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This was described in Section 3.7.
| 2 | 1 |
Please clarify the source of the helical flow of the slime, which is most likely due to the presence of the not-preserved helically arranged cell surface proteins not the arrangement or tilt of the junctional pores.
| 1 | 2 |
life4040819_perova
| 1 |
This point was corrected.
| 2 | 1 |
In the model that describes the potential activity of the junctional pores (How myxobacteria glide. Curr. Biol. 2002, 12, 369–377) it is not suggested that the nozzle actually “contracts” as mentioned in the ms. The swelling of the slime material in the nozzle fills the nozzle and eventually will generate a counter-force of the nozzle walls of the nozzle that results in the ejection of the slime. Please rephrase the text accordingly.
| 1 | 2 |
life4040819_perova
| 1 |
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 |
When the cells form spirals, there are two components to this process: the formation of spirals and their macroscopic sense of rotation: clock- or counterclockwise. In the discussion it is assumed that the rotation of the filaments is crucial for both of these components. If this would be true, then no spirals should be observed in non-rotating filamentous gliding cyanobacteria. However, this is not the case. Anabaena spec. a non-rotating species can generate spirals (see Figure X; Mucilage secretion and the movements of blue-green-algae. Protoplasm 1968, 65, 223–238). This means that the sense of rotation may only be responsible for the direction of the spiral but not for its emergence in the first place. It would be good if the authors would distinguish between these two phenomena and discuss them accordingly.
| 1 | 2 |
life4040819_perova
| 1 |
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 |
In Lines 309–315: References 17 and 22 are accurately described, however these models relate to gliding in Myxobacteria, a distinction not noted in the text. Thus, comparisons between these models (including the focal adhesion model) and the current model are only valid if the mechanism of motility in these microorganisms is the same. The authors should (briefly) explain their reasoning on the validity of comparing these two systems, and the implications of their findings to motility in Myxobacteria.
| 1 | 2 |
life4040819_perova
| 1 |
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 |
Lines 94–95: Is it standard to sequence an entire genome, but only deposit several clusters, or should the entire genome be accessible to allow other researchers access to this source?
| 1 | 2 |
life4040819_perova
| 1 |
Lines 227–230: This was corrected.
| 2 | 1 |
Lines 227–230: The interpretation of the counterclockwise spirals here is somewhat distracting. It can be left as an observation here, and the model explained later.
| 1 | 2 |
life4040819_perova
| 1 |
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 |
Lines 316–317: This model for slime secretion suggests that rather than slime being comprised of a single, long, polysaccharide, it is made of smaller subunits that are secreted in a step-wise fashion. The authors should connect this idea to the finding that pseudopilins appear to be part of the molecular machinery, as the model and the data are coherent on this point.
| 1 | 2 |
life4040819_perova
| 1 |
The three references have been noticed with gratitude and included in the MS.
| 2 | 1 |
Section 1. The place of RNA in LUCA (page 2): In search of features that are more conserved (carrying deep phylogenetic memory) than the sequence of genes, Wächtershäuser focuses on a paper of his in Systematic and Applied Microbiology (1998) that uses gene content and order of microbial genomes to make inferences about the last universal common ancestor (LUCA) of cellular life. He then mines the significance of some of the conserved chromosomal segments in light of some other evidence. The exercise is at places compelling, but forgets some recent, very global and exhaustive analyses that also use highly conserved biological features to reconstruct the makeup of LUCA (e.g., gene order, 3D molecular structure, molecular functions). See for example PubMed references PMID: 17370266, PMID: 21612591, and PMID: 17908824, which are in line with some conclusions derived from the alignment of Figure 1. The fact that these other analyses make use of hundreds of genomes to infer the ancient biochemistry of LUCA complements and strengthens the preliminary and fragmentary analysis of only 19 of them by the author, which also excludes eukaryotic genomes from the set (understandably, few sequenced genomes were available in 1998, and eukaryotes are in general “master rearrangers”).
