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: PRELIMINARY PLACEMENT AND NEW RECORDS OF AN OVERLOOKED AMAZONIAN TREE, CHRISTIANA MENNEGAE (MALVACEAE)
Review round: 3
Reviewer: 2

Output:
Basic reporting: The English writting seems to be sufficient, while the writting can be improved in some parts.

The article does not include sufficient background on broader field of knowledege. Relevant prior literature was not critically analyzed and referenced especially on morphology of Malvaceae that should aid in data interpretation and discussion of results. I consider the manuscript needs to be better conceptualized and discussed from a morphological view. I have made suggestions in the attached files including some references.

The manuscript is nicely illustrated. Figures are relevant to the content, but should be better described and labeled (some suggestions included in attached files). Nevertheless, the map should include all species occuring in that geographic region to allow comparison (as done in the tree, table, text, etc.; it is comparative biology).

Unfortunately I could not check all raw data (I do not remember seeing a data file used for GEOCAT analysis).
Experimental design: The manuscript includes original primary research. Research question is relevant and the knowledge gap was identified and the statements were made as to how the study contributes to filling that gap, but I consider the study could be better designed.

There seems to be some bias to digitalization of both herbarium specimens and literature (I think it should be one aspect to be discussed but not the main focus of the mss and the introduction). Authors focused on the absence of the focal species on generic floristic lists and recent monographs linking to digitization of both herbarium specimens and literature but they not discuss it more broadly to include the importance of specialists in Malvaceae from Brazil and taxonomy in general. It would be expected that specialists, rather than general floristic papers like ter Steege et al. or Cardoso et al., could detect this plant taxon in herbaria. Consequently, the taxon would appear in more general floristic lists.

Thus I think it would be more meaningful and helpful to discuss data in the light of the need of investment to training botanists (e.g., Ahrends et al., 2011 in Biological Conservation) and examine extant herbarium collections (e.g., Bebber et al. 2010 in PNAS) to disclose diversity of plants and avoid overlooking species.

The obtained sequences for Christiana mennegae are welcome to the phylogeny of Malvaceae but they would be unnecessary to place C. mennegae as related to C. africana (and authors should NOT state they are sister groups because sampling of the study does not allow to test such a relationship as only these two species were sampled; additional comments on attached files). This might be achieved with morphology only since both species are clearly related based on flower and fruit morphology. Moreover, the molecular focus and relative hard work on lab and analyses seem to have biased authors’ view at the expense of morphological aspects of Christiana mennegae in the context of Malvaceae. The phylogenetic framework in the mss was not used to discuss the morphology, evolution, or any other aspects.

Therefore, rigorous investigation seems to have been performed to a high technical standard regarding molecular part of the study but not the morphological part nor the conservation status assessment (two of the my main concerns). Literature on Malvaceae morphology as well as IUCN (2012) criteria should be investigated more deeply and thoroughly as well as more critically by this study. For example, I could not find justifications for the authors having considered the very similar fruits of C. africana and C. mennegae, respectively, apocarpous and syncarpous.

Also, I could not find justifications for the authors 1) having used EOO (extension of occurrence) but not AOO (Area of occupancy) which is also calculated by the tool used (GEOCAT) by them, and 2) having overlooked the need for selecting at least two of the three required conditions for IUCN criteria they used for conservation assessment (i.e., “B. Geographic range”) that can include number of locations of occurrence for which they presented data. Please see additional comments on attached files.
Validity of the findings: Some conclusions regarding, as for instance, the monophyly of Christiana, the occurrence of stomata in seeds surface and the inexistence of glandular trichomes on other four species of Christiana, are not connected to the original question investigated.

Some conclusions are fragile, like, for instance, about inexistence of sympatry between Christiana africana and C. mennegae because no (comprehensive) data on geographic distribution (from herbarium records and literature) of Christiana taxa was compiled, presented or analysed. For example, Tschá et al. (2002) cited herbarium specimens of C. africana from municipality of Rio Vermelho in the Pará state that is c. 50 km east from collections of C. mennegae in the municipality of Canaã dos Carajás.
Additional comments: I consider the manuscript is poorly conceptualized from a morphological view. Important literature on Malvaceae morphology was overlooked and not referenced in the introduction and discussion. This could contribute to a more detailed morphological description and a deeper discussion of their data. I would expect to find a critical review of the literature about the placement of Christiana in Malvaceae and Brownlowioideae. It was not clear from introduction what morphological evidence lead authors to identify material as the belonging to subfamily Brownlowioideae of the Malvaceae (e.g., fused sepals? Presence of petals? Free stamens? Staminal thecae divergent at the base? Presence of staminodes?). It would be informative and helpful to have (diagnostic) morphological characterization of this poorly known subfamily in Brazil.

Also, I have found several inconsistencies between main text and table in the manuscript regarding important morphologic aspects (including diagnostic characters) as well as main text and figure legends (e.g., classification/nomenclature of trichome types). Please see additional comments on attached files.

The IUCN conservation status asssessment was not correct. The Least Concern category applies to widespread and abundant taxa that does not qualify for Criticallly Endangered, Endangered, Vulnerable or Near Threantened. Altough authors wrote the focal taxon “spans throughout Amazon basin”, their map and specimens examined do show basically four locations sensu IUCN (2012: p. 13) based on seven collections. I think C. mennegae would qualify for VU B2ab(ii,iii).

Also, I do consider that (second-step) typification of Asterophorum is necessary to meet requirements of ICN (Turland et al., 2018). But I did not get access to the protologue and related publications. Please see comments in attached files. Authors argue strongly in favor of making available digital both literature and herbarium specimens because. I could not get literature authors stated are difficult to obtain and so I have appreciated if they had made Kubitzki (1995) and Secco (2000) available to referees to facilitate reviewing the mss.


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: PRELIMINARY PLACEMENT AND NEW RECORDS OF AN OVERLOOKED AMAZONIAN TREE, CHRISTIANA MENNEGAE (MALVACEAE)
Review round: 4
Reviewer: 1

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: VIRUS-DERIVED SEQUENCES FROM THE TRANSCRIPTOMES OF TWO SNAIL VECTORS OF SCHISTOSOMIASIS, BIOMPHALARIA PFEIFFERI AND BULINUS GLOBOSUS FROM KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study describes viruses detected in two biomedically relevant snail species known to vector human-infecting schistosomes that cause debilitating diseases affecting millions of people. Detected viruses were dominated by positive-sense single-stranded RNA (+ssRNA) viruses and represent a diversity of viruses potentially infecting the snails themselves and other organisms associated with the snail holobiont. The viruses were detected through transcriptome analysis and authors confirmed the presence of some of the viruses in snail samples through PCR assays. Authors’ conclusions were not overblown and the generated data adds to the increasing viral diversity reported from invertebrates. The study provides information regarding viruses that could be further explored for biocontrol strategies of snails harboring and transmitting parasitic schistosomes.  Is also important that we gain a better understanding of the microbiome of these snails and how it might affect schistosome infection/transmission. This study provides important baseline information regarding viruses associated with snail vectors. See general comments for the author for minor comments regarding basic reporting.
Experimental design: No major concerns, see general comments for the author
Validity of the findings: No major concerns, see general comments for the author
Additional comments: General comment:
Authors need to clarify/highlight that they used the same samples used for another study investigating snail responses to schistosome infection (Buddenborg et al 2017). Therefore, the sample set includes control snails (uninfected) and snails representing various infection treatments. This could be done briefly and authors should discuss if there were any trends (or not) in virus positive samples. For example, were viruses detected in both infected and uninfected snails?

Specific comments:
Introduction:
Line 57. Are there specific examples of viruses used as biocontrol agents of snails or is this a general comment/assumption? Please clarify and refer to previous work (either in snails or other systems).
Lines 64-66. Note that this is a general trend for eukaryotes as a whole given that +ssRNA viruses dominate the eukaryotic virome.
Line 77. Did the 1,300 transcripts represent +ssRNA viruses?

Methods:
Lines 89 – 94. Authors need to specify here that the numbers in parenthesis refer to sample numbers shown in Table 1.
Line 121. What was the quality score threshold used?
Line 124. This is where the reader would benefit from some background about the original study since suddenly authors refer to triplicate snails (triplicate of what?). In the previous paragraph authors stated that there were 3 sequencing replicates per sample but that is not the same as triplicate snails.
Lines 131-132. Authors need to specify thresholds used. For example, what was the minimum contig coverage to count a sequence as present (e.g., half the contig needed to be covered?) and what was the minimum sequence identity needed to map a given read? This is important because if most reads are only mapping to a given region they might represent related viruses that have a conserved region but are not the same viral species
Line 136. Authors need to specify what is in the local viral protein database (e.g., viral protein sequences from RefSeq? If so, which release?)
Line 137. Which version of the NCBI nr database was used?
Line 145. cDNA was synthesized using random hexamers?
Lines 147-149. Specify fragment sizes considered short (<1 kb?) or long (>1kb?).

