Structure of Non-Indigenous Fouling Assemblages and Biocontamination Levels in Portuguese Recreational Marinas Under Different Salinity Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors of this manuscript presented an interesting investigation about the role of salinity on NIS assemblages in Portuguese recreational marinas.

I would like to point out that I have previously reviewed this manuscript for submission to another journal  (in January 2025 for NeoBiota). To my surprise, the authors have not implemented any of the comments or revision requests from the previous review (even typos), nor have they provided any justification for this choice. This approach not only affects the overall quality of the manuscript but also demonstrates a lack of consideration for the peer-review process and the time that reviewers voluntarily dedicate to improving scientific research, resulting in a disrespectful professional ethic.

The purpose of peer review is to ensure the rigor, clarity, and impact of scientific work, and disregarding constructive feedback without justification undermines this fundamental principle. Failing to engage with previous reviews not only weakens the credibility of the study but also raises concerns about the authors’ commitment to academic dialogue and the advancement of knowledge in their field.

Overall, the manuscript is well written and presented. Despite this type of data are extremely relevant, and worthy of publication, the manuscript requires corrections, modifications, clarifications and more detailed explanations in places.

I strongly encourage the authors to address the feedback received in order to improve the manuscript and demonstrate a genuine commitment to scholarly discourse. It would also be advisable for them to provide a detailed point-by-point response, explaining how they have addressed each recommendation or, where relevant, justifying why specific suggestions have not been followed. This is a fundamental expectation in the peer-review process and would significantly contribute to a more transparent and constructive revision.

The Editor and Authors can find all my specific comments, suggestions and recommendations in the attached word file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The Editor and Authors can find all my specific comments, suggestions and recommendations in the attached word file.

Author Response

Comments and Suggestions for Authors

The Authors of this manuscript presented an interesting investigation about the role of salinity on NIS assemblages in Portuguese recreational marinas.

I would like to point out that I have previously reviewed this manuscript for submission to another journal (in January 2025 for NeoBiota). To my surprise, the authors have not implemented any of the comments or revision requests from the previous review (even typos), nor have they provided any justification for this choice. This approach not only affects the overall quality of the manuscript but also demonstrates a lack of consideration for the peer-review process and the time that reviewers voluntarily dedicate to improving scientific research, resulting in a disrespectful professional ethic.

The purpose of peer review is to ensure the rigor, clarity, and impact of scientific work, and disregarding constructive feedback without justification undermines this fundamental principle. Failing to engage with previous reviews not only weakens the credibility of the study but also raises concerns about the authors’ commitment to academic dialogue and the advancement of knowledge in their field.

Overall, the manuscript is well written and presented. Despite this type of data are extremely relevant, and worthy of publication, the manuscript requires corrections, modifications, clarifications and more detailed explanations in places.

I strongly encourage the authors to address the feedback received in order to improve the manuscript and demonstrate a genuine commitment to scholarly discourse. It would also be advisable for them to provide a detailed point-by-point response, explaining how they have addressed each recommendation or, where relevant, justifying why specific suggestions have not been followed. This is a fundamental expectation in the peer-review process and would significantly contribute to a more transparent and constructive revision.

The Editor and Authors can find all my specific comments, suggestions and recommendations in the attached word file.

peer-review-43987812.v1.docx

Comments on the Quality of English Language

The Editor and Authors can find all my specific comments, suggestions and recommendations in the attached word file.

Submission Date

29 January 2025

Date of this review

11 Feb 2025 14:05:04

 

REPLY

We thank your consideration and time spent in improving the quality of this manuscript. You could find in the Track Changes manuscript all the modifications and responses point by point to your specific comments, suggestions and recommendations.

As you can see, we also spent time in changing all the comments previously provided in the Neobiota manuscript to the Diversity one, even those that had already been modified according to your own previous recommendations. To our surprise, we noticed that the referee has not revised our manuscript submitted to Diversity. Instead, he/she has provided a previous revision done for a different journal (the Neobiota one). Despite that, we have made almost all the proposed modifications, including those related to formal aspects and typos.

