Key Drivers for Copepod Assemblages in a Eutrophic Coastal Brackish Lake
, Gamal El-Shabrawy 2, Mohamed E. Goher 2, Madhavapeddi N. V. Subrahmanyam 3,4, Yousef Al-Enezi 3, Mohammad Ali 3, Habib Ayadi 1 and Genuario Belmonte 5,*Round 1
Reviewer 1 Report
The presented study aimed to determine effects of eutrophication (the supply of biogenic elements) and salinity levels on the spatial distribution of Copepoda assemblages in Lake Manzalah, a eutrophic coastal brackish lake located in the Nile Delta. Another important practical aspect of the study is an analysis of the occurrence of non-indigenous species (NIS) as affected by environmental conditions, and their use as potential bioindicators. Therefore, the manuscript deserves to be published, but it should be revised before re-submission. The following points need to be addressed:
1. The trophic status index (TSI) calculated in the study is not the classical Carlson’s index; actually, it is one of the three indices proposed by Carlson (as mentioned in the Materials and Methods section). Therefore, the abbreviation TSI with the letters TP in the subscript (TSITP) should be used in this section and in lines 180-186 and 237, including the relevant description.
2. I have serious doubts regarding the correctness of PCA. The calculation procedure should be described in more detail – which software was used? were raw or transformed data analyzed? were environmental data and species abundance data analyzed simultaneously? This is an important consideration since the classical PCA is usually performed to determine the gradient of homogeneous data (e.g. environmental conditions) or to find correlated parameters. Highly diverse datasets – species abundance data are incomplete (Table 2) – should be analyzed with the use of a dedicated package containing Canonical Correspondence Analysis (CCA) algorithms. In my opinion, CCA of environmental and biological data should be performed or the analysis should be limited to differences in environmental conditions only (PCA) or this passage and Figure 8 should be deleted.
3. As shown in Figures 2 and 3, environmental conditions in the studied lake were highly variable, and the gradient of changes was very broad – e.g. in autumn and winter, ammonium concentration at Site 1 (S1) corresponded to the values typical of treated wastewater. The remaining sites could also be grouped/clustered based on habitat conditions: the northern part was influenced by the sea whereas the southern part was influenced by inland freshwaters and more affected by euthrophication. The Authors stressed that the two parts of the lake are morphometrically separated, which was also confirmed by the observed differences in the spatial distribution of copepods. Therefore, the conditional should not be used in the Abstract (lines 29 - 31) and when formulating conclusions from the study (e.g. line 358: “seems to be”).
Author Response
1st reviewer
Comments and Suggestions for Authors
The presented study aimed to determine effects of eutrophication (the supply of biogenic elements) and salinity levels on the spatial distribution of Copepoda assemblages in Lake Manzalah, a eutrophic coastal brackish lake located in the Nile Delta. Another important practical aspect of the study is an analysis of the occurrence of non-indigenous species (NIS) as affected by environmental conditions, and their use as potential bioindicators. Therefore, the manuscript deserves to be published, but it should be revised before re-submission. The following points need to be addressed:
1. The trophic status index (TSI) calculated in the study is not the classical Carlson’s index; actually, it is one of the three indices proposed by Carlson (as mentioned in the Materials and Methods section). Therefore, the abbreviation TSI with the letters TP in the subscript (TSITP) should be used in this section and in lines 180-186 and 237, including the relevant description.
Ok, we change TSI by TSITP in the manuscript and figure 4
2. I have serious doubts regarding the correctness of PCA. The calculation procedure should be described in more detail – which software was used? were raw or transformed data analyzed? were environmental data and species abundance data analyzed simultaneously? This is an important consideration since the classical PCA is usually performed to determine the gradient of homogeneous data (e.g. environmental conditions) or to find correlated parameters. Highly diverse datasets – species abundance data are incomplete (Table 2) – should be analyzed with the use of a dedicated package containing Canonical Correspondence Analysis (CCA) algorithms. In my opinion, CCA of environmental and biological data should be performed or the analysis should be limited to differences in environmental conditions only (PCA) or this passage and Figure 8 should be deleted.
The PCA was performed simultaneously on environmental and species abundance data after standardization.. Actually we first performed two separate PCA and a co-inertia analysis (using R package ade4) but we did not find any interesting results to report (weak RV value). Also CCA analysis using appropriate packages in R did not provide high correlations between the two sets of variables (species abundance and environmental parameters). Only PCA was able to give some indication on the relationship between the abundance of some species and key environmental parameters. For this reason we choose to present only PCA results. In this current version and as the 2nd reviewer suggests we performed a separate PCA on environmental variables and We then computed the correlation between species abundance (one by one) with the First and second PCA axis and we get similar conclusion as those derived from the PCA performed simultaneously on environmental and species abundance . We added two sentences in materials and method to give further details on the PCA.
