Review Reports - Fast and Reliable Identification of Ammonia Phylotypes T1, T2 and T6 Using a Stereomicroscope: Implication for Large-Scale Ecological Surveys and Monitoring Programs

Round 1

Reviewer 1 Report

Overall Evaluation

This manuscript is acceptable for publication with a few minor to moderate revisions needed, as noted in the text and figures

General Comments

This manuscript presents a new, simple and time-saving method utilizing a stereomicroscope to identify three phylotypes of Ammonia, suggesting the method can replace the use of SEMs proposed by previous studies by Richirt and others, especially in large database studies or where SEMs are not available. It presents the accuracy and time-of-use for the method and compares it to similar parameters using SEMs. The conclusions are acceptable regarding the efficacy of the method, but two of the projected uses of the methods are unclear (see #2 and #6 below).

The following are my suggestions to improve this manuscript:

Several grammatical insertions and deletions have been noted on the Word version (very poorly modified from the pdf into a Word format, I’m afraid) to improve the readability of the manuscript.
Abstract: The last sentence needs to be rewritten. The stereomicroscope method does NOTHING to assess the potential of T6 to become invasive. It may streamline the evaluation of where it is present and then implications can be made about the timing of the possible invasion or progression of the invasion in different geographic regions, but cannot help explain its potential for invading. The same is true at the end of the manuscript (somewhat of a concluding sentence).
The results section should not start with a figure. Place the figure (#2) after the text is started in this section and the figure is referred to in the text.
Be consistent whether you will use present or past tense describing the results you found (for example, “were vs. are” and others)
Approximately lines 203 on: Most of this paragraph is talking about the fact that the stereomicroscope method is not as accurate as the SEM method. Put the simple fact that the method is faster first, then go on to describe the more complicated problem with it (not being as accurate).
Approximately line 217: I don’t understand at all the point that is being made here: “Reconsider the formal names of A. tepida?” What does this sentence mean? It is not at all clear and needs to be rewritten for clarity. How does a method to identifying the three phylotypes “reconsider the formal names of A. tepida”?
Delete the “Data Availability Statement” section if it does not apply to your study.
All of your references appear in the text of your manuscript. However, the journal is not listed for #2 and sometimes you spell out the journal name and other times it is abbreviated. Be consistent and follow the format this journal (Water) requests. Part of entry #9 is capitalized and should be corrected.
The figures are good, especially Figure 1 (which is wonderful!). However, please include a location figure of your sampling sites in Gironde Estuary (should estuary be capitalized in the text? You have it as Gironde estuary’s samples).

This will be a valuable contribution to this special volume on “new methods” using foraminifera in biomonitoring. I am also aware that putting manuscripts into this journal's formatting is a major challenge. You did great.

Comments for author File: Comments.pdf

Author Response

Overall Evaluation

This manuscript is acceptable for publication with a few minor to moderate revisions needed, as noted in the text and figures

General Comments

The authors thank the reviewer#1 for his/her Overall Evaluation and General Comments and we modified the manuscript accordingly.

The following are my suggestions to improve this manuscript:

Several grammatical insertions and deletions have been noted on the Word version (very poorly modified from the pdf into a Word format, I’m afraid) to improve the readability of the manuscript.

We modified the ms accordingly.

Abstract: The last sentence needs to be rewritten. The stereomicroscope method does NOTHING to assess the potential of T6 to become invasive. It may streamline the evaluation of where it is present and then implications can be made about the timing of the possible invasion or progression of the invasion in different geographic regions but cannot help explain its potential for invading. The same is true at the end of the manuscript (somewhat of a concluding sentence).

In the abstract, we modified it as: “Finally, in the context of Ammonia phylotype T6 potentially being an alien species in Europe, this method will help to quickly identify Ammonia phylotypes; hence contribute to monitor the presence of T6 in different regions and then, offers interesting research perspectives to assess the timing and/or the progression of the possible invasion.” (lines 34, 35, 36)

At the end of the manuscript, we modified it as: “It would be quicker to identify and, as a consequence, to monitor the presence of the phylotype T6 in different regions of Europe and then, to follow the spreading pattern of T6 both at a spatial and time scales utilizing the stereomicroscope method rather than the SEM method.” (lines 265-269).

The results section should not start with a figure. Place the figure (#2) after the text is started in this section and the figure is referred to in the text.

We modified it accordingly.

