Diversity and Diet Differences of Small Mammals in Commensal Habitats
Round 1
Reviewer 1 Report
- The MS is almost well-written, in an almost intelligible language and it flows.
- Given the topic, I strongly recommend authors to read two papers:
- Bertolino S., Colangelo P., Mori E., Capizzi D. (2015). Good for management, not for conservation: an overview of research, conservation and management of Italian small mammals. Hystrix, the Italian Journal of Mammalogy, 26: 25-35.
- Mori E., Ferretti F., Fattorini N. (2019). Alien war: ectoparasite load, diet and temporal niche partitioning in a multi-species assembly of small rodents. Biological Invasions 21: 3305-3318.
- Figure 1a) The map lacks the unit of measurement and the North symbol. Please add them.
- Lines 86-88. Over 450 small mammals has been killed. Please add here the Compliance with ethical standards and all permits you have to kill animals. I definitely understand that you did not require permissions, but snap traps may kill protected species under the Habitats Directive, thus it is not in accordance as you said.
- Line 93. Where were they refrigerated? At which temperature?
- Line 94. How did you identify the species.
- Lines 90-100. Please report some more information on trapping session, as it is not sufficient to recall a Table in supplementary material.
- Line 270. Please delete “pilot”.
- Line 318. Delete “Naturally”.
Author Response
Manuscript ID: diversity-1319516
Diversity and diet differences of small mammals in commensal habitats
Comments and answers, Rev#1
Comment: Given the topic, I strongly recommend authors to read two papers: Bertolino S., Colangelo P., Mori E., Capizzi D. (2015). Good for management, not for conservation: an overview of research, conservation and management of Italian small mammals. Hystrix, the Italian Journal of Mammalogy, 26: 25-35. and Mori E., Ferretti F., Fattorini N. (2019). Alien war: ectoparasite load, diet and temporal niche partitioning in a multi-species assembly of small rodents. Biological Invasions 21: 3305-3318.
Answer: thank you for these sources, both publications are known to us. However, situation in Lithuania is very different from the more southern countries. First of all, species richness of small mammals is much smaller, secondly, there are no conservation conflicts in the agricultural lands and definitely none in the commensal habitats. Therefore, even rodent poisoning, which is common in the commercial gardens, or snap-trapping in the two sites we investigated, did not rose any conservation conflicts. We add this to the final part of discussion, where we talk about the follow-up study:
This certainly deserves dedicated and more detailed follow-up study, such as diet analysis according [63]. Differently from the more southern European countries [64], in the commensal habitats of Lithuania protected species of small mammals so far were not trapped, therefore, widening of the research will not cause conservation conflicts.
Comment: Figure 1a) The map lacks the unit of measurement and the North symbol. Please add them.
Answer: scale bar and south-north indication added
Comment: Lines 86-88. Over 450 small mammals has been killed. Please add here the Compliance with ethical standards and all permits you have to kill animals. I definitely understand that you did not require permissions, but snap traps may kill protected species under the Habitats Directive, thus it is not in accordance as you said.
Answer: Lines 105-111 we put all needed information, while the number and organization which issued approval is repeated in the back matter: “The study was approved by the Animal Welfare Committee of the Nature Research Centre, protocol No GGT-7. It was conducted in accordance with Lithuanian (the Republic of Lithuania Law on the Welfare and Protection of Animals No. XI-2271) and European legislation (Directive 2010/63/EU) on the protection of animals. In Lithuania, there is no need or legal obligation to obtain permission or approval to snap trap small mammals. This is especially relevant to the trapping of rodents on private property, which was the case.”
For the former publications we also consulted with Ministry of Environment, and were confirmed, that no permissions or approvals are needed to trap rodents. Therefore, we hardly can add more information than currently presented.
Please also note, that rodent killing with snap traps was the only measure approved by owners (they avoid poisoning and chemical treatments, keeping their crops mostly organic), therefore even if we do not use this material, all these small mammals would be killed. We also use material for the other purposes (elemental analysis, breeding analysis, pathogen analysis) – all these requiring dead animals and their organs. As the whole issue of commensal small mammals is highly understudied in the region and across northern part of Europe, we see priority for such a result.
Comment: Line 93. Where were they refrigerated? At which temperature?
Answer: we changed text, answering to this comment “All trapped specimens were put into separate bags and kept refrigerated at –20 °C in the Nature Research Centre. I, where identification of species and dissection was conducted.”