3. As shown in Figures 2 and 3, environmental conditions in the studied lake were highly variable, and the gradient of changes was very broad – e.g. in autumn and winter, ammonium concentration at Site 1 (S1) corresponded to the values typical of treated wastewater. The remaining sites could also be grouped/clustered based on habitat conditions: the northern part was influenced by the sea whereas the southern part was influenced by inland freshwaters and more affected by euthrophication. The Authors stressed that the two parts of the lake are morphometrically separated, which was also confirmed by the observed differences in the spatial distribution of copepods. Therefore, the conditional should not be used in the Abstract (lines 29 - 31) and when formulating conclusions from the study (e.g. line 358: “seems to be”).
- Ok we delete probably in abstract lines 29-31
In conclusion we change “seems to be” with “is”, line 358
Submission Date
04 December 2018
Date of this review
24 Dec 2018 09:22:07
Author Response File: 
Author Response.docx
Reviewer 2 Report
General comments:
The present study conducted seasonal sampling of the copepod community and its physical environment of the eutrophic lake Manzalah. According to physical characteristics, the lake was divided into northern and southern parts. Nine copepod species were found and related to physical parameters. The authors conclude that the heterogenous progress of eutrophication support the coexistence of the copepod species including non-indigenous species in the lake. Although the reviewer acknowledges the aim of the present study, the manuscript includes the following problems.
1. Most of Discussion is not supported by Results. The most suitable analysis, specifically statistical analysis, should be selected to classify and correlate physical and biological data. In addition, it is required to clearly and precisely describe the methods employed.
2. Results and Discussion are not well organized. Some descriptions should be summarized in tables. Division into several subsections may be useful.
3. Grammatical mistakes and ambiguous expressions are found everywhere. To avoid confusion, it is better to use a specific term for a single object throughout the manuscript: e.g., are ‘copepod assemblages’, ‘copepod community’ and ‘species assemblages’ identical or different?
Specific comments:
P1, L2-3: It is unclear of what ‘spatial distribution’ is studied. Spatial distribution of key drivers or copepod assemblages?
P1, L18: To be consistent with the title, replace ‘The copepod community’ by ‘Copepod assemblages’.
P1, L18: ‘investigated’ or ‘sampled’ is better than ‘analyzed’.
P1, L18: ‘in 2009-2010’ should be moved after the information about place.
P1, L20: ‘species composition’ or ‘community structure’ is better than ‘species assemblages’.
P1, L20-21: To be consistent with the title, replace ‘The copepod community’ by ‘Copepod assemblages’.
P1, L21: It is unclear what ‘with respect to previous years’ means. The authors seem to mean ‘for the past several decades’.
P1, 21-22: The authors seem to mean ‘possibly because of eutrophication and invasions of non-indigenous species (NIS)’.
P1, L22-23: The sentence is unclear in both structure and meaning.
P1, L23: In Results, the authors describe ‘nine copepod species … were found.
P1, L24: Replace ‘have been reported’ by ‘were reported’.
P1, L26: What does ‘selected portions’ mean?
P1, L29: Describe the present situation is how different from the past situation. Richer in species diversity or total abundance?
L1, P29: ‘Invasions of NIS’ is better than ‘The affirmation of NIS’.
L1, P30 Replace ‘added to the increasing eutrophication in parts of the lake’ by ‘in addition to the heterogeneous progress of eutrophication in the lake’.
P1-2, L35-L62: The order of the first four paragraphs is not well organized: the first and third paragraphs describe specific effects of eutrophication on copepods, whereas the second and fourth ones explain general causes and results of eutrophication. Explain eutrophication generally before describing its specific effects on copepods.
P1, L37-38: Replace ‘opportunistic-dominated’ by ‘opportunist-dominated’.
P2, L49: Replace ‘increasing’ by ‘increases’.
P2, L51: What does ‘Certain Copepoda are not conditioned by the trophic status’ mean? The authors seem to mean that several copepod species are tolerant to wide varieties of trophic status.
P2, L68: What does ‘benthos quality’ mean?
P2, L72: Replace ‘assemblages’ by ‘composition’.
P2, L73: Delete ‘among’.
P2, L79-89: Climatic and hydrographic conditions (e.g. seasonality) should be described concisely.
P2, L82-83: ‘through which pass three channels … and El-Gamil 2)’ is difficult to understand. ‘El-Boughdady’ cannot be found in Fig. 1.
P2, L88: Delete the apostrophe from ‘1970’s’.