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
Given my interest in the bioinformatics of gene content and order, I took the liberty of studying the brief Systematic & Applied Microbiology paper to check the validity of the “reconstruction” methods of Figure 1. The algorithmic implementation that is described is quite raw and does not extract important information that is embedded in the clusters of conserved gene segments. Important algorithms have been devised since the initial work of Sankoff in the sixties and seventies to do exactly that. I refer to the work of Pevzner, Tesler and Bourque as good examples, but also of Warnow. I also recommend visiting GRIMM (http://grimm.ucsd.edu/GRIMM/) and perhaps using the server to confirm or extract additional information from the alignments that are summarized in Figure 1. The Wächtershäuser algorithm makes use of conserved elements of gene content and order but discards information provided by the actual rearrangement operations that erase gene order history. The algorithm does not describe how gene homology was detected, how the limited set of genes was selected, and how the alignments were constructed (I imagine by hand). It is not clear if a guiding tree was used in the alignment (though this is mentioned in Line 65), since this is not made explicit in the 1998 publication. An alignment implies a tree but usually alignment algorithms are greedy and problematic and represent the most important limiting step of a phylogeny (the field is thus moving to the joint alignment and tree reconstruction). Therefore, I do not think the ancestors were properly reconstructed (the tracing of features in ancestor nodes of trees are not described, nor the actual trees). Regardless of all of these limitations, the tight conservation of certain segments is enough to show the existence of a core of ribosomal proteins that is universally present in cellular organisms. This in itself is valuable. Of course, a sample of 19 microbes may not be enough to encompass molecular diversity and the absence of Eukarya may also be problematic for any global evolutionary statement. In other words, the risks of sampling bias are clearly present and should be mentioned in the manuscript.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
The list of common genes in the alignment of Table 1 is enriched in small and large subunit ribosomal proteins that are the most ancient of the ribosomal set, according to the Caetano-Anolles theorem that is mentioned later in the manuscript. Interestingly, the most ancient of them are clustered toward the 5’ end of the genomic sequences that were aligned (S12, S17, S5, S4 and L2, L3 and L24). Could this imply a possible ancient segmental duplication? Also interesting is the placement of the most ancient ribosomal protein S12 between exactly two polymerase (beta and beta prime) and elongation factor (entry and translocation) genes (separated by single and much more derived ribosomal proteins). Could this be an ancient memory of the ribosome mediating translation and replication? A commentary would enhance the value of the section.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
In terms of the genome organization of LUCA (Line 105), there are numerous arguments in favor of an RNA ancestral genome and the late unfolding of DNA as genetic repository (perhaps through viruses). I cannot understand how 2'-hydroxy groups could destabilize the molecule and lead to “intramolecular self-destruction” (Line 107). RNA has the wonderful property of folding in search of energetic and kinetic minima. These processes make use of a frustrated landscape, which is powerful in terms of its biological potential (stability, function, information). It is much more versatile than the rigid DNA alternative, which is enriched in the ‘information’ capacity. None of them have “self destruction” properties, which would have been weeded out by selection and self-organization very early in “chemical” evolution. In turn, everything about LUCA should be considered quite modern and far away from an initial FeS world. I refer to the work of Daniel Lundin and colleagues in Sweden about the rather late rise of some structures of the ribonuceotide reductase enzymes needed to built the DNA polymers. The Maurel theorem stands if one thinks of single genome molecules. But what if there were many short RNA genomes, as anticipated by Woese, each perhaps linked to different and ancient tRNA-like cofactors? As the author mentions, LUCA is contemporary to a rather complex biochemistry, with numerous structures harboring a multitude of active and allosteric sites in proteins, most of which drive central enzymatic functions.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
In Line 114, the role of chromosomal rearrangement in evolution is not well described. What are the effects of genome rearrangements? Several scenarios are possible, including: (1) Genes remain linked R4 probably because it is more efficient to transcribe genes that produce interacting proteins (part of complexes) than those that are not; (2) Genes remain linked if they are of relatively recent origin (rearrangement has not had the change to split them apart); (3) Genes remain linked if they are part of functional groups historically united by genomic regions (encoding metabolic functions or rRNA? ); this includes genes sharing an operon structure for economy purposes in highly reduced organisms such as bacteria; (4) Genes remain linked because they originated when genomic rearrangements were not biochemically motivated and their sequence makeup was later refractory to rearrangement hotspots. There is a rich literature about rearrangement, hotspots and many processes related to these (including domain organization in proteins). Some discussion in this front could be clarifying.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
In relation to comments of Line 188 onwards, Di Giulio, Caetano-Anolles and others have suggested that the genetic code started to unfold prior to LUCA but continue to do so once life diversified. The corollary, is that the complete canonical set of amino acids may have not been encoded in LUCA. In terms of metabolic and biochemical competency, there are numerous and interesting studies, which have not been cited (especially in origins and evolution of modern metabolism). This includes the coevolution theory of the genetic code, the coexistence of prebiotic chemistries with modern metabolic reactions (recently studied for nucleotide metabolism), and theories about the origin of translation.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
Section 2: Thermal course of evolution (Page 6): Why a focus on thermal energy? The framework should be on thermodynamics, energy dissipation and information, all of which are linked. What is thermally upward or downward adaptation? Is it conquering niches on Earth or a process involving molecular makeup? Vocabulary and definitions are murky, especially related to the links of environmental thermal fluctuations, energy of folding and stability of polymers. Subsection (1) must be rewritten to help the reader understand the ideas. Should all adaptations comply with maximizing energy R5 dissipation? How can this be reconciled with upward and downward trends? No references are provided despite the rich literature underscoring the controversial link between physics, information theory and biology. If the focus is conquering planetary niches, then perhaps reference the contrasting views and some of their proponents (deep sea versus surface; thermophilic versus mesophilic, etc.). Further elaboration of the Wolfenden theorem appears relevant and its connections to what is known about the origin and evolution of metabolism also of importance.