Results
Lines 171 – 173. Given that EVEs are not only retroviral in nature, how did authors check for EVEs? Did they search snail genome sequences (if available) for detected viruses? Or did they simply eliminate all retroviruses from their data?
Lines 173 – 174. Authors should consider highlighting the 5 near-complete genomes on Table 2. Currently, is hard to figure out the 5 genomes until you get to the last footnote
Line 207. Refer/rephrase to ‘genome structure’ or ‘genome organization’ here
Line 242. Authors should consider a better way of highlighting the contigs of interest in Figure 3. Currently the 8 contigs do not stand out with the bold font alone.
Line 251. Were authors expecting DNA viruses in their data?

Authors should consider switching the order of the ‘confirmation of viral sequences’ and ‘viral sequences discovered from the B. globosus transcriptome’ sections. Currently, the former section discusses PCR for BG viruses that have not been discussed until you get to the next section.

Line 274. Do authors think this outcome simply reflects sampling effort? (only 3 BuG samples)
Line 281. Is BuGV2 not listed in Figure 1 because is not a near-complete genome? This is a bit confusing since it is shown on Figure 2 as one continuous contig. Should ORF1 of BuGV2 in figure 2 be partial (somehow showing an incomplete ORF) assuming the schematic is showing the 5,278 bp contig? I noticed this is stated in line 296 but it should be shown on the figure.
Line 286-296. Did authors attempt to bridge the gap between the two contigs through PCR? Seems like they predicted a 1500nt gap, which in theory should be easily amplified (Fig 5). This would support that both contigs represent the same virus. I’m not suggesting that authors do more lab work, but it would be informative to know if attempts were made to bridge the gap.
Lines 293 – 294. Based on the genome of structure of?
Lines 325-340. I believe trees shown in figures 8 and 9 are more informative than trees shown in figures 6 and 7, which seem redundant and less reliable (less information for tree building). Authors should consider eliminating current trees shown on figures 6 and 7.
Figures 8 and 9. Why authors did not distinguish between diatom (known host) and marine viruses (unknown host) on the trees?
Line 360. Do authors mean insect Nora virus?
Lines 381 – 382. How did authors come to this conclusion? The snails were sampled from freshwater. Moreover, most virome studies have been done in marine systems and there is not much data from freshwater environments. The snail viral sequences are most closely related to marine viruses, which may have to do with a database bias, and it does not mean that the identified viruses are marine (especially when considering the limited amino acid level sequence similarities).

Discussion:
Line 394. This sentence needs more information. These viruses were predicted to replicate in the snail based on their phylogenetic association with?
Lines 395 – 415. These paragraphs seem redundant with the last section of the discussion. Therefore, authors should consider merging this text with the last section.
Line 422-426. Note that Koonin et al 2008 classified iflaviruses (alongside discistroviruses and marnaviruses) under Clade 1 (which is composed of 3 subclades) based on RdRp phylogeny. Please confirm and edit accordingly.
Lines 431-437. This section reads more like results. What is the importance/function of the Rh_V and  CrPV domain (if known)? Authors mention BGV2 and BuGV2 represent a second type of dicistro-like virus but these viruses seem to have a different genome organization (Figure 2)
Line 432. ‘show no similarities to other viruses’ through BLAST?
Line 435. Rephrase to ‘genome structure of BGV3’
Line 437. Why highlight Aphis glycines virus 1 but not the ‘other insect viruses’? Is the main point here that BGV2, BGV3 and BuGV2 are most similar to insect viruses?
Line 439-454. How do these findings compare to analysis/phylogeny done by Koonin et al 2008, where all these groups were classified under the same clade?
Line 466-467. Could this be due to recombination?
Lines 474-477. Sounds promising!


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: VIRUS-DERIVED SEQUENCES FROM THE TRANSCRIPTOMES OF TWO SNAIL VECTORS OF SCHISTOSOMIASIS, BIOMPHALARIA PFEIFFERI AND BULINUS GLOBOSUS FROM KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The report by Liu S. et al 'Virus-derived sequences...Kenya" is written well, the article is clear, and its scientific English is informative and clear in its meaning and intent.

2. Generally, the report is referenced appropriately. However, the inclusion of the citation of Dheilly NM, Martínez Martínez J, Rosario K, Brindley PJ, Fichorova RN, Kaye JZ, Kohl KD, Knoll LJ, Lukeš J, Perkins SL, Poulin R, Schriml L, Thompson LR. Parasite microbiome project: Grand challenges PLoS Pathog. 2019 Oct 10;15(10):e1008028. doi: 10.1371/journal.ppat.1008028 is recommended, along with comments on the information in Dheilly et al 2019 and the authors' current findings and Discussion.

3. Professional article structure, with generally sufficient field background and context provided. Raw data evidently shared via BioProject ID PRJNA383396.

4 The report is self-contained unit with results relevant to the hypothesis that snail intermediate hosts of schistosomes are host to snail specific viruses and with associated environmental viromes.
Experimental design: Experimental designs and analysis of findings look adequate and appropriate.
Validity of the findings: Novel viruses identified in Biomphalaria pfefferi and Bulinus globosus.

Conclusions stated well.
Additional comments: Suggestions to enhance the report.

1. However, the inclusion of the citation of Dheilly NM, Martínez Martínez J, Rosario K, Brindley PJ, Fichorova RN, Kaye JZ, Kohl KD, Knoll LJ, Lukeš J, Perkins SL, Poulin R, Schriml L, Thompson LR. Parasite microbiome project: Grand challenges PLoS Pathog. 2019 Oct 10;15(10):e1008028. doi: 10.1371/journal.ppat.1008028 is recommended, along with comments on the information in Dheilly et al 2019 and the authors' current findings and Discussion.

2. L172. Provide the EVE sequences in the Supporting information as a Supplementary Table.


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: VIRUS-DERIVED SEQUENCES FROM THE TRANSCRIPTOMES OF TWO SNAIL VECTORS OF SCHISTOSOMIASIS, BIOMPHALARIA PFEIFFERI AND BULINUS GLOBOSUS FROM KENYA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The MS is well written, clear and unambiguous.

References are sufficient (although in general, many references of non primary results are somewhat old and could be updated). Snail virus references are scarce because gastropod virology literature is rare. In this context the results are novel and relevant given the medical importance of these specific snails.

The article is well structured, I provide below some suggestions to improve figures. The raw data has been shared.
Experimental design: The conducted research is within the aims and scope of the journal. The experimental design is well defined, research questions and knowledge gaps are addressed rigorously using previously validated methods, which are described in sufficient detail. I provide a few comments below to improve the M&M section.
Validity of the findings: all underlying data has been provided. No statistical analyses are included (not aplicable), conclusions are well stated.
Additional comments: Liu et al present an interesting work and valuable findings from the transcriptomes of two snail vectors of schistosomiasis, Biomphalaria pfeifferi and Bulinus globosus from Kenya. Authors have re-assessed previously reported RNAseq datasets in order to identify their RNA virus component.  To this end, authors employed a pipeline for virus discovery, which they have previously reported and validated on NGS data from insects. They provide virus like sequences, a few coding complete and several partial sequences, which appear to correspond to novel virus species associated to these snails. Given that the authors provide phylogenetic analysis, where some of these viruses cluster with four Biomphalaria glabrata viruses reported previously, there is plausible indirect evidence that these virus sequences could correspond to putative bona fide viruses of snails.

 In addition, authors identified viral sequences, which they describe as being from the entire snail holobiont, including symbionts, ingested material and organisms passively associated with the snails.

Moreover, authors have deposited the virus sequences in NCBI GenBank and raw data at NCBI SRA, reinforcing availability of reported materials, transparency, and reproducibility or their results.

The manuscript is well written and presented. I have only some suggestions for the authors and a few questions for clarification.

Main concerns:
-This reviewer feels that with little effort authors might be able to sort out many of the partial virus sequences described. I am not suggesting Sanger sequencing the gaps to complete genomes, which would be optimal but requires further wet lab. Just some additional work on the available sequences and raw data. There is a lot of redundancy and some overlapping in the partial sequences reported, which as is, do not help much in presenting a proper glimpse of these snail virome. For instance: “44 contigs of “at least” 8 viruses” as a back of the envelope calculation is not very helpful to describe virus diversity.
As an example, I provide attached to this review a nearly complete genome with a few Ns (ca. 9.25 kb) of a hypo-like virus, which was crafted just arranging BPVS8, BPVS9, BPVS45, BPSV48, BPVS49, BPSV50 and a few contigs of your reported TSA (GFMW).

- small RNA viruses (SRA): Authors keep refereeing to the detected ca. 8kb genome viruses as “small”, however, to my knowledge that would be the average size for a RNA virus genome (given the great abundance of picornavirales members linked to invertebrates, or plant potyviruses). Unless you find some literature to contrast this I usually consider small RNA viruses members of mitovirus (ca. 2.8 kb), other narnaviruses or amalgaviruses (ca. 3.4kb), for instance, and large genome RNA viruses would be nidoviruses (ca. 13-41 kb).