Fig. 1: The notation of latitude and longitude should be consistent with that in the text. If possible, it is more informative to show the locations of major islands, fish farms and reclaimed areas on the map.
P3, L100-101: How did the authors determine the four sampling dates? Do they represent the four seasons?
P4, L113-114: Does ‘TSI’ mean ‘trophic status index’ or ‘trophic state index’? Technical terms should be used consistently through the manuscript.
P4, L116-117: The break of the paragraph is unnecessary.
P4, L123-124: It is necessary to cite references used for the identification. Does ‘high-level taxa’ mean classes or higher taxonomic levels? Replace ‘according to’ by ‘by’.
P4, L128: Delete ‘analysis’.
P4, L130: Use ‘stations’ instead of ‘points’.
P4, L132-133: The sentence is unclear in both structure and meaning.
P4, L133-134: How to apply Pearson’s correlation analysis is unclear. Use caution in applying Pearson’s correlation analysis, because it is subject to outlier(s) when the data are small. In such a case, Spearman’s correlation analysis is better.
P4, L135-136: How to apply PCA is unclear. Why were copepod data included in PCA? How were different units treated in PCA?
P4, L140-141: Although temperature changes with season, it is unclear why the authors focus on spatial differences in the annual mean temperature.
P4, L145-146: Seasonality in salinity is unclear, partly because salinity at S2 changed with season differently than salinity at S4, S7 and S10. Moreover, seasonal changes in salinity are not consistent even within S4, S7 and S10.
P4, L146-147: Replace ‘The pH in Lake Manzalah’ by ‘pH’.
P4-5, L149-169: The descriptions of the result of correlation analysis seem to be sporadic. Summarizing them in a table may be better.
P5, L151: Choose one word from ‘station’, ‘point’ and ‘site’ to use it consistently through the manuscript.
Figs. 2-3: ‘Spatiotemporal variations’ is better than ‘Seasonal variations of …’.
P8, L187: Replace ‘assemblage composition’ by ‘species composition’.
P8, L188: Insert ‘the’ before ‘three orders’.
P8, L189: The meaning of ‘the most represented order’ is unclear. The authors seem to mean ‘the richest in species’ or ‘the most diverse in species’.
P8, L194: The citation of Table 1 seems unnecessary.
P8, L199: The density of nauplius are much higher than that of copepodid in summer (Fig. 5). This means that ‘nauplius/copepodid ratio’ is higher than one.
Table 2: The values in the table seem too small, given that the unit is ‘ind m^-3’. In the caption, explain what stage is included (or excluded). The explanation of H’ is missing.
Fig. 5: ‘Spatiotemporal…’ is better than ‘Seasonal and spatial…’.
Fig. 6: The same as the above. For a better visibility, use different colors for different species.
P13-14, L236-244: The descriptions of the result of PCA seem to be unprecise. It is unclear how the authors defined G1 and G2, and LS and HS. The reviewer recommends the authors to apply PCA only to physical parameters to produce a small number of combined parameters (i.e. PC1, PC2, …). Then, plot the density of each species in relation to the combined parameters.
P14, L246-255: The representativity of the data should be confirmed. Were they obtained from average years? The result of ANOVA indicated significantly higher salinities only at S2 and S7. The classification into the brackish- and fresh-water groups seems to be subjective. Multivariate analysis of physical parameters should be conducted to objectively classify the stations into several groups.
P14, L256-271: It is unclear why nitrogen is limited despite large nitrogen input from anthropogenic activities. In other words, it is necessary to explain reasons for excessive phosphorous input.
P14, L258-259: ‘the growing population in the Nile delta has increased the discharge of municipal, agricultural, and industrial wastes’ may be simpler and better.
P14, L267: Replace ‘exaggeratedly’ by ‘highly’.
P14-15, L272-291: Although the authors relate A. tonsa with eutrophication, there is no evidence concerning the link between A. tonsa and eutrophication in the lake. Indeed, A. tonsa was found mainly in the less eutrophic part of the lake. Discussion should be supported by the result of the present study.
P14, L276-277: Replace ‘confined coastal environments’ by ‘coastal embayments’.
P14, L280: Replace ‘thanks to’ by ‘due to’.
P14, L281: What is ‘equipment’?
P14, L284: Replace ‘affirmation’ by ‘occurrence’ or ‘settlement’.
P14, L286: Replace ‘affirmed itself’ by ‘settled’.
Fig. 7: Instead of annual averages, use raw values that were observed at each station in each season. Use italics for scientific names.
Fig. 8: The result is difficult to interpret. As is mentioned above, PCA should be applied only to physical parameters to produce a small number of combined parameters (i.e. PC1, PC2, …). Then, the density of copepod species should be related to the combined parameters.
