Molecular Phylogeny of the Genus Paracanthonchus (Nematoda: Chromadorida) with Description of P. yeongjongensis sp. nov. from Korea^†

Round 1

Reviewer 1 Report

This is well written and presented manuscript, with the description of the new Paracanthonchus species, updated list of valid Paracanthonchus species and molecular information about the genus. All these are of high importance in the light of Nematode taxonomy and systematics. All methods were used correctly.  This is interesting manuscript and definitely deserved to be published.  However, some changes are suggested for improvement before publication.  All my comments and suggestions are included in the pdf file that I have submitted.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

General comment: the authors provide an account of a possible new species of marine nematode from the genus Paracanthonchus, named P. yeongjongensis, found at Yeongjongdo Island, Incheon, Korea. They also revise the current number of species in the genus and provide tabular key to their morphological characteristics. The species in the genus Paracanthonchus revised by the authors are basically the ones currently listed at wormbase (nemys), so I do not see added value in this. In fact, one species is missing, Paracanthonchus wellsi Leduc & Zhao, 2023, from revised species list as well molecular comparisons. This species is recognised at nemys and has provided molecular data in NCBI database, however it is not mentioned in the manuscript, or why it was not included.

The tabular key to currently valid species is an added value, however it would be more valuable if authors performed phylogenetic/similarity analyses on morphological characters, so that when stating that there is no relationship between molecular and morphological data, that can be visualised. The main phylogenetic tree used for the discussion is provided in such poor quality that it is difficult to follow discussion and observe support values. From what I can vaguely see, you do not have good support for the separation of your species in the BI tree, it is better in ML tree, the one provided in the supplement. Generally, only strong support values are provided in figures, > 90 for BI and > 50 for ML trees. This would add to clarity.

However, the major weakness of the data is sparse species sampling in the phylogenetic tree, as well as for K2P distances.  As authors state in their discussion, not many sequences are currently available. Given that, I would not erect new species without prior detailed molecular analyses of entire genus, which is more needed than the revision of existing taxonomy based on morphological characters. Also, information from at least two markers is needed, one mitochondrial, the other nuclear.  What is also needed is to start focusing on population analyses. Two sequences do not catch species genetic diversity and it is important for judging genetic/morphological interspecific/intraspecific relationships.

Specific comments:

Lines 35 – 36 The facts cannot be verified from the literature cited (1,2) (number of genera and species); plus the literature does not state that  “Cyatholaimidae are most diverse group of marine-living nematodes”, rather relatively diverse and mostly marine species. Please rephrase the sentence for clarity and accuracy.

In fact, Cunha et al 2022 state: „Cyatholaimidae includes 211 valid species, classified in 20 genera.“

Line 40 provide full citation in reference list for the recognition of Cyatholaimidae  as a family

Lines 80 – 81 cannot verify number of species in Hodda, 2022 (ref 2?). This paper talks about trophic groupings of Nematoda, but there is: Phylum  Nematoda:  a  classification,  catalogue  and  index  of  valid  genera,  with  a census of valid species (https://doi.org/10.11646/zootaxa.5114.1.1) from 2022. Is this the correct reference?

No Tchesunov, 2015 in the reference list….but there is Tcheshunov, 2015 (n 10)?

Please recheck all your references. 

Also add how many species were in Miljutina and Miljutin, 2015.

Line 85  the sequence are not provided!

Line 103 – would ethanol affect nematode morphology? Specifically measured lengths? As stated in discussion, is it a factor in your analyses?

Lines 133 – 134 How much primer and how much template was used?

Line 138 Table 1 Why such small fragment for  mtCOI? Did you test standard Folmer primers?

mtCOI forward primer is from Bowles et al., 1992

Why two primers for 18S? There is no mention of second product in the analyses.

Line 169 Tchesunov, 2014

Line 171 delete one maybe

Line 321 Paracanthonchus wellsi Leduc & Zhao, 2023 not mentioned

Line 322 Table 3 is mentioned before Table 2, therefore table 3 should be named table 2, etc.

Line 327 I presume this is erroneous?

Line 369 males

Line 374 – 376 how are mtCOI sequences identical if there is K2P > 0? Table S1

And this is also the problem of your study. If you have identical sequences then there is no real way of assessing intraspecies variability/diversity. You need to sequence more individuals to be able to judge this parameter and propose new species (5 - 10).

Line 378 It is not clear in excel table which is Table S1 – mtCOI? Please rename the sheets to contain table names and gene names

Line 397  State the units in table caption

Line 403 Figure 4 is in very bad resolution, names and posterior probabilities are not visible. The quality of the figure must be improved!

Line 408 Check statement according to comment for line 35-36

Line 411 of genus?