-many detected viruses in the snail dataset appear to cluster close to diatoms or marine organisms of unknown hosts. One obvious reason could be the very few snails viruses available in the literature redounding in viral dark matter and a general lack of “snail viruses clades” which is in line with the very low sequence identity of the detected viruses and their closest hits (ca. 30% id). Nevertheless, in a context where most detected viruses closest counterparts correspond to non-snail viruses, at least for those, the authors could consider the possibility of shifting their proposed names to a more conservative one which won’t be affected if further studies confirm that their proper host is a snail holobiont species and not the snail itself. This reviewer suggest using for instance, “Biomphalaria pfeifferi associated virus 1” or something in that line until more direct evidence confirms that these viruses do infect the snail.

It is worth mention that most of the reported nearly complete RNA viruses, as suggested by the authors, appear not to be infecting the snails (BPV1, BPV3, BPV4 and BuGV1).

-I wonder if a very simple assessment of the primary sequence of these novel viruses could provide some clues to host assignment. For instance if you check codon usage of BPV1-BPV4 would you get something similar to gastropods codon usage??

-Here you describe many viruses probably linked to the snail holobiome. Could you try a rapid examination of the putative holobiome using the very same data? For instance running a quick rRNA assessment of each of your RNAseq libraries may show some common and rare symbionts (contaminants?) co-purified in your samples.

-consider discussing the putative virus RNA component as a share of the RNA data and some insights about potential virus abundance in the samples. E.g., what percentage of total reads correspond to virus derived reads in each sample? Which virus appear to be present at higher RNA levels in the studied samples? Etc.

Minor comments

-Introduction: Please consider defining trematodes and any literature available regarding schistosoma infecting viruses.

-Please make sure that the snail virus sequences are released by GenBank. Consider posting them as supplementary data of this article in case the seqs are not available at NCBI promptly.

-As indicated in Table 1 the RNAseq datasets used here from Buddenborg et al. 2017 correspond not only to different organisms but also to different treatments/conditions. Please consider discussing the potential effect of these treatments to the RNA levels of some of the detected viruses in terms of raw mapping differences.

Minor editorial comments (please do not hesitate to ignore all these)

Abstract:
-line 30: “using viral RNA”… add using predicted viral RNA…

Mat&Met:

-line 128: “Virus-derived contigs that encoded >800 aa…” (many (most) of the reported partial virus sequences encode proteins well below that size threshold, please clarify which sequences and using which criteria were selected for analyses and abundance.)

-131-132: “MAQ v0.5.0 (http://maq.sourceforge.net/maq-man.shtml) was used for mapping of reads to target assembled sequences.” Please provide parameters/commands used to implement MAQ.

-line 136: “against a local viral protein database” please define or provide this database in order for results to be reproducible for readers.

-line137: “Contigs that hit viral proteins were extracted” using which identity, and/or e-value and/or coverage threshold?

-line 139: how did you manually check that? Using which tools? Maybe bioedit mentioned below?

-line 142-151: could you clarify if RT-PCR confirmation of viral presence was conducted in the very same RNA which was subjected to HTS sequencing or to independent RNA extractions?

-line 154-155: “conserved domains ….were identified by blast”, this reviewer did not have access to the fully annotated sequences so could not contrast your predictions but may I suggest authors to check their predictions with additional (and perhaps more reliable) tools such as HHpred https://toolkit.tuebingen.mpg.de/tools/hhpred or NCBI CDD search https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ?

-line 159: “The domain sequence was aligned using MAFFT” please provide parameters (algorithm, scoring matrix, etc).

Results

-line 172: “endogenous viral element (EVE) sequences were not included in this analysis” how did you detect (and exclude) potential EVEs?

-line 187, line 208 and elsewhere: “ORF1 of BPV1 showed 30% protein…” please here and elsewhere clarify that the reported identity derives of comparing the ORF1 encoded predicted (poly)proteins.

-line 192-196 and elsewhere: could the authors provide some support of the predicted domains such as e-value?

-line 203 and elsewhere: “with genomes of 8,491, 8,255 and 8,253 nt” without RACE or other experimental confirmation it is not possible to predict the exact genome size only by HTS data assembly.

-Please provide sequence identity values of BPV1-BPV4 nt and predicted domains among them, which supports that the sequences correspond to distinct virus species. Also provide ICTV species demarcation criteria for picornavirales in general.

-line 360: “one insect norovirus” replace norovirus with Nora virus

-line 364: “could potentially be an ancestral virus” please rephrase, a contemporary virus should not be described as ancestral.

Discussion

-Line 388: “(-)ssRNA, dsRNA and DNA genomes” the (-)ssRNA and DNA hits on table 2 appear to be more contamination thank snail viruses.

-line 393  “predicted to replicate” I would rephrase, to my understanding there are no direct evidence of replication in this work for any virus.

-Line 456: “Not all snail-derived viruses are snail viruses” please consider discussing contamination as a potential source of putative RNA viruses.

-Table S2: row 59 The 893 aa predicted product of BuG_virus2 returns a significant hit (e-value = 1e-71) with the structural protein of Biomphalaria virus 1 (NC_032804.1).

Figure comments

Figure 1. blue (ORF1) and green (ORF2) is missing in the legend. Please provide coordinates of orfs and/or domains. You may add some symbol indicating a Poly(A) at 3’ end termini. Perhaps figure 1 and 2 could be integrated into one with a and b panels.

Figure 3. There aren´t much info here. maybe move this one to supplementary material?

Figure 6 and 7. Legends appear to be incomplete, which viruses are those? What’s the scale bar representing here? What are the numbers on the nodes?. In addition, not sure what is the general idea to generate these two trees without all available viruses as in figure 8 and 9. If the idea here is to highlight each of those corresponds to a different species, then genetic distances (nt and aa) as a plot would be more useful.

Figure 8 and 9. Please add an asterisk to highlight each of the viruses detected in this study. Also both trees are missing scale bars. The node labels are illegible. How /was this tree rooted? How did you selected which virus sequences to incorporate in these trees?


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: FIGHTING SARS-COV-2 WITH GREEN SEAWEED ULVA SP. EXTRACT: EXTRACTION PROTOCOL PREDETERMINES CRUDE ULVAN EXTRACT ANTI-SARS-COV-2 INHIBITION PROPERTIES IN IN VITRO VERO-E6 CELLS ASSAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript "Fighting SARS-CoV-2 with green seaweed Ulva sp. extract: extraction protocol predetermines crude ulvan extract anti SARS-CoV-2 inhibition properties in vitro Vero-E6 cells assay" addresses a relevant and up-to-date topic. I am of the opinion that it should be accepted for publication after the introduction of the corrections suggested below (see additional comments).
Experimental design: This manuscript is well structured, well-founded, and the results are somewhat relevant
Validity of the findings: This manuscript is well structured, well-founded, and the results are somewhat relevant
Additional comments: Corrections needed:

line 55 - Remove unnecessary dot and spaces

lines 76 and 77 - Standardize the letters of the units of measure (use "L" and not "l")

line 109 - ... Ulva sp. ("sp." is not in italics)

line 111 - ... and Ulva lactuca (formerly Ulva fasciata)

line 121 - ... Ulva sp. ("sp." is not in italics)

line 125 - ... was extracted for 2 h with ...

line 129 - ... dialysis membranes for 24 h, and ...

line 135 - ...was conducted for 2 h

line 164 - (43.6, 90, and 136.4°) with  ...

line 207 - ...humidity for 72 h, ...

line 220 - ... extract between 5 mg·mL–1 to

line 227 - ... was evaluated by adding 25 µL aliquot ... to 5 µL of ulvan ...

line 229 - ... under 90% humidity for 72 h, ...

line 259 - Ulva sp. ("sp." is not in italics)

line 260 - Ulva sp. ("sp." is not in italics)

line 2777 - after 72 h of ...

line 323 - ... which was 88% higher ...

line 356 - ... from Ulva armoricana collected ... ("armoricana" in italics)

line 397/398 - ... g·moL-1

line 431 - ... Ulva sp. ("sp." is not in italics)

lines 493/494 - ... mg·mL-1, ...

line 511 - ... Caulerpa (in italics) sp. (not in italics)

line 519 - ... Ulva sp. ("sp." is not in italics)

line 531 - ... for a thorough (remove unnecessary space)


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: FIGHTING SARS-COV-2 WITH GREEN SEAWEED ULVA SP. EXTRACT: EXTRACTION PROTOCOL PREDETERMINES CRUDE ULVAN EXTRACT ANTI-SARS-COV-2 INHIBITION PROPERTIES IN IN VITRO VERO-E6 CELLS ASSAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscritp is generally well written and understandable.There are only a few very minor points which should be improved:
Line 49: "-" is missing between COVID and 19
Line 104: "-" is too much between "under" and "regulated"
Line 130: Could you please give the respective value in mbar instead of mmHg?
Line 141: Could you please give the resp. value in x g instead of rpm (as rpm can depend on the device used).
Line 272: Should be "assay" instead of "essay"
Experimental design: no comment
Validity of the findings: No comment
Additional comments: Overall, the manuscript provides a detailed analysis and characterization of the crude extracts and I think it's no problem that the antiviral activity of the two different extracts is absent or not very strong, as this issue has been also sufficiently discussed. In my opinion, it would have been more interesting to the readers to include results of a bioassay-guided fractionation as currently, an antiviral potential of the AOx extract is still a hypothesis. On the other hand, this would have increased the size of the manuscript tremendously.

