Molecular Variation and Phylogeny within Fusarium avenaceum and Related Species

Round 1

Reviewer 1 Report

The manuscript entitled "Molecular variation within Fusarium avenaceum and related species" represents a phylogeny study using the DNA sequences of reference genes (TEF1 or/and TUB2), to test the proposed species classification of  Fusarium tricinctum species complex (FTSC) with morphological variation (if the research objective is clear and logical, while it is negative). The manuscript's writing style is acceptable, providing more recent advances focusing on the classified divergence in FTSC, particularly for the F. avenaceum and the related species. However, owing to the unspecified system used in this work, a vast proportion of overlapped data from the previous work (at least 12 self-citations included), prompting the weakness of significance and novelty. The authors need to make an effort to provide a more feasible strategy and convincing data to code the controversial situation in the identification and classification of F. avenaceum, rather than just being plausible. In addition, too many redundant comparisons were presented in the text, but very few of them supported the current data and postulation. Besides, the results and the respected conclusion presented were sketches and vague. Thus, recommend this manuscript for rejection. Authors should reconsider resubmission until reliable data and new insight into FTSC classification are provided appropriately. 

Some of a few formal comments on the manuscript as following indicated:

Specify the title by summarizing the highlight of the research. Overall, “molecular variation” is blurred and indistinct. The “F. avenaceum and related species” should be defined clearly initially.

Abstract

The abstract does not describe the study's novelty, purpose, and significance. The author should summarize the significant findings and remove the redundant description and reorder the sentences following the abstract criteria with (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article's main findings; (4) Conclusions: indicate the main conclusions or interpretations.

Materials and methods

The detailed materials used for DNA extraction, primers (TEF1 and TUB2), sequencing, and biological repeats in the current experiment should be specified, and the previous data or data used in other citations MUST be clarified.

Discussion

The authors should significantly revise this part and provide more references (except for the self-citation) to support their ideas.

Author Response

Editor Decision

Decision Ask for additional reviewer

Comments

The way your paper is structured should be improved. I feel that information is present, but it is difficult to read, and it is difficult to follow some of what you want me to understand, or appreciate, or be convinced by. Please make a specific point of telling a reader why you are presenting this material, and why it is of interest. Why will publishing your results be important?

• Thank you for your opinion. Please see below our detailed responses to the comments. We have tried to do our best to respond to the points raised.

You miss at least one crucial unit, such as bp, and that completely changed the meaning of the text: Line 163: "When the TEF1 sequences presented in Figure 1 were compared to the 515 long sequences published by Kulik et al. [35], five main groups were supported by bootstrap values of >89 % in the MP (Figure 2) and..." [This suggests 515 long sequences were used in Kulik's paper. However, I think you mean 515 nt or 515 bp long, as in length. You imply Kulik used 515 sequences, as in five hundred and fifteen sequences, in an analysis. Moreover, I read Kulik's paper and could not see use of '515' in the text. In that paper they appear not to have used anything 515 bp or nt long, or 515 sequences. Please check, and clarify.

• Thank you for your comments. We are sorry about the mistake and we really mean 515 long bp TEF1 Kulik et al. (2011) are referring to their GenBank sequences and they are 515 bp long. So, when we compare our longer DNA sequences to their sequences, we have to cut our sequences of figure 1 to fit the length of their DNA sequences in figure 2.

This leads me to another point, below...] Line 189: "When the TEF sequences used for the construction of Figure 1 were compared to the 525 bp long TEF1 sequences of the strains published by Stakheev et al. [39] representing five main clusters of PHO (phosphate permease gene) phylogenetic tree, four main groups were supported by bootstrap values of >72 % in the MP (Figure 3)..." [Here, you are still talking about Figure 1, but now you mention 525 bp long sequences used by another author. Remember, in Line 163 you had 515 (no unit) long sequences. Also, in Line 163 they are TEF1 sequences, and here in Line 189 they are TEF sequences! What are they? TEF, or TEF1? Be consistent.]

