Climate variability and parent nesting strategies influence gas exchange across avian eggshells

Embryo survival in birds depends on a controlled transfer of water vapour and respiratory gases through the eggshell, and this exchange is critically sensitive to the surrounding physical environment. As birds breed in most terrestrial habitats worldwide, we proposed that variation in eggshell conductance has evolved to optimize embryonic development under different breeding conditions. This is the first study to take a broad-scale macro-ecological view of avian eggshell conductance, encompassing all key avian taxonomic groups, to assess how life history and climate influence the evolution of this trait. Using whole eggs spanning a wide phylogenetic diversity of birds, we determine that body mass, temperature seasonality and whether both parents attend the nest are the main determinants of eggshell conductance. Birds breeding at high latitudes, where seasonal temperature fluctuations are greatest, will benefit from lower eggshell conductance to combat temporary periods of suspended embryo growth and prevent dehydration during prolonged incubation. The nest microclimate is more consistent in species where parents take turns incubating their clutch, resulting in lower eggshell conductance. This study highlights the remarkable functional qualities of eggshells and their importance for embryo survival in extreme climates.

L86: Do you mean birds that are both alpine and Arctic/Antarctic or do you mean either alpine or Arctic/Antarctic? The syntax is unclear. L168: Where exactly did the life-history data come from? If it's at all a reasonable number of sources, the original compliers of the data would probably appreciate the citations; even if listing the major sources is untenable, more information is needed. (Even just stating that the major sources are detailed in section e of the supplement would be useful to the reader!) L196-198: I understand why some would find an analysis of residuals to be intuitively easier to understand than the multivariate regression described a few sentences previously, but a regression of residuals can be statistically flawed in many circumstances. See for example https://besjournals.onlinelibrary.wiley.com/doi/full/10.1046/j. 1365-2656.2002.00618.x (Freckleton 2002. It sounds like you ran both analyses, which is okay, but tread carefully. In particular, I had trouble sometimes distinguishing which results were based on the multiple regression and which were based on the regression-of-residuals. (Moreover, if the results of these two analyses aren't similar, you have a problem with underlying correlations of your predictor variables, and would need to err on the side of your multiple regression.) L233: The interpretation of lambda < 1 is tricky (see for example https://www.carlboettiger.info/2013/10/11/is-it-time-to-retire-pagels-lambda.html for a clear explanation of some of the issues of this metric). Lambda = 1 indicates that Brownian motion is a perfect fit for the distribution of the data. Lambda < 1 could indicate a weak effect of phylogeny under Brownian motion, or it could indicate a *strong* effect of phylogeny under another macroevolutionary mode; it's impossible to tell from the analysis conducted here. L236: Is strongly constrained by phylogeny *under the assumption of Brownian motion*. (I'm not bickering with the analysis here -what you did is perfectly standard. The phrasing of the interpretation just needs to be more precise.) Figure 1: I assume these are distribution maps for breeding ranges only? Figure 2: This is a very attractive figure (as is Figure 3). How did you define near-passerine? Figure 2: Are the Creative Commons licenses all CC0 1.0? If so this should be more clearly stated; if not, the details of the licenses should be listed somewhere (in the supplement), as per the legal conditions of using phylopic. Supplement, line 93: What do you mean by "extrapolated" here? L94-S: Does the FigShare link (which didn't work for me, presumably by design) list all sources of this information? (Same question for the "monographs" in L100-S and the other sources mentioned in this paragraph.) L135-S: Missing quotation mark (or possibly an extra quotation mark, depending on what you were intending) L161-163-S: I don't understand what you're trying to convey here. Was your releveling of factors somehow dependent on the results you obtained? Table S2: Consider making the font size on this table smaller (or decreasing the number of columns); numbers and words spanning multiple lines make this very difficult to read. (Same can be said about Table S4, though to a lesser extent.)

