The Diversity of Freshwater Stygobiotic Crustaceans in the Republic of North Ossetia–Alania Provides New Evidence for the Existence of an Ancient Glacial Refugium in the North Caucasus Region

Round 1

Reviewer 1 Report

This manuscript examine the diversity of Stygobiotic amphipods in Caucasus. The scientific content is fine but should be publish after minor revision as below:

1)      Introduction – the scope of the whole introduction now focused on Caucasus caves and Caucasus amphipods. I think for example, the starting of the second paragraph on Stygobiotic crustaceans should introduce the two genera of the amphipods world wide, what are their ecological roles, what are their habitats and how many species are there in the world, then go into these amphipods in Caucasus.

For example, there are even no mention about the baisc general biology including phylogeny of Niphragus amphipods:

https://academic.oup.com/zoolinnean/article/175/4/812/2449818

2)      In Iran, there are also the discovery of Niphragis amphipod, please see the cite (https://zoolstud.sinica.edu.tw/Journals/56/56-33.pdf), this article also contains COI of the new Niphragis species. Please also use this COI sequence and add into your molecular phylogeny tree

3)      I would suggest to add in the photographs of the whole specimen as well.

ok

Author Response

Dear Reviewer,

We are very thankful for Your remarks, and herewith reply to each step-by-step

1) Introduction – the scope of the whole introduction now focused on Caucasus caves and Caucasus amphipods. I think for example, the starting of the second paragraph on Stygobiotic crustaceans should introduce the two genera of the amphipods worldwide, what are their ecological roles, what are their habitats and how many species are there in the world, then go into these amphipods in Caucasus.

For example, there are even no mention about the baisc general biology including phylogeny of Niphargus amphipods:

https://academic.oup.com/zoolinnean/article/175/4/812/2449818

In the context of this study, mentioning the general biology and the total number of species in the genus Niphargus is superfluous, and probably even excessive information. Most of the articles devoted to the genus Niphargus just begin with the words how many species are in the genus, where they are distributed and so on. In fact, these lines go from article to article without noticeable changes. We decided to avoid this and include only the information necessary for the work in the section, dedicated to the studied area and it diversity

2) In Iran, there are also the discovery of Niphargus amphipod, please see the cite (https://zoolstud.sinica.edu.tw/Journals/56/56-33.pdf), this article also contains COI of the new Niphragis species. Please also use this COI sequence and add into your molecular phylogeny tree

In our phylogenetic tree (see Fig. 1, left part) we used all available sequences of Niphargus from GenBank (NCBI) database, including sequences from Iran. At the same time, the species group studied and described in the article are not closely related to any Iranian species, that why they not used in enlarged parts. We have no ability to give name for each studied species in general phylogenetic as it includes more than 350 species. But the main idea of the tree to show that the studied area is inhabited by to unrelated phylogenetic lineages, not more ...

3) I would suggest to add in the photographs of the whole specimen as well.

Actually, we have photos of only fixed but not alive specimens. Moreover, when fixed almost all species of Niphargus, are completely similar, with some rare exceptions. In our study, these figure of the general view will not have any informative content or context.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript presents an interesting work on the hyporheic fauna of an area of the Caucasus. It is a study that covers an unusual aspect in the world of zoology and the aquatic environment, so its publication is of great importance. It also contributes to the description of new species of some taxa that are rare in the world.

The introduction presents the most recent information about the object of study and adequately justifies it with both classical and current references.

In the methodology it would be necessary to point out some additional details both in the sampling sites (A map indicating the area could be interesting), as well as details of the specific sampling point, number of points sampled, type of cavity, type of lighting, dimensions and depth of the body of water, size of the pore of the net.

In addition, it should be indicated what methodology has been used from an ecological point of view for sampling, whether it is multilevel, systematic, random, fixed-effort sampling, or whether it is sampled until the depletion curve is completed and no new taxa appear.

Regarding microscopic observation, some additional details on magnifications used and taxonomic keys used would also be of interest.

As for the results, the geographical part should be presented before the phylogenetic part (section 3.2) and a brief summary of the number of individuals found, density present, relative abundances, and taxa present.

Tables 1 and 2 and the rest of the document should show that the . as a decimal separator, and not the , as a figure. On the other hand, the parts that the authors have indicated in red must be completed (presumably these are data that should not have been available at the time).

With regard to Figure 2, as already indicated, it is proposed to place this section before the genetic part. Regarding the foot of the figure, the presence of a white triangle is indicated, but it is not observed in it. Five-pointed stars are indicated, but only one white star appears (perhaps that was the white triangle). Nor is the list of grey-hearted stars detailed at the bottom of the figure.

With regard to the names chosen for the new taxa, the authors should consider whether it is necessary to use a word as complex to write to designate a species as Niphargus tschertschesovae, since the use of four and five consonants in a row will in the future be a source of misspellings for correctly spelling the species and will contribute to making your internet search a collection of inappropriate results. In this regard, there is a locality in Switzerland, whose name is very similar to your proposal, and it will be evident that every time someone searches for its taxon, the name of that locality appears:

https://en.wikipedia.org/wiki/Tschiertschen

It is as if the proposed word, which appears to be a derivative of the gentile, comes from a mutation of the Swiss name, as is often the case in many places when name registrars misspell an original name from another language. My proposal in this regard would be either to use the adaptation of the correct geographical name (e.g. "tschiertschenovae"), or to use a simplification of it to facilitate its correct spelling (e.g. "tersesovae").

