four to eight weeks. If you cannot send it within this time, please let us know. We will be happy to consider your revision so long as nothing similar has been accepted for publication at Nature Ecology & Evolution or published elsewhere. Nature Ecology & Evolution is committed to improving transparency in authorship. As part of our efforts in this direction, we are now requesting that all authors identified as 'corresponding author' on published papers create and link their Open Researcher and Contributor Identifier (ORCID) with their account on the Manuscript Tracking System (MTS), prior to acceptance. ORCID helps the scientific community achieve unambiguous attribution of all scholarly contributions. You can create and link your ORCID from the home page of the MTS by clicking on 'Modify my Springer Nature account'. For more information please visit please visit <a href="http://www.springernature.com/orcid">www.springernature.com/orcid</a>. Please do not hesitate to contact me if you have any questions or would like to discuss these revisions further.
We look forward to seeing the revised manuscript and thank you for the opportunity to review your work.

[REDACTED]
Reviewers' comments: I thank the authors for their very thoughtful revision. After carefully reading this new version, I think that all of my criticism has been adequately addressed. In fact, I think that this new version is much clearer and easier to understand than the previous one. Especially the revised figures and descriptions of the data shown make a huge difference. Together, I think that this manuscript makes a substantial contribution to the field and fills an important gap. Thus, I recommend accepting it with minor revisions.
Below, I have one more suggestion that I think would even further enhance the manuscript: 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/.
One of the key findings of the paper is that B vitamins was the only nutrient limitation that significantly correlated with the presence of obligate symbionts. This is a very interesting observation. While the authors acknowledge that also other deficiencies might play a role alone or in combination with vitamins, they do not address the question why the vitamins seem to stand out. I think the paper would significantly benefit from an analysis or at least a more detailed discussion of why this might be the case. For example, is it that a) vitamin auxotrophies evolve first and only then other metabolic genes are lost or b) vitamin auxotrophies are simply more common. The second point could be addressed in this way: Rather than showing the relative nutrient deficiencies (e.g. Fig. 2), the authors might want to consider to also display/ analyze the overall distribution of absolute deficiencies in their data set.
Moreover, this issue receives a more in-depth treatment in the discussion section.

Minor suggestions:
Line 53: Please reword "recruit". This sounds like an active searching process.
Line 201: Consider changing the order of "negatively" and "phylogenetically" to read "are phylogenetically negatively correlated".
Reviewer #4 (Remarks to the Author): The authors have done a thorough job in addressing the reviewers' comments. They put together an enormous database and analyze it in a phylogenetic framework. While some of the conclusions are not entirely surprising, an analysis of insect symbioses at this scale is very valuable for the community as a whole and will present an important basis for the further broad-scale studies. There are a few minor comments remaining from my side, but they are mostly issue regarding wording or small errors. However, one important aspect that I would like to point out is that the comment about the consistent results of the sensitivity analyses (l. 464-465) is not true and should be carefully reworded, as amino acids repeatedly come up as significant, but in an unexpected direction. This needs to be explained. Why should symbiotic lineages be more likely to feed on eAA-rich diet, if I understand the results presented in Supplementary Tables 20, 21, and 24 correctly? As this seems to be consistent across all three "Sensitivity Analyses", it needs a thorough explanation.
Specific comments: Comment on the rebuttal: In Suppl. Table 5, there are also nonOb to nonOb and Ob to Ob transitions highlighted, which they shouldn't be. l. 80-82: Just to be clear, please specify that co-speciation and negative fitness consequences of symbiont removal were used as obligate criteria. Just a small comment here (that I know will not be relevant to the results, since you carefully selected the symbioses): Strictly speaking, your two criteria would include some associations with Wolbachia as a reproductive manipulator, because it can be fixed in certain insect species, and removal would lead to severe fitness defects under CI (sometimes 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/. even extremely so, like in Asobara wasps). Not sure how to avoid this issue, but I wanted to at least point it out, but I assume the authors are anyways aware of it… l. 139: please correct typo in "drosophilids" l. 167: Please correct typo in "lagriid" l. 181: the addition of "digestive enzymes" makes sense, but please revise, as these are not essential nutrients Fig. 3 C+D: Could you please provide statistics for the differences between groups? Fig.-3 in general: I appreciate the authors' efforts to clarify what is depicted in this figure. However, I urge them to consider whether there is a better way to display this, or at least relabel. "Non-obligate maintenance" is misleading, and it really took me a long time to grasp the figure in its full extent. In part, this was due to the use of the term "vs" in C and D, which I interpreted as divided by, which is exactly what it apparently is not supposed to mean here (rather, it's the reverse)? l. 352: would be good to mention that this is also true for essential amino acid supplementing symbionts. l. 464-465: This is not exactly true. In most of the analyses, the eAAs (and sometimes non-eAAs) also come up as significant, which does change the interpretation of the results quite a bit. I strongly recommend to add a comment on this and carefully discuss it. And it seems that they strongly go into the opposite direction than the B vitamins, indicating that symbiotic lineages are more likely to feed on amino acid-rich diet, if I interpret this correctly? This would not only be contradicting most of the accumulated symbiosis knowledge, but also be highly unlikely. This needs to be checked. l. 490: With the caveat indicated above… Don't get me wrong, I don't insist on eAAs being a general driving factor underlying the evolution of nutritional symbioses in insects, but considering the gaps in current understanding and the necessarily limited phylogenetic resolution, as well as the results of the sensitivity analyses, they may be similarly important as the B vitamins, so I think it would be wise to phrase carefully here.

