Mitochondrial Genomes of two Lycosa spiders (Araneae, Lycosidae): Genome Description and Phylogenetic Implications
Round 1
Reviewer 1 Report
The study of the mitochondrial genome is very important to understand the molecular diversity and evolution of organisms. I congratulate the authors for the effort in making this contribution and many of the results have potential to be used in further studies and to inform about mitochondrial evolution in spiders. However, I found that the manuscript needs to go through extensive revision before it is adequate to be published. Below I explain my points.
The title of the paper refers to the phylogenetic implications of the two new mitochondrial genomes assembled. The authors try to put these two genomes in the context of the whole spider phylogeny, which is interesting. But, why would the authors think that the mitogenome of these two spiders of the same genus of a single family (which already have mitogenome available) would provide any new information about the phylogeny of spiders (a group with >50000 species described)? To the best of my knowledge, these species are not involved in no single phylogenetic/taxonomic conflict that would have any implication for the whole spider phylogeny. Indeed, the whole mitogenomic results didn’t show anything new regarding the spider phylogeny. If the authors think that there is some specific hypothesis concerning the spider phylogeny that these two new mitochondrial genomes could help to address, that should be explained and based on previous evidences with appropriate citations. Some minor phylogenetic conflict which the mitochondrial genome could provide some information is related to the Oval Calamistrum Clade and the intrafamilial relationships of Lycosidae. However, this is not mentioned in the manuscript and the relevant papers are not even cited (e.g. https://doi.org/10.1016/j.ympev.2019.04.004 and https://doi.org/10.1071/IS14041 ). Nevertheless, the sampling presented in this study is also not sufficient for testing those hypotheses and any finding would be a week evidence and should be considered with caution.
The authors say (line 64) “The lack of spider mitogenome data hinders our understanding of phylogenetic relationships among spiders. Therefore, to improve the accuracy of the phylogenetic inferences among spiders”. Why do the authors think the mitogenome is essential for phylogenies? Mitogenome can be biased given its high rate of evolution, the matrimonial inheritance and because it represents the evolutionary history of only one locus. Recent methods allow the acquisition of hundreds of nuclear loci and good phylogenetic estimation without the need of mitochondrial genome, and the phylogeny of spiders have been extensively and recently studied with these hundreds of nuclear genes. The mitogenome is indeed very interesting and important to understand evolution, but if the authors think that lack of mitogenome hinders our current understanding of the phylogenetic relationships of spiders (I am not aware of any evidence that would support this argument), the reasons should be stated clearly and based on evidence with appropriate citations.
The authors also do not provide sufficient information in the methods that would allow reproducibility. The details of library construction or any reference to the protocol used for lab procedure is lacking. The details of the mitogenome assembly such as the specific functions and parameters is also not mentioned in the manuscript. The phylogenetic analyses lack how the models of evolution were chosen, why the authors decided to use only one partition which options of IQTREE were used… The journal does not have length limit, so there is no reason for the method to be oversimplified to the point that is not reproducible. Furthermore, with the lack of information it is also difficult to say if the analyses were adequate or not.
The discussion is in between the results as vague comments and it is not backed up with appropriate citations. For example (line 267) “The phylogenetic tree was in accord with the morphological classification results.”: Which morphological classification results? There are a lot of different morphological classification and phylogenies and they not always agree with each other.
The authors also need to pay attention to expression such as “pole of spider evolution”, since evolution is not linear and directional towards a better organism, and “spider tentacles”, since spiders do not have tentacles.
I suggest that the authors try and address these relevant points describing better the relevance of the study, the objectives, the methodology and the discussion. The attached file has some specific comments. I think that thoroughly addressing these points could make the paper appropriate for publication.
Best wishes.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Overall this work has a well designed methodological approach to sequence and characterize the mitogenomes of two Lycosidae spiders. The authors used adequate figures and tables to comparae their results with those obtained with other spiders. I have included several comments and edits in the main text that should be addressed. Two major concerns are:
1. The phylogenetic analysis was not properly described. I suggest to check the analytical parameters used (i.e. number of generations and the strategies used for checking convergence and stationarity), for the Bayesian analysis.
2. It is imperative a revision by a native speaker
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have sequenced two new mitogenomies of Lycosa spiders and have described these and reconstructed a phylogenetic tree using the protein-coding genes of these and other available spider mitogenomes. Below I have some suggestions for improving the manuscript.
Line 19: Stating that something was "lost" requires some more advanced phylogenetic analysis that includes some kind of trait optimization. Perhaps the trait was "gained" or was lost repeatedly? I do not think the present analysis and results provide evidence for this conclusion.