| 1 | 2 |
life4041050_makarova
| 1 |
The author expresses his gratitude for the two additional references, which have been included in the text.
| 2 | 1 |
Section 5. Place of RNA in the origin and early evolution of translation/Pre-translational coding of peptides. The view that is presented in this crucial segment posits a pre-translational mechanism (side-by-side tRNA mediated coding) that very much resembles the ribosomal entropic system, which could be very advanced. What if the pre-translational coding was assembly line-like and mediated by the aminoacyl-tRNA synthetases themselves? See PMID: 23991065 for one such alternative and a related previous model (PMID: 22210458). This would also match commentary of a possible early origin of non-ribosomal peptide synthesis machinery compared to that of the ribosome.
| 1 | 2 |
life4041050_makarova
| 1 |
The statement has been clarified.
| 2 | 1 |
Section 9. General overview. While the grand finale “Nothing in evolution makes sense except in the light of chemical predetermination” is impressive and summarizes the championing work of the author, chemical predetermination may apply to the very early stages of evolution and not to many of the stages described in the manuscript. How much chemical predetermination can there be with polymers as these explore a minute fraction of the space they make possible? In the enthalpic-entropic gradient that is proposed, there is also a gradient from “predetermination” to historical contingency. The boundaries of such a gradient is murky and the final statement may not apply to much of modern biochemistry, once proteins start to achieve stable complex structure and much earlier that the time of LUCA. In fact, the genome rearrangements that underlie Figure 1 are testament to the historical contingencies that were already at play in LUCA and not to chemical predetermination.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
Line 406. “LUCA protein cycle”. Why bring LUCA to a link with the proposed “protein cycle”. LUCA is just the last of a chain of ancestor of diversified life. If the protein cycle requires a coupling of the enthalpic activation of amino acids and the entropic biosynthetic action of a primordial ribosome, then perhaps it is better to call it “ribocellular protein cycle”, since this coupling marks the start of modern cells, not necessarily “modern diversified cells” (i.e., LUCA).
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
Please explain what do you mean with Kandler, Wolfenden etc. Theorem. Is a theorem Kandler’s theory or proposal?
| 1 | 2 |
life4041050_makarova
| 1 |
The relationship of His and Trp biosyntheses has been toned down.
| 2 | 1 |
Line 716: The biosyntheses of His and Trp are not so closely related. They only have in common that they use phosphoribosediphosphate as substrate and therefore share two related enzymes.
| 1 | 2 |
life4041050_makarova
| 1 |
Section 8 has been revised in order to address the issues involved in the last query.
| 2 | 1 |
In the iron sulfur world the surface metabolism prevents the diffusion of the fixed products into the ocean. But without compartmentation soluble intermediates would escape. Furthermore, the establishment of early bioenergetics based on electrochemical Na+ gradients would have been impossible.
| 1 | 2 |
life4041050_makarova
| 1 |
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 |
The manuscript of Eckert et al., submitted into the journal Life, concerns in principle again only one, but very important genetic criterion for evaluation of prokaryotic diversity. It should be a serious part of this complex evaluation. It is very important and useful and publication of the article is surely recommendable. However, the connection, coincidences and relations to other criteria should be better expressed.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
If we accept also strains and populations from literature or from databases to the evaluation, there is always questionable the taxonomic determination (scientific names) of various species and strains, which are based on the old nomenclature and morphology. The critical revision of identification of these strains and cited materials is necessary.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
It follows from the whole article that the authors accepted the names (and also concepts) of taxa (mostly genera) from older literature, without necessary criticism. There are included revised genera according to modern methods together with taxonomically very problematic groups. As examples is possible to mention that Aphanizomenon ovalisporum and Anabaena bergii are really very related taxa, but they were already re-classified in one special and separate genus Chrysosporum. On the other hand, the complex “Prochlorococcus/Synechococcus” is still very unclear and surely is distant from the typical genus Synechoccocus. etc.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
Numerous cited taxa (on the generic level) were already controlled and revised by most modern methods, and examples in the manuscript should be used especially and selected from these genera. It is true, that majority of the cited genera were already revised (Microcystis, Planktothrix, Aphanizomenon), but just these taxa are important as examples and should be preferred. From the presented results follows only the confirmation of the present methodological principles and a modern system. This fact should be emphasized.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
It is necessary to stress the fact that the numerical criteria (e.g., exactly limited percentage similarity) never are valid uniformly for large groups of organisms and cannot be applied as one main criterion in large groups.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
As concerns the delimitation of taxa on the level of cyanobacterial species, there exists a lot of valuable literature, which was not cited. The characterization and separation of species was not satisfactory solved, but it is particularly important (criteria are not unique for all genera) and need better discussion.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
In Line 40 is cited only DeQueiroz (2007) to the problematic of species concept. It would be useful to evaluate and mention many other authors, who discussed this question more complexly. It concerns also several other problems, e.g., problematics of horizontal gene transfers (Line 43), “bounderies between taxon units” (Line 45), species concept (Line 48), etc. About species concept in cyanobacteria exist particularly many studies from last years.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
I am not sure that cyanobacteria can be accepted as a typical example of all prokaryotes. On the contrary, they have very special position (phylogenetic, metabolic, function in nature, ultrastructural, they grow often in multicellular and differentiated thallus) and their diversity must be evaluated respecting these specificities. It is clear that the taxonomic classification must be different in different groups of organisms.