Comment: Line 94. How did you identify the species.
Answer: all trapped species are common and very common species in Lithuania (except Mediterranean water shrew), and all trapped are identifiable morphologically, with Microtus voles differing in dentition characters. Senior author has over 40-year experience of work with small mammals, therefore no keys were needed. We add short sentence to the text, “Species were identified morphologically, checking species of Microtus voles by their dental characters.”
Comment: Lines 90-100. Please report some more information on trapping session, as it is not sufficient to recall a Table in supplementary material.
Answer: we changed text as “Trapping was carried out in 2019 and 2020, trapping effort in both years was scaled for site area. Trapping sessions were 1–5 days long each, depending on owner presence; in most cases 20 medium size snap traps were used for one session. More details of trapping effort are presented in Table S1.”
Comment: Line 270. Please delete “pilot”.
Answer: deleted
Comment: Line 318. Delete “Naturally”.
Answer: deleted
Reviewer 2 Report
The authors present the results of stable isotope analyses of hair of small mammal communities at two sites in Lithuania. One site was a homestead and the other a kitchen garden. They created isotopic niches to examine potential overlap and segregation and possible differences due to provision of foods due to commensal associations with humans. They claim evidence of higher species richness due to commensal food inputs and greater niche separation among species where such inputs are less. The study is, of course, interesting but there are fundamental issues that prevents any strong inferences or interpretations. Throughout the manuscript, there are also issues with the writing that need to be addressed.
Major points:
- The main problem with this paper is the sample size of one for each treatment (homestead vs kitchen garden). This renders the study to simple speculation and the reader has no idea if differences are related to treatment, chance, or the peculiarities of site choice. A more powerful approach is to have replication of treatments or a series of paired homestead vs kitchen gardens.
- A second major problem is the simple fact that human agro-ecosystems can be quite complicated isotopically. The authors present no isotopic data on potential diets, not do they particularly describe them well. It is well known that such systems can differ tremendously isotopically depending on plant types, landuse practices, fertilizer, etc.
Other (in order as they were encountered):
Line 10. I realize “syntopic” is more correct than “sympatric” (providing you can prove they overlap in time when hair is grown?) but if you keep this word, please define it for the reader who will be more familiar with “sympatric”.
Line 12. Most people will know where Lithuania is located.
Line 14/15. Dominance and diversity are NOT methods. You mean measurements of …. The term “stable d13C and d15N isotope analysis” is redundant, that is what the delta symbol refers to….
Line 20: What do you mean by “not equally expressed”? I think you simply mean they were different.
Keywords should include “carbon-13” and “nitrogen-15”.
Line 27: should be “mammals” .. Also, the opening sentence (and the first few paragraphs) was surprising and should deal more with the ecology of rodents vs them being a “problem”… Your study is on their coexistence and subsidy from human inputs, a large area of research, so go straight to that.
Line 112. Pretreated how?
Line 120. As a general rule, please use like matrices (e.g. proteins, keratins) for your standards instead of inorganics (the result is your estimate of error is likely lower than it should be). Also, reporting the caffeine to three decimal places for δ13C and none for δ15N seems awfully strange.
Line 125-128. Please drop this. The delta formula has been published thousands of times.
Table 2 should indicate sample sizes.
Figure 2. indicate which panel is “a” and which is “b”. Center the Y-axis label.
Figure 3. Explain the complex box plot output of SIBER, what are all these layers?
Discussion:
Line 275-279. Your results in no way confirm your hypotheses due to the poor power of inference based on no treatment replication. Temporal sequences in both sites are hard to interpret if trapping removes individuals in the first year, changes in recruitment from adjacent areas, food availability etc.
Line 284-288. Well, it is almost impossible to compare your data with data from other countries or remote sites because isotope data can be so variable in these agroecosystems.
Line 309-310. You have no idea what can cause the isotopic differences (without extensive baseline foodweb sampling) and flooding may or may not have anything to do with this….
Line 319. Redundant nomenclature as indicated above.
Recommendation:
I appreciate the work involved in this study but I think the paper needs to be rewritten with emphasis on the highly preliminary results and the fact that the study in no way is a proper test of the hypotheses. This is not to say that stable isotope analyses could not ultimately be useful in trying to evaluate the effects of differential inputs of anthropogenic foods to small mammal communities. Indeed, studies that examine this question carefully put a great deal of effort into also sampling foodweb isotopic inputs (natural vs anthropogenic). One possible way of salvaging this paper for the symposium issue would be to present a clear set of guidelines and approaches to this question using stable isotopes, namely how should we use stable isotopes to investigate these questions. That rewrite would need to thoroughly downplay the hypotheses and conclusions of this paper.