Line 39: This is an oversimplification, in that there are presumably phylogenetic trees available that result from analysis of rRNA genes or partial mt genomes; i.e. yes it is probably true that mitogenomes can provide insight compared to morphological trees, but this seems to ignore other work in the mean-time. It would be useful to include some background on other molecular phylogenies.
Line 55: This is an interesting idea, but gene rearrangement and secondary structure are not used here in the phylogenetic analysis. Why not?
Line 65: As with the previous comment (line 39) this statement seems to ignore all work done on that is not mitochondrial. Are there no relevant rRNA gene phylogenies?
Line 70: Please clarify what is meant by "pole" here.
Line 92: I am not familiar with this method (med-low sensitivity stitching) so please explain in 1-2 sentences.
Line 94: Please clarify the methods here -- what does "respectively" refer to? It is unclear how (or why) the two species were handled (sequenced) differently.
Line 103 (Table 1 header): It appears these primers were used for one species, and were designed from other species (lines 89-91) this I assume only one species was sequenced with NGS? Please clarify.
Line 105: The degree of coverage is never specified and seems critical to report (read depth for NGS / coverage for Sanger). I assume there are minimum standards required by GenBank?
Line 121: There are many analyses here and the purpose is not clearly stated. For example, how does calculating the pairwise distance between 13 PCGs relate to the aims of the study (lines 71-74)?
Line 130: What are "tandem nucleotide sequences" i.e. are there other types we might expect? This was not clear to me.
Lines 134-136: How were these models selected?
Line 137: Was 1M gen enough for the analysis? Please indicate whether runs converged?
Line 142: I suggest titling this section "Results and Discussion"
Line 155: I am concerned that the mitogenome length of L singoriensis may be wrong, because the CR is very short (and notoriously difficult to PCR/sequence because of the AT-rich nature of the region). I would like to see some discussion of this, i.e., how confident are the authors that the CR was fully sequenced?
Line 183: I found the font in this figure (spider names) to be too small.
Line 197: I suggest moving this to an appendix, as it had little relevance to the study.
Lines 243-260: Same comment as above (line 197)
Line 265: I feel that BI = 0.85 is not well supported, although this only applies to a few nodes in the tree (which are not discussed).
Line 272: I disagree that this is a notable result (also in the abstract) because the Lycosidae are defined here as the clade containing Wadicosa, Pardosa, Pirata and the two newly sequenced Lycosa. This could just as easily be defined as "sister to" (rather than "nested within"). If there was another Lycosidea in the tree that was connected to all of these by a deeper branch, then one could conclude the Lycosa are "nested" but with this topology it is completely subjective.
Lines 275-277: This seems to be out of place and unrelated to the study here. Have other studies contradicted this?
Line 278: What does "this figure" refer to?
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I congratulate the authors for the effort in presenting a revised version of their manuscript. Several issues were properly tackled. Nevertheless, I found that the manuscript still needs to go through major revision. My main concern is that there is a clear disconnection between the main objetive of the manuscript and some paragraphs that are presented in the Results/Discussion section. It seems that the authors do not acknowledge that the impact of adding two newly sequenced Lycosidae to the spider tree of life is not going to drastically change the spiders tree of life backbone. I think that the authors should clearly delineate the manuscript scope and focus their results and discussion in this delimitation.
I am also attaching a file with several additional comments.
Comments for author File: Comments.pdf
Author Response
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Author Response File: 
Author Response.docx
Reviewer 3 Report
The coverage could be better addressed. In comment 9, I was asking about the coverage of each base (especially for Sanger data) not the total number of reads. I can assume that the NGS-sequenced regions had > 100-fold (or 1000-fold) coverage, but for the Sanger reads it is not specified? If it is single-coverage then it should be stated. Perhaps the PCR-fragments overlap, such that at least 2-fold coverage is obtained(?)
Author Response
Reviewer 3:
Comment: The coverage could be better addressed. In comment 9, I was asking about the coverage of each base (especially for Sanger data) not the total number of reads. I can assume that the NGS-sequenced regions had > 100-fold (or 1000-fold) coverage, but for the Sanger reads it is not specified? If it is single-coverage then it should be stated. Perhaps the PCR-fragments overlap, such that at least 2-fold coverage is obtained(?)
