eDNA Reveals the Associated Metazoan Diversity of Mediterranean Seagrass Sediments

Round 1

Reviewer 1 Report

 I have been over that MS a couple of times now.   I was concerned about between-core replicability, but au cleared that up...I am annoyed when I cannot find something to attack, but this is a good paper, an advance in the field, and ready to be accepted.  

Author Response

Review of manuscript diversity-1750027

 eDNA reveals the associated metazoan diversity of Mediterranean seagrass sediments

Reply to reviewer 1:

Comment 1: I have been over that MS a couple of times now.   I was concerned about between-core replicability, but au cleared that up...I am annoyed when I cannot find something to attack, but this is a good paper, an advance in the field, and ready to be accepted.  

Reply 1: We thank reviewer 1 for this positive review

Reviewer 2 Report

This is an interesting paper looking at changes in diversity in seagrass meadow communities over time and across species. The paper is clear and well written. I have made notes and comments directly in the PDF and I will restate my major concerns/criticisms here. 

Overall, the argument that Posidonia has higher species richness because of structural complexity lacks strong support. Only one meadow was sampled so it feels that statements to that effect should be tempered. So should statements about the loss of Posidonia meadows causing massive biodiversity loss. I think the use of eDNA is novel and the results are interesting but I would walk back some of the more speculative statements.

e.g. lines 26, 28, 127, 364 in particular, 375, 465, 487 in particular, 547 

In addition, the discussion could do with some editing, arguments are repeated several times which is unnecessary. 

Line 237 - please provide some stats in the text. 

 

Comments for author File: Comments.pdf

Author Response

Review of manuscript diversity-1750027

 

 

eDNA reveals the associated metazoan diversity of Mediterranean seagrass sediments

 

 

 

Reply to reviewer 2:

 

Comment 1: This is an interesting paper looking at changes in diversity in seagrass meadow communities over time and across species. The paper is clear and well written. I have made notes and comments directly in the PDF and I will restate my major concerns/criticisms here. Overall, the argument that Posidonia has higher species richness because of structural complexity lacks strong support. Only one meadow was sampled so it feels that statements to that effect should be tempered. So should statements about the loss of Posidonia meadows causing massive biodiversity loss. I think the use of eDNA is novel and the results are interesting but I would walk back some of the more speculative statements.e.g. lines 26, 28, 127, 364 in particular, 375, 465, 487 in particular, 547 

Reply 1: It is true than only one meadow of P. oceanica was sampled. However, it was a disturbed and impacted meadow located very close to the new port of Limassol, affected by intense human pressures (e.g. coastal constructions; Wesselmann et al., 2021). In fact, the organic carbon stocks within the sediment top 10 cm in this meadow (0.4 ± 0.1 kg Corg m^-2 Wesselmann et al., 2021) were the lowest stocks yet reported for this species (P. oceanica), lower than previous stocks of the species reported for the Eastern Mediterranean (0.5 ± 0.3 kg Corg m^-2 in 10 cm Apostolaki et al., 2019). This low organic carbon stocks could be explained because cores were taken from a disturbed and impacted meadow (Wesselmann et al., 2021), relative to the pristine meadows in previous studies where cores have typically been taken (Fourqurean et al., 2012). Similarly, the diversity of metazoan species in the sampled P. oceanica meadow might be reduced compared to non-disturbed meadows, as species diversity is reduced in sites impacted by antropogenic pressures (Reed and Hovel, 2006; Trush et al., 2001; Trush and Dayton, 2002). Thus, the metazoan diversity associated to P. oceanica reported in this study might be underestimated, and, if more meadows of P. oceanica would have been sampled (from less disturbed sites), it is expected that the metazoan diversity would be much higher than in this study and much higher than in meadows of C. nodosa and H. stipulacea. Moreover, even if only one meadow of P. oceanica was sampled, we included the number of cores per seagrass species in the statistical analysis (linear mixed effect model) and the model showed clear significant differences in species richness between the three seagrass species, with P. oceanica meadows having twice the richness of the other two seagrasses (Figure 1 and Supplementary Table S3). Therefore, we believe that the argument that Posidonia has higher species richness than the other two smaller seagrass species due to higher structural complexity is well supported, as shown by our data and the literature (see Buia et al., 2000). However, we have modified some of the speculative statements pointed out by the reviewer:

 

Action 1: In lines 27-32 we have modified the sentence “The combination of eDNA and sediment cores allowed us to reconstruct temporal patterns of metazoan community diversity and to demonstrate that the ongoing loss of P. oceanica in the Mediterranean Sea is indeed leading to major loss of biodiversity” to “The combination of eDNA and sediment cores allowed us to reconstruct temporal patterns of metazoan community diversity and provides a novel approach to follow natural communities back in time in the absence of time series and baseline data. The ongoing loss of P. oceanica meadows, likely to be compounded with future warming, might lead to a major loss of biodiversity and, the replacement by other seagrass species, whether native or exotic, does not compensate for the loss.”

 

We have deleted the sentence in line 375 “The shift from complex benthic habitats to simplified ones has been reported to lead to reduced diversity in the Mediterranean Sea (Pranovi et al., 2008; Bertolino et al., 2016; Piroddi et al., 2017; Verdura et al., 2019), and likely accounts for the step reduction in metazoan richness and biodiversity since the 1980’s.”

 

In line 444 - 450 we modified the sentence “Posidonia meadows do seem to have the highest metazoan diversity within this seagrass trio in the Mediterranean Sea” to “The metazoan diversity in P. oceanica meadows reported in this study might be underestimated, as only one meadow of P. oceanica was sampled and the core were taken from a highly disturbed and impacted meadow [27]. If more meadows of P. oceanica would have been sampled (from less disturbed sites), it is expected that the metazoan diversity would be much higher than in this study and much higher than in meadows of C. nodosa and H. stipulacea. Therefore, Posidonia meadows do seem to have the highest metazoan diversity within this seagrass trio in the Mediterranean Sea [53].

 

We have deleted the sentence in line 487 “Our results demonstrate also a high turnover of taxa during the past century, where losses of individual taxa in a seagrass meadow were compensated, in terms of taxonomic richness, by the recruitment of new taxa to the habitat, often represented by exotic species of subtropical origin introduced in the Mediterranean Sea from the Suez canal or the Gibraltar strait.”

 

We have deleted the sentence in line 547 “where taxa lost due to human pressures, such as overfishing”

 

Comment 2: In addition, the discussion could do with some editing, arguments are repeated several times which is unnecessary. 

Reply 2: We have edited the discussion and deleted several sentences (see reply to comment 1)

Comment 3: Line 237 - please provide some stats in the text. 

Reply 3: We have included the statistics in the text

Action 3: The text now reads in lines 222 - 228:

“Specifically, the linear mixed effect model detected significant differences in metazoan communities across seagrass meadows for 5 out of 15 phyla (Cnidaria, Porifera, Equinodermata, Arthropoda and Nematoda; p< 0.003) and for 6 out of 20 classes (Hydrozoa, Demospongiae, Hexanauplia, Homoscleromorpha and Echinoida; p< 0.002) after Bonferroni correction, with P. oceanica showing significantly higher richness than C. nodosa and H. stipulacea (Figure 2, Figure 3 and Supplementary Table S2).”

Comments from the PDF

 

Comment 1: Not quite. Line 27-30: The greater richness likely resulted from the more complex habitat provided by P. oceanica. The combination of eDNA and sediment cores allowed us to reconstruct temporal patterns of metazoan community diversity and to demonstrate that the ongoing loss of P.

 

Reply 1: we agree with reviewer and we have modified this sentence.

 

Action 1: the text now reads in lines 27-32 “We modified the sentence “The combination of eDNA and sediment cores allowed us to reconstruct temporal patterns of metazoan community diversity and to demonstrate that the ongoing loss of P. oceanica in the Mediterranean Sea is indeed leading to major loss of biodiversity” to “The combination of eDNA and sediment cores allowed us to reconstruct temporal patterns of metazoan community diversity and provides a novel approach to follow natural communities back in time in the absence of time series and baseline data. The ongoing loss of P. oceanica meadows, likely to be compounded with future warming, might lead to a major loss of biodiversity and, the replacement by other seagrass species, whether native or exotic, does not compensate for the loss.”

 

Comment 2: Of what? Lines 49-51: Unfortunately, most monitoring schemes started a few decades ago (Mihoub et al., 2017), well after most anthropogenic pressures began impacting biodiversity and shorter than natural climate cycles making it difficult to determine drivers (Hardesty et al., 2017).