Lines 417 – 418 incorrectly written references

Line 426 thereof? Sentence confusing

Line 437 delete being

Line 438 – 439 sentence unclear, please revise for clarity

Line 442 Maybe it was fine back in 1922, but today without molecular evidence, it is not acceptable to erect subspecies. Your argument is correct, just correct this part of the sentence

Line 482- 484  Why is this high? These are different species. What is your reference for this?  Plus you are using a really small sequence which may not capture all relevant information. 5% is the threshold, but it can be higher. This may also reflect the fact that this is not a monophyletic clade, however, that does question the integrity of genus Paracanthonchus and identification of these species.

Please corroborate all statements with references.

Line 486 you cannot judge intraspecific distance as you have only two identical sequences. This may reflect small diversity at the locus for this species, however it may also reflect small sampling. Please collect more sequences (min of 5 – 10) to be able to discuss intraspecific distances.

Line 497 – 499 Not when possible, but is a must. As you state, since vast morphological variability is possible alongside low genetic variability, entire genus requires reevaluation based on molecular and morphological dana, simultaneously.

Line 523 – 524 sentence confusing, please revise for clarity; erroneous.

Line 534 in conjunction to topology based morphological traits? Meaning not clear

Lines 563 – 564 It is of outmost importance to submit sequences generated in your research to a public database (NCBI, ENA at EBI or DDBJ) to allow future comparisons, replication, and clarification of taxonomy. Without submitted sequences (all – mtCOI, 18S, 28S), the study is not valid for publication.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear Authors,

Thank you for addressing all raised questions and complementing the description of new species Paracanthonchus yeongjongensis with more sequences. I believe conclusions are now much better supported with the data, and the adjusted narrative of the discussion well describes the issues in Cyatholaimidae taxonomy and species delimitation. While preparing the final draft to include sequence accession numbers, I have several minor corrections throughout the text.

A suggestion for accession number representation, once they are available, it would also be nice to include them in phylogenetic trees next to your species name.

Line 37 has been the subject of many studies

Line 39 - 40 sentence somewhat not clear, but it is not necessary in the text (Over the years, several…). Delete the sentence.

Line 67 – 68 and 2–4 arranged lateral differentiation – expression unclear?

did you mean: distally expanded and dentate gubernaculum?

Line 81 Tchesunov 2015 and Lee et al. 2016 are references 18 and 19 respectively in your current literature. Please recheck all the numberings, they seem to be off by one number.

Line 136 achieving 20 μl of total reaction volume

Thank you for providing volumes for primers and template, however without the concentration in stock/working solutions or DNA concentration of the template, this is not very meaningful. Please provide final concentrations/DNA yield in the reaction.

Line 139 agarose

Line 141 Table 1

Please check Amplification conditions for mtCOI; in the second line it states 72°C for 3 s – I assume this is 30 seconds?

Also is it correct that elongation was 72 °C for 3 min for 18S? This seems very long for such a small fragment.

Line 146 two strands – does this mean you sequenced PCR products in both directions? If yes, please indicate so in the previous paragraph.

Line 147 Aligned sequences were compared against NCBI GenBank database using BLAST algorithm [39].

Line 148 seven

Line 168 visualizations and modifications were made.

Line 180 larger?

and one or two pairs of smaller subventral teeth

Line 358 – 360 Sentence unclear, please rephrase for clarity

Lines 360 – 376 please English proof this section, several unclear sentences

Line 389 Table S3

Line 397 delete respectively

Line 459 60 species

Line 461 seven species? Paracanthonchus wellsi?

Line 468 delete of

Lines 472 – 477 I suggest rephrasing this as:

While subspecies may differ in minor morphological variation, it should be noted that the specimens have been fixed in ethanol for over fourteen years. Color variation can be very subjective, and ethanol samples are highly prone to discoloration. Until molecular evidence becomes available, it is our decision to consider this species synonymous with its parent species, P. steueri.

Line 481 distal of gubernaculum? Unclear

Line 484 of some species

Line 486 – 488 corroborating molecular data on existing and new species will help organize and understand species delineation of Cyatholaimidae.

Line 490 characteristics

Line 506 differs in body length

Lines 513 – 514 not clear; is this after the initial analyses or after the attempt to add P. wellsi? If you focused just on the sequences that you were able to correctly align, that is ok; you can simply state that only such sequences were used in the analyses.

Lines 513 – 515  All three markers showed very low intraspecific K2P distances (0–0.3%), indicating the seven specimens analyzed here belong to the same species (Table S1, S2, S3).

Line 517 – 519 Our results showed that mtCOI K2P distances between other congeners ranged from 29.9-40%, and from 32.8–42% between species of other genera of the family Cyatholaimidae (Table S3).

Line 529 - 530 dispersion, all whilst showing low genetic differentiation [50]. This goes to show that there is no one single data type that completely reflects inter/intra species delineation.

Line 539 mixed instead of unclean

Line 560 delete used

Line 583 begin

Line 660 Tchesunov

Author Response

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Author Response File: Author Response.docx