We appreciate the detailed explanation about how the peer review process works, we can assure you that we are familiar with it because authors have revised more than 400 papers for 90 different journals in last years. We agree with the referee that, “the purpose of peer review is to ensure the rigor, clarity, and impact of scientific work”. However, we would like to point out that this process should also be objective, preventing personal writing style from interfering with the “academic dialogue and the advancement of knowledge in their field”. In addition, it is common that different reviewers provide contradictory comments from the same manuscript. In consequence, not all changes suggested by a reviewer can be included in the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Some minor suggestions

Page 7, line 234. Multiple comparison tests are needed to identify the differences between marinas and between sites.

Page 7, line 238. I am not sure if the sentence is incomplete or if you want to refer to figure 4, in the latter case remove the parentheses.

Page 8. In Figure 4, the separation between habitats is evident, but it also seems that it could be between marine habitats. I suggest indicating each of the marinas with different symbols to determine if there are indeed differences between them and try to determine what other factor is influencing the difference. A cluster analysis could be added to identify at what level this separation between marinas occurs.

Author Response

Comments and Suggestions for Authors

Some minor suggestions

2.1. Page 7, line 234. Multiple comparison tests are needed to identify the differences between marinas and between sites.

In the data analysis section (Pages 5-6 of the Track changes Manuscript), you can check the design of our study, where Marina and Sites were considered random factors. Therefore, multiple comparison tests among the levels of these factors are not needed because we do not have any hypothesis to test about random factors (please see Underwood (1997) -reference 64-). For this reason, we focused on the comparison between Habitats (marine versus brackish) -fixed factor- and therefore the factor that allows in our experimental design to test the hypothesis of this study.

2.2. Page 7, line 238. I am not sure if the sentence is incomplete or if you want to refer to figure 4, in the latter case remove the parentheses.

Thank you for your correction, parentheses were deleted.

2.3. Page 8. In Figure 4, the separation between habitats is evident, but it also seems that it could be between marine habitats. I suggest indicating each of the marinas with different symbols to determine if there are indeed differences between them and try to determine what other factor is influencing the difference. A cluster analysis could be added to identify at what level this separation between marinas occurs.

Thank you for the suggestion, we have included the names of the different marinas in the Figure 4. The most important differences between the studied marinas are salinity conditions (please, see response to comment 2.1). You can see in the nMDS (Figure 4) that marine and brackish marinas are perfectly separated. The addition of a cluster analysis would result redundant with the nMDS already included.

Submission Date

29 January 2025

Date of this review

07 Feb 2025 22:24:34

Reviewer 3 Report

Comments and Suggestions for Authors

The MS is well written but it is trying to make too much of what is effectively a small scale study of the fouling fauna found in brackish vs. marine marinas. The major problems I have with the MS are as follows:

1. i) the relationship between salinity, NIS and biocontamination is tautologous. All of the species examined are marine, therefore salinity will have a major impact reducing the no. of species, native or NIS. The exception to this are a very few hardy species, e.g. Carcinus meanus, which can tolerate a wide range of salinities and therefore as NIS can do well in brackish environments, but these are the exceptions not the rule. In marine ports, NIS from shipping will be continually introduced and survive. In brackish ports, unless the NIS is a euryhaline species they will die and hence species diversity is low across the board. This a product of physiology nothing else. NIS and native abundance (richness/diversity/biocontamination) will always be higher in a marine environment, it is not a novel, new or unexpected finding – estuaries have fewer species than marine environments.
2. ii) the use of the word “structure” infers we are talking about the physical structure of assemblages/their organisation. However, what we are talking about in the MS is dispersion in statistical (multivariate) sense which is different and may be meaningless (an artefact).

iii) they only sampled the sites once (in May 2023) on existing established surfaces – you have no measure of temporal variation and/or the impact of initial colonisation, e.g. where all the pontons all put in the marinas at the same time (all the same age), are they pontons made of the same material?

I think the authors need to revise the paper and make it simple study of the faunal assemblages found in brackish vs. marine ports, anything more detracts from their study.

Minor correction

Line 137 – what is a multiparametric proves? I assume you mean a multiparameter probe, which you could simplify to a salinity probe as this is all you report on.