 

Reply 2: we have clarified the text

Action 2: The text now reads in lines 48-51: “Unfortunately, most monitoring schemes started a few decades ago (Mihoub et al., 2017), well after most anthropogenic pressures began impacting biodiversity and shorter than natural climate cycles, making it difficult to determine drivers of changes in communities through time (Hardesty et al., 2017).”

 

Comment 3: Words missing here? Line 120: We do so by evaluating extracellular eDNA extracted from seagrass sediment cores dated with 210 Pb (Wesselmann et al 2021a) from eastern Mediterranean Sea (two sites in Greece and two sites in Cyprus) in the and analyzing the marine sediments using the Cytochrome Oydase 1 (CO1) metazoan.

 

Reply 3: We thank the reviewer for detecting that there are words missing in this line

 

Action 3: we have completed the text in lines 112-115:

“from the eastern Mediterranean Sea (two sites in Greece and two sites in Cyprus) and assess metazoans from marine sediments using primer pairs targeting a section of the Cytochrome Oydase 1 (CO1) gene.”

 

Comment 4: No replication here. Lines 127: We sampled 12 sediment cores from 8 seagrass meadows (4 Halophila stipulacea , 3 Cymodocea nodosa  and 1 Posidonia oceanica ) from the eastern Mediterranean Sea.

 

Reply 4: It is true that only one meadow of P. oceanica was sampled. However, we included the number of cores per seagrass species in the statistical analysis (linear mixed effect model) and the model showed clear significant differences in species richness between the three seagrass species, with P. oceanica meadows having twice the richness of the other two seagrasses (Figure 1 and Supplementary Table S2).

 

Comment 5: Why? Explain briefly. Line 195 - 198: “To analyse temporal changes in metazoan communities’ in seagrass meadows we used 47 sediment samples (out of the 88 sequenced), as and 22 samples were removed after decontamination and rarefaction.”

 

Reply 5: we explained this briefly in the text.

 

Action 5: The text now reads in lines 186-191:

“To analyse temporal changes in metazoan communities’ in seagrass meadows we initially used 69 sediment samples, as 19 samples could not been reliably dated due to sediment mixing. Rarefaction to 2000 reads was performed to account for uneven sequencing depth among samples. This resulted in the removal of 22 samples and the subsequent removal of 20 ASVs no longer present after rarefaction, leaving a total of 123 ASVs and 47 samples.”

 

Comment 6: What does this do? Why are ASVs removed? Line 222: We identified a total of 143 amplicon sequence variants (ASVs), which were reduced to 123 ASVs after rarefaction .

 

Reply 6: We rarefied samples to 2000 reads per sample as some samples had many reads and others very few. The number of amplicon sequence variants (ASVs) per sample ranged from 1 to 40 before rarefaction, although most reads were dominated by a few ASVs. Some amplicon sequence variants (20 out of 143 ASVs) were present by very few reads and therefore excluded during the rarefaction process. Also, see response to comment 5

 

Action 6: we have deleted this sentence as this information is redundant and is already described in lines 186-191.

 

Comment 7: Where are the statistics for this (line 237)?

 

Reply 7: We included the statistics missing in the text.

 

Action 7: The text now reads in lines 223-228: “Specifically, the linear mixed effect model detected significant differences in metazoan communities across seagrass meadows for 5 out of 15 phyla (Cnidaria, Porifera, Equinodermata, Arthropoda and Nematoda; p< 0.003) and for 6 out of 20 classes (Hydrozoa, Demospongiae, Hexanauplia, Homoscleromorpha and Echinoida; p< 0.002) after Bonferroni correction, with P. oceanica showing significantly higher richness than C. nodosa and H. stipulacea (Figure 2, Figure 3 and Supplementary Table S3).“

 

Comment 8: What is the line black here (Figure 1)?

 

Reply 8: Black lines refers to all three seagrass species combined

 

Action 8: We have clarified this in the legend of Figure 1:

“Alpha diversity measures (mean Richness and Shannon diversity index ± SE) of metazoan communities through time in seagrass meadows (H. stipulacea in green; C. nodosa in orange; P. oceanica in blue; overall of the three seagrass species in black). Dashed dark lines indicate non-significant differences between time intervals, considering the three seagrass species combined.”

