PONE-D-23-41760Review of the genus Kora from Brazil, with description of eight new species and a new related genus Koltrora, including comparison with two Andean Neopetraeus species (Gastropoda, Eupulmonata, Orthalicoidea).PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This work contains very detailed anatomical studies of what is supposed to be a number of species of the genus Kora It contains descriptions of several new species and a new genus. Relationships of this taxa are investigated by analyses based on morphological characters; I believe the author calls these 'phenotypic analyses'.

I appreciate the exceptionally detailed documentation of anatomical/morphological features of the studied snails, which provide a large dataset. I disagree with the main conclusion of the author to have identified 8 new species and one new genus. Let me try to explain why ( i will use a single example, but I believe it to be representative of the general assessment of data in this work, which I reject)

This is an unnecessarily complex study, which to understand is challenging, because the reader is overwhelmed with large amounts of unimportant or uninformative data while at the same time relevant information is often withheld or has not been used. My review would need to be as long as the manuscript because nearly every sentence contains questionable statements, conclusions, misleading formulations, and a mishmash of unnecessary or confusing information. Sifting through all this to distill the objective truth and separate that from the interpretation by the author is too cumbersome and time consuming a task. Sorry, I can't. My live is too short ... So, I need to give a rather generic evaluation.

The English language is understandable, but it is often grammatically or stylistically imperfect. This does usually not impede the ability to understand the facts, but clarity suffers to some degree.

I've contemplated commenting on language but then I decided generally not to comment on linguistics because I do not have time for that.

I try to summarize the findings of the study:

Snails collected at 12 different sites (locations: see map in Fig. 60) have been studied and detailed recordings of anatomical characteristics have been made and collated in a data matrix. The data matrix was analyzed, and the investigated samples have been arranged in a tree shared characteristic (a presume some kind of a cladistic analysis). So, relationships between examined samples have been hypothesized based on shared characters. Equally, differences between species can be understood as diagnostic characters that underpin their distinctiveness.

My first criticism is that many recorded characters are of dubious systematic value. Now, based on my understanding of cladistics (not sure if cladistic methods were used here) is that it may not matter if some characters are uninformative, but that an analysis of all characters simultaneously will still return a plausible hypothesis on the degree of similarity / dissimilarity between the examined samples. I accept that.

My conclusion then is that irrespective of the possible flaws in some of the characters (as they have been recorded and coded), the tree shown in Fig. 61 is a plausible hypothesis of the real relationships between the examined samples as based on morphology.

Great!

Looking at this tree, I find that the sample identified as - for example - Kora ajar is most closely related to the sample identified as Kora nigra based on many shared characteristics, some of which may be synapomorphies (shared only by these two samples). Let's dive into the details:

One supposed synapomorphies of K.nigra+K.ajar is:

4 ("Length/width ratio of shell = 1.5) as opposed to the other two possible character states (2 and 3).

There is the first problem. The author has grouped continuous characters, such as size and shape variables, into classes. I think this is wrong and creates potentially misleading results. For example, for all Kora species the author classifies shells as either in the 30 mm range or the 45 mm range (Table 2). Thats so strange! Why not use real measurements? Firstly, the individuals of a species are not all identical in size. So, species will occupy a size range. I find it hard to believe that all species in a genus should be clustering around two arbitrary size classes, 30 and 45 mm each. If that was the case, I think, the most likely interpretation is that across all measured shells, we can identify two groups, not 12! In this context I also criticize the small number of measured individuals. The author has used measurements of 3 to 5 individuals per supposed species. That number is insufficient to capture the entire range of variation in a species especially as it appears that individuals of each species have only been found at single sites each. Again, it puzzles me why the author hasn't measured more shells. For example, 106 shells have been 'examined' of K. corallina, but 5 have been measured only. So, what does 'examined' actually mean? Looked at?

I conclude that all size-related shell characters are insufficiently sampled and documented.

Back to K.nigra+K. ajar.

The next supposed synapomorphy is:

14 (Umbilicus = "widely opened" ['wide' would be sufficient wording]) as opposed to alternative character "closed to narrow".

Strangely, umbilical anatomy is coded in three different characters 12 to 14 - which in my view record partly redundant features. Why not simply say "Umbilicus: 0=closed, 1=narrow, 2=wide" . Again, the problem is that a continuous character is classified. When exactly is the umbilics narrow and when is it wide? This is a completely subjective assessment. Looking through the photographs of shells, the umbilicus is often not shown or hard to recognize, so I have to rely on the author here. However, umbilical anatomy is often variable within a single species and individuals of the same species may have closed to open umbilici.

Next character:

38 (pair m1l - some buccal muscle [one out of 10]: absent). As opposed to present in all other Kora species and all outgroup taxa. Really? This is such a freaky detail. I have difficulties to believe that this is a real character. What is the likelihood that this pair of muscles is not absent but has not been observed for some other reason? Buccal muscle anatomy is highly complex and intricate, and I am not even sure that all these 10 muscles that the author proposes actually exist. I consider this a dubious character, one where I have to have blind trust in the observations of the author, which are also not sufficiently documented.