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life5010050_makarova
| 1 |
We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.
| 2 | 1 |
The correct citation of scientific names is the obvious request of serious scientific publications. Few unnecessary mistakes are in the manuscript, e.g., in the whole text and also in Figures is written “Arthospira” instead the correct Arthrospira, on other places must be Leptolyngbya (Table 1, Line 191), Fischerella (Line 207), Aphanizomenon (Line 218), Cylindrospermopsis (Line 219).
| 1 | 2 |
life5010050_makarova
| 1 |
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 do not understand why Leptolyngbya and Chamaesiphon could be “closely related genera”. It is in contraversion to the whole up to date results and taxonomic classification. The authors really found the “close” relation just between these two genera? By way, they both are polyphyletic and will be surely divided in several different taxa after the following precise studies.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
It is overall well-written and innovative work, but there are few things to consider in methods and results, which should be revised, and some formal flaws. When you refer to the Synechococcus, it is important to specify, which clade you have in mind. Honda et al. (1999) and Roberston et al. (2001) showed Synechococcus is polyphyletic genus composed of a least 5 lineages. There are more recent works showing even more clades. From an amount of sequences and relationship to Prochlorococcus, I can assume, it is a marine pelagial picoplanktic clade. However, it should be specified for unexperienced reader in the field of cyanobacteria. The other polyphyletic genera should be also specified—Phormidium, Leptolyngbya, Microcoleus and others. Moreover, although the sequences might have same name (i.e., Synechococcus), they belong to polyphyletic groups, thus they probably belong to different genera, which have not yet been described.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
They might also appear to be monophyletic, because there are missing taxa between them.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
This might also cause false positive barcoding gaps, because there would be low similarity among sequences with same name. Therefore it may largely affect results.
| 1 | 2 |
life5010050_makarova
| 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. 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 |
I suggest that the paper should be expanded with a discussion, whether barcode gaps are able to delimit described species. For example, are there two species within Cylindrospermopsis? There seems to be a barcoding gap within this clade. In this particular case, it would be beneficial to use a species epithet too to avoid confusion. Is it Cylindrospermopsis raciborskii? Does a barcoding gap in the Planktothrix clade correspond to the described species?
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
Why did you use Phormidium as an outgroup (Line 139)? Would not be more beneficial to use Gloeobacter or some other bacterium?
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
Is there any way to use barcoding gaps in a taxonomy of cyanobacteria? This might be added to discussion.
| 1 | 2 |
life5010050_makarova
| 1 |
We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.
| 2 | 1 |
Minor point: There are errors in nomenclature throughout the text, figures, and supplements, e.g. Leptolyngbya, Aphanizomenon, Arthrospira. You might check: http://www.cyanodb.cz/. It is an updated database of names of cyanobacteria.
