Revised Cornechiniscus (Heterotardigrada) and new phylogenetic analyses negate echiniscid subfamilies and tribes

Echiniscidae are undoubtedly the most thoroughly studied lineage of the class Heterotardigrada. Recently, new subfamilies and tribes grouping echiniscid genera based on traditionally recognized morphological clues have been proposed. Here, by integrative analyses of morphology and DNA sequences of numerous populations of a rare genus Cornechiniscus, we show that this hypothesized classification is artificial. Specifically, we demonstrate that Echiniscinae are diphyletic, as Bryodelphax forms a distinct phyletic lineage within Echiniscidae, and Pseudechiniscinae are polyphyletic, with Mopsechiniscus being indirectly related to Pseudechiniscus, which is closer to the Echiniscus-like genera than to other genera with pseudosegmental plates. Consequently, the subfamilies and tribes are considered as unsupported from the phylogenetic and morphological point of view. The genus Cornechiniscus is revised, and the phenotypic diagnoses of several species are updated thanks to new rich material from Africa, Asia and Europe. Cornechiniscus imperfectus sp. nov. is described from mountains of Kyrgyzstan, being the second appendaged species within the genus and the third known to exhibit dioecy. A taxonomic key to the genus is provided. Systematic positions of Acanthechiniscus and Multipseudechiniscus are also discussed. Acanthechiniscus goedeni is confirmed to be a member of the genus Acanthechiniscus.

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Reviewer comments to Author: Reviewer: 1

Comments to the Author(s) General comments
The work is well written, the critical part on previous phylogeny data is well supported and should be accepted, the descriptions are accurate, there is a lot of morphometry and the images are particularly beautiful and demonstrative. My suggestion is for minor changes: 1. Topology of Fig. 1B is not sufficiently supported and could create misunderstanding. My suggestion is to delete the values under 70%. 2. In the description of the amendments a reference to the original description is lacking. As a consequence, it is not clear what was contained in the original description, what added and what changed. In several cases the original description is well done, not in other cases. Considering that the authors have examined the type material of several species, it should be clarified what confirmed (with references), what changed and what added, in way to have a complete description of the species (and in English; in several cases the original description is in Italian). If this cannot done in Results, it could be done later in a taxonomic account. 3. A species cannot be defined parthenogenetic only because no males are found. In this case parthenogenesis and thelytoky are deductions and no data. The data is the absence of males. Parthenogenesis is highly probable only when a population with a significant number of specimens is found. If so, it must be specified. It is a pleasure to accept your manuscript entitled "Revised <em>Cornechiniscus</em> (Heterotardigrada) and new phylogenetic analyses negate echiniscid subfamilies and tribes" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. The authors have dealt thoroughly with the comments from the two reviewers (who very much approved of most of the initial submission).   (Marcus, 1936;Thulin, unpubl.;Kristensen, 1987).
There are a very few numbers of minor editorial corrections I have comment below, together with a few points that have to be discussed and corrected before the paper can be  Table 1). Please, make a Table   ("2") with all species of Cornechiniscus used and illustrated in this manuscript. I know it is very difficult to get all the information as in Table 1; however, we need "Locality" and "Collector" of all eight species of Cornechiniscus, because they all have been illustrated so nicely in the manuscript. However, where are the specimens coming from!! 5). References.
The authors cite correctly that Cornechiniscus holmeni original was described from Greenland (Petersen, 1951) and they mentioned all the other localities, where this species have been found. However, in the Table 3   7). I am not the best to criticized English language and I cannot help in this way, but it has been a great pleasure to read the manuscript; however, I think that the manuscript need a reviewer to correct the Englisha reviewer who is much better to English than me. 10). It has been a great pleasure to read the manuscript and the manuscript should be published with any doubt in "Royal Society Open Science". In fact the manuscript is outstanding. However; I will recommend a minor revision with the very few points I have written in the review and with the citations of some of the last publications.