Next character:

44 (m5 - 'covering m4'); as opposed to alternative character states 'separated from M4' and 'continuation of m4'). Yet, another buccal mass muscle... same applies to what I have said on character 38 - this is an incredible detail on a very complex and intricate structure, where I consider that observations are impossible to verify while having a high possibility of subjective error.

Next character:

67 (Carrefour bulged distal region by side of receptacle: 0= absent). As opposed to 'present'. Really? The difficulty with saying that there is a bulge next to the receptacle in the carrefour is that all the internal organs are soft tissues that are shaped by muscle contraction, preservation, dissection. I would doubt that this is a reliable character at all.

One cannot dissect the reproductive organs of a dead, preserved snail, spread them out in a petri dish and treat a little bump somewhere as a plausible anatomical feature! Anyway, not while looking at probably just a handful of specimens. This is nonsense!

SO, the aim of my exercise is that large parts of the information that 'inform' the analysis of relationships are spurious characters. These characters may be partly prone to error due to the complexity and intricacy of the structures studied. Many other characters are probably unsuitable to underpin systematic conclusions as they likley represent just variations on the level of the individual.

Let's be clear, we do not study humans this way because it makes no sense. I cannot use hair colour, skin colour, nose shape, penis size or whatever to study the relationships of human populations. The reason is that the observable variability in any of these characters is intraspecific. I could still go on and create a tree, but the tree would be an artifact of incomplete character sampling. The same applies to the snails. Many characters, which have been meticulously recorded are uninformative. I went through the whole list of characters, and I consider my statement generally true. So, the tree is at best hypothetical.

Nonetheless, let's say the tree shown in fig. 61 is correct.

There is no reason whatsoever to treat the tips of the tree as species. Basically, the tips of the tree represent individuals. The tree is entirely phenetic and records the anatomy as observed by the author. However, there is no way to tell where the species boundaries are in this tree. Essentially, what we found is that there are 12 individuals (or groups of individuals - but the author said that the individuals of each 'species' are so similar that there is virtually no variation between them, so they have all been merged into single figures). These could all be members of the same species! Or they could represent anything from between one and 12 species. To decide how many species there may be requires criteria (basically one has to conclude how much variation one allows within a species). The author has decided to not allow any variation in a species.

That means the authors is a typologist.

Typology is not science because it rejects the existence of variation. But variation is the fundamental prerequisite for evolution to happen. All the so-called species examined herein are all descendants of a single ancestor, and over time have acquired a certain amount of variation. But variation has to be there in the first place and many of the ancestral variation is likely shared by more closely related species. To not accept reasonable amounts of variation is unnecessary and unhelpful as it denies the most fundamental condition of evolution.

I consider that the author has just meticulously (and to a large extent unnecessarily) recorded random individual differences and shown how different individuals can be lined up with respect to the degree of similarity. To decide where the species limits in this tree are, one would need to try to group tips together and examine where two groups differ from each other in certain characters with statistical significance. Shell characters could be the easiest to use for that, but the author has not attempted to find a more objective way of analyzing shell characters (using real measurements rather than artificial size classes).

In biology, species are not the smallest recognizable units. The smallest recognizable unit is the individual. Individuals form populations (already a difficult term which we fail to properly define and delimitate) and species consist of populations. To delimit species correctly, is not to show that two individuals differ, but to show that populations differ in their means of certain characters. Even the sheer existence of differences in itself is not a good argument for specific distinction, in as much as the different mean hair colour of Germans and Ethiopians is not a good argument for specific distinction either! In fact, there needs to be a criterion that the differences are indicative of some sort of reproductive or genetic isolation.

The author has stopped at documenting differences between individuals. He has not done the second and third task necessary to delimit species in a plausible and scientific way.

Of course, he can still describe these species because the rules of nomenclature do not care for science. He'd be in very good company as creating meaningless species descriptions is a common occupation especially in malacology. However, while doing this we fail to adequately document biodiversity while simply inflating taxonomic catalogues that become increasingly useless records of biological diversity for their exceedingly high subjectivity and extremely limited reproducibility.

Sorry for rambling, back to the data. I tried to show what I perceive to be the main flaw of the morphological analysis. The manuscript also contains species descriptions. Yet, the challenge is to find the few informative characters in the swamp of generic descriptions. Honestly, it is confusing and unnecessary to describe the entire anatomy of an organism in the context of a systematic treatment.

Perhaps describe the anatomy in more detail at the genus level but restrict the species descriptions to the informative bits! Here you need to document the range of variation, you cannot simply treat species in a typological manner as if there was no variation. If this is the case, your so-called species is not a species! It's simply a single individual or a small group of individuals that are closely related with each other. Given that all species are closely related it is to be expected that they share many similarities, so please avoid describing the same all over again if it's just generic.

What is a 'distinctive description'? I have no idea. Your descriptions are uninformative. Where you observe differences with other species, you make statements, such as ", remarks and distinctions following" Where? It is completely impossible to follow the logic of your comparisons. Why don't you provide all the relevant information under each species instead of referring the reader to another part of the paper? The descriptions have similar statements of over the place, such as "general structures similar to preceding species" - look, it's not helpful nor necessary to swamp the few diagnostic characters in an ocean of uninformative descriptions.