• Thank you for your linguistic revision. Stakheev et al. were referring GenBank sequences, which were 525 bp long. So, again we had to cut our longer DNA sequences of Figure 1 to fit the length of their DNA sequences in Figure 3. TEF1 sequences are ”transclation elongation factor 1-alpha” sequences, but Fusarium researchers oftern refer to them only as TEF We have revised this.

Some of your paragraphs should be combined for better flow. Short paragraphs comprising just a small number of sentences each are difficult to read because the text quickly switches between different topics. It's like reading a blog, or short newspaper account of a big event! Here we need details, and convincing arguments supported by data and literature. Please work on improving consistency and flow. Consider where you really have to use a definite article ('the') or not; you use definite articles where they are not needed, and you don't use them where they are needed. I know it can be difficult to get them all in the right place, and yes... I'm a native speaker of English!

• Thank you again for your linguistic revision. We will take your comments into consideration. There are no strict rules for the use of definite articles and we do not have them in our native languages, but we think that most of them are in right places. We know that definite article is used before a noun to define it as something specific (e.g., something previously mentioned or known, something unique, or something being identified by the speaker). But we have tried to eliminate extra definite articles and add necessary ones.

Decision Date 27 May 2022

Reviewer 1:

The manuscript entitled "Molecular variation within Fusarium avenaceum and related species" represents a phylogeny study using the DNA sequences of reference genes (TEF1 or/and TUB2), to test the proposed species classification of  Fusarium tricinctum species complex (FTSC) with morphological variation (if the research objective is clear and logical, while it is negative).

• We did not quite understand the text within parentheses. Could the reviewer specify, how the research objective can be negative, while it is clear and logical?

 The manuscript's writing style is acceptable, providing more recent advances focusing on the classified divergence in FTSC, particularly for the F. avenaceum and the related species.

-Thank you

However, owing to the unspecified system used in this work, a vast proportion of overlapped data from the previous work (at least 12 self-citations included), prompting the weakness of significance and novelty.

• We are sorry about the self-citations, but we have studied this topic for a long time and we have a lot of previous molecular and morphologcal data published in well-known mycological pre-reviewed journals. So, it is necessary to discuss the results of the present work with all previous publications of this topic. Some of the strains and their DNA sequences of the present work have only been investigated in our own publications before this manuscript. So, we have to refer to them.

The authors need to make an effort to provide a more feasible strategy and convincing data to code the controversial situation in the identification and classification of F. avenaceum, rather than just being plausible.

• Thank you for your comments. We have tried to collect all known data in order to participate to the discussion dealing with molecular and molecular classification of strains closely related to avenaceum.

In addition, too many redundant comparisons were presented in the text, but very few of them supported the current data and postulation.

• We wanted to compare our combined TEF1 and beta-tubulin DNA sequences with those of other researchers, but unfortunately most of other resarchers had not used TUB2 That is why we could in most cases compare only TEF-sequences. We could find alternative ways to explain the phylogenetic relationships of Fusarium strains of Kulik et al. (Figure 2), Stakheev et al. (Figure 3) and Laraba et al. (Figure 4) related to F. avenaceum according to clusterization of their strains to the main groups of Figures 1- 5 based on MP and ML trees.

Besides, the results and the respected conclusion presented were sketches and vague. Thus, recommend this manuscript for rejection. Authors should reconsider resubmission until reliable data and new insight into FTSC classification are provided appropriately.

• We are going to continue this phylogenetic work and to use whole-genome sequencing of avenaceum and related strains. But we have already so many interesting results, which support the idea that F. avenaceum (FTSC4) can be divided into several main groups, which give new ideas to interpret phylogenetic data from previous investigations that it is necessary to publish them as soon as possible.

Some of a few formal comments on the manuscript as following indicated:

Specify the title by summarizing the highlight of the research. Overall, “molecular variation” is blurred and indistinct. The “F. avenaceum and related species” should be defined clearly initially.

1. We have defined them as morphologically described F. avenaceum, F. arthrosporioides and F. anguioides strains. Stakheev et al. (2018) used the term “F. avenaceum and related species”. Ceron-Bustamante et al. (2018) also used the term “F. avenaceum and related members of the F. tricinctum species complex”, when they described new species closely related F. avenaceum.