Is the length of the paper justified? No
Should the paper be seen by a specialist statistical reviewer? Yes Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. Yes It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author
The present study provides evidence that eggshell functionality evolves in relation to environmental pressures. The manuscript is very clear and it makes for a very interesting read. I only have some minor suggestions that I would like to see addressed before it is published.
-It would be useful to know why (or why not) would one expect that in passerines the relationship between conductance and nest type, parental incubation be different to that previously reported by Portugal et al. 2014. In other words, is there anything fundamentally different between passerine egg or nest architecture/ incubation behaviour and to non-passerine reproductive traits? -It was not obvious until reading the results that the study spanned half of the avian orders and not only passerines (which I first though, therefore the above comment). It would be useful to explain this since the introduction or methods.
-line130: change "to effect" with "to affect" (affect is the verb) -How many species were included in the measurements of conductance? (mention in the section "egg samples and preparation" -Although the sampling effort performed is commendable, sampled species account for ~4% of all bird species, this will always be a problem with comparative studies. How did the authors deal with the issue that results from having a large amount of missing data in the phylogeny, i.e. the outcome of the analyses is very sensitive to the inclusion of just a few extra species? I found the discussion slightly lengthy, perhaps some sections can be condensed. For example, last paragraph of page 13 (and first of page 14), also paragraph about calcium sources on page 14. Figure 1.-I am a little skeptical about the usefulness of information presented here, perhaps is my personal unfamiliarity with this type of mapping, how was this performed? How reliable is it? Moreover, the continental differences are not further discussed or contrasted with the finding that temperature seasonality had an effect on conductance. However, seasonality would vary with latitude but not merely differ between North America and Europe I imagine? What can one take as a message from this information? Figure 2. This figure is impressive and aesthetically appealing but it is overloaded with information and difficult to interpret. The tree for example, displays the ancestral state estimation plus 5 variables. The magnitude of the bars from the two first variables is hard to perceive. Could this figure be split?