P15-16, L300-316: The descriptions are not well organized. It is better to summarize information about the occurrence of copepod species in a table.
P16, L318-335: The reviewer generally agrees with the authors’ opinions. However, the authors’ opinions are not persuasive, because the statistical analyses employed are not enough to support them.
P16, L323: What are ‘the marine pole’ and ‘the land pole’? Definition is necessary.
P16, L332: What is ‘lake extension’?
P16, L336-346: This paragraph should be combined with the third paragraph of the discussion.
P16, L236: Replace ‘affirmation’ by ‘settlement’.
Author Response
Comments and Suggestions for Authors
General comments:
The present study conducted seasonal sampling of the copepod community and its physical environment of the eutrophic lake Manzalah. According to physical characteristics, the lake was divided into northern and southern parts. Nine copepod species were found and related to physical parameters. The authors conclude that the heterogeneous progress of eutrophication support the coexistence of the copepod species including non-indigenous species in the lake. Although the reviewer acknowledges the aim of the present study, the manuscript includes the following problems.
1. Most of Discussion is not supported by Results. The most suitable analysis, specifically statistical analysis, should be selected to classify and correlate physical and biological data. In addition, it is required to clearly and precisely describe the methods employed.
2. Results and Discussion are not well organized. Some descriptions should be summarized in tables. Division into several subsections may be useful.
3. Grammatical mistakes and ambiguous expressions are found everywhere. To avoid confusion, it is better to use a specific term for a single object throughout the manuscript: e.g., are ‘copepod assemblages’, ‘copepod community’ and ‘species assemblages’ identical or different?
Specific comments:
P1, L2-3: It is unclear of what ‘spatial distribution’ is studied. Spatial distribution of key drivers or copepod assemblages?
We delete spatial distribution. Title now is : Key drivers for copepod assemblages in an eutrophic coastal brackish lake, because we study seasonal and spatial distribution of copepods assemblages in relation to spatial and seasonal variation of physic-chemicals variables.
P1, L18: To be consistent with the title, replace ‘The copepod community’ by ‘Copepod assemblages’.
Ok , done
Also in line P1, L18: ‘investigated’ or ‘sampled’ is better than ‘analyzed’.
Ok, analyzed has been substituted with investigated
P1, L18: ‘in 2009-2010’ should be moved after the information about place.
Ok , done
P1, L20: ‘species composition’ or ‘community structure’ is better than ‘species assemblages’.
Ok we use species composition
P1, L20-21: To be consistent with the title, replace ‘The copepod community’ by ‘Copepod assemblages’.
Ok , done
P1, L21: It is unclear what ‘with respect to previous years’ means. The authors seem to mean ‘for the past several decades’.
We mean compared with previous studies in previous years (years has been changed with studies)
P1, 21-22: The authors seem to mean ‘possibly because of eutrophication and invasions of non-indigenous species (NIS)’.
Yes. The change has been inserted
P1, L22-23: The sentence is unclear in both structure and meaning.
We change the sentence : The aim of the present study is the identification of species which can be used as ecological indicators of high trophic status.
P1, L23: In Results, the authors describe ‘nine copepod species … were found.
We corrected it is 9 not 11.
P1, L24: Replace ‘have been reported’ by ‘were reported’.
OK, done.
P1, L26: What does ‘selected portions’ mean?
A. tonsa was found only at 5 stations (S2, S4, S7, S9, and S10) , thus we refer to them as “some” portions of Lake Manzalah.
P1, L29: Describe the present situation is how different from the past situation. Richer in species diversity or total abundance?
It is richer in species
L1, P29: ‘Invasions of NIS’ is better than ‘The affirmation of NIS’.
Ok , done
L1, P30 Replace ‘added to the increasing eutrophication in parts of the lake’ by ‘in addition to the heterogeneous progress of eutrophication in the lake’.
OK, done
P1-2, L35-L62: The order of the first four paragraphs is not well organized: the first and third paragraphs describe specific effects of eutrophication on copepods, whereas the second and fourth ones explain general causes and results of eutrophication. Explain eutrophication generally before describing its specific effects on copepods.
OK , done
P1, L37-38: Replace ‘opportunistic-dominated’ by ‘opportunist-dominated’.
OK, done
P2, L49: Replace ‘increasing’ by ‘increases’.
OK , done
P2, L51: What does ‘Certain Copepoda are not conditioned by the trophic status’ mean? The authors seem to mean that several copepod species are tolerant to wide varieties of trophic status.
The part has been changed: ‘Certain Copepoda are not negatively affected by high trophic levels’
P2, L68: What does ‘benthos quality’ mean?