Comment 9: How can there be a steep decrease and also no significant decrease? Also Figure 1 seems to show an increase. Line 244: “Richness of metazoan communities, showed a step decrease since the 1980’s to present, although the linear mixed effect model did not detect significant differences among time periods (Figure 1 and Supplementary Table S2).”

Reply 9: Figure 1 shows a decrease in richness through time (12.7 ± 3.5 from 1930 to 1959, 12.4 ±3.3 from 1960-1979, 10.4. ±2.1 from 1980 to 1997 and 10.9 ±1.0 from 1998 to 2017), although this decrease in richness is more noticeable in the samples of P. oceanica (23 ±7 from 1930 to 1959, 25 ±5 from 1960-1979, 21± 4 from 1980 to 1997 and 19± 5 from 1998 to 2017). Conversely, the Shannon diversity index remained stable through the different time periods (2.22 ±0.3 from 1930 to 1959, 2.16± 0.37 from 1960-1979, 2.17 ±0.17 from 1980 to 1997 and 2.29 ± 0.10) and showed a slight decrease in the samples of P. oceanica (3.10 ±0.31 from 1930 to 1959, 3.21 ±0.20 from 1960-1979, 3.04 ±0.21 from 1980 to 1997 and 2.90 ±0.26) (Figure 1).  Richness of metazoan communities (specially in P. oceanica meadows) showed a step decrease since the 1980’s to present, although the linear mixed effect model did not detect significant differences among time periods (Figure 1 and Supplementary Table S2).

 

Action 9: We have modified slightly the sentence, as most of the changes are found in P. oceanica samples, and the text now reads in lines 236-238:

“Richness of metazoan communities, especially in P. oceanica meadows, showed a step decrease since the 1980’s to present, although the linear mixed effect model did not detect significant differences among time periods (Figure 1 and Supplementary Table S3).”

 

Comment 10: Is these black lines for all seagrass species combined (line 273)?

 

Reply 10: yes, solid and dashed lines refer to all three seagrass species combined. We have clarified this in the text in the legend of Figure 1, 2 and 3.

 

Action 10: The legend of Figure 3 now reads:

“Temporal changes in richness (mean ± SE) in 10 metazoan classes within seagrass sediments (H. stipulacea in green; C. nodosa in orange; P. oceanica in blue; overall of the three seagrass species in black) from the eastern Mediterranean Sea (Greece and Cyprus). Solid dark lines indicate significant differences between time intervals and dashed dark lines indicate non-significant differences (p< 0.002 after Bonferroni correction), considering the three seagrass species combined.”

Comment 11: Explain this Kruskal stress = 0.16.

 

Reply 11: The kruskal’s stress value is used determine if the NMDS represents well the data, with values closer to zero providing a better fit of the data and values above 0.2 indicating a poor fit (Kruskal 1964).

 

Action 11: We deleted the Kruskal stress value from the text and included it in the legend of the Figure 5.

 

Comment 12: Figure 5 is unclear - some of the arrows are blocked by the words and I'm not sure what I'm supposed to be taking away from this.

 

Reply 12: We agree with reviewer and we have modified Figure 5. We have deleted panel B (Class), as the information was redundant (same as panel A “Phylum”) and Figure 5 shows now the NMDS detecting differences between the three seagrass species. Metazoan phyla’s were fitted on the ordination and their relative lengths indicate the correlation between the phyla’s and the NMDS. Samples forming a wider group (e.g. Posidonia samples) present higher variation between each other and indicate higher diversity, while samples that aggregate together show less variation between each other and indicate lower diversity.

Moreover, we have also included a new figure in the supplementary information (Figure S1) with the NMDS showing differences between the four time intervals. The period 1960-1979 shows the highest diversity as samples present higher variation between each other and form a wider group than recent time intervals (1998-2017).

 

Action 12: we have modified Figure 5 and included Figure S1 in the supplementary information.

 

Comment 13: How do you mean? Line 393-394: Therefore, this absence could also reflect more of a methodological limitation than an empirical change in the ecosystem.