Author Response
Manuscript ID: diversity-1319516
Diversity and diet differences of small mammals in commensal habitats
Comments and answers, Rev#2
Comment: The main problem with this paper is the sample size of one for each treatment (homestead vs kitchen garden). This renders the study to simple speculation and the reader has no idea if differences are related to treatment, chance, or the peculiarities of site choice. A more powerful approach is to have replication of treatments or a series of paired homestead vs kitchen gardens.
Answer: we fully agree with you, and as authors we fully understand limitations of the study. However, these are first results, we even have no literature to compare our data with – so, priority should be secured by all means. Therefore, we present the first results, hoping that investigations of small mammals in the commensal habitats not only will be continued in Lithuania, but will also burst interest in neighboring countries, and possibly, in the region. We will have an oral presentation in the international conference in Poland, this September.
We did our best to incorporate changes in the revised manuscript text to show the results as a pilot study, and to present a framework to the future investigations. We acknowledge your comments and suggestions.
Comment: A second major problem is the simple fact that human agro-ecosystems can be quite complicated isotopically. The authors present no isotopic data on potential diets, not do they particularly describe them well. It is well known that such systems can differ tremendously isotopically depending on plant types, landuse practices, fertilizer, etc.
Answer: we fully agree with you, that in general agro-ecosystems may be (not necessarily are) complicated isotopically. However, in our case, we have quite similar situation in both homestead and the kitchen garden, Cė plants are not grown in both sites – we checked list from PYANKOV, V. I.,et al. (2010). European plants with C4 photosynthesis: geographical and taxonomic distribution and relations to climate parameters. Botanical Journal of the Linnean Society, 163(3), 283–304. doi:10.1111/j.1095-8339.2010.01062.x.
In both cases there is nearly the same landuse – gardening and vegetable gardening, with difference in the scale, but in both site no hevy machinery use and no chemistry, artificial fertilizers used in minimum amounts. The only difference is related to the proximity of buildings and possibility to obtain human foods.
In the future, introducing more sites, we are going to dear with intensity of agricultural activity.
Comment: Line 10. I realize “syntopic” is more correct than “sympatric” (providing you can prove they overlap in time when hair is grown?) but if you keep this word, please define it for the reader who will be more familiar with “sympatric”.
Answer: we put explanation, so the first sentence of Abstract now reds as “The stability of diversity of syntopic (inhabiting the same habitat in the same time) small mammals in commensal habitats, such as farmsteads and kitchen gardens, and, as a proxy of their diet, their isotopic niches were investigated in Lithuania, in 2019–2020.
Comment: Line 12. Most people will know where Lithuania is located.
Answer: this text was not intended to say, where Lithuania is; most of results of investigation of small mammals in commensal habitats are from the different zones or different part of the middle latitude. However, as comparison is not done in the Introduction, we delete this part of the sentence.
Comment: Line 14/15. Dominance and diversity are NOT methods. You mean measurements of …. The term “stable d13C and d15N isotope analysis” is redundant, that is what the delta symbol refers to
Answer: we agree, that this was language problem, though it was revised by native speaker. We did not mean that diversity and dominance are methods – it was said analysis was method. Therefore, we changed text as “We analysed diversity, dominance and distribution of δ13C and δ15N values.”
Comment: Line 20: What do you mean by “not equally expressed”? I think you simply mean they were different.
Answer: we agree that “not equally expressed” means “different”, however, there should not be two times the same word in the sentence. We consulted language editor, and substitute we used is not violating language rules.
Comment: Keywords should include “carbon-13” and “nitrogen-15”.
Answer: thank you, “stable isotopes” changed with “carbon-13 and nitrogen-15 isotopes”
Comment: Line 27: should be “mammals” .. Also, the opening sentence (and the first few paragraphs) was surprising and should deal more with the ecology of rodents vs them being a “problem”… Your study is on their coexistence and subsidy from human inputs, a large area of research, so go straight to that.
Answer: thank you, indeed not “small mammals” but “rodents”, as written in [1]. We corrected this mistake.
Comment: Line 112. Pretreated how?
Answer: samples were NOT pre-treated, so there could be no “how”.