Dear Editors,
We are very grateful for the reviews and your conditional acceptance of our manuscript. We agreed with the vast majority of remarks and we provided our detailed response to the few points we did not agree with. As requested, the revised version of the manuscript was proof-read by a native English speaker.
Yours sincerely, The work is well written, the critical part on previous phylogeny data is well supported and should be accepted, the descriptions are accurate, there is a lot of morphometry and the images are particularly beautiful and demonstrative.
Thank you for your kind words and suggestions, they are all addressed below.
My suggestion is for minor changes: 1. Topology of Fig. 1B is not sufficiently supported and could create misunderstanding. My suggestion is to delete the values under 70%.
Changed as suggested.

2.
In the description of the amendments a reference to the original description is lacking. As a consequence, it is not clear what was contained in the original description, what added and what changed. In several cases the original description is well done, not in other cases. Considering that the authors have examined the type material of several species, it should be clarified what confirmed (with references), what changed and what added, in way to have a complete description of the species (and in English; in several cases the original description is in Italian). If this cannot done in Results, it could be done later in a taxonomic account.
Changed as suggested.

GENERAL. The manuscript by
Gąsiorek & Michalczyk is outstanding. It combines the classical way of tardigrade research -morphology and systematic -done with light microscopy (LM), scanning electron microscopy (SEM) and with a very sophisticated molecular dataset of the rare genus Cornechiniscus and related genera (see, Fig. 1A and Fig. 1B). The most significant is that the authors have observed all the eighth species of Cornechiniscus there are described today and illustrated them all nicely in the manuscript! That is really unique! I think that the authors have to check the "References" and citations very carefully again, especially the authors own "References". However, the list of "References" for the genus Cornechiniscus is really fantastic; perhaps the authors lack a few reports from Arctic. The last part of the manuscript is dealing with the recently published paper (Guil et al., 2019) about "An upgraded comprehensive multilocus phylogeny of the Tardigrada tree of life". This is really great that the authors "negate the recently hypothesized echiniscid subfamily and tribes", because Guil et al. 2019 nearly only have based the phylogeny of Tardigrada by morphology. However, the author's Table 12 give us not any new resolutions of "tribal classification of the family Echiniscidae". It is really great that the authors focus on the problem with the classification of the family Echiniscidae; but, how can the authors know that the Parechiniscinae is paraphyletic, when we not have published the molecular data of Novechiniscus and all the new species of Parechiniscus? Finally, both authors are the world experts in the genus Bryodelphax (Bryodelphaxini). It has been a great pleasure to read all their papers. However, why are the problem with genus Bryochoerus (sister genus to Bryodelphax, see Kristensen 1987), not mentioned in the manuscript and in Table 12? So many new species of Bryochoerus are under descriptions and the new species from China -have already been described (please cite also the papers about Bryochoerus -in the manuscript) -also when the authors think that the genus Bryochoerus Marcus, 1936 is very dubiously (Marcus, 1936;Thulin, unpubl.;Kristensen, 1987). There are a very few numbers of minor editorial corrections I have comment below, together with a few points that have to be discussed and corrected before the paper can be published with a minor revision. It is a fantastic manuscript with lot of new information about the whole phylum Tardigrada.
Thank you for your kind words and remarks -the detailed response is provided below. We double-checked the References. Table 12 summarises the drawbacks of Guil et al.'s systematic units. We clearly stated in the Discussion that, in our opinion and considering all available data, no subfamilies and, maybe, tribes, can be introduced for this family at present.
It is true that there are no available genetic data for Novechiniscus, but already at the time of publishing Guil et al. were there morphological phylogenies accessible (Jørgensen 2000, Gąsiorek et al. 2018) -all denying monophyletic character of the putative subfamily "Parechiniscinae". We clearly stated in the Discussion that these phylogenies were neglected by Guil et al. We do not know why (especially as the first work was performed by one of the co-authors of that unfortunate paper), but it non-deliberately gives an impression of cherry-picking which data the researchers like or not.
Finally, we did not elaborate on the Bryodelphax-Bryochoerus problem as it is a side issue, outside the scope of our manuscript. Nevertheless, we added additional citations signalling this conundrum. 1). Title: I would like to shorting the title -it is very long now and why to focus of a single (stupid) paper (Guil, Jørgensen and Kristensen, 2019)? Please change the title to: Revised Cornechiniscus (Echiniscidae, Heterotardigrada) and new phylogenetic analyses of echiniscid subfamilies and tribes.
We changed the title slightly "Revised Cornechiniscus (Heterotardigrada) and new phylogenetic analyses negate echiniscid subfamilies and tribes", but we cannot delete the second part of the title altogether, as the bulk of the Discussion addresses the vague analyses of Guil et al., and the present title reflects the content of the manuscript. Moreover, we did not analyse "subfamilies and tribes", as our a priori assumption was that such a grouping is artificial.
2). Keywords: I would like to include the word limno-terrestrial life cycle. Echiniscidae can now be deleted, when it is included in the title. "Echiniscidae" deleted, but the proposed terms has barely anything to do with the issues talked in this manuscript. "phylogenetic congruence" added.
Thank you. 4). The reviewer really lacks all information about what species of Cornechiniscus have been investigated "outside" the author's own collections (great Table 1). Please, make a Table ("2") with all species of Cornechiniscus used and illustrated in this manuscript. I know it is very difficult to get all the information as in Table 1; however, we need "Locality" and "Collector" of all eight species of Cornechiniscus, because they all have been illustrated so nicely in the manuscript. However, where are the specimens coming from!! Figure legends provide the information on where specimens were collected directly or indirectly by stating that they represent a type series (holotype/paratype etc.) for which collection data are provided in Table 1. We also clearly stated in the M&M section that all type series were examined, therefore a reader should refer to original descriptions in such cases. Thus, we think that the origin of populations is well-explained in the manuscript and does not require an addition of a new table.
The authors cite correctly that Cornechiniscus holmeni original was described from Greenland (Petersen, 1951) and they mentioned all the other localities, where this species have been found. However, in the Table  3 they also mention Igloolik (Turton Bay). Please, cite therefore the Igloolik paper: Cited.

