Epibenthic Harmful Marine Dinoflagellates from Fuerteventura (Canary Islands), with Special Reference to the Ciguatoxin-Producing Gambierdiscus
Round 1
Reviewer 1 Report
The objectives of this study were: 1) to know if there is any preferential associations of benthic harmful dinoflagellates, 2) advance on the knowledge of the relationships of benthic dinoflagellate assemblages with different macrophyte communities and 3) to know the most abundant species of Gambierdiscus in the benthic macrophyte communities examined during the present study from the Canary Islands. Vulcanodinium was most abundant, followed by Ostreopsis. Gambierdiscus and Sinophysis were most prevalent at a second station. Two sites were sampled – a “charco” (tidepool) at Cotillo in which the pool itself was sampled (1.5 m deep) and the platform outside the pool was also sampled (6 m deep). Similarly, a platform at Playitas was sampled at depths of 2-3 m and 6 m. Sixty seven samples were collected at both sites in September 2016.
A few minor items: Shouldn’t the mesh units be um instead of mm (lines 115-116)? Headers in Discussion need to be renumbered (4.x versus 3.x).
Major items: Many different macrophytes were collected – what about host preferences? Just because macrophyte preference is controversial doesn’t mean it should be dismissed or ignored. Abundance is clearly related to differences in structure and wet weight to surface area ratios among the different macrophyte species sampled. No real analysis of preference substrate was presented.
It is too difficult to use morphometrics (D, R1-3) in routine Gambierdiscus cell counts. Why didn’t you confirm morphometrics with molecular analyses (qPCR)?
Why were the population distributions of Gambierdiscus and Sinophysis significantly opposite to that of Ostreopsis and Vulcanodinium? How much of the site to site variability is caused by different macrophytes? What about the influence of environmental variables? A statistical analysis should be used to look at macrophyte influence and environmental (site) differences.
A good note: other studies have noted presence of these dinoflagellates, although Vulcanodinium rugosum can produce pinnatoxins – may be object of future research at these sites.
In closing - the study does not present any novel information. The morphometric results are not validated via molecular techniques. While the influence of macrophytes (or lack thereof) and environmental variables are discussed, no statistical analyses are presented to back up the claims.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
This ms addresses the distribution of benthic dinoflagellates (including potentially toxic ones) in 2 sites of Fuerteventura Island aiming to identify (i) preferential associations of benthic dinos, (ii) relationships with different macrophytes species and 3) the most abundant species of Gambierdiscus.
Although the argument is interesting, it seems that this paper is not supported by an appropriate sampling design, as (i) no environmental parameters have been considered, and (ii) the expression of cell density as cells per gram does not allow a correct comparison between data.
(i) The observed differences in terms of cell concentrations were interpreted only taking in account the macroalgal hosts, without considering that they could be better explained by environmental parameters that are very different in the 2 sampling areas. Moreover, even considering the same site (I mean in particular the charco) the environmental parameters change quickly.
(ii) In the macroalgal species list, I noted some heavy calcified species (e.g. Amphiroa, Jania, Corallina), together with non-calicied ones. This would markedly affect the cell density values, that are expressed per gram wet weight (I mean that 1 gram of Amphiroa is a very small piece of thallus compared to 1 gram of Hypnea). This heterogeneity compromises the value of a study based on the comparison of cell density between different macroalgal species. Moreover, I saw that authors do recognize that the comparison between cell density on different macroalgae is controversial and that ‘differences on macrophyte surfaces and morphologies make difficult the standardizations’, but anyway they based this paper on such a comparison.
I suggest not considering Scrippsiella as it is planktonic species and its presence over macroalgae is only casual. Even Vulcanodinium has two phases (benthic and a planktonic) that differ morphologically. If Author detected the planktonic phase, I suggest excluding it. Authors could mention and discuss their presence, but that species should not be treated as epibenthic species.
Random minor comments
L 112. Specify the sampling frequency. Only once?
L 116. In situ (italic)
L 120. Add magnification used.
L 121. genera
L 130 Rhodophyceae
L 138 Dictyota
- 144 spp. (not italic)
L 173. Calcofluor
L 300 Table
Figure 2. Control the legend. I don’t see triangles as symbols in the figure.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript presents some interesting information about benthic dinoflagelllates in Canary Islands and the associations between them and with macroalgae. It also tries to contribute to the morphological differentiation of the Gambierdiscus species found.
The information obtained is interesting but limited. Sampling was carried out only in two locations at a precise data. In general, the obtained data are interesting and the paper is worth of publication, but the manuscript is too long because it includes too much text of general character in the discussion section.
The discussion should, therefore, be shortened and better centered in the results.