Quality has been deleted, and the benthos has been considered in general as the sediment (adjacent in the text). The reference (36) contains the details of this affirmation, which refers to aquatic macrophytes.
P2, L72: Replace ‘assemblages’ by ‘composition’.
OK , done
P2, L73: Delete ‘among’.
OK , done
P2, L79-89: Climatic and hydrographic conditions (e.g. seasonality) should be described concisely.
We add :
It has been reported that the maximum mean value of salinity during winter in the lake was 9.56 ± 14.31 and it gradually declined during summer (9.40 ± 13.19 ‰) through spring (8.06 ± 10.50 ‰), and reached its minimum value during autumn (7.54 ± 7.25 ‰) (Abdel-Rasheed 2011). Lake water temperature maximum mean value was reported to reach 30.7 ± 1.57 ºC during summer, and the minimum was observed during winter (15.6 ± 0.97 ºC), while the average values of water temperature during spring and autumn, however, were nearly similar; being 22.6 ± 0.7 and 23.3 ± 1.49 ºC, respectively (Abdel-Rasheed 2011).
P2, L82-83: ‘through which pass three channels … and El-Gamil 2)’ is difficult to understand. ‘El-Boughdady’ cannot be found in Fig. 1.
Changed with: interrupted at level of three channels.
Fig 1 was corrected
P2, L88: Delete the apostrophe from ‘1970’s’.
OK, done
Fig. 1: The notation of latitude and longitude should be consistent with that in the text. If possible, it is more informative to show the locations of major islands, fish farms and reclaimed areas on the map.
Fig 1. is corrected
P3, L100-101: How did the authors determine the four sampling dates? Do they represent the four seasons?
The four dates represented four seasons and were useful for the study of seasonal variability, other than for comparisons with previous studies.
P4, L113-114: Does ‘TSI’ mean ‘trophic status index’ or ‘trophic state index’? Technical terms should be used consistently through the manuscript.
We correct : Trohic state index
P4, L116-117: The break of the paragraph is unnecessary.
Ok we delete the break of paragraph
P4, L123-124: It is necessary to cite references used for the identification. Does ‘high-level taxa’ mean classes or higher taxonomic levels? Replace ‘according to’ by ‘by’.
Copepods nauplii were considered as a single group; copepodids were counted and grouped according to the order (Calanoida, Cyclopoida, Harpacticoida), and adults were grouped according the species. Species were identified according to keys and references [44-45]. Abundances were expressed as individuals per cubic metre (ind m-3).
P4, L128: Delete ‘analysis’.
Ok, done
P4, L130: Use ‘stations’ instead of ‘points’.
Ok, done
P4, L132-133: The sentence is unclear in both structure and meaning.
We change: Prior to the ANOVA tests, the normality of all variables was checked by means of the Shapiro-wilk test and log-transform was used when necessary. Homogeneity of variance of all variables among sites was assessed using Levene's test.
P4, L133-134: How to apply Pearson’s correlation analysis is unclear. Use caution in applying Pearson’s correlation analysis, because it is subject to outlier(s) when the data are small. In such a case, Spearman’s correlation analysis is better.
We have a dataset with 44 observatons (>30) and in this condition applying Pearson correlation is possible, particularly that we checked for the Normality of distribution of the variables and log-transformed those who showed significant departure from normality. Spearman correlation coefficient values were very close the Pearson ones.
P4, L135-136: How to apply PCA is unclear. Why were copepod data included in PCA? How were different units treated in PCA?
As for the Reviewer 1: The PCA was performed simultaneously on environmental and species abundance data after standardization. Actually we first performed two separate PCA and a co-inertia analysis (using R package ade4) but we did not find any interesting results to report (weak RV value). Also CCA analysis using appropriate packages in R did not provide high correlations between the two sets of variables (species abundance and environmental parameters). Only PCA was able to give some indication on the relationship between the abundance of some species and key environmental parameters. For this reason we choose to present only PCA results. In this current version and as the 2nd reviewer suggests we performed a separate PCA on environmental variables and We then computed the correlation between species abundance (one by one) with the First and second PCA axis and we get similar conclusion as those derived from the PCA performed simultaneously on environmental and species abundance . We added two sentences in materials and method to give further details on the PCA.
P4, L140-141: Although temperature changes with season, it is unclear why the authors focus on spatial differences in the annual mean temperature.
The part has been re-written focusing both on 4 seasons and on 11 sampling stations.
P4, L145-146: Seasonality in salinity is unclear, partly because salinity at S2 changed with season differently than salinity at S4, S7 and S10. Moreover, seasonal changes in salinity are not consistent even within S4, S7 and S10.