6). Figures and
Tables. This is the most fantastic part of the whole manuscript. Both authors are known to make great SEM-illustrations of new species of tardigrades, but how can they use very old museum material in this unique way? Please, see figure 4 of Cornechiniscus holmeni from Greenland and Tables 3 and 4. A small correction: there are 3 localities in Greenland called Âta -therefore, please write Âta, Nuussuaq (Greenland).
Corrected. Fig. 1A and 1B. These two figures are what we all are waiting for: The phylogenetic relationships in the family Echiniscidae. We all use Oreella mollis as an out-group! Perhaps, this is not the best terrestrial heterotardigrade to use. Could we also use the tidal species Echiniscoides sigismundi (family Echiniscoididae) as an out-group -too? The genus Oreella in the family Oreellidae is very interesting because the females have seminal receptacles similar to the females in the order Arthrotardigrada. Seminal receptacles are never seen in the Echiniscidae and Echiniscoididae.
Phylogenies with other outgroups were provided as supplementary materials. 7). I am not the best to criticized English language and I cannot help in this way, but it has been a great pleasure to read the manuscript; however, I think that the manuscript need a reviewer to correct the English -a reviewer who is much better to English than me.
The manuscript has now been proof-read by an English native speaker. Please check all citations both in text and in the "References" very carefully, especially the German´s references.
Double-checked and adjusted to the RSOS requirements.
Corrected. 9). Acknowledgements. Please, acknowledge Asger Ken Pedersen (Museum of Natural History of Denmark, University of Copenhagen) for collecting the moss samples containing Cornechiniscus madagascariensis from Ethiopia.
Added. 10). It has been a great pleasure to read the manuscript and the manuscript should be published with any doubt in "Royal Society Open Science". In fact the manuscript is outstanding. However; I will recommend a minor revision with the very few points I have written in the review and with the citations of some of the last publications.
Thank you once again for your detailed and encouraging review.