Ocean Acidification and Mollusc Settlement in Posidonia oceanica Meadows: Does the Seagrass Buffer Lower pH Effects at CO₂ Vents?

Round 1

Reviewer 1 Report

The present manuscript examine the effect of ocean acidification on the settlement of molluscs in carbon dioxide vents in Italy. The results showed that the mollusk settlers assemblages differ among sites with different pH and microhabitats.

Overall the MS is smooth and good. There are some points need to address before publication.

• In the abstract, the genus of P. oceanica should be spelled in full.
• There are some English typos in the MS, need to check carefully.
• The author stated that possible buffering role (by reducing pH) of the Posidonia to enhance settlement of molluscs. This statement is not very convincing, as Posidonia occur in all sites from extreme pH, high pH and control. In the extreme pH site, the pH is already very low. How can P. oceania to buffer the pH in these site? If so, then the pH in these site should not be extreme pH conditions. The author need to clarify this as the content they present here now cause confusion to readers.
• I would suggest provide more photos of the sites to show the density of P. oceania and also a figure with the major mollusc settlers
• How is the original mollusc assemblages in these three sites? The author should provide the baseline survey result of original mollusk assemblages at these three sites and compared with the settlers. If there are different in settler diversity with the adult mollusc assemblages in the extreme pH sites, that means, there can be post-settlement mortality occur to remove certain portion of species in these low pH sites.
• Any morphological differences in molluscs among the sites along the pH gradient? Degree of shell erosion and shell thickness?
• In the discussion, the author should discuss whether there can be further difference in post-settlement mortality among those pH sites? Settlement differences is a starting point for assemblage difference. Post-settlement mortality is also an important factor to affect subsequent assemblage differences.
• In the discussion, the author only explain the patterns in Italy. I would suggest to cite other example of reduced pH environment from shallow vents in other parts of the world. For example, the shallow water vents in the Kueishan Island, there is also a gradient of mollusc assemblages starting from the gradient from vent to non-vent regions. I would suggest author to cite the references below and add in the comparison of patterns in other parts of the world to strengthen the discussion content.

Chan, B. K. K., Wang, T-W., Chen, P.-C., Lin, C.-W., Chan, T.-Y. and Tsang, L.M. (2016). Community structure of macrobiota and environmental parameters in shallow water hydrothermal vents off Kueishan Island, Taiwan. PLoS One, 11(2): e0148675.

Chen, C., Chan, T.-Y. and Chan, B.K.K.  (2018). Molluscan diversity in shallow water hydrothermal vents off Kueishan Island, Taiwan. Marine Biodiversity, 48(1): 709-714.

Author Response

- Comments to Reviewer #1

We want to thank Reviewer #1 for His/Her review of the manuscript, and the valuable comments and suggestions. We have revised our manuscript accordingly.

# In the abstract, the genus of P. oceanica should be spelled in full.

Done, please see line 21.

# There are some English typos in the MS, need to check carefully.

The manuscript has been carefully checked and typos have been corrected throughout the text.

# The author stated that possible buffering role (by reducing pH) of Posidonia to enhance settlement of molluscs. This statement is not very convincing as Posidonia occur in all sites from extreme pH, high pH and control. In the extreme pH site, the pH is already very low. How can P. oceania to buffer the pH in these site? If so, then the pH in these site should not be extreme pH conditions. The author need to clarify this as the content they present here now cause confusion to readers.

The definition of the pH zones in the Castello and Vullatura vents, as Ambient, Low and extreme low pH, derived from a long series of discrete and continuous measurements, some of which have been illustrated in the sampling sites of this paper in the Supplementary material (Fig. S2). Such measurements have always been carried out outside the Posidonia meadows and outside the canopy; this is due to the dimension and mooring the SeaFET in situ pH meters. Due to the characteristics of the instruments (e.g., size), it was not possible to arrange them directly into the canopy. Therefore the water outside and surrounding the Posidonia meadows has pH values which we can define extreme low (80% below 7.4 units), as showed by the pH trend at the investigated stations provided as Supplementary material, now Fig. S2. In the Discussion section (lines 543-549), we stated the probable process through which Posidonia can buffer the effects of pH reduction, inside the canopy and respect to the surrounding waters on associated fauna.

