Blood Metabolites and Profiling Stored Adipose Tissue Reveal the Differential Migratory Strategies of Eurasian Reed and Sedge Warblers

Round 1

Reviewer 1 Report

General comments:

The subject of the manuscript written by Araújó et al. is interesting and under-researched. Blood metabolites and stored adipose tissue of two closely related warbler species with similar body size and morphology but different migration strategies were compared during spring and autumn migration with different fasting conditions. The introduction is well written and logically structured, but the predictions are missing. The idea of the study promises exciting results, but unfortunately, its implementation did not succeed well. The methods section, statistical analysis, and evaluation are confusing and incomplete. In addition, it is not clear which samples were used in which analyses (see details in minor comments). The discussion is relatively well written, although there is an ambiguous, less consistent part. Publishing the manuscript is recommended only after a thorough revision and verification of the statistical results.

Minor comments:

L16 "at a Portuguese stopover site" - There are two stopover sites in the methods part.

L41 A clear description of the changes in Plasma TRIG is missing. Since TRIG between the liver and the adipose tissue is in the blood, plasma TRIG increases in the blood. Thus, plasma TRIG increases during feeding and fat deposition, whereas plasma GLYC increases during fasting (Robinson and Williamson 1980, Ramenofsky 1990). This fact is essential for the interpretability of the results.

L53 FFA- Free fatty acids? - Abbreviation without prior explanation.

L71-73 In what direction does it work? (Fatty acids that are shorter or have more double bonds are likely to increase peak performance due to their higher transport rates en route to oxidation (in Price 2010).)

L99-113 The predictions are missing.

L115-120 Does the reed warbler also breed on these stopover sites? If so, was there data on whether the caught individuals had started the migration? This question is crucial during the autumn migration to know if the non-fasting individuals had been in the migration phase.

L137 "first five individuals" Text and table do not match. Moreover, why would the authors have only taken blood from 5 individuals per species and year? Please clear this part.

L153 According to the methods (L124-126, L157-159), samples were taken for laboratory analysis only in 2014. According to the text of Table 1, only 2011 and 2012 data are included in the table; however, laboratory sampling is included. Please specify the table or the text.

What was the reason for the differences in sample sizes between TRIG + GLYC and GLUC groups?

In Table 1. "Post flying fasting," but "post-flight fasting" is written on the figures. Please use group names consistently.

In the LAB group, why is NA entered? Why not 0?

L198 Geisser-Greenhouse correction usually is used for repeated measures ANOVA when the sphericity assumption is violated. Please refer to the fact that Geisser-Greenhouse correction can also be used in case of violation of homoscedasticity in a two-way ANOVA.

L195-196 This method used to exclude the effect of the year is not appropriate because it did not include species and season as factors that may interact with the year. In addition, no post-flight fasting study was conducted in 2011, which further increased the unbalance among the sample size of the groups. I would rather suggest that where the seasonal effect is studied, data from post-flight fasting individuals should be excluded (because if the fasting condition cannot be included as a factor due to missing cells of the year 2011, it can bias the model) and put the year as a factor. Where the effect of fasting condition is tested, work only with data of the year in which both post-flight fasting and non-fasting individuals present.

L237-240 Method of analyzing fat score is missing from the Method part. However, from the t-value, I suppose t-tests are applied here. The fat score is an ordinal dependent variable; thus, it is unsuitable for parametric statistical analysis. I suggest using rank-based nonparametric statistics.

L241-266 The degrees of freedom are incorrect in all ANOVA results, not even with Geisser-Greenhouse correction. Please check the degrees of freedom or specify which individuals are included in which study.

L249-250 "Circulating blood metabolites of Sedge and Reed Warblers between post-flight fasting and non-fasting birds using portable field equipment." - This sentence requires bold highlighting.

L267-268 "Comparing fatty acid composition and average molecular structure between Sedge and Reed warblers during post-flight fasting." - This sentence requires bold highlighting.

L275 Where is the mmol / g value in the table?

L276 Please use consistently SE or SEM for "standard error of the mean" (see L549, L553, L556)

L277-278 Please use the order of the table when explaining the abbreviations.

