sash-ka/mdpi_arguments_relation · Datasets at Hugging Face

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There might be a misunderstanding in the first part. The rotation of filament does not drive macroscopic rotation or spiral formation. What we describe in the text was the switch to turn to the left was governed by the filament rotation, but the real formation of a spiral is driven by the locomotion of the filament. The cited paper described the clumping of Anabaena cylindria in a dense culture. The clumping or aggregation in Arthrospira (Ohmori group) is mediated by cAMP, but A. cylindrica might be different. I have been using Anabaena for about 40 years, but I have never seen Anabaena filaments form a spiral on agar plates. Spiral formation and clumping are different phenomena. It is difficult to discuss the relationship (if any) between clumping and rotation.	2	1	The authors’ conclusions are generally well supported and the electron microscopy provides some of the clearest images to date of the junctional pores.	null	null	life4040819_perova
The current paper is focused on Phormidium. We never used Myxobacteria, and we have no idea about the motility in Myxobacteria. To clarify the situation, the mention to Myxobacteria was added in the text.	2	1	Finally, the authors performed a detailed characterization of the motility of individual filaments of the organism by time lapse microscopy providing additional evidence that would appear to support polysaccharide secretion as the driving force for motility, and provide a model for how this motility drives the formation of supercellular structures; in this case, spirals.	null	null	life4040819_perova
Lines 94–95: We have determined genomic sequences of many organisms, but it is not easy to publish the genomes as genome paper. The sequences should be connected by PCR, and annotated. In the current study, we are interested in the genes involved in motility. We annotated the related genes but we would not connect the contigs and annotate all the genes. Nowadays, every researcher can sequence his/her own materials quite easily. It is not necessary to deposit all the raw sequence data.	2	1	Within the text, corrected words are highlighted.	null	null	life4040819_perova
Lines 227–230: This was corrected.	2	1	The authors’ conclusions are generally well supported and the electron microscopy provides some of the clearest images to date of the junctional pores.	null	null	life4040819_perova
Lines 316–317: We did not notice this point. Polysaccharide chain may not be very long. Many of the products of the hps gene cluster encode glycosyltransferases and pseudopilins, which are, respectively, involved in the synthesis and secretion of the slime. The secretion of slime is likely mediated by some molecular machinery that is otherwise involved in type II secretion/motility machinery. Reference 24 was added.	2	1	A model is presented to describe how the rotation of the filament causes left-handed sliding for the portion of the filament not encased in a slime sheath, which leads to formation of counter-clockwise spirals, the predominant macroscopic feature of colonies noted.	null	null	life4040819_perova
The three references have been noticed with gratitude and included in the MS.	2	1	In light of recent research developments and argumentation (some of it reviewed), his views should be considered a welcome addition to the many ideas that populate the “origin of life” field of inquiry that counter the dominant paradigm.	null	null	life4041050_makarova
The 1998 paper leaves much to be desired. Its deficiencies reflect the excitement of the first hour. The state of the art at that time may be gleaned from an authoritative paper that came to the opposite conclusion: A.R. Mushegian and E.V. Koonin “Gene order is not conserved in bacterial evolution”. TIG 1996, 12, 289–290. The gene cluster table of 1998 was mainly retrieved from the annotations in published genomes and constructed manually with paper and pencil. The state of information technology at that time is reflcted by the fact that the table was folded an individually pasted by hand into each issue by the publisher.	2	1	Round 1: and Author Response The manuscript comprises an exciting account on the origin of life with emphasis on the emergence and function of RNA.	null	null	life4041050_makarova
The 1998 paper contemplated speculatively a combination of small-scale gene doubling (as evidenced by the immediate neighborhood of EF-Tu/EF-G) and of a large-scale gene cluster doubling (as evidenced by the spacing between secE/secY and rpoH-A/rpoD) with the hope of a future deeper understanding based on folding structures. Now the referee makes an exciting suggestion that may, if executed successfully, go some way to satisfy that hope.	2	1	I have only a few comments, which might improve the manuscript.	null	null	life4041050_makarova
The self-cleavage of RNA by 2'-OH is a chemical textbook fact. The 2'-OH group has the proper position and orientation for a nucleophilic attack on the phosphate bridge. The kinetics of the reaction is greatly favored by the 5-membered ring structure of the resulting cyclic phospho-bisester. The length of the RNA molecule is not relevant since each nucleophilic attack causes destruction of the chain. The effect should not be confused with the length-dependent “error catastrophe” of accumulating mutations of RNA. Incidentally, some anaerobic ribonucleotide reductases are ancient, while others (aerobic ones) are later inventions.	2	1	I have only a few comments, which might improve the manuscript.	null	null	life4041050_makarova
Genome rearrangements are certainly important during later evolution of the phyla. At the level of LUCA, i.e., prior to the splitting of the domains, it is not clear, if and to what extent rearrangements of the modern style occurred. In this regard we should bear in mind that the LUCA genome may have exhibited sense-antisense coding on both strands as suggested by Rodin and Carter. This position has been adopted and discussed in the present paper. Therefore, speculations concerning possible genome rearrangements may be a bridge too far.	2	1	He reviews some of his previous work and presents an alternative to the dominant ‘Ancient RNA world’ hypothesis.	null	null	life4041050_makarova
The present analysis comes to the conclusion that the sets of canonical amino acids and bases as well as the genetic triplet code were largely complete at the level of LUCA. The Wong coevolution theory of the genetic code has been discussed in detail. The literature comprises numerous proposals concerning the origin of translation and other aspects of the genetic machinery. A review of all these proposals and many others is beyond the scope of the present paper. The present paper is a research paper and not a review paper. It aims at a comprehensive account of early evolution from the origin of life all the way to LUCA. This puts a systematic constraint on literature selection. An effort (unfortunately fallible) has been made to include all those references that integrate with the main lines of the present account into a coherent account. Contributions by others that have the character of theoretical modules that fit well into this account have been termed “theorems” with names of the main authors attached. A reference to a paper on nucleotide biosynthesis phylogeny is now cited in Section 7.	2	1	Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary	null	null	life4041050_makarova
Based on the valuable criticism Section 2 has been extensively revised. Terminology has been clarified. This Section has a rather restricted purpose. It provides chemical arguments for the proposition that the pioneer organisms could only exist at high temperature and that the subsequent forms of life remained hyperthermophilic for a long time until much later an irreversible evolution generated organisms that required lower and lower temperatures. This conclusion places severe constraints on all aspects of the evolution of the genetic machinery. The fascinating topics of thermodynamics, energy dissipation and information are outside the scope of the paper.	2	1	The alternative views he previously generated are much expanded in this manuscript.	null	null	life4041050_makarova
The author expresses his gratitude for the two additional references, which have been included in the text.	2	1	He reviews some of his previous work and presents an alternative to the dominant ‘Ancient RNA world’ hypothesis.	null	null	life4041050_makarova
The statement has been clarified.	2	1	Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary	null	null	life4041050_makarova
The author prefers the present title, because the paper is concerned with the origin of the genetic machinery. Theories on the origin of life or on other aspects of early evolution, such as cellularization, serve merely as starting points.  In the introduction the term “or proteins” has been added after “RNA” ,  and the term “retrodict” has been defined.  The term “interpolate” has been clarified.  The problem of mutational saturation is now included in the discussion of Figure 1.  The term “multiply impaired” has been replaced by a clearer wording.  LUCA is discussed only in Section 1.  The protein cycle is discussed in a separate Section 4, which is concerned with the course of evolution before LUCA.	2	1	I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.	null	null	life4041050_makarova
There are many relevant literature references. Among these there are specific proposals concerning a circumscribed problem that are included to fill a logical gap, as a theoretical module so to speak. These have been designated by the term “theorem” with the added name(s) of the main R7 author(s). This makes it clear that the account given is comprehensive in the sense that major independent contributions by other scientists integrate readily with the overall account given.	2	1	I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.	null	null	life4041050_makarova
The relationship of His and Trp biosyntheses has been toned down.	2	1	Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary	null	null	life4041050_makarova
Section 8 has been revised in order to address the issues involved in the last query.	2	1	I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.	null	null	life4041050_makarova
The concern the reviewer mentions here was also one of our main conclusions for the article. Apparently we did not formulate it well enough. It has thus been reformulated to: “However an accurate taxonomy can never be achieved by the use of a barcoding method only, since it is based on nucleotide substitutions of a single gene. Accurate classification always requires an integrative taxonomy effort R2 including characteristics from ecology, morphology and physiology, as already previously suggested for prokaryotes [9,28,29,45], and as it is becoming common also for animal taxonomy (e.g., [53,54]).” Moreover, it is necessary to mention the following aspects of this manuscript: (1)	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_makarova
We understand that our choice of naming as in the database needed clarification. We have thus added the following paragraph. We thank the reviewer for the useful example and have included it: “The names of genera and species used in this study where copied form the names given in the database. This choice was made for the reason that cyanobacterial phylogeny underlies continuous changes and genera and species are often reclassified (e.g., [34]). Thus a whole different type of work would be required in order to use all state-of-art classification of cyanobacteria. Moreover, for the aims of this study not so much the phylogenetic classification of the bacterial species, but the genetic structure of the cyanobacterial 16S rRNA genes was of importance. The use of the names provided in the database on the other hand enables other researchers to conduct a similar analysis using the same sequences. Thus some old and revised names are used, for example the species name Anabaena bergii is used throughout the study despite its revised taxonomy in the genus Crysosporum [35].” (2)	2	1	We hope that now both reviewers, and all other potential readers, will not be misled by our approach.	null	null	life5010050_makarova
We understand that particularly the case of Synechoccocus needs clarification and have thus extended the explanation there: “Sequences termed Synechococcus on the other hand were analysed together with sequences of the monophyletic clade Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included, too”.	2	1	Summary: The manuscript is surely useful and, in principle, it presents a good contribution to the recent methodological trends (polyphasic approach) in cyanobacterial taxonomy, this conclusion should be the main result from this work.	null	null	life5010050_makarova
We agree with the reviewer that we have to avoid misunderstandings of this kind and added a very clear statement to the discussion: “Moreover it has to be emphasised that a close analysis of the actual properties of the cyanobacterial groups tested is limited by the fact that we used the provided R3 names for the sequences and groups. Thus this study intends only to verify the existence of a barcoding gaps within the 16S rRNA sequences of certain cyanobacterial groups, and by no means revise or confirm the complex cyanobacterial taxonomy.”	2	1	Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.	null	null	life5010050_makarova
We agree with the reviewer that we have not stressed this fact enough and have added more sentences explaining why OTUs are used and the problem: “Enormous progress happened in prokaryote taxonomy in the recent years [8,12,24]. However, the data produced with novel methodologies, such as next generation sequencing, often requires a high throughput taxonomic classification of sequences such as fixed threshold to identify OTUs. This kind of fixed numeric classifications can always only be a vague approximation to the actual structure of relatedness of organisms.”	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_makarova
our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gap existed at all.	2	1	We thus only briefly discuss names and taxonomy in our analysis.	null	null	life5010050_makarova
we now include new citations in relationship to different problems: Butlin et al. (2009), Cohan (2011), Cohan (2013), Cohan & Aracena (2012), Diekmann et al. (2004), Nosil (2012), Vos (2011), Wiedenbeck & Cohan (2011).	2	1	Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.	null	null	life5010050_makarova
We agree with the reviewer’s concern. In fact, this is exactly the reason why we chose Cyanobacteria for this analysis. We reformulated this part of the introduction, to make this statement more clear, which now reads: “We choose Cyanobacteria as an example of prokaryotes, not because they are representative for all prokaryotes, but because there is ample phenotypic, ecological, physiological ultrastructural, and biochemical evidence of the existence of independently evolving units in this group [20,21]. Thus, the expectation is that, if a barcoding gap exists in prokaryotes, this should be more easily seen in taxa where groups can be identified also with other methods, as in Cyanobacteria.”	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_makarova
We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_makarova
We agree with the reviewer, this part was formulated clumsily. What we meant was actually exactly what the reviewer means. The two groups were polyphyletic and we could only chose a monophyletic group when analysing them together. We changed the sentence to: “… by two genera that were monophyletic only when taken together in the database used, e.g., Leptolyngbia and Chamaesiphon …” In accordance we did not check for relatedness of the taxa, neither in this one nor for any other one.	2	1	We thus only briefly discuss names and taxonomy in our analysis.	null	null	life5010050_makarova
We agree that this might be confusing to the reader. We have therefore clarified this choice and added this part to the first M&M section and the literature suggested has been cited: “Sequences termed Synechococcus on the other hand were analysed together with sequences of Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included too.”	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_makarova
The sequences we used were clustering monophyletically a-priori in the tree provided in the database used, and we chose them independently of the name. We now clarified this in the first section of M&M: “Thereby a group was considered monophyletic if it was monophyletic in the tree provided with the database, regardless of the taxonomy and the nomenclature of the organisms included in the clade (Supplemetary Figure 1), and only secondarily if there was a correspondence with known taxa.”	2	1	ISSN 2075-1729 www.mdpi.com/journal/life Peer-Review Record: Does a Barcoding Gap Exist in Prokaryotes?	null	null	life5010050_makarova
As mentioned above, we did not only choose sequences that had the same name, but groups of sequences that were monophyletic in the database tree. Thus we should not have artificially introduced a barcoding gap by sequence selection.	2	1	To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names.	null	null	life5010050_makarova
To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names. We thus only briefly discuss names and taxonomy in our analysis. Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point. We hope that now both reviewers, and all other potential readers, will not be misled by our approach. We do not deal with taxonomy at all, only with testing the presence of a barcoding gap. Similarly to the replies for the previous reviewer, our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gaps existed at all. Given this rationale and the kind of data we used, no clear statement towards the two barcoding groups within Cylindrospermopsis can be made. Nearly all sequences within the database are termed Cylindrospermopsis raciborskii by the people who deposited the sequences. However the clade does not contain the type-strain (which was not in the database). So we would rather not speculate too much on the taxonomic naming of these and other barcoded groups. However, we can clearly say that what is deposited in database SILVA 111 as Cylindrospermopsis contains two species with a barcoding gap. For Planktothrix we added a sentence: “Considering the naming of Planktothrix sequences in the database, some OTUs and ABGD units seem to correspond not only to monophyletic groups but also to named species such as P. mougeotii or similarly in the case of Fischerella muscicola (Figure 3).” R6 However we wish to remain careful on these kinds of statements since we are not sure if the underlying naming of the species used is correct or not.	2	1	Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.	null	null	life5010050_makarova
We understand why the reviewer would think that that would be beneficial. However, one has to keep in mind that we were not constructing a phylogenetic tree for all cyanobacteria but for single genera. In this case it is preferential to use a sister group that is more closely related to gain resolution within that group. e.g., Hedtke, S.M. ; Townsend, T.M. ; Hillis, D.M. Resolution of phylogenetic conflict in large data sets by increased taxon sampling. Syst. Biol. 2006, 55, 522–529. Moreover, for the sake of the barcoding analyses, the choice of the outgroup will not affect the results, given that it will not change the relationships towards the tips of the tree.	2	1	We thus only briefly discuss names and taxonomy in our analysis.	null	null	life5010050_makarova
The last paragraph of the discussion already included some comments on this issue. Now the paragraph has been expanded to provide a more detailed discussion on the possibility of using a DNA barcoding gap in cyanobacteria.	2	1	We hope that now both reviewers, and all other potential readers, will not be misled by our approach.	null	null	life5010050_makarova
We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.	2	1	We hope that now both reviewers, and all other potential readers, will not be misled by our approach.	null	null	life5010050_makarova
The concern the reviewer mentions here was also one of our main conclusions for the article. Apparently we did not formulate it well enough. It has thus been reformulated to: “However an accurate taxonomy can never be achieved by the use of a barcoding method only, since it is based on nucleotide substitutions of a single gene. Accurate classification always requires an integrative taxonomy effort R2 including characteristics from ecology, morphology and physiology, as already previously suggested for prokaryotes [9,28,29,45], and as it is becoming common also for animal taxonomy (e.g., [53,54]).”	2	1	We hope that now both reviewers, and all other potential readers, will not be misled by our approach.	null	null	life5010050_perova
We understand that our choice of naming as in the database needed clarification. We have thus added the following paragraph. We thank the reviewer for the useful example and have included it: “The names of genera and species used in this study where copied form the names given in the database. This choice was made for the reason that cyanobacterial phylogeny underlies continuous changes and genera and species are often reclassified (e.g., [34]). Thus a whole different type of work would be required in order to use all state-of-art classification of cyanobacteria. Moreover, for the aims of this study not so much the phylogenetic classification of the bacterial species, but the genetic structure of the cyanobacterial 16S rRNA genes was of importance. The use of the names provided in the database on the other hand enables other researchers to conduct a similar analysis using the same sequences. Thus some old and revised names are used, for example the species name Anabaena bergii is used throughout the study despite its revised taxonomy in the genus Crysosporum [35].” (2)	2	1	Round 2: I accept the paper in the present form.	null	null	life5010050_perova
We understand that particularly the case of Synechoccocus needs clarification and have thus extended the explanation there: “Sequences termed Synechococcus on the other hand were analysed together with sequences of the monophyletic clade Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included, too”.	2	1	Thus this study intends only to verify the existence of a barcoding gaps within the 16S rRNA sequences of certain cyanobacterial groups, and by no means revise or confirm the complex cyanobacterial taxonomy.”	null	null	life5010050_perova
We agree with the reviewer that we have to avoid misunderstandings of this kind and added a very clear statement to the discussion: “Moreover it has to be emphasised that a close analysis of the actual properties of the cyanobacterial groups tested is limited by the fact that we used the provided R3 names for the sequences and groups.	2	1	I believe that the article with proposed changes will be useful for future research.	null	null	life5010050_perova
We agree with the reviewer that we have not stressed this fact enough and have added more sentences explaining why OTUs are used and the problem: “Enormous progress happened in prokaryote taxonomy in the recent years [8,12,24]. However, the data produced with novel methodologies, such as next generation sequencing, often requires a high throughput taxonomic classification of sequences such as fixed threshold to identify OTUs. This kind of fixed numeric classifications can always only be a vague approximation to the actual structure of relatedness of organisms.”	2	1	Summary: The manuscript is surely useful and, in principle, it presents a good contribution to the recent methodological trends (polyphasic approach) in cyanobacterial taxonomy, this conclusion should be the main result from this work.	null	null	life5010050_perova
our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gap existed at all.	2	1	Of course, the complex of all these methods must be corrected with the progress of science, and especially for cyanobacteria is necessary to apply the so called “polyphasic approach” for their classification.	null	null	life5010050_perova
we now include new citations in relationship to different problems: Butlin et al. (2009), Cohan (2011), Cohan (2013), Cohan & Aracena (2012), Diekmann et al. (2004), Nosil (2012), Vos (2011), Wiedenbeck & Cohan (2011).	2	1	We hope that now both reviewers, and all other potential readers, will not be misled by our approach.	null	null	life5010050_perova
We agree with the reviewer’s concern. In fact, this is exactly the reason why we chose Cyanobacteria for this analysis. We reformulated this part of the introduction, to make this statement more clear, which now reads: “We choose Cyanobacteria as an example of prokaryotes, not because they are representative for all prokaryotes, but because there is ample phenotypic, ecological, physiological ultrastructural, and biochemical evidence of the existence of independently evolving units in this group [20,21]. Thus, the expectation is that, if a barcoding gap exists in prokaryotes, this should be more easily seen in taxa where groups can be identified also with other methods, as in Cyanobacteria.”	2	1	ISSN 2075-1729 www.mdpi.com/journal/life Peer-Review Record: Does a Barcoding Gap Exist in Prokaryotes?	null	null	life5010050_perova
We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.	2	1	Round 2: I accept the paper in the present form.	null	null	life5010050_perova
We agree with the reviewer, this part was formulated clumsily. What we meant was actually exactly what the reviewer means. The two groups were polyphyletic and we could only chose a monophyletic group when analysing them together. We changed the sentence to: “… by two genera that were monophyletic only when taken together in the database used, e.g., Leptolyngbia and Chamaesiphon …” In accordance we did not check for relatedness of the taxa, neither in this one nor for any other one.	2	1	I believe that the article with proposed changes will be useful for future research.	null	null	life5010050_perova
We agree that this might be confusing to the reader. We have therefore clarified this choice and added this part to the first M&M section and the literature suggested has been cited: “Sequences termed Synechococcus on the other hand were analysed together with sequences of Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included too.”	2	1	Of course, the complex of all these methods must be corrected with the progress of science, and especially for cyanobacteria is necessary to apply the so called “polyphasic approach” for their classification.	null	null	life5010050_perova
The sequences we used were clustering monophyletically a-priori in the tree provided in the database used, and we chose them independently of the name. We now clarified this in the first section of M&M: “Thereby a group was considered monophyletic if it was monophyletic in the tree provided with the database, regardless of the taxonomy and the nomenclature of the organisms included in the clade (Supplemetary Figure 1), and only secondarily if there was a correspondence with known taxa.”	2	1	Second Round of Evaluation Round 2: I have had the more critical remarks to the manuscript; however, I think that the presented data are useful and should be published.	null	null	life5010050_perova
As mentioned above, we did not only choose sequences that had the same name, but groups of sequences that were monophyletic in the database tree. Thus we should not have artificially introduced a barcoding gap by sequence selection.	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_perova
To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names. We thus only briefly discuss names and taxonomy in our analysis. Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point. We hope that now both reviewers, and all other potential readers, will not be misled by our approach. We do not deal with taxonomy at all, only with testing the presence of a barcoding gap. Similarly to the replies for the previous reviewer, our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gaps existed at all. Given this rationale and the kind of data we used, no clear statement towards the two barcoding groups within Cylindrospermopsis can be made. Nearly all sequences within the database are termed Cylindrospermopsis raciborskii by the people who deposited the sequences. However the clade does not contain the type-strain (which was not in the database). So we would rather not speculate too much on the taxonomic naming of these and other barcoded groups. However, we can clearly say that what is deposited in database SILVA 111 as Cylindrospermopsis contains two species with a barcoding gap. For Planktothrix we added a sentence: “Considering the naming of Planktothrix sequences in the database, some OTUs and ABGD units seem to correspond not only to monophyletic groups but also to named species such as P. mougeotii or similarly in the case of Fischerella muscicola (Figure 3).” R6 However we wish to remain careful on these kinds of statements since we are not sure if the underlying naming of the species used is correct or not.	2	1	Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.	null	null	life5010050_perova
We understand why the reviewer would think that that would be beneficial. However, one has to keep in mind that we were not constructing a phylogenetic tree for all cyanobacteria but for single genera. In this case it is preferential to use a sister group that is more closely related to gain resolution within that group. e.g., Hedtke, S.M. ; Townsend, T.M. ; Hillis, D.M. Resolution of phylogenetic conflict in large data sets by increased taxon sampling. Syst. Biol. 2006, 55, 522–529. Moreover, for the sake of the barcoding analyses, the choice of the outgroup will not affect the results, given that it will not change the relationships towards the tips of the tree.	2	1	Moreover, it is necessary to mention the following aspects of this manuscript: (1)	null	null	life5010050_perova
The last paragraph of the discussion already included some comments on this issue. Now the paragraph has been expanded to provide a more detailed discussion on the possibility of using a DNA barcoding gap in cyanobacteria.	2	1	The molecular methods must be evidently preferred in this work, but other approaches (biochemical, ecophysiological, ecological, morphological) must be included into the final evaluation.	null	null	life5010050_perova
We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.	2	1	Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.	null	null	life5010050_perova
This review does not require any action on our part.	2	1	Round 1: and Author Response The article “Emergent Chemical Behaviour in Variable-Volume Protocells” focuses on the consequences that a changing volume may have on a set of reactions encapsulated within a semipermeable vesicle; in particular, this kind of compartment can degenerate existing bistable reactions, or promote emergent bistability from very simple reactions, which are not bistable in bulk conditions.	null	null	life5010181_perova
Here, the reviewer did not like our use of the particular words “novelty” and “innovation”. o Use of the word novelty − Instances of “chemical novelty” in the manuscript replaced by “emergent chemical behaviour”. − On Page 8, after Equation 8, we removed sentence: The emergence of bistability will serve as a proxy for the emergence of other chemical novelties in the vesicle reactor model. Replaced whole paragraph, to read: In particular, in these initial stages, we will focus on the emergence of bistability in the vesicle reactor model—a dynamical feature deducible directly from the number and stability of the fixed points present (i.e., two asymptotically stable points separated by an unstable saddle point). We also expect that more complicated dynamical regimes could also be present in the model, like multi-stability or global phase space features such as limit cycles giving rise to sustained oscillations. However, investigation of these regimes will be deferred to later work: for the time being, the “emergent chemical behavior” referred to in the title will be restricted to bistability. We think this is a clearer explanation, and also uses the word “regime” suggested by the reviewer. o Use of the word innovation We kept the 2 occurrences of the word “innovation” in the abstract and the introduction. An innovation is defined as a “new method, idea or product” in the dictionary. We use the word to refer to new emergent chemical behaviour that the whole vesicle system exhibits (e.g., expanded steady states), which did not exist before. We think the use of this word is acceptable.	2	1	Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.	null	null	life5010181_perova
we added a new footnote, to explain our standing on this issue: Reference 36: In this work, concentrations outside the vesicle are set as system parameters. However, we make no commitment to the type of environment the vesicle is embedded in or how these concentrations are maintained. Our purpose is simply to show that bistability can exist in the model for certain sets of outside concentrations. Exploration of the model in explicit environments is deferred to future work.	2	1	Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.	null	null	life5010181_perova
Reference 11 has been added after Equation 10, to point the reader to where to find information about phi limits was first discussed. Repeating this information here would complicate the paper. The lines Phi = 2^(1/3) and 4^(1/3) in Figure 1d have now been explained.	2	1	Produces a dynamic model for chemical evolution rather than traditional deterministic, static, systems that requires centralised information to direct evolution, such as a constant gradient.	null	null	life5010181_perova
It is difficult to make a direct and meaningful comparison like this. Without a container, the reaction system has less parameters, and thus the sampled parameter space is smaller. The aim in the paper, was to simply show that encapsulating a Schlogl model which was bistable in bulk condition, seemed to destroy this bistability.	2	1	As the authors acknowledge, the article is the first step toward a more complete understanding of an interesting class of possible chemo-physical phenomena.	null	null	life5010181_perova
The minor issues were fixed.	2	1	Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.	null	null	life5010181_perova
This immunoquantitation method used is described in detail in the reference given (Brown et al. 2008). The reviewer is directed in particular to the Supplementary Methods section. To allow readers to get a better understanding of the method in this manuscript we have revised this part of the Experimental Section, adding extra text to increase clarity concerning the method.	2	1	The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.	null	null	life5010403_makarova
Since submitting the manuscript, we have accumulated a set of 84 parallel measurements of steady state oxygen evolution and the functional content of PSII measured using flash yields (with a solid state optode) and simultaneous FRR chlorophyll fluorescence induction curves, from which we can extract e- PSII-1 s-1, for both Synechococcus and Prochlorococcus cultures. Consistent with the long literature history of such measurements (ex. Suggett et al. 2004, 2009), we observe a good correlation between the two measures of electron transport per PSII. Plotting the FRR estimate of e- PSII-1 s-1 versus the O2 evolution/PSII content estimate of e- PSII-1 s-1 gives a slope of 1.26 and an R2 of 0.58. We have added this information to the Materials and Methods to support our use of FRR estimates of electron transport per PSII. Furthermore, our estimates of ETRmax from FRR induction curves are independently validated by the close correlation between ETRmax and 1/tau, shown in Figure 4A, since 1/tau is derived from the rate constant for the decay of fluorescence after induction, and thus does not depend (computationally) upon our estimator for ETRmax.	2	1	This is done for a number of globally significance marine microbes of Syn and Pro lineage.	null	null	life5010403_makarova
This has been addressed with the addition of text in the abstract, introduction and discussion specifying that the cultures were grown under low light conditions.	2	1	The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.	null	null	life5010403_makarova
We have removed the word “nondiazotrophic” as we agree that it is not relevant to the discussion.	2	1	The data presented is robust and worthy of publication but should be discussed more critically with regard to published literature (outlined below) I suggest some minor suggestions that should be incorporated before publications.	null	null	life5010403_makarova
We mean that they numerically dominate and we have clarified this in the introduction.	2	1	Round 1: and Author Response The authors present a dataset where they have characterised the relative abundance of components of the photosynthetic electron transfer chain and related this to the measured rate of electron transfer from PSII.	null	null	life5010403_makarova
We have clarified this statement in the text. We refer to the nitrogen cost in the form of allocation to protein per pigment bound.	2	1	Second Round of Evaluation Round 2: and Author Response	null	null	life5010403_makarova
We are referring to the number of RUBISCO active sites measured by immunoquantitation. We are not referring to measured RUBISCO activity. We use this expression to be clear that we are referring to RbcL subunits rather than oligomeric RUBISCO. We have added a parenthetic phrase to make this more clear.	2	1	Round 1: and Author Response The authors present a dataset where they have characterised the relative abundance of components of the photosynthetic electron transfer chain and related this to the measured rate of electron transfer from PSII.	null	null	life5010403_makarova
We thank the reviewer for drawing this study to our attention. We have added a paragraph to the discussion to compare and contrast the Sukenik work with that presented here. The Sukenik data support a strong positive correlation between 1/tau and the RUBISCO to PSU ratio over a series of growth irradiances. While we also see a strong positive correlation between 1/tau and RUBISCO to PSII our results differ as the molar ratios of the components of the PSU differ significantly between the strains analyzed in the current work. This allows us to pinpoint the relationship of PSII to RUBISCO rather than other subunits of the PSU as the determinant of electron transport rate.	2	1	The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.	null	null	life5010403_makarova
We have added text to the end of the discussion to address this comment and the relevant reference has been added. Thank you for this suggestion.	2	1	provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.	null	null	life5010403_makarova
We have added a supplemental figure (Supplemental Figure #2) to show this calibrations curve. As all experiments presented here were performed under iron replete conditions, no iron starvation was performed.	4	1	provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.	null	null	life5010403_makarova
We have added a supplemental figure (Supplemental Figure #1) that shows the method applied with a sample blot, calibration curve and data analysis. This work required dozens of blots, each with its own standard curve, so it would not be practical to show all of the standard curves for each determination.	4	1	provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.	null	null	life5010403_makarova
p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.	2	1	It was particularly interesting to see the differences in protein functionality among the strains of marine Synechococcus.	null	null	life5010432_makarova
p. 2, Lines 22–24; the data at the website cannot be posted as a supplementary figure because of copyright issues. I will ask them to revise the figure using English.	2	1	It was particularly interesting to see the differences in protein functionality among the strains of marine Synechococcus.	null	null	life5010432_makarova
p. 2, Line 24; I confirmed that the following website link to the data of nitrite concentration is correct. “http://www.data.jma.go.jp/gmd/kaiyou/db/vessel_obs/hq/2006spr/137e/index_line.php?id=no2” 	2	1	entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.	null	null	life5010432_makarova
p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.	2	1	CC9605 is an open ocean strain and does not encounter high nitrite concentrations, while CC9311 is a coastal strain that deals with a range of nitrate/nitrite availability.	null	null	life5010432_makarova
p5, Figure 1; Figure 1 was reproduced by using the UPGMA clustering method instead of the NJ clustering method according to the suggestion of the reviewer, then, NitM from α-cyanobacteria and β-cyanobacteria form clearly distinct groups. “using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering method of ClustalX.” was added in the legend of Figure 1.	2	1	entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.	null	null	life5010432_makarova
Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.	2	1	Such differences can be explained by habitat adaption of each strain.	null	null	life5010432_makarova
We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.	2	1	R3 Other changes (1) p. 1 line 26, “α-cyanobacteira” has been changed to “α-cyanobacteria”.	null	null	life5010432_makarova
p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.	2	1	entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.	null	null	life5010432_perova
p. 2, Lines 22–24; the data at the website cannot be posted as a supplementary figure because of copyright issues. I will ask them to revise the figure using English.	2	1	(2) p. 2 lines 2–3, “α-cyanobacteiral” has been changed to “α-cyanobacterial”.	null	null	life5010432_perova
p. 2, Line 24; I confirmed that the following website link to the data of nitrite concentration is correct. “http://www.data.jma.go.jp/gmd/kaiyou/db/vessel_obs/hq/2006spr/137e/index_line.php?id=no2” 	2	1	Round 1: and Author Response I found this research interesting, relevant and novel.	null	null	life5010432_perova
p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.	2	1	Such differences can be explained by habitat adaption of each strain.	null	null	life5010432_perova
p5, Figure 1; Figure 1 was reproduced by using the UPGMA clustering method instead of the NJ clustering method according to the suggestion of the reviewer, then, NitM from α-cyanobacteria and β-cyanobacteria form clearly distinct groups. “using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering method of ClustalX.” was added in the legend of Figure 1.	2	1	Round 1: and Author Response I found this research interesting, relevant and novel.	null	null	life5010432_perova
Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.	2	1	Functional annotation of genes through protein expression is highly needed in our time of exponentially growing sequence data.	null	null	life5010432_perova
We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.	2	1	Such differences can be explained by habitat adaption of each strain.	null	null	life5010432_perova
Multi-alignment of concatenated protein (or DNA) segments is a genome-scale, but not whole-genome approach. Its applicability depends on the scope of the phylogenetic study. When dealing with not-too-distantly related species it may yield more or less useful result. However, in a study covering many phyla it is very difficult, if not impossible, to collect a common set of conserved proteins. Moreover, the concatenation method can never lead to very convincing conclusion, as give or take a few proteins may change the result. The phylogenomics people have noticed this problem, see, e.g., O. Jeffroy, H. Brinkman, F. Delsuc, H. Philippe (2008) Phylogenomics: the beginning of incongruence? Trends in Genetics, 22(4): 225–231. An example from the Bacteria domain is the relationship of the closely related Shigella and Escherichia coli strains. Concatenation of different number of genes led to different way of mixing-up of the two groups, but CVTree gave unambiguous separation of the strains as different species in the same genus Escherichia, see: G.-H. Zuo, Z. Xu, B.L. Hao (2013) Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia. Genomics Proteomics Bioinformatics, 11: 61–65. In order to carry out multi-alignment of concatenated sequences, a postdoc or well-trained PhD student equipped with the corresponding software is required. In contrast, with genome sequencing becoming a common practice in many labs it costs no additional work for a bench-microbiologist to get phylogenetic and taxonomic information by using a convenient and publically available tool such as the CVTree web serve. Well, we would be glad to see comparison of CVTree phylogeny with multi-alignment of concatenated proteins if anyone finds a way to do it for so many diverse phyla, but we do not consider it as a doable job.	2	1	Authors present a new interactive tree-visualization tool which enables direct validation of taxonomic groups according to their monophyly.	null	null	life5010949_makarova
These points were discussed in the “Material and Method” section added at the suggestion of Reviewer 2. The following was copied from the manuscript: “Traditionally a newly generated phylogenetic tree is subject to statistical re-sampling tests such as bootstrap and jackknife. CVTree does not use sequence alignment. Consequently, there is no way to recognize informative or non-informative sites. Instead we take all the protein products encoded in a genome as a sampling pool for carrying out bootstrap or jackknife tests (citing our 2004 paper). Although it was very time-consuming, CVTrees did have well passed these tests (citing our 2010 paper). However, successfully passing statistical re-sampling tests only tells about the stability and self-consistency of the tree with respect to small variations of the input data. It is by far not a proof of objective correctness of the tree. Direct comparison of all branchings in a tree with an independent taxonomy at all ranks would provide such a proof, The 16S rRNA phylogeny cannot be verified by the Bergey's taxonomy, as the latter follows the former. However, agreement of branchings in CVTree with the Bergey's taxonomy would provide much stronger support to the tree as compared to statistical tests. This is the strategy we adopt for the CVTree approach.” “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate R3 remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values”.	2	1	Even if defined in the future, it must be lineage-dependent.	null	null	life5010949_makarova
Yes, this is an apparent discrepancy of CVTree from 16S (and 23S) analysis for the given set of 179 archaeal genomes. However, in an on-going study of ours (not published yet) using a much larger data set this violation no longer shows up; both Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes.	2	1	Results also provide additional support to recently proposed archaeal phyla and halobacterial orders.	null	null	life5010949_makarova
Highly degenerated genomes of many symbiont organisms tend to move around, in particular, to the baseline of a tree and thus distorts the overall structure of the tree. Therefore, it is better not to mix them with free-living organisms in a study. We rephrased the corresponding paragraph in the manuscript: “The nanosized archaean symbiont Nanoarchaeum equitans has a highly reduced genome (490,885 bp). It is the only described representative of a newly proposed phylum Nanoarchaeota and it cuts into the otherwise monophyletic phylum Euryarchaeota. We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees, see, e.g., Figure 4 in a 2009 microbial survey as well as (c) and (d) in our Figure 3. It has been known that tiny genomes of endosymbiont microbes often tend to move towards baseline of a tree and distort the overall picture. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny, see, e.g., (citing our 2010 paper) and a note in the home page of the CVTree Web Server. In the present case we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly reduced genome”.	2	1	present a comparative analysis of the taxonomic classification of the Archaea domain.	null	null	life5010949_makarova
Yes, there was certain disturbing effect of the tiny and lonely Nanoarchaeum genome, yet the Halobacteria is a very specific clade, forming a tightly connected group and moving around as a whole, mainly due to the biased acidity of their constituent amino acids. We anticipate that the relative placement of Halobacteria with respect to other groups may stabilize when more genomes are used to construct a tree.	2	1	Even if defined in the future, it must be lineage-dependent.	null	null	life5010949_makarova
We have A new “Material and Method” section has been added. Such issues as statistical resampling tests (bootstrap and jackknife), calibration of branch length, the meaning and choice of the peptide length K, etc. , were discussed in the new section. Figures 1 and 2 were combined to a new Figure 1; Figures 3 and 4 were combined to become a new Figure 2. Figure captions were made more detailed. The whole text was checked for language flaws and many places were rephrased.	2	1	Perhaps an additional discussion point would be the advantages of using CVtree approach with regular concatenated proteins.	null	null	life5010949_makarova
We thank the Reviewer for the detailed comments/suggestions given in the previous report and the suggestion of doing spelling-check this time. We have gone through the final manuscript carefully once more.	4	1	The latter can be assigned only after comparison with a reference taxonomy which is not a rigid framework but a modifiable system.	null	null	life5010949_makarova
A “Material and Method” section has been added where the CVTree algorithm, the interactive tree-viewer, statistical resampling tests (bootstrap, jackknife), calibration of branch lengths, etc., were discussed in slightly more detail.	2	1	are describing a comprehensive analysis of archaeal phylogeny with a genome-based alignment free method, and then comparing the findings to 16S rRNA based phylogenies.	null	null	life5010949_makarova
Yes, 16S rRNA phylogeny is quite stable and it almost defines the present taxonomy. We have given due credit for this. In general, CVTree does not challenge 16S rRNA analysis but complement it.	2	1	are describing a comprehensive analysis of archaeal phylogeny with a genome-based alignment free method, and then comparing the findings to 16S rRNA based phylogenies.	null	null	life5010949_makarova
A robust phylogenetic tree comes with a fixed branching order of leaves. One looks at the leaf names and their taxonomic lineage and tries to map the latter to the branches. To this end we added the following paragraphs in the “Material and Method” section. “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values.” “Branching order in a tree by itself does not bring about taxonomic ranks, e,g, class or order.	2	1	However, I am personally not convinced that whole genomes bring additional advantages, or if we are better off by just using a conserved set of proteins.	null	null	life5010949_makarova
We have reorganized the manuscript mainly by adding a new “Material and Method” section where discussions on branch length, statistical resampling, meaning and choice of K, etc., were given. The original Figure 1 was deleted with some related points explained in the text accompanying the original Figure 2. All figure captions have been rewritten for clarity.	2	1	— Done Second Round of Evaluation Round 2: and Author Response	null	null	life5010949_makarova
A few more sentences were added in the “Conclusion” regarding the power and achievement of the 16S rRNA analysis.	2	1	Results also provide additional support to recently proposed archaeal phyla and halobacterial orders.	null	null	life5010949_makarova
We tried to rephrase the paragraph by changing, deleting, or adding a few words as follows: “In this paper we study Archaea phylogeny across many phyla. This is in contrast with phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large, or, as Cavalier-Smith (citing cavalier-smith 2002) put it, with “megaclassificaton” of prokaryotes. Although in taxonomy the description of a newly discovered organism necessarily starts from the lower ranks, higher rank assignments are often incomplete or lacking. At present the ranks above class are not covered by the Bacteriological Code. The number of plausible microbial phyla may reach hundreds and archaeal ones are among the less studied. According to the 16S rRNA analysis, the major archaeal classes and their subordinate orders have been more or less delineated. Therefore, in order to carry out the aforementioned cross verification we make emphasis on higher ranks such as phyla, classes, and orders. A study using 179 Archaea genomes should provide a framework for further study of lower ranks.”	2	1	This kind of research should be encouraged further because old taxonomic paradigms must be systematically reviewed based on new genomic data.	null	null	life5010949_makarova
Branching order in a tree is directly related to taxonomy, while branch lengths have more to do with evolution. For large-scale phylogenetic study across many phyla the former is more important than the calibration of branch lengths. The latter is based on the assumption that mutation rate is more or less constant. This assumption cannot hold when dealing with many phyla.	2	1	— Done Second Round of Evaluation Round 2: and Author Response	null	null	life5010949_makarova
This is done in the newly added “Material and Method” section.	2	1	If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic.”	null	null	life5010949_makarova
The original Figure 1 was deleted and a few words added to the legend of the original Figure 2, now the new Figure 1.	2	1	Here monophyly must be understood in a pragmatic way restricted to the given set of input data and the reference taxonomy.	null	null	life5010949_makarova
Judging by the cluster labeled as Euryarchaeote{0+3} in Figure 2 Methanomassiliicoccus was not reclassified into Thermoplasmataceae but to an yet un-specified class.	2	1	Though a dissimilarity measure figures in the CVTree algorithm, it is not realistic to delineate taxa by using this measure at least for the time being.	null	null	life5010949_makarova

There might be a misunderstanding in the first part. The rotation of filament does not drive macroscopic rotation or spiral formation. What we describe in the text was the switch to turn to the left was governed by the filament rotation, but the real formation of a spiral is driven by the locomotion of the filament. The cited paper described the clumping of Anabaena cylindria in a dense culture. The clumping or aggregation in Arthrospira (Ohmori group) is mediated by cAMP, but A. cylindrica might be different. I have been using Anabaena for about 40 years, but I have never seen Anabaena filaments form a spiral on agar plates. Spiral formation and clumping are different phenomena. It is difficult to discuss the relationship (if any) between clumping and rotation.

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The authors’ conclusions are generally well supported and the electron microscopy provides some of the clearest images to date of the junctional pores.

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The current paper is focused on Phormidium. We never used Myxobacteria, and we have no idea about the motility in Myxobacteria. To clarify the situation, the mention to Myxobacteria was added in the text.

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Finally, the authors performed a detailed characterization of the motility of individual filaments of the organism by time lapse microscopy providing additional evidence that would appear to support polysaccharide secretion as the driving force for motility, and provide a model for how this motility drives the formation of supercellular structures; in this case, spirals.

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Lines 94–95: We have determined genomic sequences of many organisms, but it is not easy to publish the genomes as genome paper. The sequences should be connected by PCR, and annotated. In the current study, we are interested in the genes involved in motility. We annotated the related genes but we would not connect the contigs and annotate all the genes. Nowadays, every researcher can sequence his/her own materials quite easily. It is not necessary to deposit all the raw sequence data.

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Within the text, corrected words are highlighted.

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Lines 227–230: This was corrected.

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The authors’ conclusions are generally well supported and the electron microscopy provides some of the clearest images to date of the junctional pores.

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Lines 316–317: We did not notice this point. Polysaccharide chain may not be very long. Many of the products of the hps gene cluster encode glycosyltransferases and pseudopilins, which are, respectively, involved in the synthesis and secretion of the slime. The secretion of slime is likely mediated by some molecular machinery that is otherwise involved in type II secretion/motility machinery. Reference 24 was added.

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A model is presented to describe how the rotation of the filament causes left-handed sliding for the portion of the filament not encased in a slime sheath, which leads to formation of counter-clockwise spirals, the predominant macroscopic feature of colonies noted.

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The three references have been noticed with gratitude and included in the MS.

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In light of recent research developments and argumentation (some of it reviewed), his views should be considered a welcome addition to the many ideas that populate the “origin of life” field of inquiry that counter the dominant paradigm.

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The 1998 paper leaves much to be desired. Its deficiencies reflect the excitement of the first hour. The state of the art at that time may be gleaned from an authoritative paper that came to the opposite conclusion: A.R. Mushegian and E.V. Koonin “Gene order is not conserved in bacterial evolution”. TIG 1996, 12, 289–290. The gene cluster table of 1998 was mainly retrieved from the annotations in published genomes and constructed manually with paper and pencil. The state of information technology at that time is reflcted by the fact that the table was folded an individually pasted by hand into each issue by the publisher.

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Round 1: and Author Response The manuscript comprises an exciting account on the origin of life with emphasis on the emergence and function of RNA.

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The 1998 paper contemplated speculatively a combination of small-scale gene doubling (as evidenced by the immediate neighborhood of EF-Tu/EF-G) and of a large-scale gene cluster doubling (as evidenced by the spacing between secE/secY and rpoH-A/rpoD) with the hope of a future deeper understanding based on folding structures. Now the referee makes an exciting suggestion that may, if executed successfully, go some way to satisfy that hope.

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I have only a few comments, which might improve the manuscript.

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The self-cleavage of RNA by 2'-OH is a chemical textbook fact. The 2'-OH group has the proper position and orientation for a nucleophilic attack on the phosphate bridge. The kinetics of the reaction is greatly favored by the 5-membered ring structure of the resulting cyclic phospho-bisester. The length of the RNA molecule is not relevant since each nucleophilic attack causes destruction of the chain. The effect should not be confused with the length-dependent “error catastrophe” of accumulating mutations of RNA. Incidentally, some anaerobic ribonucleotide reductases are ancient, while others (aerobic ones) are later inventions.

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I have only a few comments, which might improve the manuscript.

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Genome rearrangements are certainly important during later evolution of the phyla. At the level of LUCA, i.e., prior to the splitting of the domains, it is not clear, if and to what extent rearrangements of the modern style occurred. In this regard we should bear in mind that the LUCA genome may have exhibited sense-antisense coding on both strands as suggested by Rodin and Carter. This position has been adopted and discussed in the present paper. Therefore, speculations concerning possible genome rearrangements may be a bridge too far.

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He reviews some of his previous work and presents an alternative to the dominant ‘Ancient RNA world’ hypothesis.

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life4041050_makarova

0

The present analysis comes to the conclusion that the sets of canonical amino acids and bases as well as the genetic triplet code were largely complete at the level of LUCA. The Wong coevolution theory of the genetic code has been discussed in detail. The literature comprises numerous proposals concerning the origin of translation and other aspects of the genetic machinery. A review of all these proposals and many others is beyond the scope of the present paper. The present paper is a research paper and not a review paper. It aims at a comprehensive account of early evolution from the origin of life all the way to LUCA. This puts a systematic constraint on literature selection. An effort (unfortunately fallible) has been made to include all those references that integrate with the main lines of the present account into a coherent account. Contributions by others that have the character of theoretical modules that fit well into this account have been termed “theorems” with names of the main authors attached. A reference to a paper on nucleotide biosynthesis phylogeny is now cited in Section 7.

2

1

Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary

null

life4041050_makarova

0

Based on the valuable criticism Section 2 has been extensively revised. Terminology has been clarified. This Section has a rather restricted purpose. It provides chemical arguments for the proposition that the pioneer organisms could only exist at high temperature and that the subsequent forms of life remained hyperthermophilic for a long time until much later an irreversible evolution generated organisms that required lower and lower temperatures. This conclusion places severe constraints on all aspects of the evolution of the genetic machinery. The fascinating topics of thermodynamics, energy dissipation and information are outside the scope of the paper.

2

1

The alternative views he previously generated are much expanded in this manuscript.

null

life4041050_makarova

0

The author expresses his gratitude for the two additional references, which have been included in the text.

2

1

He reviews some of his previous work and presents an alternative to the dominant ‘Ancient RNA world’ hypothesis.

null

life4041050_makarova

0

The statement has been clarified.

2

1

Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary

null

life4041050_makarova

0

The author prefers the present title, because the paper is concerned with the origin of the genetic machinery. Theories on the origin of life or on other aspects of early evolution, such as cellularization, serve merely as starting points.  In the introduction the term “or proteins” has been added after “RNA” ,  and the term “retrodict” has been defined.  The term “interpolate” has been clarified.  The problem of mutational saturation is now included in the discussion of Figure 1.  The term “multiply impaired” has been replaced by a clearer wording.  LUCA is discussed only in Section 1.  The protein cycle is discussed in a separate Section 4, which is concerned with the course of evolution before LUCA.

2

1

I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.

null

life4041050_makarova

0

There are many relevant literature references. Among these there are specific proposals concerning a circumscribed problem that are included to fill a logical gap, as a theoretical module so to speak. These have been designated by the term “theorem” with the added name(s) of the main R7 author(s). This makes it clear that the account given is comprehensive in the sense that major independent contributions by other scientists integrate readily with the overall account given.

2

1

I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.

null

life4041050_makarova

0

The relationship of His and Trp biosyntheses has been toned down.

2

1

Therefore, my comments will be slanted by my own expertise and will only serve the author as a partial devil’s advocate effort General commentary

null

life4041050_makarova

0

Section 8 has been revised in order to address the issues involved in the last query.

2

1

I must note that a careful evaluation of all facets requires expertise in a multitude of disciplines (from prebiotic chemistry and structural biology to evolutionary bioinformatics and biochemistry) and considerable time, none of which I possess.

null

life4041050_makarova

0

The concern the reviewer mentions here was also one of our main conclusions for the article. Apparently we did not formulate it well enough. It has thus been reformulated to: “However an accurate taxonomy can never be achieved by the use of a barcoding method only, since it is based on nucleotide substitutions of a single gene. Accurate classification always requires an integrative taxonomy effort R2 including characteristics from ecology, morphology and physiology, as already previously suggested for prokaryotes [9,28,29,45], and as it is becoming common also for animal taxonomy (e.g., [53,54]).” Moreover, it is necessary to mention the following aspects of this manuscript: (1)

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_makarova

0

We understand that our choice of naming as in the database needed clarification. We have thus added the following paragraph. We thank the reviewer for the useful example and have included it: “The names of genera and species used in this study where copied form the names given in the database. This choice was made for the reason that cyanobacterial phylogeny underlies continuous changes and genera and species are often reclassified (e.g., [34]). Thus a whole different type of work would be required in order to use all state-of-art classification of cyanobacteria. Moreover, for the aims of this study not so much the phylogenetic classification of the bacterial species, but the genetic structure of the cyanobacterial 16S rRNA genes was of importance. The use of the names provided in the database on the other hand enables other researchers to conduct a similar analysis using the same sequences. Thus some old and revised names are used, for example the species name Anabaena bergii is used throughout the study despite its revised taxonomy in the genus Crysosporum [35].” (2)

2

1

We hope that now both reviewers, and all other potential readers, will not be misled by our approach.

null

life5010050_makarova

0

We understand that particularly the case of Synechoccocus needs clarification and have thus extended the explanation there: “Sequences termed Synechococcus on the other hand were analysed together with sequences of the monophyletic clade Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included, too”.

2

1

Summary: The manuscript is surely useful and, in principle, it presents a good contribution to the recent methodological trends (polyphasic approach) in cyanobacterial taxonomy, this conclusion should be the main result from this work.

null

life5010050_makarova

0

We agree with the reviewer that we have to avoid misunderstandings of this kind and added a very clear statement to the discussion: “Moreover it has to be emphasised that a close analysis of the actual properties of the cyanobacterial groups tested is limited by the fact that we used the provided R3 names for the sequences and groups. Thus this study intends only to verify the existence of a barcoding gaps within the 16S rRNA sequences of certain cyanobacterial groups, and by no means revise or confirm the complex cyanobacterial taxonomy.”

2

1

Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.

null

life5010050_makarova

0

We agree with the reviewer that we have not stressed this fact enough and have added more sentences explaining why OTUs are used and the problem: “Enormous progress happened in prokaryote taxonomy in the recent years [8,12,24]. However, the data produced with novel methodologies, such as next generation sequencing, often requires a high throughput taxonomic classification of sequences such as fixed threshold to identify OTUs. This kind of fixed numeric classifications can always only be a vague approximation to the actual structure of relatedness of organisms.”

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_makarova

0

our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gap existed at all.

2

1

We thus only briefly discuss names and taxonomy in our analysis.

null

life5010050_makarova

0

we now include new citations in relationship to different problems: Butlin et al. (2009), Cohan (2011), Cohan (2013), Cohan & Aracena (2012), Diekmann et al. (2004), Nosil (2012), Vos (2011), Wiedenbeck & Cohan (2011).

2

1

Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.

null

life5010050_makarova

0

We agree with the reviewer’s concern. In fact, this is exactly the reason why we chose Cyanobacteria for this analysis. We reformulated this part of the introduction, to make this statement more clear, which now reads: “We choose Cyanobacteria as an example of prokaryotes, not because they are representative for all prokaryotes, but because there is ample phenotypic, ecological, physiological ultrastructural, and biochemical evidence of the existence of independently evolving units in this group [20,21]. Thus, the expectation is that, if a barcoding gap exists in prokaryotes, this should be more easily seen in taxa where groups can be identified also with other methods, as in Cyanobacteria.”

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_makarova

0

We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_makarova

0

We agree with the reviewer, this part was formulated clumsily. What we meant was actually exactly what the reviewer means. The two groups were polyphyletic and we could only chose a monophyletic group when analysing them together. We changed the sentence to: “… by two genera that were monophyletic only when taken together in the database used, e.g., Leptolyngbia and Chamaesiphon …” In accordance we did not check for relatedness of the taxa, neither in this one nor for any other one.

2

1

We thus only briefly discuss names and taxonomy in our analysis.

null

life5010050_makarova

0

We agree that this might be confusing to the reader. We have therefore clarified this choice and added this part to the first M&M section and the literature suggested has been cited: “Sequences termed Synechococcus on the other hand were analysed together with sequences of Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included too.”

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_makarova

0

The sequences we used were clustering monophyletically a-priori in the tree provided in the database used, and we chose them independently of the name. We now clarified this in the first section of M&M: “Thereby a group was considered monophyletic if it was monophyletic in the tree provided with the database, regardless of the taxonomy and the nomenclature of the organisms included in the clade (Supplemetary Figure 1), and only secondarily if there was a correspondence with known taxa.”

2

1

ISSN 2075-1729 www.mdpi.com/journal/life Peer-Review Record: Does a Barcoding Gap Exist in Prokaryotes?

null

life5010050_makarova

0

As mentioned above, we did not only choose sequences that had the same name, but groups of sequences that were monophyletic in the database tree. Thus we should not have artificially introduced a barcoding gap by sequence selection.

2

1

To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names.

null

life5010050_makarova

0

To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names. We thus only briefly discuss names and taxonomy in our analysis. Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point. We hope that now both reviewers, and all other potential readers, will not be misled by our approach. We do not deal with taxonomy at all, only with testing the presence of a barcoding gap. Similarly to the replies for the previous reviewer, our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gaps existed at all. Given this rationale and the kind of data we used, no clear statement towards the two barcoding groups within Cylindrospermopsis can be made. Nearly all sequences within the database are termed Cylindrospermopsis raciborskii by the people who deposited the sequences. However the clade does not contain the type-strain (which was not in the database). So we would rather not speculate too much on the taxonomic naming of these and other barcoded groups. However, we can clearly say that what is deposited in database SILVA 111 as Cylindrospermopsis contains two species with a barcoding gap. For Planktothrix we added a sentence: “Considering the naming of Planktothrix sequences in the database, some OTUs and ABGD units seem to correspond not only to monophyletic groups but also to named species such as P. mougeotii or similarly in the case of Fischerella muscicola (Figure 3).” R6 However we wish to remain careful on these kinds of statements since we are not sure if the underlying naming of the species used is correct or not.

2

1

Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.

null

life5010050_makarova

0

We understand why the reviewer would think that that would be beneficial. However, one has to keep in mind that we were not constructing a phylogenetic tree for all cyanobacteria but for single genera. In this case it is preferential to use a sister group that is more closely related to gain resolution within that group. e.g., Hedtke, S.M. ; Townsend, T.M. ; Hillis, D.M. Resolution of phylogenetic conflict in large data sets by increased taxon sampling. Syst. Biol. 2006, 55, 522–529. Moreover, for the sake of the barcoding analyses, the choice of the outgroup will not affect the results, given that it will not change the relationships towards the tips of the tree.

2

1

We thus only briefly discuss names and taxonomy in our analysis.

null

life5010050_makarova

0

The last paragraph of the discussion already included some comments on this issue. Now the paragraph has been expanded to provide a more detailed discussion on the possibility of using a DNA barcoding gap in cyanobacteria.

2

1

We hope that now both reviewers, and all other potential readers, will not be misled by our approach.

null

life5010050_makarova

0

We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.

2

1

We hope that now both reviewers, and all other potential readers, will not be misled by our approach.

null

life5010050_makarova

0

The concern the reviewer mentions here was also one of our main conclusions for the article. Apparently we did not formulate it well enough. It has thus been reformulated to: “However an accurate taxonomy can never be achieved by the use of a barcoding method only, since it is based on nucleotide substitutions of a single gene. Accurate classification always requires an integrative taxonomy effort R2 including characteristics from ecology, morphology and physiology, as already previously suggested for prokaryotes [9,28,29,45], and as it is becoming common also for animal taxonomy (e.g., [53,54]).”

2

1

We hope that now both reviewers, and all other potential readers, will not be misled by our approach.

null

life5010050_perova

0

We understand that our choice of naming as in the database needed clarification. We have thus added the following paragraph. We thank the reviewer for the useful example and have included it: “The names of genera and species used in this study where copied form the names given in the database. This choice was made for the reason that cyanobacterial phylogeny underlies continuous changes and genera and species are often reclassified (e.g., [34]). Thus a whole different type of work would be required in order to use all state-of-art classification of cyanobacteria. Moreover, for the aims of this study not so much the phylogenetic classification of the bacterial species, but the genetic structure of the cyanobacterial 16S rRNA genes was of importance. The use of the names provided in the database on the other hand enables other researchers to conduct a similar analysis using the same sequences. Thus some old and revised names are used, for example the species name Anabaena bergii is used throughout the study despite its revised taxonomy in the genus Crysosporum [35].” (2)

2

1

Round 2: I accept the paper in the present form.

null

life5010050_perova

0

We understand that particularly the case of Synechoccocus needs clarification and have thus extended the explanation there: “Sequences termed Synechococcus on the other hand were analysed together with sequences of the monophyletic clade Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included, too”.

2

1

Thus this study intends only to verify the existence of a barcoding gaps within the 16S rRNA sequences of certain cyanobacterial groups, and by no means revise or confirm the complex cyanobacterial taxonomy.”

null

life5010050_perova

0

We agree with the reviewer that we have to avoid misunderstandings of this kind and added a very clear statement to the discussion: “Moreover it has to be emphasised that a close analysis of the actual properties of the cyanobacterial groups tested is limited by the fact that we used the provided R3 names for the sequences and groups.

2

1

I believe that the article with proposed changes will be useful for future research.

null

life5010050_perova

0

We agree with the reviewer that we have not stressed this fact enough and have added more sentences explaining why OTUs are used and the problem: “Enormous progress happened in prokaryote taxonomy in the recent years [8,12,24]. However, the data produced with novel methodologies, such as next generation sequencing, often requires a high throughput taxonomic classification of sequences such as fixed threshold to identify OTUs. This kind of fixed numeric classifications can always only be a vague approximation to the actual structure of relatedness of organisms.”

2

1

Summary: The manuscript is surely useful and, in principle, it presents a good contribution to the recent methodological trends (polyphasic approach) in cyanobacterial taxonomy, this conclusion should be the main result from this work.

null

life5010050_perova

0

our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gap existed at all.

2

1

Of course, the complex of all these methods must be corrected with the progress of science, and especially for cyanobacteria is necessary to apply the so called “polyphasic approach” for their classification.

null

life5010050_perova

0

we now include new citations in relationship to different problems: Butlin et al. (2009), Cohan (2011), Cohan (2013), Cohan & Aracena (2012), Diekmann et al. (2004), Nosil (2012), Vos (2011), Wiedenbeck & Cohan (2011).

2

1

We hope that now both reviewers, and all other potential readers, will not be misled by our approach.

null

life5010050_perova

0

We agree with the reviewer’s concern. In fact, this is exactly the reason why we chose Cyanobacteria for this analysis. We reformulated this part of the introduction, to make this statement more clear, which now reads: “We choose Cyanobacteria as an example of prokaryotes, not because they are representative for all prokaryotes, but because there is ample phenotypic, ecological, physiological ultrastructural, and biochemical evidence of the existence of independently evolving units in this group [20,21]. Thus, the expectation is that, if a barcoding gap exists in prokaryotes, this should be more easily seen in taxa where groups can be identified also with other methods, as in Cyanobacteria.”

2

1

ISSN 2075-1729 www.mdpi.com/journal/life Peer-Review Record: Does a Barcoding Gap Exist in Prokaryotes?

null

life5010050_perova

0

We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.

2

1

Round 2: I accept the paper in the present form.

null

life5010050_perova

0

We agree with the reviewer, this part was formulated clumsily. What we meant was actually exactly what the reviewer means. The two groups were polyphyletic and we could only chose a monophyletic group when analysing them together. We changed the sentence to: “… by two genera that were monophyletic only when taken together in the database used, e.g., Leptolyngbia and Chamaesiphon …” In accordance we did not check for relatedness of the taxa, neither in this one nor for any other one.

2

1

I believe that the article with proposed changes will be useful for future research.

null

life5010050_perova

0

We agree that this might be confusing to the reader. We have therefore clarified this choice and added this part to the first M&M section and the literature suggested has been cited: “Sequences termed Synechococcus on the other hand were analysed together with sequences of Prochlorococcus, since Synechococcus is known to be a polyphyletic group and if all sequences (including a minimum of five lineages [36–38]) are taken together, Prochlorococcus has to be included too.”

2

1

Of course, the complex of all these methods must be corrected with the progress of science, and especially for cyanobacteria is necessary to apply the so called “polyphasic approach” for their classification.

null

life5010050_perova

0

The sequences we used were clustering monophyletically a-priori in the tree provided in the database used, and we chose them independently of the name. We now clarified this in the first section of M&M: “Thereby a group was considered monophyletic if it was monophyletic in the tree provided with the database, regardless of the taxonomy and the nomenclature of the organisms included in the clade (Supplemetary Figure 1), and only secondarily if there was a correspondence with known taxa.”

2

1

Second Round of Evaluation Round 2: I have had the more critical remarks to the manuscript; however, I think that the presented data are useful and should be published.

null

life5010050_perova

0

As mentioned above, we did not only choose sequences that had the same name, but groups of sequences that were monophyletic in the database tree. Thus we should not have artificially introduced a barcoding gap by sequence selection.

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_perova

0

To do so, we did not download named sequences from GenBank, but used a carefully annotated and checked database with a built-in phylogeny that would allow us to obtain monophyletic clades, regardless of their names. We thus only briefly discuss names and taxonomy in our analysis. Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point. We hope that now both reviewers, and all other potential readers, will not be misled by our approach. We do not deal with taxonomy at all, only with testing the presence of a barcoding gap. Similarly to the replies for the previous reviewer, our aim was not to start from known and named sequences and test whether species from DNA taxonomy matched them or not; our aim was to test whether a barcoding gaps existed at all. Given this rationale and the kind of data we used, no clear statement towards the two barcoding groups within Cylindrospermopsis can be made. Nearly all sequences within the database are termed Cylindrospermopsis raciborskii by the people who deposited the sequences. However the clade does not contain the type-strain (which was not in the database). So we would rather not speculate too much on the taxonomic naming of these and other barcoded groups. However, we can clearly say that what is deposited in database SILVA 111 as Cylindrospermopsis contains two species with a barcoding gap. For Planktothrix we added a sentence: “Considering the naming of Planktothrix sequences in the database, some OTUs and ABGD units seem to correspond not only to monophyletic groups but also to named species such as P. mougeotii or similarly in the case of Fischerella muscicola (Figure 3).” R6 However we wish to remain careful on these kinds of statements since we are not sure if the underlying naming of the species used is correct or not.

2

1

Round 1: and Author Response The authors tested novel approach to identification of species in cyanobacteria based on 16S rRNA based on identification of barcoding gaps previously used in animals.

null

life5010050_perova

0

We understand why the reviewer would think that that would be beneficial. However, one has to keep in mind that we were not constructing a phylogenetic tree for all cyanobacteria but for single genera. In this case it is preferential to use a sister group that is more closely related to gain resolution within that group. e.g., Hedtke, S.M. ; Townsend, T.M. ; Hillis, D.M. Resolution of phylogenetic conflict in large data sets by increased taxon sampling. Syst. Biol. 2006, 55, 522–529. Moreover, for the sake of the barcoding analyses, the choice of the outgroup will not affect the results, given that it will not change the relationships towards the tips of the tree.

2

1

Moreover, it is necessary to mention the following aspects of this manuscript: (1)

null

life5010050_perova

0

The last paragraph of the discussion already included some comments on this issue. Now the paragraph has been expanded to provide a more detailed discussion on the possibility of using a DNA barcoding gap in cyanobacteria.

2

1

The molecular methods must be evidently preferred in this work, but other approaches (biochemical, ecophysiological, ecological, morphological) must be included into the final evaluation.

null

life5010050_perova

0

We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.

2

1

Given that both reviewers commented on this issue, we tried to clarify our aims and included a sentence at the end of the introduction, in order to clarify the point.

null

life5010050_perova

0

This review does not require any action on our part.

2

1

Round 1: and Author Response The article “Emergent Chemical Behaviour in Variable-Volume Protocells” focuses on the consequences that a changing volume may have on a set of reactions encapsulated within a semipermeable vesicle; in particular, this kind of compartment can degenerate existing bistable reactions, or promote emergent bistability from very simple reactions, which are not bistable in bulk conditions.

null

life5010181_perova

0

Here, the reviewer did not like our use of the particular words “novelty” and “innovation”. o Use of the word novelty − Instances of “chemical novelty” in the manuscript replaced by “emergent chemical behaviour”. − On Page 8, after Equation 8, we removed sentence: The emergence of bistability will serve as a proxy for the emergence of other chemical novelties in the vesicle reactor model. Replaced whole paragraph, to read: In particular, in these initial stages, we will focus on the emergence of bistability in the vesicle reactor model—a dynamical feature deducible directly from the number and stability of the fixed points present (i.e., two asymptotically stable points separated by an unstable saddle point). We also expect that more complicated dynamical regimes could also be present in the model, like multi-stability or global phase space features such as limit cycles giving rise to sustained oscillations. However, investigation of these regimes will be deferred to later work: for the time being, the “emergent chemical behavior” referred to in the title will be restricted to bistability. We think this is a clearer explanation, and also uses the word “regime” suggested by the reviewer. o Use of the word innovation We kept the 2 occurrences of the word “innovation” in the abstract and the introduction. An innovation is defined as a “new method, idea or product” in the dictionary. We use the word to refer to new emergent chemical behaviour that the whole vesicle system exhibits (e.g., expanded steady states), which did not exist before. We think the use of this word is acceptable.

2

1

Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.

null

life5010181_perova

0

we added a new footnote, to explain our standing on this issue: Reference 36: In this work, concentrations outside the vesicle are set as system parameters. However, we make no commitment to the type of environment the vesicle is embedded in or how these concentrations are maintained. Our purpose is simply to show that bistability can exist in the model for certain sets of outside concentrations. Exploration of the model in explicit environments is deferred to future work.

2

1

Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.

null

life5010181_perova

0

Reference 11 has been added after Equation 10, to point the reader to where to find information about phi limits was first discussed. Repeating this information here would complicate the paper. The lines Phi = 2^(1/3) and 4^(1/3) in Figure 1d have now been explained.

2

1

Produces a dynamic model for chemical evolution rather than traditional deterministic, static, systems that requires centralised information to direct evolution, such as a constant gradient.

null

life5010181_perova

0

It is difficult to make a direct and meaningful comparison like this. Without a container, the reaction system has less parameters, and thus the sampled parameter space is smaller. The aim in the paper, was to simply show that encapsulating a Schlogl model which was bistable in bulk condition, seemed to destroy this bistability.

2

1

As the authors acknowledge, the article is the first step toward a more complete understanding of an interesting class of possible chemo-physical phenomena.

null

life5010181_perova

0

The minor issues were fixed.

2

1

Remarkably, even reaction systems that have no chemical species in common could become R2 indirectly coupled to each other through the volume they share, by means of a sort of osmotic coupling.

null

life5010181_perova

0

This immunoquantitation method used is described in detail in the reference given (Brown et al. 2008). The reviewer is directed in particular to the Supplementary Methods section. To allow readers to get a better understanding of the method in this manuscript we have revised this part of the Experimental Section, adding extra text to increase clarity concerning the method.

2

1

The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.

null

life5010403_makarova

0

Since submitting the manuscript, we have accumulated a set of 84 parallel measurements of steady state oxygen evolution and the functional content of PSII measured using flash yields (with a solid state optode) and simultaneous FRR chlorophyll fluorescence induction curves, from which we can extract e- PSII-1 s-1, for both Synechococcus and Prochlorococcus cultures. Consistent with the long literature history of such measurements (ex. Suggett et al. 2004, 2009), we observe a good correlation between the two measures of electron transport per PSII. Plotting the FRR estimate of e- PSII-1 s-1 versus the O2 evolution/PSII content estimate of e- PSII-1 s-1 gives a slope of 1.26 and an R2 of 0.58. We have added this information to the Materials and Methods to support our use of FRR estimates of electron transport per PSII. Furthermore, our estimates of ETRmax from FRR induction curves are independently validated by the close correlation between ETRmax and 1/tau, shown in Figure 4A, since 1/tau is derived from the rate constant for the decay of fluorescence after induction, and thus does not depend (computationally) upon our estimator for ETRmax.

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This is done for a number of globally significance marine microbes of Syn and Pro lineage.

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life5010403_makarova

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This has been addressed with the addition of text in the abstract, introduction and discussion specifying that the cultures were grown under low light conditions.

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The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.

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life5010403_makarova

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We have removed the word “nondiazotrophic” as we agree that it is not relevant to the discussion.

2

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The data presented is robust and worthy of publication but should be discussed more critically with regard to published literature (outlined below) I suggest some minor suggestions that should be incorporated before publications.

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life5010403_makarova

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We mean that they numerically dominate and we have clarified this in the introduction.

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Round 1: and Author Response The authors present a dataset where they have characterised the relative abundance of components of the photosynthetic electron transfer chain and related this to the measured rate of electron transfer from PSII.

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life5010403_makarova

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We have clarified this statement in the text. We refer to the nitrogen cost in the form of allocation to protein per pigment bound.

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Second Round of Evaluation Round 2: and Author Response

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life5010403_makarova

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We are referring to the number of RUBISCO active sites measured by immunoquantitation. We are not referring to measured RUBISCO activity. We use this expression to be clear that we are referring to RbcL subunits rather than oligomeric RUBISCO. We have added a parenthetic phrase to make this more clear.

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Round 1: and Author Response The authors present a dataset where they have characterised the relative abundance of components of the photosynthetic electron transfer chain and related this to the measured rate of electron transfer from PSII.

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life5010403_makarova

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We thank the reviewer for drawing this study to our attention. We have added a paragraph to the discussion to compare and contrast the Sukenik work with that presented here. The Sukenik data support a strong positive correlation between 1/tau and the RUBISCO to PSU ratio over a series of growth irradiances. While we also see a strong positive correlation between 1/tau and RUBISCO to PSII our results differ as the molar ratios of the components of the PSU differ significantly between the strains analyzed in the current work. This allows us to pinpoint the relationship of PSII to RUBISCO rather than other subunits of the PSU as the determinant of electron transport rate.

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The approach and methods are robust and the ability to quantify components of the photosynthetic electron transfer chain is powerful.

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life5010403_makarova

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We have added text to the end of the discussion to address this comment and the relevant reference has been added. Thank you for this suggestion.

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provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.

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life5010403_makarova

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We have added a supplemental figure (Supplemental Figure #2) to show this calibrations curve. As all experiments presented here were performed under iron replete conditions, no iron starvation was performed.

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provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.

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life5010403_makarova

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We have added a supplemental figure (Supplemental Figure #1) that shows the method applied with a sample blot, calibration curve and data analysis. This work required dozens of blots, each with its own standard curve, so it would not be practical to show all of the standard curves for each determination.

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provides us with estimates of the concentrations of various proteins representing the PSII, PSI, cytb6 and Rubisco in there different cyanobacteria.

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life5010403_makarova

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p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.

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It was particularly interesting to see the differences in protein functionality among the strains of marine Synechococcus.

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life5010432_makarova

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p. 2, Lines 22–24; the data at the website cannot be posted as a supplementary figure because of copyright issues. I will ask them to revise the figure using English.

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It was particularly interesting to see the differences in protein functionality among the strains of marine Synechococcus.

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life5010432_makarova

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p. 2, Line 24; I confirmed that the following website link to the data of nitrite concentration is correct. “http://www.data.jma.go.jp/gmd/kaiyou/db/vessel_obs/hq/2006spr/137e/index_line.php?id=no2” 

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entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.

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life5010432_makarova

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p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.

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CC9605 is an open ocean strain and does not encounter high nitrite concentrations, while CC9311 is a coastal strain that deals with a range of nitrate/nitrite availability.

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life5010432_makarova

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p5, Figure 1; Figure 1 was reproduced by using the UPGMA clustering method instead of the NJ clustering method according to the suggestion of the reviewer, then, NitM from α-cyanobacteria and β-cyanobacteria form clearly distinct groups. “using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering method of ClustalX.” was added in the legend of Figure 1.

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entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.

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life5010432_makarova

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Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.

2

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Such differences can be explained by habitat adaption of each strain.

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life5010432_makarova

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We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.

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R3 Other changes (1) p. 1 line 26, “α-cyanobacteira” has been changed to “α-cyanobacteria”.

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life5010432_makarova

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p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.

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entitled “Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria” is a very interesting piece of work since it shows the functional characterization as nitrite transporter of several nitM proteins from different picocyanobacteria, showing also fruitful comparisons of their sequences that lead to characterize the c-terminal region of a-cyanobacterial NitM proteins as an inhibitory domain of nitrite transport.

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life5010432_perova

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p. 2, Lines 22–24; the data at the website cannot be posted as a supplementary figure because of copyright issues. I will ask them to revise the figure using English.

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(2) p. 2 lines 2–3, “α-cyanobacteiral” has been changed to “α-cyanobacterial”.

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life5010432_perova

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p. 2, Line 24; I confirmed that the following website link to the data of nitrite concentration is correct. “http://www.data.jma.go.jp/gmd/kaiyou/db/vessel_obs/hq/2006spr/137e/index_line.php?id=no2” 

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Round 1: and Author Response I found this research interesting, relevant and novel.

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life5010432_perova

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p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.

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Such differences can be explained by habitat adaption of each strain.

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life5010432_perova

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p5, Figure 1; Figure 1 was reproduced by using the UPGMA clustering method instead of the NJ clustering method according to the suggestion of the reviewer, then, NitM from α-cyanobacteria and β-cyanobacteria form clearly distinct groups. “using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering method of ClustalX.” was added in the legend of Figure 1.

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Round 1: and Author Response I found this research interesting, relevant and novel.

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life5010432_perova

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Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.

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Functional annotation of genes through protein expression is highly needed in our time of exponentially growing sequence data.

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life5010432_perova

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We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.

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Such differences can be explained by habitat adaption of each strain.

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life5010432_perova

0

Multi-alignment of concatenated protein (or DNA) segments is a genome-scale, but not whole-genome approach. Its applicability depends on the scope of the phylogenetic study. When dealing with not-too-distantly related species it may yield more or less useful result. However, in a study covering many phyla it is very difficult, if not impossible, to collect a common set of conserved proteins. Moreover, the concatenation method can never lead to very convincing conclusion, as give or take a few proteins may change the result. The phylogenomics people have noticed this problem, see, e.g., O. Jeffroy, H. Brinkman, F. Delsuc, H. Philippe (2008) Phylogenomics: the beginning of incongruence? Trends in Genetics, 22(4): 225–231. An example from the Bacteria domain is the relationship of the closely related Shigella and Escherichia coli strains. Concatenation of different number of genes led to different way of mixing-up of the two groups, but CVTree gave unambiguous separation of the strains as different species in the same genus Escherichia, see: G.-H. Zuo, Z. Xu, B.L. Hao (2013) Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia. Genomics Proteomics Bioinformatics, 11: 61–65. In order to carry out multi-alignment of concatenated sequences, a postdoc or well-trained PhD student equipped with the corresponding software is required. In contrast, with genome sequencing becoming a common practice in many labs it costs no additional work for a bench-microbiologist to get phylogenetic and taxonomic information by using a convenient and publically available tool such as the CVTree web serve. Well, we would be glad to see comparison of CVTree phylogeny with multi-alignment of concatenated proteins if anyone finds a way to do it for so many diverse phyla, but we do not consider it as a doable job.

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Authors present a new interactive tree-visualization tool which enables direct validation of taxonomic groups according to their monophyly.

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life5010949_makarova

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These points were discussed in the “Material and Method” section added at the suggestion of Reviewer 2. The following was copied from the manuscript: “Traditionally a newly generated phylogenetic tree is subject to statistical re-sampling tests such as bootstrap and jackknife. CVTree does not use sequence alignment. Consequently, there is no way to recognize informative or non-informative sites. Instead we take all the protein products encoded in a genome as a sampling pool for carrying out bootstrap or jackknife tests (citing our 2004 paper). Although it was very time-consuming, CVTrees did have well passed these tests (citing our 2010 paper). However, successfully passing statistical re-sampling tests only tells about the stability and self-consistency of the tree with respect to small variations of the input data. It is by far not a proof of objective correctness of the tree. Direct comparison of all branchings in a tree with an independent taxonomy at all ranks would provide such a proof, The 16S rRNA phylogeny cannot be verified by the Bergey's taxonomy, as the latter follows the former. However, agreement of branchings in CVTree with the Bergey's taxonomy would provide much stronger support to the tree as compared to statistical tests. This is the strategy we adopt for the CVTree approach.” “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate R3 remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values”.

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Even if defined in the future, it must be lineage-dependent.

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life5010949_makarova

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Yes, this is an apparent discrepancy of CVTree from 16S (and 23S) analysis for the given set of 179 archaeal genomes. However, in an on-going study of ours (not published yet) using a much larger data set this violation no longer shows up; both Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes.

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Results also provide additional support to recently proposed archaeal phyla and halobacterial orders.

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life5010949_makarova

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Highly degenerated genomes of many symbiont organisms tend to move around, in particular, to the baseline of a tree and thus distorts the overall structure of the tree. Therefore, it is better not to mix them with free-living organisms in a study. We rephrased the corresponding paragraph in the manuscript: “The nanosized archaean symbiont Nanoarchaeum equitans has a highly reduced genome (490,885 bp). It is the only described representative of a newly proposed phylum Nanoarchaeota and it cuts into the otherwise monophyletic phylum Euryarchaeota. We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees, see, e.g., Figure 4 in a 2009 microbial survey as well as (c) and (d) in our Figure 3. It has been known that tiny genomes of endosymbiont microbes often tend to move towards baseline of a tree and distort the overall picture. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny, see, e.g., (citing our 2010 paper) and a note in the home page of the CVTree Web Server. In the present case we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly reduced genome”.

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present a comparative analysis of the taxonomic classification of the Archaea domain.

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life5010949_makarova

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Yes, there was certain disturbing effect of the tiny and lonely Nanoarchaeum genome, yet the Halobacteria is a very specific clade, forming a tightly connected group and moving around as a whole, mainly due to the biased acidity of their constituent amino acids. We anticipate that the relative placement of Halobacteria with respect to other groups may stabilize when more genomes are used to construct a tree.

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Even if defined in the future, it must be lineage-dependent.

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life5010949_makarova

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We have A new “Material and Method” section has been added. Such issues as statistical resampling tests (bootstrap and jackknife), calibration of branch length, the meaning and choice of the peptide length K, etc. , were discussed in the new section. Figures 1 and 2 were combined to a new Figure 1; Figures 3 and 4 were combined to become a new Figure 2. Figure captions were made more detailed. The whole text was checked for language flaws and many places were rephrased.

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Perhaps an additional discussion point would be the advantages of using CVtree approach with regular concatenated proteins.

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life5010949_makarova

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We thank the Reviewer for the detailed comments/suggestions given in the previous report and the suggestion of doing spelling-check this time. We have gone through the final manuscript carefully once more.

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The latter can be assigned only after comparison with a reference taxonomy which is not a rigid framework but a modifiable system.

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life5010949_makarova

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A “Material and Method” section has been added where the CVTree algorithm, the interactive tree-viewer, statistical resampling tests (bootstrap, jackknife), calibration of branch lengths, etc., were discussed in slightly more detail.

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are describing a comprehensive analysis of archaeal phylogeny with a genome-based alignment free method, and then comparing the findings to 16S rRNA based phylogenies.

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life5010949_makarova

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Yes, 16S rRNA phylogeny is quite stable and it almost defines the present taxonomy. We have given due credit for this. In general, CVTree does not challenge 16S rRNA analysis but complement it.

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are describing a comprehensive analysis of archaeal phylogeny with a genome-based alignment free method, and then comparing the findings to 16S rRNA based phylogenies.

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life5010949_makarova

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A robust phylogenetic tree comes with a fixed branching order of leaves. One looks at the leaf names and their taxonomic lineage and tries to map the latter to the branches. To this end we added the following paragraphs in the “Material and Method” section. “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values.” “Branching order in a tree by itself does not bring about taxonomic ranks, e,g, class or order.

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However, I am personally not convinced that whole genomes bring additional advantages, or if we are better off by just using a conserved set of proteins.

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life5010949_makarova

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We have reorganized the manuscript mainly by adding a new “Material and Method” section where discussions on branch length, statistical resampling, meaning and choice of K, etc., were given. The original Figure 1 was deleted with some related points explained in the text accompanying the original Figure 2. All figure captions have been rewritten for clarity.

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— Done Second Round of Evaluation Round 2: and Author Response

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life5010949_makarova

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A few more sentences were added in the “Conclusion” regarding the power and achievement of the 16S rRNA analysis.

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Results also provide additional support to recently proposed archaeal phyla and halobacterial orders.

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life5010949_makarova

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We tried to rephrase the paragraph by changing, deleting, or adding a few words as follows: “In this paper we study Archaea phylogeny across many phyla. This is in contrast with phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large, or, as Cavalier-Smith (citing cavalier-smith 2002) put it, with “megaclassificaton” of prokaryotes. Although in taxonomy the description of a newly discovered organism necessarily starts from the lower ranks, higher rank assignments are often incomplete or lacking. At present the ranks above class are not covered by the Bacteriological Code. The number of plausible microbial phyla may reach hundreds and archaeal ones are among the less studied. According to the 16S rRNA analysis, the major archaeal classes and their subordinate orders have been more or less delineated. Therefore, in order to carry out the aforementioned cross verification we make emphasis on higher ranks such as phyla, classes, and orders. A study using 179 Archaea genomes should provide a framework for further study of lower ranks.”

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This kind of research should be encouraged further because old taxonomic paradigms must be systematically reviewed based on new genomic data.

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life5010949_makarova

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Branching order in a tree is directly related to taxonomy, while branch lengths have more to do with evolution. For large-scale phylogenetic study across many phyla the former is more important than the calibration of branch lengths. The latter is based on the assumption that mutation rate is more or less constant. This assumption cannot hold when dealing with many phyla.

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— Done Second Round of Evaluation Round 2: and Author Response

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life5010949_makarova

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This is done in the newly added “Material and Method” section.

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If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic.”

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life5010949_makarova

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The original Figure 1 was deleted and a few words added to the legend of the original Figure 2, now the new Figure 1.

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Here monophyly must be understood in a pragmatic way restricted to the given set of input data and the reference taxonomy.

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life5010949_makarova

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Judging by the cluster labeled as Euryarchaeote{0+3} in Figure 2 Methanomassiliicoccus was not reclassified into Thermoplasmataceae but to an yet un-specified class.

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Though a dissimilarity measure figures in the CVTree algorithm, it is not realistic to delineate taxa by using this measure at least for the time being.

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life5010949_makarova

0