A New Species of Diacyclops (Copepoda, Cyclopoida) from the D. crassicaudis (Sars, 1863) Species Group with Critical Taxonomy Remarks^†

Round 1

Reviewer 1 Report

This study is devoted to one complex genus of Cyclopoida. In general terms, the morphology and the figures are excellent. I am not an expert on this genus; however, the comparison with D. crassicaudis seems to be ok. The description of the species is good in the book of Crustacea by Sars (1913), so the comparisons can be ok with published materials, with no need to re-visit the type material of this difficult genus. The nomenclatural act was published in Zoobank as well.

My only concern is that only one specimen was sequenced, and I can not check the trace files to see the quality of the sequences. The primers used indicate a bi-directional PCR, but I do not see if sequencing was bi-directional and how the authors edited the sequences. I suggest sequencing more specimens to see if the authors have haplotype variability and to make a more comprehensive comparison. Also. I suggest to look in BOLD (boldsystems.org) if more sequences are available. Maybe the problem of the third codon for COI discussed in the manuscript will disappear (this is the first time I have seen this problem with COI; if authors find some examples in other related animals, it will be interesting). I can see that more than one sequence was used for three species, giving more consistency to the tree. With more sequences, I would recommend other methods of species finding as ASAP:

Puillandre, N., S. Brouillet & G. Achaz, 2021. ASAP: assemble species by automatic partitioning. Molecular Ecology Resources 21(2):609-620 doi:10.1111/1755-0998.13281.

Then, authors can compare the ML tree and ASAP.

Some references need to be completed, for example, 25. Reid…..the title is incomplete; please check all the literature.

Fig 1.- I do not undersand what is B, D, E points. Are they the localities of the collection in the photographs below? The explanation could be more precise, and if authors have the other points in photographs could be interesting to show them.

All other figures are good quality.

In general terms English is good, only some explanations should be clarified.

Author Response

My only concern is that only one specimen was sequenced, and I can not check the trace files to see the quality of the sequences. The primers used indicate a bi-directional PCR, but I do not see if sequencing was bi-directional and how the authors edited the sequences. I suggest sequencing more specimens to see if the authors have haplotype variability and to make a more comprehensive comparison. Also. I suggest to look in BOLD (boldsystems.org) if more sequences are available. Maybe the problem of the third codon for COI discussed in the manuscript will disappear (this is the first time I have seen this problem with COI; if authors find some examples in other related animals, it will be interesting). I can see that more than one sequence was used for three species, giving more consistency to the tree. With more sequences, I would recommend other methods of species finding as ASAP:

The saturation effect has previously been shown for Copepoda: In the obtained set of COI gene sequences, an effect of synonymous substitution saturation is revealed (Mayor et al., 2010). COI substitutions were saturated among Eurytemora species (Lee C. 2000 Global phylogeography of a cryptic copepod species complex and reproductive isolation between genetically proximate “populations” Evolution 54(6):2014 – 2027 DOI: 10.1111/j.0014-3820.2000.tb01245.x)

The effect of saturation of nucleotide substitutions at position 3 persists, even though we changed the data set and searched for the closest sequences in BOLD. Therefore, it is not correct to use ASAP method, which distinguishes species by genetic distances. It will not be possible to add additional individuals of a new species now, because there was only one individual fixed in 96% ethanol.

We changed the values in the test for the saturation effect because the data set for other species changed.

In the text of the article, according to the comments of 1 reviewer, we wrote how we edited the sequences and how they were obtained (from two primers or one), and we also opened access to the sequences in GenBank. Trace files with sequences are posted in BOLD.

Some references need to be completed, for example, 25. Reid…..the title is incomplete; please check all the literature.

Changed

Fig 1.- I do not undersand what is B, D, E points. Are they the localities of the collection in the photographs below? The explanation could be more precise, and if authors have the other points in photographs could be interesting to show them.

Added: points B, D, E correspond to the photographs below

Reviewer 2 Report

The topic of the manuscript is the description of a new copepod species, which was collected in the northern part of Russia, in the Paleartic region. The authors join to the classical morphological description also morphometric and molecular analsys in order to validate the tassonomic classification. In addition, they critically revise some other species belonging to the genus Diacyclops. Overall the paper is well written and worth of publication, since gives new information about the complex diversity of copepods. In my opinion only minor changes are required before the publication.

- Materials and Methods: cite figures 1A and 1B before the figure 1C

- How many specimens were measured in total? How may males and how many females?

- Table 1: I suggest to divide the table in two, one for collected material and one for material deriving from previous publications. What do the acronyms (d_A1, c_c_R1...) mean?

- Line 165: explain to what the URL refers. I tried to open it in internet but it doesn’t work.

- Line 166: delete the number of figures, they are already cited in the text

- Lines 179-201: I suggest to move this part to the Materials and Methods paragraph

- Figure 6: please indicate the P6 in the figure

- Line 366: “spermatophores” instead of “speramatophores”

- Lines 372-374: move the sentence “Anabar Plateau, pH ….are known” to the Materials and Methods paragraphs.

