Demographic Traits Variation in a Pyrenean Newt (Calotriton asper) among Lacustrine and Stream Populations
Round 1
Reviewer 1 Report
Camarasa et al. studied variation in life history traits in 9 populations of the Pyrenean Newt, Calotriton asper from the southern slope of the Pyrenean Mountains in Spain. Specifically, the authors hand captured newts from six stream-dwelling populations and three lacustrine populations, measured body size (SVL), and used skeletochronology to determine ages. The authors then compared body size, age at sexual maturity, age structure, and longevity among populations and among habitat types. The authors also compared these life history traits between the two sexes. The results showed that there were differences in age structure among populations and between males and females within populations. Likewise, body size was highly variable among populations and there was support for sexual size dimorphism, with males larger than females on average in most, but not all populations. Both body size and age structure did not show clear relationships with habitat type. The minimum age at sexual maturity also varied among populations, again with no clear relationship to habitat.
This study presents interesting data on variation in life history traits for a narrowly distributed species. I have two major concerns with the manuscript. First, is that the statistical methods are much too brief and do not contain enough information for a reader to understand exactly what analyses were done with the data. This should be easy to fix by expanding the third paragraph of the Materials and Methods section to describe each ANOVA in detail, and by adding tables displaying the full ANOVA results, not just the factors that had a significant relationship with the response. Second, there are statements in the abstract about the effects of habitat on life history traits that are not supported by the data, and conflict with the conclusions of the paper that are presented in the discussion. I elaborate on both comments below in more detail. A final comment is that the English text is pretty good overall, but there are several places where the grammar could be improved or wording could be changed slightly to improve readability. I provided a few specific instances below, along with suggested changes.
Major comments
-I think the text needs more details on the statistical analyses performed. With the current description, I am not sure someone would have enough information to replicate the analysis exactly. Particularly the ANOVAs, right now there is just one sentence that says ANOVA was used to test for differences in SVL and age structure. Then in the first paragraph of the results, there is a mention of a significant interaction between sex and locality on age structure, but testing that interaction is not described at all in the methods. I would like each individual ANOVA explained clearly, along with the question or hypothesis that each ANOVA is meant to address. Then in the results, presenting ANOVA results in a table would be helpful, especially so the reader can see the non-significant results.
-Lines 24-25 - I do not think this statement, that the maximum sizes were found in lacustrine populations, is a true reflection of the results, and it conflicts with the sentence starting on line 23. Looking at Table 2, the largest maximum body sizes were found at Barranc de Acherito and Espeluchieca, which were both stream populations.
-Lines 25-27 - Again, part of this statement is not supported by the results, and conflicts with one of the concluding statements in the discussion. The text states that “The results obtained show a local variability of age and body size distributions of Calotriton asper follow a complex pattern that depend on the habitat, sex and population interaction.” The results on lines 101-102 state however that “Nevertheless, sexual effect on demography was unrelated with the habitat” and on lines 118-119 “Body size variation was not related with the kind of habitat of the populations” and lines 126-127: “As a result, the pattern of sexual dimorphism was not same for all the populations (F8 381=6.389, P<0.001) and was not related with the habitat.” Likewise, in the discussion it states on lines 182-183: “In conclusion, we reinforce the hypothesis that habitat (stream and lake) does not give a pattern to the demography in Calotriton asper.” The text of the abstract attributes patterns in body size and age structure to habitat type, but this is not supported by the results of the study.
-How do you determine sexual maturity in the newts? Is it the same examination of the external morphology of the cloacal area used to distinguish males and females?
-Lines 108-110 - Is it possible that some populations with seemingly higher ages at sexual maturity is an artifact of the small sample of newts captured and examined? For example, at the Peramola site, the minimum age at sexual maturity was 6 years for females and 7 years for males, but the sample size for females was only 16 individuals and the sample size for males was only 12 individuals. With such a small sample, I wonder if the sample just did include any younger but sexually mature newts by chance. This caveat should be addressed.
-Lines 121-122 - I think this sentence states the opposite of the result presented in Table 2. Males had larger mean body size than females in all but two populations. At Peramola and Barranc de Acherito, females hard larger mean SVL, males had larger mean SVL everywhere else.
-The largest part of the discussion devotes a lot of space to describing the specifics of the habitat and history of each individual population. If this information is important for interpreting the results, it would be helpful to add a “study area” paragraph to the Materials and Methods and provide a bit more information about the study sites up front.
-The text could use a close proof-reading to fix some grammatical issues and make the paper read better overall. I pointed out some specific issues in the minor comments below, but I did not correct everything.
Minor comments
-Line 48 - Revise to “differences in body size”
-Line 52 - Also include the latin name for the Pyrenean Newt here when the species is first introduced. Right now Calotriton asper does not appear until the start of the next paragraph. After the first use of both Pyrenean Newt and Calotriton asper, pick one name and use it throughout the rest of the manuscript.
-Lines 96–97 - What does the significant difference in age structure at the intra-population level mean? What is the comparison? I assume the difference is between males and females, but I think it should be stated clearly here.
-Figure 2 - The text on the axes and legend is very small, it would be helpful to increase the font size so readers can more easily interpret this figure.