Author Response

Comments and Suggestions for Authors

The MS is well written but it is trying to make too much of what is effectively a small scale study of the fouling fauna found in brackish vs. marine marinas. The major problems I have with the MS are as follows:

3.1. The relationship between salinity, NIS and biocontamination is tautologous. All of the species examined are marine, therefore salinity will have a major impact reducing the no. of species, native or NIS. The exception to this are a very few hardy species, e.g. Carcinus meanus, which can tolerate a wide range of salinities and therefore as NIS can do well in brackish environments, but these are the exceptions not the rule. In marine ports, NIS from shipping will be continually introduced and survive. In brackish ports, unless the NIS is a euryhaline species they will die and hence species diversity is low across the board. This a product of physiology nothing else. NIS and native abundance (richness/diversity/biocontamination) will always be higher in a marine environment, it is not a novel, new or unexpected finding – estuaries have fewer species than marine environments.

We agree with referee about the relevance of physiology about salinity tolerance of different species as included in the section 4.3 of the discussion. In the new version of the manuscript this issue was emphasized (lines 365-366 of the Track Changes manuscript).

Although the referee states that “the relationship between salinity, NIS and biocontamination is tautologous”, previous studies on this issue showed contradictory results (please, see lines 99-104 of the Track Changes manuscript). For example, references 47-51 reported a greater number of NIS in brackish habitats than in marine ones. Nevertheless, references 41,42,52,53 found the opposite pattern, a greater number of NIS in marine salinities, which is in line with our results. Thus, the influence of salinity on NIS richness is not fully understood. This gap of knowledge is still greater regarding NIS abundance, which has been rarely considered in fouling NIS studies. As far as we know, previous studies conducted in recreational marinas comparing brackish and marine salinities, have not included NIS abundance when this is of paramount importance to assess NIS impacts (see reference 59). Therefore, the role of salinity as a driver of NIS assemblages needs to be clarified.

In this way, studies like the present manuscript still seem to be necessary to confirm speculations and opinions such as that of the referee: “NIS and native abundance (richness/diversity/biocontamination) will always be higher in a marine environment, it is not a novel, new or unexpected finding – estuaries have fewer species than marine environments”. However, according to many previous studies or even the opinion of Reviewer 1: “This part could be expanded focusing also on the importance of brackish environments as bioinvasion hotspots”; it seems that the relationship between salinity, NIS and biocontamination levels is therefore not so tautologous!

About the comment that “all the species examined are marine”, this is incorrect. In our study, 24 NIS species were found, and among these, 5 were exclusively reported in brackish habitats and 6 in both marine and brackish habitats. Therefore, almost half of the total species were not exclusively marine (Please see Table S1). In this line, our results also contrast with the next referee’s statement: “The exception to this are a very few hardy species, e.g. Carcinus maenas, which can tolerate a wide range of salinities and therefore as NIS can do well in brackish environments, but these are the exceptions not the rule”. In our study, we found for instance C. caspia that never was reported in marine salinities (see Folino-Rorem and Renken 2018 https://doi.org/10.1111/ivb.12207). The example of C. maenas that referee described is similar to that of Z. holdichi as found in our study. In fact, the ability to cope with a wide range of salinities (references 87-89) was highlighted in the current manuscript (line 399 of the Track Changes manuscript). However, this is not the rule for all NIS, as pointed by our results and previous studies.

3.2. The use of the word “structure” infers we are talking about the physical structure of assemblages/their organisation. However, what we are talking about in the MS is dispersion in statistical (multivariate) sense which is different and may be meaningless (an artefact).

With the word “structure”, as the referee suggests, we refer to the organisation of the assemblage, including both the identity of species and the abundance of each species. Both are included in the PERMANOVA analyses that showed significant differences between habitats. Other different question is the dispersion. With PERMDISP analysis, we found that dispersion could contribute to differences observed between habitats with brackish samples showing higher dispersion. Despite potential contribution of dispersion to significant differences between habitats, figure 4 (nMDS) shows that samples of brackish and marine habitats are completely separated.

3.3. They only sampled the sites once (in May 2023) on existing established surfaces – you have no measure of temporal variation and/or the impact of initial colonisation, e.g. where all the pontons all put in the marinas at the same time (all the same age), are they pontons made of the same material?