Response: Thank you for your comment. Our primers were about 100 bases away from the gaps. After removing the inaccurate 30 bases on both sides, there are still more than 70 bases on each side that match the Illumina sequencing results. And, the Sanger sequencing peak maps were free of spurious peaks. All PCGs could be translated into proteins. Moreover, we looked up some articles that use Sanger sequencing and Illumina sequencing. Coverage of Sanger data was not mentioned in these articles. (Pita et al, 2017; Aleix-Mata et al, 2019; Li et al, 2021)
References:
Pita, S; Panzera, F; Vela, J; Mora, P; Palomeque, T; Lorite, P. Complete mitochondrial genome of Triatoma infestans (Hemiptera, Reduviidae, Triatominae), the main vector of Chagas disease. Infection Genetics and Evolution 2017 54: 158-163 https://doi.org/10.1016/j.meegid.2017.06.003
Aleix-Mata, G; Ruiz-Ruano, FJ; Perez, JM; Sarasa, M; Sanchez, A. Complete mitochondrial genome of the Western Capercaillie Tetrao urogallus (Phasianidae, Tetraoninae). Zootaxa 2019 4550: 585-593 https://doi.org/10.11646/zootaxa.4550.4.9
Li, F; Lv, YY; Wen, ZY; Bian, C; Zhang, XH; Guo, ST; Shi, Q; Li, DQ. The complete mitochondrial genome of the intertidal spider (Desis jiaxiangi) provides novel insights into the adaptive evolution of the mitogenome and the evolution of spiders. BMC Ecology and Evolution 2021 21:72 https://doi.org/10.1186/s12862-021-01803-y
Round 3
Reviewer 2 Report
I congratulate the authors for the effort in presenting a revised version of their manuscript. My comments are in the attached file.
Comments for author File: Comments.pdf
Author Response
Point-by-Point Responses
Please note that the changes made do not influence the content, conclusions, or framework of the paper. Although we have not listed all of the minor changes made here, these are highlighted in red in the revised manuscript.
Reviewer 2:
Comment 1：in order to explore the species phylogenetic placement and the mitogenome composition and evolution.
Response 1: Considering the reviewer’s suggestion, we have rewritten the sentence
Comment 2： Why? Provide some details
Response 2: Considering the reviewer’s suggestion, we reorganized the language of this passage. Some inaccuracies were deleted. We are sorry for the mistake.
Comment 3： What about the phylogenetic placement? This statement should agrww with the aim stated in the abstract.
Response 3: Thank you for your comment, we have rewritten the sentence
Comment 4：This is a strong extrapolation two mitochondrial genomes don’t have this impact
Response 4: Considering the reviewer’s suggestion, Some inaccuracies were deleted. We are sorry for the mistake.
Comment 5：I suggest to run at least for 10 million generations
Response 5: Thank you for your comment. At first, we used the sequences of 13 PCGs to build MrBayes. Two chains running independently for one million generations. Then, to address the reviewer’s concern, we have rebuilt MrBayes using the sequences of 13 PCGs and two rRNAs. Two chains running independently for two million generations. We compared the results of the two MrBayes trees and found that the two trees have the same phylogenetic relationship at the family level and above. The average standard deviation of split frequencies (ASDSF) has reached 0.001237. It is common practice to use ASDSF as a convergence criterion, stopping Bayesian once the ASDSF is less than 0.01. (Lakneret al, 2008; Fourment et al, 2018) In many articles, their analyses were also run for 1 or 2 million generations (Fang et al, 2016; Wang et al, 2016; Du et al 2019). Our Bayesian and ML methods yielded identical topologies. Therefore, we consider the results of our MrBayes to be credible.
Reference:
Lakner, C.; Van Der Mark, P.; Huelsenbeck, J.P.; Larget, B.; Ronquist, F. Efficiency of Markov chain Monte Carlo tree proposals in Bayesian phylogenetics. 2008 Systematic Biology 57: 86-103.
Fourment, M.; Claywell, B.C.; Dinh, V.; McCoy, C; Matsen, F.A.; Darling, A.E. Effective Online Bayesian Phylogenetics via Sequential Monte Carlo with Guided Proposals. 2018 Systematic Biology 67: 490-502.
Fang, W.Y.; Wang, Z.L.; Li, C.; Yang, X.Q.; Yu, X.P. The complete mitogenome of a jumping spider Carrhotus xanthogramma (Araneae: Salticidae) and comparative analysis in four salticid mitogenomes. 2016 Genetica 144: 699-709.
Wang, Z.L.; Li, C; Fang, W.Y.; Yu, X.P. Characterization of the complete mitogenomes of two Neoscona spiders (Araneae: Araneidae) and its phylogenetic implications. 2016 GENE 590: 298-306.
Du, Z.Y.; Hasegawa, H.; Cooley, J.R.; Simon, C.; Yoshimura, J.; Cai, W.Z.; Sota, T.; Li, H. Mitochondrial Genomics Reveals Shared Phylogeographic Patterns and Demographic History among Three Periodical Cicada Species Groups. 2019 Molecular Biology and Evolution 36: 1187-1200.
