Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 2
Reviewer: 1

Output:
Basic reporting: The current version of the manuscript by Rong Cheng and colleagues has been improved since the last time, however still there are some typos and sentences that are not phrased correctly. There are parts where the Authors describe the same statements in two consecutive sentences (see later). The "Introduction" section still needs some modifications. One point of the study was to propose the use of antifungal proteins (AFPs) in C. parapsilosis candidaemia. To properly strengthen this idea, it is important to give a consistent introduction on the currently available antifungal agents and more importantly the resistance emerging against them, as no such phenomenon has already been described against AFPs, that makes these molecules a potential alternative of the existing drugs in the treatment of fungaemia. I do not really recommend to mention 5-fluorocytosin as it is not used since at least two decades ago especially in monotherapy. In contrast, I really missed amphotericin B. I would also have expected to read a few sentences about how resistance emerges against these drugs that, as I mentioned before, would really support why AMPs could be alternative antifungals. I also missed a consistent introduction of AMPs including structure, chemical properties and their diverse sources from the nature. The Materials and methods section is detailed enough. The structure of the "Results" section has been modified, now it is easier to follow for the reader. Pictures are fine, however I would kindly ask the Authors to improve the resolution of Fig. 3-7, if it is possible. Regarding the qRT-PCR data I found it incomplete (Section 3.3). The Authors mentioned, that it had been performed, but no evaluation was included and this is also true for PPI network analysis (Section 3.4). I still did not find the "Discussion" and "Conclusion" sections appropriate. The Authors concluded that MAF-1A is involved in the inhibition of ergosterol synthesis, interferes with the cell membrane, protein synthesis and the function of the mitochondria. According to the analyses of GO terms and KEGG pathways it is hardly believable that one 26 amino acid long peptide is responsible for all these changes. I would recommend the Authors to try to put the results in context accordingly. I suppose some (more?) of the observed alterations are consequences of an interference with one or maybe two potential process(es), that however can not be supported without further work, therefore these statements should be phrased more carefully. Because of this, I think the last sentence of the „Discussion” part is an overstatement. Reconsider the „Discussion” and „Conclusion” parts according to the findings without statements not unequivocally supported by the results.  It could be me, but I could not find a link to the raw RNAseq data, that would be essentially required to keep science transparent, hence I would kindly ask the Authors to manage this issue.

I presume that the actual target is defined by the genes whose expression remains low/high at both timepoints (UG1/2). These are oxidation-reduction processes and using alternative energy sources. This is something that could be linked to the mitochondria. In my opinion all (most) of the other changes are due to this effect. Genes upregulated at six hours show that the cell is under stress (MAPK-signaling, cell cycle, meiosis) and needs alternative energy source (via peroxisome, carbon metabolism, fatty acid degradation) and components for macromolecules (autophagy). Analysis at 18h timepont shows that the metabolism has been rewired and growth curve indicates that the growth rate of the treated suspension at the later timepoints is nearly the same as that of the untreated control, meaning that the cells could have adapted to the altered circumstances. Change in the carbon utilization processes is a well-known response of C. albicans to stress (see the following publications PMID: 17158734, 31261727, 31937647).
Experimental design: no comment
Validity of the findings: I suggested the Authors to make a comparison between the response of C. albicans and that of C. parapsilosis to MAF-1A. The Authors refused this, because "it belongs to another graduate doctor" as it is written in the rebuttal letter. I am sorry, but I can not accept this explanation, because the reference to the raw data is openly available in the paper of Wang et al. (2017) (PMID: 28567034) an article that is actually cited by the Authors (Line 84). This publication says "Sequence reads have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA375109". I really urge the Authors to do this comparison, because this would serve additional valuable data on the effect of MAF-1A and moreover C. parapsilosis or C. albicans specific responses could be identified, that would be really interesting. Alternatively, rephrase the final paragraph of the introduction, because the reader will miss this comparison in the discussion, it would be so obvious.
Additional comments: I accept all the answers given to my previous questions except the one detailed at "3. Validity of findings". That analysis should either be performed or this section should be rephrased.

Grammar
Line 29 This provides new insight into the interaction between Candida parapsilosis and antimicrobial
peptides and serves as a valuable reference for future Candida parapsilosis therapies. -> This provides a new insight into the interaction between Candida parapsilosis and an antimicrobial
peptides and serves as a valuable reference for future Candida parapsilosis therapies.
Line 33-36 This sentence means the same as the following one, one of them should be deleted. And rephrase to meet proper scientific language „… (C. albicans) is the most common pathogen of Candida, its dominance has decreased as the numbers of invasive non-albicans Candida (NAC) species have increased…” -> „… (C. albicans) is the most common pathogen of Candida species, its dominance has decreased as the incidence of non-albicans Candida (NAC) species have increased…”
Line 41 their should be its (it refers to C. prapsilosis, it is singular)
Line 47 Rates of infection are high -> Rate of infection is high
Line 48-49 … and drug resistance conrast those of … -> … and drug resistance are in conrast with those of …
Line 53 „… immune response for a variety …” -> „… immune response of a variety …”
Line 54-56 This sentence is grammatically incorrect, please check it.
Line 108 triplicate -> triplicates
Line 159 A dot is missing before the word „Primers”
Line 269 „… transport process on the mitochondrial …” -> „… transport process in the mitochondrial …”
Line 281 „These processes help cells remove accumulated …” ->„These processes help cells to remove accumulated …”

Typos
Check for double spaces and missing spaces.
Line 53 The Authors write „from” is it not „form” ?
Maybe it is only the formatting of the homepage of the journal, but neither of the species names and the latin words are in italic.
Line 225. A dot is missing from the end of the senctence.

Inconsistent phrasing
Line 21-22 Instead of „DEGs” use „genes”. An upregulated/downregulated gene is a differentially expressed gene.
Line 45-46 Recent epidemiological studies (in various geographical regions) have shown that C. parapsilosis is the second to third most prevalent infection, following only C. albicans (Toth et al. 2019). -> The statement is correct if C. parapsilosis is the seconds, then C. albicans is the first. But if C. parapsilosis is the third then there must be some other species besides C. albicans. Please rephrase this sentence.
Comment on line 49: It should not be „may”, the interspecies specificities do actually affect the recognition be the host (C. parapsilosis vs C. albicans, see reference PMID: 26793173)
Line 64 Eefflux pumps are drug resistance related genes, if the latter is written, no need to mention the other.
In text UG1 was mentioned as significant (Line 201), however it is missing from Table 6.
Line 243-245 If overexpression is mentioned, point mutation should be as well (PMID: 23480635, 25385095).
Line 274-276 This sentence is misleading. The goal of ROS production is not the disruption of the electron transport chain, but it is a side effect of their presence.
Line 276 If the term „ROS” is mentioned, then no need to write hydrogen peroxide and hydroxy radicals, because these are defined as ROS.

Style
Line 51 Common Candida pathogens -> Remove „Common”
Line 54 A scientific paper should not contain such expression as „relatively small”.
Line 64 Instead of „resistant gene” I would recommend to use „drug resistance related gene”
Line 115 Time-kill curves were adapted from Li et al and Sun et al -> Instead of mentioning the complete citation in the sentence, the Authors might want to consider writing „Time-kill curves were performed according to the literature” instead, and then the citation can be included.
Citation should be at the very end of a sentence (e.g. Line 140 and 141)
Line 163-165 This sentence means the same as the following two. This part should be modified.
Line 166-167 It is not the curve that increased or remained, but the cell number. This should be rephrased properly.
Line 169 „RNA-seq results from C. parapsilosis with MAF-1A after …” -> „RNA-seq results from C. parapsilosis treated with MAF-1A after …”
Line 170 Instead of „Of the genes …” I recommend to write „Out of these genes …” or „Out of the DEGs …”
Line 171 Instead of „… 4.38% of the genes.” I suggest to write „… 4.38% of the total expressed genes.”
Line 174-176 Repharase sentence to make it grammatically correct, it has two subjects.
Line 218 „second or third commonly” -> „second or third most commonly”
Line 261 „… on ribosomes, inhibiting translation” -> „… on ribosomes therefore inhibiting translation”
Line 264 „… resulted in a loss of expression of multiple genes involved with ribosomes and translational processes.” -> „… resulted in a loss of expression of multiple genes associated with ribosomes and translational processes.”
Figure 2 „The expression of 101 genes significantly increased in contrast to 151 genes that decreased (padj < 0.05).” It is the expression that decreased not the genes. „The expression of 101 genes significantly  increased in contrast to 151 genes whose expression decreased (padj < 0.05).”
Figure 2 „Gene expression venn diagrams revealing two gene groups with opposite trends, labeled as RG1, RG2, and two gene groups with independently of time points, labeled as UG1 UG2; CPAS vs. CPAC_up: genes with increased expression after 6 hours; CPAS vs. CPAC_down: genes with decreased expression after 6 hours; CPBS vs. CPBC_up: genes with increased expression after 18 hours; CPBS vs. CPBC_down: genes with decreased expression after 18 hours.” Please repharase this sentence, maybe it should be split into more senteces. The tense is incorrect, if it is present continuous „is” is missing, but actually it should be simple present: „reveals”. The expression „two gene groups with independently of time points” does not have a meaning.