See the answer of the preceding point, and changes in the manuscript
P4, L146-147: Replace ‘The pH in Lake Manzalah’ by ‘pH’.
OK, done
P4-5, L149-169: The descriptions of the result of correlation analysis seem to be sporadic. Summarizing them in a table may be better.
Ok, done in table 2
P5, L151: Choose one word from ‘station’, ‘point’ and ‘site’ to use it consistently through the manuscript.
Ok we choose “station”
Figs. 2-3: ‘Spatiotemporal variations’ is better than ‘Seasonal variations of …’.
OK , done
P8, L187: Replace ‘assemblage composition’ by ‘species composition’.
Ok, done
P8, L188: Insert ‘the’ before ‘three orders’.
Ok, done
P8, L189: The meaning of ‘the most represented order’ is unclear. The authors seem to mean ‘the richest in species’ or ‘the most diverse in species’.
OK, done
P8, L194: The citation of Table 1 seems unnecessary.
Ok we delete the citation of Table 1
P8, L199: The density of nauplius are much higher than that of copepodid in summer (Fig. 5). This means that ‘nauplius/copepodid ratio’ is higher than one.
It is a mistake not nauplius/copepodid it is nauplius/total copepods (nauplius+copepodid+ adults)
We do the calculation of percentage of nauplii we find :
The average annual nauplius contribution was 67.4% and the maximum was about 92.8% at S5 during autumn.
Table 2: The values in the table seem too small, given that the unit is ‘ind m^-3’. In the caption, explain what stage is included (or excluded). The explanation of H’ is missing.
-The numbers are given by × 103 ind m-3
-only adults were considered
-We add in Materials and Methods: Shannon’s index [46], expressed as H’ = ∑Pi Log2Pi, where Pi = dominance of species i, computed as Pi = ni/N, ni = number of species i in sample, and N = number of samples.
Fig. 5: ‘Spatiotemporal…’ is better than ‘Seasonal and spatial…’.
Ok , done
Fig. 6: The same as the above. For a better visibility, use different colors for different species.
Ok , done
P13-14, L236-244: The descriptions of the result of PCA seem to be unprecise. It is unclear how the authors defined G1 and G2, and LS and HS. The reviewer recommends the authors to apply PCA only to physical parameters to produce a small number of combined parameters (i.e. PC1, PC2, …). Then, plot the density of each species in relation to the combined parameters.
Ok , done; and results are given in response to reviewer 1.
P14, L246-255: The representativity of the data should be confirmed. Were they obtained from average years? The result of ANOVA indicated significantly higher salinities only at S2 and S7. The classification into the brackish- and fresh-water groups seems to be subjective. Multivariate analysis of physical parameters should be conducted to objectively classify the stations into several groups.
The ANOVA was performed using a two-way classification with two factors: seasons and sites and we found that these two factors were significant (in both univariate and multivariate ANOVA) with R square value varying form 60% (salinity) to 99% (temperature).
We performed hierarchical classification analyses (see below) of site based on all environmental variables (euclidean distance): our classification based on salinity is roughly objective; in fact S2 is clearly in a separate group that also includes S7 and S10 in some seasons. Of course since there are two gradients (salinity and trophic variables), the multivariate classification is not expected to fit well with salinity classification.
* * * * * * *H I E R A R C H I C A L C L U S T E R A N A L Y S I S * * * * * * *
Dendrogram using Average Linkage (Between Groups)
Rescaled Distance Cluster Combine
C A S E 0 5 10 15 20 25
Label Num +---------+---------+---------+---------+---------+
S6 22 -+
S8 30 -+
S5 18 -+
S9 34 -+
S10 38 -+
S3 12 -+
S1 2 -+
S11 42 -+
S1 4 -+-+
S11 44 -+ |
S10 40 -+ |
S4 16 -+ |
S5 20 -+ +---+
S6 24 -+ | |
S8 32 -+ | |
S9 36 -+ | |
S3 10 -+ | |
S4 14 -+-+ |
S7 26 -+ +---+
S7 28 -+ | |
S6 21 -+ | |
S9 33 -+ | |
S1 1 -+ | |
S3 9 -+ | +-----------+
S5 17 -+-----+ | |
S11 41 -+ | |
S8 29 -+ | |
S6 23 -+ | |
S9 35 -+ | |
S5 19 -+---------+ |
S8 31 -+ +-------------------------+
S7 27 -+ | |
S10 39 -+ | |
S1 3 -+ | |
S3 11 -+ | |
S4 15 -+ | |
S11 43 -+ | |
S2 7 -----------------------+ |
S4 13 -+-+ |
S10 37 -+ +---------------------+ |
S2 5 ---+ +-----------------------+
S2 6 ---+---------------+ |
S2 8 ---+ +-----+
S7 25 -------------------+
P14, L256-271: It is unclear why nitrogen is limited despite large nitrogen input from anthropogenic activities. In other words, it is necessary to explain reasons for excessive phosphorous input.