As pointed out by the referee, temporal variation was not included in our sampling design. However, previous published studies (see reference 30 https://doi.org/10.1007/s12526-016-0563-5 and reference 31 https://doi.org/10.1016/j.marenvres.2023.106256) that tested seasonal effects on faunal assemblages associated to marinas in the Atlantic coast of the Iberian Peninsula did not find significant differences between seasons. In this way, our design (and sampling effort) focused on spatial replication (3 marinas per habitat and 4 sites per marina).

All the studied pontoons were made of the same material (fiberglass). This was clarified in the manuscript (line 157 in the Track Changes manuscript).

The aim of our study is not to test the influence of pontoon age on NIS colonisation as we are focused on the abundance, richness and NIS assemblage in long-term established fouling assemblages on pontoons. Although we do not know the exact time when the pontoons were submerged in any given marina, according to the marina staff no one was recently deployed (> 5 years). This is now clarified in the manuscript (lines 157-158 in the Track Changes manuscript).

3.4. I think the authors need to revise the paper and make it simple study of the faunal assemblages found in brackish vs. marine ports, anything more detracts from their study.

The manuscript was revised according to suggestions by three referees making it simpler.

Minor correction

3.5. Line 137 – what is a multiparametric proves? I assume you mean a multiparameter probe, which you could simplify to a salinity probe as this is all you report on.

“Multiparametric proves” was corrected and changed to “salinity probe”.

Submission Date

29 January 2025

Date of this review

09 Feb 2025 12:12:09

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the effort that the authors put into revising their work; however, after carefully examining their responses and the changes made, I have noticed that some issues remain unaddressed.

While several comments have been taken into account, some recommendations from the previous review have not been fully implemented. Furthermore, a few of the responses are not fully aligned with the issues raised and, in some cases, do not adequately explain why certain recommendations were not followed

Among the unclear modifications/unresolved issues, my main concerns are on the following:

1) Materials and Methods (Sampling and sample procedure): "All pontoons were made of fiberglass, and no one was recently deployed (> 5 years) ensuring the study of long-term established fouling assemblages.". How the Authors can ensure that sampled community are long-term established? There is a temporal cut-off in the fouling colonization for that (in case should be referenced)? Moreover, routinary cleanings of pontoons/fouling removal could have been done, resulting in more recent fouling communities. Therefore, I suggest to soft this statement accordingly.

2) Materials and Methods (Sampling and sample procedure): "Since animals can break during the scraping and sieve, colonies were counted when attached to the substrate." I do not understand to what substrate colonial were attached to, since samples were scraped and "In the laboratory, animals were sorted and identified to the lowest taxonomical level possible.". This part should be clarified.

3) Materials and Methods (Data analysis): "Prior to ANOVA, normality of data was checked by Shapiro-Wilk test and Cochran’s tests were done to check for homogeneity of variances.". Reference for Shapiro-Wilk and Cochran test should be provided.

4) Materials and Methods (Data analysis): "Multivariate patterns were illustrated by non-metric multidimensional scaling (nMDS) ordination based on centroids per marina.". As already pointed out in my previous review, reference for nMDS should be cited. Moreover, the check of stress values should be done and specified. Figure 4 just reports the stress value of the output, but this value should be checked with the maximum allowed valued calculated on the base of the number of objects (see Sturrock and Rocha, 2000).< !--EndFragment -->

5) Results: "A total of individuals belonging to taxa was found. Arthropoda was the most abundant phylum (68 taxa) followed by Annelida (53 taxa) and Cnidaria (23 taxa). A total of 39 taxa were exclusively found in brackish marinas, 145 taxa were found only in marine habitats and 33 were found in both types of marinas.". I previously suggest to the Authors to provide < !--StartFragment -->also the total list of identified species/taxa (as Supplementary Material), as they < !--StartFragment -->present data about the overall fouling community. This could help the discussion of some results, and help the reader to better understand the composition/ecology of the investigated assemblages. In light of their reply "< !--StartFragment -->This study is part of a large project and total fouling data will be provided in other studies that are being used in master and PhD thesis. As this work is focused on NIS we provide these data.", I find the first paragraph of this section quite useless as it does not provide a complete information, but an anticipation of future works that the Authors intend to publish separately.< !--EndFragment -->< !--EndFragment -->