 

Reply 13: The order Spariformes (~sparids) was absent in the samples from the most recent time periods (1980 – 2017) and present only in the older periods. This could be either due to increasing fishing pressure in the most recent years as many species of Sparid are targeted by fishing or it could also reflect a methodological limitation, as some primer pairs show amplification bias for some taxa, with the preferential capture of certain species and the lack of amplification of other (Ushio et al., 2018). Therefore, as sparids still remain some of the most abundant species of fish in and around the meadows analyzed here Azzurro et al., 2013; Harmelin-Vivien et al., 2005; kalogirou et al., 2010), the absence of sparids in the most recent time periods could also reflect more of a methodological limitation than an empirical change in the ecosystem.

 

Action 13: The text now reads in lines 377-382 “However, while many species of Sparid are indeed targeted by fishing, they remain some of the most abundant species of fish in and around the meadows analyzed here [64,65,66]. Therefore, as some primer pairs show amplification bias for some taxa, with the preferential capture of certain species and the lack of amplification of other [47], this absence might reflect more of a methodological limitation than an empirical change in the ecosystem.”

 

Comment 14: Your results show it did not. Line 462:  Therefore, it remains unclear if C. nodosa enhances metazoan diversity compared to H. stipulacea

 

Reply 14:  we agree with the reviewer, as our data indicate that the diversity is similar between H. stipulacea and C. nodosa (which is indicated in lines 454-455). Therefore, we have deleted the sentence” it remains unclear if C. nodosaenhances metazoan diversity compared to H. stipulacea”.

 

Comment 14: You only looked at one meadow. Line 465: “Posidonia meadows do seem to have the highest metazoan diversity within this seagrass trio in the Mediterranean Sea”

 

Reply 14: It is true that only one meadow of P. oceanica was sampled. However, we included the number of cores per seagrass species in the statistical analysis (linear mixed effect model) and the model showed clear significant differences in species richness between the three seagrass species, with P. oceanica meadows having twice the richness of the other two seagrasses (Figure 1 and Supplementary Table S3). In addition, see response to comment 1.

 

Comment 15: you have already said this (Line 488 – 491).

 

Reply 15: we agree with reviewer and we have deleted the sentence “Our results demonstrate also a high turnover of taxa during the past century, where losses of individual taxa in a seagrass meadow were compensated, in terms of taxonomic richness, by the recruitment of new taxa to the habitat, often represented by exotic species of subtropical origin introduced in the Mediterranean Sea from the Suez canal or the Gibraltar strait.”

 

Comment 16: This is very speculative. Line 545-547: “where taxa lost due to human pressures, such as overfishing..”

 

Reply 16: We agree with reviewer and we have modified the text

 

Action 16: The text now reads in lines 518-525:

“Our results demonstrate that species richness and diversity is higher in P. oceanica than in C. nodosa and H. stipulaceameadows and show a turnover of taxa, where taxa lost due to anthropogenic pressures are compensated by the recruitment of exotic taxa to the community.”

 

Comment 17: typographic errors are highlighted in the PDF

 

Reply 17: We corrected these typographic errors as suggested by the reviewer

 

Action 17: the text now reads in lines:

 

Line 20: we have changed “significant” to “significantly”

 

Line 23: we have changed “Phylum” to “Phyla”

 

Line 24: we have changed “Class” to “Classes”

 

Line 26: we have changed deleted “likely”

 

Line 29: we have changed “to major” to “to a major”

 

Line 40: we have changed “respond to to global warming” to “respond to global warming”

 

Line 42: we have changed “acclimation and adaptation,” to “acclimation and adaptation”

 

Line 42: we have inserted a coma after “insufficient”

 

Line 58: we have changed “cosuming” to “consuming”

 

Line 58 – 60: we included the citation and the text now reads in line 61: “and changes in observer over time [16]”.