- Lines 375-378: I suggest to move this part to the Discussion paragraph

- Line 388: put D. iranicus in italic

- In the titles of subparagraphs put the name of the species not in italic

- Line 441: delete number of figures, they are already cited in the text

- Lines 435-437: put the name of the species in italic

- Line 461-476: I suggest to move this part to the Materials and Methods paragraph

- Figure 13: put ABCDE with the lowercase letter

- Line 518: delete the number of figures, they are already cited in the text

- Lines 519-521: I suggest to move this part to the Materials and Methods paragraph

Author Response

The topic of the manuscript is the description of a new copepod species, which was collected in the northern part of Russia, in the Paleartic region. The authors join to the classical morphological description also morphometric and molecular analsys in order to validate the tassonomic classification. In addition, they critically revise some other species belonging to the genus Diacyclops. Overall the paper is well written and worth of publication, since gives new information about the complex diversity of copepods. In my opinion only minor changes are required before the publication.

- Materials and Methods: cite figures 1A and 1B before the figure 1C

- Line 366: “spermatophores” instead of “speramatophores”

- Line 388: put D. iranicus in italic

- In the titles of subparagraphs put the name of the species not in italic

- Lines 435-437: put the name of the species in italic

Changed

- How many specimens were measured in total? How may males and how many females?

Measurements were made only for females (Table 1)

- Table 1: I suggest to divide the table in two, one for collected material and one for material deriving from previous publications. What do the acronyms (d_A1, c_c_R1...) mean?

Added: d – dyabdar, c – crassicaudis, Y – Yakutia, A – Anabar plateau, T – Tatarstan, R – specimens from reference

- Line 165: explain to what the URL refers. I tried to open it in internet but it doesn’t work.

Strange, the site seems to be working.

- Line 166: delete the number of figures, they are already cited in the text

- Line 441: delete number of figures, they are already cited in the text

- Line 518: delete the number of figures, they are already cited in the text

- Lines 179-201: I suggest to move this part to the Materials and Methods paragraph

- Lines 519-521: I suggest to move this part to the Materials and Methods paragraph

- Line 461-476: I suggest to move this part to the Materials and Methods paragraph

We did not correct the text based on these comments. Figure numbers and localities in taxonomic works are given directly in the description.

- Figure 6: please indicate the P6 in the figure

There is no P6 in this figure

- Lines 372-374: move the sentence “Anabar Plateau, pH ….are known” to the Materials and Methods paragraphs.

We think that it is more logical to insert this into the ecology of a particular species, since D. crassicaudis is also considered in the work.

- Lines 375-378: I suggest to move this part to the Discussion paragraph

This directly relates to distribution and is of little significance in the discussion.

- Figure 13: put ABCDE with the lowercase letter

All figures are made in the same style

Reviewer 3 Report

The article is very valuable and can be published in its current form.

The article presents a new species, Diacyclops dyabdar sp. nov. from the Diacyclops crassicaudis species group based on an integrative taxonomy approach with very good morphological description. The manuscript gives new insight into Diacyclops taxonomy and sheds light on D. crassicaudis species group, as well as resolves the taxonomic position of several questionable taxa.

The morphological description, drawings, and photos are great. The authors could consider improving the genetic part - I lacked a slightly more precise comparison and species delimitation with D. crassicaudis. I realized that molecular markers were obtained for a single female of D. dyabdar sp. nov. It would be interesting to compare it with D. crassicaudis from Tatarstan.

Maybe you will be interested – there are a few COI sequences of Diacyclops from the groundwater of Poland (D. crassicaudis – 5 haplotypes; D. bistosus and D. bicuspidatus – 2 haplotypes) in BOLD project - DS-10867332 (open access). More info: https://doi.org/10.1016/j.ecohyd.2023.11.012; 

Lines 435-437: species names without italics

Line 657-659: different fonts

Author Response

The article presents a new species, Diacyclops dyabdar sp. nov. from the Diacyclops crassicaudis species group based on an integrative taxonomy approach with very good morphological description. The manuscript gives new insight into Diacyclops taxonomy and sheds light on D. crassicaudis species group, as well as resolves the taxonomic position of several questionable taxa.

The morphological description, drawings, and photos are great. The authors could consider improving the genetic part - I lacked a slightly more precise comparison and species delimitation with D. crassicaudis. I realized that molecular markers were obtained for a single female of D. dyabdar sp. nov. It would be interesting to compare it with D. crassicaudis from Tatarstan.

Unfortunately, individuals from Tatarstan were collected in formaldehyde. To collect you need to wait until the next season.

Maybe you will be interested – there are a few COI sequences of Diacyclops from the groundwater of Poland (D. crassicaudis – 5 haplotypes; D. bistosus and D. bicuspidatus – 2 haplotypes) in BOLD project - DS-10867332 (open access). More info: https://doi.org/10.1016/j.ecohyd.2023.11.012; 

This sequences added to tree

Lines 435-437: species names without italics

Line 657-659: different fonts

Changed