Thankfully you have a Table 2. However, corresponding to what I said already, many of the features that are stated to be diagnostic for species, based on my own experience as a land snail taxonomist, are likely more variable within a species than allowed by you. Based on three existing 'size classes' (if these are actually real), you may deal with just three species. Many of the characters listed are likely variable within a single species (bumps here and there, umbilicus closed-open etc etc). Whatever the matter, you need to demonstrate the suitability of characters that you propose to be taxonomically informative instead of simply amassing a lot of documented variation.

Bulk variation may be informative regardless of whether I consider a feature to be possibly informative, or not, but only if you have analyzed a large number of individuals from more than a single location simply because you need to sample the existing variability in each character sufficiently before you can try to recognize groups in terms of different character distribution. What you do is typology (a comparison of individuals, with each individual being treated as the essence of a species). These species exist in your imagination only. To get them out into the real world as scientifically tested hypotheses requires proper statistical analyses of objective variation.

I have made some comments in the manuscript file, but not consistently because I got tired. For what's worth check them to find some contentious lines.

These comments are by no means complete.

Reviewer #2: This is a tremendous piece of work and a benefit to Malacology. I recommend its publication, however, the writing is too difficult to understand without substantial effort and there should be a review of the English phrasing and grammar to make sure that it is as clear as possible. I would recommend a revision of the writing followed by another round of review. At present certain parts are difficult to give a fair review because it isn't entirely clear what the author is trying to say. Please see the edits and suggestions in the version of your manuscript attached. I did not attempt to revise all of the English in the paper but made my best attempt in the first 1/3.

Reviewer #3: The new species descriptions and morphological work presented in this manuscript are excellent and nicely figured. This part of the manuscript is generally clear and well written and is a very important addition to our knowledge of this group. I would very much like to see this portion of the manuscript published. My only problem with this section is that many of the species comparisons that should be part of the systematics section are presented in the Discussion section making direct species comparisons more difficult.

The phylogenetic section is much less focused and not very complete. The taxon sampling for the phylogenetic section is too incomplete to make the comparisons this manuscript attempts to make and leads to comparisons which seem to make less sense. Neopetraeus is portrayed as sister to or closely related to Kora and Koltrora, but most of the taxa needed to judge this are missing. It is not clear to me why Kora and Koltrora are compared to Neopetraeus rather than to Thaumastus. Pena et al. 2011 published at least some anatomical data for Thaumastus, which falls within the same clade as the target taxa in phylogenies generated with sequence data. Why was this group not included? The choice of outgroups also seems unnecessarily distant from the group of interest. The discussion also spends too much time talking about relationships within one of the outgroups which are not relevant to this manuscript.

The focus of the manuscript seems very narrow, especially for a journal with a diverse audience. I would like to see a little more information about the importance of the discovery of so many new species in one group. I see these discoveries as suggesting the group is diverse and relatively poorly sampled. In fact, a second paper describing a new species of Kora was published while this paper was being reviewed. Sampling is desperately needed. Unfortunately, land snails also have some of the highest extinction rates of all animals, making species discovery imperative while there is still a chance for conservation actions. I would like to see some mention of this added to the manuscript.

As mentioned above a paper describing a species of Kora was published very recently and reference to it will need to be added to this manuscript: Pena 2024. Notes on Kora and description of a new species from Minas Gerais, Southeast Brazil (Mollusca: Gastropoda: Stylommatophora) Zoologia 41: e23059 https://doi.org/10.1590/S1984-4689.v41.e23059 .

The manuscript needs some proofreading to improve English, clarity and focus. See some examples below along with other minor comments:

Page 2, paragraph 2: line 1, delete ‘the’ before ‘recent’; line 3, delete ‘truly’; line 4, ‘lands nails’ should be land snails; line 7, ‘stophocheilid’ should be strophocheilid.

Page 2, paragraph 3: line 2, change to the four described species including the type species,; line 4, add ‘are’ between ‘which also’. The second half of this paragraph needs to be edited for clarity and conciseness. Is the information about lost specimens that were studied by deceased workers that never published on them necessary?

Page 3, line 21, change exteriorized to everted and ‘the copula’ to ‘copulation’.

Page 4, line 3-4, this sentence is not clear. What do scenario, style, model and disposal refer to?

Page 6, beginning of the Systematics section. Include some higher taxonomic levels:

Class Gastropoda Cuvier, 1795

Order Stylommatophora A. Schmidt, 1855

Superfamily Orthalicoidea E. von Martens, 1860

Family ? Give family and/or explain ambiguity or why you are not giving the family.

Page 14, Distinctive Redescription. Not sure what Distinctive means here. Why not just Redescription? Also ‘Proper description in [3]. Complement:’ is not clear to me. I think 3 is a reference to the original description. This might be clearer as: ‘Complement to original description [3]:’.