We would thank Reviewer #2 for the important clarification. To this end, we have modified Materials and Methods section accordingly (please, see lines 179-183).

# I would suggest provide more photos of the sites to show the density of P. oceania and also a figure with the major mollusc settlers

We have added two more pictures showing P. oceanica meadow at the studied site (S3) and the most representative taxa found in the samples, respectively. Pictures have been provided as Supplementary materials, Fig. S1 and S5. All the other Supplementary materials have been renumbered accordingly.

# How is the original mollusc assemblages in these three sites? The author should provide the baseline survey result of original mollusk assemblages at these three sites and compared with the settlers. If there are different in settler diversity with the adult mollusc assemblages in the extreme pH sites, that means, there can be post-settlement mortality occur to remove certain portion of species in these low pH sites.

Thank you for the valuable suggestions. We have provided a brief table (Table S4), as supplementary information, to compare the post-settlement/juvenile assemblages described in the present study with the checklists reported in Foo et al. [15], Garrard [69], and Garrard et al. [54]. These studies collected and described adult assemblages of molluscs on Posidonia oceanica [54,69] and hard vegetated bottoms [15]. The Discussion section has been integrated at lines 503-514 to support the evidence of post-settlement mortality. In Table S4, stations have been identified as Control/Ambient and Acidified stations (without separating Low and Extreme Low pH stations) in order to homogenize data coming from different studies.

# Any morphological differences in molluscs among the sites along the pH gradient? Degree of shell erosion and shell thickness?

No differences in shell thickness or morphology have been highlighted in the samples. Likely, the age of the organisms found in the artificial collectors could have influenced the shell erosion pattern: since many of the collected specimens were young, they were exposed for a short period to acidified conditions (33 days only). As for the absence of shell erosion, the Discussion section has been integrated at lines 512-519.

# In the discussion, the author should discuss whether there can be further difference in post-settlement mortality among those pH sites? Settlement differences is a starting point for assemblage difference. Post-settlement mortality is also an important factor to affect subsequent assemblage differences.

We have further analyzed the post-settlement mortality at the studied sites, both under natural/control and acidified conditions (please, see lines 499-509).

# In the discussion, the authors only explain the patterns in Italy. I would suggest to cite other example of reduced pH environment from shallow vents in other parts of the world. For example, the shallow water vents in the Kueishan Island, there is also a gradient of mollusc assemblages starting from the gradient from vent to non-vent regions. I would suggest author to cite the references below and add in the comparison of patterns in other parts of the world to strengthen the discussion content.

Thank you for the valuable suggestion. We have integrated the Discussion section with the studies carried out at Kueishan Island vents (please, see lines 482-486), and added them in the references list.

Reviewer 2 Report

This is an interesting and generally well-written manuscript about an important subject – the viability of molluscs in an acidified ocean. The methodology was well designed. The authors used collectors to quantify mollusc settlement along a pH gradient at three separate CO₂ vents. Because of the replication of vents, the authors’ results are more robust than typical in situ studies of ocean acidification. The primary result, that low pH conditions reduce mollusc settlement of individuals and taxa, was supported by their data. I found the work to be a valuable contribution to Diversity.

My biggest criticism is relatively minor, that anomalies in the patterns of settlement were not more thoroughly addressed. Specifically, the highest number of individuals was observed at the site with the lowest pH (6.6). The number of taxa there were comparable to the highest number of taxa at Vullatura.  Additionally, the lowest number of individuals and taxa were observed at a site with a moderate pH (7.8, Vullatura low pH site). These suggest that a local factor has as much of an effect on settlement between sites as pH does across the pH range. You discuss storm effects, but it’s likely that even simpler drivers are more important – proximity to breeding adults, local circulation, or strength of currents. I am not an expert at this literature but these proximal drivers should be addressed.

Another example of an anomaly that should be addressed: at both Castello north and Vullatura, from low pH to extremely low pH, the number of individuals and taxa at the bottom of the canopy did not decrease. In both cases individual numbers were lower at low pH than at the lowest pH. It’s an interesting pattern.