27-Nov-2020
Dear Dr Attard: I am writing to inform you that your manuscript RSPB-2020-2685 entitled "Climate variability and mode of development influence gas exchange across avian eggshells" has, in its current form, been rejected for publication in Proceedings B.
This action has been taken on the advice of referees, who have recommended that substantial revisions are necessary. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance.
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Sincerely, Dr Sasha Dall mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Thank you for submitting your manuscript "Climate variability and mode of development influence gas exchange across avian eggshells" to Proceedings B. I have now received two reviews and evaluated the manuscript myself. While we all find the topic interesting, a number of issues have been raised that need to be addressed. Please pay particular attention to the questions regarding sampling biases made by Revewier 1 and comments by reviewer 2 about highlighting the novelty of your current work and differentiating it from your previous research.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) This manuscript, by Attard & Portugal, considers the inter-specific variation in eggshell conductance across a global sample of 364 species, finding that conductance (i.e. propensity for water loss during incubation) relates to adult body mass, temperature seasonality, and mode of development. This manuscript is very clearly written and examines an aspect of avian development not often considered at this scale.
My comments are all minor -most are superficial, with a few requests for increased clarity about the methods. I think this manuscript is very, very close to publication-ready: well done to the authors! As a general comment, though, I would be curious about the authors' views of any sampling biases underlying this dataset, given that they are making macroevolutionary conclusions on a comparatively small (albeit phylogenetically diverse) sample of species. Tring's collections are heavily biased towards former British colonies, and any Western museum collection will have better temperate sampling than tropical; the availability of eggs in the destructive collections would surely have put further taxonomic and biogeographic constraints on this study. This is of course unavoidable, but I'd be curious to see the authors' speculations about how a more phylogenetically representative sample might change their results.
I'd also be curious about the intraspecific variation in GH2O, given that the authors seem to have a large enough sample to investigate this. My understanding is that PGLS doesn't allow measurement error (though MCMCglmm does, if the authors are so inclined to test this), but even a partition of the variance in the database between intra-and inter-specific effects would reassure the reader both that this is a sensible variable to investigate at the comparative level and that the biases in comparative methods inherent in traits with high measurement error would not apply here. (See e.g. Silvestro et al. 2015ME&E, https://besjournals.onlinelibrary.wiley.com/doi/10.1111/2041-210X.12337, or Ives et al. 2007 Systematic Biology https://academic.oup.com/sysbio/article/56/2/252/1687174 ) Anyway, enough speculation. Minor comments: L31: Consider "terrestrial habitats". L70-73: This seems like an overstatement. Something along the lines of "one crucial step in understanding avian responses to environmental differences over evolution time is a better appreciation of…" would be less inflammatory but convey the same point? L84: Again, every *terrestrial* habitat. L86: Do you mean birds that are both alpine and Arctic/Antarctic or do you mean either alpine or Arctic/Antarctic? The syntax is unclear.
L168: Where exactly did the life-history data come from? If it's at all a reasonable number of sources, the original compliers of the data would probably appreciate the citations; even if listing the major sources is untenable, more information is needed. (Even just stating that the major sources are detailed in section e of the supplement would be useful to the reader!) L196-198: I understand why some would find an analysis of residuals to be intuitively easier to understand than the multivariate regression described a few sentences previously, but a regression of residuals can be statistically flawed in many circumstances. See for example https://besjournals.onlinelibrary.wiley.com/doi/full/10.1046/j. 1365-2656.2002.00618.x (Freckleton 2002. It sounds like you ran both analyses, which is okay, but tread carefully. In particular, I had trouble sometimes distinguishing which results were based on the multiple regression and which were based on the regression-of-residuals. (Moreover, if the results of these two analyses aren't similar, you have a problem with underlying correlations of your predictor variables, and would need to err on the side of your multiple regression.) L233: The interpretation of lambda < 1 is tricky (see for example https://www.carlboettiger.info/2013/10/11/is-it-time-to-retire-pagels-lambda.html for a clear explanation of some of the issues of this metric). Lambda = 1 indicates that Brownian motion is a perfect fit for the distribution of the data. Lambda < 1 could indicate a weak effect of phylogeny under Brownian motion, or it could indicate a *strong* effect of phylogeny under another macroevolutionary mode; it's impossible to tell from the analysis conducted here. L236: Is strongly constrained by phylogeny *under the assumption of Brownian motion*. (I'm not bickering with the analysis here -what you did is perfectly standard. The phrasing of the interpretation just needs to be more precise.)  The present study provides evidence that eggshell functionality evolves in relation to environmental pressures. The manuscript is very clear and it makes for a very interesting read. I only have some minor suggestions that I would like to see addressed before it is published.
-It would be useful to know why (or why not) would one expect that in passerines the relationship between conductance and nest type, parental incubation be different to that previously reported by Portugal et al. 2014. In other words, is there anything fundamentally different between passerine egg or nest architecture/ incubation behaviour and to non-passerine reproductive traits? -It was not obvious until reading the results that the study spanned half of the avian orders and not only passerines (which I first though, therefore the above comment). It would be useful to explain this since the introduction or methods.
-line130: change "to effect" with "to affect" (affect is the verb) -How many species were included in the measurements of conductance? (mention in the section "egg samples and preparation" -Although the sampling effort performed is commendable, sampled species account for ~4% of all bird species, this will always be a problem with comparative studies. How did the authors deal with the issue that results from having a large amount of missing data in the phylogeny, i.e. the outcome of the analyses is very sensitive to the inclusion of just a few extra species? I found the discussion slightly lengthy, perhaps some sections can be condensed. For example, last paragraph of page 13 (and first of page 14), also paragraph about calcium sources on page 14. Figure 1.-I am a little skeptical about the usefulness of information presented here, perhaps is my personal unfamiliarity with this type of mapping, how was this performed? How reliable is it? Moreover, the continental differences are not further discussed or contrasted with the finding that temperature seasonality had an effect on conductance. However, seasonality would vary with latitude but not merely differ between North America and Europe I imagine? What can one take as a message from this information? Figure 2. This figure is impressive and aesthetically appealing but it is overloaded with information and difficult to interpret. The tree for example, displays the ancestral state estimation plus 5 variables. The magnitude of the bars from the two first variables is hard to perceive. Could this figure be split?

General interest: Is the paper of sufficient general interest? Acceptable
Quality of the paper: Is the overall quality of the paper suitable? Excellent

Is the length of the paper justified? Yes
Should the paper be seen by a specialist statistical reviewer? No Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author
It is my opinion that after revision this paper is ready for publication. The authors did a superb job addressing all my previous comments and replying to each question in great detail. Thus, I learned something new during the reviewing process.

07-May-2021
Dear Dr Attard I am pleased to inform you that your Review manuscript RSPB-2021-0823 entitled "Climate variability and parent nesting strategies influence gas exchange across avian eggshells" has been accepted for publication in Proceedings B.
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Sincerely, Dr Sasha Dall mailto:proceedingsb@royalsociety.org Associate Editor Board Member Comments to Author: Thank you for submitting your manuscript "Climate variability and mode of development influence gas exchange across avian eggshells" to Proceedings B. I have now received feedback from a reviewer concerning the revisions of your manuscript and evaluated it myself. The reviewers and I agree that you have successfully addressed all concerns.
Reviewer(s)' Comments to Author: Referee: 2 Comments to the Author(s). It is my opinion that after revision this paper is ready for publication. The authors did a superb job addressing all my previous comments and replying to each question in great detail. Thus, I learned something new during the reviewing process.