-Figure 2 - The figure legend should explain what the symbols in the boxplot represent. For example, median, interquartile range, 2.5th and 97.5th percentiles.
-Figure 2 - I suggest ordering the populations along the y-axis to match the order presented in Table 1. That way the reader knows the first 6 populations are stream-dwelling, and the last 3 are lacustrine. With Figure 2 ordered the way it is now, I had to keep flipping back between Figure 2 and Table 1 to make the connection.
-Table 2 - Explain what “ES” is in the table legend. I am guessing standard error?
-Table 2 - Is the Age column reporting Age at sexual maturity? Or the minimum, maximum, and median ages of all newts?
-Figure 3 - My comments about Figure 2 apply to this figure as well. It would be easier to visually compare stream and lake populations if populations were sorted based on habitat.
-Line 136 - Change “especificities” to specificities.
-Line 137 - Revise to “Environmental factors have been suggested”
-Line 138 - What “Can be related to water temperature…”? This sentence is missing a subject.
-Lines 175-176 - Is there a source to back up this statement? Is this from the authors’ own data?
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Dr. Camarasa and coauthors,
I have read and reviewed the manuscript: “Demographic traits variation in a Pyrenean newt (Calotriton asper) among lacustrine and stream populations”.
I think your manuscript demonstrates a huge field effort, and data collected are interesting and worth to publish. However, I think methods should be more detailed and conclusions must be rewritten.
I made a few specific comments below.
Line 17: please, delete methods
Line 19: please, delete Perramó and Acherito, I think that specific localities are not as important as the fact of showing facultative paedomorphosis and not relevant enough to be cited in the abstract.
Line 39: delete important traits and link both sentences.
Line 40: after the 4 references a point and followed and start the next sentence.
Line 51-54: In these lines, the authors refer to Calotriton asper before introducing it formally in the next paragraph. I think will be better in this paragraph only to explain the theoretical background.
Line 52: The first time that Calotriton asper is cited must be with its scientific name after the common name.
Line 54: specific localities names are not relevant here but in methods.
Line 57. Maybe add some details about how habitat conditions affect Calotriton asper may apport more information that only the references and will make the text clearer for readers that do not know those references. Describe the different kind of habitat with more details will be also useful.
Line 62: delete model organism. Before Pyrenean newt is redundant.
Line 71: how many Calotriton were captured for scientific collection?
Line 73: the Ethics Committee consider the prophylaxis measures to avoid the expansion of Bd? (e.g. clean all material with bleach)
Line 86: how is disinfected the wound of the finger? With chlorhexidine? Please, add this information.
Line 86-89: Why there is a huge range of staining time and decalcification? Is because you use a different kind of bones? If it is like that, is not explained.
Line 91: how the age of maturity was estimated? Please, provide a brief description.
Line 92: that means that age structure was introduced as a continuous variable? It’s fine but maybe you can give some more information about how you did the analysis.
Line 94: after log-transformation, all variables were normal?
Line 107: Table 2: there is no indication of which units age is measured. As LAGs? As years? There are cases of double LAGs? It is not either detailed at methods.
Table 2: Males of Perramó show a median of 10, but has 6 to 9 years or LAGs. Please, check if there are some typing error.
Table 2: I know fit tables is not easy, but if the name of Espeluchieca could be together, the table will look better.
Line 124-125: I understand that authors refer to body size for maximum values (124) and mean values (125), but it may be better to say it in the first part of the sentence than at the end.
Line 131: please, delete “in” and add an s to amphibia.
Line 134: what evidence do you have about the link between microhabitat conditions and demographic traits? Do the authors refer to habitat or microhabitat? Please, provide specific references and explain it a bit further.
Line 137-139: link both sentences or change the start of the second one.
Line 153: are not populated by Calotriton, isn’t it? Please, specify it.
Line 154: no ideal conditions because of alien species, environmental factors or both? Maybe interesting give a bit more information about that. Moreover, I guess the presence or absence of fish can act differently on the newt populations if fish is an autochthonous species or an alien species.
Line 160-163: age structure could be biased to older ones because younger ones have died because of fish predation. It should be discussed and explain a bit further the spatial segregation.
Line 163-165: this may explain the adaptation of newt population of fish predation, but the previous one may indicate certain extinction risk. The explanation is appropriate, but some references could be added and a brief theoretical background about life-history theory could be provided.
Line 172-173: maybe there is not enough data to say that. I suggest removing this sentence.
Line 178-179: I think that microhabitat conditions were not studied in this manuscript. If the authors refer to microhabitat to rheophilous and lacustrine environments, I consider that habitat, not microhabitat. But if the authors studied microhabitat, this information must be provided and discussed.
Line 177-186: conclusions must be rewritten, as they are not supported by your data.
I hope the review will be useful. Greetings.