Response to reviewers
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/.
We are very grateful to the referees for their continued efforts in helping us improve our manuscript. Below are the details of the specific changes we have made to address the referees' comments. Briefly, we have revised the text in our paper, added the extra analyses suggested by referee 5, and have added supplementary tables to present the results of the extra analyses.
Line numbers in word documents with track changes are not always reproducible across computers. Therefore, we refer to lines numbers in the pdf version of the revised manuscript.

This is an excellent revision of the paper. The authors have taken on board comments in this new version, improving on what was already a compelling manuscript. Congratulations on a job well done!
Many thanks for the encouragement and help with our paper.

Reviewer #3 (Remarks to the Author):
I thank the authors for their very thoughtful revision. After carefully reading this new version, I think that all of my criticism has been adequately addressed. In fact, I think that this new version is much clearer and easier to understand than the previous one. Especially the revised figures and descriptions of the data shown make a huge difference. Together, I think that this manuscript makes a substantial contribution to the field and fills an important gap. Thus, I recommend accepting it with minor revisions.
Thank you for the positive feedback and continued effort in helping us improve the final details of our paper.
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/.
One of the key findings of the paper is that B vitamins was the only nutrient limitation that significantly correlated with the presence of obligate symbionts. This is a very interesting observation. While the authors acknowledge that also other deficiencies might play a role alone or in combination with vitamins, they do not address the question why the vitamins seem to stand out. I think the paper would significantly benefit from an analysis or at least a more detailed discussion of why this might be the case. For example, is it that a) vitamin auxotrophies evolve first and only then other metabolic genes are lost or b) vitamin auxotrophies are simply more common. The second point could be addressed in this way: Rather than showing the relative nutrient deficiencies (e.g. Fig. 2), the authors might want to consider to also display/ analyze the overall distribution of absolute deficiencies in their data set.

Moreover, this issue receives a more in-depth treatment in the discussion section.
This is an interesting point, and we appreciate the suggestion of adding analyses of absolute nutrient concentrations. The interpretation of such analyses is nevertheless not easy. Information on the nutrient contents of foods is given as concentrations. Therefore, to examine absolute amounts of nutrients requires data on intake rates, but such information is not available for many insect species. Furthermore, it is not clear that absolute nutrient concentrations accurately reflect the ability of species to synthesise compounds.
One possible way to address whether B-vitamin auxotrophies are more prevalent than other nutrient auxotrophies would be to conduct comparative genomic analyses. This touches upon a similar comment from referee 1 in the previous round of comments about including genomic analyses of vitamin synthesis pathways. For example, the completeness of pathways used to synthesis different nutrients could be analysed across species. However, this is a major undertaking, well beyond the current paper, but we hope our work stimulates more research in that direction. We have therefore taken the alternative suggestion made by the referee and extended the discussion of this point in the manuscript (lines 420-423).

Minor suggestions:
Line 53: Please reword "recruit". This sounds like an active searching process.