In discussion we put :
[57] suggested that the identity of the primary growth-limiting nutrient in the Mediterranean coastal systems may shift from phosphate in oligotrophic lakes towards nitrogen as eutrophication proceeds. So, it can be concluded that the N limitation in Lake Manzalah confirms its eutrophic state.
P14, L258-259: ‘the growing population in the Nile delta has increased the discharge of municipal, agricultural, and industrial wastes’ may be simpler and better.
Ok done
P14, L267: Replace ‘exaggeratedly’ by ‘highly’.
Ok , done
P14-15, L272-291: Although the authors relate A. tonsa with eutrophication, there is no evidence concerning the link between A. tonsa and eutrophication in the lake. Indeed, A. tonsa was found mainly in the less eutrophic part of the lake. Discussion should be supported by the result of the present study.
Part added:
A. tonsa is considered a euryhaline species [80], which, in the Mediterranean area, does not encounter adverse conditions and renounces in producing resting eggs as other Calanoida are obliged to do [61]. Its euryhalinity, however, does not enable the species to flourish in the southern part of Lake Manzalah (with more diluted and hypertrophic waters). In our study A. tonsa was limited to the stations with higher average salinity values (those close to the sea), i.e. S2, S4, S7, and S10.
P14, L276-277: Replace ‘confined coastal environments’ by ‘coastal embayments’.
Ok
P14, L280: Replace ‘thanks to’ by ‘due to’.
Ok
P14, L281: What is ‘equipment’?
Now L302: species and equipment for aquaculture are commonly transferred from source sites to user ones.
P14, L284: Replace ‘affirmation’ by ‘occurrence’ or ‘settlement’.
Ok we replace with settlement’
P14, L286: Replace ‘affirmed itself’ by ‘settled’.
ok
Fig. 7: Instead of annual averages, use raw values that were observed at each station in each season. Use italics for scientific names.
Ok , done
Fig. 8: The result is difficult to interpret. As is mentioned above, PCA should be applied only to physical parameters to produce a small number of combined parameters (i.e. PC1, PC2, …). Then, the density of copepod species should be related to the combined parameters.
The PCA was performed simultaneously on environmental and species abundance data after standardization. Actually we first performed two separate PCA and a co-inertia analysis (using R package ade4) but we did not find any interesting results to report (weak RV value). Also CCA analysis using appropriate packages in R did not provide high correlations between the two sets of variables (species abundance and environmental parameters). Only PCA was able to give some indication on the relationship between the abundance of some species and key environmental parameters. For this reason we choose to present only PCA results. In this current version and as the 2nd reviewer suggests we performed a separate PCA on environmental variables and We then computed the correlation between species abundance (one by one) with the First and second PCA axis and we get similar conclusion as those derived from the PCA performed simultaneously on environmental and species abundance . We added two sentences in materials and method to give further details on the PCA.
P15-16, L300-316: The descriptions are not well organized. It is better to summarize information about the occurrence of copepod species in a table.
Ok, done
P16, L318-335: The reviewer generally agrees with the authors’ opinions. However, the authors’ opinions are not persuasive, because the statistical analyses employed are not enough to support them.
Some modification in the statistical approach has been carried out.
P16, L323: What are ‘the marine pole’ and ‘the land pole’? Definition is necessary.
Marine pole : sea-affected part; Land pole : land-affected part
P16, L332: What is ‘lake extension’?
Lake area
P16, L336-346: This paragraph should be combined with the third paragraph of the discussion.
Ok, done.
P16, L236: Replace ‘affirmation’ by ‘settlement’.
Ok, done.
Submission Date
04 December 2018
Date of this review
28 Dec 2018 01:41:29
Author Response File: Author Response.docx
Reviewer 3 Report
Fig 2 and 3 histogram looks okay but unable to follow. The author should include y-axis for all histogram to understand and able to follow easily minimum and maximum values.
Author Response
Fig 2 and 3 histogram looks okay but unable to follow. The author should include y-axis for all histogram to understand and able to follow easily minimum and maximum values.
Y axis is in legend
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
General comments:
The reviewer found the manuscript improved. However, the application and interpretation of PCA are still incorrect. Figure 8 includes little information. As is mentioned at the first review, PCA should be used to combine many parameters into several representative ones (i.e. principal components). It is necessary to interpret principal components before correlating biological data with them. Of course, the result of PCA should be used to improve and/or extend discussion.