 

Line 61: we have changed “for additional“ to “of additional”

 

Line 66: we have changed “this” to “the”

 

Line 67: we have deleted “massively”

 

Line 68: we have changed “short DNA region” to “short region of DNA”

 

Line 86: we have changed “sediments stabilization” to “sediment stabilization”

 

Line 92: we have changed “produce organic matter” to “produces organic matter”

 

Line 93: we have changed “refugee” to “refuge”

 

Line 101-106: we have changed “which is home for extensive seagrass meadows, including theendemic and most widespread seagrass Posidonia oceanica (Bethoux and Copin-Montégut, 1986; Pasqualini et al., 1998) the native Cymodocea nodosa which is increasing its distribution in some areas (Montefalcone et al., 2007) as well as the exotic (Indo-Pacific origin) seagrass Halophila stipulacea , which is found mostly in the eastern Mediterranean since 1923 (Winters et al., 2020) but expanding into the western Mediterranean (Gambi et al., 2009; Orfanidis et al., 2021; Thibaut et al., 2022) and is forecasted to continue doing so in the future (Wesselmann et al., 2021b)” to “which is home to extensive seagrass meadows, including the endemic and most widespread seagrass Posidonia oceanica [36,37], the native Cymodocea nodosa which is increasing its distribution in some areas [38], as well as the exotic (Indo-Pacific origin) seagrass Halophila stipulacea, which has been found mostly in the eastern Mediterranean since 1923 [39] but is expanding into the western Mediterranean [40,41,42] and is forecasted to continue doing so [43].”

 

Line 109: we have changed “the Mediterranean is highly impacted” to “the Mediterranean has been highly impacted”

 

Line 114: we have changed “long-term changes on” to “long-term changes of”

 

Line 118: we have changed “cores in the eastern Mediterranean (1) to explore” to “cores in the eastern Mediterranean to (1) explore”

 

Line 122: we have changed “from eastern Mediterranean Sea” to “from the eastern Mediterranean Sea”

 

Line 138: we have changed “whereas” to “since”

 

Line 139: we have changed “meadows” to “meadow”

 

Line 176: we have changed “sequences were then analyzed” to “Sequences were analyzed”

 

Line 177: we have changed “(correct for errors)” to “(correcting for errors)”

 

Line 191: we have changed “presence absence” to “presence/absence”

 

Line 190-193: we have changed “uncertain” to “unreliable”.

 

Line 195: we have changed “metazoan communities’” to “metazoan communities”

 

Line 196: we have changed “could not been reliably” to “could not be reliably”

 

 

Line 201: we have changed “time period and account” to “time period and to account”

 

Line 208: we have changed “with package phyloseq” to “with the package phyloseq”

 

Line 234: we have changed “metazoan communities over seagrass” to “metazoan communities across seagrasss”

 

Line 255-257: we have changed “The Phylum Chordata did not present differences in richness through time, but at a lower taxonomic level the bony fishes (Class Actinopteri) presented significantly higher species richness” to “The Phylum Chordata did not show differences in richness through time, but at a lower taxonomic level the bony fishes (Class Actinopteri) had significantly higher species richness”

 

Line 260: we have changed deleted the coma after “and”

 

 

Line 279: we have changed “The Phylum Cnidaria did also not present differences” to “The Phylum Cnidaria also did not show differences”

 

Line 283: we have changed “as the order “ to ”as was seen for the orders”

 

Line 289: “Conversely, the Phylum Porifera presented a significant increasing” richness to “Conversely, the Phylum Porifera showed a significant increase in richness”

 

Line 295: we have changed “which also presented higher richness with time, although not significant to “ which also presented higher richness with time, though this trend was not significant”

 

Line 300: we have changed “Besides, other Arthropoda” to “Other Arthropoda”

 

Line 303: the text now reads “Mollusk richness remained constant through time (Figure 2 and 3)”.

 

Line 317: we have changed “with P. oceanica presenting the highest richness” to “with P. oceanica having the highest richness”

 

Line 318: we have changed “brittel stars” to “brittle stars”

 

Line 328: we have changed “simplified” to “simpler”

 

Line 344: we have changed “disperse” to “dispersed”

 

Line 356: we have changed “in the eastern Mediterranean seagrass meadows” to “in eastern Mediterranean seagrass meadows”

 

Line 358: we have changed “assess” to “assessing”

 

Line 365: we have changed “structural” to “structurally”

 

Line 385: we have changed “this” to “these”

 

Line 389: we have changed “increase” to “increased”

 

Line 390: we have changed “with closure” to “ with the closure”

 

 

Line 400: we have changed “therefore,” to “therefore”

 

Line 401: we have changed “increase in the trade and the globalization” to “increase in trade and globalization

 

Line 403: we have deleted “period”

 

Line 406: we have changed “typical” to “typically”

 

Line 412: we have changed “suggested” to “suggest”