14-May-2021
Dear Dr Attard I am pleased to inform you that your manuscript entitled "Climate variability and parent nesting strategies influence gas exchange across avian eggshells" has been accepted for publication in Proceedings B.
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Response to Referees
We thank the editor and both reviewers for their valuable suggestions and insight on our manuscript. The suggestions offered by the reviewers have been immensely helpful, and we also appreciate your insightful comments to better communicate the novelty of our research.
We have addressed all reviewer comments and have thoroughly revised the manuscript, incorporating most suggested changes or explaining why we have adopted a different strategy. Additionally, we have corrected a slight oversight in our R script and have set a more conservative cut-off for correlation coefficients between predictors for the phylogenetic comparative analysis. This has slightly changed the results, however, the overall message and findings of the paper remains the same. All specimen and species eggshell conductance data, conductance and life-history sources and R scripts used in this paper can now be accessed by the reviewers using the private link (https://figshare.com/s/ab01f5a20d00673018b3) on Figshare. We have added the Figshare DOI to the paper, which will become activate upon publication.
Comments from the editors and reviewers are in italics and are responses are in bold. Text that has been updated in the main document as part of our responses is highlighted in yellow. We also refer to line numbers from the revised manuscript in our responses. Amendments to the main text based on changes in our results are highlighted in green.

Associate Editor
Comments to Author: Thank you for submitting your manuscript "Climate variability and mode of development influence gas exchange across avian eggshells" to Proceedings B. I have now received two reviews and evaluated the manuscript myself. While we all find the topic interesting, a number of issues have been raised that need to be addressed. Please pay particular attention to the questions regarding: We're pleased yourself and the reviewers found our paper interesting, and we believe we have fully addressed all the issues that have been raised.

1) Sampling biases made by Reviewer 1
This has now been addressed in the manuscript. Our response can be found below and also in response to Reviewer 1, question 1 under "General comments".
The reviewer makes an excellent point, that there is potential for bias. This potential for bias in the destructive collection at the Natural History Museum was the primary driver for encompassing data from the literature. The 188 species from the literature have a worldwide distribution, along with adding phylogenetic diversity to the study also.

2) Comments by Reviewer 2 about highlighting the novelty of your current work and differentiating it from your previous research.
The novelty of this research is two-fold: it presents the first large-scale phylogenetic comparative assessment of eggshell conductance in birds (encompassing all key taxonomic groups) and its association with different life-history traits. Secondly, our phylogenetic comparative analysis uses regression-of-residuals to account for differences in adult body mass, allowing us to address the adaptive importance of specific traits of a species after allometric effects are considered.
Previous comparative research on eggshell conductance has focused on nonpasserines, ignoring 60% of bird species, given passerines make a substantial component of the avian kingdom. These prior studies on non-passerines did not correct for differences in body mass of the adults, which potentially hides critical adaptive information relating to the environment and nesting behaviour of the species (see L111-122). Moreover, previous studies have typically focused either on (i) one group of birds, e.g., gulls, with the goal to look for micro-adaptations between closely related species, or (ii) extreme examples of nest environments such as grebes and marsh-nesting black terns (Chlidonias niger). Thus, this is the first study to take a broad-scale macro-ecological holistic view of avian eggshell conductance and look for commonalties and patterns across a broad-scale of bird species, all within a phylogenetic framework.
As such, we have now updated the Abstract and Introduction (see below) to emphasise the need for large-scale comparative analysis encompassing all key avian taxonomic groups to determine how life-history and climate influence the evolution of eggshell conductance.
Abstract: "This is the first study to take a broad-scale macro-ecological view of avian eggshell conductance, encompassing all key avian taxonomic groups, to assess how life history and climate influence the evolution of this trait." (L33-36). The reviewer makes an excellent point, that there is potential for bias. This potential for bias in the destructive collection at the Natural History Museum was the primary driver for encompassing data from the literature. The 188 species from the literature have a worldwide distribution, along with adding phylogenetic diversity to the study also. Thus, eggshell conductance measures included in our study were based on specimens at the Natural History Museum that we measured in our laboratory (176 species) as well as mean species conductance values from the literature (188 species). As such, over half of the species in our dataset are derived from multiple sources worldwide, which has broadened the spatial distribution of species incorporated in this study.