It is inconsistent that Fig 5 shows enzymes by highlighting the whole box where their names are, while on Fig 6 and 7 a purple frame is used to sign the proteins encoded by genes whose expression have changed. Could it be standardized?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF THE TRANSCRIPTIONAL RESPONSE OF CANDIDA PARAPSILOSIS TO THE ANTIFUNGAL PEPTIDE MAF-1A
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think the authors have addressed the main comments expressed by the reviewers. I noticed, however, that while gene names are italicized, species names remain unchanged both in the text and in the references.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATIONS TO CURSORIALITY AND DIGIT REDUCTION IN THE FORELIMB OF THE AFRICAN WILD DOG (LYCAON PICTUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The article submitted by Smith and collaborators about the anatomy and morpho-functionality of Lycaon's leg is a relevant and useful article that I think should be published. However, there are some suggestions.
In general, the “Fossil record of Lycaon spp.” section is not relevant to the article. Only the sentence “The vestigial metacarpal I of L. pictus, discovered here, is approximately 30% the length of its metacarpal II, compared to 38% in L. sekowei (Fig. 14). This pattern indicates a functional loss, but not complete loss, of metacarpal I over time in the Lycaon lineage”, points out aspects that are based on the revised material. And about this sentence, must be taken delicately because it is only based on one measure of a single current specimen. This is not necessarily true because the article does not consider potential intra-specific variability.
I suggest migrating the section of the fossil record to the introduction if authors feel that they can use bone morphology to validate their form-functional conclusions. The article focuses on the description of the muscles and little on the description of the bones.
Finally, in some cases, the use of taxonomic nomenclature is misused. Eg., italics where it should not be italicized.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATIONS TO CURSORIALITY AND DIGIT REDUCTION IN THE FORELIMB OF THE AFRICAN WILD DOG (LYCAON PICTUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: A recent publication by Chavez et al. (2019) provides a genetic and developmental basis for the rudimentary (not lost!) MCI. This is very much in line with the ms, but authors should discuss these data in the light of their results. Currently it is not recognized in the paper.

The authors provide possible function for the first digit (dew claw) both in the introduction and discussion referring to Zink 2005. This reference is impossible to locate using standard literature engines or databases, and may not be scientifically reliable. Use of the dew claw in high speed turns as a grip is questionable.

The caput magnum m. triceps brachii needs to be defined. While it is ok to use a name not listed in Nomina Anatomica Veterinaria, it is advisable to explain the name and its use.

The ancestry of Cuon from Xenocyon is not supported by presence of vestigial MCI in latter, as the former has fully developed MCI. Please remove or rewrite.

There are some minor problems and mistakes in the figures that need to be addressed:
Fig 2. is a medial view
Fig 4 is a lateral view and deltoideus pars scapularis appears to be not right.
Fig. 5. for m. pectoralis superficialis use the same color in cranial and caudal views.
Fig 8. the long flexor tendon does not look right. Please redraw or relabel.

Ref:
Chavez, D. E., Gronau, I., Hains, T., Kliver, S., Koepfli, K. P., & Wayne, R. K. (2019). Comparative genomics provides new insights into the remarkable adaptations of the African wild dog (Lycaon pictus). Scientific reports, 9(1), 1-14.
Experimental design: The main weakness of the data is the sample size: one individual, two limbs.
While this is common and understandable when working on endangered species and does not prevent from publishing the data, the authors should make it more clear that there most likely is variation in the structures that were not revealed here. Moreover, the specimens are from a captive individual which may have slight influence on e.g. muscle masses, and this should be brought up. Also the body condition of the animal could be presented in the materials.
Validity of the findings: no comment
Additional comments: This is a very nice piece of work, and it is good to get these data and the analysis out for the African wild dog. The main problem of the paper is the specimen number which is limited to one individual. Please emphasize this uncertainty more in the discussion and interpretation. The muscle (especially the palmar!) insertion sites and numbers are known to show intraspecific variation in the domestic dog and this was also the case e.g. in the Pampas fox (Souza Junior et al 2018).

You suggest the unique strong ligaments and tendons to function as energy saving springs during fast locomotion, and this is a valid interpretation. The other aspect, the enhanced need for proprioception, is not discussed. Structures like the adductor digit 1 with its thin tendon could serve mainly a proprioceptive purpose. Please consider this when revising your manuscript.
It is tempting to compare these tendons to the equid suspensory ligament and its evolution from the mm interossei. The ligament in equids is important not only in energy saving but also for safety by providing proprioceptive feedback.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATIONS TO CURSORIALITY AND DIGIT REDUCTION IN THE FORELIMB OF THE AFRICAN WILD DOG (LYCAON PICTUS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DEMOGRAPHIC AND GENETIC CHARACTERIZATION OF HARVESTED CORBICULA FLUMINEA POPULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The use of English language needs moderate improvement, as there are numerous small spelling and grammar mistakes. Here are just a few examples:

Line 79: change "in" to "on"

Line 184: Delete "mo". On the next line, change "occurred" to "occurring".

Line 308: change "poptential" to "potential"
Experimental design: I thought the research question was not well defined. The study has basically taken genetic and demographic data from a number of locations and performed some analyses providing information on spatial variation, but the introduction and discussion text mentions many research questions only loosely related to these data and analyses:

For example, there are no data presented here from the invaded range, so these data are not closely relevant to research questions about invasion biology. Any connection between these data and invasion biology could be limited to a few sentences of speculation in the discussion.

It is stated that “The goal of this study was to test whether commercial harvest will affect demographics and genetics of C. fluminea”, but this was not really tested, or only very indirectly. Comparisons were done among "lake" habitats and "peripheral" habitats, and basic observations suggested fishing pressure differed between the habitats. However, the habitat-type, and sampling methods used, also differed between lake and periphery sites, so it is unknown if the demographic and genetic differences are a result of fishing, habitat-type, or sampling method. From Fig. 1, it seems only two sites were in the fished lake area and four were in the peripheral area, so its difficult to tell if any differences may also just be random spatial variation. A more complex experimental design than was used here is needed to answer the question of “whether commercial harvest will affect demographics and genetics of C. fluminea”.

There is mention of harvest data and numbers of dredging boats, but these data do not seem to be collected very systematically or comprehensively, are only mentioned briefly in the results, and are not analysed. If these data are to be included, they should be expanded upon, e.g. displayed graphically and analysed.

The authors aim in many cases to compare demographic and genetic variables between “lake” and “peripheral” habitats, but these comparisons are all done informally, i.e. no direct analysis involving lake/periphery as a distinct factor was done. Possibly the authors could re-analyse the data and attempt to use their lake and periphery sites or sections as replicates in direct comparisons (e.g. ANCOVA).
Validity of the findings: As mentioned above, there are considerable amounts of data collected here, but I do not think it is very robust for testing the stated aims.

Concerning the conclusions, I do not think these analyses can be used to make strong conclusions about harvest management or colonisation potential in invaded ranges, or about non-random harvest being a potential driver for population demographic and genetic characteristics. The main conclusions that can be derived from the current analyses are about spatial variation in demographic and genetic characteristics, which only may be influenced by harvesting. Possibly with some re-analysis of the data the link of the analyses with conclusions about harvesting can be made stronger. Or possibly the conclusions could be changed to better reflect the limitations of the current analyses.
Additional comments: Line 111: I am not sure that the presence of dredging boats can be used as a proxy for fishing pressure – e.g. maybe the boats were observed just passing by or doing other activities besides fishing.

Line 116: Figure 1 does not show these 84 sampling points and 28 sampling sections and eight sampling areas. The terms “sites”, “points”, “sections” and “areas” have been used without many details, which is confusing. Please provide more details on how these points, sections, sites and areas were arranged.

Line 121: The same sampling methods should have been used across all sites, not a grab sampler at one type of site and a net sampler at another. One main comparison that was attempted was between lake and peripheral sites, but as they used different methods, maybe the differences observed were simply because of the different methodologies.

Line 134: There is not much mention of these environmental data analyses in the results, please describe more in the results how the environmental variables are correlated with the demographic or genetic data. If the authors are interested, it would be my recommendation that in a re-write of this manuscript, the association of the environmental and biological data could be given greater emphasis, to make the conclusions more valid and the manuscript more interesting to a broader audience.

Line 188: Perhaps it would be better to present the fishing boat data as density, i.e. boats per km2?

Line 195-196: I think this sentence needs to be rephrased, as Figure 2 shows the highest biomass samples were from the lake habitats, and Table 1 shows the mean was also higher there.

Line 200-222: I had trouble understanding how this sentence relates to the figure 4, please provide more detailed results explanation here so the link between demographic expansion and frequencies of pairwise differences in Fig. 4 is more clear.

Line 239-240: Harvesting any individuals will reduce stock abundances, not just harvesting large ones, so this sentence does not seem to be very relevant to the hypothesis mentioned in the preceding sentence.

Overall, I found the discussion a bit difficult to read, with the subject matter changing unexpectedly and the links from one sentence to the next often not clear. Also, the links of much of the discussion text with the actual data collected in the study are also not clear. I would suggest a thorough re-write, ideally structured around some modified or extra analyses.

Line 263-265: This seems a bit obvious - larger individuals are being harvested, so you end up with populations with smaller sizes remaining. I do not think all these complex and confusing ideas about self-regulated intra-specific competition or running water affected peripheral population food delivery need to be emphasised or probably even mentioned, especially given that the data are only loosely linked to direct evidence of fishing pressure, and not closely linked at all to competition or food delivery.

Line 299: I think the world "innumerable" is an exaggeration and should be removed.

Line 303: Much of the introduction and earlier discussion talks about how data are needed to learn about how harvesting may be used to control this clam in the invaded range, but here suddenly it is stated that this should not be considered because it could "incentive deliberate long-distance introductions". This should be stated up front (e.g. in the introduction) as a reason to not consider this concept.

Figure 1: please make it more clear here which of these locations are the main lake ones and which are the peripheral ones.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DEMOGRAPHIC AND GENETIC CHARACTERIZATION OF HARVESTED CORBICULA FLUMINEA POPULATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have read and review the manuscript carefully entitled “Demographic and genetic changes in wild Corbicula fluminea populations under harvest management”. The manuscript investigated the density, biomass and genetic structure of Corbicula fluminea based on CO1 gene in Poyang Lake Basin (lake and peripheral habitats). The meaning of this manuscript is significant, but there are still many problems in the whole article due to the lack of data analysis directly related to harvest pressure (or harvest management). In a whole, I am inclined to accept this paper after a major revision or rewriting. The general comments and specific comments are as follows:

General comments:
1) The abstract section gave a lot of explanations for the reason and significance of the research, but it didn't show the main results of the study, just a few conclusions. and the same happened in the conclusion section. Rewrite is recommended. I suggest that the author rewrite these sections.
2) This manuscript hope to study the population and genetic changes of Corbicula fluminea under harvest pressure in the Poyang Lake Basin. But the results only described the production changes in Asia and China, and the general distribution of harvesting boats in the Poyang Lake Basin. There was no direct correlation between harvest pressure (or harvest management) and population changes (or genetic changes). Without the support of directly related data analysis, it is obviously insufficient to discuss the main scientific issues of this manuscript, so it is suggested to add correlation analysis.
3) The sections of introduction, discussion and conclusion contained a lot of content for Corbicula fluminea invasion, but the study was a native range research. There was no data analysis and research results about Corbicula fluminea invasion. Although this study had certain significance for Corbicula fluminea invasion, but the meaning was limited, and the relevance to the major scientific issues was weak. If the author wants to discuss this problem, please add relevant research analysis, otherwise you need to weaken this issue.
4) There were too many indirect and speculative discussions in the discussion section, and there were no directly related analysis results to support the discussions. Therefore, it is suggested that the discussion should be rewritten and discussed based on the directly related results.
5) The result for figure 5 showed that C. fluminea has had a relatively small recent demographic expansion event occurring between 1,000 -2,500 years ago. This is a large-scale issue, which may be related to geological events or climate change, and this is another scientific issue, which is less relevant to the main research issue of this paper. Suggest to delete.
6) Last but not least, there are many sentences expression and some grammar errors, so I urge the authors to polish the MS by a native English speaker.

Some specific comments are as follows:
1) When the comparative degree of adjectives is used (such as line 55), the point of comparison should be clearly indicated. If the comparison is unclear, use the positive degree of adjectives. Please check whether there is any such problem in the full text.
2) Line 92: Please change " near threatened with extinction" to " be endangered"
3) Line 132 -142: The methods section had an introduction to the methods for obtaining and analyzing the environmental data, but the results section had not any content of the environmental results. This is not appropriate. Suggest to add environmental data results or delete environmental data-related methods.
4) Line 143-144: “No significant differences … p＜0.05; results”: My understanding is that p < 0.05 is significant, which is inconsistent with the result that there is no significance in the text. Please check.
5) Line 192: “Further, it … increase occurred.”: Confused. Suggest change to “Further, it was stable from 1989 until a rapid increase occurred in 2003.”
6) Line 195-196: “The total number … lake and peripheral habitats.”: The one observation shown here or the total observation? Please clear.
7) Line 196-198: “Lake habitats … dredging boats… in peripheral habitats.”: Dredging boats here should be referring to the harvest boats of Corbicula fluminea, rather than sand dredger, are they? This needs to be clear in the methods.
8) Line 205-207: “After accounting for … higher than biomass from lake habitats”: I don't think the results here is accurate. I think figure 2 shows that the individual of C. fluminea from peripheral habitats was heavier or larger than the individual from lake habitats.
9) Line 226: “FST =0.25”: Here, FST =0.25 refers to the value among populations, while in table 3, it refers to the value within populations. Please check it.
10) Line 250-255: “In this regard, we hypothesized … reflects the increasing fishing pressure (Wang et al., 2007).”: There were too many possible reasons for the sharp drop in density, and there was no relevant data analysis. Only due to large individual have been harvested, it is not convincing to infer that the decline in density was caused by fishery pressure. Suggest adding some valid direct data analysis to support this discussion.
11) Line 268-271: There were no relevant environmental data results in the results section, but there's a related discussion going on here. Suggest to add this part of the results.
12) Line 283-284 and line 301-302: Line 283-284 showed low differentiation among populations, and Line 301-302 showed high differentiation among populations. These two conclusions seemed to be contradictory. Why did these conclusions occur? Please explain and discuss.
13) Line 287-289: Please add a reference at the end of the sentence “In general, higher genetic variation resulting from introduced populations allows the species to adapt and spread over large territories.”
14) Line 298-301: Discuss why this is the case. Were there not enough samples or something else?
15) In the conclusion section, please summarize the conclusions directly related to the research results and suggest rewriting.
16) In figure 3, there are two yellow parts in Hap1, please check for drawing errors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DEMOGRAPHIC AND GENETIC CHARACTERIZATION OF HARVESTED CORBICULA FLUMINEA POPULATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Mostly very clear and unambiguous, professional English. Literature references dominated by phytogeographic and ecological citations, few on state-of-the-are population genetics.
Article stucture good. Statistical phytogeography results (Figs. 4 and 3 - btw they are swapped) interesting but less relevant for the question of the study.
Results are relevant but cannot answer hypotheses 2 on genetic diversity change under harvest pressure.
Experimental design: Questions well-defined and relevant. Sampling very good, genetic analysis: to few specimens, unclear from which year / month - and just a single marker. This is inappropriate for such an important question that aims to guide conservation managment.
Validity of the findings: Conclusions on part 2 are not supported (population genetic analysis) as only based on few specimens per sampling (when considering the 84 samplings 216 individuals is not much). The questions is really interesting and relevant - how strong is the impact of harvesting of populations, yet a traditional phylogeographic single-marker analysis will not be of great help here; it provides very interesting first insights thought.
Conclusions on part 1 (smaller individuals under harvest pressure) is well supported but totally expected.
Additional comments: The study by Wang et al. present interesting small-scale results into harvest pressure on C. fluminea populations in the lake and the rivers connecting to it. Under strong harvest pressure, sizes of specimens are smaller as predicted. A clear strength of the study is the effort used to assess biomass x density information across years and sites. Very good data, yet it needs to be shown in a better way. The second and main part of the study addressed the question if different harvest pressures impact on population genetic diversity. Here the study presents haplotype information of different populations around Poyang Lake and suggests that no impact of harvesting on genetic diversity is found. While this may be true, the population genetic analyses are no state of the art as
i) the sample size for genetic analysis is rather small considering the 84 sampling from different time points at sites, and
ii) the whole analysis is based on a single, mitochondrial marker. It is especially the latter that clearly limits the main results of harvest pressure.
True, in the late 1990s and early 2000s single-marker analyses for population genetic inferences were often done, but it has often been shown in conservation genetic research that single marker, especially mtDNA marker analyses can be misleading. Therefore, I cannot reccommend publication as genetic results (second part of analysis) has too little explanatory power. Also, mismatch analyses, skyline plots - all these are phylgenetic methods that will assess effects over thousands of years and not recent harvest-specific impacts. My recommendation is to add a few  (e.g. 10) microsatellites (from Penarrubia et al. 2015 e.g.) and reanalyse the data, use proper population genetic statistics based on heterozygosity, allelic diversity. I would also recommend taking more from what you have - you have a temporal data set. Why not assess population parameters (haplotype diversity, allelic richness, heterozygosity) per site per sampling time point. With that you would be able to infer effective population size parameters - a much needed are rarely provided esimator for managers.

Specific comments:
- It is interesting that for 84 sampling points only 216 haplotypes were analysed - why not more? This study has an immense potential by studying genetic change over time (as you have the different time points) to also infer effective population size. What changed in populations between years? This effect can be translated to Ne using the temporal methods given in the literature.

- Better outline, which samples where obtained in which year - and what was the sample size. Perform a rarefaction analysis to see if haplotype diversity per site is saturated (take the different sampling time points as your replicates)

- Check this study for available microatellites: Peñarrubia L, Araguas RM, Pla C, Sanz N, Viñas J, Vidal O (2015) Identification of 246 microsatellites in the Asiatic clam (Corbicula fluminea). Conserv Genet Resour 7: 393–395.)

- L98: Wrong defintition of genetic drift. You mean drifting organisms. Genetic drift is random fluctuation of alleles


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DEMOGRAPHIC AND GENETIC CHARACTERIZATION OF HARVESTED CORBICULA FLUMINEA POPULATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The use of English language is improved. There were a small number of additional corrections needed:

line 77: change to "actually"

line 79: change to "Because of the"

line 185-186: change to "harvest boats were distributed"

line 239: change to "such as that described"

line 274: change to "anthropogenic"

line 276: change to "that these kinds"

line 296-297: change to "differentiation" and to "isolated".

line 309: change to "incentivize"

Figure 1 caption: change "accross" to "across"
Experimental design: no comment
Validity of the findings: no comment
Additional comments: line 36: please proved a few more details in the abstract what exactly a "peripheral" location is, so readers can understand this without having to go into the main text. On the next line it is stated that differences of the clams may be caused by harvesting, please make the link here between lake and peripheral locations, and harvesting pressure. Overall, the abstract is short with few details, it could be expanded with more sentences stating in-depth details of main results and discussion.

line 37-39: Please provide some more details about why the consequences of demographic and genetic changes presented here are especially relevant for harvest management and sustainability of the resource.

line 40: Disturbance is not measured in this study so I do not think "Disturbance ecology" should be a keyword. Also, this study is only of a native species within its native range so I do not think "Alien invasive species" should be a keyword.

line 86: most readers will not know what a "peripheral" population is. As it stands, the reader has to guess that this means the areas "surrounded by modified inland artisanal aquaculture systems" mentioned above. Please describe exactly what a "peripheral" areas is here and how it differs from a "lake" area. Also on Figure 1 it does not specify anywhere which habitats are "lake" and which are "peripheral". There are some areas marked "CR" in this figure - are they peripheral or lake? All other abbreviations are explained on this figure, please explain what "CR" stands for as well.

line 111: please provide some details or a reference about what a Surber sampler is.

line 248: change to "two quantitative sampling methods (modified Petersen grab and Surber sampler) were used"

line 290-291: please provide a reference for this statement.

line 296-297: the concept introduced in this sentence is a bit unclear, I ask the authors to expand upon this concept a bit to make it easier for the reader to understand.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DEMOGRAPHIC AND GENETIC CHARACTERIZATION OF HARVESTED CORBICULA FLUMINEA POPULATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have received the revised manuscript entitled “Demographic and genetic changes in wild Corbicula fluminea populations under harvest management” and have carefully reviewed it. The authors have rewritten the main question in this manuscript. The manuscript got a great improvement over the first draft. However, there are still some problems with the manuscript, and I suggest a further revision. The comments are as follows:

1)	Since there was no harvest data available for direct correlation analysis with C. fluminea population, and the authors also have revised the main scientific question of the manuscript to weaken the issue of harvesting pressures, so I suggest that this issue should also be weakened in the title, abstract, goal and conclusion.
2)	The PDF version of the abstract is inconsistent with the Word version. For example, line 35: “We found higher biomass…” in PDF version, but “We found lower biomass…” in Word version. Please check.
3)	Line 115-122 “Environmental data were measured in … were referenced from Li et al. (2019).”: Are physical and chemical parameters measured by yourselves or by reference? It's confusing.
4)	There is Principal component analysis (PCA) introduction in the Methods section, but there is no relevant result in the Result section. Please check. In addition, in the Methods section, statistical analysis methods of data should be put together and subtitled. Please reorganize the Methods section.
5)	There is RH region but no XH in Figure 3b, but there is XH on the corresponding line 204-207. In addition, haplotype annotations are missing in Figure 3b.
6)	It is recommended to provide R2 and p values associated with RDA analysis to understand the correlation significance and degree of environmental factors. The discussion on the effects of environmental factors is not sufficient. It is suggested to have an in-depth discussion based on the results of RDA.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have received the revised manuscript entitled “Demographic and genetic changes in wild Corbicula fluminea populations under harvest management” and have carefully reviewed it. The authors have rewritten the main question in this manuscript. The manuscript got a great improvement over the first draft. However, there are still some problems with the manuscript, and I suggest a further revision. The comments are as follows:

1)	Since there was no harvest data available for direct correlation analysis with C. fluminea population, and the authors also have revised the main scientific question of the manuscript to weaken the issue of harvesting pressures, so I suggest that this issue should also be weakened in the title, abstract, goal and conclusion.
2)	The PDF version of the abstract is inconsistent with the Word version. For example, line 35: “We found higher biomass…” in PDF version, but “We found lower biomass…” in Word version. Please check.
3)	Line 115-122 “Environmental data were measured in … were referenced from Li et al. (2019).”: Are physical and chemical parameters measured by yourselves or by reference? It's confusing.
4)	There is Principal component analysis (PCA) introduction in the Methods section, but there is no relevant result in the Result section. Please check. In addition, in the Methods section, statistical analysis methods of data should be put together and subtitled. Please reorganize the Methods section.
5)	There is RH region but no XH in Figure 3b, but there is XH on the corresponding line 204-207. In addition, haplotype annotations are missing in Figure 3b.
6)	It is recommended to provide R2 and p values associated with RDA analysis to understand the correlation significance and degree of environmental factors. The discussion on the effects of environmental factors is not sufficient. It is suggested to have an in-depth discussion based on the results of RDA.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIVE NEW PSEUDOCRYPTIC LAND PLANARIAN SPECIES OF CRATERA (PLATYHELMINTHES: TRICLADIDA) UNVEILED THROUGH INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally fine, but see the General Comments for the author.
Experimental design: Fine. See the General Comments for the author
Validity of the findings: Fine. See the General Comments for the author.
Additional comments: Reviewer: Ronald Sluys

Manuscript (#48478) for PeerJ

Five new pseudocryptic land planarian species of Cratera (Platyhelminthes: Tricladida) unveiled through integrative taxonomy

by Araujo, Carbayo, Riutort & Álvarez-Presas

General comments:
This is an exemplary manuscript on an integrative taxonomic study, combining morphological and molecular information. All figures are good and necessary. Although the writing is not bad at all, there still remains much to be desired. Although I think that this is not actually the task of a reviewer, I have inserted very many suggestions for linguistic improvements, as well as other comments (a total of 632). I hope that these suggestions are clear and self-explanatory. There are a few other, major, issues that I would like to address below.

I was disappointed to notice that the species descriptions mostly focus on describing the holotype, even in cases where there are ample paratypes. This is absolutely incorrect, as one has to describe the species on the basis of all specimens available. This means that one has to integrate all information into a single account of the species and, thus, one provides ranges and averages of measurements. In contrast, the present species accounts give absolute measurements. It does not only concern measurements, of course, but also description of the shape of structures, etc. As Mayr (1969) already wrote: ''Description of a new species is based on the entire material available to the zoologist, including the type specimen[s]. It is NOT the function of the type to serve as the exclusive or primary base of the description''. In the case that there is only one specimen available (the holotype) one cannot give ranges, of course. Nevertheless, in the diagnosis one should avoid such absolute statements as, for example, "body length 36 mm" because one knows that other specimens will differ in details. In such cases the statement is easily made more appropriate by writing "body length about 36 mm". For other characters insertion of words such as "more or less" serve the same function.

I distinctly remember that I discussed this issue several years ago with one of the authors of the manuscript. Then he appeared to be fully convinced that indeed this focus on type specimen(s) was completely wrong and he fully agreed with Mayr’s (1969) statement. The present manuscript tells differently and, therefore, should be corrected, as in this respect it fails in its integrative objective.

Second, I have some doubts about the appropriateness and correctness of the new specific epithets coined by the authors. The International Code of Zoological Nomenclature (1999) specifies in Recommendation 25C: "Authors should exercise reasonable care and consideration in forming new names to ensure that they are chosen with their subsequent users in mind and that, as far as possible, they are appropriate, compact, euphonious, memorable, and do not cause offence."

In this respect, I feel that the epithets piguaiassu, piguaiatui, piguaiaboja, and, to lesser extent, paraitinga, fail to be compact, euphonious or memorable. Certainly, the first three mentioned are unpronounceable and should be replaced. Furthermore, the first three names mentioned, as well as the epithet imbiri, do not agree in gender with the genus name Cratera, an absolute requirement of the Zoological Code. The unpronounceable names are based on word compositions and, therefore, should agree in gender with the genus (by accident one of these epithets agrees). I do strongly suggest that the authors devise new word compositions based on euphonious Latin words that agree in gender with Cratera.

The specific epithets imbiri and paraitinga concern geographical names. Derivation and declension of geographical names are complex. In general, a specific epithet derived from a geographical name should preferably be (a) an adjective (ending in e.g., -ensis), (b) a noun in the genitive case. Correct geographic epithets are, for example, orientalis, arizonicus, atlanticus, sanctaehelenae.

Next, it is strange that the manuscript uses two different codes for the specimens, i.e., personal or field codes (I guess that these are the F codes) as well as proper museum catalogue numbers. In a published paper only official registration codes of the slides/preparations should be mentioned. Evidently, the field codes will be included in the catalogue of the museum but such field codes are of no concern or value to the reader of the paper. Presumably, the field codes are also written on the labels of the slides, as well as the proper catalogue numbers. But again, this is of no importance for the reader. Personally, I do always include field codes and proper museum catalogue numbers up to the penultimate version of a manuscript (in order to ensure that no mistakes will be made in reference to the slides) but then I remove all field codes from the final version of the manuscript (but the field codes are included in the collections database). However, in case something else is implied by the F-codes, this should be made clear in the text of the manuscript.

In the legends to the figures, the various frames of a single figure are labeled with capital letters (A, B, C, etc.), whereas in the frames themselves the indications are lower case (a, b, c, etc.); this is inconsistent; replace with capitals.

Detailed comments:
For detailed comments I refer to the annotated pdf version of the manuscript (hereby attached).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIVE NEW PSEUDOCRYPTIC LAND PLANARIAN SPECIES OF CRATERA (PLATYHELMINTHES: TRICLADIDA) UNVEILED THROUGH INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: The authors provide an interesting article about the diversity of the genus Cratera (Platyhelminthes: Geoplanidae), with the description of five new species.
The descriptions of these new entities are supported by morphological features and also validated by molecular approaches. The authors selected two mitochondrial and four nuclear markers for molecular analyses, two of them (Nd4toCox1, Tnuc813) are tested for the first time in phylogenetic inferences of land planarians. The development of these new markers is very good news, mainly for those who study the phylogeny of triclads. The figures and table (plus supporting information) are informative and of good quality.
The manuscript by Araujo et al. adds more information to the genus and, according to the new phylogenetic framework, establish the relativity of some features used in the diagnosis of Cratera.
In summary, I consider that the paper can be accepted for publication in PeerJ with some minor changes.
The authors will find my comments and suggestions in the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIVE NEW PSEUDOCRYPTIC LAND PLANARIAN SPECIES OF CRATERA (PLATYHELMINTHES: TRICLADIDA) UNVEILED THROUGH INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a good manuscript, providing ample argumentation and documentation of five new species of land flatworms from South America. The authors use an integrative approach with very robust molecular and morphological data. The text is clear in its objectives, the methodology is appropriate, tables and figures are well presented, and the discussion contemplates the purposes of the work. The authors found some minor corrections and suggestions throughout the manuscript. Considering this fact, I have no further considerations and therefore my recommendation is that the article can be published with the small considerations highlighted in the text.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIVE NEW PSEUDOCRYPTIC LAND PLANARIAN SPECIES OF CRATERA (PLATYHELMINTHES: TRICLADIDA) UNVEILED THROUGH INTEGRATIVE TAXONOMY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: The authors have made a great effort in improving their manuscript by incorporating the numerous suggestions made by the reviewer. For this I wish to express my compliments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ZONULA OCCLUDENS AND NASAL EPITHELIAL BARRIER INTEGRITY IN ALLERGIC RHINITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The review has good references, is cross disciplinary and introduces the subject clearly.    However, the field has been reviewed recently:

https://www.sciencedirect.com/science/article/pii/S1323893017301594?via%3Dihub

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752131/

However, this review focuses more on HDAC inhibitors that the other reviews.
Experimental design: The review has good organization, but it needs to be edited to flow better.  Presently all of the data is there, but it is more of a list of study findings organized by topic.
Validity of the findings: The conclusions are well stated, but introductory sentences are not always present and more work is needed to identify unresolved questions and future directions.  Also the concept that ZO-1 is a scaffolding protein for the tight junction and how it may regulate the paracellular pathway should be brought up in more detail.  For example, how might it translate to barrier function?
Additional comments: I have attached some minor comments in the pdf as well


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TUBER MELANOSPORUM SHAPES NIRS-TYPE DENITRIFYING AND AMMONIA-OXIDIZING BACTERIAL COMMUNITIES IN CARYA ILLINOINENSIS ECTOMYCORRHIZOSPHERE SOILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are several methodological problems and the English needs to be completely revised (there are several sentences that are not readable and there are several verb tense errors).
Licterature references are updated.
Raw data are not shared. The gene sequence data are not deposited in a public database
Experimental design: This work compares the nirS-type denitrifying bacterial and ammonia –oxidizing bacterial communities in the soil of pecans  inoculated with Tuber melanosporum and in the soil of  not inoculated plants  in greenhouse conditions.
Several works have been  carried out on the microbial communities in truffle mycorrhizosphere in recent years. Truffle bacterial communities were mostly investigated using the 16S rRNA gene as marker.  In my opinion this research using  the nitrite reductase gene nirS and the ammonia –oxidizing gene as molecular markers, does not add new information. Morover it is not clear why the Authors decided to use these functional gene markers to analyse the bacterial communities in truffle pots.

There are several methodological problems. For example:
Line 117 -119 It is not clear which was the substrate in the pots, and how it was disinfected.
Line 122-125 why were some  plants contaminated after 3 months?
Line 128-130. How can you obtain a spore powder with soaked truffles?
Line 133- line 138 How many plants do you consider for treatment thirty or four?  The experimental design is not clear
Validity of the findings: The  experiments do not reflect the natural conditions (of the field) and thus the results are not interpretable. The soil  was disinfected  (usually the soil is steam sterilized for the production of truffle mycorrhized plants in order to avoid contaminations) so the bacteria in the uninoculated plants should be  the consequence of greenhouse contaminations due to the operators. In contrast, the plants inoculated with T. melanosporum received also the bacterial communities living inside the ascomata.

The authors do not try to interpret the biological significance of the results
Additional comments: This work could be considered only after having completely rewritten it. I am not convinced about the experimental design.
The authors should be supported by a native English speaker with mycological experience  in writing the maunuscript.

Here are some examples of scientific problems in the introduction probably due to the bad English:
Line 42- truffles are not one of the many species of Tuber! Truffles are all the species inside the genus Tuber and, for same Authors, all the hypogeous Ascomycetes.
Line 77-78 dentrifing bacteria, which are widely distributed in microbial population such as bacteria, archea……?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TUBER MELANOSPORUM SHAPES NIRS-TYPE DENITRIFYING AND AMMONIA-OXIDIZING BACTERIAL COMMUNITIES IN CARYA ILLINOINENSIS ECTOMYCORRHIZOSPHERE SOILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a number of mistakes and inconsistencies in the basic reporting of the work that need to be corrected before publishing:
- first of all, the names of the figures to which the authors refer in the text is wrong almost all over the manuscript. This makes its reviewing very tedious. For instance Fig 1 is fig 2. Fig 2 does not allow to see the average number of OTU -> this is in Table1; Fig3 is Fig4... In addition, the legend of some supplemental figures (e.g. FigS2) are missing. Please make appropriate corrections; this should have been carefully checked before submitting the manuscript for reviewing.
- secondly, raw data have been submitted to a database and made publicly available but none of the sample names are the same between the paper and the database so it is impossible to use the data that as been shared. This is make the sharing useless and is not ethical; authors must provide the correspondance between the sample name they use in the article and the one they use in their submission to SRA.
- thirdly a number of references are inappropriate while many references to the field and the work that has already been done is missing (see comments below).
- finally the aims that are given at the end of the introduction are not exactly the ones that are tackled in the work; this should be modified (see comments below)
Experimental design: There are a number of information that are missing or need to be clarified in the methods used:
- the number of replicates used is unclear: the authors mentioned that each treament had thirty replicates (l133) but at the end it seems that only four were used for mycorrhization with truffle spores (l137). Can you clarify what are the 30 replicates then ?
- the authors must describe how they verified that mycorrhiza obtained belonged to T. melanosporum. The fact that a spore inoculum of T. Melanosporum was used to inoculate the seedlings do not guarantee that mycorrhiza obtained are from this fungus. Many contaminations can occur.
- Information are missing for amplicon sequencing data analyses: at which level were the data rarefied, statistics were performed as p values are given but we have no information about which test were used, whether data were transformed or not... please add these essential information.
Validity of the findings: There are a number of missing data that do not allow the reader to assess the validity of the results:
1. Authors must provide the sequencing results; how many reads obtained after quality check and processing, how many OTUs...
2. Only a little part of the results is supported by (undescribed/unknown) statistics. All comparisons done between rhizosphere and ectomycorhizosphere must be supported by stats.
3. Steepness of rank abundance curve is not a quantitative parameter and is difficult to evaluate from the graphs provided. What do you mean by sample CK.2 was the most abundant ? Do you mean "Had the highest richness" ? If so why using a rank abundance graph, this does not provide any information about richness but the distribution of the relative abundance of different OTUs. Alpha diversity indices are more appropriate to answer this question.
4. What is the scale in Figure 5 ? It goes from -0.5 up to 4 while data described in the text are relative abundances going from 0 to 100%; none of the value matches between text and graph in figure 5. In addition, was the ordering of samples in the X axis in figure 5 organised by proximity of pattern with other samples (hierarchical clustering or similar method); if so, it demonstrate that there are a lot oh heterogeneity in the data and there very few difference between control samples and mycorrhiza samples. We can expect that none of the differences described between the most abundant OTUs are supported by statistical tests. Anyway, a barplot would be more convincing than a heatmap since differences are quite low and there are not that many OTUs to display.
5. Table 2. Please provide +/- SE and order the genera so that we can compare easily genera between themselves i.e. order by the most to the less abundant in control but not in mycorrhiza. By the way, "environment samples" is not a taxonomic rank; replace by unknown bacteria (if you are sure they are bacteria)
Last. It would be nice to know the level of mycorrhization by T. melano of the seedlings. Indeed, the importance of the finding highly depends on the level of mycorrhization by Tuber. If only a few percent of the roots have these mycorrhiza, the impact on the nitrogen cycle will be low.
Additional comments: This study is innovative and interesting to the field as there is little information about the nitrogen cycle in truffle orchards. The study does not allow to determine whether the differences measured in the relative abundance of AOB and denitrifying bacteria in the ectomycorrhizosphere vs rhizosphere has a significant impact on the nitrogen cycle. Measurements of activities in soil and nitrogen cycles intermediate would have been necessary to be able to truly conclude but it is an interesting first step. However there are a number of points that need to be corrected before publication (in addition to those describe above).

Details comments:
l23: "rarest". T. melanosporum is not the rarest truffle among the one that are commercialized
l42-43 edible fungi which can form -> remove "CAN"
l44: Gogen et al 2012; this ref is not the most appropriate to illustrate this point as it is focused on T. aestivum
l48: costing 1500 euros/kg -> reaching up to instead. Depending on the time of the year and the years, it can drop easily below 1000 €/kg
l49. T.melno contains amino acids and trace elements -> remove; all living organisms contain amino acids and trace elements that's not a specificity of truffles, those are the basal compounds of life on Earth.
l 55: study on mycorrhizal synthesis: what do you mean by mycorrhizal synthesis ?  controlled inoculation ? mechanisms of the formation of ectomycorrhiza ?
l76-78: sentence is weird; consider reformulating; not sure to understand the point you want to make saying that dentrifying bacteria are widely distributed among microbial population that include bacteria, fungi and archea.
l85: What do you mean by "bacteria of similar molecular size" ? Relative conservative morphology ? Pseudomonas are rod bacteria
l93-94: few studies have focused ... that's true for the ectomycorrhizosphere of Tuber but not for plants in general. Look for instance ate Henry at al 2008, Environmental Microbiology, many other studies have followed this one
l94: ammoxidation is not the appropriate wording, use ammonium oxidation instead
l105 Esmaeili 2017-> inappropriate citation
l105 the connection between the idea that it is not clear how microorganisms interact with each other and the plant and the fact that you infected  C. illinoensis with T. melano to observe mycorrhiza formation is not crystal clear.  You may want to rework the connections between ideas in this section.
l108-109: at the same time, we studied.... of microbial communities. This is not true; you did not look at the overall microbial communities but only the one's that have NirS or AmoA genes; this is quite different
l116: "prepared matrix": give details about what it is
L 119-120: The greenhouse... verb is missing
l121: moistened with sterile water -> to reach which level of humidity ?
l123: contamination -> control to verify the absence of ECM contamination ?
Please provide a description of the soil used; what is the content in nitrogen ?
l151 and further: please provide refs for the primers used
l179: "samples were purified" what do you mean ? remove chimera ? contaminants ? how ?
L205: How can  ACE1 and CHao index statistically different but not the richness as both are calculated on richness; the variability found in the richness data should be at least as high in CHao and ACE indxes
Lin 212 if not significant then p>0.05 not < same line 238
Fig 4 -the phylum level is not appropriate to compare the data, work at a lower taxonomic level

Discussion section
Authors should compare their results to what has already been published concerning denitrification and ammonium oxidation in other tree rhizospheres (see for instance Moreau et al 2019 Functional Ecology)
Compare also their data with what has been previously described about the microbial communities of ectomycorrhiza of T. melanosporum (Deveau et al. 2016 Mycorrhiza)
line 274: different from what ?
l279: typo in Magnetospirillum
l290: There have been a number of publication that have been made on this topic since Barbieri 2007 (which is not about T.melano by the way but T. magnatum). Similarly there is a number of articles about Pseudomonas in truffles and particularly about T. melanosporum; please expend your literature search


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TUBER MELANOSPORUM SHAPES NIRS-TYPE DENITRIFYING AND AMMONIA-OXIDIZING BACTERIAL COMMUNITIES IN CARYA ILLINOINENSIS ECTOMYCORRHIZOSPHERE SOILS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The research presented data on nirS-type denitrifying and ammonia-oxidizing bacterial populations in rhizosphere soils of pecan trees with Tuber melanosporum inoculated roots compared to uninoculated roots.  It is interesting, although there are some general issues with the paper.

In particular, the hypotheses are not well defined, and it is not clear what the expectations were. It would have been very nice to measure ecosystem processes (or rates or ammonification / denitrification), for example, to determine how measured communities related to these functions.

Overall, I would recommend improvements on the writing throughout. There are spelling inconsistencies, grammar issues, and details of the methods that need could be improved. I have tried to make note of many of these in the reviewed PDF (attached).

The figures are a bit crude, not clear to read or interpret (particularly Fig 3 & 6).

This paper from 2017 may be relevant to cite:

Mycorrhization of pecan (Carya illinoinensis) with black truffles: Tuber melanosporum and Tuber brumale would be appropriate to cite:
Mycorrhiza (2017) 27:303–309 DOI 10.1007/s00572-016-0743-y
Experimental design: The experiment is kind of minimal but there are 4 replicates of the treatment and 4 replicates of the control (so 8 samples in total).

Having some metrics for comparing control vs. treatment plants and soils would have been beneficial to include (Tuber DNA in soil; % mycorrhizas; hight or biomass of plants, denitrification / ammonification rates, etc.).

The methods are a bit sparse in places. For example, it was hard for me to follow how the nirS and ammonia-oxidizing amplicons were analyzed. That is, how were sequences filtered and clusters determined and identified? What criteria were used to determine species, or was the default 97% OTU cluster definition that is used for 16S rDNA applied to these 2 functional genes? How was taxonomy assigned to the clusters? I am not sure how complete the reference database is. It would have been nice to include 16S rDNA as a comparison for bacteria communities in total. It would also be good to include the sequence output results, total number of reads per sample / per locus post filtering.
Validity of the findings: It appears that Tuber melanosporum ectomycorrhizas lead to a change in bacterial communities (at least nir-s and AMO ones), but only these functional genes were assessed in the community.

Given some of the limitations, it is a bit challenging for me to access how novel this study is, or the implications of the results.
Additional comments: I have included a scan of my edits. I hope they are helpful.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TUBER MELANOSPORUM SHAPES NIRS-TYPE DENITRIFYING AND AMMONIA-OXIDIZING BACTERIAL COMMUNITIES IN CARYA ILLINOINENSIS ECTOMYCORRHIZOSPHERE SOILS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was considerably improved. However, the English, above all of the new parts need to be revised.  Moreover, in my opinion,  before to accept this manuscript for publication the Authors should consider that the most importance source of bacterial diversity in the mycorrhized seedlings (which are cultivated in a sterile soil) is the microbioma of the ascomata used to inoculate spores. This important aspect should be incorporated in the discussion. In the point 8 of my previous review I already have raised this point
Experimental design: no comments
Validity of the findings: The discussion need to be improved as suggeste above.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TUBER MELANOSPORUM SHAPES NIRS-TYPE DENITRIFYING AND AMMONIA-OXIDIZING BACTERIAL COMMUNITIES IN CARYA ILLINOINENSIS ECTOMYCORRHIZOSPHERE SOILS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is reporting the results of an experiment that tested the impact of black truffle on denitrifying bacteria and ammonia-oxidizing bacteria community structure. The main finding of the experiment showed that the abundance and diversity of ammonia-oxidizing bacteria decreased as a result of ectomycorrhizal colonization of the truffle, but the nirS-type denitrifying bacteria increased in abundance and diversity at the same time.

The authors supplied sufficient background, method descriptions to fully understand their motivation, significance and experimental design and set-up. The structure of the manuscript follows the requirements. Figures and tables look professional and aid the understanding of the manuscript, and seems all needed for the full story.

The English of the manuscript is clearly improved, but there could be improved a bit further (see attached document with tracking and comments for suggestions).
Experimental design: The manuscript describes a small, original experiment to examine the impact of ectomycorrhiza of truffles on nitrogen transferring bacteria in the rhizosphere, and this research is within the aims and scope of the journal.

The authors of the manuscript responded to previous reviewers' comments quite well and made significant improvements to the presentation of their research questions, methods, and explanations. There were a few responses from the authors, which made complete sense in the rebuttal letter, but their comments there did not translate into the main text. I placed comments in the attached text or suggested rewording, where I was still unclear about a few things.

I think that the methods are now described with sufficient details and it could be easily replicated.
Validity of the findings: Although the sample size was small (four replicates per treatment), the limited statistical analysis seems sound and all data analysis is free of speculations. The authors point out some of the limitations and what further studies are needed to support the outcome of this experiment.

The conclusions need a bit more clarifications with rephrasing the statement (I put a note on it in the attached text with a suggestion).
Additional comments: The manuscript significantly improved by taking the previous reviews seriously and making changes accordingly. Language editing could improve the text further. I tried my best to make suggestions in an attached file. Please consider my few comments to make the manuscript even more clear. My comments are directly inserted into the attached file.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The main conclusion is clear and supported by the results. The structure and general language of the article are also clear. I note that the authors have used an English editing and correction service, but I suggest that this service has not done its job: there are many small grammatical errors throughout the manuscript. As a native English speaker, I have taken the liberty of correcting these on the pdf (please check the attached pdf file), so this should not be a problem for a final published version. The literature is well referenced (although I have suggested one possible paper that could be relevant in the Introduction – see below), and figures are appropriate. The Supplementary materials include data for survival, mortality, life expectancy and age structure.
Experimental design: The study represents original primary (experimental) research and does fall within the aims and scope of the journal. The research question is timely and the study is a useful investigation based on a straightforward experimental design, but applied in specific circumstances. The data collection, statistics and presentation are all of a high standard and the Methods section includes details that would allow the study to be replicated.
Validity of the findings: The findings are valid and supported by the research.
Additional comments: The Introduction includes background information on the fact that plant life history traits are key to the ‘invasiveness’ of invasive species, and here there is a strong emphasis on studies that show the importance of reproductive traits. It might be useful to cite a recent study (Guo et al. (2018) Ecology Letters 21(9), 1380–1389) showing that, world-wide, the naturalization or long-term establishment of invasive species depends on the overall suite of traits that comprises the general functional strategy, and thus including vegetative traits and the integration of adaptive traits in general.

In the Results section (lines 244-245) it might be useful to add that the soil organic matter content at the plateau was extremely variable.

The full latin binomials for cattle and mule are presented in the Results section (lines 249 to 251), but they should be presented at the first mention of these organisms in the Materials and Methods section.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have presented a good background on this topic. The language use is professional, and the paper is structured conform journal guideline.
Experimental design: The manuscript is within the journal’s scope. It has well-defined research questions considering the current knowledge gap in the literature, followed by a rigorous investigation. Methods are well described however I would recommend explaining the method through the flowchart but in a more symbolic way, not just text.
Validity of the findings: Data and statistical analysis are robust and controlled.
Additional comments: Authors of this manuscript have presented a study concerning the invasive Prosopis juliflora (Fabaceae) seedling survival and growth depend on habitat and seed dispersal mode in the Caatinga dry forest. The authors have found that the plant-animal mutualism between exotic species represents a driving force to spreading P.juliflora across riparian forests, degraded river banks and sedimentary deposits, while this plant fails to dominate dryer habitats in the Caatinga dry forest. The topic is interesting, an important issue and generally well written, well structured and contributes to the existing knowledge. However, there are still some occasional grammar errors through the manuscript especially the article ‘’the’’, ‘’a’’ and ‘’an’’ is missing in many places, please make a spellchecking.

The results and discussion section needs further improvement, compare your findings with the other author's conclusions.
•	In general, the manuscript needs to revised again concerning technical and grammae errors.
•	Please provide more deep discussion about your results, compare your findings with the other author findings.
•	Please clearly state the novelty of this work.
•	Please check the reference style, some of the references are not according to the journal style, especially the journals abbreviations.
•	At present, neither the original contributions of the paper nor its practical significance is clear for me.
•	As said, it could be a failure to comprehend the main argument; and lightening the discussion would enhance comprehensibility and make the authors’ point clearer and stronger.
•	Therefore, the reviewer recommends to further improve the manuscript before accepting it for publication. Some of the specific comments are listed below.
•	The conclusion section is to short, please elaborate it a little bit more.
•	Please consider citing following literature; reviewer believes that it may help the authors to have a more holistic overview about climate impact on plants:

Rivaes, R., Rodríguez‐González, P. M., Albuquerque, A., Pinheiro, A. N., Egger, G., & Ferreira, M. T. (2013). Riparian vegetation responses to altered flow regimes driven by climate change in Mediterranean rivers. Ecohydrology, 6(3), 413-424.
Lv, X., & Zhou, G. (2018). Climatic Suitability of the Geographic Distribution of Stipa breviflora in Chinese Temperate Grassland under Climate Change. Sustainability, 10(10), 3767.
Rivaes, R. P., Rodríguez-González, P. M., Ferreira, M. T., Pinheiro, A. N., Politti, E., Egger, G.,& Francés, F. (2014). Modeling the evolution of riparian woodlands facing climate change in three European rivers with contrasting flow regimes. PloS one, 9(10), e110200.

Concluding Remarks
The work presented in this manuscript is an interesting topic, it needs some more efforts to improve it further. Reviewer recommend major revision of this manuscript and publishing it only after specific improvement of the current version are made.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 1
Reviewer: 3

Output:
Basic reporting: •	Clear and unambiguous, professional English used throughout

The authors set out to conduct an experiment looking at the effects of livestock and local geomorphology/soil chemistry on seed germination, seedling survival and a slightly ambiguous estimate of “life expectancy” of Prosopis juliflora in three different alluvial habitat’s in the São Francisco River valley of the semi-arid Caatinga region of Brazil.  This paper can be greatly improved by consulting/editing by a native/fluent English writer.  As it stands now, the introduction and stated purpose of the study are difficult to comprehend.

•	Literature references, sufficient field background/context provided

The article does include an extensive review and background of the topic.  However, most of this is contained in the discussion.  It would be good to include more background information in the introduction.  It would be good for the introduction to include a more in-depth overview of the Caatinga region in general.  What are the native plant communities that have been replaced by P. juliflora invasions?  Give specific examples, including dominant genera that have been excluded.  It would also be good to include some general information on the types and history of the human disturbance in the region.  The authors do mention that P. juliflora was introduced as a food source for livestock to the region in the 1940s.  This is good, but that is far as they go in describing the extent and degree of the disturbance.  If there has been sustained cattle grazing since the 1940s, what percent of the Sao Francisco river valley native vegetation has been replaced by invasive P. juliflora?   Lines 132:133 (word doc) states “The Caatinga vegetation has increasing human disturbance levels associated to agriculture and livestock production by low-income rural populations (da Silva et al., 2014; Leal et al., 2014; Ribeiro et al., 2015).” This suggest that other agricultural practices besides grazing are active in the area.  What are they?  Do these co-occur spatially with grazing?  Or is grazing limited to rangelands?  Are there other land management practices that occur? Is fire used?  A more complete description of disturbance and the role of grazing should be reviewed.

•	Professional article structure, figs, tables. Raw data shared.

Supplementary_material_(3,4, 5) contained counts of living P. juliflora for each sampling period at each of the habitat types.  The authors need to check that column headings are consistent across each table.  Supplementary_material 3 differs from 4 and 5.  No raw data was included for the soil data.  Only one profile example was included for one of the sites.  However, the methodology has no mentions of topographic surveys.  This data appears to have come from another source?   There is no raw data reported for soil water content either.  The methodology report that soil moisture content was measured every 2 weeks over an 11 month period.  This time series data is not shared.

The figures and tables in the main manuscript body are professional and match what is described in the main text.

•	Self-contained with relevant results to hypotheses.

The study appears to be self-contained and not inappropriately subdivided to increase publication count.
Experimental design: •	Original primary research within Aims and Scope of the journal

This question that this manuscript sets out to address is within the Aims and Scope of the journal

•	Research question well defined, relevant & meaningful.  It is stated how research fills an identified knowledge gap.

The research goals need to be more explicitly stated.  This perhaps something that can be clarified with the assistance of more fluent writing.  I am uncertain about the emphasis on “life expectancy”.  The authors need to do a better job of defining what they mean by life expectancy in the context of their study.  How is this any more relevant than seedling survival? Life expectancy is typically measured as the average number of years an individual will live.  Once established, most Prosopis are relatively long-lived species.  For example Bhojvaid et al. 1996. Reclaiming sodic soils for wheat production by Prosopis juliflora.. report on 30 year old P. juliflora plantations.  They probably live much longer.  Life expectancy seems misplaced for one year of monitoring of seedlings.

•	Rigorous investigation performed to a high technical & ethical standard

The investigation was conducted to a high technical standard.

•	Methods described with sufficient detail & information to replicate

Sampling methods could be explained with more clarity.  Line 188:191.  “The moisture content of 10 soil samples (determined using the gravimetric method) was measured twice a week at 20 cm depth from March 2014 to February 2015 and it was analyzed according to soil type and environment (Francesca et al., 2010).”    Is this 10 soil samples overall, or 10 soil samples per habitat type?  It is not clear.

Lines 193:194 No need to have the same calculation repeated twice.  Simply list which version of H was used.
Line 199:200  What are Brazilian standard protocols?  Please briefly state if these are different than others.
Line 201:202.  How were seeds collected and when?  Are they mature fruits taken off of plants or taken from the ground?  Livestock treated seeds were only searched for in manure?  Or were they purposefully fed to livestock first and then collected in manure?  Please be more explicit.
Please clarify the sowing protocol.  Lines 215:216 “One hundred seeds were adopted per plot, totaling 1,000 seeds per treatment, in mid March 2014 (i.e. raining season).”     Did each plot contain all 4 treatments?  100 seeds per plot across 120 plots would be 12,000 seeds. You have 4 treatments. That's 3,000 seeds/treatment and 1000 seeds per treatment for each habitat ( Floodplain, Alluvial, Plateau).
There should be 25 seeds for each treatment per plot.  Please state numbers more clearly

Lines 220:221  “The seeds from the fruits and in the dung were collected from several trees and animals, respectively, in the year that P. juliflora trees fruited/flowered.”  This seems to imply that P juliflora doesn’t fruit or flower every year?  If that is the case it is directly relevant to the study of seed dispersal and recruitment.  Or does this just mean that seeds were collected in the year preceding the study?  Please clarify.

Lines 238.  Equation for Tx showup on ms word document but not on pdf. Please be aware of this.

Lines 230:240 Life expectancy and survival tables.   Be mindful of your variable descriptions.  Each one contains an x which is confusing because it could mean multiply.  I’m not sure it’s intended to be used this way   Example Line 235:  number of individuals dead during the age class x (dx)
Validity of the findings: The authors have a rich dataset that contains local soil chemistry measurements as well as time series on soil moisture, germination and seedling survivorship.  The statistical analysis were fine.  However, I wonder why the authors focus on life expectancy.

Why not report or present in figures germination (#s and %) and seedling survival (#s and %)?  These are data that are buried in the supplemental material but seem to be the most relevant data to the stated research questions.
Also, survivorship across all treatments was low.

Also, soil moisture was collected as a time series.  In semi-arid environments water is often a limiting factor for survivor ship.  The timing of dry down could be directly related to timing of mortality in each of the treatments.  It would be good to visually present the soil moisture time series data in contrast to time series of seedling survivorship.

Overall, seedling survival was extremely low across all treatments.  According to the supplemental tables the highest number of living individuals at the end of the study was 4.  These were in the floodplain x cattle treatment.  4 out 1000 seeds seems like a particularly relevant point that should be made.  There are other influence besides dispersal alone that promote the expansion or maintenance of native plant communities.  For e.g., see Weltzin et al. 1997 SMALL‐MAMMAL REGULATION OF VEGETATION STRUCTURE IN A TEMPERATE SAVANNA.  Small mammals and other herbivores can maintain existing plant communities preventing colonization of Prosopis.  Is there a possibility that similar occurrences are occurring in the Caatinga?  Just something to think and perhaps address in the discussion.
Additional comments: Overall, I think the study design is valid.  Questions that move beyond the strict physical parameters of species expansions and into the biological components are important additions, and it's nice to see that folks are working on these questions.  I truly feel that all the major components of the story the authors are trying to tell are here.  I do feel that the presentation of their data can be improved.  It would be nice to see seedling survival/germination over time by habitat type and soil moisture over time by habitat type.

Also, can the spatial resolution of the soil characteristics, be tied to individual plots, or blocks of plots.  If so, this analyses would be greatly improved by adopting a mixed modelling approach.  It appears from the large drop in germination at the plateau sites that soil types are an important piece of the story.  With 120 plots you may have enough to start to tease apart the general characteristics that promote Prosopis germination and establishment.  This could further parameterize where Prosopis may be more likely to invade into the future.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 2
Reviewer: 1

Output:
Basic reporting: The quality of the English has been much improved since the last submission. The extra publication I suggested has been cited.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied that the authors have adopted all of my suggestions for improvement based on the previous submission.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is improved significantly.
Experimental design: The manuscript is improved significantly.
Validity of the findings: The manuscript is improved significantly.
Additional comments: The manuscript is improved significantly. Accept!


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEED GERMINATION AND EARLY SEEDLING SURVIVAL OF THE INVASIVE SPECIES PROSOPIS JULIFLORA (FABACEAE) DEPEND ON HABITAT AND SEED DISPERSAL MODE IN THE CAATINGA DRY FOREST
Review round: 2
Reviewer: 3

Output:
Basic reporting: The language in the manuscript has greatly improved since the previous version.  While there are still occasional errors (Please check spelling.  The focal species is reported as both Prosopis and Prosopsis multiple times) it reads reasonably well.

The authors do give a reasonable historical background and context to their experiment, and the literature references for this paper are appropriate and sufficient for the manuscript.

Please include units in your figures.  For example – Figure 3.  Is Field Capacity being reported here as %, g/g?  Gravimetric water content should be reported as θ = cm^3 cm^-3.  Are the units of life expectancy days?

I have specific concerns on what is being reported in results of the manuscript.  The author’s stated objectives are to evaluate seed germination, seedling survival, and life expectancy (lines 142 – 144).  However, seed germination is never reported in the results of the paper.  They are in the supplemental tables.  I gather that germination was the raw number of germinates and is reported as a survival rate at T=0 on the figures, however this is not clear.  A table showing the total number and percent that germinated for each experimental category would be a simple and clear way to report germination.

Why are survival rates being reported with values much higher than 1 (100%).  Is this because at each time interval it’s a step function of the total available seedlings in the previous time period?  If germination continued from 0-30 days this is why your “rate” of survival increases over time.  The figures (3-5) could more clearly express the data if % survival with survivorship curves were presented for each experimental group.  There are many examples of this.  See Salihi and Norton. 1987.  Survival of Perennial Grass Seedlings Under Intensive Grazing in Semi-Arid Rangelands. Journal of Applied Ecology, Vol. 24 (1) for a good example.

I believe the authors are correct in stating that water availability is an important factor in determining seedling survival.  The reporting could be greatly improved if soil moisture measurements were reported in time-series to match their reporting of survivorship (could include germination too).
Experimental design: This study has a simple and straightforward experimental design; to evaluate seed germination, seedling survival and life expectancy across different geomorphologies and pre-germination treatments.  The authors do a reasonable job addressing this objective in their experimental design and their experimental methods are clear enough to replicate.

The authors could be more rigorous and relate the potential covariate data they collected (soil moisture and nutrient data) in a statistical way (GLMs) to seedling survivorship.  It appears that the authors setup this study with intent to do so, but is not presented in this manuscript.
Validity of the findings: Seed germination and seedling survival are appropriate measurements to report here.  I worry about the estimates of life expectancy.  Prosopis can be long lived species.  Estimating the life expectancy based on 1 year of seedling survival might not be appropriate.

Seed germination and seedling survival are appropriate measurements to report here.  I worry about the estimates of life expectancy.  Prosopis can be long lived species.  Estimating the life expectancy based on 1 year of seedling survival might not be appropriate.

The supplemental data appears to suggest that maximum number of seedlings still surviving after 1 year was 4, that were embedded in cow manure in the floodplain.  The low overall survivorship should be addressed in the discussion.
Additional comments: Objectives of the paper are clear.  The authors seek to evaluate if geomorphological surface and the influence of livestock play a disproportionate role in seed germination and seedling survival of invasive Prosopis juliflora in the Caatinga dry forest.  Their overall experimental design and the implementation of the experiment seem to do a reasonable job addressing this objective.  I believe where this manuscript could be improved is in the reporting of their findings.  Starting with variation in seed germination.  This is a topic that may be of interest across a large number of people studying Prosopis in general, and is particularly relevant to the study in this manuscript.  I also feel that presenting simple survivorship curves for the seedlings instead of the life expectancy/survivor rate figures would be a visually strong way to present the data and tell the story.