| 1 | 2 |
life5010050_makarova
| 1 |
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 |
The manuscript of Eckert et al., submitted into the journal Life, concerns in principle again only one, but very important genetic criterion for evaluation of prokaryotic diversity. It should be a serious part of this complex evaluation. It is very important and useful and publication of the article is surely recommendable. However, the connection, coincidences and relations to other criteria should be better expressed.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
If we accept also strains and populations from literature or from databases to the evaluation, there is always questionable the taxonomic determination (scientific names) of various species and strains, which are based on the old nomenclature and morphology. The critical revision of identification of these strains and cited materials is necessary.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
It follows from the whole article that the authors accepted the names (and also concepts) of taxa (mostly genera) from older literature, without necessary criticism. There are included revised genera according to modern methods together with taxonomically very problematic groups. As examples is possible to mention that Aphanizomenon ovalisporum and Anabaena bergii are really very related taxa, but they were already re-classified in one special and separate genus Chrysosporum. On the other hand, the complex “Prochlorococcus/Synechococcus” is still very unclear and surely is distant from the typical genus Synechoccocus. etc.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
Numerous cited taxa (on the generic level) were already controlled and revised by most modern methods, and examples in the manuscript should be used especially and selected from these genera. It is true, that majority of the cited genera were already revised (Microcystis, Planktothrix, Aphanizomenon), but just these taxa are important as examples and should be preferred. From the presented results follows only the confirmation of the present methodological principles and a modern system. This fact should be emphasized.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
It is necessary to stress the fact that the numerical criteria (e.g., exactly limited percentage similarity) never are valid uniformly for large groups of organisms and cannot be applied as one main criterion in large groups.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
As concerns the delimitation of taxa on the level of cyanobacterial species, there exists a lot of valuable literature, which was not cited. The characterization and separation of species was not satisfactory solved, but it is particularly important (criteria are not unique for all genera) and need better discussion.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
In Line 40 is cited only DeQueiroz (2007) to the problematic of species concept. It would be useful to evaluate and mention many other authors, who discussed this question more complexly. It concerns also several other problems, e.g., problematics of horizontal gene transfers (Line 43), “bounderies between taxon units” (Line 45), species concept (Line 48), etc. About species concept in cyanobacteria exist particularly many studies from last years.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
I am not sure that cyanobacteria can be accepted as a typical example of all prokaryotes. On the contrary, they have very special position (phylogenetic, metabolic, function in nature, ultrastructural, they grow often in multicellular and differentiated thallus) and their diversity must be evaluated respecting these specificities. It is clear that the taxonomic classification must be different in different groups of organisms.
| 1 | 2 |
life5010050_perova
| 1 |
We apologize for our carelessness in double-checking the naming of the organisms and hope that we now eliminated all errors.
| 2 | 1 |
The correct citation of scientific names is the obvious request of serious scientific publications. Few unnecessary mistakes are in the manuscript, e.g., in the whole text and also in Figures is written “Arthospira” instead the correct Arthrospira, on other places must be Leptolyngbya (Table 1, Line 191), Fischerella (Line 207), Aphanizomenon (Line 218), Cylindrospermopsis (Line 219).
| 1 | 2 |
life5010050_perova
| 1 |
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 do not understand why Leptolyngbya and Chamaesiphon could be “closely related genera”. It is in contraversion to the whole up to date results and taxonomic classification. The authors really found the “close” relation just between these two genera? By way, they both are polyphyletic and will be surely divided in several different taxa after the following precise studies.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
When you refer to the Synechococcus, it is important to specify, which clade you have in mind. Honda et al. (1999) and Roberston et al. (2001) showed Synechococcus is polyphyletic genus composed of a least 5 lineages. There are more recent works showing even more clades. From an amount of sequences and relationship to Prochlorococcus, I can assume, it is a marine pelagial picoplanktic clade. However, it should be specified for unexperienced reader in the field of cyanobacteria. The other polyphyletic genera should be also specified—Phormidium, Leptolyngbya, Microcoleus and others. Moreover, although the sequences might have same name (i.e., Synechococcus), they belong to polyphyletic groups, thus they probably belong to different genera, which have not yet been described.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
They might also appear to be monophyletic, because there are missing taxa between them.
| 1 | 2 |
life5010050_perova
| 1 |
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 |
This might also cause false positive barcoding gaps, because there would be low similarity among sequences with same name. Therefore it may largely affect results.
| 1 | 2 |
life5010050_perova
| 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. 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 |
I suggest that the paper should be expanded with a discussion, whether barcode gaps are able to delimit described species. For example, are there two species within Cylindrospermopsis? There seems to be a barcoding gap within this clade. In this particular case, it would be beneficial to use a species epithet too to avoid confusion. Is it Cylindrospermopsis raciborskii? Does a barcoding gap in the Planktothrix clade correspond to the described species?
| 1 | 2 |
life5010050_perova
| 1 |
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 |
Why did you use Phormidium as an outgroup (Line 139)? Would not be more beneficial to use Gloeobacter or some other bacterium?
| 1 | 2 |
life5010050_perova
| 1 |
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 |
Is there any way to use barcoding gaps in a taxonomy of cyanobacteria? This might be added to discussion.
| 1 | 2 |
life5010050_perova
| 1 |
We apologize for our carelessness in double checking the naming of the organisms and hope that we now eliminated all errors.