Mar Comas
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
Authors analysed demographic traits between several populations of the Pyrenean newt Calotriton asper. They suspected phenotypic variation corresponding to an adaptation to the different habitats, especially between lakes and streams. I find the work interesting but I encourage them to better highlight the scope of their results and propose below some comments that may improve the manuscript:
First, the authors particularly insisted on the hypothesis of a strong phenotypic variation between the populations from lakes versus from streams. They considered understandably these are two different types of habitats but I have some difficulty to understand which environmental factors were suspected to drive the possible variation between these two types, and I feel authors failed at the end to identify them. Also, the chosen localities seems very different within both types especially stream one for instance regard the altitude, some are in the piedmont, from various genetic clusters (see Lucati et al 2020). The pools of sites thus does constitute neither replicants nor an gradient, nor a comparison with similar conditions between the two types (e.g. there is no stream populations around 2000m), and it was then not clear for me what the authors wanted to test exactly (if it is not only local variations). Why did you chose these populations?
In the same way, in the introduction, authors present the phenotypic plasticity as a possible process that can underlying phenotypic variations observed in different environments and later in the discussion (line 177) they conclude : “the results of this study show that the Pyrenean newt presents an important phenotypic plasticity”. However, I am convinced that they did not test that. They showed local variations but did not demonstrate that a genotype expressed different phenotypes when exposed to different environmental conditions. Mostly, comparing populations from various genetic clusters, I do not see how the authors could deal with the question.
In the Material and Methods, I understand that the sampling spent 3 years but not whether the same populations were sampled or not. Since it seems that any individual monitoring were made using capture-marking-recapture for example, and also that only individuals whom were sexed observing the cloaca were used in this study and we have any ages from individuals showing no sexual dimorphism, then I would be more careful about the interpretation of the results and the use of some expressions like for example:
“attained their maturity earlier” line 19
“in others females matured at younger ages than males”, “Maximum longevity varied”, line 21.
“Age at sexual maturity “ line 108
“In all the examined populations, age at sexual maturity was similar between sexes or the females matured at younger ages than males” line 110
I think the authors should better discuss they assumed that the youngest age among the sampled adults is a proxy of the age of sexual maturity and the older, that of longevity. In particular, since the very narrow distribution of ages observed in some populations pose the questions, possible bias would need to be removed.
I advise authors to list in the same order the localities in the box plots and tables to ease the reading.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Review of “Demographic traits variation in a Pyrenean newt (Calotriton asper) among lacustrine and stream populations”
I appreciate the authors taking the time to present more information about their ANOVA analysis and more details about their study sites. My main comment on the revised manuscript is that there are still several places where the English is confusing and awkward. I have highlighted some examples from the introduction below, but I do not have time to carefully copy-edit the entire document. I think another pass over the text with careful editing to improve readability would be valuable for helping readers understand the findings of this study. Also, in two places I think the literature cited is incomplete, there are references to “(new citation 1)” and “(new citation 2)” in the methods.
Lines 24-26 - I think the addition of “not” on line 26 was intended to address one of my comments on the first draft, but now I do not understand what this sentence means. “The results obtained show a local variability of age and body size distributions of Calotriton asper follow a complex pattern at local scale, that not depend on the habitat, sex and population interaction.” “That not depend” is not a good phrase, instead one would say “that did not depend.” But, I do not think the meaning of this sentence is correct now, because there were differences in age structure with a sex by population interaction. So this sentence needs to revised to state that age and body size distributions did not depend on habitat, but that age and body size were influenced by an interaction between sex and population.
Lines 45-47 - This sentence reads awkwardly, especially the phrase “is also affected sexual dimorphism”. I think a better statement here would be “Sexual dimorphism is determined by the balance and interaction of multiple selective forces [8].”
Line 48 - Change to “differences in body size”
Line 52 - I suggest revising the end of this sentence to “although it has secondarily colonized high mountain lakes”
Lines 53-54 - I would revise this sentence to state “In the order urodela, growth rates tend to be highest during the larval and juvenile stages [13].”
Lines 56-57 - I would delete “such as” from this sentence, and instead state “For example, Calotriton asper at some lacustrine localities exhibit facultative paedomorphosis [15,16].”
Line 64 - I suggest revising to “Another study found that that body lengths were higher for surface populations than the subterranean populations [20].”
Line 78 - “king” should be “kind”
Line 78 - The citation is not properly formatted here, it says “(new citation 1)” and I do not see what this citation is in the literature cited section.
Line 81 - Same issue as above, it says “(new citation 2)” at the end of this sentence.
Lines 87-88 - The abbreviation “LAG” is used here twice but it is not defined until later, on line 107.
Line 208 - Again, it just says “(citation 3)” here - Is that the original citation number 3 or a new one? Why isn’t this formatted as [3]?
Line 212 - You could delete “water” and just have “floods” here.
Lines 223-224 - The revised sentence here reads “In summary, the results of this study show that the Calotriton asper presents an important variability in the demography does not depend on habitat.” This is very hard to read. I think the point that is trying to me made is something like “In summary, the results of this study show that Calotriton asper exhibits inter-population variability in demography that does not depend on habitat.”
Table 3 - Age structure at the sexual level is a strange phrase. How about “Age structure by sex and population” instead? The same suggestion applies to “SVL at the sexual and population level”
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
The authors have responded convincingly to all of my remarks and have made consequential changes to the text. Congratulations for this work.
Author Response
Please see the attachment
Author Response File: Author Response.docx