L348-365 Confused, incomplete conclusion.

L559-561 Number of dots on the figures and sample size in Table 1. do not match.

Author Response

Reviewer 1

Comments and Suggestions for Authors

General comments:

The subject of the manuscript written by Araújó et al. is interesting and under-researched. Blood metabolites and stored adipose tissue of two closely related warbler species with similar body size and morphology but different migration strategies were compared during spring and autumn migration with different fasting conditions. The introduction is well written and logically structured, but the predictions are missing. The idea of the study promises exciting results, but unfortunately, its implementation did not succeed well. The methods section, statistical analysis, and evaluation are confusing and incomplete. In addition, it is not clear which samples were used in which analyses (see details in minor comments). The discussion is relatively well written, although there is an ambiguous, less consistent part. Publishing the manuscript is recommended only after a thorough revision and verification of the statistical results.

RE: First of all, we appreciate the reviewer comments. According to the reviewer advice we rephrased several parts of manuscript, including the abstract, methods and results to increase the overall understanding. We uniformized the nomenclature at entire manuscript.

Minor comments:

L16 "at a Portuguese stopover site" - There are two stopover sites in the methods part.

RE: Corrected according to reviewer advice.

L41 A clear description of the changes in Plasma TRIG is missing. Since TRIG between the liver and the adipose tissue is in the blood, plasma TRIG increases in the blood. Thus, plasma TRIG increases during feeding and fat deposition, whereas plasma GLYC increases during fasting (Robinson and Williamson 1980, Ramenofsky 1990). This fact is essential for the interpretability of the results.

RE: We explained the changes both in TRIG, GLY and GLUC later on the manuscript, as reviewer can see in line XXX (“ TRIG and GLY can be used to infer metabolic condition, although levels in the blood have been found to vary among species. For example, Jenni-Eiermann (2003) found lower TRIG levels in flying than resting Red Knots (Calidris canutus). However, Jenni-Eiermann and Jenni (1991, 1992) showed that small migratory passerines had high levels of TRIG in their blood plasma during flight. The difference among several species in TRIG levels during stopover can be attributed to metabolic differences between passerines and non-passerines, or their fasting duration due to flight distance (Jenni-Eiermann 2003). Given these differences, Jenni-Eiermann (2003) suggested an additional pathway involving TRIG and FFA to flight muscles. Therefore, levels of TRIG in flying birds should vary according to their migratory state.”

L53 FFA- Free fatty acids? - Abbreviation without prior explanation.

RE: Corrected according to reviewer advice.

L71-73 In what direction does it work? (Fatty acids that are shorter or have more double bonds are likely to increase peak performance due to their higher transport rates en route to oxidation (in Price 2010).)

RE: Corrected according to reviewer advice.

L99-113 The predictions are missing.

RE: According to both reviewers we added the predictions at introduction: “We expected that post-flight fasting birds would exhibit higher levels of TRIG + GLY due to higher TRIG mobilization from adipose tissue into glycerol. Furthermore, Sedge warblers should present, also higher TRIG + GLY levels than Reed warblers during post-flight fasting period, by TRIG hydrolysis into GLY and FFA due to their differing migratory strategy. According to Scanes and Braun (2013), migratory birds primarily use fat reserves to fuel migratory flights, and therefore we expected that GLUC concentrations in blood circulation stay constant during, or as result of a long-distance flight (Maillet and Weber 2006). Non-fasting birds that stopped naturally at our stopover sites should present higher GLUC levels due to the diet preferences at our study site and due their likely recent feeding activity. “

L115-120 Does the reed warbler also breed on these stopover sites? If so, was there data on whether the caught individuals had started the migration? This question is crucial during the autumn migration to know if the non-fasting individuals had been in the migration phase.

RE: We also ring at our stopover sites during breeding season, and we have ringed almost all the breeding individuals. By this fact we can ensure that birds used in our work are in active migration.

L137 "first five individuals" Text and table do not match. Moreover, why would the authors have only taken blood from 5 individuals per species and year? Please clear this part.

RE: We used the first 5 birds trapped to ensure that we have time to collect blood samples and run the samples or kept the blood for the later analyses before the birds start the catabolism in the net. Sampling the first five birds, we also ensure that the birds trapped are not started the feeding activity.