My major concern, which is not only relevant to this study, but generally to the antiviral activities of SPSS: Is there any source of literlature which suggests that such macromolecules can be effectively used as therapeutics? What about their bioavailability? In particular with respect to COVID-19, how would you assess their therapeutic potential? How should they be delivered to the site of infection to be able to exert their activity against the virus? As there are many reports of in vitro antiviral activites, it would be interesting to discuss this point in more detail.


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: CAN HARBOR SEALS (PHOCA VITULINA) DISCRIMINATE FAMILIAR CONSPECIFIC CALLS AFTER LONG PERIODS OF SEPARATION?
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-conducted study examining the ability of harbour seals to recognise vocalisations of pups with which they had been raised versus the vocalisations of unfamiliar pups. They were tested on re-capture some time after release and hence after exposure to the vocalizations of other pups. Probably due to the low sample size (n=3) the results were not significant.

The English expression is very good.
Experimental design: Realising the low probability of re-capture of the same individuals for which recorded vocalisations were available, I recommend publication of this study but suggest that the text be shortened to some degree. This should be possible because there is repetition at different places in the manuscript and it would allow some more figures to be added, as suggested below.
Some important details are missing from the Methods (see below).
Validity of the findings: 45-47: This reference to memory span and speech rate in humans is not particularly relevant and should be deleted.

57: Here it is suggested that long-term memory recognition could be ‘an evolutionary by-product of strong selective pressures’. This doesn’t make much sense unless you specify what those selective pressures may be.

124-127: Say exactly how old each seal was at the time of re-capture and also at the time of testing the responses to playback of the vocalisations. This should be in the text as well as in the Table.

189-220: It would be helpful to include some images from the videotapes showing the behaviour scored. Also, it would be valuable to present some sound spectrograms of the calls presented in the playbacks.

191-193: This description of the visual fields is not very clear. Perhaps adding diagrams of the binocular and monocular visual fields in the horizontal and vertical planes would help the reader.

208-209: Because I think that seals have a great deal of mobility of the head, the plane of the >900 head turning should be stated.

Footnote to Figures 3 and 5: Say exactly what is plotted (e.g. medians and type of distribution).

Error of grammar
49: Change ‘showed’ to ‘have shown’.
Additional comments: Please see above.


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: CAN HARBOR SEALS (PHOCA VITULINA) DISCRIMINATE FAMILIAR CONSPECIFIC CALLS AFTER LONG PERIODS OF SEPARATION?
Review round: 1
Reviewer: 2

Output:
Basic reporting: Varola et al. tested the hypothesis that harbor seal pups can recognize familiar from unfamiliar conspecific sounds after a long separation.

The writing is clear, the topic is well introduced.
Experimental design: Varola et al. took advantage of having 3 seals admitted for rehabilitation twice in 6 months (first as pups and then as weaners). The seals were exposed to previously recorded sounds of familiar and unfamiliar conspecifics during the second rehabilitation period.

The experimental question is well-defined and interesting.
Validity of the findings: The behavioural responses measured (lateralization patterns, number of looks, latency before looking and duration of looks) yield no sign of recognition between familiar and unfamiliar sounds. Yet, the authors cautiously argue that the data visualization could support the idea that seals differentiate familiar and unfamiliar conspecifics. However, together with the low sample size, several differences between the tested animals (sex, separation duration, rearing conditions, etc.) are present and make the result interpretation very difficult. Still, the authors reported their results with complete transparency and discussed them adequately.
Additional comments: I do not have concerns with this article and think it is worth being published as it is. I have a few comments and text issues (see later) that the authors can consider.

While the authors clearly state their hypothesis about the head turns direction for familiar calls (right turns expected), their assumptions about the other behavioural measurements are pretty unclear to me. For example, should we expect lower or higher latency to respond to familiar compared to unfamiliar calls? Ultimately, it does not make much of a difference. What is important here is whether there are behavioural differences between familiar and unfamiliar calls, but discussing your hypotheses for all behaviours measured beforehand (if you had some) could be entertaining for the readers.

The principal component analysis was new to me. Can this approach be problematic when combining behaviours for which you expect opposite direction? For example: for an animal that reacts to a call, I would expect a higher number of head turns and lower latency to respond than an animal that is not responding. Isn’t there a higher risk of information loss in using a PCA in this case?

About the lateralization pattern: Although the authors did not find any left-right correlations with the behavioural responses, it could be interesting to represent the lateralization patterns for all the recorded behaviours graphically.

Small issues:
1.	In the last paragraph of the section ‘Study site and animals’ (l.136 to l.141), the age of the animals is described for seal-1 and seal-3 as stated but not for seal-2. The authors might want to add its age in parenthesis.
2.	L.179: there might be something wrong with the reference (see ‘t)


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written, topic is relevant and important to pursue.
Experimental design: Overall, I like how the study was conducted. However, just because the BOLD databases contain 200k sequences does NOT mean the COI marker is appropriate for Antarctic biodiversity investigations. Most of the sequences in BOLD are not Antarctic...nay, the LARGE majority. Many studies are showing now that marker choice is taxonomic group-dependent and studies recommend using multiple markers (COI, 16S, cytb, etc.) to get a better overview of total biodiversity, which is the goal of this study.

One other note is that I very much like the authors comments on field and laboratory blanks/negative controls.

Why use only half the filter? Studies have shown that the DNA is heterogenous....what if youre missing the targets/more interesting species on the other side of the filter?
Validity of the findings: Valid findings. See design for comments on improving/expanding the reach of these results.

One comment: the finding of 77-80% similarity to "cyclopoida sp." is extremely variable. That difference could be the difference between cerolids and sea spiders. This circles back to my "additional markers would be of use" comment from above.
Additional comments: Overall a nice manuscript that shows eDNA should be expanded in Antarctic waters.


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see general comments. The main questions and main focus are the article are not clearly presented. They are several competing "main" points/aims.
Experimental design: Please see general comments. Experimental design is flawed. Using only one genetic marker is not up to current standards. There is also a lack of justification for use of the marker.
Validity of the findings: See General comments - Some of the experimental issues make the findings questioned
Additional comments: This manuscript uses the COI marker to explore eDNA diversity in water and sediment Vestfold Hills in the Eastern Antarctic. There are several positive aspects this study (e.g. looking at both sediment and and water column, considering hard substrate). However, there are many issues that are just not up to current standards and the how the central ideas and focus of the paper are presented can be confusing to the reader. While the topic is of broad interest, the execution of this work renders it problematic for publication. Reasons for this rather negative assessment are below.

Marker choice —One marker is not sufficient by current standards. This is a major issue and second (or ideally at least 2 more) should be used prior to publication.  All markers and primers have their issues but COI is particularly problematic for metabarcoding (which is done here) versus eDNA detection analyses for particular species. Which COI primers are used is never stated (making the work non-repeatable). This is critical in that COI primers are notorious for taxonomic bias. Also many are degenerate requiring touchdown PCR as done here. Agreed that low annealing is a problem but touchdown PCR also causes issues. Taxonomic bias is likely the reason several abundant arthropod taxa are not detected. However, that possibly of primer bias is not discussed. Moreover very little justification is provided for use of COI. At the end, the manuscript the Leray & Knowlton 2016 paper is used to argue that COI is preferred because it has better resolving ability than 18S. This statement is simply not as correct and Leray & Knowlton suggest. They are confused on the issues. Most of the 18S rDNA regions used for metabarcoding span variable regions (V1-2, V4, V9). In contracts, 18S was used for years for higher level analyses, but in those higher levels analyses these variable regions where usually excluded because they were too variable. The bottom line is that paper has to provide information and justification on the primers used, and importantly more than one marker should be employed.
Read number and sequencing depth. Only 5.45 million reads total were used. Why this is the case is not clear as even a single Miseq run produces more than 44 million paired end reads. The manuscript uses ratification to standardized read number which is good, but the number of 1000 is very low and it is not clear why.  The 5.45 million is not bad by the cutting the rarifaction curve off at 1000 (or more to the point the inflection point being at about 100 reads) is just not consistent with what others see in sediment samples (see papers from Creer lab, Halanych lab, Bik lab, etc.)
Focus of the paper is not clear. In the abstract the main aim is to develop methods, but in the introduction the aim of the paper is to develop methods, characterize Antarctic samples (line 74), and address 3 questions (line 96 - note that the first question is 3 questions by itself - so it is really 6 questions) and then the Discussion leads with main finding about the choice of sample type matters. To be direct, this manuscript does not develop new methods. Everything here has been done before. Also the results that sample type matters (sediment versus water) is not novel.  The  manuscript needs to find a coherent and novel story. Consider focusing on what taxa were found in Eastern Antarctica or comparing water and sediment (but see next point).
Quantitative statements based on metabarcoding is challenging. How can a sample size from a Niskin bottle be compared to an amount of sediment? Throughout the manuscript there are statements about more OTUs found in sediment. That is true but some caveats should be clearly stated next to such statements. Also how sediment is processed is important. The manuscript focuses on small amounts of sediment similar to what is done with bacterial metabarcoding but see Brannock and Halanych 2015 (Marine Genomics 23:67).