Be consistent whether you will use present or past tense describing the results you found (for example, “were vs. are” and oth ers)

We modified it accordingly

Approximately lines 203 on: Most of this paragraph is talking about the fact that the stereomicroscope method is not as accurate as the SEM method. Put the simple fact that the method is faster first, then go on to describe the more complicated problem with it (not being as accurate).

We modified it accordingly.

Approximately line 217: I don’t understand at all the point that is being made here: “Reconsider the formal names of A. tepida?” What does this sentence mean? It is not at all clear and needs to be rewritten for clarity. How does a method to identifying the three phylotypes “reconsider the formal names of A. tepida”?

We agree with reviewer #1 and we modified the ms accordingly, lines 240 to 244: “The method implemented here would first, help to not use anymore the formal name of the morphogroup of Ammonia tepida, still often used in large foraminiferal community surveys, and instead, result in the correct identification of numerous individuals of these pseudo-cryptic and distinct Ammonia phylotypes.”

Delete the “Data Availability Statement” section if it does not apply to your study.

We modified it accordingly.

All of your references appear in the text of your manuscript. However, the journal is not listed for #2 and sometimes you spell out the journal name and other times it is abbreviated. Be consistent and follow the format this journal (Water) requests. Part of entry #9 is capitalized and should be corrected.

We modified it accordingly.

The figures are good, especially Figure 1 (which is wonderful!). However, please include a location figure of your sampling sites in Gironde Estuary (should estuary be capitalized in the text? You have it as Gironde estuary’s samples).

We added the map as the new Figure 1 (line 97) and capitalized “Estuary” (Line 92)

Reviewer 2 Report

This paper provides very interesting methods to distinguish simply through a stereomicroscope the species belonging to the genus Ammonia that includes several cryptic species, previously uniquely distinguishable by molecular sequencing and other species morphologically discriminated only at the SEM observation.

The paper is clearly and correctly written with opportune references, except the papers related to the changes suggested below. This work is surely appealing for the Water readers.

My suggestions to improve the paper:

The authors describe the morphological characters of three phylotypes T1, T2 and T6 . To adding the importance to distinguish them in terms of ecological application (e.g., which are the ecological characters of these types? Why is it important to distinguish them?) will increase the potentiality of the paper. The authors refer only in the discussion about the fact that Ammonia sp. T6 is supposedly non-indigenous in Europe potentially replacing its congeneric autochthonous Ammonia sp. T1 and T2.

Likewise, in the Introduction something more on the advantages to distinguish the diverse types of Ammonia for environmental/ecological reconstructions and ecosystem monitoring would be appreciated. It is known that Ammonia can adapt to diverse environments and can tolerate several stress degrees, including human induced pollution, e.g., heavy metals.

Can the authors ensure that the observed morphological characters are not ecophenotypic response to the Gironde estuary’s ecological quality status? Something more about the status of the sediments at the time of sampling should be added. Is the relative percentage of the T1, T2 and T6 significant from an ecological point of view?

The authors correctly avoid analyzing broken and/or decalcified/recalcified specimens. Which is the cause(s): change in pH, pollution, low oxygen content? Couldn’t be possible that these causes may have affected at a minor degree the other specimens so influencing the pore diameter and sutures?

Are the morphological differences detectable both in forms A and B? No information about the two forms are given in the present text.

Fig. 1: In the images shown, the suture elevations are not clearly displayed: actually, they appear rather similar (flush) in the stereomicroscope views whereas in the SEM images they look raised or not depressed. I suggest keeping images more representative.

I suggest to adding a map with the location of sampling.

Line 70. Add that the types belonging to Ammonia tepida (add author and year of this species).

Line 87: explain ‘less ornamented’ for non-specialist readers.

Line 88: what do you mean with randomly picked?

Author Response

Response to reviewer #2

The paper is clearly and correctly written with opportune references, except the papers related to the changes suggested below. This work is surely appealing for the Water readers.

The authors thank the reviewer#2 for his/her comments.

My suggestions to improve the paper:

The authors describe the morphological characters of three phylotypes T1, T2 and T6. To adding the importance to distinguish them in terms of ecological application (e.g., which are the ecological characters of these types? Why is it important to distinguish them?) will increase the potentiality of the paper. The authors refer only in the discussion about the fact that Ammonia sp. T6 is supposedly non-indigenous in Europe potentially replacing its congeneric autochthonous Ammonia sp. T1 and T2.