Addressed.
Line 201: Consider changing the order of "negatively" and "phylogenetically" to read "are 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/.

phylogenetically negatively correlated".
Many thanks for the suggestion. We have changed it.

Reviewer #4 (Remarks to the Author):
The authors have done a thorough job in addressing the reviewers' comments. They put together an enormous database and analyze it in a phylogenetic framework. While some of the conclusions are not entirely surprising, an analysis of insect symbioses at this scale is very valuable for the community as a whole and will present an important basis for the further broad-scale studies. There are a few minor comments remaining from my side, but they are mostly issue regarding wording or small errors. However, one important aspect that I would like to point out is that the comment about the consistent results of the sensitivity analyses (lines 464-465) is not true and should be carefully reworded, as amino acids repeatedly come up as significant, but in an unexpected direction. This needs to be explained. Why should symbiotic lineages be more likely to feed on eAA-rich diet, if I understand the results presented in Supplementary Tables 20, 21, and 24 correctly? As this seems to be consistent across all three "Sensitivity Analyses", it needs a thorough explanation.
We are very grateful for the supportive and insightful comments.
The sensitivity analyses presented in Supplementary Tables 20, 21 and 24 were all on datasets where the data exclusion criteria removed either insects with obligate symbionts and low amino acid diets (herbivores and phloem feeders -analyses 4.6.1 and 4.6.2), or insects without obligate symbionts and high amino acid diets (omnivores and predators -analysis 4.6.4). Removing these insect families therefore results in feeding niches, such as blood feeders (high rates of symbiosis and amino acids in their diets), inflating the correlation between symbiosis and amino acids. Therefore, we believe the positive correlation between symbiosis and amino acids that emerges when restricting data illustrates the importance of broad taxonomic sampling rather than anything biologically meaningful.
We have addressed this issue by editing lines 349-368 and adding a more detailed interpretation of the sensitivity analysis section (lines 1341-1354).
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Specific comments:
Comment on the rebuttal: In Suppl. Table 5, there are also nonOb to nonOb and Ob to Ob transitions highlighted, which they shouldn't be.
Addressed l. 80-82: Just to be clear, please specify that co-speciation and negative fitness consequences of symbiont removal were used as obligate criteria. Just a small comment here (that I know will not be relevant to the results, since you carefully selected the symbioses): Strictly speaking, your two criteria would include some associations with Wolbachia as a reproductive manipulator, because it can be fixed in certain insect species, and removal would lead to severe fitness defects under CI (sometimes even extremely so, like in Asobara wasps). Not sure how to avoid this issue, but I wanted to at least point it out, but I assume the authors are anyways aware of it… Thank you for highlighting that this was unclear. The use of co-speciation and negative fitness consequences as secondary criteria is mentioned in our introduction (lines 73-76). In our data exclusion section (lines 704-713), we indicated that studies of known parasitic symbionts, such as those that manipulate host reproduction and have not evolved beneficial functions (e.g. Spiroplasma, Cardinium, Wolbachia), were excluded from our dataset. To make this clearer from the outset, we have added that these symbionts were excluded at the end of the introduction (lines 76-78).
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/. l. 181: the addition of "digestive enzymes" makes sense, but please revise, as these are not essential nutrients Addressed.  However, I urge them to consider whether there is a better way to display this, or at least relabel. "Non-obligate maintenance" is misleading, and it really took me a long time to grasp the figure in its full extent. In part, this was due to the use of the term "vs" in C and D, which I interpreted as divided by, which is exactly what it apparently is not supposed to mean here (rather, it's the reverse)?
We have clarified in the figure legend to make this clearer. l. 352: would be good to mention that this is also true for essential amino acid supplementing symbionts.
This is a good point. Given this paragraph is specifically focused on B-vitamins, we have instead included this when discussing the role of amino acids in the evolution of symbiosis (lines 399-423).
l. 464-465: This is not exactly true. In most of the analyses, the eAAs (and sometimes non-eAAs) also come up as significant, which does change the interpretation of the results quite a bit. I strongly recommend to add a comment on this and carefully discuss it. And it seems that they strongly go into the opposite direction than the B vitamins, indicating that symbiotic lineages are more likely to feed on amino acid-rich diet, if I interpret this correctly? This would not only be contradicting most of the accumulated symbiosis knowledge, but also be highly unlikely. This needs to be checked.
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Agreed. Please see response to the comment above. We have added a section to the manuscript explaining that we think this is most likely due to sampling biases introduced by removing species from specific feeding niches rather than a real effect (lines 1341-1354).
l. 490: With the caveat indicated above… Don't get me wrong, I don't insist on eAAs being a general driving factor underlying the evolution of nutritional symbioses in insects, but considering the gaps in current understanding and the necessarily limited phylogenetic resolution, as well as the results of the sensitivity analyses, they may be similarly important as the B vitamins, so I think it would be wise to phrase carefully here.
We have rephrased this sentence in line with the referee's suggestion.