L153 According to the methods (L124-126, L157-159), samples were taken for laboratory analysis only in 2014. According to the text of Table 1, only 2011 and 2012 data are included in the table; however, laboratory sampling is included. Please specify the table or the text.

RE: We rephrased the sentence of the Table 1.

What was the reason for the differences in sample sizes between TRIG + GLYC and GLUC groups?

RE: WE used the same blood sample for the analyses (TRIG+GLY and GLUC); however, some birds “give” little amount of blood and we prioritized the TRIG+GLYC analyses.

In Table 1. "Post flying fasting," but "post-flight fasting" is written on the figures. Please use group names consistently.

RE: Corrected according to reviewer advice.

In the LAB group, why is NA entered? Why not 0?

RE: Changed according to reviewer advice.  

L198 Geisser-Greenhouse correction usually is used for repeated measures ANOVA when the sphericity assumption is violated. Please refer to the fact that Geisser-Greenhouse correction can also be used in case of violation of homoscedasticity in a two-way ANOVA.

RE: We agree with reviewer comment. However, we prefer to adjust all the statistical analyzes for Linear mixed models to increase the quality of our results.

L195-196 This method used to exclude the effect of the year is not appropriate because it did not include species and season as factors that may interact with the year. In addition, no post-flight fasting study was conducted in 2011, which further increased the unbalance among the sample size of the groups. I would rather suggest that where the seasonal effect is studied, data from post-flight fasting individuals should be excluded (because if the fasting condition cannot be included as a factor due to missing cells of the year 2011, it can bias the model) and put the year as a factor. Where the effect of fasting condition is tested, work only with data of the year in which both post-flight fasting and non-fasting individuals present.

RE: We before pooled the years (2011 with 2012) we analyzed both seasons and species separately and we observed that was no statistical differences between each condition. Furthermore, we already analyzed our results with GLMM (year as random effect) and the results observed were the same. Is just the reason why we pooled these 2 years of data.

L237-240 Method of analyzing fat score is missing from the Method part. However, from the t-value, I suppose t-tests are applied here. The fat score is an ordinal dependent variable; thus, it is unsuitable for parametric statistical analysis. I suggest using rank-based nonparametric statistics.

RE: Please check the “Mist-netting” section: “All captured birds were ringed, morphological measurements were taken (tarsus length, wing length, bill length, tail length, and mass), and fat scores were assessed on a scale from 0 to 8 (where 0 is no fat and 8 is maximum fat; Kaiser 1993).”

L241-266 The degrees of freedom are incorrect in all ANOVA results, not even with Geisser-Greenhouse correction. Please check the degrees of freedom or specify which individuals are included in which study.

RE: We rephrased all the results section.

L249-250 "Circulating blood metabolites of Sedge and Reed Warblers between post-flight fasting and non-fasting birds using portable field equipment." - This sentence requires bold highlighting.

RE: Corrected according to reviewer advice.

L267-268 "Comparing fatty acid composition and average molecular structure between Sedge and Reed warblers during post-flight fasting." - This sentence requires bold highlighting.

RE: Corrected according to reviewer advice.

L275 Where is the mmol / g value in the table?

RE: We rephrased the sentence in the Table 2.

L276 Please use consistently SE or SEM for "standard error of the mean" (see L549, L553, L556)

RE: Thanks for advising me about this. We corrected to SE according to our results.

L277-278 Please use the order of the table when explaining the abbreviations.

RE: Corrected according to reviewer advice.

L348-365 Confused, incomplete conclusion.

RE: This part of discussion was rephrased.

L559-561 Number of dots on the figures and sample size in Table 1. do not match.

RE: We used the same data set for all the figures. It is possible that some dots are very close and can looks like the same dot. However, we rephrased the analyze and the figure and we obtained the same results.

Reviewer 2 Report

This study compares the circulating levels of metabolites and fatty acid composition of two Acrocephalus warblers with reportedly diverse migratory strategies, body size, morphology, migratory routes, feeding preferences and stopover schedules.  The authors designed the study to elucidate the dietary preferences – refeeding activity at a common stopover site.  The study has merit and has demonstrated some innovative and now validated techniques for field measurements with the two Roche Accutrend Chemical detection systems. Nevertheless, there are numerous questions, concerns, and structuring of the manuscript that if addressed would certainly help to improve it.