Page 15, line 11, can delete ‘(as)’.

Page 16, first paragraph, penultimate sentence is not clear.

Page 31, last line. Local should be locality.

Page 32, Measurements section. Which measure is which, add height, width or some other explanation.

Page 32-33, This new genus is similar to Kora. Make clearer how you would differentiate it from Kora.

Page 33, Etymology, change contributed ‘with’ to ‘to’. Change ‘collects’ to ‘collections’ and ‘donation’ to donations; or whole phrase to ‘by collecting and donating material’.

Page 43. The first paragraph of the Discussion describes how the previous descriptions were written. This is too late since the reader has already read the descriptions. Move this to the Methods section.

Page 44. Notorious has a negative connotation. I think you mean notable.

Page 49. The last two paragraphs are not directly related to this paper and can be deleted.

Page 52. Acknowledgements. Change ‘the collect expedition’ to collecting expeditions or the collecting expedition, depending on if singular or pleural.

Figure 60 caption. Self produced is not necessary.

Appendix 1 title is not clear, could edit to List of characters used in the phylogenetic analysis

Appendix 2 title is not clear, could edit to: Matrix of characters used in the phylogenetic analysis

In figure 61 Kota [sic] carolina should be Kora carolina.

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Reviewer #1: Yes: Frank Koehler

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-41760R1

Review of the land snails of the genus Kora from Brazil, with description of eight new species and a new related genus Koltrora, including comparison with two Andean Neopetraeus species (Gastropoda, Eupulmonata, Orthalicoidea).

PLOS ONE

Dear Dr. Simone,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically, it was noted during previous rounds of peer review that the design of the study is flawed and that there are serious issues with the usage of English language in the manuscript text. Based on the latest reviewer reports these shortcomings have not been addressed sufficiently to improve the overall quality of the work. The manuscript does thus not meet publication criteria 3 and 4 of PLOS ONE in that that the analyses are not performed to a high technical standard and that the conclusions are not supported by the reported data. In addition, given the shortcomings with English language usage, the manuscript text does not adhere to publication criterion 5 of PLOS ONE, stating that the article needs to be presented in an intelligible fashion.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Kind regards,

Michael Schubert

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #3: No

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #3: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #3: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: None of my previous points of criticism have been addressed by the author. I am not surprised because I essentially questioned that the work is scientific. The work has not changed other than for some superficial cosmetics and I uphold my assessment that the work is essentially typological and therefore unscientific. Unfortunately, this is not an unusual case in systematic taxonomy, a field which is teaming with pattern recognizers that work in a conceptual vacuum. The mistake that the author makes, is to presume that simply distinguishing something is the same as describing species. It is not! The something that the author distinguishes can be anything from a group of individuals to a population, perhaps even a species. But the complete lack of an intellectual assessment of what the documented variation means in a biological sense makes it impossible to come to a sensible conclusion.

The author goes to incredible lengths to amass information on differences and similarities between groups of individuals of land snails collected at many different sites in Brazil. My compliments for the diligence in collecting data. However, that a comparison of different individuals yields observable differences cannot be translated into a taxonomic classification without an assessment of what these differences mean. There are characters that may well vary within populations. For example, humans may have black, blond, or brown hair. To code hair colour in a matrix that is designed to find the closest relative of humans is nonsense. And even if it was not, it cannot inform us about the taxonomic rank that the closest relative should be assigned to. For that, some sort conceptual framework is required.

Now, the logic of the author is that using many, many characters will somehow miraculously filter the wheat from the chaff. Yet, looking at fig. 61 any character will do in justifying the 'distinctiveness of a species'. But this does not need to be true. I don't even question that there are differences. The tree in Fig. 61 simply shows which studied individuals are more similar with one another, and which are not. But as any other tree, it cannot inform about where the boundaries between taxa are. For that we need additional criteria. These need to be formulated and they need to make biological sense. That the individual called Kora ajar differs in a number of characters from the one labelled as Kora nigra, in itself is not an argument that they represent different species. In my previous review I provided a detailed assessment of each of these characters and concluded that they do not require that assumption to be made. I am not going to repeat myself here.

In general terms, we cannot know if the reported differences require the assumption of specific distinction in any of these cases. That can only be decided when there is at least an implicit species concept. The implicit species concept of the present work is: If there is a difference, it is a different species.

Yet, the only objectively distinguishable unit in biology is the individual. From that level on biological systems reveal fractal patterns of similarities and differences that are shaped by different evolutionary processes. In this continuum, we artificially create a hierarchical classification (an almost nonsensical idea!). The created artificial classes should be reasonably broad and allow for variation, or we run out of taxonomic categories very soon. Note also: Artificial does not mean arbitrary. We need to accept that classifications are models of the objective truth, but we need to adhere to sensible, generally accepted criteria to create this model, otherwise we go out of business.