Introduction: "A study across 141 non-passerine species detected differences in GH
We agree that there are some biogeographical constraints by incorporating a large portion of British species. It is worth noting, however, that many British species have substantial geographical distributions (barn owls, peregrine falcons, house sparrows, ospreys etc), even though the eggs themselves (in the present study) for those species where gathered from the United Kingdom. We thank Reviewer 1 for their suggestion to run a MCMCglmm to assess variation in the dataset between intra-and inter-specific effects. We agree that this would greatly benefit the paper, and clarify the contribution that intraspecific variation is making to our overall findings. Unfortunately, we do not have specimen-specific values for any species obtained for the literature, as only mean species values were presented within the paper and thus available to us. As such, we are unable to run such an analysis on the full data set. We reviewed the papers from the literature, and many were from decades ago with authors who are no longer with us. Due to this, we decided it wouldn't be fruitful to try and track down these full data sets from contacting the authors.

2) I'd also be curious about the intraspecific variation in GH2O, given that the authors seem to have a large enough sample to investigate this. My understanding is that PGLS doesn't allow measurement error (though MCMCglmm does, if the authors are so inclined to test this), but even a partition of the variance in the database between intra-and inter-specific effects would reassure the reader both that this is a sensible variable to investigate at the comparative level and that the biases in comparative methods inherent in traits with
Of course, however, we do have all values in full for our species that we directly measured in the laboratory. We have applied MCMCglmm to 176 species from our dataset, where we measured conductance of eggs directly.

When accounting for intra-specific variation in eggshell conductance, body mass was the only significant predictor of log(GH 2 O) in passerines based on MCMCglmm. This concurs with our findings based on PGLS on 364 species.
As the MCMCglmm results do not cover a broad taxonomic range due to the limited number of non-passerines with specimen-specific values (n=1), using these samples alone does not meet the main objective of our study. Therefore, we decided not to include MCMCglmm results in the revised manuscript. Minor comments: 1) L31: Consider "terrestrial habitats".

Done. Reworded to "terrestrial habitats" (L31), as suggested.
2) L70-73: This seems like an overstatement. Something along the lines of "one crucial step in understanding avian responses to environmental differences over evolution time is a better appreciation of…" would be less inflammatory but convey the same point?
A very good point, and we have amended accordingly as follows:

"One crucial step in understanding avian responses to environmental differences over evolutionary time is a better appreciation of factors shaping avian incubation and their subsequent influence on the embryo [7]." (L70-73)
3) L84: Again, every *terrestrial* habitat.

4) L86: Do you mean birds that are both alpine and Arctic/Antarctic or do you mean either alpine or Arctic/Antarctic? The syntax is unclear.
Thank you for spotting this, as we had been ambiguous. We have corrected the syntax to "Among these are ground nesting birds in alpine or Arctic/Antarctic regions" (L86-87). The Figshare DOI will be activated once the paper is published. Reviewers can download all data and R scripts directly using the private Figshare link (https://figshare.com/s/ab01f5a20d00673018b3). Every source used to extract life-history data for each species can be found in the following spreadsheet:  "species_eggshell_conductance_data_references.xlsx" We have also uploaded this spreadsheet in our manuscript resubmission for the reviewers. Please refer to tab "Life-history ref list" and "Conductance literature ref list" in the spreadsheet for the full list of references for lifehistory and conductance values from the literature. The tab "species_data" contains all species-specific conductance and life-history data. The column "source_lifehistory" (column AV) cites all sources used to obtain life-history traits for each individual species.

5) L168
6) L196-198: I understand why some would find an analysis of residuals to be intuitively easier to understand than the multivariate regression described a few sentences previously, but a regression of residuals can be statistically flawed in many circumstances. See for example https: //besjournals.onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2656.2002.00618.x (Freckleton 2002. It sounds like you ran both analyses, which is okay, but tread carefully. In particular, I had trouble sometimes distinguishing which results were based on the multiple regression and which were based on the regression-of-residuals. (Moreover, if the results of these two analyses aren't similar, you have a problem with underlying correlations of your predictor variables, and would need to err on the side of your multiple regression.) Thank you for your suggestions. We ran both analyses, and found that the same predictors were present in the top-ranked models (AIC<2). Body mass was highly influential (Table S1 and S2) in predicting GH 2 O, so we ran PGLS on the regression-of-residuals to account for allometric effects, and to determine what contributed to he observed variation in eggshell conductance, after accounting for body mass.
To help distinguish between our results, the main predictors of log(GH 2 O) and residual GH 2 O are now presented as two separate figures. Figure 1 now shows the most influential predictors for log(GH 2 O), while figure 2 shows the most influential predictors for residual GH 2 O. Hopefully this is much clearer now, and avoids any ambiguity.