 

Line 414: we have changed “in the 1998” to “in 1998”

 

Line 417: we have changed “detect” to “detected”

 

Line 421: we have changed “play” to “playing”

 

Line 422: we have changed “developing” to “with”

 

Line 424: we have changed “associated to” to “associated with”

 

Line 426: we have changed “the higher species” to “higher species”

 

Line 431: we deleted the sentence “Our results show metazoan groups to be richer and more diverse in P. oceanicameadows”

 

Line 437: we have changed “the surface area” to “surface area”

 

Line 444: we have changed “turn-over leaves” to “turn-over of leaves”

 

Line 481: we have changed “meadows that were from P. oceanica or C. nodosa” to “meadows that were P. oceanica or C. nodosa”

 

Line 486: we have changed “warmer thermal affinity with ocean warming” to “warmer thermal affinity to ocean warming”

 

Line 502: we have changed “diversity patterns at order level” to “diversity patterns at the order level”

 

Line 535: we have changed “presence absence” to “presence/absence”

 

Bibliography:

 

Apostolaki, E. T., Vizzini, S., Santinelli, V., Kaberi, H., Andolina, C., & Papathanassiou, E. (2019). Exotic Halophila stipulacea is an introduced carbon sink for the Eastern Mediterranean Sea. Scientific reports, 9(1), 1-13.

 

Azzurro, E., La Mesa, G., & Fanelli, E. (2013). The rocky-reef fish assemblages of Malta and Lampedusa islands (Strait of Sicily, Mediterranean Sea): a visual census study in a changing biogeographical sector. J. Mar. Biolog. Assoc. U.K., 93(8), 2015-2026.

 

Buia, M. C., Gambi, M. C., & Zupo, V. (2000). Structure and functioning of Mediterranean seagrass ecosystems: an overview. Biologia Marina Mediterranea, 7(2), 167-190.

 

Fourqurean, J. W., Duarte, C. M., Kennedy, H., Marbà, N., Holmer, M., Mateo, M. A., ... & Serrano, O. (2012). Seagrass ecosystems as a globally significant carbon stock. Nature geoscience, 5(7), 505-509.

 

Harmelin-Vivien, M. L., Bitar, G., Harmelin, J. G., & Monestiez, P. (2005). The littoral fish community of the Lebanese rocky coast (eastern Mediterranean Sea) with emphasis on Red Sea immigrants. Biol. Invasions, 7(4), 625-637.

 

Kalogirou, S., Corsini‐Foka, M., Sioulas, A., Wennhage, H., & Pihl, L. (2010). Diversity, structure and function of fish assemblages associated with Posidonia oceanica beds in an area of the eastern Mediterranean Sea and the role of non‐indigenous species. J. Fish Biol., 77(10), 2338-2357.

 

Kruskal, J. B. (1964). Nonmetric multidimensional scaling: a numerical method. Psychometrika, 29(2), 115-129.

 

Reed, B. J., & Hovel, K. A. (2006). Seagrass habitat disturbance: how loss and fragmentation of eelgrass Zostera marinainfluences epifaunal abundance and diversity. Marine Ecology Progress Series, 326, 133-143.

 

Thrush, S. F., & Dayton, P. K. (2002). Disturbance to marine benthic habitats by trawling and dredging: implications for marine biodiversity. Annual review of ecology and systematics, 449-473.

 

Thrush, S. F., Hewitt, J. E., Funnell, G. A., Cummings, V. J., Ellis, J., Schultz, D., ... & Norkko, A. (2001). Fishing disturbance and marine biodiversity: role of habitat structure in simple soft-sediment systems. Marine ecology progress series, 221, 255-264.

 

Ushio, M., Murakami, H., Masuda, R., Sado, T., Miya, M., Sakurai, S., ... & Kondoh, M. (2018). Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing. Metabarcoding Metagenom., 2, e23297.

 

Wesselmann, M., Geraldi, N. R., Duarte, C. M., Garcia‐Orellana, J., Díaz‐Rúa, R., Arias‐Ortiz, A., ... & Marbà, N. (2021). Seagrass (Halophila stipulacea) invasion enhances carbon sequestration in the Mediterranean Sea. Global Change Biology, 27(11), 2592-2607.

 

Author Response File: Author Response.pdf