Reviewer #5 (Remarks to the Author):
I appreciate the authors have considered my comments. However, I still have some considerable concerns around these issues. I will go through them here.
We are grateful to the referee for once again helping us clarify important issues in our manuscript.
With regard to the tree uncertainty the authors added the following sentence in the methods 'For information on the dating methods and topological uncertainties see Rainford et al. 201412". This is not sufficient and certainly does not describe the dating and uncertainty in the tree. I think the authors need to be honest that the results could be dependent on this tree of insects and that there is lost uncertainty and polytomies in the tree itself that is not considered. I know the paper has been used beforebut I think it is the authors duty to be honest that it is not clear what would happen in the case that these results were integrated across the uncertainty. However, I don't think this, in itself, should preclude publication of this work. It just needs to be made clear in the main text.
Thank you for this suggestion. We have now extended our description of how the Rainford tree was constructed and how analyses that account for tree uncertainty are an important extension of this work (lines 837-845).
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The authors response about the fact that number of species estimated in each family will be highly correlated with research effort is not at all satisfactoryand rather strange. In the figure 1 of their rebuttal they plot raw species richness verses raw number of species examined. As expected, they would not find a strong corelation! It is well established and obvious that these data should be transformed (the are bounded at zero!) for this demonstration. If one plots the transformed data (below) it is clear the corelation is very strong! It is also evident that the data are very singleton inflated (an over preponderance of ones in the dataset). Thus, this need very careful attention in the reanalyses for the data for another submission -I suggest the authors work with a statistician on this issue as there is some considerable confusion evident here! We realise that our response to the referee's original comment was confusing and are grateful for the opportunity to clarify. With regards to the above issues, the plot we included of species richness and the number of symbiont species studied does not help resolve the issues raised by the referee: a positive correlation between number of species examined for symbionts and species richness is expected both with and without biases in research effort, making it rather redundant.
Going back to the referee's original concern: "I think it will be quite likely that the number of species estimated in each family will be highly correlated with research effort, which will in turn is likely to be associated with occurrences of symbiosis and other covariates important here. So given this, I think the author need to control for research effort (associated with each family) in their analyses." The main objective of our species richness analyses was to test if obligate symbiosis is related to the number of species within families. The referee's concern is that variation in research effort will generate a spurious correlation between the number of species in each family and occurrences of symbiosis. There are three main reasons why this is not the case: First, there was no correlation between species richness and obligate symbiosis as expected if research effort confounds our results. Instead, we found obligate symbiosis was associated with both extreme highs and lows of species richness.
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/. Second, we analysed the proportion of species within families rather than the number of species with symbionts. Increased research effort (number of species checked for symbionts) is expected to increase the accuracy of the estimated proportion of species with obligate symbionts per family, but not systematically bias the mean proportion. It is therefore difficult to see how variation in research effort will induce correlations between the mean proportion of species with obligate symbionts and species richness.
Third, our analyses explicitly control for variation in the effort used to measure obligate symbiosis (e.g. the number of species examined for obligate symbionts). Specifically, we model the binomial outcome of the number of species with obligate symbionts out of the total number of species examined for symbionts (binomial BPMM presented in analysis 4.5.1). This approach accounts for variation in the number of species studied (research effort) across insect families. We have consulted with three experts about the approaches we use that are at the forefront of phylogenetic comparative analyses (Jarrod Hadfield, Shinichi Nakagawa and Dan Noble).
We agree with the referee that it would be ideal to account for the effort used to estimate species richness, but this is extremely difficult. The estimates of species richness come from Rainford et al 2014, who compiled information from field guides, encyclopaedias, museum collections, phylogenetic studies and online databases. Species richness was typically estimated by summing up numbers of species from these sources, which do not have quantitative measures of research effort that could be included in analyses (we do not know of any previous studies on diversification across insect families that have managed to do this). It is also difficult to see how variation in estimates of species richness would systematically bias our results. Estimates of species richness may be higher for some clades because they have received more attention (e.g. the attractiveness of butterflies means they are well studied), but symbionts are invisible to observers and so are unlikely to suffer from the same biases.