7) L233: The interpretation of lambda < 1 is tricky (see for example https://www.carlboettiger.info/2013/10/11/is-it-time-to-retire-pagelslambda.html for a clear explanation of some of the issues of this metric). Lambda = 1 indicates that Brownian motion is a perfect fit for the distribution of the data. Lambda < 1 could indicate a weak effect of phylogeny under Brownian motion, or it could indicate a *strong* effect of phylogeny under another macroevolutionary mode; it's impossible to tell from the analysis conducted here.
Thank you for raising this point. We agree with the reviewer that we cannot tell from the analysis whether the effect is "strong" or "weak". Following the reviewer's suggestion, we have reworded L264-271 to: "Phylogenetic signal was high for Log(GH 2 O) (λ=0.96), showing that closely related species exhibit similar eggshell conductance prior to accounting for differences in body mass, and this biological similarity decreases as the evolutionary distance between species increases. Phylogenetic signal was intermediate for RGH 2 O (λ=0.55), suggesting that phylogeny and other selective pressures (e.g., those associated with species life-history or climate) are important in determining eggshell conductance, after accounting for differences in species body mass." Additionally, we have added in the manuscript how we tested if Pagel's lambda is significantly different from 0 and 1: "The phylosig function was used to test the hypothesis that Pagel's λ is different from 0. To test the alternative hypothesis (that Pagel's λ is less than 1), we computed the difference in the log-likelihood ratio of the lambda model (phylosig function) and Brownian motion model (brownie.lite function), then compared it to a chi-squared (χ 2 ) distribution with 1 degree of freedom." (L229-233).

8) L236: Is strongly constrained by phylogeny *under the assumption of Brownian motion*. (I'm not bickering with the analysis herewhat you did is perfectly standard. The phrasing of the interpretation just needs to be more precise.)
We agree, and thank you for highlighting this. We have made changes in the manuscript to clarify our interpretation, pasted below: "Phylogenetic signal was high for Log(GH 2 O) (λ=0.96), showing that closely related species exhibit similar eggshell conductance prior to accounting for differences in body mass, and this biological similarity decreases as the evolutionary distance between species increases. Phylogenetic signal was intermediate for RGH 2 O (λ=0.55), suggesting that phylogeny and other selective pressures (e.g., those associated with species life-history or climate) are important in determining eggshell conductance, after accounting for differences in species body mass." (L264-271) 9) Figure 1: I assume these are distribution maps for breeding ranges only?
The distribution maps were created using spatial data compiled by BirdLife International and Handbook of birds of the World. This encompasses the total range of each species, as unfortunately shapefiles of species breeding range alone are not available.
We have provided a description of how the global spatial distribution maps for eggshell conductance were created in section (i) of Supplementary Information and have upload the R script and all shapefiles used to create the eggshell conductance distribution map to Figshare. These files are compressed into the folder "spatial distribution traits", and can be downloaded by reviewers using our private Figshare link (https://figshare.com/s/ab01f5a20d00673018b3). As the distribution map is not pivotal to our study, we have moved this figure to Supplementary information ( Figure S2). Figure 2: This is a very attractive figure (as is Figure 3). How did you define nearpasserine?

10)
We have added a description of near-passerines in L192-206 of the methods to be absolutely clear (see below). We do agree that these categories are somewhat contentious. However, we were still keen to explore these three groups separately due to possibility that eggshell conductance in nearpasserines may more closely resemble passerines than non-passerines because of their ecological similarities.
"Prior to updated avian phylogenies based on genomic DNA, near-passerines was a term given to tree-dwelling birds (within the conventional nonpasserines) that were traditionally believed to be related to Passeriformes due to ecological similarities. In this study Pterocliformes (sandgrouse), This is correct. By design, the Figshare DOI will not work until the paper is published. We are happy to share these data with the referees prior to publication so they can be assessed. The life-history spreadsheet (titled "species_eggshell_conductance_data_references.xlsx") has been uploaded in the re-submission. This spreadsheet can also be downloaded by reviewers using our private Figshare link (https://figshare.com/s/ab01f5a20d00673018b3).