| 2 | 1 |
There are errors in nomenclature throughout the text, figures, and supplements, e.g. Leptolyngbya, Aphanizomenon, Arthrospira. You might check: http://www.cyanodb.cz/. It is an updated database of names of cyanobacteria.
| 1 | 2 |
life5010050_perova
| 1 |
This review does not require any action on our part.
| 2 | 1 |
Overall a beautifully written, wonderfully presented, paper with clear concepts, methods and proposals that significantly contribute to original knowledge in the field.
| 1 | 2 |
life5010181_perova
| 1 |
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 |
The authors write: “In these initial stages, the emergence of bistability will serve as a proxy for the emergence of other chemical novelties in the vesicle reactor model.” The authors probably means that the existence of more than one stable regime could consent regime changes, but this situation do not indicates how these changes appear: in the article, the change is stimulated by means of a deliberated injection of chemical substances from outside. So, I think that the appearance of novelties requires a different order of considerations, whereas the bistability plays possibly the role of novelties amplification and consolidation (in particular conditions). The same observation can be done for the word “innovation”, used on the abstract and at Line 45. In order to avoid misunderstandings, I think that the authors should avoid these expressions, ad use the words “regime change”, or similar concepts.
| 1 | 2 |
life5010181_perova
| 1 |
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 |
Note 4 presents a very important issue: why the inner chemical environment is different from the external one, if the running chemical reactions are the same? The presence of particular features of the vesicle interior or of entrapped catalysts seem a too easy and ad hoc hypothesis. A useful reference proposing an explanation of this symmetry breaking (without requiring different conditions in internal and external environments) could be “R. Serra, M. Villani Mechanism for the formation of density gradients through semipermeable membranes Physical Review E 87, 2013”.
| 1 | 2 |
life5010181_perova
| 1 |
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 |
How the authors derive the range [0.9, 101/3] for the so called “reduced surface” index? In Figure 1d, Lines 21/3 and 41/3 have some particular meanings, or are plotted mainly to ease the figure comprehension? Could the authors add some indications about the derivation of these upper limits?
| 1 | 2 |
life5010181_perova
| 1 |
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 |
The authors write (case 1): “of the 5000 parameter set tested under constant surface area, 82% (4098) gave a single fixed point, 321 15.7% (785) gave two fixed points, and 2.3% (117) no fixed points.” How this statistics compare with a similar one, performed on a similar chemical situation without container? Could the authors add the results of the same within a not confined environment?
| 1 | 2 |
life5010181_perova
| 1 |
The minor issues were fixed.
| 2 | 1 |
Adamala, K.; Szostak, J. ompetition betweenmodel protocells driven by an encapsulated catalyst. Nat. Chem. 2013, 5, 495–501. → “ompetition” should be: “Competition”.
| 1 | 2 |
life5010181_perova
| 1 |
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 data are interesting and worth publishing but… (1) I don’t understand how they calculated the protein molar ratios. In the methodology section we learn that they extracted the crude proteins and then performed western analyses using specific antibodies, followed by image analyses. I don’t understand how they converted the western data to read the amount of specific proteins, in fmol, in the various organisms as shown in Figure 2; and used for the calculations in the rest of the paper. (Why are the units provided again in Line 220?). Please provide the reader with the methodology used for the quantification.
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
PSII activity is derived from fluorescence measurement, as ETR. I am missing calibration with real measurements of PSII activity such as O2 evolution. You have got to show it for each organism you are examining otherwise it is worthless. There are many reports in the literature showing dramatic decline in fluorescence but hardly any change in O2 evolution.
| 1 | 2 |
life5010403_makarova
| 1 |
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 cells were grown under a relatively low light intensity and thus the data presented here only apply to these conditions. Please make sure the reader is aware of it.
| 1 | 2 |
life5010403_makarova
| 1 |
We have removed the word “nondiazotrophic” as we agree that it is not relevant to the discussion.
| 2 | 1 |
Line 19 – I am not sure why stating the cyanobacteria are nondiazatrophic is relevant?
| 1 | 2 |
life5010403_makarova
| 1 |
We mean that they numerically dominate and we have clarified this in the introduction.
| 2 | 1 |
Line 46 – (and throughout) be clear if you mean numerically dominate or dominate production – they are not always the same thing.
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
Introduction paragraph 2 – I am unsure we know that physbobilisomes require more resource than pcb proteins – this depends on the ratio of antenna to reaction centre? Please include references if R3 this has been shown. Also, not all Pcb proteins are constitutively expressed (e.g., see Bibby et al. Nature 2003).