Manuscript Structure:  The authors rely on previous publication of the two subspecies that point out differences.  Using this information and the background material on lipid and glucose metabolism in migrants, restructuring the paper with a hypothesis at the outset to distinguish the two warblers would clarify the work.  This could be done by using the recognized (published) differences and hypothesizing that there would or could be differences in their metabolic profiles.  Following this, offer predictions in the direction of how they may differ.   This approach can strengthen the results and expand on the published work.  Another issue is that I surmise from the manuscript and geographic breeding ranges of the two subspecies, is that breeding locations for Reed warblers are located mainly in Portuguese wetlands; whereas, Sedge subspecies tend to spread throughout central and northern Europe,  If this is the general pattern then the migratory distances  as well as the routes of the two may vary to a large extent and have an impact on the stopover scheduling of the two. So that the selected site for trapping the birds would favor the Reed Warblers who may be closer to their destination than that of the Sedge providing a smaller sample size and possibly less accurate data for the  Sedge.  This was in fact confirmed by the comment (Lines 166 – 168) that trapping enough Sedge warblers in the spring was difficult in terms of amassing enough numbers. Does this raise the possibility that these stopover sites may not be on a major routing for the Sedge warblers that would make these sites less optimal than the more distant central routing through Europe? If this is a major difference, the ecology and scheduling of the two subspecies could well differ.  But given this, it is evident that the migratory strategies of the two differ.  Lines 85 – 88  state that Sedge warblers carry large fat loads at least while departing from wintering grounds than do Reeds during spring migration.  If this is correct, then Sedge warblers would be considered “time minimizers”  - those that reduce the time spent on stopovers or have few options to stop and feed in the early phases of spring migration (Alerstam and Hedenstrom, 1998). By contrast, Reed warblers are departing the wintering sites as “energy minimizers” and carry less fuel but rely on more stops per trip. Yet, by the time the Sedge warblers reach the Portuguese sites they are few in number and lean (Lines 165-166).  So doesn’t this indicate a change in migration tactic or compete loss of quality stopover sites. By Fall however both subspecies appear to be well fueled and as described in the manuscript Sedge warblers have access to rich aphid resources.  The interesting thing here is that two are examples of diverse migratory strategies that the authors are keying in on.

Specific points

It was not clear that your nonlured birds were feeding and not part of the lured cohort.  Did you observe them feeding, resting, or whatever? Please describe more fully how you separated these from the lured birds.

How much fat (mg) did you collect for your samples?

Lines 244- 245, this is not a complete sentence.  Is it a heading of some sort?

Results:

First two paragraphs show no correlations however do the functions show anything of interest – perhaps curvilinear or other relationships that might be elucidating? 

The Trig+glyc data favor Sedge over Reed warblers showing consistent results across both subspecies and seasons and fed conditions that emphasize the distinctions between these cohorts and can contribute to your designed hypothesis and prediction that would give you more options for discussion of how different the migratory strategies are of these two cohorts that would contribute to additional studies.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This study compares the circulating levels of metabolites and fatty acid composition of two Acrocephalus warblers with reportedly diverse migratory strategies, body size, morphology, migratory routes, feeding preferences and stopover schedules. The authors designed the study to elucidate the dietary preferences – refeeding activity at a common stopover site.  The study has merit and has demonstrated some innovative and now validated techniques for field measurements with the two Roche Accutrend Chemical detection systems. Nevertheless, there are numerous questions, concerns, and structuring of the manuscript that if addressed would certainly help to improve it.