The manuscript would also be improved if the authors would explicitly quantify the decrease in settlement of individuals and taxa. From your figure 2, it looks like individuals decreased by 0 to 25% and taxa decreased by 10 to 40% from ambient to extremely-low pH. It would be useful to report the correct numbers in the abstract, and to add a paragraph of the discussion to compare these decreases to decreases in settlement observed in other studies.

Otherwise I found the Discussion well-written with interesting observations on the sensitivity of molluscs to acidification. 

I recommend other more minor changes as follows: 

Line 92: Please include more background information on mollusc settlement. This could be useful in the discussion. Drivers of settlement include proximity to adults, bottom type, and currents. Are there other important factors that are relevant? 

Line 172. It’s not clear from your statement that this type of artificial collector has been used previously, only that artificial collectors in general have been used previously. You may also want to state that this style of collector is effective for collection of juveniles and adults and give evidence for it. 

Line 254: you state that a total of 19613 molluscs were collected, but I think it would be more accurate to state that this many “invertebrates were collected.” The big number appears to include Polychaeta and Amphipoda, which are not molluscs.  

Line 283: the first time you refer to microhabitats as “B” or “C” include the words “bottom” or “canopy”

Figure 2: I recommend including an axis break from 200 to 300 for the anomalously high error bar on number of individuals at Castello South. This would let you almost double the height of all of the bars in the figure.

There are a few grammatical issues in the manuscript. I believe a grammar check – like Microsoft Word - will find most of them, but I’ve addressed the ones on the first page below. These are most likely to be noticed.  

Line 2, Title, I recommend changing “molluscs” to “mollusc”. “Molluscs settlement” without an apostrophe looks like a grammatical error. 

Line 19, “to assess the effect of decrease” should be “to assess the effect of a decrease”

Line 26: instead of “Simplified mollusc assemblages” I recommend “Reduced mollusc assemblages”

Line 30-31 “...were the species making the differences observed along pH gradient, and were all relatively robust to OA conditions.” This sounds like a contradiction. If these species made the differences along the pH gradient, I’d expect them to be absent at low pH, yet they are robust to OA.

Line 34-36: I made a few grammatical edits, and edits for clarity to the final sentence of the Abstract “Our study provides new evidence of shifts in marine benthic communities due to ocean acidification and evidence of how P. oceanica meadows could mitigate its effects on associated biota in light of future climate change.”

Line 43: I suggest changing “reported nowadays” to “today”.

Thank you for conducting this work. I found it interesting and valuable. I look forward to seeing it published.

Author Response

- Comments to Reviewer #2

We want to thank Reviewer #2, He/She raises important issues and His/Her inputs were constructive for improving the manuscript. We have considered all the comments and suggestions, revising our manuscript accordingly.

# My biggest criticism is relatively minor, that anomalies in the patterns of settlement were not more thoroughly addressed. Specifically, the highest number of individuals was observed at the site with the lowest pH (6.6). The number of taxa there were comparable to the highest number of taxa at Vullatura.  Additionally, the lowest number of individuals and taxa were observed at a site with a moderate pH (7.8, Vullatura low pH site). These suggest that a local factor has as much of an effect on settlement between sites as pH does across the pH range. You discuss storm effects, but it’s likely that even simpler drivers are more important – proximity to breeding adults, local circulation, or strength of currents. I am not an expert at this literature but these proximal drivers should be addressed.

Thanks for your observation, we have inserted it now and better discussed this issue in the Discussion section (see lines 469-473)

# Another example of an anomaly that should be addressed: at both Castello north and Vullatura, from low pH to extremely low pH, the number of individuals and taxa at the bottom of the canopy did not decrease. In both cases individual numbers were lower at low pH than at the lowest pH. It’s an interesting pattern.

Thanks for this observation. We have now addressed this apparent anomaly with a statement in the Discussion section (see lines 458-463), at least explaining the possible trend of the number of individuals, probably favored by the reduced competition under extreme low pH conditions, respect to low pH stations.

# The manuscript would also be improved if the authors would explicitly quantify the decrease in settlement of individuals and taxa. From your figure 2, it looks like individuals decreased by 0 to 25% and taxa decreased by 10 to 40% from ambient to extremely-low pH. It would be useful to report the correct numbers in the abstract, and to add a paragraph of the discussion to compare these decreases to decreases in settlement observed in other studies.