5) Discussion: "4.3 NIS multivariate assemblage". As I alredy pointed out in my previous review, even richness and abundance are a part of an assemblage structure. Multivariate analyses just take into account all the single taxa identified and its variation among investigated factors. Therefore, sections 4.1 and 4.3 should be combined and synthetized/restructured accordingly, as they both discuss the stucture of NIS assemblage. The subdivision of uni- and multivariate analyses is just a logistic process in data analysis, but they are required togheter for the pverall discussion of community structure.< !--EndFragment -->

Given these considerations, I leave to the Editor discretion to determine whether the manuscript, in its current form, meets the journal’s standards or whether further revisions should be requested.

Author Response

Comments and Suggestions for Authors

I appreciate the effort that the authors put into revising their work; however, after carefully examining their responses and the changes made, I have noticed that some issues remain unaddressed.

While several comments have been taken into account, some recommendations from the previous review have not been fully implemented. Furthermore, a few of the responses are not fully aligned with the issues raised and, in some cases, do not adequately explain why certain recommendations were not followed

Among the unclear modifications/unresolved issues, my main concerns are on the following:

1.1. Materials and Methods (Sampling and sample procedure): "All pontoons were made of fiberglass, and no one was recently deployed (> 5 years) ensuring the study of long-term established fouling assemblages.". How the Authors can ensure that sampled community are long-term established? There is a temporal cut-off in the fouling colonization for that (in case should be referenced)? Moreover, routinary cleanings of pontoons/fouling removal could have been done, resulting in more recent fouling communities. Therefore, I suggest to soft this statement accordingly.

This statement was modified to “All pontoons were made of fiberglass, and, according to marina staff, no one was recently deployed (> 5 years) or cleaned, avoiding the sampling of incipient or very disturbed assemblages” following referee’s recommendation.

1.2. Materials and Methods (Sampling and sample procedure): "Since animals can break during the scraping and sieve, colonies were counted when attached to the substrate." I do not understand to what substrate colonial were attached to, since samples were scraped and "In the laboratory, animals were sorted and identified to the lowest taxonomical level possible.". This part should be clarified.

In the lab, during sorting under dissection microscope we are still able to distinguish colonial species attached to different substrates. These substrates include mussel shells and byssus, barnacles, algae, ascidians, sponges or even litter. This was clarified in the manuscript by including the following sentences.

“Although animals can break during the scraping and sieving procedures, colonial species were still individually recognized during sorting under dissection microscope since they were attached to different substrates (mussel shells and byssus, barnacles, algae, ascidians, sponges or even litter). Thus, colonies were only counted when attached to the substrate”.

1.3. Materials and Methods (Data analysis): "Prior to ANOVA, normality of data was checked by Shapiro-Wilk test and Cochran’s tests were done to check for homogeneity of variances.". Reference for Shapiro-Wilk and Cochran test should be provided.

References were included.

1.4. Materials and Methods (Data analysis): "Multivariate patterns were illustrated by non-metric multidimensional scaling (nMDS) ordination based on centroids per marina.". As already pointed out in my previous review, reference for nMDS should be cited. Moreover, the check of stress values should be done and specified. Figure 4 just reports the stress value of the output, but this value should be checked with the maximum allowed valued calculated on the base of the number of objects (see Sturrock and Rocha, 2000).”

Reference for nMDS was included. The following sentence was added: “The stress value was checked using Sturrock and Rocha (2000)”, following referee’s recommendation.

1.5. Results: "A total of individuals belonging to taxa was found. Arthropoda was the most abundant phylum (68 taxa) followed by Annelida (53 taxa) and Cnidaria (23 taxa). A total of 39 taxa were exclusively found in brackish marinas, 145 taxa were found only in marine habitats and 33 were found in both types of marinas.". I previously suggest to the Authors to provide also the total list of identified species/taxa (as Supplementary Material), as they present data about the overall fouling community. This could help the discussion of some results, and help the reader to better understand the composition/ecology of the investigated assemblages. In light of their reply "This study is part of a large project and total fouling data will be provided in other studies that are being used in master and PhD thesis. As this work is focused on NIS we provide these data.", I find the first paragraph of this section quite useless as it does not provide a complete information, but an anticipation of future works that the Authors intend to publish separately.