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
Line 250 – This is estimated number of RUBISCO active sites – or is there evidence all the RUBSCIO you quantify is active?
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
Figure 4 – The crunch of this paper is Figure 4e which show the Rubisco:PSII ratio is correlated to ETR. While I’m convinced in the presented data only three data-points are shown – I’m most convinced in that this same relationship is presented in in “Aquatic Photosynthesis (edition 2) Figure 7.9 – Falkowski” based on work of Sukenik (1986?). A greater discussion should be given in the text to how these datasets compare and what is significantly new in this dataset? References 35 and 36 are discussed in relation to the potential control of cytb6f on limiting electron transfer from PSII but more should be made of Falkowski’s observations which support this data.
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
Line 308 – The data presented is used to support potential cyclic electron flow around PSI – however other alternative terminal electron sinks could also be up-regulated – these concepts are discussed in a recent review by Milligan and Behrenfeld annual review 2013 – the implications of this should be discussed unless the authors can show specifically enhanced flow around PSI.
| 1 | 2 |
life5010403_makarova
| 1 |
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 |
“In line 178 onward we read about the calibration curve. Please presnt it in the paper. Once you apply stress such as iron starvation you should recalibrate.” R4 Response:
| 3 | 2 |
life5010403_makarova
| 1 |
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 |
“Please present the calibration curves for the protein levels.” Response:
| 3 | 2 |
life5010403_makarova
| 1 |
p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.
| 2 | 1 |
In Figure 3, formate formula should be better symbolized as HCOO- instead of COOH- as indicated.
| 1 | 2 |
life5010432_makarova
| 1 |
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 |
Lines 59–60: Instead of links, a figure (may be included in supplementary document) will be more helpful. The links lead to a non-English website, and also they may become outdated in year or two.
| 1 | 2 |
life5010432_makarova
| 1 |
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 |
Line 60: Link to NO2− concentrations shows temperature profiles.
| 1 | 2 |
life5010432_makarova
| 1 |
p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.
| 2 | 1 |
Line 118: Was the analysis done on all FNT-proteins available at NCBI database? Please clarify.
| 1 | 2 |
life5010432_makarova
| 1 |
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 |
Line 40: Line 120: From the tree as it is, one can see that NitM from β-cyanobacteria form a distinct clade. However, NitM from α‐cyanobacteria are not grouped in one clade. It is possible that another outgroup sequence or different methods for alignment and tree construction will help. Line 122: What methods were used for creating alignment and phylogenetic tree (for example, neighbor-joining)?
| 1 | 2 |
life5010432_makarova
| 1 |
Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.
| 2 | 1 |
Was formate inhibition test also done on strains with nitM from CC9311, CC9605 and MIT9313?
| 1 | 2 |
life5010432_makarova
| 1 |
We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.
| 2 | 1 |
It would be especially exciting to see the uptake of nitrite at environmentally relevant concentrations (less than 50 nM)? Considering the genetic constructs available, I was wondering if the authors have attempted such experiments and what were the results.
| 1 | 2 |
life5010432_makarova
| 1 |
p. 7, Figure 3, formate formula has been changed to “HCOO-” from “COOH-”.
| 2 | 1 |
In Figure 3, formate formula should be better symbolized as HCOO- instead of COOH- as indicated.
| 1 | 2 |
life5010432_perova
| 1 |
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 |
Lines 59–60: Instead of links, a figure (may be included in supplementary document) will be more helpful. The links lead to a non-English website, and also they may become outdated in year or two.
| 1 | 2 |
life5010432_perova
| 1 |
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 |
Line 60: Link to NO2− concentrations shows temperature profiles.
| 1 | 2 |
life5010432_perova
| 1 |
p. 4, line 16; Six NitM proteins registered recently were added to Figure 1 and Figure 5.
| 2 | 1 |
Line 118: Was the analysis done on all FNT-proteins available at NCBI database? Please clarify.
| 1 | 2 |
life5010432_perova
| 1 |
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 |
Line 40: Line 120: From the tree as it is, one can see that NitM from β-cyanobacteria form a distinct clade. However, NitM from α‐cyanobacteria are not grouped in one clade. It is possible that another outgroup sequence or different methods for alignment and tree construction will help. Line 122: What methods were used for creating alignment and phylogenetic tree (for example, neighbor-joining)?
| 1 | 2 |
life5010432_perova
| 1 |
Formate inhibition experiments of the NitM from CC9311, CC9605 and MIT9313 have not been carried out yet.
| 2 | 1 |
Was formate inhibition test also done on strains with nitM from CC9311, CC9605 and MIT9313?
| 1 | 2 |
life5010432_perova
| 1 |
We will consider the nitrite uptake experiments at environmentally relevant concentrations of nitrite (~50 nM) in the future.