Comment: Line 120. As a general rule, please use like matrices (e.g. proteins, keratins) for your standards instead of inorganics (the result is your estimate of error is likely lower than it should be). Also, reporting the caffeine to three decimal places for δ13C and none for δ15N seems awfully strange.
Answer: we refer to the provider, namely IAEA. International Atomic Energy Agency (IAEA) is providing reference materials characterized for stable isotope ratios: https://nucleus.iaea.org/sites/ReferenceMaterials/Pages/Stable-Isotopes.aspx. For the caffeine IAEA-600, its recommended δ13C value was determined in an international calibration, which propose to use value δ13C= -27.771 ‰VPDB. https://nucleus.iaea.org/sites/ReferenceMaterials/Pages/IAEA-600.aspx. Three decimal places are given here, however, on your request we skip the third decimal for all standards referred.
As for the use of proteins or keratins as a standard, Coffeine is the only organic material available from the IAEA for the nitrogen isotope ratios. The others, like IAEA-NO-3 are inorganic substances, but despite that, the very good agreement on delta values was measured in our laboratory between substances used for the isotope scale normalisation.
It must be noted, that reference materials used in this research for the isotope scale normalisation are secondary reference materials (primary materials are not available anymore). Any kind of third reference materials (i.e. proteins, keratins) can be isotopically normalised using secondary reference materials, but the precision and accuracy for third (or internal laboratory) reference materials became worse.
Comment: Line 125-128. Please drop this. The delta formula has been published thousands of times.
Answer: OK, deleted.
Comment: Table 2 should indicate sample sizes.
Answer: requires sample sizes were added, Table 2 re-formatted
Comment: Figure 2. indicate which panel is “a” and which is “b”. Center the Y-axis label.
Answer: done as requested
Comment: Figure 3. Explain the complex box plot output of SIBER, what are all these layers?
Answer: Thank you for pointing this out, we failed to explain. According Jackson, A.L.; Inger, R.; Parnell, A.C.; Bearhop, S. Comparing isotopic niche widths among and within communities: SI-BER—Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 2011, 80, 595–602, doi:10.1111/j.1365-2656.2011.01806.x. [40], in the inset statistics of the TA for analysed species is shown, including confidence intervals. We supplied this text:
“Statistics of the total areas of isotopic spaces for these species is shown as insets for both habitats, dots representing their mode and the shaded boxes representing the 50%, 75%and 95% CI, shown from dark to light grey.”
Comment: Line 275-279. Your results in no way confirm your hypotheses due to the poor power of inference based on no treatment replication. Temporal sequences in both sites are hard to interpret if trapping removes individuals in the first year, changes in recruitment from adjacent areas, food availability etc.
Answer: we did changes to hypotheses (now called predictions), removing third one –text now is “We also checked, if diets of small mammals have seasonal trends.”, and discussion, text now is “Temporal changes of δ13C and δ15N values were not equally expressed in all species (see Fig. 4). However, we need additional investigations to check, if differences are related to the availability of plant production from gardening practices in the commensal habitats.”
We plan to introduce replications and treatment analysis with new data from the other homesteads, and add this to the end of Discussion, just before Conclusions.
Comment: Line 284-288. Well, it is almost impossible to compare your data with data from other countries or remote sites because isotope data can be so variable in these agroecosystems.
Answer: we may agree with you, however, available data are of totally different species, characterized with different diets and biology. Therefore, not only variability of isotopic data in the agroecosystems may be the reason. By the way, variability of isotopic niches or rodents in agroecosystems is not the rule. For example, in Balčiauskas, L., Skipitytė, R., Garbaras, A., Stirkė, V., Balčiauskienė, L., & Remeikis, V. (2021). Stable Isotopes Reveal the Dominant Species to Have the Widest Trophic Niche of Three Syntopic Microtus Voles. Animals, 11(6), 1814. We show, that
“The cumulative influence … of the year and season on the distribution of δ13C and δ15N values in the three vole species … explained only a very small part of the variance (year: T2 = 0.21, F4,740 = 19.4, eta2 = 0.095; season: T2 = 0.02, F2,371 = 3.5, eta2 = 0.019). Given the fact that the cumulative effect of the time factor on the distribution of δ13C and δ15N values is about 10%, this showing a relative stability of the diets”
Comment: Line 309-310. You have no idea what can cause the isotopic differences (without extensive baseline foodweb sampling) and flooding may or may not have anything to do with this…
Answer: well, here we cite Forbes et al., [51], not stating our position as for the influence of the diet. According to your comment, and also suggested paper of Rev#1, we added diet analysis into the framework of our future investigations (this including the baseline) into the end of discussion.