We have eliminated that section of the manuscript according to referee´s suggestion.

1.6. Discussion: "4.3 NIS multivariate assemblage". As I alredy pointed out in my previous review, even richness and abundance are a part of an assemblage structure. Multivariate analyses just take into account all the single taxa identified and its variation among investigated factors. Therefore, sections 4.1 and 4.3 should be combined and synthetized/restructured accordingly, as they both discuss the stucture of NIS assemblage. The subdivision of uni- and multivariate analyses is just a logistic process in data analysis, but they are required togheter for the pverall discussion of community structure.

Sections 4.1 and 4.3 were combined according to referee’s suggestion.

Given these considerations, I leave to the Editor discretion to determine whether the manuscript, in its current form, meets the journal’s standards or whether further revisions should be requested.

Submission Date

29 January 2025

Reviewer 3 Report

Comments and Suggestions for Authors

The authors dispute my statement that: “the relationship between salinity, NIS and biocontamination is tautologous. All of the species examined are marine, therefore salinity will have a major impact reducing the no. of species, native or NIS.”  stating that some of the  species found are considered estuarine or brackish water species. Apart from a very few examples, estuarine and brackish water species are marine in origin just inferior competitors in the marine environment. Furthermore, all brackish water NIS have arrived from the marine environment by vessels.

 

I still have concerns about the experimental design which have been increased by re-reading the MS and some of the comments for other reviewers in the edited MS. Specifically:

1. the exact age and time of placement of the pontoons utilised in the study is not known. Hence this is not an experimental study, rather an observational study with a very low level of replication. I would be very hesitant abut making any strong conclusions;
2. I am concerned by the use of ANOVAs (lines 352 to 355) where the data lacks homogeneity of variance.  The authors posit, that they deal with this by picking the 0.01 significance level over a 0.05 significance level but that is not appropriate and certainly not without any literature to support it. The lack of homoscedasticity, is the bane of parametric statistics and in reality we have no way to overcome it, even permutational approaches are affected by it. I would remove these analyses or use a non-parametric or permutational equivalent at the 0.05 level of significance.  Yes, I would be unhappy with this, but it is an accepted solution.

My opinion is that the authors should revise the MS and present it as a short descriptive study.

Author Response

Comments and Suggestions for Authors

3.1. The authors dispute my statement that: “the relationship between salinity, NIS and biocontamination is tautologous. All of the species examined are marine, therefore salinity will have a major impact reducing the no. of species, native or NIS.”  stating that some of the  species found are considered estuarine or brackish water species. Apart from a very few examples, estuarine and brackish water species are marine in origin just inferior competitors in the marine environment. Furthermore, all brackish water NIS have arrived from the marine environment by vessels.

 We still disagree about this point. There is not scientific evidence about the introduction by vessel of the NIS species that we found in the estuaries. In fact, to the date, the way to introduction of these species in north Portugal estuaries is unknown. Moreover, there are other alternative ways of introduction as plausible (or even more) than vessel traffic (see, for example, Minchin et al. 2009; Anderson et al. 2015; Grosholz et al. 2015; Williams et al. 2015; Zieritz et al. 2017). The introduction of NIS due to oyster importation from estuaries in many countries have been proved to be an important vector of introduction of NIS species (Grosholz et al. 2015; Zieritz et al. 2017; Di Blasio et al. 2023), and in the Lima estuary (in the North of Portugal) there is an important oyster farming. Moreover, many NIS species in north Portugal estuaries come from introduction in the river, then later colonise estuarine habitats. Here we provided some examples: Corbicula fluminea (Sousa et al. 2007; Franco et al. 2012; Ferreira-Rodríguez et al. 2022), Procambarus clarkii in north Portugal and Italy (Martín-Torrijos et al. 2021; Nota et al. 2024) or Xenostrobus securis in Spain (Gestoso et al. 2014; Sanz-Latorre et al. 2023). All these NIS have not arrived from the marine environment, as referee considers. Therefore, all this discussion is based on speculations and may occur in other parts of the world but not necessarily in our study area as referee suggests. Moreover, the origin or the way of introduction of the NIS species is completely out of the scope of our study. Our aim is to explore and compare the biocontamination levels and NIS assemblage structure between marinas in marine and brackish water independently of their origin or introduction vector. Therefore, despite the scientific interest of this debate it is completely sterile to improve the manuscript.