Other points include:

Line 39 - Clitellates are derived polychaete - that is the node on the tree of life that defines polychaete is the same as the one that defines annelids. Thus the 10% comment is likely not correct.

Line 191 - creating a phylogenetic tree using Geneious -This is not a proper methodological description. Also a tree across metazoans with 300bp of COI.  Not sure why this adds anything.

There is no discussion of DNA from live taxa versus historic DNA (mainly a sediment issue).

For the water samples one of the primary environmental variables is likely to current regimes, especially given the narrow network of channels and fjords in the Vestfold Hills region.

There is no comment on the non-metazoan taxa which compose roughly half the sample. The fact that so many sequences are non-metazoan raised further questions about the primers.

Main discussion point does not track main questions. Second sentence a question

Line 341 Water or sediment is hardly a “niche;” it is a habitat.


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written. A couple of recent relevant references could be cited. The raw data is shared in the SRA repository, but it is missing some crucial metadata (sample tags) which prevent the reader from reproducing the pipeline. This metadata could be added as supplementary material.
Experimental design: The research questions are well defined, But some methods could be described in higher detail. The bioinformatic procedures could be improved by performing a minimal abundance filtering step.
Validity of the findings: The findings are generally valid and the implications of the results are well discussed. There is no speculation.
Additional comments: This manuscript by Clarke et al. is an interesting contribution to our scarce knowledge of molecular biodiversity of Antarctic benthic ecosystems. However, I think that there are a few significant issues that need to be addressed before this manuscript is ready to be publishable in PeerJ.
- First, the materials and methods are not completely clear and some details should be clarified (see below).
- Second, the data availability can be improved. Although the raw reads datasets (2x250 bp) have been uploaded to the NCBI SRA repository, the information in this BioProject is not enough to reproduce the bioinformatic procedures. The authors have used 6-bp sample tags, but the information of these tags can not be found anywhere. I suggest that the authors should upload a table of the tags used for every sample as supplementary material to this paper. Otherwise, all the data shared in the SRA repository would be mosly useless. Moreover, the final table of zOTUs (after denoising) and the final curated table of species (after taxonomic clustering) should also be uploaded and shared as supplementary material to this paper.
- Third, I am a little concerned that the authors did not mention to use any filtering step based on minimal read abundance by sample in their bioinformatic pipeline. But then they are performing calculations (beta diversity) based on presence/absence. For this calculations to be robust, it is crucial to establish a minimal read abundance filter to consider a true presence. Otherwise, false positives are very easily spread along the dataset (for example, due to tag jumping), which can significantly interfere with presence/absence metrics. I would be more comfortable if a minimal read abundance filtering had been used before the ecological analyses. This minimal abundance filtering should be ideally done in a sample-by-sample basis, in order to remove low-frequency MOTUs in every sample, before calculating the rarefied species table.

Other suggestions and issues that should be addressed:

INTRODUCTION
L81: While water eDNA can -potentially- be used to study both soft- and hard-bottom communities, two recent studies (which could be cited here) have highlighted the limitations of this approach. (1) Antich et al. (2020, doi:10.1111/mec.15641) proved that less than 5% of hard-bottom benthic organisms from Mediterranean shallow communities could be detected from water eDNA, even when water was taken very closely to the bottom. (2) Brandt et al. (2021 doi:10.1038/s41598-021-86396-8) showed that eDNA from deep-sea water did not either reflect the composition of the benthos (3-8% benthic MOTUs detected in water), even when high volumes of water were filtered. Both papers conclude that sampling water is not a useful alternative for characterising benthic diversity.

MATERIALS AND METHODS
L141: Change "(approximately 300 bp)" to "(313 bp for most metazoan species)".

L144. The sequences of the metabarcoding primers used for the amplifications should be written here explicitly. My guess is that the original Leray et al. (2013) primers were used, with inosines in the reverse jgHCO2198 primer and with no inosines in the forward mlCOIintF primer. But it is important to state this detail clearly, for future reproducibility.

L166: Are the authors sure that they used a V2 Nano kit? This kit yields barely a maximum of 1 million raw reads. It is impossible to get 8 millions of paired-end raw reads (as deposited in BioProject PRJNA720767) or 5.45 million paired-end reads (after filtering and QC) using a Nano kit. I think that they used either a V2 full kit (maximum yield ~15 million raw reads) or several Nano kits (less probably, since this is not economically optimal). Please double-check this.

L180-205: The authors are using a hybrid system to get their diversity matrices, which is not totally clear to me, and should be further clarified here. First, they generate the zOTU table with UNOISE3, and this is clear. Then, I understand that taxonomic clustering is used to delimite species and generate a table at the species level. This is a reasonable way to go, as most ecological analyses with COI should be done at the species (cluster) level, rather than at the zOTU level (Antich et al. BMC Bioinformatics, 2021). But the procedure to perform the taxonomic clustering should be explained more clearly. Authors mention that they are using BLASTN against the NCBI, then they use MEGAN to assign taxonomy. Why using BLASTN then? Was BLASTN used to filter non-metazoans sequences, and then MEGAN used only to assign the remaining metazoan sequences? This should be clarified. Finally they generate "a phylogenetic tree" with Geneious for further clustering other sequences at >97% identity. They should explicitly mention which phylogenetic algorithm for tree generation was used, since Geneious has many different possibilities. Moreover, It would be a good idea to include in the supplementary materials the raw table at the zOTU level and the final curated table at the species (taxonomic cluster) level. Also, a summary of the total number of zOTUs in the initial table and the total number of species-level clusters in the final table should be more clearly mentioned in the Results (currently there are two sentences that are contradictory). This would help to better understand the results of the non-orthodox taxonomy clustering procedure used.

L214: Change "nut" to "but".

L208-209: It is not clear to me is this "species richness" rarefaction and the following analyses was tested at the zOTU level or at the species cluster level. Which of the two biodiversity tables was used for these analyses? Only the 99 assigned Metazoan table? Or the whole zOTU table? Please specify.

L219-222: The same is true for the beta-diversity analyses. The results of both Jaccard and Bray-Curtis dissimilarities can be very different if they are performed at the zOTU level or at the "species" level. The ecological analyses for COI should be done using the species-level matrix, and not the zOTU matrix. I guess that the authors used this. But, please, specify.

RESULTS
L245: Between the 5.45 million raw reads and the 3950 zOTUs, it is also interesting to know how many unique sequences were there before denoising. I think this number should be mentioned here.

L245-246. "518 zOTUs" containing 2.16 million reads are assigned here to 99 metazoan taxa. However in L254-255, it is said that the 99 metazoan taxa contained "479 zOTUs" (and 2.16 million reads). These two sentences are contradictory and contribute to generate confusion. Please double-check the numbers.

L279 and L309-310: In my experience, using sqrt-transformed (or even fourth-root transformed) read abundance data can significantly improve the ordination of Bray-Curtis dissimilarities for COI metabarcoding data. Have you tried if you could get more logical ordinations of the replicates after any of these transformations? Just out of curiosity...

DISCUSSION:
L324: You can also cite Antich et al (2020) and Brandt et al. (2021) here. Both papers are very relevant for this issue.

FIGURES:
FIG5: The caption of Fig. 5 is incomplete. (A) is described but (B) is not.