Specific comments:
P1, L22: It is still unclear what ‘changed with respect to previous studies’ means.
P1, L30: ‘… , the present copepod assemblage of Lake Manzalah is richer in species.’ is better. Except for species richness, it is still unclear how the present copepod assemblage is different from the past one.
P2, L46: Replace ‘elective sites for’ by ‘susceptible to’ or ‘subject to’.
P2, L53: Delete ‘species’.
P2, L56: Delete ‘-body’.
P2, L73-75: The sentence is almost the same as the sentence L50-51. Avoid such a repetition.
P5, L158-159: No dissolved oxygen at S1 through the year is incredible. As oxygen is likely dissolved in the water surface, it is necessary to explain why the dissolved oxygen is totally consumed at S1 through the year.
P6, L176: ‘Physicochemical parameters observed in Lake Manzalah. Annual mean ± SD at each station and seasonal mean ± SD in the whole lake are shown.’ is better.
Table 1: ‘Stations’ and ‘Seasons’ should be aligned to the left.
Table 2: It is unnecessary to duplicate Pearson’s correlation coefficient. Delete the upper or lower part.
P9, L189: Delete ‘at each station’.
P9, L191: The same as the above.
P11, L235: A. tonsa also seems tolerant to salinity.
P11, L236: Relationships with anoxic conditions are uncertain in Fig.7.
P12, L238: ‘Density of adult copepods observed in Lake Manzalah. Annual mean ± SD at each station and seasonal mean ± SD in the whole lake are shown.’ is better.
P16, L252-263: Replace Fig. 7 by Fig. 8. In PCA, the first and second principal components are usually abbreviated as PC1 and PC2, respectively. As is mentioned in the general comments, the application and interpretation of PCA should be corrected.
P16, L269-271: It is necessary to cite references relevant to water circulation in the lake.
P16, L291-292: As is mentioned at the first review, it is not persuasive to emphasize nitrogen loading before suggesting nitrogen limitation. Why is phosphorus excessive?
P16, L293: ‘The copepod assemblage’ seems better.
Fig. 7: Increase the visibility of the titles of each panel and axis.
Table 4: It is more informative to explain what the shade means.
P18, L340-341: The sentence is difficult to understand. It may be better to use the past perfect tense.
L18, L345: As is mentioned in L265-266, it is better to use ‘brackish part’ and ‘freshwater part’, instead of ‘marine pole’ and ‘land pole’.
P18, L349: ‘benthic communities’ is unclear. Describe it more concretely.
P19, L354: What is ‘lake extension’? This was already pointed out at the first review.
P19, L362: ‘it is considered to be…’ is better.
P19, L363-364: The clause is difficult to understand.
P19, L367, L380: It seems unnecessary to break the paragraphs.
Author Response
Thank you very much for the accuracy and the time spent in suggesting amelioration of our ms.
We all (the Authors) really appreciate your availability in suggesting clarifications.
As a member of the Editorial Board of Water (section Water Quality and Ecosystems), in addition, I really appreciate your effort in maintaining high the quality level of the journal.
Thank you again,
Best regards,
Genuario Belmonte
P.S. the system does not allow me to upload also the manuscript with all corrections.
I'll do it in a separate action.
Author Response File: Author Response.docx
Round 3
Reviewer 2 Report
General comments:
Despite the reviewer’s comments provided at the first and second review rounds, the application and interpretation of PCA have not fully corrected. Bianchi et al. (2003), which is included in the reference list, may be useful to improve the manuscript.
Figure 8 shows eigenvalues of environmental parameter at the first and second principal components (PC1 and PC2), indicating that PC1 is correlated positively with nutrient concentrations and negatively with DO and pH. In contrast, PC2 is correlated positively with electric conductivity and salinity. These results suggest that PC1 and PC2 represent the eutrophication and salinity gradients, respectively.
It is much better to show biological data in relation to PC1 and PC2 graphically. As is shown in Fig. 5 in Bianchi et al. (2003), every sample (i.e. each station in each season) can be distributed on the PC1-PC2 plain. If dots of samples are replaced by babbles (or rings) that indicate the density of a specific copepod species in each sample, the occurrence pattern of the species is shown on the plot, namely in relation to the eutrophication and salinity gradients. Such graphs are more informative than Fig. 7.
Author Response
Reply
The PCA was done now including environmental variables and the five dominant species abundance (naupli copepodites and adults). Fig 8 now shows two parts of the analyses: in the first case, the species have been arranged in the plot; in the second case the samples (stations/seasons) have been arranged in the plot