The morphological discrimination presents the problem that there is no positive controls in this study. I suppose that typical specimens are used as a reference (not having PCR confirmation), but this should be made clear in the text.
An objective seems to be missing from the introduction (morphological differentiation of Gambierdiscus species)
The figures and the table are misplaced. The should be placed near their first citation.
In text citations, when a direct reference is made, should include authors (e.g. following Author et al [37] instead of following [37]).
Check the units used and replace in the text and the figures gr by g.
Fig.3 presents several problems:
Some Y-axis labels are incorrect (30,000 is duplicated)
Some other labels in the same axis are odd and seem to be inconsistent with others (40, 90 instead of 50 100, apparently)
There is no differentiation between major and minor tick marks which makes impossible to know to which mark is associated each label.
Preferably one of the symbols should be replaced (not using the same geometric symbol for the two series).
Adding the mean (or the mean and the SE) would be useful.
L47 Higher that what?
L65 Replace O.cf. ovata by one Ostreopsis species
L106, L19 probably shelf would be more correct, or at least usual, than platform, if the authors refer to the "continental" self.
L115 "in situ" use italics
Table 2 "mean+sd" ± instead +
L178 explained
Fig 2. The figure and caption are unclear. There are not triangles. The meaning of the crosses are not explained. Components in PC Analysis cannot be rotated (factors, in factorial analysis can). I suppose that the crosses are the ends of the autovectors or the factor loadings corresponding to each taxonomic group.
L195 Scrippsiella was only
L209 what does p<0.01 mean referring to PCA??
L213-215 this sentence cannot be understood. It should be reworded (and corrected: "from" instead of "of")
L220-221 forementioned
L231, L238 insert the numerical citation
L252-253 this sentence cannot be understood. It should be reworded.
Fig 6 thousand separators are missing in the Y-axis labels
L282 a few
L301 I suppose that there is a misplaced citation
L329 cite the authors because they have not been cited.
L343-346 reword this sentences
L362 produces
L366 "...symptoms in mice and mass spectrometry". Explain this a little better.
L372-374 reword this sentence
L373 presence instead of appearance
L376-380 reword the sentences. They are completely unclear
L396-398 This sentence cannot be understood
Other comments:
It would be interesting to try linear or quadratic discriminant if, as it seems, some cells can be unequivocally identified.
It would also be interesting to give at least a rough estimation of the surface/weight of the macroalgae in the study, as could focus additionally the discussion about the dinoflagellate preferences.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors have adequately addressed my concerns and the manuscript is now acceptable for publication.
Author Response
All done, thanks.
Reviewer 2 Report
The ms has been substantially improved.
The part about Gambierdiscus is ok. The other part requires minor revision.
L 24. Change ‘filiforms’ with ‘filiforms morphotypes. Moreover, the term ’filiform’ is never used in describing macroalgal morphotypes. I suggest changing ‘filiform’ with ‘filamentous’ throughout the text.
L 335 Perhaps you should change ‘These genera’ with ‘The concentrations observed in this study’.
L 363-369. The part in red that you added have been partly written above (L 342-344). Avoid to repeat the same concepts.
L 364. Change ‘equivalent’ with ‘comparable’.
L 370. Change ‘our abundances’ with ‘The abundances of Ostreopsis found in the present study were lower than ....
L 400. Remove ‘preferential’ from the paragraph title.
L 407-408. Take in account that a number of studies report the Ostreopsis concentrations are higher in sheltered sites. You should discuss appropriately your results that agree with some studies but disagree with other ones. Moreover, you cannot derive any conclusion as you studied only 2 sites and you should mention that other parameters, different that those addressed in this study, could have had a major role.
L 415. write 'elements'.
L 426-430. In the red part, add some considerations about the fact that a number of environmental parameters other than macrophytes, could have had a major role in determining the distribution of benthic dinoflagellates (cite here some appropriate literature that addressed the role of other environmental parameters).
L 428. The sentence ‘it cannot be ruled out other kind of preferential host macrophytes’ is unclear. Perhaps you mean ‘it cannot be ruled out any preferential epiphyte/host relationships’? Anyway, in the following paragraph, you seem to restart again speaking about macrophytes and their epiphytes.
L 441. Change ‘macrophyte taxonomic groups’ with ‘macrophyte taxa’.
L 464. Preferred substrates.
Table 1.
Write Cyanophyceae not in italic.
Add a column explaining which morphotypes (Type 1, 2, 3, 4) was assigned to each macroalgal species.
Figure 5. spp.
Author Response
The corresponding changes have been carried out following all the comments. Thank you.
Reviewer 3 Report
The manuscript is now acceptable for publication.
Author Response
Thank you for all the suggestions