Thanks for your suggestion; we have inserted the range of reduction that you provided into a statement in the Discussion section (see lines 469-480). However no comparison could be made with other studies on settlement since they are not available for molluscs under low pH conditions, except the data synthesized by Foo et al. [15] and Gaglioti et al. [73].

Minor changes

# Line 92: Please include more background information on mollusc settlement […]

Information on mollusc settlement have been included in the Introduction section (please see lines 99-106).

# Line 172. It’s not clear from your statement that this type of artificial collector has been used previously, only that artificial collectors in general have been used previously [...]

Thank you for the observation. We have stressed the effectiveness of the type of artificial collectors we used, and their large use in previous similar studies (please see lines 188-192).

# Line 254: you state that a total of 19613 molluscs were collected […]

The sentence has been corrected (please, check at line 273).

# Line 283: the first time you refer to microhabitat […]

Done (see lines 304-305).

# Figure 2: I recommend including an axis break from 200 to 300 for [..]

Figure 2 has been modified following Reviewer #2 suggestion.

# There are a few grammatical issues in the manuscript. I believe a grammar check

We have carefully checked and corrected throughout the text.

# Line 2, Title, I recommend changing “molluscs” to “mollusc”. “Molluscs settlement” without an apostrophe looks like a grammatical error.

Title has been changed following Reviewer #2 suggestion. The same grammatical error has been corrected throughout the manuscript.

# Line 19, “to assess the effect of decrease” should be “to assess the effect of a decrease”

Done (please, see line 20).

# Line 26: instead of “Simplified mollusc assemblages” I recommend “Reduced mollusc assemblages”

The sentence at line 27 has been corrected.

# Line 30-31 “...were the species making the differences observed along pH gradient, and were all relatively robust to OA conditions.” This sounds like a contradiction […]

The sentence at lines 31-32 has been changed as follows “were the species mostly found in low and extreme low pH stations, and were all relatively robust to OA acidified conditions.”

# Line 34-36: I made a few grammatical edits, and edits for clarity to the final sentence of the Abstract […]

The final sentence of the Abstract has been changed accordingly.

# Line 43: I suggest changing “reported nowadays” to “today”.

Done (please, see line 47).

Round 2

Reviewer 1 Report

The MS can be accepted

Author Response

Dear Prof. Lina Mtwana Nordlund, Diversity Guest Editor,

Thank you for the valuable suggestions. All the minor issues have been addressed. Track changes have been accepted, so the line numbers herein indicated refer to this new version of the manuscript. The corrections have been highlighted in yellow for easier comprehension.

• The sentence at lines 26-27 has been changed as follows “The number of taxa was lower in low and extreme low pH conditions”.
• The sentence at line 185-186 has been corrected.
• The comma has been deleted at line 188 and the sentence corrected following Editor’s comment.
• The word “bar” has been changed with the plural “bars” (line 191).
• Sentences at lines 192-194 have been rephrased.
• The word "without" has been spelled correctly at line 196.
• The sentence at lines 263-264 has been modified.
• The sentence at lines 270-273 has been changed as follows: “…the former showed lower abundance at the acidified stations (from 37% to 33%) if compared to the ambient pH stations. On the other hand, the Amphipoda were more abundant at low and extreme low pH stations (from 19% to 31% and 25%).”.
• We have stated, throughout the manuscript, that artificial collectors were collected after 33 days.
• The Figs 3 and 5 have been integrated with the values of PC1 and PC2.
• The shape and size of the scouring pads have been stated at line 181; the number of pads positioned at each sampling station has been specified in the Material and Methods section (line 192).
• The two paragraphs have been merged at line 70.
• We have avoided using the verb “decrease” throughout the whole manuscript when referring to mollusc assemblage because we did not measure before and after changes.
• Some of the sentences in the Discussion section have been modified to improve readability (lines 463-472; 507-511)
• Sentences at lines 518-523 have been modified to improve readability.
• Sentences at lines 540-542 have been modified to improve readability