In line with the referee's suggestion, we have now added a discussion of the issues of sampling effort to the section on measuring species richness (lines 827-833).
The authors comments about Stochastic mapping and Discrete are not correct. The discrete model is for correlated models but there is another element of the program BayesTraits called Multistate that is far more appropriate for ancestral reconstruction -Stochastic mapping should only really be used for visualisation. The Bayesian implementation of Multistate was introduced in 2004 (Systematic Biology, Volume 53, Issue 5, October 2004, Pages 673-684) and has been cited over 1000 times.
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The referee previously asked about the use of stochastic character mapping (SCM) and Discrete, not Multistate. As a result, our reply was focused on justifying why we used Discrete and Stochastic character mapping. To recap, we used Discrete to test for correlated evolution between obligate symbiosis and Bvitamins, which we think the referee agrees is appropriate here. SCM was used to reconstruct ancestral states of feeding niches. The new issue raised in this comment is that a different module of BayesTraits -Multistate -is more appropriate for this purpose than SCM.
SCM and Multistate can be used for similar analytical purposesancestral state reconstruction of multistate categorical traits. SCM was designed for ancestral state reconstruction not just visualisation (details are given in 1,2 , and as Bollback 2006 describes "SIMMAP can be used to calculate the posterior distribution of ancestral states"). SCM has been used to infer the ancestral states of a variety of categorical traits, including feeding niches, across a variety of organisms (e.g. [3][4][5][6], which is why we choose to use this technique. However, we have added extra analyses that reconstruct the ancestral states of feeding niches using Multistate that show very similar results to our SCM analysis: 94% of nodes are predicted to have the same ancestral feeding niche by SCM and BayesTraits, which are presented in new supplementary table 20 and lines 1037-1054.
The authors suggest that they are talking the point of view that they use many techniques it makes there results more robustbut this is not true. As all these techniques have different assumption which need to be considered. And the most appropriate should be used. Again, I think some advice from a statistician might help here.
We agree with the referee that some analystical techniques are more appropriate for some purposes than others. However, some methods have been designed to address similar types of questions, but make different assumptions. For example, SCM and Multistate have been used to address similar problems, and in some cases have even be used on the same data to check the robustness of results to the analystical approach used 5,7 . Therefore there are siutations where different techniques with different assumptions are equally appropreiate. We believe, as has been argued by others 7 , that in such cases conducting analyses with multiple techniques helps verify that results are robust to different analytical assumptions.

Unfortunately, the authors interpretation of the reversible-jump procedure available in
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Reviewer #5 (Remarks to the Author): I thank the authors for their consideration of my comments. I think it is clear that I and they do not see eye-to-eye on some of the technical issues associated with the analyses. However, I don't think there is much value in prolonging this discussion here. The authors have been reasonable in their responses and largely considered my comments in the text. I enjoyed the manuscript from the outset, and congratulate the authors on an interesting piece of work. Thank you for your patience as we've prepared the guidelines for final submission of your Nature Ecology & Evolution manuscript, "Symbioses shape feeding niches and diversification across insects" (NATECOLEVOL-220616867B). Please carefully follow the step-by-step instructions provided in the attached file, and add a response in each row of the table to indicate the changes that you have made. Please also check and comment on any additional marked-up edits we have proposed within 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 text. Ensuring that each point is addressed will help to ensure that your revised manuscript can be swiftly handed over to our production team. **We would like to start working on your revised paper, with all of the requested files and forms, as soon as possible (preferably within two weeks). Please get in contact with us immediately if you anticipate it taking more than two weeks to submit these revised files.** When you upload your final materials, please include a point-by-point response to any remaining reviewer comments.
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