We have planned a second paper contributing to understand the ecology of these phylotypes in the Gironde estuary. Here, we want to focus only on the methodological aspect to discriminate the three phylotypes occurring in Europe using a stereomicroscope to make a methodological paper only. We think that mixing the methodological aspects and the ecological ones would confuse the reader. This might explain the lack of details in terms of ecological explanations.

However, to help the reader and meet the reviewer#2 comment, we added some information on the ecology of Ammonia in the introduction to emphasize on the need for accurate identification procedure:

lines 59-65: “It is known that Ammonia can adapt to diverse environments and tolerate a wide range of different stresses such as salinity, organic matter enrichment, hypoxia or trace metals [6–10]. However, these species may have different optimal requirements in those ranges and reducing taxonomical uncertainties is then crucial for every aspect of biology and ecology of the Ammonia species in order to better define requirements for each species.”.

Phylotypes T1, T2 and T6 where shown to represent different species. The morphological variability of these three phylotypes was extensively investigated in a very large range of locations (e.g., Hayward et al., 2004; 2021; Richirt et al., 2019a; 2021; Schönfeld et al., 2021). Although morphological variability exists for these three phylotypes, as pointed out by the reviewer in his commentary, the used characters (average pore diameter and suture elevation on the spiral side) where shown to be highly reliable to recognize them correctly (with almost no overlap between phylotypes). This is strongly suggesting that in the case where there is ecophenotypic variation on either or both average pore diameter and suture elevation, these variations occur inside the range of each separated phylotype, but not between phylotypes.

In another paper in preparation at this moment, we will discuss from an ecological point of view the relative abundances of T1 T2 and T6.The scope of the present methodological paper is to discuss on their identification using a stereomicroscope.

Broken chambers or decalcification/recalcification observations are not unusual and might be the result of a different causes (mechanical break due to high energy systems such as estuaries or manipulation with the brush in the lab, dissolution, or re-precipitation of shell material due to geochemistry of the sediment…). As a consequence, it is difficult to say what is(are) the cause(s).

The reviewer is right when arguing that environmental conditions, more specifically oxygen concentration or dissolution of the test, might have an effect on the pore diameter of the individuals.

Average pore diameter is expected to vary with oxygen content in the pore waters (Richirt et al., 2019b), but this was never demonstrated experimentally in our knowledge. However, the range of variability regarding pore diameter is different between phylotypes T2 and T1/T6, meaning that if oxygen content influences pore diameter, this might be the case at the intra phylotype level, but very unlikely at the inter phylotype level.

For suture elevation, dissolution might make them less clear or even flush when they were actually raised (and inversely with re-precipitation of material on the shell). This could also artificially increase (or decrease in case of re-calcification) average pore diameter.

For these reasons we preferred to not include these damaged specimens in our study, as noted by the reviewer.

Are the morphological differences detectable both in forms A and B? No information about the two forms are given in the present text.

We did not understand this comment about forms A and B. Does the reviewer#2 refer to phylotypes? If he/she does, the question has been answered in the previous answer above.

We agree with reviewer#2. The original image in the word version of the ms are of better quality than in the pdf one. There was an obvious loss of quality in the conversion from word to pdf. This may explain the reviewer#2 comment.

May the journal provide a technical solution for this?

I suggest to adding a map with the location of sampling.

We added the map as the new Figure 1 (Lines 112-133).

Line 70. Add that the types belonging to Ammonia tepida (add author and year of this species).

We modified accordingly (lines 69-72): “A recent method based on SEM morphometry was proposed to discriminate three Ammonia phylotypes, belonging to Ammonia tepida [11], morphogroup, occurring along the North East Atlantic coasts (phylotypes T1, T2 and T6) without the need for molecular analysis [12].”

Line 87: explain ‘less ornamented’ for non-specialist readers.

We detailed this term on lines 101, 102: “(i.e., without or with a few ornaments around the sutures, such as beads, grooves, pustules, bosses or secondary calcite)”

Line 88: what do you mean with randomly picked?

We modified the manuscript to explain this further (lines 100-104): “Among the Ammonia tepida-like picked specimens, three-hundred specimens belonging to the less ornamented (i.e., without or with a few ornaments around the sutures, such as beads, grooves, pustules, bosses or secondary calcite) Ammonia tepida morphogroup (containing phylotypes T1, T2 and T6 following [3]) were randomly selected for the purpose of this study.”