Manuscript Structure:  The authors rely on previous publication of the two subspecies that point out differences.  Using this information and the background material on lipid and glucose metabolism in migrants, restructuring the paper with a hypothesis at the outset to distinguish the two warblers would clarify the work.  This could be done by using the recognized (published) differences and hypothesizing that there would or could be differences in their metabolic profiles.  Following this, offer predictions in the direction of how they may differ.   This approach can strengthen the results and expand on the published work.  Another issue is that I surmise from the manuscript and geographic breeding ranges of the two subspecies, is that breeding locations for Reed warblers are located mainly in Portuguese wetlands; whereas, Sedge subspecies tend to spread throughout central and northern Europe,  If this is the general pattern then the migratory distances  as well as the routes of the two may vary to a large extent and have an impact on the stopover scheduling of the two. So that the selected site for trapping the birds would favor the Reed Warblers who may be closer to their destination than that of the Sedge providing a smaller sample size and possibly less accurate data for the Sedge.  This was in fact confirmed by the comment (Lines 166 – 168) that trapping enough Sedge warblers in the spring was difficult in terms of amassing enough numbers. Does this raise the possibility that these stopover sites may not be on a major routing for the Sedge warblers that would make these sites less optimal than the more distant central routing through Europe? If this is a major difference, the ecology and scheduling of the two subspecies could well differ.  But given this, it is evident that the migratory strategies of the two differ.  Lines 85 – 88 state that Sedge warblers carry large fat loads at least while departing from wintering grounds than do Reeds during spring migration.  If this is correct, then Sedge warblers would be considered “time minimizers” - those that reduce the time spent on stopovers or have few options to stop and feed in the early phases of spring migration (Alerstam and Hedenstrom, 1998). By contrast, Reed warblers are departing the wintering sites as “energy minimizers” and carry less fuel but rely on more stops per trip. Yet, by the time the Sedge warblers reach the Portuguese sites they are few in number and lean (Lines 165-166).  So doesn’t this indicate a change in migration tactic or compete loss of quality stopover sites. By Fall however both subspecies appear to be well fueled and as described in the manuscript Sedge warblers have access to rich aphid resources.  The interesting thing here is that two are examples of diverse migratory strategies that the authors are keying in on.

RE: We appreciate all the comments provided by the reviewer. The comments were carefully analyzed, and we rephrased several parts of manuscript to incorporate the comments of both reviewers.

Specific points

It was not clear that your nonlured birds were feeding and not part of the lured cohort. Did you observe them feeding, resting, or whatever? Please describe more fully how you separated these from the lured birds.

RE: We do not observe the birds before the captures. However, the nonlured birds were captured in days that we do not used the calls. By this reason we are confident that birds stopped naturally in our study areas.

How much fat (mg) did you collect for your samples?

RE: About 10mg.

Lines 244- 245, this is not a complete sentence. Is it a heading of some sort?

RE: Indeed, it is a heading. We already Bolted this sentence.

Results:

First two paragraphs show no correlations however do the functions show anything of interest – perhaps curvilinear or other relationships that might be elucidating?

The Trig+glyc data favor Sedge over Reed warblers showing consistent results across both subspecies and seasons and fed conditions that emphasize the distinctions between these cohorts and can contribute to your designed hypothesis and prediction that would give you more options for discussion of how different the migratory strategies are of these two cohorts that would contribute to additional studies.

RE: We appreciate reviewer comment, and according to both reviewers we incorporate the predictions on introduction, and we rephrased several parts in the discussion, to increase the overall quality of the manuscript.

Round 2

Reviewer 2 Report

I would advise that they do address the basic differences of the two stopover sites, the distances from each species breeding areas and that the Sedge Warbler site might not be optimal given the difficulties they describe obtaining sufficient numbers of birds for sampling.  Such differences could support the different strategies and physiology observed in the two species.  

Author Response

Comments Reviewers Round 2

I thank you for submitting the revised version of your ms with response letter. I have responded quite well for the remarks of the R1, but check once again that your good replies in your response letter are also added in the manuscript itself.

RE:

R2 was not fully satisfied your responses for her/his remarks. Therefore, you should seriously consider her/his remarks, e.g. she/he advise that authors should address the basic differences of the two stopover sites, the distances from each species breeding areas and that the Sedge Warbler site might not be optimal given the difficulties they describe obtaining sufficient numbers of birds for sampling.  Such differences could support the different strategies and physiology observed in the two species.

RE: We answered to the reviewer 2 to improve the clarity and added more information in the manuscript according to its suggestions.