Above the level of an individual, the first layer is probably a family. The members of a family are closely related, yet not morphologically identical. Using the method of the author on ten human siblings and two cousins would create essentially the same tree. Because we will find shared similarities between these individuals as well as differences. (Look at those noses!) So why do we not classify these individuals as distinct species?! Of course, this is utter nonsense and exaggerated. But my point is, this essentially is what the author does - piling up observed differences with no regard of whether these differences are useful to address the problem at hand. This mix of similarities and differences continues to shape populations, metapopulations, which eventually become perhaps genetically isolated so we can call them species. The whole process is utterly arbitrary and inadequate since we try to please our human desire to categorize stuff. In reality there are no distinct borders unless shaped by extinction of intermediate forms.

Whatever conceptual consideration there may be, it has all been relegated to the discussion chapter. Here, the observed differences are summarized for each taxon. I suspect, however, that each description is based on a single individual (and even if not, it is based on few individuals from the same population). Who says that (for example) "the radular sac bulging in posterior end" is a useful character to distinguish anything? Any character is influenced by individual variation relating to genetics, nutritional status, reproductive status, age, preservation of the animal etc. So, a bulging radular sac may mean NOTHING! Unless you confirm this character is consistent in several specimens (more than 5 please) - and equally consistently different in what you perceive to be a different species. Yet, even then, this finding would not necessitate distinct species status. But at least, I could understand where you are coming from. My previous assessment of how evidence was treated was more comprehensive and has remained completely ignored. I won't provide further details to support my claim but refer you to my previous review.

Now, as inevitably the taxonomic endeavor is highly subjective, one may argue that the present work is just another way of distinguishing stuff and that stuff is called by THIS author species and by MYSELF just a group of individuals. However, if we accept that level of subjectivity, we have left the realm of science and entered the realm of populism.

To avoid of getting to a place where taxonomy is completely uncoupled from scientific theory (the work makes a big step in this direction) we need to have a concept of what constitutes a species. That means, we need to make a reasoned assessment of which differences are meaningful and which are not. Many of the differences that are compiled in tables 1 and 2 are not biologically meaningful. That is because the author does not allow for any variation among a species. Starting with shell dimensions. A species does not have 45 mm long shells - no variation present. The author claims to have studied 146 specimens, but does not document the range of variation among these specimens even in such basic measures as shell dimensions. Similarly, no variation is documented in any character. Now, I wouldn't expect a huge deal of variation among individuals collected at exactly the same site. But I would start to question my own work (and in fact I have done this!) when every snail from a new place belongs to a new species. Species have ranges. They are not restricted to a single site. Even if the geographic spread of the work is huge, it is impossible that all species are single-site endemics. If this is the case, what you call species are at best populations. If every population you study appears to represent a distinct species then there are two possible scenarios. Either your species are not species but simply populations, or you have entered an area that is utterly understudied. It may well be that the second scenario is true. But then you are prematurely elevating everything to species. What you need to do in this case is work much harder. Go back and collect and spend another 5-10 years in documenting biodiversity patterns. You just take the easy way out and leave it to the those that come after you to validate your shamanic taxonomy.

How should it be done? Let's say that to distinguish species means at the least to search for discontinuous distributions in character states. However, discontinuous distributions can only be identified if the sampling allows for that (you need to have a continuous sampling, so you can distinguish between discontinuity in characters based on discontinuous sampling [your case] and discontinuity that is real). So, for each presumed species, you would need to sample additional populations to study the amount of variation between close populations and how this variation spreads across spatial and environmental gradients. Ideally, we would seek to identify regions where purported species overlap or contact and document the discontinuity in the distribution of characteristics that are unlikely to be varying a lot between individuals, such as hair colour.

If we cannot do that because our sampling is sparse, we cannot just go ahead and speculate that every population we study belongs to a different species, simply because we observed some sort of difference.

Anyway.. why do I feel the need to explain how systematic research should be done to an accomplished author, who will simply be annoyed by my condemnation and shrug it off? I know these taxa will be described simply because the way 'scientific publishing works. It seems that everybody is entitled to their own truth. But not if I can help it. Hence, I assess this work as NOT WORTH BEING PUBLISHED in its present form. what is definitely worth being published is the rich data. Just take away the new names, and I congratulate the author to a very important piece of work! Also, you need to pay much more attention to document observed variation. Variation is the food for evolutionary change. At some stage evolutionary change deserves recognition in a taxonomic classification, but not at any level.

Reviewer #3: This manuscript provides a great deal of morphological information on this relatively poorly known group of snails and includes useful data that is worthy of publication. However, the author has failed to address the more important critiques of this manuscript. Most of my criticisms focus on the ease of comparison of species in the taxonomic section and the sparse taxon coverage in the phylogenetic portion.

As mentioned in my earlier review. I dislike the organization of the taxonomic discussion. The descriptions are very hard to compare between species because they do not contain the same information. The manuscript seems to avoid clear statements like Koltrora differs from Koro in x, y, z or this species differs from that species in x, y , z. Clear, unambiguous comparisons need to be presented in a taxonomic remarks section under every species. Each description should include the exact same data for each taxon in the same order to allow readers to make easy comparisons. The tables of species comparisons include measurement data without any measures of variation making the comparisons between species meaningless. I do not see strong data for recognizing Koltrora as a separate genus from Kora. The number of morphological differences between Koltrora and Koro do not seem very different from the differences within Kora species. I don’t think it makes sense to further subdivide a group which is clearly not well sampled and where relationships are not clearly resolved.