Columbiformes (pigeons), Cuculiformes (cuckoos), Caprimulgiformes (nightjars), and Apodiformes (swifts, hummingbirds) were defined as near
In the life-history spreadsheet, we list every source used in a separate column next to each species so readers can find the original source of information.

14) L135-S: Missing quotation mark (or possibly an extra quotation mark, depending on what you were intending)
We thank the reviewer for pointing this out. Extra quotation mark added.

15) L161-163-S: I don't understand what you're trying to convey here. Was your releveling of factors somehow dependent on the results you obtained?
We have now clarified this in the supplementary text. If there were more than 2 levels in a factor (e.g., the category "nest location" has three levels: ground, tree, cliff), the PGLS needs to be run again, whereby the levels of this factor are re-ordered so that the level specified by "ref" is first and the others are moved down. In doing so, each level becomes the reference to be compared with the remaining levels in that factor. This allows pair-wise comparisons within each level to be assessed, as shown in the conditional averaged outputs. This does not influence the results of other factors included in the analysis. All R scripts used in this publication, including PGLS, will be available on Figshare. The private link to download R scripts prior to publication is https://figshare.com/s/ab01f5a20d00673018b3.
We have pasted our amendment to Supplementary Information below: "When a multi-level predictor (i.e., a factor with more than two levels) was significant for conditional averaging, the "relevel" function was used to reorder the levels of that predictor and thereby change the reference variable for that predictor. The PGLS models were then rerun to obtain conditional averaging results for all multi-level predictor pair-wise comparisons. For example, the category "nest location" had three levels (ground, tree and cliff), thus the PGLS needs to be run again, whereby the levels of this factor are reordered so that the level specified as the reference is first, and the others are moved down. In doing so, each level becomes the reference to be compared with the remaining levels in that factor. This allows pair-wise comparisons within each level to be assessed, as shown in the conditional averaged outputs. This does not influence the results of other factors included in the analysis." (L177-188-S) We have now used 9 font size and single line spacing in all tables in supplementary to make it easier to read. All supplementary tables now fit on a single page each.

Reviewer 2
The present study provides evidence that eggshell functionality evolves in relation to environmental pressures. The manuscript is very clear and it makes for a very interesting read. I only have some minor suggestions that I would like to see addressed before it is published.
We're pleased the reviewer felt the manuscript was clear and interesting. We believe we've addressed all the minor issues, and thank the reviewer for the improvements to our paper.