Abstract

The abstract does not describe the study's novelty, purpose, and significance. The author should summarize the significant findings and remove the redundant description and reorder the sentences following the abstract criteria with (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article's main findings; (4) Conclusions: indicate the main conclusions or interpretations.

• We have marked the sentences corresponding to the specified sections.

Materials and methods

The detailed materials used for DNA extraction, primers (TEF1 and TUB2), sequencing, and biological repeats in the current experiment should be specified, and the previous data or data used in other citations MUST be clarified.

• We refer the previous studies containing the detailed information on primers, sequencing et al.

Discussion

The authors should significantly revise this part and provide more references (except for the self-citation) to support their ideas.

• We already have as many references as we have found including the most recent ones, but there has not been many references dealing with this topic. We have tried to find more references and we have revised the discussion.

Reviewer 2 Report

In this paper, strains of Fusarium tricinctum species complex (FTSC) were analyzed by phylogenetic analysis of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2), including strains of Fusarium avenaceum, Fusarium arthrosporioides and Fusarium anguioides, due to these species have similar formation of macroconidia. 5 phylogenetic trees were constructed with different outgroups and showed many diversity in genetics. According to this paper, F. acuminatum and F. torulosum can distinguished from the FTSC complex, but only Fig 1 and Fig 4 constructed with F. acuminatum as outgroup. I suggest other trees can reconstructed using F. acuminatum as outgroup to see if the topology of these trees is similar.

Author Response

Reviewer 2:

In this paper, strains of Fusarium tricinctum species complex (FTSC) were analyzed by phylogenetic analysis of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2), including strains of F, avenaceum, F.arthrosporioides and F. anguioides, due to these species have similar formation of macroconidia. 5 phylogenetic trees were constructed with different outgroups and showed many diversity in genetics. According to this paper, F. acuminatum and F. torulosum can distinguished from the FTSC complex, but only Fig 1 and Fig 4 constructed with F. acuminatum as outgroup. I suggest other trees can reconstructed using F. acuminatum as outgroup to see if the topology of these trees is similar.

• Thank you for your comments. We think that we used acuminatum strains together with F. tricinctum and F. torulosum strains as outgroups in all five phylogenetic MP and ML trees. As you can see F. acuminatum, F. tricinctum and F. torulosum strains are clearly separated from F. avenaceum and closely related strains including main groups I-V in all phylogenetic MP and ML trees. We also used PHYLIP program with the same results (not shown). Should we include these PHYLIP trees?

Reviewer 3 Report

Fusarium spp. are important phytopathogens, but their classification is difficult. In this study, FTSC strains were morphologically identified as Fusarium avenaceum, Fusarium arthrosporioides and Fusarium anguioides, the phylogenetic analysis of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2), supported the existence of at least five main groups among these strains. So, the study is meaningful to identify FTSC strains at the species level. However, there some questions in the manuscript, as follow:

Line 20 add a comma before the word “including”.

Full name of TEF1 sequences should be added in footnote of Fig. 1-5. what’s meaning of FTSC9, 30 and 22. The classification of FTSC should be described in the “Introduction”.

Line 149 the sentence “bootstrap values of 61 % and 75 % in MP and MP trees” is confused.

Author Response

Reviewer 3:

Fusarium spp. are important phytopathogens, but their classification is difficult. In this study, FTSC strains were morphologically identified as Fusarium avenaceum, Fusarium arthrosporioides and Fusarium anguioides, the phylogenetic analysis of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2), supported the existence of at least five main groups among these strains. So, the study is meaningful to identify FTSC strains at the species level. However, there some questions in the manuscript, as follow:

Line 20 add a comma before the word “including”.

Full name of TEF1 sequences should be added in footnote of Fig. 1-5.

what’s meaning of FTSC9, 30 and 22. The classification of FTSC should be described in the “Introduction”.

• FTSC species classification is based on the paper of Laraba et al. (2022). We can describe the FTSC classification in the introduction.

Line 149 the sentence “bootstrap values of 61 % and 75 % in MP and MP trees” is confused.

• We revised it.

Round 2

Reviewer 1 Report

The revision is acceptable

Author Response

Thank you.