The author makes claims about the monophyly of Neopetraeus, Koro and Koltrora but has not sampled Thaumastus even though a recent analysis based on sequence data suggests Thaumastus may be sister to Kora and a fair amount of morphological data is available for Thaumastus. The lack of sampling does not really allow the authors to make strong claims about relationships.

The English usage while much better, still needs editing. I have not copy edited extensively. A few examples are given below along with some other mostly minor comments.

Abstract line 1, The definite arcticle ‘The’ should be deleted from the beginning of the second sentence.

Third sentence change north and south to ‘northern Gerais state to southern Bahia’.

Delete the sentence that starts ‘However, I remember…’ from the last paragraph of the Introduction.

At the end of page 3 change ‘shell last whorl’ to ‘last whorl of the shell’.

At end of page 3 what do you mean by material? Make this clearer. Radulae were extracted from buccal masses using potassium hypochlorite…

Beginning of page 4. Only the penis is everted. Also, the sentence including ‘despite it is recognized that’ can be simplified to make it clearer.

Table 2. The shell measurements are not meaningful without some measure of variation.

Table 2, ‘longer than wide’? All are longer than wide, I think this should be height to width ratio.

Page 37 Distribution section. Sentence needs editing “Known only from the for region”.

Page 37. Etymology section. Last sentence is not clear. Could rewrite as Color easily seen through translucent shell.

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Reviewer #1: No

Reviewer #3: No

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For journal use only: PONEDEC3

PONE-D-23-41760R2Review of the land snails of the genus Kora from Brazil, with description of eight new species and a new related genus Koltrora, including comparison with two Andean Neopetraeus species (Gastropoda, Eupulmonata, Orthalicoidea).PLOS ONE

Dear Dr. Simone,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I have carefully reviewed the submission history of your manuscript, including the suggestions made by various reviewers and the editor in previous rounds. First and foremost, I would like to apologize for the lengthy and demanding review process, as well as for the time it has required on your part. In recognition of the rigor of this process, I requested two additional reviewers, who had no prior knowledge of your work, to assess the manuscript anew. For this current round of review, both the reviewers and I, as the editor, are distinct from those previously involved with the manuscript.

With the completion of these two reviews, along with my own assessment, I am pleased to inform you that I believe your manuscript is indeed suitable for publication. However, it is clear that there are still some revisions and suggestions that need to be addressed, with particular attention to the comments made by Reviewer 4. Reviewer 4 has provided critical feedback that, in my assessment, should be implemented to meet PLOS ONE’s publication standards.

Please let me know if you have any questions regarding the feedback or the next steps in the revision process. I look forward to seeing the revised version of your work.

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Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Partly

Reviewer #5: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: No

Reviewer #5: N/A

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: The manuscript deals with the revision of a previously created genus of land snails with the addition of a set of new species and the creation of another new genus with a single species. All this information is presented in a cladistic analysis with a particular methodology. This work follows the same scheme and methodology as another contribution by the same author previously published in PLOS ONE. Although this is an indisputable fact, contrary to what the author suggests, criticisms can still be made of the method used and the format employed. The author of the present manuscript is a malacologist renowned for his extensive experience in marine gastropods. On this occasion, the manuscript he wrote deals with species of terrestrial pulmonate gastropods and this is evident in the framework used and the selection of characters for the description of species.

As has been stated for some time and only to cite a single author on this subject: Morphology-based cladistics, the birthplace of cladistic methods, has been attacked by molecular systematists as hopelessly ambiguous and inevitably dispensable in an era increasingly awash in molecular data (Sereno, 2009). In complete agreement with Sereno, I consider that morphological studies as well as cladistics based on morphology are enormously useful tool and should not be discredited to achieve the advancement of science.

At the same time, this kind of study must be rigorous and repeatable, as well as they have to provide predictable hypotheses. The present manuscript, after having already gone through revisions, has been improved since its initial version. It denotes a detailed study of the species involved, with extensive descriptions of organs not generally used after the publication of Tiller (1989). Tillier studied morphological characters from the nervous, pallial and digestive systems in a comparative way of different Pulmonate gastropod groups. This mentioned work is a classic, but received many criticisms for the phylogenetic method used to address its results, in addition to revealing that these characters are highly homoplasic, so in general, only those of the reproductive system continued to be used in taxonomy and phylogeny because are more informative.

Following there is a list of some points for the author to address:

Abstract:

-“a calcified epiphragm, unique in South American snails.” This is incorrect as there are other genera with calcified epiphragm inhabiting South America, please change the sentence.

Results:

All the species descriptions are very detailed, but I found the following problems:

1. The diameter of glandular organs is cited as a distinguishing species characteristic. However, glandular organs undergo significant changes due to seasonality, making this trait unreliable for species comparisons—unless a possibility would be that all specimens were collected and preserved during the same season, which is not specified in this work.