Major comments:
1) It would be useful to know why (or why not) would one expect that in passerines the relationship between conductance and nest type, parental incubation be different to that previously reported by Portugal et al. 2014. In other words, is there anything fundamentally different between passerine egg or nest architecture/ incubation behaviour and to non-passerine reproductive traits? Table 2 describes how different life-history traits may influence eggshell conductance. We predicted that passerines and near-passerines would have lower eggshell conductance than non-passerines, as passerines and nearpasserines typically have altricial young and use exposed nests, while nonpasserines have altricial and precocial young and are less likely to use exposed nests. The key differences between development mode and nest architecture between passerines, near-passerines and non-passerines are described in L197-206 of the Methods (pasted below), which could potentially be responsible for differences in eggshell conductance between these taxonomic groups. . Although these groups are no longer recognised as near-passerines, this definition was used here to distinguish between ecologically profound differences among birds." Differences in embryo development and incubation period between altricial and precocial species, and how this may influence eggshell conductance in passerines and near-passerines versus non-passerines is described the last paragraph of the discussion (pasted below). "Variation in incubation period across the altricial-precocial spectrum reflects a trade-off between embryo growth rate and degree of maturity when hatched. Precocial species take up to 2 times longer to incubate an egg of the same size as altricial species, but are far more developed when they hatch [75]. For eggs of the same mass, precocial species incur a higher total energy cost than altricial species because the embryo is larger for a longer period during incubation [76]. Consequently, eggs of species with fast (precocial) growing offspring had significantly higher RGH 2 O than those of species with slow (altricial) growing offspring based on top-ranked models. As higher conductance enables greater gas exchange, this may optimise embryo access to high energy content in precocial eggs [77], thus resulting in a more developed chick at birth. RGH 2 O in passerines was found here to be particularly low, likely because they have altricial young, whereas non-passerines consist of precocial and altricial species." (L372-384) 2) It was not obvious until reading the results that the study spanned half of the avian orders and not only passerines (which I first thought, therefore the above comment). It would be useful to explain this since the introduction or methods.
Thank you for highlighting this. We have made additions to the Introduction to clarify this. We have added "spanning across 28 avian orders" to our study aims in the Introduction (L145) to increase clarity. We have also added a paragraph in the Methods describing the key avian taxonomic groups explored in this study (L192-206), pasted below. "Prior to updated avian phylogenies based on genomic DNA, near-passerines was a term given to tree-dwelling birds (within the conventional nonpasserines) that were traditionally believed to be related to Passeriformes due to ecological similarities. In this study Pterocliformes (sandgrouse), Columbiformes (pigeons), Cuculiformes (cuckoos), Caprimulgiformes (nightjars), and Apodiformes (swifts, hummingbirds) were defined as nearpasserines. All passerines and near-passerines are land birds and have altricial and nidicolous (stay within the nest) chicks, while non-passerine chicks vary in their mode of development and include water and land birds [38]. Sandgrouse are an exception, as they have precocial young and are not tree-dwelling [39]. In respect to nest architecture, most passerines build opencup nests, though some build more elaborate dome structures with roofs [40]. Dome nests, however, are more common among passerines than nonpasserines, and are particularly frequent among very small passerines [41]. Although these groups are no longer recognised as near-passerines, this definition was used here to distinguish between ecologically profound differences among birds." 3) Although the sampling effort performed is commendable, sampled species account for ~4% of all bird species, this will always be a problem with comparative studies. How did the authors deal with the issue that results from having a large amount of missing data in the phylogeny, i.e. the outcome of the analyses is very sensitive to the inclusion of just a few extra species?
We agree that our study, as with most comparative studies, will have missing data in the phylogeny. Although we did not account for missing data directly in our analysis, we were extremely careful in selecting categorical predictors, ensuring a sufficient sample size for all factor levels.
Our top-ranked models identified multiple factors that influence eggshell conductance across a broad-taxonomic range of species, incorporating 28 avian orders. We acknowledge that incorporating a larger number of species (preferably by adding representatives from other avian orders, or additional species from extreme nesting environments to capture the full spectrum of life-histories) would improve the robustness of our statistical analysis, and help support our findings.
It is possible that the factors that were present but not significant (p>0.05) in our top-ranked models (e.g., eggshell maculation) may be important if eggs from other avian families or orders were available. However, we do not expect that the inclusion or exclusion of a few species from our current dataset would drastically change our results.
We would also expect the outcome to vary depending on the taxonomic scale. Thank you for your advice on this matter. We agree that the discussion needs to be condense, so we have removed these sections from the discussion, as suggested.

5) L130: change "to effect" with "to affect" (affect is the verb)
This has been corrected. See L139.

6) How many species were included in the measurements of conductance? (mention in the section "egg samples and preparation".
We have added at the start of Materials and Methods (L145): "In total, 365 bird species were included in this study." Figure 1: I am a little skeptical about the usefulness of information presented here, perhaps is my personal unfamiliarity with this type of mapping, how was this performed? How reliable is it? Moreover, the continental differences are not further discussed or contrasted with the finding that temperature seasonality had an effect on conductance. However, seasonality would vary with latitude but not merely differ between North America and Europe I imagine? What can one take as a message from this information?

Figures 7)
This figure is provided to give a general indication of geographical variation in eggshell conductance based on the spatial distribution of species included in this study, and is not intended to be used for any analytical purposes. As this distribution map is not paramount to this study, we have moved Figure 1 to Supplementary Information. We used a modified R script provided on Dr Bruno Vilea's website (link: https://rmacroecology.netlify.app/2018/01/23/a-guide-to-transform-speciesshapefiles-into-a-presence-absence-matrix-based-on-a-user-defined-gridsystem/ and https://rmacroecology.netlify.app/2018/01/30/mapping-speciestraits/) to create the spatial distribution maps. Further information about the "letsR" package is available in their publication (Vilela and Villalobos 2015). We have provided the R script in Figshare (titled "R markdown_Species Distribution GH20 map.Rmd" in Spatial Distribution Traits folder), and we have described the process in section (