| 2 | 1 |
It would be especially exciting to see the uptake of nitrite at environmentally relevant concentrations (less than 50 nM)? Considering the genetic constructs available, I was wondering if the authors have attempted such experiments and what were the results.
| 1 | 2 |
life5010432_perova
| 1 |
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 |
I think the alignment-free methodology is interesting, however I would like to see a comparison with at least one regular alignment/treeing method, based on the same genomes the authors used, and not just a visual topological comparison with other published trees.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
As far as I can see, statistical support on the branches is missing, so I have no way of assessing if this branching order is valid. Bootstrapping or jackknifing are by no means the final word on the significance of branches, however an explanation as to how the user should assess the significance of braches would be good, i.e., branch length.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
The fact that Thermofilum is placed outside Crenarchaeota in Figures 3 and 4 is a little disturbing. I haven’t come across such a placement in other phylogenetic analyses of Archaea, for example in Brochier-Armanet et al 2008 Nat Rev Microb, or Rinke et al. 2013 Nature. I believe this needs to better explained in the manuscript, rather than just saying “this fact is noted”.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
I am also not very convinced with the placement of Nanoarchaeota. It seems like this phylum is moving around with the addition of new sequence data (for example in Rinke et al. Figure 2 tree, they are on an entirely different branch than Euryarchaeota). Though the authors also rightfully point out that the reduced genome size may have something to do with this placement.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
The placement of Halobacteria (due to interfering Nanoarchaeota, I presume) is also a little disturbing. I would recommend that the authors provide a discussion of this. Especially with regards to other archaeal trees. For instance, in the tree of Armanet et al 2011 that the authors also refer to, the placement of Halobacteria with respect to Nanoarchaeota is very different.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
In summary, I find the piece interesting, but parts of the discussion are rather weak, therefore I am suggesting a major revision. Another reason for major revision is the style that the manuscript is written. I am not a native speaker, but given that I had to read sentences several times, I suspect the manuscript can benefit from an English language check.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
I found the revised version of this manuscript quite good, and I thank the authors for responding thoroughly to all my comments.
| 3 | 2 |
life5010949_makarova
| 1 |
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 |
I would really appreciate a “Methods” part where the CVTree is explained shortly, and the tree-viewer is explained with more detail. The absence of branch lengths and bootstraps should be discussed here. Other technical aspects of the paper (e.g., sequence dataset), parameters, criteria ... all could be well organized in this part.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
Archaeal phylogeny has already been studied in detail, with 16S and other marker genes, and with genomic approaches too. Some of the undersigning authors had already published on this before, although with smaller input datasets. Therefore, the fact that 16S topology is quite stable and comparable with other approaches is already known.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
Taxonomists have traditionally circumscribed the high taxa (specially orders and classes) with great subjectivity, i.e., without well accepted criteria. In terms of phylogenetic trees one premise has always been clear, a taxon must be monophyletic. This principle has been used in the present work to reconsider the status of some high taxa. However, authors do not explain objective criteria to properly interpret the rank of the clades, which impedes making a profound evaluation of the archaeal classification. Therefore, although authors have strong tools and dataset, they just achieved a small revision of the high taxa which is, indeed, quite biased by the underlying 16S guidelines.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
I have noted some lack of scientific rigor according to: many wrong taxonomic names and typos, scarce figure legends, few comments about the missing branch lengths or bootstraps (! ), redundancy in text and figures and fragments which are really difficult to understand. Authors should pay attention to language, explanations and text organization.
| 1 | 2 |
life5010949_makarova
| 1 |
A few more sentences were added in the “Conclusion” regarding the power and achievement of the 16S rRNA analysis.
| 2 | 1 |
Authors shouldn't forget (particularly in conclusion) that the resolution power of 16S for high ranks (genus and above) is currently well accepted. And the number of non-redundant 16S entries available is much much larger than that of archaeal genomes. 16s data offer a much comprehensive view of the archaeal diversity, including deep branches.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
- L40 “Since at present (…) are not covered (...)” is a weak reason for choosing high ranks. I recommend to shortly summarize why high ranks are so important, and why do you choose order as the lowest considered rank.
| 1 | 2 |
life5010949_makarova
| 1 |
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 |
Part 3.1 The figure legends require more rigorous explanation. Would be interesting to remember that the branch lengths are not taken into account, or were omitted.
| 1 | 2 |
life5010949_makarova
| 1 |
This is done in the newly added “Material and Method” section.
| 2 | 1 |
In Line 80—authors write some explanations to understand the tree figures. I would suggest to put this text before the first tree figure.
| 1 | 2 |
life5010949_makarova
| 1 |
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