2. The Sperm inner longitudinal fold is a character described in different species and shown through a drawing of a transverse section of the spermoviduct. However, since histology has not been carried out I wonder how he managed to see such a detailed aspect in the interior glandular organ. Not only its morphology but also how it is inserted and connected to the albumen gland.

3. In the same way, there is apparently a problem in the identification/delimitation of organs and the terminology used by the author since he describes and equals the “Carrefour” with the fertilization pouch-spermathecal complex organ. The Carrefour is a portion basal to that structure and usually it is embedded into the albumen gland, for which only with histological methods is possible to see how it is connected either to the hermaphroditic duct or to the albumen gland/spermividuct. See Tompa (1984) for a better description of this area. Figure 11C for example shows a fertilization pouch-spermathecal complex term as “Carrefour” by the author with a long basal tube described as “simple, wide, ~twice longer than receptacle and 3-times its width”. It is not clear to me if this an inner duct (embedded into the albumen gland) or if it is external. Is it consider as part of the “carrefour” in the present manuscript?

4. Taxonomic remarks are excessively long, often repeating characters already described for each species. They should be more concise highlighting the differences of main characters.

5. Pag. 46: author said: …because it lacks the reticulated sculpturing seen in the protoconchs of Bulimulus. This is incorrect since species of Bulimulus lack of a reticulate Protoconch.

6. It is not explained why or for what purpose two cladistic softwares such as TNT and PAUP were used or if differences were found between them. Nor is it indicated which commands and strategy were used for the phylogenetic analysis performed. Regardless of establishing that a method already published is used, the author must indicate which particular strategy was used for the analysis of this data.

7. Another interesting feature is the presence of a thick muscular region in the basal portion of the duct of the bursa copulatrix (e.g., Fig. 6A: bu)” A better drawing or photo is needed here because Fig. 6A is not clear in indicating the basal area of the duct.

8. “A second diverticulum-like projection, usually balloon-like, also filled with albumen and interpreted as an accessory albumen chamber (as), is a novelty in the presently studied species, being absent only in Koltrora”. The terminology used is confusing since this area of union between the albumen gland and the spermatoviduct is present in many Pulmonate snails, but does not have a projection shape nor is it a diverticulum.

Discussion:

Taxonomic discussion

The first and second paragraphs of the discussion should be compacted because they are repetitive and seem to be more of a justification of what was done in the work. Throughout the text, there are also many repetitions of what was already said in Results and at the same time in the same Discussion they are repeated at least twice. The text should be more concise discussing the results obtained concerning what was previously known. In turn, I find it difficult to follow the excessively detailed comparisons of the musculature of the buccal mass and odontophore whose importance is lost when the information is not hierarchical.

The author sustained that “Importantly, no exclusive diagnostic character was obtained, neither at the genus nor at the species levels”, Does it refer to not having identified autapomorphies? How do you interpret the set of diagnostic characters detailed in each case? This is contradictory to the cladistic analysis carried out as well as to the identification of a set of particular characters for each genus.

Please avoid using the following words as they are totally subjective and only meaningful in a narrative context: Importantly, interesting (used several times), relatively, mostly, extraordinary, impressively, Notably, enigmatic.

-“The ureter is totally closed (tubular) in Kora and Koltrora, as apparently is the usual pattern of Orthalicoideans but Neopetraeus has the ureter opened in a larger degree”. This is incorrect since there are other genera classified in Orthalicoidea with this same characteristic.

-“Koltrora differs from Kora….by the thick glandular uterine walls (Fig. 47E: ut); by the epiphallus being widely open to the penis”. The first character is inappropriate since a glandular reproductive organ varies seasonally in its diameter/thickness. The second mentioned character is not clear since the relationship of the epiphallus with the penis is not clarified nor is it detectable in the figures.

-In diagnosing Kora, the author includes characters shared with Koltrora and Neopetraeus. Only those characters unique to Kora that justify its difference from the other genera should be mentioned.

-“Concerning the odontophore, all species exhibit fused cartilages along the ventral edge, but the degree of fusion varies. Koltrora has approximately 100%, while K. uhlei has around 90%; K. nigra has the smallest fusion value of approximately 60%, and the remaining species are about 75% fused.” How is the degree of fusion in % of the cartilages of the odontophore measured? The differences in % are very subtle and merit an explanation of how they were obtained.

-What does it mean that the m8 muscles are relatively uniform??

-The author sustained that “The carrefour region, for example, has a unique arrangement in each species (Figs 6B, 11C, 15A, 20E, 23E, 27E, 33E, 38A, 47C, 53D, 57B).” However, the mentioned figures show significant similarities in the morphology of the organ between Fig.11C and 15A, between 20E and 33E, also between Fig 23E and 27E making this assertion doubtful or at least these figures are not sufficiently clear to show the difference that the author maintains.

The title “Biogeographic analysis” should be changed to “Distribution” since no analysis was performed.

