Compensatory Base Changes in ITS2 Secondary Structure Alignment, Modelling, and Molecular Phylogeny: An Integrated Approach to Improve Species Delimitation in Tulasnella (Basidiomycota)
, Oscar Vivanco-Galván 2, DarÃo Cruz 2, Angelo Armijos-Carrión 3 and Juan Pablo Suárez 2Round 1
Reviewer 1 Report
The ms “Compensatory Base Changes in ITS2 secondary structure alignment, modelling, and molecular phylogeny: an integrated approach to improve species delimitation in Tulasnella (Basidiomycota)” is dealing with species delimitation in Tulasnella, a taxonomically difficult group. By using advanced methodologies as combination of align-based and align-free phylogenetic approaches, and identification of CBCs between the 2D structural helices, the authors are aiming to improving species recognition.
It was an interesting reading, although in some parts it was not easy to follow the logic of the authors. Thus, here are some comments:
- Since the results of this work should be possible to replicate, it would be necessary to make it easy to access the programs/packages that were used. It would be good to include links to the sources (on-line), the version/s of the program/s, including all necessary settings applied etc. Thus, to add all basic information for making a reproduction of this work possible.
- The Figure text should be easy to follow per se, i.e. without having to search the text for important details. Thus, in Figure 1. “Flowchart summary”, it is not easy to follow, since the figure includes orange circles that are not explained in the figure text (but i, ii etc is used). I would strongly suggest to make the figure, figure text and text consistent.
- Figures are not easy to read (a bigger font size needed), and the markings should be consistent (like those above the clades etc). In addition, it would be good if the specific (important) clades with max support were marked with bold lines. All important clades should be marked with appropriate letters and numbers.
- If would seem be more appropriate in Figure 2b to compare with the secondary structure of ITS2 transcript of a fungus rather than Fagus (tree).
- For some conclusions I do disagree – T. albida B2-II is NOT monophyletic in Figure 5. Thus parts of the conclusions have to be rewritten.
- Links for the files, sequences, trees, matrices results, and dataset analyzed during the study were NOT possible to access and thus not to assess; this vital information should be available.
For some details please read the comments in the ms.
Comments for author File: 
Comments.pdf
Author Response
Comment 1
Since the results of this work should be possible to replicate, it would be necessary to make it easy to access the programs/packages that were used. It would be good to include links to the sources (on-line), the version/s of the program/s, including all necessary settings applied etc. Thus, to add all basic information for making a reproduction of this work possible.
Answer:
Thanks, the results and links to the programs/packages, sources and all basic informatic used in this work is shared in the next link https://drive.google.com/drive/folders/1Fs7ewUhC_LJa0kSFAWdJ7G4XXPcKerJ1?usp=sharing, and in the appendix A.
Comment 2
The Figure text should be easy to follow per se, i.e. without having to search the text for important details. Thus, in Figure 1. “Flowchart summary”, it is not easy to follow, since the figure includes orange circles that are not explained in the figure text (but i, ii etc is used). I would strongly suggest making the figure, figure text and text consistent.
Answer: The figure 1 was changed such that the figure text and text are consistent.
Comment 3
Figures are not easy to read (a bigger font size needed), and the markings should be consistent (like those above the clades etc). In addition, it would be good if the specific (important) clades with max support were marked with bold lines. All-important clades should be marked with appropriate letters and numbers.
Answer:
All figures are attached as pdf file in the Figures folder https://drive.google.com/drive/folders/1Fs7ewUhC_LJa0kSFAWdJ7G4XXPcKerJ1?usp=sharing , for easy reading.
Comment 4
If would seem be more appropriate in Figure 2b to compare with the secondary structure of ITS2 transcript of a fungus rather than Fagus (tree).
Answer:
The figure 2b was change from fagus to fungus secondary structure of ITS2 transcript of Colletotrichum gloeosporioides (Genbank AF444327) modeled by ITS2 database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/).
Comment 5
For some conclusions I do disagree – T. albida B2-II is NOT monophyletic in Figure 5. Thus parts of the conclusions have to be rewritten.
Answer:
Thanks, you have reason T. albida B2-II is not monophyletic clade in Figure 5, we have only seven monophyletic clades A1, A2, B1,B2-I, B2-II, B2-III and B3 as main monophyletic clades, this was rewritten. We appreciate the comment.
Comment 6
Links for the files, sequences, trees, matrices results, and dataset analyzed during the study were NOT possible to access and thus not to assess; this vital information should be available.
Answer:
Thanks, the link was update to http://purl.org/phylo/treebase/phylows/study/TB2:S30176?x-access-code=9d9bc5fb651f6b5d18b25a1ac244f4dd&format=html
Reviewer 2 Report
Q1: This article focuses on the study of a complex genus such as Tulasnella, in which the analysis of secondary structures and nucleotide substitution events in ITS2 is used as support in the delimitation of species in this genus. The manuscript is direct, well-structured and with comprehensive literature on the methodology used. I recommend the job for publication, however, authors are asked to answer some minor questions.
Q2: Why was Puccinia boraniae chosen as an outgroup sequence?
Q3: Add reference to figure 4 in the text.
Q4: Webpage addresses should have the same font size as the text (lines 125, 148).
Q5: Add webpage address of RaxML HPC2 (line 152).
Q6: Add webpage address of LocARNA software (line 199).
Q7: Why did the Tulasnella ITS2 models in this work not coincide with the consensus models reported in the literature?
Author Response
Q1: This article focuses on the study of a complex genus such as Tulasnella, in which the analysis of secondary structures and nucleotide substitution events in ITS2 is used as support in the delimitation of species in this genus. The manuscript is direct, well-structured and with comprehensive literature on the methodology used. I recommend the job for publication, however, authors are asked to answer some minor questions.
Q2: Why was Puccinia boraniae chosen as an outgroup sequence?
Answer:
We know that generally an outgroup is the most informative sister group. However, we selected the sequence of Puccinia boroniae because this group does not share close characters with Tulasnella spp. and allowed us to strongly mark the formation of phylogenetic clades in this highly variable group.
Q3: Add reference to figure 4 in the text.
Answer:
The figure 4 was created by us, the software references was added.
Q4: Webpage addresses should have the same font size as the text (lines 125, 148).
Answer:
It was modified, thanks.
Q5: Add webpage address of RaxML HPC2 (line 152).
Answer:
It was added, thanks.
Q6: Add webpage address of LocARNA software (line 199).
Answer:
It was added, thanks.
Q7: Why did the Tulasnella ITS2 models in this work not coincide with the consensus models reported in the literature?
Answer:
The species of the Tulasnella group have truly divergent ITS-5.8S regions ranging from 600 to 900 bp for the different species, allowing for the formation of ingroups, as reported by Cruz et al [7,50] and Freitas et al.[53]. The presence of indels in this group represent interspecific variability displaying different consensus secondary structure prototypes which revealed three types of models.
Some molecular phylogenetic studies on nrITS-5.8S sequences of Tulasnella species previously isolated from mycorrhizas of epiphytic orchids showed genomic variability among clones which was difficult to interpret as intra or interspecific variations or to correlate with described Tulasnella species. Thus, it could be attribute to the apparent variability in the number of helices and structural details, for example four and six helices were modelled in this study.
Because of diversity of mycorrhizal Tulasnella associated with epiphytic orchids show morphological variability.
As we mentioned in discussion section (line 418)
“In Eukaryotics that also have a predefined pattern of four helices the slippage of RNA polymerase during transcription may result in production of mononucleotide repeats (“UUUU”) in the RNA sequences [53,54]. These inadvertent errors in transcription may lead to an increase in the number of detected ITS2 ribotypes [55]. Also, it could be attribute to the apparent variability in the number of helices and structural details that occur in the ITS1 transcript among Eukaryotics.”
