Two New Free-Living Marine Nematode Species of the Genus Haliplectus Cobb, 1913 (Haliplectidae) from Mangroves of Shenzhen, Guangdong Province, China
Round 1
Reviewer 1 Report
The manuscript is devoted to the description and molecular characterisation of two new species of nematodes belonging to the genus with uncertain phylogenic position.
The description itself is adequate and appropriate.
The main problem is the figures. Line drawings are of average quality. Photos are of low quality. The later too small and most of peculiarities are not distinguishable. Photos are with low resolution, not in the focus. For example, Fig 2A - lateral view of male head end, showing amphidial fovea - in fact, not amphid is visible on the photo. And the rest of the photo are with same problems. So, the photos must be redone.
All main structures that distinguish described species from congeners must be clearly visible on both photos and drawings. For example, supplement in H. major description are not visible at the photo and hardly depicted in the drawings.
The name of the second species H. futian does not correspond Latin rules. Adjective derives from toponym must have special ending, so it should be either futianensis or futianisus.
The most expecting and intriguing part of the manuscript was molecular phylogeny. Because position on the phylogeny tree of the family Haliplectidae is still unclear phylogeny tree provided in the manuscript could enlighten this. But authors restricted themself with one genus, while not taking into account recent paper by
Gharahkhani, A., Pourjam, E., Leduc, D., & Pedram, M. (2022). The nematode genus Haliplectus Cobb, 1913 (Chromadorea: Haliplectidae): phylogenetic relationships, description of a new species from the Persian Gulf, southern Iran, and a tabular key to valid species. Nematology, 1(aop), 1-17. My recommendation is to rewrite discussion part with taking into account new paper.
Author Response
1、The main problem is the figures. Line drawings are of average quality. Photos are of low quality. The later too small and most of peculiarities are not distinguishable. Photos are with low resolution, not in the focus. For example, Fig 2A - lateral view of male head end, showing amphidial fovea - in fact, not amphid is visible on the photo. And the rest of the photo are with same problems. So, the photos must be redone.
Response：Thanks for your reminder, all of the photos have been redone. And added arrows to indicate morphological feature points.
2、All main structures that distinguish described species from congeners must be clearly visible on both photos and drawings. For example, supplement in H. major description are not visible at the photo and hardly depicted in the drawings.
Response：Thanks for your reminder. The supplements in H.major are not obvious. It has been indicated by arrows in figure 1D and figure 2C.
3、The name of the second species H. futian does not correspond Latin rules. Adjective derives from toponym must have special ending, so it should be either futianensis or futianisus.
Response：The problem has been corrected.
4、The most expecting and intriguing part of the manuscript was molecular phylogeny. Because position on the phylogeny tree of the family Haliplectidae is still unclear phylogeny tree provided in the manuscript could enlighten this. But authors restricted themself with one genus, while not taking into account recent paper by “Gharahkhani, A., Pourjam, E., Leduc, D., & Pedram, M. (2022). The nematode genus Haliplectus Cobb, 1913 (Chromadorea: Haliplectidae): phylogenetic relationships, description of a new species from the Persian Gulf, southern Iran” and a tabular key to valid species. Nematology, 1(aop), 1-17. My recommendation is to rewrite discussion part with taking into account new paper.
Response：Thanks for your suggestion. In this study, we focus on the established new species. Gharahkhani et al. (2022) just published a review paper on Haliplectidae, which inspired our research. In this case we have considered the different genes are suitable for resolution at different taxonomic levels. In order to further enrich the database resources, we mainly supplemented the sequence of new species, and added one more cladogram result by COI gene. The results and discussion were rewritten. The new sentences were following:
Materials and Methods:
Add “And the primer JB2F (5’-ATGTTTTGATTTTACCWGCWTTYGGTGT-3’) and JB5GEDR (5’-AGCACCTAAACTTAAAACATARTGRAARTG-3’) [20] was choosen to amplify COI gene sequences.”
Result:
3.3 Sequences Information and Caladogram Analyses
According to the NCBI database, 18S rDNA and COI gene are more completed resource than 28S rDNA sequences. Therefore, two fragments of gene were chosen and analyzation respectively in this study. The Genbank accession numbers of 18S rDNA were as follows: ON688994 - ON689005 and ON689013 - ON689024, and COI gene were ON660976 – ON660979. A 630 basepairs (bp) sequence of COI, and a 310 bp sequence of COI were used in this study. The Maximum Likelihood (ML) topology was used in this study (Fig. 5 and 6). Both 18S rDNA and COI cladogram indicated that morphological evidence of H. futianisus sp. nov. and H. major sp. nov. being a distinct species. Interspecific and intrageneric thresholds of Kimura 2-parameter (K2P) distance divergences were as follows: 0.1 – 3.1 % and 6.7 – 9.0% for 18S rDNA; 0 – 0.3 % and 19.1% – 29.5% for COI, respectively.
Discussion:
Regarding phylogenetic analyses, the genes are suitable for resolution at different taxonomic levels. 18S rDNA sequences possess good resolution for identification at higher taxonomic rankings such as to separate the allied genera, while COI is a useful gene for identification at the species level [21,24-25]. Gharahkhani et al. (2022) [10] had constructed a consensus cladogram by 18S rDNA and gave a viewpoint that the classi-fication of the family Haliplectidae might be considered as incerta sedis within the class Chromadorea. However, the current molecular sequence database of marine nematodes was remains incomplete, also misidentified or unidentified organisms is a huge prob-lem [26]. Those reasons were restricted to clarity the systematic of family Haliplectidae. In present work, we provided two different sequence fragments which were 18S rDNA and COI to established the new species and rich the source of database for further study.
Reviewer 2 Report
Dear Author,
The study “Two new species of free-living marine nematode of the genus Haliplectus Cobb, 1913 (Haliplectidae) from mangroves of Shenzhen, Guangdong Province, China” contains a description of two new Haliplectus marine nematodes. These two new species described are distinct from other known species in the genus. Validity of the new species is no problems. Quality of the text description and illustrations is generally appropriate. But there are some errors in the manuscript, especially in the discussion section for molecular results, which should be modified by people with high English level or native English. For the errors, see the comments on the manuscript. Please, see the attached file.
Comments for author File: 
Comments.pdf
Author Response
The study “Two new species of free-living marine nematode of the genus Haliplectus Cobb, 1913 (Haliplectidae) from mangroves of Shenzhen, Guangdong Province, China” contains a description of two new Haliplectus marine nematodes. These two new species described are distinct from other known species in the genus. Validity of the new species is no problems. Quality of the text description and illustrations is generally appropriate. But there are some errors in the manuscript, especially in the discussion section for molecular results, which should be modified by people with high English level or native English. For the errors, see the comments on the manuscript. Please, see the attached file.
Response：Thank the reviewer for their detailed review and modification of the full text. Most of the errors (on line 2, 5, 16, 20-23, 31, 41, 46, 48, 49, 52, 53, 66, 67, 99, 114, 122, 125, 126, 128, 130, 133, 136-138, 140, 148, 157, 165, 174, 205, 209, 212, 214, 216, 219, 221, 222, 229, 238, 244-246, 253, 255, 260, 261, 269-274, 288-290, 292-295, 298, 302, 306, 325, 352, 364, 377.) have been corrected. The description of molecular results in the discussion section has been revised by a scholar who has been engaged in scientific research in Singapore for a long time. Please see the article for details.
As for the length of the holotype specimen you mentioned in Table 2, after checking and re measuring the specimen, the data is correct. Because its morphological characteristics are the clearest among all our specimens, it has been selected as the holotype specimen.