The referee may say that this study is not original, and results were expected but, as we pointed out with published studies in the manuscript and in our reply for Round 1, the statement of the referee 3 suggesting that NIS species use to be more abundant in brackish than in marine water is completely opposite to referee’s position. This lack of consensus among specialists, even among previous studies (see please our previous reply) justify our study with conclusions based on data instead opinions and personal theories.

3.2. I still have concerns about the experimental design which have been increased by re-reading the MS and some of the comments for other reviewers in the edited MS. Specifically:

The exact age and time of placement of the pontoons utilised in the study is not known. Hence this is not an experimental study, rather an observational study with a very low level of replication. I would be very hesitant about making any strong conclusions.

We agree in the point that this is an observational study, it has always been, nor in the first version of the manuscript or in the corrected one was suggested that this study was an experimental study. Indeed, you will not find the word “experiment” or “experimental” in any of the versions. Considering the objective of comparing marine vs brackish water marinas, the analyses to explore significant differences of the fixed main term “Habitat” was based on 36 replicates per each habitat (72 replicates in total). In fact, our replication is similar to that of O’Shaughnessy et al. (2023) and higher than that used in other published studies in marinas: 54 replicates (Guerra-García et al. 2021; Saenz-Arias et al. 2022ab) or 15 replicates (Ferrario et al. 2024). If referee considers this sampling effort insufficient, most of the published papers about this topic should be invalid as their sampling effort is lower than ours.

Regarding the age and time of placement of the pontoons, we ensured that all the pontoons were at least 5-10 years old and thus, supported well established assemblages (please, see our response to point 1.1 of referee 1). Anyway, some potential variability due to the time of placement of the pontoons should be irrelevant with our sampling design by considering different marinas within each habitat, different sites with each marina and different quadrats within each site. By including in the analysis all these sources of variability we ensure that differences were not due to time of placement, or other natural or anthropogenic disturbance related to a single, marina, pontoon, site or quadrat.

3.3. I am concerned by the use of ANOVAs (lines 352 to 355) where the data lacks homogeneity of variance.  The authors posit, that they deal with this by picking the 0.01 significance level over a 0.05 significance level but that is not appropriate and certainly not without any literature to support it. The lack of homoscedasticity, is the bane of parametric statistics and in reality we have no way to overcome it, even permutational approaches are affected by it. I would remove these analyses or use a non-parametric or permutational equivalent at the 0.05 level of significance.  Yes, I would be unhappy with this, but it is an accepted solution.

It is well known that in the study of the benthic assemblages the only constant is the variability. Therefore, the lack of homoscedasticity is very frequent in benthic studies. The heterogeneity of variances in a study of benthos can be as interesting as differences among means but, many ecologists view heterogeneity of variance as a problem. Sometimes, transformations cannot solve this “problem”, but if you check tables 2 and 3 you can see that there are not significant differences for the interest factor “Habitat” for the variables (N_NIS and ACI) that were the only with heterogeneous variances.

The main problem with the heterogeneity of variance is that increases the probability of type I error (i.e., to find significant differences when they do not exist). In our study the ANOVA did not find significant differences between marine and brackish marinas for these variables (N_NIS and ACI), despite the lack of homoscedasticity, and, therefore, the result is still valid.

Moreover, in analyses of variance balanced designs with proper replication (as this one) are robust to departures from the assumptions and by picking the 0.01 significance level over the classical 0.05 level, we ensure the validity of results obtained with analysis of variance. This solution is widely used in benthic ecology, both in marinas (e.g. Revanales et al. 2022; Saenz-Arias et al. 2022a; Ruiz-Velasco et al. 2023): and natural habitats (e.g. Fraschetti et al. 2005; Bertocci et al. 2019) it is widely “accepted solution”.

 

REFERENCES

Anderson et al. (2015). https://doi.org/10.1371/journal.pone.0140833

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