Congratulations to the authors for a nicely written, highly interesting, paper.
Owen S. Wangensteen


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors note in their text that COI is the 'best' marker for use in this work. However, as reviewed previously, COI fails to detect many species and multiple marker studies are commonplace in the eDNA literature. Simply stating the authors believe COI is the best without testing is insufficient.
Experimental design: Single marker eDNA studies, particularly using COI as their only marker, is not sufficient to make any biodiversity conclusions.  See for example: https://www.mdpi.com/2073-4441/13/13/1767.  This is one of many papers out that discuss this. While in a different system, COI alone will not work as was noted in previous reviews. Other markers could be either added to the databases or simply used and considered as operational taxonomic units (OTUs).
Validity of the findings: With only a single marker, as previously reviewed, these findings are not meaningful, are speculative, and cannot be considered robust or sound.
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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The original manuscript received three reviews with some agreement on the strengths and weaknesses of the work.  There is considerable excitement for the topic.  Some of the comments made focused on design flaws in the analyses (e.g., one marker, read depth, rarefaction).   The revision was far more superficial than these comments deserved. There are limited wording changes to the manuscript to try and explain a particular view rather than address the underlying problem or presenting a fair presentation of the weaknesses of the approaches used.
Experimental design: see below
Validity of the findings: see below
Additional comments: The use of the single COI marker is still problematic and rather than be direct about some of the short comings, the manuscript tries to defend this weakness. For example, CO1 is defended because it is has been the most sequenced in terms of the number of taxa…agreed. However, that does not make it good enough to use by itself. The explanation of the 18S variable regions is more complex than just saying Leray and Knowleton say,  (more or less) “18S variable regions do not work”. Variable regions due underestimate diversity, but so does COI. That is one reason people use more than one marker. Also, the degree of underestimation is taxon specific for both markers, and for some taxa 18S variable regions work just fine. The explanation presented does not justify why COI alone is enough. As another example, the Yang et al paper is mentioned as showing that wet lab and bioinformatics can be used to limit OTU drop out. However, that paper employed a pool of only arthropod sequences with different primers. The limited drop out by Yang et al. is not comparable given the methods used here. Also, the over reliance on the Leray et al primers has not been adequately discussed. As a comparison point, Folmer et al 1994 primer were claimed to work over across animals, but that turned out not to be true.  There are about three or four other sets of COI primer papers that have made similar suggestions, none were correct. The manuscript should embrace and discuss the issues and challenges with this marker instead of trying to explain it away or ignore it altogether.

To be clear just because “we feel that COI is by far the best target” does not justify its use as the only marker.

The Leray et al and the Leray and Knowlton papers are generally very good, but they over interpret some issues which are be amplified here.

Most of the other suggestions were adequately addressed.


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript has been significantly improved with the additional changes made by the authors. I am now fully satisfied with this version, and I strongly recommend it to be published in PeerJ.
I would like to add that I strongly disagree with the views expressed by Reviewer 2, who is asking for the use of at least two markers, and questioning the validity of these findings only because they are exclusively based on COI. In my opinion, we can indeed extract highly valuable taxonomical and ecological information from metabarcoding data sets based exclusively on COI. We should never reject a paper based only on this fact. I fully agree with the authors' rebuttal. In my experience, in most cases, 18S metabarcoding provides only low-resolution data for metazoans (typically at the family or order level), which does not add much value to the information that you can retrieve from COI. 18S is probably more important when targeting whole Eukaryotic diversity patterns at coarse taxonomic resolution (e.g. groups of protists), but it is definitely not needed for metazoans at the species level. Moreover, samples from Antarctica are always precious, and any new information that we may retrieve from them, even from a single COI marker, is definitely worth publishing!
Again, congratulations to the authors.


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: ENVIRONMENTAL DNA METABARCODING FOR MONITORING METAZOAN BIODIVERSITY IN ANTARCTIC NEARSHORE ECOSYSTEMS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I am completely satisfied with this version of the manuscript. I think that the additional ecoPCR analyses performed by the authors are justifying the choice of metabarcoding primer. This additional information may be useful for other researchers to design metabarcoding projects in the future.
I am happy with the editor's decision of not rejecting this work based only on the false premise that using a single marker completely invalidates any retrieved information. The presented results are indeed novel, interesting, and unique. And they contribute to increase our scarce knowledge on molecular biodiversity of Antarctic marine ecosystems.
I strongly recommend the publication of this work in PeerJ.


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: The research question is relevant and well stated. As the authors argue, an Integrated Population Model (IPM) is a an appropriate tool for addressing the question (identifying mechanisms and drivers of population decline), not least because of the somewhat fragmentary nature of the available data. However, some aspects of the methods are not described in adequate detail, and some analytical choices can be questioned. Specifically:

1. The populations modeled are not clearly defined. It is not clear whether the study areas can be considered self-contained populations, or represent a sample of many other sites that also hold breeding snowy plovers. In either case, dispersal in to and out of the study sites is surely also possible, although this aspect is not included in the IPM. I think that a better description of the study sites, their importance for the species, and the arguments for including these specific sites is needed.
2. Some assumptions of the model are not explicitly mentioned. For instance, it appears from the very simple population model (l. 188-189) that all birds are assumed to breed when one year old. This assumption should be clearly stated and arguments provided. Generally, a bit more information about the study species and its life cycle should be provided. For instance, it is not necessarily clear to the reader that plover chicks are nidifugous, and that nest success thus equates hatching success, whereas post-hatching mortality must be estimated with capture-recapture methods.
3. The way detection probability is handled in the population surveys is inconsistent. In Texas and New Mexico, it appears that counts are taken as unbiased estimates of population size, i.e. detection probability is assumed to be 1.There is no information on what is actually counted in these surveys, e.g. whether all birds seen are included, regardless of distance to the observer? This point interacts with point 1 - defining the study area and population more clearly would be helpful. In this context, it is strange that  some counts are up to ~10 times as high as the estimated population size (Fig. 3). How is this possible - do counts include transient birds? In Oklahoma, detection probability is included in the model, although it is not at all clear how this parameter is estimated in years without distance sampling. Please provide more details.
Validity of the findings: All the underlying data are available as R data sets. However, this means that the only way to access these data is to run the provided R script. Unfortunately, this script is not well commented, and it is difficult to get an overview of the data. Therefore:

1. I strongly suggest that the authors provide an overview of the data included in the manuscript (e.g. as a table). How many surveys were carried out, and how many birds were counted? How many nests were followed? How many birds were marked (by age class), and how many recaptures and resightings were obtained? Without this information, it is near impossible to assess the work.
2. I think the conclusions are somewhat overstated, particularly regarding the environmental covariates. In most cases, the 80% credible intervals overlap zero, which in my view indicates that there is no evidence to support the importance of the covariate in question. Some sort of assessment of the importance of the covariates would be helpful. I'm not completely up to date about how this is best done in a Bayesian context, but some sort of comparison of the performance of models with and without (i.e with constant demographic parameters) covariates might be helpful. Also, I suggest plotting some of the identified relationships before claiming e.g. that you have shown 'strong positive effects' of the Palmer drought index (l. 361).
3. You say that 'reasonable precision of demographic estimates' was achieved (l. 407). I would dispute that this is the case for survival (Fig. 5), where the 80% credible intervals in many cases almost span the entire possible range (0-1). It would be interesting to see how much the CMR data actually contribute to the likelihood - would you get similar estimates without including these data?
Additional comments: Figs. 6-8: please explain exactly what is shown in the violin plots (median, interquartile range, or whatever).


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) The literature is well referenced and mostly relevant. However, reference to previous work also estimating demographic parameters using IPMs with limited data is missing. Most importantly, the paper titled “Use of Integrated Modeling to Enhance Estimates of Population Dynamics Obtained from Limited Data” from Schaub et al. 2007 is not cited but highly relevant. Other reference dealing with this issue: Davis et al. 2014 “An integrated modeling approach to estimating Gunnison sage-grouse population dynamics: combining index and demographic data”.

2) The raw data is supplied and I commend the authors for also providing their R script, which runs smoothly on the provided data. I think it would be very useful to provide a “readme” file of some sort that annotates/explains the raw data. Similarly, for the R script more annotations to explain the different steps and the IPM would be highly useful. For example, the formatting of the capture-recapture data (and the way it is modelled in the IPM) are not standard, so explanations would be particularly useful there. I would also suggest providing the script for the post modelling analysis (hierarchical partitioning analysis).

The paper is clearly written and well organised. Very nice and easy to read.

The figures are of exceptional quality.
Experimental design: 3) I definitely think that the research fills an identified knowledge gap (a better understanding of the ecological drivers of the population dynamics of the study species. However, I do not see how the study fills a knowledge gap notably implied in the title, that “Integrated population modelling improves the utility of partially aligned data” as this is already known (see work mentioned above). As far as I can see, the proposed model has no particular feature that allows it to deal with missing/partially aligned data (all Bayesian IPMs will automatically deal with missing data by treating it as a parameter and simulating values from the likelihood). If I am wrong and the model proposed does include novelty that allows dealing with partially aligned data, then it should be better emphasised.

Otherwise, I suggest that the authors either:
i) change the focus for the biological aspect of the study, that is, assessment of ecological factors influencing the demography of the study population and mention the missing data as a feature of the data.
ii) keep the focus on the use of partially aligned data, and in this case explicitly assess the gain/loss in parameters identifiability and precision in their estimation when adding/deleting years for the different types of data, using the real dataset as a case study. Then the authors could provide more precise recommendations regarding which and how much data to collect in priority.

The research questions are otherwise well defined, relevant & meaningful.

4) The investigation performed is overall rigorous and to a high technical standard. However a goodness of fit assessment of the sub models is crucially needed (the authors should perform and provide some posterior predictive checks).