And lastly, I noted that you have NOT responded for my own remarks in any way. I will repeat them below. In addition, please see the Journal guidelines for the authors and flow them carefully before you submit the revised version (e.g. numbering of sections is lacking, citation style is wrong..).

Summary summarum: your ms will still need a minor revision before the possible final acceptance.

And below is my remarks from the 1st revision round:
add a Simple Summary before the Abstract

RE: Summary was added as suggested by Editor: “ SUMMARY. Through the examination of several blood metabolites in two migratory warblers at two stopover sites in Portugal, Sedge warblers showed higher values of triglycerides (TRIG) + glycerol (GLY) than Reed warblers, both during post-flight fasting and when stopping naturally. The higher values of TRIG + GLY in circulation should reflect a higher fat reserve mobilization by Sedge warblers, attributed to the fewer possibilities to refuel, relying more on fat reserves during their migratory journeys than those of Reed warblers. Sedge warblers use more lipids or more efficiently the reserves in each migratory step, due to longer fasting periods causing lower glycaemia levels. Our study revealed differences in the level of unsaturation subcutaneous fatty acids, mainly the higher levels of polyunsaturated fatty acids in Sedge warbler. Differences in preying habits may trigger a series of adaptations that results in different migratory strategies between Sedge and Reed warblers.”

-follow up the Journal guidelines for the authors (e.g. citation style (use numbering [1,23,25]; reference list style)

RE:

-some sections were lacking (see guidelines); e.g. ethical statement is needed since blood samples were collected
*Author Contributions: Each author is expected to have made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; or have drafted the work or substantively revised it; AND has approved the submitted version (and version substantially edited by journal staff that involves the author’s contribution to the study); AND agrees to be personally accountable for the author’s own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature.
For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used "Conceptualization, X.X. and Y.Y.; Methodology, X.X.; Software, X.X.; Validation, X.X., Y.Y. and Z.Z.; Formal Analysis, X.X.; Investigation, X.X.; Resources, X.X.; Data Curation, X.X.; Writing – Original Draft Preparation, X.X.; Writing – Review & Editing, X.X.; Visualization, X.X.; Supervision, X.X.; Project Administration, X.X.; Funding Acquisition, Y.Y.”, please turn to the CRediT taxonomy for the term explanation. For more background on CRediT, see here. "Authorship must include and be limited to those who have contributed substantially to the work. Please read the section concerning the criteria to qualify for authorship carefully".
*Institutional Review Board Statement: In this section, please add the Institutional Review Board Statement and approval number for studies involving humans or animals. Please note that the Editorial Office might ask you for further information. Please add “The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of NAME OF INSTITUTE (protocol code XXX and date of approval).” OR “Ethical review and approval were waived for this study, due to REASON (please provide a detailed justification).” OR “Not applicable” for studies not involving humans or animals. You might also choose to exclude this statement if the study did not involve humans or animals.

RE: First of all, we appreciate the positive comments addressed to the manuscript, and we now addressed all the questions made previously by the Editor, as well as the comments made previously by reviewer 2.

According to the Editor comments we added

“Author contributions

P.M.A. and J.A.R. conceived and designed the experiment; P.M.A. organized fieldwork. P.M.A., I.V. and L.C. performed the lab work; P.M.A., L.P.S., and P.B.L. performed the field captures; P.M.A. led the writing of the manuscript with substantial inputs from all other authors.

Competing interests

The authors declare no competing or financial interests.

Compliance with ethical standards

All sampling procedures and/or experimental manipulations were reviewed and approved by the relevant authorities (Instituto da Conservação da Natureza e da Biodiversidade, research permits 107/2006, 116/2007, 333/2007/CAPT).”

Furthermore, we reviewed all the references list and changed the citation style using numbers. Please check the List of References.

Revision 2 (major comments of first revision):

This study compares the circulating levels of metabolites and fatty acid composition of two Acrocephalus warblers with reportedly diverse migratory strategies, body size, morphology, migratory routes, feeding preferences and stopover schedules.