The supplementary material is correct and necessary for the description of new taxa, and I believe that it should be published in the same document as an annex, not as supplementary material. In my opinion, I believe that drawings should be marked with an arrow to indicate the details of interest in the drawing.

Review the wording of the "Data Availability Statement" section.

As for the bibliography, it is presented correctly, but 11 self-citations of each author are observed. Although this may seem like an excessive number, they have been reviewed and are appropriate because they are part of previous studies by the two authors, both individually and together. Therefore, this aspect should not be taken into account.

Author Response

Dear Reviewer,

Thank You for the valuable comments. We used some of them and modified the manuscript. A number of changes have been made to the text, which are highlighted in red in the new revised manuscript

Herewith, we give the answers step-by-step

In the methodology it would be necessary to point out some additional details both in the sampling sites (A map indicating the area could be interesting), as well as details of the specific sampling point, number of points sampled, type of cavity, type of lighting, dimensions and depth of the body of water, size of the pore of the net.

Amphipods were sampled with plastic hand net with pore size about 1 mm. In our study, we conducted a random sample in all available subterranean water sources (springs, wells, cave reservoirs and river hyporhea). In fact, this is the first search study in this area, which could not be carried out according to some scheme. The specific type of biotope where stygobiotic amphipods were found is indicated under the descriptions of individual species.

In addition, it should be indicated what methodology has been used from an ecological point of view for sampling, whether it is multilevel, systematic, random, fixed-effort sampling, or whether it is sampled until the depletion curve is completed and no new taxa appear.

We represent all available ecological information under the description of each species. This is the first search study in presently completely unstudied area, and not all ecological parameters were studied. The main question of the study is to estimate the diversity of the genus Niphargus in the area and describe the discovered species.

As for the results, the geographical part should be presented before the phylogenetic part (section 3.2) and a brief summary of the number of individuals found, density present, relative abundances, and taxa present.

We represent all available ecological information under the description of each species. This is the first search study in presently completely unstudied area, and not all ecological parameters were studied. The main question of the study is to estimate the diversity of the genus Niphargus in the area and describe the discovered species.

Tables 1 and 2 and the rest of the document should show that the . as a decimal separator, and not the , as a figure.

Changed in the manuscript

On the other hand, the parts that the authors have indicated in red must be completed (presumably these are data that should not have been available at the time).

These data will be added after acceptance of the manuscript as their deposition in the Museum collection and GenBank (NCBI) database needed the verified species name and the title of the publication

 With regard to Figure 2, as already indicated, it is proposed to place this section before the genetic part. Regarding the foot of the figure, the presence of a white triangle is indicated, but it is not observed in it. Five-pointed stars are indicated, but only one white star appears (perhaps that was the white triangle). Nor is the list of grey-hearted stars detailed at the bottom of the figure.

The Legend for the Figure is Changed in the manuscript

As for the order of the pictures and parts of the article. First of all, we conducted a genetic analysis of all the collected samples and understood the geographical distribution of the species, and then indicated them on the map. In the present, the arrangement of pictures and parts is the logic of a sequential work process. At the same time, most of the articles usually give a general geographical picture of the place of work, but currently it is almost meaningless since everyone has a portable gadget (phone) where you can enter the coordinates and understand which area of work is being discussed. We considered giving two different geographical images to be excessive

With regard to the names chosen for the new taxa, the authors should consider whether it is necessary to use a word as complex to write to designate a species as Niphargus tschertschesovae, since the use of four and five consonants in a row will in the future be a source of misspellings for correctly spelling the species and will contribute to making your internet search a collection of inappropriate results. In this regard, there is a locality in Switzerland, whose name is very similar to your proposal, and it will be evident that every time someone searches for its taxon, the name of that locality appears:

https://en.wikipedia.org/wiki/Tschiertschen

It is as if the proposed word, which appears to be a derivative of the gentile, comes from a mutation of the Swiss name, as is often the case in many places when name registrars misspell an original name from another language. My proposal in this regard would be either to use the adaptation of the correct geographical name (e.g. "tschiertschenovae"), or to use a simplification of it to facilitate its correct spelling (e.g. "tersesovae").

We understand the reviewer's concern, but the names of the species were not chosen by chance. First of all, if it concerns toponyms, we coordinated the name with the locals so as not to violate any local traditions. If the name is given in honor of a person (the person, who greatly helped in sampling, for example), then this name is agreed with him personally, and we would not like to change it. There are many similar species names in zoology, and this is not always a problem, especially since there are currently no consonant or similar scientific names among the species of the genus Niphargus. We are forced to reject the proposal to simplify the names of species and leave the previously proposed ones, according to the International Code

The supplementary material is correct and necessary for the description of new taxa, and I believe that it should be published in the same document as an annex, not as supplementary material. In my opinion, I believe that drawings should be marked with an arrow to indicate the details of interest in the drawing.

This was exactly the case in the original text of the article. But the inclusion in the Appendix made it possible to avoid the revision of the article on a large number of borrowed signs that are found in universal descriptions of species. In fact, the article can only pass the examination of the journal in this form. If the editor allows us to change this, then we will be happy to do it.

Review the wording of the "Data Availability Statement" section.

Changed in the manuscript

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have made the modifications proposed in my review and you have also answered those doubts that I had raised, so I consider that it is correct and can be published.