The methods are described with sufficient detail &information to replicate (but see comment above regarding a more detailed annotation of the data and script).
Validity of the findings: 5) Conclusions are well stated and linked to original research question. That being said, I think that the confidence in the results seem often too strong given the high uncertainty in the estimates. Except from the effects of the PDSI and minimum temperature on clutch fate, there is always a non-negligible proportion of the posteriors that is below zero, suggesting statistically unclear effects. For example, given figure 8, I would not say that “The Palmer drought severity index had positive effects on … apparent survival”. I would recommend being more careful in the abstract, result section and discussion given this uncertainty. That being said I appreciated where the authors were careful (e.g. regarding the results on wind speed lines 388-389).

All underlying data have been provided; they seem sound as far as I can tell.
Additional comments: Line1: “improves” Although very likely, this was not explicitly investigated in the paper (compare with utility of non-integrated datasets (modelled independently)).

Lines 20-21: “we developed an IPM for partially aligned population and demographic data” it is not clear that/how this model is specifically developed for dealing with partially aligned data (not more than any other Bayesian IPM).

Line 34-37: I would specify that these effects are only clear on clutch fate.

Line 41: “Our modelling approach lays a foundation of allocating limited conservation resources…” I would rather say that it follows/confirm previous work on this (see above-mentioned references).

Line 87: perhaps use a transition word (e.g. “Additionally, “Finally”) to make it clearer that wind speed is newly introduced here.

Method section: it would be useful to provide information on sample size for the different datasets.

Line 143: was brood reduction assumed inexistent in the model? If so, is there some evidence that “partial nest mortality” is low in this species? It is a very common assumption to make and I think it is OK, it would just strengthen the validity of this assumption.

Line 188: State that demographic stochasticity was considered negligible, and that Immigration was considered absent.

Line 213: why “t-1” and not “t”? Typo?

Line 257: how is this method better/different than the more classically used LTRE contribution approach (Koons et al. 2016 and 2017)?

Line 271: “CI” Define somewhere that these are Credible Intervals and not Confidence Intervals.

Line 278: Can this decline be estimated? It is not so clear from the figures given the important temporal random variation.

Line 290: I would not call it “substantial” since the important overlap of the posterior samples with zero (Fig. 6). I would say uncertain.

Line 300: I would rather say high uncertainty (but is there any rule of thumb used to define low/moderate/high uncertainty here?). Similar comment for the description of the other effects and see general comment above regarding this issue.

Lines 325-328: I do not see how this show that CR and count should be continuous while productivity data should be segmented without further investigation (e.g. simulations). This statement is misleading.

Lines 330-331: Was it compared with non-IPM estimates? I do not see this.

Line 346: Another strategy that allows both borrowing info and flexibility would be to have random locality effects? It may be more useful with more than three populations but it could be suggested here (with existing references)?

Lines 358: what are the demographic mechanisms of the decline? It would require looking more specifically at the drops in population growth rates and see if they are mostly associated with e.g. drops in productivity or survival (e.g. using year-to-year transient LTRE contributions)?

Line 364: is wetland habitat proxi (PDSI) declining throughout the study period? It would be useful to provide a graph of the yearly variation of the three studied ecological factors. Also similarly to the comment just above, perhaps only increases (not decreases) in population growth are associated with increased PDSI?

Line 395: There are studies on small insectivorous birds that looked at the contributions of demographic rates to changes in growth rate so some could be cited rather than unpublished data.

Lines 404-407 “Furthermore, it would be ideal to jointly analyse count and demographic data to achieve comprehensive understanding and robust inference of population dynamics and underlying demographic processes (Zhao 2020).” Isn't it precisely was it done when using IPMs? I do not see what is meant here (and the relevance of the cited reference). However, I think I just do not understand.

Line 435 and 437: I would only say “IPMs” and not our IPM as it is the case for any IPM. Similarly, “out modelling approach” can be replaced with “IPMs” more generally.

Figures 4-5: it would be useful to show on these figures when the associated data collection was present for each pop.


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is mostly OK. I do (below) comment on a number of things where I think a revision will help the paper to become better.
Experimental design: There is no experiment involved, so this is not an issue.
Validity of the findings: Seem all OK
Additional comments: Review of the PeerJ ms by Zhao et al.
(Please note that my comments are not in decreasing order of importance. )
Zhao et al. build an IPM for the snowy plower in three regions of three Southern states using a variety of available data sets that cover a total of 21 years. They study the effects on demography of various environmental covariates. Importantly, as so often, these data sets do not cover the exact same range of years and they come from different areas. Thus, they are at least in part spatially and temporally misaligned. Such misalignment occurs in virtually all instances of integrated models, where multiple data sets are combined in a model to (generally) inform on the same underlying process. Hence, they deal with questions that are important in general (i.e., for any type of integrated statistical models) as well as for one species that appears to have become a sort of conservation flagship in the US.
	I liked the ms very much and don’t have any major complaints about it. Some comments that may be useful during the revision are here:
1.	A key part of the story told by this ms is about misaligned data. I agree that this is an important topic in integrated models, but I think that the authors should discuss it a little bit more, since they made it one of their main topics. I suggest they highlight a little more those parts of the model where they accommodate this and add some material on this in the Discussion.
2.	In the population submodel the authors fix the variance of initial population size at some small value. Please comment on why you did this? Usually, placing a very wide prior on this parameter makes the population size estimates in the initial year extremely wide, but then does not (so much) affect results in subsequent years. Was this different in your case so that you felt you had to essentially ignore this uncertainty in N[1] ?
3.	Around the same place, where you use a Normal distribution throughout to represent the variability of population size rather than a discrete valued distribution such as a Poisson and binomial, can you also comment on that. Clearly, the Normal makes for better mixing of the MCMC, but it really assumes that your N's are not too close to the boundary of zero. Please comment on that, too.
4.	Starting on line 403 the authors say that several recent studies have shown that count data carry information about demographic rates and then cite the Dail-Madsen developmental line of models. This is fine, as these authors formalized the estimation of demographic rates from counts, but of course this idea is much older and indeed lies at the heart of the whole business of IPMs right from the start (i.e. Besbeas et al. 2002): that the annual change in numbers of unmarked individuals can be expressed as a function of last year’s numbers and the demographic rates.

And here are some more minor comments:
-	Fig. 3: can you say that the CRIs are shown as credible bands ? I would rather call them density strips, since a band to me implies constant density or color hue.
-	Lines 55-56: so true. And you can perhaps mention that this data deficiency is the rule rather than the exception in demographic population assessments.
-	line 66: could also cite Schaub et al. 2007 who particularly emphasize the (perhaps not so surprising) fact that data integration is particularly useful when data sets are very small.
-	Line 196: might it be better to say that the counts were assumed to follow a Poisson log-normal distribution ? To clarify right away the feature of overdispersion that you allow for there.
-	Line 206: that’s a strange detection function. Does it have a recognized name in the Distance sampling universe ? Why not take a more traditional choice such as a half-normal ?
-	Line 264-265: nice ! Use of the full set of MCMC draws produced from a Bayesian analysis in some secondary analysis, to obtain new results with full error propagation, is done not nearly often enough.
-	Line 290: yes, actually I find it rather surprising how similar the contribution of all the demographic components were estimated to be.

Marc Kéry, 6 September 2021


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have done an admirable job of revising the manuscript in accordance with the suggestions of the three reviewers. I have no further comments at this stage.


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: 1) The authors have addressed my main comments. However, (regarding a previous minor comment) I still think that the authors consider immigration to be absent. I agree that the process error term would include variation in “net immigration” in cases where i) “true” (not apparent) survival is estimated (see e.g. Fay et al. 2019 Integrated population model reveals that kestrels breeding in nest boxes operate as a source population) AND ii) immigration is assumed to equal emigration overall, so that the mean net immigration is assumed to be zero.
I do not see where Schaub et al. 2013 refer to “net immigration” (immigration - emigration). They refer to “immigration” and clearly state that permanent emigration and mortality are both accounted for (and confounded) when estimating apparent survival.
In the present study, apparent (not true) survival is estimated. Therefore, emigration is already accounted for when estimating apparent survival and immigration can only be positive. If the authors wish to allow immigration to occur, then they should include it explicitly in the population model, for example as a constant positive integer (then I agree that its temporal variation can be accounted for by the process error). Otherwise, they should state that it is assumed to be absent.

2) Apologies for not asking earlier but I don’t quite understand the prior for “pcount” (gamma prior on theta). The fact that the posterior for this parameter virtually equals 1 (no uncertainty due to no chain fluctuation) seems driven by this prior choice. Why not a Uniform(0,1) on “pcount” (in the main text it is stated that Uniform priors between 0 and 1 were used for probabilities)?
Validity of the findings: 3) Although I appreciate the effort, I disagree with the use of the “p-tail” statistics to describe the strength of the effects. There is a confusion here between “strong” and statistically clear effects. The strength of an effect should refer to its effect size, or its “biological significance” only. Please see the paper from Dushoff et al. (2019). “I can see clearly now: reinterpreting statistical significance” that is a nice commentary about this common misinterpretation of p values and how to address it.
If the authors want to refer to the strength of effects, they should report standardised effects sizes and state which values will be considered small, medium and large effects throughout the manuscript (e.g. for Cohen’s d, d = 0.2 is considered a 'small' effect size, 0.5 represents a 'medium' effect size and 0.8 a 'large' effect size).
Also I strongly agree with Reviewer 1 that plotting estimated relationships (point estimates and credible intervals) between demographic rates (y axis on natural scale) and the covariates of interest (x axis showing the natural range of values) would be highly helpful for the reader to evaluate (and estimate for themselves) the strength of the relationships and the uncertainty around them (such as in Zhao et al. “Land‐use change increases climatic vulnerability of migratory birds: Insights from integrated population modelling” Figure 5).

Dushoff, J., Kain, M. P., & Bolker, B. M. (2019). I can see clearly now: reinterpreting statistical significance. Methods in Ecology and Evolution, 10(6), 756-759.
Cohen, J. (1992). A power primer. Psychological bulletin, 112(1), 155.

4) Perhaps rather than saying that survival parameters were estimated with greater uncertainty in years without data (e.g. in the abstract line 31), it may be more correct to state that they could not be estimated in those years? Often, posterior chains just seem to represent the prior, with modes at 0 and 1, likely due to the fact that the prior is flat on the logit scale but then not on the natural scale (this could be changed).

5) Make sure that the file names in the R code correspond to the names of the data provided (there are underscores missing, file 3 is file 2 and file 2 is 3). The constant “narea” is never used in the model so it does not need to figure in the “jags.data” list.

6) It seems like population size for the New Mexico population cannot be estimated properly (and R hat are higher than 1.15). Is it the case for the posterior used by the authors? It may be bad luck when I ran the models, but if not it may be due to the recent changes in the code (addition of the pcount parameter).
Additional comments: Line 1 Title “reveals drivers”: add “potential”, or say instead e.g. “reveals ecological covariates of demography”. Best to avoid causal language since causality was not evidenced.

Line 301: “Indicating nearly perfect detection”. Not necessarily. This merely means that double counting is as likely as no detection (false positive and negative cancel out on average). Imperfect detection is already accounted for with the Poisson observation error. It accounts for both double counting and no detection which are assumed to be as likely on average. It may be best to not include this pcount parameter (except for the distance sampling) to avoid over-parameterisation (also see previous comment 2) regarding the prior).

Lines 308-315: “adult female (…) and adult male (…) has greater survival than juvenile female (…) and juvenile male”. Either write “on average greater survival” or provide the posterior differences and their credible intervals.

Line 352 and 355: “Impacted” “influenced”, best to change for non-causal statements (here and throughout the manuscript. Or at least use “hedging” (e.g. may influence).

Lines 355-356: “Based on these results, we recommend to prioritize data collection on population and capture-recapture surveys” it is not straightforward for me how this is based on the abovementioned results. Please clarify, or delete "based on these results".

Line 390: “drivers” ditto about causal statements.


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 2
Reviewer: 3

Output:
Basic reporting: I had previously thought that the reporting was good and now it has gotten even better.
Experimental design: The authors have made a good job a improving the descriptions of the design of their field studies, as asked for by the two other referees. This, together with the analytical methods, are much improved now.
Validity of the findings: The findings appear all valid.
Additional comments: Here are some minor comments by line:

-	line 94: 'foundation' sounds a bit grand. Why not 'causes' ?
-	119-120: but not among years ?
-	166: I must say that drought severety index is a clear misnomer ! Palmer should have multiplied his index with -1.
-	171: 'We also considered' twice in two subsequent sentences
-	213: why did you not use one of the more traditional detection functions, such as a half-normal ?
-	326: since you said this already perhaps you can precede the half-sentence in the parentheses by "recall that"
-	461: "IPMs lay ..."


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: INTEGRATED POPULATION MODELLING REVEALS POTENTIAL DRIVERS OF DEMOGRAPHY FROM PARTIALLY ALIGNED DATA: A CASE STUDY OF SNOWY PLOVER DECLINES UNDER HUMAN STRESSORS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: I am really sorry about the ongoing confusion regarding effects sizes, and I really don’t mean to slow down the publication process of this paper any further, but I am afraid I still don’t find the “standardised effect sizes” suggested as an appropriate measure of effect size. The authors divide the posterior mean effects by the width of the 80% credible interval. I am completely unfamiliar with this type of effect size (and there was no reference provided), but such effect size runs into exactly the same issue as the one I mentioned earlier for the p-tail, that is, it directly depends on the uncertainty around the estimate. For a given effect size, effects estimated with more precision will be considered stronger.
There are several possibilities for estimating effect sizes in the literature and I am not a statistician so may not know of the best options, but one option that seem reasonable and meaningful would be to divide each posterior estimate of the slopes by the associated posterior standard deviation of the (transformed) response variable, e.g. the temporal standard deviation of the logit of survival for effects modelled on survival. For example the effect size of PDSI on juvenile male survival of the Oklahoma population would be (for each posterior iteration):
Effect_size_sjf_ok <- logit_sjf_palm/sd(logit_sjf_ok[])
This should provide an estimation of the proportion of variance of each vital rate explained by each effect (and their credible interval).
But I leave it up to the authors to choose what measure they think is most appropriate and useful.

I really appreciate the addition of Figure 8, thank you!
Additional comments: No more comment, and apologies again, this is a nice and clear paper.


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: MORPHOLOGICAL AND MOLECULAR ANALYSES OF PARASITIC BARNACLES (CRUSTACEA: CIRRIPEDIA: RHIZOCEPHALA) IN KOREA: PRELIMINARY DATA FOR THE TAXONOMY AND HOST RANGES OF KOREAN SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript examine the diversity and taxonomy of Rhizocephalan barnacles in Korea
Experimental design: The molecular analysis and morphological examination is fine.
Validity of the findings: This is an important paper to provide baseline Rhizocephalan barnacles in Korea and I would support accept after major revision as my comments below.
Additional comments: The MS can be accepted after major revision.

1) Introduction, first paragraph, the author describe the basic biology of Rhizocephalan. I would suggest the author also cite the lastest molecular based taxonomy of Rhizocephalans which included in Chan et al. (2021).  This can be added in line 34: Latest molecular based taxonomy of Rhizocephalan barnacles was included in Chan et al. (2021).

Chan, B. K. K., N. Dreyer, A. S. Gale, H. Glenner, C. Ewers-Saucedo, M. Pérez-Losada, G. A. Kolbasov, K. A. Crandall, and J. T. Høeg. 2021. The evolutionary diversity of barnacles, with an updated classification of fossil and living forms. Zoological Journal of the Linnean Society zlaa 160. https://doi.org/10.1093/zoolinnean/zlaa160

2) Line 45, The authors reported diversity of Rhizocepahala was reported in different geographical regions. There is also diversity of rhizcocephala in Taiwan. I would suggest to cite cite Yoshida et al. 2012.

Yoshida R, Hirose M, Mok HK, Hirose E. 2012. The first records
of peltogastrid rhizocephalans (Crustacea: Cirripedia:
Rhizocephala) on hermit crabs (Paguroidea) in Taiwan and
differences in prevalences among collection sites. Zool Stud
51:1027–1039

3) Line 46, the author stated “little is known about the species diversity, distribution, taxonomy, and host range of rhizocephalan parasitic barnacles in Korea.” The author should add one more sentence to state “Jung et al. (2009) described xx species of Peltogastrid barnacles from xx hermit crabs in Korea.

4) Line 47, I would suggest to add that in Korea, there are three major oceanographic ecoregions, including the Yellow Sea, East Sea and East China Sea ecoregions, in which thoracican barnacles have different assemblages among these ecoregions (Kim et al. 2021). Then the author can follow that to survey Rhizocephalans in different ecoregions in Korea.

Kim HK, Chan BKK, Lee S-K, Kim W (2020). Biogeography of intertidal and
subtidal native and invasive barnacles in Korea in relation to oceanographic current
ecoregions and global climatic changes. Journal of the Marine Biological Association of the United Kingdom 100, 1079–1091. https://doi.org/10.1017/S0025315420001009

5) Line 54, the author should state the 16 sites in different ecoregions in Korea following Kim et al. 2021. This can be Figure 1A, then Figure 1B can be the distribution map. The sites where no rhizocephalan was found should also be indicated.

6) In discussion, the author should state their molecular tree pattern which showed three major monophyletic clades, the Polyascidae, Sacculinidae and Peltogastidiae. The author should state the Polyascidae is characterized by multiple externa and can reproductive by asexusal reproduction (Glenner et al. 2003), Sacculinidae – single externa, sexual reproduction. Peltogastridiae – mainly in hermit crabs, elaborate more on its features.

7) The authors should plot the vertical bar charts as additional figure, each bar represent one Rhizocephala species, the bar showing different percentage proportion of decapod or hermit crab species. This can provide a good ecological figure on host usage of different species.

8) Since Jung et al 2019 has conducted the survey of Peltogastid barnacles in Korea, this pattern should be also added together in the map in Figure 1 with the present data (but use different colour for the data in Jung et al 2019), to give an overall distribution pattern of Rhizocephalan barnacles in Korea.