The authors designed the study to elucidate the dietary preferences – refeeding activity at a common stopover site.  The study has merit and has demonstrated some innovative and now validated techniques for field measurements with the two Roche Accutrend Chemical detection systems. Nevertheless, there are numerous questions, concerns, and structuring of the manuscript that if addressed would certainly help to improve it.

Manuscript Structure:  The authors rely on previous publication of the two subspecies that point out differences.  Using this information and the background material on lipid and glucose metabolism in migrants, restructuring the paper with a hypothesis at the outset to distinguish the two warblers would clarify the work.  This could be done by using the recognized (published) differences and hypothesizing that there would or could be differences in their metabolic profiles.  Following this, offer predictions in the direction of how they may differ.   This approach can strengthen the results and expand on the published work. 

RE: We provided predictions in the end of the introduction as solicited by both reviewers.

Another issue is that I surmise from the manuscript and geographic breeding ranges of the two subspecies, is that breeding locations for Reed warblers are located mainly in Portuguese wetlands; whereas, Sedge subspecies tend to spread throughout central and northern Europe,  If this is the general pattern then the migratory distances  as well as the routes of the two may vary to a large extent and have an impact on the stopover scheduling of the two. So that the selected site for trapping the birds would favor the Reed Warblers who may be closer to their destination than that of the Sedge providing a smaller sample size and possibly less accurate data for the Sedge.  This was in fact confirmed by the comment (Lines 166 – 168) that trapping enough Sedge warblers in the spring was difficult in terms of amassing enough numbers. Does this raise the possibility that these stopover sites may not be on a major routing for the Sedge warblers that would make these sites less optimal than the more distant central routing through Europe? If this is a major difference, the ecology and scheduling of the two subspecies could well differ.  But given this, it is evident that the migratory strategies of the two differ.  Lines 85 – 88 state that Sedge warblers carry large fat loads at least while departing from wintering grounds than do Reeds during spring migration.  If this is correct, then Sedge warblers would be considered “time minimizers” - those that reduce the time spent on stopovers or have few options to stop and feed in the early phases of spring migration (Alerstam and Hedenstrom, 1998). By contrast, Reed warblers are departing the wintering sites as “energy minimizers” and carry less fuel but rely on more stops per trip. Yet, by the time the Sedge warblers reach the Portuguese sites they are few in number and lean (Lines 165-166).  So doesn’t this indicate a change in migration tactic or compete loss of quality stopover sites. By Fall however both subspecies appear to be well fueled and as described in the manuscript Sedge warblers have access to rich aphid resources. The interesting thing here is that two are examples of diverse migratory strategies that the authors are keying in on.

RE: Reed warblers do not breed mainly in Portuguese reedbeds, but across all European reedbeds, from the Iberian Peninsula to Northern Europe. Yes, Sedge warblers, breed mainly throughout central to northern Europe. However, we ringed birds in our study sites in central Portugal also during the breeding season, and have a good knowledge of the breeding pairs and the juveniles that fledge in our study area. So, by avoiding ringed birds we increased the probability of catching birds from other breeding areas. As the reviewer stated, Sedge warblers would be considered “time minimizers”, and we considered that they just stop to rest and not to re-feed. We also stated that Sedge warblers would depart from breeding sites with higher amounts of fat, due to their dietary preferences on aphides. Actually, when Sedge warblers stop at our stopovers the peak of aphides had already happened. Despite the fact that the number of Sedge warblers captured in our study sites were smaller than those of Reed warblers, the study sites present good conditions for both species, and the number of Sedge warblers caught was still high.

During spring we caught more Reed warblers mainly because this species can breed in the surroundings or closer of our study site, whereas Sedge warblers need to reach their breeding grounds in central and northern Europe as soon as possible, and therefore the majority do not stop in Portugal during spring.

However, we add predictions of FA profiles of Reed and Sedge Warblers:

“Finally, we expected that Sedge Warblers should present higher PUFAs and n-3 FA, given that do not refuel along their migratory route benefiting of their feeding habits on reed aphids at the Northern stopover sites ^[30]. By other side, Reed warblers would present highest proportion of SFA, once birds store and mobilize preferentially shorter and more saturated FAs in their adipose tissues, mainly species whose major fattening occurs later in the migration route ^[30].

Author Response File: Author Response.docx