Cladistic Analysis

The goal in cladistics is not always to find a single tree but to identify the most likely and robust relationships, which may involve accepting that there are multiple equally valid phylogenetic hypotheses. Finding multiple equally parsimonious trees is a common situation in cladistics, and although it can present some challenges, this does not imply that the analysis is flawed or useless. Parsimony is one of the criteria for evaluating phylogeny, and finding multiple parsimonious trees means that there are several equally valid hypotheses based on the current data. It may reflect the reality of the evolutionary data. The key is how to interpret and handle this situation.

The tree obtained in the phylogenetic analysis is described in the Discussion, not in the Results section. This should be migrated to Results and only a discussion of the relationships found in the proposed hypothesis should be made.

Some characters that result in synapomorphies in the analysis carried out are doubtful due to what was previously explained about "carrefour" and its delimitation, for example. Character64.

The author sustained that in node 12, 11 synapomorphies support its monophyly and then mentioned the “interesting” ones which in fact are the total set of synapomorphies. Why they are “interestings”? It is not informative to indicate that they are interesting, since having resulted in synapomorphic characters they support the monophyly of the clade. If you wanted to say something more about each of them, you should specify it in another way.

In page 62 the author mentioned that Koltrora pyrostoma has “13 homoplastic autapomorphies, being 4 of them reversions in the present context; two of them are noteworthy: the loss of the accessory albumen chamber (ch. 70) and of the penis

shield (ch. 80). This set of autapomorphies, and the lack of the Kora synapomorphies, are

sufficient reasons for the generic separations of both taxa, despite they share more characters

than Neopetraeus. Please explain why the absence of characters are enough to define the monophyly of a genus. Again in the same page, the author says: The Kora branch (node 13) has the extraordinary support of 25 synapomorphies. The more interesting are: characters 6,7,15,18,29, 36,37,45,47,57,59,62,74,78,83,94”. Why is that? Are there synapomorphies? Which criteria follows the author to consider them “interesting” characters?

Explanations are given in the Discussion section of phylogenetic relationships of non-Orthalicoidean groups (such as Solaropsidae) considered as outgroups in the analysis performed, although the author himself clarifies that his phylogeny does not attempt to resolve relationships outside his ingroup. Inferences are also made about relationships of genera such as Drymaeus and Bulimulus that have few or single representatives in the cladogram. These inferences should be removed because they may be speculative and based on little evidence. The author should focus on discussing only his results with previous phylogenies.

In page 65 the author sustains that: “With such fluidity, maintaining taxonomic stability becomes challenging, and it poses a significant obstacle to achieving mental coherence in the classification trustworthiness”. I would like to suggest to the author another point of view explained by Dominguez & Wheeler (1987) in their paper “Taxonomic stability is Ignorance”. They argued that stability should not be prioritised over accuracy and that sticking to outdated classifications for the sake of consistency reflects ignorance of evolving scientific knowledge. Therefore, the ultimate goal of taxonomy should be to reflect the best possible understanding of evolutionary relationships, even if it means frequent revisions.

Main evolutive processes

Page 66: “Despite being preliminary, this marks the first attempt to construct an orthalicoid

phylogeny based solely on phenotypic features—a crucial initial step toward a broader

understanding” Although I agree with the disconnection of morphology with the latest published molecular analyses, considerations of phylogenetic relationships cannot be made using a restricted morphological phylogeny of such an hyperdiverse superfamily as Orthalicoidea using a low number of representative species.

I would suggest to eliminate this title from the Discussion section.

Reviewer #5: I commend the author for the work done, the detail in the descriptions, and the composure in responding to each of the reviewers' suggestions.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #4: No

Reviewer #5: No

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Review of the land snails of the genus Kora from Brazil, with description of eight new species and a new related genus Koltrora, including comparison with two Andean Neopetraeus species (Gastropoda, Eupulmonata, Orthalicoidea).

PONE-D-23-41760R3

Dear Dr. Simone,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Wesley Dondoni Colombo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I have carefully analyzed your revised manuscript, particularly given its long and complex revision history. This in-depth assessment allowed me to appreciate the scientific rigor and persistence you have demonstrated throughout the review process.

I wholeheartedly agree with your statement that morphological studies, along with morphology-based cladistics, remain invaluable tools for advancing scientific understanding. Despite the increasing focus on molecular data, your work highlights the critical role that morphological and anatomical studies play in deepening our knowledge of biodiversity. This is especially significant in taxonomic and cladistic frameworks, which often face unwarranted criticism in contemporary scientific discourse.

Upon review, I find that the majority of the reviewers' comments have been addressed comprehensively. For the points where you diverged from the reviewers' suggestions, your responses and justifications were clear, well-reasoned, and sufficient to substantiate your choices.

Regarding the taxonomic remarks, which you left to my discretion, I agree with Reviewer 3. I find these remarks to be a valuable addition to your paper and consider them important for maintaining the depth and comprehensiveness of your taxonomic work. I recommend that they be retained in the final manuscript.

In conclusion, I am pleased to inform you that I consider your manuscript ready for publication in PLOS ONE. Congratulations on this accomplishment, which represents a significant contribution to malacology and the study of land snails.

Reviewers' comments: