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Thank you for your clarification of those few extra points. I think that the paper reads very well and will make an interesting contribution to the literature.
I have identified a few more minor, mostly grammatical and typographical, errors for you to fix. I will email you the tracked changes file directly.
Please note also that one of the reviewers has uploaded a pdf file with comments in it.
I found this to be a very interesting paper detailing a well thought out and conducted study. I therefore have only minor comments to improve some aspects of your writing. Grammatical errors in several places make it hard to understand some of what you are saying.
Abstract, line 7: “cited until 2007.”
Specimen collection section: For clarity, I suggest stating what your sampling area was in the text at the beginning of this section.
Line 92: “…preserved in 70% alcohol...”
Morphological studies section: Did you use any publications that readers can refer to for morphological identification of these taxa?
Line 107: “…rest of the tissue…”
Line 142: Perhaps better wording: “…detect potentially at risk areas…”
Line 146: It would help to state what these coordinates were. I assume they were presence observations.
Line 149: “…selected non-correlated…”
Lines 150-153: This sentence doesn’t make sense to me and I can’t understand what you did here. In what way did these 9 variables best explain your data? Do you mean you correlated the climatic variables with your presence data? If your presence data were binomial, why is a Pearson correlation appropriate?
Results section (morphological: I recommend avoiding bullet points. Just use separate paragraphs with full sentences to describe your species.
Line 269, sentence beginning “In all the plant…”. This sentence doesn’t make sense and needs to be reworded.
Line 270: “…where six species were found…”
Line 284: “…in the IP, based on…”
Line 302: “Phylogenetic analysis of these sequences…”
Line 307: This is not a full sentence.
Line 313: “…three species that are distinguishable…”
Line 323: delete the extra space next to Jones, 1998
Line 336: This is not a full sentence.
Line 336: “…it was assimilated..”. Is this the right word to use here? I’m not sure what you mean exactly.
Line 337: “…external similarity; however, molecular…”
Line 339: “Sampling performed…”
Line 340: “…sp. 6), which is…and that, in the molecular trees, is…”
Line 342: “…case, a morphological…”
Line 343, sentence beginning “The two quite…”. This sentence doesn’t make sense and needs to be reworded.
Lines 346-348: This sentence doesn’t make sense and needs to be reworded.
Line 400: “…these types of habitats…”
Line 405: “…in recently restored areas…”
Line 415: “…climatic databases, it is possible…”
Line 416, sentence beginning “The knowledge of biological…”. This sentence doesn’t make sense and needs to be reworded.
Line 419: “…since it is the only…”
Line 424: “…feed on arthropods, but on…”
Line 425: “…arthropods have their[?] food…environments and, as a consequence, have a higher…established or even invasive.”
Line 428 sub-heading: This doesn’t make sense. Can it read something more like “What consequences might the introduction of flatworms have on human economies and biodiversity”?
Line 429, sentence beginning “Another important question…”. This sentence doesn’t make sense and needs to be reworded.
Line 439: “No reference has been made to…”
Line 446: “…voracious, have more aggressive behaviour, and sometimes appear to have a generalist diet (pers. obs.), they may be more resistant to…”
Line 450: “…observations is whether governments…”
Line 451: “…restrictive rules on the trade…”
Line 452: “…or alternatively, to establish…”
Line 454: “However, it is probably too late…”
Line 455: “…may still have enough time…”
Table 1: The formatting is inconsistent: fonts and spacing.
Table 2: Some symbols may not have worked properly because you have quotation marks in the table.
Table 3: It is hard to distinguish which entries are in the same row. Please add some spaces. Fonts are inconsistent.
Figure 1: The resolution is quite low.
Figure 9: The legend values should use ‘.’ instead of a comma for the decimal point.
This is an excellent paper based on sound data and analyses that highlight the inherent problems of international and domestic plant trade in the dispersal of terrestrial planarians especially the potential to disperse non-indigenous flatworms to restored areas that border native ecosystems in the Spainish Iberian Peninsula.
I am unable to comment on the molecular methodology.
It is highly desirable that voucher specimens of the species terrestrial planarians in this study, particularly the locally obtained specimens of Caenoplana bicolor and Caenoplana coerulea have been lodged in a museum or elsewhere; there is no indication of this (no voucher registration numbers and repository). Accurate morphological identification of these taxa validates their molecular data.
From a technical aspect, I would question the reliability and utility of histological sections cut at 10-15um and stained by the H&E method. Step sectioning at 10-15um would be reasonable with thinner sections at 7-8um maximum thickness at intervals would be more helpful, especially in any retrospective study.
Synonymy of Kontikia ventrolineata and Parakontikia ventrolineata.
Jones, Johns and Winsor (1998) proposed that the genus Parakontikia was a junior synonym Kontikia. However this synonymy has not been universally accepted (including by two of the three original authors), as in publications cited in this paper. For example, Winsor L, Johns PM, Yeates GW. 1998, and Winsor L, Johns PM, Barker GM. 2004, refer to Parakontikia ventrolineata , and Sluys R, Kawakatsu M, Riutort M, Baguna J. list both Kontikia and Parakontikia in the Caenoplanini. Some explanation may be required.
Caenoplana bicolor – is there anatomical evidence supporting this identification? If not, this should be stated.
Caenoplana coerulea. Given that Caenoplana coerulea is recognized as a species complex (line 310) it is suggested that the species is referred to in this paper as Caenoplana coerulea sensu lato. The Australian distribution data is for Caenoplana coerulea sensu lato and does not differentiate between members of this complex.
Minor typos and suggested minor changes to improve the clarity of the text:
Title: The title refers to “A cautionary tale”. (singular). However there are two cautionary tales presented.
Line 223: Rhynchodeminae
Lines 257-258: The for K. ventrolineata specimens … a low variability, the French representative being the more divergent.
Line 269: In all the plant nurseries, only one species of terrestrial planarian … was found except in Bordils ..
Line 284: … based on data ..
Line 428 Heading: What are the consequences of the introduction of terrestrial planarians on human economy and for biodiversity?
Line 429: Another important question is what are the negative effects of the spread of these species?
Line 449 and following: Two cautionary tales: Plant trading and landscape restoration. Only the second cautionary tale is mentioned. The following changes are suggested:
An important question raised by all these observations is the fact whether governments in
Europe should be asked to propose new, more restrictive rules to the trade of plants coming from
outside, or alternatively establish better controls or protocols to avoid the introduction of
unwanted organisms together with the plants. The first cautionary tale is that it is probably too late now to have an impact on the transport of species around the world. Nonetheless, the following second cautionary tale indicates that we may still be on time to avoid invasions of terrestrial planarians.
The restoration of degraded areas includes planting native plant species. These plants ….
Line 464 – paragraph. This paragraph is confusing, and two alternative interpretations and changes to improve clarity of the text are provided.
Do you mean this:
Some simple, easy to perform sanitizing procedures such as heating the soil (EPPO, 2000a; 2000b;SEERAD, 2000; Sugiura, 2008) before transplanting the nursery plants to the natural environment must be sufficiently effective and reliable to ensure that there is no concomitant dispersal of flatworms. A periodic analysis of the introduced species present in garden centers and nurseries together with a study of the potential areas of flatworm distribution would also help avoid the introduction of terrestrial planarians in areas where they are more likely to become invasive (DEFRA, 2005; DOVE, 2012).
Some simple, easy to perform sanitizing procedures such as heating the soil (EPPO, 2000a; 2000b;SEERAD, 2000; Sugiura, 2008) before transplanting the nursery plants to the natural environment must be sufficiently effective and reliable to ensure that there is no concomitant dispersal of flatworms. Such procedures, together with a periodic analysis of the introduced species present in garden centers and nurseries and a study of the potential areas of flatworm distribution would also help avoid the introduction of terrestrial planarians in areas where they are more likely to become invasive (DEFRA, 2005; DOVE, 2012).
Line 537: delete inverted commas
This is an excellent study, including a number of new sequences and a simple but efficient analysis of the molecular data. The data are excellent, the discussion is clear and the conclusions are sound. In addition, certain specimens have been serial-sectioned: although the results are not presented here (and they don’t need to), this represents a significant work.
The figures are good, but my advice is to expand and improve the photographs of animals presented in Figure 3, and to improve all the trees with more explicit labelling: this is detailed in the section “general comment”.
All molecular methods are clearly explained.
Line 120-121, a few comments about “PCR products that yielded double bands” would be welcome.
The data are sound, there are many new sequences (all with GenBank numbers), and the phylogenetic analyses are good.
I see a few issues with species identifications or names. These will probably need a few additional explanations in the text.
a. Kontikia ventrolineata and the synonymy with Parakontikia ventrolineata.
I understand that this binomial corresponds to the conclusion of the paper by Jones, Johns & Winsor (1998). However, more recently, Sluys, Kawakatsu, Riutort & Baguña (2009) accepted Parakontikia Winsor, 1991, suggesting that the current name of this animal should be Parakontikia ventrolineata (Dendy, 1892) Winsor, 1991. Some explanation is needed here.
b. Caenoplana bicolor.
The species was probably identified by the authors from its aspect, which is typical. However, no sectioning is mentioned and no sequence from C. bicolor from its place of origin was used to confirm the identification – indeed, a single sequence of C. bicolor is listed in Table 2 and Figure 7. I am not saying that the specimen was not C. bicolor, but I believe that the authors should clearly state the limitations explained above.
c. Caenoplana coerulea.
I understand well that this binomial designates a complex of species, as shown in Figure 7, and that future work will probably result in the erection of several additional species names. However, in our present state of knowledge, all these taxa should be designated as Caenoplana coerulea (perhaps as Caenoplana coerulea sensus lato?). In Figure 7, there are 3 clades (a, b, and c) which correspond to this species. I suggest to label them with “Caenoplana coerulea” (in inverted commas) while the clade in the top (with CcoTal1, etc.) could be labelled Caenoplana coerulea (no inverted commas).
d. Bipalium kewense
The authors show that none of the Cox1 sequences already in GenBank is close to their sequence labelled Bipalium kewense, suggesting that this is the first sequence of this species. From my personal experience with sequences of Bipalium spp. found in France, I am inclined to agree with this (none of our Cox1 sequences was close to sequences already in GenBank). Bipalium is not a simple genus, and since specimens in Europe are often asexual, identifications should only rely on external colour pattern and/or sequences. Since B. kewense is a worldwide invasive, it would be good to have some material deposited in a Museum of national collection, and/or a few excellent photographs of the specimens. There is a single photograph in Figure 3, showing only the head. Add photographs (see also my general remarks about photographs).
e. Obama sp.
Since Figure 6 shows that there are two clades, it would be good to have photographs of at least one specimen from each clade.
These comments should not be understood as a criticism of the work of the authors, but instead as constructive suggestions to increase the impact of this excellent work on readers.
Most trees seem to come directly from the phylogeny software, with minimal or no graphical improvement. They would be much more pleasant to read with some additional effort.
1.1. Names of taxa: The authors take several lines in the caption of Table 2 to explain their complex naming code. The readers who want to understand the trees have to understand the code, look at Table 2, then go back to the trees. All this, for each species. I strongly suggest to edit all codes in trees with complete names.
Example: In Figure 4, “621BkeCan” can be “Bipalium kewense (Cantabria) 620”. Much easier to read, more informative, and finally not so long. The additional place taken could be easily found by making the branches 20% shorter.
A minimum effort is needed to change all codes into understandable names.
1.2. Additional labelling at the right part of trees.
Example in Figure 4, instead of A, B, C, D, simply label each clade with its name (Bipaliinae, etc.). Easier to read, more informative. By the way, colours could be used for each clade (currently with shaded grey levels).
These remarks are also valid for Figures 5-8.
In Figure 6, label the two clades of “Obama sp.” with appropriate labels on the right, and Obama sp. 6 with its own label.
For Figure 7, see remarks above about Caenoplana coerulea with or without inverted commas. Change a, b, c, in the right to complete names. Caenoplana bicolor should be clearly labelled with a label of the same importance as the current a, b, c.
In addition to my remarks above about additional photographs needed for Bipalium kewense and Obama sp.
PeerJ is an open access journal, meaning that all figures can be re-used by others, including in Wikipedia (it is likely that these figures WILL be used in Wikipedia). In addition, composite “plates” prepared for journals printed on paper, as we used to do before, are not needed any more for journals which are now read in various formats (HTML, PDF, ePub and others – see PubMed Central) including future formats that we do not even know now. For both these reasons, I suggest to split all photographs in Figure 3 into separate photographs for each species. That would allow more freedom to choose appropriate photographs, with higher resolution, and to add detailed information about the geographic origin of each specimen shown.
Finally, less work than a composite plate, with much better results and impact.
(3) Details in the text
3.1. Title. Why is the title about the “Iberian Peninsula”, which is composed of Spain and Portugal, when there is not even a single sample from Portugal? This is a study about Spain.
3.2. Line 44, the authorship date for the taxon Platydemus manokwari is 1963, not 1962. See Justine et al (2014), note on nomenclature.
3.3. Line 45, “worm farm” is right, but I would prefer “earthworm farm” here, just to make it clear that these farms do not produce flatworms.
3.4. Mention of Obama sp. in Spain.
A simple research of “Obama marmorata” (with inverted commas to straighten the research) in Google finds a 2013 congress record. This abstract should probably be cited, since it is available from the web (but see the journal policy). This is better than “Lisandro Negrete personal communication”, in my opinion.
(4) Minor details and typos
Abstract, 5 lines before the end “its phylogenetic range” – what is “its”?
Line 223, Rhynchodeminae
Line 228, “is” very small.
Line 241, after Cox1 tree genus, add (Figure 7).
Line 455, I do not understand what is “the second cautionary tale”.
Line 484, J Journal.
Line 533, Biological
Line 537, delete inverted commas
The manuscript is of great interest for several reasons. Mainly because it is a taxon yet little studied, whose diversity is underestimated in many countries. Moreover, in recent years there have been known introduced species in many regions, with possible negative impacts on the economy and wildlife.
This contribution provides new and valuable information on alien species (at least ten) in the Iberian Peninsula, whose potential risk may be underestimated by the competent authorities. Therefore, I see such contributions as indispensable. The authors used a model species distribution to evaluate the spread risk of a species, which is novelty for this group. So, I strongly support the publication of the article. I just have some comments/suggestions and minor corrections, which appear throughout the ms (in attachment).
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