Comment: Line 319. Redundant nomenclature as indicated above.
Answer: as per your proposal above, changed to “carbon-13 and nitrogen-15 isotopes”
Recommendation: I appreciate the work involved in this study but I think the paper needs to be rewritten with emphasis on the highly preliminary results and the fact that the study in no way is a proper test of the hypotheses. This is not to say that stable isotope analyses could not ultimately be useful in trying to evaluate the effects of differential inputs of anthropogenic foods to small mammal communities. Indeed, studies that examine this question carefully put a great deal of effort into also sampling foodweb isotopic inputs (natural vs anthropogenic). One possible way of salvaging this paper for the symposium issue would be to present a clear set of guidelines and approaches to this question using stable isotopes, namely how should we use stable isotopes to investigate these questions. That rewrite would need to thoroughly downplay the hypotheses and conclusions of this paper.
Answer: authors fully understand limitations of the study. However, these are first results, we even have no literature to compare our data with – so, priority should be secured by all means. Therefore we are publishing results and setting framework for the further investigations (end of Discussion). In 2021 we are collecting more data from the same Site 1 and Site 2, and have plans to analyse baseline plants and seeds not only from commensal habitats but also from commercial orchards. We also will add new homesteads. It is not so easy work, as homesteads are personal properties near the homes of owners.
According your comments we made other changes in the text to show limitations of the study. Thank you for the suggestions.
Round 2
Reviewer 2 Report
Line 15-16. Should read “We analyzed small mammal diversity, dominance and distribution of hair δ13C and δ15N values”
Line 16. Change “domination” to “dominance”.
Line 23: change to “Thus, small mammal trophic ecology is likely related to intensity of agricultural activ- ities in the limited space of commensal habitats.”
Line 101: Change “refrigerated” to “frozen”. Same on Line 119.
Lines 294-295 do not really embrace the point that readers need to understand how future studies really need to address the questions raised here. I think recognition of how complex agroecosystems can be isotopically and mention of the need for replication is needed. See the following, especially as related to δ15N.
Pardo LH, Nadelhoffer KJ (2010) Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales. In: Isoscapes: Understanding Movement, Pattern, and Process on Earth through Isotope Mapping. Springer Netherlands, Dordrecht, pp 221–249.
Author Response
Manuscript ID: diversity-1319516
Diversity and diet differences of small mammals in commensal habitats
Rev#2 comments and answers, round 2
Comment: Line 15-16. Should read “We analyzed small mammal diversity, dominance and distribution of hair δ13C and δ15N values”
Answer: changed as proposed
Comment: Line 16. Change “domination” to “dominance”.
Answer: changed as proposed
Comment Line 23: change to “Thus, small mammal trophic ecology is likely related to intensity of agricultural activities in the limited space of commensal habitats.”
Answer: changed as proposed
Comment Line 101: Change “refrigerated” to “frozen”. Same on Line 119.
Answer: changed as proposed
Comment Lines 294-295 do not really embrace the point that readers need to understand how future studies really need to address the questions raised here. I think recognition of how complex agroecosystems can be isotopically and mention of the need for replication is needed. See the following, especially as related to δ15N.
Pardo LH, Nadelhoffer KJ (2010) Using nitrogen isotope ratios to assess terrestrial ecosystems at regional and global scales. In: Isoscapes: Understanding Movement, Pattern, and Process on Earth through Isotope Mapping. Springer Netherlands, Dordrecht, pp 221–249.
Answer: text and reference added. We found that these changes in the text should be related to the part where we talk about future prospects, therefore text was expanded at Lines 358-360:
We recognize, that agroecosystems may be quite complex isotopically. Most complex situation is with nitrogen-15 isotope, as δ15N values are influenced by many internal and external fluxes [64], such as atmospheric deposition, fixation, loss of denitrification products, hydrologic leaching, ammonification, nitrification, denitrification, immobilization of inorganic and organic N, uptake by plants, etc. Therefore, we need to replicate our study in other commensal habitats and in different sites, yielding much larger dataset. Fortunately, differently from the more southern European countries [65], in the commensal habitats of Lithuania protected species of small mammals so far were not trapped, therefore, widening of the research will not cause conservation conflicts.
Thank you for pointing this out and proposing reference.
